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Sample records for col4a5 mutation detection

  1. Mutation analysis of COL4A3 and COL4A4 genes in a Chinese

    Indian Academy of Sciences (India)

    Autosomal dominant Alport syndrome (ADAS) accounts for 5% of all cases of Alport syndrome (AS), a primary basement membrane disorder arising from mutations in genes encoding the type IV collagen protein family.Mutationsin COL4A3 and COL4A4 genes were reported to be associated with ADAS. In this study, clinical ...

  2. Detection of mutations in the COL4A5 gene by SSCP in X-linked Alport syndrome

    DEFF Research Database (Denmark)

    Hertz, Jens Michael; Juncker, I; Persson, U

    2001-01-01

    , three in-frame deletions, four nonsense mutations, and six splice site mutations. Twenty-two of the mutations have not previously been reported. Furthermore, we found one non-pathogenic amino acid substitution, one rare variant in a non-coding region, and one polymorphism with a heterozygosity of 28...... of type IV-collagen. We performed mutation analysis of the COL4A5 gene by PCR-SSCP analysis of each of the 51 exons with flanking intronic sequences in 81 patients suspected of X-linked Alport syndrome including 29 clear X-linked cases, 37 cases from families with a pedigree compatible with X...

  3. Mutation analysis of COL4A3 and COL4A4 genes in a Chinese ...

    Indian Academy of Sciences (India)

    2016-10-24

    ,. People's Republic of China ... mutations in COL4A3 and COL4A4 genes which encode type IV collagen α3 and α4 chainsrespectively can .... hematuria and evidence for activation of the unfolded protein response. Focal and ...

  4. Childhood presentation of COL4A1 mutations

    NARCIS (Netherlands)

    Shah, S.; Ellard, S.; Kneen, R.; Lim, M.; Osborne, N.; Rankin, J.; Stoodley, N.; van der Knaap, M.S.; Whitney, A.; Jardine, P.

    2012-01-01

    Aim To describe the clinical and radiological features of four new families with a childhood presentation of COL4A1 mutation. Method We retrospectively reviewed the clinical presentation. Investigations included radiological findings and COL4A1 mutation analysis of the four cases. Affected family

  5. Childhood presentation of COL4A1 mutations

    NARCIS (Netherlands)

    Shah, Siddharth; Ellard, Sian; Kneen, Rachel; Lim, Ming; Osborne, Nigel; Rankin, Julia; Stoodley, Neil; van der Knaap, Marjo; Whitney, Andrea; Jardine, Philip

    2012-01-01

    To describe the clinical and radiological features of four new families with a childhood presentation of COL4A1 mutation. We retrospectively reviewed the clinical presentation. Investigations included radiological findings and COL4A1 mutation analysis of the four cases. Affected family members were

  6. Col4a1 mutations cause progressive retinal neovascular defects and retinopathy.

    Science.gov (United States)

    Alavi, Marcel V; Mao, Mao; Pawlikowski, Bradley T; Kvezereli, Manana; Duncan, Jacque L; Libby, Richard T; John, Simon W M; Gould, Douglas B

    2016-01-27

    Mutations in collagen, type IV, alpha 1 (COL4A1), a major component of basement membranes, cause multisystem disorders in humans and mice. In the eye, these include anterior segment dysgenesis, optic nerve hypoplasia and retinal vascular tortuosity. Here we investigate the retinal pathology in mice carrying dominant-negative Col4a1 mutations. To this end, we examined retinas longitudinally in vivo using fluorescein angiography, funduscopy and optical coherence tomography. We assessed retinal function by electroretinography and studied the retinal ultrastructural pathology. Retinal examinations revealed serous chorioretinopathy, retinal hemorrhages, fibrosis or signs of pathogenic angiogenesis with chorioretinal anastomosis in up to approximately 90% of Col4a1 mutant eyes depending on age and the specific mutation. To identify the cell-type responsible for pathogenesis we generated a conditional Col4a1 mutation and determined that primary vascular defects underlie Col4a1-associated retinopathy. We also found focal activation of Müller cells and increased expression of pro-angiogenic factors in retinas from Col4a1(+/Δex41)mice. Together, our findings suggest that patients with COL4A1 and COL4A2 mutations may be at elevated risk of retinal hemorrhages and that retinal examinations may be useful for identifying patients with COL4A1 and COL4A2 mutations who are also at elevated risk of hemorrhagic strokes.

  7. Severe Hemolytic Jaundice in a Neonate with a Novel COL4A1 Mutation.

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    Tomotaki, Seiichi; Mizumoto, Hiroshi; Hamabata, Takayuki; Kumakura, Akira; Shiota, Mitsutaka; Arai, Hiroshi; Haginoya, Kazuhiro; Hata, Daisuke

    2016-12-01

    We report our experience with a preterm infant with severe hemolytic jaundice who required exchange transfusion just after birth. The patient was negative for alloimmune hemolysis as a result of maternal-fetal blood type incompatibility, and tests for inherited defects in erythrocyte metabolism, membrane function, and hemoglobin synthesis were normal. We also performed a bone marrow examination, but could not identify the cause of hemolysis. The patient had several other complications, including porencephaly, epilepsy, elevated serum levels of creatine kinase, and persistent microscopic hematuria. Later, we detected a genetic mutation in COL4A1, which was recently found to be associated with hemolytic anemia. We therefore believe that all of the patient's clinical features, including hemolytic anemia, were due to the mutation in COL4A1. Genetic testing for COL4A1 mutations is recommended in neonates who exhibit hemolytic disease of unknown etiology, especially when other complications compatible with COL4A1-related disorders are present. Copyright © 2014. Published by Elsevier B.V.

  8. A novel COL4A3 mutation causes autosomal-recessive Alport syndrome in a large Turkish family.

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    Uzak, Asli Subasioglu; Tokgoz, Bulent; Dundar, Munis; Tekin, Mustafa

    2013-03-01

    Alport syndrome (AS) is a genetically heterogeneous disorder that is characterized by hematuria, progressive renal failure typically resulting in end-stage renal disease, sensorineural hearing loss, and variable ocular abnormalities. Only 15% of cases with AS are autosomal recessive and are caused by mutations in the COL4A3 or COL4A4 genes, encoding type IV collagen. Clinical data in a large consanguineous family with four affected members were reviewed, and genomic DNA was extracted. For mapping, 15 microsatellite markers flanking COL4A3, COL4A4, and COL4A5 in 16 family members were typed. For mutation screening, all coding exons of COL4A3 were polymerase chain reaction- amplified and Sanger-sequenced from genomic DNA. The disease locus was mapped to chromosome 2q36.3, where COL4A3 and COL4A4 reside. Sanger sequencing revealed a novel mis-sense mutation (c.2T>C; p.M1T) in exon 1 of COL4A3. The identified nucleotide change was not found in 100 healthy ethnicity-matched controls via Sanger sequencing. We present a large consanguineous Turkish family with AS that was found to have a COL4A3 mutation as the cause of the disease. Although the relationship between the various genotypes and phenotypes in AS has not been fully elucidated, detailed clinical and molecular analyses are helpful for providing data to be used in genetic counseling. It is important to identify new mutations to clarify their clinical importance, to assess the prognosis of the disease, and to avoid renal biopsy for final diagnosis.

  9. Alport Syndrome: De Novo Mutation in the COL4A5 Gene Converting Glycine 1205 to Valine

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    Pilar Antón-Martín

    2012-01-01

    Full Text Available Background Alport syndrome is a primary basement membrane disorder arising from mutations in genes encoding the type IV collagen protein family. It is a genetically heterogeneous disease with different mutations and forms of inheritance that presents with renal affection, hearing loss and eye defects. Several new mutations related to X-linked forms have been previously determined. Methods We report the case of a 12 years old male and his family diagnosed with Alport syndrome after genetic analysis was performed. Result Anew mutation determining a nucleotide change C.3614G > T (p. Gly1205Val in hemizygosis in the COL4A5 gene was found. This molecular defect has not been previously described. Conclusion Molecular biology has helped us to comprehend the mechanisms of pathophysiology in Alport syndrome. Genetic analysis provides the only conclusive diagnosis of the disorder at the moment. Our contribution with a new mutation further supports the need of more sophisticated molecular methods to increase the mutation detection rates with lower costs and less time.

  10. Fetal origin of brain damage in 2 infants with a COL4A1 mutation: fetal and neonatal MRI

    NARCIS (Netherlands)

    Vermeulen, R. J.; Peeters-Scholte, C.; van Vugt, J. J. M.; van Vught, J. J. M. G.; Barkhof, F.; Rizzu, P.; van der Schoor, S. R. D.; van der Knaap, M. S.

    2011-01-01

    Mutations in the gene COL4A1, encoding collagen IV A1, are associated with familial porencephaly. Previously, COL4A1 mutation-associated antenatal hemorrhages have been suggested by early post-natal imaging. We describe 2 children with fetal intracerebral hemorrhages and a COL4A1 mutation. There was

  11. Mutation Analysis of COL1A1 and COL1A2 in Fetuses with Osteogenesis Imperfecta Type II/III.

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    Wang, Wenbo; Wu, Qichang; Cao, Lin; Sun, Li; Xu, Yasong; Guo, Qiwei

    2015-01-27

    Aim: To analyze COL1A1/2 mutations in prenatal-onset OI for determine the proportion of mutations in type I collagen genes among prenatal onset OI and to provide additional data for genotype-phenotype analyses. Material and Methods: Ten cases of severe fetal short-limb dwarfism detected by antenatal ultrasonography were referred to our center. Before the termination of pregnancy, cordocentesis was performed for fetal karyotype and COL1A1/2 gene sequencing analysis. Postmortem radiographic examination was performed at all instances for definitive diagnosis. Results: COL1A1 and COL1A2 SNP and mutations were identified in all the cases. Among these, one synonymous SNP and four synonymous SNPs were recognized in COL1A1/2, respectively, seven cases have distinct heterozygous mutations and six new COL1A1/2 gene mutations were identified. Conclusion: There has been substantial progress in the identification of the molecular defects responsible for skeletal dysplasias. With the constant increase in the number of identified mutations in COL1A1 and COL1A2, genotype-phenotype correlation is becoming increasingly pertinent. © 2015 S. Karger AG, Basel.

  12. Mutational analysis of COL1A1 and COL1A2 genes among Estonian osteogenesis imperfecta patients.

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    Zhytnik, Lidiia; Maasalu, Katre; Reimann, Ene; Prans, Ele; Kõks, Sulev; Märtson, Aare

    2017-08-15

    Osteogenesis imperfecta (OI) is a rare bone disorder. In 90% of cases, OI is caused by mutations in the COL1A1/2 genes, which code procollagen α1 and α2 chains. The main aim of the current research was to identify the mutational spectrum of COL1A1/2 genes in Estonian patients. The small population size of Estonia provides a unique chance to explore the collagen I mutational profile of 100% of OI families in the country. We performed mutational analysis of peripheral blood gDNA of 30 unrelated Estonian OI patients using Sanger sequencing of COL1A1 and COL1A2 genes, including all intron-exon junctions and 5'UTR and 3'UTR regions, to identify causative OI mutations. We identified COL1A1/2 mutations in 86.67% of patients (26/30). 76.92% of discovered mutations were located in the COL1A1 (n = 20) and 23.08% in the COL1A2 (n = 6) gene. Half of the COL1A1/2 mutations appeared to be novel. The percentage of quantitative COL1A1/2 mutations was 69.23%. Glycine substitution with serine was the most prevalent among missense mutations. All qualitative mutations were situated in the chain domain of pro-α1/2 chains. Our study shows that among the Estonian OI population, the range of collagen I mutations is quite high, which agrees with other described OI cohorts of Northern Europe. The Estonian OI cohort differs due to the high number of quantitative variants and simple missense variants, which are mostly Gly to Ser substitutions and do not extend the chain domain of COL1A1/2 products.

  13. ER stress and basement membrane defects combine to cause glomerular and tubular renal disease resulting from Col4a1 mutations in mice

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    Frances E. Jones

    2016-02-01

    Full Text Available Collagen IV is a major component of basement membranes, and mutations in COL4A1, which encodes collagen IV alpha chain 1, cause a multisystemic disease encompassing cerebrovascular, eye and kidney defects. However, COL4A1 renal disease remains poorly characterized and its pathomolecular mechanisms are unknown. We show that Col4a1 mutations in mice cause hypotension and renal disease, including proteinuria and defects in Bowman's capsule and the glomerular basement membrane, indicating a role for Col4a1 in glomerular filtration. Impaired sodium reabsorption in the loop of Henle and distal nephron despite elevated aldosterone levels indicates that tubular defects contribute to the hypotension, highlighting a novel role for the basement membrane in vascular homeostasis by modulation of the tubular response to aldosterone. Col4a1 mutations also cause diabetes insipidus, whereby the tubular defects lead to polyuria associated with medullary atrophy and a subsequent reduction in the ability to upregulate aquaporin 2 and concentrate urine. Moreover, haematuria, haemorrhage and vascular basement membrane defects confirm an important vascular component. Interestingly, although structural and compositional basement membrane defects occurred in the glomerulus and Bowman's capsule, no tubular basement membrane defects were detected. By contrast, medullary atrophy was associated with chronic ER stress, providing evidence for cell-type-dependent molecular mechanisms of Col4a1 mutations. These data show that both basement membrane defects and ER stress contribute to Col4a1 renal disease, which has important implications for the development of treatment strategies for collagenopathies.

  14. Case of Small Vessel Disease Associated with COL4A1 Mutations following Trauma

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    Joao McONeil Plancher

    2015-06-01

    Full Text Available With this case report, we would like to heighten the awareness of clinicians about COL4A1 as a single-gene disorder causing cerebral small vessel disease and describe a previously unreported pathogenic missense substitution in COL4A1 (p.Gly990Val and a new clinical presentation. We identified a heterozygous putatively pathogenic mutation of COL4A1 in a 50-year-old female with a history of congenital cataracts and glaucoma who presented with multiple diffusion-positive infarcts and areas of contrast enhancement following mild head trauma. We believe that this presentation of multiple areas of acute brain and vascular injury in the setting of mild head trauma is a new manifestation of this genetic disorder. Imaging findings of multiple acute infarcts and regions of contrast enhancement with associated asymptomatic old deep microhemorrhages and leukomalacia in adults after head trauma should raise a high suspicion for a COL4A1 genetic disorder. Radiographic patterns of significant leukoaraiosis and deep microhemorrhages can also be seen in patients with long-standing vasculopathy associated with hypertension, which our patient lacked. Our findings demonstrate the utility of genetic screening for COL4A1 mutations in young patients who have small vessel vasculopathy on brain imaging but who do not have significant cardiovascular risk factors.

  15. Novel COL4A1 mutations associated with HANAC syndrome: a role for the triple helical CB3[IV] domain.

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    Plaisier, Emmanuelle; Chen, Zhiyong; Gekeler, Florian; Benhassine, Safa; Dahan, Karine; Marro, Béatrice; Alamowitch, Sonia; Paques, Michel; Ronco, Pierre

    2010-10-01

    The COL4A1 gene encodes the α1-chain of type IV collagen, which is ubiquitously expressed in basement membranes. Mutations in COL4A1 have been reported in autosomal-dominant porencephaly and in patients with symptomatic small vessel brain disease, inconstantly associated with eye defects. We have previously reported three COL4A1 mutations associated with a systemic phenotype that we called HANAC (Hereditary Angiopathy, Nephropathy, Aneurysms, and Cramps). We carried out a clinical and genetic study of three families presenting with characteristic features of HANAC syndrome. Common systemic signs included arterial retinal tortuosity and muscle cramps, with a variable combination of small vessel brain disease, Raynaud phenomena, and kidney defects. Three novel COL4A1 missense substitutions are described, which affect highly conserved glycine residues within the collagenous domain of the protein. All six known mutations associated with the HANAC phenotype are localized within the CB3[IV] fragment of COL4A1, which encompasses major integrin-binding sites. Our results confirm that HANAC syndrome is a distinct clinical entity within the COL4A1-related disorders, which is characterized by systemic involvement and usually asymptomatic brain disease. The restricted distribution of COL4A1 mutations within the CB3[IV] region is a characteristic of the reports of patients with HANAC, which suggests that abnormal cell-type IV collagen interactions may underlie the systemic defects observed in this syndrome. Copyright © 2010 Wiley-Liss, Inc.

  16. A novel COL11A1 missense mutation in siblings with non-ocular Stickler syndrome.

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    Kohmoto, Tomohiro; Tsuji, Atsumi; Morita, Kei-Ichi; Naruto, Takuya; Masuda, Kiyoshi; Kashimada, Kenichi; Enomoto, Keisuke; Morio, Tomohiro; Harada, Hiroyuki; Imoto, Issei

    2016-01-01

    Stickler syndrome (STL) is an autosomal, dominantly inherited, clinically variable and genetically heterogeneous connective tissue disorder characterized by ocular, auditory, orofacial and skeletal abnormalities. We conducted targeted resequencing using a next-generation sequencer for molecular diagnosis of a 2-year-old girl who was clinically suspected of having STL with Pierre Robin sequence. We detected a novel heterozygous missense mutation, NM_001854.3:n.4838G>A [NM_001854.3 (COL11A1_v001):c.4520G>A], in COL11A1, resulting in a Gly to Asp substitution at position 1507 [NM_001854.3(COL11A1_i001)] within one of the collagen-like domains of the triple helical region. The same mutation was detected in her 4-year-old brother with cleft palate and high-frequency sensorineural hearing loss.

  17. A COL11A2 mutation in Labrador retrievers with mild disproportionate dwarfism.

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    Frischknecht, Mirjam; Niehof-Oellers, Helena; Jagannathan, Vidhya; Owczarek-Lipska, Marta; Drögemüller, Cord; Dietschi, Elisabeth; Dolf, Gaudenz; Tellhelm, Bernd; Lang, Johann; Tiira, Katriina; Lohi, Hannes; Leeb, Tosso

    2013-01-01

    We describe a mild form of disproportionate dwarfism in Labrador Retrievers, which is not associated with any obvious health problems such as secondary arthrosis. We designate this phenotype as skeletal dysplasia 2 (SD2). It is inherited as a monogenic autosomal recessive trait with incomplete penetrance primarily in working lines of the Labrador Retriever breed. Using 23 cases and 37 controls we mapped the causative mutation by genome-wide association and homozygosity mapping to a 4.44 Mb interval on chromosome 12. We re-sequenced the genome of one affected dog at 30x coverage and detected 92 non-synonymous variants in the critical interval. Only two of these variants, located in the lymphotoxin A (LTA) and collagen alpha-2(XI) chain gene (COL11A2), respectively, were perfectly associated with the trait. Previously described COL11A2 variants in humans or mice lead to skeletal dysplasias and/or deafness. The dog variant associated with disproportionate dwarfism, COL11A2:c.143G>C or p.R48P, probably has only a minor effect on collagen XI function, which might explain the comparatively mild phenotype seen in our study. The identification of this candidate causative mutation thus widens the known phenotypic spectrum of COL11A2 mutations. We speculate that non-pathogenic COL11A2 variants might even contribute to the heritable variation in height.

  18. A novel missense mutation of COL5A2 in a patient with Ehlers-Danlos syndrome.

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    Watanabe, Miki; Nakagawa, Ryuji; Naruto, Takuya; Kohmoto, Tomohiro; Suga, Ken-Ichi; Goji, Aya; Kagami, Shoji; Masuda, Kiyoshi; Imoto, Issei

    2016-01-01

    Ehlers-Danlos syndrome (EDS) is a group of inherited connective tissue disorders characterized by hyperextensible skin, joint hypermobility and soft tissue fragility. For molecular diagnosis, targeted exome sequencing was performed on a 9-year-old male patient who was clinically suspected to have EDS. The patient presented with progressive kyphoscoliosis, joint hypermobility and hyperextensible skin without scars. Ultimately, classical EDS was diagnosed by identifying a novel, mono-allelic mutation in COL5A2 [NM_000393.3(COL5A2_v001):c.682G>A, p.Gly228Arg].

  19. A novel missense mutation of COL5A2 in a patient with Ehlers–Danlos syndrome

    Science.gov (United States)

    Watanabe, Miki; Nakagawa, Ryuji; Naruto, Takuya; Kohmoto, Tomohiro; Suga, Ken-ichi; Goji, Aya; Kagami, Shoji; Masuda, Kiyoshi; Imoto, Issei

    2016-01-01

    Ehlers–Danlos syndrome (EDS) is a group of inherited connective tissue disorders characterized by hyperextensible skin, joint hypermobility and soft tissue fragility. For molecular diagnosis, targeted exome sequencing was performed on a 9-year-old male patient who was clinically suspected to have EDS. The patient presented with progressive kyphoscoliosis, joint hypermobility and hyperextensible skin without scars. Ultimately, classical EDS was diagnosed by identifying a novel, mono-allelic mutation in COL5A2 [NM_000393.3(COL5A2_v001):c.682G>A, p.Gly228Arg]. PMID:27656288

  20. A COL11A2 mutation in Labrador retrievers with mild disproportionate dwarfism.

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    Mirjam Frischknecht

    Full Text Available We describe a mild form of disproportionate dwarfism in Labrador Retrievers, which is not associated with any obvious health problems such as secondary arthrosis. We designate this phenotype as skeletal dysplasia 2 (SD2. It is inherited as a monogenic autosomal recessive trait with incomplete penetrance primarily in working lines of the Labrador Retriever breed. Using 23 cases and 37 controls we mapped the causative mutation by genome-wide association and homozygosity mapping to a 4.44 Mb interval on chromosome 12. We re-sequenced the genome of one affected dog at 30x coverage and detected 92 non-synonymous variants in the critical interval. Only two of these variants, located in the lymphotoxin A (LTA and collagen alpha-2(XI chain gene (COL11A2, respectively, were perfectly associated with the trait. Previously described COL11A2 variants in humans or mice lead to skeletal dysplasias and/or deafness. The dog variant associated with disproportionate dwarfism, COL11A2:c.143G>C or p.R48P, probably has only a minor effect on collagen XI function, which might explain the comparatively mild phenotype seen in our study. The identification of this candidate causative mutation thus widens the known phenotypic spectrum of COL11A2 mutations. We speculate that non-pathogenic COL11A2 variants might even contribute to the heritable variation in height.

  1. A novel COL11A1 mutation affecting splicing in a patient with Stickler syndrome.

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    Kohmoto, Tomohiro; Naruto, Takuya; Kobayashi, Haruka; Watanabe, Miki; Okamoto, Nana; Masuda, Kiyoshi; Imoto, Issei; Okamoto, Nobuhiko

    2015-01-01

    Stickler syndrome is a clinically and genetically heterogeneous collagenopathy characterized by ocular, auditory, skeletal and orofacial abnormalities, commonly occurring as an autosomal dominant trait. We conducted target resequencing to analyze candidate genes associated with known clinical phenotypes from a 4-year-old girl with Stickler syndrome. We detected a novel heterozygous intronic mutation (NM_001854.3:c.3168+5G>A) in COL11A1 that may impair splicing, which was suggested by in silico prediction and a minigene assay.

  2. Whole exome analysis identifies dominant COL4A1 mutations in patients with complex ocular phenotypes involving microphthalmia.

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    Deml, B; Reis, L M; Maheshwari, M; Griffis, C; Bick, D; Semina, E V

    2014-11-01

    Anophthalmia/microphthalmia (A/M) is a developmental ocular malformation defined as complete absence or reduction in size of the eye. A/M is a heterogenous disorder with numerous causative genes identified; however, about half the cases lack a molecular diagnosis. We undertook whole exome sequencing in an A/M family with two affected siblings, two unaffected siblings, and unaffected parents; the ocular phenotype was isolated with only mild developmental delay/learning difficulties reported and a normal brain magnetic resonance imaging (MRI) in the proband at 16 months. No pathogenic mutations were identified in 71 known A/M genes. Further analysis identified a shared heterozygous mutation in COL4A1, c.2317G>A, p.(Gly773Arg) that was not seen in the unaffected parents and siblings. Analysis of 24 unrelated A/M exomes identified a novel c.2122G>A, p.(Gly708Arg) mutation in an additional patient with unilateral microphthalmia, bilateral microcornea and Peters anomaly; the mutation was absent in the unaffected mother and the unaffected father was not available. Mutations in COL4A1 have been linked to a spectrum of human disorders; the most consistent feature is cerebrovascular disease with variable ocular anomalies, kidney and muscle defects. This study expands the spectrum of COL4A1 phenotypes and indicates screening in patients with A/M regardless of MRI findings or presumed inheritance pattern. © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  3. Mutation analysis of the COL1A1 and COL1A2 genes in Vietnamese patients with osteogenesis imperfecta.

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    Ho Duy, Binh; Zhytnik, Lidiia; Maasalu, Katre; Kändla, Ivo; Prans, Ele; Reimann, Ene; Märtson, Aare; Kõks, Sulev

    2016-08-12

    The genetics of osteogenesis imperfecta (OI) have not been studied in a Vietnamese population before. We performed mutational analysis of the COL1A1 and COL1A2 genes in 91 unrelated OI patients of Vietnamese origin. We then systematically characterized the mutation profiles of these two genes which are most commonly related to OI. Genomic DNA was extracted from EDTA-preserved blood according to standard high-salt extraction methods. Sequence analysis and pathogenic variant identification was performed with Mutation Surveyor DNA variant analysis software. Prediction of the pathogenicity of mutations was conducted using Alamut Visual software. The presence of variants was checked against Dalgleish's osteogenesis imperfecta mutation database. The sample consisted of 91 unrelated osteogenesis imperfecta patients. We identified 54 patients with COL1A1/2 pathogenic variants; 33 with COL1A1 and 21 with COL1A2. Two patients had multiple pathogenic variants. Seventeen novel COL1A1 and 10 novel COL1A2 variants were identified. The majority of identified COL1A1/2 pathogenic variants occurred in a glycine substitution (36/56, 64.3 %), usually serine (23/36, 63.9 %). We found two pathogenic variants of the COL1A1 gene c.2461G > A (p.Gly821Ser) in four unrelated patients and one, c.2005G > A (p.Ala669Thr), in two unrelated patients. Our data showed a lower number of collagen OI pathogenic variants in Vietnamese patients compared to reported rates for Asian populations. The OI mutational profile of the Vietnamese population is unique and related to the presence of a high number of recessive mutations in non-collagenous OI genes. Further analysis of OI patients negative for collagen mutations, is required.

  4. Scoliosis in osteogenesis imperfecta caused by COL1A1/COL1A2 mutations - genotype-phenotype correlations and effect of bisphosphonate treatment.

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    Sato, Atsuko; Ouellet, Jean; Muneta, Takeshi; Glorieux, Francis H; Rauch, Frank

    2016-05-01

    Bisphosphonates are widely used to treat children with osteogenesis imperfecta (OI), a bone fragility disorder that is most often caused by mutations in COL1A1 or COL1A2. However, it is unclear whether this treatment decreases the risk of developing scoliosis. We retrospectively evaluated spine radiographs and charts of 437 patients (227 female) with OI caused by mutations in COL1A1 or COL1A2 and compared the relationship between scoliosis, genotype and bisphosphonate treatment history. At the last follow-up (mean age 11.9 [SD: 5.9] years), 242 (55%) patients had scoliosis. The prevalence of scoliosis was highest in OI type III (89%), followed by OI type IV (61%) and OI type I (36%). Moderate to severe scoliosis (Cobb angle ≥25°) was rare in individuals with COL1A1 haploinsufficiency mutations but was present in about two fifth of patients with triple helical glycine substitutions or C-propeptide mutations. During the first 2 to 4years of bisphosphonate therapy, patients with OI type III had lower Cobb angle progression rates than before bisphosphonate treatment, whereas in OI types I and IV bisphosphonate treatment was not associated with a change in Cobb angle progression rates. At skeletal maturity, the prevalence of scoliosis (Cobb angle >10°) was similar in patients who had started bisphosphonate treatment early in life (before 5.0years of age) and in patients who had started therapy later (after the age of 10.0years) or had never received bisphosphonate therapy. Bisphosphonate treatment decreased progression rate of scoliosis in OI type III but there was no evidence of a positive effect on scoliosis in OI types I and IV. The prevalence of scoliosis at maturity was not influenced by the bisphosphonate treatment history in any OI type. Copyright © 2016 Elsevier Inc. All rights reserved.

  5. Embryo genome profiling by single-cell sequencing for successful preimplantation genetic diagnosis in a family harboring COL4A1 c.1537G>A; p.G513S mutation

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    Nayana H Patel

    2016-01-01

    Full Text Available CONTEXT: Genetic profiling of embryos (also known as preimplantation genetic diagnosis before implantation has dramatically enhanced the success quotient of in vitro fertilization (IVF in recent times. The technology helps in avoiding selective pregnancy termination since the baby is likely to be free of the disease under consideration. AIM: Screening of embryos free from c.1537G>A; p.G513S mutation within the COL4A1 gene for which the father was known in before be in heterozygous condition. SUBJECTS AND METHODS: Processing of trophectoderm biopsies was done from twelve embryos for c.1537G>A; p.G513S mutation within the COL4A1 gene. DNA extracted from isolated cells were subjected to whole genome amplification using an isothermal amplification and strand displacement technology. Oligonucleotide primers bracketing the mutation were synthesized and used to amplify 162 base pairs (bp polymerase chain reaction amplicons originating from each embryo which were subsequently sequenced to detect the presence or absence of the single base polymorphism. RESULTS: Three out of 12 embryos interrogated in this study were found to be normal while 9 were found to harbor the mutation in heterozygous condition. Implantation of one of the normal embryos following by chorionic villus sampling at 11 th week of pregnancy indicated that the baby was free from c.1537G>A; p.G513S mutation within the COL4A1 gene. CONCLUSIONS: Single-cell sequencing is a helpful tool for preimplantation embryo profiling. This is the first report from India describing the birth of a normal child through IVF procedure where a potential pathogenic COL4A1 allele was avoided using this technology.

  6. X-linked Alport syndrome associated with a synonymous p.Gly292Gly mutation alters the splicing donor site of the type IV collagen alpha chain 5 gene.

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    Fu, Xue Jun; Nozu, Kandai; Eguchi, Aya; Nozu, Yoshimi; Morisada, Naoya; Shono, Akemi; Taniguchi-Ikeda, Mariko; Shima, Yuko; Nakanishi, Koichi; Vorechovsky, Igor; Iijima, Kazumoto

    2016-10-01

    X-linked Alport syndrome (XLAS) is a progressive hereditary nephropathy caused by mutations in the type IV collagen alpha chain 5 gene (COL4A5). Although many COL4A5 mutations have previously been identified, pathogenic synonymous mutations have not yet been described. A family with XLAS underwent mutational analyses of COL4A5 by PCR and direct sequencing, as well as transcript analysis of potential splice site mutations. In silico analysis was also conducted to predict the disruption of splicing factor binding sites. Immunohistochemistry (IHC) of kidney biopsies was used to detect α2 and α5 chain expression. We identified a hemizygous point mutation, c.876A>T, in exon 15 of COL4A5 in the proband and his brother, which is predicted to result in a synonymous amino acid change, p.(Gly292Gly). Transcript analysis showed that this mutation potentially altered splicing because it disrupted the splicing factor binding site. The kidney biopsy of the proband showed lamellation of the glomerular basement membrane (GBM), while IHC revealed negative α5(IV) staining in the GBM and Bowman's capsule, which is typical of XLAS. This is the first report of a synonymous COL4A5 substitution being responsible for XLAS. Our findings suggest that transcript analysis should be conducted for the future correct assessment of silent mutations.

  7. Identification of a novel COL1A1 frameshift mutation, c.700delG, in a Chinese osteogenesis imperfecta family

    Science.gov (United States)

    Wang, Xiran; Pei, Yu; Dou, Jingtao; Lu, Juming; Li, Jian; Lv, Zhaohui

    2015-01-01

    Osteogenesis imperfecta (OI) is a family of genetic disorders associated with bone loss and fragility. Mutations associated with OI have been found in genes encoding the type I collagen chains. People with OI type I often produce insufficient α1-chain type I collagen because of frameshift, nonsense, or splice site mutations in COL1A1 or COL1A2. This report is of a Chinese daughter and mother who had both experienced two bone fractures. Because skeletal fragility is predominantly inherited, we focused on identifying mutations in COL1A1 and COL1A2 genes. A novel mutation in COL1A1, c.700delG, was detected by genomic DNA sequencing in the mother and daughter, but not in their relatives. The identification of this mutation led to the conclusion that they were affected by mild OI type I. Open reading frame analysis indicated that this frameshift mutation would truncate α1-chain type I collagen at residue p263 (p.E234KfsX264), while the wild-type protein would contain 1,464 residues. The clinical data were consistent with the patients’ diagnosis of mild OI type I caused by haploinsufficiency of α1-chain type I collagen. Combined with previous reports, identification of the novel mutation COL1A1-c.700delG in these patients suggests that additional genetic and environmental factors may influence the severity of OI. PMID:25983617

  8. Tissue-specific mosaicism for a lethal osteogenesis imperfecta COL1A1 mutation causes mild OI/EDS overlap syndrome.

    Science.gov (United States)

    Symoens, Sofie; Steyaert, Wouter; Demuynck, Lynn; De Paepe, Anne; Diderich, Karin E M; Malfait, Fransiska; Coucke, Paul J

    2017-04-01

    Type I collagen is the predominant protein of connective tissues such as skin and bone. Mutations in the type I collagen genes (COL1A1 and COL1A2) mainly cause osteogenesis imperfecta (OI). We describe a patient with clinical signs of Ehlers-Danlos syndrome (EDS), including fragile skin, easy bruising, recurrent luxations, and fractures resembling mild OI. Biochemical collagen analysis of the patients' dermal fibroblasts showed faint overmodification of the type I collagen bands, a finding specific for structural defects in type I collagen. Bidirectional Sanger sequencing detected an in-frame deletion in exon 44 of COL1A1 (c.3150_3158del), resulting in the deletion of three amino acids (p.Ala1053_Gly1055del) in the collagen triple helix. This COL1A1 mutation was hitherto identified in four probands with lethal OI, and never in EDS patients. As the peaks on the electropherogram corresponding to the mutant allele were decreased in intensity, we performed next generation sequencing of COL1A1 to study mosaicism in skin and blood. While approximately 9% of the reads originating from fibroblast gDNA harbored the COL1A1 deletion, the deletion was not detected in gDNA from blood. Most likely, the mild clinical symptoms observed in our patient can be explained by the mosaic state of the mutation. © 2017 Wiley Periodicals, Inc.

  9. Mutations in COL1A1 and COL1A2 and dental aberrations in children and adolescents with osteogenesis imperfecta – A retrospective cohort study

    Science.gov (United States)

    Dahllöf, Göran; Lindahl, Katarina; Kindmark, Andreas; Grigelioniene, Giedre; Åström, Eva; Malmgren, Barbro

    2017-01-01

    Osteogenesis imperfecta (OI) is a heterogeneous group of disorders of connective tissue, caused mainly by mutations in the collagen I genes (COL1A1 and COL1A2). Dentinogenesis imperfecta (DGI) and other dental aberrations are common features of OI. We investigated the association between collagen I mutations and DGI, taurodontism, and retention of permanent second molars in a retrospective cohort of 152 unrelated children and adolescents with OI. The clinical examination included radiographic evaluations. Teeth from 81 individuals were available for histopathological evaluation. COL1A1/2 mutations were found in 104 individuals by nucleotide sequencing. DGI was diagnosed clinically and radiographically in 29% of the individuals (44/152) and through isolated histological findings in another 19% (29/152). In the individuals with a COL1A1 mutation, 70% (7/10) of those with a glycine substitution located C-terminal of p.Gly305 exhibited DGI in both dentitions while no individual (0/7) with a mutation N-terminal of this point exhibited DGI in either dentition (p = 0.01). In the individuals with a COL1A2 mutation, 80% (8/10) of those with a glycine substitution located C terminal of p.Gly211 exhibited DGI in both dentitions while no individual (0/5) with a mutation N-terminal of this point (p = 0.007) exhibited DGI in either dentition. DGI was restricted to the deciduous dentition in 20 individuals. Seventeen had missense mutations where glycine to serine was the most prevalent substitution (53%). Taurodontism occurred in 18% and retention of permanent second molars in 31% of the adolescents. Dental aberrations are strongly associated with qualitatively changed collagen I. The varying expressivity of DGI is related to the location of the collagen I mutation. Genotype information may be helpful in identifying individuals with OI who have an increased risk of dental aberrations. PMID:28498836

  10. Persistent Notochord in a Fetus with COL2A1 Mutation

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    Elisabeth Codsi

    2015-01-01

    Full Text Available Multiple anomalies including micromelia, poor mineralization of the vertebrae, and a persistent notochord were identified on second trimester ultrasound in a fetus with a COL2A1 mutation. To our knowledge, this represents the first case of a persistent notochord associated with a COL2A1 mutation in humans. In this case report, we describe ultrasound and postmortem findings and review the pathogenesis associated with a persistent notochord.

  11. Mutations in COL1A1 and COL1A2 and dental aberrations in children and adolescents with osteogenesis imperfecta - A retrospective cohort study.

    Directory of Open Access Journals (Sweden)

    Kristofer Andersson

    Full Text Available Osteogenesis imperfecta (OI is a heterogeneous group of disorders of connective tissue, caused mainly by mutations in the collagen I genes (COL1A1 and COL1A2. Dentinogenesis imperfecta (DGI and other dental aberrations are common features of OI. We investigated the association between collagen I mutations and DGI, taurodontism, and retention of permanent second molars in a retrospective cohort of 152 unrelated children and adolescents with OI. The clinical examination included radiographic evaluations. Teeth from 81 individuals were available for histopathological evaluation. COL1A1/2 mutations were found in 104 individuals by nucleotide sequencing. DGI was diagnosed clinically and radiographically in 29% of the individuals (44/152 and through isolated histological findings in another 19% (29/152. In the individuals with a COL1A1 mutation, 70% (7/10 of those with a glycine substitution located C-terminal of p.Gly305 exhibited DGI in both dentitions while no individual (0/7 with a mutation N-terminal of this point exhibited DGI in either dentition (p = 0.01. In the individuals with a COL1A2 mutation, 80% (8/10 of those with a glycine substitution located C terminal of p.Gly211 exhibited DGI in both dentitions while no individual (0/5 with a mutation N-terminal of this point (p = 0.007 exhibited DGI in either dentition. DGI was restricted to the deciduous dentition in 20 individuals. Seventeen had missense mutations where glycine to serine was the most prevalent substitution (53%. Taurodontism occurred in 18% and retention of permanent second molars in 31% of the adolescents. Dental aberrations are strongly associated with qualitatively changed collagen I. The varying expressivity of DGI is related to the location of the collagen I mutation. Genotype information may be helpful in identifying individuals with OI who have an increased risk of dental aberrations.

  12. Evaluation of a patient with classical Ehlers-Danlos syndrome due to a 9q34 duplication affecting COL5A1.

    Science.gov (United States)

    Kuroda, Yukiko; Ohashi, Ikuko; Naruto, Takuya; Ida, Kazumi; Enomoto, Yumi; Saito, Toshiyuki; Nagai, Jun-Ichi; Kurosawa, Kenji

    2018-03-09

    Ehlers-Danlos syndrome classical type is a connective tissue disorder characterized by skin hyperextensibility, atrophic scarring, and joint hypermobility. The condition typically results from mutations in COL5A1 or COL5A2 leading to the functional haploinsufficiency. Here, we report of a 24-year-old male with mild intellectual disability, dysmorphic features, and a phenotype consistent with Ehlers-Danlos syndrome classical type. A copy number variant-calling algorithm from panel sequencing data identified the deletions exons 2-11 and duplications of exons 12-67 within COL5A1. Array comparative genomic hybridization confirmed a 94 kb deletion at 9q34.3 involving exons 2-11 of COL5A1, and a 3.4 Mb duplication at 9q34.3 involving exons 12-67 of COL5A1. © 2018 Japanese Teratology Society.

  13. High mutation detection rate in the COL4A5 collagen gene in suspected Alport syndrome using PCR and direct DNA sequencing

    DEFF Research Database (Denmark)

    Martin, P; Heiskari, N; Zhou, J

    1998-01-01

    -amplified and sequenced from DNA of 50 randomly chosen patients with suspected Alport syndrome. Mutations were found in 41 patients, giving a mutation detection rate of 82%. Retrospective analysis of clinical data revealed that two of the cases might be autosomal. Although it could not be determined whether the remaining...

  14. A novel splice variant in the N-propeptide of COL5A1 causes an EDS phenotype with severe kyphoscoliosis and eye involvement.

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    Sofie Symoens

    Full Text Available BACKGROUND: The Ehlers-Danlos Syndrome (EDS is a heritable connective tissue disorder characterized by hyperextensible skin, joint hypermobility and soft tissue fragility. The classic subtype of EDS is caused by mutations in one of the type V collagen genes (COL5A1 and COL5A2. Most mutations affect the type V collagen helical domain and lead to a diminished or structurally abnormal type V collagen protein. Remarkably, only two mutations were reported to affect the extended, highly conserved N-propeptide domain, which plays an important role in the regulation of the heterotypic collagen fibril diameter. We identified a novel COL5A1 N-propeptide mutation, resulting in an unusual but severe classic EDS phenotype and a remarkable splicing outcome. METHODOLOGY/PRINCIPAL FINDINGS: We identified a novel COL5A1 N-propeptide acceptor-splice site mutation (IVS6-2A>G, NM_000093.3_c.925-2A>G in a patient with cutaneous features of EDS, severe progressive scoliosis and eye involvement. Two mutant transcripts were identified, one with an exon 7 skip and one in which exon 7 and the upstream exon 6 are deleted. Both transcripts are expressed and secreted into the extracellular matrix, where they can participate in and perturb collagen fibrillogenesis, as illustrated by the presence of dermal collagen cauliflowers. Determination of the order of intron removal and computational analysis showed that simultaneous skipping of exons 6 and 7 is due to the combined effect of delayed splicing of intron 7, altered pre-mRNA secondary structure, low splice site strength and possibly disturbed binding of splicing factors. CONCLUSIONS/SIGNIFICANCE: We report a novel COL5A1 N-propeptide acceptor-splice site mutation in intron 6, which not only affects splicing of the adjacent exon 7, but also causes a splicing error of the upstream exon 6. Our findings add further insights into the COL5A1 splicing order and show for the first time that a single COL5A1 acceptor-splice site

  15. A New COL3A1 Mutation in Ehlers-Danlos Syndrome Vascular Type With Different Phenotypes in the Same Family.

    Science.gov (United States)

    Cortini, Francesca; Marinelli, Barbara; Romi, Silvia; Seresini, Agostino; Pesatori, Angela Cecilia; Seia, Manuela; Montano, Nicola; Bassotti, Alessandra

    2017-04-01

    Vascular Ehlers-Danlos syndrome (vEDS) is a rare and severe connective tissue disorder caused by mutations in the collagen type III alpha I chain ( COL3A1) gene. We describe a pathogenetic heterozygous COL3A1 mutation c.3140 G>A, p. Gly1047Asp, identified using next-generation sequencing, in a 40-year-old Italian female. The genetic test performed on her relatives, which present different clinical phenotypes, confirmed that they carry the same mutation in heterozygous state. This finding confirms that mutations causing vEDS have an incomplete penetrance.

  16. Delineation of Ehlers-Danlos syndrome phenotype due to the c.934C>T, p.(Arg312Cys) mutation in COL1A1: Report on a three-generation family without cardiovascular events, and literature review.

    Science.gov (United States)

    Colombi, Marina; Dordoni, Chiara; Venturini, Marina; Zanca, Arianna; Calzavara-Pinton, Piergiacomo; Ritelli, Marco

    2017-02-01

    Classical Ehlers-Danlos syndrome (cEDS) is a rare connective tissue disorder primarily characterized by hyperextensible skin, defective wound healing, abnormal scars, easy bruising, and generalized joint hypermobility; arterial dissections are rarely observed. Mutations in COL5A1 and COL5A2 encoding type V collagen account for more than 90% of the patients so far characterized. In addition, cEDS phenotype was reported in a small number of patients carrying the c.934C>T mutation in COL1A1 that results in an uncommon substitution of a non-glycine residue in one Gly-Xaa-Yaa repeat of the pro-α1(I)-chain p.(Arg312Cys), which leads to disturbed collagen fibrillogenesis due to delayed removal of the type I procollagen N-propeptide. This specific mutation has been associated with propensity to arterial rupture in early adulthood; indeed, in literature the individuals harboring this mutation are also referred to as "(classic) vascular-like" EDS patients. Herein, we describe a three-generation cEDS family with six adults carrying the p.(Arg312Cys) substitution, which show a variable and prevalent cutaneous involvement without any major vascular event. These data, together with those available in literature, suggest that vascular events are not a diagnostic handle to differentiate patients with the p.(Arg312Cys) COL1A1 mutation from those with COL5A1 and COL5A2 defects, and highlight that during the diagnostic process the presence of at least the p.(Arg312Cys) substitution in COL1A1 should be investigated in cEDS patients without type V collagen mutations. Nevertheless, for these patients, as well as for those affected with cEDS, a periodical vascular surveillance should be carried out together with cardiovascular risk factors monitoring. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

  17. A Gene Gun-mediated Nonviral RNA trans-splicing Strategy for Col7a1 Repair

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    Patricia Peking

    2016-01-01

    Full Text Available RNA trans-splicing represents an auspicious option for the correction of genetic mutations at RNA level. Mutations within COL7A1 causing strong reduction or absence of type VII collagen are associated with the severe skin blistering disease dystrophic epidermolysis bullosa. The human COL7A1 mRNA constitutes a suitable target for this RNA therapy approach, as only a portion of the almost 9 kb transcript has to be delivered into the target cells. Here, we have proven the feasibility of 5′ trans-splicing into the Col7a1 mRNA in vitro and in vivo. We designed a 5′ RNA trans-splicing molecule, capable of replacing Col7a1 exons 1–15 and verified it in a fluorescence-based trans-splicing model system. Specific and efficient Col7a1 trans-splicing was confirmed in murine keratinocytes. To analyze trans-splicing in vivo, we used gene gun delivery of a minicircle expressing a FLAG-tagged 5′ RNA trans-splicing molecule into the skin of wild-type mice. Histological and immunofluorescence analysis of bombarded skin sections revealed vector delivery and expression within dermis and epidermis. Furthermore, we have detected trans-spliced type VII collagen protein using FLAG-tag antibodies. In conclusion, we describe a novel in vivo nonviral RNA therapy approach to restore type VII collagen expression for causative treatment of dystrophic epidermolysis bullosa.

  18. COL1A2 gene analysis in a Czech osteogenesis imperfecta patient: a candidate novel mutation in a patient affected by osteogenesis imperfecta type 3

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    Hrušková L

    2015-08-01

    Full Text Available Lucie Hrušková,1 Ivo Mařík,2,3 Stella Mazurová,1 Pavel Martásek,1 Ivan Mazura1 1Department of Pediatrics and Adolescent Medicine, First Faculty of Medicine, Charles University in Prague, Prague, Czech Republic; 2Ambulant Centre for Defects of Locomotor Apparatus 1.1.c., Prague, Czech Republic; 3Faculty of Medical Studies, West Bohemia University, Pilsen, Czech RepublicAbstract: Osteogenesis imperfecta is a heritable bone fragility disease with a heterogenic genetic origin. Most cases result from mutations of either the COL1A1 gene or the COL1A2 gene. We identified a novel COL1A2 gene mutation in a Czech patient, born to unaffected parents, who was diagnosed according to clinical and anthropometric findings and radiographic features as having type 3 osteogenesis imperfecta, which is a severe form of this disease. The identified Gly814Trp mutation was predicted by a number of complementary bioinformatic programs to result in functional alteration of the protein. This case report provides both evidence of a novel COL1A2 mutation resulting in type 3 osteogenesis imperfecta and a genotype:phenotype correlation in this affected individual. Keywords: osteogenesis imperfecta type 3, collagen, alpha-2 (I chain, substitution, sequencing 

  19. A familial case of achondrogenesis type II caused by a dominant COL2A1 mutation and "patchy" expression in the mosaic father.

    Science.gov (United States)

    Forzano, F; Lituania, M; Viassolo, A; Superti-Furga, V; Wildhardt, G; Zabel, B; Faravelli, F

    2007-12-01

    Achondrogenesis type II (ACG2) is the most severe disorder that can be produced by dominant mutations in COL2A1. We report on four pregnancies of an apparently healthy, nonconsanguineous young couple. The father had scoliosis as a child, and has slight body disproportion with short trunk. The first child was born at 32 weeks and died neonatally. In the second pregnancy, short limbs and fetal hygroma were noted on ultrasound at 17 weeks' gestation. Similar findings were observed in the third fetus. Clinical, radiological, and histological evaluation of the fetuses after termination of the pregnancies showed findings consistent with ACG2. Molecular analysis of genomic DNA extracted from amniotic cells of the second and third fetuses revealed heterozygosity for a 10370G > T missense mutation (G346V) in the COL2A1 gene. This mutation was also found in the father, as a mosaic. The couple had a fourth pregnancy, and at 11 weeks fetal hydrops with a septated cystic hygroma were obvious. DNA from CVS demonstrated the same COL2A1 mutation. (c) 2007 Wiley-Liss, Inc.

  20. Asymptomatic parental mosaicism for osteogenesis imperfect associated with a new splice site mutation in COL1A2

    DEFF Research Database (Denmark)

    Frederiksen, Anja Lisbeth; Dunø, Morten; Johnsen, Iben Birgit Gade

    2016-01-01

    Recurrent lethal perinatal osteogenesis imperfecta may result from asymptomatic parental mosaicism. A previously unreported mutation in COL1A2 leads to recurrent cases of fetal osteogenesis imperfecta Sillence type IIA, which emphasizes the importance of clinical and genetic evaluation of mosaicism...

  1. CHARACTERIZATION AND NUCLEOTIDE SEQUENCE DETERMINATION OF A REPEAT ELEMENT ISOLATED FROM A 2,4,5,-T DEGRADING STRAIN OF PSEUDOMONAS CEPACIA

    Science.gov (United States)

    Pseudomonas cepacia strain AC1100, capable of growth on 2,4,5-trichlorophenoxyacetic acid (2,4,5-T), was mutated to the 2,4,5-T− strain PT88 by a ColE1 :: Tn5 chromosomal insertion. Using cloned DNA from the region flanking the insertion, a 1477-bp sequence (designated RS1100) wa...

  2. Common variants in the COL4A4 gene confer susceptibility to lattice degeneration of the retina.

    Science.gov (United States)

    Meguro, Akira; Ideta, Hidenao; Ota, Masao; Ito, Norihiko; Ideta, Ryuichi; Yonemoto, Junichi; Takeuchi, Masaki; Uemoto, Riyo; Nishide, Tadayuki; Iijima, Yasuhito; Kawagoe, Tatsukata; Okada, Eiichi; Shiota, Tomoko; Hagihara, Yuta; Oka, Akira; Inoko, Hidetoshi; Mizuki, Nobuhisa

    2012-01-01

    Lattice degeneration of the retina is a vitreoretinal disorder characterized by a visible fundus lesion predisposing the patient to retinal tears and detachment. The etiology of this degeneration is still uncertain, but it is likely that both genetic and environmental factors play important roles in its development. To identify genetic susceptibility regions for lattice degeneration of the retina, we performed a genome-wide association study (GWAS) using a dense panel of 23,465 microsatellite markers covering the entire human genome. This GWAS in a Japanese cohort (294 patients with lattice degeneration and 294 controls) led to the identification of one microsatellite locus, D2S0276i, in the collagen type IV alpha 4 (COL4A4) gene on chromosome 2q36.3. To validate the significance of this observation, we evaluated the D2S0276i region in the GWAS cohort and in an independent Japanese cohort (280 patients and 314 controls) using D2S0276i and 47 single nucleotide polymorphisms covering the region. The strong associations were observed in D2S0276i and rs7558081 in the COL4A4 gene (Pc = 5.8 × 10(-6), OR = 0.63 and Pc = 1.0 × 10(-5), OR = 0.69 in a total of 574 patients and 608 controls, respectively). Our findings suggest that variants in the COL4A4 gene may contribute to the development of lattice degeneration of the retina.

  3. Common variants in the COL4A4 gene confer susceptibility to lattice degeneration of the retina.

    Directory of Open Access Journals (Sweden)

    Akira Meguro

    Full Text Available Lattice degeneration of the retina is a vitreoretinal disorder characterized by a visible fundus lesion predisposing the patient to retinal tears and detachment. The etiology of this degeneration is still uncertain, but it is likely that both genetic and environmental factors play important roles in its development. To identify genetic susceptibility regions for lattice degeneration of the retina, we performed a genome-wide association study (GWAS using a dense panel of 23,465 microsatellite markers covering the entire human genome. This GWAS in a Japanese cohort (294 patients with lattice degeneration and 294 controls led to the identification of one microsatellite locus, D2S0276i, in the collagen type IV alpha 4 (COL4A4 gene on chromosome 2q36.3. To validate the significance of this observation, we evaluated the D2S0276i region in the GWAS cohort and in an independent Japanese cohort (280 patients and 314 controls using D2S0276i and 47 single nucleotide polymorphisms covering the region. The strong associations were observed in D2S0276i and rs7558081 in the COL4A4 gene (Pc = 5.8 × 10(-6, OR = 0.63 and Pc = 1.0 × 10(-5, OR = 0.69 in a total of 574 patients and 608 controls, respectively. Our findings suggest that variants in the COL4A4 gene may contribute to the development of lattice degeneration of the retina.

  4. Next-generation sequencing and a novel COL3A1 mutation associated with vascular Ehlers-Danlos syndrome with severe intestinal involvement: a case report.

    Science.gov (United States)

    Cortini, Francesca; Marinelli, Barbara; Seia, Manuela; De Giorgio, Barbara; Pesatori, Angela Cecilia; Montano, Nicola; Bassotti, Alessandra

    2016-10-31

    The vascular type of Ehlers-Danlos syndrome is an autosomal dominant connective tissue disorder caused by a mutation in the COL3A1 gene encoding pro-alpha1 chain of type III collagen. The vascular type of Ehlers-Danlos syndrome causes severe fragility of connective tissues with arterial and intestinal ruptures and complications in surgical and radiological treatments. We present a case of a 38-year-old Italian woman who was diagnosed as having the vascular type of Ehlers-Danlos syndrome. Genetic testing, conducted by Target Enrichment approach (Agilent Technologies), identified a new mutation c.1493G>A, p.G498D in exon 21 of COL3A1 gene (heterozygous state). This mutation disrupts the normal glycine-X-Y repetitions of type III procollagen by converting glycine to aspartic acid. We report a new genetic mutation associated with the vascular type of Ehlers-Danlos syndrome. We also describe clinical and genetic findings that are important to understand the genotype/phenotype correlation in patients with the vascular type of Ehlers-Danlos syndrome.

  5. Confirmation of the pathogenicity of a mutation p.G337C in the COL1A2 gene associated with osteogenesis imperfecta

    Science.gov (United States)

    Jia, Mingrui; Shi, Ranran; Zhao, Xuli; Fu, Zhijian; Bai, Zhijing; Sun, Tao; Zhao, Xuejun; Wang, Wenbo; Xu, Chao; Yan, Fang

    2017-01-01

    Abstract Mutation analysis as the gold standard is particularly important in diagnosis of osteogenesis imperfecta (OI) and it may be preventable upon early diagnosis. In this study, we aimed to analyze the clinical and genetic materials of an OI pedigree as well as to confirm the deleterious property of the mutation. A pedigree with OI was identified. All family members received careful clinical examinations and blood was drawn for genetic analyses. Genes implicated in OI were screened for mutation. The function and structure of the mutant protein were predicted using bioinformatics analysis. The proband, a 9-month fetus, showed abnormal sonographic images. Disproportionately short and triangular face with blue sclera was noticed at birth. She can barely walk and suffered multiple fractures till 2-year old. Her mother appeared small stature, frequent fractures, blue sclera, and deformity of extremities. A heterozygous missense mutation c.1009G>T (p.G337C) in the COL1A2 gene was identified in her mother and her. Bioinformatics analysis showed p.G337 was well-conserved among multiple species and the mutation probably changed the structure and damaged the function of collagen. We suggest that the mutation p.G337C in the COL1A2 gene is pathogenic for OI by affecting the protein structure and the function of collagen. PMID:28953610

  6. Upregulated expression of integrin α1 in mesangial cells and integrin α3 and vimentin in podocytes of Col4a3-null (Alport mice.

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    Brooke M Steenhard

    Full Text Available Alport disease in humans, which usually results in proteinuria and kidney failure, is caused by mutations to the COL4A3, COL4A4, or COL4A5 genes, and absence of collagen α3α4α5(IV networks found in mature kidney glomerular basement membrane (GBM. The Alport mouse harbors a deletion of the Col4a3 gene, which also results in the lack of GBM collagen α3α4α5(IV. This animal model shares many features with human Alport patients, including the retention of collagen α1α2α1(IV in GBMs, effacement of podocyte foot processes, gradual loss of glomerular barrier properties, and progression to renal failure. To learn more about the pathogenesis of Alport disease, we undertook a discovery proteomics approach to identify proteins that were differentially expressed in glomeruli purified from Alport and wild-type mouse kidneys. Pairs of cy3- and cy5-labeled extracts from 5-week old Alport and wild-type glomeruli, respectively, underwent 2-dimensional difference gel electrophoresis. Differentially expressed proteins were digested with trypsin and prepared for mass spectrometry, peptide ion mapping/fingerprinting, and protein identification through database searching. The intermediate filament protein, vimentin, was upregulated ∼2.5 fold in Alport glomeruli compared to wild-type. Upregulation was confirmed by quantitative real time RT-PCR of isolated Alport glomeruli (5.4 fold over wild-type, and quantitative confocal immunofluorescence microscopy localized over-expressed vimentin specifically to Alport podocytes. We next hypothesized that increases in vimentin abundance might affect the basement membrane protein receptors, integrins, and screened Alport and wild-type glomeruli for expression of integrins likely to be the main receptors for GBM type IV collagen and laminin. Quantitative immunofluorescence showed an increase in integrin α1 expression in Alport mesangial cells and an increase in integrin α3 in Alport podocytes. We conclude that

  7. Somatic mosaicism for the COL7A1 mutation p.Gly2034Arg in the unaffected mother of a patient with dystrophic epidermolysis bullosa pruriginosa

    NARCIS (Netherlands)

    van den Akker, P. C.; Pasmooij, A. M. G.; Meijer, R.; Scheffer, H.; Jonkman, M. F.

    Dystrophic epidermolysis bullosa (DEB) is a heritable blistering disorder caused by mutations in the type VII collagen gene, COL7A1. Although revertant mosaicism is well known in DEB, 'forward' somatic mosaicism, in which a pathogenic mutation arises on a wild-type (WT) background, extending beyond

  8. Somatic mosaicism for the COL7A1 mutation p.Gly2034Arg in the unaffected mother of a patient with dystrophic epidermolysis bullosa pruriginosa

    NARCIS (Netherlands)

    Akker, P.C. van den; Pasmooij, A.M.; Meijer, R.; Scheffer, H.; Jonkman, M.F.

    2015-01-01

    Dystrophic epidermolysis bullosa (DEB) is a heritable blistering disorder caused by mutations in the type VII collagen gene, COL7A1. Although revertant mosaicism is well known in DEB, 'forward' somatic mosaicism, in which a pathogenic mutation arises on a wild-type (WT) background, extending beyond

  9. Analysis of four families with the Stickler syndrome by linkage studies. Identification of a new premature stop codon in the COL2A1 gene in a family

    Energy Technology Data Exchange (ETDEWEB)

    Bonaventure, J.; Lasselin, C. [Hopital Necker, Paris (France); Toutain, A. [CHU Bretonneau, Tours (France)] [and others

    1994-09-01

    The Stickler syndrome is an arthro-ophthalmopathy which associates progressive myopia with vitreal degeneration and retinal detachment. Cleft palate, cranio-facial abnormalities, deafness and osteoarthritis are often associated symptoms. Genetic heterogeneity of this autosomal dominant disease was consistent with its large clinical variability. Linkage studies have provided evidence for cosegregation of the disease with COL2A1, the gene coding for type II collagen, in about 50% of the families. Four additional families are reported here. Linkage analyses by using a VNTR located in the 3{prime} region of the gene were achieved. In three families, positive lod scores were obtained with a cumulative maximal value of 3.5 at a recombination fraction of 0. In one of these families, single strand conformation analysis of 25 exons disclosed a new mutation in exon 42. Codon for glutamic acid at position a1-803 was converted into a stop codon. The mutation was detected in DNA samples from all the affected members of the family but not in the unaffected. This result confirms that most of the Stickler syndromes linked to COL2A1 are due to premature stop codons. In a second family, an abnormal SSCP pattern of exon 34 was detected in all the affected individuals. The mutation is likely to correspond to a splicing defect in the acceptor site of intron 33. In one family the disease did not segregate with the COL2A1 locus. Further linkage studies with intragenic dimorphic sites in the COL10A1 gene and highly polymorphic markers close to the COL9A1 locus indicated that this disorder did not result from defects in these two genes.

  10. Osteogenesis imperfecta Type IV: a newly identified variant at position c.560 (G > T; p.Gly187Val) in the COL1A2 gene.

    Science.gov (United States)

    Usta, Akin; Karademir, Dilay; Sen, Eylem; Yazici, Selcuk; Adali, Ertan; Erdem, Erkan; Karacan, Meric

    2017-01-01

    Osteogenesis imperfecta is a clinically heterogenous disease caused by defective collagen syntesis associated with a mutation in the COL1A1 or COL1A2 genes. In this report, we present a case of osteogenesis imperfecta (OI) type IV, seen in a female fetus with incurved femurs at 18 weeks of gestation. Molecular analysis of the newborn revealed a novel mutation at position c.560 (c.560 G > T) of the exon 12 in the COL1A2 gene; which lead to the glycine modification with valine (p.Gly187Val) at codon 187. The pregnancy follow-up was uneventful. After delivery, the newborn underwent biphosponat therapy and no fracture was detected until 1 year old.

  11. Genetic screening of a pedigree with osteogenesis imperfecta type Ⅰand identification of a novel mutation in COL1A2 pathogenic gene%成骨不全Ⅰ型家系的基因检测和COL1A2基因新突变的致病性鉴定

    Institute of Scientific and Technical Information of China (English)

    李荣; 郭源平; 潘敬新; 郭奕斌

    2015-01-01

    To uncover the molecular pathogenic mechanism of congenital osteogenesis imperfecta (OI) type I, all the 103 exons of the COL1A1 (Collagen, type Ⅰ, alpha 1) and COL1A2 (Collagen, type Ⅰ, alpha 2) genes in a child with OI type Ⅰ were screened using PCR-DNA direct sequencing. The results showed no pathological mutation in COL1A1 gene, but a novel mutation c.946G>T/p.G316C in the exon 19 of COL1A2 gene, which was inherited from her father. This mutation was not found in her mother and other six phenotypically normal relatives. By denaturing high perfor-mance liquid chromatography (DHPLC) screening, the abnormal double-peak was visualized in PCR products of exon 19 of COL1A2 gene in the proband and her father, while the normal single-peak was shown in those of her mother and all the healthy controls. Using allele specific amplification (ASA) screening, a specific band of 391 bp in COL1A2 exon 19 was amplified only in the proband and her father, but not in other samples. The amino acid encoded by the mutation site is evolutionarily highly conserved, and this mutation was a“damaging”or“probably damaging”factor to OI type Ⅰ, based on the predicting results using SIFT and Polyphen-2 softwares. In conclusion, the novel c.946G>T/p.G316C mutation in COL1A2 gene is a pathogenic mutation that could result in OI type Ⅰ. If the couple wants to get pregnant again, it is necessary to screen the mutation site in COL1A2 gene through the prenatal genetic diagnosis in the first trimester or through preimplantation genetic diagnosis (PGD) in the progestation.%为了揭示成骨不全(Osteogenesis imperfecta, OI)Ⅰ型家系的分子遗传学发生机制,文章采用PCR-DNA直接测序法,对患儿 COL1A1和 COL1A2基因共103个外显子(E)进行突变检测。结果显示:患儿 COL1A1基因未发现任何病理性突变,而在 COL1A2基因 E19内发现一新的杂合错义突变(p.G316C),该突变来自其父,而其母正常,其他表型正常的6

  12. Genetic Variations of COL4A1 Gene and Intracerebral Hemorrhage Outcome: A Cohort Study in a Chinese Han Population.

    Science.gov (United States)

    Xia, Chao; Lin, Sen; Yang, Jie; He, Sha; Li, Hao; Liu, Ming; You, Chao

    2018-05-01

    To investigate the relationship between single nucleotide polymorphisms or haplotypes of COL4A1 gene and the outcome of intracerebral hemorrhage (ICH). In our study, 181 patients with hypertensive ICH were enrolled and followed up at 3 and 6 months. Outcome data included any cause of death and disability. Genomic DNA was extracted by DNA extraction kit, and the 6 single nucleotide polymorphism genotyping of the COL4A1 gene was detected through MassARRAY Analyzer. Unphased 3.1.4 and SPSS 19.0 were used to analyze the association between alleles, genotypes, and haplotypes of the COL4A1 gene and the outcomes of ICH. Of the 181 patients with hypertensive ICH, 12 were lost in follow-up, which accounted for 6.6%. Our association analysis showed that the rs532625 AA genotype of the COL4A1 gene may increase risk of disability at 3 months; the rs532625 A allele and AA genotype were association factors of the risk of disability at 6 months; the rs532625 AA genotype was an association factor of the risk of death/disability at 6 months. After adjusting for gender, age, coma, and severe neurologic deficits, only the rs532625 AA genotype was independently associated with the risk of disability at 3 and 6 months and the risk of death/disability at 6 months. Our study found that the rs532625 AA genotype in the COL4A1 gene was independently associated with the risk of disability at 3 and 6 months and death/disability at 6 months in a Chinese Han population. These conclusions need to be verified in future studies with larger samples. Copyright © 2018 Elsevier Inc. All rights reserved.

  13. siRNA-mediated Allele-specific Silencing of a COL6A3 Mutation in a Cellular Model of Dominant Ullrich Muscular Dystrophy

    Directory of Open Access Journals (Sweden)

    Véronique Bolduc

    2014-01-01

    Full Text Available Congenital muscular dystrophy type Ullrich (UCMD is a severe disorder of early childhood onset for which currently there is no effective treatment. UCMD commonly is caused by dominant-negative mutations in the genes coding for collagen type VI, a major microfibrillar component of the extracellular matrix surrounding the muscle fibers. To explore RNA interference (RNAi as a potential therapy for UCMD, we designed a series of small interfering RNA (siRNA oligos that specifically target the most common mutations resulting in skipping of exon 16 in the COL6A3 gene and tested them in UCMD-derived dermal fibroblasts. Transcript analysis by semiquantitative and quantitative reverse transcriptase PCR showed that two of these siRNAs were the most allele-specific, i.e., they efficiently knocked down the expression from the mutant allele, without affecting the normal allele. In HEK293T cells, these siRNAs selectively suppressed protein expression from a reporter construct carrying the mutation, with no or minimal suppression of the wild-type (WT construct, suggesting that collagen VI protein levels are as also reduced in an allele-specific manner. Furthermore, we found that treating UCMD fibroblasts with these siRNAs considerably improved the quantity and quality of the collagen VI matrix, as assessed by confocal microscopy. Our current study establishes RNAi as a promising molecular approach for treating dominant COL6-related dystrophies.

  14. Transcriptome analysis of skin fibroblasts with dominant negative COL3A1 mutations provides molecular insights into the etiopathology of vascular Ehlers-Danlos syndrome.

    Science.gov (United States)

    Chiarelli, Nicola; Carini, Giulia; Zoppi, Nicoletta; Ritelli, Marco; Colombi, Marina

    2018-01-01

    Vascular Ehlers-Danlos syndrome (vEDS) is a dominantly inherited connective tissue disorder caused by mutations in the COL3A1 gene that encodes type III collagen (COLLIII), which is the major expressed collagen in blood vessels and hollow organs. The majority of disease-causing variants in COL3A1 are glycine substitutions and in-frame splice mutations in the triple helix domain that through a dominant negative effect are associated with the severe clinical spectrum potentially lethal of vEDS, characterized by fragility of soft connective tissues with arterial and organ ruptures. To shed lights into molecular mechanisms underlying vEDS, we performed gene expression profiling in cultured skin fibroblasts from three patients with different structural COL3A1 mutations. Transcriptome analysis revealed significant changes in the expression levels of several genes involved in maintenance of cell redox and endoplasmic reticulum (ER) homeostasis, COLLs folding and extracellular matrix (ECM) organization, formation of the proteasome complex, and cell cycle regulation. Protein analyses showed that aberrant COLLIII expression is associated with the disassembly of many structural ECM constituents, such as fibrillins, EMILINs, and elastin, as well as with the reduction of the proteoglycans perlecan, decorin, and versican, all playing an important role in the vascular system. Furthermore, the altered distribution of the ER marker protein disulfide isomerase PDI and the strong reduction of the COLLs-modifying enzyme FKBP22 are consistent with the disturbance of ER-related homeostasis and COLLs biosynthesis and post-translational modifications, indicated by microarray analysis. Our findings add new insights into the pathophysiology of this severe vascular disorder, since they provide a picture of the gene expression changes in vEDS skin fibroblasts and highlight that dominant negative mutations in COL3A1 also affect post-translational modifications and deposition into the ECM of

  15. Transcriptome analysis of skin fibroblasts with dominant negative COL3A1 mutations provides molecular insights into the etiopathology of vascular Ehlers-Danlos syndrome.

    Directory of Open Access Journals (Sweden)

    Nicola Chiarelli

    Full Text Available Vascular Ehlers-Danlos syndrome (vEDS is a dominantly inherited connective tissue disorder caused by mutations in the COL3A1 gene that encodes type III collagen (COLLIII, which is the major expressed collagen in blood vessels and hollow organs. The majority of disease-causing variants in COL3A1 are glycine substitutions and in-frame splice mutations in the triple helix domain that through a dominant negative effect are associated with the severe clinical spectrum potentially lethal of vEDS, characterized by fragility of soft connective tissues with arterial and organ ruptures. To shed lights into molecular mechanisms underlying vEDS, we performed gene expression profiling in cultured skin fibroblasts from three patients with different structural COL3A1 mutations. Transcriptome analysis revealed significant changes in the expression levels of several genes involved in maintenance of cell redox and endoplasmic reticulum (ER homeostasis, COLLs folding and extracellular matrix (ECM organization, formation of the proteasome complex, and cell cycle regulation. Protein analyses showed that aberrant COLLIII expression is associated with the disassembly of many structural ECM constituents, such as fibrillins, EMILINs, and elastin, as well as with the reduction of the proteoglycans perlecan, decorin, and versican, all playing an important role in the vascular system. Furthermore, the altered distribution of the ER marker protein disulfide isomerase PDI and the strong reduction of the COLLs-modifying enzyme FKBP22 are consistent with the disturbance of ER-related homeostasis and COLLs biosynthesis and post-translational modifications, indicated by microarray analysis. Our findings add new insights into the pathophysiology of this severe vascular disorder, since they provide a picture of the gene expression changes in vEDS skin fibroblasts and highlight that dominant negative mutations in COL3A1 also affect post-translational modifications and deposition

  16. Mutations in the codon for a conserved arginine-1563 in the COL4A5 collagen gene in Alport syndrome

    DEFF Research Database (Denmark)

    Zhou, J; Gregory, M C; Hertz, Jens Michael

    1993-01-01

    for arginine to the translation stop codon TGA. In Utah kindred 2123 and in the Danish kindred A13, there was a C-->T mutation in the noncoding strand changing the same codon to CAA for glutamine. Both mutations were confirmed by allele-specific hybridization on PCR-amplified DNA from other family members....

  17. Unique mutation portraits and frequent COL2A1 gene alteration in chondrosarcoma

    Science.gov (United States)

    Totoki, Yasushi; Yoshida, Akihiko; Hosoda, Fumie; Nakamura, Hiromi; Hama, Natsuko; Ogura, Koichi; Yoshida, Aki; Fujiwara, Tomohiro; Arai, Yasuhito; Toguchida, Junya; Tsuda, Hitoshi; Miyano, Satoru; Kawai, Akira

    2014-01-01

    Chondrosarcoma is the second most frequent malignant bone tumor. However, the etiological background of chondrosarcomagenesis remains largely unknown, along with details on molecular alterations and potential therapeutic targets. Massively parallel paired-end sequencing of whole genomes of 10 primary chondrosarcomas revealed that the process of accumulation of somatic mutations is homogeneous irrespective of the pathological subtype or the presence of IDH1 mutations, is unique among a range of cancer types, and shares significant commonalities with that of prostate cancer. Clusters of structural alterations localized within a single chromosome were observed in four cases. Combined with targeted resequencing of additional cartilaginous tumor cohorts, we identified somatic alterations of the COL2A1 gene, which encodes an essential extracellular matrix protein in chondroskeletal development, in 19.3% of chondrosarcoma and 31.7% of enchondroma cases. Epigenetic regulators (IDH1 and YEATS2) and an activin/BMP signal component (ACVR2A) were recurrently altered. Furthermore, a novel FN1-ACVR2A fusion transcript was observed in both chondrosarcoma and osteochondromatosis cases. With the characteristic accumulative process of somatic changes as a background, molecular defects in chondrogenesis and aberrant epigenetic control are primarily causative of both benign and malignant cartilaginous tumors. PMID:25024164

  18. Clinical phenotype of 5 females with a CDKL5 mutation.

    Science.gov (United States)

    Stalpers, Xenia L; Spruijt, Liesbeth; Yntema, Helger G; Verrips, Aad

    2012-01-01

    Mutations in the X-linked cyclin dependent kinase like 5 (CDKL5) gene have been reported in approximately 80 patients since the first description in 2003. The clinical presentation partly corresponds with Rett syndrome, considering clinical features as intellectual disability, hypotonia, and poor visual, language, and motor development. However, these patients do not meet the consensus criteria for Rett syndrome since they lack the clear period of regression. Furthermore, in contrast to Rett syndrome, patients with CDKL5 mutations, have seizures or infantile spasms starting in the first weeks of life. We present clinical phenotype of 5 girls having a mutation in the CDKL5 gene. All mutations are novel and are pathogenic since they either lead to a frameshift in the reading frame or affect a consensus splice site. Four of the mutations are detected de novo in the affected girl.

  19. Evaluation the COL9A2 gene with high myopia

    Science.gov (United States)

    Zhang, Dingding; Huang, Maomin

    2017-11-01

    This paper investigates the association of the COL9A2 gene between high myopia and normal controls in the Han Chinese population. It shows that the frameshift mutation (D281fs) in the COL9A2 gene is not associated with high myopia in the Han Chinese population, and the two novel variants(c.143G>C and c.884G>A) may contribute to the development of high myopia.

  20. Loss of Col3a1, the gene for Ehlers-Danlos syndrome type IV, results in neocortical dyslamination.

    Directory of Open Access Journals (Sweden)

    Sung-Jin Jeong

    Full Text Available It has recently been discovered that Collagen III, the encoded protein of the type IV Ehlers-Danlos Syndrome (EDS gene, is one of the major constituents of the pial basement membrane (BM and serves as the ligand for GPR56. Mutations in GPR56 cause a severe human brain malformation called bilateral frontoparietal polymicrogyria, in which neurons transmigrate through the BM causing severe mental retardation and frequent seizures. To further characterize the brain phenotype of Col3a1 knockout mice, we performed a detailed histological analysis. We observed a cobblestone-like cortical malformation, with BM breakdown and marginal zone heterotopias in Col3a1⁻/⁻ mouse brains. Surprisingly, the pial BM appeared intact at early stages of development but starting as early as embryonic day (E 11.5, prominent BM defects were observed and accompanied by neuronal overmigration. Although collagen III is expressed in meningeal fibroblasts (MFs, Col3a1⁻/⁻ MFs present no obvious defects. Furthermore, the expression and posttranslational modification of α-dystroglycan was undisturbed in Col3a1⁻/⁻ mice. Based on the previous finding that mutations in COL3A1 cause type IV EDS, our study indicates a possible common pathological pathway linking connective tissue diseases and brain malformations.

  1. A novel nonsteroidal antifibrotic oligo decoy containing the TGF-beta element found in the COL1A1 gene which regulates murine schistosomiasis liver fibrosis.

    Science.gov (United States)

    Boros, D L; Singh, K P; Gerard, H C; Hudson, A P; White, S L; Cutroneo, K R

    2005-08-01

    Schistosomiasis mansoni disseminated worm eggs in mice and humans induce granulomatous inflammations and cumulative fibrosis causing morbidity and possibly mortality. In this study, intrahepatic and I.V. injections of a double-stranded oligodeoxynucleotide decoy containing the TGF-beta regulatory element found in the distal promoter of the COL1A1 gene into worm-infected mice suppressed TGF-beta1, COL1A1, tissue inhibitor of metalloproteinase-1, and decreased COL3A1 mRNAs to a lesser extent. Sequence comparisons within the mouse genome found homologous sequences within the COL3A1, TGF-beta1, and TIMP-1 5' flanking regions. Cold competition gel mobility shift assays using these homologous sequences with 5' and 3' flanking regions found in the natural COL1A1 gene showed competition. Competitive gel mobility assays in a separate experiment showed no competition using a 5-base mutated or scrambled sequence. Explanted liver granulomas from saline-injected mice incorporated 10.45 +/- 1.7% (3)H-proline into newly synthesized collagen, whereas decoy-treated mice showed no collagen synthesis. Compared with the saline control schistosomiasis mice phosphorothioate double-stranded oligodeoxynucleotide treatment decreased total liver collagen content (i.e. hydroxy-4-proline) by 34%. This novel molecular approach has the potential to be employed as a novel antifibrotic treatment modality. (c) 2005 Wiley-Liss, Inc.

  2. [Identification of an ideal noninvasive method to detect A3243G gene mutation in MELAS syndrome].

    Science.gov (United States)

    Ma, Yi-nan; Fang, Fang; Yang, Yan-ling; Zhang, Ying; Wang, Song-tao; Xu, Yu-feng; Pei, Pei; Yuan, Yun; Bu, Ding-fang; Qi, Yu

    2008-12-16

    To identify a better non-invasive method to detect the carrier of mitochondrial A3243G mutation, a cause of mitochondrial encephalopathy-lactic acidosis-stroke like episode (MELAS) syndrome. DNA was extracted from the peripheral blood, urine, hair follicle, and saliva of 25 MELAS syndrome patients carrying A3243G mutation and their mothers and other maternal relatives, 33 persons in number, and the muscle tissues from 5 patients obtained by biopsy. A3243G mutation was detected by PCR-RFLP method, and the A3243G mutation ratio was identified by measuring the density of each band and calculation with the software AlphaEase 5.0. A3243G mutations were detected in all tissues of the 25 MELAS patients. The A3243G mutation ratio in urine was 62% +/- 9%, significantly higher than that in the blood [(36% +/- 10%), t = -11.13, P < 0.01]. A3243G mutations were detected in at least one tissue of the 28 maternal relatives. The A3243G mutation rates in their urine samples was 33.0% (5.0% - 70.4%), significantly higher than that in their blood samples [8.0% (0 - 33.3%), z = -4.197, P < 0.01]. There was no significant difference in A3243G mutation ratio among the samples of hair follicle, saliva, and blood. The A3243G mutation ratio in urine is significantly higher than those in blood samples of the patients and their maternal relatives. A noninvasive method, A3243G mutation ratio analysis of urine is superior to that in blood.

  3. Osteogenesis imperfecta type I: second-trimester diagnosis and incidental identification of a dominant COL1A1 deletion mutation in the paucisymptomatic father.

    Science.gov (United States)

    Chen, Chih-Ping; Su, Yi-Ning; Chang, Tung-Yao; Chern, Schu-Rern; Chen, Chen-Yu; Su, Jun-Wei; Wang, Wayseen

    2012-06-01

    To present second-trimester ultrasound and molecular diagnosis for osteogenesis imperfecta (OI) type I in a female fetus and incidental identification of a dominant COL1A1 deletion mutation in her paucisymptomatic father. A 30-year-old, primigravid woman was referred for genetic counseling in the second trimester because of bowing of the fetal lower limbs. She and her husband were non-consanguineous, and there was no family history of skeletal dysplasias. Prenatal ultrasound at 22 weeks of gestation revealed short and curved right femur and left tibia, and a short left fibula. The lengths of other long bones were normal. The husband was 158 cm tall, had blue sclerae, a history of habitual subluxation and dislocation of bilateral elbows and left knee, and an episode of left ulna fracture, and was not aware of his being affected with OI type I. The woman underwent amniocentesis. Cytogenetic analysis revealed a karyotype of 46,XX. Molecular analysis of the amniocytes revealed a heterozygous deletion mutation of c.1064_1068delCTGGT in exon 17 of the COL1A1 gene. By genetic testing the husband was found to carry the same mutation. Despite counseling of favorable outcome for OI type I with the parents, the woman elected to terminate the pregnancy. Postnatal skeletal X-ray findings were consistent with OI type I. Prenatal ultrasound diagnosis of mild forms of OI should include molecular analysis of type I collagen genes in both fetus and parents. Molecular genetic analysis of the family may incidentally identify a collagen gene mutation in the paucisymptomatic affected parent. Copyright © 2012. Published by Elsevier B.V.

  4. Genetics Home Reference: COL4A1-related brain small-vessel disease

    Science.gov (United States)

    ... hemorrhage Johns Hopkins Medicine Department of Neurology and Neurosurgery: Intracerebral Hemorrhage Johns Hopkins Medicine Department of Neurology and Neurosurgery: Stroke MalaCards: col4a1-related brain small-vessel disease ...

  5. Mutation analysis of COL4A3 and COL4A4 genes in a Chinese ...

    Indian Academy of Sciences (India)

    Liwei Guo

    2017-06-17

    Jun 17, 2017 ... IV collagen α5 chain are generally believed to cause X- linked AS (XLAS, OMIM: ... and male patients showed a high clinical variability, and a clear-cut ... serum creatinine was 146 μmol/L and blood urea nitro- gen was 8.64 ...

  6. ColVI myopathies: where do we stand, where do we go?

    Directory of Open Access Journals (Sweden)

    Allamand Valérie

    2011-09-01

    Full Text Available Abstract Collagen VI myopathies, caused by mutations in the genes encoding collagen type VI (ColVI, represent a clinical continuum with Ullrich congenital muscular dystrophy (UCMD and Bethlem myopathy (BM at each end of the spectrum, and less well-defined intermediate phenotypes in between. ColVI myopathies also share common features with other disorders associated with prominent muscle contractures, making differential diagnosis difficult. This group of disorders, under-recognized for a long time, has aroused much interest over the past decade, with important advances made in understanding its molecular pathogenesis. Indeed, numerous mutations have now been reported in the COL6A1, COL6A2 and COL6A3 genes, a large proportion of which are de novo and exert dominant-negative effects. Genotype-phenotype correlations have also started to emerge, which reflect the various pathogenic mechanisms at play in these disorders: dominant de novo exon splicing that enables the synthesis and secretion of mutant tetramers and homozygous nonsense mutations that lead to premature termination of translation and complete loss of function are associated with early-onset, severe phenotypes. In this review, we present the current state of diagnosis and research in the field of ColVI myopathies. The past decade has provided significant advances, with the identification of altered cellular functions in animal models of ColVI myopathies and in patient samples. In particular, mitochondrial dysfunction and a defect in the autophagic clearance system of skeletal muscle have recently been reported, thereby opening potential therapeutic avenues.

  7. Genetic Variations of the COL4A1 Gene and Intracerebral Hemorrhage Risk: A Case-Control Study in a Chinese Han Population.

    Science.gov (United States)

    Lin, Sen; Xia, Chao; He, Sha; Yang, Jie; Li, Hao; Zheng, Jun; Liu, Ming; You, Chao

    2018-04-01

    To investigate the association between single nucleotide polymorphisms or haplotypes of the COL4A1 gene and the risk of intracerebral hemorrhage (ICH). We conducted a case-control study that included 181 patients from the Chinese Han population with hypertensive ICH and 197 hypertension patients without ICH. Genomic DNA was extracted by DNA extraction kit, and the 6 single nucleotide polymorphism genotypes of the COL4A1 gene were detected with a MassARRAY Analyzer. Unphased 3.1.4 and SPSS 19.0 were used to analyze the association between alleles, genotypes, and haplotypes of the COL4A1 gene and the risk of ICH. Compared with the control group, patients in the ICH group were significantly younger. There were no differences in gender, diabetes, hyperlipidemia, current smoking, and alcohol consumption between the 2 groups. Our association analysis showed that the rs3742207 A, rs11069830 A, and rs679505 A alleles were association factors of the risks of ICH; rs11069830 AA, rs544012 AC, and rs679505 AA genotypes were association factors of the risk of ICH; AA haplotype (rs3742207-rs11069830) was an association factor of the risk of ICH. After adjusting age and gender by multivariate logistic regression, the rs544012 AC and rs679505 AA genotypes were independently associated with the risk of ICH. Our study showed that the rs544012 AC and rs679505 AA genotypes were independently associated with the risk of ICH in the Chinese Han population and that the AA haplotype (rs3742207-rs11069830) in the COL4A1 gene may be related to the risk of ICH in the Chinese Han population; these conclusions need further confirmation in future studies with larger samples. Copyright © 2018. Published by Elsevier Inc.

  8. Mutation analysis of the human CYP3A4 gene 5' regulatory region: population screening using non-radioactive SSCP.

    Science.gov (United States)

    Hamzeiy, Hossein; Vahdati-Mashhadian, Nasser; Edwards, Helen J; Goldfarb, Peter S

    2002-03-20

    Human CYP3A4 is the major cytochrome P450 isoenzyme in adult human liver and is known to metabolise many xenobiotic and endogenous compounds. There is substantial inter-individual variation in the hepatic levels of CYP3A4. Although, polymorphic mutations have been reported in the 5' regulatory region of the CYP3A4 gene, those that have been investigated so far do not appear to have any effect on gene expression. To determine whether other mutations exist in this region of the gene, we have performed a new population screen on a panel of 101 human DNA samples. A 1140 bp section of the 5' proximal regulatory region of the CYP3A4 gene, containing numerous regulatory motifs, was amplified from genomic DNA as three overlapping segments. The 300 bp distal enhancer region at -7.9kb containing additional regulatory motifs was also amplified. Mutation analysis of the resulting PCR products was carried out using non-radioactive single strand conformation polymorphism (SSCP) and confirmatory sequencing of both DNA strands in those samples showing extra SSCP bands. In addition to detection of the previously reported CYP3A4*1B allele in nine subjects, three novel alleles were found: CYP3A4*1E (having a T-->A transversion at -369 in one subject), CYP3A4*1F (having a C-->G tranversion at -747 in 17 subjects) and CYP3A4*15B containing a nine-nucleotide insertion between -845 and -844 linked to an A-->G transition at -392 and a G-->A transition in exon 6 (position 485 in the cDNA) in one subject. All the novel alleles were heterozygous. No mutations were found in the upstream distal enhancer region. Our results clearly indicate that this rapid and simple SSCP approach can reveal mutant alleles in drug metabolising enzyme genes. Detection and determination of the frequency of novel alleles in CYP3A4 will assist investigation of the relationship between genotype, xenobiotic metabolism and toxicity in the CYP3A family of isoenzymes.

  9. A nonsense mutation in the COL4A5 collagen gene in a family with X-linked juvenile Alport syndrome

    DEFF Research Database (Denmark)

    Hertz, Jens Michael; Heiskari, N; Zhou, J

    1995-01-01

    diagnosis on chorionic villi tissue, obtained from one of the female carriers in the family, revealed a male fetus hemizygous for the mutated allele. A subsequent prenatal test in her next pregnancy revealed a normal male fetus. Prenatal diagnosis of Alport syndrome has not previously been reported....

  10. Mutation Spectrum of the ABCA4 Gene in a Greek Cohort with Stargardt Disease: Identification of Novel Mutations and Evidence of Three Prevalent Mutated Alleles

    Directory of Open Access Journals (Sweden)

    Kamakari Smaragda

    2018-01-01

    Full Text Available Aim. To evaluate the frequency and pattern of disease-associated mutations of ABCA4 gene among Greek patients with presumed Stargardt disease (STGD1. Materials and Methods. A total of 59 patients were analyzed for ABCA4 mutations using the ABCR400 microarray and PCR-based sequencing of all coding exons and flanking intronic regions. MLPA analysis as well as sequencing of two regions in introns 30 and 36 reported earlier to harbor deep intronic disease-associated variants was used in 4 selected cases. Results. An overall detection rate of at least one mutant allele was achieved in 52 of the 59 patients (88.1%. Direct sequencing improved significantly the complete characterization rate, that is, identification of two mutations compared to the microarray analysis (93.1% versus 50%. In total, 40 distinct potentially disease-causing variants of the ABCA4 gene were detected, including six previously unreported potentially pathogenic variants. Among the disease-causing variants, in this cohort, the most frequent was c.5714+5G>A representing 16.1%, while p.Gly1961Glu and p.Leu541Pro represented 15.2% and 8.5%, respectively. Conclusions. By using a combination of methods, we completely molecularly diagnosed 48 of the 59 patients studied. In addition, we identified six previously unreported, potentially pathogenic ABCA4 mutations.

  11. An RNA-splicing mutation (G{sup +51VS20}) in the Type II collagen gene (COL2A1) in a family with spondyloepiphyseal dysplasia congenita

    Energy Technology Data Exchange (ETDEWEB)

    Tiller, G.E.; Polumbo, P.A. [Vanderbilt Univ. School of Medicine, Nashville, TN (United States); Weis, M.A.; Eyre, D.R. [Univ. of Washington, Seattle, WA (United States); Gruber, H.E.; Rimoin, D.L.; Cohn, D.H. [Cedars-Sinai Medical Center, Los Angeles, CA (United States)]|[Univ. of California School of Medicine, Los Angeles, CA (United States)

    1995-02-01

    Defects in type II collagen have been demonstrated in a phenotypic continuum of chondrodysplasias that includes achondrogenesis II, hypochondrogenesis, spondyloepiphyseal dysplasia congenita (SEDC), Kniest dysplasia, and Stickler syndrome. We have determined that cartilage from a terminated fetus with an inherited form of SEDC contained both normal {alpha}1(II) collagen chains and chains that lacked amino acids 256-273 of the triple-helical domain. PCR amplification of this region of COL2A1, from genomic DNA, yielded products of normal size, while amplification of cDNA yielded a normal sized species and a shorter fragment missing exon 20. Sequence analysis of genomic DNA from the fetus revealed a G{yields}T transversion at position +5 of intron 20; the affected father was also heterozygous for the mutation. Allele-specific PCR and heteroduplex analysis of a VNTR in COL2A1 independently confirmed the unaffected status of a fetus in a subsequent pregnancy. Thermodynamic calculations suggest that the mutation prevents normal splicing of exon 20 by interfering with binding of U{sub 1} small-nuclear RNA to pre-mRNA, thus leading to skipping of exon 20 in transcripts from the mutant allele. Electron micrographs of diseased cartilage showed intracellular inclusion bodies, which were stained by an antibody to {alpha}1(II) procollagen. Our findings support the hypothesis that {alpha}-chain length alterations that preserve the Gly-X-Y repeat motif of the triple helix result in partial intracellular retention of {alpha}1(II) procollagen and produce mild to moderate chondrodysplasia phenotypes. 50 refs., 6 figs., 1 tab.

  12. TUMOR-SPECIFIC EXPRESSION AND ALTERNATIVE SPLICING OF THE COL6A3 GENE IN PANCREATIC CANCER

    Science.gov (United States)

    Arafat, Hwyda; Lazar, Melissa; Salem, Khalifa; Chipitsyna, Galina; Gong, Qiaoke; Pan, Te-Cheng; Zhang, Rui-Zhu; Yeo, Charles J.; Chu, Mon-Li

    2011-01-01

    Introduction Pancreatic ductal adenocarcinoma (PDA) is a highly lethal disease in which a prominent desmoplastic reaction is a defining characteristic. Fibrillar collagens, such as collagen I and to a lesser extent, collagen III and V comprise the majority of this stromal fibrosis. Type VI collagen (COL6) forms a microfibrillar network associated with type I collagen fibrils. The expression of COL6 has been linked to inflammation and survival. Importantly, tumor-specific alternative splicing in COL6A3 has been identified in several cancers by genome exon arrays. We evaluated the expression and localization of COL6A3 in PDA and premalignant lesions and explored the presence of alternative splicing events. Methods We analyzed paired PDA-normal (n=18), IPMN (n=5), pancreatic cystadenoma (n=5), and eight PDA cell lines with RT-PCR, using unique primers that identify total COL6A3 gene and alternative splicing sites in several of its exons. Western blot analysis and immunohistochemistry were used to analyze the expression levels and localization of COL6A3 protein in the different lesions, and in two animal models of PDA. Results COL6A3 protein levels were significantly upregulated in 77% of the paired PDA-adjacent tissue examined. COL6A3 was mainly present in the desmoplastic stroma of PDA, with high deposition around the malignant ducts and in between the sites of stromal fatty infiltration. Analysis of the COL6A3 splice variants showed tumor-specific consistent inclusion of exons 3 and 6 in 17 of the 18 (94%) paired PDA-adjacent tissues. Inclusion of exon 4 was exclusively tumor-specific, with barely detectable expression in the adjacent tissues. IPMN and pancreatic cystadenomas showed no expression of any of the examined exons. Total COL6A3 mRNA and exon 6 were identified in six PDA cell lines, but only two cell lines (MIA PACA-2 and ASPC-1) expressed exons 3 and 4. In both the xenograft and transgenic models of PDA, COL6A3 immunoreactivity was present in the stroma

  13. Detection of COL III in Parchment by Amino Acid Analysis

    DEFF Research Database (Denmark)

    Vestergaard Poulsen Sommer, Dorte; Larsen, René

    2016-01-01

    Cultural heritage parchments made from the reticular dermis of animals have been subject to studies of deterioration and conservation by amino acid analysis. The reticular dermis contains a varying mixture of collagen I and III (COL I and III). When dealing with the results of the amino acid...... analyses, till now the COL III content has not been taken into account. Based on the available amino acid sequences we present a method for determining the amount of COL III in the reticular dermis of new and historical parchments calculated from the ratio of Ile/Val. We find COL III contents between 7...... and 32 % in new parchments and between 0.2 and 40 % in the historical parchments. This is consistent with results in the literature. The varying content of COL III has a significant influence on the uncertainty of the amino acid analysis. Although we have not found a simple correlation between the COL...

  14. Detection of MPL mutations by a novel allele-specific PCR-based strategy.

    Science.gov (United States)

    Furtado, Larissa V; Weigelin, Helmut C; Elenitoba-Johnson, Kojo S J; Betz, Bryan L

    2013-11-01

    MPL mutation testing is recommended in patients with suspected primary myelofibrosis or essential thrombocythemia who lack the JAK2 V617F mutation. MPL mutations can occur at allelic levels below 15%, which may escape detection by commonly used mutation screening methods such as Sanger sequencing. We developed a novel multiplexed allele-specific PCR assay capable of detecting most recurrent MPL exon 10 mutations associated with primary myelofibrosis and essential thrombocythemia (W515L, W515K, W515A, and S505N) down to a sensitivity of 2.5% mutant allele. Test results were reviewed from 15 reference cases and 1380 consecutive specimens referred to our laboratory for testing. Assay performance was compared to Sanger sequencing across a series of 58 specimens with MPL mutations. Positive cases consisted of 45 with W515L, 6 with S505N, 5 with W515K, 1 with W515A, and 1 with both W515L and S505N. Seven cases had mutations below 5% that were undetected by Sanger sequencing. Ten additional cases had mutation levels between 5% and 15% that were not consistently detected by sequencing. All results were easily interpreted in the allele-specific test. This assay offers a sensitive and reliable solution for MPL mutation testing. Sanger sequencing appears insufficiently sensitive for robust MPL mutation detection. Our data also suggest the relative frequency of S505N mutations may be underestimated, highlighting the necessity for inclusion of this mutation in MPL test platforms. Copyright © 2013 American Society for Investigative Pathology and the Association for Molecular Pathology. Published by Elsevier Inc. All rights reserved.

  15. Detection of resistance mutations and CD4 slopes in individuals experiencing sustained virological failure

    DEFF Research Database (Denmark)

    Schultze, Anna; Paredes, Roger; Sabin, Caroline

    2014-01-01

    during the episode were included. Mutations were identified using the IAS-US (2013) list, and were presumed to be present from detection until the end of an episode. Multivariable linear mixed models with a random intercept and slope adjusted for age, baseline CD4 count, hepatitis C, drug type, RNA (log...... mutations on CD4 slopes in patients undergoing episodes of viral failure. MATERIALS AND METHODS: Patients from the EuroSIDA and UK CHIC cohorts undergoing at least one episode of virological failure (>3 consecutive RNA measurements >500 on ART) with at least three CD4 measurements and a resistance test......-scale), risk group and subtype were used to estimate CD4 slopes. Individual mutations with a population prevalence of >10% were tested for their effect on the CD4 slope. RESULTS: A total of 2731 patients experiencing a median of 1 (range 1-4) episodes were included in this analysis. The prevalence of any...

  16. Hot-spot KIF5A mutations cause familial ALS

    Science.gov (United States)

    Yilmaz, Rüstem; Müller, Kathrin; Grehl, Torsten; Petri, Susanne; Meyer, Thomas; Grosskreutz, Julian; Weydt, Patrick; Ruf, Wolfgang; Neuwirth, Christoph; Weber, Markus; Pinto, Susana; Claeys, Kristl G; Schrank, Berthold; Jordan, Berit; Knehr, Antje; Günther, Kornelia; Hübers, Annemarie; Zeller, Daniel; Kubisch, Christian; Jablonka, Sibylle; Klopstock, Thomas; de Carvalho, Mamede; Sperfeld, Anne; Borck, Guntram; Volk, Alexander E; Dorst, Johannes; Weis, Joachim; Otto, Markus; Schuster, Joachim; Del Tredici, Kelly; Braak, Heiko; Danzer, Karin M; Freischmidt, Axel; Meitinger, Thomas; Strom, Tim M; Ludolph, Albert C; Andersen, Peter M; Weishaupt, Jochen H; Weyen, Ute; Hermann, Andreas; Hagenacker, Tim; Koch, Jan Christoph; Lingor, Paul; Göricke, Bettina; Zierz, Stephan; Baum, Petra; Wolf, Joachim; Winkler, Andrea; Young, Peter; Bogdahn, Ulrich; Prudlo, Johannes; Kassubek., Jan

    2018-01-01

    Abstract Heterozygous missense mutations in the N-terminal motor or coiled-coil domains of the kinesin family member 5A (KIF5A) gene cause monogenic spastic paraplegia (HSP10) and Charcot-Marie-Tooth disease type 2 (CMT2). Moreover, heterozygous de novo frame-shift mutations in the C-terminal domain of KIF5A are associated with neonatal intractable myoclonus, a neurodevelopmental syndrome. These findings, together with the observation that many of the disease genes associated with amyotrophic lateral sclerosis disrupt cytoskeletal function and intracellular transport, led us to hypothesize that mutations in KIF5A are also a cause of amyotrophic lateral sclerosis. Using whole exome sequencing followed by rare variant analysis of 426 patients with familial amyotrophic lateral sclerosis and 6137 control subjects, we detected an enrichment of KIF5A splice-site mutations in amyotrophic lateral sclerosis (2/426 compared to 0/6137 in controls; P = 4.2 × 10−3), both located in a hot-spot in the C-terminus of the protein and predicted to affect splicing exon 27. We additionally show co-segregation with amyotrophic lateral sclerosis of two canonical splice-site mutations in two families. Investigation of lymphoblast cell lines from patients with KIF5A splice-site mutations revealed the loss of mutant RNA expression and suggested haploinsufficiency as the most probable underlying molecular mechanism. Furthermore, mRNA sequencing of a rare non-synonymous missense mutation (predicting p.Arg1007Gly) located in the C-terminus of the protein shortly upstream of the splice donor of exon 27 revealed defective KIF5A pre-mRNA splicing in respective patient-derived cell lines owing to abrogation of the donor site. Finally, the non-synonymous single nucleotide variant rs113247976 (minor allele frequency = 1.00% in controls, n = 6137), also located in the C-terminal region [p.(Pro986Leu) in exon 26], was significantly enriched in familial amyotrophic lateral sclerosis patients (minor

  17. Genetic associations of body composition, flexibility and injury risk with ACE, ACTN3 and COL5A1 polymorphisms in Korean ballerinas

    Science.gov (United States)

    Kim, Jun Ho; Jung, Eun Sun; Kim, Chul-Hyun; Youn, Hyeon; Kim, Hwa Rye

    2014-01-01

    [Purpose] The purpose of this study was to exam the association of body composition, flexibility, and injury risk to genetic polymorphisms including ACE ID, ACTN3 RX, and COL5A1 polymorphisms in ballet dancers in Korea. [Methods] For the purpose of this study, elite ballerinas (n = 97) and normal female adults (n = 203) aged 18 to 39 were recruited and these participants were tested for body weight, height, body fat, fat free mass, flexibility, injury risks on the joints and gene polymorphisms (ACE, ACTN3, COL5A1 polymorphism). [Results] As results, the ACE DD genotype in ballerinas was associated with higher body fat and percentage of body fat than the ACE II and ID genotypes (p sports injuries, the ankle injury of the XX-genotyped ballerinas was in significantly more prevalence than the RR and XX-genotyped ballerinas (p < 0.05). According to the odd ratio analysis, XX-genotyped ballerinas have the injury risk on the ankle about 4.7 (95% CI: 1.6~13.4, p < 0.05) times more than the RR and RX-genotyped ballerinas. Meanwhile, the COL5A1 polymorphism in ballerinas has no association with any factors including flexibility and injury risks. [Conclusion] In conclusion, ACE polymorphism and ACTN3 polymorphism were associated with ballerinas' performance capacity; COL5A1 was not associated with any factors of performance of Ballerinas. The results suggested that the ACE DD genotype is associated with high body fat, the ACTN3 XX genotype is associated with low fat-free mass, low flexibility, and higher risk of ankle-joint injury. PMID:25566457

  18. A novel approach to detect KRAS/BRAF mutation for colon cancer: Highly sensitive simultaneous detection of mutations and simple pre-treatment without DNA extraction.

    Science.gov (United States)

    Suzuki, Shun-Ichi; Matsusaka, Satoshi; Hirai, Mitsuharu; Shibata, Harumi; Takagi, Koichi; Mizunuma, Nobuyuki; Hatake, Kiyohiko

    2015-07-01

    It has been reported that colon cancer patients with KRAS and BRAF mutations that lie downstream of epidermal growth factor receptor (EGFR) acquire resistance against therapy with anti‑EGFR antibodies, cetuximab and panitumumab. On the other hand, some reports say KRAS codon 13 mutation (p.G13D) has lower resistance against anti-EGFR antibodies, thus there is a substantial need for detection of specific KRAS mutations. We have established a state-of-the-art measurement system using QProbe (QP) method that allows simultaneous measurement of KRAS codon 12/13, p.G13D and BRAF mutation, and compared this method against Direct Sequencing (DS) using 182 specimens from colon cancer patients. In addition, 32 biopsy specimens were processed with a novel pre-treatment method without DNA purification in order to detect KRAS/BRAF. As a result of KRAS mutation measurement, concordance rate between the QP method and DS method was 81.4% (144/177) except for the 5 specimens that were undeterminable. Among them, 29 specimens became positive with QP method and negative with DS method. BRAF was measured with QP method only, and the mutation detection rate was 3.9% (6/153). KRAS measurement using a simple new pre-treatment method without DNA extraction resulted in 31 good results out of 32, all of them matching with the DS method. We have established a simple but highly sensitive simultaneous detection system for KRAS/BRAF. Moreover, introduction of the novel pre-treatment technology eliminated the inconvenient DNA extraction process. From this research achievement, we not only anticipate quick and accurate results returned in the clinical field but also contribution in improving the test quality and work efficiency.

  19. RNase H and replication of ColE1 DNA in Escherichia coli.

    OpenAIRE

    Naito, S; Uchida, H

    1986-01-01

    Amber mutations within the rnh (RNase H) gene of Escherichia coli K-12 were isolated by selecting for bacteria capable of replicating in a sup+ background replication-defective cer-6 mutant of the ColE1 replicon. The cer-6 mutation is an alteration of one base pair located 160 nucleotides upstream of the unique replication origin of this plasmid. Subsequently, we determined the DNA alterations present within these mutants. ColE1 DNA replicated in rnh(Am) recA cells, indicating that (i) RNase ...

  20. A nonsense mutation in cGMP-dependent type II protein kinase (PRKG2) causes dwarfism in American Angus cattle.

    Science.gov (United States)

    Koltes, James E; Mishra, Bishnu P; Kumar, Dinesh; Kataria, Ranjit S; Totir, Liviu R; Fernando, Rohan L; Cobbold, Rowland; Steffen, David; Coppieters, Wouter; Georges, Michel; Reecy, James M

    2009-11-17

    Historically, dwarfism was the major genetic defect in U.S. beef cattle. Aggressive culling and sire testing were used to minimize its prevalence; however, neither of these practices can eliminate a recessive genetic defect. We assembled a 4-generation pedigree to identify the mutation underlying dwarfism in American Angus cattle. An adaptation of the Elston-Steward algorithm was used to overcome small pedigree size and missing genotypes. The dwarfism locus was fine-mapped to BTA6 between markers AFR227 and BM4311. Four candidate genes were sequenced, revealing a nonsense mutation in exon 15 of cGMP-dependant type II protein kinase (PRKG2). This C/T transition introduced a stop codon (R678X) that truncated 85 C-terminal amino acids, including a large portion of the kinase domain. Of the 75 mutations discovered in this region, only this mutation was 100% concordant with the recessive pattern of inheritance in affected and carrier individuals (log of odds score = 6.63). Previous research has shown that PRKG2 regulates SRY (sex-determining region Y) box 9 (SOX9)-mediated transcription of collagen 2 (COL2). We evaluated the ability of wild-type (WT) or R678X PRKG2 to regulate COL2 expression in cell culture. Real-time PCR results confirmed that COL2 is overexpressed in cells that overexpressed R678X PRKG2 as compared with WT PRKG2. Furthermore, COL2 and COL10 mRNA expression was increased in dwarf cattle compared with unaffected cattle. These experiments indicate that the R678X mutation is functional, resulting in a loss of PRKG2 regulation of COL2 and COL10 mRNA expression. Therefore, we present PRKG2 R678X as a causative mutation for dwarfism cattle.

  1. Osteogenesis imperfecta with ectopic mineralizations in dentin and cementum and a COL1A2 mutation.

    Science.gov (United States)

    Kantaputra, Piranit Nik; Sirirungruangsarn, Yuddhasert; Intachai, Worrachet; Ngamphiw, Chumpol; Tongsima, Sissades; Dejkhamron, Prapai

    2018-04-10

    We report a Thai father (patient 1) and his daughter (patient 2) affected with osteogenesis imperfecta type IV and dentinogenesis imperfecta. Both were heterozygous for the c.1451G>A (p.Gly484Glu) mutation in COL1A2. The father, a Thai boxer, had very mild osteogenesis imperfecta with no history of low-trauma bone fractures. Scanning electron micrography of the primary teeth with DI of the patient 2, and the primary teeth with DI of another OI patient with OI showed newly recognized dental manifestations of teeth with DI. Normal dentin and cementum might have small areas of ectopic mineralizations. Teeth affected with DI have well-organized ectopic mineralizations in dentin and cementum. The "French-fries-appearance" of the crystals at the cemento-dentinal junction and abnormal cementum have never been reported to be associated with dentinogenesis imperfecta, either isolated or osteogenesis imperfecta-associated. Our study shows for the first time that abnormal collagen fibers can lead to ectopic mineralization in dentin and cementum and abnormal cementum can be a part of osteogenesis imperfecta.

  2. Hot-spot KIF5A mutations cause familial ALS.

    Science.gov (United States)

    Brenner, David; Yilmaz, Rüstem; Müller, Kathrin; Grehl, Torsten; Petri, Susanne; Meyer, Thomas; Grosskreutz, Julian; Weydt, Patrick; Ruf, Wolfgang; Neuwirth, Christoph; Weber, Markus; Pinto, Susana; Claeys, Kristl G; Schrank, Berthold; Jordan, Berit; Knehr, Antje; Günther, Kornelia; Hübers, Annemarie; Zeller, Daniel; Kubisch, Christian; Jablonka, Sibylle; Sendtner, Michael; Klopstock, Thomas; de Carvalho, Mamede; Sperfeld, Anne; Borck, Guntram; Volk, Alexander E; Dorst, Johannes; Weis, Joachim; Otto, Markus; Schuster, Joachim; Del Tredici, Kelly; Braak, Heiko; Danzer, Karin M; Freischmidt, Axel; Meitinger, Thomas; Strom, Tim M; Ludolph, Albert C; Andersen, Peter M; Weishaupt, Jochen H

    2018-01-12

    Heterozygous missense mutations in the N-terminal motor or coiled-coil domains of the kinesin family member 5A (KIF5A) gene cause monogenic spastic paraplegia (HSP10) and Charcot-Marie-Tooth disease type 2 (CMT2). Moreover, heterozygous de novo frame-shift mutations in the C-terminal domain of KIF5A are associated with neonatal intractable myoclonus, a neurodevelopmental syndrome. These findings, together with the observation that many of the disease genes associated with amyotrophic lateral sclerosis disrupt cytoskeletal function and intracellular transport, led us to hypothesize that mutations in KIF5A are also a cause of amyotrophic lateral sclerosis. Using whole exome sequencing followed by rare variant analysis of 426 patients with familial amyotrophic lateral sclerosis and 6137 control subjects, we detected an enrichment of KIF5A splice-site mutations in amyotrophic lateral sclerosis (2/426 compared to 0/6137 in controls; P = 4.2 × 10-3), both located in a hot-spot in the C-terminus of the protein and predicted to affect splicing exon 27. We additionally show co-segregation with amyotrophic lateral sclerosis of two canonical splice-site mutations in two families. Investigation of lymphoblast cell lines from patients with KIF5A splice-site mutations revealed the loss of mutant RNA expression and suggested haploinsufficiency as the most probable underlying molecular mechanism. Furthermore, mRNA sequencing of a rare non-synonymous missense mutation (predicting p.Arg1007Gly) located in the C-terminus of the protein shortly upstream of the splice donor of exon 27 revealed defective KIF5A pre-mRNA splicing in respective patient-derived cell lines owing to abrogation of the donor site. Finally, the non-synonymous single nucleotide variant rs113247976 (minor allele frequency = 1.00% in controls, n = 6137), also located in the C-terminal region [p.(Pro986Leu) in exon 26], was significantly enriched in familial amyotrophic lateral sclerosis patients (minor allele

  3. COL11A1 in FAP polyps and in sporadic colorectal tumors

    International Nuclear Information System (INIS)

    Fischer, Heléne; Salahshor, Sima; Stenling, Roger; Björk, Jan; Lindmark, Gudrun; Iselius, Lennart; Rubio, Carlos; Lindblom, Annika

    2001-01-01

    We previously reported that the α-1 chain of type 11 collagen (COL11A1), not normally expressed in the colon, was up-regulated in stromal fibroblasts in most sporadic colorectal carcinomas. Patients with germline mutations in the APC gene show, besides colonic polyposis, symptoms of stromal fibroblast involvement, which could be related to COL11A1 expression. Most colorectal carcinomas are suggested to be a result of an activated Wnt- pathway, most often involving an inactivation of the APC gene or activation of β-catenin. We used normal and polyp tissue samples from one FAP patient and a set of 37 sporadic colorectal carcinomas to find out if the up-regulation of COL11A1 was associated with an active APC/β-catenin pathway. In this study we found a statistically significant difference in COL11A1 expression between normal tissue and adenomas from one FAP patient, and all adenomas gave evidence for an active APC/β-catenin pathway. An active Wnt pathway has been suggested to involve stromal expression of WISP-1. We found a strong correlation between WISP-1 and COL11A1 expression in sporadic carcinomas. Our results suggest that expression of COL11A1 in colorectal tumors could be associated with the APC/β-catenin pathway in FAP and sporadic colorectal cancer

  4. Mutation in the alpha 5(IV) collagen chain in juvenile-onset Alport syndrome without hearing loss or ocular lesions

    DEFF Research Database (Denmark)

    Zhou, J; Hertz, Jens Michael; Tryggvason, K

    1992-01-01

    A single base mutation was identified in the type IV collagen alpha 5 chain gene (COL4A5) of a Danish kindred with Alport syndrome. The 27-year-old male proband developed hematuria in childhood and terminal renal failure at the age of 25 years. He has no hearing loss or ocular lesions. Electron...... microscopy demonstrated splitting of the lamina densa of the glomerular basement membrane. The proband's mother has had persistent microscopic hematuria since the age of 40 years, but no other manifestations. Southern analysis of MspI-digested genomic DNA from the proband showed the absence of 1.3-kb and 0...

  5. Partial isodisomy for maternal chromosome 7 and short stature in an individual with a mutation at the COL1A2 locus

    Energy Technology Data Exchange (ETDEWEB)

    Spotila, L.D.; Sereda, L.; Prockop, D.J. (Jefferson Medical College, Philadelphia, PA (United States))

    1992-12-01

    Uniparental disomy for chromosome 7 has been described previously in two individuals with cystic fibrosis. Here, the authors describe a third case that was discovered because the proband was homozygous for a mutation in the COL1A2 gene for type I procollagen, although his mother was heterozygous and his father did not have the mutation. Phenotypically, the proband was similar to the two previously reported cases with uniparental disomy for chromosome 7, in that he was short in stature and growth retarded. Paternity was assessed with five polymorphic markers. Chromosome 7 inheritance in the proband was analyzed using 12 polymorphic markers distributed along the entire chromosome. Similar analysis of the proband's two brothers established the phase of the alleles at the various loci, assuming minimal recombination. The proband inherited only maternal alleles at five loci and was homozygous at all loci examined, except one. He was heterozygous for an RFLP at the IGBP-1 locus at 7p13-p12. The results suggest that the isodisomy was not complete because of a recombination event involving the proximal short arms of two maternal chromosomes. In addition, the phenotype of proportional dwarfism in the proband suggests imprinting of one or more growth-related genes on chromosome 7. 42 refs., 5 figs., 3 tabs.

  6. Critical early roles for col27a1a and col27a1b in zebrafish notochord morphogenesis, vertebral mineralization and post-embryonic axial growth.

    Science.gov (United States)

    Christiansen, Helena E; Lang, Michael R; Pace, James M; Parichy, David M

    2009-12-29

    Fibrillar collagens are well known for their links to human diseases, with which all have been associated except for the two most recently identified fibrillar collagens, type XXIV collagen and type XXVII collagen. To assess functions and potential disease phenotypes of type XXVII collagen, we examined its roles in zebrafish embryonic and post-embryonic development. We identified two type XXVII collagen genes in zebrafish, col27a1a and col27a1b. Both col27a1a and col27a1b were expressed in notochord and cartilage in the embryo and early larva. To determine sites of type XXVII collagen function, col27a1a and col27a1b were knocked down using morpholino antisense oligonucleotides. Knockdown of col27a1a singly or in conjunction with col27a1b resulted in curvature of the notochord at early stages and formation of scoliotic curves as well as dysmorphic vertebrae at later stages. These defects were accompanied by abnormal distributions of cells and protein localization in the notochord, as visualized by transmission electron microscopy, as well as delayed vertebral mineralization as detected histologically. Together, our findings indicate a key role for type XXVII collagen in notochord morphogenesis and axial skeletogenesis and suggest a possible human disease phenotype.

  7. Overexpression of COL11A1 by cancer-associated fibroblasts: clinical relevance of a stromal marker in pancreatic cancer.

    Directory of Open Access Journals (Sweden)

    Carmen García-Pravia

    Full Text Available BACKGROUND: The collagen11A1 (COL11A1 gene is overexpressed in pancreatic cancer. The expression of COL11A1 protein could be involved in desmoplastic events in pancreatic cancer, but an antibody that specifically stains the COL11A1 protein is not currently available. METHODS AND FINDINGS: A total of 54 pancreatic ductal adenocarcinomas (PDAC, 23 chronic pancreatitis (CP samples, and cultured peritumoral stromal cells of PDAC (passages 3-6 were studied. Normal human pancreas tissue samples were obtained through a cadaveric organ donation program. 1 Validation of COL11A1 gene overexpression by q-RT-PCR. FINDINGS: the expression of COL11A1 gene is significantly increased in PDAC samples vs. normal and CP samples. 2 Analysis of COL11A1 by immunohistochemistry using highly specific anti-proCOL11A1 antibodies. FINDINGS: anti-proCOL11A1 stains stromal cells/cancer-associated fibroblasts (CAFs of PDAC but it does not stain chronic benign condition (chronic pancreatitis stromal cells, epithelial cells, or normal fibroblasts. 3 Evaluation of the discrimination ability of the antibody. FINDINGS: anti-proCOL11A1 immunostaining accurately discriminates between PDAC and CP (AUC 0.936, 95% CI 0.851, 0.981. 4 Phenotypic characterization of proCOL11A1+ stromal cells co-staining with mesenchymal, epithelial and stellate cell markers on pancreatic tissue samples and cultured peritumoral pancreatic cancer stromal cells. FINDINGS: ProCOL11A1+ cells present co-staining with mesenchymal, stellate and epithelial markers (EMT phenotype in different proportions. CONCLUSIONS/SIGNIFICANCE: Detection of proCOL11A1 through immunostaining with this newly-developed antibody allows for a highly accurate distinction between PDAC and CP. Unlike other available antibodies commonly used to detect CAFs, anti-proCOL11A1 is negative in stromal cells of the normal pancreas and almost absent in benign inflammation. These results strongly suggest that proCOL11A1 is a specific marker for

  8. Genome-first approach diagnosed Cabezas syndrome via novel CUL4B mutation detection.

    Science.gov (United States)

    Okamoto, Nobuhiko; Watanabe, Miki; Naruto, Takuya; Matsuda, Keiko; Kohmoto, Tomohiro; Saito, Masako; Masuda, Kiyoshi; Imoto, Issei

    2017-01-01

    Cabezas syndrome is a syndromic form of X-linked intellectual disability primarily characterized by a short stature, hypogonadism and abnormal gait, with other variable features resulting from mutations in the CUL4B gene. Here, we report a clinically undiagnosed 5-year-old male with severe intellectual disability. A genome-first approach using targeted exome sequencing identified a novel nonsense mutation [NM_003588.3:c.2698G>T, p.(Glu900*)] in the last coding exon of CUL4B , thus diagnosing this patient with Cabezas syndrome.

  9. Novel mutations in the TBX5 gene in patients with Holt-Oram Syndrome

    Directory of Open Access Journals (Sweden)

    Marianna P.R. Porto

    2010-01-01

    Full Text Available The Holt-Oram syndrome (HOS is an autosomal dominant condition characterized by upper limb and cardiac malformations. Mutations in the TBX5 gene cause HOS and have also been associated with isolated heart and arm defects. Interactions between the TBX5, GATA4 and NKX2.5 proteins have been reported in humans. We screened the TBX5, GATA4, and NKX2.5 genes for mutations, by direct sequencing, in 32 unrelated patients presenting classical (8 or atypical HOS (1, isolated congenital heart defects (16 or isolated upper-limb malformations (7. Pathogenic mutations in the TBX5 gene were found in four HOS patients, including two new mutations (c.374delG; c.678G > T in typical patients, and the hotspot mutation c.835C > T in two patients, one of them with an atypical HOS phenotype involving lower-limb malformations. Two new mutations in the GATA4 gene were found in association with isolated upper-limb malformations, but their clinical significance remains to be established. A previously described possibly pathogenic mutation in the NKX2.5 gene (c.73C > 7 was detected in a patient with isolated heart malformations and also in his clinically normal father.

  10. Targeted exome sequencing integrated with clinicopathological information reveals novel and rare mutations in atypical, suspected and unknown cases of Alport syndrome or proteinuria.

    Directory of Open Access Journals (Sweden)

    Rajshekhar Chatterjee

    Full Text Available We applied customized targeted next-generation exome sequencing (NGS to determine if mutations in genes associated with renal malformations, Alport syndrome (AS or nephrotic syndrome are a potential cause of renal abnormalities in patients with equivocal or atypical presentation. We first sequenced 4,041 exons representing 292 kidney disease genes in a Caucasian woman with a history of congenital vesicoureteral reflux (VUR, recurrent urinary tract infections and hydronephrosis who presented with nephrotic range proteinuria at the age of 45. Her biopsy was remarkable for focal segmental glomerulosclerosis (FSGS, a potential complication of longstanding VUR. She had no family history of renal disease. Her proteinuria improved initially, however, several years later she presented with worsening proteinuria and microhematuria. NGS analysis revealed two deleterious COL4A3 mutations, one novel and the other previously reported in AS, and a novel deleterious SALL2 mutation, a gene linked to renal malformations. Pedigree analysis confirmed that COL4A3 mutations were nonallelic and compound heterozygous. The genomic results in conjunction with subsequent abnormal electron microscopy, Collagen IV minor chain immunohistochemistry and progressive sensorineural hearing loss confirmed AS. We then modified our NGS approach to enable more efficient discovery of variants associated with AS or a subset of FSGS by multiplexing targeted exome sequencing of 19 genes associated with AS or FSGS in 14 patients. Using this approach, we found novel or known COL4A3 or COL4A5 mutations in a subset of patients with clinically diagnosed or suspected AS, APOL1 variants associated with FSGS in African Americans and novel mutations in genes associated with nephrotic syndrome. These studies demonstrate the successful application of targeted capture-based exome sequencing to simultaneously evaluate genetic variations in many genes in patients with complex renal phenotypes and

  11. Distinct mutations in STXBP2 are associated with variable clinical presentations in patients with familial hemophagocytic lymphohistiocytosis type 5 (FHL5)

    DEFF Research Database (Denmark)

    Pagel, Julia; Beutel, Karin; Lehmberg, Kai

    2012-01-01

    the genetic and clinical spectrum of FHL5, we analyzed a cohort of 185 patients with suspected FHL for mutations in STXBP2. We detected biallelic mutations in 37 patients from 28 families of various ethnic origins. Missense mutations and mutations affecting 1 of the exon 15 splice sites were the predominant...... changes detectable in this cohort. Patients with exon 15 splice-site mutations (n = 13) developed clinical manifestations significantly later than patients with other mutations (median age, 4.1 year vs 2 months) and showed less severe impairment of degranulation and cytotoxic function of NK cells and CTLs....... Patients with FHL5 showed several atypical features, including sensorineural hearing deficit, abnormal bleeding, and, most frequently, severe diarrhea that was only present in early-onset disease. In conclusion, we report the largest cohort of patients with FHL5 so far, describe an extended disease...

  12. Dietary Lecithin Decreases Skeletal Muscle COL1A1 and COL3A1 Gene Expression in Finisher Gilts

    Directory of Open Access Journals (Sweden)

    Henny Akit

    2016-06-01

    Full Text Available The purpose of this study was to investigate the effect of dietary lecithin on skeletal muscle gene expression of collagen precursors and enzymes involved in collagen synthesis and degradation. Finisher gilts with an average start weight of 55.9 ± 2.22 kg were fed diets containing either 0, 4, 20 or 80 g/kg soybean lecithin prior to harvest for six weeks and the rectus abdominis muscle gene expression profile was analyzed by quantitative real-time PCR. Lecithin treatment down-regulated Type I (α1 procollagen (COL1A1 and Type III (α1 procollagen (COL3A1 mRNA expression ( p < 0.05, respectively, indicating a decrease in the precursors for collagen synthesis. The α-subunit of prolyl 4-hydroxylase (P4H mRNA expression also tended to be down-regulated ( p = 0.056, indicating a decrease in collagen synthesis. Decreased matrix metalloproteinase-1 (MMP-1 mRNA expression may reflect a positive regulatory response to the reduced collagen synthesis in muscle from the pigs fed lecithin ( p = 0.035. Lecithin had no effect on tissue inhibitor metalloproteinase-1 (TIMP-1, matrix metalloproteinase-13 (MMP-13 and lysyl oxidase mRNA expression. In conclusion, lecithin down-regulated COL1A1 and COL3A1 as well as tended to down-regulate α-subunit P4H expression. However, determination of muscle collagen content and solubility are required to support the gene functions.

  13. Analysis of Hungarian patients with Rett syndrome phenotype for MECP2, CDKL5 and FOXG1 gene mutations.

    Science.gov (United States)

    Hadzsiev, Kinga; Polgar, Noemi; Bene, Judit; Komlosi, Katalin; Karteszi, Judit; Hollody, Katalin; Kosztolanyi, Gyorgy; Renieri, Alessandra; Melegh, Bela

    2011-03-01

    Rett syndrome (RTT) is characterized by a relatively specific clinical phenotype. We screened 152 individuals with RTT phenotype. A total of 22 different known MECP2 mutations were identified in 42 subjects (27.6%). Of the 22 mutations, we identified 7 (31.8%) frameshift-causing deletions, 4 (18.2%) nonsense, 10 (45.5%) missense mutations and one insertion (4.5%). The most frequent pathologic changes were: p.Thr158Met (14.2%) and p.Arg133Cys (11.9%) missense, and p.Arg255Stop (9.5%) and p.Arg294Stop (9.5%) nonsense mutations. We also detected the c.925C >T (p.Arg309Trp) mutation in an affected patient, whose role in RTT pathogenesis is still unknown. Patients without detectable MECP2 defects were screened for mutations of cyclin-dependent kinase-like 5 (CDKL5) gene, responsible for the early-onset variant of RTT. We discovered two novel mutations: c.607G >T resulting in a termination codon at aa203, disrupting the catalytic domain, and c.1708G >T leading to a stop at aa570 of the C terminus. Both patients with CDKL5 mutation presented therapy-resistant epilepsy and a phenotype fitting with the diagnosis of early-onset variant of RTT. No FOXG1 mutation was detected in any of the remaining patients. A total of 110 (72.5%) patients remained without molecular genetic diagnosis that necessitates further search for novel gene mutations in this phenotype. Our results also suggest the need of screening for CDKL5 mutations in patients with Rett phenotype tested negative for MECP2 mutations.

  14. Bone mineral properties in growing Col1a2(+/G610C) mice, an animal model of osteogenesis imperfecta.

    Science.gov (United States)

    Masci, Marco; Wang, Min; Imbert, Laurianne; Barnes, Aileen M; Spevak, Lyudmila; Lukashova, Lyudmila; Huang, Yihe; Ma, Yan; Marini, Joan C; Jacobsen, Christina M; Warman, Matthew L; Boskey, Adele L

    2016-06-01

    was noted in 10-day-old trabecular bone in vertebrae (31%) and in 2-month old trabecular bone in both tibia (31%) and vertebrae (4%). There was also a 16% lower carbonate-to-phosphate ratio in vertebral trabeculae and a correspondingly higher (22%) carbonate-to-phosphate ratio in 2month-old vertebral cortices. At age 3-months-of-age, male femurs with both a Col1a2(+/G610C) allele and a Lrp5 high bone mass allele (Lrp5+/A214V) showed an improvement in bone composition, presenting higher trabecular carbonate-to-phosphate ratio (18%) and lower trabecular and cortical acid-phosphate substitutions (8% and 18%, respectively). Together, these results indicate that mutant collagen α2(I) chain affects both bone quantity and composition, and the usefulness of this model for studies of potential OI therapies such as anti-sclerostin treatments. Copyright © 2016 Elsevier Inc. All rights reserved.

  15. Prenatal diagnosis of autosomal dominant hereditary spastic paraplegia (SPG4) using direct mutation detection

    DEFF Research Database (Denmark)

    Nielsen, Jørgen E; Koefoed, Pernille; Kjaergaard, Susanne

    2004-01-01

    OBJECTIVE: To present a report on prenatal diagnosis using direct SPG4 gene analysis in a family with autosomal dominant hereditary spastic paraplegia (AD-HSP). METHODS: Genetic linkage and haplotype analysis were previously carried out with chromosome 2p markers. DNA was obtained from affected...... individuals, the affected father, the mother, and fetal DNA from an ongoing pregnancy by chorionic villus sampling (CVS) in the first trimester. The spastin gene (SPG4) was completely sequenced. RESULTS: A novel 832insGdelAA frameshift mutation, predicted to cause loss of functional protein, was identified...... in the affected father and in the fetal DNA. CONCLUSIONS: This is the first report on direct prenatal diagnosis of chromosome 2p-linked AD-HSP (SPG4). In addition, we report a novel SPG4-combined small insertion/deletion mutation in exon 5, which may be the first SPG4 mutational hot spot....

  16. The Variants Within the COL5A1 Gene are Associated with Reduced Risk of Anterior Cruciate Ligament Injury in Skiers

    Directory of Open Access Journals (Sweden)

    Stępień-Słodkowska Marta

    2015-03-01

    Full Text Available The purpose of this study was to examine the association of the BstUI RFLP C/T (rs 12722 and DpnII RFLP C/T (rs 13946 COL5A1 polymorphisms, individually and as haplotypes, with anterior cruciate ligament ruptures in recreational skiers. Subjects were 138 male recreational skiers with surgically diagnosed primary anterior cruciate ligament ruptures. The control group consisted of 183 apparently healthy male recreational skiers, who were without any self-reported history of ligament or tendon injury. DNA was extracted from buccal cells donated by the subjects and genotyping was carried out using real-time PCR. The genotype distributions for both polymorphisms met Hardy- Weinberg expectations in both groups. There were no significant differences in genotype distribution of allele frequencies of COL5A1 BstUI RFLP C/T and COL5A1 DpnII RFLP C/T polymorphisms between the ACL rupture and control groups. The T-T (BstUI RFLP T, DpnII RFLP T haplotype was the most common (55.6%. The haplotype T-C was not present in any of the subjects. There was an underrepresentation tendency of the C-T haplotype in the study group compared to controls under recessive mode of inheritance. Higher frequency of the COL5A1 BstUI RFLP C/T and COL5A1DpnII RFLP C/T polymorphisms haplotype is associated with reduced risk of anterior cruciate ligament injury in a group of apparently healthy male recreational skiers.

  17. ABNORMAL TYPE-III COLLAGEN PRODUCED BY AN EXON-17-SKIPPING MUTATION OF THE COL3A1 GENE IN EHLERS-DANLOS SYNDROME TYPE-IV IS NOT INCORPORATED INTO THE EXTRACELLULAR-MATRIX

    NARCIS (Netherlands)

    CHIODO, AA; SILLENCE, DO; COLE, WG; BATEMAN, JF

    1995-01-01

    A novel heterozygous mutation of the COL3Al gene that encodes the alpha 1(III) chains of type III collagen was identified in a family with the: acrogeric form of Ehlers-Danlos syndrome type IV (EDS-IV). Cultured dermal fibroblasts produced normal and shortened alpha 1(III) chains. The triple helix

  18. SIRT1 deacetylates RFX5 and antagonizes repression of collagen type I (COL1A2) transcription in smooth muscle cells

    Energy Technology Data Exchange (ETDEWEB)

    Xia, Jun [Department of Respiratory Medicine, The First Affiliated Hospital of Nanjing Medical University (China); Department of Respiratory Medicine, Jiangsu Provincial Hospital of Chinese Traditional Medicine (China); Wu, Xiaoyan; Yang, Yuyu; Zhao, Yuhao [Atherosclerosis Research Center, Key Laboratory of Cardiovascular Disease and Molecular Intervention, Department of Pathophysiology, Nanjing Medical University (China); Fang, Mingming [Jiangsu Jiankang Vocational Institute (China); Xie, Weiping, E-mail: wpxienjmu@gmail.com [Department of Respiratory Medicine, The First Affiliated Hospital of Nanjing Medical University (China); Wang, Hong, E-mail: hwangnjmu@gmail.com [Department of Respiratory Medicine, The First Affiliated Hospital of Nanjing Medical University (China); Xu, Yong [Atherosclerosis Research Center, Key Laboratory of Cardiovascular Disease and Molecular Intervention, Department of Pathophysiology, Nanjing Medical University (China)

    2012-11-16

    Highlights: Black-Right-Pointing-Pointer SIRT1 interacts with and deacetylates RFX5. Black-Right-Pointing-Pointer SIRT1 activation attenuates whereas SIRT1 inhibition enhances collagen repression by RFX5 in vascular smooth muscle cells. Black-Right-Pointing-Pointer SIRT1 promotes cytoplasmic localization and proteasomal degradation of RFX5 and cripples promoter recruitment of RFX5. Black-Right-Pointing-Pointer IFN-{gamma} represses SIRT1 expression in vascular smooth muscle cells. Black-Right-Pointing-Pointer SIRT1 agonist alleviates collagen repression by IFN-{gamma} in vascular smooth muscle cells. -- Abstract: Decreased expression of collagen by vascular smooth muscle cells (SMCs) within the atherosclerotic plaque contributes to the thinning of the fibrous cap and poses a great threat to plaque rupture. Elucidation of the mechanism underlying repressed collagen type I (COL1A2) gene would potentially provide novel solutions that can prevent rupture-induced complications. We have previously shown that regulatory factor for X-box (RFX5) binds to the COL1A2 transcription start site and represses its transcription. Here we report that SIRT1, an NAD-dependent, class III deacetylase, forms a complex with RFX5. Over-expression of SIRT1 or NAMPT, which synthesizes NAD+ to activate SIRT1, or treatment with the SIRT1 agonist resveratrol decreases RFX5 acetylation and disrupts repression of the COL1A2 promoter activity by RFX5. On the contrary, knockdown of SIRT1 or treatment with SIRT1 inhibitors induces RFX5 acetylation and enhances the repression of collagen transcription. SIRT1 antagonizes RFX5 activity by promoting its nuclear expulsion and proteasomal degradation hence dampening its binding to the COL1A2 promoter. The pro-inflammatory cytokine IFN-{gamma} represses COL1A2 transcription by down-regulating SIRT1 expression in SMCs. Therefore, our data have identified as novel pathway whereby SIRT1 maintains collagen synthesis in SMCs by modulating RFX5 activity.

  19. Radiation-induced germ-line mutations detected by a direct comparison of parents and children DNA sequences containing SNPs

    International Nuclear Information System (INIS)

    Morimyo, M.; Hongo, E.; Higashi, T.; Wu, J.; Matsumoto, I.; Okamoto, M.; Kawano, A.; Tsuji, S.

    2003-01-01

    Full text: Germ-line mutation is detected in mice but not in humans. To estimate genetic risk of humans, a new approach to extrapolate from animal data to humans or to directly detect radiation-induced mutations in man is expected. We have developed a new method to detect germ-line mutations by directly comparing DNA sequences of parents and children. The nucleotide sequences among mouse strains are almost identical except SNP markers that are detected at 1/1000 frequency. When gamma-irradiated male mice are mated with female mice, heterogeneous nucleotide sequences induced in children DNA are a candidate of mutation, whose assignment can be done by SNP analysis. This system can easily detect all types of mutations such as transition, transversion, frameshift and deletion induced by radiation and can be applied to humans having genetically heterogeneous nucleotide sequences and many SNP markers. C3H male mice of 8 weeks of gestation were irradiated with gamma rays of 3 and 1 Gy and after 3 weeks, they were mated with the same aged C57BL female mice. After 3 weeks breeding, DNA was extracted from parents and children mice. The nucleotide sequences of 150 STS markers containing 300-900 bp and SNPs of parents and children DNA were determined by a direct sequencing; amplification of STS markers by Taq DNA polymerase, purification of PCR products, and DNA sequencing with a dye-terminator method. At each radiation dose, a total amount of 5 Mb DNA sequences were examined to detect radiation-induced mutations. We could find 6 deletions in 3 Gy irradiated mice but not in 1 Gy and control mice. The mutation frequency was about 4.0 x 10 -7 /bp/ Gy or 1.6 x 10 -4 /locus/Gy, and suggested the non-linear increase of mutation rate with dose

  20. Detection of EGFR somatic mutations in non-small cell lung cancer (NSCLC) using a novel mutant-enriched liquidchip (MEL) technology.

    Science.gov (United States)

    Zhang, Li; Yang, Huiyi; Zhao, Yanwei; Liu, Wenchao; Wu, Shiyang; He, Jiaying; Luo, Xiaodi; Zhu, Zeyao; Xu, Jiasen; Zhou, Qinghua; Ren-Heidenreich, Lifen

    2012-09-01

    We have developed and standardized a novel technology, mutant-enriched liquidchip (MEL), for clinical detection of EGFR mutations. The MEL integrates a mutant-enriched PCR procedure with liquidchip technology for detections of EGFR exon 19 deletions and L858R mutation on both formalin-fixed and paraffin-embedded (FFPE) slides and plasma samples from patients with non-small cell lung cancer (NSCLC). The detection sensitivity was 0.1% of mutant DNA in the presence of its wild-type DNA. The cross-reaction rate was lower than 5%. To evaluate the MEL platform, the EGFR mutation status of 59 patients with advanced NSCLC treated with EGFRTKIs (Tyrosine Kinase Inhibitors) were tested on their FFPE samples. EGFR exon 19 deletions and L858R were detected in 21 patients (21/59) and 76.2% (16/21) of them had partial response to the EGFR-TKIs, while by sequencing method, only 4 (4/59) mutations were detected. Plasma samples from 627 patients with various stages of NSCLC were examined with the MEL and 22% of EGFR exon 19 deletions and L858R were detected. Furthermore, in patients with advanced disease there are more mutations detected in plasma samples than in patients with less advanced disease. In conclusion, the MEL is a sensitive, stable, and robust technology for detecting EGFR DNA mutations from both FFPE and plasma samples from patients with NSCLC and is now routinely used for clinical diagnosis.

  1. Cerebral infarction and femoral venous thrombosis detected in a patient with diabetic ketoacidosis and heterozygous factor V Leiden G1691A and PAI-1 4G/5G mutations.

    Science.gov (United States)

    Yaroglu Kazanci, Selcen; Yesilbas, Osman; Ersoy, Melike; Kihtir, Hasan Serdar; Yildirim, Hamdi Murat; Sevketoglu, Esra

    2015-09-01

    Cerebral infarction is one of the serious neurological complications of diabetic ketoacidosis (DKA). Especially in patients who are genetically prone to thrombosis, cerebral infarction may develop due to inflammation, dehydration, and hyperviscocity secondary to DKA. A 6-year-old child with DKA is diagnosed with cerebral infarction after respiratory insufficiency, convulsion, and altered level of consciousness. Femoral and external iliac venous thrombosis also developed in a few hours after central femoral catheter had been inserted. Heterozygous type of factor V Leiden and PAI-14G/5G mutation were detected. In patients with DKA, cerebral infarction may be suspected other than cerebral edema when altered level of consciousness, convulsion, and respiratory insufficiency develop and once cerebral infarction occurs the patients should also be evaluated for factor V Leiden and PAI-14G/5G mutation analysis in addition to the other prothrombotic risk factors.

  2. A founder mutation in LEPRE1 carried by 1.5% of West Africans and 0.4% of African Americans causes lethal recessive osteogenesis imperfecta.

    Science.gov (United States)

    Cabral, Wayne A; Barnes, Aileen M; Adeyemo, Adebowale; Cushing, Kelly; Chitayat, David; Porter, Forbes D; Panny, Susan R; Gulamali-Majid, Fizza; Tishkoff, Sarah A; Rebbeck, Timothy R; Gueye, Serigne M; Bailey-Wilson, Joan E; Brody, Lawrence C; Rotimi, Charles N; Marini, Joan C

    2012-05-01

    Deficiency of prolyl 3-hydroxylase 1, encoded by LEPRE1, causes recessive osteogenesis imperfecta (OI). We previously identified a LEPRE1 mutation exclusively in African Americans and contemporary West Africans. We hypothesized that this allele originated in West Africa and was introduced to the Americas with the Atlantic slave trade. We aimed to determine the frequency of carriers for this mutation among African Americans and West Africans, and the mutation origin and age. Genomic DNA was screened for the mutation using PCR and restriction digestion, and a custom TaqMan genomic single-nucleotide polymorphism assay. The mutation age was estimated using microsatellites and short tandem repeats spanning 4.2 Mb surrounding LEPRE1 in probands and carriers. Approximately 0.4% (95% confidence interval: 0.22-0.68%) of Mid-Atlantic African Americans carry this mutation, estimating recessive OI in 1/260,000 births in this population. In Nigeria and Ghana, 1.48% (95% confidence interval: 0.95-2.30%) of unrelated individuals are heterozygous carriers, predicting that 1/18,260 births will be affected with recessive OI, equal to the incidence of de novo dominant OI. The mutation was not detected in Africans from surrounding countries. All carriers shared a haplotype of 63-770 Kb, consistent with a single founder for this mutation. Using linkage disequilibrium analysis, the mutation was estimated to have originated between 650 and 900 years before present (1100-1350 CE). We identified a West African founder mutation for recessive OI in LEPRE1. Nearly 1.5% of Ghanians and Nigerians are carriers. The estimated age of this allele is consistent with introduction to North America via the Atlantic slave trade (1501-1867 CE).

  3. Hybridization alters spontaneous mutation rates in a parent-of-origin-dependent fashion in Arabidopsis.

    Science.gov (United States)

    Bashir, Tufail; Sailer, Christian; Gerber, Florian; Loganathan, Nitin; Bhoopalan, Hemadev; Eichenberger, Christof; Grossniklaus, Ueli; Baskar, Ramamurthy

    2014-05-01

    Over 70 years ago, increased spontaneous mutation rates were observed in Drosophila spp. hybrids, but the genetic basis of this phenomenon is not well understood. The model plant Arabidopsis (Arabidopsis thaliana) offers unique opportunities to study the types of mutations induced upon hybridization and the frequency of their occurrence. Understanding the mutational effects of hybridization is important, as many crop plants are grown as hybrids. Besides, hybridization is important for speciation and its effects on genome integrity could be critical, as chromosomal rearrangements can lead to reproductive isolation. We examined the rates of hybridization-induced point and frameshift mutations as well as homologous recombination events in intraspecific Arabidopsis hybrids using a set of transgenic mutation detector lines that carry mutated or truncated versions of a reporter gene. We found that hybridization alters the frequency of different kinds of mutations. In general, Columbia (Col)×Cape Verde Islands and Col×C24 hybrid progeny had decreased T→G and T→A transversion rates but an increased C→T transition rate. Significant changes in frameshift mutation rates were also observed in some hybrids. In Col×C24 hybrids, there is a trend for increased homologous recombination rates, except for the hybrids from one line, while in Col×Cape Verde Islands hybrids, this rate is decreased. The overall genetic distance of the parents had no influence on mutation rates in the progeny, as closely related accessions on occasion displayed higher mutation rates than accessions that are separated farther apart. However, reciprocal hybrids had significantly different mutation rates, suggesting parent-of-origin-dependent effects on the mutation frequency.

  4. Clinical features and gene mutational spectrum of CDKL5-related diseases in a cohort of Chinese patients.

    Science.gov (United States)

    Zhao, Ying; Zhang, Xiaoying; Bao, Xinhua; Zhang, Qingping; Zhang, Jingjing; Cao, Guangna; Zhang, Jie; Li, Jiarui; Wei, Liping; Pan, Hong; Wu, Xiru

    2014-02-25

    Mutations in the cyclin-dependent kinase-like 5 (CDKL5) (NM_003159.2) gene have been associated with early-onset epileptic encephalopathies or Hanefeld variants of RTT(Rett syndrome). In order to clarify the CDKL5 genotype-phenotype correlations in Chinese patients, CDKL5 mutational screening in cases with early-onset epileptic encephalopathies and RTT without MECP2 mutation were performed. The detailed clinical information including clinical manifestation, electroencephalogram (EEG), magnetic resonance imaging (MRI), blood, urine amino acid and organic acid screening of 102 Chinese patients with early-onset epileptic encephalopathies and RTT were collected. CDKL5 gene mutations were analyzed by PCR, direct sequencing and multiplex ligation-dependent probe amplification (MLPA). The patterns of X-chromosome inactivation (XCI) were studied in the female patients with CDKL5 gene mutation. De novo CDKL5 gene mutations were found in ten patients including one missense mutation (c.533G > A, p.R178Q) which had been reported, two splicing mutations (ISV6 + 1A > G, ISV13 + 1A > G), three micro-deletions (c.1111delC, c.2360delA, c.234delA), two insertions (c.1791 ins G, c.891_892 ins TT in a pair of twins) and one nonsense mutation (c.1375C > T, p.Q459X). Out of ten patients, 7 of 9 females with Hanefeld variants of RTT and the remaining 2 females with early onset epileptic encephalopathy, were detected while only one male with infantile spasms was detected. The common features of all female patients with CDKL5 gene mutations included refractory seizures starting before 4 months of age, severe psychomotor retardation, Rett-like features such as hand stereotypies, deceleration of head growth after birth and poor prognosis. In contrast, the only one male patient with CDKL5 mutation showed no obvious Rett-like features as females in our cohort. The X-chromosome inactivation patterns of all the female patients were random. Mutations in CDKL5 gene are responsible for 7 with

  5. Detecting β-thalassaemia mutations from a single cell by PEP and RDB

    Institute of Scientific and Technical Information of China (English)

    YI Ping; LI Li; YAO Hong; ZHOU Yuan-guo; DENG Bing; CHEN Zhu-qin

    2006-01-01

    Objective:To evaluate the possibility of the technology involving PEP and RDB for detecting β-thalassaemia multipoint mutations from a single cell simultaneously. Methods: A set of allele specific oligonucleotide (ASO) probes used for detecting 8 familiar β-thalassaemia mutations (CD41-42, IVS- Ⅱ -654, CD17, TATA box nt-28, CD71-72, TATA box nt-29, CD26, IVS- Ⅰ -5) were immobilized on a strip of nylon membrane. The genome of a individual cell was amplified by primer extension preamplification (PEP) with the mixture of15-base random oligonucleotides. The aliquots from PEP were used to amplify the objective gene fractions of β-thalassaemia gene by nested or semi-nested PCR. The membrane was hybridized with the final amplified products and then treated with Streptavidin-HRP and color development.Results :Totally 30 lymphocytes were picked up from blood samples of 1 healthy female and 4 patients with known β-thalassaemia mutations respectively. Each single lymphocyte was lysed in the proteinase K buffer. The amplification efficacy was 94.0% and alle drop-out(ADO) rate was 8.0%. Revert dot blot (RDB) was applied to the final amplified products from the 5 participants. The results of diagnosis were the same to the expected, and their genotypes were N/N, CD17 (A→T)/N, IVS- Ⅱ -654(C→T)/CD17(A → T), CD41-42 (-CTTT)/N and TATA box nt-28 (A→G)/N, respectively. Conclusion: The technology involving PEP and RDB could detectmultiple β-thalassaemia mutations from a single cell simultaneously,and the research provides experimental evidences for the feasibility of applying PEP and DNA array technology to screening multiple genetic mutations from a single cell, and will be applied to preimplantation genetic diagnosis and non-invasive prenatal diagnosis for β-thalassaemia.

  6. Alport syndrome: impact of digenic inheritance in patients management.

    Science.gov (United States)

    Fallerini, C; Baldassarri, M; Trevisson, E; Morbidoni, V; La Manna, A; Lazzarin, R; Pasini, A; Barbano, G; Pinciaroli, A R; Garosi, G; Frullanti, E; Pinto, A M; Mencarelli, M A; Mari, F; Renieri, A; Ariani, F

    2017-07-01

    Alport syndrome (ATS) is a genetically heterogeneous nephropathy with considerable phenotypic variability and different transmission patterns, including monogenic (X-linked/autosomal) and digenic inheritance (DI). Here we present a new series of families with DI and we discuss the consequences for genetic counseling and risk assessment. Out of five families harboring variants in more than one COL4 gene detected by next generation sequencing (NGS), minigene-splicing assay allowed us to identify four as true digenic. Two families showed COL4A3/A4 mutations in cis, mimicking an autosomal dominant inheritance with a more severe phenotype and one showed COL4A3/A4 mutations in trans, mimicking an autosomal recessive inheritance with a less severe phenotype. In a fourth family, a de novo mutation (COL4A5) combined with an inherited mutation (COL4A3) triggered a more severe phenotype. A fifth family, predicted digenic on the basis of silico tools, rather showed monogenic X-linked inheritance due to a hypomorphic mutation, in accordance with a milder phenotype. In conclusion, this study highlights the impact of DI in ATS and explains the associated atypical presentations. More complex inheritance should be therefore considered when reviewing prognosis and recurrence risks. On the other side, these findings emphasize the importance to accompany NGS with splicing assays in order to avoid erroneous identification of at risk members. © 2016 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  7. [Rapid detection of hot spot mutations of FGFR3 gene with PCR-high resolution melting assay].

    Science.gov (United States)

    Li, Shan; Wang, Han; Su, Hua; Gao, Jinsong; Zhao, Xiuli

    2017-08-10

    To identify the causative mutations in five individuals affected with dyschondroplasia and develop an efficient procedure for detecting hot spot mutations of the FGFR3 gene. Genomic DNA was extracted from peripheral blood samples with a standard phenol/chloroform method. PCR-Sanger sequencing was used to analyze the causative mutations in the five probands. PCR-high resolution melting (HRM) was developed to detect the identified mutations. A c.1138G>A mutation in exon 8 was found in 4 probands, while a c.1620C>G mutation was found in exon 11 of proband 5 whom had a mild phenotype. All patients were successfully distinguished from healthy controls with the PCR-HRM method. The results of HRM analysis were highly consistent with that of Sanger sequencing. The Gly380Arg and Asn540Lys are hot spot mutations of the FGFR3 gene among patients with ACH/HCH. PCR-HRM analysis is more efficient for detecting hot spot mutations of the FGFR3 gene.

  8. Genetic and Epigenetic Factors at COL2A1 and ABCA4 Influence Clinical Outcome in Congenital Toxoplasmosis

    Science.gov (United States)

    Jamieson, Sarra E.; de Roubaix, Lee-Anne; Cortina-Borja, Mario; Tan, Hooi Kuan; Mui, Ernest J.; Cordell, Heather J.; Kirisits, Michael J.; Miller, E. Nancy; Peacock, Christopher S.; Hargrave, Aubrey C.; Coyne, Jessica J.; Boyer, Kenneth; Bessieres, Marie-Hélène; Buffolano, Wilma; Ferret, Nicole; Franck, Jacqueline; Kieffer, François; Meier, Paul; Nowakowska, Dorota E.; Paul, Malgorzata; Peyron, François; Stray-Pedersen, Babill; Prusa, Andrea-Romana; Thulliez, Philippe; Wallon, Martine; Petersen, Eskild; McLeod, Rima; Gilbert, Ruth E.; Blackwell, Jenefer M.

    2008-01-01

    Background Primary Toxoplasma gondii infection during pregnancy can be transmitted to the fetus. At birth, infected infants may have intracranial calcification, hydrocephalus, and retinochoroiditis, and new ocular lesions can occur at any age after birth. Not all children who acquire infection in utero develop these clinical signs of disease. Whilst severity of disease is influenced by trimester in which infection is acquired by the mother, other factors including genetic predisposition may contribute. Methods and Findings In 457 mother-child pairs from Europe, and 149 child/parent trios from North America, we show that ocular and brain disease in congenital toxoplasmosis associate with polymorphisms in ABCA4 encoding ATP-binding cassette transporter, subfamily A, member 4. Polymorphisms at COL2A1 encoding type II collagen associate only with ocular disease. Both loci showed unusual inheritance patterns for the disease allele when comparing outcomes in heterozygous affected children with outcomes in affected children of heterozygous mothers. Modeling suggested either an effect of mother's genotype, or parent-of-origin effects. Experimental studies showed that both ABCA4 and COL2A1 show isoform-specific epigenetic modifications consistent with imprinting. Conclusions These associations between clinical outcomes of congenital toxoplasmosis and polymorphisms at ABCA4 and COL2A1 provide novel insight into the molecular pathways that can be affected by congenital infection with this parasite. PMID:18523590

  9. Genetic and epigenetic factors at COL2A1 and ABCA4 influence clinical outcome in congenital toxoplasmosis.

    Directory of Open Access Journals (Sweden)

    Sarra E Jamieson

    2008-06-01

    Full Text Available Primary Toxoplasma gondii infection during pregnancy can be transmitted to the fetus. At birth, infected infants may have intracranial calcification, hydrocephalus, and retinochoroiditis, and new ocular lesions can occur at any age after birth. Not all children who acquire infection in utero develop these clinical signs of disease. Whilst severity of disease is influenced by trimester in which infection is acquired by the mother, other factors including genetic predisposition may contribute.In 457 mother-child pairs from Europe, and 149 child/parent trios from North America, we show that ocular and brain disease in congenital toxoplasmosis associate with polymorphisms in ABCA4 encoding ATP-binding cassette transporter, subfamily A, member 4. Polymorphisms at COL2A1 encoding type II collagen associate only with ocular disease. Both loci showed unusual inheritance patterns for the disease allele when comparing outcomes in heterozygous affected children with outcomes in affected children of heterozygous mothers. Modeling suggested either an effect of mother's genotype, or parent-of-origin effects. Experimental studies showed that both ABCA4 and COL2A1 show isoform-specific epigenetic modifications consistent with imprinting.These associations between clinical outcomes of congenital toxoplasmosis and polymorphisms at ABCA4 and COL2A1 provide novel insight into the molecular pathways that can be affected by congenital infection with this parasite.

  10. Mutations in the collagen XII gene define a new form of extracellular matrix-related myopathy.

    Science.gov (United States)

    Hicks, Debbie; Farsani, Golara Torabi; Laval, Steven; Collins, James; Sarkozy, Anna; Martoni, Elena; Shah, Ashoke; Zou, Yaqun; Koch, Manuel; Bönnemann, Carsten G; Roberts, Mark; Lochmüller, Hanns; Bushby, Kate; Straub, Volker

    2014-05-01

    Bethlem myopathy (BM) [MIM 158810] is a slowly progressive muscle disease characterized by contractures and proximal weakness, which can be caused by mutations in one of the collagen VI genes (COL6A1, COL6A2 and COL6A3). However, there may be additional causal genes to identify as in ∼50% of BM cases no mutations in the COL6 genes are identified. In a cohort of -24 patients with a BM-like phenotype, we first sequenced 12 candidate genes based on their function, including genes for known binding partners of collagen VI, and those enzymes involved in its correct post-translational modification, assembly and secretion. Proceeding to whole-exome sequencing (WES), we identified mutations in the COL12A1 gene, a member of the FACIT collagens (fibril-associated collagens with interrupted triple helices) in five individuals from two families. Both families showed dominant inheritance with a clinical phenotype resembling classical BM. Family 1 had a single-base substitution that led to the replacement of one glycine residue in the triple-helical domain, breaking the Gly-X-Y repeating pattern, and Family 2 had a missense mutation, which created a mutant protein with an unpaired cysteine residue. Abnormality at the protein level was confirmed in both families by the intracellular retention of collagen XII in patient dermal fibroblasts. The mutation in Family 2 leads to the up-regulation of genes associated with the unfolded protein response (UPR) pathway and swollen, dysmorphic rough-ER. We conclude that the spectrum of causative genes in extracellular matrix (ECM)-related myopathies be extended to include COL12A1.

  11. Synthesis and Characterization of Hydroxyapatite-Collagen-Chitosan (HA/Col/Chi) Composite Coated on Ti6Al4V

    Science.gov (United States)

    Charlena; Bikharudin, Ahmad; Wahyudi, Setyanto Tri

    2018-01-01

    HA-collagen-chitosan (HA/col/chi) composite is developed to increase bioactivity adhesiveness between the metal and the material composite and to improve corrosion resistance. The Ti6Al4V alloy was coated by soaking in HA/col/chi composite at room temperature and then allowed to stand for 5, 6, and 7 days. Diffraction pattern analysis of the coated Ti6Al4V alloy showed that the dominant phase were HA and Ti6Al4V alloy. Corrosion resistance test in media by using 0.9% NaCl showed the corrosion rate at the level of 0.3567 mpy, which was better than that of the uncoated Ti6Al4V alloy (0.4152 mpy). In vitro cytocompatibility assay on endothelial cell of calf pulmonary artery endothelium (CPAE) (ATCC-CCL 209) showed there was no toxicity in the cell culture with the percent inhibition of 33.33% after 72 hours of incubation.

  12. EuroCirCol: A key to New Physics

    CERN Multimedia

    Johannes Gutleber

    2015-01-01

    Monday 1 June saw the start of EuroCirCol, the EC-funded part of the FCC study that will develop the conceptual design for an energy-frontier hadron collider.   Attendees at the EuroCirCol meeting at CERN. The EuroCirCol kick-off event at CERN on 2 to 4 June brought together 62 participants to constitute governance bodies, commit to the project plan and align the organisation, structures and processes of 16 institutions from 10 countries. The goal of the project is to conceive a post-LHC research infrastructure around a 100 km circular energy-frontier hadron collider capable of reaching 100 TeV collisions. The project officially started on 1 June and will run for four years. The total estimated budget of 11.2 MEUR includes a 2.99 MEUR contribution from the Horizon 2020 programme dedicated to the development of new world-class research infrastructures. EuroCirCol will deliver a design for a hadron collider as part of the broader Future Circular Collider (FCC) study. It will provide input to an...

  13. Male Hypogonadism and Germ Cell Loss Caused by a Mutation in Polo-Like Kinase 4

    Science.gov (United States)

    Harris, Rebecca M.; Weiss, Jeffrey

    2011-01-01

    The genetic etiologies of male infertility remain largely unknown. To identify genes potentially involved in spermatogenesis and male infertility, we performed genome-wide mutagenesis in mice with N-ethyl-N-nitrosourea and identified a line with dominant hypogonadism and patchy germ cell loss. Genomic mapping and DNA sequence analysis identified a novel heterozygous missense mutation in the kinase domain of Polo-like kinase 4 (Plk4), altering an isoleucine to asparagine at residue 242 (I242N). Genetic complementation studies using a gene trap line with disruption in the Plk4 locus confirmed that the putative Plk4 missense mutation was causative. Plk4 is known to be involved in centriole formation and cell cycle progression. However, a specific role in mammalian spermatogenesis has not been examined. PLK4 was highly expressed in the testes both pre- and postnatally. In the adult, PLK4 expression was first detected in stage VIII pachytene spermatocytes and was present through step 16 elongated spermatids. Because the homozygous Plk4I242N/I242N mutation was embryonic lethal, all analyses were performed using the heterozygous Plk4+/I242N mice. Testis size was reduced by 17%, and histology revealed discrete regions of germ cell loss, leaving only Sertoli cells in these defective tubules. Testis cord formation (embryonic day 13.5) was normal. Testis histology was also normal at postnatal day (P)1, but germ cell loss was detected at P10 and subsequent ages. We conclude that the I242N heterozygous mutation in PLK4 is causative for patchy germ cell loss beginning at P10, suggesting a role for PLK4 during the initiation of spermatogenesis. PMID:21791561

  14. High resolution melting analysis: a rapid and accurate method to detect CALR mutations.

    Directory of Open Access Journals (Sweden)

    Cristina Bilbao-Sieyro

    Full Text Available The recent discovery of CALR mutations in essential thrombocythemia (ET and primary myelofibrosis (PMF patients without JAK2/MPL mutations has emerged as a relevant finding for the molecular diagnosis of these myeloproliferative neoplasms (MPN. We tested the feasibility of high-resolution melting (HRM as a screening method for rapid detection of CALR mutations.CALR was studied in wild-type JAK2/MPL patients including 34 ET, 21 persistent thrombocytosis suggestive of MPN and 98 suspected secondary thrombocytosis. CALR mutation analysis was performed through HRM and Sanger sequencing. We compared clinical features of CALR-mutated versus 45 JAK2/MPL-mutated subjects in ET.Nineteen samples showed distinct HRM patterns from wild-type. Of them, 18 were mutations and one a polymorphism as confirmed by direct sequencing. CALR mutations were present in 44% of ET (15/34, 14% of persistent thrombocytosis suggestive of MPN (3/21 and none of the secondary thrombocytosis (0/98. Of the 18 mutants, 9 were 52 bp deletions, 8 were 5 bp insertions and other was a complex mutation with insertion/deletion. No mutations were found after sequencing analysis of 45 samples displaying wild-type HRM curves. HRM technique was reproducible, no false positive or negative were detected and the limit of detection was of 3%.This study establishes a sensitive, reliable and rapid HRM method to screen for the presence of CALR mutations.

  15. Modified Proofreading PCR for Detection of Point Mutations, Insertions and Deletions Using a ddNTP-Blocked Primer

    Science.gov (United States)

    Chen, Qianqian; Chen, Xiaoxiang; Zhang, Sichao; Lan, Ke; Lu, Jian; Zhang, Chiyu

    2015-01-01

    The development of simple, accurate, rapid and cost-effective technologies for mutation detection is crucial to the early diagnosis and prevention of numerous genetic diseases, pharmacogenetics, and drug resistance. Proofreading PCR (PR-PCR) was developed for mutation detection in 1998 but is rarely applied due to its low efficiency in allele discrimination. Here we developed a modified PR-PCR method using a ddNTP-blocked primer and a mixture of DNA polymerases with and without the 3'-5' proofreading function. The ddNTP-blocked primer exhibited the best blocking efficiency to avoid nonspecific primer extension while the mixture of a tiny amount of high-fidelity DNA polymerase with a routine amount of Taq DNA polymerase provided the best discrimination and amplification effects. The modified PR-PCR method is quite capable of detecting various mutation types, including point mutations and insertions/deletions (indels), and allows discrimination amplification when the mismatch is located within the last eight nucleotides from the 3'-end of the ddNTP-blocked primer. The modified PR-PCR has a sensitivity of 1-5 × 102 copies and a selectivity of 5 × 10-5 mutant among 107 copies of wild-type DNA. It showed a 100% accuracy rate in the detection of P72R germ-line mutation in the TP53 gene among 60 clinical blood samples, and a high potential to detect rifampin-resistant mutations at low frequency in Mycobacterium tuberculosis using an adaptor and a fusion-blocked primer. These results suggest that the modified PR-PCR technique is effective in detection of various mutations or polymorphisms as a simple, sensitive and promising approach. PMID:25915410

  16. Abortive phage-infection and UV-protection markers on ColI plasmids from epidemic strains of Salmonella

    International Nuclear Information System (INIS)

    Barker, R.M.

    1988-01-01

    Cultures of Escherichia coli carrying ColI plasmids received in conjugation from strains of Salmonella typhimurium and S. agona were examined for abortive infection (Abi) of phage BF23 and for enhanced resistance to the lethal action of UV-irradiation (Uvr). The Abi character of stored cultures of E. coli was also compared with the reaction of the same stock culture tested 5 years before. Seven of the eight potential types differentiated by three characters were represented among 160 ColI plasmids: ColIa Abi + Uvr + (3 plasmids), ColIa Abi - Uvr + (1), ColIa Abi - Uvr-> (2), ColIb Abi + Uvr + (85), ColIb Abi + Uvr - (5), ColIb Abi - Uvr + (4), ColIb Abi-? Uvr - (60). Recognition that different plasmid types could be carried by strains of a clone proved useful in the interpretation of the epidemic spread of strains of S. typhimurium of phage type/biotype 141/9f in Scotland and in tracing the ancestry of a recently emerged rhamnose non-fermenting mutant strain of S. agona. (author)

  17. SLC26A4 mutations are associated with a specific inner ear malformation.

    Science.gov (United States)

    Fitoz, Suat; Sennaroğlu, Levent; Incesulu, Armağan; Cengiz, Filiz Başak; Koç, Yasemin; Tekin, Mustafa

    2007-03-01

    Inner ear anomalies have been reported in approximately 30% of children with early onset deafness. Identification of causative genetic factors in a large proportion of these patients was not successful. Mutations in the SLC26A4 gene have been detected in individuals with enlarged vestibular aqueduct (EVA) or Mondini dysplasia. We aimed to characterize the inner ear anomalies associated with SLC26A4 mutations. The SLC26A4 gene has been screened for mutations in 16 subjects from 14 unrelated Turkish families with a variety of inner ear anomalies ranging from Michel aplasia to incomplete partition-II and EVA. None of the patients was diagnosed to have a recognizable genetic syndrome. Additional four patients with Pendred syndrome from three families were included. Only one patient with EVA was found to have a heterozygous mutation (c.1586delT) in SLC26A4. All patients with Pendred syndrome had homozygous mutations and were noted to have either EVA or EVA associated with incomplete partition-II on the computed tomography of the temporal bone. SLC26A4 mutations are not associated with a large spectrum of inner ear anomalies. They, instead, result in a specific morphological appearance consistent with EVA or incomplete partition-II.

  18. Repair of ultraviolet-light damaged ColE1 factor carrying Escherichia coli genes for guanine synthesis

    International Nuclear Information System (INIS)

    Kibe, A.; Shimada, K.; Tagaki, Y.

    1979-01-01

    Hybrid ColE1 plasmids called ColE1-cos lambda-guaA or ColE1-cos lambda-gal can be efficiently transduced into various E.coli K-12 cells through packaging into lambda phage particles. Using these plasmids, repair of ultraviolet-light (UV) damaged ColE1 DNAs was studied in various UV sensitive E.coli K-12 mutants. The host mutations uvrA and uvrB markedly reduced host-cell reactivation of UV-irradiated ColE1-cos lambda-guaA. Pre-existing hybrid ColE1 plasmids had no effect on the frequency of lambda phage-mediated transduction of another differentially marked hybrid ColE1 DNAs. ColE1-cos lambda-guaA and ColE1-cos lambda-gal DNAs could temporarily but not stably co-exist in E.coli K-12 recA cells. The presence of ColE1-cos lambda-gal in uvrB cells promoted the repair of super-infected UV-irradiated ColE1-cos lambda-guaA about 7-fold. The same ColE1-cos lambda-gal plasmid in a uvrB recA double mutant did not have this promoting effect. These results indicate that the effect of resident hybrid ColE1 plasmids is manifested by the host recA + gene function(s) and suggest that ColE1 plasmit itself provides no recA + -like functions. (orig.) [de

  19. Repair of ultraviolet-light damaged ColE1 factor carrying Escherichia coli genes for guanine synthesis

    Energy Technology Data Exchange (ETDEWEB)

    Kibe, A; Shimada, K; Tagaki, Y [Kyushu Univ., Fukuoka (Japan). Dept. of Biochemistry

    1979-01-01

    Hybrid ColE1 plasmids called ColE1-cos lambda-guaA or ColE1-cos lambda-gal can be efficiently transduced into various E.coli K-12 cells through packaging into lambda phage particles. Using these plasmids, repair of ultraviolet-light (UV) damaged ColE1 DNAs was studied in various UV sensitive E.coli K-12 mutants. The host mutations uvrA and uvrB markedly reduced host-cell reactivation of UV-irradiated ColE1-cos lambda-guaA. Pre-existing hybrid ColE1 plasmids had no effect on the frequency of lambda phage-mediated transduction of another differentially marked hybrid ColE1 DNAs. ColE1-cos lambda-guaA and ColE1-cos lambda-gal DNAs could temporarily but not stably co-exist in E.coli K-12 recA cells. The presence of ColE1-cos lambda-gal in uvrB cells promoted the repair of super-infected UV-irradiated ColE1-cos lambda-guaA about 7-fold. The same ColE1-cos lambda-gal plasmid in a uvrB recA double mutant did not have this promoting effect. These results indicate that the effect of resident hybrid ColE1 plasmids is manifested by the host recA/sup +/ gene function(s) and suggest that ColE1 plasmit itself provides no recA/sup +/-like functions.

  20. Targeting the LRP5 pathway improves bone properties in a mouse model of osteogenesis imperfecta.

    Science.gov (United States)

    Jacobsen, Christina M; Barber, Lauren A; Ayturk, Ugur M; Roberts, Heather J; Deal, Lauren E; Schwartz, Marissa A; Weis, MaryAnn; Eyre, David; Zurakowski, David; Robling, Alexander G; Warman, Matthew L

    2014-10-01

    The cell surface receptor low-density lipoprotein receptor-related protein 5 (LRP5) is a key regulator of bone mass and bone strength. Heterozygous missense mutations in LRP5 cause autosomal dominant high bone mass (HBM) in humans by reducing binding to LRP5 by endogenous inhibitors, such as sclerostin (SOST). Mice heterozygous for a knockin allele (Lrp5(p.A214V) ) that is orthologous to a human HBM-causing mutation have increased bone mass and strength. Osteogenesis imperfecta (OI) is a skeletal fragility disorder predominantly caused by mutations that affect type I collagen. We tested whether the LRP5 pathway can be used to improve bone properties in animal models of OI. First, we mated Lrp5(+/p.A214V) mice to Col1a2(+/p.G610C) mice, which model human type IV OI. We found that Col1a2(+/p.G610C) ;Lrp5(+/p.A214V) offspring had significantly increased bone mass and strength compared to Col1a2(+/p.G610C) ;Lrp5(+/+) littermates. The improved bone properties were not a result of altered mRNA expression of type I collagen or its chaperones, nor were they due to changes in mutant type I collagen secretion. Second, we treated Col1a2(+/p.G610C) mice with a monoclonal antibody that inhibits sclerostin activity (Scl-Ab). We found that antibody-treated mice had significantly increased bone mass and strength compared to vehicle-treated littermates. These findings indicate increasing bone formation, even without altering bone collagen composition, may benefit patients with OI. © 2014 American Society for Bone and Mineral Research.

  1. CNV analysis in Tourette syndrome implicates large genomic rearrangements in COL8A1 and NRXN1.

    Directory of Open Access Journals (Sweden)

    Abhishek Nag

    Full Text Available Tourette syndrome (TS is a neuropsychiatric disorder with a strong genetic component. However, the genetic architecture of TS remains uncertain. Copy number variation (CNV has been shown to contribute to the genetic make-up of several neurodevelopmental conditions, including schizophrenia and autism. Here we describe CNV calls using SNP chip genotype data from an initial sample of 210 TS cases and 285 controls ascertained in two Latin American populations. After extensive quality control, we found that cases (N = 179 have a significant excess (P = 0.006 of large CNV (>500 kb calls compared to controls (N = 234. Amongst 24 large CNVs seen only in the cases, we observed four duplications of the COL8A1 gene region. We also found two cases with ∼400 kb deletions involving NRXN1, a gene previously implicated in neurodevelopmental disorders, including TS. Follow-up using multiplex ligation-dependent probe amplification (and including 53 more TS cases validated the CNV calls and identified additional patients with rearrangements in COL8A1 and NRXN1, but none in controls. Examination of available parents indicates that two out of three NRXN1 deletions detected in the TS cases are de-novo mutations. Our results are consistent with the proposal that rare CNVs play a role in TS aetiology and suggest a possible role for rearrangements in the COL8A1 and NRXN1 gene regions.

  2. CNV analysis in Tourette syndrome implicates large genomic rearrangements in COL8A1 and NRXN1.

    Science.gov (United States)

    Nag, Abhishek; Bochukova, Elena G; Kremeyer, Barbara; Campbell, Desmond D; Muller, Heike; Valencia-Duarte, Ana V; Cardona, Julio; Rivas, Isabel C; Mesa, Sandra C; Cuartas, Mauricio; Garcia, Jharley; Bedoya, Gabriel; Cornejo, William; Herrera, Luis D; Romero, Roxana; Fournier, Eduardo; Reus, Victor I; Lowe, Thomas L; Farooqi, I Sadaf; Mathews, Carol A; McGrath, Lauren M; Yu, Dongmei; Cook, Ed; Wang, Kai; Scharf, Jeremiah M; Pauls, David L; Freimer, Nelson B; Plagnol, Vincent; Ruiz-Linares, Andrés

    2013-01-01

    Tourette syndrome (TS) is a neuropsychiatric disorder with a strong genetic component. However, the genetic architecture of TS remains uncertain. Copy number variation (CNV) has been shown to contribute to the genetic make-up of several neurodevelopmental conditions, including schizophrenia and autism. Here we describe CNV calls using SNP chip genotype data from an initial sample of 210 TS cases and 285 controls ascertained in two Latin American populations. After extensive quality control, we found that cases (N = 179) have a significant excess (P = 0.006) of large CNV (>500 kb) calls compared to controls (N = 234). Amongst 24 large CNVs seen only in the cases, we observed four duplications of the COL8A1 gene region. We also found two cases with ∼400 kb deletions involving NRXN1, a gene previously implicated in neurodevelopmental disorders, including TS. Follow-up using multiplex ligation-dependent probe amplification (and including 53 more TS cases) validated the CNV calls and identified additional patients with rearrangements in COL8A1 and NRXN1, but none in controls. Examination of available parents indicates that two out of three NRXN1 deletions detected in the TS cases are de-novo mutations. Our results are consistent with the proposal that rare CNVs play a role in TS aetiology and suggest a possible role for rearrangements in the COL8A1 and NRXN1 gene regions.

  3. Knock-in human GDF5 proregion L373R mutation as a mouse model for proximal symphalangism.

    Science.gov (United States)

    Zhang, Xinxin; Xing, Xuesha; Liu, Xing; Hu, Yu; Qu, Shengqiang; Wang, Heyi; Luo, Yang

    2017-12-26

    Proximal symphalangism (SYM1) is an autosomal dominant disorder, mainly characterized by bony fusions of the proximal phalanges of the hands and feet. GDF5 and NOG were identified to be responsible for SYM1. We have previously reported on a p.Leu373Arg mutation in the GDF5 proregion present in a Chinese family with SYM1. Here, we investigated the effects of the GDF-L373R mutation. The variant caused proteolysis efficiency of GDF5 increased in ATDC5 cells. The variant also caused upregulation of SMAD1/5/8 phosphorylation and increased expression of target genes SMURF1 , along with COL2A1 and SOX9 which are factors associated with chondrosis. Furthermore, we developed a human-relevant SYM1 mouse model by making a Gdf5 L367R (the orthologous position for L373R in humans) knock-in mouse. Gdf5 L367R/+ and Gdf5 L367R/L367R mice displayed stiffness and adhesions across the proximal phalanx joint which were in complete accord with SYM1. It was also confirmed the joint formation and development was abnormal in Gdf5 L367R/+ and Gdf5 L367R/L367R mice, including the failure to develop the primary ossification center and be hypertrophic chondrocytes during embryonic development. This knock-in mouse model offers a tool for assessing the pathogenesis of SYM1 and the function of the GDF5 proregion.

  4. Novel mutations of CYP3A4 in Chinese.

    Science.gov (United States)

    Hsieh, K P; Lin, Y Y; Cheng, C L; Lai, M L; Lin, M S; Siest, J P; Huang, J D

    2001-03-01

    Human cytochrome P450 3A4 is a major P450 enzyme in the liver and gastrointestinal tract. It plays important roles in the metabolism of a wide variety of drugs, some endogenous steroids, and harmful environmental contaminants. CYP3A4 exhibits a remarkable interindividual activity variation as high as 20-fold. To investigate whether the interindividual variation in CYP3A4 levels can be partly explained by genetic polymorphism, we analyzed DNA samples from 102 Chinese subjects by polymerase chain reaction (PCR)-single-strand conformation polymorphism analysis for novel point mutation in the CYP3A4 coding sequence and promoter region. Using PCR and directed sequencing method to establish the complete intron sequence of CYP3A4 from leukocytes, the complete genomic sequence from exon 1 through 13 of CYP3A4 was determined and published in the GenBank database (accession no. AF209389). CYP3A4-specific primers were designed accordingly. After PCR-single-strand conformation polymorphism and restriction fragment length polymorphism screening, we found three novel mutations; two are point mutations and one is insertion. The first variant allele (CYP3A4*4), an Ile118Val change, was found in 3 of 102 Chinese subjects. The next allele (CYP3A4*5), which causes a Pro218Arg amino acid change, was found in 2 of 102 subjects. We found an insertion in A(17776), designated as CYP3A4*6, which causes frame shift and an early stop codon in exon 9, in one heterozygous subject. We also investigated the CYP3A4 activity in these mutant subjects by measuring the morning spot urinary 6beta-hydroxycortisol to free cortisol ratio with the enzyme-linked immunosorbent assay method. When compared with healthy Chinese population data, the 6beta-hydroxycortisol to free cortisol ratio data suggested that these alleles (CYP3A4*4, CYP3A4*5, and CYP3A4*6) may decrease the CYP3A4 activity. Incidences of these mutations in Chinese subjects are rare. The prevalence of these point mutations in other ethnic

  5. Validation of high-resolution DNA melting analysis for mutation scanning of the CDKL5 gene: identification of novel mutations.

    Science.gov (United States)

    Raymond, Laure; Diebold, Bertrand; Leroux, Céline; Maurey, Hélène; Drouin-Garraud, Valérie; Delahaye, Andre; Dulac, Olivier; Metreau, Julia; Melikishvili, Gia; Toutain, Annick; Rivier, François; Bahi-Buisson, Nadia; Bienvenu, Thierry

    2013-01-01

    Mutations in the cyclin-dependent kinase-like 5 gene (CDKL5) have been predominantly described in epileptic encephalopathies of female, including infantile spasms with Rett-like features. Up to now, detection of mutations in this gene was made by laborious, expensive and/or time consuming methods. Here, we decided to validate high-resolution melting analysis (HRMA) for mutation scanning of the CDKL5 gene. Firstly, using a large DNA bank consisting to 34 samples carrying different mutations and polymorphisms, we validated our analytical conditions to analyse the different exons and flanking intronic sequences of the CDKL5 gene by HRMA. Secondly, we screened CDKL5 by both HRMA and denaturing high performance liquid chromatography (dHPLC) in a cohort of 135 patients with early-onset seizures. Our results showed that point mutations and small insertions and deletions can be reliably detected by HRMA. Compared to dHPLC, HRMA profiles are more discriminated, thereby decreasing unnecessary sequencing. In this study, we identified eleven novel sequence variations including four pathogenic mutations (2.96% prevalence). HRMA appears cost-effective, easy to set up, highly sensitive, non-toxic and rapid for mutation screening, ideally suited for large genes with heterogeneous mutations located along the whole coding sequence, such as the CDKL5 gene. Copyright © 2012 Elsevier B.V. All rights reserved.

  6. Mutational characterization of the P3H1/CRTAP/CypB complex in recessive osteogenesis imperfecta.

    Science.gov (United States)

    Barbirato, C; Trancozo, M; Almeida, M G; Almeida, L S; Santos, T O; Duarte, J C G; Rebouças, M R G O; Sipolatti, V; Nunes, V R R; Paula, F

    2015-12-03

    Osteogenesis imperfecta (OI) is a genetic disease characterized by bone deformities and fractures. Most cases are caused by autosomal dominant mutations in the type I collagen genes COL1A1 and COL1A2; however, an increasing number of recessive mutations in other genes have been reported. The LEPRE1, CRTAP, and PPIB genes encode proteins that form the P3H1/CRTAP/CypB complex, which is responsible for posttranslational modifications of type I collagen. In general, mutations in these genes lead to severe and lethal phenotypes of recessive OI. Here, we describe sixteen genetic variations detected in LEPRE1, CRTAP, and PPIB from 25 Brazilian patients with OI. Samples were screened for mutations on single-strand conformation polymorphism gels and variants were determined by automated sequencing. Seven variants were detected in patients but were absent in control samples. LEPRE1 contained the highest number of variants, including the previously described West African allele (c.1080+1G>T) found in one patient with severe OI as well as a previously undescribed p.Trp675Leu change that is predicted to be disease causing. In CRTAP, one patient carried the c.558A>G homozygous mutation, predicted as disease causing through alteration of a splice site. Genetic variations detected in the PPIB gene are probably not pathogenic due to their localization or because of their synonymous effect. This study enhances our knowledge about the mutational pattern of the LEPRE1, CRTAP, and PPIB genes. In addition, the results strengthen the proposition that LEPRE1 should be the first gene analyzed in mutation detection studies in patients with recessive OI.

  7. Mutation spectrum of genes associated with steroid-resistant nephrotic syndrome in Chinese children.

    Science.gov (United States)

    Wang, Ying; Dang, Xiqiang; He, Qingnan; Zhen, Yan; He, Xiaoxie; Yi, Zhuwen; Zhu, Kuichun

    2017-08-20

    Approximately 20% of children with idiopathic nephrotic syndrome do not respond to steroid therapy. More than 30 genes have been identified as disease-causing genes for the steroid-resistant nephrotic syndrome (SRNS). Few reports were from the Chinese population. The coding regions of genes commonly associated with SRNS were analyzed to characterize the gene mutation spectrum in children with SRNS in central China. The first phase study involved 38 children with five genes (NPHS1, NPHS2, PLCE1, WT1, and TRPC6) by Sanger sequencing. The second phase study involved 33 children with 17 genes by next generation DNA sequencing (NGS. 22 new patients, and 11 patients from first phase study but without positive findings). Overall deleterious or putatively deleterious gene variants were identified in 19 patients (31.7%), including four NPHS1 variants among five patients and three PLCE1 variants among four other patients. Variants in COL4A3, COL4A4, or COL4A5 were found in six patients. Eight novel variants were identified, including two in NPHS1, two in PLCE1, one in NPHS2, LAMB2, COL4A3, and COL4A4, respectively. 55.6% of the children with variants failed to respond to immunosuppressive agent therapy, while the resistance rate in children without variants was 44.4%. Our results show that screening for deleterious variants in some common genes in children clinically suspected with SRNS might be helpful for disease diagnosis as well as prediction of treatment efficacy and prognosis. Copyright © 2017 Elsevier B.V. All rights reserved.

  8. Evolution of Regions Containing Antibiotic Resistance Genes in FII-2-FIB-1 ColV-Colla Virulence Plasmids.

    Science.gov (United States)

    Moran, Robert A; Hall, Ruth M

    2018-05-01

    Three ColV virulence plasmids carrying antibiotic resistance genes were assembled from draft genome sequences of commensal ST95, ST131, and ST2705 Escherichia coli isolates from healthy Australians. Plasmids pCERC4, pCERC5, and pCERC9 include almost identical backbones containing FII-2 and FIB-1 replicons and the conserved ColV virulence region with an additional ColIa determinant. Only pCERC5 includes a complete, uninterrupted F-like transfer region and was able to conjugate. pCERC5 and pCERC9 contain Tn1721, carrying the tet(A) tetracycline resistance determinant in the same location, with Tn2 (bla TEM ; ampicillin resistance) interrupting the Tn1721 in pCERC5. pCERC4 has a Tn1721/Tn21 hybrid transposon carrying dfrA5 (trimethoprim resistance) and sul1 (sulfamethoxazole resistance) in a class 1 integron. Four FII-2:FIB-1 ColV-ColIa plasmids in the GenBank nucleotide database have a related transposon in the same position, but an IS26 has reshaped the resistance gene region, deleting 2,069 bp of the integron 3'-CS, including sul1, and serving as a target for IS26 translocatable units containing bla TEM , sul2 and strAB (streptomycin resistance), or aphA1 (kanamycin/neomycin resistance). Another ColV-ColIa plasmid containing a related resistance gene region has lost the FII replicon and acquired a unique transfer region via recombination within the resistance region and at oriT. Eighteen further complete ColV plasmid sequences in GenBank contained FIB-1, but the FII replicons were of three types, FII-24, FII-18, and a variant of FII-36.

  9. A de novo deletion mutation in SOX10 in a Chinese family with Waardenburg syndrome type 4.

    Science.gov (United States)

    Wang, Xiong; Zhu, Yaowu; Shen, Na; Peng, Jing; Wang, Chunyu; Liu, Haiyi; Lu, Yanjun

    2017-01-27

    Waardenburg syndrome type 4 (WS4) or Waardenburg-Shah syndrome is a rare genetic disorder with a prevalence of <1/1,000,000 and characterized by the association of congenital sensorineural hearing loss, pigmentary abnormalities, and intestinal aganglionosis. There are three types of WS4 (WS4A-C) caused by mutations in endothelin receptor type B, endothelin 3, and SRY-box 10 (SOX10), respectively. This study investigated a genetic mutation in a Chinese family with one WS4 patient in order to improve genetic counselling. Genomic DNA was extracted, and mutation analysis of the three WS4 related genes was performed using Sanger sequencing. We detected a de novo heterozygous deletion mutation [c.1333delT (p.Ser445Glnfs*57)] in SOX10 in the patient; however, this mutation was absent in the unaffected parents and 40 ethnicity matched healthy controls. Subsequent phylogenetic analysis and three-dimensional modelling of the SOX10 protein confirmed that the c.1333delT heterozygous mutation was pathogenic, indicating that this mutation might constitute a candidate disease-causing mutation.

  10. A Novel Missense Mutation in SLC5A5 Gene in a Sudanese Family with Congenital Hypothyroidism.

    Science.gov (United States)

    Watanabe, Yui; Ebrhim, Reham Shareef; Abdullah, Mohamed A; Weiss, Roy E

    2018-05-15

    Thyroid hormone synthesis requires the presence of iodide. The sodium iodide symporter (NIS) is a glycoprotein which mediates the active uptake of iodide from the blood stream into the thyroid grand. NIS defects due to SLC5A5 gene mutations are known to cause congenital hypothyroidism (CH). The proposita is a 28-year-old female whose origin is the North Sudan where neonatal screening for CH is not available. She presented with severe constipation and a goiter at the age of 40 days. Laboratory testing confirmed CH and she was started on levothyroxine (L-T4). Presumably due to the delayed treatment the patient developed mental retardation. Her younger sister presented with a goiter, tongue protrusion and umbilical hernia and the youngest brother was also diagnosed with CH based on the TSH >100 µIU/mL at the age of 22 days and 8 days, respectively. Two siblings were treated with L-T4 and had normal development. Their consanguineous parents had no history of thyroid disorders. We performed whole exome sequencing (WES) on the proposita. WES identified a novel homozygous missense mutation in the SLC5A5 gene: c.1042T>G, p.Tyr348Asp, which was subsequently confirmed by Sanger sequencing. All affected children were homozygous for the same mutation and their unaffected mother was heterozygous. The NIS protein is composed of 13 transmembrane segments (TMS), an extracellular amino-terminus and an intracellular carboxyl terminus. The mutation is located in the TMS IX which has the most β-OH group-containing amino acids (serine and threonine) which is implicated in Na+ binding and translocation. In conclusion, a novel homozygous missense mutation in the SLC5A5 gene was identified in the Sudanese family with CH. The mutation is located in the TMS IX of the NIS protein which is essential for NIS function. Low iodine intake in Sudan is considered to affect severity of hypothyroidism in the patients.

  11. Mutation Analysis in Classical Phenylketonuria Patients Followed by Detecting Haplotypes Linked to Some PAH Mutations.

    Science.gov (United States)

    Dehghanian, Fatemeh; Silawi, Mohammad; Tabei, Seyed M B

    2017-02-01

    Deficiency of phenylalanine hydroxylase (PAH) enzyme and elevation of phenylalanine in body fluids cause phenylketonuria (PKU). The gold standard for confirming PKU and PAH deficiency is detecting causal mutations by direct sequencing of the coding exons and splicing involved sequences of the PAH gene. Furthermore, haplotype analysis could be considered as an auxiliary approach for detecting PKU causative mutations before direct sequencing of the PAH gene by making comparisons between prior detected mutation linked-haplotypes and new PKU case haplotypes with undetermined mutations. In this study, 13 unrelated classical PKU patients took part in the study detecting causative mutations. Mutations were identified by polymerase chain reaction (PCR) and direct sequencing in all patients. After that, haplotype analysis was performed by studying VNTR and PAHSTR markers (linked genetic markers of the PAH gene) through application of PCR and capillary electrophoresis (CE). Mutation analysis was performed successfully and the detected mutations were as follows: c.782G>A, c.754C>T, c.842C>G, c.113-115delTCT, c.688G>A, and c.696A>G. Additionally, PAHSTR/VNTR haplotypes were detected to discover haplotypes linked to each mutation. Mutation detection is the best approach for confirming PAH enzyme deficiency in PKU patients. Due to the relatively large size of the PAH gene and high cost of the direct sequencing in developing countries, haplotype analysis could be used before DNA sequencing and mutation detection for a faster and cheaper way via identifying probable mutated exons.

  12. High Resolution Melting Analysis for Detecting p53 Gene Mutations in Patients with Non-small Cell Lung Cancer

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    Zhihong CHEN

    2011-10-01

    Full Text Available Background and objective It has been proven that p53 gene was related to many human cancers. The mutations in p53 gene play an important role in carcinogensis and mostly happened in exon 5-8. The aim of this study is to establish a high resolution melting (HRM assay to detect p53 mutations from patients with non-small cell lung cancer (NSCLC, to investigate the characteristics of p53 gene mutations, and to analyze the relationship between p53 mutations and evolution regularity of pathogenesis. Methods p53 mutations in exon 5-8 were detected by HRM assay on DNA insolated from 264 NSCLC samples derived from tumor tissues and 54 control samples from pericancerous pulmonary tissues. The mutation samples by the HRM assay were confirmed by sequencing technique. Samples which were positive by HRM but wild type by sequencing were further confirmed by sub-clone and sequencing. Results No mutation was found in 54 pericancerous pulmonary samples by HRM assay. 104 of the 264 tumor tissues demonstrated mutation curves by HRM assay, 102 samples were confirmed by sequencing, including 95 point mutations and 7 frame shift mutations by insertion or deletion. The mutation rate of p53 gene was 39.4%. The mutation rate from exon 5-8 were 11.7%, 8%, 12.5% and 10.6%, respectively and there was no statistically significant difference between them (P=0.35. p53 mutations were significantly more frequent in males than that in females, but not related to the other clinicopathologic characteristics. Conclusion The results indicate that HRM is a sensitive in-tube methodology to detect for mutations in clinical samples. The results suggest that the arising p53 mutations in NSCLC may be due to spontaneous error in DNA synthesis and repair.

  13. Detection of HIV drug resistance mutations in pregnant women receiving single dose Nevirapine in south India

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    Mini S Jacob

    2011-01-01

    Full Text Available Background: Single dose of Nevirapine to prevent mother to child transmission of HIV is the commonest preventive regimen in resource-limited countries. Objectives: The objective of this study was to detect drug-resistant virus after single dose of Nevirapine (sdNVP provided to delivering HIV seropositive (HIV+ve women and to evaluate the time taken for its decay. Results: Of the 36 consenting HIV+ve pregnant women enrolled into the study, the mean hemoglobin and total lymphocyte counts were 10.8 g/dl and 1843 cells/mm 3 , respectively. Mean CD4 counts in 64% of women was 363 cells/mm 3 and mean viral load for 16/36 women was 28,143 copies/ml of plasma. Nevirapine-resistance mutations were detected in 28% of women at delivery; using OLA (Oligonucleotide Ligation Assay. K103N mutations were seen in 19.4% of women while the Y181C mutation was seen in 5%. Both the mutations were detected in 2.7% of women. Sequential blood samples collected at delivery, 7-10 days, 6 weeks, 4 months, 6 months and one year postpartum showed that 81% of K103N mutations and 66.7% of Y181C mutations were detected at 6 weeks postpartum . Wild-type virus had replaced the mutants by one year postpartum in all women except one. Conclusion : These observations are relevant for future treatment with antiretroviral therapy in these women for their HIV disease.

  14. Vascular Ehlers-Danlos Syndrome With a Novel Missense COL3A1 Mutation Present With Pulmonary Complications and Iliac Arterial Dissection.

    Science.gov (United States)

    Gu, Guangchao; Yang, Hang; Cui, Lijia; Fu, Yuanyuan; Li, Fangda; Zhou, Zhou; Zheng, Yuehong

    2018-02-01

    Vascular Ehlers-Danlos syndrome (vEDS) is a life-threatening connective tissue disorder due to its high tendency of arterial and organ rupture. Pulmonary complications in vEDS are rare. We present a young male patient with vEDS who developed severe pulmonary complications and severe rupture of the iliac artery at different stages of his life. Vascular Ehlers-Danlos syndrome was diagnosed based on clinical manifestations and confirmed by the identification of COL3A1 gene mutation. Due to high bleeding tendency and weak cardiopulmonary capacity, conservative treatment was taken for him. To our knowledge, this is the first report of vEDS case in which the patient developed both pulmonary complications and dissection of large arteries. Our report emphasizes the importance of considering vEDS when an adolescent develops unexplained pulmonary cysts with fragility of lung tissues. Genetic counseling and close monitoring should be performed for earlier diagnosis and prevention of severe complications of large arteries. The typical presentations of vEDS were also discussed by means of a review of case reports on vEDS with pulmonary complications.

  15. Linkage analysis in a family with Stickler syndrome leads to the exclusion of the COL2A1 locus

    Energy Technology Data Exchange (ETDEWEB)

    Mottes, M.; Zolezzi, F.; Pignatti, P.F. [Univ. of Verona (Italy)

    1994-09-01

    Hereditary arthro-ophtalmopathy (AO) or Stickler Syndrome (MIM No. 10830) is a dominantly inherited disorder characterized by vitro-retinal degeneration and other connective tissue disturbances. Mutations in the COL2A1 gene, coding for type II collagen chains, have been described in a few patients. The wide spectrum of clinical manifestations is presumably due to genetic heterogeneity, since only about 50% of the Stickler families so far studied show cosegregation of the disease with the COL2A1 locus. We have investigated a large pedigree (19 individuals of whom 9 are affected) in which severe myopia with vitro-retinal degeneration consegregated with joint laxity, recurrent inguinal hernias, and degenerative changes of the hip and the knee. The 3{prime} end COL2A1 VNTR polymorphism was utilized for linkage analysis. In order to get the maximum informativity, we have analyzed the allelic microheterogeneity of this VNTR, due to the repeat sequence variation, by means of a single strand polymorphism. Mendelian inheritance of the different single strands was observed as expected. Discordance of segregation between the disease and the COL2A1 locus was thus established inequivocally in this family.

  16. Cisto gigante de colédoco Giant choledochal cyst

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    Olival Cirilo Lucena da Fonseca-Neto

    2007-12-01

    Full Text Available INTRODUÇÃO: A doença cística das vias biliares é anomalia congênita que pode acometer as vias biliares intra e/ou extra-hepáticas. A extra-hepática inclui os cistos de colédoco e a intra-hepática é conhecida por Doença de Caroli. Os cistos de colédoco de tamanho gigante são muito raros. OBJETIVO: Relatar o caso de um cisto de colédoco de tamanho gigante em uma paciente feminina. RELATO DE CASO: Mulher de 19 anos foi admitida com história de icterícia e acolia fecal há sete dias. Referia dor epigástrica associada com ingestão de dieta rica em gordura. Nos antecedentes pessoais relatou dois episódios de icterícia, aos 8 e 14 anos, que progrediram espontaneamente. No exame físico apresentava icterícia (+3/+4 e uma massa palpável indolor em mesogástrio foram os únicos achados. A ultrassonografia demonstrou grande formação cística de paredes finas adjacente ao hepatocolédoco, pâncreas e rim direito que media 18,5 x 10,2 cm. A colangioressonância confirmou o grande cisto de colédoco e hepatojejunoanastomose em "Y" de Roux após excisão do cisto e colecistectomia foi realizada. A formação cística media 20 x 15,5 x 12,5 cm e com um volume médio de 1000 mL. A paciente encontra-se em acompanhamento ambulatorial sem alterações hepatobiliares após o sétimo mês da operação. CONCLUSÃO: O cisto de colédoco deve fazer parte do diagnóstico diferencial em pacientes adultos jovens com icterícia e massa palpável; no entanto, a diferenciação entre ele e neoplasia maligna deve ser pesquisada.BACKGROUND: Choledochal cyst represents a rare congenital anomaly, eventually associated with intra and extrahepatic biliary tract disorders. Extrahepatic diseases include choledochal cysts and congenital dilation of the lower intrahepatic bile duct is known as Caroli's disease. Giant choledochal cyst constitutes a very rare abnormality. AIM:To report a giant choledochal cyst in a female patient. CASE REPORT: A 19-year

  17. DETECTION OF RECESSIVE MUTATIONS (CVM, BLAD AND RED FACTOR INHOLSTEIN BULLS IN SLOVENIA

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    Betka LOGAR

    2008-07-01

    Full Text Available Detection of recessive mutations that causes complex vertebral malformation (CVM and bovine leukocyte adhesion defi ciency (BLAD in Holstein cattle is especially required for bulls, which are used for artifi cial insemination (A.I.; these enable elimination of carriers from the A.I. programs and therefore prevent transmission of unwanted mutations to a large number of offspring. Some breeders are also interested in the identifi cation of carriers of recessive allele for red and white coat colour (Red factor. Here, we performed genetic tests for detection of mutations associated with CVM, BLAD and Red factor using methods previously reported or modifi ed methods. Analysis of Holstein bulls, which were recommended for A.I in Slovenia in the years 2007 and 2008, revealed four (10 % carriers of CVM, and two (5.4 % carriers of red gene, while all bulls were non-carriers of BLAD.

  18. Mosaicism for dominant collagen 6 mutations as a cause for intrafamilial phenotypic variability.

    Science.gov (United States)

    Donkervoort, Sandra; Hu, Ying; Stojkovic, Tanya; Voermans, Nicol C; Foley, A Reghan; Leach, Meganne E; Dastgir, Jahannaz; Bolduc, Véronique; Cullup, Thomas; de Becdelièvre, Alix; Yang, Lin; Su, Hai; Meilleur, Katherine; Schindler, Alice B; Kamsteeg, Erik-Jan; Richard, Pascale; Butterfield, Russell J; Winder, Thomas L; Crawford, Thomas O; Weiss, Robert B; Muntoni, Francesco; Allamand, Valérie; Bönnemann, Carsten G

    2015-01-01

    Collagen 6-related dystrophies and myopathies (COL6-RD) are a group of disorders that form a wide phenotypic spectrum, ranging from severe Ullrich congenital muscular dystrophy, intermediate phenotypes, to the milder Bethlem myopathy. Both inter- and intrafamilial variable expressivity are commonly observed. We present clinical, immunohistochemical, and genetic data on four COL6-RD families with marked intergenerational phenotypic heterogeneity. This variable expression seemingly masquerades as anticipation is due to parental mosaicism for a dominant mutation, with subsequent full inheritance and penetrance of the mutation in the heterozygous offspring. We also present an additional fifth simplex patient identified as a mosaic carrier. Parental mosaicism was confirmed in the four families through quantitative analysis of the ratio of mutant versus wild-type allele (COL6A1, COL6A2, and COL6A3) in genomic DNA from various tissues, including blood, dermal fibroblasts, and saliva. Consistent with somatic mosaicism, parental samples had lower ratios of mutant versus wild-type allele compared with the fully heterozygote offspring. However, there was notable variability of the mutant allele levels between tissues tested, ranging from 16% (saliva) to 43% (fibroblasts) in one mosaic father. This is the first report demonstrating mosaicism as a cause of intrafamilial/intergenerational variability of COL6-RD, and suggests that sporadic and parental mosaicism may be more common than previously suspected. © 2014 WILEY PERIODICALS, INC.

  19. Collagen XVIII Mutation in Knobloch Syndrome with Acute Lymphoblastic Leukemia

    Science.gov (United States)

    Mahajan, Vinit B.; Olney, Ann Haskins; Garrett, Penny; Chary, Ajit; Dragan, Ecaterina; Lerner, Gary; Murray, Jeffrey; Bassuk, Alexander G.

    2010-01-01

    Knobloch syndrome (KNO) is caused by mutations in the collagen XIII gene (COL18A1) and patients develop encephalocele and vitreoretinal degeneration. Here we report an El Salvadorian family where two sisters showed features of KNO. One of the siblings also developed acute lymphoblastic leukemia. DNA sequencing of COL18A1revealed a homozygous, 2-base pair deletion (c3514-3515delCT) in exon 41, which leads to abnormal collagen XVIII and deficiency of its proteolytic cleavage product endostatin. KNO patients with mutations in COL18A1 may be at risk for endostatin-related conditions including malignancy. PMID:20799329

  20. Real-time PCR-based method for the rapid detection of extended RAS mutations using bridged nucleic acids in colorectal cancer.

    Science.gov (United States)

    Iida, Takao; Mizuno, Yukie; Kaizaki, Yasuharu

    2017-10-27

    Mutations in RAS and BRAF are predictors of the efficacy of anti-epidermal growth factor receptor (EGFR) therapy in patients with metastatic colorectal cancer (mCRC). Therefore, simple, rapid, cost-effective methods to detect these mutations in the clinical setting are greatly needed. In the present study, we evaluated BNA Real-time PCR Mutation Detection Kit Extended RAS (BNA Real-time PCR), a real-time PCR method that uses bridged nucleic acid clamping technology to rapidly detect mutations in RAS exons 2-4 and BRAF exon 15. Genomic DNA was extracted from 54 formalin-fixed paraffin-embedded (FFPE) tissue samples obtained from mCRC patients. Among the 54 FFPE samples, BNA Real-time PCR detected 21 RAS mutations (38.9%) and 5 BRAF mutations (9.3%), and the reference assay (KRAS Mutation Detection Kit and MEBGEN™ RASKET KIT) detected 22 RAS mutations (40.7%). The concordance rate of detected RAS mutations between the BNA Real-time PCR assay and the reference assays was 98.2% (53/54). The BNA Real-time PCR assay proved to be a more simple, rapid, and cost-effective method for detecting KRAS and RAS mutations compared with existing assays. These findings suggest that BNA Real-time PCR is a valuable tool for predicting the efficacy of early anti-EGFR therapy in mCRC patients. Copyright © 2017 Elsevier B.V. All rights reserved.

  1. Case Report: Whole exome sequencing reveals a novel frameshift deletion mutation p.G2254fs in COL7A1 associated with autosomal recessive dystrophic epidermolysis bullosa [version 2; referees: 2 approved, 1 approved with reservations

    Directory of Open Access Journals (Sweden)

    Shamsudheen Karuthedath Vellarikkal

    2016-07-01

    Full Text Available Dystrophic epidermolysis bullosa simplex (DEB is a phenotypically diverse inherited skin fragility disorder. It is majorly manifested by appearance of epidermal bullae upon friction caused either by physical or environmental trauma. The phenotypic manifestations also include appearance of milia, scarring all over the body and nail dystrophy. DEB can be inherited in a recessive or dominant form and the recessive form of DEB (RDEB is more severe. In the present study, we identify a novel p.G2254fs mutation in COL7A1 gene causing a sporadic case of RDEB by whole exome sequencing (WES. Apart from adding a novel frameshift Collagen VII mutation to the repertoire of known mutations reported in the disease, to the best of our knowledge, this is the first report of a genetically characterized case of DEB from India.

  2. Case Report: Whole exome sequencing reveals a novel frameshift deletion mutation p.G2254fs in COL7A1 associated with autosomal recessive dystrophic epidermolysis bullosa [version 1; referees: 2 approved, 1 approved with reservations

    Directory of Open Access Journals (Sweden)

    Shamsudheen Karuthedath Vellarikkal

    2016-05-01

    Full Text Available Dystrophic epidermolysis bullosa simplex (DEB is a phenotypically diverse inherited skin fragility disorder. It is majorly manifested by appearance of epidermal bullae upon friction caused either by physical or environmental trauma. The phenotypic manifestations also include appearance of milia, scarring all over the body and nail dystrophy. DEB can be inherited in a recessive or dominant form and the recessive form of DEB (RDEB is more severe. In the present study, we identify a novel p.G2254fs mutation in COL7A1 gene causing a sporadic case of RDEB by whole exome sequencing (WES. Apart from adding a novel frameshift Collagen VII mutation to the repertoire of known mutations reported in the disease, to the best of our knowledge, this is the first report of a genetically characterized case of DEB from India.

  3. HABP2 p.G534E variant in patients with family history of thyroid and breast cancer

    DEFF Research Database (Denmark)

    Pinheiro, Maísa; Drigo, Sandra Aparecida; Tonhosolo, Renata

    2017-01-01

    Familial Papillary Thyroid Carcinoma (PTC) has been described as a hereditary predisposition cancer syndrome associated with mutations in candidate genes including HABP2. Two of 20 probands from families with history of PTC and breast carcinoma (BC) were evaluated by whole exome sequencing (WES...... familial PTC cases. Genes potentially associated with deregulation of the extracellular matrix organization pathway (CTSB, TNXB, COL4A3, COL16A1, COL24A1, COL5A2, NID1, LOXL2, MMP11, TRIM24 and MUSK) and DNA repair function (NBN and MSH2) were detected by WES, suggesting that other cancer-associated genes...

  4. Amelogenesis Imperfecta: 1 Family, 2 Phenotypes, and 2 Mutated Genes.

    Science.gov (United States)

    Prasad, M K; Laouina, S; El Alloussi, M; Dollfus, H; Bloch-Zupan, A

    2016-12-01

    Amelogenesis imperfecta (AI) is a clinically and genetically heterogeneous group of diseases characterized by enamel defects. The authors have identified a large consanguineous Moroccan family segregating different clinical subtypes of hypoplastic and hypomineralized AI in different individuals within the family. Using targeted next-generation sequencing, the authors identified a novel heterozygous nonsense mutation in COL17A1 (c.1873C>T, p.R625*) segregating with hypoplastic AI and a novel homozygous 8-bp deletion in C4orf26 (c.39_46del, p.Cys14Glyfs*18) segregating with hypomineralized-hypoplastic AI in this family. This study highlights the phenotypic and genotypic heterogeneity of AI that can exist even within a single consanguineous family. Furthermore, the identification of novel mutations in COL17A1 and C4orf26 and their correlation with distinct AI phenotypes can contribute to a better understanding of the pathophysiology of AI and the contribution of these genes to amelogenesis. © International & American Associations for Dental Research 2016.

  5. Performance of mitochondrial DNA mutations detecting early stage cancer

    International Nuclear Information System (INIS)

    Jakupciak, John P; Srivastava, Sudhir; Sidransky, David; O'Connell, Catherine D; Maragh, Samantha; Markowitz, Maura E; Greenberg, Alissa K; Hoque, Mohammad O; Maitra, Anirban; Barker, Peter E; Wagner, Paul D; Rom, William N

    2008-01-01

    Mutations in the mitochondrial genome (mtgenome) have been associated with cancer and many other disorders. These mutations can be point mutations or deletions, or admixtures (heteroplasmy). The detection of mtDNA mutations in body fluids using resequencing microarrays, which are more sensitive than other sequencing methods, could provide a strategy to measure mutation loads in remote anatomical sites. We determined the mtDNA mutation load in the entire mitochondrial genome of 26 individuals with different early stage cancers (lung, bladder, kidney) and 12 heavy smokers without cancer. MtDNA was sequenced from three matched specimens (blood, tumor and body fluid) from each cancer patient and two matched specimens (blood and sputum) from smokers without cancer. The inherited wildtype sequence in the blood was compared to the sequences present in the tumor and body fluid, detected using the Affymetrix Genechip ® Human Mitochondrial Resequencing Array 1.0 and supplemented by capillary sequencing for noncoding region. Using this high-throughput method, 75% of the tumors were found to contain mtDNA mutations, higher than in our previous studies, and 36% of the body fluids from these cancer patients contained mtDNA mutations. Most of the mutations detected were heteroplasmic. A statistically significantly higher heteroplasmy rate occurred in tumor specimens when compared to both body fluid of cancer patients and sputum of controls, and in patient blood compared to blood of controls. Only 2 of the 12 sputum specimens from heavy smokers without cancer (17%) contained mtDNA mutations. Although patient mutations were spread throughout the mtDNA genome in the lung, bladder and kidney series, a statistically significant elevation of tRNA and ND complex mutations was detected in tumors. Our findings indicate comprehensive mtDNA resequencing can be a high-throughput tool for detecting mutations in clinical samples with potential applications for cancer detection, but it is

  6. Sensitivity of the ViroSeq HIV-1 Genotyping System for Detection of the K103N Resistance Mutation in HIV-1 Subtypes A, C, and D

    Science.gov (United States)

    Church, Jessica D.; Jones, Dana; Flys, Tamara; Hoover, Donald; Marlowe, Natalia; Chen, Shu; Shi, Chanjuan; Eshleman, James R.; Guay, Laura A.; Jackson, J. Brooks; Kumwenda, Newton; Taha, Taha E.; Eshleman, Susan H.

    2006-01-01

    The US Food and Drug Administration-cleared ViroSeq HIV-1 Genotyping System (ViroSeq) and other population sequencing-based human immunodeficiency virus type 1 (HIV-1) genotyping methods detect antiretroviral drug resistance mutations present in the major viral population of a test sample. These assays also detect some mutations in viral variants that are present as mixtures. We compared detection of the K103N nevirapine resistance mutation using ViroSeq and a sensitive, quantitative point mutation assay, LigAmp. The LigAmp assay measured the percentage of K103N-containing variants in the viral population (percentage of K103N). We analyzed 305 samples with HIV-1 subtypes A, C, and D collected from African women after nevirapine administration. ViroSeq detected K103N in 100% of samples with >20% K103N, 77.8% of samples with 10 to 20% K103N, 71.4% of samples with 5 to 10% K103N, and 16.9% of samples with 1 to 5% K103N. The sensitivity of ViroSeq for detection of K103N was similar for subtypes A, C, and D. These data indicate that the ViroSeq system reliably detects the K103N mutation at levels above 20% and frequently detects the mutation at lower levels. Further studies are needed to compare the sensitivity of different assays for detection of HIV-1 drug resistance mutations and to determine the clinical relevance of HIV-1 minority variants. PMID:16931582

  7. Views of Astronaut (Col.) Joe Engle and son Jon with L-5 Piper Cub

    Science.gov (United States)

    1981-01-01

    Views of Astronaut (Col.) Joe Engle and son Jon with L-5 Piper Cub at Clover Airport. Photos include Engle turning propeller while his son sits in the cockpit (34323); both Engle and son examine propeller (34324); Engle works on engine while his son sits in cockpit (34325).

  8. Molecular methods for the detection of mutations.

    Science.gov (United States)

    Monteiro, C; Marcelino, L A; Conde, A R; Saraiva, C; Giphart-Gassler, M; De Nooij-van Dalen, A G; Van Buuren-van Seggelen, V; Van der Keur, M; May, C A; Cole, J; Lehmann, A R; Steinsgrimsdottir, H; Beare, D; Capulas, E; Armour, J A

    2000-01-01

    We report the results of a collaborative study aimed at developing reliable, direct assays for mutation in human cells. The project used common lymphoblastoid cell lines, both with and without mutagen treatment, as a shared resource to validate the development of new molecular methods for the detection of low-level mutations in the presence of a large excess of normal alleles. As the "gold standard, " hprt mutation frequencies were also measured on the same samples. The methods under development included i) the restriction site mutation (RSM) assay, in which mutations lead to the destruction of a restriction site; ii) minisatellite length-change mutation, in which mutations lead to alleles containing new numbers of tandem repeat units; iii) loss of heterozygosity for HLA epitopes, in which antibodies can be used to direct selection for mutant cells; iv) multiple fluorescence-based long linker arm nucleotides assay (mf-LLA) technology, for the detection of substitutional mutations; v) detection of alterations in the TP53 locus using a (CA) array as the target for the screening; and vi) PCR analysis of lymphocytes for the presence of the BCL2 t(14:18) translocation. The relative merits of these molecular methods are discussed, and a comparison made with more "traditional" methods.

  9. Utility of BRAF V600E mutation detection in cytologically indeterminate thyroid nodules

    Directory of Open Access Journals (Sweden)

    Rowe Leslie R

    2006-04-01

    Full Text Available Abstract Background Fine needle aspiration (FNA is widely utilized for evaluation of patients with thyroid nodules. However, approximately 30% are indeterminate for malignancy. Recently, a mutation in the BRAF gene has been reported to be the most common genetic event in papillary thyroid carcinoma (PTC. In this retrospective study, we assessed the utility of BRAF V600E mutation detection for refining indeterminate preoperative cytologic diagnoses in patients with PTC. Methods Archival indeterminate thyroid FNAs and corresponding formalin-fixed, paraffin-embedded (FFPE surgical samples with PTC were identified in our patient files. DNA extracted from slide scape lysates and 5 μm FFPE sections were evaluated for the BRAF V600E mutation using LightCycler PCR and fluorescent melting curve analysis (LCPCR. Amplification products that showed deviation from the wild-type genomic DNA melting peak, discordant FNA and FFPE matched pairs, and all benign control samples, underwent direct DNA sequencing. Results A total of 19 indeterminate thyroid FNAs demonstrating PTC on FFPE surgical samples were included in the study. Using BRAF mutation analysis, the preoperative diagnosis of PTC was confirmed in 3/19 (15.8% FNA samples that could not be conclusively diagnosed on cytology alone. However, 9/19 (47.4% FFPE tissue samples were positive for the V600E mutation. Of the discordant pairs, 5/6 FNAs contained less than 50% tumor cells. Conclusion When used with indeterminate FNA samples, BRAF mutation analysis may be a useful adjunct technique for confirming the diagnosis of malignancy in an otherwise equivocal case. However, overall tumor cell content of some archival FNA smear slides is a limiting factor for mutation detection.

  10. CHROMagar COL-APSE: a selective bacterial culture medium for the isolation and differentiation of colistin-resistant Gram-negative pathogens

    DEFF Research Database (Denmark)

    Abdul Momin, Muhd Haziq F; Bean, David C; Hendriksen, Rene S.

    2017-01-01

    medium (SuperPolymyxin). Methodology. The medium was challenged with 84 isolates, including polymyxin B (POL B)-susceptible and -resistant type strains and colistin (COL)-resistant organisms recovered from human and animal samples. Susceptibility to COL and POL B was determined by agar dilution and broth...... microtitre dilution. The lower limit for the detection of COL-resistant organisms was also calculated for both CHROMagar COL-APSE and SuperPolymyxin media. The ability to isolate and correctly differentiate COL-resistant organisms within mixed cultures was also assessed and compared using both media. Results...

  11. Characterization of the factor VIII defect in 147 patients with sporadic hemophilia A: Family studies indicate a mutation type-dependent sex ratio of mutation frequencies

    Energy Technology Data Exchange (ETDEWEB)

    Becker, J.; Schmidt, W.; Olek, K. [Univ. of Bonn (Germany)] [and others

    1996-04-01

    The clinical manifestation of hemophilia A is caused by a wide range of different mutations. In this study the factor VIII genes of 147 severe hemophilia A patients-all exclusively from sporadic families-were screened for mutations by use of the complete panel of modern DNA techniques. The pathogenous defect could be characterized in 126 patients (85.7%). Fifty-five patients (37.4%) showed a F8A-gene inversion, 47 (32.0%) a point mutation, 14 (9.5%) a small deletion, 8 (5.4%) a large deletion, and 2 (1.4%) a small insertion. Further, four (2.7%) mutations were localized but could not be sequenced yet. No mutation could be identified in 17 patients (11.6%). Sixteen (10.9%) of the P identified mutations occurred in the B domain. Four of these were located in an adenosine nucleotide stretch at codon 1192, indicating a mutation hotspot. Somatic mosaicisms were detected in 3 (3.9%) of 76 patients` mothers, comprising 3 of 16 de novo mutations in the patients` mothers. Investigation of family relatives allowed detection of a de novo mutation in 16 of 76 two-generation and 28 of 34 three-generation families. On the basis of these data, the male:female ratio of mutation frequencies (k) was estimated as k = 3.6. By use of the quotients of mutation origin in maternal grandfather to patient`s mother or to maternal grandmother, k was directly estimated as k = 15 and k = 7.5, respectively. Considering each mutation type separately, we revealed a mutation type-specific sex ratio of mutation frequencies. Point mutations showed a 5-to-10-fold-higher and inversions a >10-fold- higher mutation rate in male germ cells, whereas deletions showed a >5-fold-higher mutation rate in female germ cells. Consequently, and in accordance with the data of other diseases like Duchenne muscular dystrophy, our results indicate that at least for X-chromosomal disorders the male:female mutation rate of a disease is determined by its proportion of the different mutation types. 68 refs., 1 fig., 5 tabs.

  12. X-Linked and Autosomal Recessive Alport Syndrome

    DEFF Research Database (Denmark)

    Savige, Judith; Storey, Helen; Il Cheong, Hae

    2016-01-01

    Alport syndrome results from mutations in the COL4A5 (X-linked) or COL4A3/COL4A4 (recessive) genes. This study examined 754 previously- unpublished variants in these genes from individuals referred for genetic testing in 12 accredited diagnostic laboratories worldwide, in addition to all published...... COL4A5, COL4A3 and COL4A4 variants in the LOVD databases. It also determined genotype-phenotype correlations for variants where clinical data were available. Individuals were referred for genetic testing where Alport syndrome was suspected clinically or on biopsy (renal failure, hearing loss...

  13. Polymorphisms within the COL5A1 gene and regulators of the extracellular matrix modify the risk of Achilles tendon pathology in a British case-control study.

    Science.gov (United States)

    Brown, Karryn L; Seale, Kirsten B; El Khoury, Louis Y; Posthumus, Michael; Ribbans, William J; Raleigh, Stuart M; Collins, Malcolm; September, Alison V

    2017-08-01

    Several genetic loci have been associated with risk of Achilles tendon pathology (ATP) within South African and Australian populations. The aim of this study was, therefore, to evaluate eight previously implicated genetic variants in an independent British population. A total of 130 asymptomatic controls (CON) and 112 participants clinically diagnosed with ATP comprising 87 individuals with chronic Achilles tendinopathy (TEN) and 25 with Achilles tendon ruptures (RUP) were included. All participants were genotyped for variants within the COL5A1, MIR608, IL-1β, IL-6 and CASP8 genes. Primary findings implicated COL5A1 and CASP8. Three inferred allele combinations constructed from COL5A1 rs12722, rs3196378 and rs71746744 were identified as risk modifiers. The T-C-D combination was associated with increased risk of ATP (P = 0.023) and RUP (P RUP (P = 0.011) and the C-C-D combination was associated with decreased risk of ATP (P = 0.011) and RUP (P = 0.004). The CASP8 rs3834129 DD genotype was associated with decreased risk of TEN (P = 0.020, odds ratio: 0.45, 95% confidence interval: 0.22-0.90) and the CASP8 I-G (rs3834129-rs1045485) inferred allele combination was associated with increased risk of TEN (P = 0.031). This study further highlights the importance of polymorphisms within COL5A1 and CASP8 in the aetiology of ATP.

  14. Epilepsy caused by CDKL5 mutations.

    Science.gov (United States)

    Castrén, Maija; Gaily, Eija; Tengström, Carola; Lähdetie, Jaana; Archer, Hayley; Ala-Mello, Sirpa

    2011-01-01

    Mutations in the cyclin-dependent kinase-like 5 gene (CDKL5) have been identified in female patients with early onset epileptic encephalopathy and severe mental retardation with a Rett-like phenotype. Subsequently CDKL5 mutations were shown to be associated with more diverse phenotypes including mild epilepsy and autism without epilepsy. Furthermore, CDKL5 mutations were found in patients with Angelman-like phenotype. The severity of epilepsy associated with CDKL5 mutations was recently shown to correlate with the type of CDKL5 mutations and epilepsy was identified to involve three distinct sequential stages. Here, we describe the phenotype of a severe form of neurodevelopmental disease in a female patient with a de novo nonsense mutation of the CDKL5 gene c.175C > T (p.R59X) affecting the catalytic domain of CDKL5 protein. Mutations in the CDKL5 gene are less common in males and can be associated with a genomic deletion as found in our male patient with a deletion of 0.3 Mb at Xp22.13 including the CDKL5 gene. We review phenotypes associated with CDKL5 mutations and examine putative relationships between the clinical epilepsy phenotype and the type of the mutation in the CDKL5 gene. © 2010 European Paediatric Neurology Society. Published by Elsevier Ltd. All rights reserved.

  15. Disruption of ERBB2IP is not associated with dystrophic epidermolysis bullosa in both father and son carrying a balanced 5;13 translocation.

    Science.gov (United States)

    Stefanova, Margarita; Zemke, Katrin; Dimitrov, Boyan; Has, Christina; Kern, Johannes S; Bruckner-Tuderman, Leena; Kutsche, Kerstin

    2005-10-01

    Mutations in the type VII collagen gene (COL7A1) cause autosomal recessive and autosomal dominant inherited dystrophic epidermolysis bullosa (DEB). We report a family with three individuals who present blistering, scarring, hypo- and hyperpigmentation, and nail dystrophy suggestive for DEB. Whereas father and son carry a 5;13 translocation, the daughter shows a normal karyotype. Segregation analysis revealed that all affected family members inherited the same COL7A1 allele. Mutation analysis disclosed a heterozygous missense mutation, c.6227G > A (p.G2076D), in COL7A1 in all affected individuals. Delineation of the translocation breakpoints showed that the ERBB2IP (erbb2 interacting protein or Erbin) gene is disrupted in 5q13.1 and GPC6 in 13q32. GPC6 encodes glypican 6 belonging to a family of cell surface heparan sulfate proteoglycans. The binding partners of Erbin, BP230 (BPAG1) and the integrin beta4 subunit, both involved in hemidesmosome (HD) function, and the presence of Erbin in HD suggested that it plays a role in establishment and maintenance of cell-basement membrane adhesions. However, loss of function of one ERBB2IP copy or expression of a putative novel ERBB2IP fusion protein did not apparently modulate the DEB phenotype in both translocation patients. Nonetheless, one cannot yet exclude that ERBB2IP is a candidate for human blistering disorders such as epidermolysis bullosa.

  16. Patients with acephalic spermatozoa syndrome linked to SUN5 mutations have a favorable pregnancy outcome from ICSI.

    Science.gov (United States)

    Fang, Jianzheng; Zhang, Jingjing; Zhu, Fuxi; Yang, Xiaoyu; Cui, Yugui; Liu, Jiayin

    2018-01-10

    Are Sad1 and UNC84 domain containing 5 (SUN5) mutations associated with the outcomes of ICSI in patients with acephalic spermatozoa syndrome (ASS)? Despite highly abnormal sperm morphology, ASS patients with SUN5 mutations have a favorable pregnancy outcome following ICSI. ASS is a rare cause of infertility characterized by the production of a majority of headless spermatozoa, along with a small proportion of intact spermatozoa with an abnormal head-tail junction. Previous studies have demonstrated that SUN5 mutations may cause ASS. Several studies showed that ICSI could help patients with ASS father children. This retrospective cohort study included 11 infertile ASS males with SUN5 mutations. Five of them underwent five ICSI cycles. Their ICSI results were compared to men with ASS without SUN5 mutations (n = 3) and to men with multiple morphological abnormalities of the sperm flagella (MMAF) (n = 9). All ICSI treatments were completed between Jan 2011 and May 2017. Sanger DNA sequencing was used to detect mutations in SUN5. Clinical and biological data were collected from patients at the fertility center. Sanger sequencing validated 11 patients with SUN5 mutations. Three novel mutations in SUN5 (c.829C>T [p.Q277*]; c.1067G>A [p.R356H]; c.211+1 insGT [p.S71Cfs11*]) were identified in three patients. The rates of fertilization, good-quality embryos and pregnancy for five patients with SUN5 mutations following ICSI were 81.5%, 81.8% and 100%, respectively. The rates of fertilization and good-quality embryos in patients with MMAF were significantly lower compared with ASS patients (65.6 versus 82.4%, P = 0.039 and 53.6 versus 85.2%, P = 0.031, respectively). There were no differences in ICSI results between ASS patients with and without SUN5 mutations. Only a small number patients with SUN5 mutations was available because of its rare incidence. Patients with ASS can be effectively treated with ICSI. SUN5 mutations may be one of the genetic causes of ASS. This study

  17. Pyrosequencing, a method approved to detect the two major EGFR mutations for anti EGFR therapy in NSCLC

    Directory of Open Access Journals (Sweden)

    Richard Marie-Jeanne

    2011-05-01

    Full Text Available Abstract Background Epidermal Growth Factor Receptor (EGFR mutations, especially in-frame deletions in exon 19 (ΔLRE and a point mutation in exon 21 (L858R predict gefitinib sensitivity in patients with non-small cell lung cancer. Several methods are currently described for their detection but the gold standard for tissue samples remains direct DNA sequencing, which requires samples containing at least 50% of tumor cells. Methods We designed a pyrosequencing assay based on nested PCR for the characterization of theses mutations on formalin-fixed and paraffin-embedded tumor tissue. Results This method is highly specific and permits precise characterization of all the exon 19 deletions. Its sensitivity is higher than that of "BigDye terminator" sequencing and enabled detection of 3 additional mutations in the 58 NSCLC tested. The concordance between the two methods was very good (97.4%. In the prospective analysis of 213 samples, 7 (3.3% samples were not analyzed and EGFR mutations were detected in 18 (8.7% patients. However, we observed a deficit of mutation detection when the samples were very poor in tumor cells. Conclusions pyrosequencing is then a highly accurate method for detecting ΔLRE and L858R EGFR mutations in patients with NSCLC when the samples contain at least 20% of tumor cells.

  18. Extremely discrepant mutation spectrum of SLC26A4 between Chinese patients with isolated Mondini deformity and enlarged vestibular aqueduct.

    Science.gov (United States)

    Huang, Shasha; Han, Dongyi; Yuan, Yongyi; Wang, Guojian; Kang, Dongyang; Zhang, Xin; Yan, Xiaofei; Meng, Xiaoxiao; Dong, Min; Dai, Pu

    2011-09-30

    Mutations in SLC26A4 cause Pendred syndrome (hearing loss with goiter) or DFNB4 (non-syndromic hearing loss with inner ear malformation, such as enlarged vestibular aqueduct or Mondini deformity). The relationship between mutations in SLC26A4 and Mondini deformity without enlarged vestibular aqueduct has not been studied in any Chinese deaf population. The purpose of this study was to assess whether mutations in the SLC26A4 gene cause Mondini deformity without an enlarged vestibular aqueduct (isolated Mondini deformity) in a Chinese population. In total, 144 patients with sensorineural hearing loss were included and subjected to high-resolution temporal bone CT. Among them, 28 patients with isolated Mondini dysplasia (MD group), 50 patients with enlarged vestibular aqueduct with Mondini dysplasia (EVA with MD group), 50 patients with enlarged vestibular aqueduct without Mondini dysplasia (EVA group), and 16 patients with other types of inner ear malformations (IEM group) were identified. The coding exons of SLC26A4 were analyzed in all subjects. DNA sequence analysis of SLC26A4 was performed in all 144 patients. In the different groups, the detection rate of the SLC26A4 mutation differed. In the isolated MD group, only one single allelic mutation in SLC26A4 was found in one patient (1/28, 3.6%). In the EVA with MD group, biallelic and monoallelic SLC26A4 mutations were identified in 46 patients (46/50, 92.0%) and three patients (3/50, 6.0%), respectively. Also, in the EVA group, biallelic and monoallelic SLC26A4 mutations were identified in 46 patients (46/50, 92.0%) and three patients (3/50, 6.0%), respectively. These percentages were identical to those in the EVA plus MD group. Only two patients carried monoallelic mutations of the SLC26A4 gene in the IEM group (2/16, 12.5%). There were significant differences in the frequency of SLC26A4 mutation among the groups (P0.5). Although mutations in the SLC26A4 gene were frequently found in Chinese EVA patients with and

  19. Allelic heterogeneity of FGF5 mutations causes the long-hair phenotype in dogs.

    Science.gov (United States)

    Dierks, C; Mömke, S; Philipp, U; Distl, O

    2013-08-01

    Hitherto, the only known mutant gene leading to the long-hair phenotype in mammals is the fibroblast growth factor 5 (FGF5). In many dog breeds, the previously discovered FGF5:p.Cys95Phe mutation appeared completely concordant with the long-hair phenotype, but for some breeds, the long-hair phenotype could not be resolved. First, we studied the role of the FGF5:p.Cys95Phe and FGF5:g.145_150dupACCAGC mutations in 268 dogs descending from 27 breeds and seven wolves. As these mutations did not explain all the long-hair phenotypes, all exons and their neighbouring regions of FGF5 were re-sequenced. We detected three novel mutations in the coding sequence and one novel non-coding splice-site mutation in FGF5 associated with the long-hair phenotype. The FGF5:p.Ala193Val polymorphism was perfectly consistent with long hair in Akitas and probably in Siberian huskies, too. Dogs of the long-hair breed Samoyed were either homozygous or compound heterozygous for the FGF5:p.Ala193Val or the FGF5:p.Cys95Phe polymorphisms respectively. The two newly detected polymorphisms FGF5:c.559_560dupGG and FGF5:g.8193T>A and the known mutation FGF5:p.Cys95Phe explained the long-hair phenotype of all Afghan hounds analysed. An FGF5:c.556_571del16 mutation was found in one longhaired Eurasier. All long-hair-associated mutations follow a recessive mode of inheritance, and allelic heterogeneity was a common finding in breeds other than Akita. © 2013 The Authors, Animal Genetics © 2013 Stichting International Foundation for Animal Genetics.

  20. Heteroduplex analysis of the dystrophin gene: Application to point mutation and carrier detection

    Energy Technology Data Exchange (ETDEWEB)

    Prior, T.W.; Papp, A.C.; Snyder, P.J.; Sedra, M.S.; Western, L.M.; Bartolo, C.; Mendell, J.R. [Ohio State Univ., Columbus, OH (United States); Moxley, R.T. [Univ. of Rochester Medical Center, NY (United States)

    1994-03-01

    Approximately one-third of Duchenne muscular dystrophy patients have undefined mutations in the dystrophin gene. For carrier and prenatal studies in families without detectable mutations, the indirect restriction fragment length polymorphism linkage approach is used. Using a multiplex amplification and heteroduplex analysis of dystrophin exons, the authors identified nonsense mutations in two DMD patients. Although the nonsense mutations are predicted to severely truncate the dystrophin protein, both patients presented with mild clinical courses of the disease. As a result of identifying the mutation in the affected boys, direct carrier studies by heteroduplex analysis were extended to other relatives. The authors conclude that the technique is not only ideal for mutation detection but is also useful for diagnostic testing. 29 refs., 4 figs.

  1. The mutational spectrum in Treacher Collins syndrome reveals a predominance of mutations that create a premature-termination codon

    Energy Technology Data Exchange (ETDEWEB)

    Edwards, S.J.; Gladwin, A.J.; Dixon, M.J. [Univ. of Manchester (United Kingdom)

    1997-03-01

    Treacher Collins syndrome (TCS) is an autosomal dominant disorder of craniofacial development, the features of which include conductive hearing loss and cleft palate. The TCS locus has been mapped to human chromosome 5q31.3-32 and the mutated gene identified. In the current investigation, 25 previously undescribed mutations, which are spread throughout the gene, are presented. This brings the total reported to date to 35, which represents a detection rate of 60%. Of the mutations that have been reported to date, all but one result in the introduction of a premature-termination codon into the predicted protein, treacle. Moreover, the mutations are largely family specific, although a common 5-bp deletion in exon 24 (seven different families) and a recurrent splicing mutation in intron 3 (two different families) have been identified. This mutational spectrum supports the hypothesis that TCS results from haploin-sufficiency. 49 refs., 4 figs., 3 tabs.

  2. The transcription factor Lc-Maf participates in Col27a1 regulation during chondrocyte maturation

    Energy Technology Data Exchange (ETDEWEB)

    Mayo, Jaime L.; Holden, Devin N. [Department of Microbiology and Molecular Biology, Brigham Young University, 591 WIDB, Provo, UT 84602 (United States); Barrow, Jeffery R. [Department of Physiology and Developmental Biology, Brigham Young University, Provo, UT 84602 (United States); Bridgewater, Laura C., E-mail: laura_bridgewater@byu.edu [Department of Microbiology and Molecular Biology, Brigham Young University, 591 WIDB, Provo, UT 84602 (United States)

    2009-08-01

    The transcription factor Lc-Maf, which is a splice variant of c-Maf, is expressed in cartilage undergoing endochondral ossification and participates in the regulation of type II collagen through a cartilage-specific Col2a1 enhancer element. Type XXVII and type XI collagens are also expressed in cartilage during endochondral ossification, and so enhancer/reporter assays were used to determine whether Lc-Maf could regulate cartilage-specific enhancers from the Col27a1 and Col11a2 genes. The Col27a1 enhancer was upregulated over 4-fold by Lc-Maf, while the Col11a2 enhancer was downregulated slightly. To confirm the results of these reporter assays, rat chondrosarcoma (RCS) cells were transiently transfected with an Lc-Maf expression plasmid, and quantitative RT-PCR was performed to measure the expression of endogenous Col27a1 and Col11a2 genes. Endogenous Col27a1 was upregulated 6-fold by Lc-Maf overexpression, while endogenous Col11a2 was unchanged. Finally, in situ hybridization and immunohistochemistry were performed in the radius and ulna of embryonic day 17 mouse forelimbs undergoing endochondral ossification. Results demonstrated that Lc-Maf and Col27a1 mRNAs are coexpressed in proliferating and prehypertrophic regions, as would be predicted if Lc-Maf regulates Col27a1 expression. Type XXVII collagen protein was also most abundant in prehypertrophic and proliferating chondrocytes. Others have shown that mice that are null for Lc-Maf and c-Maf have expanded hypertrophic regions with reduced ossification and delayed vascularization. Separate studies have indicated that Col27a1 may serve as a scaffold for ossification and vascularization. The work presented here suggests that Lc-Maf may affect the process of endochondral ossification by participating in the regulation of Col27a1 expression.

  3. NF2 tumor suppressor gene: a comprehensive and efficient detection of somatic mutations by denaturing HPLC and microarray-CGH.

    Science.gov (United States)

    Szijan, Irene; Rochefort, Daniel; Bruder, Carl; Surace, Ezequiel; Machiavelli, Gloria; Dalamon, Viviana; Cotignola, Javier; Ferreiro, Veronica; Campero, Alvaro; Basso, Armando; Dumanski, Jan P; Rouleau, Guy A

    2003-01-01

    The NF2 tumor suppressor gene, located in chromosome 22q12, is involved in the development of multiple tumors of the nervous system, either associated with neurofibromatosis 2 or sporadic ones, mainly schwannomas and meningiomas. In order to evaluate the role of the NF2 gene in sporadic central nervous system (CNS) tumors, we analyzed NF2 mutations in 26 specimens: 14 meningiomas, 4 schwannomas, 4 metastases, and 4 other histopathological types of neoplasms. Denaturing high performance liquid chromatography (denaturing HPLC) and comparative genomic hybridization on a DNA microarray (microarray- CGH) were used as scanning methods for small mutations and gross rearrangements respectively. Small mutations were identified in six out of seventeen meningiomas and schwannomas, one mutation was novel. Large deletions were detected in six meningiomas. All mutations were predicted to result in truncated protein or in the absence of a large protein domain. No NF2 mutations were found in other histopathological types of CNS tumors. These results provide additional evidence that mutations in the NF2 gene play an important role in the development of sporadic meningiomas and schwannomas. Denaturing HPLC analysis of small mutations and microarray-CGH of large deletions are complementary, fast, and efficient methods for the detection of mutations in tumor tissues.

  4. Purification of supercoiled DNA of plasmid Col E1 by RPC-5 chromatography

    Energy Technology Data Exchange (ETDEWEB)

    Best, A.N.; Allison, D.P.; Novelli, G.D.

    1981-07-01

    Col E1 DNA can be purified to a high degree by RPC-5 chromatography of a partially purified cell lysate with a very shallow linear NaC1 gradient at pH 7.8. Electron micrographs demonstrated that the purest fractions were composed of 93% supercoiled (form I) DNA and 7% open circular (form II) DNA. The actual chromatography can be accomplished in 13 to 14 h and is designed for the production of several milligrams of plasmid DNA.

  5. Detection of Anti-Hepatitis B Virus Drug Resistance Mutations Based on Multicolor Melting Curve Analysis.

    Science.gov (United States)

    Mou, Yi; Athar, Muhammad Ammar; Wu, Yuzhen; Xu, Ye; Wu, Jianhua; Xu, Zhenxing; Hayder, Zulfiqar; Khan, Saeed; Idrees, Muhammad; Nasir, Muhammad Israr; Liao, Yiqun; Li, Qingge

    2016-11-01

    Detection of anti-hepatitis B virus (HBV) drug resistance mutations is critical for therapeutic decisions for chronic hepatitis B virus infection. We describe a real-time PCR-based assay using multicolor melting curve analysis (MMCA) that could accurately detect 24 HBV nucleotide mutations at 10 amino acid positions in the reverse transcriptase region of the HBV polymerase gene. The two-reaction assay had a limit of detection of 5 copies per reaction and could detect a minor mutant population (5% of the total population) with the reverse transcriptase M204V amino acid mutation in the presence of the major wild-type population when the overall concentration was 10 4 copies/μl. The assay could be finished within 3 h, and the cost of materials for each sample was less than $10. Clinical validation studies using three groups of samples from both nucleos(t)ide analog-treated and -untreated patients showed that the results for 99.3% (840/846) of the samples and 99.9% (8,454/8,460) of the amino acids were concordant with those of Sanger sequencing of the PCR amplicon from the HBV reverse transcriptase region (PCR Sanger sequencing). HBV DNA in six samples with mixed infections consisting of minor mutant subpopulations was undetected by the PCR Sanger sequencing method but was detected by MMCA, and the results were confirmed by coamplification at a lower denaturation temperature-PCR Sanger sequencing. Among the treated patients, 48.6% (103/212) harbored viruses that displayed lamivudine monoresistance, adefovir monoresistance, entecavir resistance, or lamivudine and adefovir resistance. Among the untreated patients, the Chinese group had more mutation-containing samples than did the Pakistani group (3.3% versus 0.56%). Because of its accuracy, rapidness, wide-range coverage, and cost-effectiveness, the real-time PCR assay could be a robust tool for the detection if anti-HBV drug resistance mutations in resource-limited countries. Copyright © 2016, American Society for

  6. Detection and Analysis of EGFR and KRAS Mutations 
in the Patients with Lung Squamous Cell Carcinomas

    Directory of Open Access Journals (Sweden)

    Hui ZHANG

    2015-10-01

    Full Text Available Background and objective Activating mutations in epidermal growth factor receptor (EGFR and KRAS are important markers in non-small cell lung cancer. However, EGFR and KRAS gene mutations in lung squamous cell carcinoma are rarely reported. The aim of this study was to analyze EGFR and KRAS gene mutation rate and their relationship with clinical features in patients with lung squamous cell carcinomas. Methods A total of 139 patients undergoing treatment for naïve lung squamous cell carcinomas with tumor tissue samples available for testing were recruited. EGFR and KRAS mutation statuses of the tumor samples were detected using a mutant enriched liquid chip. Results Of the 139 cases of lung squamous cell carcinoma, EGFR mutations were detected in 25 cases (18%, KRAS mutations were detected in 7 cases (5%, and the presence of both EGFR and KRAS mutations was detected in 1 case (0.7%. EGFR mutations occurred more often in females than in males (33.3% vs 16.5% and in patients that never smoked than in those who smoke (29.6% vs 16.1%. However, the difference did not reach statistical significance (P>0.05. No significant differences were observed in age, stage, and different biopsy type. KRAS mutations occurred more often in males than in females (5.5% vs 0%, but the difference did not reach statistical significance (P>0.05. No significant differences were observed in age, stage, different biopsy type, and smoking status (P>0.05. Conclusion EGFR and KRAS mutations were low in lung squamous cell carcinomas, and had no significant correlation with clinical features. Before using tyrosine kinase inhibitor targeted therapy, EGFR and KRAS mutations should be detected in patients with lung squamous cell carcinomas.

  7. Mutations in the ABCA4 (ABCR) gene are the major cause of autosomal recessive cone-rod dystrophy.

    Science.gov (United States)

    Maugeri, A; Klevering, B J; Rohrschneider, K; Blankenagel, A; Brunner, H G; Deutman, A F; Hoyng, C B; Cremers, F P

    2000-10-01

    The photoreceptor cell-specific ATP-binding cassette transporter gene (ABCA4; previously denoted "ABCR") is mutated, in most patients, with autosomal recessive (AR) Stargardt disease (STGD1) or fundus flavimaculatus (FFM). In addition, a few cases with AR retinitis pigmentosa (RP) and AR cone-rod dystrophy (CRD) have been found to have ABCA4 mutations. To evaluate the importance of the ABCA4 gene as a cause of AR CRD, we selected 5 patients with AR CRD and 15 patients from Germany and The Netherlands with isolated CRD. Single-strand conformation-polymorphism analysis and sequencing revealed 19 ABCA4 mutations in 13 (65%) of 20 patients. In six patients, mutations were identified in both ABCA4 alleles; in seven patients, mutations were detected in one allele. One complex ABCA4 allele (L541P;A1038V) was found exclusively in German patients with CRD; one patient carried this complex allele homozygously, and five others were compound heterozygous. These findings suggest that mutations in the ABCA4 gene are the major cause of AR CRD. A primary role of the ABCA4 gene in STGD1/FFM and AR CRD, together with the gene's involvement in an as-yet-unknown proportion of cases with AR RP, strengthens the idea that mutations in the ABCA4 gene could be the most frequent cause of inherited retinal dystrophy in humans.

  8. A novel NaV1.5 voltage sensor mutation associated with severe atrial and ventricular arrhythmias.

    Science.gov (United States)

    Wang, Hong-Gang; Zhu, Wandi; Kanter, Ronald J; Silva, Jonathan R; Honeywell, Christina; Gow, Robert M; Pitt, Geoffrey S

    2016-03-01

    Inherited autosomal dominant mutations in cardiac sodium channels (NaV1.5) cause various arrhythmias, such as long QT syndrome and Brugada syndrome. Although dozens of mutations throughout the protein have been reported, there are few reported mutations within a voltage sensor S4 transmembrane segment and few that are homozygous. Here we report analysis of a novel lidocaine-sensitive recessive mutation, p.R1309H, in the NaV1.5 DIII/S4 voltage sensor in a patient with a complex arrhythmia syndrome. We expressed the wild type or mutant NaV1.5 heterologously for analysis with the patch-clamp and voltage clamp fluorometry (VCF) techniques. p.R1309H depolarized the voltage-dependence of activation, hyperpolarized the voltage-dependence of inactivation, and slowed recovery from inactivation, thereby reducing the channel availability at physiologic membrane potentials. Additionally, p.R1309H increased the "late" Na(+) current. The location of the mutation in DIIIS4 prompted testing for a gating pore current. We observed an inward current at hyperpolarizing voltages that likely exacerbates the loss-of-function defects at resting membrane potentials. Lidocaine reduced the gating pore current. The p.R1309H homozygous NaV1.5 mutation conferred both gain-of-function and loss-of-function effects on NaV1.5 channel activity. Reduction of a mutation-induced gating pore current by lidocaine suggested a therapeutic mechanism. Copyright © 2016 Elsevier Ltd. All rights reserved.

  9. A novel fully automated molecular diagnostic system (AMDS for colorectal cancer mutation detection.

    Directory of Open Access Journals (Sweden)

    Shiro Kitano

    Full Text Available BACKGROUND: KRAS, BRAF and PIK3CA mutations are frequently observed in colorectal cancer (CRC. In particular, KRAS mutations are strong predictors for clinical outcomes of EGFR-targeted treatments such as cetuximab and panitumumab in metastatic colorectal cancer (mCRC. For mutation analysis, the current methods are time-consuming, and not readily available to all oncologists and pathologists. We have developed a novel, simple, sensitive and fully automated molecular diagnostic system (AMDS for point of care testing (POCT. Here we report the results of a comparison study between AMDS and direct sequencing (DS in the detection of KRAS, BRAF and PI3KCA somatic mutations. METHODOLOGY/PRINCIPAL FINDING: DNA was extracted from a slice of either frozen (n = 89 or formalin-fixed and paraffin-embedded (FFPE CRC tissue (n = 70, and then used for mutation analysis by AMDS and DS. All mutations (n = 41 among frozen and 27 among FFPE samples detected by DS were also successfully (100% detected by the AMDS. However, 8 frozen and 6 FFPE samples detected as wild-type in the DS analysis were shown as mutants in the AMDS analysis. By cloning-sequencing assays, these discordant samples were confirmed as true mutants. One sample had simultaneous "hot spot" mutations of KRAS and PIK3CA, and cloning assay comfirmed that E542K and E545K were not on the same allele. Genotyping call rates for DS were 100.0% (89/89 and 74.3% (52/70 in frozen and FFPE samples, respectively, for the first attempt; whereas that of AMDS was 100.0% for both sample sets. For automated DNA extraction and mutation detection by AMDS, frozen tissues (n = 41 were successfully detected all mutations within 70 minutes. CONCLUSIONS/SIGNIFICANCE: AMDS has superior sensitivity and accuracy over DS, and is much easier to execute than conventional labor intensive manual mutation analysis. AMDS has great potential for POCT equipment for mutation analysis.

  10. Extremely discrepant mutation spectrum of SLC26A4 between Chinese patients with isolated Mondini deformity and enlarged vestibular aqueduct

    Directory of Open Access Journals (Sweden)

    Yan Xiaofei

    2011-09-01

    Full Text Available Abstract Background Mutations in SLC26A4 cause Pendred syndrome (hearing loss with goiter or DFNB4 (non-syndromic hearing loss with inner ear malformation, such as enlarged vestibular aqueduct or Mondini deformity. The relationship between mutations in SLC26A4 and Mondini deformity without enlarged vestibular aqueduct has not been studied in any Chinese deaf population. The purpose of this study was to assess whether mutations in the SLC26A4 gene cause Mondini deformity without an enlarged vestibular aqueduct (isolated Mondini deformity in a Chinese population. Methods In total, 144 patients with sensorineural hearing loss were included and subjected to high-resolution temporal bone CT. Among them, 28 patients with isolated Mondini dysplasia (MD group, 50 patients with enlarged vestibular aqueduct with Mondini dysplasia (EVA with MD group, 50 patients with enlarged vestibular aqueduct without Mondini dysplasia (EVA group, and 16 patients with other types of inner ear malformations (IEM group were identified. The coding exons of SLC26A4 were analyzed in all subjects. Results DNA sequence analysis of SLC26A4 was performed in all 144 patients. In the different groups, the detection rate of the SLC26A4 mutation differed. In the isolated MD group, only one single allelic mutation in SLC26A4 was found in one patient (1/28, 3.6%. In the EVA with MD group, biallelic and monoallelic SLC26A4 mutations were identified in 46 patients (46/50, 92.0% and three patients (3/50, 6.0%, respectively. Also, in the EVA group, biallelic and monoallelic SLC26A4 mutations were identified in 46 patients (46/50, 92.0% and three patients (3/50, 6.0%, respectively. These percentages were identical to those in the EVA plus MD group. Only two patients carried monoallelic mutations of the SLC26A4 gene in the IEM group (2/16, 12.5%. There were significant differences in the frequency of SLC26A4 mutation among the groups (P SLC26A4 mutation in the isolated MD group was

  11. Laser desorption mass spectrometry for point mutation detection

    Energy Technology Data Exchange (ETDEWEB)

    Taranenko, N.I.; Chung, C.N.; Zhu, Y.F. [Oak Ridge National Lab., TN (United States)] [and others

    1996-12-31

    A point mutation can be associated with the pathogenesis of inherited or acquired diseases. Laser desorption mass spectrometry coupled with allele specific polymerase chain reaction (PCR) was first used for point mutation detection. G551D is one of several mutations of the cystic fibrosis transmembrane conductance regulator (CFTR) gene present in 1-3% of the mutant CFTR alleles in most European populations. In this work, two different approaches were pursued to detect G551D point mutation in the cystic fibrosis gene. The strategy is to amplify the desired region of DNA template by PCR using two primers that overlap one base at the site of the point mutation and which vary in size. If the two primers based on the normal sequence match the target DNA sequence, a normal PCR product will be produced. However, if the alternately sized primers that match the mutant sequence recognize the target DNA, an abnormal PCR product will be produced. Thus, the mass spectrometer can be used to identify patients that are homozygous normal, heterozygous for a mutation or homozygous abnormal at a mutation site. Another approach to identify similar mutations is the use of sequence specific restriction enzymes which respond to changes in the DNA sequence. Mass spectrometry is used to detect the length of the restriction fragments by digestion of a PCR generated target fragment. 21 refs., 10 figs., 2 tabs.

  12. Quantum dots immunofluorescence histochemical detection of EGFR gene mutations in the non-small cell lung cancers using mutation-specific antibodies

    Directory of Open Access Journals (Sweden)

    Qu YG

    2014-12-01

    Full Text Available Yan-Gang Qu,1 Qian Zhang,2 Qi Pan,3 Xian-Da Zhao,4 Yan-Hua Huang,2 Fu-Chun Chen,3 Hong-Lei Chen41Department of Pathology, The Central Hospital of Enshi Autonomous Prefecture, Enshi, 2Department of Molecular Pathology, Wuhan Nano Tumor Diagnosis Engineering Research Center, Wuhan, Hubei, People’s Republic of China; 3Department of Thoracosurgery, Traditional Chinese Medical Hospital of Wenling, Wenling, Zhejiang, People’s Republic of China; 4Department of Pathology, School of Basic Medical Science, Wuhan University, Wuhan, Hubei, People’s Republic of ChinaBackground: Epidermal growth factor receptor (EGFR mutation status plays an important role in therapeutic decision making for non-small cell lung cancer (NSCLC patients. Since EGFR mutation-specific antibodies (E746-A750del and L858R have been developed, EGFR mutation detection by immunohistochemistry (IHC is a suitable screening test. On this basis, we want to establish a new screening test, quantum dots immunofluorescence histochemistry (QDs-IHC, to assess EGFR gene mutation in NSCLC tissues, and we compared it to traditional IHC and amplification refractory mutation system (ARMS.Materials and methods: EGFR gene mutations were detected by QDs-IHC, IHC, and ADx-ARMS in 65 cases of NSCLC composed of 55 formalin-fixed, paraffin-embedded specimens and ten pleural effusion cell blocks, including 13 squamous cell carcinomas, two adenosquamous carcinomas, and 50 adenocarcinomas.Results: Positive rates of EGFR gene mutations detected by QDs-IHC, IHC, and ADx-ARMS were 40.0%, 36.9%, and 46.2%, respectively, in 65 cases of NSCLC patients. The sensitivity of QDs-IHC when detecting EGFR mutations, as compared to ADx-ARMS, was 86.7% (26/30; the specificity for both antibodies was 100.0% (26/26. IHC sensitivity was 80.0% (24/30 and the specificity was 92.31% (24/26. When detecting EGFR mutations, QDs-IHC and ADx-ARMS had perfect consistency (κ=0.882; P<0.01. Excellent agreement was observed

  13. Analysis of SLX4/FANCP in non-BRCA1/2-mutated breast cancer families

    Directory of Open Access Journals (Sweden)

    Fernández-Rodríguez Juana

    2012-03-01

    Full Text Available Abstract Background Genes that, when mutated, cause Fanconi anemia or greatly increase breast cancer risk encode for proteins that converge on a homology-directed DNA damage repair process. Mutations in the SLX4 gene, which encodes for a scaffold protein involved in the repair of interstrand cross-links, have recently been identified in unclassified Fanconi anemia patients. A mutation analysis of SLX4 in German or Byelorussian familial cases of breast cancer without detected mutations in BRCA1 or BRCA2 has been completed, with globally negative results. Methods The genomic region of SLX4, comprising all exons and exon-intron boundaries, was sequenced in 94 Spanish familial breast cancer cases that match a criterion indicating the potential presence of a highly-penetrant germline mutation, following exclusion of BRCA1 or BRCA2 mutations. Results This mutational analysis revealed extensive genetic variation of SLX4, with 21 novel single nucleotide variants; however, none could be linked to a clear alteration of the protein function. Nonetheless, genotyping 10 variants (nine novel, all missense amino acid changes in a set of controls (138 women and 146 men did not detect seven of them. Conclusions Overall, while the results of this study do not identify clearly pathogenic mutations of SLX4 contributing to breast cancer risk, further genetic analysis, combined with functional assays of the identified rare variants, may be warranted to conclusively assess the potential link with the disease.

  14. Analysis of SLX4/FANCP in non-BRCA1/2-mutated breast cancer families

    International Nuclear Information System (INIS)

    Fernández-Rodríguez, Juana; Schindler, Detlev; Capellá, Gabriel; Brunet, Joan; Lázaro, Conxi; Pujana, Miguel Angel; Quiles, Francisco; Blanco, Ignacio; Teulé, Alex; Feliubadaló, Lídia; Valle, Jesús del; Salinas, Mónica; Izquierdo, Àngel; Darder, Esther

    2012-01-01

    Genes that, when mutated, cause Fanconi anemia or greatly increase breast cancer risk encode for proteins that converge on a homology-directed DNA damage repair process. Mutations in the SLX4 gene, which encodes for a scaffold protein involved in the repair of interstrand cross-links, have recently been identified in unclassified Fanconi anemia patients. A mutation analysis of SLX4 in German or Byelorussian familial cases of breast cancer without detected mutations in BRCA1 or BRCA2 has been completed, with globally negative results. The genomic region of SLX4, comprising all exons and exon-intron boundaries, was sequenced in 94 Spanish familial breast cancer cases that match a criterion indicating the potential presence of a highly-penetrant germline mutation, following exclusion of BRCA1 or BRCA2 mutations. This mutational analysis revealed extensive genetic variation of SLX4, with 21 novel single nucleotide variants; however, none could be linked to a clear alteration of the protein function. Nonetheless, genotyping 10 variants (nine novel, all missense amino acid changes) in a set of controls (138 women and 146 men) did not detect seven of them. Overall, while the results of this study do not identify clearly pathogenic mutations of SLX4 contributing to breast cancer risk, further genetic analysis, combined with functional assays of the identified rare variants, may be warranted to conclusively assess the potential link with the disease

  15. Physiological and pathophysiological insights of Nav1.4 and Nav1.5 comparison

    Directory of Open Access Journals (Sweden)

    Gildas eLoussouarn

    2016-01-01

    Full Text Available Mutations in Nav1.4 and Nav1.5 α-subunits have been associated with muscular and cardiac channelopathies, respectively. Despite intense research on the structure and function of these channels, a lot of information is still missing to delineate the various physiological and pathophysiological processes underlying their activity at the molecular level. Nav1.4 and Nav1.5 sequences are similar, suggesting structural and functional homologies between the two orthologous channels. This also suggests that any characteristics described for one channel subunit may shed light on the properties of the counterpart channel subunit. In this review article, after a brief clinical description of the muscular and cardiac channelopathies related to Nav1.4 and Nav1.5 mutations, respectively, we compare the knowledge accumulated in different aspects of the expression and function of Nav1.4 and Nav1.5 α-subunits: the regulation of the two encoding genes (SCN4A and SCN5A, the associated/regulatory proteins and at last, the functional effect of the same missense mutations detected in Nav1.4 and Nav1.5. First, it appears that more is known on Nav1.5 expression and accessory proteins. Because of the high homologies of Nav1.5 binding sites and equivalent Nav1.4 sites, Nav1.5-related results may guide future investigations on Nav1.4. Second, the analysis of the same missense mutations in Nav1.4 and Nav1.5 revealed intriguing similarities regarding their effects on membrane excitability and alteration in channel biophysics. We believe that such comparison may bring new cues to the physiopathology of cardiac and muscular diseases.

  16. Detection of MPLW515L/K mutations and determination of allele frequencies with a single-tube PCR assay.

    Science.gov (United States)

    Takei, Hiraku; Morishita, Soji; Araki, Marito; Edahiro, Yoko; Sunami, Yoshitaka; Hironaka, Yumi; Noda, Naohiro; Sekiguchi, Yuji; Tsuneda, Satoshi; Ohsaka, Akimichi; Komatsu, Norio

    2014-01-01

    A gain-of-function mutation in the myeloproliferative leukemia virus (MPL) gene, which encodes the thrombopoietin receptor, has been identified in patients with essential thrombocythemia and primary myelofibrosis, subgroups of classic myeloproliferative neoplasms (MPNs). The presence of MPL gene mutations is a critical diagnostic criterion for these diseases. Here, we developed a rapid, simple, and cost-effective method of detecting two major MPL mutations, MPLW515L/K, in a single PCR assay; we termed this method DARMS (dual amplification refractory mutation system)-PCR. DARMS-PCR is designed to produce three different PCR products corresponding to MPLW515L, MPLW515K, and all MPL alleles. The amplicons are later detected and quantified using a capillary sequencer to determine the relative frequencies of the mutant and wild-type alleles. Applying DARMS-PCR to human specimens, we successfully identified MPL mutations in MPN patients, with the exception of patients bearing mutant allele frequencies below the detection limit (5%) of this method. The MPL mutant allele frequencies determined using DARMS-PCR correlated strongly with the values determined using deep sequencing. Thus, we demonstrated the potential of DARMS-PCR to detect MPL mutations and determine the allele frequencies in a timely and cost-effective manner.

  17. Detection of MPLW515L/K mutations and determination of allele frequencies with a single-tube PCR assay.

    Directory of Open Access Journals (Sweden)

    Hiraku Takei

    Full Text Available A gain-of-function mutation in the myeloproliferative leukemia virus (MPL gene, which encodes the thrombopoietin receptor, has been identified in patients with essential thrombocythemia and primary myelofibrosis, subgroups of classic myeloproliferative neoplasms (MPNs. The presence of MPL gene mutations is a critical diagnostic criterion for these diseases. Here, we developed a rapid, simple, and cost-effective method of detecting two major MPL mutations, MPLW515L/K, in a single PCR assay; we termed this method DARMS (dual amplification refractory mutation system-PCR. DARMS-PCR is designed to produce three different PCR products corresponding to MPLW515L, MPLW515K, and all MPL alleles. The amplicons are later detected and quantified using a capillary sequencer to determine the relative frequencies of the mutant and wild-type alleles. Applying DARMS-PCR to human specimens, we successfully identified MPL mutations in MPN patients, with the exception of patients bearing mutant allele frequencies below the detection limit (5% of this method. The MPL mutant allele frequencies determined using DARMS-PCR correlated strongly with the values determined using deep sequencing. Thus, we demonstrated the potential of DARMS-PCR to detect MPL mutations and determine the allele frequencies in a timely and cost-effective manner.

  18. Congenital syndactyly in cattle: four novel mutations in the low density lipoprotein receptor-related protein 4 gene (LRP4

    Directory of Open Access Journals (Sweden)

    Höltershinken Martin

    2007-02-01

    Full Text Available Abstract Background Isolated syndactyly in cattle, also known as mulefoot, is inherited as an autosomal recessive trait with variable penetrance in different cattle breeds. Recently, two independent mutations in the bovine LRP4 gene have been reported as the primary cause of syndactyly in the Holstein and Angus cattle breeds. Results We confirmed the previously described LRP4 exon 33 two nucleotide substitution in most of the affected Holstein calves and revealed additional evidence for allelic heterogeneity by the identification of four new LRP4 non-synonymous point mutations co-segregating in Holstein, German Simmental and Simmental-Charolais families. Conclusion We confirmed a significant role of LRP4 mutations in the pathogenesis of congenital syndactyly in cattle. The newly detected missense mutations in the LRP4 gene represent independent mutations affecting different conserved protein domains. However, the four newly described LRP4 mutations do still not explain all analyzed cases of syndactyly.

  19. Linkage mapping of the gene for Type III collagen (COL3A1) to human chromosome 2q using a VNTR polymorphism

    Energy Technology Data Exchange (ETDEWEB)

    Tiller, G.E.; Polumbo, P.A.; Summar, M.L. (Vanderbilt Univ. Medical Center, Nashville, TN (United States))

    1994-03-15

    The gene for the [alpha]1(III) chain of type III collagen, COL3A1, has been previously mapped to human chromosome 2q24.3-q31 by in situ hybridization. Physical mapping by pulsed-field gel electrophoresis has demonstrated that COL3A1 lies within 35 kb of COL5A2. The authors genotyped the CEPH families at the COL3A2 locus using a pentanucleotide repeat polymorphism within intron 25. They demonstrated significant linkage to 18 anonymous markers as well as the gene for carbamyl phosphate synthetase (CPSI), which had been previously mapped to this region. No recombination was seen between COL3A1 and COL5A2 (Z = 9.93 at [theta] = 0) or D2S24 (Z = 10.55 at [theta] = 0). The locus order is (D2S32-D2S138-D2S148)-(D2S24-COL5A2-COL3A1)-(D2S118-D2S161), with odds of 1:2300 for the next most likely order. These relationships are consistent with the physical mapping of COL3A1 to the distal portion of 2q and place it proximal to CPSI by means of multipoint analysis. These linkage relationships should prove useful in further studies of Ehlers-Danlos syndrome type IV and carbamyl phosphate synthetase I deficiency and provide an additional framework for localizing other genes in this region. 13 refs., 2 figs., 1 tab.

  20. Laser desorption mass spectrometry for point mutation detection

    Energy Technology Data Exchange (ETDEWEB)

    Taranenko, N.I.; Chung, C.N.; Zhu, Y.F. [Oak Ridge National Lab., TN (United States)] [and others

    1996-10-01

    A point mutation can be associated with the pathogenesis of inherited or acquired diseases. Laser desorption mass spectrometry coupled with allele specific polymerase chain reaction (PCR) was first used for point mutation detection. G551D is one of several mutations of the cystic fibrosis transmembrane conductance regulator (CFTR) gene present in 1-3% of the mutant CFTR alleles in most European populations. In this work, two different approaches were pursued to detect G551D point mutation in the cystic fibrosis gene. The strategy is to amplify the desired region of DNA template by PCR using two primers that overlap one base at the site of the point mutation and which vary in size. If the two primers based on the normal sequence match the target DNA sequence, a normal PCR product will be produced. However, if the alternately sized primers that match the mutant sequence recognize the target DNA, an abnormal PCR product will be produced. Thus, the mass spectrometer can be used to identify patients that are homozygous normal, heterozygous for a mutation or homozygous abnormal at a mutation site. Another approach to identify similar mutations is the use of sequence specific restriction enzymes which respond to changes in the DNA sequence. Mass spectrometry is used to detect the length of the restriction fragments generated by digestion of a PCR generated target fragment. 21 refs., 10 figs., 2 tabs.

  1. A parylene-based dual channel microelectrophoresis system for rapid mutation detection via heteroduplex analysis

    NARCIS (Netherlands)

    Sukas, S.; Erson, Ayse Elif; Sert, Cuneyt; Kulah, Haluk

    2008-01-01

    A new dual channel micro-electrophoresis system for rapid mutation detection based on heteroduplex analysis was designed and implemented. Mutation detection was successfully achieved in a total separation length of 250 μm in less than 3 min for a 590 bp DNA sample harboring a 3 bp mutation causing

  2. [Study of gene mutation in 62 hemophilia A children].

    Science.gov (United States)

    Hu, Q; Liu, A G; Zhang, L Q; Zhang, A; Wang, Y Q; Wang, S M; Lu, Y J; Wang, X

    2017-11-02

    Objective: To analyze the mutation type of FⅧ gene in children with hemophilia A and to explore the relationship among hemophilia gene mutation spectrum, gene mutation and clinical phenotype. Method: Sixty-two children with hemophilia A from Department of Pediatric Hematology, Tongji Hospital of Tongji Medical College, Huazhong University of Science and Technology between January 2015 and March 2017 were enrolled. All patients were male, aged from 4 months to 7 years and F Ⅷ activity ranged 0.2%-11.0%. Fifty cases had severe, 10 cases had moderate and 2 cases had mild hemophilia A. DNA was isolated from peripheral blood in hemophilia A children and the target gene fragment was amplified by PCR, in combination with the second generation sequencing, 22 and 1 introns were detected. Negative cases were detected by the second generation sequencing and results were compared with those of the international FⅧ gene mutation database. Result: There were 20 cases (32%) of intron 22 inversion, 2 cases (3%) of intron 1 inversion, 18 cases (29%) of missense mutation, 5 cases (8%) of nonsense mutation, 7 cases (11%) of deletion mutation, 1 case(2%)of splice site mutation, 2 cases (3%) of large fragment deletion and 1 case of insertion mutation (2%). No mutation was detected in 2 cases (3%), and 4 cases (7%) failed to amplify. The correlation between phenotype and genotype showed that the most common gene mutation in severe hemophilia A was intron 22 inversion (20 cases), accounting for 40% of severe patients, followed by 11 cases of missense mutation (22%). The most common mutation in moderate hemophilia A was missense mutation (6 cases), accounting for 60% of moderate patients. Conclusion: The most frequent mutation type in hemophilia A was intron 22 inversion, followed by missense mutation, again for missing mutation. The relationship between phenotype and genotype: the most frequent gene mutation in severe hemophilia A is intron 22 inversion, followed by missense

  3. IFITM5 mutations and osteogenesis imperfecta.

    Science.gov (United States)

    Hanagata, Nobutaka

    2016-03-01

    Interferon-induced transmembrane protein 5 (IFITM5) is an osteoblast-specific membrane protein that has been shown to be a positive regulatory factor for mineralization in vitro. However, Ifitm5 knockout mice do not exhibit serious bone abnormalities, and thus the function of IFITM5 in vivo remains unclear. Recently, a single point mutation (c.-14C>T) in the 5' untranslated region of IFITM5 was identified in patients with osteogenesis imperfecta type V (OI-V). Furthermore, a single point mutation (c.119C>T) in the coding region of IFITM5 was identified in OI patients with more severe symptoms than patients with OI-V. Although IFITM5 is not directly involved in the formation of bone in vivo, the reason why IFITM5 mutations cause OI remains a major mystery. In this review, the current state of knowledge of OI pathological mechanisms due to IFITM5 mutations will be reviewed.

  4. Molecular Etiology of Hearing Impairment in Inner Mongolia: mutations in SLC26A4 gene and relevant phenotype analysis

    Directory of Open Access Journals (Sweden)

    Wu Bailin

    2008-11-01

    Full Text Available Abstract Background The molecular etiology of hearing impairment in Chinese has not been thoroughly investigated. Study of GJB2 gene revealed that 30.4% of the patients with hearing loss in Inner Mongolia carried GJB2 mutations. The SLC26A4 gene mutations and relevant phenotype are analyzed in this study. Methods One hundred and thirty-five deaf patients were included. The coding exons of SLC26A4 gene were sequence analyzed in 111 patients, not including 22 patients carrying bi-allelic GJB2 mutations or one patient carrying a known GJB2 dominant mutation as well as one patient with mtDNA 1555A>G mutation. All patients with SLC26A4 mutations or variants were subjected to high resolution temporal bone CT scan and those with confirmed enlarged vestibular aqueduct and/or other inner ear malformation were then given further ultrasound scan of thyroid and thyroid hormone assays. Results Twenty-six patients (19.26%, 26/135 were found carrying SLC26A4 mutation. Among them, 17 patients with bi-allelic SLC26A4 mutations were all confirmed to have EVA or other inner ear malformation by CT scan. Nine patients were heterozygous for one SLC26A4 mutation, including 3 confirmed to be EVA or EVA and Mondini dysplasia by CT scan. The most common mutation, IVS7-2A>G, accounted for 58.14% (25/43 of all SLC26A4 mutant alleles. The shape and function of thyroid were confirmed to be normal by thyroid ultrasound scan and thyroid hormone assays in 19 of the 20 patients with EVA or other inner ear malformation except one who had cystoid change in the right side of thyroid. No Pendred syndrome was diagnosed. Conclusion In Inner Mongolia, China, mutations in SLC26A4 gene account for about 12.6% (17/135 of the patients with hearing loss. Together with GJB2 (23/135, SLC26A4 are the two most commonly mutated genes causing deafness in this region. Pendred syndrome is not detected in this deaf population. We established a new strategy that detects SLC26A4 mutations prior to the

  5. Molecular Etiology of Hearing Impairment in Inner Mongolia: mutations in SLC26A4 gene and relevant phenotype analysis

    Science.gov (United States)

    Dai, Pu; Yuan, Yongyi; Huang, Deliang; Zhu, Xiuhui; Yu, Fei; Kang, Dongyang; Yuan, Huijun; Wu, Bailin; Han, Dongyi; Wong, Lee-Jun C

    2008-01-01

    Background The molecular etiology of hearing impairment in Chinese has not been thoroughly investigated. Study of GJB2 gene revealed that 30.4% of the patients with hearing loss in Inner Mongolia carried GJB2 mutations. The SLC26A4 gene mutations and relevant phenotype are analyzed in this study. Methods One hundred and thirty-five deaf patients were included. The coding exons of SLC26A4 gene were sequence analyzed in 111 patients, not including 22 patients carrying bi-allelic GJB2 mutations or one patient carrying a known GJB2 dominant mutation as well as one patient with mtDNA 1555A>G mutation. All patients with SLC26A4 mutations or variants were subjected to high resolution temporal bone CT scan and those with confirmed enlarged vestibular aqueduct and/or other inner ear malformation were then given further ultrasound scan of thyroid and thyroid hormone assays. Results Twenty-six patients (19.26%, 26/135) were found carrying SLC26A4 mutation. Among them, 17 patients with bi-allelic SLC26A4 mutations were all confirmed to have EVA or other inner ear malformation by CT scan. Nine patients were heterozygous for one SLC26A4 mutation, including 3 confirmed to be EVA or EVA and Mondini dysplasia by CT scan. The most common mutation, IVS7-2A>G, accounted for 58.14% (25/43) of all SLC26A4 mutant alleles. The shape and function of thyroid were confirmed to be normal by thyroid ultrasound scan and thyroid hormone assays in 19 of the 20 patients with EVA or other inner ear malformation except one who had cystoid change in the right side of thyroid. No Pendred syndrome was diagnosed. Conclusion In Inner Mongolia, China, mutations in SLC26A4 gene account for about 12.6% (17/135) of the patients with hearing loss. Together with GJB2 (23/135), SLC26A4 are the two most commonly mutated genes causing deafness in this region. Pendred syndrome is not detected in this deaf population. We established a new strategy that detects SLC26A4 mutations prior to the temporal bone CT scan to

  6. Knockdown resistance (kdr)-like mutations in the voltage-gated sodium channel of a malaria vector Anopheles stephensi and PCR assays for their detection.

    Science.gov (United States)

    Singh, Om P; Dykes, Cherry L; Lather, Manila; Agrawal, Om P; Adak, Tridibes

    2011-03-14

    Knockdown resistance (kdr) in insects, resulting from mutation(s) in the voltage-gated sodium channel (vgsc) gene is one of the mechanisms of resistance against DDT and pyrethroid-group of insecticides. The most common mutation(s) associated with knockdown resistance in insects, including anophelines, has been reported to be present at residue Leu1014 in the IIS6 transmembrane segment of the vgsc gene. This study reports the presence of two alternative kdr-like mutations, L1014S and L1014F, at this residue in a major malaria vector Anopheles stephensi and describes new PCR assays for their detection. Part of the vgsc (IIS4-S5 linker-to-IIS6 transmembrane segment) of An. stephensi collected from Alwar (Rajasthan, India) was PCR-amplified from genomic DNA, sequenced and analysed for the presence of deduced amino acid substitution(s). Analysis of DNA sequences revealed the presence of two alternative non-synonymous point mutations at L1014 residue in the IIS6 transmembrane segment of vgsc, i.e., T>C mutation on the second position and A>T mutation on the third position of the codon, leading to Leu (TTA)-to-Ser (TCA) and -Phe (TTT) amino acid substitutions, respectively. Polymerase chain reaction (PCR) assays were developed for identification of each of these two point mutations. Genotyping of An. stephensi mosquitoes from Alwar by PCR assays revealed the presence of both mutations, with a high frequency of L1014S. The PCR assays developed for detection of the kdr mutations were specific as confirmed by DNA sequencing of PCR-genotyped samples. Two alternative kdr-like mutations, L1014S and L1014F, were detected in An. stephensi with a high allelic frequency of L1014S. The occurrence of L1014S is being reported for the first time in An. stephensi. Two specific PCR assays were developed for detection of two kdr-like mutations in An. stephensi.

  7. Genetic Screening of WNT4 and WNT5B in Two Populations with Deviating Bone Mineral Densities

    DEFF Research Database (Denmark)

    Hendrickx, Gretl; Boudin, Eveline; Steenackers, Ellen

    2017-01-01

    A role for WNT4 and WNT5B in bone metabolism was indicated by genome-wide association studies (GWAS) and a Wnt4 knockout mouse model. The aim of this study was therefore to replicate and further investigate the causality between genetic variation in WNT4 and WNT5B and deviating bone mineral density...... (BMD) values. A WNT4 and WNT5B mutation screening was performed in patients with craniotubular hyperostosis using Sanger sequencing. Here, no putative causal mutations were detected. Moreover, a high and low BMD cohort was selected from the Odense Androgen Study population for re-sequencing. In WNT4 we...

  8. Mutations in the C-terminus of CDKL5: proceed with caution.

    Science.gov (United States)

    Diebold, Bertrand; Delépine, Chloé; Gataullina, Svetlana; Delahaye, Andrée; Nectoux, Juliette; Bienvenu, Thierry

    2014-02-01

    Mutations in the cyclin-dependent kinase-like 5 (CDKL5) gene have been described in girls with Rett-like features and early-onset epileptic encephalopathy including infantile spasms. Milder phenotypes have been associated with sequence variations in the 3'-end of the CDKL5 gene. Identification of novel CDKL5 transcripts coding isoforms characterized by an altered C-terminal region strongly questions the eventual pathogenicity of sequence variations located in the 3'-end of the gene. We investigated a group of 30 female patients with a clinically heterogeneous phenotype ranging from nonspecific intellectual disability to a severe neonatal encephalopathy and identified two heterozygous CDKL5 missense mutations, the previously reported p.Val999Met and the novel mutation p.Pro944Thr. However, these mutations have also been detected in their healthy father. Considering our results and all data from the literature, we suggest that genetic variations beyond the codon 938 in human CDKL5115 protein may have minor or no significance. It is probable that screening of exons 19-21 of the CDKL5 gene is not useful in practical molecular diagnosis of atypical Rett syndrome.

  9. Detecting negative selection on recurrent mutations using gene genealogy

    Science.gov (United States)

    2013-01-01

    Background Whether or not a mutant allele in a population is under selection is an important issue in population genetics, and various neutrality tests have been invented so far to detect selection. However, detection of negative selection has been notoriously difficult, partly because negatively selected alleles are usually rare in the population and have little impact on either population dynamics or the shape of the gene genealogy. Recently, through studies of genetic disorders and genome-wide analyses, many structural variations were shown to occur recurrently in the population. Such “recurrent mutations” might be revealed as deleterious by exploiting the signal of negative selection in the gene genealogy enhanced by their recurrence. Results Motivated by the above idea, we devised two new test statistics. One is the total number of mutants at a recurrently mutating locus among sampled sequences, which is tested conditionally on the number of forward mutations mapped on the sequence genealogy. The other is the size of the most common class of identical-by-descent mutants in the sample, again tested conditionally on the number of forward mutations mapped on the sequence genealogy. To examine the performance of these two tests, we simulated recurrently mutated loci each flanked by sites with neutral single nucleotide polymorphisms (SNPs), with no recombination. Using neutral recurrent mutations as null models, we attempted to detect deleterious recurrent mutations. Our analyses demonstrated high powers of our new tests under constant population size, as well as their moderate power to detect selection in expanding populations. We also devised a new maximum parsimony algorithm that, given the states of the sampled sequences at a recurrently mutating locus and an incompletely resolved genealogy, enumerates mutation histories with a minimum number of mutations while partially resolving genealogical relationships when necessary. Conclusions With their

  10. Spondyloenchondrodysplasia Due to Mutations in ACP5: A Comprehensive Survey.

    Science.gov (United States)

    Briggs, Tracy A; Rice, Gillian I; Adib, Navid; Ades, Lesley; Barete, Stephane; Baskar, Kannan; Baudouin, Veronique; Cebeci, Ayse N; Clapuyt, Philippe; Coman, David; De Somer, Lien; Finezilber, Yael; Frydman, Moshe; Guven, Ayla; Heritier, Sébastien; Karall, Daniela; Kulkarni, Muralidhar L; Lebon, Pierre; Levitt, David; Le Merrer, Martine; Linglart, Agnes; Livingston, John H; Navarro, Vincent; Okenfuss, Ericka; Puel, Anne; Revencu, Nicole; Scholl-Bürgi, Sabine; Vivarelli, Marina; Wouters, Carine; Bader-Meunier, Brigitte; Crow, Yanick J

    2016-04-01

    Spondyloenchondrodysplasia is a rare immuno-osseous dysplasia caused by biallelic mutations in ACP5. We aimed to provide a survey of the skeletal, neurological and immune manifestations of this disease in a cohort of molecularly confirmed cases. We compiled clinical, genetic and serological data from a total of 26 patients from 18 pedigrees, all with biallelic ACP5 mutations. We observed a variability in skeletal, neurological and immune phenotypes, which was sometimes marked even between affected siblings. In total, 22 of 26 patients manifested autoimmune disease, most frequently autoimmune thrombocytopenia and systemic lupus erythematosus. Four patients were considered to demonstrate no clinical autoimmune disease, although two were positive for autoantibodies. In the majority of patients tested we detected upregulated expression of interferon-stimulated genes (ISGs), in keeping with the autoimmune phenotype and the likely immune-regulatory function of the deficient protein tartrate resistant acid phosphatase (TRAP). Two mutation positive patients did not demonstrate an upregulation of ISGs, including one patient with significant autoimmune disease controlled by immunosuppressive therapy. Our data expand the known phenotype of SPENCD. We propose that the OMIM differentiation between spondyloenchondrodysplasia and spondyloenchondrodysplasia with immune dysregulation is no longer appropriate, since the molecular evidence that we provide suggests that these phenotypes represent a continuum of the same disorder. In addition, the absence of an interferon signature following immunomodulatory treatments in a patient with significant autoimmune disease may indicate a therapeutic response important for the immune manifestations of spondyloenchondrodysplasia.

  11. Enzyme-activity mutations detected in mice after paternal fractionated irradiation

    International Nuclear Information System (INIS)

    Charles, D.J.; Pretsch, W.

    1986-01-01

    (101/E1 X C3H/E1)F 1 -hybrid male mice were exposed in a 24-h fractionation interval to either 3.0 + 3.0-Gy or 5.1 + 5.1-Gy X-irradiation, and mated to untreated Test-stock females. The offspring were examined for mutations at 7 recessive specific loci and for activity alterations of erythrocyte enzymes controlled presumably by 12 loci. No enzyme-activity mutant was found in 3610 F 1 -offspring of the control group. In the experimental groups, no mutant was detected in 533 (3.0 + 3.0 Gy) and 173 (5.1 + 5.1 Gy) offspring from postspermatogonial germ cells treated. After treatment of spermatogonia, 1 mutant in 3388 F 1 -offspring of the 3.0 + 3.0-Gy group, and 5 mutants in 3187 F 1 offspring of the 5.1 + 5.1-Gy group were found. The mutants were all genetically confirmed. The frequency (expressed as mutants/locus/gamete) of enzyme-activity mutations is 2 (5.1 + 5.1-Gy group) to 10 (3.0 + 3.0-Gy group) times lower than the frequency of recessive specific-locus mutations. (Auth.)

  12. 21 CFR 864.7280 - Factor V Leiden DNA mutation detection systems.

    Science.gov (United States)

    2010-04-01

    ... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Factor V Leiden DNA mutation detection systems....7280 Factor V Leiden DNA mutation detection systems. (a) Identification. Factor V Leiden deoxyribonucleic acid (DNA) mutation detection systems are devices that consist of different reagents and...

  13. Microarray-based mutation analysis of the ABCA4 (ABCR) gene in autosomal recessive cone-rod dystrophy and retinitis pigmentosa.

    Science.gov (United States)

    Klevering, B Jeroen; Yzer, Suzanne; Rohrschneider, Klaus; Zonneveld, Marijke; Allikmets, Rando; van den Born, L Ingeborgh; Maugeri, Alessandra; Hoyng, Carel B; Cremers, Frans P M

    2004-12-01

    Mutations in the ABCA4 gene have been associated with autosomal recessive Stargardt disease (STGD1), cone-rod dystrophy (CRD), and retinitis pigmentosa (RP). We employed a recently developed genotyping microarray, the ABCR400-chip, to search for known ABCA4 mutations in patients with isolated or autosomal recessive CRD (54 cases) or RP (90 cases). We performed detailed ophthalmologic examinations and identified at least one ABCA4 mutation in 18 patients (33%) with CRD and in five patients (5.6%) with RP. Single-strand conformation polymorphism (SSCP) analysis and subsequent DNA sequencing revealed four novel missense mutations (R24C, E161K, P597S, G618E) and a novel 1-bp deletion (5888delG). Ophthalmoscopic abnormalities in CRD patients ranged from minor granular pigmentary changes in the posterior pole to widespread atrophy. In 12 patients with recordable electroretinogram (ERG) tracings, a cone-rod pattern was detected. Three patients demonstrated progression from a retinal dystrophy resembling STGD1 to a more widespread degeneration, and were subsequently diagnosed as CRD. In addition to a variable degree of atrophy, all RP patients displayed ophthalmologic characteristics of classic RP. When detectable, ERG recordings in these patients demonstrated rod-cone patterns of photoreceptor degeneration. In conclusion, in this study, we show that the ABCA4 mutation chip is an efficient first screening tool for arCRD.

  14. NDUFA4 Mutations Underlie Dysfunction of a Cytochrome c Oxidase Subunit Linked to Human Neurological Disease

    Directory of Open Access Journals (Sweden)

    Robert D.S. Pitceathly

    2013-06-01

    Full Text Available The molecular basis of cytochrome c oxidase (COX, complex IV deficiency remains genetically undetermined in many cases. Homozygosity mapping and whole-exome sequencing were performed in a consanguineous pedigree with isolated COX deficiency linked to a Leigh syndrome neurological phenotype. Unexpectedly, affected individuals harbored homozygous splice donor site mutations in NDUFA4, a gene previously assigned to encode a mitochondrial respiratory chain complex I (NADH:ubiquinone oxidoreductase subunit. Western blot analysis of denaturing gels and immunocytochemistry revealed undetectable steady-state NDUFA4 protein levels, indicating that the mutation causes a loss-of-function effect in the homozygous state. Analysis of one- and two-dimensional blue-native polyacrylamide gels confirmed an interaction between NDUFA4 and the COX enzyme complex in control muscle, whereas the COX enzyme complex without NDUFA4 was detectable with no abnormal subassemblies in patient muscle. These observations support recent work in cell lines suggesting that NDUFA4 is an additional COX subunit and demonstrate that NDUFA4 mutations cause human disease. Our findings support reassignment of the NDUFA4 protein to complex IV and suggest that patients with unexplained COX deficiency should be screened for NDUFA4 mutations.

  15. Tooth agenesis in osteogenesis imperfecta related to mutations in the collagen type I genes.

    Science.gov (United States)

    Malmgren, B; Andersson, K; Lindahl, K; Kindmark, A; Grigelioniene, G; Zachariadis, V; Dahllöf, G; Åström, E

    2017-01-01

    Osteogenesis imperfecta (OI) is a heterogeneous group of disorders of connective tissue, mainly caused by mutations in the collagen type I genes (COL1A1 and COL1A2). Tooth agenesis is a common feature of OI. We investigated the association between tooth agenesis and collagen type I mutations in individuals with OI. In this cohort study, 128 unrelated individuals with OI were included. Panoramic radiographs were analyzed regarding dentinogenesis imperfecta (DGI) and congenitally missing teeth. The collagen I genes were sequenced in all individuals, and in 25, multiplex ligation-dependent probe amplification was performed. Mutations in the COL1A1 and COL1A2 genes were found in 104 of 128 individuals. Tooth agenesis was diagnosed in 17% (hypodontia 11%, oligodontia 6%) and was more frequent in those with DGI (P = 0.016), and in those with OI type III, 47%, compared to those with OI types I, 12% (P = 0.003), and IV, 13% (P = 0.017). Seventy-five percent of the individuals with oligodontia (≥6 missing teeth) had qualitative mutations, but there was no association with OI type, gender, or presence of DGI. The prevalence of tooth agenesis is high (17%) in individuals with OI, and OI caused by a qualitative collagen I mutation is associated with oligodontia. © 2016 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  16. A Comprehensive Strategy for Accurate Mutation Detection of the Highly Homologous PMS2.

    Science.gov (United States)

    Li, Jianli; Dai, Hongzheng; Feng, Yanming; Tang, Jia; Chen, Stella; Tian, Xia; Gorman, Elizabeth; Schmitt, Eric S; Hansen, Terah A A; Wang, Jing; Plon, Sharon E; Zhang, Victor Wei; Wong, Lee-Jun C

    2015-09-01

    Germline mutations in the DNA mismatch repair gene PMS2 underlie the cancer susceptibility syndrome, Lynch syndrome. However, accurate molecular testing of PMS2 is complicated by a large number of highly homologous sequences. To establish a comprehensive approach for mutation detection of PMS2, we have designed a strategy combining targeted capture next-generation sequencing (NGS), multiplex ligation-dependent probe amplification, and long-range PCR followed by NGS to simultaneously detect point mutations and copy number changes of PMS2. Exonic deletions (E2 to E9, E5 to E9, E8, E10, E14, and E1 to E15), duplications (E11 to E12), and a nonsense mutation, p.S22*, were identified. Traditional multiplex ligation-dependent probe amplification and Sanger sequencing approaches cannot differentiate the origin of the exonic deletions in the 3' region when PMS2 and PMS2CL share identical sequences as a result of gene conversion. Our approach allows unambiguous identification of mutations in the active gene with a straightforward long-range-PCR/NGS method. Breakpoint analysis of multiple samples revealed that recurrent exon 14 deletions are mediated by homologous Alu sequences. Our comprehensive approach provides a reliable tool for accurate molecular analysis of genes containing multiple copies of highly homologous sequences and should improve PMS2 molecular analysis for patients with Lynch syndrome. Copyright © 2015 American Society for Investigative Pathology and the Association for Molecular Pathology. Published by Elsevier Inc. All rights reserved.

  17. High resolution melting for mutation scanning of TP53 exons 5–8

    International Nuclear Information System (INIS)

    Krypuy, Michael; Dobrovic, Alexander; Ahmed, Ahmed Ashour; Etemadmoghadam, Dariush; Hyland, Sarah J; Australian Ovarian Cancer Study Group; Fazio, Anna de; Fox, Stephen B; Brenton, James D; Bowtell, David D

    2007-01-01

    p53 is commonly inactivated by mutations in the DNA-binding domain in a wide range of cancers. As mutant p53 often influences response to therapy, effective and rapid methods to scan for mutations in TP53 are likely to be of clinical value. We therefore evaluated the use of high resolution melting (HRM) as a rapid mutation scanning tool for TP53 in tumour samples. We designed PCR amplicons for HRM mutation scanning of TP53 exons 5 to 8 and tested them with DNA from cell lines hemizygous or homozygous for known mutations. We assessed the sensitivity of each PCR amplicon using dilutions of cell line DNA in normal wild-type DNA. We then performed a blinded assessment on ovarian tumour DNA samples that had been previously sequenced for mutations in TP53 to assess the sensitivity and positive predictive value of the HRM technique. We also performed HRM analysis on breast tumour DNA samples with unknown TP53 mutation status. One cell line mutation was not readily observed when exon 5 was amplified. As exon 5 contained multiple melting domains, we divided the exon into two amplicons for further screening. Sequence changes were also introduced into some of the primers to improve the melting characteristics of the amplicon. Aberrant HRM curves indicative of TP53 mutations were observed for each of the samples in the ovarian tumour DNA panel. Comparison of the HRM results with the sequencing results revealed that each mutation was detected by HRM in the correct exon. For the breast tumour panel, we detected seven aberrant melt profiles by HRM and subsequent sequencing confirmed the presence of these and no other mutations in the predicted exons. HRM is an effective technique for simple and rapid scanning of TP53 mutations that can markedly reduce the amount of sequencing required in mutational studies of TP53

  18. Collagen type I induces EGFR-TKI resistance in EGFR-mutated cancer cells by mTOR activation through Akt-independent pathway.

    Science.gov (United States)

    Yamazaki, Shota; Higuchi, Youichi; Ishibashi, Masayuki; Hashimoto, Hiroko; Yasunaga, Masahiro; Matsumura, Yasuhiro; Tsuchihara, Katsuya; Tsuboi, Masahiro; Goto, Koichi; Ochiai, Atsushi; Ishii, Genichiro

    2018-06-01

    Primary resistance to epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs) is a serious problem in lung adenocarcinoma patients harboring EGFR mutations. The aim of this study was to examine whether and how collagen type I (Col I), the most abundantly deposited matrix in tumor stroma, affects EGFR-TKI sensitivity in EGFR-mutant cells. We evaluated the EGFR-TKI sensitivity of EGFR-mutated cancer cells cultured with Col I. Changes in the activation of downstream signaling molecules of EGFR were analyzed. We also examined the association between the Col I expression in tumor stroma in surgical specimens and EGFR-TKI response of postoperative recurrence patients with EGFR mutations. Compared to cancer cells without Col I, the survival rate of cancer cells cultured with Col I was significantly higher after EGFR-TKI treatment. In cancer cells cultured with and without Col I, EGFR-TKI suppressed the levels of phosphorylated (p-)EGFR, p-ERK1/2, and p-Akt. When compared to cancer cells without Col I, expression of p-P70S6K, a hallmark of mTOR activation, was dramatically upregulated in cancer cells with Col I. This activation was maintained even after EGFR-TKI treatment. Simultaneous treatment with EGFR-TKI and mTOR inhibitor abrogated Col I-induced resistance to EGFR-TKI. Patients with Col I-rich stroma had a significantly shorter progression-free survival time after EGFR-TKI therapy (238 days vs 404 days; P Cancer Science published by John Wiley & Sons Australia, Ltd on behalf of Japanese Cancer Association.

  19. Assessment of SLX4 Mutations in Hereditary Breast Cancers.

    Directory of Open Access Journals (Sweden)

    Sohela Shah

    Full Text Available SLX4 encodes a DNA repair protein that regulates three structure-specific endonucleases and is necessary for resistance to DNA crosslinking agents, topoisomerase I and poly (ADP-ribose polymerase (PARP inhibitors. Recent studies have reported mutations in SLX4 in a new subtype of Fanconi anemia (FA, FA-P. Monoallelic defects in several FA genes are known to confer susceptibility to breast and ovarian cancers.To determine if SLX4 is involved in breast cancer susceptibility, we sequenced the entire SLX4 coding region in 738 (270 Jewish and 468 non-Jewish breast cancer patients with 2 or more family members affected by breast cancer and no known BRCA1 or BRCA2 mutations. We found a novel nonsense (c.2469G>A, p.W823* mutation in one patient. In addition, we also found 51 missense variants [13 novel, 23 rare (MAF1%], of which 22 (5 novel and 17 rare were predicted to be damaging by Polyphen2 (score = 0.65-1. We performed functional complementation studies using p.W823* and 5 SLX4 variants (4 novel and 1 rare cDNAs in a human SLX4-null fibroblast cell line, RA3331. While wild type SLX4 and all the other variants fully rescued the sensitivity to mitomycin C (MMC, campthothecin (CPT, and PARP inhibitor (Olaparib the p.W823* SLX4 mutant failed to do so.Loss-of-function mutations in SLX4 may contribute to the development of breast cancer in very rare cases.

  20. COL4A1 mutations associated with a characteristic pattern of intracranial calcification

    NARCIS (Netherlands)

    Livingston, J.; Doherty, D.; Orcesi, S.; Tonduti, D.; Piechiecchio, A.; La Piana, R.; Tournier-Lasserve, E.; Majumdar, A.; Tomkins, S.; Rice, G.; Kneen, R.; van der Knaap, M.; Crow, Y.

    2011-01-01

    Intracranial calcification (ICC) is a relatively common radiological finding in children undergoing investigation for neurological disorders. Many causes are recognised, and ICC is often regarded as a non-specific sign.From an ongoing study of ICC, we identified 5 patients with characteristic

  1. Mutations detected in the repetitive sequences in the children of the atomic bomb survivors

    International Nuclear Information System (INIS)

    Satoh, Chiyoko; Kodaira, Mieko

    1994-01-01

    We have been examining genetic effects of radiation in the children of the atomic bomb survivors. In a pilot study, 50 exposed families with 64 children and 50 control families with 60 children were examined for trinucleotide repeat expansion mutations at 3 loci and mutations at 6 minisatellite loci. Average dose of the 51 exposed parents was 1.8 Sv. By examining 124 children of 100 families, 65 germ cells derived from exposed parents and 183 germ cells of non-exposed parents were examined. The trinucleotide repeat expansions in genes of certain human genetic diseases show remarkable variation both within the cells of a single individual and among affected members of a single family which have been interpreted as mitotic and meiotic instability. We examined the regions with triplet repeats in the FMR-1, AR and DM genes causative for fragile X syndrome, spinobulbar muscular atrophy and myotonic dystrophy. No mutations were detected in 177 regions derived from 65 germ cells of exposed parents and 443 regions from 183 germ cells of non-exposed parents. No effects on the instability of the triplet repeats in the germ cells derived from exposed or unexposed individuals were observed. In the examinations of the 6 minisatellite loci of Pc-1, λTM-18, ChdTC-15, pλg3, λMS-1, and CEB-1, we detected single mutations at each of the pλg3 and λMS-1, and 4 mutations at the CEB-1 locus which had occurred in the 65 gametes in the exposed parents. Thus, mutation rates per gamete at the pλg3, λMS-1 and CEB-1 were 1.5%, 1.5% and 6.2%. On the other hand, mutations in these 3 loci in the 183 gametes of non-exposed parents were 0, 11 and 11, that is, the mutation rates per gamete were 0%, 6.0% and 6.0%. No significant difference was observed in the mutation rate at each of the 3 loci between 2 groups of parents. These preliminary results suggest that A-bomb exposure seems not to affect the germline instability at these 3 loci. (J.P.N)

  2. Detection of mutation in isoniazid-resistant Mycobacterium tuberculosis isolates from tuberculosis patients in Belarus

    Directory of Open Access Journals (Sweden)

    Bostanabad S

    2008-01-01

    Full Text Available The aim of this study was to investigate the frequency, location and type of katG mutations in Mycobacterium tuberculosis strains isolated from patients in Belarus. Forty two isoniazid-resistant isolates were identified from sputum of 163 patients with active pulmonary tuberculosis. Drug susceptibility testing was determined by using CDC standard conventional proportional method and BACTEC system. Standard PCR method for detection of isoniazid resistance associated mutations was performed by katG gene amplification and DNA sequencing. Most mutations were found in katG gene codons 315, 316 and 309. Four types of mutations were identified in codon 315: AGC→ACC ( n = 36 85%, AGC→AGG ( n = 1 2.3%, AGC→AAC ( n = 2 4.7%, AGC→GGC ( n = 1 2.3%. One type of mutation was found in codon 316: GGC→AGC ( n = 1841.4%, four types of mutations were detected in codon 309: GGT→GGT ( n = 716.1%, GGT→GCT ( n = 49.2%, GGT→GTC ( n = 36.9%, GGT→GGG ( n = 12.7%. The highest frequency of mutations sharing between primary and secondary infections was found in codon 315.

  3. Development of a human somatic mutation detection method--GPA assay

    International Nuclear Information System (INIS)

    Mao Jianping; Dong Yan; Liu Bin; Lin Ruxian; Sun Zhixian

    2000-01-01

    Objective: To study the damage to human body caused by environmental radiation, and supervise the somatic mutations. Methods: Three monoclonal antibodies specific to M-type(3G4), N-type(6A8), and MN-type (3C5) of glycophorin A, respectively, were prepared. Fluorescence or biotin conjugated antibodies were bound specifically to formalin and/or dimethyl suber-imidate fixed erythrocytes. M, MN and N type cells were divided by cytometry to demonstrate the erythrocyte mutation characteristics (MN→MO, MM, NO, NN) and give out the variant frequency. Results: 1Wa, 1Wb and 2Wa methods of GPA assay were developed. Erythrocytes of MN type individuals could be separated to normal and single locus variant groups by 1W methods and they could be sorted as normal (MN), single gene deletion mutants (MO, NO), homozygous mutants (MM, NN) cell groups by 2Wa method. Conclusion: The assay is applicable to evaluating the frequency of variant erythrocytes from human somatic mutation

  4. Tumor‐associated DNA mutation detection in individuals undergoing colonoscopy

    OpenAIRE

    Fleshner, Phillip; Braunstein, Glenn D.; Ovsepyan, Gayane; Tonozzi, Theresa R.; Kammesheidt, Anja

    2017-01-01

    Abstract The majority of colorectal cancers (CRC) harbor somatic mutations and epigenetic modifications in the tumor tissue, and some of these mutations can be detected in plasma as circulating tumor DNA (ctDNA). Precancerous colorectal lesions also contain many of these same mutations. This study examined plasma for ctDNA from patients undergoing a screening or diagnostic colonoscopy to determine the sensitivity and specificity of the ctDNA panel for detecting CRC and precancerous lesions. T...

  5. Detection of genetic mutations associated with macrolide resistance of Mycoplasma pneumoniae

    Directory of Open Access Journals (Sweden)

    Chi Eun Oh

    2010-02-01

    Full Text Available Purpose : The aim of this study was to identify mutations associated with macrolide resistance in Mycoplasma pneumoniae (MP and to establish a cultural method to determine antimicrobial susceptibility. Methods : Nasopharyngeal aspirates (NPAs were collected from 62 children diagnosed with MP pneumonia by a serologic method or polymerase chain reaction. The 23S rRNA and L4 ribosomal protein genes of MP were amplified and sequenced. To identify mutations in these 2 genes, their nucleotide sequences were compared to those of the reference strain M129. MP cultivation was carried out for 32 (28 frozen and 5 refrigerated NPAs and M129 strain using Chanock’s glucose broth and agar plate in a 5% CO2 incubator at 37?#608;and examined at 2-3 day intervals for 6 weeks. Results : Among the 62 specimens, 17 had M144V mutations in ribosomal protein L4. The A2064G mutation was observed in 1 specimen; its 23S rRNA gene was successfully sequenced. Culture for MP was successful from the M129 strain and 2 of the 5 NPAs that were refrigerated for no longer than 3 days. However, MP did not grow from the 28 NPAs that were kept frozen at -80?#608;since 2003. Conclusion : We found the M144V mutation of L4 protein to be common and that of domain V of 23S rRNA gene was relatively rare among MP. Studies on the prevalence of macrolide-resistant MP and the relationship between the mutations of 23S rRNA gene and ribosomal protein L4 will aid in understanding the mechanism of macrolide resistance in MP.

  6. Screening for duplications, deletions and a common intronic mutation detects 35% of second mutations in patients with USH2A monoallelic mutations on Sanger sequencing.

    Science.gov (United States)

    Steele-Stallard, Heather B; Le Quesne Stabej, Polona; Lenassi, Eva; Luxon, Linda M; Claustres, Mireille; Roux, Anne-Francoise; Webster, Andrew R; Bitner-Glindzicz, Maria

    2013-08-08

    23 (35%) of 'missing' mutations in Usher type 2 probands with only a single heterozygous USH2A mutation detected with Sanger sequencing could be attributed to deletions, duplications or a pathogenic deep intronic variant. Future mutation detection strategies and genetic counselling will need to take into account the prevalence of these types of mutations in order to provide a more comprehensive diagnostic service.

  7. Efficient Detection of Copy Number Mutations in PMS2 Exons with a Close Homolog.

    Science.gov (United States)

    Herman, Daniel S; Smith, Christina; Liu, Chang; Vaughn, Cecily P; Palaniappan, Selvi; Pritchard, Colin C; Shirts, Brian H

    2018-07-01

    Detection of 3' PMS2 copy-number mutations that cause Lynch syndrome is difficult because of highly homologous pseudogenes. To improve the accuracy and efficiency of clinical screening for these mutations, we developed a new method to analyze standard capture-based, next-generation sequencing data to identify deletions and duplications in PMS2 exons 9 to 15. The approach captures sequences using PMS2 targets, maps sequences randomly among regions with equal mapping quality, counts reads aligned to homologous exons and introns, and flags read count ratios outside of empirically derived reference ranges. The method was trained on 1352 samples, including 8 known positives, and tested on 719 samples, including 17 known positives. Clinical implementation of the first version of this method detected new mutations in the training (N = 7) and test (N = 2) sets that had not been identified by our initial clinical testing pipeline. The described final method showed complete sensitivity in both sample sets and false-positive rates of 5% (training) and 7% (test), dramatically decreasing the number of cases needing additional mutation evaluation. This approach leveraged the differences between gene and pseudogene to distinguish between PMS2 and PMS2CL copy-number mutations. These methods enable efficient and sensitive Lynch syndrome screening for 3' PMS2 copy-number mutations and may be applied similarly to other genomic regions with highly homologous pseudogenes. Copyright © 2018 American Society for Investigative Pathology and the Association for Molecular Pathology. Published by Elsevier Inc. All rights reserved.

  8. A novel CDKL5 mutation in a 47,XXY boy with the early-onset seizure variant of Rett syndrome.

    Science.gov (United States)

    Sartori, Stefano; Di Rosa, Gabriella; Polli, Roberta; Bettella, Elisa; Tricomi, Giovanni; Tortorella, Gaetano; Murgia, Alessandra

    2009-02-01

    Mutations of the cyclin-dependent kinase-like 5 gene (CDKL5), reported almost exclusively in female subjects, have been recently found to be the cause of a phenotype overlapping Rett syndrome with early-onset epileptic encephalopathy. We describe the first CDKL5 mutation detected in a male individual with 47,XXY karyotype. This previously unreported, de novo, mutation truncates the large CDKL5 COOH-terminal region, thought to be crucial for the proper sub-cellular localization of the CDKL5 protein. The resulting phenotype is characterized by a severe early-onset epileptic encephalopathy, global developmental delay, and profound intellectual and motor impairment with features reminiscent of Rett syndrome. In light of the data presented we discuss the possible phenotypic modulatory effects of the supernumerary wild type X allele and pattern of X chromosome inactivation and stress the importance of considering the causal involvement of CDKL5 in developmentally delayed males with early-onset seizures. (c) 2009 Wiley-Liss, Inc.

  9. Overview of the mutation spectrum in familial exudative vitreoretinopathy and Norrie disease with identification of 21 novel variants in FZD4, LRP5, and NDP.

    Science.gov (United States)

    Nikopoulos, Konstantinos; Venselaar, Hanka; Collin, Rob W J; Riveiro-Alvarez, Rosa; Boonstra, F Nienke; Hooymans, Johanna M M; Mukhopadhyay, Arijit; Shears, Deborah; van Bers, Marleen; de Wijs, Ilse J; van Essen, Anthonie J; Sijmons, Rolf H; Tilanus, Mauk A D; van Nouhuys, C Erik; Ayuso, Carmen; Hoefsloot, Lies H; Cremers, Frans P M

    2010-06-01

    Wnt signaling is a crucial component of the cell machinery orchestrating a series of physiological processes such as cell survival, proliferation, and migration. Among the plethora of roles that Wnt signaling plays, its canonical branch regulates eye organogenesis and angiogenesis. Mutations in the genes encoding the low density lipoprotein receptor protein 5 (LRP5) and frizzled 4 (FZD4), acting as coreceptors for Wnt ligands, cause familial exudative vitreoretinopathy (FEVR). Moreover, mutations in the gene encoding NDP, a ligand for these Wnt receptors, cause Norrie disease and FEVR. Both FEVR and Norrie disease share similar phenotypic characteristics, including abnormal vascularization of the peripheral retina and formation of fibrovascular masses in the eye that can lead to blindness. In this mutation update, we report 21 novel variants for FZD4, LRP5, and NDP, and discuss the putative functional consequences of missense mutations. In addition, we provide a comprehensive overview of all previously published variants in the aforementioned genes and summarize the phenotypic characteristics in mouse models carrying mutations in the orthologous genes. The increasing molecular understanding of Wnt signaling, related to ocular development and blood supply, offers more tools for accurate disease diagnosis that may be important in the development of therapeutic interventions.

  10. Consortium for Osteogenesis Imperfecta Mutations in the Helical Domain of Type I Collagen: Regions Rich in Lethal Mutations Align With Collagen Binding Sites for Integrins and Proteoglycans

    Science.gov (United States)

    Marini, Joan C.; Forlino, Antonella; Cabral, Wayne A.; Barnes, Aileen M.; San Antonio, James D.; Milgrom, Sarah; Hyland, James C.; Körkkö, Jarmo; Prockop, Darwin J.; De Paepe, Anne; Coucke, Paul; Symoens, Sofie; Glorieux, Francis H.; Roughley, Peter J.; Lund, Alan M.; Kuurila-Svahn, Kaija; Hartikka, Heini; Cohn, Daniel H.; Krakow, Deborah; Mottes, Monica; Schwarze, Ulrike; Chen, Diana; Yang, Kathleen; Kuslich, Christine; Troendle, James; Dalgleish, Raymond; Byers, Peter H.

    2014-01-01

    Osteogenesis imperfecta (OI) is a generalized disorder of connective tissue characterized by fragile bones and easy susceptibility to fracture. Most cases of OI are caused by mutations in type I collagen. We have identified and assembled structural mutations in type I collagen genes (COL1A1 and COL1A2, encoding the proα1(I) and proα2(I) chains, respectively) that result in OI. Quantitative defects causing type I OI were not included. Of these 832 independent mutations, 682 result in substitution for glycine residues in the triple helical domain of the encoded protein and 150 alter splice sites. Distinct genotype–phenotype relationships emerge for each chain. One-third of the mutations that result in glycine substitutions in α1(I) are lethal, especially when the substituting residues are charged or have a branched side chain. Substitutions in the first 200 residues are nonlethal and have variable outcome thereafter, unrelated to folding or helix stability domains. Two exclusively lethal regions (helix positions 691–823 and 910–964) align with major ligand binding regions (MLBRs), suggesting crucial interactions of collagen monomers or fibrils with integrins, matrix metalloproteinases (MMPs), fibronectin, and cartilage oligomeric matrix protein (COMP). Mutations in COL1A2 are predominantly nonlethal (80%). Lethal substitutions are located in eight regularly spaced clusters along the chain, supporting a regional model. The lethal regions align with proteoglycan binding sites along the fibril, suggesting a role in fibril–matrix interactions. Recurrences at the same site in α2(I) are generally concordant for outcome, unlike α1(I). Splice site mutations comprise 20% of helical mutations identified in OI patients, and may lead to exon skipping, intron inclusion, or the activation of cryptic splice sites. Splice site mutations in COL1A1 are rarely lethal; they often lead to frameshifts and the mild type I phenotype. In α2(I), lethal exon skipping events are

  11. Detection of Ultra-Rare Mitochondrial Mutations in Breast Stem Cells by Duplex Sequencing.

    Directory of Open Access Journals (Sweden)

    Eun Hyun Ahn

    Full Text Available Long-lived adult stem cells could accumulate non-repaired DNA damage or mutations that increase the risk of tumor formation. To date, studies on mutations in stem cells have concentrated on clonal (homoplasmic mutations and have not focused on rarely occurring stochastic mutations that may accumulate during stem cell dormancy. A major challenge in investigating these rare mutations is that conventional next generation sequencing (NGS methods have high error rates. We have established a new method termed Duplex Sequencing (DS, which detects mutations with unprecedented accuracy. We present a comprehensive analysis of mitochondrial DNA mutations in human breast normal stem cells and non-stem cells using DS. The vast majority of mutations occur at low frequency and are not detectable by NGS. The most prevalent point mutation types are the C>T/G>A and A>G/T>C transitions. The mutations exhibit a strand bias with higher prevalence of G>A, T>C, and A>C mutations on the light strand of the mitochondrial genome. The overall rare mutation frequency is significantly lower in stem cells than in the corresponding non-stem cells. We have identified common and unique non-homoplasmic mutations between non-stem and stem cells that include new mutations which have not been reported previously. Four mutations found within the MT-ND5 gene (m.12684G>A, m.12705C>T, m.13095T>C, m.13105A>G are present in all groups of stem and non-stem cells. Two mutations (m.8567T>C, m.10547C>G are found only in non-stem cells. This first genome-wide analysis of mitochondrial DNA mutations may aid in characterizing human breast normal epithelial cells and serve as a reference for cancer stem cell mutation profiles.

  12. DHPLC technology for high-throughput detection of mutations in a durum wheat TILLING population.

    Science.gov (United States)

    Colasuonno, Pasqualina; Incerti, Ornella; Lozito, Maria Luisa; Simeone, Rosanna; Gadaleta, Agata; Blanco, Antonio

    2016-02-17

    Durum wheat (Triticum turgidum L.) is a cereal crop widely grown in the Mediterranean regions; the amber grain is mainly used for the production of pasta, couscous and typical breads. Single nucleotide polymorphism (SNP) detection technologies and high-throughput mutation induction represent a new challenge in wheat breeding to identify allelic variation in large populations. The TILLING strategy makes use of traditional chemical mutagenesis followed by screening for single base mismatches to identify novel mutant loci. Although TILLING has been combined to several sensitive pre-screening methods for SNP analysis, most rely on expensive equipment. Recently, a new low cost and time saving DHPLC protocol has been used in molecular human diagnostic to detect unknown mutations. In this work, we developed a new durum wheat TILLING population (cv. Marco Aurelio) using 0.70-0.85% ethyl methane sulfonate (EMS). To investigate the efficiency of the mutagenic treatments, a pilot screening was carried out on 1,140 mutant lines focusing on two target genes (Lycopene epsilon-cyclase, ε-LCY, and Lycopene beta-cyclase, β-LCY) involved in carotenoid metabolism in wheat grains. We simplify the heteroduplex detection by two low cost methods: the enzymatic cleavage (CelI)/agarose gel technique and the denaturing high-performance liquid chromatography (DHPLC). The CelI/agarose gel approach allowed us to identify 31 mutations, whereas the DHPLC procedure detected a total of 46 mutations for both genes. All detected mutations were confirmed by direct sequencing. The estimated overall mutation frequency for the pilot assay by the DHPLC methodology resulted to be of 1/77 kb, representing a high probability to detect interesting mutations in the target genes. We demonstrated the applicability and efficiency of a new strategy for the detection of induced variability. We produced and characterized a new durum wheat TILLING population useful for a better understanding of key gene functions

  13. Two Siblings With a CDKL5 Mutation: Genotype and Phenotype Evaluation

    NARCIS (Netherlands)

    Hagebeuk, Eveline E. O.; Marcelis, Carlo L.; Alders, Mariëlle; Kaspers, Ageeth; de Weerd, Al W.

    2015-01-01

    This is the second report of a family with a recurrence of a CDKL5 mutation (c. 283-3_290del) in 2 sisters. Both parents tested negative for the mutation in all tissues, but germline mosaicism is likely. Clinically CDKL5 patients resemble those with Rett syndrome, caused by a MECP2 mutation, who

  14. RADIA: RNA and DNA integrated analysis for somatic mutation detection.

    Directory of Open Access Journals (Sweden)

    Amie J Radenbaugh

    Full Text Available The detection of somatic single nucleotide variants is a crucial component to the characterization of the cancer genome. Mutation calling algorithms thus far have focused on comparing the normal and tumor genomes from the same individual. In recent years, it has become routine for projects like The Cancer Genome Atlas (TCGA to also sequence the tumor RNA. Here we present RADIA (RNA and DNA Integrated Analysis, a novel computational method combining the patient-matched normal and tumor DNA with the tumor RNA to detect somatic mutations. The inclusion of the RNA increases the power to detect somatic mutations, especially at low DNA allelic frequencies. By integrating an individual's DNA and RNA, we are able to detect mutations that would otherwise be missed by traditional algorithms that examine only the DNA. We demonstrate high sensitivity (84% and very high precision (98% and 99% for RADIA in patient data from endometrial carcinoma and lung adenocarcinoma from TCGA. Mutations with both high DNA and RNA read support have the highest validation rate of over 99%. We also introduce a simulation package that spikes in artificial mutations to patient data, rather than simulating sequencing data from a reference genome. We evaluate sensitivity on the simulation data and demonstrate our ability to rescue back mutations at low DNA allelic frequencies by including the RNA. Finally, we highlight mutations in important cancer genes that were rescued due to the incorporation of the RNA.

  15. Effect of KCNJ5 Mutations on Gene Expression in Aldosterone-Producing Adenomas and Adrenocortical Cells

    Science.gov (United States)

    Monticone, Silvia; Hattangady, Namita G.; Nishimoto, Koshiro; Mantero, Franco; Rubin, Beatrice; Cicala, Maria Verena; Pezzani, Raffaele; Auchus, Richard J.; Ghayee, Hans K.; Shibata, Hirotaka; Kurihara, Isao; Williams, Tracy A.; Giri, Judith G.; Bollag, Roni J.; Edwards, Michael A.; Isales, Carlos M.

    2012-01-01

    Context: Primary aldosteronism is a heterogeneous disease that includes both sporadic and familial forms. A point mutation in the KCNJ5 gene is responsible for familial hyperaldosteronism type III. Somatic mutations in KCNJ5 also occur in sporadic aldosterone producing adenomas (APA). Objective: The objective of the study was to define the effect of the KCNJ5 mutations on gene expression and aldosterone production using APA tissue and human adrenocortical cells. Methods: A microarray analysis was used to compare the transcriptome profiles of female-derived APA samples with and without KCNJ5 mutations and HAC15 adrenal cells overexpressing either mutated or wild-type KCNJ5. Real-time PCR validated a set of differentially expressed genes. Immunohistochemical staining localized the KCNJ5 expression in normal adrenals and APA. Results: We report a 38% (18 of 47) prevalence of KCNJ5 mutations in APA. KCNJ5 immunostaining was highest in the zona glomerulosa of NA and heterogeneous in APA tissue, and KCNJ5 mRNA was 4-fold higher in APA compared with normal adrenals (P APA with and without KCNJ5 mutations displayed slightly different gene expression patterns, notably the aldosterone synthase gene (CYP11B2) was more highly expressed in APA with KCNJ5 mutations. Overexpression of KCNJ5 mutations in HAC15 increased aldosterone production and altered expression of 36 genes by greater than 2.5-fold (P APA, and our data suggest that these mutations increase expression of CYP11B2 and NR4A2, thus increasing aldosterone production. PMID:22628608

  16. Pathogenic substitution of IVS15 + 5G > A in SLC26A4 in patients of Okinawa Islands with enlarged vestibular aqueduct syndrome or Pendred syndrome.

    Science.gov (United States)

    Ganaha, Akira; Kaname, Tadashi; Yanagi, Kumiko; Naritomi, Kenji; Tono, Tetsuya; Usami, Shin-ichi; Suzuki, Mikio

    2013-05-24

    Pendred syndrome (PS) and nonsyndromic hearing loss associated with enlarged vestibular aqueduct (EVA) are caused by SLC26A4 mutations. The Okinawa Islands are the southwestern-most islands of the Japanese archipelago. And ancestral differences have been reported between people from Okinawa Island and those from the main islands of Japan. To confirm the ethnic variation of the spectrum of SLC26A4 mutations, we investigated the frequencies of SLC26A4 mutations and clinical manifestations of patients with EVA or PS living in the Okinawa Islands. We examined 22 patients with EVA or PS from 21 unrelated families in Okinawa Islands. The patient's clinical history, findings of physical and otoscopic examinations, hearing test, and computed tomography (CT) scan of the temporal bones were recorded. To detect mutations, all 21 exons and the exon-intron junctions of SLC26A4 were sequenced for all subjects. Quantitative reverse-transcription polymerase chain reaction (qRT-PCR) for SLC26A4 and calculations using the comparative CT (2(-ΔΔCT)) method were used to determine the pathogenicity associated with gene substitutions. SLC26A4 mutations were identified in 21 of the 22 patients. We found a compound heterozygous mutation for IVS15 + 5G > A/H723R in nine patients (41%), a homozygous substitution of IVS15 + 5G > A in six patients (27%), and homozygous mutation for H723R in five patients (23%). The most prevalent types of SLC26A4 alleles were IVS15 + 5G > A and H723R, which both accounted for 15/22 (68%) of the patients. There were no significant correlations between the types of SLC26A4 mutation and clinical manifestations. Based on qRT-PCR results, expression of SLC26A4 was not identified in patients with the homozygous substitution of IVS15 + 5G > A. The substitution of IVS15 + 5G > A in SLC26A4 was the most common mutation in uniquely found in patients with PS and EVA in Okinawa Islands. This suggested that the spectrum of SLC26A4 mutation differed

  17. New mutations affecting induced mutagenesis in yeast.

    Science.gov (United States)

    Lawrence, C W; Krauss, B R; Christensen, R B

    1985-01-01

    Previously isolated mutations in baker's yeast, Saccharomyces cerevisiae, that impair induced mutagenesis were all identified with the aid of tests that either exclusively or predominantly detect base-pair substitutions. To avoid this bias, we have screened 11 366 potentially mutant clones for UV-induced reversion of the frameshift allele, his4-38, and have identified 10 mutants that give much reduced yields of revertants. Complementation and recombination tests show that 6 of these carry mutations at the previously known REV1, REV1 and REV3 loci, while the remaining 4 define 3 new genes, REV4 (2 mutations), REV5 and REV6. The rev4 mutations are readily suppressed in many genetic backgrounds and, like the rev5 mutation, impart only a limited deficiency for induced mutagenesis: it is likely, therefore that the REV4+ and REV5+ gene functions are only remotely concerned with this process. The rev6 mutants have a more general deficiency, however, as well as marked sensitivity to UV and an increased spontaneous mutation rate, properties that suggest the REV6 gene is directly involved in mutation induction. The REV5 gene is located about 1 cM proximal to CYC1 on chromosome X.

  18. [Analysis the relationship between SLC26A4 mutation and current diagnosis of inner ear malformation in children with sensorineural hearing loss].

    Science.gov (United States)

    Sun, Baochun; Zhou, Chengyong; Dai, Zhiyao

    2014-11-01

    Explore the relationship between the pathogenic mutations of SLC26A4 gene and inner ear malformation, and analyze the feasibility of genetic testing to help current diagnosis in part of children with sensorineural hearing loss. 2094 cases of children were detected by SLC26A4 with the method of DNA sequence. CT phenotypes of those children were classified according to the method proposed by Sennaroglu. We analyzed the relationship between the pathogenic mutations of gene and the CT phenotypes. (1) 685 cases of inner ear malformations were found in 2094 cases of children with sensorineural hearing loss by CT examination (371 cases of cochlea malformation were consisted of the follow types of malformation. Michel deformity was 6 cases, cochlea aplasia was 8 cases, common cavity deformity was 12 cases, incomplete partition type I was 27 cases, cochlea hypoplasia was 30 cases and Mondini malformation was 288 cases); Vestibular aqueduct was 265 cases; Vestibular/semicircular canal/internal auditory canal were 49 cases, normal was 1409 cases. (2) The DNA sequence results revealed that 465 cases carried pathogenic mutations (Bi-allelic mutations) of SLC26A4 gene, among which 135 cases were homozygous, 330 cases were compound heterozygous. (3) Pathogenic mutations of SLC26A4 gene detected 100% (465/465) in the group related to vestibular aqueduct malformation. The results suggest that pathogenic mutation of SLC26A4 gene is closely related to the CT phenotype of vestibular aqueduct malformation. Detecting of pathogenic mutations for hearing loss is binging the possibility to identify children with inner malformations at an early stage. As a consequence, it will improve the current diagnosis and therapeutical option.

  19. Fragment length analysis screening for detection of CEBPA mutations in intermediate-risk karyotype acute myeloid leukemia.

    Science.gov (United States)

    Fuster, Oscar; Barragán, Eva; Bolufer, Pascual; Such, Esperanza; Valencia, Ana; Ibáñez, Mariam; Dolz, Sandra; de Juan, Inmaculada; Jiménez, Antonio; Gómez, Maria Teresa; Buño, Ismael; Martínez, Joaquín; Cervera, José; Montesinos, Pau; Moscardó, Federico; Sanz, Miguel Ángel

    2012-01-01

    During last years, molecular markers have been increased as prognostic factors routinely screened in acute myeloid leukemia (AML). Recently, an increasing interest has been reported in introducing to clinical practice screening for mutations in the CCAAT/enhancer-binding protein α (CEBPA) gene in AML, as it seems to be a good prognostic factor. However, there is no reliable established method for assessing CEBPA mutations during the diagnostic work-up of AMLs. We describe here a straightforward and reliable fragment analysis method based in PCR capillary electrophoresis (PCR-CE) for screening of CEBPA mutations; moreover, we present the results obtained in 151 intermediate-risk karyotype AML patients (aged 16-80 years). The method gave a specificity of 100% and sensitivity of 93% with a lower detection limit of 1-5% for CEBPA mutations. The series found 19 mutations and four polymorphisms in 12 patients, seven of whom (58%) presented two mutations. The overall frequency of CEBPA mutations in AML was 8% (n = 12). CEBPA mutations showed no coincidence with FLT3-ITD or NPM1 mutations. CEBPA mutation predicted better disease-free survival in the group of patients without FLT3-ITD, NPM, or both genes mutated (HR 3.6, IC 95%; 1.0-13.2, p = 0.05) and better overall survival in patients younger than 65 of this group without molecular markers (HR 4.0, IC 95%; 1.0-17.4, p = 0.05). In conclusion, the fragment analysis method based in PCR-CE is a rapid, specific, and sensitive method for CEBPA mutation screening and our results confirm that CEBPA mutations can identify a subgroup of patients with favorable prognosis in AML with intermediate-risk karyotype.

  20. Clinical phenotype of 5 females with a CDKL5 mutation

    NARCIS (Netherlands)

    Stalpers, X.L.; Spruijt, L.; Yntema, H.G.; Verrips, A.

    2012-01-01

    Mutations in the X-linked cyclin dependent kinase like 5 (CDKL5) gene have been reported in approximately 80 patients since the first description in 2003. The clinical presentation partly corresponds with Rett syndrome, considering clinical features as intellectual disability, hypotonia, and poor

  1. The three stages of epilepsy in patients with CDKL5 mutations.

    Science.gov (United States)

    Bahi-Buisson, Nadia; Kaminska, Anna; Boddaert, Nathalie; Rio, Marlène; Afenjar, Alexandra; Gérard, Marion; Giuliano, Fabienne; Motte, Jacques; Héron, Delphine; Morel, Marie Ange N'guyen; Plouin, Perrine; Richelme, Christian; des Portes, Vincent; Dulac, Olivier; Philippe, Christophe; Chiron, Catherine; Nabbout, Rima; Bienvenu, Thierry

    2008-06-01

    Mutations in the X-linked cyclin-dependent kinase-like 5 (CDKL5) gene are responsible for a severe encephalopathy with early epilepsy. So far, the electroclinical phenotype remains largely unknown and no clear genotype-phenotype correlations have been established. To characterize the epilepsy associated with CDKL5 mutations and to look for a relationship between the genotype and the course of epilepsy. We retrospectively analyzed the electroclinical phenotypes of 12 patients aged from 2.5 to 19 years diagnosed with pathogenic CDKL5 mutations and one patient with a novel intronic sequence variation of uncertain pathogenicity and examined whether the severity of the epilepsy was linked to the type and location of mutations. The epilepsy course reveals three successive stages: (Stage I) early epilepsy (onset 1-10 weeks) with normal interictal electroencephalogram (EEG) (10/13) despite frequent convulsive seizures; (Stage II) epileptic encephalopathy with infantile spasms (8/8) and hypsarrhythmia (8/8). At the age of evaluation, seven patients were seizure free and six had developed refractory epilepsy (stage III) with tonic seizures and myoclonia (5/6). Interestingly, the patients carrying a CDKL5 mutations causing a truncation of the catalytic domain tended to develop a more frequent refractory epilepsy than patients with mutations located downstream (4/6, 66.6% versus 1/6, 16%) although, these trends are not yet significant. Our data contribute to a better definition of the epileptic phenotype in CDKL5 mutations, and might give some clues to a potential relationship between the phenotype and the genotype in these patients.

  2. Validation and comparison of two NGS assays for the detection of EGFR T790M resistance mutation in liquid biopsies of NSCLC patients.

    Science.gov (United States)

    Vollbrecht, Claudia; Lehmann, Annika; Lenze, Dido; Hummel, Michael

    2018-04-06

    Analysis of circulating cell-free DNA (cfDNA) derived from peripheral blood ("liquid biopsy") is an attractive alternative to identify non-small cell lung cancer (NSCLC) patients with the EGFR T790M mutation eligible for 3rd generation tyrosine kinase inhibitor therapy. We evaluated two PCR-based next generation sequencing (NGS) approaches, one including unique molecular identifiers (UMI), with focus on highly sensitive EGFR T790M mutation detection. Therefore, we extracted and sequenced cfDNA from synthetic plasma samples spiked with mutated DNA at decreasing allele frequencies and from 21 diagnostic NSCLC patients. Data evaluation was performed to determine the limit of detection (LoD), accuracy, specificity and sensitivity of both assays. Considering all tested reference dilutions and mutations the UMI assay performed best in terms of LoD (1% vs. 5%), sensitivity (95.8% vs. 81.3%), specificity (100% vs. 93.8%) and accuracy (96.9% vs. 84.4%). Comparing mutation status of diagnostic samples with both assays showed 81.3% concordance with primary mutation verifiable in 52% of cases. EGFR T790M was detected concordantly in 6/7 patients with allele frequencies from 0.1% to 27%. In one patient, the T790M mutation was exclusively detectable with the UMI assay. Our data demonstrate that both assays are applicable as multi-biomarker NGS tools enabling the simultaneous detection of primary EGFR driver and resistance mutations. However, for mutations with low allelic frequencies the use of NGS panels with UMI facilitates a more sensitive and reliable detection.

  3. Spectrum of MECP2 gene mutations in a cohort of Indian patients with Rett syndrome: report of two novel mutations.

    Science.gov (United States)

    Das, Dhanjit Kumar; Raha, Sarbani; Sanghavi, Daksha; Maitra, Anurupa; Udani, Vrajesh

    2013-02-15

    Rett syndrome (RTT) is an X-linked neurodevelopmental disorder, primarily affecting females and characterized by developmental regression, epilepsy, stereotypical hand movements, and motor abnormalities. Its prevalence is about 1 in 10,000 female births. Rett syndrome is caused by mutations within methyl CpG-binding protein 2 (MECP2) gene. Over 270 individual nucleotide changes which cause pathogenic mutations have been reported. However, eight most commonly occurring missense and nonsense mutations account for almost 70% of all patients. We screened 90 individuals with Rett syndrome phenotype. A total of 19 different MECP2 mutations and polymorphisms were identified in 27 patients. Of the 19 mutations, we identified 7 (37%) frameshift, 6 (31%) nonsense, 14 (74%) missense mutations and one duplication (5%). The most frequent pathogenic changes were: missense p.T158M (11%), p.R133C (7.4%), and p.R306C (7.4%) and nonsense p.R168X (11%), p.R255X (7.4%) mutations. We have identified two novel mutations namely p.385-388delPLPP present in atypical patients and p.Glu290AlafsX38 present in a classical patient of Rett syndrome. Sequence homology for p.385-388delPLPP mutation revealed that these 4 amino acids were conserved across mammalian species. This indicated the importance of these 4 amino acids in structure and function of the protein. A novel variant p.T479T has also been identified in a patient with atypical Rett syndrome. A total of 62 (69%) patients remained without molecular genetics diagnosis that necessitates further search for mutations in other genes like CDKL5 and FOXG1 that are known to cause Rett phenotype. The majority of mutations are detected in exon 4 and only one mutation was present in exon 3. Therefore, our study suggests the need for screening exon 4 of MECP2 as first line of diagnosis in these patients. Copyright © 2012 Elsevier B.V. All rights reserved.

  4. Volume da gota dos colírios lubrificantes: estudo farmacoeconômico

    Directory of Open Access Journals (Sweden)

    Alexandre Xavier da Costa

    2015-12-01

    Full Text Available RESUMO Objetivo: Determinar o volume médio das gotas produzidas pelos colírios lubrificantes em diferentes ângulos de inclinação e determinar o custo médio do tratamento. Métodos: Determinação do volume da gota de 3 frascos originais dos colírios lubrificantes Artelac®, Hylo Comod®, Lacrima® Plus, Systane® UL, Lacrifilm®, Hyabak®, Lacribell®, Ecofilm®, Mirugell®, Plenigell®, Fresh Tears®, Optive® e Endura® à inclinação de 90º e 45º. Determinou-se o número médio de gotas em cada frasco e foi feita avaliação farmacoeconômica dos colírios. Resultados: O volume das gotas variou de 32,2 a 64,0 µL a 45o e de 29,1 a 65,1 µL a 90o. A diferença entre as gotas em cada inclinação foi de 2 a 24% e o custo anual dos colírios de acordo com a inclinação variou de R$2,73 a R$130,73. A Duração Máxima de Tratamento (DMT foi de 29,3 a 51,4 dias na inclinação de 45o, e de 28,8 a 48,4 dias a 90º, sendo que a diferença na DMT foi de 0,5 até 8 dias a mais ou a menos, de acordo com a marca. Conclusão: Nenhum dos colírios estudados apresentou gotas ideais para o olho humano, levando a um desperdício do produto e maior custo para o fabricante e para o consumidor. Percebemos que existe uma variação significativa no volume da gota de acordo com a inclinação do frasco, e que uma variação maior do que 10% traria impactos financeiros para o paciente.

  5. 21 CFR 866.5900 - Cystic fibrosis transmembrane conductance regulator (CFTR) gene mutation detection system.

    Science.gov (United States)

    2010-04-01

    ... regulator (CFTR) gene mutation detection system. 866.5900 Section 866.5900 Food and Drugs FOOD AND DRUG...) gene mutation detection system. (a) Identification. The CFTR gene mutation detection system is a device... Guidance Document: CFTR Gene Mutation Detection System.” See § 866.1(e) for the availability of this...

  6. Ultrarapid mutation detection by multiplex, solid-phase chemical cleavage

    Energy Technology Data Exchange (ETDEWEB)

    Rowley, G.; Saad, S.; Giannelli, F.; Green, P.M. [Guy`s & St. Thomas`s Hospitals, London (United Kingdom)

    1995-12-10

    The chemical cleavage of mismatches in heteroduplexes formed by probe and test DNA detects and locates any sequence change in long DNA segments ({approximately}1.8 kb), and its efficiency has been well tested in the analysis of both average (e.g., coagulation factor IX) and large, complex genes (e.g., coagulation factor VIII and dystrophin). In the latter application RT/PCR products allow the examination of all essential sequences of the gene in a minimum number of reactions. We use two specific chemical reactants (hydroxylamine and osmium tetroxide) and piperidine cleavage of the above procedure to develop a very fast mutation screening method. This is based on: (1) 5{prime} or internal fluorescent labeling to allow concurrent screening of three to four DNA fragments and (2) solid-phase chemistry to use a microliter format and reduce the time required for the procedure, from amplification of sequence to gel loading inclusive, to one person-working-day. We test the two variations of the method, one entailing 5{prime} labeling of probe DNA and the other uniform labeling of both probe and target DNA, by detecting 114 known hemophilia B (coagulation factor IX) mutations and by analyzing 129 new patients. Uniform labeling of both probe and target DNA prior to formation of the heteroduplexes leads to almost twofold redundancy in the ability to detect mutations. Alternatively, the latter procedure may offer very efficient though less than 100% screening for sequence changes with only hydroxylamine. The full method with two chemical reactions (hydroxylamine and osmium tetroxide) should allow one person to screen with virtually 100% accuracy more than 300 kb of sequence in three ABI 373 gels in 1 day. 26 refs., 7 figs., 1 tab.

  7. CDKL5 mutations in boys with severe encephalopathy and early-onset intractable epilepsy.

    Science.gov (United States)

    Elia, M; Falco, M; Ferri, R; Spalletta, A; Bottitta, M; Calabrese, G; Carotenuto, M; Musumeci, S A; Lo Giudice, M; Fichera, M

    2008-09-23

    To search for CDKL5 gene mutations in boys presenting with severe early-onset encephalopathy and intractable epilepsy, a clinical picture very similar to that already described in girls with CDKL5 mutations. Eight boys (age range 3-16 years, mean age 8.5 years, SD 4.38) with severe or profound mental retardation and early-onset intractable seizures were selected for CDKL5 gene mutation screening by denaturing high-performance liquid chromatography analysis. We found three unrelated boys carrying three different missense mutations of the CDKL5 gene: c.872G>A (p.C291Y), c.863C>T (p.T288I), and c.533G>C (p.R178P). They presented early-onset, polymorphous, and drug-resistant seizures, mostly myoclonic and tonic or spasms. EEG showed epileptiform abnormalities which were multifocal during wakefulness, and pseudoperiodic bisynchronous during sleep. This study describes three boys carrying CDKL5 missense mutations and their detailed clinical and EEG data, and indicates that CDKL5 gene mutations may represent a cause of severe or profound mental retardation and early-onset intractable seizures, also in boys. Screening for CDKL5 mutations is strongly recommended in individuals with these clinical features.

  8. Detection of DNA oligonucleotides with base mutations by terahertz spectroscopy and microstructures.

    Directory of Open Access Journals (Sweden)

    Mingjie Tang

    Full Text Available DNA oligonucleotides with a 5-base mutation at the 3'-terminus were investigated by terahertz (THz spectroscopy in a marker-free manner. The four single-stranded oligonucleotides with 17nt have been detected with specificity on a microfluidic chip, and corroborated by spectral measurements with split-ring resonators. The number of hydrogen bonds formed between the oligonucleotide and its surrounding water molecules, deemed a key contribution to the THz absorption of biological solutions, was explored by molecular dynamics simulations to explain the experimental findings. Our work underlies the feasibility of THz spectroscopy combined with microstructures for marker-free detection of DNA, which may form the basis of a prospective diagnostic tool for studying genic mutation.

  9. SCN5A mutations and polymorphisms in patients with ventricular fibrillation during acute myocardial infarction.

    Science.gov (United States)

    Boehringer, Tim; Bugert, Peter; Borggrefe, Martin; Elmas, Elif

    2014-10-01

    Mutations in the SCN5A gene encoding the Nav1.5 channel α-subunit are known to be risk factors of arrhythmia, including Brugada Syndrome and Long QT syndrome subtype 3. The present study focused on the role of SCN5A variants in the development of ventricular fibrillation (VF) during acute myocardial infarction (AMI). Since VF during AMI is the major cause of sudden death in the Western world, SCN5A mutations represent genetic risk factors for sudden death. By exon re-sequencing, the entire coding region and flanking intron regions were sequenced in 46 AMI/VF+ patients. In total, nine single nucleotide variants were identified of which four represented common single nucleotide polymorphisms (SNPs; 87G>A, 1673A>G, IVS16‑6C>T and 5457T>A). Only five rare variants were identified, each in only one patient. Only two of the rare variants represented missense mutations (3578G>A and 4786T>A). The common SNPs and the missense mutations were also genotyped using polymerase chain reaction methods in 79 AMI/VF‑ patients and 480 healthy controls. The SNPs did not demonstrate significant differences in allele and genotype frequencies between the study groups. The 3578G>A mutation was identified in one out of the 480 controls, whereas the 4786T>A mutation was not present in AMI/VF- patients and controls. In conclusion, the majority of AMI/VF+ patients demonstrated a wild type sequence or common SNPs in SCN5A. Only two out of 46 (4.3%) AMI/VF+ patients revealed mutations that may be involved in Nav1.5 dysfunction and VF. However, this requires further functional validation.

  10. KRAS and BRAF Mutation Detection: Is Immunohistochemistry a Possible Alternative to Molecular Biology in Colorectal Cancer?

    Directory of Open Access Journals (Sweden)

    Nicolas Piton

    2015-01-01

    Full Text Available KRAS genotyping is mandatory in metastatic colorectal cancer treatment prior to undertaking antiepidermal growth factor receptor (EGFR monoclonal antibody therapy. BRAF V600E mutation is often present in colorectal carcinoma with CpG island methylator phenotype and microsatellite instability. Currently, KRAS and BRAF evaluation is based on molecular biology techniques such as SNaPshot or Sanger sequencing. As molecular testing is performed on formalin-fixed paraffin-embedded (FFPE samples, immunodetection would appear to be an attractive alternative for detecting mutations. Thus, our objective was to assess the validity of KRAS and BRAF immunodetection of mutations compared with the genotyping reference method in colorectal adenocarcinoma. KRAS and BRAF genotyping was assessed by SNaPshot. A rabbit anti-human KRAS polyclonal antibody was tested on 33 FFPE colorectal tumor samples with known KRAS status. Additionally, a mouse anti-human BRAF monoclonal antibody was tested on 30 FFPE tumor samples with known BRAF status. KRAS immunostaining demonstrated both poor sensitivity (27% and specificity (64% in detecting KRAS mutation. Conversely, BRAF immunohistochemistry showed perfect sensitivity (100% and specificity (100% in detecting V600E mutation. Although molecular biology remains the reference method for detecting KRAS mutation, immunohistochemistry could be an attractive method for detecting BRAF V600E mutation in colorectal cancer.

  11. KRAS and BRAF Mutation Detection: Is Immunohistochemistry a Possible Alternative to Molecular Biology in Colorectal Cancer?

    Science.gov (United States)

    Borrini, Francesco; Bolognese, Antonio; Lamy, Aude; Sabourin, Jean-Christophe

    2015-01-01

    KRAS genotyping is mandatory in metastatic colorectal cancer treatment prior to undertaking antiepidermal growth factor receptor (EGFR) monoclonal antibody therapy. BRAF V600E mutation is often present in colorectal carcinoma with CpG island methylator phenotype and microsatellite instability. Currently, KRAS and BRAF evaluation is based on molecular biology techniques such as SNaPshot or Sanger sequencing. As molecular testing is performed on formalin-fixed paraffin-embedded (FFPE) samples, immunodetection would appear to be an attractive alternative for detecting mutations. Thus, our objective was to assess the validity of KRAS and BRAF immunodetection of mutations compared with the genotyping reference method in colorectal adenocarcinoma. KRAS and BRAF genotyping was assessed by SNaPshot. A rabbit anti-human KRAS polyclonal antibody was tested on 33 FFPE colorectal tumor samples with known KRAS status. Additionally, a mouse anti-human BRAF monoclonal antibody was tested on 30 FFPE tumor samples with known BRAF status. KRAS immunostaining demonstrated both poor sensitivity (27%) and specificity (64%) in detecting KRAS mutation. Conversely, BRAF immunohistochemistry showed perfect sensitivity (100%) and specificity (100%) in detecting V600E mutation. Although molecular biology remains the reference method for detecting KRAS mutation, immunohistochemistry could be an attractive method for detecting BRAF V600E mutation in colorectal cancer. PMID:25983749

  12. Detection of epidermal growth factor receptor mutation in lung cancer by droplet digital polymerase chain reaction

    Directory of Open Access Journals (Sweden)

    Xu Q

    2015-06-01

    patient samples that the qPCR method failed to detect. About 49% of this patient cohort had EGFR mutations (L858R, 15.4%; ex19del, 29.5%; T790M, 6.4%. Two patients with the ex19del mutation also had a naïve T790M mutation.Conclusion: These data suggest that the ddPCR method could be useful in the personalized treatment of patients with lung cancer.Keywords: ddPCR, companion diagnostics, L858R, ex19del, T790M

  13. MutScan: fast detection and visualization of target mutations by scanning FASTQ data.

    Science.gov (United States)

    Chen, Shifu; Huang, Tanxiao; Wen, Tiexiang; Li, Hong; Xu, Mingyan; Gu, Jia

    2018-01-22

    Some types of clinical genetic tests, such as cancer testing using circulating tumor DNA (ctDNA), require sensitive detection of known target mutations. However, conventional next-generation sequencing (NGS) data analysis pipelines typically involve different steps of filtering, which may cause miss-detection of key mutations with low frequencies. Variant validation is also indicated for key mutations detected by bioinformatics pipelines. Typically, this process can be executed using alignment visualization tools such as IGV or GenomeBrowse. However, these tools are too heavy and therefore unsuitable for validating mutations in ultra-deep sequencing data. We developed MutScan to address problems of sensitive detection and efficient validation for target mutations. MutScan involves highly optimized string-searching algorithms, which can scan input FASTQ files to grab all reads that support target mutations. The collected supporting reads for each target mutation will be piled up and visualized using web technologies such as HTML and JavaScript. Algorithms such as rolling hash and bloom filter are applied to accelerate scanning and make MutScan applicable to detect or visualize target mutations in a very fast way. MutScan is a tool for the detection and visualization of target mutations by only scanning FASTQ raw data directly. Compared to conventional pipelines, this offers a very high performance, executing about 20 times faster, and offering maximal sensitivity since it can grab mutations with even one single supporting read. MutScan visualizes detected mutations by generating interactive pile-ups using web technologies. These can serve to validate target mutations, thus avoiding false positives. Furthermore, MutScan can visualize all mutation records in a VCF file to HTML pages for cloud-friendly VCF validation. MutScan is an open source tool available at GitHub: https://github.com/OpenGene/MutScan.

  14. Naturally occurring resistance mutations within the core and NS5B regions in hepatitis C genotypes, particularly genotype 5a, in South Africa.

    Science.gov (United States)

    Prabdial-Sing, N; Blackard, J T; Puren, A J; Mahomed, A; Abuelhassan, W; Mahlangu, J; Vermeulen, M; Bowyer, S M

    2016-03-01

    Approximately 1 million South Africans are infected with Hepatitis C virus (HCV). The standard of care (SOC) in South Africa is combination therapy (pegylated interferon and ribavirin). HCV genotypes and/or mutations in the core/non-structural regions have been associated with response to therapy and/or disease progression. This study examines mutations in the core (29-280 amino acids, including ∼ 90 E1 amino acids) and NS5B (241-306 amino acids) regions on pre-treatment isolates from patients attending Johannesburg hospitals or asymptomatic South African blood donors. Diversity within known CD4+ and CD8+ T-cell epitopes was also explored. Samples grouped into subtypes 1a(N = 10) 1b(N = 12), 3a(N = 5), 4a(N = 3) and 5a(N = 61). Two mutations, associated with interferon resistance-R70Q and T110N-were present in 29 genotype 5a core sequences. No resistance mutation to NS5B nucleotide inhibitors, sofosbuvir was found. Six putative CD8+ and one CD4+ T-cell epitope sequence in the core region showed binding scores of <300 IC50nM to HLA alleles frequently observed in the South African population. No known CD8+ and CD4+ T-cell epitopes were mapped in the NS5B region. The analysis begs the question whether those infected with genotype 5a will benefit better on interferon-free combination therapies. This study provides new insight into one of the lesser studied HCV genotypes and compares the diversity seen in a large pre-treatment cohort with other subtypes. Copyright © 2015 Elsevier B.V. All rights reserved.

  15. Diagnostic markers for the detection of ovarian cancer in BRCA1 mutation carriers.

    Directory of Open Access Journals (Sweden)

    Daphne Gschwantler-Kaulich

    Full Text Available Screening for ovarian cancer (OC in women at high risk consists of a combination of carbohydrate antigen 125 (CA125 and transvaginal ultrasound, despite their low sensitivity and specificity. This could be improved by the combination of several biomarkers, which has been shown in average risk patients but has not been investigated until now in female BRCA mutation carriers.Using a multiplex, bead-based, immunoassay system, we analyzed the concentrations of leptin, prolactin, osteopontin, insulin-like growth factor II, macrophage inhibitory factor, CA125 and human epididymis antigen 4 in 26 healthy wild type women, 26 healthy BRCA1 mutation carriers, 28 wildtype OC patients and 26 OC patients with BRCA1 mutation.Using the ROC analysis, we found a high overall sensitivity of 94.3% in differentiating healthy controls from OC patients with comparable results in the wildtype subgroup (sensitivity 92.8%, AUC = 0.988; p = 5.2e-14 as well as in BRCA1 mutation carriers (sensitivity 95.2%, AUC = 0.978; p = 1.7e-15 at an overall specificity of 92.3%. The used algorithm also allowed to identify healthy BRCA1 mutation carriers when compared to healthy wildtype women (sensitivity 88.4%, specificity 80.7%, AUC = 0.895; p = 6e-08, while this was less pronounced in patients with OC (sensitivity 66.7%, specificity 67.8%, AUC = 0.724; p = 0.00065.We have developed an algorithm, which can differentiate between healthy women and OC patients and have for the first time shown, that such an algorithm can also be used in BRCA mutation carriers. To clarify a suggested benefit to the existing early detection program, large prospective trials with mainly early stage OC cases are warranted.

  16. Meta-analysis diagnostic accuracy of SNP-based pathogenicity detection tools: a case of UTG1A1 gene mutations.

    Science.gov (United States)

    Galehdari, Hamid; Saki, Najmaldin; Mohammadi-Asl, Javad; Rahim, Fakher

    2013-01-01

    Crigler-Najjar syndrome (CNS) type I and type II are usually inherited as autosomal recessive conditions that result from mutations in the UGT1A1 gene. The main objective of the present review is to summarize results of all available evidence on the accuracy of SNP-based pathogenicity detection tools compared to published clinical result for the prediction of in nsSNPs that leads to disease using prediction performance method. A comprehensive search was performed to find all mutations related to CNS. Database searches included dbSNP, SNPdbe, HGMD, Swissvar, ensemble, and OMIM. All the mutation related to CNS was extracted. The pathogenicity prediction was done using SNP-based pathogenicity detection tools include SIFT, PHD-SNP, PolyPhen2, fathmm, Provean, and Mutpred. Overall, 59 different SNPs related to missense mutations in the UGT1A1 gene, were reviewed. Comparing the diagnostic OR, PolyPhen2 and Mutpred have the highest detection 4.983 (95% CI: 1.24 - 20.02) in both, following by SIFT (diagnostic OR: 3.25, 95% CI: 1.07 - 9.83). The highest MCC of SNP-based pathogenicity detection tools, was belong to SIFT (34.19%) followed by Provean, PolyPhen2, and Mutpred (29.99%, 29.89%, and 29.89%, respectively). Hence the highest SNP-based pathogenicity detection tools ACC, was fit to SIFT (62.71%) followed by PolyPhen2, and Mutpred (61.02%, in both). Our results suggest that some of the well-established SNP-based pathogenicity detection tools can appropriately reflect the role of a disease-associated SNP in both local and global structures.

  17. Mutations in the NKX2-5 gene in patients with stroke and patent foramen ovale.

    Science.gov (United States)

    Belvís, Robert; Tizzano, Eduardo F; Martí-Fàbregas, Joan; Leta, Rubén G; Baena, Manel; Carreras, Francesc; Pons-Lladó, Guillem; Baiget, Montserrat; Martí-Vilalta, Josep Lluis

    2009-09-01

    Patent foramen ovale (PFO) has been related to stroke but its existence has not been explained to date. NKX2-5 is the most implicated gene in fetal atrial septation. We studied NKX2-5 with respect to the presence or absence of PFO in stroke patients. A prospective analysis of NKX2-5 regarding age, gender, PFO, right-to-left shunt (RLS) size and atrial septal aneurysm (ASA) was performed in consecutive stroke patients and in 50 controls. The entire coding region and intron-exon boundaries of NKX2-5 gene were analyzed by PCR and sequencing of DNA from peripheral lymphocytes. One hundred patients participated in the study (mean age 56.5+/-12.4 years, 58% males) and PFO was diagnosed in 34% of them by transesophageal echocardiography. RLS was small (12%), moderate (2%) and large (20%). ASA was present in four patients. DNA revealed a novel c.2357G>A change in one PFO patient with cryptogenic stroke. Furthermore, c.182C>T, a mutation previously described in patients with cardiac defects, was detected in two non-PFO women with cryptogenic stroke. None of these changes were detected in our controls. The c.172A>G polymorphism was found in 21% of controls. It appeared more frequently in ASA patients (p=0.084), in cryptogenic PFO stroke patients (p=0.097) and in patients with known causes of stroke (p=0.037). The c.2850C>A polymorphism was also detected in our series with no differences in PFO, RLS size or ASA. Despite the fact that the NKX2-5 could account for the persistence of PFO, mutations of this gene in peripheral blood DNA were barely detected in our study.

  18. Highly sensitive detection of the PIK3CAH1047R mutation in colorectal cancer using a novel PCR-RFLP method

    International Nuclear Information System (INIS)

    Li, Wan-Ming; Hu, Ting-Ting; Zhou, Lin-Lin; Feng, Yi-Ming; Wang, Yun-Yi; Fang, Jin

    2016-01-01

    The PIK3CA H1047R mutation is considered to be a potential predictive biomarker for EGFR-targeted therapies. In this study, we developed a novel PCR-PFLP approach to detect the PIK3CA H1047R mutation in high effectiveness. A 126-bp fragment of PIK3CA exon-20 was amplified by PCR, digested with FspI restriction endonuclease and separated by 3 % agarose gel electrophoresis for the PCR-RFLP analysis. The mutant sequence of the PIK3CA H1047R was spiked into the corresponding wild-type sequence in decreasing ratios for sensitivity analysis. Eight-six cases of formalin-fixed paraffin-embedded colorectal cancer (CRC) specimens were subjected to PCR-RFLP to evaluate the applicability of the method. The PCR-RFLP method had a capability to detect as litter as 0.4 % of mutation, and revealed 16.3 % of the PIK3CA H1047R mutation in 86 CRC tissues, which was significantly higher than that discovered by DNA sequencing (9.3 %). A positive association between the PIK3CA H1047R mutation and the patients’ age was first found, except for the negative relationship with the degree of tumor differentiation. In addition, the highly sensitive detection of a combinatorial mutation of PIK3CA, KRAS and BRAF was achieved using individual PCR-RFLP methods. We developed a sensitive, simple and rapid approach to detect the low-abundance PIK3CA H1047R mutation in real CRC specimens, providing an effective tool for guiding cancer targeted therapy

  19. Deciphering KRAS and NRAS mutated clone dynamics in MLL-AF4 paediatric leukaemia by ultra deep sequencing analysis.

    Science.gov (United States)

    Trentin, Luca; Bresolin, Silvia; Giarin, Emanuela; Bardini, Michela; Serafin, Valentina; Accordi, Benedetta; Fais, Franco; Tenca, Claudya; De Lorenzo, Paola; Valsecchi, Maria Grazia; Cazzaniga, Giovanni; Kronnie, Geertruy Te; Basso, Giuseppe

    2016-10-04

    To induce and sustain the leukaemogenic process, MLL-AF4+ leukaemia seems to require very few genetic alterations in addition to the fusion gene itself. Studies of infant and paediatric patients with MLL-AF4+ B cell precursor acute lymphoblastic leukaemia (BCP-ALL) have reported mutations in KRAS and NRAS with incidences ranging from 25 to 50%. Whereas previous studies employed Sanger sequencing, here we used next generation amplicon deep sequencing for in depth evaluation of RAS mutations in 36 paediatric patients at diagnosis of MLL-AF4+ leukaemia. RAS mutations including those in small sub-clones were detected in 63.9% of patients. Furthermore, the mutational analysis of 17 paired samples at diagnosis and relapse revealed complex RAS clone dynamics and showed that the mutated clones present at relapse were almost all originated from clones that were already detectable at diagnosis and survived to the initial therapy. Finally, we showed that mutated patients were indeed characterized by a RAS related signature at both transcriptional and protein levels and that the targeting of the RAS pathway could be of beneficial for treatment of MLL-AF4+ BCP-ALL clones carrying somatic RAS mutations.

  20. A mutation in canine CLN5 causes neuronal ceroid lipofuscinosis in Border collie dogs.

    Science.gov (United States)

    Melville, Scott A; Wilson, Carmen L; Chiang, Chiu S; Studdert, Virginia P; Lingaas, Frode; Wilton, Alan N

    2005-09-01

    Neuronal ceroid lipofuscinosis (NCL) is a neurodegenerative disease found in Border collie dogs, humans, and other animals. Disease gene studies in humans and animals provided candidates for the NCL gene in Border collies. A combination of linkage analysis and comparative genomics localized the gene to CFA22 in an area syntenic to HSA13q that contains the CLN5 gene responsible for the Finnish variant of human late infantile NCL. Sequencing of CLN5 revealed a nonsense mutation (Q206X) within exon 4 that correlated with NCL in Border collies. This truncation mutation should result in a protein product of a size similar to that of some mutations identified in human CLN5 and therefore the Border collie may make a good model for human NCL. A simple test was developed to enable screening of the Border collie population for carriers so the disease can be eliminated as a problem in the breed.

  1. An activating mutation of interferon regulatory factor 4 (IRF4) in adult T cell leukemia.

    Science.gov (United States)

    Cherian, Mathew A; Olson, Sydney; Sundaramoorthi, Hemalatha; Cates, Kitra; Cheng, Xiaogang; Harding, John; Martens, Andrew; Challen, Grant A; Tyagi, Manoj; Ratner, Lee; Rauch, Daniel

    2018-03-14

    The human T cell leukemia virus-1 (HTLV-1) oncoprotein Tax drives cell proliferation and resistance to apoptosis early in the pathogenesis of adult T-cell leukemia (ATL). Subsequently, likely as a result of specific immuno-editing, Tax expression is downregulated and functionally replaced by somatic driver mutations of the host genome. Both amplification and point mutations of interferon regulatory factor 4 (IRF4) have been previously detected in ATL, and the K59R mutation is the most common single-nucleotide variation in IRF4 and is found exclusively in ATL. Here high throughput whole-exome sequencing revealed recurrent activating genetic alterations in the T cell receptor, CD28, and NF-kB pathways. Moreover, we found that IRF4, which is transcriptionally activated downstream of these pathways, is frequently mutated in ATL. IRF4 RNA, protein, and IRF4 transcriptional targets are uniformly elevated in HTLV transformed cells and ATL cell lines, and IRF4 was bound to genomic regulatory DNA of many of these transcriptional targets in HTLV-1 transformed cell lines. We further noted that the K59R IRF4 mutant is expressed at higher levels in the nucleus than is wild-type IRF4, and is transcriptionally more active. Expression of both wild-type and the K59R mutant of IRF4 from a constitutive promoter in retrovirally transduced murine bone marrow cells increased the abundance of T lymphocytes but not myeloid cells or B lymphocytes in mice. IRF4 may represent a therapeutic target in ATL since ATL cells select for a mutant of IRF4 with higher nuclear expression and transcriptional activity, and over-expression of IRF4 induces the expansion of T lymphocytes in vivo. Published under license by The American Society for Biochemistry and Molecular Biology, Inc.

  2. Comprehensive detection of diverse exon 19 deletion mutations of EGFR in lung Cancer by a single probe set.

    Science.gov (United States)

    Bae, Jin Ho; Jo, Seong-Min; Kim, Hak-Sung

    2015-12-15

    Detection of exon 19 deletion mutation of EGFR, one of the most frequently occurring mutations in lung cancer, provides the crucial information for diagnosis and treatment guideline in non-small-cell lung cancer (NSCLC). Here, we demonstrate a simple and efficient method to detect various exon 19 deletion mutations of EGFR using a single probe set comprising of an oligo-quencher (oligo-Q) and a molecular beacon (MB). While the MB hybridizes to both the wild and mutant target DNA, the oligo-Q only binds to the wild target DNA, leading to a fluorescent signal in case of deletion mutation. This enables the comprehensive detection of the diverse exon 19 deletion mutations using a single probe set. We demonstrated the utility and efficiency of the approach by detecting the frequent exon 19 deletion mutations of EGFR through a real-time PCR and in situ fluorescence imaging. Our approach enabled the detection of genomic DNA as low as 0.02 ng, showing a detection limit of 2% in a heterogeneous DNA mixture, and could be used for detecting mutations in a single cell level. The present MB and oligo-Q dual probe system can be used for diagnosis and treatment guideline in NSCLC. Copyright © 2015 Elsevier B.V. All rights reserved.

  3. CDKL5 mutations as a cause of severe epilepsy in infancy: clinical and electroencephalographic long-term course in 4 patients.

    Science.gov (United States)

    Jähn, Johanna; Caliebe, Almuth; von Spiczak, Sarah; Boor, Rainer; Stefanova, Irina; Stephani, Ulrich; Helbig, Ingo; Muhle, Hiltrud

    2013-07-01

    CDKL5 mutations cause severe epilepsy in infancy with subsequent epileptic encephalopathy. As yet, few studies report on long-term observations in patients with CDKL5-related epileptic encephalopathy. In this study, we describe the evolution of the epilepsy phenotype and the electroencephalographic (EEG) features in 4 patients during a maximum observation period of 22 years. All 4 patients had epilepsy starting with focal seizures in the first 3 months of life, evolving to epileptic spasms between the ages of 2 and 6 years and later on to tonic seizures. In 3 patients, epilepsy was resistant to antiepileptic therapy. Although there was no common EEG pattern in all patients, late hypsarrhythmia until the age of 9 years was observed in 2 patients. CDKL5-related epileptic encephalopathies are a group of refractory seizure disorders starting in early infancy. The phenomenon of late hypsarrhythmia may help define a subgroup of patients with severe and adverse outcomes.

  4. ERBB4 Mutations that Disrupt the Neuregulin-ErbB4 Pathway Cause Amyotrophic Lateral Sclerosis Type 19

    Science.gov (United States)

    Takahashi, Yuji; Fukuda, Yoko; Yoshimura, Jun; Toyoda, Atsushi; Kurppa, Kari; Moritoyo, Hiroyoko; Belzil, Veronique V.; Dion, Patrick A.; Higasa, Koichiro; Doi, Koichiro; Ishiura, Hiroyuki; Mitsui, Jun; Date, Hidetoshi; Ahsan, Budrul; Matsukawa, Takashi; Ichikawa, Yaeko; Moritoyo, Takashi; Ikoma, Mayumi; Hashimoto, Tsukasa; Kimura, Fumiharu; Murayama, Shigeo; Onodera, Osamu; Nishizawa, Masatoyo; Yoshida, Mari; Atsuta, Naoki; Sobue, Gen; Fifita, Jennifer A.; Williams, Kelly L.; Blair, Ian P.; Nicholson, Garth A.; Gonzalez-Perez, Paloma; Brown, Robert H.; Nomoto, Masahiro; Elenius, Klaus; Rouleau, Guy A.; Fujiyama, Asao; Morishita, Shinichi; Goto, Jun; Tsuji, Shoji

    2013-01-01

    Amyotrophic lateral sclerosis (ALS) is a devastating neurological disorder characterized by the degeneration of motor neurons and typically results in death within 3–5 years from onset. Familial ALS (FALS) comprises 5%–10% of ALS cases, and the identification of genes associated with FALS is indispensable to elucidating the molecular pathogenesis. We identified a Japanese family affected by late-onset, autosomal-dominant ALS in which mutations in genes known to be associated with FALS were excluded. A whole- genome sequencing and parametric linkage analysis under the assumption of an autosomal-dominant mode of inheritance with incomplete penetrance revealed the mutation c.2780G>A (p. Arg927Gln) in ERBB4. An extensive mutational analysis revealed the same mutation in a Canadian individual with familial ALS and a de novo mutation, c.3823C>T (p. Arg1275Trp), in a Japanese simplex case. These amino acid substitutions involve amino acids highly conserved among species, are predicted as probably damaging, and are located within a tyrosine kinase domain (p. Arg927Gln) or a C-terminal domain (p. Arg1275Trp), both of which mediate essential functions of ErbB4 as a receptor tyrosine kinase. Functional analysis revealed that these mutations led to a reduced autophosphorylation of ErbB4 upon neuregulin-1 (NRG-1) stimulation. Clinical presentations of the individuals with mutations were characterized by the involvement of both upper and lower motor neurons, a lack of obvious cognitive dysfunction, and relatively slow progression. This study indicates that disruption of the neuregulin-ErbB4 pathway is involved in the pathogenesis of ALS and potentially paves the way for the development of innovative therapeutic strategies such using NRGs or their agonists to upregulate ErbB4 functions. PMID:24119685

  5. HDAC4: a key factor underlying brain developmental alterations in CDKL5 disorder.

    Science.gov (United States)

    Trazzi, Stefania; Fuchs, Claudia; Viggiano, Rocchina; De Franceschi, Marianna; Valli, Emanuele; Jedynak, Paulina; Hansen, Finn K; Perini, Giovanni; Rimondini, Roberto; Kurz, Thomas; Bartesaghi, Renata; Ciani, Elisabetta

    2016-09-15

    Cyclin-dependent kinase-like 5 (CDKL5) is a Ser/Thr protein kinase predominantly expressed in the brain. Mutations of the CDKL5 gene lead to CDKL5 disorder, a neurodevelopmental pathology that shares several features with Rett Syndrome and is characterized by severe intellectual disability. The phosphorylation targets of CDKL5 are largely unknown, which hampers the discovery of therapeutic strategies for improving the neurological phenotype due to CDKL5 mutations. Here, we show that the histone deacetylase 4 (HDAC4) is a direct phosphorylation target of CDKL5 and that CDKL5-dependent phosphorylation promotes HDAC4 cytoplasmic retention. Nuclear HDAC4 binds to chromatin as well as to MEF2A transcription factor, leading to histone deacetylation and altered neuronal gene expression. By using a Cdkl5 knockout (Cdkl5 -/Y) mouse model, we found that hypophosphorylated HDAC4 translocates to the nucleus of neural precursor cells, thereby reducing histone 3 acetylation. This effect was reverted by re-expression of CDKL5 or by inhibition of HDAC4 activity through the HDAC4 inhibitor LMK235. In Cdkl5 -/Y mice treated with LMK235, defective survival and maturation of neuronal precursor cells and hippocampus-dependent memory were fully normalized. These results demonstrate a critical role of HDAC4 in the neurodevelopmental alterations due to CDKL5 mutations and suggest the possibility of HDAC4-targeted pharmacological interventions. © The Author 2016. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

  6. Novel mutations of endothelin-B receptor gene in Pakistani patients with Waardenburg syndrome.

    Science.gov (United States)

    Jabeen, Raheela; Babar, Masroor Ellahi; Ahmad, Jamil; Awan, Ali Raza

    2012-01-01

    Mutations in EDNRB gene have been reported to cause Waardenburg-Shah syndrome (WS4) in humans. We investigated 17 patients with WS4 for identification of mutations in EDNRB gene using PCR and direct sequencing technique. Four genomic mutations were detected in four patients; a G to C transversion in codon 335 (S335C) in exon 5 and a transition of T to C in codon (S361L) in exon 5, a transition of A to G in codon 277 (L277L) in exon 4, a non coding transversion of T to A at -30 nucleotide position of exon 5. None of these mutations were found in controls. One of the patients harbored two novel mutations (S335C, S361L) in exon 5 and one in Intronic region (-30exon5 A>G). All of the mutations were homozygous and novel except the mutation observed in exon 4. In this study, we have identified 3 novel mutations in EDNRB gene associated with WS4 in Pakistani patients.

  7. Significance of combined detection of JAK2V617F, MPL and CALR gene mutations in patients with essential thrombocythemia.

    Science.gov (United States)

    Ji, Liying; Qian, Mengyao; Wu, Nana; Wu, Jianmin

    2017-03-01

    The aim of this study was to analyze the mutation rate of JAK2V617F, MPLW515L/K and CALR genes in adult patients with essential thrombocythemia (ET) and the accuracy of the combined detection by the receiver operating curve. Three hundred and forty-two cases with high-platelets (≥300×10 9 /l) were consecutively selected. The patients were analyzed for routine blood examination, bone marrow biopsy and genetic testing. One hundred and fifty-four cases (45.03%) were diagnosed with ET and 188 cases of secondary thrombocythemia according to the hematopoietic and lymphoid tissue tumor classification standards of 2008. It was found that the mutant type of three genes showed three bands, whereas only one band for wild-type. The JAK2V617F and MPL mutations did not cause a change in the open reading frame and the CALR mutation resulted in its change. The mutation rate of JAK2V617F and CALR in ET group was significantly higher than that in the secondary thrombocythemia group (p<0.05). The positive mutation rate of MPL was only 4.55%. JAK2V617F-positive mutation alone was used to diagnose with ET. The area under the curve (AUC) was 0.721. The sensitivity was 72.4%, the specificity was 79.5% and the cut-off value was 0.25. When CALR-positive mutation alone was used to diagnose ET, the AUC, sensitivity, specificity and cut-off value were 0.664, 68.4, 82.4 and 0.09%, respectively. JAK2V617F combined with CALR mutation were used for diagnosis of ET. The AUC was 0.862, the sensitivity was 85.9%, the specificity was 87.8%, and the cut-off values were 0.21 and 0.07. In conclusion, the positive mutation rate of JAK2V617F and CALR in ET was higher, and the sensitivity, specificity and accuracy of the diagnosis of ET were significantly improved using the detection of JAK2V617F and CALR.

  8. Generation of Col2a1-EGFP iPS cells for monitoring chondrogenic differentiation.

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    Taku Saito

    Full Text Available Induced pluripotent stem cells (iPSC are a promising cell source for cartilage regenerative medicine; however, the methods for chondrocyte induction from iPSC are currently developing and not yet sufficient for clinical application. Here, we report the establishment of a fluorescent indicator system for monitoring chondrogenic differentiation from iPSC to simplify screening for effective factors that induce chondrocytes from iPSC. We generated iPSC from embryonic fibroblasts of Col2a1-EGFP transgenic mice by retroviral transduction of Oct4, Sox2, Klf4, and c-Myc. Among the 30 clones of Col2a1-EGFP iPSC we established, two clones showed high expression levels of embryonic stem cell (ESC marker genes, similar to control ESC. A teratoma formation assay showed that the two clones were pluripotent and differentiated into cell types from all three germ layers. The fluorescent signal was observed during chondrogenic differentiation of the two clones concomitant with the increase in chondrocyte marker expression. In conclusion, Col2a1-EGFP iPSC are useful for monitoring chondrogenic differentiation and will contribute to research in cartilage regenerative medicine.

  9. Mutation spectrum of RB1 mutations in retinoblastoma cases from Singapore with implications for genetic management and counselling.

    Directory of Open Access Journals (Sweden)

    Swati Tomar

    Full Text Available Retinoblastoma (RB is a rare childhood malignant disorder caused by the biallelic inactivation of RB1 gene. Early diagnosis and identification of carriers of heritable RB1 mutations can improve disease outcome and management. In this study, mutational analysis was conducted on fifty-nine matched tumor and peripheral blood samples from 18 bilateral and 41 unilateral unrelated RB cases by a combinatorial approach of Multiplex Ligation-dependent Probe Amplification (MLPA assay, deletion screening, direct sequencing, copy number gene dosage analysis and methylation assays. Screening of both blood and tumor samples yielded a mutation detection rate of 94.9% (56/59 while only 42.4% (25/59 of mutations were detected if blood samples alone were analyzed. Biallelic mutations were observed in 43/59 (72.9% of tumors screened. There were 3 cases (5.1% in which no mutations could be detected and germline mutations were detected in 19.5% (8/41 of unilateral cases. A total of 61 point mutations were identified, of which 10 were novel. There was a high incidence of previously reported recurrent mutations, occurring at 38.98% (23/59 of all cases. Of interest were three cases of mosaic RB1 mutations detected in the blood from patients with unilateral retinoblastoma. Additionally, two germline mutations previously reported to be associated with low-penetrance phenotypes: missense-c.1981C>T and splice variant-c.607+1G>T, were observed in a bilateral and a unilateral proband, respectively. These findings have implications for genetic counselling and risk prediction for the affected families. This is the first published report on the spectrum of mutations in RB patients from Singapore and shows that further improved mutation screening strategies are required in order to provide a definitive molecular diagnosis for every case of RB. Our findings also underscore the importance of genetic testing in supporting individualized disease management plans for patients and

  10. Some mutations of exon-7 in cytochrome P450 gene 3A4 and their effect on 6beta-hydroxylation of cortisol.

    Science.gov (United States)

    Shchepotina, E G; Vavilin, V A; Goreva, O B; Lyakhovich, V V

    2006-06-01

    Analysis of variants of exon 7 sequences in cytochrome P450 gene 3A4 in a sample of Caucasoid persons was carried out. The effect of these variants on activity of CYP3A was assessed by the level of cortisol 6beta-hydroxylation. Alleles CYP3A4*5 and *17 were not detected: probably, these mutations are rare and consequently they have little effect on the character of polymorphic distribution of CYP3A4 activity in this population. The incidence of CYP3A4*2 was 5.26%. The 6betaOH-cortisol/cortisol ratio in an individual with CYP3A4*2/*2 genotype was 7.408, which corresponded to "slow metabolizer" phenotype in this sample.

  11. Rapid detection of drug resistance and mutational patterns of extensively drug-resistant strains by a novel GenoType® MTBDRsl assay

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    A K Singh

    2013-01-01

    Full Text Available Background: The emergence of extensively drug-resistant tuberculosis (XDR-TB is a major concern in the India. The burden of XDR-TB is increasing due to inadequate monitoring, lack of proper diagnosis, and treatment. The GenoType ® Mycobacterium tuberculosis drug resistance second line (MTBDRsl assay is a novel line probe assay used for the rapid detection of mutational patterns conferring resistance to XDR-TB. Aim: The aim of this study was to study the rapid detection of drug resistance and mutational patterns of the XDR-TB by a novel GenoType ® MTBDRsl assay. Materials and Methods: We evaluated 98 multidrug-resistant (MDR M. tuberculosis isolates for second line drugs susceptibility testing by 1% proportion method (BacT/ALERT 3D system and GenoType ® MTBDRsl assay for rapid detection of conferring drug resistance to XDR-TB. Results: A total of seven (17.4% were identified as XDR-TB by using standard phenotypic method. The concordance between phenotypic and GenoType ® MTBDRsl assay was 91.7-100% for different antibiotics. The sensitivity and specificity of the MTBDRsl assay were 100% and 100% for aminoglycosides; 100% and 100% for fluoroquinolones; 91.7% and 100% for ethambutol. The most frequent mutations and patterns were gyrA MUT1 (A90V in seven (41.2% and gyrA + WT1-3 + MUT1 in four (23.5%; rrs MUT1 (A1401G in 11 (64.7%, and rrs WT1-2 + MUT1 in eight (47.1%; and embB MUT1B (M306V in 11 (64.7% strains. Conclusions: These data suggest that the GenoType ® MTBDRsl assay is rapid, novel test for detection of resistance to second line anti-tubercular drugs. This assay provides additional information about the frequency and mutational patterns responsible for XDR-TB resistance.

  12. An apple B-box protein, MdCOL11, is involved in UV-B- and temperature-induced anthocyanin biosynthesis.

    Science.gov (United States)

    Bai, Songling; Saito, Takanori; Honda, Chikako; Hatsuyama, Yoshimichi; Ito, Akiko; Moriguchi, Takaya

    2014-11-01

    Our studies showed that an apple B-box protein, MdCOL11, the homolog of AtBBX22, is involved in UV-B- and temperature-induced anthocyanin biosynthesis in apple peel. Anthocyanin is responsible for the red pigmentation in apple peel and a R2R3 MYB gene, MdMYBA/1/10, a homolog of MdMYBA, controls its accumulation. Arabidopsis PAP1 is under the control of a series of upstream factors involved in light signal transduction and photomorphogenesis, such as ELONGATED HYPOCOTYL 5 (HY5) and B-box family (BBX) proteins. In this study, we identified and characterized the homolog of Arabidopsis BBX22 in apple, designated as MdCOL11. Overexpression of MdCOL11 in Arabidopsis enhanced the accumulation of anthocyanin. In apples, MdCOL11 was differentially expressed in all tissues, with the highest expression in petals and the lowest expression in the xylem. Transcripts of MdCOL11 noticeably accumulated at the ripening stage, concomitant with increases in the expressions of anthocyanin biosynthesis-related genes. In an in vitro treatment experiment, MdCOL11 was upregulated in an ultra-violet (UV)-B- and temperature-dependent manner, together with the inductions of anthocyanin biosynthesis-related genes and anthocyanin accumulation in apple peel. Furthermore, a dual-luciferase assay indicated that (1) MdCOL11 regulated the expression of MdMYBA and (2) MdCOL11 was a target of MdHY5. Taken together, our results suggest that MdCOL11 is involved in MdHY5-mediated signal transduction and regulates anthocyanin accumulation in apple peel, which sheds new light on anthocyanin accumulation in apples.

  13. The frequency of CCR5 promoter polymorphisms and CCR5 32 mutation in Iranian populations

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    Mohammad Zare-Bidaki

    2015-04-01

    Full Text Available Evidence showed that chemokines serve as pro-migratory factors for immune cells. CCL3, CCL4 and CCL5, as the main CC  chemokines subfamily members, activate immune cells through binding to CC chemokine receptor 5 or CCR5. Macrophages, NK cells and T lymphocytes express CCR5 and thus, affected CCR5 expression or functions could be associated with altered immune responses. Deletion of 32 base pairs (D 32 in the exon 1 of the CCR5 gene, which is known as CCR5 D 32 mutation causes down regulation and malfunction of the molecule. Furthermore, it has been evidenced that three polymorphisms in the promoter region of CCR5 modulate its expression. Altered CCR5 expression in microbial infection and immune related diseases have been reported by several researchers but the role of CCR5 promoter polymorphisms and CCR5 D 32 mutation in Iranian patients suffering from these diseases are controversial. Due to the fact that Iranian people have different genetic backgrounds compared to other ethnics, hence, CCR5 promoter polymorphisms and CCR5 D 32 mutation association with the diseases may be different in Iranian patients. Therefore, this review addresses the most recent information regarding the prevalence as well as association of the mutation and polymorphisms in Iranian patients with microbial infection and immune related diseases as along with normal population.

  14. Functional analysis of the novel TBX5 c.1333delC mutation resulting in an extended TBX5 protein

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    Ekman-Joelsson Britt-Marie

    2008-10-01

    Full Text Available Abstract Background Autosomal dominant Holt-Oram syndrome (HOS is caused by mutations in the TBX5 gene and is characterized by congenital heart and preaxial radial ray upper limb defects. Most of the TBX5 mutations found in patients with HOS cause premature truncation of the primary TBX5 transcript. TBX5 missense mutations alter the three-dimensional structure of the protein and result in failed nuclear localization or reduced binding to target DNA. In this study we present our functional analyses of the novel and unusual c.1333delC mutation found in a patient with classical HOS. Methods The functional impact of this novel mutation was assessed by investigating the intracellular localization of the resulting TBX5 protein and its ability to activate the expression of its downstream target ANF. Results The deletion of the cytosine is the first TBX5 frameshift mutation predicted to result in an elongated TBX5 protein with 74 miscoding amino acids and 62 supernumerary C-terminal amino acids. The c.1333delC mutation affects neither the nuclear localization, nor its colocalization with SALL4, but severely affects the activation of the ANF promoter. Conclusion The mutation c.1333delC does not locate within functional domains, but impairs the activation of the downstream target. This suggests that misfolding of the protein prevents its biological function.

  15. JP-HHT phenotype in Danish patients with SMAD4 mutations

    DEFF Research Database (Denmark)

    Jelsig, A M; Tørring, P M; Kjeldsen, A D

    2016-01-01

    Patients with germline mutations in SMAD4 can present symptoms of both juvenile polyposis syndrome (JPS) and hereditary hemorrhagic telangiectasia (HHT): the JP-HHT syndrome. The complete phenotypic picture of this syndrome is only just emerging. We describe the clinical characteristics of 14......, aortopathy and family history were noted. We detected 14 patients with SMAD4 mutations. All patients had polyps removed and 11 of 14 fulfilled the diagnostic criteria for JPS. Eight patients were screened for HHT-symptoms and seven of these fulfilled the Curaçao criteria. One patient had aortic root dilation...

  16. [Optimization and assessment of a reverse hybridization system for the detection of HBV drug-resistant mutations].

    Science.gov (United States)

    Liu, Yan-chen; Huang, Ai-long; Hu, Yuan; Hu, Jie-li; Lai, Guo-qi; Zhang, Wen-lu

    2011-12-01

    To establish a detection method for HBV drug-resistant mutations related to lamivudine, adefovir and entecavir by optimization and assessment of reverse hybridization system. 26 degenerated probes covering 10 drug-resistant hotspots of 3 drugs were synthesized and immobilized on the same positively charged nylon membrane. PCR products labeled with digoxigenin were hybridized with corresponding probes. To improve the sensitivity and specificity, 4 reaction steps of reverse hybridization were optimized including the number of labeled digoxigenin, the energy intensity of UV cross-linking, hybridization and stringency wash conditions. To prove the feasibility, the specificity, sensitivity and accuracy of this system were assessed respectively. Sensitive and specific results are obtained by the optimization of the following 4 reaction steps: the primers labeled with 3 digoxigenin, energy intensity of UV cross-linking for 1500 x 0.1 mJ/cm², hybridization at 42 degrees C and stringency wash with 0.5 x SSC and 0.1% SDS solution at 44 degrees C for 30 min. In the assessment of system, the majority of probes have high specificity. The quantity of PCR product with a concentration of 10 ng/μl or above can be detected by this method. The concordant rate between reverse hybridization and direct sequencing is 93.9% in the clinical sample test. Though the specificity of several probes needs to be improved further, it is a simple, rapid and sensitive method which can detect HBV resistant mutations related to lamivudine, adefovir and entecavir simultaneously. Due to the short distance between 180 and 181, likewise 202 and 204, the sequence of the same probe covers two codon positions, and hybridization will be interfered by each other. To avoid such interference, the possible solution is that probes are designed by arranging and combining various forms of two near codons.

  17. Detection of EGFR mutations with mutation-specific antibodies in stage IV non-small-cell lung cancer

    Directory of Open Access Journals (Sweden)

    Viteri Santiago

    2010-12-01

    Full Text Available Abstract Background Immunohistochemistry (IHC with mutation-specific antibodies may be an ancillary method of detecting EGFR mutations in lung cancer patients. Methods EGFR mutation status was analyzed by DNA assays, and compared with IHC results in five non-small-cell lung cancer (NSCLC cell lines and tumor samples from 78 stage IV NSCLC patients. Results IHC correctly identified del 19 in the H1650 and PC9 cell lines, L858R in H1975, and wild-type EGFR in H460 and A549, as well as wild-type EGFR in tumor samples from 22 patients. IHC with the mAb against EGFR with del 19 was highly positive for the protein in all 17 patients with a 15-bp (ELREA deletion in exon 19, whereas in patients with other deletions, IHC was weakly positive in 3 cases and negative in 9 cases. IHC with the mAb against the L858R mutation showed high positivity for the protein in 25/27 (93% patients with exon 21 EGFR mutations (all with L858R but did not identify the L861Q mutation in the remaining two patients. Conclusions IHC with mutation-specific mAbs against EGFR is a promising method for detecting EGFR mutations in NSCLC patients. However these mAbs should be validated with additional studies to clarify their possible role in routine clinical practice for screening EGFR mutations in NSCLC patients.

  18. Simplifying the detection of MUTYH mutations by high resolution melting analysis

    International Nuclear Information System (INIS)

    López-Villar, Isabel; Martínez-López, Joaquín; Ayala, Rosa; Wesselink, Jan; Morillas, Juan Diego; López, Elena; Marín, José Carlos; Díaz-Tasende, José; González, Sara; Robles, Luis

    2010-01-01

    MUTYH-associated polyposis (MAP) is a disorder caused by bi-allelic germline MUTYH mutation, characterized by multiple colorectal adenomas. In order to identify mutations in MUTYH gene we applied High Resolution Melting (HRM) genotyping. HRM analysis is extensively employed as a scanning method for the detection of heterozygous mutations. Therefore, we applied HRM to show effectiveness in detecting homozygous mutations for these clinically important and frequent patients. In this study, we analyzed phenotype and genotype data from 82 patients, with multiple (>= 10) synchronous (19/82) or metachronous (63/82) adenomas and negative APC study (except one case). Analysis was performed by HRM-PCR and direct sequencing, in order to identify mutations in MUTYH exons 7, 12 and 13, where the most prevalent mutations are located. In monoallelic mutation carriers, we evaluated entire MUTYH gene in search of another possible alteration. HRM-PCR was performed with strict conditions in several rounds: the first one to discriminate the heteroduplex patterns and homoduplex patterns and the next ones, in order to refine and confirm parameters. The genotypes obtained were correlated to phenotypic features (number of adenomas (synchronous or metachronous), colorectal cancer (CRC) and family history). MUTYH germline mutations were found in 15.8% (13/82) of patients. The hot spots, Y179C (exon 7) and G396D (exon 13), were readily identified and other mutations were also detected. Each mutation had a reproducible melting profile by HRM, both heterozygous mutations and homozygous mutations. In our study of 82 patients, biallelic mutation is associated with being a carrier of ≥10 synchronous polyps (p = 0.05) and there is no association between biallelic mutation and CRC (p = 0.39) nor family history (p = 0.63). G338H non-pathogenic polymorphism (exon 12) was found in 23.1% (19/82) of patients. In all cases there was concordance between HRM (first and subsequent rounds) and sequencing

  19. Complement factor 5 (C5) p.A252T mutation is prevalent in, but not restricted to, sub-Saharan Africa: implications for the susceptibility to meningococcal disease.

    Science.gov (United States)

    Franco-Jarava, C; Comas, D; Orren, A; Hernández-González, M; Colobran, R

    2017-08-01

    Complement C5 deficiency (C5D) is a rare primary immunodeficiency associated with recurrent infections, particularly meningitis, by Neisseria species. To date, studies to elucidate the molecular basis of hereditary C5D have included fewer than 40 families, and most C5 mutations (13 of 17) have been found in single families. However, the recently described C5 p.A252T mutation is reported to be associated with approximately 7% of meningococcal disease cases in South Africa. This finding raises the question of whether the mutation may be prevalent in other parts of Africa or other continental regions. The aim of this study was to investigate the prevalence of C5 p.A252T in Africa and other regions and discuss the implications for prophylaxis against meningococcal disease. In total, 2710 samples from healthy donors within various populations worldwide were analysed by quantitative polymerase chain reaction (qPCR) assay to detect the C5 p.A252T mutation. Eleven samples were found to be heterozygous for p.A252T, and nine of these samples were from sub-Saharan African populations (allele frequency 0·94%). Interestingly, two other heterozygous samples were from individuals in populations outside Africa (Israel and Pakistan). These findings, together with data from genomic variation databases, indicate a5-2% prevalence of the C5 p.A252T mutation in heterozygosity in sub-Saharan Africa. Therefore, this mutation may have a relevant role in meningococcal disease susceptibility in this geographical area. © 2017 British Society for Immunology.

  20. Novel Missense Mitochondrial ND4L Gene Mutations in Friedreich's Ataxia

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    Mohammad Mehdi Heidari

    2011-05-01

    Full Text Available AbstractObjective(sThe mitochondrial defects in Friedreich's ataxia have been reported in many researches. Mitochondrial DNA is one of the candidates for defects in mitochondrion, and complex I is the first and one of the largest catalytic complexes of oxidative phosphorylation (OXPHOS system. Materials and MethodsWe searched the mitochondrial ND4L gene for mutations by TTGE and sequencing on 30 FRDA patients and 35 healthy controls.ResultsWe found 3 missense mutations [m.10506A>G (T13A, m.10530G>A (V21M, and m.10653G>A (A62T] in four patients whose m.10530G>A and m.10653G>A were not reported previously. In two patients, heteroplasmic m.10530G>A mutation was detected. They showed a very early ataxia syndrome. Our results showed that the number of mutations in FRDA patients was higher than that in the control cases (P= 0.0287.ConclusionAlthough this disease is due to nuclear gene mutation, the presence of these mutations might be responsible for further mitochondrial defects and the increase of the gravity of the disease. Thus, it should be considered in patients with this disorder.

  1. Altered Pre-mRNA Splicing Caused by a Novel Intronic Mutation c.1443+5G>A in the Dihydropyrimidinase (DPYS) Gene.

    Science.gov (United States)

    Nakajima, Yoko; Meijer, Judith; Zhang, Chunhua; Wang, Xu; Kondo, Tomomi; Ito, Tetsuya; Dobritzsch, Doreen; Van Kuilenburg, André B P

    2016-01-12

    Dihydropyrimidinase (DHP) deficiency is an autosomal recessive disease caused by mutations in the DPYS gene. Patients present with highly elevated levels of dihydrouracil and dihydrothymine in their urine, blood and cerebrospinal fluid. The analysis of the effect of mutations in DPYS on pre-mRNA splicing is hampered by the fact that DHP is primarily expressed in liver and kidney cells. The minigene approach can detect mRNA splicing aberrations using cells that do not express the endogenous mRNA. We have used a minigene-based approach to analyze the effects of a presumptive pre-mRNA splicing mutation in two newly identified Chinese pediatric patients with DHP deficiency. Mutation analysis of DPYS showed that both patients were compound heterozygous for a novel intronic mutation c.1443+5G>A in intron 8 and a previously described missense mutation c.1001A>G (p.Q334R) in exon 6. Wild-type and the mutated minigene constructs, containing exons 7, 8 and 9 of DPYS, yielded different splicing products after expression in HEK293 cells. The c.1443+5G>A mutation resulted in altered pre-mRNA splicing of the DPYS minigene construct with full skipping of exon 8. Analysis of the DHP crystal structure showed that the deletion of exon 8 severely affects folding, stability and homooligomerization of the enzyme as well as disruption of the catalytic site. Thus, the analysis suggests that the c.1443+5G>A mutation results in aberrant splicing of the pre-mRNA encoding DHP, underlying the DHP deficiency in two unrelated Chinese patients.

  2. Identification of drug resistance and immune-driven variations in hepatitis C virus (HCV) NS3/4A, NS5A and NS5B regions reveals a new approach toward personalized medicine.

    Science.gov (United States)

    Ikram, Aqsa; Obaid, Ayesha; Awan, Faryal Mehwish; Hanif, Rumeza; Naz, Anam; Paracha, Rehan Zafar; Ali, Amjad; Janjua, Hussnain Ahmed

    2017-01-01

    Cellular immune responses (T cell responses) during hepatitis C virus (HCV) infection are significant factors for determining the outcome of infection. HCV adapts to host immune responses by inducing mutations in its genome at specific sites that are important for HLA processing/presentation. Moreover, HCV also adapts to resist potential drugs that are used to restrict its replication, such as direct-acting antivirals (DAAs). Although DAAs have significantly reduced disease burden, resistance to these drugs is still a challenge for the treatment of HCV infection. Recently, drug resistance mutations (DRMs) observed in HCV proteins (NS3/4A, NS5A and NS5B) have heightened concern that the emergence of drug resistance may compromise the effectiveness of DAAs. Therefore, the NS3/4A, NS5A and NS5B drug resistance variations were investigated in this study, and their prevalence was examined in a large number of protein sequences from all HCV genotypes. Furthermore, potential CD4 + and CD8 + T cell epitopes were predicted and their overlap with genetic variations was explored. The findings revealed that many reported DRMs within NS3/4A, NS5A and NS5B are not drug-induced; rather, they are already present in HCV strains, as they were also detected in HCV-naïve patients. This study highlights several hot spots in which HLA and drug selective pressure overlap. Interestingly, these overlapping mutations were frequently observed among many HCV genotypes. This study implicates that knowledge of the host HLA type and HCV subtype/genotype can provide important information in defining personalized therapy. Copyright © 2016 Elsevier B.V. All rights reserved.

  3. A Multipoint Method for Detecting Genotyping Errors and Mutations in Sibling-Pair Linkage Data

    OpenAIRE

    Douglas, Julie A.; Boehnke, Michael; Lange, Kenneth

    2000-01-01

    The identification of genes contributing to complex diseases and quantitative traits requires genetic data of high fidelity, because undetected errors and mutations can profoundly affect linkage information. The recent emphasis on the use of the sibling-pair design eliminates or decreases the likelihood of detection of genotyping errors and marker mutations through apparent Mendelian incompatibilities or close double recombinants. In this article, we describe a hidden Markov method for detect...

  4. Suitability of two-dimensional electrophoretic protein separations for quantitative detection of mutations

    International Nuclear Information System (INIS)

    Taylor, J.; Anderson, N.L.; Anderson, N.G.; Gemmell, A.; Giometti, C.S.; Nance, S.L.; Tollaksen, S.L.

    1986-01-01

    Separation of proteins by two-dimensional electrophoresis (2DE) provides a powerful method for mutagenesis studies, since hundreds of proteins can be monitored simultaneously. In previous mutation studies in which 2DE has been used, only qualitative protein differences were monitored; quantitative protein variations were not evaluated. Although significant differences in protein abundance can be detected by eye, the large number of protein spots present in 2DE patterns together with the large number of individual patterns required for a mutagenesis study would necessitate the use of a computerized analysis system to detect the rare quantitative protein changes indicative of gene deletions or inactivation of genes by point mutations in regulatory genes. A pilot study to search for heritable mutations induced by treatment of mice with either ethylnitrosourea or gamma radiation is underway. Samples are being monitored for quantitative changes that reduce the amount of protein by about 50%. The results of this study indicate that the key methods to improve the application of 2DE to mutation screening are to increase the number of measurable spots (i.e., improve stain sensitivity) and to decrease the spread of values for the volume measurements. Even small improvements in these areas could greatly increase the number of monitorable spots. 9 refs., 4 figs

  5. Multiplex fluorescence melting curve analysis for mutation detection with dual-labeled, self-quenched probes.

    Directory of Open Access Journals (Sweden)

    Qiuying Huang

    2011-04-01

    Full Text Available Probe-based fluorescence melting curve analysis (FMCA is a powerful tool for mutation detection based on melting temperature generated by thermal denaturation of the probe-target hybrid. Nevertheless, the color multiplexing, probe design, and cross-platform compatibility remain to be limited by using existing probe chemistries. We hereby explored two dual-labeled, self-quenched probes, TaqMan and shared-stem molecular beacons, in their ability to conduct FMCA. Both probes could be directly used for FMCA and readily integrated with closed-tube amplicon hybridization under asymmetric PCR conditions. Improved flexibility of FMCA by using these probes was illustrated in three representative applications of FMCA: mutation scanning, mutation identification and mutation genotyping, all of which achieved improved color-multiplexing with easy probe design and versatile probe combination and all were validated with a large number of real clinical samples. The universal cross-platform compatibility of these probes-based FMCA was also demonstrated by a 4-color mutation genotyping assay performed on five different real-time PCR instruments. The dual-labeled, self-quenched probes offered unprecedented combined advantage of enhanced multiplexing, improved flexibility in probe design, and expanded cross-platform compatibility, which would substantially improve FMCA in mutation detection of various applications.

  6. The combination of heteroduplex analysis and protein truncation test for exact detection of the APC gene mutations

    International Nuclear Information System (INIS)

    Tomka, M.; Kirchhoff, T.; Stefurkova, V.; Zajac, V.; Kulcsar, L.

    1998-01-01

    Familial adenomatous polyposis (FAP) is usually associated with mutation in the adenomatous polyposis coli (APC) gene. To examine the occurrence of these mutations in the number of FAP suspected families from the whole Slovakia effectively, we have applied heteroduplex analysis (HDA) and protein truncation test (PTT) for the analyses of 2-5 base pair deletions and point mutations of the APC gene. In the analyzed exon 15 of the APC gene determined by the primers 15Efor-15Grev for HDA and 15ET7-15J3 for PTT more than 70% of mutations should be deletions [3, 12], which are detectable by HDA. In our collection of 5 FAP families mutations in the APC gene were found in families 10, 27 and 41 using HDA. By PTT test the formation of truncated APC protein in FAP families 2, 10, 16 and 27 were revealed. The necessity of combination of at least HDA and PTT techniques for exact detection of APC mutations in analyzed APC region is discussed. (authors)

  7. A novel CDKL5 mutation in a Japanese patient with atypical Rett syndrome.

    Science.gov (United States)

    Christianto, Antonius; Katayama, Syouichi; Kameshita, Isamu; Inazu, Tetsuya

    2016-08-01

    Rett syndrome (RTT) is a severe X-linked dominant inheritance disorder with a wide spectrum of clinical manifestations. Mutations in Methyl CpG binding protein 2 (MECP2), Cyclin dependent kinase-like 5 (CDKL5) and Forkhead box G1 (FOXG1) have been associated with classic and/or variant RTT. This study was conducted to identify the responsible gene(s) in atypical RTT patient, and to examine the effect of the mutation on protein function. DNA sequence analysis showed a novel heterozygous mutation in CDKL5 identified as c.530A>G which resulted in an amino acid substitution at position 177, from tyrosine to cysteine. Genotyping analysis indicated that the mutation was not merely a single nucleotide polymorphism (SNP). We also revealed that patient's blood lymphocytes had random X-chromosome inactivation (XCI) pattern. Further examination by bioinformatics analysis demonstrated the mutation caused damage or deleterious in its protein. In addition, we demonstrated in vitro kinase assay of mutant protein showed impairment of its activity. Taken together, the results suggested the mutant CDKL5 was responsible for the disease. Copyright © 2016 Elsevier B.V. All rights reserved.

  8. Electrophysiological characteristics of a SCN5A voltage sensors mutation R1629Q associated with Brugada syndrome.

    Directory of Open Access Journals (Sweden)

    Zhipeng Zeng

    Full Text Available Brugada syndrome (BrS is an inherited arrhythmogenic syndrome leading to sudden cardiac death, partially associated with autosomal dominant mutations in SCN5A, which encodes the cardiac sodium channel alpha-subunit (Nav1.5. To date some SCN5A mutations related with BrS have been identified in voltage sensor of Nav1.5. Here, we describe a dominant missense mutation (R1629Q localized in the fourth segment of domain IV region (DIV-S4 in a Chinese Han family. The mutation was identified by direct sequencing of SCN5A from the proband's DNA. Co-expression of Wild-type (WT or R1629Q Nav1.5 channel and hβ1 subunit were achieved in human embryonic kidney cells by transient transfection. Sodium currents were recorded using whole cell patch-clamp protocols. No significant changes between WT and R1629Q currents were observed in current density or steady-state activation. However, hyperpolarized shift of steady-state inactivation curve was identified in cells expressing R1629Q channel (WT: V1/2 = -81.1 ± 1.3 mV, n = 13; R1629Q: V1/2 = -101.7 ± 1.2 mV, n = 18. Moreover, R1629Q channel showed enhanced intermediate inactivation and prolonged recovery time from inactivation. In summary, this study reveals that R1629Q mutation causes a distinct loss-of-function of the channel due to alter its electrophysiological characteristics, and facilitates our understanding of biophysical mechanisms of BrS.

  9. Establishment and application of a multiplex genetic mutation-detection method of lung cancer based on MassARRAY platform

    International Nuclear Information System (INIS)

    Tian, Hong-Xia; Zhang, Xu-Chao; Wang, Zhen; Chen, Jian-Guang; Chen, Shi-Liang; Guo, Wei-Bang; Wu, Yi-Long

    2016-01-01

    Objective: This study aims to establish a method for highly parallel multiplexed detection of genetic mutations in Chinese lung cancer samples through Agena iPLEX chemistry and matrix-assisted laser desorption ionization time-of-flight analysis on MassARRAY mass spectrometry platform. Methods: We reviewed the related literature and data on lung cancer treatments. We also identified 99 mutation hot spots in 13 target genes closely related to the pathogenesis, drug resistance, and metastasis of lung cancer. A total of 297 primers, composed of 99 paired forward and reverse amplification primers and 99 matched extension primers, were designed using Assay Design software. The detection method was established by analyzing eight cell lines and six lung cancer specimens. The proposed method was then validated through comparisons by using a LungCarta TM kit. The sensitivity and specificity of the proposed method were evaluated by directly sequencing EGFR and KRAS genes in 100 lung cancer cases. Results: The proposed method was able to detect multiplex genetic mutations in lung cancer cell lines. This finding was consistent with the observations on previously reported mutations. The proposed method can also detect such mutations in clinical lung cancer specimens. This result was consistent with the observations with LungCarta TM kit. However, an FGFR2 mutation was detected only through the proposed method. The measured sensitivity and specificity were 100% and 96.3%, respectively. Conclusions: The proposed MassARRAY technology-based multiplex method can detect genetic mutations in Chinese lung cancer patients. Therefore, the proposed method can be applied to detect mutations in other cancer tissues

  10. Identification of a novel mutation in RIPK4 in a kindred with phenotypic features of Bartsocas-Papas and CHAND syndromes.

    Science.gov (United States)

    Gollasch, Benjamin; Basmanav, Fitnat Buket; Nanda, Arti; Fritz, Günter; Mahmoudi, Hassnaa; Thiele, Holger; Wehner, Maria; Wolf, Sabrina; Altmüller, Janine; Nürnberg, Peter; Frank, Jorge; Betz, Regina C

    2015-11-01

    Three children from an expanded consanguineous Kuwaiti kindred presented with ankyloblepharon, sparse and curly hair, and hypoplastic nails, suggestive of CHAND syndrome (OMIM 214350) that belongs to the heterogeneous spectrum of ectodermal dysplasias. After exclusion of pathogenic mutations in TP63 we performed homozygosity mapping, followed by exome sequencing of one affected individual. We initially identified three homozygous mutations in the linked region, located in PWP2, MX2 and RIPK4. Recently, mutations in RIPK4 have been reported in Bartsocas-Papas syndrome (OMIM 263650) that shows overlapping clinical symptoms with the phenotype observed in the affected individuals studied here. Subsequent analysis of affected and non-affected family members showed that mutation c.850G>A (p.Glu284Lys) in RIPK4 was in complete segregation with the disease phenotype, in accordance with an autosomal recessive inheritance pattern, thus supporting pathogenicity of this variant. Interestingly, however, our patients did not have cleft lip/palate, a common feature encountered in Bartsocas-Papas syndrome. Whereas in Bartsocas-Papas syndromes missense mutations are usually located within the serin/threonin kinase of RIPK4, the mutation detected in our family resides just outside of the kinase domain, which could explain the milder phenotype. Our data raise the question if CHAND syndrome indeed is a distinct entity. Alternatively, CHAND and Bartsocas-Papas syndrome might be allelic disorders or RIPK4 mutations could confer varying degrees of phenotypic severity, depending on their localization within or outside functionally important domains. Our findings indicate that making an accurate diagnosis based only on the prevailing clinical symptoms is challenging. © 2015 Wiley Periodicals, Inc.

  11. Mutations in pseudohypoparathyroidism 1a and pseudopseudohypoparathyroidism in ethnic Chinese.

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    Yi-Lei Wu

    Full Text Available An inactivating mutation in the GNAS gene causes either pseudohypoparathyroidism 1a (PHP1A when it is maternally inherited or pseudopseudohypoparathyroidism (PPHP when it is paternally inherited. We investigated clinical manifestations and mutations of the GNAS gene in ethnic Chinese patients with PHP1A or PPHP. Seven patients from 5 families including 4 girls and 2 boys with PHP1A and 1 girl with PPHP were studied. All PHP1A patients had mental retardation. They were treated with calcitriol and CaCO3 with regular monitoring of serum Ca levels, urinary Ca/Cr ratios, and renal sonography. Among them, 5 patients also had primary hypothyroidism suggesting TSH resistance. One female patient had a renal stone which was treated with extracorporeal shockwave lithotripsy. She had an increased urinary Ca/Cr ratio of 0.481 mg/mg when the stone was detected. We detected mutations using PCR and sequencing as well as analysed a splice acceptor site mutation using RT-PCR, sequencing, and minigene construct. We detected 5 mutations: c.85C>T (Q29*, c.103C>T (Q35*, c.840-2A>G (R280Sfs*21, c.1027_1028delGA (D343*, and c.1174G>A (E392K. Mutations c.840-2A>G and c.1027_1028delGA were novel. The c.840-2A>G mutation at the splice acceptor site of intron 10 caused retention of intron 10 in the minigene construct but skipping of exon 11 in the peripheral blood cells. The latter was the most probable mechanism which caused a frameshift, changing Arg to Ser at residue 280 and invoking a premature termination of translation at codon 300 (R280Sfs*21. Five GNAS mutations in ethnic Chinese with PHP1A and PPHP were reported. Two of them were novel. Mutation c.840-2A>G destroyed a spice acceptor site and caused exon skipping. Regular monitoring and adjustment in therapy are mandatory to achieve optimal therapeutic effects and avoid nephrolithiasis in patients with PHP1A.

  12. Historic, clinical, and prognostic features of epileptic encephalopathies caused by CDKL5 mutations.

    Science.gov (United States)

    Moseley, Brian D; Dhamija, Radhika; Wirrell, Elaine C; Nickels, Katherine C

    2012-02-01

    Mutations within the X-linked cyclin-dependent kinase-like 5 (CDKL5) gene are important causes of early-onset epileptic encephalopathies. We sought to determine the historic, clinical, and prognostic features of epilepsy secondary to CDKL5 mutations. We performed retrospective chart reviews of children at our institution with epilepsy and CDKL5 mutations. Six children were identified. One manifested a deletion in exons 10-15 of the CDKL5 gene, another manifested a single base-pair duplication in exon 3, and the rest manifested base-pair exchanges. The mean age of seizure onset was 1.8 months (range, 1-3 months). Although the majority (4/6, 67%) presented with partial-onset seizures, all children developed infantile spasms. All children demonstrated developmental delay and visual impairment. Although such mutations are X-linked, two children were boys. They did not present with more severe phenotypes than their female counterparts. Despite trials of antiepileptic drugs (mean, 5; range, 3-7), steroids/adrenocorticotropic hormone (4/6; 67%), and the ketogenic diet (6/6; 100%), all children manifested refractory seizures at last follow-up. Although no treatment eliminated seizures, topiramate, vigabatrin, and the ketogenic diet were most helpful at reducing seizure frequency. Copyright © 2012 Elsevier Inc. All rights reserved.

  13. The p16INK4alpha/p19ARF gene mutations are infrequent and are mutually exclusive to p53 mutations in Indian oral squamous cell carcinomas.

    Science.gov (United States)

    Kannan, K; Munirajan, A K; Krishnamurthy, J; Bhuvarahamurthy, V; Mohanprasad, B K; Panishankar, K H; Tsuchida, N; Shanmugam, G

    2000-03-01

    Eighty-seven untreated primary oral squamous cell carcinomas (SCCs) associated with betel quid and tobacco chewing from Indian patients were analysed for the presence of mutations in the commonly shared exon 2 of p16INK4alpha/p19ARF genes. Polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) and sequencing analysis were used to detect mutations. SSCP analysis indicated that only 9% (8/87) of the tumours had mutation in p16INK4alpha/p19ARF genes. Seventy-two tumours studied here were previously analysed for p53 mutations and 21% (15/72) of them were found to have mutations in p53 gene. Only one tumour was found to have mutation at both p53 and p16INK4alpha/p19ARF genes. Thus, the mutation rates observed were 21% for p53, 9% for p16INK4alpha/p19ARF, and 1% for both. Sequencing analysis revealed two types of mutations; i) G to C (GCAG to CCAG) transversion type mutation at intron 1-exon 2 splice junction and ii) another C to T transition type mutation resulting in CGA to TGA changing arginine to a termination codon at p16INK4alpha gene codon 80 and the same mutation will alter codon 94 of p19ARF gene from CCG to CTG (proline to leucine). These results suggest that p16INK4alpha/p19ARF mutations are less frequent than p53 mutations in Indian oral SCCs. The p53 and p16INK4alpha/p19ARF mutational events are independent and are mutually exclusive suggesting that mutational inactivation of either p53 or p16INK4alpha/p19ARF may alleviate the need for the inactivation of the other gene.

  14. Identification of functional mutations in GATA4 in patients with congenital heart disease.

    Directory of Open Access Journals (Sweden)

    Erli Wang

    Full Text Available Congenital heart disease (CHD is one of the most prevalent developmental anomalies and the leading cause of noninfectious morbidity and mortality in newborns. Despite its prevalence and clinical significance, the etiology of CHD remains largely unknown. GATA4 is a highly conserved transcription factor that regulates a variety of physiological processes and has been extensively studied, particularly on its role in heart development. With the combination of TBX5 and MEF2C, GATA4 can reprogram postnatal fibroblasts into functional cardiomyocytes directly. In the past decade, a variety of GATA4 mutations were identified and these findings originally came from familial CHD pedigree studies. Given that familial and sporadic CHD cases allegedly share a basic genetic basis, we explore the GATA4 mutations in different types of CHD. In this study, via direct sequencing of the GATA4 coding region and exon-intron boundaries in 384 sporadic Chinese CHD patients, we identified 12 heterozygous non-synonymous mutations, among which 8 mutations were only found in CHD patients when compared with 957 controls. Six of these non-synonymous mutations have not been previously reported. Subsequent functional analyses revealed that the transcriptional activity, subcellular localization and DNA binding affinity of some mutant GATA4 proteins were significantly altered. Our results expand the spectrum of GATA4 mutations linked to cardiac defects. Together with the newly reported mutations, approximately 110 non-synonymous mutations have currently been identified in GATA4. Our future analysis will explore why the evolutionarily conserved GATA4 appears to be hypermutable.

  15. Two novel COLVI long chains in zebrafish that are essential for muscle development.

    Science.gov (United States)

    Ramanoudjame, Laetitia; Rocancourt, Claire; Lainé, Jeanne; Klein, Arnaud; Joassard, Lucette; Gartioux, Corine; Fleury, Marjory; Lyphout, Laura; Kabashi, Edor; Ciura, Sorana; Cousin, Xavier; Allamand, Valérie

    2015-12-01

    Collagen VI (COLVI), a protein ubiquitously expressed in connective tissues, is crucial for structural integrity, cellular adhesion, migration and survival. Six different genes are recognized in mammalians, encoding six COLVI-chains that assemble as two 'short' (α1, α2) and one 'long' chain (theoretically any one of α3-6). In humans, defects in the most widely expressed heterotrimer (α123), due to mutations in the COL6A1-3 genes, cause a heterogeneous group of neuromuscular disorders, collectively termed COLVI-related muscle disorders. Little is known about the function(s) of the recently described α4-6 chains and no mutations have been detected yet. In this study, we characterized two novel COLVI long chains in zebrafish that are most homologous to the mammalian α4 chain; therefore, we named the corresponding genes col6a4a and col6a4b. These orthologues represent ancestors of the mammalian Col6a4-6 genes. By in situ hybridization and RT-qPCR, we unveiled a distinctive expression kinetics for col6a4b, compared with the other col6a genes. Using morpholino antisense oligonucleotides targeting col6a4a, col6a4b and col6a2, we modelled partial and complete COLVI deficiency, respectively. All morphant embryos presented altered muscle structure and impaired motility. While apoptosis was not drastically increased, autophagy induction was defective in all morphants. Furthermore, motoneuron axon growth was abnormal in these morphants. Importantly, some phenotypical differences emerged between col6a4a and col6a4b morphants, suggesting only partial functional redundancy. Overall, our results further confirm the importance of COLVI in zebrafish muscle development and may provide important clues for potential human phenotypes associated with deficiency of the recently described COLVI-chains. © The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

  16. Altered Pre-mRNA Splicing Caused by a Novel Intronic Mutation c.1443+5G>A in the Dihydropyrimidinase (DPYS Gene

    Directory of Open Access Journals (Sweden)

    Yoko Nakajima

    2016-01-01

    Full Text Available Dihydropyrimidinase (DHP deficiency is an autosomal recessive disease caused by mutations in the DPYS gene. Patients present with highly elevated levels of dihydrouracil and dihydrothymine in their urine, blood and cerebrospinal fluid. The analysis of the effect of mutations in DPYS on pre-mRNA splicing is hampered by the fact that DHP is primarily expressed in liver and kidney cells. The minigene approach can detect mRNA splicing aberrations using cells that do not express the endogenous mRNA. We have used a minigene-based approach to analyze the effects of a presumptive pre-mRNA splicing mutation in two newly identified Chinese pediatric patients with DHP deficiency. Mutation analysis of DPYS showed that both patients were compound heterozygous for a novel intronic mutation c.1443+5G>A in intron 8 and a previously described missense mutation c.1001A>G (p.Q334R in exon 6. Wild-type and the mutated minigene constructs, containing exons 7, 8 and 9 of DPYS, yielded different splicing products after expression in HEK293 cells. The c.1443+5G>A mutation resulted in altered pre-mRNA splicing of the DPYS minigene construct with full skipping of exon 8. Analysis of the DHP crystal structure showed that the deletion of exon 8 severely affects folding, stability and homooligomerization of the enzyme as well as disruption of the catalytic site. Thus, the analysis suggests that the c.1443+5G>A mutation results in aberrant splicing of the pre-mRNA encoding DHP, underlying the DHP deficiency in two unrelated Chinese patients.

  17. Molecular characteristics of the KCNJ5 mutated aldosterone-producing adenomas.

    Science.gov (United States)

    Murakami, Masanori; Yoshimoto, Takanobu; Nakabayashi, Kazuhiko; Nakano, Yujiro; Fukaishi, Takahiro; Tsuchiya, Kyoichiro; Minami, Isao; Bouchi, Ryotaro; Okamura, Kohji; Fujii, Yasuhisa; Hashimoto, Koshi; Hata, Ken-Ichiro; Kihara, Kazunori; Ogawa, Yoshihiro

    2017-10-01

    The pathophysiology of aldosterone-producing adenomas (APAs) has been investigated via genetic approaches and the pathogenic significance of a series of somatic mutations, including KCNJ5 , has been uncovered. However, how the mutational status of an APA is associated with its molecular characteristics, including its transcriptome and methylome, has not been fully understood. This study was undertaken to explore the molecular characteristics of APAs, specifically focusing on APAs with KCNJ5 mutations as opposed to those without KCNJ5 mutations, by comparing their transcriptome and methylome status. Cortisol-producing adenomas (CPAs) were used as reference. We conducted transcriptome and methylome analyses of 29 APAs with KCNJ5 mutations, 8 APAs without KCNJ5 mutations and 5 CPAs. Genome-wide gene expression and CpG methylation profiles were obtained from RNA and DNA samples extracted from these 42 adrenal tumors. Cluster analysis of the transcriptome and methylome revealed molecular heterogeneity in APAs depending on their mutational status. DNA hypomethylation and gene expression changes in Wnt signaling and inflammatory response pathways were characteristic of APAs with KCNJ5 mutations. Comparisons between transcriptome data from our APAs and that from normal adrenal cortex obtained from the Gene Expression Omnibus suggested similarities between APAs with KCNJ5 mutations and zona glomerulosa. The present study, which is based on transcriptome and methylome analyses, indicates the molecular heterogeneity of APAs depends on their mutational status. Here, we report the unique characteristics of APAs with KCNJ5 mutations. © 2017 Society for Endocrinology.

  18. Tuberculose du col utérin simulant un cancer du col utérin : à propos ...

    African Journals Online (AJOL)

    Tuberculose du col utérin simulant un cancer du col utérin : à propos d'un cas, au Centre Hospitalier Universitaire Souro Sanou de Bobo-Dioulasso, Burkina Faso. A. Dembélé, V. Konségré, E. Birba, D.A. Somé, H. Zamané, A.S. Ouédraogo, S. Kiemtoré, S. Ouattara, A. Lamien-Sanou, M. Bambara, B. Bonané/Thiéba ...

  19. Gradual Loss of ACTH Due to a Novel Mutation in LHX4: Comprehensive Mutation Screening in Japanese Patients with Congenital Hypopituitarism

    Science.gov (United States)

    Takagi, Masaki; Ishii, Tomohiro; Inokuchi, Mikako; Amano, Naoko; Narumi, Satoshi; Asakura, Yumi; Muroya, Koji; Hasegawa, Yukihiro; Adachi, Masanori; Hasegawa, Tomonobu

    2012-01-01

    Mutations in transcription factors genes, which are well regulated spatially and temporally in the pituitary gland, result in congenital hypopituitarism (CH) in humans. The prevalence of CH attributable to transcription factor mutations appears to be rare and varies among populations. This study aimed to define the prevalence of CH in terms of nine CH-associated genes among Japanese patients. We enrolled 91 Japanese CH patients for DNA sequencing of POU1F1, PROP1, HESX1, LHX3, LHX4, SOX2, SOX3, OTX2, and GLI2. Additionally, gene copy numbers for POU1F1, PROP1, HESX1, LHX3, and LHX4 were examined by multiplex ligation-dependent probe amplification. The gene regulatory properties of mutant LHX4 proteins were characterized in vitro. We identified two novel heterozygous LHX4 mutations, namely c.249-1G>A, p.V75I, and one common POU1F1 mutation, p.R271W. The patient harboring the c.249-1G>A mutation exhibited isolated growth hormone deficiency at diagnosis and a gradual loss of ACTH, whereas the patient with the p.V75I mutation exhibited multiple pituitary hormone deficiency. In vitro experiments showed that both LHX4 mutations were associated with an impairment of the transactivation capacities of POU1F1 andαGSU, without any dominant-negative effects. The total mutation prevalence in Japanese CH patients was 3.3%. This study is the first to describe, a gradual loss of ACTH in a patient carrying an LHX4 mutation. Careful monitoring of hypothalamic–pituitary -adrenal function is recommended for CH patients with LHX4 mutations. PMID:23029363

  20. De novo mutations of GCK, HNF1A and HNF4A may be more frequent in MODY than previously assumed.

    Science.gov (United States)

    Stanik, Juraj; Dusatkova, Petra; Cinek, Ondrej; Valentinova, Lucia; Huckova, Miroslava; Skopkova, Martina; Dusatkova, Lenka; Stanikova, Daniela; Pura, Mikulas; Klimes, Iwar; Lebl, Jan; Gasperikova, Daniela; Pruhova, Stepanka

    2014-03-01

    MODY is mainly characterised by an early onset of diabetes and a positive family history of diabetes with an autosomal dominant mode of inheritance. However, de novo mutations have been reported anecdotally. The aim of this study was to systematically revisit a large collection of MODY patients to determine the minimum prevalence of de novo mutations in the most prevalent MODY genes (i.e. GCK, HNF1A, HNF4A). Analysis of 922 patients from two national MODY centres (Slovakia and the Czech Republic) identified 150 probands (16%) who came from pedigrees that did not fulfil the criterion of two generations with diabetes but did fulfil the remaining criteria. The GCK, HNF1A and HNF4A genes were analysed by direct sequencing. Mutations in GCK, HNF1A or HNF4A genes were detected in 58 of 150 individuals. Parents of 28 probands were unavailable for further analysis, and in 19 probands the mutation was inherited from an asymptomatic parent. In 11 probands the mutations arose de novo. In our cohort of MODY patients from two national centres the de novo mutations in GCK, HNF1A and HNF4A were present in 7.3% of the 150 families without a history of diabetes and 1.2% of all of the referrals for MODY testing. This is the largest collection of de novo MODY mutations to date, and our findings indicate a much higher frequency of de novo mutations than previously assumed. Therefore, genetic testing of MODY could be considered for carefully selected individuals without a family history of diabetes.

  1. A novel -192c/g mutation in the proximal P2 promoter of the hepatocyte nuclear factor-4 alpha gene (HNF4A) associates with late-onset diabetes

    DEFF Research Database (Denmark)

    Ek, Jakob; Hansen, Sara P; Lajer, Maria

    2006-01-01

    Recently, it has been shown that mutations in the P2 promoter of the hepatocyte nuclear factor (HNF)-4 alpha gene (HNF4A) cause maturity-onset diabetes of the young (MODY), while single nucleotide polymorphisms in this locus are associated with type 2 diabetes. In this study, we examined 1,189 bp...... of the P2 promoter and the associated exon 1D of HNF4A for variations associated with diabetes in 114 patients with type 2 diabetes, 72 MODYX probands, and 85 women with previous gestational diabetes mellitus. A -192c/g mutation was found in five patients. We screened 1,587 diabetic subjects and 4......,812 glucose-tolerant subjects for the -192c/g mutation and identified 5 diabetic and 1 glucose-tolerant mutation carriers (P=0.004). Examination of the families showed that carriers of the -192c/g mutation had a significantly impaired glucose-stimulated insulin release and lower levels of serum total...

  2. Effects of the umuC36 mutation on ultraviolet-radiation-induced base-change and frameshift mutations in Escherichia coli

    International Nuclear Information System (INIS)

    Kato, T.; Nakano, E.

    1981-01-01

    The effects of the umuC36 mutation on the induction of base-change and frameshift mutations were studied. An active umuC gene was necessary in either the uvr + or uvr - strains of Escherichia coli K12 for UV- and X-ray-induced mutations to His + , ColE and Spc, which are presumably base-change mutations, but it was not essential for ethyl methanesulphonate or N-methyl-N'-nitro-N-nitrosoguanidine-induced His + mutations. In contrast, only 1 out of 13 trp - frameshift mutations examined was UV reversible, and the process of mutagenesis was umuC + -dependent, whereas a potent frameshift mutagen, ICR191, effectively induced Trp + mutations in most of the strains regardless of the umu + or umuC genetic background. These results suggest that base substitutions are a major mutational type derived from the umuC + -dependent pathway of error-prone repair. (orig.)

  3. Detecting selection signatures between Duroc and Duroc synthetic pig populations using high-density SNP chip.

    Science.gov (United States)

    Edea, Z; Hong, J-K; Jung, J-H; Kim, D-W; Kim, Y-M; Kim, E-S; Shin, S S; Jung, Y C; Kim, K-S

    2017-08-01

    The development of high throughput genotyping techniques has facilitated the identification of selection signatures of pigs. The detection of genomic selection signals in a population subjected to differential selection pressures may provide insights into the genes associated with economically and biologically important traits. To identify genomic regions under selection, we genotyped 488 Duroc (D) pigs and 155 D × Korean native pigs (DKNPs) using the Porcine SNP70K BeadChip. By applying the F ST and extended haplotype homozygosity (EHH-Rsb) methods, we detected genes under directional selection associated with growth/stature (DOCK7, PLCB4, HS2ST1, FBP2 and TG), carcass and meat quality (TG, COL14A1, FBXO5, NR3C1, SNX7, ARHGAP26 and DPYD), number of teats (LOC100153159 and LRRC1), pigmentation (MME) and ear morphology (SOX5), which are all mostly near or at fixation. These results could be a basis for investigating the underlying mutations associated with observed phenotypic variation. Validation using genome-wide association analysis would also facilitate the inclusion of some of these markers in genetic evaluation programs. © 2017 Stichting International Foundation for Animal Genetics.

  4. A new scintillation proximity assay-based approach for the detection of KRAS mutations

    Energy Technology Data Exchange (ETDEWEB)

    Lee, So-Young; Lim, Jae-Cheong; Cho, Eun-Ha; Jung, Sung-Hee [Korea Atomic Energy Research Institute (KAERI), Daejeon (Korea, Republic of). Radioisotope Research Div.

    2016-04-01

    KRAS is very commonly mutated resulting in a constitutively activated protein, which is independent of epidermal growth factor receptor (EGFR) ligand binding and resistant to anti-EGFR therapy. Although KRAS is frequently studied, there is still no uniform standard for detecting of KRAS mutations. In this report, a new scintillation proximity assay-based approach is described that determines the relative affinities of wild-type and mutated KRAS to the anti-KRAS antibody. We performed in vitro experiments using normal human colonic cells (CCD18Co), KRAS wild type (Caco-2) and KRAS mutant (HCT 116) cell lines to determine the relative affinities of wild type or mutated KRAS toward an anti-KRAS monoclonal antibody. The process consists of two primary steps: immunoprecipitation from cell lysate to enrich the KRAS protein and the scintillation proximity assay of the immunoprecipitant to determine the relative affinity against the antibody. A fixed concentration of cell lysates was purified by the immunoprecipitation method. The expressions of the KRAS protein in all cell lines was quantitatively confirmed by western blot analysis. For the scintillation proximity assay, the KRAS standard protein was radiolabeled with {sup 125}I by a simple mixing process in the iodogen tube immediately at room temperature immediately before use. The obtained CPM (count per minute) values of were used to calculate the KRAS concentration using purified KRAS as the standard. The calculated relative affinities of 7 μg of Caco-2 and HCT 116 immunoprecipitants for the anti-KRAS antibody were 77 and 0%, respectively. The newly developed scintillation proximity assay-based strategy determines the relative affinities of wild-type or mutated KRAS towards the anti-KRAS monoclonal antibody. This determination can help distinguish mutated KRAS from the wild type protein. The new SPA based approach for detecting KRAS mutations is applicable to many other cancer-related mutations.

  5. A new scintillation proximity assay-based approach for the detection of KRAS mutations

    International Nuclear Information System (INIS)

    Lee, So-Young; Lim, Jae-Cheong; Cho, Eun-Ha; Jung, Sung-Hee

    2016-01-01

    KRAS is very commonly mutated resulting in a constitutively activated protein, which is independent of epidermal growth factor receptor (EGFR) ligand binding and resistant to anti-EGFR therapy. Although KRAS is frequently studied, there is still no uniform standard for detecting of KRAS mutations. In this report, a new scintillation proximity assay-based approach is described that determines the relative affinities of wild-type and mutated KRAS to the anti-KRAS antibody. We performed in vitro experiments using normal human colonic cells (CCD18Co), KRAS wild type (Caco-2) and KRAS mutant (HCT 116) cell lines to determine the relative affinities of wild type or mutated KRAS toward an anti-KRAS monoclonal antibody. The process consists of two primary steps: immunoprecipitation from cell lysate to enrich the KRAS protein and the scintillation proximity assay of the immunoprecipitant to determine the relative affinity against the antibody. A fixed concentration of cell lysates was purified by the immunoprecipitation method. The expressions of the KRAS protein in all cell lines was quantitatively confirmed by western blot analysis. For the scintillation proximity assay, the KRAS standard protein was radiolabeled with 125 I by a simple mixing process in the iodogen tube immediately at room temperature immediately before use. The obtained CPM (count per minute) values of were used to calculate the KRAS concentration using purified KRAS as the standard. The calculated relative affinities of 7 μg of Caco-2 and HCT 116 immunoprecipitants for the anti-KRAS antibody were 77 and 0%, respectively. The newly developed scintillation proximity assay-based strategy determines the relative affinities of wild-type or mutated KRAS towards the anti-KRAS monoclonal antibody. This determination can help distinguish mutated KRAS from the wild type protein. The new SPA based approach for detecting KRAS mutations is applicable to many other cancer-related mutations.

  6. Identification of two novel mutations in the SLC45A2 gene in a Hungarian pedigree affected by unusual OCA type 4.

    Science.gov (United States)

    Tóth, Lola; Fábos, Beáta; Farkas, Katalin; Sulák, Adrienn; Tripolszki, Kornélia; Széll, Márta; Nagy, Nikoletta

    2017-03-15

    Oculocutaneous albinism (OCA) is a clinically and genetically heterogenic group of pigmentation abnormalities. OCA type IV (OCA4, OMIM 606574) develops due to homozygous or compound heterozygous mutations in the solute carrier family 45, member 2 (SLC45A2) gene. This gene encodes a membrane-associated transport protein, which regulates tyrosinase activity and, thus, melanin content by changing melanosomal pH and disrupting the incorporation of copper into tyrosinase. Here we report two Hungarian siblings affected by an unusual OCA4 phenotype. After genomic DNA was isolated from peripheral blood of the patients, the coding regions of the SLC45A2 gene were sequenced. In silico tools were applied to identify the functional impact of the newly detected mutations. Direct sequencing of the SLC45A2 gene revealed two novel, heterozygous mutations, one missense (c.1226G > A, p.Gly409Asp) and one nonsense (c.1459C > T, p.Gln437*), which were present in both patients, suggesting the mutations were compound heterozygous. In silico tools suggest that these variations are disease causing mutations. The newly identified mutations may affect the transmembrane domains of the protein, and could impair transport function, resulting in decreases in both melanosomal pH and tyrosinase activity. Our study provides expands on the mutation spectrum of the SLC45A2 gene and the genetic background of OCA4.

  7. Mutations of CDKL5 Cause a Severe Neurodevelopmental Disorder with Infantile Spasms and Mental Retardation

    Science.gov (United States)

    Weaving, Linda S.; Christodoulou, John; Williamson, Sarah L.; Friend, Kathie L.; McKenzie, Olivia L. D.; Archer, Hayley; Evans, Julie; Clarke, Angus; Pelka, Gregory J.; Tam, Patrick P. L.; Watson, Catherine; Lahooti, Hooshang; Ellaway, Carolyn J.; Bennetts, Bruce; Leonard, Helen; Gécz, Jozef

    2004-01-01

    Rett syndrome (RTT) is a severe neurodevelopmental disorder caused, in most classic cases, by mutations in the X-linked methyl-CpG-binding protein 2 gene (MECP2). A large degree of phenotypic variation has been observed in patients with RTT, both those with and without MECP2 mutations. We describe a family consisting of a proband with a phenotype that showed considerable overlap with that of RTT, her identical twin sister with autistic disorder and mild-to-moderate intellectual disability, and a brother with profound intellectual disability and seizures. No pathogenic MECP2 mutations were found in this family, and the Xq28 region that contains the MECP2 gene was not shared by the affected siblings. Three other candidate regions were identified by microsatellite mapping, including 10.3 Mb at Xp22.31-pter between Xpter and DXS1135, 19.7 Mb at Xp22.12-p22.11 between DXS1135 and DXS1214, and 16.4 Mb at Xq21.33 between DXS1196 and DXS1191. The ARX and CDKL5 genes, both of which are located within the Xp22 region, were sequenced in the affected family members, and a deletion of nucleotide 183 of the coding sequence (c.183delT) was identified in CDKL5 in the affected family members. In a screen of 44 RTT cases, a single splice-site mutation, IVS13-1G→A, was identified in a girl with a severe phenotype overlapping RTT. In the mouse brain, Cdkl5 expression overlaps—but is not identical to—that of Mecp2, and its expression is unaffected by the loss of Mecp2. These findings confirm CDKL5 as another locus associated with epilepsy and X-linked mental retardation. These results also suggest that mutations in CDKL5 can lead to a clinical phenotype that overlaps RTT. However, it remains to be determined whether CDKL5 mutations are more prevalent in specific clinical subgroups of RTT or in other clinical presentations. PMID:15492925

  8. Mutational analysis uncovers monogenic bone disorders in women with pregnancy-associated osteoporosis: three novel mutations in LRP5, COL1A1, and COL1A2.

    Science.gov (United States)

    Butscheidt, S; Delsmann, A; Rolvien, T; Barvencik, F; Al-Bughaili, M; Mundlos, S; Schinke, T; Amling, M; Kornak, U; Oheim, R

    2018-03-29

    Pregnancy was found to be a skeletal risk factor promoting the initial onset of previously unrecognized monogenic bone disorders, thus explaining a proportion of cases with pregnancy-associated osteoporosis. Therapeutic measures should focus in particular on the normalization of the disturbed calcium homeostasis in order to enable the partial skeletal recovery. Pregnancy-associated osteoporosis (PAO) is a rare skeletal condition, which is characterized by a reduction in bone mineral density (BMD) in the course of pregnancy and lactation. Typical symptoms include vertebral compression fractures and transient osteoporosis of the hip. Since the etiology is not well understood, this prospective study was conducted in order to elucidate the relevance of pathogenic gene variants for the development of PAO. Seven consecutive cases with the diagnosis of PAO underwent a skeletal assessment (blood tests, DXA, HR-pQCT) and a comprehensive genetic analysis using a custom-designed gene panel. All cases showed a reduced BMD (DXA T-score, lumbar spine - 3.2 ± 1.0; left femur - 2.2 ± 0.5; right femur - 1.9 ± 0.5), while the spine was affected more severely (p Pregnancy should be considered a skeletal risk factor, which can promote the initial clinical onset of such skeletal disorders. The underlying increased calcium demand is essential in terms of prophylactic and therapeutic measures, which are especially required in individuals with a genetically determined low bone mass. The implementation of this knowledge in clinical practice can enable the partial recovery of the skeleton. Consistent genetic studies are needed to analyze the frequency of pathogenic variants in women with PAO.

  9. Diagnostic screening identifies a wide range of mutations involving the SHOX gene, including a common 47.5 kb deletion 160 kb downstream with a variable phenotypic effect.

    Science.gov (United States)

    Bunyan, David J; Baker, Kevin R; Harvey, John F; Thomas, N Simon

    2013-06-01

    Léri-Weill dyschondrosteosis (LWD) results from heterozygous mutations of the SHOX gene, with homozygosity or compound heterozygosity resulting in the more severe form, Langer mesomelic dysplasia (LMD). These mutations typically take the form of whole or partial gene deletions, point mutations within the coding sequence, or large (>100 kb) 3' deletions of downstream regulatory elements. We have analyzed the coding sequence of the SHOX gene and its downstream regulatory regions in a cohort of 377 individuals referred with symptoms of LWD, LMD or short stature. A causative mutation was identified in 68% of the probands with LWD or LMD (91/134). In addition, a 47.5 kb deletion was found 160 kb downstream of the SHOX gene in 17 of the 377 patients (12% of the LWD referrals, 4.5% of all referrals). In 14 of these 17 patients, this was the only potentially causative abnormality detected (13 had symptoms consistent with LWD and one had short stature only), but the other three 47.5 kb deletions were found in patients with an additional causative SHOX mutation (with symptoms of LWD rather than LMD). Parental samples were available on 14/17 of these families, and analysis of these showed a more variable phenotype ranging from apparently unaffected to LWD. Breakpoint sequence analysis has shown that the 47.5 kb deletion is identical in all 17 patients, most likely due to an ancient founder mutation rather than recurrence. This deletion was not seen in 471 normal controls (P<0.0001), providing further evidence for a phenotypic effect, albeit one with variable penetration. Copyright © 2013 Wiley Periodicals, Inc.

  10. Epidural Analgesia with Ropivacaine during Labour in a Patient with a SCN5A Gene Mutation

    Directory of Open Access Journals (Sweden)

    A. L. M. J. van der Knijff-van Dortmont

    2016-01-01

    Full Text Available SCN5A gene mutations can lead to ion channel defects which can cause cardiac conduction disturbances. In the presence of specific ECG characteristics, this mutation is called Brugada syndrome. Many drugs are associated with adverse events, making anesthesia in patients with SCN5A gene mutations or Brugada syndrome challenging. In this case report, we describe a pregnant patient with this mutation who received epidural analgesia using low dose ropivacaine and sufentanil during labour.

  11. Nephrolithiasis, kidney failure and bone disorders in Dent disease patients with and without CLCN5 mutations.

    Science.gov (United States)

    Anglani, Franca; D'Angelo, Angela; Bertizzolo, Luisa Maria; Tosetto, Enrica; Ceol, Monica; Cremasco, Daniela; Bonfante, Luciana; Addis, Maria Antonietta; Del Prete, Dorella

    2015-01-01

    Dent disease (DD) is a rare X-linked recessive renal tubulopathy characterised by low-molecular-weight proteinuria (LMWP), hypercalciuria, nephrocalcinosis and/or nephrolithiasis. DD is caused by mutations in both the CLCN5 and OCRL genes. CLCN5 encodes the electrogenic chloride/proton exchanger ClC-5 which is involved in the tubular reabsorption of albumin and LMW proteins, OCRL encodes the inositol polyphosphate 5-phosphatase, and was initially associated with Lowe syndrome. In approximately 25 % of patients, no CLCN5 and OCRL mutations were detected. The aim of our study was to evaluate whether calcium phosphate metabolism disorders and their clinical complications are differently distributed among DD patients with and without CLCN5 mutations. Sixty-four male subjects were studied and classified into three groups: Group I (with CLCN5 mutations), Group II (without CLCN5 mutations) and Group III (family members with the same CLCN5 mutation). LMWP, hypercalciuria and phosphaturic tubulopathy and the consequent clinical complications nephrocalcinosis, nephrolithiasis, bone disorders, and chronic kidney disease (CKD) were considered present or absent in each patient. We found that the distribution of nephrolithiasis, bone disorders and CKD differs among patients with and without CLCN5 mutations. Only in patients harbouring CLCN5 mutations was age-independent nephrolithiasis associated with hypercalciuria, suggesting that nephrolithiasis is linked to altered proximal tubular function caused by a loss of ClC-5 function, in agreement with ClC-5 KO animal models. Similarly, only in patients harbouring CLCN5 mutations was age-independent kidney failure associated with nephrocalcinosis, suggesting that kidney failure is the consequence of a ClC-5 dysfunction, as in ClC-5 KO animal models. Bone disorders are a relevant feature of DD phenotype, as patients were mainly young males and this complication occurred independently of age. The triad of symptoms, LMWP

  12. Recurrent mutations in the CDKL5 gene: genotype-phenotype relationships.

    Science.gov (United States)

    Bahi-Buisson, Nadia; Villeneuve, Nathalie; Caietta, Emilie; Jacquette, Aurélia; Maurey, Helene; Matthijs, Gert; Van Esch, Hilde; Delahaye, Andrée; Moncla, Anne; Milh, Mathieu; Zufferey, Flore; Diebold, Bertrand; Bienvenu, Thierry

    2012-07-01

    Mutations in the cyclin-dependent kinase-like 5 gene (CDKL5) have been described in epileptic encephalopathies in females with infantile spasms with features that overlap with Rett syndrome. With more than 80 reported patients, the phenotype of CDKL5-related encephalopathy is well-defined. The main features consist of seizures starting before 6 months of age, severe intellectual disability with absent speech and hand stereotypies and deceleration of head growth, which resembles Rett syndrome. However, some clinical discrepancies suggested the influence of genetics and/or environmental factors. No genotype-phenotype correlation has been defined and thus there is a need to examine individual mutations. In this study, we analyzed eight recurrent CDKL5 mutations to test whether the clinical phenotype of patients with the same mutation is similar and whether patients with specific CDKL5 mutations have a milder phenotype than those with other CDKL5 mutations. Patients bearing missense mutations in the ATP binding site such as the p.Ala40Val mutation typically walked unaided, had normocephaly, better hand use ability, and less frequent refractory epilepsy when compared to girls with other CDKL5 mutations. In contrast, patients with mutations in the kinase domain (such as p.Arg59X, p.Arg134X, p.Arg178Trp/Pro/Gln, or c.145 + 2T > C) and frameshift mutations in the C-terminal region (such as c.2635_2636delCT) had a more severe phenotype with infantile spasms, refractory epileptic encephalopathy, absolute microcephaly, and inability to walk. It is important for clinicians to have this information when such patients are diagnosed. Copyright © 2012 Wiley Periodicals, Inc.

  13. Development of a PCR/LDR/capillary electrophoresis assay with potential for the detection of a beta-thalassemia fetal mutation in maternal plasma.

    Science.gov (United States)

    Yi, Ping; Chen, Zhuqin; Yu, Lili; Zheng, Yingru; Liu, Guodong; Xie, Haichang; Zhou, Yuanguo; Zheng, Xiuhui; Han, Jian; Li, Li

    2010-08-01

    Analysis of fetal DNA in maternal plasma has recently been introduced for non-invasive prenatal diagnosis. We have now investigated the feasibility of polymerase chain reaction (PCR)/ligase detection reaction (LDR)/capillary electrophoresis for the detection of fetal point mutations, such as the beta-thalassemia mutation, IVS2 654(C --> T), in maternal plasma DNA. The sensitivity of LDR/capillary electrophoresis was examined by quantifying the mutant PCR products in the presence of a vast excess of non-mutant competitor template, a situation that mimics the detection of rare fetal mutations in the presence of excess maternal DNA. PCR/LDR/capillary electrophoresis was applied to detect the mutation, IVS2 654(C --> T), in an experimental model at different sensitivity levels and from 10 maternal plasma samples. Our results demonstrated that this approach to detect a low abundance IVS2 654(C --> T) mutation achieved a sensitivity of approximately 1:10,000. The approach was applied to maternal plasma DNA to detect the paternally inherited fetal IVS2 654(C --> T) mutation, and the results were equivalent to those obtained by PCR/reverse dot blot of amniotic fluid cell DNA. PCR/LDR/capillary electrophoresis has a very high sensitivity that can distinguish low abundance single nucleotide differences and can detect paternally inherited fetal point mutations in maternal plasma.

  14. Genetically Targeted Dipeptidyl Peptidase-4 Inhibitor Use in a Patient with a Novel Mutation of MODY type 4

    Directory of Open Access Journals (Sweden)

    Christian Mangrum

    2015-01-01

    Full Text Available Maturity onset diabetes of the young (MODY is a rare form of diabetes mellitus typically seen in young adults that results from pancreatic beta-cell dysfunction. MODY4 is a rare subtype caused by a PDX1 mutation. In this case, we present a nonobese 26-year-old male with polyuria and polydipsia. Lab work showed a blood glucose of 511 mg/dL, no ketones or antibodies (insulin, islet cell, and glutamic acid decarboxylase [GAD], C-peptide of 1.6 ng/mL, and A1c 9.3%. Genetic analysis revealed a novel nonsense mutation in the PDX1 gene, consistent with MODY type 4. Given this patient's particular genetic mutation affecting the incretin pathway, sitagliptin was substituted for glyburide, which led to significant improvement in glycemic control. Our case report identifies a unique mutation in a rare form of MODY and outlines management of ensuing diabetes through targeting its inherent genetic mutation.

  15. Thermodynamic framework to assess low abundance DNA mutation detection by hybridization

    Science.gov (United States)

    Willems, Hanny; Jacobs, An; Hadiwikarta, Wahyu Wijaya; Venken, Tom; Valkenborg, Dirk; Van Roy, Nadine; Vandesompele, Jo; Hooyberghs, Jef

    2017-01-01

    The knowledge of genomic DNA variations in patient samples has a high and increasing value for human diagnostics in its broadest sense. Although many methods and sensors to detect or quantify these variations are available or under development, the number of underlying physico-chemical detection principles is limited. One of these principles is the hybridization of sample target DNA versus nucleic acid probes. We introduce a novel thermodynamics approach and develop a framework to exploit the specific detection capabilities of nucleic acid hybridization, using generic principles applicable to any platform. As a case study, we detect point mutations in the KRAS oncogene on a microarray platform. For the given platform and hybridization conditions, we demonstrate the multiplex detection capability of hybridization and assess the detection limit using thermodynamic considerations; DNA containing point mutations in a background of wild type sequences can be identified down to at least 1% relative concentration. In order to show the clinical relevance, the detection capabilities are confirmed on challenging formalin-fixed paraffin-embedded clinical tumor samples. This enzyme-free detection framework contains the accuracy and efficiency to screen for hundreds of mutations in a single run with many potential applications in molecular diagnostics and the field of personalised medicine. PMID:28542229

  16. Loss of function mutations in EPHB4 are responsible for vein of Galen aneurysmal malformation.

    Science.gov (United States)

    Vivanti, Alexandre; Ozanne, Augustin; Grondin, Cynthia; Saliou, Guillaume; Quevarec, Loic; Maurey, Helène; Aubourg, Patrick; Benachi, Alexandra; Gut, Marta; Gut, Ivo; Martinovic, Jelena; Sénat, Marie Victoire; Tawk, Marcel; Melki, Judith

    2018-04-01

    See Meschia (doi:10.1093/brain/awy066) for a scientific commentary on this article.Vein of Galen aneurysmal malformation is a congenital anomaly of the cerebral vasculature representing 30% of all paediatric vascular malformations. We conducted whole exome sequencing in 19 unrelated patients presenting this malformation and subsequently screened candidate genes in a cohort of 32 additional patients using either targeted exome or Sanger sequencing. In a cohort of 51 patients, we found five affected individuals with heterozygous mutations in EPHB4 including de novo frameshift (p.His191Alafs*32) or inherited deleterious splice or missense mutations predicted to be pathogenic by in silico tools. Knockdown of ephb4 in zebrafish embryos leads to specific anomalies of dorsal cranial vessels including the dorsal longitudinal vein, which is the orthologue of the median prosencephalic vein and the embryonic precursor of the vein of Galen. This model allowed us to investigate EPHB4 loss-of-function mutations in this disease by the ability to rescue the brain vascular defect in knockdown zebrafish co-injected with wild-type, but not truncated EPHB4, mimicking the p.His191Alafs mutation. Our data showed that in both species, loss of function mutations of EPHB4 result in specific and similar brain vascular development anomalies. Recently, EPHB4 germline mutations have been reported in non-immune hydrops fetalis and in cutaneous capillary malformation-arteriovenous malformation. Here, we show that EPHB4 mutations are also responsible for vein of Galen aneurysmal malformation, indicating that heterozygous germline mutations of EPHB4 result in a large clinical spectrum. The identification of EPHB4 pathogenic mutations in patients presenting capillary malformation or vein of Galen aneurysmal malformation should lead to careful follow-up of pregnancy of carriers for early detection of anomaly of the cerebral vasculature in order to propose optimal neonatal care. Endovascular

  17. Mutational analysis of EGFR and related signaling pathway genes in lung adenocarcinomas identifies a novel somatic kinase domain mutation in FGFR4.

    Directory of Open Access Journals (Sweden)

    Jenifer L Marks

    2007-05-01

    Full Text Available Fifty percent of lung adenocarcinomas harbor somatic mutations in six genes that encode proteins in the EGFR signaling pathway, i.e., EGFR, HER2/ERBB2, HER4/ERBB4, PIK3CA, BRAF, and KRAS. We performed mutational profiling of a large cohort of lung adenocarcinomas to uncover other potential somatic mutations in genes of this signaling pathway that could contribute to lung tumorigenesis.We analyzed genomic DNA from a total of 261 resected, clinically annotated non-small cell lung cancer (NSCLC specimens. The coding sequences of 39 genes were screened for somatic mutations via high-throughput dideoxynucleotide sequencing of PCR-amplified gene products. Mutations were considered to be somatic only if they were found in an independent tumor-derived PCR product but not in matched normal tissue. Sequencing of 9MB of tumor sequence identified 239 putative genetic variants. We further examined 22 variants found in RAS family genes and 135 variants localized to exons encoding the kinase domain of respective proteins. We identified a total of 37 non-synonymous somatic mutations; 36 were found collectively in EGFR, KRAS, BRAF, and PIK3CA. One somatic mutation was a previously unreported mutation in the kinase domain (exon 16 of FGFR4 (Glu681Lys, identified in 1 of 158 tumors. The FGFR4 mutation is analogous to a reported tumor-specific somatic mutation in ERBB2 and is located in the same exon as a previously reported kinase domain mutation in FGFR4 (Pro712Thr in a lung adenocarcinoma cell line.This study is one of the first comprehensive mutational analyses of major genes in a specific signaling pathway in a sizeable cohort of lung adenocarcinomas. Our results suggest the majority of gain-of-function mutations within kinase genes in the EGFR signaling pathway have already been identified. Our findings also implicate FGFR4 in the pathogenesis of a subset of lung adenocarcinomas.

  18. Identification of a de novo mutation of SOX10 in a Chinese patient with Waardenburg syndrome type IV.

    Science.gov (United States)

    Liang, Fenghe; Zhao, Min; Fan, Lynn; Zhang, Hongyan; Shi, Yang; Han, Rui; Qu, Chunyan

    2016-12-01

    Waardenburg syndrome is a rare genetic disorder, characterized by the association of sensorineural hearing loss and pigmentation abnormalities. Four subtypes have been classified. The present study aimed to analyze the clinical feature and investigate the genetic cause for a Chinese case of Waardenburg type IV (WS4). The patient and his family members were subjected to mutation detection in the candidate gene SOX10 by Sanger sequencing. The patient has the clinical features of WS4, including sensorineural hearing loss, bright blue irides, premature graying of the hair and Hirschsprung disease. A novel heterozygous frameshift mutation, c.752_753ins7 (p.Gly252Alafs*31) in the exon 5 of SOX10 was detected in the patient, but not found in the unaffected family members and 100 normal controls. This mutation results in a premature stop codon 31 amino acid downstream. The novel mutation c.752_753ins7 (p.Gly252Alafs*31) arose de novo and was considered as the cause of WS4 in the proband. This study further characterized the molecular complexity of WS4 and provided a clinical case for genotype-phenotype correlation studies of different phenotypes caused by SOX10 mutations. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

  19. Melanocortin 4 receptor mutations in obese Czech children

    DEFF Research Database (Denmark)

    Hainerová, Irena; Larsen, Lesli H; Holst, Birgitte

    2007-01-01

    Mutations in the melanocortin 4 receptor gene (MC4R) represent the most common known cause of monogenic human obesity.......Mutations in the melanocortin 4 receptor gene (MC4R) represent the most common known cause of monogenic human obesity....

  20. Clinical and mutational characteristics of Duchenne muscular dystrophy patients based on a comprehensive database in South China.

    Science.gov (United States)

    Wang, Dan-Ni; Wang, Zhi-Qiang; Yan, Lei; He, Jin; Lin, Min-Ting; Chen, Wan-Jin; Wang, Ning

    2017-08-01

    The development of clinical trials for Duchenne muscular dystrophy (DMD) in China faces many challenges due to limited information about epidemiological data, natural history and clinical management. To provide these detailed data, we developed a comprehensive database based on registered DMD patients from South China and analysed their clinical and mutational characteristics. The database included DMD registrants confirmed by clinical presentation, family history, genetic detection, prognostic outcome, and/or muscle biopsy. Clinical data were collected by a registry form. Mutations of dystrophin were detected by multiplex ligation-dependent probe amplification (MLPA) and Sanger sequencing. Currently, 132 DMD patients from 128 families in South China have been registered, and 91.7% of them were below 10 years old. In mutational detection, large deletions were the most frequent type (57.8%), followed by small deletion/insertion mutations (14.1%), nonsense mutations (13.3%), large duplications (10.9%), and splice site mutations (3.1%). Clinical analysis revealed that most patients reported initial symptoms between 1 and 3 years of age, but the diagnostic age was more frequently between 6 and 8 years. 81.4% of patients were ambulatory. Baseline cardiac assessments at diagnosis were conducted in 39.4% and 29.5% of patients by echocardiograms and electrocardiograms, respectively. Only 22.7% of registrants performed baseline respiratory assessments. A small numbers of patients (20.5%) were treated with glucocorticoids. 13.3% of patients were eligible for stop codon read-through therapy, and 48.4% of patients would potentially benefit from exon skipping. The top five exon skips applicable to the largest group of registrants were skipping of exons 51 (14.8% of total mutations), 53 (12.5%), 45 (7.0%), 55 (4.7%), and 44 (3.9%). In conclusion, our database provided information on the natural history, diagnosis and management status of DMD in South China, as well as potential

  1. Sensitive detection of point mutation by electrochemiluminescence and DNA ligase-based assay

    Science.gov (United States)

    Zhou, Huijuan; Wu, Baoyan

    2008-12-01

    The technology of single-base mutation detection plays an increasingly important role in diagnosis and prognosis of genetic-based diseases. Here we reported a new method for the analysis of point mutations in genomic DNA through the integration of allele-specific oligonucleotide ligation assay (OLA) with magnetic beads-based electrochemiluminescence (ECL) detection scheme. In this assay the tris(bipyridine) ruthenium (TBR) labeled probe and the biotinylated probe are designed to perfectly complementary to the mutant target, thus a ligation can be generated between those two probes by Taq DNA Ligase in the presence of mutant target. If there is an allele mismatch, the ligation does not take place. The ligation products are then captured onto streptavidin-coated paramagnetic beads, and detected by measuring the ECL signal of the TBR label. Results showed that the new method held a low detection limit down to 10 fmol and was successfully applied in the identification of point mutations from ASTC-α-1, PANC-1 and normal cell lines in codon 273 of TP53 oncogene. In summary, this method provides a sensitive, cost-effective and easy operation approach for point mutation detection.

  2. Simultaneous detection of hepatitis B virus genotypes and mutations associated with resistance to lamivudine, adefovir, and telbivudine by the polymerase chain reaction-ligase detection reaction

    Directory of Open Access Journals (Sweden)

    Yong-Zhong Wang

    Full Text Available OBJECTIVES: Detection of mutations associated to nucleos(tide analogs and hepatitis B virus (HBV genotyping are essential for monitoring treatment of HBV infection. We developed a multiplex polymerase chain reaction-ligase detection reaction (PCR-LDR assay for the rapid detection of HBV genotypes and mutations associated with lamivudine, adefovir, and telbivudine resistance in HBV-infected patients. METHODS: HBV templates were amplified by PCR, followed by LDR and electrophoresis on a sequencer. The assay was evaluated using plasmids that contained wild-type or mutant HBV sequences and 216 clinical samples. RESULTS: The PCR-LDR assay and sequencing gave comparable results for 158 of the 216 samples (73.1% with respect to mutation detection and genotyping. Complete agreement between the two methods was observed for all the samples (100% at codon 180 and codon 204. Concordant results were observed for 99.4% of the 158 samples at codon 181 and 98.7% at codon 236. The genotyping results were completely concordant between the PCR-LDR assay and sequencing. The PCR-LDR assay could detect a proportion of 1% mutant plasmid in a background of wild-type plasmid. CONCLUSION: The PCR-LDR assay is sensitive and specific for detection of HBV genotypes and drug resistance mutations, and could be helpful for decision making in the treatment of HBV infection.

  3. THE EXON 5, 6, 7, 8 OF P53 MUTATIONS IN ORAL SQUAMOUS CELLS CARCINOMA

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    Retno P Rahayu

    2012-04-01

    Full Text Available Genetic instability may underlie the etiology of multistep carcinogenesis. The altered p53 gene observed in tumors may represent the expression of such instability and may allow the accumulation of other gene alterations caused by multiple mechanism. p53 gene is the guardian of the genome, that is why we pay more attention to this gene. In this study, we evaluated the significance of p53 mutation in 55 patient with oral squamous carcinoma. Thirty among them underwent well-differentiated carcinoma, while the remaining 25 patients underwent poorly differentiated carcinoma. The mutations were detected by PCR-SSCP (Single strand Conformational Polymorphism analysis in the region between exon 5 and exon 8. The results indicated that the p53 mutation in exon 5 (40%, exon 6 (28%, exon 7 (24% and exon 8 (8% were associated with poorly differentiated carcinoma, whereas mutation in exon 5 (10%, exon 6 (30%, exon 7 (40% and exon 8 (20% were associated with well-differentiated carcinoma. These observations suggest that p53 mutation in exon 5, 6, and 7 have strong correlation with poorly differentiated in oral squamous carcinoma while well-differentiated level was related with mutation in exon 6,7 and 8.

  4. Detection of high frequency of mutations in a breast and/or ovarian cancer cohort: implications of embracing a multi-gene panel in molecular diagnosis in India.

    Science.gov (United States)

    Mannan, Ashraf U; Singh, Jaya; Lakshmikeshava, Ravikiran; Thota, Nishita; Singh, Suhasini; Sowmya, T S; Mishra, Avshesh; Sinha, Aditi; Deshwal, Shivani; Soni, Megha R; Chandrasekar, Anbukayalvizhi; Ramesh, Bhargavi; Ramamurthy, Bharat; Padhi, Shila; Manek, Payal; Ramalingam, Ravi; Kapoor, Suman; Ghosh, Mithua; Sankaran, Satish; Ghosh, Arunabha; Veeramachaneni, Vamsi; Ramamoorthy, Preveen; Hariharan, Ramesh; Subramanian, Kalyanasundaram

    2016-06-01

    Breast and/or ovarian cancer (BOC) are among the most frequently diagnosed forms of hereditary cancers and leading cause of death in India. This emphasizes on the need for a cost-effective method for early detection of these cancers. We sequenced 141 unrelated patients and families with BOC using the TruSight Cancer panel, which includes 13 genes strongly associated with risk of inherited BOC. Multi-gene sequencing was done on the Illumina MiSeq platform. Genetic variations were identified using the Strand NGS software and interpreted using the StrandOmics platform. We were able to detect pathogenic mutations in 51 (36.2%) cases, out of which 19 were novel mutations. When we considered familial breast cancer cases only, the detection rate increased to 52%. When cases were stratified based on age of diagnosis into three categories, ⩽40 years, 40-50 years and >50 years, the detection rates were higher in the first two categories (44.4% and 53.4%, respectively) as compared with the third category, in which it was 26.9%. Our study suggests that next-generation sequencing-based multi-gene panels increase the sensitivity of mutation detection and help in identifying patients with a high risk of developing cancer as compared with sequential tests of individual genes.

  5. MSH6 Mutations are Frequent in Hereditary Nonpolyposis Colorectal Cancer Families With Normal pMSH6 Expression as Detected by Immunohistochemistry

    DEFF Research Database (Denmark)

    Okkels, Henrik; Larsen, K.L.; Thorlacius-Ussing, O.

    2012-01-01

    INTRODUCTION:: Hereditary nonpolyposis colorectal cancer (HNPCC) is an autosomal dominant condition accounting for 2% to 4% of all colorectal cancer cases worldwide. Families with germ line mutations in 1 of 6 mismatch repair genes are known as Lynch syndrome families. The largest number...... this approach in Lynch families carrying mutations in MSH6. MATERIALS AND METHODS:: Results of the screening of the MSH6 gene in HNPCC families were compared with those obtained on immunohistochemical protein analysis. RESULTS:: In 56 (7%) of 815 families, at least 1 MSH6 mutation, 23 definitively pathogenic...... be detected, whereas in 34.5% pMSH6 was present and pMLH1/pPMS2 was absent. CONCLUSIONS:: If genetic screening of HNPCC families depended on immunohistochemical results, a substantial number of families harboring a pathogenic mutation in MSH6 and the vast majority of families harboring an MSH6 unclassified...

  6. Nkx2-5 Mutations in Patients With Nonsyndromic Congenital Heart Disease

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    Fariborz Soheili

    2016-01-01

    Full Text Available Background Congenital heart diseases (CHD are the most common of all birth defects, affecting nearly 0.9% of all live births. Nkx2-5 mutations were reported to cause CHD but data in Kurdish populations of Iran are limited. Objectives In this experimental study, we performed high resolution melt (HRM mutation scanning of Nkx2-5 exons of non-syndrome patients. Patients and Methods Thirty nine patients with atrial septal defect and 57 patients with ventricular septal defect, 4 patients possessing both defects as case groups and 50 healthy controls. Then we grouped samples according to HRM graph and sequenced several samples from each group. Results HRM analysis showed 2 deviated curves for exon 1 and one group for exon 2A and exon 2B. Then, 2 samples of exon 1 that showed different HRM curves, 3 samples of another group from this exon and 5 samples of exon 2A, 2B and healthy controls were randomly sequenced. The results of sequencing confirmed the HRM analysis, and one polymorphism (A65G was identified in 2 atrial septal defects with deviated curves. Conclusions The environmental and effective factors on the heart development within embryonic evolution as well as the possibility of the existence of the mutation in coding genes of the other cardiac transcription factors such as GATA4 and TBX5 can be the reasons for the lack of the pathogenic mutation in this study. It is suggested in further related studies to investigate normal and abnormal cardiac tissue samples of these studied patients and coding genes of the other cardiac transcription factors.

  7. Rapid detection of most frequent Slovenian germ-line mutations in BRCA1 gene using real-time PCR and melting curve analysis

    International Nuclear Information System (INIS)

    Novakovic, S.; Stegel, V.

    2005-01-01

    Background. Detection of inherited mutations in cancer susceptibility genes is of great importance in some types of cancers including the colorectal cancer (mutations of APC gene in familial adenomatous polyposis - FAP, mutations in mismatch repair genes in hereditary nonpolyposis colorectal cancer - HNPCC), malignant melanoma (mutations in CDKN2A and CDK4 genes) and breast cancer (mutations in BRCA1 and BRCA2 genes). Methods. This article presents the technical data for the detection of five mutations in BRCA1 gene in breast cancer patients and their relatives. The mutations - 1806C>T, 300T>G, 300T>A, 310G>A, 5382insC - were determined by the real-time PCR and the melting curve analysis. Results and conclusion. In comparison to direct sequencing, this method proved to be sensitive and rapid enough for the routine daily determination of mutations in DNA isolated from the peripheral blood. (author)

  8. A novel homozygous AP4B1 mutation in two brothers with AP-4 deficiency syndrome and ocular anomalies.

    Science.gov (United States)

    Accogli, Andrea; Hamdan, Fadi F; Poulin, Chantal; Nassif, Christina; Rouleau, Guy A; Michaud, Jacques L; Srour, Myriam

    2018-04-01

    Adaptor protein complex-4 (AP-4) is a heterotetrameric protein complex which plays a key role in vesicle trafficking in neurons. Mutations in genes affecting different subunits of AP-4, including AP4B1, AP4E1, AP4S1, and AP4M1, have been recently associated with an autosomal recessive phenotype, consisting of spastic tetraplegia, and intellectual disability (ID). The overlapping clinical picture among individuals carrying mutations in any of these genes has prompted the terms "AP-4 deficiency syndrome" for this clinically recognizable phenotype. Using whole-exome sequencing, we identified a novel homozygous mutation (c.991C>T, p.Q331*, NM_006594.4) in AP4B1 in two siblings from a consanguineous Pakistani couple, who presented with severe ID, progressive spastic tetraplegia, epilepsy, and microcephaly. Sanger sequencing confirmed the mutation was homozygous in the siblings and heterozygous in the parents. Similar to previously reported individuals with AP4B1 mutations, brain MRI revealed ventriculomegaly and white matter loss. Interestingly, in addition to the typical facial gestalt reported in other AP-4 deficiency cases, the older brother presented with congenital left Horner syndrome, bilateral optic nerve atrophy and cataract, which have not been previously reported in this condition. In summary, we report a novel AP4B1 homozygous mutation in two siblings and review the phenotype of AP-4 deficiency, speculating on a possible role of AP-4 complex in eye development. © 2018 Wiley Periodicals, Inc.

  9. The KRAS Strip Assay for detection of KRAS mutation in Egyptian patients with colorectal cancer (CRC): A pilot study

    International Nuclear Information System (INIS)

    Abd El Kader, Y.; Safwat, E.; Kassem, H.A.; Kassem, N.M.; Emera, G.

    2013-01-01

    Background: Epidermal growth factor receptor (EGFR) and its downstream factors KRAS and BRAF are mutated in several types of cancer, affecting the clinical response to EGFR inhibitors. Mutations in the EGFR kinase domain predict sensitivity to the tyrosine kinase inhibitors gefltinib and erlotinib in lung adenocarcinoma, while activating point mutations in KRAS and BRAF confer resistance to the anti-EGFR monoclonal antibody cetuximab in colorectal cancer. The development of new generation methods for systematic mutation screening of these genes will allow more appropriate therapeutic choices. Purpose: Detection of KRAS mutation in Egyptian colorectal cancer (CRC) patients by the KRAS Strip Assay. Methods: Examination of 20 colorectal cancer (CRC) patients is done to detect KRAS mutations by KRAS Strip Assay. For the Strip Assay, a mutant-enriched PCR was followed by hybridization to KRAS-specific probes bound to a nitrocellulose strip. Results: Among 20 patients, KRAS mutations were identified in 80% of patients by the KRAS Strip Assay. Conclusions: Our preliminary results suggest that KRAS Strip Assay is an alternative to protocols currently in use for KRAS mutation detection

  10. Breed distribution of the nt230(del4) MDR1 mutation in dogs.

    Science.gov (United States)

    Gramer, Irina; Leidolf, Regina; Döring, Barbara; Klintzsch, Stefanie; Krämer, Eva-Maria; Yalcin, Ebru; Petzinger, Ernst; Geyer, Joachim

    2011-07-01

    A 4-bp deletion mutation associated with multiple drug sensitivity exists in the canine multidrug resistance (MDR1) gene. This mutation has been detected in more than 10 purebred dog breeds as well as in mixed breed dogs. To evaluate the breed distribution of this mutation in Germany, 7378 dogs were screened, including 6999 purebred and 379 mixed breed dogs. The study included dog breeds that show close genetic relationship or share breeding history with one of the predisposed breeds but in which the occurrence of the MDR1 mutation has not been reported. The breeds comprised Bearded Collies, Anatolian Shepherd Dog, Greyhound, Belgian Tervuren, Kelpie, Borzoi, Australian Cattle Dog and the Irish Wolfhound. The MDR1 mutation was not detected is any of these breeds, although it was found as expected in the Collie, Longhaired Whippet, Shetland Sheepdog, Miniature Australian Shepherd, Australian Shepherd, Wäller, White Swiss Shepherd, Old English Sheepdog and Border Collie with varying allelic frequencies for the mutant MDR1 allele of 59%, 45%, 30%, 24%, 22%, 17%, 14%, 4% and 1%, respectively. Allelic frequencies of 8% and 2% were determined in herding breed mixes and unclassified mixed breeds, respectively. Because of its widespread breed distribution and occurrence in many mixed breed dogs, it is difficult for veterinarians and dog owners to recognise whether MDR1-related drug sensitivity is relevant for an individual animal. This study provides a comprehensive overview of all affected dog breeds and many dog breeds that are probably unaffected on the basis of ∼15,000 worldwide MDR1 genotyping data. Copyright © 2010 Elsevier Ltd. All rights reserved.

  11. Phytochrome-interacting factors PIF4 and PIF5 negatively regulate anthocyanin biosynthesis under red light in Arabidopsis seedlings.

    Science.gov (United States)

    Liu, Zhongjuan; Zhang, Yongqiang; Wang, Jianfeng; Li, Ping; Zhao, Chengzhou; Chen, Yadi; Bi, Yurong

    2015-09-01

    Light is an important environmental factor inducing anthocyanin accumulation in plants. Phytochrome-interacting factors (PIFs) have been shown to be a family of bHLH transcription factors involved in light signaling in Arabidopsis. Red light effectively increased anthocyanin accumulation in wild-type Col-0, whereas the effects were enhanced in pif4 and pif5 mutants but impaired in overexpression lines PIF4OX and PIF5OX, indicating that PIF4 and PIF5 are both negative regulators for red light-induced anthocyanin accumulation. Consistently, transcript levels of several genes involved in anthocyanin biosynthesis and regulatory pathway, including CHS, F3'H, DFR, LDOX, PAP1 and TT8, were significantly enhanced in mutants pif4 and pif5 but decreased in PIF4OX and PIF5OX compared to in Col-0, indicating that PIF4 and PIF5 are transcriptional repressor of these gene. Transient expression assays revealed that PIF4 and PIF5 could repress red light-induced promoter activities of F3'H and DFR in Arabidopsis protoplasts. Furthermore, chromatin immunoprecipitation-quantitative PCR (ChIP-qPCR) test and electrophoretic mobility shift assay (EMSA) showed that PIF5 could directly bind to G-box motifs present in the promoter of DFR. Taken together, these results suggest that PIF4 and PIF5 negatively regulate red light-induced anthocyanin accumulation through transcriptional repression of the anthocyanin biosynthetic genes in Arabidopsis. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

  12. Macrosomia and hyperinsulinaemic hypoglycaemia in patients with heterozygous mutations in the HNF4A gene.

    Directory of Open Access Journals (Sweden)

    Ewan R Pearson

    2007-04-01

    Full Text Available Macrosomia is associated with considerable neonatal and maternal morbidity. Factors that predict macrosomia are poorly understood. The increased rate of macrosomia in the offspring of pregnant women with diabetes and in congenital hyperinsulinaemia is mediated by increased foetal insulin secretion. We assessed the in utero and neonatal role of two key regulators of pancreatic insulin secretion by studying birthweight and the incidence of neonatal hypoglycaemia in patients with heterozygous mutations in the maturity-onset diabetes of the young (MODY genes HNF4A (encoding HNF-4alpha and HNF1A/TCF1 (encoding HNF-1alpha, and the effect of pancreatic deletion of Hnf4a on foetal and neonatal insulin secretion in mice.We examined birthweight and hypoglycaemia in 108 patients from families with diabetes due to HNF4A mutations, and 134 patients from families with HNF1A mutations. Birthweight was increased by a median of 790 g in HNF4A-mutation carriers compared to non-mutation family members (p < 0.001; 56% (30/54 of HNF4A-mutation carriers were macrosomic compared with 13% (7/54 of non-mutation family members (p < 0.001. Transient hypoglycaemia was reported in 8/54 infants with heterozygous HNF4A mutations, but was reported in none of 54 non-mutation carriers (p = 0.003. There was documented hyperinsulinaemia in three cases. Birthweight and prevalence of neonatal hypoglycaemia were not increased in HNF1A-mutation carriers. Mice with pancreatic beta-cell deletion of Hnf4a had hyperinsulinaemia in utero and hyperinsulinaemic hypoglycaemia at birth.HNF4A mutations are associated with a considerable increase in birthweight and macrosomia, and are a novel cause of neonatal hypoglycaemia. This study establishes a key role for HNF4A in determining foetal birthweight, and uncovers an unanticipated feature of the natural history of HNF4A-deficient diabetes, with hyperinsulinaemia at birth evolving to decreased insulin secretion and diabetes later in life.

  13. Two Siblings With a CDKL5 Mutation: Genotype and Phenotype Evaluation.

    Science.gov (United States)

    Hagebeuk, Eveline E O; Marcelis, Carlo L; Alders, Mariëlle; Kaspers, Ageeth; de Weerd, Al W

    2015-10-01

    This is the second report of a family with a recurrence of a CDKL5 mutation (c. 283-3_290del) in 2 sisters. Both parents tested negative for the mutation in all tissues, but germline mosaicism is likely. Clinically CDKL5 patients resemble those with Rett syndrome, caused by a MECP2 mutation, who experience a regression, after an initial normal development. Even though both siblings showed a typical CDKL5 phenotype, their presentation is different. From birth, the oldest daughter had a severe developmental delay, feeding problems, and hypotonia and experienced daily refractory seizures. The youngest daughter appeared to be normal until age 3 months. At that age seizures started, deterioration and regression became evident, and an epileptic encephalopathy developed. This report of familial recurrence, with suspected germline mosaicism in a healthy parent, has important consequences for genetic counseling. Although it is not possible to predict an exact recurrence risk, it is likely to be increased. © The Author(s) 2015.

  14. A unique TBX5 microdeletion with microinsertion detected in patient with Holt-Oram syndrome.

    Science.gov (United States)

    Morine, Mikio; Kohmoto, Tomohiro; Masuda, Kiyoshi; Inagaki, Hidehito; Watanabe, Miki; Naruto, Takuya; Kurahashi, Hiroki; Maeda, Kazuhisa; Imoto, Issei

    2015-12-01

    Holt-Oram syndrome (HOS) is an autosomal dominant condition characterized by upper limb and congenital heart defects and caused by numerous germline mutations of TBX5 producing preterminal stop codons. Here, we report on a novel and unusual heterozygous TBX5 microdeletion with microinsertion (microindel) mutation (c.627delinsGTGACTCAGGAAACGCTTTCCTGA), which is predicted to synthesize a truncated TBX5 protein, detected in a sporadic patient with clinical features of HOS prenatally diagnosed by ultrasonography. This uncommon and relatively large inserted sequence contains sequences derived from nearby but not adjacent templates on both sense and antisense strands, suggesting two possible models, which require no repeat sequences, causing this complex microindel through the bypass of large DNA adducts via an error-prone DNA polymerase-mediated translesion synthesis. © 2015 Wiley Periodicals, Inc.

  15. CDKL5 and ARX mutations in males with early-onset epilepsy.

    Science.gov (United States)

    Mirzaa, Ghayda M; Paciorkowski, Alex R; Marsh, Eric D; Berry-Kravis, Elizabeth M; Medne, Livija; Alkhateeb, Asem; Grix, Art; Wirrell, Elaine C; Powell, Berkley R; Nickels, Katherine C; Burton, Barbara; Paras, Andrea; Kim, Katherine; Chung, Wendy; Dobyns, William B; Das, Soma

    2013-05-01

    Mutations in CDKL5 and ARX are known causes of early-onset epilepsy and severe developmental delay in males and females. Although numerous males with ARX mutations associated with various phenotypes have been reported in the literature, the majority of CDKL5 mutations have been identified in females with a phenotype characterized by early-onset epilepsy, severe global developmental delay, absent speech, and stereotypic hand movements. To date, only 10 males with CDKL5 mutations have been reported. Our retrospective study reports on the clinical, neuroimaging, and molecular findings of 18 males with early-onset epilepsy caused by either CDKL5 or ARX mutations. These 18 patients include eight new males with CDKL5 mutations and 10 with ARX mutations identified through sequence analysis of 266 and 346 males, respectively, at our molecular diagnostic laboratory. Our large dataset therefore expands on the number of reported males with CDKL5 mutations and highlights that aberrations of CDKL5 and ARX combined are an important consideration in the genetic forms of early-onset epilepsy in boys. Copyright © 2013 Elsevier Inc. All rights reserved.

  16. Key clinical features to identify girls with CDKL5 mutations.

    Science.gov (United States)

    Bahi-Buisson, Nadia; Nectoux, Juliette; Rosas-Vargas, Haydeé; Milh, Mathieu; Boddaert, Nathalie; Girard, Benoit; Cances, Claude; Ville, Dorothée; Afenjar, Alexandra; Rio, Marlène; Héron, Delphine; N'guyen Morel, Marie Ange; Arzimanoglou, Alexis; Philippe, Christophe; Jonveaux, Philippe; Chelly, Jamel; Bienvenu, Thierry

    2008-10-01

    mutations. In conclusion, our report show that search for mutations in CDKL5 is indicated in girls with early onset of a severe intractable seizure disorder or infantile spasms with severe hypotonia, and in girls with RTT-like phenotype and early onset seizures, though, in our cohort, mutations in CDKL5 account for about 10% of the girls affected by these disorders.

  17. A PLK4 mutation causing azoospermia in a man with Sertoli cell-only syndrome.

    Science.gov (United States)

    Miyamoto, T; Bando, Y; Koh, E; Tsujimura, A; Miyagawa, Y; Iijima, M; Namiki, M; Shiina, M; Ogata, K; Matsumoto, N; Sengoku, K

    2016-01-01

    About 15% of couples wishing to have children are infertile; approximately half these cases involve a male factor. Polo-like kinase 4 (PLK-4) is a member of the polo protein family and a key regulator of centriole duplication. Male mice with a point mutation in the Plk4 gene show azoospermia associated with germ cell loss. Mutational analysis of 81 patients with azoospermia and Sertoli cell-only syndrome (SCOS) identified one man with a heterozygous 13-bp deletion in the Ser/Thr kinase domain of PLK4. Division of centrioles occurred in wild-type PLK4-transfected cells, but was hampered in PLK-4-mutant transfectants, which also showed abnormal nuclei. Thus, this PLK4 mutation might be a cause of human SCOS and nonobstructive azoospermia. © 2015 American Society of Andrology and European Academy of Andrology.

  18. Análise da expressão do colágeno VI na distrofia muscular congênita Analysis of the expression of collagen VI in congenital muscular dystrophy

    Directory of Open Access Journals (Sweden)

    Regina Toni Loureiro de Freitas

    2005-06-01

    Full Text Available A distrofia muscular congênita (DMC compõe um grupo de miopatias caracterizadas por hipotonia e fraqueza muscular notadas já no primeiro ano de vida. A forma de Ullrich é caracterizada por retrações musculares proximais e hiperextensibilidade distal. Cerca de 40% destes pacientes apresentam mutações em um dos genes que codificam as três sub-unidades do colágeno VI (COL6, acarretando deficiência total ou parcial na marcação da proteína. Analisamos, através de imunofluorescência, a marcação do COL6 em fragmentos musculares de 50 pacientes com DMC, 20 deles com ausência da marcação para merosina. Identificamos 4 casos com deficiência total da marcação do COL6 (8% do total, representando 13% dos casos com marcação normal para merosina. As alterações histológicas musculares dos pacientes com COL6 deficiente eram indistinguíveis das outras formas de DMC, porém mais brandas que as observadas na DMC com deficiência de merosina. Em três dos pacientes com COL6 deficiente observou-se hipotonia e fraqueza muscular, notadas já no período neonatal, atraso do desenvolvimento motor, retrações musculares em joelhos e cotovelos, hiperextensibilidade distal e luxação congênita do quadril (dois pacientes. Um paciente perdeu a capacidade para a marcha, e outro faleceu por problemas respiratórios. A análise da marcação do COL6, assim como da merosina, no tecido muscular de pacientes com DMC pode auxiliar na identificação e caracterização fenotípica dos diversos subtipos de DMC.Congenital muscular dystrophy (CMD composes a group of disorders characterized by hypotonia and muscular weakness noticed in the first year of life. The Ullrich's form is characterized by proximal joint contractures and distal hiperextensibility. About 40% of these patients present mutations in one of the genes that codify the sub-units of the collagen VI protein (COL6, producing total or partial deficiency of the protein expression. We analyzed

  19. The mutational spectrum of Lynch syndrome in cyprus.

    Directory of Open Access Journals (Sweden)

    Maria A Loizidou

    Full Text Available Lynch syndrome is the most common form of hereditary colorectal cancer and is caused by germline mutations in the mismatch repair (MMR genes MLH1, MSH2, MSH6 and PMS2. Mutation carriers have an increased lifetime risk of developing colorectal cancer as well as other extracolonic tumours. The aim of the current study was to evaluate the frequency and distribution of mutations in the MLH1, MSH2 and MSH6 genes within a cohort of Cypriot families that fulfilled the revised Bethesda guidelines. The study cohort included 77 patients who fulfilled at least one of the revised Bethesda guidelines. Mutational analysis revealed the presence of 4 pathogenic mutations, 3 in the MLH1 gene and 1 in the MSH2 gene, in 5 unrelated individuals. It is noted that out of the 4 pathogenic mutations detected, one is novel (c.1610delG in exon 14 of the MLH1 and has been detected for the first time in the Cypriot population. Overall, the pathogenic mutation detection rate in our patient cohort was 7%. This percentage is relatively low but could be explained by the fact that the sole criterion for genetic screening was compliance to the revised Bethesda guidelines. Larger numbers of Lynch syndrome families and screening of the two additional predisposition genes, PMS2 and EPCAM, are needed in order to decipher the full spectrum of mutations associated with Lynch syndrome predisposition in Cyprus.

  20. Altered Gene-Regulatory Function of KDM5C by a Novel Mutation Associated With Autism and Intellectual Disability.

    Science.gov (United States)

    Vallianatos, Christina N; Farrehi, Clara; Friez, Michael J; Burmeister, Margit; Keegan, Catherine E; Iwase, Shigeki

    2018-01-01

    Intellectual disability (ID) affects up to 2% of the population world-wide and often coincides with other neurological conditions such as autism spectrum disorders. Mutations in KDM5C cause Mental Retardation, X-linked, Syndromic, Claes-Jensen type (MRXSCJ, OMIM #300534) and are one of the most common causes of X-linked ID. KDM5C encodes a histone demethylase for di- and tri-methylated histone H3 lysine 4 (H3K4me2/3), which are enriched in transcriptionally engaged promoter regions. KDM5C regulates gene transcription; however, it remains unknown whether removal of H3K4me is fully responsible for KDM5C-mediated gene regulation. Most mutations functionally tested to date result in reduced enzymatic activity of KDM5C, indicating loss of demethylase function as the primary mechanism underlying MRXSCJ. Here, we report a novel KDM5C mutation, R1115H, identified in an individual displaying MRXSCJ-like symptoms. The carrier mother's cells exhibited a highly skewed X-inactivation pattern. The KDM5C-R1115H substitution does not have an impact on enzymatic activity nor protein stability. However, when overexpressed in post-mitotic neurons, KDM5C-R1115H failed to fully suppress expression of target genes, while the mutant also affected expression of a distinct set of genes compared to KDM5C-wildtype. These results suggest that KDM5C may have non-enzymatic roles in gene regulation, and alteration of these roles contributes to MRXSCJ in this patient.

  1. The transmembrane collagen COL-99 guides longitudinally extending axons in C. elegans.

    Science.gov (United States)

    Taylor, Jesse; Unsoeld, Thomas; Hutter, Harald

    2018-06-01

    We have identified the transmembrane collagen, COL-99, in a genetic screen for novel genes involved in axon guidance in the nematode C. elegans. COL-99 is similar to transmembrane collagens type XIII, XXIII and XXV in vertebrates. col-99 mutants exhibit guidance defects in axons extending along the major longitudinal axon tracts, most prominently the left ventral nerve cord (VNC). COL-99 is expressed in the hypodermis during the time of axon outgrowth. We provide evidence that a furin cleavage site in COL-99 is essential for function, suggesting that COL-99 is released from the cells producing it. Vertebrate homologs of COL-99 have been shown to be expressed in mammalian nervous systems and linked to various neurological disease but have not been associated with guidance of extending neurons. col-99 acts genetically with the discoidin domain receptors ddr-1 and ddr-2, which are expressed by neurons affected in col-99 mutants. Discoidin domain receptors are activated by collagens in vertebrates. DDR-1 and DDR-2 may function as receptors for COL-99. Our results establish a novel role for a transmembrane collagen in axonal guidance and asymmetry establishment of the VNC. Copyright © 2018 Elsevier Inc. All rights reserved.

  2. Deep sequencing of uveal melanoma identifies a recurrent mutation in PLCB4

    DEFF Research Database (Denmark)

    Johansson, Peter; Aoude, Lauren G; Wadt, Karin

    2016-01-01

    Next generation sequencing of uveal melanoma (UM) samples has identified a number of recurrent oncogenic or loss-of-function mutations in key driver genes including: GNAQ, GNA11, EIF1AX, SF3B1 and BAP1. To search for additional driver mutations in this tumor type we carried out whole......, instead, a BRCA mutation signature predominated. In addition to mutations in the known UM driver genes, we found a recurrent mutation in PLCB4 (c.G1888T, p.D630Y, NM_000933), which was validated using Sanger sequencing. The identical mutation was also found in published UM sequence data (1 of 56 tumors......-genome or whole-exome sequencing of 28 tumors or primary cell lines. These samples have a low mutation burden, with a mean of 10.6 protein changing mutations per sample (range 0 to 53). As expected for these sun-shielded melanomas the mutation spectrum was not consistent with an ultraviolet radiation signature...

  3. Unmasking of a hemizygous WFS1 gene mutation by a chromosome 4p deletion of 8.3 Mb in a patient with Wolf-Hirschhorn syndrome.

    Science.gov (United States)

    Flipsen-ten Berg, Klara; van Hasselt, Peter M; Eleveld, Marc J; van der Wijst, Suzanne E; Hol, Frans A; de Vroede, Monique A M; Beemer, Frits A; Hochstenbach, P F Ron; Poot, Martin

    2007-11-01

    The Wolf-Hirschhorn syndrome (WHS (MIM 194190)), which is characterized by growth delay, mental retardation, epilepsy, facial dysmorphisms, and midline fusion defects, shows extensive phenotypic variability. Several of the proposed mutational and epigenetic mechanisms in this and other chromosomal deletion syndromes fail to explain the observed phenotypic variability. To explain the complex phenotype of a patient with WHS and features reminiscent of Wolfram syndrome (WFS (MIM 222300)), we performed extensive clinical evaluation and classical and molecular cytogenetic (GTG banding, FISH and array-CGH) and WFS1 gene mutation analyses. We detected an 8.3 Mb terminal deletion and an adjacent 2.6 Mb inverted duplication in the short arm of chromosome 4, which encompasses a gene associated with WFS (WFS1). In addition, a nonsense mutation in exon 8 of the WFS1 gene was found on the structurally normal chromosome 4. The combination of the 4p deletion with the WFS1 point mutation explains the complex phenotype presented by our patient. This case further illustrates that unmasking of hemizygous recessive mutations by chromosomal deletions represents an additional explanation for the phenotypic variability observed in chromosomal deletion disorders.

  4. Determining mutations in G6PC and SLC37A4 genes in a sample of Brazilian patients with glycogen storage disease types Ia and Ib

    Directory of Open Access Journals (Sweden)

    Marcelo Paschoalete Carlin

    2013-01-01

    Full Text Available Glycogen storage disease (GSD comprises a group of autosomal recessive disorders characterized by deficiency of the enzymes that regulate the synthesis or degradation of glycogen. Types Ia and Ib are the most prevalent; while the former is caused by deficiency of glucose-6-phosphatase (G6Pase, the latter is associated with impaired glucose-6-phosphate transporter, where the catalytic unit of G6Pase is located. Over 85 mutations have been reported since the cloning of G6PC and SLC37A4 genes. In this study, twelve unrelated patients with clinical symptoms suggestive of GSDIa and Ib were investigated by using genetic sequencing of G6PC and SLC37A4 genes, being three confirmed as having GSD Ia, and two with GSD Ib. In seven of these patients no mutations were detected in any of the genes. Five changes were detected in G6PC, including three known point mutations (p.G68R, p.R83C and p.Q347X and two neutral mutations (c.432G > A and c.1176T > C. Four changes were found in SLC37A4: a known point mutation (p.G149E, a novel frameshift insertion (c.1338_1339insT, and two neutral mutations (c.1287G > A and c.1076-28C > T. The frequency of mutations in our population was similar to that observed in the literature, in which the mutation p.R83C is also the most frequent one. Analysis of both genes should be considered in the investigation of this condition. An alternative explanation to the negative results in this molecular study is the possibility of a misdiagnosis. Even with a careful evaluation based on laboratory and clinical findings, overlap with other types of GSD is possible, and further molecular studies should be indicated.

  5. Coffin-Siris syndrome: phenotypic evolution of a novel SMARCA4 mutation.

    Science.gov (United States)

    Tzeng, Michael; du Souich, Christèle; Cheung, Helen Wing-Hong; Boerkoel, Cornelius F

    2014-07-01

    Coffin-Siris Syndrome (CSS) is an intellectual disability disorder caused by mutation of components of the SWI/SNF chromatin-remodeling complex. We describe the evolution of the phenotypic features for a male patient with CSS from birth to age 7 years and 9 months and by review of reported CSS patients, we expand the phenotype to include neonatal and infantile hypertonia and upper airway obstruction. The propositus had a novel de novo heterozygous missense mutation in exon 17 of SMARCA4 (NM_001128849.1:c.2434C>T (NP_001122321.1:p.Leu812Phe)). This is the first reported mutation within motif Ia of the SMARCA4 SNF2 domain. In summary, SMARCA4-associated CSS is a pleiotropic disorder in which the pathognomic clinical features evolve and for which the few reported individuals do not demonstrate a clear genotype-phenotype correlation. © 2014 Wiley Periodicals, Inc.

  6. Sensitive and fast mutation detection by solid phase chemical cleavage

    DEFF Research Database (Denmark)

    Hansen, Lise Lotte; Justesen, Just; Kruse, Torben A

    1996-01-01

    We have developed a solid phase chemical cleavage method (SpCCM) for screening large DNA fragments for mutations. All reactions can be carried out in microtiterwells from the first amplification of the patient (or test) DNA through the search for mutations. The reaction time is significantly...... reduced compared to the conventional chemical cleavage method (CCM), and even by using a uniformly labelled probe, the exact position and nature of the mutation can be revealed. The SpCCM is suitable for automatization using a workstation to carry out the reactions and a fluorescent detection-based DNA...

  7. Ehlers-Danlos Syndrome type IV: a multi-exon deletion in one of the two COL3A1 alleles affecting structure, stability, and processing of type III procollagen

    International Nuclear Information System (INIS)

    Superti-Furga, A.; Gugler, E.; Gitzelmann, R.; Steinmann, B.

    1988-01-01

    The authors have studied a patient with severe, dominantly inherited Ehlers-Danlos syndrome type IV. The results indicate that this patient carries a deletion of 3.3 kilobase pairs in the triple helical coding domain of one of the two alleles for the pro-α-chains of type III collagen (COL3A1). His cultured skin fibroblasts contain equal amounts of normal length mRNA and of mRNA shortened by approximately 600 bases, and synthesize both normal and shortened pro-α1(III)-chains. In procollagen molecules containing one or more shortened chains, a triple helix is formed with a length of only about 780 amino acids. The mutant procollagen molecules have decreased thermal stability, are less efficiently secreted, and are not processed as their normal counterpart. The deletion in this family is the first mutation to be described in COL3A1

  8. Mutation analysis of the ERCC4/FANCQ gene in hereditary breast cancer.

    Directory of Open Access Journals (Sweden)

    Sandra Kohlhase

    Full Text Available The ERCC4 protein forms a structure-specific endonuclease involved in the DNA damage response. Different cancer syndromes such as a subtype of Xeroderma pigmentosum, XPF, and recently a subtype of Fanconi Anemia, FA-Q, have been attributed to biallelic ERCC4 gene mutations. To investigate whether monoallelic ERCC4 gene defects play some role in the inherited component of breast cancer susceptibility, we sequenced the whole ERCC4 coding region and flanking untranslated portions in a series of 101 Byelorussian and German breast cancer patients selected for familial disease (set 1, n = 63 or for the presence of the rs1800067 risk haplotype (set 2, n = 38. This study confirmed six known and one novel exonic variants, including four missense substitutions but no truncating mutation. Missense substitution p.R415Q (rs1800067, a previously postulated breast cancer susceptibility allele, was subsequently screened for in a total of 3,698 breast cancer cases and 2,868 controls from Germany, Belarus or Russia. The Gln415 allele appeared protective against breast cancer in the German series, with the strongest effect for ductal histology (OR 0.67; 95%CI 0.49; 0.92; p = 0.003, but this association was not confirmed in the other two series, with the combined analysis yielding an overall Mantel-Haenszel OR of 0.94 (95% CI 0.81; 1.08. There was no significant effect of p.R415Q on breast cancer survival in the German patient series. The other three detected ERCC4 missense mutations included two known rare variants as well as a novel substitution, p.E17V, that we identified on a p.R415Q haplotype background. The p.E17V mutation is predicted to be probably damaging but was present in just one heterozygous patient. We conclude that the contribution of ERCC4/FANCQ coding mutations to hereditary breast cancer in Central and Eastern Europe is likely to be small.

  9. Plasmid ColE1 as a Molecular Vehicle for Cloning and Amplification of DNA

    Science.gov (United States)

    Hershfield, Vickers; Boyer, Herbert W.; Yanofsky, Charles; Lovett, Michael A.; Helinski, Donald R.

    1974-01-01

    DNA fragments obtained from EcoRI endonuclease digestion of bacteriophage ϕ80pt190 (trp+) and the plasmid ColE1 were covalently joined with polynucleotide ligase. Transformation of Escherichia coli trp- strains to tryptophan independence with the recombined DNA selected for reconstituted ColE1 plasmids containing the tryptophan operon and the ϕ80 immunity region. Similarly, an EcoRI endonuclease generated fragment of plasmid pSC105 DNA containing the genetic determinant of kanamycin resistance was inserted into the ColE1 plasmid and recovered in E. coli. The plasmids containing the trp operon (ColE1-trp) and the kanamycin resistance gene were maintained under logarithmic growth conditions at a level of 25-30 copies per cell and accumulate to the extent of several hundred copies per cell in the presence of chloramphenicol. Cells carrying the ColE1-trp plasmid determined the production of highly elevated levels of trp operon-specific mRNA and tryptophan biosynthetic enzymes. Images PMID:4610576

  10. Biochip-Based Detection of KRAS Mutation in Non-Small Cell Lung Cancer

    Directory of Open Access Journals (Sweden)

    Barbara Ziegler

    2011-11-01

    Full Text Available This study is aimed at evaluating the potential of a biochip assay to sensitively detect KRAS mutation in DNA from non-small cell lung cancer (NSCLC tissue samples. The assay covers 10 mutations in codons 12 and 13 of the KRAS gene, and is based on mutant-enriched PCR followed by reverse-hybridization of biotinylated amplification products to an array of sequence-specific probes immobilized on the tip of a rectangular plastic stick (biochip. Biochip hybridization identified 17 (21% samples to carry a KRAS mutation of which 16 (33% were adenocarcinomas and 1 (3% was a squamous cell carcinoma. All mutations were confirmed by DNA sequencing. Using 10 ng of starting DNA, the biochip assay demonstrated a detection limit of 1% mutant sequence in a background of wild-type DNA. Our results suggest that the biochip assay is a sensitive alternative to protocols currently in use for KRAS mutation testing on limited quantity samples.

  11. Highly sensitive detection of ESR1 mutations in cell-free DNA from patients with metastatic breast cancer using molecular barcode sequencing.

    Science.gov (United States)

    Masunaga, Nanae; Kagara, Naofumi; Motooka, Daisuke; Nakamura, Shota; Miyake, Tomohiro; Tanei, Tomonori; Naoi, Yasuto; Shimoda, Masafumi; Shimazu, Kenzo; Kim, Seung Jin; Noguchi, Shinzaburo

    2018-01-01

    We aimed to develop a highly sensitive method to detect ESR1 mutations in cell-free DNA (cfDNA) using next-generation sequencing with molecular barcode (MB-NGS) targeting the hotspot segment (c.1600-1713). The sensitivity of MB-NGS was tested using serially diluted ESR1 mutant DNA and then cfDNA samples from 34 patients with metastatic breast cancer were analyzed with MB-NGS. The results of MB-NGS were validated in comparison with conventional NGS and droplet digital PCR (ddPCR). MB-NGS showed a higher sensitivity (0.1%) than NGS without barcode (1%) by reducing background errors. Of the cfDNA samples from 34 patients with metastatic breast cancer, NGS without barcode revealed seven mutations in six patients (17.6%) and MB-NGS revealed six additional mutations including three mutations not reported in the COSMIC database of breast cancer, resulting in total 13 ESR1 mutations in ten patients (29.4%). Regarding the three hotspot mutations, all the patients with mutations detected by MB-NGS had identical mutations detected by droplet digital PCR (ddPCR), and mutant allele frequency correlated very well between both (r = 0.850, p < 0.01). Moreover, all the patients without these mutations by MB-NGS were found to have no mutations by ddPCR. In conclusion, MB-NGS could successfully detect ESR1 mutations in cfDNA with a higher sensitivity of 0.1% than conventional NGS and was considered as clinically useful as ddPCR.

  12. Non-invasive prenatal diagnosis for cystic fibrosis: detection of paternal mutations, exploration of patient preferences and cost analysis.

    Science.gov (United States)

    Hill, Melissa; Twiss, Philip; Verhoef, Talitha I; Drury, Suzanne; McKay, Fiona; Mason, Sarah; Jenkins, Lucy; Morris, Stephen; Chitty, Lyn S

    2015-10-01

    We aim to develop non-invasive prenatal diagnosis (NIPD) for cystic fibrosis (CF) and determine costs and implications for implementation. A next-generation sequencing assay was developed to detect ten common CF mutations for exclusion of the paternal mutation in maternal plasma. Using uptake data from a study exploring views on NIPD for CF, total test-related costs were estimated for the current care pathway and compared with those incorporating NIPD. The assay reliably predicted mutation status in all control and maternal plasma samples. Of carrier or affected adults with CF (n = 142) surveyed, only 43.5% reported willingness to have invasive testing for CF with 94.4% saying they would have NIPD. Using these potential uptake data, the incremental costs of NIPD over invasive testing per 100 pregnancies at risk of CF are £9025 for paternal mutation exclusion, and £26,510 for direct diagnosis. We have developed NIPD for risk stratification in around a third of CF families. There are economic implications due to potential increased test demand to inform postnatal management rather than to inform decisions around termination of an affected pregnancy. © 2015 The Authors. Prenatal Diagnosis published by John Wiley & Sons, Ltd.

  13. Direct detection of common and rare inversion mutations in the genetic diagnosis of severe hemophilia A

    Energy Technology Data Exchange (ETDEWEB)

    Windsor, A.S.; Lillicrap, D.P.; Taylor, S.A.M. [Queen`s Univ., Ontario (Canada)

    1994-09-01

    Approximately 50% of the cases of severe hemophilia A (factor VIII:C < 0.01 units/ml) may be due to gross rearrangements of the factor VIII gene. The mutation involves homologous sequences upstream of the factor VIII locus and within intron 22 in an intrachromosomal recombination, inversion, event. The rearrangements can readily be detected on a Southern blot using a probe that is complementary to sequences from within intron 22. We describe here the analysis of this mutation in 71 severe hemophilia A patients. Thirty two of the patients (45%) showed evidence of a rearrangement. Five different patterns of rearrangements were seen, two of which have previously been described and account for the majority of cases (pattern 1, 70% and pattern 2, 16%). Three other abnormal patterns were observed. The inversion mechanism does not usually result in the loss or gain of any genetic material, but in one patient, in whom a unique rearrangement pattern was observed (pattern 3), we have previously documented a gross deletion which removes exons 1-22 of the factor VII gene as well as sequences 5{prime} to the gene. In another individual a fourth pattern in which an extra 19.0 kb band is present was detected. In this case it is unclear as to whether the rearrangement is responsible for the disease or is simply coincident normal variation. A fifth pattern, in which an extra 16.0 kb band was detected, was observed in a family with a new mutation causing hemophilia A. The affected individual and his mother inherited a de novo rearrangement of the factor VIII gene from his unaffected grandfather, implicating it as the cause of the disease. In conclusion, testing for the factor VIII inversion mutation was positive in approximately 45% of severe hemophiliacs, 72% of whom were isolated cases, and as such should constitute the initial stage in the genetic testing protocol for these patients` families.

  14. Novel phenotypes of pyridox(am)ine-5'-phosphate oxidase deficiency and high prevalence of c.445_448del mutation in Chinese patients.

    Science.gov (United States)

    Xue, Jiao; Chang, Xingzhi; Zhang, Yuehua; Yang, Zhixian

    2017-08-01

    To analyze the clinical and genetic characteristics of Chinese patients with pyridox(am)ine-5'-phosphate oxidase (PNPO) deficiency. The clinical presentations and the responses to treatments were analyzed in 4 patients. Blood and urinary metabolic screenings, electroencephalogram (EEG), brain magnetic resonance imaging (MRI) and epilepsy-related genes detection were performed in all patients. Patient 1 and 2 were identical twin brothers, who were born at 35 +5 w gestation with a sign of encephalopathy. Their seizures started within the first day and could not be controlled by pyridoxine or pyridoxal-5'-phosphate (PLP) completely. Patient 3 presented seizures at 5 months, responding well to pyridoxine. Seizures in patient 4 began at 40 days after birth and were controlled by valproic acid and topiramate. EEG showed atypical hypsarrhythmia or multifocal epileptiform discharges in 3 patients, and showed normality in patient 4. MRI showed nonspecific abnormality or normality. Blood metabolic screening showed multiple amino acids level abnormalities in all cases. Urinary metabolic screening showed vanillactic acid prominently elevated in 3 patients. Genetic analysis revealed 5 mutations of PNPO, three of which were novel. The mutation c.445_448del was carried by the twins and patient 3. Assessment of psychomotor development indicated severe delay in 3 patients and borderline to mild delay in patient 3. This is the first time to report patients with PNPO deficiency diagnosed by gene analysis in China. The novel clinical characteristics and novel mutations found here expanded the phenotypes and genotypes of this disease. Further, the frameshift mutation c.445_448del might be high prevalence in PNPO deficiency in Chinese patients.

  15. PCR-Based Analysis of ColE1 Plasmids in Clinical Isolates and Metagenomic Samples Reveals Their Importance as Gene Capture Platforms

    Directory of Open Access Journals (Sweden)

    Manuel Ares-Arroyo

    2018-03-01

    determinants, supporting the previous results achieved with the collections of bacterial isolates. In addition, we detected cryptic ColE1 plasmids in both families with no known genes in their variable region, which we have named sentinel plasmids. In conclusion, in this work we present a useful genetic tool for the detection and analysis of ColE1 plasmids, and confirm their important role in the dissemination of antibiotic resistance, especially in the Pasteurellaceae family of bacteria.

  16. Splice, insertion-deletion and nonsense mutations that perturb the phenylalanine hydroxylase transcript cause phenylketonuria in India.

    Science.gov (United States)

    Bashyam, Murali D; Chaudhary, Ajay K; Kiran, Manjari; Nagarajaram, Hampapathalu A; Devi, Radha Rama; Ranganath, Prajnya; Dalal, Ashwin; Bashyam, Leena; Gupta, Neerja; Kabra, Madhulika; Muranjan, Mamta; Puri, Ratna D; Verma, Ishwar C; Nampoothiri, Sheela; Kadandale, Jayarama S

    2014-03-01

    Phenylketonuria (PKU) is an autosomal recessive metabolic disorder caused by mutational inactivation of the phenylalanine hydroxylase (PAH) gene. Missense mutations are the most common PAH mutation type detected in PKU patients worldwide. We performed PAH mutation analysis in 27 suspected Indian PKU families (including 7 from our previous study) followed by structure and function analysis of specific missense and splice/insertion-deletion/nonsense mutations, respectively. Of the 27 families, disease-causing mutations were detected in 25. A total of 20 different mutations were identified of which 7 "unique" mutations accounted for 13 of 25 mutation positive families. The unique mutations detected exclusively in Indian PKU patients included three recurrent mutations detected in three families each. The 20 mutations included only 5 missense mutations in addition to 5 splice, 4 each nonsense and insertion-deletion mutations, a silent variant in coding region and a 3'UTR mutation. One deletion and two nonsense mutations were characterized to confirm significant reduction in mutant transcript levels possibly through activation of nonsense mediated decay. All missense mutations affected conserved amino acid residues and sequence and structure analysis suggested significant perturbations in the enzyme activity of respective mutant proteins. This is probably the first report of identification of a significantly low proportion of missense PAH mutations from PKU families and together with the presence of a high proportion of splice, insertion-deletion, and nonsense mutations, points to a unique PAH mutation profile in Indian PKU patients. © 2013 Wiley Periodicals, Inc.

  17. GluD1 is a common altered player in neuronal differentiation from both MECP2-mutated and CDKL5-mutated iPS cells.

    Science.gov (United States)

    Livide, Gabriella; Patriarchi, Tommaso; Amenduni, Mariangela; Amabile, Sonia; Yasui, Dag; Calcagno, Eleonora; Lo Rizzo, Caterina; De Falco, Giulia; Ulivieri, Cristina; Ariani, Francesca; Mari, Francesca; Mencarelli, Maria Antonietta; Hell, Johannes Wilhelm; Renieri, Alessandra; Meloni, Ilaria

    2015-02-01

    Rett syndrome is a monogenic disease due to de novo mutations in either MECP2 or CDKL5 genes. In spite of their involvement in the same disease, a functional interaction between the two genes has not been proven. MeCP2 is a transcriptional regulator; CDKL5 encodes for a kinase protein that might be involved in the regulation of gene expression. Therefore, we hypothesized that mutations affecting the two genes may lead to similar phenotypes by dysregulating the expression of common genes. To test this hypothesis we used induced pluripotent stem (iPS) cells derived from fibroblasts of one Rett patient with a MECP2 mutation (p.Arg306Cys) and two patients with mutations in CDKL5 (p.Gln347Ter and p.Thr288Ile). Expression profiling was performed in CDKL5-mutated cells and genes of interest were confirmed by real-time RT-PCR in both CDKL5- and MECP2-mutated cells. The only major change in gene expression common to MECP2- and CDKL5-mutated cells was for GRID1, encoding for glutamate D1 receptor (GluD1), a member of the δ-family of ionotropic glutamate receptors. GluD1 does not form AMPA or NMDA glutamate receptors. It acts like an adhesion molecule by linking the postsynaptic and presynaptic compartments, preferentially inducing the inhibitory presynaptic differentiation of cortical neurons. Our results demonstrate that GRID1 expression is downregulated in both MECP2- and CDKL5-mutated iPS cells and upregulated in neuronal precursors and mature neurons. These data provide novel insights into disease pathophysiology and identify possible new targets for therapeutic treatment of Rett syndrome.

  18. Gain-of-function somatic mutations contribute to inflammation and blood vessel damage that lead to Alzheimer dementia: a hypothesis.

    Science.gov (United States)

    Marchesi, Vincent T

    2016-02-01

    Amyloid deposits are a characteristic feature of advanced Alzheimer dementia (AD), but whether they initiate the disease or are a consequence of it remains an unsettled question. To explore an alternative pathogenic mechanism, I propose that the triggering events that begin the pathogenic cascade are not amyloid deposits but damaged blood vessels caused by inflammatory reactions that lead to ischemia, amyloid accumulation, axonal degeneration, synaptic loss, and eventually irreversible neuronal cell death. Inflammation and blood vessel damage are well recognized complications of AD, but what causes them and why the cerebral microvasculature is affected have never been adequately addressed. Because heritable autosomal dominant mutations of NLRP3, APP, TREX1, NOTCH3, and Col4A1 are known to provoke inflammatory reactions and damage the brain in a wide variety of diseases, I propose that one or more low abundant, gain-of-function somatic mutations of the same 5 gene families damage the microvasculature of the brain that leads to dementia. This implies that the pathogenic triggers that lead to AD are derived not from external invaders or amyloid but from oxidative damage of our own genes. © FASEB.

  19. Rapid detection of single nucleotide mutation in p53 gene based on ...

    Indian Academy of Sciences (India)

    mutation.27 Nevertheless, more than 50% of all human tumors contain p53 mutation; ... gene mutation detection in various fields of biology and medicine persuaded us to find ..... Yola M L, Eren T and Atar N 2014 Electrochim. Acta. 125 38. 26.

  20. Novel TGM5 mutations in acral peeling skin syndrome.

    Science.gov (United States)

    van der Velden, Jaap J A J; van Geel, Michel; Nellen, Ruud G L; Jonkman, Marcel F; McGrath, John A; Nanda, Arti; Sprecher, Eli; van Steensel, Maurice A M; McLean, W H Irwin; Cassidy, Andrew J

    2015-04-01

    Acral peeling skin syndrome (APSS, MIM #609796) is a rare autosomal recessive disorder characterized by superficial exfoliation and blistering of the volar and dorsal aspects of hands and feet. The level of separation is at the junction of the stratum granulosum and stratum corneum. APSS is caused by mutations in the TGM5 gene encoding transglutaminase-5, which is important for structural integrity of the outermost epidermal layers. The majority of patients originate from Europe and carry a p.(Gly113Cys) mutation in TGM5. In this study, we report both European and non-European families carrying other mutations in the TGM5 gene. In 5 patients, we found 3 novel mutations: c.1001+2_1001+3del, c.1171G>A and c.1498C>T. To confirm their pathogenicity, we performed functional analyses with a transglutaminase activity assay, determined alternative splicing by reverse-transcribed PCR analysis and used databases and in silico prediction tools. © 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  1. Pitt-Hopkins syndrome: report of a case with a TCF4 gene mutation

    Directory of Open Access Journals (Sweden)

    Orsini Alessandro

    2010-02-01

    Full Text Available Abstract Aims We will discuss the clinical and genetic diagnosis of a child with severe psychomotor delay, who at 3 years of age presented with paroxysms of hyperpnea-apnea and seizures unrelated to breathing anomalies. Methods The child underwent genetic (karyotype, FISH telomeres and neuroradiological (cranial CT and MRI tests, which proved to be normal. He came under our clinical observation at 3 years and 5 months of age. Due to severe psychomotor delay and facial dysmorphisms we completed the genetic investigations based on his clinical feature and analysis of the available literature. Results The presence of severe mental retardation associated with anomalous breathing pattern may suggest the Joubert and Rett syndrome, however these were excluded on the basis of clinical and genetic examination. Angelman syndrome, suspected for facial dysmorphisms and absent language, was also excluded because of the presence of a normal pattern of methylation at SNRPN locus. Another possible diagnosis was the Pitt-Hopkins Syndrome (PHS, characterized by severe mental retardation, breathing anomalies (paroxisms of hyperpnea-apnea, dysmorphisms and sometimes epilepsy. Haploinsufficiency of TCF4 gene located at 18q21.2 region has been recently identified as causative of this syndrome. In our patient the research of TCF4 mutation by the Institute of Human Genetics, University Hospital Erlangen (Germany, showed a de novo mutation. Conclusions The diagnosis of Pitt-Hopkins syndrome, an underdiagnosed cause of mental retardation, was based on clinical and genetic findings. Searching for TCF4 mutations is highly recommended when others overlapping syndromes was excluded. At our knowledge our patient is the first italian case of PHS diagnosed at molecular level.

  2. Evaluation of Lama5 as a candidate for the mouse ragged (Ra) mutation

    DEFF Research Database (Denmark)

    Durkin, M E; Albrechtsen, R; Chambers, D M

    1998-01-01

    The laminin alpha5 chain is a component of the basement membranes of many developing and adult tissues. The mouse laminin alpha5 chain gene (Lama5) has been mapped close to the locus of the semidominant ragged (Ra) mutation on distal chromosome 2. The cause of the Ra mutation, which is usually...... lethal in the homozygous state, has not been determined. We have investigated whether a defect in Lama5 is responsible for the ragged mutation, using the RaJ strain. No differences in the level of the laminin alpha5 chain transcript were found in placental RNA from homozygous RaJ mutant embryos compared...... to normal littermates. Antiserum raised against a recombinant laminin alpha5 chain polypeptide stained the basement membranes of both normal and homozygous mutant embryos to a similar extent. More precise mapping of Lama5 on an interspecific Ra backcross indicated that Lama5 is proximal to the Ra locus...

  3. Detection of CALR and MPL Mutations in Low Allelic Burden JAK2 V617F Essential Thrombocythemia.

    Science.gov (United States)

    Usseglio, Fabrice; Beaufils, Nathalie; Calleja, Anne; Raynaud, Sophie; Gabert, Jean

    2017-01-01

    Myeloproliferative neoplasms are clonal hematopoietic stem cell disorders characterized by aberrant proliferation and an increased tendency toward leukemic transformation. The genes JAK2, MPL, and CALR are frequently altered in these syndromes, and their mutations are often a strong argument for diagnosis. We analyzed the mutational profiles of these three genes in a cohort of 164 suspected myeloproliferative neoplasms. JAK2 V617F mutation was detected by real-time PCR, whereas high-resolution melting analysis followed by Sanger sequencing were used for searching for mutations in JAK2 exon 12, CALR, and MPL. JAK2 V617F mutation was associated with CALR (n = 4) and MPL (n = 4) mutations in 8 of 103 essential thrombocytosis patients. These cases were harboring a JAK2 V617F allelic burden of MPL genes in myeloproliferative neoplasms and suggest that CALR and MPL should be analyzed not only in JAK2-negative patients but also in low V617F mutation patients. Follow-up of these double-mutation cases will be important for determining whether this group of patients presents particular evolution or complications. Copyright © 2017 American Society for Investigative Pathology and the Association for Molecular Pathology. Published by Elsevier Inc. All rights reserved.

  4. BAP1 missense mutation c.2054 A>T (p.E685V completely disrupts normal splicing through creation of a novel 5' splice site in a human mesothelioma cell line.

    Directory of Open Access Journals (Sweden)

    Arianne Morrison

    Full Text Available BAP1 is a tumor suppressor gene that is lost or deleted in diverse cancers, including uveal mela¬noma, malignant pleural mesothelioma (MPM, clear cell renal carcinoma, and cholangiocarcinoma. Recently, BAP1 germline mutations have been reported in families with combinations of these same cancers. A particular challenge for mutation screening is the classification of non-truncating BAP1 sequence variants because it is not known whether these subtle changes can affect the protein function sufficiently to predispose to cancer development. Here we report mRNA splicing analysis on a homozygous substitution mutation, BAP1 c. 2054 A&T (p.Glu685Val, identified in an MPM cell line derived from a mesothelioma patient. The mutation occurred at the 3rd nucleotide from the 3' end of exon 16. RT-PCR, cloning and subsequent sequencing revealed several aberrant splicing products not observed in the controls: 1 a 4 bp deletion at the end of exon 16 in all clones derived from the major splicing product. The BAP1 c. 2054 A&T mutation introduced a new 5' splice site (GU, which resulted in the deletion of 4 base pairs and presumably protein truncation; 2 a variety of alternative splicing products that led to retention of different introns: introns 14-16; introns 15-16; intron 14 and intron 16; 3 partial intron 14 and 15 retentions caused by activation of alternative 3' splice acceptor sites (AG in the introns. Taken together, we were unable to detect any correctly spliced mRNA transcripts in this cell line. These results suggest that aberrant splicing caused by this mutation is quite efficient as it completely abolishes normal splicing through creation of a novel 5' splice site and activation of cryptic splice sites. These data support the conclusion that BAP1 c.2054 A&T (p.E685V variant is a pathogenic mutation and contributes to MPM through disruption of normal splicing.

  5. Carrier status for the common R501X and 2282del4 filaggrin mutations is not associated with hearing phenotypes in 5,377 children from the ALSPAC cohort.

    Directory of Open Access Journals (Sweden)

    Santiago Rodriguez

    2009-06-01

    Full Text Available Filaggrin is a major protein in the epidermis. Several mutations in the filaggrin gene (FLG have been associated with a number of conditions. Filaggrin is expressed in the tympanic membrane and could alter its mechanical properties, but the relationship between genetic variation in FLG and hearing has not yet been tested.We examined whether loss-of function mutations R501X and 2282del4 in the FLG gene affected hearing in children. Twenty eight hearing variables representing five different aspects of hearing at age nine years in 5,377 children from the Avon Longitudinal Study of Parents and Children (ALSPAC cohort were tested for association with these mutations. No evidence of association was found between R501X or 2282del4 (or overall FLG mutation carrier status and any of the hearing phenotypes analysed.In conclusion, carrier status for common filaggrin mutations does not affect hearing in children.

  6. A Comparison Between Denaturing Gradient Gel Electrophoresis and Denaturing High Performance Liquid Chromatography in Detecting Mutations in Genes Associated with Hereditary Non-Polyposis Colorectal Cancer (HNPCC and the Identification of 9 New Mutations Previously Unidentified by DGGE

    Directory of Open Access Journals (Sweden)

    Meldrum Cliff J

    2003-12-01

    Full Text Available Abstract Denaturing high performance liquid chromatography is a relatively new method by which heteroduplex structures formed during the PCR amplification of heterozygote samples can be rapidly identified. The use of this technology for mutation detection in hereditary non-polyposis colorectal cancer (HNPCC has the potential to appreciably shorten the time it takes to analyze genes associated with this disorder. Prior to acceptance of this method for screening genes associated with HNPCC, assessment of the reliability of this method should be performed. In this report we have compared mutation and polymorphism detection by denaturing gradient gel electrophoresis (DGGE with denaturing high performance liquid chromatography (DHPLC in a set of 130 families. All mutations/polymorphisms representing base substitutions, deletions, insertions and a 23 base pair inversion were detected by DHPLC whereas DGGE failed to identify four single base substitutions and a single base pair deletion. In addition, we show that DHPLC has been used for the identification of 5 different mutations in exon 7 of hMSH2 that could not be detected by DGGE. From this study we conclude that DHPLC is a more effective and rapid alternative to the detection of mutations in hMSH2 and hMLH1 with the same or better accuracy than DGGE. Furthermore, this technique offers opportunities for automation, which have not been realised for the majority of other methods of gene analysis.

  7. Novel mutations in cyclin-dependent kinase-like 5 (CDKL5) gene in Indian cases of Rett syndrome.

    Science.gov (United States)

    Das, Dhanjit Kumar; Mehta, Bhakti; Menon, Shyla R; Raha, Sarbani; Udani, Vrajesh

    2013-03-01

    Rett syndrome is a severe neurodevelopmental disorder, almost exclusively affecting females and characterized by a wide spectrum of clinical manifestations. Both the classic and atypical forms of Rett syndrome are primarily due to mutations in the methyl-CpG-binding protein 2 (MECP2) gene. Mutations in the X-linked cyclin-dependent kinase-like 5 (CDKL5) gene have been identified in patients with atypical Rett syndrome, X-linked infantile spasms sharing common features of generally early-onset seizures and mental retardation. CDKL5 is known as serine/threonine protein kinase 9 (STK9) and is mapped to the Xp22 region. It has a conserved serine/threonine kinase domain within its amino terminus and a large C-terminal region. Disease-causing mutations are distributed in both the amino terminal domain and in the large C-terminal domain. We have screened the CDKL5 gene in 44 patients with atypical Rett syndrome who had tested negative for MECP2 gene mutations and have identified 6 sequence variants, out of which three were novel and three known mutations. Two of these novel mutations p.V966I and p.A1011V were missense and p.H589H a silent mutation. Other known mutations identified were p.V999M, p.Q791P and p.T734A. Sequence homology for all the mutations revealed that the two mutations (p.Q791P and p.T734A) were conserved across species. This indicated the importance of these residues in structure and function of the protein. The damaging effects of these mutations were analysed in silico using PolyPhen-2 online software. The PolyPhen-2 scores of p.Q791P and p.T734A were 0.998 and 0.48, revealing that these mutations could be deleterious and might have potential functional effect. All other mutations had a low score suggesting that they might not alter the activity of CDKL5. We have also analysed the position of the mutations in the CDKL5 protein and found that all the mutations were present in the C-terminal domain of the protein. The C-terminal domain is required for

  8. Mutations in noncoding regions of GJB1 are a major cause of X-linked CMT

    Science.gov (United States)

    Tomaselli, Pedro J.; Rossor, Alexander M.; Horga, Alejandro; Jaunmuktane, Zane; Carr, Aisling; Saveri, Paola; Piscosquito, Giuseppe; Pareyson, Davide; Laura, Matilde; Blake, Julian C.; Poh, Roy; Polke, James; Houlden, Henry

    2017-01-01

    Objective: To determine the prevalence and clinical and genetic characteristics of patients with X-linked Charcot-Marie-Tooth disease (CMT) due to mutations in noncoding regions of the gap junction β-1 gene (GJB1). Methods: Mutations were identified by bidirectional Sanger sequence analysis of the 595 bases of the upstream promoter region, and 25 bases of the 3′ untranslated region (UTR) sequence in patients in whom mutations in the coding region had been excluded. Clinical and neurophysiologic data were retrospectively collected. Results: Five mutations were detected in 25 individuals from 10 kindreds representing 11.4% of all cases of CMTX1 diagnosed in our neurogenetics laboratory between 1996 and 2016. Four pathogenic mutations, c.-17G>A, c.-17+1G>T, c.-103C>T, and c.-146-90_146-89insT were detected in the 5′UTR. A novel mutation, c.*15C>T, was detected in the 3′ UTR of GJB1 in 2 unrelated families with CMTX1 and is the first pathogenic mutation in the 3′UTR of any myelin-associated CMT gene. Mutations segregated with the phenotype, were at sites predicted to be pathogenic, and were not present in the normal population. Conclusions: Mutations in noncoding DNA are a major cause of CMTX1 and highlight the importance of mutations in noncoding DNA in human disease. Next-generation sequencing platforms for use in inherited neuropathy should therefore include coverage of these regions. PMID:28283593

  9. Quantitative and molecular analyses of mutation in a pSV2gpt transformed CHO cell line

    International Nuclear Information System (INIS)

    Stankowski, L.F. Jr.; Tindall, K.R.; Hsie, A.W.

    1983-01-01

    Following NDA-mediated gene transfer we have isolated a cell line useful for studying gene mutation at the molecular level. This line, AS52, derived from a hypoxanthine-guanine phosphoribosyl transferase (HGPRT) deficient Chinese hamster ovary (CHO) cell line, carries a single copy of the E. coli xanthine-guanine phosphoribosyl transferase (XGPRT) gene (gpt) and exhibits a spontaneous mutant frequency of 20 TG/sup r/ mutants/10 6 clonable cells. As with HGPRT - mutants, XGPRT - mutants can be selected in 6-thioguanine. AS52 (XGPRT + ) and wild type CHO (HGPRT + ) cell exhibit almost identical cytotoxic responses to various agents. We observed significant differences in mutation induction by UV light and ethyl methanesulfonate (EMS). Ratios of XGPRT - to HGPRT - mutants induced per unit dose (J/m 2 for UV light and μg/ml for EMS) are 1.4 and 0.70, respectively. Preliminary Southern blot hybridization analyses has been performed on 30 XGPRT - AS52 mutants. A majority of spontaneous mutants have deletions ranging in size from 1 to 4 kilobases (9/19) to complete loss of gpt sequences (4/19); the remainder have no detectable (5/19) or only minor (1/19) alterations. 5/5 UV-induced and 5/6 EMS-induced mutants do not show a detectable change. Similar analyses are underway for mutations induced by x-irradiation and ICR 191 treatment

  10. Genotype-phenotype correlation of Coffin-Siris syndrome caused by mutations in SMARCB1, SMARCA4, SMARCE1, and ARID1A.

    Science.gov (United States)

    Kosho, Tomoki; Okamoto, Nobuhiko

    2014-09-01

    Coffin-Siris syndrome (CSS) is a rare congenital malformation syndrome, recently found to be caused by mutations in several genes encoding components of the BAF complex. To date, 109 patients have been reported with their mutations: SMARCB1 (12%), SMARCA4 (11%), SMARCE1 (2%), ARID1A (7%), ARID1B (65%), and PHF6 (2%). We review genotype-phenotype correlation of all previously reported patients with mutations in SMARCB1, SMARCA4, SMARCE1, and ARID1A through reassessment of their clinical and molecular findings. Cardinal features of CSS included variable degrees of intellectual disability (ID) predominantly affecting speech, sucking/feeding difficulty, and craniofacial (thick eyebrows, long eyelashes), digital (hypoplastic 5th fingers or toes, hypoplastic 5th fingernails or toenails), and other characteristics (hypertrichosis). In addition, patients with SMARCB1 mutations had severe neurodevelopmental deficits including severe ID, seizures, CNS structural abnormalities, and no expressive words as well as scoliosis. Especially, those with a recurrent mutation "p.Lys364del" represented strikingly similar phenotypes including characteristic facial coarseness. Patients with SMARCA4 mutations had less coarse craniofacial appearances and behavioral abnormalities. Patients with SMARCE1 mutations had a wide spectrum of manifestations from severe to moderate ID. Patients with ARID1A also had a wide spectrum of manifestations from severe ID and serous internal complications that could result in early death to mild ID. Mutations in SMARCB1, SMARCA4, and SMARCE1 are expected to exert dominant-negative or gain-of-function effects, whereas those in ARID1A are expected to exert loss-of-function effects. © 2014 Wiley Periodicals, Inc.

  11. Molecular Evolution and Phylogenetic Analysis of Eight COL Superfamily Genes in Group I Related to Photoperiodic Regulation of Flowering Time in Wild and Domesticated Cotton (Gossypium) Species

    Science.gov (United States)

    Zhang, Rui; Ding, Jian; Liu, Chunxiao; Cai, Caiping; Zhou, Baoliang; Zhang, Tianzhen; Guo, Wangzhen

    2015-01-01

    Flowering time is an important ecological trait that determines the transition from vegetative to reproductive growth. Flowering time in cotton is controlled by short-day photoperiods, with strict photoperiod sensitivity. As the CO-FT (CONSTANS-FLOWER LOCUS T) module regulates photoperiodic flowering in several plants, we selected eight CONSTANS genes (COL) in group I to detect their expression patterns in long-day and short-day conditions. Further, we individually cloned and sequenced their homologs from 25 different cotton accessions and one outgroup. Finally, we studied their structures, phylogenetic relationship, and molecular evolution in both coding region and three characteristic domains. All the eight COLs in group I show diurnal expression. In the orthologous and homeologous loci, each gene structure in different cotton species is highly conserved, while length variation has occurred due to insertions/deletions in intron and/or exon regions. Six genes, COL2 to COL5, COL7 and COL8, exhibit higher nucleotide diversity in the D-subgenome than in the A-subgenome. The Ks values of 98.37% in all allotetraploid cotton species examined were higher in the A-D and At-Dt comparison than in the A-At and D-Dt comparisons, and the Pearson’s correlation coefficient (r) of Ks between A vs. D and At vs. Dt also showed positive, high correlations, with a correlation coefficient of at least 0.797. The nucleotide polymorphism in wild species is significantly higher compared to G. hirsutum and G. barbadense, indicating a genetic bottleneck associated with the domesticated cotton species. Three characteristic domains in eight COLs exhibit different evolutionary rates, with the CCT domain highly conserved, while the B-box and Var domain much more variable in allotetraploid species. Taken together, COL1, COL2 and COL8 endured greater selective pressures during the domestication process. The study improves our understanding of the domestication-related genes/traits during cotton

  12. SMARCA4 inactivating mutations cause concomitant Coffin–Siris syndrome, microphthalmia and small‐cell carcinoma of the ovary hypercalcaemic type

    Science.gov (United States)

    Mustafa, Noor; Vetro, Annalisa; Notarangelo, Lucia Dora; de Jonge, Hugo; Rinaldi, Berardo; Vergani, Debora; Giglio, Sabrina Rita; Morbini, Patrizia; Zuffardi, Orsetta

    2017-01-01

    Abstract SMARCA4 chromatin remodelling factor is mutated in 11% of Coffin–Siris syndrome (CSS) patients and in almost all small‐cell carcinoma of the ovary hypercalcaemic type (SCCOHT) tumours. Missense mutations with gain‐of‐function or dominant‐negative effects are associated with CSS, whereas inactivating mutations, leading to loss of SMARCA4 expression, have been exclusively found in SCCOHT. We applied whole‐exome sequencing to study a 15‐year‐old patient with mild CSS who concomitantly developed SCCOHT at age 13 years. Interestingly, our patient also showed congenital microphthalmia, which has never previously been reported in CSS patients. We detected a de novo germline heterozygous nonsense mutation in exon 19 of SMARCA4 (c.2935C > T;p.Arg979*), and a somatic frameshift mutation in exon 6 (c.1236_1236delC;p.Gln413Argfs*88), causing complete loss of SMARCA4 immunostaining in the tumour. The immunohistochemical findings are supported by the observation that the c.2935C > T mutant transcript was detected by reverse transcription polymerase chain reaction at a much lower level than the wild‐type allele in whole blood and the lymphoblastoid cell line of the proband, confirming nonsense‐mediated mRNA decay. Accordingly, immunoblotting demonstrated that there was approximately half the amount of SMARCA4 protein in the proband's cells as in controls. This study suggests that SMARCA4 constitutional mutations associated with CSS are not necessarily non‐truncating, and that haploinsufficiency may explain milder CSS phenotypes, as previously reported for haploinsufficient ARID1B. In addition, our case supports the dual role of chromatin remodellers in developmental disorders and cancer, as well as the involvement of SMARCA4 in microphthalmia, confirming previous findings in mouse models and the DECIPHER database. Finally, we speculate that mild CSS might be under‐recognized in a proportion of SCCOHT patients harbouring SMARCA4 mutations

  13. Molecular Analysis of Collagen XVIII Reveals Novel Mutations, Presence of a Third Isoform, and Possible Genetic Heterogeneity in Knobloch Syndrome

    Science.gov (United States)

    Suzuki, O. T.; Sertié, A. L.; Der Kaloustian, V. M.; Kok, F.; Carpenter, M.; Murray, J.; Czeizel, A. E.; Kliemann, S. E.; Rosemberg, S.; Monteiro, M.; Olsen, B. R.; Passos-Bueno, M. R.

    2002-01-01

    Knobloch syndrome (KS) is a rare disease characterized by severe ocular alterations, including vitreoretinal degeneration associated with retinal detachment and occipital scalp defect. The responsible gene, COL18A1, has been mapped to 21q22.3, and, on the basis of the analysis of one family, we have demonstrated that a mutation affecting only one of the three COL18A1 isoforms causes this phenotype. We report here the results of the screening of both the entire coding region and the exon-intron boundaries of the COL18A1 gene (which includes 43 exons), in eight unrelated patients with KS. Besides 20 polymorphic changes, we identified 6 different pathogenic changes in both alleles of five unrelated patients with KS (three compound heterozygotes and two homozygotes). All are truncating mutations leading to deficiency of one or all collagen XVIII isoforms and endostatin. We have verified that, in exon 41, the deletion c3514-3515delCT, found in three unrelated alleles, is embedded in different haplotypes, suggesting that this mutation has occurred more than once. In addition, our results provide evidence of nonallelic genetic heterogeneity in KS. We also show that the longest human isoform (NC11-728) is expressed in several tissues (including the human eye) and that lack of either the short variant or all of the collagen XVIII isoforms causes similar phenotypes but that those patients who lack all forms present more-severe ocular alterations. Despite the small sample size, we found low endostatin plasma levels in those patients with mutations leading to deficiency of all isoforms; in addition, it seems that absence of all collagen XVIII isoforms causes predisposition to epilepsy. PMID:12415512

  14. Limited copy number-high resolution melting (LCN-HRM) enables the detection and identification by sequencing of low level mutations in cancer biopsies.

    Science.gov (United States)

    Do, Hongdo; Dobrovic, Alexander

    2009-10-08

    Mutation detection in clinical tumour samples is challenging when the proportion of tumour cells, and thus mutant alleles, is low. The limited sensitivity of conventional sequencing necessitates the adoption of more sensitive approaches. High resolution melting (HRM) is more sensitive than sequencing but identification of the mutation is desirable, particularly when it is important to discriminate false positives due to PCR errors or template degradation from true mutations.We thus developed limited copy number - high resolution melting (LCN-HRM) which applies limiting dilution to HRM. Multiple replicate reactions with a limited number of target sequences per reaction allow low level mutations to be detected. The dilutions used (based on Ct values) are chosen such that mutations, if present, can be detected by the direct sequencing of amplicons with aberrant melting patterns. Using cell lines heterozygous for mutations, we found that the mutations were not readily detected when they comprised 10% of total alleles (20% tumour cells) by sequencing, whereas they were readily detectable at 5% total alleles by standard HRM. LCN-HRM allowed these mutations to be identified by direct sequencing of those positive reactions.LCN-HRM was then used to review formalin-fixed paraffin-embedded (FFPE) clinical samples showing discordant findings between sequencing and HRM for KRAS exon 2 and EGFR exons 19 and 21. Both true mutations present at low levels and sequence changes due to artefacts were detected by LCN-HRM. The use of high fidelity polymerases showed that the majority of the artefacts were derived from the damaged template rather than replication errors during amplification. LCN-HRM bridges the sensitivity gap between HRM and sequencing and is effective in distinguishing between artefacts and true mutations.

  15. Environmentally sensitive molecular probes reveal mutations and epigenetic 5-methyl cytosine in human oncogenes

    DEFF Research Database (Denmark)

    Taskova, M.; Barducci, M. C.; Astakhova, K.

    2017-01-01

    There is currently an unmet need for reliable tools that allow for direct detection and quantification of modifications in genomic DNA. For example, in cancer research and clinical diagnostics, target DNA has to be amplified and sequenced in order to reveal mutations. For 5-methylcytosine detecti...

  16. Collagen Type I alpha1 (COL1A1 Gene Polymorphism and Bone Mineral Density in Postmenopausal Kazakh Women

    Directory of Open Access Journals (Sweden)

    Akbota Aitkulova

    2014-12-01

    Full Text Available Introduction: Single nucleotide polymorphism (SNP at the collagen type I alpha 1 gene (COL1A1 rs1800012 has been widely studied and has shown an association with bone mineral density (BMD and fractures. A minor allele TT of this SNP was found to be greatly overrepresented in individuals with fractures compared to controls, thus becoming a good predictor of  increased fracture risk. The aim of this investigation was to evaluate potential association between COL1A1 gene polymorphism and osteoporosis in Kazakh postmenopausal women.Methods: The study population included 103 postmenopausal women recruited from Pavlodar and Almaty clinics. BMD was measured using DEXA. Genomic DNA was extracted from peripheral venous blood of study participants with Wizard® Genomic DNA Purification Kit (Promega, USA. Detection of COL1A1 +1245G/T (Sp1 polymorphism was done by the TaqMan® SNP Genotyping Assay of real-time PCR.Results: Densitometry results revealed 36 osteoporotic, 42 osteopenic, and 25 normal postmenopausal women. Data analysis of 1245G>T polymorphism in COL1A1 gene in the group of women with osteopenia and osteoporosis revealed deviation from Hardy-Weinberg equilibrium. The mutant TT genotype was prevalent compared to the heterozygous genotype GT in both groups. Distributions were 83% GG, 3% GT, and 14% TT in the group with osteopenia and 80% GG, 6% GT, and 14% TT in the group with osteoporosis. The distribution of genotypes frequency in the group of normal postmenopausal women was 76% GG, 16% GT, and 8% TT.Conclusion: These results suggest that TT genotype of COL1A1 +1245G/T (Sp1 polymorphism is associated with risk of postmenopausal osteoporosis in Kazakh women. Further studies involving a larger number of women are needed to clarify the relationship of this polymorphism with risk of osteoporosis. 

  17. Molecular characterization of a novel HEXA mutation at the +3 position of intron 8 in a Tay-Sachs disease patient

    Energy Technology Data Exchange (ETDEWEB)

    Richard, M.; Triggs-Raine, B. [Univ. of Manitoba, Winnipeg (Canada); Natowicz, M. [E.K. Shriver Center, Waltham, MA (United States)

    1994-09-01

    Tay-Sachs disease is an autosomal recessive lysosomal storage disorder resulting from mutations in the HEXA gene that cause a deficiency in the activity of that enzyme {beta}-hexosaminidase A (Hex A). This deficiency leads to the build-up of G{sub M2} ganglioside, resulting in neurodegeneration and death. Biochemical analysis of a non-Jewish patient with a late-infantile form of Tay-Sachs disease revealed a substantial level of Hex A activity (38.4%) when 4-MUG was used as the substrate. However, when a substrate (4-MUGS) specific for the {alpha}-subunit of Hex A ({alpha}{beta}) was used, almost no activity was detected in the HEXA gene of the patient using SSCP analysis followed by sequencing. The first mutation, a G533A substitution in exon 5, is previously described and associated with the B1 form of Tay-Sachs disease. The second mutation is a novel a-to-g base change at the +3 position of intron 8. This was confirmed using the AIRS method, whereby a MaeIII site was created in the presence of the mutation. Normal and patient mRNA was reverse transcribed and exons 7 to 9 were PCR-amplified from the cDNA. An abnormally sized amplification product detected only in the patient cDNA was sequenced; exon 8 had been deleted and exons 7 and 9 were spliced together. A substantial level of normally-sized PCR product was also detected in the patient`s cDNA. Experiments are in progress to determine if this is produced from the allele harboring the G533A mutation. Given that previous mutations of this type have been associated with 97-100% abnormal splicing, this mutation is likely to be the cause, together with the G533A mutation, of Tay-Sachs disease in this patient.

  18. Frameshift mutation in the APOA5 gene causing hypertriglyceridemia in a Pakistani family: Management and considerations for cardiovascular risk.

    Science.gov (United States)

    Thériault, Sébastien; Don-Wauchope, Andrew; Chong, Michael; Lali, Ricky; Morrison, Katherine M; Paré, Guillaume

    2016-01-01

    We report a novel homozygous apolipoprotein A5 (APOA5) frameshift mutation (c.G425del-C, p.Arg143AlafsTer57) identified in a 12-year-old boy of Pakistani origin with severe hypertriglyceridemia (up to 35 mmol/L) and type V hyperlipoproteinemia. The patient did not respond to fibrate therapy, but his condition improved under a very low fat diet, although compliance was suboptimal. Heterozygous status was detected in both parents (consanguineous union) and one sibling, all showing moderate hypertriglyceridemia (between 5 and 10 mmol/L). There was a significant family history of premature cardiovascular disease. The index case was also diagnosed with a coronary artery anomaly. Considering the recently reported association of rare mutations in APOA5 with the risk of early myocardial infarction, we discuss the implications of these findings for the young man and his family. Copyright © 2016 National Lipid Association. Published by Elsevier Inc. All rights reserved.

  19. Comparison of Detection Rate and Mutational Pattern of Drug-Resistant Mutations Between a Large Cohort of Genotype B and Genotype C Hepatitis B Virus-Infected Patients in North China.

    Science.gov (United States)

    Li, Xiaodong; Liu, Yan; Xin, Shaojie; Ji, Dong; You, Shaoli; Hu, Jinhua; Zhao, Jun; Wu, Jingjing; Liao, Hao; Zhang, Xin-Xin; Xu, Dongping

    2017-06-01

    The study aimed to investigate the association of prevalent genotypes in China (HBV/C and HBV/B) with HBV drug-resistant mutations. A total of 13,847 nucleos(t)ide analogue (NA)-treated patients with chronic HBV infection from North China were enrolled. HBV genotypes and resistant mutations were determined by direct sequencing and confirmed by clonal sequencing if necessary. HBV/B, HBV/C, and HBV/D occupied 14.3%, 84.9%, and 0.8% across the study population, respectively. NA usage had no significant difference between HBV/B- and HBV/C-infected patients. Lamivudine-resistant mutations were more frequently detected in HBV/C-infected patients, compared with HBV/B-infected patients (31.67% vs. 25.26%, p M250 V/I/L substitution (0.67% vs. 1.46%, p < 0.01). Multidrug-resistant mutations (defined as coexistence of mutation to nucleoside and nucleotide analogues) were detected in 104 patients. HBV/C-infected patients had a higher detection rate of multidrug-resistant mutation than HBV/B-infected patients (0.83% vs. 0.35%, p < 0.05). The study for the first time clarified that HBV/C-infected patients had a higher risk to develop multidrug-resistant mutations, compared with HBV/B-infected patients; and HBV/C- and HBV/B-infected patients had different inclinations in the ETV-resistant mutational pattern.

  20. p16 mutation spectrum in the premalignant condition Barrett's esophagus.

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    Thomas G Paulson

    Full Text Available BACKGROUND: Mutation, promoter hypermethylation and loss of heterozygosity involving the tumor suppressor gene p16 (CDKN2a/INK4a have been detected in a wide variety of human cancers, but much less is known concerning the frequency and spectrum of p16 mutations in premalignant conditions. METHODS AND FINDINGS: We have determined the p16 mutation spectrum for a cohort of 304 patients with Barrett's esophagus, a premalignant condition that predisposes to the development of esophageal adenocarcinoma. Forty seven mutations were detected by sequencing of p16 exon 2 in 44 BE patients (14.5% with a mutation spectrum consistent with that caused by oxidative damage and chronic inflammation. The percentage of patients with p16 mutations increased with increasing histologic grade. In addition, samples from 3 out of 19 patients (15.8% who underwent esophagectomy were found to have mutations. CONCLUSIONS: The results of this study suggest the environment of the esophagus in BE patients can both generate and select for clones with p16 mutations.

  1. Characterization of beta-thalassemia mutations in patients from the state of Rio Grande do Norte, Brazil

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    Zama Messala Luna da Silveira

    2011-01-01

    Full Text Available 35 unrelated individuals were studied for characterization as either heterozygous or homozygous for beta-thalassemia. Molecular analysis was done by PCR/RFLP to detect the mutations most commonly associated with beta-thalassemia (β0IVS-I-1, β+IVS-I-6, and β039. In the patients who showed none of these mutations, the beta-globin genes were sequenced. Of the 31 heterozygous patients, 13 (41.9% had the β+IVS-I-6 mutation, 15 (48.4% the β0IVS-I-1 mutation, 2 (6.5% the β+IVS-I-110 mutation and 1 (3.2% the β+IVS-I-5 mutation. IVS-I-6 was detected in the four homozygotes. The mutation in codon 39, often found in previous studies in Brazil, was not detected in the present case. This is the first study aiming at identifying mutations that determine beta-thalassemia in the state of Rio Grande do Norte.

  2. Rapid and Simple Detection of Hot Spot Point Mutations of Epidermal Growth Factor Receptor, BRAF, and NRAS in Cancers Using the Loop-Hybrid Mobility Shift Assay

    Science.gov (United States)

    Matsukuma, Shoichi; Yoshihara, Mitsuyo; Kasai, Fumio; Kato, Akinori; Yoshida, Akira; Akaike, Makoto; Kobayashi, Osamu; Nakayama, Haruhiko; Sakuma, Yuji; Yoshida, Tsutomu; Kameda, Yoichi; Tsuchiya, Eiju; Miyagi, Yohei

    2006-01-01

    A simple and rapid method to detect the epidermal growth factor receptor hot spot mutation L858R in lung adenocarcinoma was developed based on principles similar to the universal heteroduplex generator technology. A single-stranded oligonucleotide with an internal deletion was used to generate heteroduplexes (loop-hybrids) bearing a loop in the complementary strand derived from the polymerase chain reaction product of the normal or mutant allele. By placing deletion in the oligonucleotide adjacent to the mutational site, difference in electrophoretic mobility between loop-hybrids with normal and mutated DNA was distinguishable in a native polyacrylamide gel. The method was also modified to detect in-frame deletion mutations of epidermal growth factor receptor in lung adenocarcinomas. In addition, the method was adapted to detect hot spot mutations in the B-type Raf kinase (BRAF) at V600 and in a Ras-oncogene (NRAS) at Q61, the mutations commonly found in thyroid carcinomas. Our mutation detection system, designated the loop-hybrid mobility shift assay was sensitive enough to detect mutant DNA comprising 7.5% of the total DNA. As a simple and straightforward mutation detection technique, loop-hybrid mobility shift assay may be useful for the molecular diagnosis of certain types of clinical cancers. Other applications are also discussed. PMID:16931592

  3. PCR-based methods for the detection of L1014 kdr mutation in Anopheles culicifacies sensu lato

    Science.gov (United States)

    Singh, Om P; Bali, Prerna; Hemingway, Janet; Subbarao, Sarala K; Dash, Aditya P; Adak, Tridibes

    2009-01-01

    Background Anopheles culicifacies s.l., a major malaria vector in India, has developed widespread resistance to DDT and is becoming resistant to pyrethroids–the only insecticide class recommended for the impregnation of bed nets. Knock-down resistance due to a point mutation in the voltage gated sodium channel at L1014 residue (kdr) is a common mechanism of resistance to DDT and pyrethroids. The selection of this resistance may pose a serious threat to the success of the pyrethroid-impregnated bed net programme. This study reports the presence of kdr mutation (L1014F) in a field population of An. culicifacies s.l. and three new PCR-based methods for kdr genotyping. Methods The IIS4-IIS5 linker to IIS6 segments of the para type voltage gated sodium channel gene of DDT and pyrethroid resistant An. culicifacies s.l. population from the Surat district of India was sequenced. This revealed the presence of an A-to-T substitution at position 1014 leading to a leucine-phenylalanine mutation (L1014F) in a few individuals. Three molecular methods viz. Allele Specific PCR (AS-PCR), an Amplification Refractory Mutation System (ARMS) and Primer Introduced Restriction Analysis-PCR (PIRA-PCR) were developed and tested for kdr genotyping. The specificity of the three assays was validated following DNA sequencing of the samples genotyped. Results The genotyping of this An. culicifacies s.l. population by the three PCR based assays provided consistent result and were in agreement with DNA sequencing result. A low frequency of the kdr allele mostly in heterozygous condition was observed in the resistant population. Frequencies of the different genotypes were in Hardy-Weinberg equilibrium. Conclusion The Leu-Phe mutation, which generates the kdr phenotype in many insects, was detected in a pyrethroid and DDT resistant An. culicifacies s.l. population. Three PCR-based methods were developed for kdr genotyping. All the three assays were specific. The ARMS method was refractory to non

  4. PCR-based methods for the detection of L1014 kdr mutation in Anopheles culicifacies sensu lato

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    Dash Aditya P

    2009-07-01

    Full Text Available Abstract Background Anopheles culicifacies s.l., a major malaria vector in India, has developed widespread resistance to DDT and is becoming resistant to pyrethroids–the only insecticide class recommended for the impregnation of bed nets. Knock-down resistance due to a point mutation in the voltage gated sodium channel at L1014 residue (kdr is a common mechanism of resistance to DDT and pyrethroids. The selection of this resistance may pose a serious threat to the success of the pyrethroid-impregnated bed net programme. This study reports the presence of kdr mutation (L1014F in a field population of An. culicifacies s.l. and three new PCR-based methods for kdr genotyping. Methods The IIS4-IIS5 linker to IIS6 segments of the para type voltage gated sodium channel gene of DDT and pyrethroid resistant An. culicifacies s.l. population from the Surat district of India was sequenced. This revealed the presence of an A-to-T substitution at position 1014 leading to a leucine-phenylalanine mutation (L1014F in a few individuals. Three molecular methods viz. Allele Specific PCR (AS-PCR, an Amplification Refractory Mutation System (ARMS and Primer Introduced Restriction Analysis-PCR (PIRA-PCR were developed and tested for kdr genotyping. The specificity of the three assays was validated following DNA sequencing of the samples genotyped. Results The genotyping of this An. culicifacies s.l. population by the three PCR based assays provided consistent result and were in agreement with DNA sequencing result. A low frequency of the kdr allele mostly in heterozygous condition was observed in the resistant population. Frequencies of the different genotypes were in Hardy-Weinberg equilibrium. Conclusion The Leu-Phe mutation, which generates the kdr phenotype in many insects, was detected in a pyrethroid and DDT resistant An. culicifacies s.l. population. Three PCR-based methods were developed for kdr genotyping. All the three assays were specific. The ARMS method

  5. Malignant pleural effusion cell blocks are substitutes for tissue in EML4-ALK rearrangement detection in patients with advanced non-small-cell lung cancer.

    Science.gov (United States)

    Zhong, J; Li, X; Bai, H; Zhao, J; Wang, Z; Duan, J; An, T; Wu, M; Wang, Y; Wang, S; Wang, J

    2016-12-01

    To evaluate the feasibility of malignant pleural effusions (MPE) as surrogate samples for the detection of echinoderm microtubule-associated protein-like4 (EML4)-anaplastic lymphoma kinase (ALK) and to investigate the prognostic and predictive value of EML4-ALK in MPE of non-small-cell lung cancer (NSCLC). One hundred and nine NSCLC patients were retrospectively analysed. EML4-ALK was identified using paraffin-embedded tumour cells in MPE samples by immunohistochemistry (IHC, Ventana) and confirmed by fluorescence using in situ hybridisation (FISH) and qRT-PCR. The EGFR mutation was determined by MPE, using denaturing high-performance liquid chromatography (DHPLC). A total of 5 out of 109 (4.58%) patients were identified as EML4-ALK rearrangement in MPE by IHC.; In addition to two metachronous samples, the consistency of MPE and tissue for EML4-ALK detection was 100% (21/21), and the sensitivity and specificity were 100% (2/2) and 100% (19/19), respectively. EML4-ALK rearrangement cases were confirmed by FISH and qRT-PCR; the sensitivity were both 100% (2/2) when compared with tissue, and it was 60% (3/5) and 100% (5/5), respectively, when compared with MPE by IHC. The overall response rate (ORR) was 100% (2/2) for patients with EML4-ALK in MPE. Moreover, the PFS of these patients appeared to be prolonged in chemotherapy (9.27 versus 6.53 and versus 4.67 months, P = 0.122), compared with the EGFR mutation and the EGFR/ALK double negative group, respectively. EML4-ALK rearrangement detection in malignant pleural effusions is a complementary method for EML4-ALK detection. VETANA and qRT-PCR are more appropriate for MPE detection. EML4-ALK rearrangement in pleural effusions has a predictive value for treatment. © 2016 John Wiley & Sons Ltd.

  6. Novel Mutations Detected in Avirulence Genes Overcoming Tomato Cf Resistance Genes in Isolates of a Japanese Population of Cladosporium fulvum.

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    Yuichiro Iida

    Full Text Available Leaf mold of tomato is caused by the biotrophic fungus Cladosporium fulvum which complies with the gene-for-gene system. The disease was first reported in Japan in the 1920s and has since been frequently observed. Initially only race 0 isolates were reported, but since the consecutive introduction of resistance genes Cf-2, Cf-4, Cf-5 and Cf-9 new races have evolved. Here we first determined the virulence spectrum of 133 C. fulvum isolates collected from 22 prefectures in Japan, and subsequently sequenced the avirulence (Avr genes Avr2, Avr4, Avr4E, Avr5 and Avr9 to determine the molecular basis of overcoming Cf genes. Twelve races of C. fulvum with a different virulence spectrum were identified, of which races 9, 2.9, 4.9, 4.5.9 and 4.9.11 occur only in Japan. The Avr genes in many of these races contain unique mutations not observed in races identified elsewhere in the world including (i frameshift mutations and (ii transposon insertions in Avr2, (iii point mutations in Avr4 and Avr4E, and (iv deletions of Avr4E, Avr5 and Avr9. New races have developed by selection pressure imposed by consecutive introductions of Cf-2, Cf-4, Cf-5 and Cf-9 genes in commercially grown tomato cultivars. Our study shows that molecular variations to adapt to different Cf genes in an isolated C. fulvum population in Japan are novel but overall follow similar patterns as those observed in populations from other parts of the world. Implications for breeding of more durable C. fulvum resistant varieties are discussed.

  7. Structural and functional analysis of APOA5 mutations identified in patients with severe hypertriglyceridemia[S

    Science.gov (United States)

    Mendoza-Barberá, Elena; Julve, Josep; Nilsson, Stefan K.; Lookene, Aivar; Martín-Campos, Jesús M.; Roig, Rosa; Lechuga-Sancho, Alfonso M.; Sloan, John H.; Fuentes-Prior, Pablo; Blanco-Vaca, Francisco

    2013-01-01

    During the diagnosis of three unrelated patients with severe hypertriglyceridemia, three APOA5 mutations [p.(Ser232_Leu235)del, p.Leu253Pro, and p.Asp332ValfsX4] were found without evidence of concomitant LPL, APOC2, or GPIHBP1 mutations. The molecular mechanisms by which APOA5 mutations result in severe hypertriglyceridemia remain poorly understood, and the functional impairment/s induced by these specific mutations was not obvious. Therefore, we performed a thorough structural and functional analysis that included follow-up of patients and their closest relatives, measurement of apoA-V serum concentrations, and sequencing of the APOA5 gene in 200 nonhyperlipidemic controls. Further, we cloned, overexpressed, and purified both wild-type and mutant apoA-V variants and characterized their capacity to activate LPL. The interactions of recombinant wild-type and mutated apoA-V variants with liposomes of different composition, heparin, LRP1, sortilin, and SorLA/LR11 were also analyzed. Finally, to explore the possible structural consequences of these mutations, we developed a three-dimensional model of full-length, lipid-free human apoA-V. A complex, wide array of impairments was found in each of the three mutants, suggesting that the specific residues affected are critical structural determinants for apoA-V function in lipoprotein metabolism and, therefore, that these APOA5 mutations are a direct cause of hypertriglyceridemia. PMID:23307945

  8. [Analysis of gene mutation in a Chinese family with Norrie disease].

    Science.gov (United States)

    Zhang, Tian-xiao; Zhao, Xiu-li; Hua, Rui; Zhang, Jin-song; Zhang, Xue

    2012-09-01

    To detect the pathogenic mutation in a Chinese family with Norrie disease. Clinical diagnosis was based on familial history, clinical sign and B ultrasonic examination. Peripheral blood samples were obtained from all available members in a Chinese family with Norrie disease. Genomic DNA was extracted from lymphocytes by the standard SDS-proteinase K-phenol/chloroform method. Two coding exons and all intron-exon boundaries of the NDP gene were PCR amplified using three pairs of primers and subjected to automatic DNA sequence. The causative mutation was confirmed by restriction enzyme analysis and genotyping analysis in all members. Sequence analysis of NDP gene revealed a missense mutation c.220C > T (p.Arg74Cys) in the proband and his mother. Further mutation identification by restriction enzyme analysis and genotyping analysis showed that the proband was homozygote of this mutation. His mother and other four unaffected members (III3, IV4, III5 and II2) were carriers of this mutation. The mutant amino acid located in the C-terminal cystine knot-like domain, which was critical motif for the structure and function of NDP. A NDP missense mutation was identified in a Chinese family with Norrie disease.

  9. A 7666-bp genomic deletion is frequent in Chinese Han deaf patients with non-syndromic enlarged vestibular aqueduct but without bi-allelic SLC26A4 mutations.

    Science.gov (United States)

    Pang, Xiuhong; Chai, Yongchuan; He, Longxia; Chen, Penghui; Wang, Xiaowen; Li, Lei; Jia, Huan; Wu, Hao; Yang, Tao

    2015-12-01

    To investigate the genetic cause of the patients with non-syndromic enlarged vestibular aqueduct (EVA) but without bi-allelic SLC26A4 mutations. Presence of a homozygous genomic deletion was detected in a Chinese Han deaf patient (D1467-1) who failed to amplify the first three exons of SLC26A4. The breakpoints of the deletion were fine-mapped and revealed by PCR amplification and sequencing. This deletion was subsequently screened in 22 Chinese Han EVA probands with mono-allelic SLC26A4 mutations. The possible founder effect of the newly identified genomic deletion was evaluated by haplotype analysis. A homozygous c.-2071_307+3801del7666 deletion of SLC26A4 was identified in patient D1467-1. This novel genomic deletion was subsequently identified in 18% (4/22) of the Chinese Han EVA probands with mono-allelic SLC26A4 mutations. Haplotype analysis showed that this genomic deletion is likely a founder mutation in Chinese Hans. Our results suggested that the cryptic c.-2071_307+3801del7666 deletion of SLC26A4 is relatively frequent in Chinese Han non-syndromic EVA patients without bi-allelic SLC26A4 mutations. Screening of this genomic deletion should be incorporated into the routine DNA testing of SLC26A4 in Chinese Hans. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

  10. Photobiological activity of 4-methylpsoralen and 4-methyl-4', 5'-dihydropsoralen with respect to lethal and mutagenic effects on E. coli, and prophage induction

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    Fujita, H. (Tokai Univ., Isehara, Kanagawa (Japan). School of Medicine)

    1984-06-01

    The lethal and mutagenic effects on E. coli as well as the induction of prophage lambda were determined after treatment with 4-methylpsoralen, 8-methoxypsoralen, psoralen or 4-methyl-4',5'-dihydropsoralen and UV-A irradiation. All psoralens used caused photokilling and photomutagenesis of strains H/r30R and Hs30R. 4-Methylpsoralen was more efficient for killing and for the induced mutation than 8-methoxypsoralen or psoralen in view of the dose modification factor. This finding can be explained by the methylation effect of psoralen. 4-Methylpsoralen induced more mutation in Hs30R than in H/r30R. Monofunctional 4-methyl-4',5'-dihydropsoralen required much higher fluence than bifunctional psoralens to kill cells and to induce the mutation. When the induced mutation frequency was expressed as a function of survival, mutagenic efficiency ranked in the following order: 8-methoxypsoralen > psoralen > 4-methylpsoralen > 4-methyl-4',5'-dihydropsoralen. 4-Methylpsoralen was 3-4-fold less mutagenic than 8-methoxypsoralen in this plot. Lytic growth of prophage in E. coli AB1157 (lambda) was induced by the treatment. When the bifunctional psoralens were used, the maximum induced fraction was larger than 20%. However, it was only 2% when 4-methyl-4',5'-dihydropsoralen was used.

  11. Application of PCR-LDR-nucleic acid detection strip in detection of YMDD mutation in hepatitis B patients treated with lamivudine.

    Science.gov (United States)

    Xu, Gaolian; You, Qimin; Pickerill, Sam; Zhong, Huayan; Wang, Hongying; Shi, Jian; Luo, Ying; You, Paul; Kong, Huimin; Lu, Fengmin; Hu, Lin

    2010-07-01

    Chronic hepatitis B virus (CHBV) infection causes cirrhosis and hepatocellular carcinoma. Lamivudine (LAM) has been successfully used to treat CHBV infections but prolonged use leads to the emergence of drug-resistant variants. This is primarily linked to a mutation in the tyrosine-methionine-aspartate-aspartate (YMDD) motif of the HBV polymerase gene at position 204. Rapid diagnosis of drug-resistant HBV is necessary for a prompt treatment response. Common diagnostic methods such as sequencing and restriction fragment length polymorphism (RFLP) analysis lack sensitivity and require significant processing. The aim of this study was to demonstrate the usefulness of a novel diagnostic method that combines polymerase chain reaction (PCR), ligase detection reaction (LDR) and a nucleic acid detection strip (NADS) in detecting site-specific mutations related to HBV LAM resistance. We compared this method (PLNA) to direct sequencing and RFLP analysis in 50 clinical samples from HBV infected patients. There was 90% concordance between all three results. PLNA detected more samples containing mutant variants than both sequencing and RFLP analysis and was more sensitive in detecting mixed variant populations. Plasmid standards indicated that the sensitivity of PLNA is at or below 3,000 copies per ml and that it can detect a minor variant at 5% of the total viral population. This warrants its further development and suggests that the PLNA method could be a useful tool in detecting LAM resistance. (c) 2010 Wiley-Liss, Inc.

  12. Highly sensitive KRAS mutation detection from formalin-fixed paraffin-embedded biopsies and circulating tumour cells using wild-type blocking polymerase chain reaction and Sanger sequencing.

    Science.gov (United States)

    Huang, Meggie Mo Chao; Leong, Sai Mun; Chua, Hui Wen; Tucker, Steven; Cheong, Wai Chye; Chiu, Lily; Li, Mo-Huang; Koay, Evelyn Siew-Chuan

    2014-08-01

    Among patients with colorectal cancer (CRC), KRAS mutations were reported to occur in 30-51 % of all cases. CRC patients with KRAS mutations were reported to be non-responsive to anti-epidermal growth factor receptor (EGFR) monoclonal antibody (MoAb) treatment in many clinical trials. Hence, accurate detection of KRAS mutations would be critical in guiding the use of anti-EGFR MoAb therapies in CRC. In this study, we carried out a detailed investigation of the efficacy of a wild-type (WT) blocking real-time polymerase chain reaction (PCR), employing WT KRAS locked nucleic acid blockers, and Sanger sequencing, for KRAS mutation detection in rare cells. Analyses were first conducted on cell lines to optimize the assay protocol which was subsequently applied to peripheral blood and tissue samples from patients with CRC. The optimized assay provided a superior sensitivity enabling detection of as little as two cells with mutated KRAS in the background of 10(4) WT cells (0.02 %). The feasibility of this assay was further investigated to assess the KRAS status of 45 colorectal tissue samples, which had been tested previously, using a conventional PCR sequencing approach. The analysis showed a mutational discordance between these two methods in 4 of 18 WT cases. Our results present a simple, effective, and robust method for KRAS mutation detection in both paraffin embedded tissues and circulating tumour cells, at single-cell level. The method greatly enhances the detection sensitivity and alleviates the need of exhaustively removing co-enriched contaminating lymphocytes.

  13. Cancer risk and clinicopathological characteristics of thyroid nodules harboring thyroid-stimulating hormone receptor gene mutations.

    Science.gov (United States)

    Mon, Sann Y; Riedlinger, Gregory; Abbott, Collette E; Seethala, Raja; Ohori, N Paul; Nikiforova, Marina N; Nikiforov, Yuri E; Hodak, Steven P

    2018-05-01

    Thyroid-stimulating hormone receptor (TSHR) gene mutations play a critical role in thyroid cell proliferation and function. They are found in 20%-82% of hyperfunctioning nodules, hyperfunctioning follicular thyroid cancers (FTC), and papillary thyroid cancers (PTC). The diagnostic importance of TSHR mutation testing in fine needle aspiration (FNA) specimens remains unstudied. To examine the association of TSHR mutations with the functional status and surgical outcomes of thyroid nodules, we evaluated 703 consecutive thyroid FNA samples with indeterminate cytology for TSHR mutations using next-generation sequencing. Testing for EZH1 mutations was performed in selected cases. The molecular diagnostic testing was done as part of standard of care treatment, and did not require informed consent. TSHR mutations were detected in 31 (4.4%) nodules and were located in exons 281-640, with codon 486 being the most common. Allelic frequency ranged from 3% to 45%. Of 16 cases (12 benign, 3 FTC, 1 PTC) with surgical correlation, 15 had solitary TSHR mutations and 1 PTC had comutation with BRAF V600E. Hyperthyroidism was confirmed in all 3 FTC (2 overt, 1 subclinical). Of 5 nodules with solitary TSHR mutations detected at high allelic frequency, 3 (60%) were FTC. Those at low allelic frequency (3%-22%) were benign. EZH1 mutations were detected in 2 of 4 TSHR-mutant malignant nodules and neither of 2 benign nodules. We report that TSHR mutations occur in ∼5% thyroid nodules in a large consecutive series with indeterminate cytology. TSHR mutations may be associated with an increased cancer risk when present at high allelic frequency, even when the nodule is hyperfunctioning. Benign nodules were however most strongly correlated with TSHR mutations at low allelic frequency. © 2018 Wiley Periodicals, Inc.

  14. Patients with Obesity Caused by Melanocortin-4 Receptor Mutations Can Be Treated with a Glucagon-like Peptide-1 Receptor Agonist

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    Iepsen, Eva W; Zhang, Jinyi; Thomsen, Henrik S

    2018-01-01

    Pathogenic mutations in the appetite-regulating melanocortin-4 receptor (MC4R) represent the most common cause of monogenic obesity with limited treatment options. Glucagon-like peptide-1 receptor agonists (GLP-1 RAs) cause weight loss by reducing appetite. We assessed the effect of the GLP-1 RA...... liraglutide 3.0 mg for 16 weeks in 14 obese individuals with pathogenic MC4R mutations (BMI 37.5 ± 6.8) and 28 matched control participants without MC4R mutation (BMI 36.8 ± 4.8). Liraglutide decreased body weight by 6.8 kg ± 1.8 kg in individuals with pathogenic MC4R mutations and by 6.1 kg ± 1.2 kg...... in control participants. Total body fat, waist circumference, and fasting and postprandial glucose concentrations similarly decreased in both groups. Thus, liraglutide induced an equal, clinically significant weight loss of 6% in both groups, indicating that the appetite-reducing effect of liraglutide...

  15. Recurrent occurrences of CDKL5 mutations in patients with epileptic encephalopathy.

    Science.gov (United States)

    Yamamoto, Toshiyuki; Shimojima, Keiko; Kimura, Nobusuke; Mogami, Yukiko; Usui, Daisuke; Takayama, Rumiko; Ikeda, Hiroko; Imai, Katsumi

    2015-01-01

    The cyclin-dependent kinase-like 5 gene (CDKL5) is recognized as one of the genes responsible for epileptic encephalopathy. We identified CDKL5 mutations in five Japanese patients (one male and four female) with epileptic encephalopathy. Although all mutations were of de novo origin, they were located in the same positions as previously reported pathogenic mutations. These recurrent occurrences of de novo mutations in the same loci may indicate hot spots of nucleotide alteration.

  16. Clinical flow cytometric screening of SAP and XIAP expression accurately identifies patients with SH2D1A and XIAP/BIRC4 mutations.

    Science.gov (United States)

    Gifford, Carrie E; Weingartner, Elizabeth; Villanueva, Joyce; Johnson, Judith; Zhang, Kejian; Filipovich, Alexandra H; Bleesing, Jack J; Marsh, Rebecca A

    2014-07-01

    X-linked lymphoproliferative disease is caused by mutations in two genes, SH2D1A and XIAP/BIRC4. Flow cytometric methods have been developed to detect the gene products, SAP and XIAP. However, there is no literature describing the accuracy of flow cytometric screening performed in a clinical lab setting. We reviewed the clinical flow cytometric testing results for 656 SAP and 586 XIAP samples tested during a 3-year period. Genetic testing was clinically performed as directed by the managing physician in 137 SAP (21%) and 115 XIAP (20%) samples. We included these samples for analyses of flow cytometric test accuracy. SH2D1A mutations were detected in 15/137 samples. SAP expression was low in 13/15 (sensitivity 87%, CI 61-97%). Of the 122 samples with normal sequencing, SAP was normal in 109 (specificity 89%, CI 82-94%). The positive predictive values (PPVs) and the negative predictive values (NPVs) were 50% and 98%, respectively. XIAP/BIRC4 mutations were detected in 19/115 samples. XIAP expression was low in 18/19 (sensitivity 95%, CI 73-100%). Of the 96 samples with normal sequencing, 59 had normal XIAP expression (specificity 61%, CI 51-71%). The PPVs and NPVs were 33% and 98%, respectively. Receiver-operating characteristic analysis was able to improve the specificity to 75%. Clinical flow cytometric screening tests for SAP and XIAP deficiencies offer good sensitivity and specificity for detecting genetic mutations, and are characterized by high NPVs. We recommend these tests for patients suspected of having X-linked lymphoproliferative disease type 1 (XLP1) or XLP2. © 2014 Clinical Cytometry Society.

  17. BRAF, KIT, NRAS, GNAQ and GNA11 mutation analysis in cutaneous melanomas in Turkish population

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    Ismail Yilmaz

    2015-01-01

    Full Text Available Background: KIT and mitogen-activated protein kinase cascade are important for melanomagenesis. In the present study, we analyzed the frequency of BRAF, NRAS, KIT, GNAQ and GNA11 gene mutations and investigated their association with clinicopathological features of melanomas in Turkish population. Materials and Methods: Forty-seven primary cutaneous melanomas were included in our study. Sanger sequencing method was used for mutation analysis in all cases. Results: Mean age was 62.1 (29-101 years. Female:male ratio was 17:30. Among 47 melanomas, 14 (29.8% BRAF, 10 (21.3% NRAS, 4 (8.5% KIT and 1(2.1% GNAQ gene mutations were detected. Two of the KIT mutations were found in acral lentiginous melanoma (ALM. In the head and neck region, mutation frequency was significantly lower than in other locations (P = 0.035. The only GNAQ gene mutation (p.Q209L was detected in a melanoma arising from blue nevus located on the scalp. None of the melanomas harbored NRAS exon 2, KIT exon 13/17/18, GNAQ exon 4 and GNA11 exon 4/5 mutations. Overall mutation frequency did not show significant difference between metastatic (8/14, 57.1% and nonmetastatic (18/33, 54.5% patients. We did not observe any significant association between mutation status and gender or age of various patients. Conclusions: Our results support that BRAF and NRAS gene mutations are common in cutaneous melanomas. The activating mutations of KIT gene are rare and especially seen in ALM. GNAQ and GNA11 mutations are infrequent in cutaneous melanomas and may be associated only with melanomas arising from blue nevus.

  18. Clone-specific MYD88 L265P and CXCR4 mutation status can provide clinical utility in suspected Waldenström macroglobulinemia/lymphoplasmacytic lymphoma.

    Science.gov (United States)

    Burnworth, Bettina; Wang, Zhixing; Singleton, Timothy P; Bennington, Angela; Fritschle, Wayne; Bennington, Richard; Brodersen, Lisa Eidenschink; Wells, Denise A; Loken, Michael R; Zehentner, Barbara K

    2016-12-01

    MYD88 L265P, a diagnostic marker for lymphoplasmacytic lymphoma (LPL)/Waldenström macroglobulinemia (WM) can also be detected in other hematopoietic malignancies. We demonstrate a novel approach to increase the specificity of this marker for WM/LPL diagnosis by combining flow cytometric cell sorting with molecular analysis. Clonal B-lymphocyte and co-occurring clonal plasma cell populations of low-grade B-cell lymphomas were sorted by flow cytometry and analyzed for immunoglobulin gene rearrangements (PCR), and for MYD88 and CXCR4 mutations. Identical clonal origin was confirmed by PCR for 21 LPL/WM cases and MYD88 L265P was detected in both B-cell and plasma cell fractions. 9/20 other B-cell lymphomas with identical light chain restriction on B-cells and plasma cells were genotypically identical by PCR and MYD88 L265P was detected in both cell fractions in 7/9 whereas in 11/20 specimens with different clonal origin, MYD88 L265P was absent (5/11), or only found in B-lymphocytes (4/11), or plasma cells (2/11). CXCR4 mutations were detected in 17/39 cases, but missed in 63% of these without cell sorting. Confirming MYD88L265P in both B-cells and plasma cell fractions can provide a novel and powerful discriminator to distinguish LPL/WM from phenotypically similar disorders. Furthermore, this approach significantly increases CXCR4 detection sensitivity. Copyright © 2016 Elsevier Ltd. All rights reserved.

  19. A trade-off in replication in mosquito versus mammalian systems conferred by a point mutation in the NS4B protein of dengue virus type 4

    International Nuclear Information System (INIS)

    Hanley, Kathryn A.; Manlucu, Luella R.; Gilmore, Lara E.; Blaney, Joseph E.; Hanson, Christopher T.; Murphy, Brian R.; Whitehead, Stephen S.

    2003-01-01

    An acceptable live-attenuated dengue virus vaccine candidate should have low potential for transmission by mosquitoes. We have identified and characterized a mutation in dengue virus type 4 (DEN4) that decreases the ability of the virus to infect mosquitoes. A panel of 1248 mutagenized virus clones generated previously by chemical mutagenesis was screened for decreased replication in mosquito C6/36 cells but efficient replication in simian Vero cells. One virus met these criteria and contained a single coding mutation: a C-to-U mutation at nucleotide 7129 resulting in a Pro-to-Leu change in amino acid 101 of the nonstructural 4B gene (NS4B P101L). This mutation results in decreased replication in C6/36 cells relative to wild-type DEN4, decreased infectivity for mosquitoes, enhanced replication in Vero and human HuH-7 cells, and enhanced replication in SCID mice implanted with HuH-7 cells (SCID-HuH-7 mice). A recombinant DEN4 virus (rDEN4) bearing this mutation exhibited the same set of phenotypes. Addition of the NS4B P101L mutation to rDEN4 bearing a 30 nucleotide deletion (Δ30) decreased the ability of the double-mutant virus to infect mosquitoes but increased its ability to replicate in SCID-HuH-7 mice. Although the NS4B P101L mutation decreases infectivity of DEN4 for mosquitoes, its ability to enhance replication in SCID-HuH-7 mice suggests that it might not be advantageous to include this specific mutation in an rDEN4 vaccine. The opposing effects of the NS4B P101L mutation in mosquito and vertebrate systems suggest that the NS4B protein is involved in maintaining the balance between efficient replication in the mosquito vector and the human host

  20. Levels of H-ras codon 61 CAA to AAA mutation: response to 4-ABP-treatment and Pms2-deficiency.

    Science.gov (United States)

    Parsons, Barbara L; Delongchamp, Robert R; Beland, Frederick A; Heflich, Robert H

    2006-01-01

    DNA mismatch repair (MMR) deficiencies result in increased frequencies of spontaneous mutation and tumor formation. In the present study, we tested the hypothesis that a chemically-induced mutational response would be greater in a mouse with an MMR-deficiency than in the MMR-proficient mouse models commonly used to assay for chemical carcinogenicity. To accomplish this, the induction of H-ras codon 61 CAA-->AAA mutation was examined in Pms2 knockout mice (Pms2-/-, C57BL/6 background) and sibling wild-type mice (Pms2+/+). Groups of five or six neonatal male mice were treated with 0.3 micromol 4-aminobiphenyl (4-ABP) or the vehicle control, dimethylsulfoxide. Eight months after treatment, liver DNAs were isolated and analysed for levels of H-ras codon 61 CAA-->AAA mutation using allele-specific competitive blocker-PCR. In Pms2-proficient and Pms2-deficient mice, 4-ABP treatment caused an increase in mutant fraction (MF) from 1.65x10(-5) to 2.91x10(-5) and from 3.40x10(-5) to 4.70x10(-5), respectively. Pooling data from 4-ABP-treated and control mice, the approximately 2-fold increase in MF observed in Pms2-deficient as compared with Pms2-proficient mice was statistically significant (P=0.0207) and consistent with what has been reported previously in terms of induction of G:C-->T:A mutation in a Pms2-deficient background. Pooling data from both genotypes, the increase in H-ras MF in 4-ABP-treated mice, as compared with control mice, did not reach the 95% confidence level of statistical significance (P=0.0606). The 4-ABP treatment caused a 1.76-fold and 1.38-fold increase in average H-ras MF in Pms2-proficient and Pms2-deficient mice, respectively. Furthermore, the levels of induced mutation in Pms2-proficient and Pms2-deficient mice were nearly identical (1.26x10(-5) and 1.30x10(-5), respectively). We conclude that Pms2-deficiency does not result in an amplification of the H-ras codon 61 CAA-->AAA mutational response induced by 4-ABP.

  1. [Mutation analysis of the PAH gene in children with phenylketonuria from the Qinghai area of China].

    Science.gov (United States)

    He, Jiang; Wang, Hui-Zhen; Xu, Fa-Liang; Yang, Xi; Wang, Rui; Zou, Hong-Yun; Yu, Wu-Zhong

    2015-11-01

    To study the mutation characteristics of the phenylalanine hydroxylase (PAH) gene in children with phenylketonuria (PKU) from the Qinghai area of China, in order to provide basic information for genetic counseling and prenatal diagnosis. Mutations of the PAH gene were detected in the promoter and exons 1-13 and their flanking intronic sequences of PAH gene by PCR and DNA sequencing in 49 children with PKU and their parents from the Qinghai area of China. A total of 30 different mutations were detected in 80 out of 98 mutant alleles (82%), including 19 missense (63%), 5 nonsense (17%), 3 splice-site (10%) and 3 deletions (10%). Most mutations were detected in exons 3, 6, 7, 11 and intron 4 of PAH gene. The most frequent mutations were p.R243Q (19%), IVS4-1G>A (9%), p.Y356X (7%) and p.EX6-96A>G(5%). Two novel mutations p.N93fsX5 (c.279-282delCATC) and p.G171E (c.512G>A) were found. p.H64fsX9(c.190delC) was documented for the second time in Chinese PAH gene. The mutation spectrum of the gene PAH in the Qinghai population was similar to that in other populations in North China while significantly different from that in the populations from some provinces in southern China, Japan and Europe. The mutations of PAH gene in the Qinghai area of China demonstrate a unique diversity, complexity and specificity.

  2. SMARCA4 inactivating mutations cause concomitant Coffin-Siris syndrome, microphthalmia and small-cell carcinoma of the ovary hypercalcaemic type.

    Science.gov (United States)

    Errichiello, Edoardo; Mustafa, Noor; Vetro, Annalisa; Notarangelo, Lucia Dora; de Jonge, Hugo; Rinaldi, Berardo; Vergani, Debora; Giglio, Sabrina Rita; Morbini, Patrizia; Zuffardi, Orsetta

    2017-09-01

    SMARCA4 chromatin remodelling factor is mutated in 11% of Coffin-Siris syndrome (CSS) patients and in almost all small-cell carcinoma of the ovary hypercalcaemic type (SCCOHT) tumours. Missense mutations with gain-of-function or dominant-negative effects are associated with CSS, whereas inactivating mutations, leading to loss of SMARCA4 expression, have been exclusively found in SCCOHT. We applied whole-exome sequencing to study a 15-year-old patient with mild CSS who concomitantly developed SCCOHT at age 13 years. Interestingly, our patient also showed congenital microphthalmia, which has never previously been reported in CSS patients. We detected a de novo germline heterozygous nonsense mutation in exon 19 of SMARCA4 (c.2935C > T;p.Arg979*), and a somatic frameshift mutation in exon 6 (c.1236_1236delC;p.Gln413Argfs*88), causing complete loss of SMARCA4 immunostaining in the tumour. The immunohistochemical findings are supported by the observation that the c.2935C > T mutant transcript was detected by reverse transcription polymerase chain reaction at a much lower level than the wild-type allele in whole blood and the lymphoblastoid cell line of the proband, confirming nonsense-mediated mRNA decay. Accordingly, immunoblotting demonstrated that there was approximately half the amount of SMARCA4 protein in the proband's cells as in controls. This study suggests that SMARCA4 constitutional mutations associated with CSS are not necessarily non-truncating, and that haploinsufficiency may explain milder CSS phenotypes, as previously reported for haploinsufficient ARID1B. In addition, our case supports the dual role of chromatin remodellers in developmental disorders and cancer, as well as the involvement of SMARCA4 in microphthalmia, confirming previous findings in mouse models and the DECIPHER database. Finally, we speculate that mild CSS might be under-recognized in a proportion of SCCOHT patients harbouring SMARCA4 mutations. © 2017 The Authors. The

  3. Detection of Hepatitis B Virus M204I Mutation by Quantum Dot-Labeled DNA Probe

    Directory of Open Access Journals (Sweden)

    Cheng Zhang

    2017-04-01

    Full Text Available Quantum dots (QDs are semiconductor nanoparticles with a diameter of less than 10 nm, which have been widely used as fluorescent probes in biochemical analysis and vivo imaging because of their excellent optical properties. Sensitive and convenient detection of hepatitis B virus (HBV gene mutations is important in clinical diagnosis. Therefore, we developed a sensitive, low-cost and convenient QDs-mediated fluorescent method for the detection of HBV gene mutations in real serum samples from chronic hepatitis B (CHB patients who had received lamivudine or telbivudine antiviral therapy. We also evaluated the efficiency of this method for the detection of drug-resistant mutations compared with direct sequencing. In CHB, HBV DNA from the serum samples of patients with poor response or virological breakthrough can be hybridized to probes containing the M204I mutation to visualize fluorescence under fluorescence microscopy, where fluorescence intensity is related to the virus load, in our method. At present, the limits of the method used to detect HBV genetic variations by fluorescence quantum dots is 103 IU/mL. These results show that QDs can be used as fluorescent probes to detect viral HBV DNA polymerase gene variation, and is a simple readout system without complex and expensive instruments, which provides an attractive platform for the detection of HBV M204I mutation.

  4. Detection of new MHC mutations in mice by skin grafting, tumor transplantation and monoclonal antibodies: a comparison

    International Nuclear Information System (INIS)

    Egorov, I.K.; Egorov, O.S.

    1988-01-01

    Two mechanisms of major histocompatibility complex (MHC) mutations have been described in mice: gene conversion and homologous but unequal recombination. However, the knowledge of mutations in MHC is incomplete because studies have been limited almost exclusively to two haplotypes, H-2/sup b/ and H-2/sup d/, while hundreds of haplotypes exist in nature; it has been biased by the use of only one procedure of screening for mutation, skin grafting. The authors used three procedures to screen for MHC mutations: (1) conventional techniques of skin grafting, (2) syngeneic tumor transplantation and (3) typing with monoclonal anti-MHC antibodies (mAbs) and complement. The faster technique of tumor transplantation detected mutants similar to those discovered by skin grafting technique. Screening with mAbs allowed us to detect both mutants that are capable of rejecting standard skin grafts and those that are silent in skin grafting tests, and which therefore resulted in a higher apparent mutation frequency. Two mutants of the H-2/sup a/ haplotype were found that carry concomitant class I and class II antigenic alterations. Both MHC mutants silent in skin grafting tests and mutants carrying concomitant class I and class II alterations have never been studied before and are expected to reveal new mechanisms of generating MHC mutations. 1-Ethyl-1-nitrosourea (ENU) failed to induce de novo MHC mutations in our skin grafting series

  5. Mucopolysaccharidosis IVA: Four new exonic mutations in patients with N-acetylgalactosamine-6-sulfate sulfatase deficiency

    Energy Technology Data Exchange (ETDEWEB)

    Tomatsu, Shunji; Fukuda, Seiji; Yamagishi, Atsushi [Gifu Univ. (Japan)] [and others

    1996-05-01

    We report four new mutations in Japanese patients with mucopolysaccharidosis IVA (MPSIVA) who were heterozygous for a common double gene deletion. A nonsense mutation of CAG to TAG at codon 148 in exon 4 was identified, resulting in a change of Q to a stop codon and three missense mutations: V (GTC) to A (GCC) at codon 138 in exon 4, P (CCC) to S (TCC) at codon 151 in exon 5, and P (CCC) to L (CTC) at codon 151 in exon 5. Introduction of these mutations into the normal GALNS cDNA and transient expression in cultured fibroblasts resulted in a significant decrease in the enzyme activity. V138A and Q148X mutations result in changes of restriction site, which were analyzed by restriction-enzyme assay. P151S and P151L mutations that did not alter the restriction site were detected by direct sequencing or allele specific oligohybridization. Detection of the double gene deletion was initially done using Southern blots and was confirmed by PCR. Haplotypes were determined using seven polymorphisms to the GALNS locus in families with the double gene deletion. Haplotype analysis showed that the common double gene deletion occurred on a single haplotype, except for some variation in a VNTR-like polymorphism. This finding is consistent with a common founder for all individuals with this mutation. 48 refs., 5 figs., 1 tab.

  6. Cross-Sectional Study for the Detection of Mutations in the Beta-Globin Gene Among Patients with Hemoglobinopathies in the Bengali Population.

    Science.gov (United States)

    Panja, Amrita; Chowdhury, Prosanto; Chakraborty, Sharmistha; Ghosh, Tapan Kumar; Basu, Anupam

    2017-01-01

    Thalassemia is a common autosomal recessive blood disorder, which is most prevalent in South East Asian and Mediterranean populations. It is considered as a major health burden in the Indian population. The aims of the present study were to investigate the common, as well as uncommon, mutations responsible for thalassemia in the Bengali population. The Bengali state was divided into four sampling zones. Mutation detection was done using Sanger sequencing of the HBB gene. A total of 14 different mutations were observed, including rare mutations IVS1-130(G>C), IVS1-129(A>C), -90(T>C), CD16(-C), -30(T>C), CD15(-T), and a novel mutation CD53(C>T). The frequencies of IVS1-5(G>C) and CD26(G>A) mutations were higher than other mutations. There were also some silent polymorphisms found in the studied group, CD3(T>C), CD10(C>A), IVSII-16(G>C), IVSII-74(T>G), -42(C>G). The present study is the first attempt to screen for β-thalassemia-causing mutations by direct sequencing in different districts of West Bengal. The information obtained from the present study may be helpful for thalassemia management and prenatal mutation detection.

  7. Support of positive association in family-based genetic analysis between COL27A1 and Tourette syndrome.

    Science.gov (United States)

    Liu, Shiguo; Yu, Xiaoxia; Xu, Quanchen; Cui, Jiajia; Yi, Mingji; Zhang, Xinhua; Ge, Yinlin; Ma, Xu

    2015-08-03

    Recently, a genome-wide association study has indicated associations between single nucleotide polymorphisms in the Collagen Type XXVII Alpha 1 gene (COL27A1) and Tourette syndrome in several ethnic populations. To clarify the global relevance of the previously identified SNPs in the development of Tourette syndrome, the associations between polymorphisms in COL27A1 and Tourette syndrome were assessed in Chinese trios. PCR-directed sequencing was used to evaluate the genetic contributions of three SNPs in COL27A1(rs4979356, rs4979357 and rs7868992) using haplotype relative risk (HRR) and transmission disequilibrium tests (TDT) with a total of 260 Tourette syndrome trios. The family-based association was significant between Tourette syndrome and rs4979356 (TDT: χ2 = 4.804, P = 0.033; HRR = 1.75, P = 0.002; HHRR = 1.32, P = 0.027), and transmission disequilibrium was suspected for rs4979357 (TDT: χ2 = 3.969, P = 0.053; HRR = 1.84, P = 0.001; HHRR = 1.29, P = 0.044). No statistically significant allele transfer was found for rs7868992 (TDT: χ2 = 2.177, P = 0.158). Although the TDT results did not remain significant after applying the conservative Bonferroni correction (p = 0.005), the significant positive HRR analysis confirmed the possibility of showing transmission disequilibrium, which provides evidence for an involvement of COL27A1in the development of TS. However, these results need to be verified with larger datasets from different populations.

  8. A Biofunctional Molecular Beacon for Detecting Single Base Mutations in Cancer Cells

    Directory of Open Access Journals (Sweden)

    Haiyan Dong

    2016-01-01

    Full Text Available The development of a convenient and sensitive biosensing system to detect specific DNA sequences is an important issue in the field of genetic disease therapy. As a classic DNA detection technique, molecular beacon (MB is often used in the biosensing system. However, it has intrinsic drawbacks, including high assay cost, complicated chemical modification, and operational complexity. In this study, we developed a simple and cost-effective label-free multifunctional MB (LMMB by integrating elements of polymerization primer, template, target recognition, and G-quadruplex into one entity to detect target DNA. The core technique was accomplished by introducing a G-hairpin that features fragments of both G-quadruplex and target DNA recognition in the G-hairpin stem. Hybridization between LMMB and target DNA triggered conformational change between the G-hairpin and the common C-hairpin, resulting in significant SYBR-green signal amplification. The hybridization continues to the isothermal circular strand-displacement polymerization and accumulation of the double-stranded fragments, causing the uninterrupted extension of the LMMB without a need of chemical modification and other assistant DNA sequences. The novel and programmable LMMB could detect target DNA with sensitivity at 250 pmol/l with a linear range from 2 to 100 nmol/l and the relative standard deviation of 7.98%. The LMMB could sense a single base mutation from the normal DNA, and polymerase chain reaction (PCR amplicons of the mutant-type cell line from the wild-type one. The total time required for preparation and assaying was only 25 minutes. Apparently, the LMMB shows great potential for detecting DNA and its mutations in biosamples, and therefore it opens up a new prospect for genetic disease therapy.

  9. Predictive efficacy of low burden EGFR mutation detected by next-generation sequencing on response to EGFR tyrosine kinase inhibitors in non-small-cell lung carcinoma.

    Directory of Open Access Journals (Sweden)

    Hye Sook Kim

    Full Text Available Direct sequencing remains the most widely used method for the detection of epidermal growth factor receptor (EGFR mutations in lung cancer; however, its relatively low sensitivity limits its clinical use. The objective of this study was to investigate the sensitivity of detecting an epidermal growth factor receptor (EGFR mutation from peptide nucleic acid-locked nucleic acid polymerase chain reaction (PNA-LNA PCR clamp and Ion Torrent Personal Genome Machine (PGM techniques compared to that by direct sequencing. Furthermore, the predictive efficacy of EGFR mutations detected by PNA-LNA PCR clamp was evaluated. EGFR mutational status was assessed by direct sequencing, PNA-LNA PCR clamp, and Ion Torrent PGM in 57 patients with non-small cell lung cancer (NSCLC. We evaluated the predictive efficacy of PNA-LNA PCR clamp on the EGFR-TKI treatment in 36 patients with advanced NSCLC retrospectively. Compared to direct sequencing (16/57, 28.1%, PNA-LNA PCR clamp (27/57, 47.4% and Ion Torrent PGM (26/57, 45.6% detected more EGFR mutations. EGFR mutant patients had significantly longer progressive free survival (14.31 vs. 21.61 months, P = 0.003 than that of EGFR wild patients when tested with PNA-LNA PCR clamp. However, no difference in response rate to EGFR TKIs (75.0% vs. 82.4%, P = 0.195 or overall survival (34.39 vs. 44.10 months, P = 0.422 was observed between the EGFR mutations by direct sequencing or PNA-LNA PCR clamp. Our results demonstrate firstly that patients with EGFR mutations were detected more frequently by PNA-LNA PCR clamp and Ion Torrent PGM than those by direct sequencing. EGFR mutations detected by PNA-LNA PCR clamp may be as a predicative factor for EGFR TKI response in patients with NSCLC.

  10. Diferença no tamanho de cascas de ovos de colônias de Panstrongylus megistus

    Directory of Open Access Journals (Sweden)

    Rosa João A da

    2003-01-01

    Full Text Available O objetivo do estudo foi avaliar as mensurações de cascas de ovos de duas colônias laboratoriais e uma silvestre de P. megistus. De cada uma das colônias, 50 cascas de ovos foram mensuradas por meio de microscópio estereoscópico e sistema de análise de imagem QWin. A análise estatística foi efetuada utilizando o teste t-Student, não pareado no programa INSTAT. A largura, o comprimento e o diâmetro da abertura opercular das cascas de ovos de uma colônia silvestre foram maiores que de duas colônias laboratoriais de P. megistus.

  11. Novel Munc13–4 mutations in children and young adult patients with haemophagocytic lymphohistiocytosis

    Science.gov (United States)

    Santoro, A; Cannella, S; Bossi, G; Gallo, F; Trizzino, A; Pende, D; Dieli, F; Bruno, G; Stinchcombe, J C; Micalizzi, C; De Fusco, C; Danesino, C; Moretta, L; Notarangelo, L D; Griffiths, G M; Aricò, M

    2006-01-01

    Familial haemophagocytic lymphohistiocytosis (FHL) is a genetically heterogeneous disorder characterised by constitutive defects in cellular cytotoxicity resulting in fever, hepatosplenomegaly and cytopenia, and the outcome is fatal unless treated by chemoimmunotherapy followed by haematopoietic stem‐cell transplantation. Since 1999, mutations in the perforin gene giving rise to this disease have been identified; however, these account only for 40% of cases. Lack of a genetic marker hampers the diagnosis, suitability for transplantation, selection of familial donors, identification of carriers, genetic counselling and prenatal diagnosis. Mutations in the Munc13–4 gene have recently been described in patients with FHL. We sequenced the Munc13–4 gene in all patients with haemophagocytic lymphohistiocytosis not due to PRF1 mutations. In 15 of the 30 families studied, 12 novel and 4 known Munc13–4 mutations were found, spread throughout the gene. Among novel mutations, 2650C→T introduced a stop codon; 441del A, 532del C, 3082del C and 3226ins G caused a frameshift, and seven were mis sense mutations. Median age of diagnosis was 4 months, but six patients developed the disease after 5 years of age and one as a young adult of 18 years. Involvement of central nervous system was present in 9 of 15 patients, activity of natural killer cells was markedly reduced or absent in 13 of 13 tested patients. Chemo‐immunotherapy was effective in all patients. Munc13–4 mutations were found in 15 of 30 patients with FHL without PRF1 mutations. Because these patients may develop the disease during adolescence or even later, haematologists should include FHL2 and FHL3 in the differential diagnosis of young adults with fever, cytopenia, splenomegaly and hypercytokinaemia. PMID:16825436

  12. Efficient detection of factor IX mutations by denaturing high-performance liquid chromatography in Taiwanese hemophilia B patients, and the identification of two novel mutations

    Directory of Open Access Journals (Sweden)

    Pei-Chin Lin

    2014-04-01

    Full Text Available Hemophilia B (HB is an X-linked recessive disorder characterized by mutations in the clotting factor IX (FIX gene that result in FIX deficiency. Previous studies have shown a wide variation of FIX gene mutations in HB. Although the quality of life in HB has greatly improved mainly because of prophylactic replacement therapy with FIX concentrates, there exists a significant burden on affected families and the medical care system. Accurate detection of FIX gene mutations is critical for genetic counseling and disease prevention in HB. In this study, we used denaturing high-performance liquid chromatography (DHPLC, which has proved to be a highly informative and practical means of detecting mutations, for the molecular diagnosis of our patients with HB. Ten Taiwanese families affected by HB were enrolled. We used the DHPLC technique followed by direct sequencing of suspected segments to detect FIX gene mutations. In all, 11 FIX gene mutations (8 point mutations, 2 small deletions/insertions, and 1 large deletion, including two novel mutations (exon6 c.687–695, del 9 mer and c.460–461, ins T were found. According to the HB pedigrees, 25% and 75% of our patients were defined as familial and sporadic HB cases, respectively. We show that DHPLC is a highly sensitive and cost-effective method for FIX gene analysis and can be used as a convenient system for disease prevention.

  13. A Homozygous Missense Mutation in TGM5 Abolishes Epidermal Transglutaminase 5 Activity and Causes Acral Peeling Skin Syndrome

    Science.gov (United States)

    Cassidy, Andrew J.; van Steensel, Maurice A. M.; Steijlen, Peter M.; van Geel, Michel; Velden, Jaap van der; Morley, Susan M.; Terrinoni, Alessandro; Melino, Gerry; Candi, Eleonora; McLean, W. H. Irwin

    2005-01-01

    Peeling skin syndrome is an autosomal recessive genodermatosis characterized by the shedding of the outer epidermis. In the acral form, the dorsa of the hands and feet are predominantly affected. Ultrastructural analysis has revealed tissue separation at the junction between the granular cells and the stratum corneum in the outer epidermis. Genomewide linkage analysis in a consanguineous Dutch kindred mapped the gene to 15q15.2 in the interval between markers D15S1040 and D15S1016. Two homozygous missense mutations, T109M and G113C, were found in TGM5, which encodes transglutaminase 5 (TG5), in all affected persons in two unrelated families. The mutation was present on the same haplotype in both kindreds, indicating a probable ancestral mutation. TG5 is strongly expressed in the epidermal granular cells, where it cross-links a variety of structural proteins in the terminal differentiation of the epidermis to form the cornified cell envelope. An established, in vitro, biochemical cross-linking assay revealed that, although T109M is not pathogenic, G113C completely abolishes TG5 activity. Three-dimensional modeling of TG5 showed that G113C lies close to the catalytic domain, and, furthermore, that this glycine residue is conserved in all known transglutaminases, which is consistent with pathogenicity. Other families with more-widespread peeling skin phenotypes lacked TGM5 mutations. This study identifies the first causative gene in this heterogeneous group of skin disorders and demonstrates that the protein cross-linking function performed by TG5 is vital for maintaining cell-cell adhesion between the outermost layers of the epidermis. PMID:16380904

  14. Chimeric proteins for detection and quantitation of DNA mutations, DNA sequence variations, DNA damage and DNA mismatches

    Science.gov (United States)

    McCutchen-Maloney, Sandra L.

    2002-01-01

    Chimeric proteins having both DNA mutation binding activity and nuclease activity are synthesized by recombinant technology. The proteins are of the general formula A-L-B and B-L-A where A is a peptide having DNA mutation binding activity, L is a linker and B is a peptide having nuclease activity. The chimeric proteins are useful for detection and identification of DNA sequence variations including DNA mutations (including DNA damage and mismatches) by binding to the DNA mutation and cutting the DNA once the DNA mutation is detected.

  15. KRAS mutation detection in colorectal cancer by a commercially available gene chip array compares well with Sanger sequencing.

    Science.gov (United States)

    French, Deborah; Smith, Andrew; Powers, Martin P; Wu, Alan H B

    2011-08-17

    Binding of a ligand to the epidermal growth factor receptor (EGFR) stimulates various intracellular signaling pathways resulting in cell cycle progression, proliferation, angiogenesis and apoptosis inhibition. KRAS is involved in signaling pathways including RAF/MAPK and PI3K and mutations in this gene result in constitutive activation of these pathways, independent of EGFR activation. Seven mutations in codons 12 and 13 of KRAS comprise around 95% of the observed human mutations, rendering monoclonal antibodies against EGFR (e.g. cetuximab and panitumumab) useless in treatment of colorectal cancer. KRAS mutation testing by two different methodologies was compared; Sanger sequencing and AutoGenomics INFINITI® assay, on DNA extracted from colorectal cancers. Out of 29 colorectal tumor samples tested, 28 were concordant between the two methodologies for the KRAS mutations that were detected in both assays with the INFINITI® assay detecting a mutation in one sample that was indeterminate by Sanger sequencing and a third methodology; single nucleotide primer extension. This study indicates the utility of the AutoGenomics INFINITI® methodology in a clinical laboratory setting where technical expertise or access to equipment for DNA sequencing does not exist. Copyright © 2011 Elsevier B.V. All rights reserved.

  16. Clinical application of antenatal genetic diagnosis of osteogenesis imperfecta type IV.

    Science.gov (United States)

    Yuan, Jing; Li, Song; Xu, YeYe; Cong, Lin

    2015-04-02

    Clinical analysis and genetic testing of a family with osteogenesis imperfecta type IV were conducted, aiming to discuss antenatal genetic diagnosis of osteogenesis imperfecta type IV. Preliminary genotyping was performed based on clinical characteristics of the family members and then high-throughput sequencing was applied to rapidly and accurately detect the changes in candidate genes. Genetic testing of the III5 fetus and other family members revealed missense mutation in c.2746G>A, pGly916Arg in COL1A2 gene coding region and missense and synonymous mutation in COL1A1 gene coding region. Application of antenatal genetic diagnosis provides fast and accurate genetic counseling and eugenics suggestions for patients with osteogenesis imperfecta type IV and their families.

  17. FATP4 missense and nonsense mutations cause similar features in Ichthyosis Prematurity Syndrome

    Directory of Open Access Journals (Sweden)

    Dahl Niklas

    2011-03-01

    Full Text Available Abstract Background Ichthyosis Prematurity Syndrome (IPS is an autosomal recessive disorder characterized by premature birth, non-scaly ichthyosis and atopic manifestations. The disease was recently shown to be caused by mutations in the gene encoding the fatty acid transport protein 4 (FATP4 and a specific reduction in the incorporation of very long chain fatty acids (VLCFA into cellular lipids. Findings We screened probands from five families segregating IPS for mutations in the FATP4 gene. Four probands were compound heterozygous for four different mutations of which three are novel. Four patients were heterozygous and one patient homozygous for the previously reported non-sense mutation p.C168X (c.504c > a. All patients had clinical characteristics of IPS and a similar clinical course. Conclusions Missense mutations and non-sense mutations in FATP4 are associated with similar clinical features suggesting that missense mutations have a severe impact on FATP4 function. The results broaden the mutational spectrum in FATP4 associated with IPS for molecular diagnosis of and further functional analysis of FATP4.

  18. MLPA based detection of mutations in the dystrophin gene of 180 Polish families with Duchenne/Becker muscular dystrophy.

    Science.gov (United States)

    Zimowski, Janusz G; Massalska, Diana; Holding, Mariola; Jadczak, Sylwia; Fidziańska, Elżbieta; Lusakowska, Anna; Kostera-Pruszczyk, Anna; Kamińska, Anna; Zaremba, Jacek

    2014-01-01

    Duchenne/Becker muscular dystrophy (DMD/BMD) is a recessive, X-linked disorder caused by a mutation in the dystrophin gene. Deletions account for approximately 60-65% of mutations, duplications for 5-10%. The remaining cases are mainly point mutations. According to Monaco theory clinical form of the disease depends on maintaining or disrupting the reading frame. The purpose of the study was to determine frequency and location of deletions and duplications in the dystrophin gene, to determine the compliance between maintaining/disrupting the reading frame and clinical form of the disease and to check the effectiveness of MLPA (multiplex ligation-dependent probe amplification) in the detection of these mutations in hemizygous patients and heterozygous female carriers. The material is composed of combined results of molecular diagnosis carried out in years 2009-2012 in 180 unrelated patients referred with the diagnosis of DMD/BMD tested by use of MLPA. We identified 110 deletions, 22 duplication (in one patient two different duplications were detected) and 2 point mutations. Deletions involved mainly exons 45-54 and 3-21, whereas most duplications involved exons 3-18. The compliance with Monaco theory was 95% for deletions and 76% for duplications. Most of mutations in the dystrophin gene were localized in the hot spots - different for deletions and duplications. MLPA enabled their quick identification, exact localization and determination whether or not they maintained or disrupted the reading frame. MLPA was also effective in detection of deletions and duplications in female carriers. Copyright © 2014 Polish Neurological Society. Published by Elsevier Urban & Partner Sp. z o.o. All rights reserved.

  19. Pyrosequencing-Based Assays for Rapid Detection of HER2 and HER3 Mutations in Clinical Samples Uncover an E332E Mutation Affecting HER3 in Retroperitoneal Leiomyosarcoma

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    Paula González-Alonso

    2015-08-01

    Full Text Available Mutations in Human Epidermal Growth Factor Receptors (HER are associated with poor prognosis of several types of solid tumors. Although HER-mutation detection methods are currently available, such as Next-Generation Sequencing (NGS, alternative pyrosequencing allow the rapid characterization of specific mutations. We developed specific PCR-based pyrosequencing assays for identification of most prevalent HER2 and HER3 mutations, including S310F/Y, R678Q, L755M/P/S/W, V777A/L/M, 774-776 insertion, and V842I mutations in HER2, as well as M91I, V104M/L, D297N/V/Y, and E332E/K mutations in HER3. We tested 85 Formalin Fixed and Paraffin Embbeded (FFPE samples and we detected three HER2-V842I mutations in colorectal carcinoma (CRC, ovarian carcinoma, and pancreatic carcinoma patients, respectively, and a HER2-L755M mutation in a CRC specimen. We also determined the presence of a HER3-E332K mutation in an urothelial carcinoma sample, and two HER3-D297Y mutations, in both gastric adenocarcinoma and CRC specimens. The D297Y mutation was previously detected in breast and gastric tumors, but not in CRC. Moreover, we found a not-previously-described HER3-E332E synonymous mutation in a retroperitoneal leiomyosarcoma patient. The pyrosequencing assays presented here allow the detection and characterization of specific HER2 and HER3 mutations. These pyrosequencing assays might be implemented in routine diagnosis for molecular characterization of HER2/HER3 receptors as an alternative to complex NGS approaches.

  20. Detecting BRAF Mutations in Formalin-Fixed Melanoma: Experiences with TwoState-of-the-Art Techniques

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    Nicola L. Schoenewolf

    2012-06-01

    Full Text Available Background: Melanoma is characterized by a high frequency of BRAF mutations. It is unknown if the BRAF mutation status has any predictive value for therapeutic approaches such as angiogenesis inhibition. Patients and Methods: We used 2 methods to analyze the BRAF mutation status in 52 of 62 melanoma patients. Method 1 (mutation-specific real-time PCR specifically detects the most frequent BRAF mutations, V600E and V600K. Method 2 (denaturing gel gradient electrophoresis and direct sequencing identifies any mutations affecting exons 11 and 15. Results: Eighteen BRAF mutations and 15 wild-type mutations were identified with both methods. One tumor had a double mutation (GAA in codon 600. Results of 3 samples were discrepant. Additional mutations (V600M, K601E were detected using method 2. Sixteen DNA samples were analyzable with either method 1 or method 2. There was a significant association between BRAF V600E mutation and survival. Conclusion: Standardized tissue fixation protocols are needed to optimize BRAF mutation analysis in melanoma. For melanoma treatment decisions, the availability of a fast and reliable BRAF V600E screening method may be sufficient. If other BRAF mutations in exons 11 and 15 are found to be of predictive value, a combination of the 2 methods would be useful.

  1. Mutation in an alternative transcript of CDKL5 in a boy with early-onset seizures.

    Science.gov (United States)

    Bodian, Dale L; Schreiber, John M; Vilboux, Thierry; Khromykh, Alina; Hauser, Natalie S

    2018-06-01

    Infantile-onset epilepsies are a set of severe, heterogeneous disorders for which clinical genetic testing yields causative mutations in ∼20%-50% of affected individuals. We report the case of a boy presenting with intractable seizures at 2 wk of age, for whom gene panel testing was unrevealing. Research-based whole-genome sequencing of the proband and four unaffected family members identified a de novo mutation, NM_001323289.1:c.2828_2829delGA in CDKL5, a gene associated with X-linked early infantile epileptic encephalopathy 2. CDKL5 has multiple alternative transcripts, and the mutation lies in an exon in the brain-expressed forms. The mutation was undetected by gene panel sequencing because of its intronic location in the CDKL5 transcript typically used to define the exons of this gene for clinical exon-based tests (NM_003159). This is the first report of a patient with a mutation in an alternative transcript of CDKL5 This finding suggests that incorporating alternative transcripts into the design and variant interpretation of exon-based tests, including gene panel and exome sequencing, could improve the diagnostic yield. © 2018 Bodian et al.; Published by Cold Spring Harbor Laboratory Press.

  2. Demarcação e Divulgação dos Limites entre o Brasil e a Colômbia

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    Novaes, André Reyes; Palacios, David Ramírez

    2015-01-01

    Os documentos aqui reproduzidos expressam um momento importante no processo de demarcação dos limites entre o Brasil e a Colômbia. O primeiro é a “Acta da vigésima primeira e última conferência da Comissão Mixta Brasileira Colombiana Demarcadora de Limites”, realizada em 5 de janeiro de 1937 na cidade de Manaus. O segundo é a “Carta geográfica da fronteira do Alto Solimões entre Brasil, Perú e Colômbia”, publicada em 1940 com base nos trabalhos da citada comissão, e disponível na Biblioteca D...

  3. Loss of the BMP antagonist USAG-1 ameliorates disease in a mouse model of the progressive hereditary kidney disease Alport syndrome.

    Science.gov (United States)

    Tanaka, Mari; Asada, Misako; Higashi, Atsuko Y; Nakamura, Jin; Oguchi, Akiko; Tomita, Mayumi; Yamada, Sachiko; Asada, Nariaki; Takase, Masayuki; Okuda, Tomohiko; Kawachi, Hiroshi; Economides, Aris N; Robertson, Elizabeth; Takahashi, Satoru; Sakurai, Takeshi; Goldschmeding, Roel; Muso, Eri; Fukatsu, Atsushi; Kita, Toru; Yanagita, Motoko

    2010-03-01

    The glomerular basement membrane (GBM) is a key component of the filtering unit in the kidney. Mutations involving any of the collagen IV genes (COL4A3, COL4A4, and COL4A5) affect GBM assembly and cause Alport syndrome, a progressive hereditary kidney disease with no definitive therapy. Previously, we have demonstrated that the bone morphogenetic protein (BMP) antagonist uterine sensitization-associated gene-1 (USAG-1) negatively regulates the renoprotective action of BMP-7 in a mouse model of tubular injury during acute renal failure. Here, we investigated the role of USAG-1 in renal function in Col4a3-/- mice, which model Alport syndrome. Ablation of Usag1 in Col4a3-/- mice led to substantial attenuation of disease progression, normalization of GBM ultrastructure, preservation of renal function, and extension of life span. Immunohistochemical analysis revealed that USAG-1 and BMP-7 colocalized in the macula densa in the distal tubules, lying in direct contact with glomerular mesangial cells. Furthermore, in cultured mesangial cells, BMP-7 attenuated and USAG-1 enhanced the expression of MMP-12, a protease that may contribute to GBM degradation. These data suggest that the pathogenetic role of USAG-1 in Col4a3-/- mice might involve crosstalk between kidney tubules and the glomerulus and that inhibition of USAG-1 may be a promising therapeutic approach for the treatment of Alport syndrome.

  4. Mutational pattern of TP53 tumor suppressor gene in human lung cells exposed to air pollution PM{sub 2.5}; Spectre mutationnel de TP53 en reponse a une exposition in vitro a un aerosol atmospherique particulaire PM{sub 2,} {sub 5}

    Energy Technology Data Exchange (ETDEWEB)

    Billet, Sylvain; Paget, Vincent [Universite Lille Nord de France, Lille (France); Unite de Chimie Environnementale et Interactions sur le Vivant, Maison de la Recherche en Environnement Industriel, Universite du Littoral Cote d' Opale, Dunkerque (France); GRECAN, Universite de Caen Basse-Normandie et Centre Regional de Lutte Contre le Cancer Francois Baclesse, Caen (France); Garcon, Guillaume; Verdin, Anthony; Shirali, Pirouz [Universite Lille Nord de France, Lille (France); Unite de Chimie Environnementale et Interactions sur le Vivant, Maison de la Recherche en Environnement Industriel, Universite du Littoral Cote d' Opale, Dunkerque (France); Andre, Veronique; Heutte, Natacha; Sichel, Francois [GRECAN, Universite de Caen Basse-Normandie et Centre Regional de Lutte Contre le Cancer Francois Baclesse, Caen (France)

    2012-01-15

    Environmental exposure to fine airborne particulate matter (PM 2.5) is thought to be responsible for cardiopulmonary diseases, including lung cancer. However, the mechanisms of action potentially involved in PM{sub 2.5} toxicity are not yet fully described. Mutations in the TP53 gene are the most common alterations in human solid tumors. TP53 mutational patterns have sometimes been linked to carcinogen exposure. The purpose of this study was to determine the mutations that alter the functionality of this transcription factor in a model of human epithelial lung cells (A549) exposed to the fine particulate fraction (PM{sub 2.5}) of an atmospheric aerosol sampled under urban and industrial influences. PM{sub 2.5} was collected in Dunkerque City by cascade impaction. Its physicochemical characterization revealed the presence of many inorganic and organic compounds, including some that are known for their toxicity. The search for mutations altering the functionality of the P53 protein was performed 72 h after exposure of A549 cells to PM{sub 2.5} at its lethal concentration at 50% (LC{sub 50}, 118.60 {mu}g/mL = 31.63 {mu}g/cm{sup 2}), using the Functional Analysis of Separated Alleles in Yeast (FASAY). Sixteen mutations altering P53 function were detected after A549 cells exposure to the collected PM{sub 2.5}: eight deletions of one or two nucleotides and eight nucleotide substitutions, mainly transitions A > G and G > A. These mutations are described in the literature as possibly caused by endogenous mechanisms, such as oxidative stress. This kind of alteration can be induced by metal content of the PM{sub 2.5}, as well as by metabolic activation of the organic compounds coated onto its surface. Involvement of oxidative stress in TP53 mutations was confirmed by the detection of an oxidative DNA adduct, 8-hydroxy-2'-deoxyguanosine (8-OHdG), in A549 cells exposed to the collected PM. (authors)

  5. Development of a Targeted Next-Generation Sequencing Assay to Detect Diagnostically Relevant Mutations of JAK2, CALR, and MPL in Myeloproliferative Neoplasms.

    Science.gov (United States)

    Frawley, Thomas; O'Brien, Cathal P; Conneally, Eibhlin; Vandenberghe, Elisabeth; Percy, Melanie; Langabeer, Stephen E; Haslam, Karl

    2018-02-01

    The classical Philadelphia chromosome-negative myeloproliferative neoplasms (MPNs), consisting of polycythemia vera, essential thrombocythemia, and primary myelofibrosis, are a heterogeneous group of neoplasms that harbor driver mutations in the JAK2, CALR, and MPL genes. The detection of mutations in these genes has been incorporated into the recent World Health Organization (WHO) diagnostic criteria for MPN. Given a pressing clinical need to screen for mutations in these genes in a routine diagnostic setting, a targeted next-generation sequencing (NGS) assay for the detection of MPN-associated mutations located in JAK2 exon 14, JAK2 exon 12, CALR exon 9, and MPL exon 10 was developed to provide a single platform alternative to reflexive, stepwise diagnostic algorithms. Polymerase chain reaction (PCR) primers were designed to target mutation hotspots in JAK2 exon 14, JAK2 exon 12, MPL exon 10, and CALR exon 9. Multiplexed PCR conditions were optimized by using qualitative PCR followed by NGS. Diagnostic genomic DNA from 35 MPN patients, known to harbor driver mutations in one of the target genes, was used to validate the assay. One hundred percent concordance was observed between the previously-identified mutations and those detected by NGS, with no false positives, nor any known mutations missed (specificity = 100%, CI = 0.96, sensitivity = 100%, CI = 0.89). Improved resolution of mutation sequences was also revealed by NGS analysis. Detection of diagnostically relevant driver mutations of MPN is enhanced by employing a targeted multiplex NGS approach. This assay presents a robust solution to classical MPN mutation screening, providing an alternative to time-consuming sequential analyses.

  6. CDKL5 gene status in female patients with epilepsy and Rett-like features: two new mutations in the catalytic domain.

    Science.gov (United States)

    Maortua, Hiart; Martínez-Bouzas, Cristina; Calvo, María-Teresa; Domingo, Maria-Rosario; Ramos, Feliciano; García-Ribes, Ainhoa; Martínez, María-Jesús; López-Aríztegui, María-Asunción; Puente, Nerea; Rubio, Izaskun; Tejada, María-Isabel

    2012-08-06

    Mutations in the cyclin-dependent kinase-like 5 gene (CDKL5) located in the Xp22 region have been shown to cause a subset of atypical Rett syndrome with infantile spasms or early seizures starting in the first postnatal months. We performed mutation screening of CDKL5 in 60 female patients who had been identified as negative for the methyl CpG-binding protein 2 gene (MECP2) mutations, but who had current or past epilepsy, regardless of the age of onset, type, and severity. All the exons in the CDKL5 gene and their neighbouring sequences were examined, and CDKL5 rearrangements were studied by multiplex ligation-dependent probe amplification (MLPA). Six previously unidentified DNA changes were detected, two of which were disease-causing mutations in the catalytic domain: a frameshift mutation (c.509_510insGT; p.Glu170GlyfsX36) and a complete deletion of exon 10. Both were found in patients with seizures that started in the first month of life. This study demonstrated the importance of CDKL5 mutations as etiological factors in neurodevelopmental disorders, and indicated that a thorough analysis of the CDKL5 gene sequence and its rearrangements should be considered in females with Rett syndrome-like phenotypes, severe encephalopathy and epilepsy with onset before 5 months of age. This study also confirmed the usefulness of MLPA as a diagnostic screening method for use in clinical practice.

  7. CDKL5 gene status in female patients with epilepsy and Rett-like features: two new mutations in the catalytic domain

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    Maortua Hiart

    2012-08-01

    Full Text Available Abstract Background Mutations in the cyclin-dependent kinase-like 5 gene (CDKL5 located in the Xp22 region have been shown to cause a subset of atypical Rett syndrome with infantile spasms or early seizures starting in the first postnatal months. Methods We performed mutation screening of CDKL5 in 60 female patients who had been identified as negative for the methyl CpG-binding protein 2 gene (MECP2 mutations, but who had current or past epilepsy, regardless of the age of onset, type, and severity. All the exons in the CDKL5 gene and their neighbouring sequences were examined, and CDKL5 rearrangements were studied by multiplex ligation-dependent probe amplification (MLPA. Results Six previously unidentified DNA changes were detected, two of which were disease-causing mutations in the catalytic domain: a frameshift mutation (c.509_510insGT; p.Glu170GlyfsX36 and a complete deletion of exon 10. Both were found in patients with seizures that started in the first month of life. Conclusions This study demonstrated the importance of CDKL5 mutations as etiological factors in neurodevelopmental disorders, and indicated that a thorough analysis of the CDKL5 gene sequence and its rearrangements should be considered in females with Rett syndrome-like phenotypes, severe encephalopathy and epilepsy with onset before 5 months of age. This study also confirmed the usefulness of MLPA as a diagnostic screening method for use in clinical practice.

  8. A family with hereditary hemochromatosis carrying HFE gene splice site mutation: a case report

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    NING Huibin

    2017-01-01

    Full Text Available ObjectiveTo investigate a new type of HFE gene mutation in a family with hereditary hemochromatosis (HH. MethodsThe analysis of HFE gene was performed for one patient with a confirmed diagnosis of HH and five relatives. Blood genomic DNA was extracted and PCR multiplication was performed for the exon and intron splice sequences of related HFE, HJV, HAMP, transferrin receptor 2 (TfR2, and SLC40A1 genes. After agarose gel electrophoresis and purification, bi-directional direct sequencing was performed to detect mutation sites. ResultsThe proband had abnormal liver function and increases in serum iron, total iron binding capacity, serum ferritin, and transferrin saturation, as well as T→C homozygous mutation in the fourth base of intron 2 in the intervening sequence of the exon EXON2 of HFE gene (IVs 2+4T→C, C/C homozygous, splicing, abnormal. There were no abnormalities in HJV, HAMP, TfR2, and SLC40A1 genes. The proband′s son had the same homozygous mutation, three relatives had heterozygous mutations, and one relative had no abnormal mutations. ConclusionGene detection plays an important role in the diagnosis of hemochromatosis, and IVs 2+4T→C mutation may be a new pathogenic mutation for HH in China.

  9. Macrolides for KCNJ5-mutated aldosterone-producing adenoma (MAPA): design of a study for personalized diagnosis of primary aldosteronism.

    Science.gov (United States)

    Maiolino, Giuseppe; Ceolotto, Giulio; Battistel, Michele; Barbiero, Giulio; Cesari, Maurizio; Amar, Laurence; Caroccia, Brasilina; Padrini, Roberto; Azizi, Michel; Rossi, Gian Paolo

    2018-02-06

    Aldosterone-producing adenoma (APA) is the main curable cause of endocrine hypertension cause of primary aldosteronism (PA) and it is in up to 66% of all cases investigated with adrenal vein sampling (AVS). Mutations in the KCNJ5 potassium channel involve up to 70% of APA and cause the most florid PA phenotypes. The recent finding that macrolide antibiotics specifically inhibit in vitro the altered function of mutated KCNJ5 channels has opened new horizons for the diagnosis and treatment of APA with KCNJ5 mutations in that it can allow identification and target treatment of PA patients harbouring a mutated APA. Thus, we aimed at investigating if clarithromycin and roxithromycin, two macrolides that potently blunt mutated Kir3.4 channel function in vitro, affect plasma aldosterone concentration in adrenal vein blood during AVS and in peripheral blood, respectively, in PA patients with a mutated APA. We designed two proof of concept studies. In study A: consecutive patients with an unambiguous biochemical evidence of PA will be exposed to a single dose of 250 mg clarithromycin during AVS, to assess its effect on the relative aldosterone secretion index in adrenal vein blood from the gland with and without APA. In study B: consecutive hypertensive patients submitted to the work-up for hypertension will receive a single oral dose of 150 mg roxithromycin. The experimental endpoints will be the change induced by roxithromycin of plasma aldosterone concentration and other steroids, direct active renin concentration, serum K + , systolic and diastolic blood pressure. We expect to prove that: (i) clarithromycin allows identification of mutated APA before adrenalectomy and sequencing of tumour DNA; (ii) the acute changes of plasma aldosterone concentration, direct active renin concentration, and blood pressure in peripheral venous blood after roxithromycin can be a proxy for the presence of an APA with somatic mutations.

  10. Highly sensitive chemiluminescent point mutation detection by circular strand-displacement amplification reaction.

    Science.gov (United States)

    Shi, Chao; Ge, Yujie; Gu, Hongxi; Ma, Cuiping

    2011-08-15

    Single nucleotide polymorphism (SNP) genotyping is attracting extensive attentions owing to its direct connections with human diseases including cancers. Here, we have developed a highly sensitive chemiluminescence biosensor based on circular strand-displacement amplification and the separation by magnetic beads reducing the background signal for point mutation detection at room temperature. This method took advantage of both the T4 DNA ligase recognizing single-base mismatch with high selectivity and the strand-displacement reaction of polymerase to perform signal amplification. The detection limit of this method was 1.3 × 10(-16)M, which showed better sensitivity than that of most of those reported detection methods of SNP. Additionally, the magnetic beads as carrier of immobility was not only to reduce the background signal, but also may have potential apply in high through-put screening of SNP detection in human genome. Copyright © 2011 Elsevier B.V. All rights reserved.

  11. Distinct Clinicopathological Patterns of Mismatch Repair Status in Colorectal Cancer Stratified by KRAS Mutations.

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    Wenbin Li

    Full Text Available In sporadic colorectal cancer (CRC, the BRAFV600E mutation is associated with deficient mismatch repair (MMR status and inversely associated with to KRAS mutations. In contrast to deficient MMR (dMMR CRC, data on the presence of KRAS oncogenic mutations in proficient MMR (pMMR CRC and their relationship with tumor progression are scarce. We therefore examined the MMR status in combination with KRAS mutations in 913 Chinese patients and correlated the findings obtained with clinical and pathological features. The MMR status was determined based on detection of MLH1, MSH2, MSH6 and PMS2 expression. KRAS mutation and dMMR status were detected in 36.9% and 7.5% of cases, respectively. Four subtypes were determined by MMR and KRAS mutation status: KRAS (+/pMMR (34.0%, KRAS (+/dMMR (2.9%, KRAS (-/pMMR (58.5% and KRAS (-/dMMR (4.6%. A higher percentage of pMMR tumors with KRAS mutation were most likely to be female (49.0%, proximal located (45.5%, a mucinous histology (38.4%, and to have increased lymph node metastasis (60.3%, compared with pMMR tumors without BRAFV600E and KRAS mutations (36.0%, 29.3%, 29.4% and 50.7%, respectively; all P < 0.01. To the contrary, compared with those with KRAS(-/dMMR tumors, patients with KRAS(+/dMMR tumors demonstrated no statistically significant differences in gender, tumor location, pT depth of invasion, lymph node metastasis, pTNM stage, and histologic grade. This study revealed that specific epidemiologic and clinicopathologic characteristics are associated with MMR status stratified by KRAS mutation. Knowledge of MMR and KRAS mutation status may enhance molecular pathologic staging of CRC patients and metastatic progression in CRC can be estimated based on the combination of these biomarkers.

  12. Mutation spectrum of β-globin gene in thalassemia patients at Hasan Sadikin Hospital - West Java Indonesia.

    Science.gov (United States)

    Maskoen, Ani Melani; Rahayu, Nurul S; Reniarti, Lelani; Susanah, Susi; Laksono, Bremmy; Fauziah, Prima Nanda; Zada, Almira; Hidayat, Dadang S

    2017-12-30

    Thalassemia is the most common hereditary haemolytic anemia in Southeast Asia, in which Indonesia is among countries that are at a high risk for thalassemia. It has been reported that mutation in the beta-globin gene is responsible in severe Thalassemia. However, the spectrum of beta-globin gene mutations in Indonesian population varies in different regions . Thus, this study aimed to identify the most prevalent mutation of Thalassemia patients from the Hasan Sadikin Hospital, Bandung, using this as a reference hospital for Thalassemia in West Java. The three most prevalent mutations of beta globin (IVS1nt5, Cd26 (HbE), and IVS1nt1), were conducted in the beginning of this study. Mutations of 291 samples were detected by PCR-RFLP in the Molecular Genetic Laboratory, Faculty of Medicine Universitas Padjadjaran, Bandung. The prevalence of the beta globin gene mutation types were 47.4% IVS1nt5 homozygote, 9.9% compound heterozygote IVS1nt5/HbE, 5.4% compound heterozygote IVS1nt5/IVS1nt1, 1.4% compound heterozygote HbE/IVS1nt1, 1% HbE homozygote, 14.4% Compound heterzygote IVS1nt5/… (no paired mutation), 2.06% compound heterozygote HbE/… (no paired mutation), 1.3% compound heterozygote IVS1nt1/… (no paired mutation), and 7 samples were unidentified. The thalassemia mutation IVS1nt5 homozygote is the most common mutation found in Thalassemia patients at Hasan Sadikin Hospital, Bandung. The samples with unidentified results might carry mutations other than the three that are observed in the present study.

  13. Epidermal growth factor receptor mutations in lung adenocarcinoma in Malaysian patients.

    Science.gov (United States)

    Liam, Chong-Kin; Wahid, Mohamed Ibrahim A; Rajadurai, Pathmanathan; Cheah, Yoke-Kqueen; Ng, Tiffany Shi-Yeen

    2013-06-01

    Despite available data from other Asian countries, the prevalence of epidermal growth factor receptor (EGFR) mutations among lung adenocarcinoma patients has not been reported in Malaysia. This study sought to determine the frequency of EGFR mutations among multiethnic Malaysian patients diagnosed with lung adenocarcinoma. Demographic and clinical information of patients whose lung adenocarcinoma biopsy specimens were submitted for EGFR mutation testing at Sime Darby Medical Center from 2009 to 2011 were analyzed. EGFR mutations at exons 18, 19, 20, and 21 were detected either through bidirectional sequencing or real-time polymerase chain reaction. Among 812 patients in the study, 49% were female, 63.7% were ethnic Chinese, 29.4% Malay, 4.8% Indian, and 2.1% other ethnic groups. Mutations were present in the tumors of 321 patients (39.5%), with mutations at exons 19 (23.5%) and 21 (14.9%) being the most common. Mutations were significantly more frequent among women than in men (52.5% versus 27.8%, p < 0.001). Although mutations were more common among Chinese (40.8%) compared with Malay (37.2%) or Indian (33.3%) patients, the difference was not statistically significant (p = 0.591). Of 211 patients with smoking history records, never-smokers had a higher mutation rate compared with ever-smokers (54.8% versus 20.7%, p < 0.001). EGFR mutations were present in 39.5% of patients. Mutations were more common in women and never-smokers with no differences in mutation frequency between different ethnicities. Because of the high mutation rates, reflex testing for EGFR mutation should be a routine practice for advanced lung adenocarcinoma patients in Malaysia.

  14. Microarray-based mutation detection and phenotypic characterization in Korean patients with retinitis pigmentosa

    Science.gov (United States)

    Kim, Cinoo; Kim, Kwang Joong; Bok, Jeong; Lee, Eun-Ju; Kim, Dong-Joon; Oh, Ji Hee; Park, Sung Pyo; Shin, Joo Young; Lee, Jong-Young

    2012-01-01

    Purpose To evaluate microarray-based genotyping technology for the detection of mutations responsible for retinitis pigmentosa (RP) and to perform phenotypic characterization of patients with pathogenic mutations. Methods DNA from 336 patients with RP and 360 controls was analyzed using the GoldenGate assay with microbeads containing 95 previously reported disease-associated mutations from 28 RP genes. Mutations identified by microarray-based genotyping were confirmed by direct sequencing. Segregation analysis and phenotypic characterization were performed in patients with mutations. The disease severity was assessed by visual acuity, electroretinography, optical coherence tomography, and kinetic perimetry. Results Ten RP-related mutations of five RP genes (PRP3 pre-mRNA processing factor 3 homolog [PRPF3], rhodopsin [RHO], phosphodiesterase 6B [PDE6B], peripherin 2 [PRPH2], and retinitis pigmentosa 1 [RP1]) were identified in 26 of the 336 patients (7.7%) and in six of the 360 controls (1.7%). The p.H557Y mutation in PDE6B, which was homozygous in four patients and heterozygous in nine patients, was the most frequent mutation (2.5%). Mutation segregation was assessed in four families. Among the patients with missense mutations, the most severe phenotype occurred in patients with p.D984G in RP1; less severe phenotypes occurred in patients with p.R135W in RHO; a relatively moderate phenotype occurred in patients with p.T494M in PRPF3, p.H557Y in PDE6B, or p.W316G in PRPH2; and a mild phenotype was seen in a patient with p.D190N in RHO. Conclusions The results reveal that the GoldenGate assay may not be an efficient method for molecular diagnosis in RP patients with rare mutations, although it has proven to be reliable and efficient for high-throughput genotyping of single-nucleotide polymorphisms. The clinical features varied according to the mutations. Continuous effort to identify novel RP genes and mutations in a population is needed to improve the efficiency and

  15. Development and inter-laboratory validation of unlabeled probe melting curve analysis for detection of JAK2 V617F mutation in polycythemia vera.

    Science.gov (United States)

    Wu, Zhiyuan; Yuan, Hong; Zhang, Xinju; Liu, Weiwei; Xu, Jinhua; Zhang, Wei; Guan, Ming

    2011-01-01

    JAK2 V617F, a somatic point mutation that leads to constitutive JAK2 phosphorylation and kinase activation, has been incorporated into the WHO classification and diagnostic criteria of myeloid neoplasms. Although various approaches such as restriction fragment length polymorphism, amplification refractory mutation system and real-time PCR have been developed for its detection, a generic rapid closed-tube method, which can be utilized on routine genetic testing instruments with stability and cost-efficiency, has not been described. Asymmetric PCR for detection of JAK2 V617F with a 3'-blocked unlabeled probe, saturate dye and subsequent melting curve analysis was performed on a Rotor-Gene® Q real-time cycler to establish the methodology. We compared this method to the existing amplification refractory mutation systems and direct sequencing. Hereafter, the broad applicability of this unlabeled probe melting method was also validated on three diverse real-time systems (Roche LightCycler® 480, Applied Biosystems ABI® 7500 and Eppendorf Mastercycler® ep realplex) in two different laboratories. The unlabeled probe melting analysis could genotype JAK2 V617F mutation explicitly with a 3% mutation load detecting sensitivity. At level of 5% mutation load, the intra- and inter-assay CVs of probe-DNA heteroduplex (mutation/wild type) covered 3.14%/3.55% and 1.72%/1.29% respectively. The method could equally discriminate mutant from wild type samples on the other three real-time instruments. With a high detecting sensitivity, unlabeled probe melting curve analysis is more applicable to disclose JAK2 V617F mutation than conventional methodologies. Verified with the favorable inter- and intra-assay reproducibility, unlabeled probe melting analysis provided a generic mutation detecting alternative for real-time instruments.

  16. Development and inter-laboratory validation of unlabeled probe melting curve analysis for detection of JAK2 V617F mutation in polycythemia vera.

    Directory of Open Access Journals (Sweden)

    Zhiyuan Wu

    Full Text Available BACKGROUND: JAK2 V617F, a somatic point mutation that leads to constitutive JAK2 phosphorylation and kinase activation, has been incorporated into the WHO classification and diagnostic criteria of myeloid neoplasms. Although various approaches such as restriction fragment length polymorphism, amplification refractory mutation system and real-time PCR have been developed for its detection, a generic rapid closed-tube method, which can be utilized on routine genetic testing instruments with stability and cost-efficiency, has not been described. METHODOLOGY/PRINCIPAL FINDINGS: Asymmetric PCR for detection of JAK2 V617F with a 3'-blocked unlabeled probe, saturate dye and subsequent melting curve analysis was performed on a Rotor-Gene® Q real-time cycler to establish the methodology. We compared this method to the existing amplification refractory mutation systems and direct sequencing. Hereafter, the broad applicability of this unlabeled probe melting method was also validated on three diverse real-time systems (Roche LightCycler® 480, Applied Biosystems ABI® 7500 and Eppendorf Mastercycler® ep realplex in two different laboratories. The unlabeled probe melting analysis could genotype JAK2 V617F mutation explicitly with a 3% mutation load detecting sensitivity. At level of 5% mutation load, the intra- and inter-assay CVs of probe-DNA heteroduplex (mutation/wild type covered 3.14%/3.55% and 1.72%/1.29% respectively. The method could equally discriminate mutant from wild type samples on the other three real-time instruments. CONCLUSIONS: With a high detecting sensitivity, unlabeled probe melting curve analysis is more applicable to disclose JAK2 V617F mutation than conventional methodologies. Verified with the favorable inter- and intra-assay reproducibility, unlabeled probe melting analysis provided a generic mutation detecting alternative for real-time instruments.

  17. One-step isothermal detection of multiple KRAS mutations by forming SNP specific hairpins on a gold nanoshell.

    Science.gov (United States)

    Chung, Chan Ho; Kim, Joong Hyun

    2018-04-24

    We developed a one-step isothermal method for typing multiple KRAS mutations using a designed set of primers to form a hairpin on a gold nanoshell upon being ligated by a SNP specific DNA ligase after binding of targets. As a result, we could detect as low as 20 attomoles of KRAS mutations within 1 h.

  18. A Restricted Spectrum of Mutations in the SMAD4 Tumor-Suppressor Gene Underlies Myhre Syndrome

    Science.gov (United States)

    Caputo, Viviana; Cianetti, Luciano; Niceta, Marcello; Carta, Claudio; Ciolfi, Andrea; Bocchinfuso, Gianfranco; Carrani, Eugenio; Dentici, Maria Lisa; Biamino, Elisa; Belligni, Elga; Garavelli, Livia; Boccone, Loredana; Melis, Daniela; Andria, Generoso; Gelb, Bruce D.; Stella, Lorenzo; Silengo, Margherita; Dallapiccola, Bruno; Tartaglia, Marco

    2012-01-01

    Myhre syndrome is a developmental disorder characterized by reduced growth, generalized muscular hypertrophy, facial dysmorphism, deafness, cognitive deficits, joint stiffness, and skeletal anomalies. Here, by performing exome sequencing of a single affected individual and coupling the results to a hypothesis-driven filtering strategy, we establish that heterozygous mutations in SMAD4, which encodes for a transducer mediating transforming growth factor β and bone morphogenetic protein signaling branches, underlie this rare Mendelian trait. Two recurrent de novo SMAD4 mutations were identified in eight unrelated subjects. Both mutations were missense changes altering Ile500 within the evolutionary conserved MAD homology 2 domain, a well known mutational hot spot in malignancies. Structural analyses suggest that the substituted residues are likely to perturb the binding properties of the mutant protein to signaling partners. Although SMAD4 has been established as a tumor suppressor gene somatically mutated in pancreatic, gastrointestinal, and skin cancers, and germline loss-of-function lesions and deletions of this gene have been documented to cause disorders that predispose individuals to gastrointestinal cancer and vascular dysplasias, the present report identifies a previously unrecognized class of mutations in the gene with profound impact on development and growth. PMID:22243968

  19. A case of lung adenocarcinoma harboring EGFR mutation and EML4-ALK fusion gene

    International Nuclear Information System (INIS)

    Tanaka, Hisashi; Hayashi, Akihito; Morimoto, Takeshi; Taima, Kageaki; Tanaka, Yoshihito; Shimada, Michiko; Kurose, Akira; Takanashi, Shingo; Okumura, Ken

    2012-01-01

    Lung cancer is the leading cause of cancer-related death worldwide. Epidermal growth factor receptor (EGFR) - tyrosine kinase inhibitor (TKI) is used for the patients with EGFR-mutant lung cancer. Recently, phase III studies in the patients with EGFR-mutant demonstrated that EGFR-TKI monotherapy improved progression-free survival compared with platinum-doublet chemotherapy. The echinoderm microtubule-associated protein-like 4 (EML4) - anaplastic lymphoma kinase (ALK) fusion oncogene represents one of the newest molecular targets in non-small cell lung cancer (NSCLC). Patients who harbor EML4-ALK fusions have been associated with a lack of EGFR or KRAS mutations. We report a 39-year-old patient diagnosed as adenocarcinoma harboring EGFR mutation and EML4-ALK fusion gene. We treated this patient with erlotinib as the third line therapy, but no clinical benefit was obtained. We experienced a rare case with EGFR mutation and EML4-ALK. Any clinical benefit using EGFR-TKI was not obtained in our case. The therapeutic choice for the patients with more than one driver mutations is unclear. We needs further understanding of the lung cancer molecular biology and the biomarker infomation

  20. Can Genetics Predict Sports Injury? The Association of the Genes GDF5, AMPD1, COL5A1 and IGF2 on Soccer Player Injury Occurrence

    Directory of Open Access Journals (Sweden)

    Kiah McCabe

    2018-03-01

    Full Text Available Genetics plays an integral role in athletic performance and is increasingly becoming recognised as an important risk factor for injury. Ankle and knee injuries are the most common injuries sustained by soccer players. Often these injuries result in players missing training and matches, which can incur significant costs to clubs. This study aimed to identify genotypes associated with ankle and knee injuries in soccer players and how these impacted the number of matches played. 289 soccer players, including 46 professional, 98 semi-professional and 145 amateur players, were genetically tested. Ankle and knee injuries and the number of matches played were recorded during the 2014/15 season. Four genes were assessed in relation to injury. Genotypes found to be associated with injury included the TT (nucleobase genotype of the GDF5 gene, TT and CT (nucleobase genotypes of AMPD1 gene, TT genotype of COL5A1 and GG (nucleobase genotype of IGF2 gene. These genes were also associated with a decrease in the number of matches played.

  1. COL-3, a chemically modified tetracycline, inhibits lipopolysaccharide-induced microglia activation and cytokine expression in the brain.

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    Rawan Abdulhameed Edan

    Full Text Available Microglia activation results in release of proinflammatory molecules including cytokines, which contribute to neuronal damage in the central nervous system (CNS if not controlled. Tetracycline antibiotics such as minocycline inhibit microglial activation and cytokine expression during CNS inflammation. In the present study we found that administration of chemically modified tetracycline-3 (COL-3, inhibits lipopolysaccharide (LPS-induced microglial and p38 MAPK activation, as well as the increase in TNF-α, but not IL-1β expression, in the brains of BALB/c mice. COL-3 has been described to have no antibacterial activity. We observed that COL-3 had no activity against a Gram-negative bacteria, Escherichia coli; however surprisingly, COL-3 had antibacterial activity against a Gram-positive bacteria Staphylococcus aureus, with a minimum inhibitory concentration of 1 mg/ml. Our data show that COL-3 has some antibacterial activity against S. aureus, inhibits LPS-induced neuroinflammation, and displays potential as a therapeutic agent for treatment of conditions involving CNS inflammation.

  2. Association of a homozygous nonsense mutation in the ABCA4 (ABCR) gene with cone-rod dystrophy phenotype in an Italian family.

    Science.gov (United States)

    Simonelli, Francesca; Testa, Francesco; Zernant, Jana; Nesti, Anna; Rossi, Settimio; Rinaldi, Ernesto; Allikmets, Rando

    2004-01-01

    Genetic variation in the ABCA4 (ABCR) gene has been associated with several distinct retinal phenotypes, including Stargardt disease/fundus flavimaculatus (STGD/FFM), cone-rod dystrophy (CRD), retinitis pigmentosa (RP) and age-related macular degeneration. The current model of genotype/phenotype association suggests that patients harboring deleterious mutations in both ABCR alleles would develop RP-like retinal pathology. Here we describe ABCA4-associated phenotypes, including a proband with a homozygous nonsense mutation in a family from Southern Italy. The proband had been originally diagnosed with STGD. Ophthalmologic examination included kinetic perimetry, electrophysiological studies and fluorescein angiography. DNA of the affected individual and family members was analyzed for variants in all 50 exons of the ABCA4 gene by screening on the ABCR400 microarray. A homozygous nonsense mutation 2971G>T (G991X) was detected in a patient initially diagnosed with STGD based on funduscopic evidence, including bull's eye depigmentation of the fovea and flecks at the posterior pole extending to the mid-peripheral retina. Since this novel nucleotide substitution results in a truncated, nonfunctional, ABCA4 protein, the patient was examined in-depth for the severity of the disease phenotype. Indeed, subsequent electrophysiological studies determined severely reduced cone amplitude as compared to the rod amplitude, suggesting the diagnosis of CRD. ABCR400 microarray is an efficient tool for determining causal genetic variation, including new mutations. A homozygous protein-truncating mutation in ABCA4 can cause a phenotype ranging from STGD to CRD as diagnosed at an early stage of the disease. Only a combination of comprehensive genotype/phenotype correlation studies will determine the proper diagnosis and prognosis of ABCA4-associated pathology. Copyright 2004 S. Karger AG, Basel

  3. Common Β- Thalassaemia Mutations in

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    P Azarfam

    2005-01-01

    Full Text Available Introduction: β –Thalassaemia was first explained by Thomas Cooly as Cooly’s anaemia in 1925. The β- thalassaemias are hereditary autosomal disorders with decreased or absent β-globin chain synthesis. The most common genetic defects in β-thalassaemias are caused by point mutations, micro deletions or insertions within the β-globin gene. Material and Methods: In this research , 142 blood samples (64 from childrens hospital of Tabriz , 15 samples from Shahid Gazi hospital of Tabriz , 18 from Urumia and 45 samples from Aliasghar hospital of Ardebil were taken from thalassaemic patients (who were previously diagnosed .Then 117 non-familial samples were selected . The DNA of the lymphocytes of blood samples was extracted by boiling and Proteinase K- SDS procedure, and mutations were detected by ARMS-PCR methods. Results: From the results obtained, eleven most common mutations,most of which were Mediterranean mutations were detected as follows; IVS-I-110(G-A, IVS-I-1(G-A ،IVS-I-5(G-C ,Frameshift Codon 44 (-C,( codon5(-CT,IVS-1-6(T-C, IVS-I-25(-25bp del ,Frameshift 8.9 (+G ,IVS-II-1(G-A ,Codon 39(C-T, Codon 30(G-C the mutations of the samples were defined. The results showed that Frameshift 8.9 (+G, IVS-I-110 (G-A ,IVS-II-I(G-A, IVS-I-5(G-C, IVS-I-1(G-A , Frameshift Codon 44(-C , codon5(-CT , IVS-1-6(T-C , IVS-I-25(-25bp del with a frequency of 29.9%, 25.47%,17.83%, 7.00%, 6.36% , 6.63% , 3.8% , 2.5% , 0.63% represented the most common mutations in North - west Iran. No mutations in Codon 39(C-T and Codon 30(G-C were detected. Cunclusion: The frequency of the same mutations in patients from North - West of Iran seems to be different as compared to other regions like Turkey, Pakistan, Lebanon and Fars province of Iran. The pattern of mutations in this region is more or less the same as in the Mediterranean region, but different from South west Asia and East Asia.

  4. Upper bound on the non-colorability threshold of the 2+p-COL problem

    International Nuclear Information System (INIS)

    Cruz, R.

    2004-10-01

    The 2p-COL problem introduced by Walsh, smoothly interpolates between P and NP by mixing together the polynornial 2-coloring problem and the NP complete 3-coloring problem. A natural upper bound on the non colorability of the 2+p-COL problem in min {r-bar 2 /(1 - p)r-bar 3 }, where r-bar 2 and r-bar 3 are the upper bounds on 2-COL and 3-COL thresholds respectively. In this paper we improve this upper-bound for each 0.73 ≤ p 1. This means that for p ≥ 0.73 the 2+p-COL problem does not behave like the 2-COL problem. We use the method developed by Kaporis et al., which combines the concept of legal rigid colorings introduced by Achlioptas and Molloy with the occupancy problem for random allocations of balls into bins. (author)

  5. Prognostic implications of c-Ki-ras2 mutations in patients with advanced colorectal cancer treated with 5-fluorouracil and interferon: a study of the eastern cooperative oncology group (EST 2292)

    Science.gov (United States)

    Wadler, S; Bajaj, R; Neuberg, D; Agarwal, V; Haynes, H; Benson, A B

    1997-01-01

    Mutations in c-Ki-ras2 (ras) occur in about 40% of patients with colorectal cancers and occur early in the pathogenesis of this disease. To evaluate the prognostic value of mutations in ras, the Eastern Cooperative Oncology Group (ECOG) conducted a retrospective study (EST 2292) to determine the frequency of mutations in patients with advanced colorectal cancer, and to determine whether ras mutations were associated with altered response to therapy and survival. Patients were enrolled from four studies: P-Z289, an ECOG phase II trial of 5-fluorouracil (5-FU) and interferon (IFN) in patients with advanced colorectal cancer; P-Z991, an ECOG phase I trial of 5-FU and IFN in patients with advanced malignancies; and two trials from the Albert Einstein College of Medicine in patients with advanced colorectal cancer treated with 5-FU and either IFN-alpha or IFN-beta. All patients had advanced colorectal carcinoma and had sufficient histologic material available for analysis for the presence and type of ras, using polymerase chain reaction and dot-blot analysis with sets of probes sufficient to detect all the common mutations of ras at codons 12, 13, and 61. Seventy-two patients were enrolled in this trial. Mutations in ras were detected in 25 (35%), including 17 (23%) in codon 12, four (6%) in codon 13, and four (6%) in codon 61. There was no correlation between the presence of a ras mutation and age, sex, Dukes' stage, histology, or tumor markers. Thirty-one of 72 patients (43%) responded to therapy with 5-FU and IFN, and 10 of 31 responders (32%) and 15 of 41 nonresponders (37%) had mutations in ras. There was no difference in response rates or overall survival between the groups with and without ras mutations. It is unlikely that ras mutations will have significant prognostic value for either response to therapy or survival in patients with colorectal carcinomas treated with 5-FU and IFN.

  6. The ColM Family, Polymorphic Toxins Breaching the Bacterial Cell Wall

    Directory of Open Access Journals (Sweden)

    Maarten G. K. Ghequire

    2018-02-01

    Full Text Available Bacteria host an arsenal of antagonism-mediating molecules to combat for ecologic space. Bacteriocins represent a pivotal group of secreted antibacterial peptides and proteins assisting in this fight, mainly eliminating relatives. Colicin M, a model for peptidoglycan-interfering bacteriocins in Gram-negative bacteria, appears to be part of a set of polymorphic toxins equipped with such a catalytic domain (ColM targeting lipid II. Diversifying recombination has enabled parasitism of different receptors and has also given rise to hybrid bacteriocins in which ColM is associated with another toxin module. Remarkably, ColM toxins have recruited a diverse array of immunity partners, comprising cytoplasmic membrane-associated proteins with different topologies. Together, these findings suggest that different immunity mechanisms have evolved for ColM, in contrast to bacteriocins with nuclease activities.

  7. The 95ΔG mutation in the 5'untranslated region of the norA gene increases efflux activity in Staphylococcus epidermidis isolates.

    Science.gov (United States)

    García-Gómez, Elizabeth; Jaso-Vera, Marcos E; Juárez-Verdayes, Marco A; Alcántar-Curiel, María D; Zenteno, Juan C; Betanzos-Cabrera, Gabriel; Peralta, Humberto; Rodríguez-Martínez, Sandra; Cancino-Díaz, Mario E; Jan-Roblero, Janet; Cancino-Diaz, Juan C

    2017-02-01

    In the Staphylococcus aureus ATCC25923 strain, the flqB mutation in the 5'untranslated region (5'UTR) of the norA gene causes increased norA mRNA expression and high efflux activity (HEA). The involvement of the norA gene 5'UTR in HEA has not been explored in S. epidermidis; therefore, we examined the function of this region in S. epidermidis clinical isolates. The selection of isolates with HEA was performed based on ethidium bromide (EtBr) MIC values and efflux efficiency (EF) using the semi-automated fluorometric method. The function of the 5'UTR was studied by quantifying the levels of norA expression (RT-qPCR) and by identifying 5'UTR mutations by sequence analysis. Only 10 isolates from a total of 165 (6.1%) had HEA (EtBr MIC = 300 μg/ml and EF ranged from 48.4 to 97.2%). Eight of 10 isolates with HEA had the 5'UTR 95 Δ G mutation. Isolates carrying the 95 Δ G mutation had higher levels of norA expression compared with those that did not. To corroborate that the 95 Δ G mutation is involved in HEA, a strain adapted to EtBr was obtained in vitro. This strain also presented the 95 Δ G mutation and had a high level of norA expression and EF, indicating that the 95 Δ G mutation is important for the HEA phenotype. The 95 Δ G mutation produces a different structure in the Shine-Dalgarno region, which may promote better translation of norA mRNA. To our knowledge, this is the first report to demonstrate the participation of the 5'UTR 95 Δ G mutation of the norA gene in the HEA phenotype of S. epidermidis isolates. Here, we propose that the efflux of EtBr is caused by an increment in the transcription and/or translation of the norA gene. Copyright © 2016 Elsevier Ltd. All rights reserved.

  8. Whole-exome sequencing of muscle-invasive bladder cancer identifies recurrent mutations of UNC5C and prognostic importance of DNA repair gene mutations on survival.

    Science.gov (United States)

    Yap, Kai Lee; Kiyotani, Kazuma; Tamura, Kenji; Antic, Tatjana; Jang, Miran; Montoya, Magdeline; Campanile, Alexa; Yew, Poh Yin; Ganshert, Cory; Fujioka, Tomoaki; Steinberg, Gary D; O'Donnell, Peter H; Nakamura, Yusuke

    2014-12-15

    Because of suboptimal outcomes in muscle-invasive bladder cancer even with multimodality therapy, determination of potential genetic drivers offers the possibility of improving therapeutic approaches and discovering novel prognostic indicators. Using pTN staging, we case-matched 81 patients with resected ≥pT2 bladder cancers for whom perioperative chemotherapy use and disease recurrence status were known. Whole-exome sequencing was conducted in 43 cases to identify recurrent somatic mutations and targeted sequencing of 10 genes selected from the initial screening in an additional 38 cases was completed. Mutational profiles along with clinicopathologic information were correlated with recurrence-free survival (RFS) in the patients. We identified recurrent novel somatic mutations in the gene UNC5C (9.9%), in addition to TP53 (40.7%), KDM6A (21.0%), and TSC1 (12.3%). Patients who were carriers of somatic mutations in DNA repair genes (one or more of ATM, ERCC2, FANCD2, PALB2, BRCA1, or BRCA2) had a higher overall number of somatic mutations (P = 0.011). Importantly, after a median follow-up of 40.4 months, carriers of somatic mutations (n = 25) in any of these six DNA repair genes had significantly enhanced RFS compared with noncarriers [median, 32.4 vs. 14.8 months; hazard ratio of 0.46, 95% confidence interval (CI), 0.22-0.98; P = 0.0435], after adjustment for pathologic pTN staging and independent of adjuvant chemotherapy usage. Better prognostic outcomes of individuals carrying somatic mutations in DNA repair genes suggest these mutations as favorable prognostic events in muscle-invasive bladder cancer. Additional mechanistic investigation into the previously undiscovered role of UNC5C in bladder cancer is warranted. ©2014 American Association for Cancer Research.

  9. Circular RNA profiling reveals that circCOL3A1-859267 regulate type I collagen expression in photoaged human dermal fibroblasts

    International Nuclear Information System (INIS)

    Peng, Yating; Song, Xiaojing; Zheng, Yue; Wang, Xinyi; Lai, Wei

    2017-01-01

    Production of type I collagen declines is a main characteristic during photoaging, but the mechanism is still not fully understood. Circular RNAs (circRNAs) are a class of newly identified non-coding RNAs with regulatory potency by sequestering miRNAs like a sponge. It's more stable than linear RNAs, and would be a useful tool for regulation of gene expression. However, the role of circRNAs in collagen expression during photoaging is still unclear. Here we performed deep sequencing of RNA generated from UVA irradiated and no irradiated human dermal fibroblasts (HDFs) and identified 29 significantly differentially expressed circRNAs (fold change ≥ 1.5, P < 0.05), 12 circRNAs were up-regulated and 17 circRNAs were down-regulated.3 most differentially expressed circRNAs were verified by qRT-PCR and the down-regulated circCOL3A1-859267 exhibited the most significantly altered in photoaged HDFs. Overexpression of circCOL3A1-859267 inhibited UVA-induced decrease of type I collagen expression and silencing of it reduced type I collagen intensity. Via a bioinformatic method, 44 miRNAs were predicted to binding with circCOL3A1-859267, 5 of them have been confirmed or predicted to interact with type I collagen. This study show that circCOL3A1-859267 regulate type I collagen expression in photoaged HDFs, suggesting it may be a novel target for interfering photoaging.

  10. Hearing-loss-associated gene detection in neonatal intensive care unit.

    Science.gov (United States)

    Yang, S M; Liu, Ying; Liu, C; Yin, A H; Wu, Y F; Zheng, X E; Yang, H M; Yang, J

    2018-02-01

    To investigate the frequency and mutation spectrum of hearing loss-associated gene mutation in Neonatal Intensive Care Unit (NICU). Neonates (n=2305) admitted to NICU were enrolled in this study. Nine prominent hearing loss-associated genes, GJB2 (35 del G, 176 del 16,235 del C, 299 del AT), GJB3 (538 C > T), SLC26A4 (IVS7-2A > G, 2168 A > G) and mtDNA 12S rRNA(1555 A > G, 1494 C > T), were detected. There were 73 cases hearing-loss-associated gene mutation among 2305 cases, the mutation frequency was 3.1%, with 40 cases GJB2 (235del C) mutation (54.8%), 6 cases GJB2 (299 del AT) mutation (8.2%), 21 cases SLC26A4 (IVS 7-2 A > G) mutation (28.7%), 4 cases SLC26A4 (2168 A > G) mutation (5.5%), 2 cases of GJB2 (235del C) combined SLC26A4 (IVS 7-2 A > G, 2168 A > G) mutation (2.8%). Among 73 gene mutation cases, preterm neonates presented in 18 cases, accounting for 24.7% (18/73); hyperbilirubinemia in 13 cases, accounting for 17.8% (13/73); Torch Syndrome in 15 cases, with 12 cases CMV, 2 cases rubella, 1 case toxoplasm, respectively, totally accounting for 20.54% (15/73); neonatal pneumonia in 12 cases, accounting for 16.4% (12/73); birth asphyxia in 5 cases, accounting for 6.9% (5/73); sepsis in 5 cases, accounting for 6.9% (5/73); others in 5 cases, accounting for 6.8% (5/73) . The frequency of hearing loss-associated gene mutation was higher in NICU.There were hearing loss-associated gene mutations in the NICU, suggesting this mutation may complicate with perinatal high-risk factors.

  11. Presymptomatic breast cancer in Egypt: role of BRCA1 and BRCA2 tumor suppressor genes mutations detection

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    Hashishe Mervat M

    2010-06-01

    Full Text Available Abstract Background Breast cancer is one of the most common diseases affecting women. Inherited susceptibility genes, BRCA1 and BRCA2, are considered in breast, ovarian and other common cancers etiology. BRCA1 and BRCA2 genes have been identified that confer a high degree of breast cancer risk. Objective Our study was performed to identify germline mutations in some exons of BRCA1 and BRCA2 genes for the early detection of presymptomatic breast cancer in females. Methods This study was applied on Egyptian healthy females who first degree relatives to those, with or without a family history, infected with breast cancer. Sixty breast cancer patients, derived from 60 families, were selected for molecular genetic testing of BRCA1 and BRCA2 genes. The study also included 120 healthy first degree female relatives of the patients, either sisters and/or daughters, for early detection of presymptomatic breast cancer mutation carriers. Genomic DNA was extracted from peripheral blood lymphocytes of all the studied subjects. Universal primers were used to amplify four regions of the BRCA1 gene (exons 2,8,13 and 22 and one region (exon 9 of BRCA2 gene using specific PCR. The polymerase chain reaction was carried out. Single strand conformation polymorphism assay and heteroduplex analysis were used to screen for mutations in the studied exons. In addition, DNA sequencing of the normal and mutated exons were performed. Results Mutations in both BRCA1 and BRCA2 genes were detected in 86.7% of the families. Current study indicates that 60% of these families were attributable to BRCA1 mutations, while 26.7% of them were attributable to BRCA2 mutations. Results showed that four mutations were detected in the BRCA1 gene, while one mutation was detected in the BRCA2 gene. Asymptomatic relatives, 80(67% out of total 120, were mutation carriers. Conclusions BRCA1 and BRCA2 genes mutations are responsible for a significant proportion of breast cancer. BRCA mutations

  12. Application of a New Genetic Deafness Microarray for Detecting Mutations in the Deaf in China.

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    Hong Wu

    Full Text Available The aim of this study was to evaluate the GoldenGate microarray as a diagnostic tool and to elucidate the contribution of the genes on this array to the development of both nonsyndromic and syndromic sensorineural hearing loss in China.We developed a microarray to detect 240 mutations underlying syndromic and nonsyndromic sensorineural hearing loss. The microarray was then used for analysis of 382 patients with nonsyndromic sensorineural hearing loss (including 15 patients with enlarged vestibular aqueduct syndrome, 21 patients with Waardenburg syndrome, and 60 unrelated controls. Subsequently, we analyzed the sensitivity, specificity, and reproducibility of this new approach after Sanger sequencing-based verification, and also determined the contribution of the genes on this array to the development of distinct hearing disorders.The sensitivity and specificity of the microarray chip were 98.73% and 98.34%, respectively. Genetic defects were identified in 61.26% of the patients with nonsyndromic sensorineural hearing loss, and 9 causative genes were identified. The molecular etiology was confirmed in 19.05% and 46.67% of the patients with Waardenburg syndrome and enlarged vestibular aqueduct syndrome, respectively.Our new mutation-based microarray comprises an accurate and comprehensive genetic tool for the detection of sensorineural hearing loss. This microarray-based detection method could serve as a first-pass screening (before next-generation-sequencing screening for deafness-causing mutations in China.

  13. CHROMagar COL-APSE: a selective bacterial culture medium for the isolation and differentiation of colistin-resistant Gram-negative pathogens

    DEFF Research Database (Denmark)

    Abdul Momin, Muhd Haziq F; Bean, David C; Hendriksen, Rene S.

    2017-01-01

    Purpose. A selective chromogenic culture medium for the laboratory isolation and differentiation of colistin resistant Acinetobacter, Pseudomonas, Stenotrophomonas and Enterobacteriaceae spp. (CHROMagar COL-APSE) was developed, evaluated and compared to an existing selective bacterial culture......-resistant non-fermentative bacteria (Acinetobacter, Pseudomonas and Stenotrophomonas). CHROMagar COL-APSE was also more sensitive in supporting the growth of Enterobacteriaceae with COL resistance associated with the carriage of mcr-1. Conclusion. CHROMagar COL-APSE is a sensitive and specific medium...

  14. Screening for germline mutations of MLH1, MSH2, MSH6 and PMS2 genes in Slovenian colorectal cancer patients: implications for a population specific detection strategy of Lynch syndrome.

    Science.gov (United States)

    Berginc, Gasper; Bracko, Matej; Ravnik-Glavac, Metka; Glavac, Damjan

    2009-01-01

    Microsatellite instability (MSI) is present in more than 90% of colorectal cancers of patients with Lynch syndrome, and is therefore a feasible marker for the disease. Mutations in MLH1, MSH2, MSH6 and PMS2, which are one of the main causes of deficient mismatch repair and subsequent MSI, have been linked to the disease. In order to establish the role of each of the 4 genes in Slovenian Lynch syndrome patients, we performed MSI analysis on 593 unselected CRC patients and subsequently searched for the presence of point mutations, larger genomic rearrangements and MLH1 promoter hypermethylation in patients with MSI-high tumours. We detected 43 (7.3%) patients with MSI-H tumours, of which 7 patients (1.3%) harboured germline defects: 2 in MLH1, 4 in MSH2, 1 in PMS2 and none in MSH6. Twenty-nine germline sequence variations of unknown significance and 17 deleterious somatic mutations were found. MLH1 promoter methylation was detected in 56% of patients without detected germline defects and in 1 (14%) suspected Lynch syndrome. Due to the minor role of germline MSH6 mutations, we adapted the Lynch syndrome detection strategy for the Slovenian population of CRC patients, whereby germline alterations should be first sought in MLH1 and MSH2 followed by a search for larger genomic rearrangements in these two genes. When no germline mutations are found tumors should be further tested for the presence of germline defects in PMS2 and MSH6. The choice about which gene should be tested first can be guided more accurately by the immunohistochemical analysis. Our study demonstrates that the incidence of MMR mutations in a population should be known prior to the application of one of several suggested strategies for detection of Lynch syndrome.

  15. Sick sinus syndrome, progressive cardiac conduction disease, atrial flutter and ventricular tachycardia caused by a novel SCN5A mutation

    DEFF Research Database (Denmark)

    Holst, Anders G; Liang, Bo; Jespersen, Thomas

    2010-01-01

    father carried the same mutation, but had a milder phenotype, presenting with progressive cardiac conduction later in life. The mutation was found to result in a loss-of-function in the sodium current. In conclusion, the same SCN5A mutation can result in a wide array of clinical phenotypes and perhaps......Mutations in the cardiac sodium channel encoded by the gene SCN5A can result in a wide array of phenotypes. We report a case of a young male with a novel SCN5A mutation (R121W) afflicted by sick sinus syndrome, progressive cardiac conduction disorder, atrial flutter and ventricular tachycardia. His...

  16. The use of FTA cards for transport and detection of gyrA mutation of Campylobacter jejuni from poultry.

    Science.gov (United States)

    Sierra-Arguello, Y M; Faulkner, O; Tellez, G; Hargis, B M; Pinheiro do Nascimento, V

    2016-04-01

    The purpose of the present study was to evaluate a technique involving the use of commercially available FTA classic card (Whatman) for transporting and detection of DNA to use in PCR analysis and genetic sequencing of Campylobacter jejuni of poultry origin. Fifty isolates of Campylobacter jejuni were obtained from broiler carcasses in Rio Grande do Sul, Brazil. Antimicrobial susceptibility testing to ciprofloxacin revealed that all 50 isolates were resistant to ciprofloxacin. Each isolate was transferred to Brucella broth tubes and incubated overnight at 41.5°C. Cell cultures were diluted to match a McFarland Turbidity Standard 0.5, and 110 μL of the cell suspension were applied to one circle on Whatman FTA classic cards. The samples were then covered and allowed to dry at room temperature. Cards were identified and stored at room temperature until further use (3 mo after collection). FTA cards were shipped for analysis to the Department of Poultry Science, University of Arkansas. Amplification of the Campylobacter gyrA gene was successful and demonstrated strong bands for a large amplicon for all 50 samples preserved on FTA cards. Mutations present in each gene were confirmed by DNA sequencing. Then, 7 samples were chosen for the sequencing. The detection of a mutation regarding ciprofloxacin-resistant isolates revealed that 7 samples had a mutation in the gyrA gene. In conclusion, the characteristics of the profiles suggest that the DNA has maintained its integrity after 3 mo of storage at room temperature and is a suitable template for PCR and sequencing from Campylobacter samples. The application of this technology has potential in numerous methodologies, especially when working in remote areas and in developing countries where access to laboratory facilities and equipment is limited. © 2016 Poultry Science Association Inc.

  17. Naturally occurring mutations associated with resistance to HCV NS5B polymerase and NS3 protease inhibitors in treatment-naïve patients with chronic hepatitis C.

    Science.gov (United States)

    Costantino, Angela; Spada, Enea; Equestre, Michele; Bruni, Roberto; Tritarelli, Elena; Coppola, Nicola; Sagnelli, Caterina; Sagnelli, Evangelista; Ciccaglione, Anna Rita

    2015-11-14

    The detection of baseline resistance mutations to new direct-acting antivirals (DAAs) in HCV chronically infected treatment-naïve patients could be important for their management and outcome prevision. In this study, we investigated the presence of mutations, which have been previously reported to be associated with resistance to DAAs in HCV polymerase (NS5B) and HCV protease (NS3) regions, in sera of treatment-naïve patients. HCV RNA from 152 naïve patients (84 % Italian and 16 % immigrants from various countries) infected with different HCV genotypes (21,1a; 21, 1b; 2, 2a; 60, 2c; 22, 3a; 25, 4d and 1, 4k) was evaluated for sequence analysis. Amplification and sequencing of fragments in the NS5B (nt 8256-8640) and NS3 (nt 3420-3960) regions of HCV genome were carried out for 152 and 28 patients, respectively. The polymorphism C316N/H in NS5B region, associated with resistance to sofosbuvir, was detected in 9 of the 21 (43 %) analysed sequences from genotype 1b-infected patients. Naturally occurring mutations V36L, and M175L in the NS3 protease region were observed in 100 % of patients infected with subtype 2c and 4. A relevant proportion of treatment naïve genotype 1b infected patients evaluated in this study harboured N316 polymorphism and might poorly respond to sofosbuvir treatment. As sofosbuvir has been approved for treatment of HCV chronic infection in USA and Europe including Italy, pre-treatment testing for N316 polymorphism on genotype 1b naïve patients should be considered for this drug.

  18. The DNA-mimic antirestriction proteins ArdA ColIB-P9, Arn T4, and Ocr T7 as activators of H-NS-dependent gene transcription.

    Science.gov (United States)

    Melkina, Olga E; Goryanin, Ignatiy I; Zavilgelsky, Gennadii B

    2016-11-01

    The antirestriction proteins ArdA ColIb-P9, Arn T4 and Ocr T7 specifically inhibit type I and type IV restriction enzymes and belong to the family of DNA-mimic proteins because their three-dimensional structure is similar to the double-helical B-form DNA. It is proposed that the DNA-mimic proteins are able to bind nucleoid protein H-NS and alleviate H-NS-silencing of the transcription of bacterial genes. Escherichia coli lux biosensors were constructed by inserting H-NS-dependent promoters into a vector, thereby placing each fragment upstream of the promoterless Photorhabdus luminescens luxCDABE operon. It was demonstrated that the DNA-mimic proteins ArdA, Arn and Ocr activate the transcription of H-NS-dependent promoters of the lux operon of marine luminescent bacteria (mesophilic Aliivibrio fischeri and psychrophilic Aliivibrio logei), and the dps gene from E. coli. It was also demonstrated that the ArdA antirestriction protein, the genes of which are located on transmissive plasmids ColIb-P9, R64, PK101, decreases levels of H-NS silencing of the PluxC promoter during conjugation in the recipient bacteria. Copyright © 2016 Elsevier GmbH. All rights reserved.

  19. Occurrence of mutations in the epidermal growth factor receptor gene in X-ray-induced rat lung tumors

    International Nuclear Information System (INIS)

    Kitahashi, Tsukasa; Takahashi, Mami; Yamada, Yutaka

    2008-01-01

    Epidermal growth factor receptor (EGFR) gene alterations have been found in human lung cancers. However, there is no information on the factors inducing EGFR mutations. In rodents, K-ras mutations are frequently found in many lung carcinogenesis models, but hitherto, Egfr mutations have not been reported. Their presence was therefore investigated in representative lung carcinogenesis models with 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), N-nitrosobis(2-hydroxypropyl)amine (BHP), 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MelQx) and ethyl carbamate (urethane), as well as X-ray irradiation. With the chemical carcinogenesis models, no mutations were detected in Egfr, which is in clear contrast to the high rates observed in either codon 12 or 61 of K-ras (21/23 of the lung tumors induced with NNK, 4/5 with MelQx, 1/4 with urethane and 7/18 with BHP). However, in the X-ray-induced lung tumors, Egfr mutations with amino acid substitution were observed in exons 18 and 21 (4/12, 33%), but no activating mutation of K-ras was detected. In addition, one and four silent mutations were identified in K-ras (exon 1) and Egfr (exons 18, 20 and 21), respectively. Most mutations in both Egfr and K-ras were G/C→A/T transitions (7/8, 88% and 31/34, 91%, respectively). Although, the mutational patterns in equivalent human lesions were not completely coincident, this first report of Egfr mutations in an experimental lung tumor model suggests that X-rays or other factors producing oxygen radicals could cause EGFR mutations in some proportion of lung cancers in humans. (author)

  20. Detection of EGFR and KRAS mutations in fine-needle aspirates stored on Whatman FTA cards: is this the tool for biobanking cytological samples in the molecular era?

    Science.gov (United States)

    da Cunha Santos, Gilda; Liu, Ni; Tsao, Ming-Sound; Kamel-Reid, Suzanne; Chin, Kayu; Geddie, William R

    2010-12-25

    The aims of this study were to compare the quality of DNA recovered from fine-needle aspirates (FNAs) stored on Whatman FTA cards with that retrieved from corresponding cell blocks and to determine whether the DNA extracted from the cards is suitable for multiple mutation analyses. FNAs collected from 18 resected lung tumors and cell suspensions from 4 lung cancer cell lines were placed on FTA Indicating Micro Cards and further processed to produce paired formalin-fixed paraffin-embedded (FFPE) cell blocks. Fragment analysis was used for the detection of EGFR exon 19 deletion, and direct sequencing for detection of EGFR exon 21 L858R mutation and exon 2 deletion of KRAS. Corresponding FFPE tissue sections from 2 resection specimens were also tested. Analyses were successful with all FNAs and lung cancer-derived cell lines collected on cards. Polymerase chain reaction failed in 2 cell blocks. For FNAs collected on cards, 5 cases showed EGFR and 3 showed KRAS mutations. Eleven cases were wild type. With cell blocks, 4 cases were found to harbor KRAS and 4 harbored EGFR mutations. All lung cancer-derived cell lines tested positive for their respective mutations, and there was complete agreement between card and cell block FNA samples for EGFR exon 21. For EGFR exon 19, 1 of 18 cases showed discordant results between the card and cell block, and for KRAS 1 of 17. The two resection specimens tested gave concordant results with the FTA card. Storage of cytologic material on FTA cards can maximize and simplify sample procurement for multiple mutational analyses with results similar to those from cell blocks.

  1. A maladaptive role for EP4 receptors in mouse mesangial cells.

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    Guang-xia Yang

    Full Text Available Roles of the prostaglandin E2 E-prostanoid 4 receptor (EP4 on extracellular matrix (ECM accumulation induced by TGF-β1 in mouse glomerular mesangial cells (GMCs remain unknown. Previously, we have identified that TGF-β1 stimulates the expression of FN and Col I in mouse GMCs. Here we asked whether stimulation of EP4 receptors would exacerbate renal fibrosis associated with enhanced glomerular ECM accumulation. We generated EP4(Flox/Flox and EP4(+/- mice, cultured primary WT, EP4(Flox/Flox and EP4(+/- GMCs, AD-EP4 transfected WT GMCs (EP4 overexpression and AD-Cre transfected EP4(Flox/Flox GMCs (EP4 deleted. We found that TGF-β1-induced cAMP and PGE2 synthesis decreased in EP4 deleted GMCs and increased in EP4 overexpressed GMCs. Elevated EP4 expression in GMCs augmented the coupling of TGF-β1 to FN, Col I expression and COX2/PGE2 signaling, while TGF-β1 induced FN, Col I expression and COX2/PGE2 signaling were down-regulated in EP4 deficiency GMCs. 8 weeks after 5/6 nephrectomy (Nx, WT and EP4(+/- mice exhibited markedly increased accumulation of ECM compared with sham-operated controls. Albuminuria, blood urea nitrogen and creatinine (BUN and Cr concentrations were significantly increased in WT mice as compared to those of EP4(+/- mice. Urine osmotic pressure was dramatically decreased after 5/6 Nx surgery in WT mice as compared to EP4(+/- mice. The pathological changes in kidney of EP4(+/- mice was markedly alleviated compared with WT mice. Immunohistochemical analysis showed significant reductions of Col I and FN in the kidney of EP4(+/- mice compared with WT mice. Collectively, this investigation established EP4 as a potent mediator of the pro-TGF-β1 activities elicited by COX2/PGE2 in mice GMCs. Our findings suggested that prostaglandin E2, acting via EP4 receptors contributed to accumulation of ECM in GMCs and promoted renal fibrosis.

  2. Frequencies of CCR5-D32, CCR2-64I and SDF1-3’A mutations in Human Immunodeficiency Virus (HIV seropositive subjects and seronegative individuals from the state of Pará in Brazilian Amazonia

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    Fernanda Andreza de Pinho Lott Carvalhaes

    2005-12-01

    Full Text Available The distribution of genetic polymorphisms of chemokine receptors CCR5-delta32, CCR2-64I and chemokine (SDF1-3’A mutations were studied in 110 Human Immunodeficiency Virus type 1 (HIV-1 seropositive individuals (seropositive group and 139 seronegative individuals (seronegative group from the population of the northern Brazilian city of Belém which is the capital of the state of Pará in the Brazilian Amazon. The CCR5-delta32 mutation was found in the two groups at similar frequencies, i.e. 2.2% for the seronegative group and 2.7% for the seropositive group. The frequencies of the SDF1-3’A mutation were 21.0% for the seronegative group and 15.4% for the seropositive group, and the CCR2-64I allele was found at frequencies of 12.5% for the seronegative group and 5.4% for the seropositive group. Genotype distributions were consistent with Hardy-Weinberg expectations in both groups, suggesting that none of the three mutations has a detectable selective effect. Difference in the allelic and genotypic frequencies was statistically significant for the CCR2 locus, the frequency in the seronegative group being twice that found in the seropositive group. This finding may indicate a protective effect of the CCR2-64I mutation in relation to HIV transmission. However, considering that the CCR2-64I mutation has been more strongly associated with a decreased risk for progression for AIDS than to the resistance to the HIV infection, this could reflect an aspect of population structure or a Type I error.

  3. Mutation Detection in Patients with Retinal Dystrophies Using Targeted Next Generation Sequencing.

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    Nicole Weisschuh

    Full Text Available Retinal dystrophies (RD constitute a group of blinding diseases that are characterized by clinical variability and pronounced genetic heterogeneity. The different nonsyndromic and syndromic forms of RD can be attributed to mutations in more than 200 genes. Consequently, next generation sequencing (NGS technologies are among the most promising approaches to identify mutations in RD. We screened a large cohort of patients comprising 89 independent cases and families with various subforms of RD applying different NGS platforms. While mutation screening in 50 cases was performed using a RD gene capture panel, 47 cases were analyzed using whole exome sequencing. One family was analyzed using whole genome sequencing. A detection rate of 61% was achieved including mutations in 34 known and two novel RD genes. A total of 69 distinct mutations were identified, including 39 novel mutations. Notably, genetic findings in several families were not consistent with the initial clinical diagnosis. Clinical reassessment resulted in refinement of the clinical diagnosis in some of these families and confirmed the broad clinical spectrum associated with mutations in RD genes.

  4. [MPLW515L point mutation in patients with myeloproliferative disease].

    Science.gov (United States)

    Xia, Jun; Xu, Wei; Zhang, Su-Jiang; Fan, Lei; Qiao, Chun; Li, Jian-Yong

    2008-12-01

    In order to investigate the frequency of MPLW515L and JAK2V617F point mutations of the patients with myeloproliferative disease (MPD) in Nanjing area, MPLW515L and JAK2V617F point mutations were simultaneously detected by alleles specific polymerase chain reaction (AS-PCR) and sequencing in 190 MPD patients. The results showed that MPLW515L point mutation was detected in 1 out of 102 essential thrombocythemia (ET) patients (1.0%) and was not detected in 32 polycythemia vera (PV) patients, 13 idiopathic myelofibrosis (IMF) patients, 43 chronic myelogenous leukemia (CML) patients. JAK2V617F point mutation was detected in 20 out of 32 PV patients (62.5%), 43 out of 102 ET patients (42.2%), 5 out of 13 IMF patients (38.5%), and was not detected in 43 CML patients. It is concluded that MPLW515L point mutation exists in ET patient, but is not found in PV, IMF and CML. JAK2V617F point mutation exists in PV, ET and IMF, but not in CML.

  5. Mutations in the NHEJ component XRCC4 cause primordial dwarfism.

    Science.gov (United States)

    Murray, Jennie E; van der Burg, Mirjam; IJspeert, Hanna; Carroll, Paula; Wu, Qian; Ochi, Takashi; Leitch, Andrea; Miller, Edward S; Kysela, Boris; Jawad, Alireza; Bottani, Armand; Brancati, Francesco; Cappa, Marco; Cormier-Daire, Valerie; Deshpande, Charu; Faqeih, Eissa A; Graham, Gail E; Ranza, Emmanuelle; Blundell, Tom L; Jackson, Andrew P; Stewart, Grant S; Bicknell, Louise S

    2015-03-05

    Non-homologous end joining (NHEJ) is a key cellular process ensuring genome integrity. Mutations in several components of the NHEJ pathway have been identified, often associated with severe combined immunodeficiency (SCID), consistent with the requirement for NHEJ during V(D)J recombination to ensure diversity of the adaptive immune system. In contrast, we have recently found that biallelic mutations in LIG4 are a common cause of microcephalic primordial dwarfism (MPD), a phenotype characterized by prenatal-onset extreme global growth failure. Here we provide definitive molecular genetic evidence supported by biochemical, cellular, and immunological data for mutations in XRCC4, encoding the obligate binding partner of LIG4, causing MPD. We report the identification of biallelic mutations in XRCC4 in five families. Biochemical and cellular studies demonstrate that these alterations substantially decrease XRCC4 protein levels leading to reduced cellular ligase IV activity. Consequently, NHEJ-dependent repair of ionizing-radiation-induced DNA double-strand breaks is compromised in XRCC4 cells. Similarly, immunoglobulin junctional diversification is impaired in cells. However, immunoglobulin levels are normal, and individuals lack overt signs of immunodeficiency. Additionally, in contrast to individuals with LIG4 mutations, pancytopenia leading to bone marrow failure has not been observed. Hence, alterations that alter different NHEJ proteins give rise to a phenotypic spectrum, from SCID to extreme growth failure, with deficiencies in certain key components of this repair pathway predominantly exhibiting growth deficits, reflecting differential developmental requirements for NHEJ proteins to support growth and immune maturation. Copyright © 2015 The American Society of Human Genetics. Published by Elsevier Inc. All rights reserved.

  6. Colágeno na cartilagem osteoartrótica

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    Ana Paula P. Velosa

    2003-06-01

    Full Text Available A cartilagem articular é um tecido altamente especializado, composto por células, os condrócitos, e um conjunto de macromoléculas, como o colágeno e os proteoglicanos. O colágeno é uma proteína fibrilar que garante resistência ao tecido, enquanto os proteoglicanos têm a função de mola biológica, sendo responsáveis pela compressibilidade da cartilagem. A complexa interação entre estas duas proteínas garante a elasticidade. Estas características específicas da cartilagem são essenciais para amortecer as grandes forças de impacto a que as articulações diartrodiais estão submetidas, sem muito gasto de energia, visto tratar-se de um tecido avascular. Em processos artrósicos ocorre um desequilíbrio entre a produção de componentes da matriz extracelular e destruição pelas metaloproteases, levando à degradação e perda do tecido cartilaginoso. A fase inicial da osteoartrose é marcada por perda de fragmentos de proteoglicanos para o líquido sinovial, aumento dos colágenos tipo II e tipo VI, aparecimento dos colágenos I e III, não típicos da cartilagem, e diminuição do colágeno tipo IX, que é importante para manter a integridade da matriz extracelular, além do entumescimento da cartilagem. Como conseqüência, a cartilagem perde suas características específicas, levando a alterações na função articular. A evolução da doença promove diminuição significativa das proteínas, até mesmo do colágeno tipo XI, que tem localização mais interna na estrutura da fibrila heterotípica, e, portanto levando até a exposição do osso. Até o momento, o tratamento da osteoartrose está baseado principalmente no controle da dor e/ou inflamação, não diminuindo ou impedindo a degradação da cartilagem articular. Neste aspecto a perspectiva de tratamento futuro da osteoartrose estaria na utilização de inibidores das metaloproteases associadas a condroprotetores interferindo no "turnover" da cartilagem e

  7. Hybrid Modified K-Means with C4.5 for Intrusion Detection Systems in Multiagent Systems.

    Science.gov (United States)

    Laftah Al-Yaseen, Wathiq; Ali Othman, Zulaiha; Ahmad Nazri, Mohd Zakree

    2015-01-01

    Presently, the processing time and performance of intrusion detection systems are of great importance due to the increased speed of traffic data networks and a growing number of attacks on networks and computers. Several approaches have been proposed to address this issue, including hybridizing with several algorithms. However, this paper aims at proposing a hybrid of modified K-means with C4.5 intrusion detection system in a multiagent system (MAS-IDS). The MAS-IDS consists of three agents, namely, coordinator, analysis, and communication agent. The basic concept underpinning the utilized MAS is dividing the large captured network dataset into a number of subsets and distributing these to a number of agents depending on the data network size and core CPU availability. KDD Cup 1999 dataset is used for evaluation. The proposed hybrid modified K-means with C4.5 classification in MAS is developed in JADE platform. The results show that compared to the current methods, the MAS-IDS reduces the IDS processing time by up to 70%, while improving the detection accuracy.

  8. Brain Malformations Associated with Knobloch Syndrome – Review of Literature, Expanding Clinical Spectrum and Identification of Novel Mutations

    Science.gov (United States)

    Caglayan, Ahmet Okay; Baranoski, Jacob F.; Aktar, Fesih; Han, Wengi; Tuysuz, Beyhan; Guzel, Aslan; Guclu, Bulent; Kaymakcalan, Hande; Aktekin, Berrin; Akgumus, Gozde Tugce; Murray, Phillip B.; Omay, E. Zeynep Erson; Caglar, Caner; Bakircioglu, Mehmet; Sakalar, Yildirim Bayezit; Guzel, Ebru; Demir, Nihat; Tuncer, Oguz; Senturk, Senem; Ekici, Baris; Minja, Frank J.; Šestan, Nenad; Yasuno, Katsuhito; Bilguvar, Kaya; Caksen, Huseyin; Gunel, Murat

    2014-01-01

    BACKGROUND Knobloch syndrome is a rare, autosomal recessive, developmental disorder characterized by stereotyped ocular abnormalities with or without occipital skull deformities (encephalocele, bone defects, cutis aplasia). Although there is clear heterogeneity in clinical presentation, central nervous system malformations, aside from the characteristic encephalocele, have not typically been considered a component of the disease phenotype. METHODS Four patients originally presented for genetic evaluation of symptomatic structural brain malformations. Whole-genome genotyping, whole-exome sequencing, and confirmatory Sanger sequencing were performed. Using immunohistochemical analysis, we investigated the protein expression pattern of COL18A1 in the mid-fetal and adult human cerebral cortex and then analyzed the spatial and temporal changes in the expression pattern of COL18A1 during human cortical development using the Human Brain Transcriptome database. RESULTS We identified two novel homozygous deleterious frame-shift mutations in the COL18A1 gene. Upon further investigation of these patients and their families, we found that many exhibited certain characteristics of Knobloch syndrome, including pronounced ocular defects. Our data strongly support an important role for COL18A1 in brain development and this report contributes to an enhanced characterization of the brain malformations that can result from deficiencies of collagen XVIII. CONCLUSIONS This case series highlights the diagnostic power and clinical utility of whole-exome sequencing technology – allowing clinicians and physician scientists to better understand the pathophysiology and presentations of rare diseases. We suggest that patients who are clinically diagnosed with Knobloch syndrome and/or found to have COL18A1 mutations via genetic screening should be investigated for potential structural brain abnormalities even in the absence of encephaloceles. PMID:25456301

  9. A novel mutation in the X-linked cyclin-dependent kinase-like 5 (CDKL5) gene associated with a severe Rett phenotype.

    Science.gov (United States)

    Sprovieri, T; Conforti, F L; Fiumara, A; Mazzei, R; Ungaro, C; Citrigno, L; Muglia, M; Arena, A; Quattrone, A

    2009-02-15

    Mutations in the X-linked cyclin-dependent kinase-like 5 (CDKL5) gene have recently been reported in patients with severe neurodevelopmental disorder characterized by early-onset seizures, infantile spasms, severe psychomotor impairment and very recently, in patients with Rett syndrome (RTT)-like phenotype. Although the involvement of CDKL5 in specific biological pathways and its neurodevelopmental role have not been completely elucidated, the CDKL5 appears to be physiologically related to the MECP2 gene. Here we report on the clinical and CDKL5 molecular investigation in a very unusual RTT case, with severe, early-neurological involvement in which we have shown in a previous report, a novel P388S MECP2 mutation [Conforti et al. (2003); Am J Med Genet A 117A: 184-187]. The patient has had severe psychomotor delay since the first month of life and infantile spasms since age 5 months. Moreover, at age 5 years the patient suddenly presented with renal failure. The severe pattern of symptoms in our patient, similar to a CDKL5 phenotype, prompted us to perform an analysis of the CDKL5, which revealed a novel missense mutation never previously described. The X-inactivation assay was non-informative. In conclusion, this report reinforces the observation that the CDKL5 phenotype overlaps with RTT and that CDKL5 analysis is recommended in patients with a seizure disorder commencing during the first months of life.

  10. Mutations in CDK5RAP2 cause Seckel syndrome.

    Science.gov (United States)

    Yigit, Gökhan; Brown, Karen E; Kayserili, Hülya; Pohl, Esther; Caliebe, Almuth; Zahnleiter, Diana; Rosser, Elisabeth; Bögershausen, Nina; Uyguner, Zehra Oya; Altunoglu, Umut; Nürnberg, Gudrun; Nürnberg, Peter; Rauch, Anita; Li, Yun; Thiel, Christian Thomas; Wollnik, Bernd

    2015-09-01

    Seckel syndrome is a heterogeneous, autosomal recessive disorder marked by prenatal proportionate short stature, severe microcephaly, intellectual disability, and characteristic facial features. Here, we describe the novel homozygous splice-site mutations c.383+1G>C and c.4005-9A>G in CDK5RAP2 in two consanguineous families with Seckel syndrome. CDK5RAP2 (CEP215) encodes a centrosomal protein which is known to be essential for centrosomal cohesion and proper spindle formation and has been shown to be causally involved in autosomal recessive primary microcephaly. We establish CDK5RAP2 as a disease-causing gene for Seckel syndrome and show that loss of functional CDK5RAP2 leads to severe defects in mitosis and spindle organization, resulting in cells with abnormal nuclei and centrosomal pattern, which underlines the important role of centrosomal and mitotic proteins in the pathogenesis of the disease. Additionally, we present an intriguing case of possible digenic inheritance in Seckel syndrome: A severely affected child of nonconsanguineous German parents was found to carry heterozygous mutations in CDK5RAP2 and CEP152. This finding points toward a potential additive genetic effect of mutations in CDK5RAP2 and CEP152.

  11. Mutations in CDK5RAP2 cause Seckel syndrome

    Science.gov (United States)

    Yigit, Gökhan; Brown, Karen E; Kayserili, Hülya; Pohl, Esther; Caliebe, Almuth; Zahnleiter, Diana; Rosser, Elisabeth; Bögershausen, Nina; Uyguner, Zehra Oya; Altunoglu, Umut; Nürnberg, Gudrun; Nürnberg, Peter; Rauch, Anita; Li, Yun; Thiel, Christian Thomas; Wollnik, Bernd

    2015-01-01

    Seckel syndrome is a heterogeneous, autosomal recessive disorder marked by prenatal proportionate short stature, severe microcephaly, intellectual disability, and characteristic facial features. Here, we describe the novel homozygous splice-site mutations c.383+1G>C and c.4005-9A>G in CDK5RAP2 in two consanguineous families with Seckel syndrome. CDK5RAP2 (CEP215) encodes a centrosomal protein which is known to be essential for centrosomal cohesion and proper spindle formation and has been shown to be causally involved in autosomal recessive primary microcephaly. We establish CDK5RAP2 as a disease-causing gene for Seckel syndrome and show that loss of functional CDK5RAP2 leads to severe defects in mitosis and spindle organization, resulting in cells with abnormal nuclei and centrosomal pattern, which underlines the important role of centrosomal and mitotic proteins in the pathogenesis of the disease. Additionally, we present an intriguing case of possible digenic inheritance in Seckel syndrome: A severely affected child of nonconsanguineous German parents was found to carry heterozygous mutations in CDK5RAP2 and CEP152. This finding points toward a potential additive genetic effect of mutations in CDK5RAP2 and CEP152. PMID:26436113

  12. Cancel all Hollidays for SLX4 mutations: identification of a new Fanconi anemia subtype, FANCP.

    Science.gov (United States)

    Kang, M H

    2011-07-01

    SLX4, a coordinator of structure-specific endo-nucleases, is mutated in a new Fanconi anemia subtype Stoepker et al. (2011) Nature Genetics 43:138-141. Mutations of the SLX4 gene in Fanconi anemia Kim et al. (2011) Nature Genetics 43:142-146. © 2011 John Wiley & Sons A/S.

  13. Mutations in the coding regions of the hepatocyte nuclear factor 4 alpha in Iranian families with maturity onset diabetes of the young

    Directory of Open Access Journals (Sweden)

    Tavakolafshari Jalil

    2009-12-01

    Full Text Available Abstract Hepatocyte nuclear factor 4α (HNF4α is a nuclear receptor involved in glucose homeostasis and is required for normal β cell function. Mutations in the HNF4α gene are associated with maturity onset diabetes of the young type 1 (MODY1. The aim of the present study was to determine the prevalence and nature of mutations in HNF4α gene in Iranian patients with a clinical diagnosis of MODY and their family members. Twelve families including 30 patients with clinically MODY diagnosis and 21 members of their family were examined using PCR-RFLP method and in case of mutation confirmed by sequencing techniques. Fifty age and sex matched subjects with normal fasting blood sugar (FBS and Glucose tolerance test (GTT were constituted the control group and investigated in the similar pattern. Single mutation of V255M in the HNF4α gene was detected. This known mutation was found in 8 of 30 patients and 3 of 21 individuals in relatives. Fifty healthy control subjects did not show any mutation. Here, it is indicated that the prevalence of HNF4α mutation among Iranian patients with clinical MODY is considerable. This mutation was present in 26.6% of our patients, but nothing was found in control group. In the family members, 3 subjects with the age of ≤25 years old carried this mutation. Therefore, holding this mutation in this range of age could be a predisposing factor for developing diabetes in future.

  14. Visual function in patients with cone-rod dystrophy (CRD) associated with mutations in the ABCA4(ABCR) gene.

    Science.gov (United States)

    Birch, D G; Peters, A Y; Locke, K L; Spencer, R; Megarity, C F; Travis, G H

    2001-12-01

    Mutations in the ABCA4(ABCR) gene cause autosomal recessive Stargardt disease (STGD). ABCR mutations were identified in patients with cone-rod dystrophy (CRD) and retinitis pigmentosa (RP) by direct sequencing of all 50 exons in 40 patients. Of 10 patients with RP, one contained two ABCR mutations suggesting a compound heterozygote. This patient had a characteristic fundus appearance with attenuated vessels, pale disks and bone-spicule pigmentation. Rod electroretinograms (ERGs) were non-detectable, cone ERGs were greatly reduced in amplitude and delayed in implicit time, and visual fields were constricted to 10 degrees diameter. Eleven of 30 (37%) patients with CRD had mutations in ABCR. In general, these patients showed reduced but detectable rod ERG responses, reduced and delayed cone responses, and poor visual acuity. Rod photoresponses to high intensity flashes were of reduced maximum amplitude but showed normal values for the gain of phototransduction. Most CRD patients with mutations in ABCR showed delayed recovery of sensitivity (dark adaptation) following exposure to bright light. Pupils were also significantly smaller in these patients compared to controls at 30 min following light exposure, consistent with a persistent 'equivalent light' background due to the accumulation of a tentatively identified 'noisy' photoproduct. Copyright 2001 Academic Press.

  15. Toxicidade córneo-conjuntival do colírio de iodo-povidona: estudo experimental

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    Santos Namir Clementino

    2003-01-01

    Full Text Available OBJETIVO: Avaliar a toxicidade ocular do colírio de iodo-povidona a 2,5% e a 0,5% sobre a superfície ocular, na regeneração do epitélio corneal e as alterações histopatológicas da córnea. MÉTODOS: Realizaram-se estudos experimentais consecutivos em coelhos albinos, nos quais se fez a ablação do epitélio de uma área circular central da córnea de 6,5 mm de diâmetro. Em cada experimento foram utilizados 20 animais (40 olhos, sendo que no olho direito foi instilado o colírio de iodo-povidona (caso e no olho esquerdo água destilada (controle, em intervalos de uma hora, durante três dias consecutivos. Durante o experimento, os animais foram submetidos a exames biomicroscópicos diários para avaliação da superfície córneo-conjuntival e realização de fotografias seriadas da área sem epitélio, corada com fluoresceína, para medida da área projetada da lesão com auxílio de analisador de imagem computadorizado. No final do experimento, os animais foram sacrificados para avaliação histopatológica das córneas. RESULTADOS: O colírio de iodo-povidona a 2,5% comprometeu a regeneração epitelial, causou conjuntivite em 100% dos olhos, com produção de secreção de aspecto mucoso em 80%, ceratite ponteada em 40% e edema estrômico leve em 10% dos casos. Os achados histopatológicos foram úlcera de córnea, degeneração hidrópica das células endoteliais e infiltrado inflamatório com predomínio de eosinófilos em 100% dos casos. Nos olhos em que se instilou iodo-povidona a 0,5%, assim como nos controles, observou-se completa regeneração da lesão epitelial (p<0,001 após 72 horas do início do experimento. Do ponto de vista histopatológico, epitelização normal em todos os casos e controles, em apenas um caso observou-se discreto infiltrado de leucócitos perilímbicos. CONCLUSÃO: A toxicidade ocular do colírio de iodo-povidona é dependente da concentração da solução, sendo que o colírio a 2,5% mostrou

  16. APOA5 Q97X Mutation Identified through homozygosity mapping causes severe hypertriglyceridemia in a Chilean consanguineous family

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    Dussaillant Catalina

    2012-11-01

    Full Text Available Abstract Background Severe hypertriglyceridemia (HTG has been linked to defects in LPL, APOC2, APOA5, LMF1 and GBIHBP1 genes. However, a number of severe HTG cases are probably caused by as yet unidentified mutations. Very high triglyceride plasma levels (>112 mmol/L at diagnosis were found in two sisters of a Chilean consanguineous family, which is strongly suggestive of a recessive highly penetrant mutation. The aim of this study was to determine the genetic locus responsible for the severe HTG in this family. Methods We carried out a genome-wide linkage study with nearly 300,000 biallelic markers (Illumina Human CytoSNP-12 panel. Using the homozygosity mapping strategy, we searched for chromosome regions with excess of homozygous genotypes in the affected cases compared to non-affected relatives. Results A large homozygous segment was found in the long arm of chromosome 11, with more than 2,500 consecutive homozygous SNP shared by the proband with her affected sister, and containing the APOA5/A4/C3/A1 cluster. Direct sequencing of the APOA5 gene revealed a known homozygous nonsense Q97X mutation (p.Gln97Ter found in both affected sisters but not in non-affected relatives nor in a sample of unrelated controls. Conclusion The Q97X mutation of the APOA5 gene in homozygous status is responsible for the severe hypertriglyceridemia in this family. We have shown that homozygosity mapping correctly pinpointed the genomic region containing the gene responsible for severe hypertriglyceridemia in this consanguineous Chilean family.

  17. APOA5 Q97X mutation identified through homozygosity mapping causes severe hypertriglyceridemia in a Chilean consanguineous family.

    Science.gov (United States)

    Dussaillant, Catalina; Serrano, Valentina; Maiz, Alberto; Eyheramendy, Susana; Cataldo, Luis Rodrigo; Chavez, Matías; Smalley, Susan V; Fuentes, Marcela; Rigotti, Attilio; Rubio, Lorena; Lagos, Carlos F; Martinez, José Alfredo; Santos, José Luis

    2012-11-15

    Severe hypertriglyceridemia (HTG) has been linked to defects in LPL, APOC2, APOA5, LMF1 and GBIHBP1 genes. However, a number of severe HTG cases are probably caused by as yet unidentified mutations. Very high triglyceride plasma levels (>112 mmol/L at diagnosis) were found in two sisters of a Chilean consanguineous family, which is strongly suggestive of a recessive highly penetrant mutation. The aim of this study was to determine the genetic locus responsible for the severe HTG in this family. We carried out a genome-wide linkage study with nearly 300,000 biallelic markers (Illumina Human CytoSNP-12 panel). Using the homozygosity mapping strategy, we searched for chromosome regions with excess of homozygous genotypes in the affected cases compared to non-affected relatives. A large homozygous segment was found in the long arm of chromosome 11, with more than 2,500 consecutive homozygous SNP shared by the proband with her affected sister, and containing the APOA5/A4/C3/A1 cluster. Direct sequencing of the APOA5 gene revealed a known homozygous nonsense Q97X mutation (p.Gln97Ter) found in both affected sisters but not in non-affected relatives nor in a sample of unrelated controls. The Q97X mutation of the APOA5 gene in homozygous status is responsible for the severe hypertriglyceridemia in this family. We have shown that homozygosity mapping correctly pinpointed the genomic region containing the gene responsible for severe hypertriglyceridemia in this consanguineous Chilean family.

  18. The use of high resolution melting analysis to detect Fabry mutations in heterozygous females via dry bloodspots.

    Science.gov (United States)

    Tai, Chang-Long; Liu, Mei-Ying; Yu, Hsiao-Chi; Chiang, Chiang-Chuan; Chiang, Hung; Suen, Jeng-Hung; Kao, Shu-Min; Huang, Yu-Hsiu; Wu, Tina Jui-Ting; Yang, Chia-Feng; Tsai, Fang-Chih; Lin, Ching-Yuang; Chang, Jan-Gowth; Chen, Hong-Duo; Niu, Dau-Ming

    2012-02-18

    As an X-linked genetic disorder, Fabry disease was first thought to affect males only, and females were generally considered to be asymptomatic carriers. However, recent research suggests that female carriers of Fabry disease may still develop vital organ damage causing severe morbidity and mortality. In the previous newborn screening, from 299,007 newborns, we identified a total of 20 different Fabry mutations and 121 newborns with Fabry mutations. However, we found that most female carriers are not detected by enzyme assays. A streamlined method for high resolution melting (HRM) analysis was designed to screen for GLA gene mutations using a same PCR and melting program. Primer sets were designed to cover the 7 exons and the Chinese common intronic mutation, IVS4+919G>A of GLA gene. The HRM analysis was successful in identifying heterozygous and hemizygous patients with the 20 surveyed mutations. We were also successful in using this method to test dry blood spots of newborns afflicted with Fabry mutations without having to determine DNA concentration before PCR amplification. The results of this study show that HRM could be a reliable and sensitive method for use in the rapid screening of females for GLA mutations. Copyright © 2011 Elsevier B.V. All rights reserved.

  19. Isolation and sequence analysis of pCS36-4CPA, a small plasmid from Citrobacter sp. 36-4CPA.

    Science.gov (United States)

    Zharikova, Natalia V; Iasakov, Timur R; Bumazhkin, Boris K; Patutina, Ekaterina O; Zhurenko, Evgeniia I; Korobov, Vladislav V; Sagitova, Alina I; Kuznetsov, Boris B; Markusheva, Tatiana V

    2018-05-01

    A small plasmid designated pCS36-4CPA with a size of 5217 base pairs and G-C content of 50.74% was isolated from Citrobacter sp. 36-4CPA. The origin of replication ( ori ) of the plasmid was identified as a region of about 800 bp in length with an identity of 67.1% to the ColE1 plasmid at the nucleotide level. The replication region contained typical elements of ColE1-like plasmids: RNA I and RNA II with their corresponding -10 and -35 boxes, a single-strand initiation site ( ssi ), and a lagging-strand termination site ( terH ). As seen in other ColE1-like plasmids, pCS36-4CPA carried mobilisation machinery that include mobABCD genes but it did not possess the rom gene. Analysis of the multimer resolution site ( mrs ) was performed and XerC and XerD binding sites were identified. Also, the 70-nt transcript Rcd of pCS36-4CPA was predicted and similarity of the transcript's secondary structure with those of the ColE1-family was shown. The cargo module of pCS36-4CPA contained three open reading frames (ORFs). Two of them (ORF5 and ORF6) showed no significant homology to any known gene sequences but contained putative THAP DNA-binding (DBD) and type II restriction endonuclease Eco O109I domains. The seventh open reading frame (ORF7) encodes YhdJ-like DNA modification methylase. The region highly homologous to pCS36-4CPA was found in the Salmonella phage SE2 genome.

  20. Direct detection of hemophilia B F9 gene mutation using multiplex PCR and conformation sensitive gel electrophoresis

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    Ki Young Yoo

    2010-03-01

    Full Text Available Purpose : The F9 gene is known to be the causative gene for hemophilia B, but unfortunately the detection rate for restriction fragment length polymorphism-based linkage analysis is only 55.6%. Direct DNA sequencing can detect 98% of mutations, but this alternative procedure is very costly. Here, we conducted multiplex polymerase chain reactions (PCRs and conformation sensitive gel electrophoresis (CSGE to perform a screened DNA sequencing for the F9 gene, and we compared the results with direct sequencing in terms of accuracy, cost, simplicity, and time consumption. Methods : A total of 27 unrelated hemophilia B patients were enrolled. Direct DNA sequencing was performed for 27 patients by a separate institute, and multiplex PCR-CSGE screened sequencing was done in our laboratory. Results of the direct DNA sequencing were used as a reference, to which the results of the multiplex PCR-CSGE screened sequencing were compared. For the patients whose mutation was not detected by the 2 methods, multiplex ligation-dependent probe amplification (MLPA was conducted. Results : With direct sequencing, the mutations could be identified from 26 patients (96.3%, whereas for multiplex PCR- CSGE screened sequencing, the mutations could be detected in 23 (85.2%. One patient’s mutation was identified by MLPA. A total of 21 different mutations were found among the 27 patients. Conclusion : Multiplex PCR-CSGE screened DNA sequencing detected 88.9% of mutations and reduced costs by 55.7% compared with direct DNA sequencing. However, it was more labor-intensive and time-consuming.

  1. Detection of haplotypes associated with prenatal death in dairy cattle and identification of deleterious mutations in GART, SHBG and SLC37A2.

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    Sébastien Fritz

    Full Text Available The regular decrease of female fertility over time is a major concern in modern dairy cattle industry. Only half of this decrease is explained by indirect response to selection on milk production, suggesting the existence of other factors such as embryonic lethal genetic defects. Genomic regions harboring recessive deleterious mutations were detected in three dairy cattle breeds by identifying frequent haplotypes (>1% showing a deficit in homozygotes among Illumina Bovine 50k Beadchip haplotyping data from the French genomic selection database (47,878 Holstein, 16,833 Montbéliarde, and 11,466 Normande animals. Thirty-four candidate haplotypes (p<10(-4 including previously reported regions associated with Brachyspina, CVM, HH1, and HH3 in Holstein breed were identified. Haplotype length varied from 1 to 4.8 Mb and frequencies from 1.7 up to 9%. A significant negative effect on calving rate, consistent in heifers and in lactating cows, was observed for 9 of these haplotypes in matings between carrier bulls and daughters of carrier sires, confirming their association with embryonic lethal mutations. Eight regions were further investigated using whole genome sequencing data from heterozygous bull carriers and control animals (45 animals in total. Six strong candidate causative mutations including polymorphisms previously reported in FANCI (Brachyspina, SLC35A3 (CVM, APAF1 (HH1 and three novel mutations with very damaging effect on the protein structure, according to SIFT and Polyphen-2, were detected in GART, SHBG and SLC37A2 genes. In conclusion, this study reveals a yet hidden consequence of the important inbreeding rate observed in intensively selected and specialized cattle breeds. Counter-selection of these mutations and management of matings will have positive consequences on female fertility in dairy cattle.

  2. Detection of Haplotypes Associated with Prenatal Death in Dairy Cattle and Identification of Deleterious Mutations in GART, SHBG and SLC37A2

    Science.gov (United States)

    Fritz, Sébastien; Capitan, Aurelien; Djari, Anis; Rodriguez, Sabrina C.; Barbat, Anne; Baur, Aurélia; Grohs, Cécile; Weiss, Bernard; Boussaha, Mekki; Esquerré, Diane; Klopp, Christophe; Rocha, Dominique; Boichard, Didier

    2013-01-01

    The regular decrease of female fertility over time is a major concern in modern dairy cattle industry. Only half of this decrease is explained by indirect response to selection on milk production, suggesting the existence of other factors such as embryonic lethal genetic defects. Genomic regions harboring recessive deleterious mutations were detected in three dairy cattle breeds by identifying frequent haplotypes (>1%) showing a deficit in homozygotes among Illumina Bovine 50k Beadchip haplotyping data from the French genomic selection database (47,878 Holstein, 16,833 Montbéliarde, and 11,466 Normande animals). Thirty-four candidate haplotypes (p<10−4) including previously reported regions associated with Brachyspina, CVM, HH1, and HH3 in Holstein breed were identified. Haplotype length varied from 1 to 4.8 Mb and frequencies from 1.7 up to 9%. A significant negative effect on calving rate, consistent in heifers and in lactating cows, was observed for 9 of these haplotypes in matings between carrier bulls and daughters of carrier sires, confirming their association with embryonic lethal mutations. Eight regions were further investigated using whole genome sequencing data from heterozygous bull carriers and control animals (45 animals in total). Six strong candidate causative mutations including polymorphisms previously reported in FANCI (Brachyspina), SLC35A3 (CVM), APAF1 (HH1) and three novel mutations with very damaging effect on the protein structure, according to SIFT and Polyphen-2, were detected in GART, SHBG and SLC37A2 genes. In conclusion, this study reveals a yet hidden consequence of the important inbreeding rate observed in intensively selected and specialized cattle breeds. Counter-selection of these mutations and management of matings will have positive consequences on female fertility in dairy cattle. PMID:23762392

  3. DETECTION OF K-RAS AND P53 MUTATIONS IN SPUTUM SAMPLES OF LUNG CANCER PATIENTS USING LASER CAPTURE MICRODISSECTION MICROSCOPE AND MUTATION ANALYSIS

    Science.gov (United States)

    Detection of K-ras and p53 Mutations in Sputum Samples of Lung Cancer Patients Using Laser Capture Microdissection Microscope and Mutation AnalysisPhouthone Keohavong a,*, Wei-Min Gao a, Kui-Cheng Zheng a, Hussam Mady b, Qing Lan c, Mona Melhem b, and Judy Mumford d.<...

  4. DNA sequence analysis of the mutational specificity of u.v. light in the SUP4-o gene of yeast

    International Nuclear Information System (INIS)

    Kunz, B.A.; Mis, J.R.A.; Pierce, M.K.; Giroux, C.N.

    1987-01-01

    Mutations induced in the SUP4-o gene of Saccharomyces cerevisiae by u.v. irradiation have been characterized. DNA sequence analysis of 120 mutants revealed that u.v. induced all types of base substitutions, although transitions, in particular G:C → A:T events predominated. In addition, a small number of single base pair deletions and double mutations, occurring in tandem or separated by a few base pairs, were recovered. The base pair substitutions were not distributed randomly in the SUP4-o gene and, with one exception, were all located at sites of adjacent pyrimidines, suggesting they were targeted by u.v. photolesions. A substantial fraction of the mutations were detected at hotspots for u.v. mutagenesis. The majority of changes occurred at the 3' base of dipyrimidine sequences where both cyclobutane dimers and [6-4]-photoproducts could form. Approximately one-third of the induced base substitutions were found at potential pyrimidine dimer sites where [6-4]-photoproducts would be expected to occur rarely. Possible origins of the induced mutations and the role of cyclobutane dimers as premutational u.v. lesions in yeast are considered. (author)

  5. Calreticulin Mutations in Myeloproliferative Neoplasms

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    Noa Lavi

    2014-10-01

    Full Text Available With the discovery of the JAK2V617F mutation in patients with Philadelphia chromosome-negative (Ph− myeloproliferative neoplasms (MPNs in 2005, major advances have been made in the diagnosis of MPNs, in understanding of their pathogenesis involving the JAK/STAT pathway, and finally in the development of novel therapies targeting this pathway. Nevertheless, it remains unknown which mutations exist in approximately one-third of patients with non-mutated JAK2 or MPL essential thrombocythemia (ET and primary myelofibrosis (PMF. At the end of 2013, two studies identified recurrent mutations in the gene encoding calreticulin (CALR using whole-exome sequencing. These mutations were revealed in the majority of ET and PMF patients with non-mutated JAK2 or MPL but not in polycythemia vera patients. Somatic 52-bp deletions (type 1 mutations and recurrent 5-bp insertions (type 2 mutations in exon 9 of the CALR gene (the last exon encoding the C-terminal amino acids of the protein calreticulin were detected and found always to generate frameshift mutations. All detected mutant calreticulin proteins shared a novel amino acid sequence at the C-terminal. Mutations in CALR are acquired early in the clonal history of the disease, and they cause activation of JAK/STAT signaling. The CALR mutations are the second most frequent mutations in Ph− MPN patients after the JAK2V617F mutation, and their detection has significantly improved the diagnostic approach for ET and PMF. The characteristics of the CALR mutations as well as their diagnostic, clinical, and pathogenesis implications are discussed in this review.

  6. Genetic study of the PAH locus in the Iranian population: familial gene mutations and minihaplotypes.

    Science.gov (United States)

    Razipour, Masoumeh; Alavinejad, Elaheh; Sajedi, Seyede Zahra; Talebi, Saeed; Entezam, Mona; Mohajer, Neda; Kazemi-Sefat, Golnaz-Ensieh; Gharesouran, Jalal; Setoodeh, Aria; Mohaddes Ardebili, Seyyed Mojtaba; Keramatipour, Mohammad

    2017-10-01

    Phenylketonuria (PKU), one of the most common inborn errors of amino acid metabolism, is caused by mutations in the phenylalanine hydroxylase (PAH) gene (PAH). PKU has wide allelic heterogeneity, and over 600 different disease-causing mutations in PAH have been detected to date. Up to now, there have been no reports on the minihaplotype (VNTR/STR) analysis of PAH locus in the Iranian population. The aims of the present study were to determine PAH mutations and minihaplotypes in Iranian families with PAH deficiency and to investigate the correlation between them. A total of 81 Iranian families with PAH deficiency were examined using PCR-sequencing of all 13 PAH exons and their flanking intron regions to identify sequence variations. Fragment analysis of the PAH minihaplotypes was performed by capillary electrophoresis for 59 families. In our study, 33 different mutations were found accounting for 95% of the total mutant alleles. The majority of these mutations (72%) were distributed across exons 7, 11, 2 and their flanking intronic regions. Mutation c.1066-11G > A was the most common with a frequency of 20.37%. The less frequent mutations, p.Arg261Gln (8%), p.Arg243Ter (7.4%), p.Leu48Ser (7.4%), p.Lys363Asnfs*37 (6.79%), c.969 + 5G > A (6.17%), p.Pro281Leu (5.56), c.168 + 5G > C (5.56), and p.Arg261Ter (4.94) together comprised about 52% of all mutant alleles. In this study, a total of seventeen PAH gene minihaplotypes were detected, six of which associated exclusively with particular mutations. Our findings indicate a broad PAH mutation spectrum in the Iranian population, which is consistent with previous studies reporting a wide range of PAH mutations, most likely due to ethnic heterogeneity. High prevalence of c.1066-11G > A mutation linked to minihaplotype 7/250 among both Iranian and Mediterranean populations is indicative of historical and geographical links between them. Also, strong association between particular mutations and minihaplotypes

  7. Mutations in Splicing Factor Genes Are a Major Cause of Autosomal Dominant Retinitis Pigmentosa in Belgian Families

    Science.gov (United States)

    Coppieters, Frauke; Roels, Dimitri; De Jaegere, Sarah; Flipts, Helena; De Zaeytijd, Julie; Walraedt, Sophie; Claes, Charlotte; Fransen, Erik; Van Camp, Guy; Depasse, Fanny; Casteels, Ingele; de Ravel, Thomy

    2017-01-01

    Purpose Autosomal dominant retinitis pigmentosa (adRP) is characterized by an extensive genetic heterogeneity, implicating 27 genes, which account for 50 to 70% of cases. Here 86 Belgian probands with possible adRP underwent genetic testing to unravel the molecular basis and to assess the contribution of the genes underlying their condition. Methods Mutation detection methods evolved over the past ten years, including mutation specific methods (APEX chip analysis), linkage analysis, gene panel analysis (Sanger sequencing, targeted next-generation sequencing or whole exome sequencing), high-resolution copy number screening (customized microarray-based comparative genomic hybridization). Identified variants were classified following American College of Medical Genetics and Genomics (ACMG) recommendations. Results Molecular genetic screening revealed mutations in 48/86 cases (56%). In total, 17 novel pathogenic mutations were identified: four missense mutations in RHO, five frameshift mutations in RP1, six mutations in genes encoding spliceosome components (SNRNP200, PRPF8, and PRPF31), one frameshift mutation in PRPH2, and one frameshift mutation in TOPORS. The proportion of RHO mutations in our cohort (14%) is higher than reported in a French adRP population (10.3%), but lower than reported elsewhere (16.5–30%). The prevalence of RP1 mutations (10.5%) is comparable to other populations (3.5%-10%). The mutation frequency in genes encoding splicing factors is unexpectedly high (altogether 19.8%), with PRPF31 the second most prevalent mutated gene (10.5%). PRPH2 mutations were found in 4.7% of the Belgian cohort. Two families (2.3%) have the recurrent NR2E3 mutation p.(Gly56Arg). The prevalence of the recurrent PROM1 mutation p.(Arg373Cys) was higher than anticipated (3.5%). Conclusions Overall, we identified mutations in 48 of 86 Belgian adRP cases (56%), with the highest prevalence in RHO (14%), RP1 (10.5%) and PRPF31 (10.5%). Finally, we expanded the molecular

  8. The discovery and the structural basis of an imidazo[4,5-b]pyridine-based p21-activated kinase 4 inhibitor.

    Science.gov (United States)

    Park, Jeung Kuk; Kim, Sunmin; Han, Yu Jin; Kim, Seong Hwan; Kang, Nam Sook; Lee, Hyuk; Park, SangYoun

    2016-06-01

    p21-Activated kinases (PAKs) which belong to the family of ste20 serine/threonine protein kinases regulate cytoskeletal reorganization, cell motility, cell proliferation, and oncogenic transformation which are all related to the cellular functions during cancer induction and metastasis. The fact that PAK mutations are detected in multiple tumor tissues makes PAKs a novel therapeutic drug target. In this study, an imidazo[4,5-b]pyridine-based PAK4 inhibitor, KY-04045 (6-Bromo-2-(3-isopropyl-1-methyl-1H-pyrazol-4-yl)-1H-imidazo[4,5-b]pyridine), was discovered using a virtual site-directed fragment-based drug design and was validated using an inhibition assay. Although PAK4 affinity to KY-04045 seems much weaker than that of the reported PAK4 inhibitors, the location of KY-04045 is clearly defined in the structure of PAK4 co-crystallized with KY-04045. The crystal structure illustrates that the pyrazole and imidazopyridine rings of KY-04045 are sufficient for mediating PAK4 hinge loop interaction. Hence, we believe that KY-04045 can be exploited as a basic building block in designing novel imidazo[4,5-b]pyridine-based PAK4 inhibitors. Copyright © 2016 Elsevier Ltd. All rights reserved.

  9. Adição de colágeno em pão sem glúten elaborado com farinha de arroz

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    Carolina da Silva Graça

    2017-02-01

    Full Text Available Resumo Os portadores da doença celíaca, uma enteropatia, apresentam intolerância ao glúten e a restrição de alimentos que o contenham mostra-se eficaz na normalização da função intestinal e das manifestações clínicas. O objetivo do presente estudo foi avaliar o efeito da adição de colágeno em pó e em fibra nas características estruturais, tecnológicas e sensoriais de pães sem glúten, elaborados com farinha de arroz. Primeiramente, foram realizadas adições de 2%, 4%, 6% e 8% de colágeno, sob as formas em pó e em fibra, em pães elaborados com farinha de arroz, sendo estes avaliados quanto às suas características tecnológicas: volume específico, firmeza do miolo e cor da crosta e do miolo. O pão que apresentou as melhores características tecnológicas foi caracterizado quanto à sua composição química (proteínas, lipídios, cinzas, umidade e carboidratos, avaliado quanto às perdas decorrentes da cocção e submetido à análise sensorial. As massas cruas dos pães adicionados com o colágeno em fibra, assim como a massa do pão padrão (sem adição de colágeno, foram avaliadas através de reologia e microscopia eletrônica de varredura. A adição de colágeno aos pães elaborados com farinha de arroz não colaborou para a formação de uma rede semelhante à formada pelo glúten. O pão da formulação com 4% de colágeno em fibra apresentou as características tecnológicas mais próximas ao esperado, não diferindo significativamente quanto à composição química, do pão padrão. Por meio da análise sensorial, constatou-se que os pães sem glúten (padrão e com 4% de colágeno em fibra obtiveram aceitação sensorial superior a 75%. Conclui-se, com o estudo, que é possível adicionar colágeno em pães sem glúten, sendo que o pão adicionado de 4% de colágeno em fibra apresentou características mais próximas às do pão padrão testado.

  10. Detection of mismatch repair gene germline mutation carrier among Chinese population with colorectal cancer

    International Nuclear Information System (INIS)

    Jin, Hei-Ying; Zhao, Ronghua; Liu, Xiufang; Li, Vicky Ka Ming; Ding, Yijiang; Yang, Bolin; Geng, Jianxiang; Lai, Rensheng; Ding, Shuqing; Ni, Min

    2008-01-01

    Hereditary nonpolyposis colorectal cancer (HNPCC) is an autosomal dominant syndrome. The National Cancer Institute (NCI) has recommended the Revised Bethesda guidelines for screening HNPCC. There has been a great deal of research on the value of these tests in other countries. However, literature about the Chinese population is scarce. Our objective is to detect and study microsatellite instability (MSI) and mismatch repair (MMR) gene germline mutation carriers among a Chinese population with colorectal cancer. In 146 prospectively recruited consecutive patients with clinically proven colorectal cancer, MSI carriers were identified by analysis of tumor tissue using multiplex fluorescence polymerase chain reaction (PCR) using the NCI recommended panel and classified into microsatellite instability-low (MSI-L), microsatellite instability-high (MSI-H) and microsatellite stable (MSS) groups. Immunohistochemical staining for MSH2, MSH6 and MLH1 on tissue microarrays (TMAs) was performed, and methylation of the MLH1 promoter was analyzed by quantitative methylation specific PCR (MSP). Germline mutation analysis of blood samples was performed for MSH2, MSH6 and MLH1 genes. Thirty-four out of the 146 colorectal cancers (CRCs, 23.2%) were MSI, including 19 MSI-H CRCs and 15 MSI-L CRCS. Negative staining for MSH2 was found in 8 CRCs, negative staining for MSH6 was found in 6 CRCs. One MSI-H CRC was negative for both MSH6 and MSH2. Seventeen CRCs stained negatively for MLH1. MLH1 promoter methylation was determined in 34 MSI CRCs. Hypermethylation of the MLH1 promoter occurred in 14 (73.7%) out of 19 MSI-H CRCs and 5 (33.3%) out of 15 MSI-L CRCs. Among the 34 MSI carriers and one MSS CRC with MLH1 negative staining, 8 had a MMR gene germline mutation, which accounted for 23.5% of all MSI colorectal cancers and 5.5% of all the colorectal cancers. Five patients harbored MSH2 germline mutations, and three patients harbored MSH6 germline mutations. None of the patients had an MLH

  11. TGM5 mutations impact epidermal differentiation in acral peeling skin syndrome.

    Science.gov (United States)

    Pigors, Manuela; Kiritsi, Dimitra; Cobzaru, Cristina; Schwieger-Briel, Agnes; Suárez, Jose; Faletra, Flavio; Aho, Heikki; Mäkelä, Leeni; Kern, Johannes S; Bruckner-Tuderman, Leena; Has, Cristina

    2012-10-01

    Acral peeling skin syndrome (APSS) is an autosomal recessive skin disorder characterized by acral blistering and peeling of the outermost layers of the epidermis. It is caused by mutations in the gene for transglutaminase 5, TGM5. Here, we report on clinical and molecular findings in 11 patients and extend the TGM5 mutation database by four, to our knowledge, previously unreported mutations: p.M1T, p.L41P, p.L214CfsX15, and p.S604IfsX9. The recurrent mutation p.G113C was found in 9 patients, but also in 3 of 100 control individuals in a heterozygous state, indicating that APSS might be more widespread than hitherto expected. Using quantitative real-time PCR, immunoblotting, and immunofluorescence analysis, we demonstrate that expression and distribution of several epidermal differentiation markers and corneodesmosin (CDSN) is altered in APSS keratinocytes and skin. Although the expression of transglutaminases 1 and 3 was not changed, we found an upregulation of keratin 1, keratin 10, involucrin, loricrin, and CDSN, probably as compensatory mechanisms for stabilization of the epidermal barrier. Our results give insights into the consequences of TGM5 mutations on terminal epidermal differentiation.

  12. A 20 bp Duplication in Exon 2 of the Aristaless-Like Homeobox 4 Gene (ALX4 Is the Candidate Causative Mutation for Tibial Hemimelia Syndrome in Galloway Cattle.

    Directory of Open Access Journals (Sweden)

    Bertram Brenig

    Full Text Available Aristaless-like homeobox 4 (ALX4 gene is an important transcription regulator in skull and limb development. In humans and mice ALX4 mutations or loss of function result in a number of skeletal and organ malformations, including polydactyly, tibial hemimelia, omphalocele, biparietal foramina, impaired mammary epithelial morphogenesis, alopecia, coronal craniosynostosis, hypertelorism, depressed nasal bridge and ridge, bifid nasal tip, hypogonadism, and body agenesis. Here we show that a complex skeletal malformation of the hind limb in Galloway cattle together with other developmental anomalies is a recessive autosomal disorder most likely caused by a duplication of 20 bp in exon 2 of the bovine ALX4 gene. A second duplication of 34 bp in exon 4 of the same gene has no known effect, although both duplications result in a frameshift and premature stop codon leading to a truncated protein. Genotyping of 1,688 Black/Red/Belted/Riggit Galloway (GA and 289 White Galloway (WGA cattle showed that the duplication in exon 2 has allele frequencies of 1% in GA and 6% in WGA and the duplication in exon 4 has frequencies of 23% in GA and 38% in WGA. Both duplications were not detected in 876 randomly selected German Holstein Friesian and 86 cattle of 21 other breeds. Hence, we have identified a candidate causative mutation for tibial hemimelia syndrome in Galloway cattle and selection against this mutation can be used to eliminate the mutant allele from the breed.

  13. Avaliação do custo de colírios lubrificantes a base exclusivamente de carboximetilcelulose no mercado brasileiro

    Directory of Open Access Journals (Sweden)

    Paulo Estacia

    2013-10-01

    Full Text Available OBJETIVO: Avaliar o custo do uso dos colírios lubrificantes mais utilizados no mercado nacional que apresentam na formulação o mesmo princípio ativo - carboximetilcelulose, considerando o número total de gotas presente em cada frasco dos colírios. MÉTODOS: Foi realizado um estudo experimental utilizando três frascos de cada um dos colírios Lacrifilm® (colírio 1 e Fresh Tears® (colírio 2. Para análise do custo dos colírios contou-se o número de gotas de cada frasco correlacionando os preços dos mesmos. O preço considerado para cada medicação foi o mínimo ao consumidor com alíquota de 17% publicado para o mês de janeiro de 2012. A análise estatística foi efetuada em SPSS® 18. A comparação das variáveis quantitativas analisadas foi procedida através do teste não paramétrico Mann-Whitney e correlação linear de Spearman, sendo considerada uma diferença estatisticamente significante um valor de probabilidade inferior a 0,05. RESULTADOS: Verificou-se que há diferença estatisticamente significativa entre as marcas de colírios avaliados com relação ao número total de gotas. O colírio 1 apresentou o maior número de gotas. CONCLUSÃO: O colírio 1, que tem o maior número de gotas por frasco, também é o que tem o preço mais acessível. Portanto, verifica-se que é o produto mais econômico, ou seja, o paciente pagará menos por cada gota.

  14. The association between detected drug resistance mutations and CD4(+) T-cell decline in HIV-positive individuals maintained on a failing treatment regimen

    DEFF Research Database (Denmark)

    Schultze, Anna; Paredes, Roger; Sabin, Caroline

    2018-01-01

    BACKGROUND: To analyse the effect of drug resistance mutations (DRM) on CD4 cell trends in HIV-positive people maintained on virologically failing antiretroviral therapy (ART). METHODS: Individuals from two large cohorts experiencing virological failure (VF) while maintained on ART with >1 CD4...

  15. Waardenburg syndrome type 4: report of two new cases caused by SOX10 mutations in Spain.

    Science.gov (United States)

    Fernández, Raquel M; Núñez-Ramos, Raquel; Enguix-Riego, M Valle; Román-Rodríguez, Francisco José; Galán-Gómez, Enrique; Blesa-Sánchez, Emilio; Antiñolo, Guillermo; Núñez-Núñez, Ramón; Borrego, Salud

    2014-02-01

    Shah-Waardenburg syndrome or Waardenburg syndrome type 4 (WS4) is a neurocristopathy characterized by the association of deafness, depigmentation and Hirschsprung disease. Three disease-causing genes have been identified so far for WS4: EDNRB, EDN3, and SOX10. SOX10 mutations, found in 45-55% of WS4 patients, are inherited in autosomal dominant way. In addition, mutations in SOX10 are also responsible for an extended syndrome involving peripheral and central neurological phenotypes, referred to as PCWH (peripheral demyelinating neuropathy, central dysmyelinating leucodystrophy, Waardenburg syndrome, Hirschsprung disease). Such mutations are mostly private, and a high intra- and inter-familial variability exists. In this report, we present a patient with WS4 and a second with PCWH due to SOX10 mutations supporting again the genetic and phenotypic heterogeneity of these syndromes. Interestingly, the WS4 family carries an insertion of 19 nucleotides in exon 5 of SOX10, which results in distinct phenotypes along three different generations: hypopigmentation in the maternal grandmother, hearing loss in the mother, and WS4 in the proband. Since mosaicism cannot explain the three different related-WS features observed in this family, we propose as the most plausible explanation the existence of additional molecular events, acting in an additive or multiplicative fashion, in genes or regulatory regions unidentified so far. On the other hand, the PCWH case was due to a de novo deletion in exon 5 of the gene. Efforts should be devoted to unravel the mechanisms underlying the intrafamilial phenotypic variability observed in the families affected, and to identify new genes responsible for the still unsolved WS4 cases. © 2013 Wiley Periodicals, Inc.

  16. COL5A1 gene variants previously associated with reduced soft tissue injury risk are associated with elite athlete status in rugby.

    Science.gov (United States)

    Heffernan, Shane M; Kilduff, Liam P; Erskine, Robert M; Day, Stephen H; Stebbings, Georgina K; Cook, Christian J; Raleigh, Stuart M; Bennett, Mark A; Wang, Guan; Collins, Malcolm; Pitsiladis, Yannis P; Williams, Alun G

    2017-11-14

    Two common single nucleotide polymorphisms within the COL5A1 gene (SNPs; rs12722 C/T and rs3196378 C/A) have previously been associated with tendon and ligament pathologies. Given the high incidence of tendon and ligament injuries in elite rugby athletes, we hypothesised that both SNPs would be associated with career success. In 1105 participants (RugbyGene project), comprising 460 elite rugby union (RU), 88 elite rugby league athletes and 565 non-athlete controls, DNA was collected and genotyped for the COL5A1 rs12722 and rs3196378 variants using real-time PCR. For rs12722, the injury-protective CC genotype and C allele were more common in all athletes (21% and 47%, respectively) and RU athletes (22% and 48%) than in controls (16% and 41%, P ≤ 0.01). For rs3196378, the CC genotype and C allele were overrepresented in all athletes (23% and 48%) and RU athletes (24% and 49%) compared with controls (16% and 41%, P ≤ 0.02). The CC genotype in particular was overrepresented in the back and centres (24%) compared with controls, with more than twice the odds (OR = 2.25, P = 0.006) of possessing the injury-protective CC genotype. Furthermore, when considering both SNPs simultaneously, the CC-CC SNP-SNP combination and C-C inferred allele combination were higher in all the athlete groups (≥18% and ≥43%) compared with controls (13% and 40%; P = 0.01). However, no genotype differences were identified for either SNP when RU playing positions were compared directly with each other. It appears that the C alleles, CC genotypes and resulting combinations of both rs12722 and rs3196378 are beneficial for rugby athletes to achieve elite status and carriage of these variants may impart an inherited resistance against soft tissue injury, despite exposure to the high-risk environment of elite rugby. These data have implications for the management of inter-individual differences in injury risk amongst elite athletes.

  17. Mutations in the ABCA4 (ABCR) Gene Are the Major Cause of Autosomal Recessive Cone-Rod Dystrophy

    OpenAIRE

    Maugeri, Alessandra; Klevering, B. Jeroen; Rohrschneider, Klaus; Blankenagel, Anita; Brunner, Han G.; Deutman, August F.; Hoyng, Carel B.; Cremers, Frans P. M.

    2000-01-01

    The photoreceptor cell–specific ATP-binding cassette transporter gene (ABCA4; previously denoted “ABCR”) is mutated in most patients with autosomal recessive (AR) Stargardt disease (STGD1) or fundus flavimaculatus (FFM). In addition, a few cases with AR retinitis pigmentosa (RP) and AR cone-rod dystrophy (CRD) have been found to have ABCA4 mutations. To evaluate the importance of the ABCA4 gene as a cause of AR CRD, we selected 5 patients with AR CRD and 15 patients with isolated CRD, all fro...

  18. A novel HSF4 gene mutation (p.R405X causing autosomal recessive congenital cataracts in a large consanguineous family from Pakistan

    Directory of Open Access Journals (Sweden)

    Cheema Abdul

    2008-11-01

    Full Text Available Abstract Background Hereditary cataracts are most frequently inherited as autosomal dominant traits, but can also be inherited in an autosomal recessive or X-linked fashion. To date, 12 loci for autosomal recessive cataracts have been mapped including a locus on chromosome 16q22 containing the disease-causing gene HSF4 (Genbank accession number NM_001040667. Here, we describe a family from Pakistan with the first nonsense mutation in HSF4 thus expanding the mutational spectrum of this heat shock transcription factor gene. Methods A large consanguineous Pakistani family with autosomal recessive cataracts was collected from Quetta. Genetic linkage analysis was performed for the common known autosomal recessive cataracts loci and linkage to a locus containing HSF4 (OMIM 602438 was found. All exons and adjacent splice sites of the heat shock transcription factor 4 gene (HSF4 were sequenced. A mutation-specific restriction enzyme digest (HphI was performed for all family members and unrelated controls. Results The disease phenotype perfectly co-segregated with markers flanking the known cataract gene HSF4, whereas other autosomal recessive loci were excluded. A maximum two-point LOD score with a Zmax = 5.6 at θ = 0 was obtained for D16S421. Direct sequencing of HSF4 revealed the nucleotide exchange c.1213C > T in this family predicting an arginine to stop codon exchange (p.R405X. Conclusion We identified the first nonsense mutation (p.R405X in exon 11 of HSF4 in a large consanguineous Pakistani family with autosomal recessive cataract.

  19. Detection of p53 gene mutations in bronchial biopsy samples of patients with lung cancer

    International Nuclear Information System (INIS)

    Irshad, S.; Nawaz, T.

    2008-01-01

    Lung cancer is the malignant transformation and expansion of lung tissue. It is the most lethal of all cancers worldwide, responsible for 1.2 million deaths annually. The goal of this study was to detect the p53 gene mutations in lung cancer, in local population of Lahore, Pakistan. These mutations were screened in the bronchial biopsy lung cancer tissue samples. For this purpose microtomed tissue sections were collected. Following DNA extraction from tissue sections, the p53 mutations were detected by amplifying Exon 7 (145 bp) and Exon 8 (152 bp) of the p53 gene. PCR then followed by single-strand conformation polymorphism analysis for screening the p53 gene mutations. This results of SSCP were visualized of silver staining. The results showed different banding pattern indicating the presence of mutation. Majority of the mutations were found in Exon 7. Exon 7 of p53 gene may be the mutation hotspot in lung cancer. In lung cancer, the most prevalent mutations of p53 gene are G -> T transversions; other types of insertions and deletions are also expected, however, the exact nature of mutations in presented work could be confirmed by direct sequencing. (author)

  20. Analyses of a novel SCN5A mutation (C1850S): conduction vs. repolarization disorder hypotheses in the Brugada syndrome

    DEFF Research Database (Denmark)

    Petitprez, Séverine; Jespersen, Thomas; Pruvot, Etienne

    2008-01-01

    S SCN5A mutation. METHODS AND RESULTS: SCN5A was screened for mutations in a male patient with type-1 BrS pattern ECG. Wild-type (WT) and mutant Na(v)1.5 channels were expressed in HEK293 cells. Sodium currents (I(Na)) were analysed using the whole-cell patch-clamp technique at 37 degrees C......AIMS: Brugada syndrome (BrS) is characterized by arrhythmias leading to sudden cardiac death. BrS is caused, in part, by mutations in the SCN5A gene, which encodes the sodium channel alpha-subunit Na(v)1.5. Here, we aimed to characterize the biophysical properties and consequences of a novel Br....... The electrophysiological effects of the mutation were simulated using the Luo-Rudy model, into which the transient outward current (I(to)) was incorporated. A new mutation (C1850S) was identified in the Na(v)1.5 C-terminal domain. In HEK293 cells, mutant I(Na) density was decreased by 62% at -20 mV. Inactivation of mutant...

  1. Late-onset Stargardt disease is associated with missense mutations that map outside known functional regions of ABCR (ABCA4).

    Science.gov (United States)

    Yatsenko, A N; Shroyer, N F; Lewis, R A; Lupski, J R

    2001-04-01

    Based on recent studies of the photoreceptor-specific ABC transporter gene ABCR (ABCA4) in Stargardt disease (STGD1) and other retinal dystrophies, we and others have developed a model in which the severity of retinal disease correlates inversely with residual ABCR activity. This model predicts that patients with late-onset STGDI may retain partial ABCR activity attributable to mild missense alleles. To test this hypothesis, we used late-onset STGDI patients (onset: > or =35 years) to provide an in vivo functional analysis of various combinations of mutant alleles. We sequenced directly the entire coding region of ABCR and detected mutations in 33/50 (66%) disease chromosomes, but surprisingly, 11/33 (33%) were truncating alleles. Importantly, all 22 missense mutations were located outside the known functional domains of ABCR (ATP-binding or transmembrane), whereas in our general cohort of STGDI subjects, alterations occurred with equal frequency across the entire protein. We suggest that these missense mutations in regions of unknown function are milder alleles and more susceptible to modifier effects. Thus, we have corroborated a prediction from the model of ABCR pathogenicity that (1) one mutant ABCR allele is always missense in late-onset STGD1 patients, and (2) the age-of-onset is correlated with the amount of ABCR activity of this allele. In addition, we report three new pseudodominant families that now comprise eight of 178 outbred STGD1 families and suggest a carrier frequency of STGD1-associated ABCR mutations of about 4.5% (approximately 1/22).

  2. High-resolution melting curve analysis for rapid detection of mutations in a Medaka TILLING library

    Directory of Open Access Journals (Sweden)

    Deguchi Tomonori

    2010-09-01

    Full Text Available Abstract Background During the last two decades, DNA sequencing has led to the identification of numerous genes in key species; however, in most cases, their functions are still unknown. In this situation, reverse genetics is the most suitable method to assign function to a gene. TILLING (Targeting Induced Local Lesions IN Genomes is a reverse-genetic strategy that combines random chemical mutagenesis with high-throughput discovery of the induced mutations in target genes. The method has been applied to a variety of plant and animal species. Screening of the induced mutations is the most important step in TILLING. Currently, direct sequencing or nuclease-mediated screening of heteroduplexes is widely used for detection of mutations in TILLING. Both methods are useful, but the costs are substantial and turnaround times are relatively long. Thus, there is a need for an alternative method that is of higher throughput and more cost effective. Results In this study, we developed a high resolution melting (HRM assay and evaluated its effectiveness for screening ENU-induced mutations in a medaka TILLING library. We had previously screened mutations in the p53 gene by direct sequencing. Therefore, we first tested the efficiency of the HRM assay by screening mutations in p53, which indicated that the HRM assay is as useful as direct sequencing. Next, we screened mutations in the atr and atm genes with the HRM assay. Nonsense mutations were identified in each gene, and the phenotypes of these nonsense mutants confirmed their loss-of-function nature. Conclusions These results demonstrate that the HRM assay is useful for screening mutations in TILLING. Furthermore, the phenotype of the obtained mutants indicates that medaka is an excellent animal model for investigating genome stability and gene function, especially when combined with TILLING.

  3. Autoradiographic detection of mutation to exotoxin-A resistance in mouse fibroblasts treated with ethyl methanesulfonate, X-rays and ultraviolet light

    International Nuclear Information System (INIS)

    Tiah, M.; Ronen, A.

    1989-01-01

    P. aeruginosa exotoxin-A (PE) blocks protein synthesis in mammalian cells by inactivating elongation factor 2 (EF-2). Toxin-resistant mutant cells can be detected autoradioraphically, in cultures grown on microscope coverslips in the presence of PE, and exposed to [ 3 H]leucine. The frequency of PE-resistant cells detected by the autoradiographic assay in non-mutagenized cells of the established mouse cell line LTKA is 9.7 j 0.6 x 10 -5 . Upon treatment with ethyl methanesulfonate (EMS), X-rays of ultraviolet (UV) light it increases in a dose-dependent fashion. The mutational nature of the resistance detected by the assay is indicated by its clonal inheritance, and by the dose-dependent increase in the frequency of resistant cells after utagenesis. On the basis of the high frequency of PE-resistant cells detected by the autoradiographic assay, and their cross-resistance to diphteria toxin (DT), the authors suggest that the PE-resistant mutants detected by the autoradiographic assay are of class II, i.e., they are altered in the structural gene for EF-2. (author). 27 refs.; 5 figs.; 3 tabs

  4. A male case with CDKL5-associated encephalopathy manifesting transient methylmalonic acidemia.

    Science.gov (United States)

    Akamine, Satoshi; Ishizaki, Yoshito; Sakai, Yasunari; Torisu, Hiroyuki; Fukai, Ryoko; Miyake, Noriko; Ohkubo, Kazuhiro; Koga, Hiroshi; Sanefuji, Masafumi; Sakata, Ayumi; Kimura, Masahiko; Yamaguchi, Seiji; Sakamoto, Osamu; Hara, Toshiro; Saitsu, Hirotomo; Matsumoto, Naomichi; Ohga, Shouichi

    2018-03-03

    Mutations in the X-linked gene CDKL5 cause early-onset epileptic encephalopathy and severe developmental delay. Because this disorder predominantly affects females, the full clinical spectrum of male patients remains elusive. We herein report a 16-year-old boy, who suffered from intractable seizures 20 days after birth. Serial electroencephalograms detected recurrent focal epileptiform discharges from age 4 months, which evolved to hypsarrhythmia later in infancy. Mass-spectrometric analyses revealed increase in urinary excretion of methylmalonic acid without perturbed concentrations of propionic acid, homocystein and methionine. Whole-exome sequencing identified a de novo, truncating mutation in CDKL5 (NM_003159.2:c.419dupA, p.Asn140Lysfs*8). Targeted sequencing excluded concomitant mutations in methylmalonic academia-associated genes. No methylmalonic acidemia has been reported in children with CDKL5 disorder. Extensive analyses on organic acid metabolism for males with CDKL5 mutations will gain more insight into their biochemical profiles in infancy. Copyright © 2018. Published by Elsevier Masson SAS.

  5. Tempo de reepitelização corneana com a instilação de colírio contendo hialuronato de sódio e carboximetilcelulose

    Directory of Open Access Journals (Sweden)

    Luciane Bugman Moreira

    2013-10-01

    Full Text Available OBJETIVO: Avaliar o tempo de reepitelização corneana pós abrasão usando colírios comercialmente disponíveis, um contendo hialuronato de sódio a 0,4%, outro contendo carboximetilcelulose a 1%, e comparar com a reepitelização sem instilação de colírio. MÉTODOS: Foram utilizados 24 coelhos, nos quais foi feita a abrasão mecânica da córnea nos 8 mm centrais. Esses animais foram divididos em três grupos. O primeiro grupo recebeu um colírio disponível comercialmente contendo hialuronato de sódio 0,4%, o segundo recebeu um colírio contendo carboximetilcelulose 1% e o terceiro não recebeu nenhuma droga. A avaliação foi feita a cada 24 horas por meio da análise de fotografias digitais sob luz azul de cobalto e coramento das córneas com fluoresceína a 2%. O estudo das imagens foi feito pelo sistema de análise de imagens do Autocad 2009®. A análise dos dados foi feita comparando o tempo total de reepitelização da córnea e a cada 24 horas entre os três grupos. RESULTADOS: A velocidade de reepitelização do grupo que usou colírio contendo hialuronato de sódio foi em média 90 horas; o grupo que usou carboximetilcelulose apresentou média de 105 horas; e o grupo que não usou nenhum tipo de lubrificante apresentou média de 108 horas para total reepitelização. Houve uma melhor performance na reepitelização após 96 horas nas córneas dos coelhos que usaram os colírios lubrificantes, sendo essa diferença estatisticamente comprovada. CONCLUSÃO: O colírio contendo hialuronato de sódio 0,4% mostrou índice de eficácia maior que aquele contendo carboximetilcelulose 1%, e este maior eficácia que o controle. Os resultados encontrados neste estudo mostram que o uso de lubrificantes no processo de reepitelização são de extrema valia e devem ser usados de rotina na clínica oftalmológica.

  6. Key Clinical Features to Identify Girls with "CDKL5" Mutations

    Science.gov (United States)

    Bahi-Buisson, Nadia; Nectoux, Juliette; Rosas-Vargas, Haydee; Milh, Mathieu; Boddaert, Nathalie; Girard, Benoit; Cances, Claude; Ville, Dorothee; Afenjar, Alexandra; Rio, Marlene; Heron, Delphine; Morel, Marie Ange N'Guyen; Arzimanoglou, Alexis; Philippe, Christophe; Jonveaux, Philippe; Chelly, Jamel; Bienvenu, Thierry

    2008-01-01

    Mutations in the human X-linked cyclin-dependent kinase-like 5 ("CDKL5") gene have been shown to cause infantile spasms as well as Rett syndrome (RTT)-like phenotype. To date, less than 25 different mutations have been reported. So far, there are still little data on the key clinical diagnosis criteria and on the natural history of…

  7. Mutation Detection in Patients With Advanced Cancer by Universal Sequencing of Cancer-Related Genes in Tumor and Normal DNA vs Guideline-Based Germline Testing.

    Science.gov (United States)

    Mandelker, Diana; Zhang, Liying; Kemel, Yelena; Stadler, Zsofia K; Joseph, Vijai; Zehir, Ahmet; Pradhan, Nisha; Arnold, Angela; Walsh, Michael F; Li, Yirong; Balakrishnan, Anoop R; Syed, Aijazuddin; Prasad, Meera; Nafa, Khedoudja; Carlo, Maria I; Cadoo, Karen A; Sheehan, Meg; Fleischut, Megan H; Salo-Mullen, Erin; Trottier, Magan; Lipkin, Steven M; Lincoln, Anne; Mukherjee, Semanti; Ravichandran, Vignesh; Cambria, Roy; Galle, Jesse; Abida, Wassim; Arcila, Marcia E; Benayed, Ryma; Shah, Ronak; Yu, Kenneth; Bajorin, Dean F; Coleman, Jonathan A; Leach, Steven D; Lowery, Maeve A; Garcia-Aguilar, Julio; Kantoff, Philip W; Sawyers, Charles L; Dickler, Maura N; Saltz, Leonard; Motzer, Robert J; O'Reilly, Eileen M; Scher, Howard I; Baselga, Jose; Klimstra, David S; Solit, David B; Hyman, David M; Berger, Michael F; Ladanyi, Marc; Robson, Mark E; Offit, Kenneth

    2017-09-05

    Guidelines for cancer genetic testing based on family history may miss clinically actionable genetic changes with established implications for cancer screening or prevention. To determine the proportion and potential clinical implications of inherited variants detected using simultaneous sequencing of the tumor and normal tissue ("tumor-normal sequencing") compared with genetic test results based on current guidelines. From January 2014 until May 2016 at Memorial Sloan Kettering Cancer Center, 10 336 patients consented to tumor DNA sequencing. Since May 2015, 1040 of these patients with advanced cancer were referred by their oncologists for germline analysis of 76 cancer predisposition genes. Patients with clinically actionable inherited mutations whose genetic test results would not have been predicted by published decision rules were identified. Follow-up for potential clinical implications of mutation detection was through May 2017. Tumor and germline sequencing compared with the predicted yield of targeted germline sequencing based on clinical guidelines. Proportion of clinically actionable germline mutations detected by universal tumor-normal sequencing that would not have been detected by guideline-directed testing. Of 1040 patients, the median age was 58 years (interquartile range, 50.5-66 years), 65.3% were male, and 81.3% had stage IV disease at the time of genomic analysis, with prostate, renal, pancreatic, breast, and colon cancer as the most common diagnoses. Of the 1040 patients, 182 (17.5%; 95% CI, 15.3%-19.9%) had clinically actionable mutations conferring cancer susceptibility, including 149 with moderate- to high-penetrance mutations; 101 patients tested (9.7%; 95% CI, 8.1%-11.7%) would not have had these mutations detected using clinical guidelines, including 65 with moderate- to high-penetrance mutations. Frequency of inherited mutations was related to case mix, stage, and founder mutations. Germline findings led to discussion or initiation of

  8. Stimulation of Escherichia coli F-18Col- Type-1 fimbriae synthesis by leuX

    DEFF Research Database (Denmark)

    Newman, Joseph V.; Burghoff, Robert L.; Pallesen, Lars

    1994-01-01

    Escherichia coli F-18, a normal human fecal isolate, is an excellent colonizer of the streptomycin-treated mouse large intestine. E. coli F-18Col-, a derivative of E. coli F-18 which no longer makes the E. coli F-18 colicin, colonizes the large intestine as well as E. coli F-18 when fed to mice...... alone but is eliminated when fed together with E. coli F-18. Recently we randomly cloned E. coli F-18 DNA into E. coli F-18Col- and let the mouse intestine select the best colonizer. In this way, we isolated a 6.5-kb E. coli F-18 DNA sequence that simultaneously stimulated synthesis of type 1 fimbriae...... and enhanced E. coli F-18Col- colonizing ability. In the present investigation we show that the gene responsible for stimulation of type 1 fimbriae synthesis appears to be leuX, which encodes a tRNA specific for the rare leucine codon UUG. Moreover, it appears that expression of leuX may be regulated by two...

  9. RFLP analysis of rice semi-dwarf mutation induced by high energy argon ion radiation

    International Nuclear Information System (INIS)

    Zhuang Chuxiong; Hu Weimin; Mei Mantong

    1997-01-01

    Two Indica rice varieties, Bianpizhan and Xiangzhan, and their semi-dwarf mutants induced by high energy argon ion radiation, Ar-10, and Xiang-Ar-1, were examined with restriction fragment length polymorphism (RFLP) analysis by using 97 rice single copy genomic clones mapped on 12 chromosomes of molecular genetic map, combined with 5 restriction enzymes. Among the markers screened, 9 detected polymorphism were between Bianpizhen and Ar-10, and 11 detected polymorphism were between Xiangzhan and Xiang-Ar-1. Moreover, two or more restriction enzymes could generate RFLP patterns when screened with a given marker for several polymorphic markers. Based on the polymorphic allelic loci, the mutation frequencies were estimated as 5.15% and 6.39% for Ar-10 and Xiang-Ar-1 respectively. These results suggested that the nature of mutation on the DNA level was probably large genetic changes rather than point mutation. Genetic analysis and gene tagging of semi-dwarf mutation in one of the mutant line, Ar-10, indicated that this mutation was controlled by a major recessive gene, which was preliminary located on chromosome 4

  10. RFLP Analysis of rice semi dwarf mutation induced by high energy argon ion radiation

    International Nuclear Information System (INIS)

    Zhuang Chuxiong; Hu Weimin; Mei Mantong

    1997-01-01

    Two Indica rice varieties, Bianpizhan and Xiangzhan, and their semi dwarf mutants induced by high energy argon ion radiation, Ar 10, and Xiang Ar 1, were examined with restriction fragment length polymorphism(RFLP)analysis by using 97 rice single copy genomic clones mapped on 12 chromosomes of molecular genetic map, combined with 5 restriction enzymes.Among the markers screened, 9 detected polymorphism were between Bianpizhan and Ar 10, and 11 detected polymorphism were between Xiangzhan and Xiang Ar 1.Moreover, two or more restriction enzymes could generate RFLP patterns when screened with a given marker for several polymorphic markers. Based on the polymorphic allelic loci, the mutation frequencies were estimated as 5 15% and 6 39% for Ar 10 and Xiang Ar 1 respectively.These results suggested that the nature of mutation on the DNA level was probably large genetic changes rather than point mutation.Genetic analysis and gene tagging of semi dwarf mutation in one of the mutant line, Ar 10, indicated that this mutation was controlled by a major recessive gene, which was preliminary located on chromosome 4. (author)

  11. Germline CDKN2A/P16INK4A mutations contribute to genetic determinism of sarcoma.

    Science.gov (United States)

    Jouenne, Fanélie; Chauvot de Beauchene, Isaure; Bollaert, Emeline; Avril, Marie-Françoise; Caron, Olivier; Ingster, Olivier; Lecesne, Axel; Benusiglio, Patrick; Terrier, Philippe; Caumette, Vincent; Pissaloux, Daniel; de la Fouchardière, Arnaud; Cabaret, Odile; N'Diaye, Birama; Velghe, Amélie; Bougeard, Gaelle; Mann, Graham J; Koscielny, Serge; Barrett, Jennifer H; Harland, Mark; Newton-Bishop, Julia; Gruis, Nelleke; Van Doorn, Remco; Gauthier-Villars, Marion; Pierron, Gaelle; Stoppa-Lyonnet, Dominique; Coupier, Isabelle; Guimbaud, Rosine; Delnatte, Capucine; Scoazec, Jean-Yves; Eggermont, Alexander M; Feunteun, Jean; Tchertanov, Luba; Demoulin, Jean-Baptiste; Frebourg, Thierry; Bressac-de Paillerets, Brigitte

    2017-09-01

    Sarcomas are rare mesenchymal malignancies whose pathogenesis is poorly understood; both environmental and genetic risk factors could contribute to their aetiology. We performed whole-exome sequencing (WES) in a familial aggregation of three individuals affected with soft-tissue sarcoma (STS) without TP53 mutation (Li-Fraumeni-like, LFL) and found a shared pathogenic mutation in CDKN2A tumour suppressor gene. We searched for individuals with sarcoma among 474 melanoma-prone families with a CDKN2A -/+ genotype and for CDKN2A mutations in 190 TP53 -negative LFL families where the index case was a sarcoma. Including the initial family, eight independent sarcoma cases carried a germline mutation in the CDKN2A /p16 INK4A gene. In five out of seven formalin-fixed paraffin-embedded sarcomas, heterozygosity was lost at germline CDKN2A mutations sites demonstrating complete loss of function. As sarcomas are rare in CDKN2A /p16 INK4A carriers, we searched in constitutional WES of nine carriers for potential modifying rare variants and identified three in platelet-derived growth factor receptor ( PDGFRA ) gene. Molecular modelling showed that two never-described variants could impact the PDGFRA extracellular domain structure. Germline mutations in CDKN2A /P16 INK4A , a gene known to predispose to hereditary melanoma, pancreatic cancer and tobacco-related cancers, account also for a subset of hereditary sarcoma. In addition, we identified PDGFRA as a candidate modifier gene. © Article author(s) (or their employer(s) unless otherwise stated in the text of the article) 2017. All rights reserved. No commercial use is permitted unless otherwise expressly granted.

  12. MPL515 mutations in myeloproliferative and other myeloid disorders: a study of 1182 patients.

    Science.gov (United States)

    Pardanani, Animesh D; Levine, Ross L; Lasho, Terra; Pikman, Yana; Mesa, Ruben A; Wadleigh, Martha; Steensma, David P; Elliott, Michelle A; Wolanskyj, Alexandra P; Hogan, William J; McClure, Rebecca F; Litzow, Mark R; Gilliland, D Gary; Tefferi, Ayalew

    2006-11-15

    Recently, a gain-of-function MPL mutation, MPLW515L, was described in patients with JAK2V617F-negative myelofibrosis with myeloid metaplasia (MMM). To gain more information on mutational frequency, disease specificity, and clinical correlates, genomic DNA from 1182 patients with myeloproliferative and other myeloid disorders and 64 healthy controls was screened for MPL515 mutations, regardless of JAK2V617F mutational status: 290 with MMM, 242 with polycythemia vera, 318 with essential thrombocythemia (ET), 88 with myelodysplastic syndrome, 118 with chronic myelomonocytic leukemia, and 126 with acute myeloid leukemia (AML). MPL515 mutations, either MPLW515L (n = 17) or a previously undescribed MPLW515K (n = 5), were detected in 20 patients. The diagnosis of patients with mutant MPL alleles at the time of molecular testing was de novo MMM in 12 patients, ET in 4, post-ET MMM in 1, and MMM in blast crisis in 3. Six patients carried the MPLW515L and JAK2V617F alleles concurrently. We conclude that MPLW515L or MPLW515K mutations are present in patients with MMM or ET at a frequency of approximately 5% and 1%, respectively, but are not observed in patients with polycythemia vera (PV) or other myeloid disorders. Furthermore, MPL mutations may occur concurrently with the JAK2V617F mutation, suggesting that these alleles may have functional complementation in myeloproliferative disease.

  13. Characterizations of a loss-of-function mutation in the Kir3.4 channel subunit

    DEFF Research Database (Denmark)

    Calloe, Kirstine; Ravn, Lasse Steen; Schmitt, Nicole

    2007-01-01

    and two-electrode voltage-clamp revealed that Kir3.4-G247R basal current was reduced compared to wild-type Kir3.4 and co-expression with the muscarinic acetylcholine receptor type 2 showed that also the acetylcholine induced current was severely reduced in Kir3.4-G247R, indicating that the mutation...... the lack of clear clinical manifestations and further studies are necessary to elucidate if mutations in Kir3.4 are predisposing AF. Udgivelsesdato: 2007-Dec-28...

  14. Clinical and genetic investigation of a Japanese family with cardiac fabry disease. Identification of a novel α-galactosidase A missense mutation (G195V).

    Science.gov (United States)

    Nakagawa, Naoki; Maruyama, Hiroki; Ishihara, Takayuki; Seino, Utako; Kawabe, Jun-ichi; Takahashi, Fumihiko; Kobayashi, Motoi; Yamauchi, Atsushi; Sasaki, Yukie; Sakamoto, Naka; Ota, Hisanobu; Tanabe, Yasuko; Takeuchi, Toshiharu; Takenaka, Toshihiro; Kikuchi, Kenjiro; Hasebe, Naoyuki

    2011-01-01

    Fabry disease is an X-linked lysosomal storage disorder caused by mutations of the α-galactosidase A gene (GLA), and the disease is a relatively prevalent cause of left ventricular hypertrophy mimicking idiopathic hypertrophic cardiomyopathy. We assessed clinically 5 patients of a three-generation family and also searched for GLA mutations in 10 family members. The proband had left ventricular hypertrophy with localized thinning in the basal posterior wall and late gadolinium enhancement (LGE) in the near-circumferential wall in cardiovascular magnetic resonance images and her sister had vasospastic angina pectoris without organic stenosis of the coronary arteries. LGE notably appeared in parallel with decreased α-galactosidase A activity and increased NT-pro BNP in our patients. We detected a new GLA missense mutation (G195V) in exon 4, resulting in a glycine-to-valine substitution. Of the 10 family members, 5 family members each were positive and negative for this mutation. These new data extend our clinical and molecular knowledge of GLA gene mutations and confirm that a novel missense mutation in the GLA gene is important not only for a precise diagnosis of heterozygous status, but also for confirming relatives who are negative for this mutation.

  15. Complex mosaic CDKL5 deletion with two distinct mutant alleles in a 4-year-old girl.

    Science.gov (United States)

    Boutry-Kryza, Nadia; Ville, Dorothée; Labalme, Audrey; Calender, Alain; Dupont, Jean-Michel; Touraine, Renaud; Edery, Patrick; des Portes, Vincent; Sanlaville, Damien; Lesca, Gaetan

    2014-08-01

    Mutations of the CDKL5 gene cause early epileptic encephalopathy. Patients manifest refractory epilepsy, beginning before the age of 3 months, which is associated with severe psychomotor delay and features that overlap with Rett syndrome. We report here a patient with mosaicism for CDKL5 exonic deletion, with the presence of two mutant alleles. The affected 4-year-old girl presented with infantile spasms, beginning at the age of 9 months, but subsequent progression of the disease was consistent with the classical CDKL5-related phenotype. A deletion of exons 17 and 18 was suspected on the basis of Multiplex Ligation Probe Amplification analysis, but unexpected results for cDNA analysis, which showed the presence of an abnormal transcript with the deletion of exon 18 only, led us to suspect that two distinct events might have occurred. We used custom array-CGH to determine the size and breakpoints of these deletions. Exon 18 was deleted from one of the abnormal alleles, and exon 17 was deleted from the other. A Fork Stalling and Template Switching (FoSTeS) mechanism was proposed to explain the two events, given the presence of regions of microhomology at the breakpoints. We propose here an original involvement of the FoSTeS mechanism to explain the co-occurrence of these two events in the CDKL5 gene in a single patient. This patient highlights the difficulties involved in the detection of such abnormalities, particularly when they occur in a mosaic state and involve two distinct mutational events in a single gene. © 2014 Wiley Periodicals, Inc.

  16. Synthesis of diadenosine 5', 5'''-P1, P4-tetraphosphate (AP4A) in sea urchin embryos

    International Nuclear Information System (INIS)

    Morioka, Mizue; Shimada, Hiraku

    1984-01-01

    Sea urchin embryos were labeled with ( 3 H)adenosine at two developmental stages (morula and prism) and the labeled acid-soluble nucleotides were fractionated successively by column chromatography with DEAE-Sephadex and DEAE-cellulose, and by thin-layer chromatography on a PEI-cellulose plate. Significant radioactivity was detected on the PEI-cellulose plate at the region of diadenosine 5', 5'''-P 1 , P 4 -tetraphosphate (AP 4 A). After treatment of this fraction with phosphodiesterase, the radioactivity was all recovered in the AMP region, while alkaline phosphatase had no effect on the AP 4 A fraction. The present result suggests that AP 4 A is actively synthesized in the sea urchin embryos. (author)

  17. Complete COL1A1 allele deletions in osteogenesis imperfecta

    NARCIS (Netherlands)

    van Dijk, Fleur S.; Huizer, Margriet; Kariminejad, Ariana; Marcelis, Carlo L.; Plomp, Astrid S.; Terhal, Paulien A.; Meijers-Heijboer, Hanne; Weiss, Marjan M.; van Rijn, Rick R.; Cobben, Jan M.; Pals, Gerard

    Purpose: To identify a molecular genetic cause in patients with a clinical diagnosis of osteogenesis imperfecta (OI) type I/IV. Methods: The authors performed multiplex ligation-dependent probe amplification analysis of the COL1A1 gene in a group of 106 index patients. Results: In four families with

  18. Complete COL1A1 allele deletions in osteogenesis imperfecta

    NARCIS (Netherlands)

    van Dijk, Fleur S.; Huizer, Margriet; Kariminejad, Ariana; Marcelis, Carlo L.; Plomp, Astrid S.; Terhal, Paulien A.; Meijers-Heijboer, Hanne; Weiss, Marjan M.; van Rijn, Rick R.; Cobben, Jan M.; Pals, Gerard

    2010-01-01

    To identify a molecular genetic cause in patients with a clinical diagnosis of osteogenesis imperfecta (OI) type I/IV. The authors performed multiplex ligation-dependent probe amplification analysis of the COL1A1 gene in a group of 106 index patients. In four families with mild osteogenesis

  19. Validation of an NGS mutation detection panel for melanoma.

    Science.gov (United States)

    Reiman, Anne; Kikuchi, Hugh; Scocchia, Daniela; Smith, Peter; Tsang, Yee Wah; Snead, David; Cree, Ian A

    2017-02-22

    Knowledge of the genotype of melanoma is important to guide patient management. Identification of mutations in BRAF and c-KIT lead directly to targeted treatment, but it is also helpful to know if there are driver oncogene mutations in NRAS, GNAQ or GNA11 as these patients may benefit from alternative strategies such as immunotherapy. While polymerase chain reaction (PCR) methods are often used to detect BRAF mutations, next generation sequencing (NGS) is able to determine all of the necessary information on several genes at once, with potential advantages in turnaround time. We describe here an Ampliseq hotspot panel for melanoma for use with the IonTorrent Personal Genome Machine (PGM) which covers the mutations currently of most clinical interest. We have validated this in 151 cases of skin and uveal melanoma from our files, and correlated the data with PCR based assessment of BRAF status. There was excellent agreement, with few discrepancies, though NGS does have greater coverage and picks up some mutations that would be missed by PCR. However, these are often rare and of unknown significance for treatment. PCR methods are rapid, less time-consuming and less expensive than NGS, and could be used as triage for patients requiring more extensive diagnostic workup. The NGS panel described here is suitable for clinical use with formalin-fixed paraffin-embedded (FFPE) samples.

  20. EuroCirCol kick-off event

    CERN Multimedia

    Hardre, Julie

    2015-01-01

    The EuroCirCol (http://cern.ch/eurocircol) kick-off event at CERN on June 2-4 brought together 62 participants to constitute governance bodies, commit to the project plan and align the organisation, structures and processes of 16 institutions from 10 countries. The goal of the project is to conceive a post-LHC research infrastructure around a 100 km circular energy-frontier hadron collider capable of reaching 100 TeV collisions. The project officially started on June 1 and will run for four years. The total estimated budget of 11.2 million Euros includes a 2.99 million Euro contribution from the Horizon 2020 programme on developing new world-class research infrastructures (http://cordis.europa.eu/project/rcn/194962_en.html).