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Sample records for rna-based mutation screening

  1. A siRNA-based screen for genes involved in chromosome end protection.

    Directory of Open Access Journals (Sweden)

    Daniel H Lackner

    Full Text Available Telomeres are nucleoprotein complexes which protect the ends of linear chromosomes from detection as DNA damage and provide a sequence buffer against replication-associated shortening. In mammals, telomeres consist of repetitive DNA sequence (TTAGGG and associated proteins. The telomeric core complex is called shelterin and is comprised of the proteins TRF1, TRF2, POT1, TIN2, TPP1 and RAP1. Excessive telomere shortening or de-protection of telomeres through the loss of shelterin subunits allows the detection of telomeres as DNA damage, which can be visualized as DNA damage protein foci at chromosome ends called TIF (Telomere Dysfunction-Induced Foci. We sought to exploit the TIF phenotype as marker for telomere dysfunction to identify novel genes involved in telomere protection by siRNA-mediated knock-down of a set of 386 candidates. Here we report the establishment, specificity and feasibility of such a screen and the results of the genes tested. Only one of the candidate genes showed a unique TIF phenotype comparable to the suppression of the main shelterin components TRF2 or TRF1 and that gene was identified as a TRF1-like pseudogene. We also identified a weak TIF phenotype for SKIIP (SNW1, a splicing factor and transcriptional co-activator. However, the knock-down of SKIIP also induced a general, not telomere-specific DNA damage response, which complicates conclusions about a telomeric role. In summary, this report is a technical demonstration of the feasibility of a cell-based screen for telomere deprotection with the potential of scaling it to a high-throughput approach.

  2. An siRNA-based functional genomics screen for the identification of regulators of ciliogenesis and ciliopathy genes

    NARCIS (Netherlands)

    Wheway, Gabrielle; Schmidts, Miriam; Mans, Dorus A; Szymanska, Katarzyna; Nguyen, Thanh-Minh T; Racher, Hilary; Phelps, Ian G; Toedt, Grischa; Kennedy, Julie; Wunderlich, Kirsten A; Sorusch, Nasrin; Abdelhamed, Zakia A; Natarajan, Subaashini; Herridge, Warren; van Reeuwijk, Jeroen; Horn, Nicola; Boldt, Karsten; Parry, David A; Letteboer, Stef J F; Roosing, Susanne; Adams, Matthew; Bell, Sandra M; Bond, Jacquelyn; Higgins, Julie; Morrison, Ewan E; Tomlinson, Darren C; Slaats, Gisela G; van Dam, Teunis J P; Huang, Lijia; Kessler, Kristin; Giessl, Andreas; Logan, Clare V; Boyle, Evan A; Shendure, Jay; Anazi, Shamsa; Aldahmesh, Mohammed; Al Hazzaa, Selwa; Hegele, Robert A; Ober, Carole; Frosk, Patrick; Mhanni, Aizeddin A; Chodirker, Bernard N; Chudley, Albert E; Lamont, Ryan; Bernier, Francois P; Beaulieu, Chandree L; Gordon, Paul; Pon, Richard T; Donahue, Clem; Barkovich, A James; Wolf, Louis; Toomes, Carmel; Thiel, Christian T; Boycott, Kym M; McKibbin, Martin; Inglehearn, Chris F; Stewart, Fiona; Omran, Heymut; Huynen, Martijn A; Sergouniotis, Panagiotis I; Alkuraya, Fowzan S; Parboosingh, Jillian S; Innes, A Micheil; Willoughby, Colin E; Giles, Rachel H; Webster, Andrew R; Ueffing, Marius; Blacque, Oliver; Gleeson, Joseph G; Wolfrum, Uwe; Beales, Philip L; Gibson, Toby; Doherty, Dan; Mitchison, Hannah M; Roepman, Ronald; Johnson, Colin A

    Defects in primary cilium biogenesis underlie the ciliopathies, a growing group of genetic disorders. We describe a whole-genome siRNA-based reverse genetics screen for defects in biogenesis and/or maintenance of the primary cilium, obtaining a global resource. We identify 112 candidate ciliogenesis

  3. Screening of three Mediterranean phenylketonuria mutations in ...

    Indian Academy of Sciences (India)

    Kaabachi N. 2012 Screening of three Mediterranean phenylketonuria mutations in Tunisian families. J. Genet. 91, 91–94]. Introduction. Phenylketonuria (PKU; OMIM 261600) is an autoso- mal recessive disease caused by the liver phenylalanine hydroxylase (PAH) enzyme (EC1.14.16.1) deficiency. If untreated, causes ...

  4. mRNA-based detection of rare CFTR mutations improves genetic diagnosis of cystic fibrosis in populations with high genetic heterogeneity.

    Science.gov (United States)

    Felício, V; Ramalho, A S; Igreja, S; Amaral, M D

    2017-03-01

    Even with advent of next generation sequencing complete sequencing of large disease-associated genes and intronic regions is economically not feasible. This is the case of cystic fibrosis transmembrane conductance regulator (CFTR), the gene responsible for cystic fibrosis (CF). Yet, to confirm a CF diagnosis, proof of CFTR dysfunction needs to be obtained, namely by the identification of two disease-causing mutations. Moreover, with the advent of mutation-based therapies, genotyping is an essential tool for CF disease management. There is, however, still an unmet need to genotype CF patients by fast, comprehensive and cost-effective approaches, especially in populations with high genetic heterogeneity (and low p.F508del incidence), where CF is now emerging with new diagnosis dilemmas (Brazil, Asia, etc). Herein, we report an innovative mRNA-based approach to identify CFTR mutations in the complete coding and intronic regions. We applied this protocol to genotype individuals with a suspicion of CF and only one or no CFTR mutations identified by routine methods. It successfully detected multiple intronic mutations unlikely to be detected by CFTR exon sequencing. We conclude that this is a rapid, robust and inexpensive method to detect any CFTR coding/intronic mutation (including rare ones) that can be easily used either as primary approach or after routine DNA analysis. © 2016 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  5. Amelogenesis Imperfecta and Screening of Mutation in Amelogenin Gene

    Directory of Open Access Journals (Sweden)

    Fernanda Veronese Oliveira

    2014-01-01

    Full Text Available The aim of this study was to report the clinical findings and the screening of mutations of amelogenin gene of a 7-year-old boy with amelogenesis imperfecta (AI. The genomic DNA was extracted from saliva of patient and his family, followed by PCR and direct DNA sequencing. The c.261C>T mutation was found in samples of mother, father, and brother, but the mutation was not found in the sequence of the patient. This mutation is a silent mutation and a single-nucleotide polymorphism (rs2106416. Thus, it is suggested that the mutation found was not related to the clinical presence of AI. Further research is necessary to examine larger number of patients and genes related to AI.

  6. IRF6 mutation screening in non-syndromic orofacial clefting

    DEFF Research Database (Denmark)

    Leslie, Elizabeth J; Koboldt, Daniel C; Kang, C. J.

    2016-01-01

    Van der Woude syndrome (VWS) is an autosomal dominant malformation syndrome characterized by orofacial clefting (OFC) and lower lip pits. The clinical presentation of VWS is variable and can present as an isolated OFC, making it difficult to distinguish VWS cases from individuals with non......-syndromic OFCs. About 70% of causal VWS mutations occur in IRF6, a gene that is also associated with non-syndromic OFCs. Screening for IRF6 mutations in apparently non-syndromic cases has been performed in several modestly sized cohorts with mixed results. In this study, we screened 1521 trios with presumed non......-syndromic OFCs to determine the frequency of causal IRF6 mutations. We identified seven likely causal IRF6 mutations, although a posteriori review identified two misdiagnosed VWS families based on the presence of lip pits. We found no evidence for association between rare IRF6 polymorphisms and non...

  7. Transposon-mediated Generation of Cellular and Mouse Models of Splicing Mutations to Assess the Efficacy of snRNA-based Therapeutics

    Directory of Open Access Journals (Sweden)

    Elena Barbon

    2016-01-01

    Full Text Available Disease-causing splicing mutations can be rescued by variants of the U1 small nuclear RNA (U1snRNAs. However, the evaluation of the efficacy and safety of modified U1snRNAs as therapeutic tools is limited by the availability of cellular and animal models specific for a given mutation. Hence, we exploited the hyperactive Sleeping Beauty transposon system (SB100X to integrate human factor IX (hFIX minigenes into genomic DNA in vitro and in vivo. We generated stable HEK293 cell lines and C57BL/6 mice harboring splicing-competent hFIX minigenes either wild type (SChFIX-wt or mutated (SChFIXex5-2C. In both models the SChFIXex5-2C variant, found in patients affected by Hemophilia B, displayed an aberrant splicing pattern characterized by exon 5 skipping. This allowed us to test, for the first time in a genomic DNA context, the efficacy of the snRNA U1-fix9, delivered with an adeno-associated virus (AAV vector. With this approach, we showed rescue of the correct splicing pattern of hFIX mRNA, leading to hFIX protein expression. These data validate the SB100X as a versatile tool to quickly generate models of human genetic mutations, to study their effect in a stable DNA context and to assess mutation-targeted therapeutic strategies.

  8. Screening for familial APP mutations in sporadic cerebral amyloid angiopathy.

    Directory of Open Access Journals (Sweden)

    Alessandro Biffi

    2010-11-01

    Full Text Available Advances in genetic technology have revealed that variation in the same gene can cause both rare familial and common sporadic forms of the same disease. Cerebral amyloid angiopathy (CAA, a common cause of symptomatic intracerebral hemorrhage (ICH in the elderly, can also occur in families in an autosomal dominant pattern. The majority of affected families harbor mutations in the Beta amyloid Peptide (Aβ coding region of the gene for amyloid precursor protein (APP or have duplications of chromosomal segments containing APP.A total of 58 subjects with a diagnosis of probable or definite CAA according to validated criteria were included in the present study. We sequenced the Aβ coding region of APP in 58 individuals and performed multiplex ligation-dependent probe amplification to determine APP gene dosage in 60. No patient harbored a known or novel APP mutation or gene duplication. The frequency of mutations investigated in the present study is estimated to range from 0% to 8% in individuals with probable CAA in the general population, based on the ascertained sample size.We found no evidence that variants at loci associated with familial CAA play a role in sporadic CAA. Based on our findings, these rare highly-penetrant mutations are unlikely to be seen in sporadic CAA patients. Therefore, our results do not support systematic genetic screening of CAA patients who lack a strong family history of hemorrhage or dementia.

  9. Screening of Duchenne muscular dystrophy (DMD) mutations and investigating its mutational mechanism in Chinese patients.

    Science.gov (United States)

    Chen, Chen; Ma, Hongwei; Zhang, Feng; Chen, Lu; Xing, Xuesha; Wang, Shusen; Zhang, Xue; Luo, Yang

    2014-01-01

    Duchenne muscular dystrophy (DMD) is a common X-linked recessive disease of muscle degeneration and death. In order to provide accurate and reliable genetic counseling and prenatal diagnosis, we screened DMD mutations in a cohort of 119 Chinese patients using multiplex ligation-dependent probe amplification (MLPA) and denaturing high performance liquid chromatography (DHPLC) followed by Sanger sequencing. In these unrelated DMD patients, we identified 11 patients with DMD small mutations (9.2%) and 81 patients with DMD deletions/duplications (del/dup) (68.1%), of which 64 (79.0%) were deletions, 16 (19.8%) were duplications, and one (1.2%) was both deletion and duplication. Furthermore, we analyzed the frequency of DMD breakpoint in the 64 deletion cases by calculating exon-deletion events of certain exon interval that revealed a novel mutation hotspot boundary. To explore why DMD rearrangement breakpoints were predisposed to specific regions (hotspot), we precisely characterized junction sequences of breakpoints at the nucleotide level in 21 patients with exon deleted/duplicated in DMD with a high-resolution SNP microarray assay. There were no exactly recurrent breakpoints and there was also no significant difference between single-exon del/dup and multiple-exon del/dup cases. The data from the current study provided a comprehensive strategy to detect DMD mutations for clinical practice, and identified two deletion hotspots at exon 43-55 and exon 10-23 by calculating exon-deletion events of certain exon interval. Furthermore, this is the first study to characterize DMD breakpoint at the nucleotide level in a Chinese population. Our observations provide better understanding of the mechanism for DMD gene rearrangements.

  10. Screening of Duchenne muscular dystrophy (DMD mutations and investigating its mutational mechanism in Chinese patients.

    Directory of Open Access Journals (Sweden)

    Chen Chen

    Full Text Available Duchenne muscular dystrophy (DMD is a common X-linked recessive disease of muscle degeneration and death. In order to provide accurate and reliable genetic counseling and prenatal diagnosis, we screened DMD mutations in a cohort of 119 Chinese patients using multiplex ligation-dependent probe amplification (MLPA and denaturing high performance liquid chromatography (DHPLC followed by Sanger sequencing. In these unrelated DMD patients, we identified 11 patients with DMD small mutations (9.2% and 81 patients with DMD deletions/duplications (del/dup (68.1%, of which 64 (79.0% were deletions, 16 (19.8% were duplications, and one (1.2% was both deletion and duplication. Furthermore, we analyzed the frequency of DMD breakpoint in the 64 deletion cases by calculating exon-deletion events of certain exon interval that revealed a novel mutation hotspot boundary. To explore why DMD rearrangement breakpoints were predisposed to specific regions (hotspot, we precisely characterized junction sequences of breakpoints at the nucleotide level in 21 patients with exon deleted/duplicated in DMD with a high-resolution SNP microarray assay. There were no exactly recurrent breakpoints and there was also no significant difference between single-exon del/dup and multiple-exon del/dup cases. The data from the current study provided a comprehensive strategy to detect DMD mutations for clinical practice, and identified two deletion hotspots at exon 43-55 and exon 10-23 by calculating exon-deletion events of certain exon interval. Furthermore, this is the first study to characterize DMD breakpoint at the nucleotide level in a Chinese population. Our observations provide better understanding of the mechanism for DMD gene rearrangements.

  11. Genetic screening for a single common LRRK2 mutation in familial Parkinson's disease.

    Science.gov (United States)

    Nichols, William C; Pankratz, Nathan; Hernandez, Dena; Paisán-Ruíz, Coro; Jain, Shushant; Halter, Cheryl A; Michaels, Veronika E; Reed, Terry; Rudolph, Alice; Shults, Clifford W; Singleton, Andrew; Foroud, Tatiana

    Mutations in the leucine-rich repeat kinase 2 (LRRK2) gene cause some forms of autosomal dominant Parkinson's disease. We measured the frequency of a novel mutation (Gly2019 ser) in familial Parkinson's disease by screening genomic DNA of patients and controls. Of 767 affected individuals from 358 multiplex families, 35 (5%) individuals were either heterozygous (34) or homozygous (one) for the mutation, and had typical clinical findings of idiopathic Parkinson's disease. Thus, our results suggest that a single LRRK2 mutation causes Parkinson's disease in 5% of individuals with familial disease. Screening for this mutation should be a component of genetic testing for Parkinson's disease.

  12. Screening of sarcomere gene mutations in young athletes with abnormal findings in electrocardiography: identification of a MYH7 mutation and MYBPC3 mutations.

    Science.gov (United States)

    Kadota, Chika; Arimura, Takuro; Hayashi, Takeharu; Naruse, Taeko K; Kawai, Sachio; Kimura, Akinori

    2015-10-01

    There is an overlap between the physiological cardiac remodeling associated with training in athletes, the so-called athlete's heart, and mild forms of hypertrophic cardiomyopathy (HCM), the most common hereditary cardiac disease. HCM is often accompanied by unfavorable outcomes including a sudden cardiac death in the adolescents. Because one of the initial signs of HCM is abnormality in electrocardiogram (ECG), athletes may need to monitor for ECG findings to prevent any unfavorable outcomes. HCM is caused by mutations in genes for sarcomere proteins, but there is no report on the systematic screening of gene mutations in athletes. One hundred and two genetically unrelated young Japanese athletes with abnormal ECG findings were the subjects for the analysis of four sarcomere genes, MYH7, MYBPC3, TNNT2 and TNNI3. We found that 5 out of 102 (4.9%) athletes carried mutations: a heterozygous MYH7 Glu935Lys mutation, a heterozygous MYBPC3 Arg160Trp mutation and another heterozygous MYBPC3 Thr1046Met mutation, all of which had been reported as HCM-associated mutations, in 1, 2 and 2 subjects, respectively. This is the first study of systematic screening of sarcomere gene mutations in a cohort of athletes with abnormal ECG, demonstrating the presence of sarcomere gene mutations in the athlete's heart.

  13. Screening of 99 Danish patients with congenital heart disease for GATA4 mutations

    DEFF Research Database (Denmark)

    Zhang, Litu; Tümer, Zeynep; Jacobsen, Joes Ramsøe

    2006-01-01

    Congenital heart disease (CHD) affects nearly 1% of the population, but only few genes involved in human CHD are presently known. Germ-line mutations in the zinc finger transcription factor GATA4 have been associated with familial cases of atrial and ventricular septal defects and pulmonary...... stenosis. We have screened 99 unrelated Danish patients with different CHD phenotypes to evaluate the prevalence of GATA4 mutations in CHD. No pathogenic mutations were found among the patients, suggesting that GATA4 mutations are relatively rare among CHD patients. Thus, the diagnostic importance of GATA4...... mutations may be confined to familial cases or specific subgroups of CHD phenotypes....

  14. High-resolution melting facilitates mutation screening of PYGM in patients with McArdle disease

    DEFF Research Database (Denmark)

    Duno, M.; Quinlivan, R.; Vissing, J.

    2009-01-01

    Mutations in PYGM, encoding the muscle-specific glycogen phosphorylase (myophosphorylase), are responsible for McArdle disease. Among Caucasians, a large proportion of patients are homozygous for the R50X mutation, but other mutations can affect all the 20 exons of PYGM, making mutation detection...... variations. Thirteen of these are pathogenic, and three were classified as polymorphisms. Nine variations had not previously been described. One of the novel mutations, c.2430C > T, was initially predicted to result in a silent G810G change, but cDNA analysis demonstrated that the mutation led to abnormal m...... laborious. We have developed a high-resolution melting (HRM) assay for mutation detection in PYGM. Twelve McArdle patients were investigated, in whom pre-screening had ruled out homozygosity or compound heterozygosity for the two common G205S and R50X mutations. In total, we identified 16 different...

  15. Screening for calreticulin mutations in a cohort of patients suspected ...

    African Journals Online (AJOL)

    Background. The discovery of calreticulin (CALR) has shown it to be the second most frequent mutation after the Janus Kinase 2 (JAK2) mutation in myeloproliferative neoplasms (MPNs). Its structure indicates various functions, of which two are to ensure calcium homeostasis and proper folding of other target proteins.

  16. Head-to-Head Comparison of the RNA-Based Aptima Human Papillomavirus (HPV) Assay and the DNA-Based Hybrid Capture 2 HPV Test in a Routine Screening Population of Women Aged 30 to 60 Years in Germany.

    Science.gov (United States)

    Iftner, Thomas; Becker, Sven; Neis, Klaus-Joachim; Castanon, Alejandra; Iftner, Angelika; Holz, Barbara; Staebler, Annette; Henes, Melanie; Rall, Katharina; Haedicke, Juliane; von Weyhern, Claus Hann; Clad, Andreas; Brucker, Sara; Sasieni, Peter

    2015-08-01

    Testing for E6/E7 mRNA in cells infected with high-risk (HR) human papillomavirus (HPV) might improve the specificity of HPV testing for the identification of cervical precancerous lesions. Here we compared the RNA-based Aptima HPV (AHPV) assay (Hologic) and the DNA-based Hybrid Capture 2 (HC2) HPV test (Qiagen) to liquid-based cytology (LBC) for women undergoing routine cervical screening. A total of 10,040 women, 30 to 60 years of age, were invited to participate in the study, 9,451 of whom were included in the analysis. Specimens were tested centrally by LBC, the AHPV test, and the HC2 test, and women who tested positive on any test were referred for colposcopy. Genotyping was performed on all HR-HPV-positive samples. Test characteristics were calculated based on histological review. As a result, we identified 90 women with cervical intraepithelial neoplasia grade 2+ (CIN2+), including 43 women with CIN3+. Sensitivity differences between the AHPV test and the HC2 test in detecting CIN2+ (P = 0.180) or CIN3+ (P = 0.0625) lesions were statistically nonsignificant. Of three CIN3 cases that were missed with the AHPV test, two cases presented lesion-free cones and one had a non-HR HPV67 infection. The specificity (detection of high-grade precancerous lesions and may be used in primary cervical cancer screening for women ≥30 years of age. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

  17. 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

    Usher Syndrome is the leading cause of inherited deaf-blindness. It is divided into three subtypes, of which the most common is Usher type 2, and the USH2A gene accounts for 75-80% of cases. Despite recent sequencing strategies, in our cohort a significant proportion of individuals with Usher type 2 have just one heterozygous disease-causing mutation in USH2A, or no convincing disease-causing mutations across nine Usher genes. The purpose of this study was to improve the molecular diagnosis in these families by screening USH2A for duplications, heterozygous deletions and a common pathogenic deep intronic variant USH2A: c.7595-2144A>G. Forty-nine Usher type 2 or atypical Usher families who had missing mutations (mono-allelic USH2A or no mutations following Sanger sequencing of nine Usher genes) were screened for duplications/deletions using the USH2A SALSA MLPA reagent kit (MRC-Holland). Identification of USH2A: c.7595-2144A>G was achieved by Sanger sequencing. Mutations were confirmed by a combination of reverse transcription PCR using RNA extracted from nasal epithelial cells or fibroblasts, and by array comparative genomic hybridisation with sequencing across the genomic breakpoints. Eight mutations were identified in 23 Usher type 2 families (35%) with one previously identified heterozygous disease-causing mutation in USH2A. These consisted of five heterozygous deletions, one duplication, and two heterozygous instances of the pathogenic variant USH2A: c.7595-2144A>G. No variants were found in the 15 Usher type 2 families with no previously identified disease-causing mutations. In 11 atypical families, none of whom had any previously identified convincing disease-causing mutations, the mutation USH2A: c.7595-2144A>G was identified in a heterozygous state in one family. All five deletions and the heterozygous duplication we report here are novel. This is the first time that a duplication in USH2A has been reported as a cause of Usher syndrome. We found that 8 of

  18. Screening for mutations in rhodopsin and peripherin/RDS in patients with autosomal dominant retinitis pigmentosa

    Energy Technology Data Exchange (ETDEWEB)

    Rodriguez, J.A.; Gannon, A.M.; Daiger, S.P. [Univ. of Texas HSC, Houston, TX (United States)] [and others

    1994-09-01

    Mutations in rhodopsin account for approximately 30% of all cases of autosomal dominant retinits pigmentosa (adRP) and mutations in peripherin/RDS account for an additional 5% of cases. Also, mutations in rhodopsin can cause autosomal recessive retinitis pigmentosa and mutations in peripherin/RDS can cause dominant macular degeneration. Most disease-causing mutations in rhodopsin and peripherin/RDS are unique to one family or, at most, to a few families within a limited geographic region, though a few mutations are found in multiple, unrelated families. To further determine the spectrum of genetic variation in these genes, we screened DNA samples from 134 unrelated patients with retinitis pigmentosa for mutations in both rhodopsin and peripherin/RDS using SSCP followed by genomic sequencing. Of the 134 patients, 86 were from families with apparent adRP and 48 were either isolated cases or were from families with an equivocal mode of inheritance. Among these patients we found 14 distinct rhodopsin mutations which are likely to cause retinal disease. Eleven of these mutations were found in one individual or one family only, whereas the Pro23His mutation was found in 14 {open_quotes}unrelated{close_quotes}individuals. The splice-site mutation produces dominant disease though with highly variable expression. Among the remaining patients were found 6 distinct peripherin/RDS mutations which are likely to cause retinal disease. These mutations were also found in one patient or family only, except the Gly266Asp mutation which was found in two unrelated patients. These results confirm the expected frequency and broad spectrum of mutations causing adRP.

  19. Mutational screening of the USH2A gene in Spanish USH patients reveals 23 novel pathogenic mutations

    Directory of Open Access Journals (Sweden)

    Diaz-Llopis Manuel

    2011-10-01

    Full Text Available Abstract Background Usher Syndrome type II (USH2 is an autosomal recessive disorder, characterized by moderate to severe hearing impairment and retinitis pigmentosa (RP. Among the three genes implicated, mutations in the USH2A gene account for 74-90% of the USH2 cases. Methods To identify the genetic cause of the disease and determine the frequency of USH2A mutations in a cohort of 88 unrelated USH Spanish patients, we carried out a mutation screening of the 72 coding exons of this gene by direct sequencing. Moreover, we performed functional minigene studies for those changes that were predicted to affect splicing. Results As a result, a total of 144 DNA sequence variants were identified. Based upon previous studies, allele frequencies, segregation analysis, bioinformatics' predictions and in vitro experiments, 37 variants (23 of them novel were classified as pathogenic mutations. Conclusions This report provide a wide spectrum of USH2A mutations and clinical features, including atypical Usher syndrome phenotypes resembling Usher syndrome type I. Considering only the patients clearly diagnosed with Usher syndrome type II, and results obtained in this and previous studies, we can state that mutations in USH2A are responsible for 76.1% of USH2 disease in patients of Spanish origin.

  20. Screening for circulating RAS/RAF mutations by multiplex digital PCR

    DEFF Research Database (Denmark)

    Andersen, Rikke Fredslund; Jakobsen, Anders

    2016-01-01

    by technical challenges primarily due to the low levels of ctDNA in patients with localized disease and in patients responding to therapy. The approach presented here is a multiplex digital PCR method of screening for 31 mutations in the KRAS, NRAS, BRAF, and PIK3CA genes in the plasma. The upper level...... of the limit of blank, which defines the specificity of the multiplexes, was 0.006%-0.06%. Mutations found by multiplex analyses were identified and quantified by duplex analyses. The method was tested on samples from cholangiocarcinoma patients with known tumor mutational status. Mutations found in the tumor...... were also found in plasma samples in all cases with analyses for all other mutations being negative. There was a perfect agreement as to wild type status in tumor and plasma. The method combines a high sensitivity with the ability to analyze for several mutations at a time and could be a step towards...

  1. Screening for Pancreatic Adenocarcinoma in BRCA2 Mutation Carriers: Results of a Disease Simulation Model.

    Science.gov (United States)

    Pandharipande, Pari V; Jeon, Alvin; Heberle, Curtis R; Dowling, Emily C; Kong, Chung Yin; Chung, Daniel C; Brugge, William R; Hur, Chin

    2015-12-01

    BRCA2 mutation carriers are at increased risk for multiple cancers including pancreatic adenocarcinoma (PAC). Our goal was to compare the effectiveness of different PAC screening strategies in BRCA2 mutation carriers, from the standpoint of life expectancy. A previously published Markov model of PAC was updated and extended to incorporate key aspects of BRCA2 mutation carrier status, including competing risks of breast- and ovarian-cancer specific mortality. BRCA2 mutation carriers were modeled and analyzed as the primary cohort for the analysis. Additional higher risk BRCA2 cohorts that were stratified according to the number of first-degree relatives (FDRs) with PAC were also analyzed. For each cohort, one-time screening and annual screening were evaluated, with screening starting at age 50 in both strategies. The primary outcome was net gain in life expectancy (LE) compared to no screening. Sensitivity analysis was performed on key model parameters, including surgical mortality and MRI test performance. One-time screening at age 50 resulted in a LE gain of 3.9 days for the primary BRCA2 cohort, and a gain of 5.8 days for those with BRCA2 and one FDR. Annual screening resulted in LE loss of 12.9 days for the primary cohort and 1.3 days for BRCA2 carriers with 1 FDR, but resulted in 20.6 days gained for carriers with 2 FDRs and 260 days gained for those with 3 FDRs. For patients with ≥ 3 FDRs, annual screening starting at an earlier age (i.e. 35-40) was optimal. Among BRCA2 mutation carriers, aggressive screening regimens may be ineffective unless additional indicators of elevated risk (e.g., 2 or more FDRs) are present. More clinical studies are needed to confirm these findings. American Cancer Society - New England Division - Ellison Foundation Research Scholar Grant (RSG-15-129-01-CPHPS).

  2. Risk modeling and screening for BRCA1 mutations among Filipino breast cancer patients

    CERN Document Server

    Nato, A Q J

    2003-01-01

    Breast cancer susceptibility gene, type 1(BRCA1) has been thought to be responsible for approx 45% of families with multiple breast carcinomas and for approx 80% of breast and ovarian cancer families. In this study, we investigated 34 familial Filipino breast cancer (BC) patients to: (a) estimate breast cancer risks and BRCA1/2 mutation carrier probabilities using risk assessment and prior probability models, respectively; (b) screen for putative polymorphisms at selected smaller exons of BRCA1 by single-strand conformation polymorphism (SSCP) analysis; (c) screen for truncated mutations at BRCA1 exon 11 by radioactive protein truncation test (PTT); and (d) estimate posterior probabilities upon incorporation of screening results. SSCP analysis revealed 8 unique putative polymorphisms. Low prevalence of unique putative polymorphisms at exon 2, 5, 17, and 22 may indicate probable mutations. Contrastingly, high prevalence of unique putative polymorphisms at exons 13, 15, and 16 may suggest true polymorphisms whi...

  3. All azoospermic males should be screened for cystic fibrosis mutations before intracytoplasmic sperm injection.

    LENUS (Irish Health Repository)

    Mocanu, Edgar

    2012-02-01

    We assessed the frequency of CFTR mutations in groups with varying degrees of sub-fertility and compared these groups to a fertile male group with proven paternity. Screening for CFTR mutations should be routine for all azoospermic males, irrespective of obstructive or non-obstructive etiology, prior to proposing ICSI treatment. CFTR testing has no value in the investigation of non-azoospermic infertile males.

  4. All azoospermic males should be screened for cystic fibrosis mutations before intracytoplasmic sperm injection.

    LENUS (Irish Health Repository)

    Mocanu, Edgar

    2010-11-01

    We assessed the frequency of CFTR mutations in groups with varying degrees of sub-fertility and compared these groups to a fertile male group with proven paternity. Screening for CFTR mutations should be routine for all azoospermic males, irrespective of obstructive or non-obstructive etiology, prior to proposing ICSI treatment. CFTR testing has no value in the investigation of non-azoospermic infertile males.

  5. Screening of RET gene mutations in Chinese patients with medullary thyroid carcinoma and their relatives.

    Science.gov (United States)

    Wang, Junyi; Zhang, Bin; Liu, Wensheng; Zhang, Yongxia; Di, Xuebing; Yang, Yanmei; Yan, Dangui

    2016-01-01

    The rearranged during transfection (RET) gene is a proto-oncogene; active mutations frequently occur in medullary thyroid carcinoma (MTC). This study investigated the spectrum of germline RET mutations and clinical features in Chinese hereditary MTC patients. A total of 53 family members from 11 different hereditary MTC families were recruited for detection of RET exon 8, 10, 11, 13, 14, 15, and 16 mutations, in genomic DNA from peripheral blood leucocytes using polymerase chain reaction (PCR) and direct DNA sequencing. Of the 53 participants, eight different germline RET mutations were detected in 37 individuals. These RET mutations were distributed in exons 10, 11, 13, and 16. The most frequent RET mutation was localized at exon 11 codon 634 (67.6 %; 25/37) and the most prevalent mutation was C634R (37.8 %; 14/37). The most frequent phenotype was multiple endocrine neoplasia type 2A (MEN2A). The incidences of MTC, pheochromocytoma, and hyperparathyroidism in the MEN2A patients were 100, 36.4 and 18.2 %, respectively. The phenotype of families with Y606C or L790F mutation was categorized as familial medullary thyroid carcinoma. Moreover, one proband was identified with multiple endocrine neoplasia type 2B and carried a de novo mutation of M918T. Two families with C618S/Y mutation were categorized as unclassified multiple endocrine neoplasia type 2. Our results further substantiate that most germline mutations of the RET proto-oncogene were localized at codon 634 in Chinese hereditary MTC patients and carriers. RET mutation at codon 634 was always associated to the phenotype of MEN2A. Screening of RET mutations should be probably limited to exons 10, 11, 13 and 16 initially to be cost-effective in China.

  6. Genetic mutation screen in early non--small-cell lung cancer (NSCLC) specimens.

    Science.gov (United States)

    Bar, Jair; Damianovich, Maya; Hout Siloni, Goni; Dar, Erel; Cohen, Yoram; Perelman, Marina; Ben Nun, Alon; Simansky, David; Yellin, Alon; Urban, Damien; Onn, Amir

    2014-03-01

    Testing for genetic abnormalities in epithelial growth factor receptor (EGFR), anaplastic lymphoma receptor tyrosine kinase (ALK), and potentially additional genes is a critical tool in the care of advanced NSCLC. There is conflicting evidence for the role of such tests in early NSCLC. We report a single-institute Sequenom testing for a wide range of mutations and their clinical correlations in early-resected NSCLC specimens. Early NSCLC paraffin-embedded, formalin-fixed (FFPE) specimens were collected, DNA extracted, and using Sequenom-based matrix-assisted laser desorption/ionization-time of flight analysis, mutations in 22 oncogenes and tumor suppressor genes were evaluated. Clinical data was collected retrospectively. The technique was found to be feasible. Thirty-six of 96 patients (37.5%) had any genetic abnormality identified, and 8 (8.3%) had 2 or more mutations. Kirsten rat sarcoma viral oncogene homolog (KRAS) and EGFR were the most common genes to appear mutated (15.6%); phosphatidylinositol-4,5-bisphosphate 3-kinase, catalytic subunit alpha (PIK3CA) was the gene to be found most commonly in tumors with co-mutations. Transversions were found mostly in KRAS gene mutations and to be nonprognostic. No difference in the spectrum of mutations was found between squamous-cell and non-squamous-cell lung cancers. Ever-smokers showed a trend for worse prognosis, with a similar spectrum of mutations. Sequenom-based mutation screen is feasible using FFPE samples. More than a third of the patients were found to harbor some genetic abnormality, and 8% were found to have more than a single mutated gene. Wide-range gene screens using large sample depositories are required for further insight into the important genes at play in early NSCLC. Copyright © 2014 Elsevier Inc. All rights reserved.

  7. Mutational screening of LCA genes emphasizing RPE65 in South Indian cohort of patients.

    Directory of Open Access Journals (Sweden)

    Anshuman Verma

    Full Text Available BACKGROUND: Leber congenital amaurosis (LCA is the most severe form of inherited retinal visual impairment in children. So far, mutations in more than 20 genes have been known to cause LCA and among them, RPE65 is a suitable candidate for gene therapy. The mutational screenings of RPE65 and other LCA genes are requisite in support of emerging gene specific therapy for LCA. Therefore, we have carried out a comprehensive LCA genes screening using a combined approach of direct sequencing and DNA microarray based Asper chip analysis. METHODOLOGY/PRINCIPAL FINDINGS: Thirty clinically diagnosed index LCA cases from Southern India were screened for coding and flanking intronic regions of RPE65 through direct sequencing. Among thirty, 25 cases excluded from RPE65 mutations were subjected to Asper chip analysis, testing 784 known pathogenic variations in 15 major LCA genes. In RPE65 screening, four different pathogenic variations including two novel (c.361insT & c.939T>A and two known (c.394G>A & c.361delT mutations were identified in five index cases. In the chip analysis, seven known pathogenic mutations were identified in six index cases, involving genes GUCY2D, RPGRIP1, AIPL1, CRX and IQCB1. Overall, 11 out of 30 LCA cases (36.6% revealed pathogenic variations with the involvement of RPE65 (16.6%, GUCY2D (10%, RPGRIP1 (3.3%, AIPL1 (3.3% and CRX & IQCB1 (3.3%. CONCLUSIONS/SIGNIFICANCE: Our study suggests that such combined screening approach is productive and cost-effective for mutation detection and can be applied in Indian LCA cohort for molecular diagnosis and genetic counselling.

  8. Mutational screening of LCA genes emphasizing RPE65 in South Indian cohort of patients.

    Science.gov (United States)

    Verma, Anshuman; Perumalsamy, Vijayalakshmi; Shetty, Shashikant; Kulm, Maigi; Sundaresan, Periasamy

    2013-01-01

    Leber congenital amaurosis (LCA) is the most severe form of inherited retinal visual impairment in children. So far, mutations in more than 20 genes have been known to cause LCA and among them, RPE65 is a suitable candidate for gene therapy. The mutational screenings of RPE65 and other LCA genes are requisite in support of emerging gene specific therapy for LCA. Therefore, we have carried out a comprehensive LCA genes screening using a combined approach of direct sequencing and DNA microarray based Asper chip analysis. Thirty clinically diagnosed index LCA cases from Southern India were screened for coding and flanking intronic regions of RPE65 through direct sequencing. Among thirty, 25 cases excluded from RPE65 mutations were subjected to Asper chip analysis, testing 784 known pathogenic variations in 15 major LCA genes. In RPE65 screening, four different pathogenic variations including two novel (c.361insT & c.939T>A) and two known (c.394G>A & c.361delT) mutations were identified in five index cases. In the chip analysis, seven known pathogenic mutations were identified in six index cases, involving genes GUCY2D, RPGRIP1, AIPL1, CRX and IQCB1. Overall, 11 out of 30 LCA cases (36.6%) revealed pathogenic variations with the involvement of RPE65 (16.6%), GUCY2D (10%), RPGRIP1 (3.3%), AIPL1 (3.3%) and CRX & IQCB1 (3.3%). Our study suggests that such combined screening approach is productive and cost-effective for mutation detection and can be applied in Indian LCA cohort for molecular diagnosis and genetic counselling.

  9. Cost-effectiveness of MRI for breast cancer screening in BRCA1/2 mutation carriers.

    Science.gov (United States)

    Pataky, Reka; Armstrong, Linlea; Chia, Stephen; Coldman, Andrew J; Kim-Sing, Charmaine; McGillivray, Barbara; Scott, Jenna; Wilson, Christine M; Peacock, Stuart

    2013-07-10

    Women with mutations in BRCA1 or BRCA2 are at high risk of developing breast cancer and, in British Columbia, Canada, are offered screening with both magnetic resonance imaging (MRI) and mammography to facilitate early detection. MRI is more sensitive than mammography but is more costly and produces more false positive results. The purpose of this study was to calculate the cost-effectiveness of MRI screening for breast cancer in BRCA1/2 mutation carriers in a Canadian setting. We constructed a Markov model of annual MRI and mammography screening for BRCA1/2 carriers, using local data and published values. We calculated cost-effectiveness as cost per quality-adjusted life-year gained (QALY), and conducted one-way and probabilistic sensitivity analysis. The incremental cost-effectiveness ratio (ICER) of annual mammography plus MRI screening, compared to annual mammography alone, was $50,900/QALY. After incorporating parameter uncertainty, MRI screening is expected to be a cost-effective option 86% of the time at a willingness-to-pay of $100,000/QALY, and 53% of the time at a willingness-to-pay of $50,000/QALY. The model is highly sensitive to the cost of MRI; as the cost is increased from $200 to $700 per scan, the ICER ranges from $37,100/QALY to $133,000/QALY. The cost-effectiveness of using MRI and mammography in combination to screen for breast cancer in BRCA1/2 mutation carriers is finely balanced. The sensitivity of the results to the cost of the MRI screen itself warrants consideration: in jurisdictions with higher MRI costs, screening may not be a cost-effective use of resources, but improving the efficiency of MRI screening will also improve cost-effectiveness.

  10. Mutation screening and association analysis of six candidate genes for autism on chromosome 7q

    DEFF Research Database (Denmark)

    Bonora, Elena; Lamb, Janine A; Barnby, Gabrielle

    2005-01-01

    Genetic studies have provided evidence for an autism susceptibility locus (AUTS1) on chromosome 7q. Screening for mutations in six genes mapping to 7q, CUTL1, SRPK2, SYPL, LAMB1, NRCAM and PTPRZ1 in 48 unrelated individuals with autism led to the identification of several new coding variants in t...

  11. RPE65 gene: multiplex PCR and mutation screening in patients from ...

    Indian Academy of Sciences (India)

    Unknown

    Medical and Vision Research Foundations, Sankara Nethralaya, Chennai 600 006, India. Abstract. We used multiplex PCR followed by sequencing to screen for mutations in the 14 exons of the RPE65 gene in early- childhood-onset autosomal recessive retinitis pigmentosa (arRP) and Leber's congenital amaurosis (LCA) ...

  12. RPE65 gene: multiplex PCR and mutation screening in patients from ...

    Indian Academy of Sciences (India)

    We used multiplex PCR followed by sequencing to screen for mutations in the 14 exons of the RPE65 gene in early-childhood-onset autosomal recessive retinitis pigmentosa (arRP) and Leber's congenital amaurosis (LCA) patients. The RPE65 protein is believed to play an important role in the metabolism of vitamin A in ...

  13. Myosin VIIA mutation screening in 189 Usher syndrome type 1 patients

    Energy Technology Data Exchange (ETDEWEB)

    Weston, M.D.; Kelley, P.M.; Overbeck, L.D. [Univ. of Nebraska Medical Center, Omaha, NE (United States)] [and others

    1996-11-01

    Usher syndrome type 1b (USH1B) is an autosomal recessive disorder characterized by congenital profound hearing loss, vestibular abnormalities, and retinitis pigmentosa. The disorder has recently been shown to be caused by mutations in the myosin VIIa gene (MYO7A) located on 11q14. In the current study, a panel of 189 genetically independent Usher I cases were screened for the presence of mutations in the N-terminal coding portion of the motor domain of MYO7A by heteroduplex analysis of 14 exons. Twenty-three mutations were found segregating with the disease in 20 families. Of the 23 mutations, 13 were unique, and 2 of the 13 unique mutations (Arg212His and Arg212Cys) accounted for the greatest percentage of observed mutant alleles (8/23, 31%). Six of the 13 mutations caused premature stop codons, 6 caused changes in the amino acid sequence of the myosin VIIa protein, and 1 resulted in a splicing defect. Three patients were homozygotes or compound heterozygotes for mutant alleles; these three cases were Tyr333Stop/Tyr333Stop, Arg212His-Arg302His/Arg212His-Arg302His, and IVS13nt-8c{r_arrow}g/ G1u450Gln. All the other USH1B mutations observed were simple heterozygotes, and it is presumed that the mutation on the other allele is present in the unscreened regions of the gene. None of the mutations reported here were observed in 96 unrelated control samples, although several polymorphisms were detected. These results add three patients to a single case reported previously where mutations have been found in both alleles and raises the total number of unique mutations in MYO7A to 16. 22 refs., 4 figs., 3 tabs.

  14. No efficacy of annual gynaecological screening in BRCA1/2 mutation carriers; an observational follow-up study

    NARCIS (Netherlands)

    B.B.J. Hermsen; R.I. Olivier; R.H.M. Verheijen (René); M. van Beurden (Marc); J.A. de Hullu (Joanne); L.F. Massuger (Leon); C.W. Burger (Curt); C.T. Brekelmans (Cecile); M.J. Mourits; G.H. de Bock (Geertruida); K.N. Gaarenstroom (Katja); H.H. van Boven (Hester); T.M. Mooij (Thea); M.A. Rookus (Matti)

    2007-01-01

    textabstractBRCA1/2 mutation carriers are offered gynaecological screening with the intention to reduce mortality by detecting ovarian cancer at an early stage. We examined compliance and efficacy of gynaecological screening in BRCA1/2 mutation carriers. In this multicentre, observational, follow-up

  15. No efficacy of annual gynaecological screening in BRCA1/2 mutation carriers; an observational follow-up study

    NARCIS (Netherlands)

    Hermsen, B. B. J.; Olivier, R. I.; Verheijen, R. H. M.; van Beurden, M.; de Hullu, J. A.; Massuger, L. F.; Burger, C. W.; Brekelmans, C. T.; Mourits, M. J.; de Bock, G. H.; Gaarenstroom, K. N.; van Boven, H. H.; Mooij, T. M.; Rookus, M. A.

    2007-01-01

    BRCA1/ 2 mutation carriers are offered gynaecological screening with the intention to reduce mortality by detecting ovarian cancer at an early stage. We examined compliance and efficacy of gynaecological screening in BRCA1/ 2 mutation carriers. In this multicentre, observational, follow-up study we

  16. Screening of 1331 Danish breast and/or ovarian cancer families identified 40 novel BRCA1 and BRCA2 mutations

    DEFF Research Database (Denmark)

    Hansen, Thomas V O; Jønson, Lars; Steffensen, Ane Y

    2011-01-01

    and BRCA2 in high risk breast and/or ovarian cancer families. The mutations were detected via pre-screening using dHPLC or high-resolution melting and direct sequencing. We identified 16 variants in BRCA1, including 9 deleterious frame-shift mutations, 2 intronic variants, 4 missense mutations, and 1......Germ-line mutations in the tumour suppressor genes BRCA1 and BRCA2 predispose to breast and ovarian cancer. Since 1999 we have performed mutational screening of breast and/or ovarian cancer patients in East Denmark. During this period we have identified 40 novel sequence variations in BRCA1...

  17. Screening for Ataxia-Telangiectasia Mutations in a Population-Based Sample of Women with Early-Onset Breast Cancer

    Science.gov (United States)

    1999-09-01

    G, Itnyre J (1997) Prevalence and contribution of BRCA1 mutations in breast and ovarian cancer: results from three U.S. population-based case-control...GTTAAGAA two of these cases, AT1ILA and AT119LA, the families were consanguineous , and the mutations were homo- zygous, simplifying the interpretation...screening efficiency and expand significantly the number of cases to be screened. Assessment of the prevalence of ATM mutations in breast cancer

  18. Comprehensive mutational screening in a cohort of Danish families with hereditary congenital cataract

    DEFF Research Database (Denmark)

    Hansen, Lars; Mikkelsen, Annemette; Nürnberg, Peter

    2009-01-01

    PURPOSE: Identification of the causal mutations in 28 unrelated families and individuals with hereditary congenital cataract identified from a national Danish register of hereditary eye diseases. Seven families have been published previously, and the data of the remaining 21 families are presented...... together with an overview of the results in all families. METHODS: A combined screening approach of linkage analysis and sequencing of 17 cataract genes were applied to mutation analyses of total 28 families. RESULTS: The study revealed a disease locus in seven of eight families that were amenable......, and a gene conversion is the most likely mutational event causing this variant. Ten families had microcornea cataract, and a mutation was identified in eight of those. Most families displayed mixed phenotypes with nuclear, lamellar, and polar opacities and no apparent genotype-phenotype correlation emerged...

  19. Screening for mutations in the uroporphyrinogen decarboxylase gene using denaturing gradient gel electrophoresis

    DEFF Research Database (Denmark)

    Christiansen, L; Ged, C; Hombrados, I

    1999-01-01

    , confirming the heterogeneity of the underlying genetic defects of these diseases. We have established a denaturing gradient gel electrophoresis (DGGE) assay for mutation detection in the UROD gene, enabling the simultaneous screening for known and unknown mutations. The established assay has proved able...... improves the genetic diagnosis of fPCT and HEP, thereby facilitating the detection of familial UROD deficient patients as well as the discrimination between familial and sporadic PCT cases.......The two porphyrias, familial porphyria cutanea tarda (fPCT) and hepatoerythropoietic porphyria (HEP), are associated with mutations in the gene encoding the enzyme uroporphyrinogen decarboxylase (UROD). Several mutations, most of which are private, have been identified in HEP and fPCT patients...

  20. Screening of Two Neighboring CFTR Mutations in Iranian Infertile Men with Non-Obstructive Azoospermia

    Directory of Open Access Journals (Sweden)

    Somayeh Heidari

    2016-11-01

    Full Text Available The genetic association between cystic fibrosis transmembrane conductance regulator (CFTR gene mutations and male infertility due to congenital bilateral absence of vas deferens (CBAVD is well established. Mutant CFTR, however may also be involved in the etiology of male infertility in non-CBAVD cases. The present study was conducted to estimate the frequency of ΔI507 and ΔF508 CFTR gene mutations in Iranian infertile males. We undertook the first study of association between these CFTR mutations and non-obstructive azoospermia in Iran. In this case-control study, 100 fertile healthy fathers and 100 non-obstructive azoospermia’s men were recruited from Isfahan Infertility Center (IIC and Sari Saint Mary’s Infertility Center, between 2008 and 2009. Screening of F508del and I507del mutations was carried out by the multiplex-ARMS-PCR. Significance of differences in mutation frequencies between the patient and control groups was assessed by Fisher’s exact test. The ΔF508 was detected in three patients. However there are no significant association was found between the presence of this mutated allele and infertility [OR=9.2 (allele-based and 7.2 (individual-based, P=0.179]. None of the samples carried the ΔI507 mutation. Altogether, we show that neither ΔI507 nor ΔF508 is involved in this population of Iranian infertile males with non-obstructive azoospermia.

  1. [Screening and analysis of the mutations on beta-myosin heavy chain gene in 3 Chinese families with hypertrophic cardiomyopathy].

    Science.gov (United States)

    Feng, Xiu-li; Fan, Xin-ping; Yang, Zhong-wei; Yang, Fu-hui

    2011-02-01

    To detect gene mutations on beta-myosin heavy chain gene MYH7 in 3 Chinese families with hypertrophic cardiomyopathy (HCM), and to analyze the correlation between genotype and phenotype. A denaturing high-performance liquid chromatography (DHPLC) and sequencing mutation screening of the exons (exon3-23) coding for MYH7 gene were performed in 3 Chinese families with HCM. In this study, we identified several mutations in MYH7. A mutation of Thr441Met previously reported in a patient with Laing distal myopathy was first identified in one Chinese pedigree. This study illustrated the high frequency of mutation in MYH7 gene in Chinese HCM families. Different mutations and carriers of the MYH7 gene present phenotypic heterogeneity. Mutation screening and analysis in HCM family could therefore facilitate the early HCM diagnosis and would be helpful for the prediction, prevention and early treatment of HCM linked with MYH7 gene mutation.

  2. [Screening for genetic mutations in hyperphenylalaninemia using Ion Torrent PGM sequencing].

    Science.gov (United States)

    Cao, Yanyan; Qu, Yujin; Song, Fang; Bai, Jinli; Jin, Yuwei; Wang, Hong

    2015-02-01

    To establish a hyperphenylalaninemia related genes screening method using Ion Torrent Personal Genome Machine (PGM) for early detection and differential diagnosis of hyperphenylalaninemia (HPA). Three children with known HPA mutations and a healthy control were used for setting up the method. Ten children with HPA with known mutations were recruited for validating the method. Ion Ampliseq PCR was used to amplify the 5' and 3' untranslated region, coding sequence, and flanking introns of PAH, GCH1, PTS, QDPR, and PCBD1 genes. After the enrichment with the Ion OneTouch system, the products were sequenced by PGM. Data from the PGM were processed with Torrent Suite v2.2 software package. All variations were confirmed by Sanger sequencing. For the 4 samples, the PGM output was 94.22 Mb, with approximately 99.5% of reads mapping to the target regions. Among these samples, we detected 74 variations (28 positions) including 6 known mutations. Compared with database and results of Sanger sequencing, 55 (18 positions) polymorphisms and 13 (4 positions) false positive calls were confirmed. For the 10 samples, all the known mutations were successfully identified. Ion Torrent PGM sequencing is suitable for screening genetic mutation underlying HPA from the perspective of metabolic pathways, which can meet the clinical demand for individualized diagnosis and treatment.

  3. BRCA1 and BRCA2 Gene Mutations Screening In Sporadic Breast Cancer Patients In Kazakhstan.

    Directory of Open Access Journals (Sweden)

    Ainur R. Akilzhanova

    2013-05-01

    Full Text Available Background: A large number of distinct mutations in the BRCA1 and BRCA2 genes have been reported worldwide, but little is known regarding the role of these inherited susceptibility genes in breast cancer risk among Kazakhstan women. Aim: To evaluate the role of BRCA1/2 mutations in Kazakhstan women presenting with sporadic breast cancer. Methods: We investigated the distribution and nature of polymorphisms in BRCA1 and BRCA2 entire coding regions in 156 Kazakhstan sporadic breast cancer cases and 112 age-matched controls using automatic direct sequencing. Results: We identified 22 distinct variants, including 16 missense mutations and 6 polymorphisms in BRCA1/2 genes. In BRCA1, 9 missense mutations and 3 synonymous polymorphisms were observed. In BRCA2, 7 missense mutations and 3 polymorphisms were detected. There was a higher prevalence of observed mutations in Caucasian breast cancer cases compared to Asian cases (p<0.05; higher frequencies of sequence variants were observed in Asian controls. No recurrent or founder mutations were observed in BRCA1/2 genes. There were no statistically significant differences in age at diagnosis, tumor histology, size of tumor, and lymph node involvement between women with breast cancer with or without the BRCA sequence alterations. Conclusions: Considering the majority of breast cancer cases are sporadic, the present study will be helpful in the evaluation of the need for the genetic screening of BRCA1/2 mutations and reliable genetic counseling for Kazakhstan sporadic breast cancer patients. Evaluation of common polymorphisms and mutations and breast cancer risk in families with genetic predisposition to breast cancer is ongoing in another current investigation. 

  4. NMD Microarray Analysis for Rapid Genome-Wide Screen of Mutated Genes in Cancer

    Directory of Open Access Journals (Sweden)

    Maija Wolf

    2005-01-01

    Full Text Available Gene mutations play a critical role in cancer development and progression, and their identification offers possibilities for accurate diagnostics and therapeutic targeting. Finding genes undergoing mutations is challenging and slow, even in the post-genomic era. A new approach was recently developed by Noensie and Dietz to prioritize and focus the search, making use of nonsense-mediated mRNA decay (NMD inhibition and microarray analysis (NMD microarrays in the identification of transcripts containing nonsense mutations. We combined NMD microarrays with array-based CGH (comparative genomic hybridization in order to identify inactivation of tumor suppressor genes in cancer. Such a “mutatomics” screening of prostate cancer cell lines led to the identification of inactivating mutations in the EPHB2 gene. Up to 8% of metastatic uncultured prostate cancers also showed mutations of this gene whose loss of function may confer loss of tissue architecture. NMD microarray analysis could turn out to be a powerful research method to identify novel mutated genes in cancer cell lines, providing targets that could then be further investigated for their clinical relevance and therapeutic potential.

  5. Diagnostic yield, interpretation, and clinical utility of mutation screening of sarcomere encoding genes in Danish hypertrophic cardiomyopathy patients and relatives

    DEFF Research Database (Denmark)

    Andersen, Paal Skytt; Havndrup, Ole; Hougs, Lotte

    2008-01-01

    The American Heart Association (AHA) recommends family screening for hypertrophic cardiomyopathy (HCM). We assessed the outcome of family screening combining clinical evaluation and screening for sarcomere gene mutations in a cohort of 90 Danish HCM patients and their close relatives, in all 451......, and in 37.5% of relatives without a mutation, one or more criteria was fulfilled. A total of 60% of family members had no mutation and could be reassured and further follow-up ceased. Genetic diagnosis may be established in approximately 40% of families with the highest yield in familial HCM with clinical...

  6. Mutation Screening of Her-2, N-ras and Nf1 Genes in Brain Tumor Biopsies.

    Science.gov (United States)

    Yapijakis, Christos; Adamopoulou, Maria; Tasiouka, Konstantina; Voumvourakis, Costas; Stranjalis, George

    2016-09-01

    A deeper understanding of the complex molecular pathology of brain malignancies is needed in order to develop more effective and targeted therapies of these highly lethal disorders. In an effort to further enlighten the molecular pathology of brain oncogenesis involving the her-2 (erbB-2/neu/ngl)/N-ras/nf1 pathway, we screened the genotypes of specimens from various types of brain tumors. The studied specimens included 35 biopsies of four general categories: 13 neuroglial tumors (4 astrocytomas, 2 oligodendrogliomas, 7 glioblastomas multiforme), 14 meningiomas, 3 other nervous system tumors (2 schwannomas, 1 craniopharyngioma) and 5 metastatic tumors (such as lung carcinomas and chronic myelocytic leukemia). Screening for most common mutations in oncogenes her-2, N-ras and tumor suppressor gene nf1 was conducted with molecular hybridization techniques (Southern blotting, dot blot and single-strand conformational polymorphism (SSCP) analysis, respectively), and was confirmed by DNA sequencing. Gene amplification of her-2 was observed in only two cases (6%), namely in one glioblastoma and in one meningioma. Screening of 3 hot spot codons of the N-ras gene (12, 13 and 61) and subsequent DNA sequencing revealed mutations in 19 biopsies encompassing all categories (54%). Screening for mutations in exons of the nf1 gene by SSCP analysis detected a novel nonsense mutation in exon 31 in a unique case of a glioblastoma biopsy (3%) taken from a patient without neurofibromatosis type I. Activated N-ras appears to be a major oncogene in brain oncogenesis, exhibiting the most important role in the her-2/N-ras/nf1 pathway. Copyright© 2016 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved.

  7. Screening for mtDNA diabetes mutations in Pima Indians with NIDDM

    Energy Technology Data Exchange (ETDEWEB)

    Sepehrnia, B.; Prezant, T.R.; Rotter, J.I. [UCLA School of Medicine, Los Angeles, CA (United States)] [and others

    1995-03-27

    More than half of the Pima Indians over age 35 years have non-insulin-dependent (type II) diabetes mellitus (NIDDM). Extensive data indicate the importance of maternal diabetes in determining their risk for diabetes. Generally, the risk of having NIDDM is higher in patients with affected mothers than affected fathers. This has been attributed to intrauterine factors, but recently mitochondrial inheritance has been raised as an alternative hypothesis. In other populations, several families and individuals with diabetes due to a mitochondrial DNA point mutation at nucleotide 3243 in the tRNA{sup leu(UUR)} gene have been described, as has one family with a 10.4 kb mitochondrial DNA duplication/deletion. We tested whether these specific mitochondrial gene mutations could explain a portion of the excess maternal transmission seen in the Pima Indians. Mitochondrial DNA obtained from blood lymphocytes of 148 Pima Indians with NIDDM was screened both for the point mutation at nt 3243, and the 10.4 kb duplication/deletion. Neither of these mutations was detected, and although a small proportion of the excess maternal transmission in Pima Indians could still be due to yet undescribed mitochondrial mutations or imprinted nuclear genes, our data support the role of the intrauterine environment in this population. 32 refs, 21 figs.

  8. Mutation Frequency of Three Neurodegenerative Lysosomal Storage Diseases: From Screening to Treatment?

    Science.gov (United States)

    Duarte, Ana Joana; Ribeiro, Diogo; Oliveira, Pedro; Amaral, Olga

    2017-04-01

    The ascertainment of mutation frequencies in the general population may have impact on the population's wellbeing and respective healthcare services. Furthermore, it may help define which approaches will be more effective for certain patients based on the genetic cause of disease. Determine the frequency of three mutations, known to be a major cause of three distinct Lysosomal Storage Diseases (LSDs). The following pre-requisites were met: each mutation accounted for over 55% of the disease alleles among previously reported unrelated patients, all three diseases were among the most prevalent LSDs in the population under study, they all involved devastating deterioration of the nervous system, lacked curative treatment and may be fatal in childhood or adolescence. The anonymous samples used in this study were representative of the whole population; mutations were tested by PCR based methods, positive results were further confirmed. The diseases studied were Mucopolysaccharidosis type I (Hurler, MIM 607014), Tay Sachs disease variant B1 (TS, MIM 272800) and Metachromatic Leukodystrophy (MLD, MIM 250100); the mutations were, respectively, p.W402X, p.R178C and c.465+1G>A. Increased carrier frequencies were found for Tay Sachs disease variant B1 HEXA p.R178C mutation (1:340) and for the infantile MLD ARSA c.465+1G> A mutation (1:350) denoting higher risk for these sub-types of disease in Portugal and possibly in individuals of Iberian ancestry. Carrier screening in target populations may provide the foundations for more effective approaches to precision medicine. Copyright © 2017 IMSS. Published by Elsevier Inc. All rights reserved.

  9. Breast cancer screening, outside the population-screening program, of women from breast cancer families without proven BRCA1/BRCA2 mutations : a simulation study

    NARCIS (Netherlands)

    Jacobi, C.E.; Nagelkerke, N.J.D.; van Houwelingen, J.C.; de Bock, G.H.

    Purpose: We assessed the cost-effectiveness of mammography screening for women under the age of 50, from breast cancer families without proven BRCA1./BRCA2 mutations, because current criteria for screening healthy women from breast cancer families are not evidence-based. Methods: We did simulation

  10. Breast cancer screening, outside the population-screening program, of women from breast cancer families without proven BRCA1/BRCA2 mutations: a simulation study

    NARCIS (Netherlands)

    Jacobi, C.E.; Nagelkerke, N.J.D.; van Houwelingen, J.C.; de Bock, Truuske

    2006-01-01

    Purpose: We assessed the cost-effectiveness of mammography screening for women under the age of 50, from breast cancer families without proven BRCA1./BRCA2 mutations, because current criteria for screening healthy women from breast cancer families are not evidence-based. Methods: We did simulation

  11. Microfluidic screening and whole-genome sequencing identifies mutations associated with improved protein secretion by yeast

    DEFF Research Database (Denmark)

    Huang, Mingtao; Bai, Yunpeng; Sjostrom, Staffan L.

    2015-01-01

    interest in improving its protein secretion capacity. Due to the complexity of the secretory machinery in eukaryotic cells, it is difficult to apply rational engineering for construction of improved strains. Here we used high-throughput microfluidics for the screening of yeast libraries, generated by UV...... mutagenesis. Several screening and sorting rounds resulted in the selection of eight yeast clones with significantly improved secretion of recombinant a-amylase. Efficient secretion was genetically stable in the selected clones. We performed whole-genome sequencing of the eight clones and identified 330...... to construct efficient cell factories for protein secretion. The combined use of microfluidics screening and whole-genome sequencing to map the mutations associated with the improved phenotype can easily be adapted for other products and cell types to identify novel engineering targets, and this approach could...

  12. Molecular screening of 980 cases of suspected hereditary optic neuropathy with a report on 77 novel OPA1 mutations

    DEFF Research Database (Denmark)

    Ferré, Marc; Bonneau, Dominique; Milea, Dan

    2009-01-01

    We report the results of molecular screening in 980 patients carried out as part of their work-up for suspected hereditary optic neuropathies. All the patients were investigated for Leber's hereditary optic neuropathy (LHON) and autosomal dominant optic atrophy (ADOA), by searching for the ten...... primary LHON-causing mtDNA mutations and examining the entire coding sequences of the OPA1 and OPA3 genes, the two genes currently identified in ADOA. Molecular defects were identified in 440 patients (45% of screened patients). Among these, 295 patients (67%) had an OPA1 mutation, 131 patients (30%) had...... an mtDNA mutation, and 14 patients (3%), belonging to three unrelated families, had an OPA3 mutation. Interestingly, OPA1 mutations were found in 157 (40%) of the 392 apparently sporadic cases of optic atrophy. The eOPA1 locus-specific database now contains a total of 204 OPA1 mutations, including 77...

  13. Screening of 1331 Danish breast and/or ovarian cancer families identified 40 novel BRCA1 and BRCA2 mutations

    DEFF Research Database (Denmark)

    Hansen, Thomas V O; Jønson, Lars; Steffensen, Ane Y

    2011-01-01

    Germ-line mutations in the tumour suppressor genes BRCA1 and BRCA2 predispose to breast and ovarian cancer. Since 1999 we have performed mutational screening of breast and/or ovarian cancer patients in East Denmark. During this period we have identified 40 novel sequence variations in BRCA1...... and BRCA2 in high risk breast and/or ovarian cancer families. The mutations were detected via pre-screening using dHPLC or high-resolution melting and direct sequencing. We identified 16 variants in BRCA1, including 9 deleterious frame-shift mutations, 2 intronic variants, 4 missense mutations, and 1...... synonymous variant. The remaining 24 variants were identified in BRCA2, including 10 deleterious mutants (6 frame-shift and 4 nonsense), 2 intronic variants, 10 missense mutations and 2 synonymous variants. The frequency of the variants of unknown significance was examined in control individuals. Moreover...

  14. High Resolution Melt analysis for mutation screening in PKD1 and PKD2

    Directory of Open Access Journals (Sweden)

    Fontes Michel

    2011-10-01

    Full Text Available Abstract Background Autosomal dominant polycystic kidney disease (ADPKD is the most common hereditary kidney disorder. It is characterized by focal development and progressive enlargement of renal cysts leading to end-stage renal disease. PKD1 and PKD2 have been implicated in ADPKD pathogenesis but genetic features and the size of PKD1 make genetic diagnosis tedious. Methods We aim to prove that high resolution melt analysis (HRM, a recent technique in molecular biology, can facilitate molecular diagnosis of ADPKD. We screened for mutations in PKD1 and PKD2 with HRM in 37 unrelated patients with ADPKD. Results We identified 440 sequence variants in the 37 patients. One hundred and thirty eight were different. We found 28 pathogenic mutations (25 in PKD1 and 3 in PKD2 within 28 different patients, which is a diagnosis rate of 75% consistent with literature mean direct sequencing diagnosis rate. We describe 52 new sequence variants in PKD1 and two in PKD2. Conclusion HRM analysis is a sensitive and specific method for molecular diagnosis of ADPKD. HRM analysis is also costless and time sparing. Thus, this method is efficient and might be used for mutation pre-screening in ADPKD genes.

  15. High Resolution Melt analysis for mutation screening in PKD1 and PKD2.

    Science.gov (United States)

    Bataille, Stanislas; Berland, Yvon; Fontes, Michel; Burtey, Stéphane

    2011-10-18

    Autosomal dominant polycystic kidney disease (ADPKD) is the most common hereditary kidney disorder. It is characterized by focal development and progressive enlargement of renal cysts leading to end-stage renal disease. PKD1 and PKD2 have been implicated in ADPKD pathogenesis but genetic features and the size of PKD1 make genetic diagnosis tedious. We aim to prove that high resolution melt analysis (HRM), a recent technique in molecular biology, can facilitate molecular diagnosis of ADPKD. We screened for mutations in PKD1 and PKD2 with HRM in 37 unrelated patients with ADPKD. We identified 440 sequence variants in the 37 patients. One hundred and thirty eight were different. We found 28 pathogenic mutations (25 in PKD1 and 3 in PKD2 ) within 28 different patients, which is a diagnosis rate of 75% consistent with literature mean direct sequencing diagnosis rate. We describe 52 new sequence variants in PKD1 and two in PKD2. HRM analysis is a sensitive and specific method for molecular diagnosis of ADPKD. HRM analysis is also costless and time sparing. Thus, this method is efficient and might be used for mutation pre-screening in ADPKD genes.

  16. NGS-based reverse genetic screen for common embryonic lethal mutations compromising fertility in livestock.

    Science.gov (United States)

    Charlier, Carole; Li, Wanbo; Harland, Chad; Littlejohn, Mathew; Coppieters, Wouter; Creagh, Frances; Davis, Steve; Druet, Tom; Faux, Pierre; Guillaume, François; Karim, Latifa; Keehan, Mike; Kadri, Naveen Kumar; Tamma, Nico; Spelman, Richard; Georges, Michel

    2016-10-01

    We herein report the result of a large-scale, next generation sequencing (NGS)-based screen for embryonic lethal (EL) mutations in Belgian beef and New Zealand dairy cattle. We estimated by simulation that cattle might carry, on average, ∼0.5 recessive EL mutations. We mined exome sequence data from >600 animals, and identified 1377 stop-gain, 3139 frame-shift, 1341 splice-site, 22,939 disruptive missense, 62,399 benign missense, and 92,163 synonymous variants. We show that cattle have a comparable load of loss-of-function (LoF) variants (defined as stop-gain, frame-shift, or splice-site variants) as humans despite having a more variable exome. We genotyped >40,000 animals for up to 296 LoF and 3483 disruptive missense, breed-specific variants. We identified candidate EL mutations based on the observation of a significant depletion in homozygotes. We estimated the proportion of EL mutations at 15% of tested LoF and 6% of tested disruptive missense variants. We confirmed the EL nature of nine candidate variants by genotyping 200 carrier × carrier trios, and demonstrating the absence of homozygous offspring. The nine identified EL mutations segregate at frequencies ranging from 1.2% to 6.6% in the studied populations and collectively account for the mortality of ∼0.6% of conceptuses. We show that EL mutations preferentially affect gene products fulfilling basic cellular functions. The resulting information will be useful to avoid at-risk matings, thereby improving fertility. © 2016 Charlier et al.; Published by Cold Spring Harbor Laboratory Press.

  17. Mutation screening of the glutamate cysteine ligase modifier (GCLM) gene in patients with schizophrenia

    DEFF Research Database (Denmark)

    Butticaz, Christophe; Werge, Thomas; Beckmann, Jacques S

    2009-01-01

    relationship of any of these DNA variants with schizophrenia. CONCLUSION: It is unlikely that functional mutations in the GCLM gene could play a major role in genetic predisposition to schizophrenia and further studies will be required to assess its etiological function in the disease....... in noncoding regions of glutamate cysteine ligase modifier (GCLM) gene, which specifies for the modifier subunit of GCL and schizophrenia. OBJECTIVE: We wanted to investigate the presence of GCLM true functional mutations, likely in linkage disequilibrium with the previously identified single nucleotide...... polymorphism alleles, in the same set of cases that allowed the detection of the original association signal. METHODS: We screened all the coding regions of GCLM and their intronic flanking vicinities in 353 patients with schizophrenia by direct DNA sequencing. RESULTS: Ten sequence variations were identified...

  18. Breast cancer screening in BRCA1 and BRCA2 mutation carriers after risk reducing salpingo-oophorectomy

    NARCIS (Netherlands)

    Fakkert, I.E.; Jansen, L.; Meijer, K.; Kok, Theo; Oosterwijk, J.C.; Mourits, M.J.E.; de Bock, G.H.

    Breast cancer screening is offered to BRCA1 and BRCA2 mutation carriers from the age of 25 years because of their increased risk of breast cancer. As ovarian cancer screening is not effective, risk-reducing salpingho-oophorectomy (RRSO) is offered after child bearing age. RRSO before menopause

  19. Social Health Insurance-Based Simultaneous Screening for 154 Mutations in 19 Deafness Genes Efficiently Identified Causative Mutations in Japanese Hearing Loss Patients.

    Directory of Open Access Journals (Sweden)

    Kentaro Mori

    Full Text Available Sensorineural hearing loss is one of the most common neurosensory disorders in humans. The incidence of SNHL is estimated to be 1 in 500-1000 newborns. In more than half of these patients, the hearing loss is associated with genetic causes. In Japan, genetic testing for the patients with SNHL using the Invader assay to screen for 46 mutations in 13 deafness genes was approved by the Ministry of Health, Labour and Welfare for inclusion in social health insurance coverage in 2012. Furthermore, from August 2015, this genetic testing has been expanded to screen for 154 mutations in 19 deafness genes using targeted genomic enrichment with massively parallel DNA sequencing combined with the Invader assay and TaqMan genotyping. For this study we analyzed 717 unrelated Japanese hearing loss patients. The total allele frequency of 154 mutations in 19 deafness genes was 32.64% (468/1434 and the total numbers of cases associated with at least one mutation was 44.07% (316/717. Among these, we were able to diagnose 212 (30% patients, indicating that the present screening could efficiently identify causative mutations in hearing loss patients. It is noteworthy that 27 patients (3.8% had coexistent multiple mutations in different genes. Five of these 27 patients (0.7%, 5/717 overall were diagnosed with genetic hearing loss affected by concomitant with responsible mutations in more than two different genes. For patients identified with multiple mutations in different genes, it is necessary to consider that several genes might have an impact on their phenotypes.

  20. Screening for mutations in human alpha-globin genes by nonradioactive single-strand conformation polymorphism

    Directory of Open Access Journals (Sweden)

    Jorge S.B.

    2003-01-01

    Full Text Available Point mutations and small insertions or deletions in the human alpha-globin genes may produce alpha-chain structural variants and alpha-thalassemia. Mutations can be detected either by direct DNA sequencing or by screening methods, which select the mutated exon for sequencing. Although small (about 1 kb, 3 exons and 2 introns, the alpha-globin genes are duplicate (alpha2 and alpha1 and highy G-C rich, which makes them difficult to denature, reducing sequencing efficiency and causing frequent artifacts. We modified some conditions for PCR and electrophoresis in order to detect mutations in these genes employing nonradioactive single-strand conformation polymorphism (SSCP. Primers previously described by other authors for radioactive SSCP and phast-SSCP plus denaturing gradient gel electrophoresis were here combined and the resultant fragments (6 new besides 6 original per alpha-gene submitted to silver staining SSCP. Nine structural and one thalassemic mutations were tested, under different conditions including two electrophoretic apparatus (PhastSystem(TM and GenePhor(TM, Amersham Biosciences, different polyacrylamide gel concentrations, run temperatures and denaturing agents, and entire and restriction enzyme cut fragments. One hundred percent of sensitivity was achieved with four of the new fragments formed, using the PhastSystem(TM and 20% gels at 15ºC, without the need of restriction enzymes. This nonradioactive PCR-SSCP approach showed to be simple, rapid and sensitive, reducing the costs involved in frequent sequencing repetitions and increasing the reliability of the results. It can be especially useful for laboratories which do not have an automated sequencer.

  1. Detection of five rare cystic fibrosis mutations peculiar to Southern Italy: implications in screening for the disease and phenotype characterization for patients with homozygote mutations.

    Science.gov (United States)

    Castaldo, G; Fuccio, A; Cazeneuve, C; Picci, L; Salvatore, D; Raia, V; Scarpa, M; Goossens, M; Salvatore, F

    1999-07-01

    The search for the eight most frequent mutations (i.e., DeltaF508, G542X, W1282X, N1303K, 1717-1G-->A, R553X, 2183AA-->G, and I148T) by allele-specific oligonucleotide dot-blot analysis revealed 78% of 396 cystic fibrosis alleles in Southern Italy. The observation of frequent haplotypes on the unidentified cystic fibrosis alleles suggested that a few mutations could account for a large number of unidentified alleles. We screened most of the coding sequence of the cystic fibrosis transmembrane regulator gene by denaturing gradient gel electrophoresis to determine the spectrum of these mutations in 68 unrelated cystic fibrosis patients bearing one or both unidentified mutations. The screening revealed five mutations, R1158X, 711+1G-->T, 4016insT, L1065P, and G1244E, each of which had a frequency of 1.3-1.8% (7% collectively). The 7% increase in the detection rate (85% vs 78%) reduces by >50% the residual risk of being cystic fibrosis carriers for couples who had tested negative by molecular analysis. We therefore designed a second allele-specific oligonucleotide set to analyze the five mutations. Among the patients analyzed, one patient homozygous for the L1065P mutation expressed a mild pulmonary and intestinal form of the disease with pancreatic insufficiency. Two other patients, homozygous for mutations R1158X and 4016insT, both expressed a severe cystic fibrosis phenotype. Five cystic fibrosis mutations are peculiar to patients from Southern Italy. The method described for their analysis is efficient, inexpensive, and can be semi-automated by use of a robotic workstation. The results obtained in patients from Southern Italy may have an impact on laboratories in other countries, given the large migrations of populations from Southern Italy to other countries in the last two centuries.

  2. A whole mitochondrial genome screening in a MELAS patient: A novel mitochondrial tRNA{sup Val} mutation

    Energy Technology Data Exchange (ETDEWEB)

    Mezghani, Najla [Laboratoire de Genetique Moleculaire Humaine, Faculte de Medecine de Sfax, Universite de Sfax (Tunisia); Mnif, Mouna [Service d' endocrinologie, C.H.U. Habib Bourguiba de Sfax (Tunisia); Kacem, Maha [Service de Medecine interne, C.H.U. Fattouma Bourguiba de Monastir (Tunisia); Mkaouar-Rebai, Emna, E-mail: emna_mkaouar@mail2world.com [Laboratoire de Genetique Moleculaire Humaine, Faculte de Medecine de Sfax, Universite de Sfax (Tunisia); Hadj Salem, Ikhlass [Laboratoire de Genetique Moleculaire Humaine, Faculte de Medecine de Sfax, Universite de Sfax (Tunisia); Kallel, Nozha; Charfi, Nadia; Abid, Mohamed [Service d' endocrinologie, C.H.U. Habib Bourguiba de Sfax (Tunisia); Fakhfakh, Faiza [Laboratoire de Genetique Moleculaire Humaine, Faculte de Medecine de Sfax, Universite de Sfax (Tunisia)

    2011-04-22

    Highlights: {yields} We report a young Tunisian patient with clinical features of MELAS syndrome. {yields} Reported mitochondrial mutations were absent after a mutational screening of the whole mtDNA. {yields} We described a novel m.1640A>G mutation in the tRNA{sup Val} gene which was absent in 150 controls. {yields} Mitochondrial deletions and POLG1 gene mutations were absent. {yields} The m.1640A>G mutation could be associated to MELAS syndrome. -- Abstract: Mitochondrial encephalopathy, lactic acidosis and strokelike episodes (MELAS) syndrome is a mitochondrial disorder characterized by a wide variety of clinical presentations and a multisystemic organ involvement. In this study, we report a Tunisian girl with clinical features of MELAS syndrome who was negative for the common m.3243A>G mutation, but also for the reported mitochondrial DNA (mtDNA) mutations and deletions. Screening of the entire mtDNA genome showed several known mitochondrial variants besides to a novel transition m.1640A>G affecting a wobble adenine in the anticodon stem region of the tRNA{sup Val}. This nucleotide was conserved and it was absent in 150 controls suggesting its pathogenicity. In addition, no mutations were found in the nuclear polymerase gamma-1 gene (POLG1). These results suggest further investigation nuclear genes encoding proteins responsible for stability and structural components of the mtDNA or to the oxidative phosphorylation machinery to explain the phenotypic variability in the studied family.

  3. Simultaneous screening of multiple mutations by invader assay improves molecular diagnosis of hereditary hearing loss: a multicenter study.

    Directory of Open Access Journals (Sweden)

    Shin-ichi Usami

    Full Text Available Although etiological studies have shown genetic disorders to be a common cause of congenital/early-onset sensorineural hearing loss, there have been no detailed multicenter studies based on genetic testing. In the present report, 264 Japanese patients with bilateral sensorineural hearing loss from 33 ENT departments nationwide participated. For these patients, we first applied the Invader assay for screening 47 known mutations of 13 known deafness genes, followed by direct sequencing as necessary. A total of 78 (29.5% subjects had at least one deafness gene mutation. Mutations were more frequently found in the patients with congenital or early-onset hearing loss, i.e., in those with an awareness age of 0-6 years, mutations were significantly higher (41.8% than in patients with an older age of awareness (16.0%. Among the 13 genes, mutations in GJB2 and SLC26A4 were mainly found in congenital or early-onset patients, in contrast with mitochondrial mutations (12S rRNA m.1555A>G, tRNA(Leu(UUR m.3243A>G, which were predominantly found in older-onset patients. The present method of simultaneous screening of multiple deafness mutations by Invader assay followed by direct sequencing will enable us to detect deafness mutations in an efficient and practical manner for clinical use.

  4. Identification of six pathogenic RAD51C mutations via mutational screening of 1228 Danish individuals with increased risk of hereditary breast and/or ovarian cancer.

    Science.gov (United States)

    Jønson, Lars; Ahlborn, Lise B; Steffensen, Ane Y; Djursby, Malene; Ejlertsen, Bent; Timshel, Susanne; Nielsen, Finn C; Gerdes, Anne-Marie; Hansen, Thomas V O

    2016-01-01

    Germ-line mutations in the RAD51C gene have recently been identified in families with breast and ovarian cancer and have been associated with an increased risk of ovarian cancer. In this study, we describe the frequency of pathogenic RAD51C mutations identified in Danish breast and/or ovarian cancer families. We screened the RAD51C gene in 1228 Danish hereditary breast and/or ovarian cancer families by next-generation sequencing analysis. The frequency of the identified variants was examined in the exome sequencing project database and in data from 2000 Danish exomes and the presumed significance of missense and intronic variants was predicted by in silico analysis. We identified six families with a pathogenic mutation in RAD51C, including three frameshift mutations, one nonsense mutation, and 2 missense mutations. Overall, pathogenic RAD51C mutations were identified in 0.5 % of Danish families with increased risk of hereditary breast and/or ovarian cancer. Moreover, we identified 24 additional RAD51C variants of which 14 have not been previously reported in the literature. In this study, we determine the prevalence of RAD51C mutations in Danish breast and/or ovarian cancer families. We identified six pathogenic RAD51C mutations as well as 23 variants of uncertain clinical significance and one benign variant. Together, the study extends our knowledge of the RAD51C mutation spectrum and supports that RAD51C should be included in gene panel testing of individuals with high risk of breast and ovarian cancer.

  5. Mutation screening of the TPO gene in a cohort of 192 Chinese patients with congenital hypothyroidism.

    Science.gov (United States)

    Fu, Chunyun; Xie, Bobo; Zhang, Shujie; Wang, Jin; Luo, Shiyu; Zheng, Haiyang; Su, Jiasun; Hu, Xuyun; Chen, Rongyu; Fan, Xin; Luo, Jingsi; Gu, Xuefan; Chen, Shaoke

    2016-05-12

    Defects in the human thyroid peroxidase (TPO) gene are reported to be one of the causes of congenital hypothyroidism (CH) due to dyshormonogenesis. The aim of this study was to examine the TPO mutation spectrum and prevalence among patients with CH in the Guangxi Zhuang Autonomous Region of China and to define the relationships between TPO genotypes and clinical phenotypes. Blood samples were collected from 192 patients with CH in the Guangxi Zhuang Autonomous Region, China and genomic DNA was extracted from peripheral blood leucocytes. All exons of the 10 common CH-associated genes including TPO together with their exon-intron boundaries were screened by next-generation sequencing (NGS). The effect of the novel TPO mutation was investigated by 'in silico' studies. NGS analysis of TPO in 192 patients with CH revealed 3 different variations in 2 individuals (2/192, 1%). Sequencing other CH candidate genes in the patients with TPO variants revealed that patient 1 was homozygous for c.2422delT TPO mutation combined with double heterozygous DUOX2 pathogenic variants (p.R683L/p.L1343F) and patient 2 was triallelic for TPO pathogenic variants (p.R648Q/p.T561M/p.T561M). The present study identified a novel TPO variation c.1682C>T/p.T561M; and four known mutations: c.2422delT/p.C808Afs×24 and c.1943C>T/p.R648Q in TPO, c.2048G>T/p.R683L and c.4027C>T/p.L1343F in DUOX2. Our study indicated that the prevalence of TPO mutations was 1% among studied Chinese patients with CH. More than two variations in one or more CH-associated genes can be found in a single patient, and may, in combination, affect the phenotype of the individual. A novel TPO variation c.1682C>T/p.T561M was found, thereby expanding the mutational spectrum of the gene. Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://www.bmj.com/company/products-services/rights-and-licensing/

  6. Mutation screening of melatonin-related genes in patients with autism spectrum disorders

    Directory of Open Access Journals (Sweden)

    Thuresson Kent

    2010-04-01

    Full Text Available Abstract Background One consistent finding in autism spectrum disorders (ASD is a decreased level of the pineal gland hormone melatonin and it has recently been demonstrated that this decrease to a large extent is due to low activity of the acetylserotonin O-methyltransferase (ASMT, the last enzyme in the melatonin synthesis pathway. Moreover, mutations in the ASMT gene have been identified, including a splice site mutation, that were associated with low ASMT activity and melatonin secretion, suggesting that the low ASMT activity observed in autism is, at least partly, due to variation within the ASMT gene. Methods In the present study, we have investigated all the genes involved in the melatonin pathway by mutation screening of AA-NAT (arylalkylamine N-acetyltransferase, ASMT, MTNR1A, MTNR1B (melatonin receptor 1A and 1B and GPR50 (G protein-coupled receptor 50, encoding both synthesis enzymes and the three main receptors of melatonin, in 109 patients with autism spectrum disorders (ASD. A cohort of 188 subjects from the general population was used as a comparison group and was genotyped for the variants identified in the patient sample. Results Several rare variants were identified in patients with ASD, including the previously reported splice site mutation in ASMT (IVS5+2T>C. Of the variants affecting protein sequence, only the V124I in the MTNR1B gene was absent in our comparison group. However, mutations were found in upstream regulatory regions in three of the genes investigated, ASMT, MTNR1A, and MTNR1B. Conclusions Our report of another ASD patient carrying the splice site mutation IVS5+2T>C, in ASMT further supports an involvement of this gene in autism. Moreover, our results also suggest that other melatonin related genes might be interesting candidates for further investigation in the search for genes involved in autism spectrum disorders and related neurobehavioral phenotypes. However, further studies of the novel variants identified

  7. Targeted prostate cancer screening in BRCA1 and BRCA2 mutation carriers: results from the initial screening round of the IMPACT study.

    Science.gov (United States)

    Bancroft, Elizabeth K; Page, Elizabeth C; Castro, Elena; Lilja, Hans; Vickers, Andrew; Sjoberg, Daniel; Assel, Melissa; Foster, Christopher S; Mitchell, Gillian; Drew, Kate; Mæhle, Lovise; Axcrona, Karol; Evans, D Gareth; Bulman, Barbara; Eccles, Diana; McBride, Donna; van Asperen, Christi; Vasen, Hans; Kiemeney, Lambertus A; Ringelberg, Janneke; Cybulski, Cezary; Wokolorczyk, Dominika; Selkirk, Christina; Hulick, Peter J; Bojesen, Anders; Skytte, Anne-Bine; Lam, Jimmy; Taylor, Louise; Oldenburg, Rogier; Cremers, Ruben; Verhaegh, Gerald; van Zelst-Stams, Wendy A; Oosterwijk, Jan C; Blanco, Ignacio; Salinas, Monica; Cook, Jackie; Rosario, Derek J; Buys, Saundra; Conner, Tom; Ausems, Margreet G; Ong, Kai-ren; Hoffman, Jonathan; Domchek, Susan; Powers, Jacquelyn; Teixeira, Manuel R; Maia, Sofia; Foulkes, William D; Taherian, Nassim; Ruijs, Marielle; Helderman-van den Enden, Apollonia T; Izatt, Louise; Davidson, Rosemarie; Adank, Muriel A; Walker, Lisa; Schmutzler, Rita; Tucker, Kathy; Kirk, Judy; Hodgson, Shirley; Harris, Marion; Douglas, Fiona; Lindeman, Geoffrey J; Zgajnar, Janez; Tischkowitz, Marc; Clowes, Virginia E; Susman, Rachel; Ramón y Cajal, Teresa; Patcher, Nicholas; Gadea, Neus; Spigelman, Allan; van Os, Theo; Liljegren, Annelie; Side, Lucy; Brewer, Carole; Brady, Angela F; Donaldson, Alan; Stefansdottir, Vigdis; Friedman, Eitan; Chen-Shtoyerman, Rakefet; Amor, David J; Copakova, Lucia; Barwell, Julian; Giri, Veda N; Murthy, Vedang; Nicolai, Nicola; Teo, Soo-Hwang; Greenhalgh, Lynn; Strom, Sara; Henderson, Alex; McGrath, John; Gallagher, David; Aaronson, Neil; Ardern-Jones, Audrey; Bangma, Chris; Dearnaley, David; Costello, Philandra; Eyfjord, Jorunn; Rothwell, Jeanette; Falconer, Alison; Gronberg, Henrik; Hamdy, Freddie C; Johannsson, Oskar; Khoo, Vincent; Kote-Jarai, Zsofia; Lubinski, Jan; Axcrona, Ulrika; Melia, Jane; McKinley, Joanne; Mitra, Anita V; Moynihan, Clare; Rennert, Gad; Suri, Mohnish; Wilson, Penny; Killick, Emma; Moss, Sue; Eeles, Rosalind A

    2014-09-01

    Men with germline breast cancer 1, early onset (BRCA1) or breast cancer 2, early onset (BRCA2) gene mutations have a higher risk of developing prostate cancer (PCa) than noncarriers. IMPACT (Identification of Men with a genetic predisposition to ProstAte Cancer: Targeted screening in BRCA1/2 mutation carriers and controls) is an international consortium of 62 centres in 20 countries evaluating the use of targeted PCa screening in men with BRCA1/2 mutations. To report the first year's screening results for all men at enrollment in the study. We recruited men aged 40-69 yr with germline BRCA1/2 mutations and a control group of men who have tested negative for a pathogenic BRCA1 or BRCA2 mutation known to be present in their families. All men underwent prostate-specific antigen (PSA) testing at enrollment, and those men with PSA >3 ng/ml were offered prostate biopsy. PSA levels, PCa incidence, and tumour characteristics were evaluated. The Fisher exact test was used to compare the number of PCa cases among groups and the differences among disease types. We recruited 2481 men (791 BRCA1 carriers, 531 BRCA1 controls; 731 BRCA2 carriers, 428 BRCA2 controls). A total of 199 men (8%) presented with PSA >3.0 ng/ml, 162 biopsies were performed, and 59 PCas were diagnosed (18 BRCA1 carriers, 10 BRCA1 controls; 24 BRCA2 carriers, 7 BRCA2 controls); 66% of the tumours were classified as intermediate- or high-risk disease. The positive predictive value (PPV) for biopsy using a PSA threshold of 3.0 ng/ml in BRCA2 mutation carriers was 48%-double the PPV reported in population screening studies. A significant difference in detecting intermediate- or high-risk disease was observed in BRCA2 carriers. Ninety-five percent of the men were white, thus the results cannot be generalised to all ethnic groups. The IMPACT screening network will be useful for targeted PCa screening studies in men with germline genetic risk variants as they are discovered. These preliminary results support the

  8. Targeted Prostate Cancer Screening in BRCA1 and BRCA2 Mutation Carriers : Results from the Initial Screening Round of the IMPACT Study

    NARCIS (Netherlands)

    Bancroft, Elizabeth K.; Page, Elizabeth C.; Castro, Elena; Lilja, Hans; Vickers, Andrew; Sjoberg, Daniel; Assel, Melissa; Foster, Christopher S.; Mitchell, Gillian; Drew, Kate; Maehle, Lovise; Axcrona, Karol; Evans, D. Gareth; Bulman, Barbara; Eccles, Diana; McBride, Donna; van Asperen, Christi; Vasen, Hans; Kiemeney, Lambertus A.; Ringelberg, Janneke; Cybulski, Cezary; Wokolorczyk, Dominika; Selkirk, Christina; Hulick, Peter J.; Bojesen, Anders; Skytte, Anne-Bine; Lam, Jimmy; Taylor, Louise; Oldenburg, Rogier; Cremers, Ruben; Verhaegh, Gerald; van Zelst-Stams, Wendy A.; Oosterwijk, Jan C.; Blanco, Ignacio; Salinas, Monica; Cook, Jackie; Rosario, Derek J.; Buys, Saundra; Conner, Tom; Ausems, Margreet G.; Ong, Kai-ren; Hoffman, Jonathan; Domchek, Susan; Powers, Jacquelyn; Teixeira, Manuel R.; Maia, Sofia; Foulkes, William D.; Taherian, Nassim; Ruijs, Marielle; Helderman-van den Enden, Apollonia T.; Izatt, Louise; Davidson, Rosemarie; Adank, Muriel A.; Walker, Lisa; Schmutzler, Rita; Tucker, Kathy; Kirk, Judy; Hodgson, Shirley; Harris, Marion; Douglas, Fiona; Lindeman, Geoffrey J.; Zgajnar, Janez; Tischkowitz, Marc; Clowes, Virginia E.; Susman, Rachel; Ramon y Cajal, Teresa; Patcher, Nicholas; Gadea, Neus; Spigelman, Allan; van Os, Theo; Liljegren, Annelie; Side, Lucy; Brewer, Carole; Brady, Angela F.; Donaldson, Alan; Stefansdottir, Vigdis; Friedman, Eitan; Chen-Shtoyerman, Rakefet; Amor, David J.; Copakova, Lucia; Barwell, Julian; Giri, Veda N.; Murthy, Vedang; Nicolai, Nicola; Teo, Soo-Hwang; Greenhalgh, Lynn; Strom, Sara; Henderson, Alex; McGrath, John; Gallagher, David; Aaronson, Neil; Ardern-Jones, Audrey; Bangma, Chris; Dearnaley, David; Costello, Philandra; Eyfjord, Jorunn; Rothwell, Jeanette; Falconer, Alison; Gronberg, Henrik; Hamdy, Freddie C.; Johannsson, Oskar; Khoo, Vincent; Kote-Jarai, Zsofia; Lubinski, Jan; Axcrona, Ulrika; Melia, Jane; McKinley, Joanne; Mitra, Anita V.; Moynihan, Clare; Rennert, Gad; Suri, Mohnish; Wilson, Penny; Killick, Emma; Moss, Sue; Eeles, Rosalind A.

    Background: Men with germline breast cancer 1, early onset (BRCA1) or breast cancer 2, early onset (BRCA2) gene mutations have a higher risk of developing prostate cancer (PCa) than noncarriers. IMPACT (Identification of Men with a genetic predisposition to ProstAte Cancer: Targeted screening in

  9. Targeted Prostate Cancer Screening in BRCA1 and BRCA2 Mutation Carriers: Results from the Initial Screening Round of the IMPACT Study

    NARCIS (Netherlands)

    Bancroft, Elizabeth K.; Page, Elizabeth C.; Castro, Elena; Lilja, Hans; Vickers, Andrew; Sjoberg, Daniel; Assel, Melissa; Foster, Christopher S.; Mitchell, Gillian; Drew, Kate; Mæhle, Lovise; Axcrona, Karol; Evans, D. Gareth; Bulman, Barbara; Eccles, Diana; McBride, Donna; van Asperen, Christi; Vasen, Hans; Kiemeney, Lambertus A.; Ringelberg, Janneke; Cybulski, Cezary; Wokolorczyk, Dominika; Selkirk, Christina; Hulick, Peter J.; Bojesen, Anders; Skytte, Anne-Bine; Lam, Jimmy; Taylor, Louise; Oldenburg, Rogier; Cremers, Ruben; Verhaegh, Gerald; van Zelst-Stams, Wendy A.; Oosterwijk, Jan C.; Blanco, Ignacio; Salinas, Monica; Cook, Jackie; Rosario, Derek J.; Buys, Saundra; Conner, Tom; Ausems, Margreet G.; Ong, Kai-Ren; Hoffman, Jonathan; Domchek, Susan; Powers, Jacquelyn; Teixeira, Manuel R.; Maia, Sofia; Foulkes, William D.; Taherian, Nassim; Ruijs, Marielle; Helderman-van den Enden, Apollonia T.; Izatt, Louise; Davidson, Rosemarie; Adank, Muriel A.; Walker, Lisa; Schmutzler, Rita; Tucker, Kathy; Kirk, Judy; Hodgson, Shirley; Harris, Marion; Douglas, Fiona; Lindeman, Geoffrey J.; Zgajnar, Janez; Tischkowitz, Marc; Clowes, Virginia E.; Susman, Rachel; Ramón Y Cajal, Teresa; Patcher, Nicholas; Gadea, Neus; Spigelman, Allan; van Os, Theo; Liljegren, Annelie; Side, Lucy; Brewer, Carole; Brady, Angela F.; Donaldson, Alan; Stefansdottir, Vigdis; Friedman, Eitan; Chen-Shtoyerman, Rakefet; Amor, David J.; Copakova, Lucia; Barwell, Julian; Giri, Veda N.; Murthy, Vedang; Nicolai, Nicola; teo, Soo-Hwang; Greenhalgh, Lynn; Strom, Sara; Henderson, Alex; McGrath, John; Gallagher, David; Aaronson, Neil; Ardern-Jones, Audrey; Bangma, Chris; Dearnaley, David; Costello, Philandra; Eyfjord, Jorunn; Rothwell, Jeanette; Falconer, Alison; Gronberg, Henrik; Hamdy, Freddie C.; Johannsson, Oskar; Khoo, Vincent; Kote-Jarai, Zsofia; Lubinski, Jan; Axcrona, Ulrika; Melia, Jane; McKinley, Joanne; Mitra, Anita V.; Moynihan, Clare; Rennert, Gad; Suri, Mohnish; Wilson, Penny; Killick, Emma; Moss, Sue; Eeles, Rosalind A.; Eeles, Rosalind; Bancroft, Elizabeth; Page, Elizabeth; Taylor, Natalie; Pope, Jenny; Saya, Sibel; Martin, Sue; Keating, Diana; Petelin, Lara; Murphy, Morgan; Doherty, Rebecca; Pratt, Sarah; Murphy, Declan; Cleeve, Laurence; Miller, Cathy; Stapleton, Alan; Chong, Michael; Suthers, Graeme; Tucker, Katherine; Andrews, Lesley; Duffy, Jessica; Millard, Richard; Ward, Robyn; Williams, Rachel; Stricker, Phillip; Bowman, Michelle; Patel, Manish; O'Connell, Shona; Hunt, Clare; Smyth, Courtney; Frydenberg, Mark; Lindeman, Geoffrey; Shackleton, Kylie; McGaughran, Julie; Boon, Melanie; Pachter, Nicholas; Townshend, Sharron; Schofield, Lyn; Gleeson, Margaret; Scott, Rodney; Amor, David; Burke, Jo; Patterson, Briony; Bacic, Sonya; Swindle, Peter; Foulkes, William; Aprikian, Armen; Bojeson, Anders; Cruger, Dorthe; Osther, Palle; Gerdes, Anne-Marie; Rhiem, Kerstin; Luedtke-Heckenkamp, Kerstin; Ochsendorf, Nicole; Fiddike, Kerstin; Sarin, Rajiv; Awatagiri, Kasturi; Ghonge, Sujata; Kowtal, Pradnya; Mulgund, Gouri; Bambury, Richard; Farrell, Michael; Gallagher, Fergal; Ben-Yehoshua, Sagi Josefsberg; Nissani, Rachel; Appelman, Zvi; Moriel, Evyatar; Radice, Paolo; Valdagni, Riccardo; Magnani, Tiziana; teo, Soo Hwang; Tan, Hui Meng; Yoon, Sook-Yee; Thong, Meow Keong; Kiemeney, Bart; van Zelst-Stams, Wendy; Ausems, Margreet G. E. M.; van der Luijt, Rob B.; Oldenburg, Rogier A.; Maehle, Lovise; Moller, Pal; Brennhovd, Bjorn; Medvik, Heidi; Hanslien, Eldbjørg; Ruijs, Mariëlle W. G.; Helderman- van den Enden, A. Paula T. J. M.; Teixeira, Manuel; Peixoto, Ana; Henrique, Rui; Oliveira, Jorge; Gonçalves, Nuno; Araújo, Luís; Seixas, Manuela; Souto, João Paulo; Nogueira, Pedro; Krajc, Mateja; Vrecar, Alenka; Salinas, Mónica; Capella, Gabriel; Fisas, David; Balmaña, Judith; Morote, Juan; Hjälm-Eriksson, Marie; Ekdahl, Karl-Johan; Carlsson, Stefan; Hanson, Helen; Shanley, Susan; Goh, Chee; Wiggins, Jennifer; Kohut, Kelly; van As, Nicholas; Thompson, Alan; Ogden, Chris; Borley, Nigel; Woodhouse, Christopher; Kumar, Pardeep; Mercer, Catherine; Paterson, Joan; Clowes, Virginia; Taylor, Amy; Newcombe, Barbara; Halliday, Dorothy; Stayner, Barbara; Fleming-Brown, D.; Brice, Glen; Homfray, Tessa; Hammond, Carrie; Potter, Alison; Renton, Caroline; Searle, Anne; Hill, Kathryn; Goodman, Selina; Garcia, Lynda; Devlin, Gemma; Everest, Sarah; Nadolski, Maria; Jobson, Irene; Paez, Edgar; Tomkins, Sue; Pichert, Gabriella; Jacobs, Chris; Langman, Caroline; Weston, Michelle; Brady, Angela; Dorkins, Huw; Melville, Athalie; Kosicka-Slawinska, Monika; Cummings, Carole; Kiesel, Vicki; Bartlett, Marion; Randhawa, Kashmir; Ellery, Natalie; Male, Alison; Simon, Kate; Rees, Katie; Compton, Cecilia; Tidey, Lizzie; Nevitt, Louise; Ingram, Stuart; Rosario, Derek; Catto, James; Howson, Joanne; Chapman, Cyril; Cole, Trevor; Heaton, Tricia; Burgess, Lucy; Longmuir, Mark; Watt, Cathy; Duncan, Alexis; Kockelbergh, Roger; Sattar, Ayisha; Kaemba, Beckie; Sidat, Zahirah; Patel, Nafisa; Siguake, Kas; Birt, Angela; Poultney, Una; Umez-Eronini, Nkem; Mom, Jaswant; Roberts, Gillian; Woodward, Anthony; Sutton, Vivienne; Cornford, Philip; Treherne, Katy; Griffiths, Julie; Cogley, Lyn; Hulick, Peter; Rubinstein, Wendy; Brendler, Charles; Helfand, Brian; McGuire, Michael; Kaul, Karen; Shevrin, Daniel; Weissman, Scott; Newlin, Anna; Vogel, Kristen; Weiss, Shelly; Goldgar, David; Venne, Vickie; Stephenson, Robert; Dechet, Christopher; Arun, Banu; Davis, John W.; Yamamura, Yuko; Giri, Veda; Gross, Laura

    2014-01-01

    Background: Men with germline breast cancer 1, early onset (BRCA1) or breast cancer 2, early onset (BRCA2) gene mutations have a higher risk of developing prostate cancer (PCa) than noncarriers. IMPACT (Identification of Men with a genetic predisposition to ProstAte Cancer: Targeted screening in

  10. Screening for hotspot mutations in PI3K, JAK2, FLT3 and NPM1 in patients with myelodysplastic syndromes

    Directory of Open Access Journals (Sweden)

    João Agostinho Machado-Neto

    2011-01-01

    Full Text Available INTRODUCTION: Myelodysplastic syndromes encompass a heterogeneous group of clonal hematopoietic stem cell disorders characterized by ineffective hematopoiesis, refractory cytopenia and a tendency to progress toward acute myeloid leukemia. The accumulation of genetic alterations is closely associated with the progression of myelodysplastic syndromes toward acute myeloid leukemia. OBJECTIVE: To investigate the presence of mutations in the points most frequent for mutations (hotspot mutations in phosphatidylinositol-3-kinase (PI3K, Janus kinase 2 (JAK2, FMS-like tyrosine kinase 3 (FLT3 and nucleophosmin (NPM1, which are involved in leukemia and other cancers, in a population of Brazilian MDS patients. METHODS: Fifty-one myelodysplastic syndromes patients were included in the study. According to French-American-British classification, the patients were distributed as follows: 31 with refractory anemia, 8 with refractory anemia with ringed sideroblasts, 7 with refractory anemia with excess blasts, 3 with refractory anemia with excess blasts in transformation and 2 with chronic myelomonocytic leukemia. Bone marrow samples were obtained and screened for the presence of hotspot mutations using analysis based on amplification with the polymerase chain reaction, sequencing, fragment size polymorphisms or restriction enzyme digestion. All patients were screened for mutations at the time of diagnosis, and 5 patients were also screened at the time of disease progression. RESULTS: In the genes studied, no mutations were detected in the patients at the time of diagnosis. One patient with chronic myelomonocytic leukemia was heterozygous for a Janus kinase 2 mutation after disease progression. CONCLUSIONS: These results show that hotspot mutations in the PI3K, JAK2, FLT3 and NPM1 genes are not common in MDS patients; nevertheless, JAK2 mutations may be present in myelodysplasia during disease progression.

  11. Screening for mutations in RPGR and RP2 genes in Jordanian families with X-linked retinitis pigmentosa.

    Science.gov (United States)

    Haddad, M F; Khabour, O F; Abuzaideh, K A Y; Shihadeh, W

    2016-06-03

    Retinitis pigmentosa (RP) is a clinically and genetically heterogeneous disease causing progressive degeneration of retinal photoreceptor cells. X-linked RP (XLRP), in which photoreceptor degeneration begins in early childhood and complete blindness often occurs by the fourth decade of life, constitutes the most severe form of this disease. Two genes commonly associated with XLRP have previously been cloned: retinitis pigmentosa GTPase regulator (RPGR) and retinitis pigmentosa 2 (RP2). We sought to identify mutations in these genes in Jordanian families suffering from this disease. Five unrelated Jordanian families with confirmed XLRP were screened for such mutations using direct sequencing. Three mutations were identified in the ORF15 exon of RPGR. The silent g.ORF15+470G>A substitution and the g.ORF15+1822insA insertion in the 3ꞌ-untranslated region were found in both normal and affected male family members at comparable frequencies, and thus were considered normal variants. The third mutation, g.ORF15+588G>A, in which alanine is substituted by threonine, was found in all affected men and one unaffected man in the two families harboring this variant. Thus, this mutation may be pathogenic, but with incomplete penetrance. No RP2 mutations were found among the examined families. Mutation screening of RP patients is essential to understand the mechanism behind this disease and develop treatments. A complete family history is required to identify its inheritance pattern and provide genetic counseling for patients and their families.

  12. Screening the three LHON primary mutations in the general Chinese population by using an optimized multiplex allele-specific PCR.

    Science.gov (United States)

    Bi, Rui; Zhang, A-Mei; Yu, Dandan; Chen, Diana; Yao, Yong-Gang

    2010-11-11

    Leber hereditary optic neuropathy (LHON) is one of the most common mitochondrial diseases, which is mainly caused by three mitochondrial DNA (mtDNA) mutations (m.3460G>A, m.11778G>A and m.14484T>C). Incomplete penetrance suggests that there might be asymptomatic carriers in general populations. These asymptomatic carriers are clinically important as they are potential future patients and the female carriers could transfer the pathogenic mutations to their offspring. Thus, screening the three LHON primary mutations in general populations is important for genetic counseling. We optimized a multiplex allele-specific PCR method based on previous studies, and the sensitivity was evaluated. The three LHON primary mutations were screened by using this MAS-PCR method in 1571 subjects from general Chinese populations that are without symptoms or family history of optic neuropathy. The optimized MAS-PCR approach can detect a heteroplasmy level at 5%, 5%, and 20% for m.3460G>A, m.11778G>A and m.14484T>C, respectively. None of the three LHON primary mutations was detected in the 1571 subjects. The three LHON primary mutations are rare in general Chinese populations. The optimized MAS-PCR assay provides an easier, faster and more cost-effective method for detection of the three LHON primary mutations, making it practical for clinical diagnosis. 2010 Elsevier B.V. All rights reserved.

  13. Mutation screening and association analysis of six candidate genes for autism on chromosome 7q

    DEFF Research Database (Denmark)

    Bonora, E.; Lamb, J.A.; Barnby, G.

    2005-01-01

    Genetic studies have provided evidence for an autism susceptibility locus (AUTS1) on chromosome 7q. Screening for mutations in six genes mapping to 7q, CUTL1, SRPK2, SYPL, LAMB1, NRCAM and PTPRZ1 in 48 unrelated individuals with autism led to the identification of several new coding variants...... in the genes CUTL1, LAMB1 and PTPRZ1. Analysis of genetic variants provided evidence for association with autism for one of the new missense changes identified in LAMB1; this effect was stronger in a subgroup of affected male sibling pair families, implying a possible specific sex-related effect...... for this variant. Association was also detected for several polymorphisms in the promoter and untranslated region of NRCAM, suggesting that alterations in expression of this gene may be linked to autism susceptibility....

  14. A rapid and effective method for screening, sequencing and reporter verification of engineered frameshift mutations in zebrafish

    Directory of Open Access Journals (Sweden)

    Sergey V. Prykhozhij

    2017-06-01

    Full Text Available Clustered regularly interspaced palindromic repeats (CRISPR/Cas-based adaptive immunity against pathogens in bacteria has been adapted for genome editing and applied in zebrafish (Danio rerio to generate frameshift mutations in protein-coding genes. Although there are methods to detect, quantify and sequence CRISPR/Cas9-induced mutations, identifying mutations in F1 heterozygous fish remains challenging. Additionally, sequencing a mutation and assuming that it causes a frameshift does not prove causality because of possible alternative translation start sites and potential effects of mutations on splicing. This problem is compounded by the relatively few antibodies available for zebrafish proteins, limiting validation at the protein level. To address these issues, we developed a detailed protocol to screen F1 mutation carriers, and clone and sequence identified mutations. In order to verify that mutations actually cause frameshifts, we created a fluorescent reporter system that can detect frameshift efficiency based on the cloning of wild-type and mutant cDNA fragments and their expression levels. As proof of principle, we applied this strategy to three CRISPR/Cas9-induced mutations in pycr1a, chd7 and hace1 genes. An insertion of seven nucleotides in pycr1a resulted in the first reported observation of exon skipping by CRISPR/Cas9-induced mutations in zebrafish. However, of these three mutant genes, the fluorescent reporter revealed effective frameshifting exclusively in the case of a two-nucleotide deletion in chd7, suggesting activity of alternative translation sites in the other two mutants even though pycr1a exon-skipping deletion is likely to be deleterious. This article provides a protocol for characterizing frameshift mutations in zebrafish, and highlights the importance of checking mutations at the mRNA level and verifying their effects on translation by fluorescent reporters when antibody detection of protein loss is not possible.

  15. A rapid and effective method for screening, sequencing and reporter verification of engineered frameshift mutations in zebrafish.

    Science.gov (United States)

    Prykhozhij, Sergey V; Steele, Shelby L; Razaghi, Babak; Berman, Jason N

    2017-06-01

    Clustered regularly interspaced palindromic repeats (CRISPR)/Cas-based adaptive immunity against pathogens in bacteria has been adapted for genome editing and applied in zebrafish (Danio rerio) to generate frameshift mutations in protein-coding genes. Although there are methods to detect, quantify and sequence CRISPR/Cas9-induced mutations, identifying mutations in F1 heterozygous fish remains challenging. Additionally, sequencing a mutation and assuming that it causes a frameshift does not prove causality because of possible alternative translation start sites and potential effects of mutations on splicing. This problem is compounded by the relatively few antibodies available for zebrafish proteins, limiting validation at the protein level. To address these issues, we developed a detailed protocol to screen F1 mutation carriers, and clone and sequence identified mutations. In order to verify that mutations actually cause frameshifts, we created a fluorescent reporter system that can detect frameshift efficiency based on the cloning of wild-type and mutant cDNA fragments and their expression levels. As proof of principle, we applied this strategy to three CRISPR/Cas9-induced mutations in pycr1a, chd7 and hace1 genes. An insertion of seven nucleotides in pycr1a resulted in the first reported observation of exon skipping by CRISPR/Cas9-induced mutations in zebrafish. However, of these three mutant genes, the fluorescent reporter revealed effective frameshifting exclusively in the case of a two-nucleotide deletion in chd7, suggesting activity of alternative translation sites in the other two mutants even though pycr1a exon-skipping deletion is likely to be deleterious. This article provides a protocol for characterizing frameshift mutations in zebrafish, and highlights the importance of checking mutations at the mRNA level and verifying their effects on translation by fluorescent reporters when antibody detection of protein loss is not possible. © 2017. Published by

  16. Mutation screening of patients with Alzheimer disease identifies APP locus duplication in a Swedish patient

    Science.gov (United States)

    2011-01-01

    Background Missense mutations in three different genes encoding amyloid-β precursor protein, presenilin 1 and presenilin 2 are recognized to cause familial early-onset Alzheimer disease. Also duplications of the amyloid precursor protein gene have been shown to cause the disease. At the Dept. of Geriatric Medicine, Karolinska University Hospital, Sweden, patients are referred for mutation screening for the identification of nucleotide variations and for determining copy-number of the APP locus. Methods We combined the method of microsatellite marker genotyping with a quantitative real-time PCR analysis to detect duplications in patients with Alzheimer disease. Results In 22 DNA samples from individuals diagnosed with clinical Alzheimer disease, we identified one patient carrying a duplication on chromosome 21 which included the APP locus. Further mapping of the chromosomal region by array-comparative genome hybridization showed that the duplication spanned a maximal region of 1.09 Mb. Conclusions This is the first report of an APP duplication in a Swedish Alzheimer patient and describes the use of quantitative real-time PCR as a tool for determining copy-number of the APP locus. PMID:22044463

  17. Screening for and verification of novel mutations associated with drug resistance in the HIV type 1 subtype B(' in China.

    Directory of Open Access Journals (Sweden)

    Hanping Li

    Full Text Available OBJECTIVE: Mutations associated with HIV drug resistance have been extensively characterized at the HIV-1 polymerase domain, but more studies have verified that mutations outside of the polymerase domain also results in resistance to antiviral drugs. In this study, mutations were identified in 354 patients experiencing antiretroviral therapy (ART failure and in 97 naïve-therapy patients. Mutations whose impact on antiviral drugs was unknown were verified by phenotypic testing. METHODS: Pol sequences of HIV subtype B(' obtained from patients experiencing ART failure and from naïve-therapy patients were analyzed for mutations distinct between two groups. Mutations that occurred at a significantly higher frequency in the ART failure than the naïve-therapy group were submitted to the Stanford HIV Drug Resistance Database (SHDB to analyze the correlation between HIV mutations and drug resistance. For mutations whose impact on the antiviral drug response is unknown, the site-directed mutagenesis approach was applied to construct plasmids containing the screened mutations. 50% inhibitory concentration (IC(50 to AZT, EFV and NVP was measured to determine the response of the genetically constructed viruses to antiviral drugs. RESULTS: 7 mutations at 6 positions of the RT region, D123E, V292I, K366R, T369A, T369V, A371V and I375V, occurred more frequently in the ART failure group than the naïve-therapy group. Phenotypic characterization of these HIV mutants revealed that constructed viruses with mutations A371V and T369V exhibited dual resistance to AZT and EFV respectively, whereas the other 5 mutations showed weak resistance. Although the impact of the other six mutations on response to NVP was minimal, mutation T369V could enhance resistance to NVP. CONCLUSIONS: This study demonstrated that mutations at the RT C-terminal in subtype B' could result in resistance to RT inhibitors if the mutations occurred alone, but that some mutations could promote

  18. RNA-based therapies for genodermatoses

    NARCIS (Netherlands)

    Bornert, Olivier; Peking, Patricia; Bremer, Jeroen; Koller, Ulrich; van den Akker, Peter C.; Aartsma-Rus, Annemieke; Pasmooij, Anna M. G.; Murauer, Eva M.; Nystroem, Alexander

    Genetic disorders affecting the skin, genodermatoses, constitute a large and heterogeneous group of diseases, for which treatment is generally limited to management of symptoms. RNA-based therapies are emerging as a powerful tool to treat genodermatoses. In this review, we discuss in detail RNA

  19. Novel mutations causing biotinidase deficiency in individuals identified by newborn screening in Michigan including an unique intronic mutation that alters mRNA expression of the biotinidase gene.

    Science.gov (United States)

    Li, H; Spencer, L; Nahhas, F; Miller, J; Fribley, A; Feldman, G; Conway, R; Wolf, B

    2014-07-01

    Biotinidase deficiency (BD) is an autosomal recessive disorder resulting in the inability to recycle the vitamin biotin. Individuals with biotinidase deficiency can develop neurological and cutaneous symptoms if they are not treated with biotin. To date, more than 165 mutations in the biotinidase gene (BTD) have been reported. Essentially all the mutations result in enzymatic activities with less than 10% of mean normal serum enzyme activity (profound biotinidase deficiency) with the exception of the c.1330G>C (p.D444H) mutation, which results in an enzyme having 50% of mean normal serum activity and causes partial biotinidase deficiency (10-30% of mean normal serum biotinidase activity) if there is a mutation for profound biotinidase deficiency on the second allele. We now reported eight novel mutations in ten children identified by newborn screening in Michigan from 1988 to the end of 2012. Interestingly, one intronic mutation, c.310-15delT, results in an approximately two-fold down-regulation of BTD mRNA expression by Quantitative real-time reverse-transcription PCR (qRT-PCR). This is the first report of an intronic mutation in the BTD gene with demonstration of its effect on enzymatic activity by altering mRNA expression. This study identified three other mutations likely to cause partial biotinidase deficiency. These results emphasize the importance of full gene sequencing of BTD on patients with biotinidase deficiency to better understand the genotype and phenotype correlation in the future. Copyright © 2014 Elsevier Inc. All rights reserved.

  20. Identification of six pathogenic RAD51C mutations via mutational screening of 1228 Danish individuals with increased risk of hereditary breast and/or ovarian cancer

    DEFF Research Database (Denmark)

    Jønson, Lars; Ahlborn, Lise B; Steffensen, Ane Y

    2016-01-01

    families with increased risk of hereditary breast and/or ovarian cancer. Moreover, we identified 24 additional RAD51C variants of which 14 have not been previously reported in the literature. In this study, we determine the prevalence of RAD51C mutations in Danish breast and/or ovarian cancer families. We......Germ-line mutations in the RAD51C gene have recently been identified in families with breast and ovarian cancer and have been associated with an increased risk of ovarian cancer. In this study, we describe the frequency of pathogenic RAD51C mutations identified in Danish breast and/or ovarian...... cancer families. We screened the RAD51C gene in 1228 Danish hereditary breast and/or ovarian cancer families by next-generation sequencing analysis. The frequency of the identified variants was examined in the exome sequencing project database and in data from 2000 Danish exomes and the presumed...

  1. Low-density lipoprotein receptor gene mutations and cardiovascular risk in a large genetic cascade screening population

    NARCIS (Netherlands)

    Umans-Eckenhausen, Marina A. W.; Sijbrands, Eric J. G.; Kastelein, John J. P.; Defesche, Joep C.

    2002-01-01

    Background-A large cohort of patients with familial hypercholesterolemia (FH), free from selection for cardiovascular disease (CVD), and their unaffected relatives was collected by genetic cascade screening and examined for the influence of different mutations of the LDL receptor gene on lipoprotein

  2. Predictors of prophylactic bilateral salpingo-oophorectomy compared with gynecologic screening use in BRCA1/2 mutation carriers

    NARCIS (Netherlands)

    Madalinska, Joanna B.; van Beurden, Marc; Bleiker, Eveline M. A.; Valdimarsdottir, Heiddis B.; Lubsen-Brandsma, Lottie; Massuger, Leon F.; Mourits, Marian J. E.; Gaarenstroom, Katja N.; van Dorst, Eleonora B. L.; van der Putten, Hans; Boonstra, Henk; Aaronson, Neil K.

    2007-01-01

    Women with BRCA1/2 gene mutations who have completed their childbearing are strong candidates for risk-reducing prophylactic bilateral salpingo-oophorectomy (PBSO). The aim of the current study was to identify baseline predictors of PBSO versus gynecologic screening (GS) in this group of high-risk

  3. Predictors of prophylactic bilateral salpingo-oophorectomy compared with gynecologic screening use in BRCA1/2 mutation carriers.

    NARCIS (Netherlands)

    Madalinska, J.B.; Beurden, M. van; Bleiker, E.M.A.; Valdimarsdottir, H.B.; Lubsen-Brandsma, L.; Massuger, L.F.A.G.; Mourits, M.J.E.; Gaarenstroom, K.N.; Dorst, E.B.L. van; Putten, H. van der; Boonstra, H.; Aaronson, N.K.

    2007-01-01

    PURPOSE: Women with BRCA1/2 gene mutations who have completed their childbearing are strong candidates for risk-reducing prophylactic bilateral salpingo-oophorectomy (PBSO). The aim of the current study was to identify baseline predictors of PBSO versus gynecologic screening (GS) in this group of

  4. Predictors of prophylactic bilateral salpingo-oophorectomy compared with gynecologic screening use in BRCA1/2 mutation carriers

    NARCIS (Netherlands)

    Madalinska, Joanna B.; van Beurden, Marc; Bleiker, Eveline M. A.; Valdimarsdottir, Heiddis B.; Lubsen-Brandsma, Lottie; Massuger, Leon F.; Mourits, Marian J. E.; Gaarenstroom, Katja N.; van Dorst, Eleonora B. L.; van der Putten, Hans; Boonstra, Henk; Aaronson, Neil K.

    2007-01-01

    Purpose Women with BRCA1/2 gene mutations who have completed their childbearing are strong candidates for risk-reducing prophylactic bilateral salpingo-oophorectomy (PBSO). The aim of the current study was to identify baseline predictors of PBSO versus gynecologic screening (GS) in this group of

  5. Screen for IDH1, IDH2, IDH3, D2HGDH and L2HGDH mutations in glioblastoma.

    Directory of Open Access Journals (Sweden)

    Daniel Krell

    Full Text Available Isocitrate dehydrogenases (IDHs catalyse oxidative decarboxylation of isocitrate to α-ketoglutarate (α-KG. IDH1 functions in the cytosol and peroxisomes, whereas IDH2 and IDH3 are both localized in the mitochondria. Heterozygous somatic mutations in IDH1 occur at codon 132 in 70% of grade II-III gliomas and secondary glioblastomas (GBMs, and in 5% of primary GBMs. Mutations in IDH2 at codon 172 are present in grade II-III gliomas at a low frequency. IDH1 and IDH2 mutations cause both loss of normal enzyme function and gain-of-function, causing reduction of α-KG to D-2-hydroxyglutarate (D-2HG which accumulates. Excess hydroxyglutarate (2HG can also be caused by germline mutations in D- and L-2-hydroxyglutarate dehydrogenases (D2HGDH and L2HGDH. If loss of IDH function is critical for tumourigenesis, we might expect some tumours to acquire somatic IDH3 mutations. Alternatively, if 2HG accumulation is critical, some tumours might acquire somatic D2HGDH or L2HGDH mutations. We therefore screened 47 glioblastoma samples looking for changes in these genes. Although IDH1 R132H was identified in 12% of samples, no mutations were identified in any of the other genes. This suggests that mutations in IDH3, D2HGDH and L2HGDH do not occur at an appreciable frequency in GBM. One explanation is simply that mono-allelic IDH1 and IDH2 mutations occur more frequently by chance than the bi-allelic mutations expected at IDH3, D2HGDH and L2HGDH. Alternatively, both loss of IDH function and 2HG accumulation might be required for tumourigenesis, and only IDH1 and IDH2 mutations have these dual effects.

  6. Mutations in THAP1 (DYT6) and generalised dystonia with prominent spasmodic dysphonia: a genetic screening study

    DEFF Research Database (Denmark)

    Djarmati, Ana; Schneider, Susanne A; Lohmann, Katja

    2009-01-01

    and these and an additional 75 controls were screened for a rare non-coding mutation. FINDINGS: We identified two mutations in THAP1 (388_389delTC and 474delA), respectively, in two (1%) German patients from the 160 patients with dystonia. Both mutation carriers had laryngeal dystonia that started in childhood and both went......-onset generalised dystonia with spasmodic dysphonia. This combination of symptoms might be a characteristic feature of DYT6 dystonia and could be useful in the differential diagnosis of DYT1, DYT4, DYT12, and DYT17 dystonia. In addition to the identified mutations, a rare non-coding substitution in THAP1 might...

  7. Screening of SCN1A gene mutation in 10 families with generalized epilepsy accompanied by febrile seizure

    Directory of Open Access Journals (Sweden)

    Guang-sheng WU

    2014-10-01

    Full Text Available Objective To analyze the phenotypes and to identify SCN1A gene mutation in families with generalized epilepsy accompanied by febrile seizures (GEFS+. Methods The clinical data were collected, and genomic DNA was extracted from peripheral blood lymphocytes of the proband and available members in the GEFS+ families. The phenotypes of the related members were analyzed. The coding regions and flanking intronic regions of the SCN1A gene were screened for mutations using PCR and direct DNA sequencing. Results Thirty-three related members from 10 GEFS+ families, ranged from 2 to 7 members in each family received the examination. Their phenotypes included febrile seizures (FS in 11 persons (33.3%, febrile seizures plus (Fs+ in 11 (33.3%, FS with partial seizures in 1 (3.0%, afebrile generalized tonic-clonic seizures (AGTCS in 3 (9.1%, Dravet syndrome in 1 (3.0%, childhood absence epilepsy in 1 (3.0%, and unclassified seizures in 5 members (15.2%. SCN1A mutations were found in 3 families, including one family with missense mutation (glu1444-lys, and silent mutation: synonymous mutation existed in exon 15 (c.2592G>A, with synonymous mutation in exon 9 (c.1212 G>A and exon 26 (c.5385 G>A in two families. Conclusions A new missense mutation and three synonymous mutation have been found in the coding regions of SCN1A gene in three families. One of the families has synonymous mutation in two exons. Genetic mechanism is complicated in GEFS+ families. DOI: 10.11855/j.issn.0577-7402.2014.10.09

  8. Sensitive High-Resolution Melting Analysis for Screening of KRAS and BRAF Mutations in Iranian Human Metastatic Colorectal Cancers

    Science.gov (United States)

    Niya, Mohammad Hadi Karbalaie; Basi, Ali; Koochak, Aghigh; Tameshkel, Fahimeh Safarnezhad; Rakhshani, Nasser; Zamani, Farhad; Imanzade, Farid; Rezvani, Hamid; sereshki, Mohammad Mahdi Adib; Sohrabi, Masoud Reza

    2016-01-01

    Background: Investigations of methods for detection of mutations have uncovered major weaknesses of direct sequencing and pyrosequencing, with their high costs and low sensitivity in screening for both known and unknown mutations. High resolution melting (HRM) analysis is an alternative tool for the rapid detection of mutations. Here we describe the accuracy of HRM in screening for KRAS and BRAF mutations in metastatic colorectal cancer (mCRCs) samples. Materials and Methods: A total of 1000 mCRC patients in Mehr Hospital, Tehran, Iran, from Feb 2008 to May 2012 were examined for KRAS mutations and 242 of them were selected for further assessment of BRAF mutations by HRM analysis. In order to calculate the sensitivity and specificity, HRM results were checked by pyrosequencing as the golden standard and Dxs Therascreen as a further method. Results: In the total of 1,000 participants, there were 664 (66.4%) with wild type and 336 (33.6%) with mutant codons 12 and/or 13 of the KRAS gene. Among 242 samples randomly checked for the BRAF gene, all were wild type by HRM. Pyrosequencing and Dxs Therascreen results were in line with those of the HRM. In this regard, the sensitivity and specificity of HRM were evaluated as 100%. Conclusion: The findings suggest that the HRM, in comparison with DNA sequencing, is a more appropriate method for precise scanning of KRAS and BRAF mutations. It is also possible to state that HRM may be an attractive technique for the detection of known or unknown somatic mutations in other genes. PMID:28122448

  9. Molecular screening of the LPCAT1 gene in patients with retinitis pigmentosa without defined mutations in known retinitis pigmentosa genes.

    Science.gov (United States)

    Wu, Juan; Wang, Hong-Ting; Huang, Xiu-Feng; Lei, Xin-Lan; Lu, Qin-Kang; Jin, Zi-Bing

    2015-10-01

    Retinitis pigmentosa (RP) is an inherited retinopathy, which affects the photoreceptors in the retina. Lysophosphatidylcholine acyltransferase (LPCAT) is a critical phospholipid biosynthesis enzyme, which promotes the conversion of lysophosphatidylcholine into phosphatidylcholine in the remodeling pathway of PC biosynthesis. A previous study reported a homozygous insertion in the LPCAT1 gene in mice exhibiting retinal degeneration (rd11). However, whether genetic mutations in LPCAT1 predispose individuals to RP remains to be elucidated. Therefore, the aim of the present study was to investigate whether LPCAT1 mutations exist in patients with RP. A total of 50 unrelated patients diagnosed with either a sporadic or recessive inheritance pattern of RP were recruited in the present study. All of the patients were comprehensively screened for genes associated with the predisposition of RP, and no pathogenic mutations were identified. Reverse transcription-polymerase chain reaction and Sanger sequencing were performed to investigate the coding regions and exon‑intron boundaries of the LPCAT1 gene in the recruited patients. In total, three genetic variations in the coding regions, which lead to amino acid changes, were identified. Although two of these mutations were predicted to be pathogenic, co‑segregation analysis in the pedigrees excluded these as disease‑causing mutations. In addition, the LPCAT1 gene was screen in a panel of RP patients who exhibited no identifiable mutations in any of the known RP‑associated genes. No disease‑causing mutations in the LPCAT1 gene were identified, indicating that LPCAT1 either does not confer a genetic predisposition to RP, or that the incidence of mutations in LPCAT1 is particularly rare in patients with RP.

  10. Screening and analysis of mutation hot-spots in deafness-associated genes among adolescents with hearing loss.

    Science.gov (United States)

    Jiang, Hong; Liu, Qizhen; Chen, Lihong

    2015-12-01

    The present study aimed to screen the hot-spot deafness gene mutations of adolescents with non‑syndromic hearing loss in Yongchuan, Chongqing (CQ-YC ANSHL), aiming to preliminarily understand the region's spectrum and occurrence frequency of deafness gene mutation hot‑spots. A total of 60 CQ‑YC ANSHL were selected from the Special Education School of Yongchuan, Chongqing and the nine most common mutations of four deafness genes among the Chinese population were detected and associated with the patients' medical history as well as family history of deafness. Deafness gene mutations were detected in 22 cases, among which the detection rates of GJB2, mitochondrial 12S ribosomal ribonucleic acid and SLC26A4 mutations were 23.73% (14/59), 10.17% (6/59) and 5.08% (3/59), respectively, while no GJB3 mutation was detected. The carrying rate of deafness gene mutations in CQ‑YC ANSHL was high; therefore, based on the deafness gene diagnosis, the combination of medication guidance, pre‑natal diagnosis and clinical interventions may be able to effectively reduce the incidence of deafness in this region.

  11. Mutational screening of VSX1 in keratoconus patients from the European population.

    Science.gov (United States)

    Dash, D P; George, S; O'Prey, D; Burns, D; Nabili, S; Donnelly, U; Hughes, A E; Silvestri, G; Jackson, J; Frazer, D; Héon, E; Willoughby, C E

    2010-06-01

    To perform mutational screening of the visual system homeobox gene 1 (VSX1; MIM#605020) in patients with sporadic and familial keratoconus (MIM#148300) in a European population and, for the first time, report the mutational analysis of the two newly identified VSX1exons. VSX1sequence variants in patients with keratoconus were evaluated by direct sequencing of the entire coding region, including two novel exons. In familial keratoconus cases, segregation of potentially pathogenic VSX1variants was assessed to determine pathogenicity. Transcript analysis was carried out on splice site and synonymous sequence variants not detected in controls. A total of 66 unrelated patients with keratoconus from the European population (27 with familial keratoconus; 39 with sporadic keratoconus) were analysed for VSX1 mutations. Four sequence variants were not observed in 100 healthy control individuals: c.432C>G (p.D144E), c.479G>A (p.G160D), c.789C>T (p.S263S), and an intronic change c.844-13T>A (numbered with respect to NM_014588). Segregation was not detected for p.D144E and c.844-13T>A. The change in p.G160D was observed in two patients with sporadic keratoconus. Although predicted to alter VSX1 splicing, p.S263S had no effect on transcript processing. Four known SNPs were detected and the following polymorphic variants were observed in keratoconus patients and controls: c.711T>A (NM_199425; p.P237P), c.844-5_-6insT (NM_014588), c.*28G>T (DQ854811/DQ854812), and c.*50G>A (DQ854809/DQ854810). VSX1has a minor role in keratoconus pathogenesis. The pathogenicity of p.G160D remains controversial and this change may represent a rare polymorphism or genetic modifier. Further evidence is provided that the previously reported variant, p.D144E, is a polymorphism.

  12. Screening for PS1 mutations in a referral-based series of AD cases: 21 novel mutations

    NARCIS (Netherlands)

    Rogaeva, E. A.; Fafel, K. C.; Song, Y. Q.; Medeiros, H.; Sato, C.; Liang, Y.; Richard, E.; Rogaev, E. I.; Frommelt, P.; Sadovnick, A. D.; Meschino, W.; Rockwood, K.; Boss, M. A.; Mayeux, R.; St George-Hyslop, P.

    2001-01-01

    Mutations in the presenilin-1 gene (PS1) account for a majority of patients with early-onset familial AD. However, the clinical indications and algorithms for genetic testing in dementia are still evolving. The entire open reading frame of the PS1 gene was sequenced in a series of 414 consecutive

  13. Mutations in THAP1 (DYT6) and generalised dystonia with prominent spasmodic dysphonia: a genetic screening study.

    Science.gov (United States)

    Djarmati, Ana; Schneider, Susanne A; Lohmann, Katja; Winkler, Susen; Pawlack, Heike; Hagenah, Johann; Brüggemann, Norbert; Zittel, Simone; Fuchs, Tania; Raković, Aleksandar; Schmidt, Alexander; Jabusch, Hans-Christian; Wilcox, Robert; Kostić, Vladimir S; Siebner, Hartwig; Altenmüller, Eckart; Münchau, Alexander; Ozelius, Laurie J; Klein, Christine

    2009-05-01

    DYT6 is a primary, early-onset torsion dystonia; however, unlike in DYT1 dystonia, the symptoms of DYT6 dystonia frequently involve the craniocervical region. Recently, two mutations in THAP1, the gene that encodes THAP (thanatos-associated protein) domain-containing apoptosis-associated protein 1 (THAP1), have been identified as a cause of DYT6 dystonia. We screened THAP1 by sequence analysis and quantitative real-time polymerase chain reaction (PCR) in 160 white patients of European ancestry who had dystonia with an early age at onset (n=64), generalised dystonia (n=35), a positive family history of dystonia (n=56), or facial or laryngeal dystonia. Another 160 patients with dystonia were screened for reported and novel variants in THAP1. 280 neurologically healthy controls were screened for the newly identified and previously reported changes in THAP1 and these and an additional 75 controls were screened for a rare non-coding mutation. We identified two mutations in THAP1 (388_389delTC and 474delA), respectively, in two (1%) German patients from the 160 patients with dystonia. Both mutation carriers had laryngeal dystonia that started in childhood and both went on to develop generalised dystonia. Thus, two of three patients with early-onset generalised dystonia with orobulbar involvement had mutations in THAP1. One of the identified patients with DYT6 dystonia had two family members with subtle motor signs who also carried the same mutation. A rare substitution in the 5'untranslated region (-236_235GA-->TT) was found in 20 of 320 patients and in seven of 355 controls (p=0.0054). Although mutations in THAP1 might have only a minor role in patients with different, but mainly focal, forms of dystonia, they do seem to be associated with early-onset generalised dystonia with spasmodic dysphonia. This combination of symptoms might be a characteristic feature of DYT6 dystonia and could be useful in the differential diagnosis of DYT1, DYT4, DYT12, and DYT17 dystonia. In

  14. Full-exon pyrosequencing screening of BRCA germline mutations in Mexican women with inherited breast and ovarian cancer.

    Directory of Open Access Journals (Sweden)

    Felipe Vaca-Paniagua

    Full Text Available Hereditary breast cancer comprises 10% of all breast cancers. The most prevalent genes causing this pathology are BRCA1 and BRCA2 (breast cancer early onset 1 and 2, which also predispose to other cancers. Despite the outstanding relevance of genetic screening of BRCA deleterious variants in patients with a history of familial cancer, this practice is not common in Latin American public institutions. In this work we assessed mutations in the entire exonic and splice-site regions of BRCA in 39 patients with breast and ovarian cancer and with familial history of breast cancer or with clinical features suggestive for BRCA mutations by massive parallel pyrosequencing. First we evaluated the method with controls and found 41-485 reads per sequence in BRCA pathogenic mutations. Negative controls did not show deleterious variants, confirming the suitability of the approach. In patients diagnosed with cancer we found 4 novel deleterious mutations (c.2805_2808delAGAT and c.3124_3133delAGCAATATTA in BRCA1; c.2639_2640delTG and c.5114_5117delTAAA in BRCA2. The prevalence of BRCA mutations in these patients was 10.2%. Moreover, we discovered 16 variants with unknown clinical significance (11 in exons and 5 in introns; 4 were predicted as possibly pathogenic by in silico analyses, and 3 have not been described previously. This study illustrates how massive pyrosequencing technology can be applied to screen for BRCA mutations in the whole exonic and splice regions in patients with suspected BRCA-related cancers. This is the first effort to analyse the mutational status of BRCA genes on a Mexican-mestizo population by means of pyrosequencing.

  15. BRAFV600 mutations in solid tumors, other than metastatic melanoma and papillary thyroid cancer, or multiple myeloma: a screening study

    Directory of Open Access Journals (Sweden)

    Cohn AL

    2017-02-01

    Full Text Available Allen L Cohn,1 Bann-Mo Day,2 Sarang Abhyankar,3 Edward McKenna,2 Todd Riehl,4 Igor Puzanov5 1Medical Research, Rocky Mountain Cancer Centers, Denver, CO, 2US Medical Affairs, 3Global Safety and Risk Management, 4Product Development Oncology, Genentech, Inc., South San Francisco, CA, 5Melanoma Section, Division of Hematology-Oncology, Department of Medicine, Vanderbilt University Medical Center, Nashville, TN, USA Background: Mutations in the BRAF gene have been implicated in several human cancers. The objective of this screening study was to identify patients with solid tumors (other than metastatic melanoma or papillary thyroid cancer or multiple myeloma harboring activating BRAFV600 mutations for enrollment in a vemurafenib clinical study.Methods: Formalin-fixed, paraffin-embedded tumor samples were collected and sent to a central laboratory to identify activating BRAFV600 mutations by bidirectional direct Sanger sequencing.Results: Overall incidence of BRAFV600E mutation in evaluable patients (n=548 was 3% (95% confidence interval [CI], 1.7–4.7: 11% in colorectal tumors (n=75, 6% in biliary tract tumors (n=16, 3% in non-small cell lung cancers (n=71, 2% in other types of solid tumors (n=180, and 3% in multiple myeloma (n=31. There were no BRAFV600 mutations in this cohort of patients with ovarian tumors (n=68, breast cancer (n=86, or prostate cancer (n=21.Conclusion: This multicenter, national screening study confirms previously reported incidences of BRAFV600 mutations from single-center studies. Patients identified with BRAFV600 mutations were potentially eligible for enrollment in the VE-BASKET study. Keywords: genetic testing, proto-oncogene proteins B-raf, PLX4032

  16. [Screening of common deafness gene mutations in 17 000 Chinese newborns from Chengdu based on microarray analysis].

    Science.gov (United States)

    Lyu, Kangmo; Xiong, Yehua; Yu, Hao; Zou, Ling; Ran, Longrong; Liu, Deshun; Yin, Qin; Xu, Yingwen; Fang, Xue; Song, Zuling; Huang, Lijia; Tan, Dayong; Zhang, Zhiwei

    2014-10-01

    mutation rate of mitochondrial DNA 12S rRNA is 2.71‰, which may have deafness induced by aminoglycoside antibiotics. Newborn screening for mutation of genes related to hereditary deafness plays an important role in the early detection and proper management for neonatal deafness as well as genetic counseling for premarital, prenatal and postnatal diagnosis.

  17. Screening for SLC7A14 gene mutations in patients with autosomal recessive or sporadic retinitis pigmentosa.

    Science.gov (United States)

    Sugahara, Masako; Oishi, Maho; Oishi, Akio; Ogino, Ken; Morooka, Satoshi; Gotoh, Norimoto; Kang, Inyeop; Yoshimura, Nagahisa

    2017-01-01

    In this study, we aimed to detect mutations in the SLC7A14 cationic transporter gene, which has recently been reported as a causative gene for retinitis pigmentosa (RP), in Japanese patients with autosomal recessive (AR) or sporadic RP. We included 146 unrelated Japanese patients with AR or sporadic RP who lacked mutations in genes known to be associated with RP despite next-generation sequencing-based screening. We sequenced the seven SLC7A14 coding exons along with their flanking intronic DNA using the Sanger method. The detected polymorphisms were assessed for their pathogenicity with in silico prediction tools. For those who had heterozygous, nonsynonymous variants, we performed multiplex ligation-dependent probe amplification (MLPA) to search for additional deletion/duplication. We detected four distinct SLC7A14 polymorphisms excluding synonymous polymorphisms. Two of these polymorphisms were assessed as detrimental by in silico prediction tools. However, all of the mutations were heterozygous. Neither homozygous polymorphisms nor compound heterozygous polymorphisms, which are considered detrimental variants, were detected. Neither deletion nor duplication was found with MLPA in patients with heterozygous variants. The four SLC7A14 mutations detected herein were unlikely to be pathogenic in this Japanese cohort. The frequency and pathogenicity of SLC7A14 mutations may vary depending on ethnicity, and these mutations may be rare in Japanese patients.

  18. Screening of ARHSP-TCC patients expands the spectrum of SPG11 mutations and includes a large scale gene deletion.

    Science.gov (United States)

    Denora, Paola S; Schlesinger, David; Casali, Carlo; Kok, Fernando; Tessa, Alessandra; Boukhris, Amir; Azzedine, Hamid; Dotti, Maria Teresa; Bruno, Claudio; Truchetto, Jeremy; Biancheri, Roberta; Fedirko, Estelle; Di Rocco, Maja; Bueno, Clarissa; Malandrini, Alessandro; Battini, Roberta; Sickl, Elisabeth; de Leva, Maria Fulvia; Boespflug-Tanguy, Odile; Silvestri, Gabriella; Simonati, Alessandro; Said, Edith; Ferbert, Andreas; Criscuolo, Chiara; Heinimann, Karl; Modoni, Anna; Weber, Peter; Palmeri, Silvia; Plasilova, Martina; Pauri, Flavia; Cassandrini, Denise; Battisti, Carla; Pini, Antonella; Tosetti, Michela; Hauser, Erwin; Masciullo, Marcella; Di Fabio, Roberto; Piccolo, Francesca; Denis, Elodie; Cioni, Giovanni; Massa, Roberto; Della Giustina, Elvio; Calabrese, Olga; Melone, Marina A B; De Michele, Giuseppe; Federico, Antonio; Bertini, Enrico; Durr, Alexandra; Brockmann, Knut; van der Knaap, Marjo S; Zatz, Mayana; Filla, Alessandro; Brice, Alexis; Stevanin, Giovanni; Santorelli, Filippo M

    2009-03-01

    Autosomal recessive spastic paraplegia with thinning of corpus callosum (ARHSP-TCC) is a complex form of HSP initially described in Japan but subsequently reported to have a worldwide distribution with a particular high frequency in multiple families from the Mediterranean basin. We recently showed that ARHSP-TCC is commonly associated with mutations in SPG11/KIAA1840 on chromosome 15q. We have now screened a collection of new patients mainly originating from Italy and Brazil, in order to further ascertain the spectrum of mutations in SPG11, enlarge the ethnic origin of SPG11 patients, determine the relative frequency at the level of single Countries (i.e., Italy), and establish whether there is one or more common mutation. In 25 index cases we identified 32 mutations; 22 are novel, including 9 nonsense, 3 small deletions, 4 insertions, 1 in/del, 1 small duplication, 1 missense, 2 splice-site, and for the first time a large genomic rearrangement. This brings the total number of SPG11 mutated patients in the SPATAX collection to 111 cases in 44 families and in 17 isolated cases, from 16 Countries, all assessed using homogeneous clinical criteria. While expanding the spectrum of mutations in SPG11, this larger series also corroborated the notion that even within apparently homogeneous population a molecular diagnosis cannot be achieved without full gene sequencing. 2008 Wiley-Liss, Inc.

  19. A focused and efficient genetic screening strategy in the mouse: identification of mutations that disrupt cortical development.

    Directory of Open Access Journals (Sweden)

    Konstantinos Zarbalis

    2004-08-01

    Full Text Available Although the mechanisms that regulate development of the cerebral cortex have begun to emerge, in large part through the analysis of mutant mice (Boncinelli et al. 2000; Molnar and Hannan 2000; Walsh and Goffinet 2000, many questions remain unanswered. To provide resources for further dissecting cortical development, we have carried out a focused screen for recessive mutations that disrupt cortical development. One aim of the screen was to identify mutants that disrupt the tangential migration of interneurons into the cortex. At the same time, we also screened for mutations that altered the growth or morphology of the cerebral cortex. We report here the identification of thirteen mutants with defects in aspects of cortical development ranging from the establishment of epithelial polarity to the invasion of thalamocortical axons. Among the collection are three novel alleles of genes for which mutant alleles had already been used to explore forebrain development, and four mutants with defects in interneuron migration. The mutants that we describe here will aid in deciphering the molecules and mechanisms that regulate cortical development. Our results also highlight the utility of focused screens in the mouse, in addition to the large-scale and broadly targeted screens that are being carried out at mutagenesis centers.

  20. Screening the 3{prime} region of the polycystic kidney disease 1 (PKD1) gene reveals six novel mutations

    Energy Technology Data Exchange (ETDEWEB)

    Peral, B.; San Millan, J.L.; Ong, A.C.M. [John Radcliffe Hospital, Oxford (United Kingdom)] [and others

    1996-01-01

    Recently, the gene for the most common form of autosomal dominant polycystic kidney disease (ADPKD), PKD1 (polycystic kidney disease 1), has been fully characterized and shown to encode an integral membrane protein, polycystin, involved in cell-cell and/or cell-matrix interactions. Study of the PKD1 gene has been complicated because most of the gene lies in a genomic region reiterated several times elsewhere on the same chromosome, and consequently only seven mutations have been described so far. Here we report a systematic screen covering {approximately}80% of the {approximately}-2.75 kb of translated transcript that is encoded by single-copy DNA. We have identified and characterized six novel mutations that, together with the previously described changes, amount to a detection rate of 10%-15% in the population studied. The newly described mutations are two deletions, an insertion of a T-nucleotide causing a frameshift, two single-base-pair substitutions resulting in premature stop codons, and a G{yields}C transversion that may be a missense mutation. These results have important implications for genetic diagnosis of PKD1 because they indicate that the majority of mutations lie within the duplicated area, which is difficult to study. The regions of polycystin removed in each mutation so far described are assessed for their functional significance; an area disrupted by two new small in-frame changes is highlighted. PKD1 mutations are contrasted with those in the PKD1/TSC2 contiguous-gene syndrome, and the likely mutational mechanism in PKD1 is considered. 36 refs., 6 figs., 2 tabs.

  1. Combined mutation and rearrangement screening by quantitative PCR high-resolution melting: is it relevant for hereditary recurrent Fever genes?

    Directory of Open Access Journals (Sweden)

    Nathalie Pallares-Ruiz

    2010-11-01

    Full Text Available The recent identification of genes implicated in hereditary recurrent fevers has allowed their specific diagnosis. So far however, only punctual mutations have been identified and a significant number of patients remain with no genetic confirmation of their disease after routine molecular approaches such as sequencing. The possible involvement of sequence rearrangements in these patients has only been examined in familial Mediterranean fever and was found to be unlikely. To assess the existence of larger genetic alterations in 3 other concerned genes, MVK (Mevalonate kinase, NLRP3 (Nod like receptor family, pyrin domain containing 3 and TNFRSF1A (TNF receptor superfamily 1A, we adapted the qPCR-HRM method to study possible intragenic deletions and duplications. This single-tube approach, combining both qualitative (mutations and quantitative (rearrangement screening, has proven effective in Lynch syndrome diagnosis. Using this approach, we studied 113 unselected (prospective group and 88 selected (retrospective group patients and identified no intragenic rearrangements in the 3 genes. Only qualitative alterations were found with a sensitivity similar to that obtained using classical molecular techniques for screening punctual mutations. Our results support that deleterious copy number alterations in MVK, NLRP3 and TNFRSF1A are rare or absent from the mutational spectrum of hereditary recurrent fevers, and demonstrate that a routine combined method such as qPCR-HRM provides no further help in genetic diagnosis. However, quantitative approaches such as qPCR or SQF-PCR did prove to be quick and effective and could still be useful after non contributory punctual mutation screening in the presence of clinically evocative signs.

  2. Large cohort screening of G6PD deficiency and the mutational spectrum in the Dongguan District in Southern China.

    Science.gov (United States)

    Peng, Qi; Li, Siping; Ma, Keze; Li, Wenrui; Ma, Qiang; He, Xiaoguang; He, Yuejing; He, Ting; Lu, Xiaomei

    2015-01-01

    Glucose-6-phosphate dehydrogenase (G6PD) deficiency is a common enzymatic disorder of the erythrocytes that affects 400 million people worldwide. We developed a PCR-reverse dot blot (RDB) assay to screen twenty genotypes of seventeen Chinese G6PD mutations and investigate the spectrum of G6PD deficiency mutations in Dongguan District, Guangdong Province, in southern China. The PCR-RDB assay consists of multiplex PCR amplification of seven fragments in the G6PD target sequence of wild-type and mutant genomic DNA samples followed by hybridization to a test strip containing allele-specific oligonucleotide probes. A total of 16,464 individuals were analyzed by a combination of phenotypic screening and genotypic detection using the PCR-RDB assay and DNA sequence analysis. The PCR-RDB assay had a detection rate of 98.1%, which was validated by direct sequencing in a blind study with 100% concordance. The G6PD deficiency incidence rate in Dongguan District is 4.08%. Thirty-two genotypes from 469 individuals were found. The two most common variants were c.1376G>T and c.1388G>A, followed by c.95A>G, c.871G>A, c.392G>T, and c.1024 C>T. In addition, two rare mutations (c.703C>A and c.406C>T) were detected by DNA sequencing analysis. In our study, 65 cases harbored the C1311T/IVS polymorphism and 67 cases were homozygote. The PCR-RDB assay we established is a reliable and effective method for screening G6PD mutations in the Chinese population. Data on the spectrum of mutations in the Dongguan District is beneficial to the clinical diagnosis and prevention of G6PD deficiency.

  3. Frequency of Fanconi anemia in Brazil and efficacy of screening for the FANCA 3788-3790del mutation

    Directory of Open Access Journals (Sweden)

    N. Magdalena

    2005-05-01

    Full Text Available Fanconi anemia (FA is an autosomal recessive genetic disease characterized by progressive bone marrow failure, susceptibility to cancer and multiple congenital anomalies. There is important clinical variability among patients and the knowledge of factors which might predict outcome would greatly help the decision making regarding the choices of treatment and the appropriate time to start it. Future studies of the possible correlation between specific mutations with specific clinical presentations will provide the answer to one of these factors. At our Center we standardized a rapid and precise screening test using a mismatch PCR assay for a specific mutation (3788-3790del in exon 38 of gene FANCA in Brazilian FA patients. We present the results obtained after screening 80 non-consanguineous FA patients referred from all regions of Brazil with a clinical diagnosis of FA supported by cellular hypersensitivity to diepoxybutane. We were able to detect the 3788-3790del allele in 24 of the 80 (30% FA patients studied. Thirteen of the 80 (16.25% were homozygotes and 11 of the 80 (13.75% were compound heterozygotes, thus confirming the high frequency of the FANCA 3788-3790del mutation in Brazilian FA patients. The identification of patients with specific mutations in the FA genes may lead to a better clinical description of this condition, also providing data for genotype-phenotype correlations, to a better understanding of the interaction of this specific mutation with other mutations in compound heterozygote patients, and ultimately to the right choices of treatment for each patient with improvement of the prognosis on future studies.

  4. Cost-effectiveness of population screening for BRCA mutations in Ashkenazi jewish women compared with family history-based testing.

    Science.gov (United States)

    Manchanda, Ranjit; Legood, Rosa; Burnell, Matthew; McGuire, Alistair; Raikou, Maria; Loggenberg, Kelly; Wardle, Jane; Sanderson, Saskia; Gessler, Sue; Side, Lucy; Balogun, Nyala; Desai, Rakshit; Kumar, Ajith; Dorkins, Huw; Wallis, Yvonne; Chapman, Cyril; Taylor, Rohan; Jacobs, Chris; Tomlinson, Ian; Beller, Uziel; Menon, Usha; Jacobs, Ian

    2015-01-01

    Population-based testing for BRCA1/2 mutations detects the high proportion of carriers not identified by cancer family history (FH)-based testing. We compared the cost-effectiveness of population-based BRCA testing with the standard FH-based approach in Ashkenazi Jewish (AJ) women. A decision-analytic model was developed to compare lifetime costs and effects amongst AJ women in the UK of BRCA founder-mutation testing amongst: 1) all women in the population age 30 years or older and 2) just those with a strong FH (≥10% mutation risk). The model assumes that BRCA carriers are offered risk-reducing salpingo-oophorectomy and annual MRI/mammography screening or risk-reducing mastectomy. Model probabilities utilize the Genetic Cancer Prediction through Population Screening trial/published literature to estimate total costs, effects in terms of quality-adjusted life-years (QALYs), cancer incidence, incremental cost-effectiveness ratio (ICER), and population impact. Costs are reported at 2010 prices. Costs/outcomes were discounted at 3.5%. We used deterministic/probabilistic sensitivity analysis (PSA) to evaluate model uncertainty. Compared with FH-based testing, population-screening saved 0.090 more life-years and 0.101 more QALYs resulting in 33 days' gain in life expectancy. Population screening was found to be cost saving with a baseline-discounted ICER of -£2079/QALY. Population-based screening lowered ovarian and breast cancer incidence by 0.34% and 0.62%. Assuming 71% testing uptake, this leads to 276 fewer ovarian and 508 fewer breast cancer cases. Overall, reduction in treatment costs led to a discounted cost savings of £3.7 million. Deterministic sensitivity analysis and 94% of simulations on PSA (threshold £20000) indicated that population screening is cost-effective, compared with current NHS policy. Population-based screening for BRCA mutations is highly cost-effective compared with an FH-based approach in AJ women age 30 years and older. © The Author

  5. Cost-effectiveness of Population Screening for BRCA Mutations in Ashkenazi Jewish Women Compared With Family History–Based Testing

    Science.gov (United States)

    Manchanda, Ranjit; Legood, Rosa; Burnell, Matthew; McGuire, Alistair; Raikou, Maria; Loggenberg, Kelly; Wardle, Jane; Sanderson, Saskia; Gessler, Sue; Side, Lucy; Balogun, Nyala; Desai, Rakshit; Kumar, Ajith; Dorkins, Huw; Wallis, Yvonne; Chapman, Cyril; Taylor, Rohan; Jacobs, Chris; Tomlinson, Ian; Beller, Uziel; Menon, Usha

    2015-01-01

    Background: Population-based testing for BRCA1/2 mutations detects the high proportion of carriers not identified by cancer family history (FH)–based testing. We compared the cost-effectiveness of population-based BRCA testing with the standard FH-based approach in Ashkenazi Jewish (AJ) women. Methods: A decision-analytic model was developed to compare lifetime costs and effects amongst AJ women in the UK of BRCA founder-mutation testing amongst: 1) all women in the population age 30 years or older and 2) just those with a strong FH (≥10% mutation risk). The model assumes that BRCA carriers are offered risk-reducing salpingo-oophorectomy and annual MRI/mammography screening or risk-reducing mastectomy. Model probabilities utilize the Genetic Cancer Prediction through Population Screening trial/published literature to estimate total costs, effects in terms of quality-adjusted life-years (QALYs), cancer incidence, incremental cost-effectiveness ratio (ICER), and population impact. Costs are reported at 2010 prices. Costs/outcomes were discounted at 3.5%. We used deterministic/probabilistic sensitivity analysis (PSA) to evaluate model uncertainty. Results: Compared with FH-based testing, population-screening saved 0.090 more life-years and 0.101 more QALYs resulting in 33 days’ gain in life expectancy. Population screening was found to be cost saving with a baseline-discounted ICER of -£2079/QALY. Population-based screening lowered ovarian and breast cancer incidence by 0.34% and 0.62%. Assuming 71% testing uptake, this leads to 276 fewer ovarian and 508 fewer breast cancer cases. Overall, reduction in treatment costs led to a discounted cost savings of £3.7 million. Deterministic sensitivity analysis and 94% of simulations on PSA (threshold £20000) indicated that population screening is cost-effective, compared with current NHS policy. Conclusion: Population-based screening for BRCA mutations is highly cost-effective compared with an FH-based approach in AJ

  6. Mutation screening in a cohort of patients with lissencephaly and subcortical band heterotopia.

    Science.gov (United States)

    Torres, F R; Montenegro, M A; Marques-De-Faria, A P; Guerreiro, M M; Cendes, F; Lopes-Cendes, I

    2004-03-09

    The authors describe clinical, neuroimaging and molecular findings in a group of 15 patients with classic lissencephaly (LIS) and subcortical band heterotopia (SBH). A 1385A-->C mutation was found in the LIS1 gene in one patient with LIS more severe than expected for individuals with missense mutations in LIS1. The authors believe that the site of the mutation, present in a functionally critical region of the protein, could explain the unusual severe phenotype found in this patient.

  7. Mutation screening in a cohort of patients with lissencephaly and subcortical band heterotopia

    OpenAIRE

    Torres, FR; Montenegro, MA; Marques-de-Faria, AP; Guerreiro, MM; Cendes, F.; Lopes-Cendes, I

    2004-01-01

    The authors describe clinical, neuroimaging and molecular findings in a group of 15 patients with classic lissencephaly ( LIS) and subcortical band heterotopia (SBH). A 1385A-->C mutation was found in the LIS1 gene in one patient with LIS more severe than expected for individuals with missense mutations in LIS1. The authors believe that the site of the mutation, present in a functionally critical region of the protein, could explain the unusual severe phenotype found in this patient.

  8. Screening mutations in myosin binding protein C3 gene in a cohort of patients with Hypertrophic Cardiomyopathy

    Directory of Open Access Journals (Sweden)

    Maneiro Emilia

    2010-04-01

    Full Text Available Abstract Background MyBPC3 mutations are amongst the most frequent causes of hypertrophic cardiomyopathy, however, its prevalence varies between populations. They have been associated with mild and late onset disease expression. Our objectives were to establish the prevalence of MyBPC3 mutations and determine their associated clinical characteristics in our patients. Methods Screening by Single Strand Conformation Polymorphisms (SSCP and sequencing of the fragments with abnormal motility of the MyBPC3 gene in 130 unrelated consecutive HCM index cases. Genotype-Phenotype correlation studies were done in positive families. Results 16 mutations were found in 20 index cases (15%: 5 novel [D75N, V471E, Q327fs, IVS6+5G>A (homozygous, and IVS11-9G>A] and 11 previously described [A216T, R495W, R502Q (2 families, E542Q (3 families, T957S, R1022P (2 families, E1179K, K504del, K600fs, P955fs and IVS29+5G>A]. Maximum wall thickness and age at time of diagnosis were similar to patients with MYH7 mutations [25(7 vs. 27(8, p = 0.16], [46(16 vs. 44(19, p = 0.9]. Conclusions Mutations in MyBPC3 are present in 15% of our hypertrophic cardiomyopathy families. Severe hypertrophy and early expression are compatible with the presence of MyBPC3 mutations. The genetic diagnosis not only allows avoiding clinical follow up of non carriers but it opens new possibilities that includes: to take preventive clinical decisions in mutation carriers than have not developed the disease yet, the establishment of genotype-phenotype relationship, and to establish a genetic diagnosis routine in patients with familial HCM.

  9. Ductal Carcinoma in situ Detected during Prospective MR Imaging Screening of a Woman with a BRCA2 Mutation: The First Case Report in Japan.

    Science.gov (United States)

    Tozaki, Mitsuhiro; Nakamura, Seigo; Kitagawa, Dai; Iwase, Takuji; Horii, Rie; Akiyama, Futoshi; Arai, Masami

    2017-07-10

    The present case is the first report in Japan in which a breast cancer was discovered as a result of prospective magnetic resonance imaging (MRI) screening study for BRCA1/2 mutation carriers who were free of breast or ovarian cancer. This case is significant and it verifies the importance of MRI screening in breast or ovarian cancer-free BRCA1/2 mutation carriers who do not exhibit positive mammographic or ultrasonographic findings.

  10. Phenotype and frequency of STUB1 mutations: next-generation screenings in Caucasian ataxia and spastic paraplegia cohorts.

    Science.gov (United States)

    Synofzik, Matthis; Schüle, Rebecca; Schulze, Martin; Gburek-Augustat, Janina; Schweizer, Roland; Schirmacher, Anja; Krägeloh-Mann, Ingeborg; Gonzalez, Michael; Young, Peter; Züchner, Stephan; Schöls, Ludger; Bauer, Peter

    2014-04-17

    Mutations in the gene STUB1, encoding the protein CHIP (C-terminus of HSC70-interacting protein), have recently been suggested as a cause of recessive ataxia based on the findings in few Chinese families. Here we aimed to investigate the phenotypic and genotypic spectrum of STUB1 mutations, and to assess their frequency in different Caucasian disease cohorts. 300 subjects with degenerative ataxia (n = 167) or spastic paraplegia (n = 133) were screened for STUB1 variants by whole-exome-sequencing (n = 204) or shotgun-fragment-library-sequencing (n = 96). To control for the specificity of STUB1 variants, we screened an additional 1707 exomes from 891 index families with other neurological diseases. We identified 3 ataxia patients (3/167 = 1.8%) with 4 novel missense mutations in STUB1, including 3 mutations in its tetratricopeptide-repeat domain. All patients showed evidence of pyramidal tract damage. Cognitive impairment was present only in one and hypogonadism in none of them. Ataxia did not start before age 48 years in one subject. No recessive STUB1 variants were identified in families with other neurological diseases, demonstrating that STUB1 variants are not simply rare polymorphisms ubiquitous in neurodegenerative disease. STUB1-disease occurs also in Caucasian ataxia populations (1.8%). Our results expand the genotypic spectrum of STUB1-disease, showing that pathogenic mutations affect also the tetratricopeptide-repeat domain, thus providing clinical evidence for the functional importance of this domain. Moreover, they further delineate the phenotypic core features of STUB1-ataxia. Pyramidal tract damage is a common accompanying feature and can include lower limb spasticity, thus adding STUB1-ataxia to the differential diagnosis of "spastic ataxias". However, STUB1 is rare in subjects with predominant spastic paraplegia (0/133). In contrast to previous reports, STUB1-ataxia can start even above age 40 years, and neither hypogonadism nor prominent cognitive

  11. Proteomics screen to reveal molecular changes mediated by C722G missense mutation in CHRM2 gene.

    Science.gov (United States)

    Hou, Dongyan; Chen, Ying; Liu, Jiamei; Xu, Lin; Zhang, Zhiyong; Zhang, Juan; Wang, Hua; Wang, Xin; Chen, Jin; Zhao, Rongrui; Hu, Aihua; Hakonarson, Hakon; Zhang, Lin; Shen, Yan

    2013-08-26

    Previously, we reported a missense mutation (C722G) in the M2-muscarinic acetylcholine receptor (CHRM2) gene associated with familial dilated cardiomyopathy. However, the exact molecular mechanisms by the related protein changes of CHRM2-C722G mutation induced are still unclear. CHRM2 and CHRM2-C722G lentiviral vector was infected to CHO cells. Proteomic analysis by label-free shotgun strategy and the STRING 9.0 software were performed. A total of 102 proteins with at least 2-fold change in the CHRM2-C722G group were identified, 42 proteins were up-regulated, whereas 57 were down-regulated. These altered proteins belong to three broad functional categories: (i) metabolic (e.g. Cytosolic acyl coenzyme A thioester hydrolase, Malate dehydrogenase); (ii) cytoskeletal (e.g. Actin-related protein, Myosin light polypeptide 6 and Alpha-actinin-1) and (iii) stress response (e.g. heat shock protein 70, Ras-related protein Rab-10). Interestingly, the marked differences in the expression of selected eight proteins (change >4.0-fold), were connected with many proteins related to apoptosis and immune/inflammatory response such as: FOS, BAX, MYC, TP53 and IL6. This novel study demonstrated for the first time a full-scale screening of the proteomics research by CHRM2-C722G mutation and profiled 102 changed proteins, of which, eight might be critical in cardiac dysfunction for future mapping. It was a full-scale screening of the proteomics research by CHRM2-C722G mutation. These proteins might serve as valuable biomarkers that could predict the presence of a precursor field. These proteins might serve to further explore the pathophysiological mechanisms in familial DCM patients with C176W mutation. Copyright © 2013 Elsevier B.V. All rights reserved.

  12. A Panel of High Resolution Melting (HRM Technology-Based Assays with Direct Sequencing Possibility for Effective Mutation Screening of EGFR and K-ras Genes

    Directory of Open Access Journals (Sweden)

    D. A. M. Heideman

    2009-01-01

    Full Text Available Background: Increasing data from clinical trials support EGFR and K-ras mutation status as predictive markers of tumour response to EGFR-targeted therapies. Consequently, rapid and reliable mutation screening assays are demanded to guide rational use of EGFR-targeted therapies.

  13. Targeted prostate cancer screening in BRCA1 and BRCA2 mutation carriers

    DEFF Research Database (Denmark)

    Bancroft, Elizabeth K; Page, Elizabeth C; Castro, Elena

    2014-01-01

    AND PARTICIPANTS: We recruited men aged 40-69 yr with germline BRCA1/2 mutations and a control group of men who have tested negative for a pathogenic BRCA1 or BRCA2 mutation known to be present in their families. All men underwent prostate-specific antigen (PSA) testing at enrollment, and those men with PSA >3 ng...

  14. Genetic screening of the FLCN gene identify six novel variants and a Danish founder mutation

    DEFF Research Database (Denmark)

    Rossing, Maria; Albrechtsen, Anders; Skytte, Anne-Bine

    2017-01-01

    Pathogenic germline mutations in the folliculin (FLCN) tumor suppressor gene predispose to Birt-Hogg-Dubé (BHD) syndrome, a rare disease characterized by the development of cutaneous hamartomas (fibrofolliculomas), multiple lung cysts, spontaneous pneumothoraces and renal cell cancer. In this study...... families exhibited the c.1062+2T>G mutation. Combined single nucleotide polymorphism array-haplotype analysis showed that these families share a 3-Mb genomic fragment containing the FLCN gene, revealing that the c.1062+2T>G mutation is a Danish founder mutation. On the basis of in silico prediction...... and functional splicing assays, we classify the 16 identified variants in the FLCN gene as follows: nine as pathogenic, one as likely pathogenic, three as likely benign and three as polymorphisms. In conclusion, the study describes the FLCN mutation spectrum in Danish BHD patients, and contributes to a better...

  15. [Application of gene capture technology on mutation screening of RB1 gene in retinoblastoma patients].

    Science.gov (United States)

    Meng, Q Y; Huang, L Z; Wang, B; Li, X X; Liang, J H

    2017-06-11

    Objectives: To analyze RB1 gene mutation in retinoblastoma (RB) patients using gene capture technology. Methods: Experimental research. The clinical data of 17 RB patients were collected at Department of Ophthalmology, Peking University People's Hospital from June 2010 to Jun 2014. Peripheral blood samples of seventeen RB patients and their parents were collected and genomic DNA were extracted. DNA library from RB patients was mixed with designed gene capture probe of RB1 exons and its flanking sequences. The data were analyzed using bioinformatics software. To avoid the false positive, the abnormal sites were verified using the Sanger sequencing method. Results: Totally, there were 17 RB patients, including 12 males and 5 females, from 0.5 to 23 years old, average ages were (3.2±5.2) years old. Both eyes were involved in 6 patients. The other 11 cases were only one eye was attacked. Four RB patients were found to have germline mutations, among whom 2 had bilateral tumors and 2 had unilateral tumors. 2 novel missense mutations were identified, including 15(th) exon c.1408A>T (p. Ile470Phe) and c.1960G>C (p. Val654Leu) at 19(th) exon. No RB1 mutation was identified in any of their parents. We also identified 2 mutations reported previously. One is c.1030C>T termination mutation at 10(th) exon in a bilateral RB patients and his father, who was diagnosed with unilateral RB. The other is c.371-372delTA frame shift mutation at 3(rd) exon. No mutation was found in their parents. Conclusions: Two novel germline RB1 mutations were found using gene capture technology, which enriched RB1 mutations library.(Chin J Ophthalmol, 2017, 53: 455-459).

  16. Screening of WT1 mutations in exon 8 and 9 in children with steroid resistant nephrotic syndrome from a single centre and establishment of a rapid screening assay using high-resolution melting analysis in a clinical setting.

    Science.gov (United States)

    Siji, Annes; Pardeshi, Varsha Chhotusing; Ravindran, Shilpa; Vasudevan, Ambily; Vasudevan, Anil

    2017-01-10

    Mutations in Wilm's tumor 1 (WT1) gene is one of the commonly reported genetic mutations in children with steroid resistant nephrotic syndrome (SRNS). We report the results of direct sequencing of exons 8 and 9 of WT1 gene in 100 children with SRNS from a single centre. We standardized and validated High Resolution Melt (HRM) as a rapid and cost effective screening step to identify individuals with normal sequence and distinguish it from those with a potential mutation. Since only mutation positive samples identified by HRM will be further processed for sequencing it will help in reducing the sequencing burden and speed up the screening process. One hundred SRNS children were screened for WT1 mutations in Exon 8 and 9 using Sanger sequencing. HRM assay was standardized and validated by performing analysis for exon 8 and 9 on 3 healthy control and 5 abnormal variants created by site directed mutagenesis and verified by sequencing. To further test the clinical applicability of the assay, we screened additional 91 samples for HRM testing and performed a blinded assessment. WT1 mutations were not observed in the cohort of children with SRNS. The results of HRM analysis were concordant with the sequencing results. The WT1 gene mutations were not observed in the SRNS cohort indicating it has a low prevalence. We propose applying this simple, rapid and cost effective assay using HRM technique as the first step for screening the WT1 gene hot spot region in a clinical setting.

  17. Cystic fibrosis screening in neonates--measurement of immunoreactive trypsin and direct genotype analysis for delta F508 mutation.

    Science.gov (United States)

    Larsen, J; Campbell, S; Faragher, E B; Götz, M; Eichler, I; Waldherr, S; Dobianer, K; Spona, J

    1994-08-01

    This study investigated the clinical usefulness of screening for cystic fibrosis (CF) in 19,992 newborns, over 39 months, in an Austrian population. Immunoreactive serum trypsin (IRT) determination was followed by sweat chloride analysis (sweat test) to establish diagnosis. In a retrospective analysis covering 6 months of the study period, individuals who were considered to be at risk after IRT estimation (n = 22) were analysed for delta F508 mutation, using a new method of DNA extraction from the initial dried blood specimens. A total of 119 infants (0.6%) had values greater than 750 ng trypsin/ml whole blood. In 88 babies sweat tests were performed, leading to the diagnosis of CF in 11 cases. One patient was not initially identified by screening but was later discovered due to his clinical status. Three infants were noted to carry the delta F508 mutation (1 homozygous, 2 heterozygous). Two of these babies already had CF. The second heterozygote was a carrier. A highly efficient three tier screening strategy is presented in which IRT estimation, determination of delta F508 status and sweat chloride testing could lead to a high sensitivity analysis of this population.

  18. Identification of a Neonate with Thalassemia Intermedia Despite Premarital Screening Program in Mazandaran Province (Co-inheritance of Hb Knossos and IVS II-1 G> A Mutations

    Directory of Open Access Journals (Sweden)

    Hossein Karami

    2017-05-01

    Conclusion: The presented case shows that using CBC and hemoglobin electrophoresis in premarital screening program for detecting β-thalassemia carriers is not a valid approach and individuals who are carriers of silent β-globin gene mutations are missed in this procedure. Hence, in premarital screening program precise molecular investigation especially when the partner is a typical β-thalassemia carrier is recommended.

  19. Which screening strategy should be offered to women with BRCA1 or BRCA2 mutations? A simulation of comparative cost-effectiveness

    NARCIS (Netherlands)

    de Bock, G.H.; Vermeulen, K.M.; Jansen, L.; Oosterwijk, J.C.; Siesling, S.; Dorrius, M.D.; Feenstra, Talitha; Houssami, N.; Greuter, M.J.W.

    2013-01-01

    Background: There is no consensus on the most effective strategy (mammography or magnetic resonance imaging (MRI)) for screening women with BRCA1 or BRCA2 mutations. The effectiveness and cost-effectiveness of the Dutch, UK and US screening strategies, which involve mammography and MRI at different

  20. Screening for TMEM230 mutations in young-onset Parkinson's disease.

    Science.gov (United States)

    Ma, Dongrui; Foo, Jia Nee; Yulin Ng, Ebonne; Zhao, Yi; Liu, Jian-Jun; Tan, Eng King

    2017-10-01

    TMEM230 gene mutations have been reported to be linked with Parkinson's disease (PD) recently. To investigate the prevalence of this gene in southeastern Chinese patients with PD, whole exome sequencing was performed in young-onset and familial PD patients and healthy controls in our Asian population. One heterozygous missense p.Phe121Ser mutation was detected in a healthy 76-year-old control subject and no other TMEM230 mutations were found in PD patients and controls. These data suggest that TMEM230 mutation might be a rare cause of Chinese familial and sporadic PD patients and a larger sample size will be needed to evaluate the association of TMEM230 polymorphic variants with PD. Copyright © 2017 Elsevier Inc. All rights reserved.

  1. Screening of Two Neighboring CFTR Mutations in Iranian Infertile Men with Non-Obstructive Azoospermia

    OpenAIRE

    Somayeh Heidari; Zohreh Hojati; Majid Motovali-Bashi

    2016-01-01

    The genetic association between cystic fibrosis transmembrane conductance regulator (CFTR) gene mutations and male infertility due to congenital bilateral absence of vas deferens (CBAVD) is well established. Mutant CFTR, however may also be involved in the etiology of male infertility in non-CBAVD cases. The present study was conducted to estimate the frequency of ?I507 and ?F508 CFTR gene mutations in Iranian infertile males. We undertook the first study of association between these CFTR mut...

  2. Targeted prostate cancer screening in men with mutations in BRCA1 and BRCA2 detects aggressive prostate cancer: preliminary analysis of the results of the IMPACT study

    DEFF Research Database (Denmark)

    Mitra, Anita V; Bancroft, Elizabeth K; Barbachano, Yolanda

    2011-01-01

    in BRCA1/2 mutation carriers and controls), was established. This is the first multicentre screening study targeted at men with a known genetic predisposition to prostate cancer. A preliminary analysis of the data is reported. MATERIALS AND METHODS: Men aged 40-69 years from families with BRCA1 or BRCA2...... mutations were offered annual prostate specific antigen (PSA) testing, and those with PSA >3 ng/mL, were offered a prostate biopsy. Controls were men age-matched (± 5 years) who were negative for the familial mutation. RESULTS: In total, 300 men were recruited (205 mutation carriers; 89 BRCA1, 116 BRCA2...

  3. Fabry disease: incidence of the common later-onset α-galactosidase A IVS4+919G→A mutation in Taiwanese newborns--superiority of DNA-based to enzyme-based newborn screening for common mutations.

    Science.gov (United States)

    Chien, Yin-Hsiu; Lee, Ni-Chung; Chiang, Shu-Chuan; Desnick, Robert J; Hwu, Wuh-Liang

    2012-07-18

    Fabry disease is a panethnic, X-linked, inborn error of glycosphingolipid metabolism resulting from mutations in the α-galactosidase A gene (GLA) that lead to the deficient activity of the lysosomal enzyme, α-galactosidase A (α-Gal A). Affected males with no α-Gal A activity have the early-onset classic phenotype, whereas those with residual activity present with the later-onset subtype. Recently, we reported that newborn enzyme-based screening using dried blood spots (DBS) in Taiwan revealed a high incidence of newborn males who had the GLA c.936+919G→A (IVS4+919G→A) mutation. This lesion causes cryptic splicing, markedly reducing the amount of wild-type GLA mRNA, and has been found in males with the later-onset Fabry phenotype, manifesting as cardiac, renal and/or cerebrovascular disease. To more accurately determine the incidence of the IVS4+919G→A mutation, 20,063 consecutive newborns were screened by a deoxyribonucleic acid (DNA)-based assay. Of the 10,499 males, 12 (1/875) and 24 of the 9,564 females (1/399) had the mutation. On the basis of these frequencies, the previous newborn enzyme-based DBS screening (cutoff: mutation-positive males and females, respectively. The mean DBS α-Gal A activities in the mutation-positive males and females were 23% (1.54 U) and 55% (3.63 U) of normal mean male/female values, respectively. These studies confirm the high incidence of the IVS4+919G→A mutation in the Taiwanese population and indicate that its detectability by enzyme-based DBS screening is unreliable, especially in females. These studies emphasize the superiority of DNA-based newborn screening for common mutations, particularly for X-linked diseases.

  4. Screening of mutations affecting protein stability and dynamics of FGFR1—A simulation analysis

    Directory of Open Access Journals (Sweden)

    C. George Priya Doss

    2012-12-01

    Full Text Available Single amino acid substitutions in Fibroblast Growth Factor Receptor 1 (FGFR1 destabilize protein and have been implicated in several genetic disorders like various forms of cancer, Kallamann syndrome, Pfeiffer syndrome, Jackson Weiss syndrome, etc. In order to gain functional insight into mutation caused by amino acid substitution to protein function and expression, special emphasis was laid on molecular dynamics simulation techniques in combination with in silico tools such as SIFT, PolyPhen 2.0, I-Mutant 3.0 and SNAP. It has been estimated that 68% nsSNPs were predicted to be deleterious by I-Mutant, slightly higher than SIFT (37%, PolyPhen 2.0 (61% and SNAP (58%. From the observed results, P722S mutation was found to be most deleterious by comparing results of all in silico tools. By molecular dynamics approach, we have shown that P722S mutation leads to increase in flexibility, and deviated more from the native structure which was supported by the decrease in the number of hydrogen bonds. In addition, biophysical analysis revealed a clear insight of stability loss due to P722S mutation in FGFR1 protein. Majority of mutations predicted by these in silico tools were in good concordance with the experimental results.

  5. A Short History and Description of Drosophila melanogaster Classical Genetics: Chromosome Aberrations, Forward Genetic Screens, and the Nature of Mutations.

    Science.gov (United States)

    Kaufman, Thomas C

    2017-06-01

    The purpose of this chapter in FlyBook is to acquaint the reader with the Drosophila genome and the ways in which it can be altered by mutation. Much of what follows will be familiar to the experienced Fly Pusher but hopefully will be useful to those just entering the field and are thus unfamiliar with the genome, the history of how it has been and can be altered, and the consequences of those alterations. I will begin with the structure, content, and organization of the genome, followed by the kinds of structural alterations (karyotypic aberrations), how they affect the behavior of chromosomes in meiotic cell division, and how that behavior can be used. Finally, screens for mutations as they have been performed will be discussed. There are several excellent sources of detailed information on Drosophila husbandry and screening that are recommended for those interested in further expanding their familiarity with Drosophila as a research tool and model organism. These are a book by Ralph Greenspan and a review article by John Roote and Andreas Prokop, which should be required reading for any new student entering a fly lab for the first time. Copyright © 2017 by the Genetics Society of America.

  6. Phenotype and frequency of STUB1 mutations: next-generation screenings in Caucasian ataxia and spastic paraplegia cohorts

    Science.gov (United States)

    2014-01-01

    Background Mutations in the gene STUB1, encoding the protein CHIP (C-terminus of HSC70-interacting protein), have recently been suggested as a cause of recessive ataxia based on the findings in few Chinese families. Here we aimed to investigate the phenotypic and genotypic spectrum of STUB1 mutations, and to assess their frequency in different Caucasian disease cohorts. Methods 300 subjects with degenerative ataxia (n = 167) or spastic paraplegia (n = 133) were screened for STUB1 variants by whole-exome-sequencing (n = 204) or shotgun-fragment-library-sequencing (n = 96). To control for the specificity of STUB1 variants, we screened an additional 1707 exomes from 891 index families with other neurological diseases. Results We identified 3 ataxia patients (3/167 = 1.8%) with 4 novel missense mutations in STUB1, including 3 mutations in its tetratricopeptide-repeat domain. All patients showed evidence of pyramidal tract damage. Cognitive impairment was present only in one and hypogonadism in none of them. Ataxia did not start before age 48 years in one subject. No recessive STUB1 variants were identified in families with other neurological diseases, demonstrating that STUB1 variants are not simply rare polymorphisms ubiquitous in neurodegenerative disease. Conclusions STUB1-disease occurs also in Caucasian ataxia populations (1.8%). Our results expand the genotypic spectrum of STUB1-disease, showing that pathogenic mutations affect also the tetratricopeptide-repeat domain, thus providing clinical evidence for the functional importance of this domain. Moreover, they further delineate the phenotypic core features of STUB1-ataxia. Pyramidal tract damage is a common accompanying feature and can include lower limb spasticity, thus adding STUB1-ataxia to the differential diagnosis of “spastic ataxias”. However, STUB1 is rare in subjects with predominant spastic paraplegia (0/133). In contrast to previous reports, STUB1-ataxia can start even above age 40

  7. Rapid screening of β-Globin gene mutations by Real-Time PCR in ...

    African Journals Online (AJOL)

    Introduction of the real time PCR has made a revolution in the time taken for the PCR reactions. We present a method for the diagnosis of the common mutations of the B-thalassemia in Egyptian children & families. The procedure depends on the real-time PCR using specific fluorescently labeled hybridization probes.

  8. Expanding sialidosis spectrum by genome-wide screening: NEU1 mutations in adult-onset myoclonus.

    Science.gov (United States)

    Canafoglia, Laura; Robbiano, Angela; Pareyson, Davide; Panzica, Ferruccio; Nanetti, Lorenzo; Giovagnoli, Anna Rita; Venerando, Anna; Gellera, Cinzia; Franceschetti, Silvana; Zara, Federico

    2014-06-03

    To identify the genetic cause of a familial form of late-onset action myoclonus in 2 unrelated patients. Both probands had 2 siblings displaying a similar disorder. Extensive laboratory examinations, including biochemical assessment for urine sialic acid in the 2 probands, were negative. Exome sequencing was performed in the probands using an Illumina platform. Segregation analysis of putative mutations was performed in all family members by standard Sanger sequencing protocols. NEU1 mutations were detected in 3 siblings of each family with prominent cortical myoclonus presenting in the third decade of life and having a mild and slowly progressive course. They did not have macular cherry-red spot and their urinary sialic acid excretion was within normal values. Genetic analysis demonstrated a homozygous mutation in family 1 (c.200G>T, p.S67I) and 2 compound heterozygous mutations in family 2 (c.679G>A, p.G227R; c.913C>T, p.R305C). Our observation indicates that sialidosis should be suspected and the NEU1 gene analyzed in patients with isolated action myoclonus presenting in adulthood in the absence of other typical clinical and laboratory findings. © 2014 American Academy of Neurology.

  9. Mutational screening in genes related with porto-pulmonary hypertension: An analysis of 6 cases.

    Science.gov (United States)

    Pousada, Guillermo; Baloira, Adolfo; Valverde, Diana

    2017-04-07

    Portopulmonary hypertension (PPH) is a rare disease with a low incidence and without a clearly-identified genetic component. The aim of this work was to check genes and genetic modifiers related to pulmonary arterial hypertension in patients with PPH in order to clarify the molecular basis of the pathology. We selected a total of 6 patients with PPH and amplified the exonic regions and intronic flanking regions of the relevant genes and regions of interest of the genetic modifiers. Six patients diagnosed with PPH were analyzed and compared to 55 healthy individuals. Potentially-pathogenic mutations were identified in the analyzed genes of 5 patients. None of these mutations, which are highly conserved throughout evolution, were detected in the control patients nor different databases analyzed (1000 Genomes, ExAC and DECIPHER). After analyzing for genetic modifiers, we found different variations that could favor the onset of the disease. The genetic analysis carried out in this small cohort of patients with PPH revealed a large number of mutations, with the ENG gene showing the greatest mutational frequency. Copyright © 2017 Elsevier España, S.L.U. All rights reserved.

  10. Screening for Fabry Disease in Left Ventricular Hypertrophy: Documentation of a Novel Mutation

    Energy Technology Data Exchange (ETDEWEB)

    Baptista, Ana, E-mail: baptista-ana@hotmail.com; Magalhães, Pedro; Leão, Sílvia; Carvalho, Sofia; Mateus, Pedro; Moreira, Ilídio [Centro Hospitalar de Trás-os-Montes e Alto Douro, Unidade de Vila Real (Portugal)

    2015-08-15

    Fabry disease is a lysosomal storage disease caused by enzyme α-galactosidase A deficiency as a result of mutations in the GLA gene. Cardiac involvement is characterized by progressive left ventricular hypertrophy. To estimate the prevalence of Fabry disease in a population with left ventricular hypertrophy. The patients were assessed for the presence of left ventricular hypertrophy defined as a left ventricular mass index ≥ 96 g/m{sup 2} for women or ≥ 116 g/m{sup 2} for men. Severe aortic stenosis and arterial hypertension with mild left ventricular hypertrophy were exclusion criteria. All patients included were assessed for enzyme α-galactosidase A activity using dry spot testing. Genetic study was performed whenever the enzyme activity was decreased. A total of 47 patients with a mean left ventricular mass index of 141.1 g/m{sup 2} (± 28.5; 99.2 to 228.5 g/m{sup 2}] were included. Most of the patients were females (51.1%). Nine (19.1%) showed decreased α-galactosidase A activity, but only one positive genetic test − [GLA] c.785G>T; p.W262L (exon 5), a mutation not previously described in the literature. This clinical investigation was able to establish the association between the mutation and the clinical presentation. In a population of patients with left ventricular hypertrophy, we documented a Fabry disease prevalence of 2.1%. This novel case was defined in the sequence of a mutation of unknown meaning in the GLA gene with further pathogenicity study. Thus, this study permitted the definition of a novel causal mutation for Fabry disease - [GLA] c.785G>T; p.W262L (exon 5)

  11. In silico screening of mutational effects on enzyme-proteic inhibitor affinity: a docking-based approach.

    Science.gov (United States)

    Dell'Orco, Daniele; De Benedetti, Pier Giuseppe; Fanelli, Francesca

    2007-06-08

    mutants compared to the native state.Finally, the results of this study corroborate and extend a previously developed quantitative model for in silico predictions of absolute protein-protein binding affinities spanning a wide range of values, i.e. from -10 up to -21 kcal/mol. The computational approach is simple and fast and can be used for structure-based design of protein-protein complexes and for in silico screening of mutational effects on protein-protein recognition.

  12. In silico screening of mutational effects on enzyme-proteic inhibitor affinity: a docking-based approach

    Directory of Open Access Journals (Sweden)

    De Benedetti Pier

    2007-06-01

    on the thermodynamics and kinetics of binding of protein mutants compared to the native state. Finally, the results of this study corroborate and extend a previously developed quantitative model for in silico predictions of absolute protein-protein binding affinities spanning a wide range of values, i.e. from -10 up to -21 kcal/mol. The computational approach is simple and fast and can be used for structure-based design of protein-protein complexes and for in silico screening of mutational effects on protein-protein recognition.

  13. Expanding the spectrum of mutations in GH1 and GHRHR: genetic screening in a large cohort of patients with congenital isolated growth hormone deficiency

    DEFF Research Database (Denmark)

    Alatzoglou, Kyriaki S; Turton, James P; Kelberman, Daniel

    2009-01-01

    CONTEXT: It is estimated that 3-30% of cases with isolated GH deficiency (IGHD) have a genetic etiology, with a number of mutations being reported in GH1 and GHRHR. The aim of our study was to genetically characterize a cohort of patients with congenital IGHD and analyze their characteristics....... CONCLUSIONS: IGHD patients with severe growth failure and a positive family history should be screened for genetic mutations; the evolving endocrinopathy observed in some of these patients suggests the need for long-term follow-up........ PATIENTS AND METHODS: A total of 224 patients (190 pedigrees) with IGHD and a eutopic posterior pituitary were screened for mutations in GH1 and GHRHR. To explore the possibility of an association of GH1 abnormalities with multiple pituitary hormone deficiencies, we have screened 62 patients with either...

  14. BRCA1 and BRCA2 Germline Mutations Screening in Algerian Breast/Ovarian Cancer Families

    Directory of Open Access Journals (Sweden)

    Farid Cherbal

    2010-01-01

    Full Text Available Background: Breast cancer is the leading cause of cancer death in women in Algeria. The contribution of BRCA1 and BRCA2 mutations to hereditary breast/ovarian cancer in Algerian population is largely unknown. Here, we describe analysis of BRCA1 and BRCA2 genes in 86 individuals from 70 families from an Algerian cohort with a personal and family history suggestive of genetic predisposition to breast cancer.

  15. CHO/HGPRT mutation assay. IV. Progress with quantitative mutagenesis and mutagen screening

    Energy Technology Data Exchange (ETDEWEB)

    Hsie, Abraham W.; O& #x27; Neill, J. Patrick; San Sebastian, Juan R.; Brimer, Patricia A.

    1979-01-01

    Work is summarized on development and application of their mutation system to study mutagenicity of various physical and chemical agents which bear profound environmental concerns. Eleven alkylating chemicals, nineteen heterocyclic nitrogen mustards, nineteen metallic compounds, nineteen miscellaneous compounds were reviewed for mutagencity. Some results remain to be confirmed. Mutagencity of various classes of promutagens were also discussed; including 27 polycyclic aromatic hydrocarbons, 16 nitrosamines, 5 quinoline compounds; 5 aromatic amines and 4 miscellaneous promutagens. (PCS)

  16. Targeted pharmacotherapy after somatic cancer mutation screening [version 2; referees: 2 approved

    Directory of Open Access Journals (Sweden)

    Thomas M. Polasek

    2016-09-01

    Full Text Available Many patients with solid tumours are treated with targeted pharmacotherapy based on the results of genetic testing (‘precision medicine’. This study investigated the use of targeted drugs after OncoFOCUS™+KIT screening in patients with malignant melanoma, non-small cell lung cancer and metastatic colorectal cancer, and then audited the results against the National Comprehensive Cancer Network (NCCN guidelines. Patients who were not indicated for targeted pharmacotherapy did not receive such treatment (99%, 100/101. Of the patients indicated for targeted drugs, 79% (33/42 received treatment according to NCCN guidelines. In 48% (20/42 of these patients the results from OncoFOCUS™+KIT screening were required for targeted drug selection, with the remaining 52% (22/42 prescribed drugs independent of the screening results for various reasons. This study highlights the growing importance of precision medicine approaches in directing pharmacotherapy in medical oncology.

  17. Mutational screening of VSX1, SPARC, SOD1, LOX, and TIMP3 in keratoconus.

    Science.gov (United States)

    De Bonis, Patrizia; Laborante, Antonio; Pizzicoli, Costantina; Stallone, Raffaella; Barbano, Raffaela; Longo, Costanza; Mazzilli, Emilio; Zelante, Leopoldo; Bisceglia, Luigi

    2011-01-01

    To evaluate the involvement of Visual System Homeobox 1 (VSX1), Secreted Protein Acidic and Rich in Cysteine (SPARC), Superoxide Dismutase 1 (SOD1), Lysyl Oxidase (LOX), and Tissue Inhibitor of Metalloproteinase 3 (TIMP3) in sporadic and familial keratoconus. Mutational analysis of the five genes was performed by sequencing and fragment analysis in a large cohort of 302 Italian patients, with a diagnosis of keratoconus based on clinical examination and corneal topography. The variants identified in VSX1 and SPARC were also assessed in the available relatives of the probands. A novel mutation p.G239R and previously reported mutations were found in VSX1. Novel and already reported variants were identified in SPARC and SOD1, whose pathogenic significance has not been established. No pathogenic variants have been identified in LOX and TIMP3. Molecular analysis of the five genes in a cohort of 225 sporadic and 77 familial keratoconus cases confirms the possible pathogenic role of VSX1 though in a small number of patients; a possible involvement of LOX and TIMP3 could be excluded; and the role played by SOD1 and SPARC in determining the disease as not been definitively clarified. Further studies are required to identify other important genetic factors involved in the pathogenesis and progression of the disease that in the authors' opinion, and according with several authors, should be considered as a complex disease.

  18. Screening of BRCA1/2 Mutations Using Direct Sequencing in Indonesian Familial Breast Cancer Cases.

    Science.gov (United States)

    Anwar, Sumadi Lukman; Haryono, Samuel J; Aryandono, Teguh; Datasena, I Gusti Bagus

    2016-01-01

    Breast cancer has emerged as the most prevalent cancer among women worldwide, including in Indonesia. The contribution of genes associated with high-risk breast-ovarian cancers, BRCA1 and BRCA2, in the Indonesian population is relatively unknown. We have characterized family history of patients with moderate- to high-risk of breast cancer predisposition in 26 unrelated cases from Indonesia for BRCA1/2 mutation analyses using direct sequencing. Known deleterious mutations were not found in either BRCA1 or BRCA2 genes. Seven variants in BRCA2 were documented in 10 of 26 patients (38%). All variants were categorized as unclassified (VUSs). Two synonymous variants, c.3623A>G and c.4035T>C, were found in 5 patients. One variant, c4600T>C, was found in a 38 year old woman with a family history of breast cancer. We have found 4 novel variants in BRCA2 gene including c.6718C>G, c.3281A>G, c.10176C>G, and c4490T>C in 4 unrelated patients, all of them having a positive family history of breast cancer. In accordance to other studies in Asian population, our study showed more frequent variants in BRCA2 compared to BRCA1. Further studies involving larger numbers of hereditary breast cancer patients are required to reveal contribution of BRCA1/2 mutations and/or other predisposing genes among familial breast cancer patients in Indonesia.

  19. Mutational screening of VSX1, SPARC, SOD1, LOX, and TIMP3 in keratoconus

    Science.gov (United States)

    De Bonis, Patrizia; Laborante, Antonio; Pizzicoli, Costantina; Stallone, Raffaella; Barbano, Raffaela; Longo, Costanza; Mazzilli, Emilio; Zelante, Leopoldo

    2011-01-01

    Purpose To evaluate the involvement of Visual System Homeobox 1 (VSX1), Secreted Protein Acidic and Rich in Cysteine (SPARC), Superoxide Dismutase 1 (SOD1), Lysyl Oxidase (LOX), and Tissue Inhibitor of Metalloproteinase 3 (TIMP3) in sporadic and familial keratoconus. Methods Mutational analysis of the five genes was performed by sequencing and fragment analysis in a large cohort of 302 Italian patients, with a diagnosis of keratoconus based on clinical examination and corneal topography. The variants identified in VSX1 and SPARC were also assessed in the available relatives of the probands. Results A novel mutation p.G239R and previously reported mutations were found in VSX1. Novel and already reported variants were identified in SPARC and SOD1, whose pathogenic significance has not been established. No pathogenic variants have been identified in LOX and TIMP3. Conclusions Molecular analysis of the five genes in a cohort of 225 sporadic and 77 familial keratoconus cases confirms the possible pathogenic role of VSX1 though in a small number of patients; a possible involvement of LOX and TIMP3 could be excluded; and the role played by SOD1 and SPARC in determining the disease as not been definitively clarified. Further studies are required to identify other important genetic factors involved in the pathogenesis and progression of the disease that in the authors’ opinion, and according with several authors, should be considered as a complex disease. PMID:21976959

  20. Screening individuals with intellectual disability, autism and Tourette's syndrome for KCNK9 mutations and aberrant DNA methylation within the 8q24 imprinted cluster

    DEFF Research Database (Denmark)

    Delgado, Marta Sánchez; Camprubí, Cristina; Tümer, Zeynep

    2014-01-01

    The phenotype overlap between autism spectrum disorders (ASD) & intellectual disabilities (ID) is mirrored at the genetic level, with common genes being reported mutated in variety of developmental disabilities. However despite widespread genetic screening for mutations, in approximately 40......-60% of childhood developmental disorders the genetic cause remains unknown. Several genome-wide linkage screens in ASD have identified a locus mapping to distal 8q. We have recently identified a novel brain-specific imprinted cluster at this location, which contains the reciprocally expressed maternal KCNK9...

  1. Functional genome-wide siRNA screen identifies KIAA0586 as mutated in Joubert syndrome

    NARCIS (Netherlands)

    Roosing, S.; Hofree, M.; Kim, S.; Scott, E.; Copeland, B.; Romani, M.; Silhavy, J.L.; Rosti, R.O.; Schroth, J.; Mazza, T.; Miccinilli, E.; Zaki, M.S.; Swoboda, K.J.; Milisa-Drautz, J.; Dobyns, W.B.; Mikati, M.A.; Incecik, F.; Azam, M.; Borgatti, R.; Romaniello, R.; Boustany, R.M.; Clericuzio, C.L.; D'Arrigo, S.; Stromme, P.; Boltshauser, E.; Stanzial, F.; Mirabelli-Badenier, M.; Moroni, I.; Bertini, E.; Emma, F.; Steinlin, M.; Hildebrandt, F.; Johnson, C.A.; Freilinger, M.; Vaux, K.K.; Gabriel, S.B.; Aza-Blanc, P.; Heynen-Genel, S.; Ideker, T.; Dynlacht, B.D.; Lee, J.E.; Valente, E.M.; Kim, J.; Gleeson, J.G.

    2015-01-01

    Defective primary ciliogenesis or cilium stability forms the basis of human ciliopathies, including Joubert syndrome (JS), with defective cerebellar vermis development. We performed a high-content genome-wide small interfering RNA (siRNA) screen to identify genes regulating ciliogenesis as

  2. Screening for SH3TC2 gene mutations in a series of demyelinating recessive Charcot-Marie-Tooth disease (CMT4).

    Science.gov (United States)

    Piscosquito, Giuseppe; Saveri, Paola; Magri, Stefania; Ciano, Claudia; Gandioli, Claudia; Morbin, Michela; Bella, Daniela D; Moroni, Isabella; Taroni, Franco; Pareyson, Davide

    2016-09-01

    Charcot-Marie-Tooth disease type 4C (CMT4C) is an autosomal recessive (AR) demyelinating neuropathy associated to SH3TC2 mutations, characterized by early onset, spine deformities, and cranial nerve involvement. We screened 43 CMT4 patients (36 index cases) with AR inheritance, demyelinating nerve conductions, and negative testing for PMP22 duplication, GJB1 and MPZ mutations, for SH3TC2 mutations. Twelve patients (11 index cases) had CMT4C as they carried homozygous or compound heterozygous mutations in SH3TC2. We found six mutations: three nonsense (p.R1109*, p.R954*, p.Q892*), one splice site (c.805+2T>C), one synonymous variant (p.K93K) predicting altered splicing, and one frameshift (p.F491Lfs*32) mutation. The splice site and the frameshift mutations are novel. Mean onset age was 7 years (range: 1-14). Neuropathy was moderate-to-severe. Scoliosis was present in 11 patients (severe in 4), and cranial nerve deficits in 9 (hearing loss in 7). Scoliosis and cranial nerve involvement are frequent features of this CMT4 subtype, and their presence should prompt the clinician to look for SH3TC2 gene mutations. In our series of undiagnosed CMT4 patients, SH3TC2 mutation frequency is 30%, confirming that CMT4C may be the most common AR-CMT type. © 2016 Peripheral Nerve Society.

  3. A mutation screening of oncogenes, tumor suppressor gene TP53 and nuclear encoded mitochondrial complex I genes in oncocytic thyroid tumors.

    Science.gov (United States)

    Evangelisti, Cecilia; de Biase, Dario; Kurelac, Ivana; Ceccarelli, Claudio; Prokisch, Holger; Meitinger, Thomas; Caria, Paola; Vanni, Roberta; Romeo, Giovanni; Tallini, Giovanni; Gasparre, Giuseppe; Bonora, Elena

    2015-03-21

    Thyroid neoplasias with oncocytic features represent a specific phenotype in non-medullary thyroid cancer, reflecting the unique biological phenomenon of mitochondrial hyperplasia in the cytoplasm. Oncocytic thyroid cells are characterized by a prominent eosinophilia (or oxyphilia) caused by mitochondrial abundance. Although disruptive mutations in the mitochondrial DNA (mtDNA) are the most significant hallmark of such tumors, oncocytomas may be envisioned as heterogeneous neoplasms, characterized by multiple nuclear and mitochondrial gene lesions. We investigated the nuclear mutational profile of oncocytic tumors to pinpoint the mutations that may trigger the early oncogenic hit. Total DNA was extracted from paraffin-embedded tissues from 45 biopsies of oncocytic tumors. High-resolution melting was used for mutation screening of mitochondrial complex I subunits genes. Specific nuclear rearrangements were investigated by RT-PCR (RET/PTC) or on isolated nuclei by interphase FISH (PAX8/PPARγ). Recurrent point mutations were analyzed by direct sequencing. In our oncocytic tumor samples, we identified rare TP53 mutations. The series of analyzed cases did not include poorly- or undifferentiated thyroid carcinomas, and none of the TP53 mutated cases had significant mitotic activity or high-grade features. Thus, the presence of disruptive TP53 mutations was completely unexpected. In addition, novel mutations in nuclear-encoded complex I genes were identified. These findings suggest that nuclear genetic lesions altering the bioenergetics competence of thyroid cells may give rise to an aberrant mitochondria-centered compensatory mechanism and ultimately to the oncocytic phenotype.

  4. RAD51C mutation screening in high-risk patients from Serbian hereditary breast/ovarian cancer families.

    Science.gov (United States)

    Krivokuca, Ana; Yanowski, Kira; Rakobradovic, Jelena; Benitez, Javier; Brankovic-Magic, Mirjana

    2015-01-01

    In 2010 an important finding was published showing that heterozygous mutations in RAD51C were highly penetrant and were able to confer an increased risk for breast and ovarian cancers. The role of possible third high penetrance breast cancer susceptibility gene was assigned to RAD51C. Because of its rising importance in breast cancer development and the lack of information about RAD51C in Slavic populations, our goal was to identify potential population specific mutations in this gene in order to determine more detailed genetic screening strategy and breast cancer risk assessment. The study included 55 females from Serbian hereditary breast/ovarian cancer families negative for sequence alterations and large genomic rearrangements in BRCA1/2 genes. Whole coding region and exon-intron boundaries of RAD51C were analyzed by dHPLC. All mutations were confirmed by Sanger sequencing. SIFT and Polyphen were used to predict possible impact of non-synonymous variants. We found 5 variants in RAD51C including two missense, one intronic, one in the 5'UTR and one variant in the promoter region of the gene. Three detected variants are common - c.1-118G>A (rs16943176, MAF = 0,203); c.1-26C>T (rs12946397, MAF = 0,207) and c.904+34T>C (rs28363318, MAF = 0,186). We detected two missense variants, c.790G>A (p.Gly264Ser) in exon 5 and c.859A>G (p.Thr287Ala) in exon 6. Both of them were previously shown to exhibit reduced protein function but their contribution to cancer risk is still unknown. Although the initial reports implied that RAD51C might be promising candidate for next high penetrance breast cancer susceptibility gene, lack of confirmation suggested that RAD51C mutations are not as common as expected. Our study did not reveal truncating mutations in RAD51C suggesting that other breast cancer susceptibility genes may account for the increased susceptibility in our cohort of high-risk BRCA1/2 negative families.

  5. Screening of point mutations by multiple SSCP analysis in the dystrophin gene

    Energy Technology Data Exchange (ETDEWEB)

    Lasa, A.; Baiget, M.; Gallano, P. [Hospital Sant Pau, Barcelona (Spain)

    1994-09-01

    Duchenne muscular dystrophy (DMD) is a lethal, X-linked neuromuscular disorder. The population frequency of DMD is one in approximately 3500 boys, of which one third is thought to be a new mutant. The DMD gene is the largest known to date, spanning over 2,3 Mb in band Xp21.2; 79 exons are transcribed into a 14 Kb mRNA coding for a protein of 427 kD which has been named dystrophin. It has been shown that about 65% of affected boys have a gene deletion with a wide variation in localization and size. The remaining affected individuals who have no detectable deletions or duplications would probably carry more subtle mutations that are difficult to detect. These mutations occur in several different exons and seem to be unique to single patients. Their identification represents a formidable goal because of the large size and complexity of the dystrophin gene. SSCP is a very efficient method for the detection of point mutations if the parameters that affect the separation of the strands are optimized for a particular DNA fragment. The multiple SSCP allows the simultaneous study of several exons, and implies the use of different conditions because no single set of conditions will be optimal for all fragments. Seventy-eight DMD patients with no deletion or duplication in the dystrophin gene were selected for the multiple SSCP analysis. Genomic DNA from these patients was amplified using the primers described for the diagnosis procedure (muscle promoter and exons 3, 8, 12, 16, 17, 19, 32, 45, 48 and 51). We have observed different mobility shifts in bands corresponding to exons 8, 12, 43 and 51. In exons 17 and 45, altered electrophoretic patterns were found in different samples identifying polymorphisms already described.

  6. Systematic screening for mutations in the human serotonin 1F receptor gene in patients with bipolar affective disorder and schizophrenia

    Energy Technology Data Exchange (ETDEWEB)

    Shimron-Abarbanell, D.; Harms, H.; Erdmann, J.; Propping, P.; Noethen, M.M. [Univ. of Bonn (Germany)] [and others

    1996-04-09

    Using single strand conformational analysis we screened the complete coding sequence of the serotonin 1F (5-HT{sub 1F}) receptor gene for the presence of DNA sequence variation in a sample of 137 unrelated individuals including 45 schizophrenic patients, 46 bipolar patients, as well as 46 healthy controls. We detected only three rare sequence variants which are characterized by single base pair substitutions, namely a silent T{r_arrow}A transversion in the third position of codon 261 (encoding isoleucine), a silent C{r_arrow}T transition in the third position of codon 176 (encoding histidine), and a C{r_arrow}T transition in position -78 upstream from the start codon. The lack of significant mutations in patients suffering from schizophrenia and bipolar affective disorder indicates that the 5-HT{sub 1F} receptor is not commonly involved in the etiology of these diseases. 12 refs., 1 fig., 2 tabs.

  7. Screening of WT1 mutations in exon 8 and 9 in children with steroid resistant nephrotic syndrome from a single centre and establishment of a rapid screening assay using high-resolution melting analysis in a clinical setting

    OpenAIRE

    Siji, Annes; Pardeshi, Varsha Chhotusing; Ravindran, Shilpa; Vasudevan, Ambily; Vasudevan, Anil

    2017-01-01

    Background Mutations in Wilm?s tumor 1 (WT1) gene is one of the commonly reported genetic mutations in children with steroid resistant nephrotic syndrome (SRNS). We report the results of direct sequencing of exons 8 and 9 of WT1 gene in 100 children with SRNS from a single centre. We standardized and validated High Resolution Melt (HRM) as a rapid and cost effective screening step to identify individuals with normal sequence and distinguish it from those with a potential mutation. Since only ...

  8. A novel high-throughput in vivo molecular screen for shade avoidance mutants identifies a novel phyA mutation.

    Science.gov (United States)

    Wang, Xuewen; Roig-Villanova, Irma; Khan, Safina; Shanahan, Hugh; Quail, Peter H; Martinez-Garcia, Jaime F; Devlin, Paul F

    2011-05-01

    The shade avoidance syndrome (SAS) allows plants to anticipate and avoid shading by neighbouring plants by initiating an elongation growth response. The phytochrome photoreceptors are able to detect a reduction in the red:far red ratio in incident light, the result of selective absorption of red and blue wavelengths by proximal vegetation. A shade-responsive luciferase reporter line (PHYB::LUC) was used to carry out a high-throughput screen to identify novel SAS mutants. The dracula 1 (dra1) mutant, that showed no avoidance of shade for the PHYB::LUC response, was the result of a mutation in the PHYA gene. Like previously characterized phyA mutants, dra1 showed a long hypocotyl in far red light and an enhanced hypocotyl elongation response to shade. However, dra1 additionally showed a long hypocotyl in red light. Since phyB levels are relatively unaffected in dra1, this gain-of-function red light phenotype strongly suggests a disruption of phyB signalling. The dra1 mutation, G773E within the phyA PAS2 domain, occurs at a residue absolutely conserved among phyA sequences. The equivalent residue in phyB is absolutely conserved as a threonine. PAS domains are structurally conserved domains involved in molecular interaction. Structural modelling of the dra1 mutation within the phyA PAS2 domain shows some similarity with the structure of the phyB PAS2 domain, suggesting that the interference with phyB signalling may be the result of non-functional mimicry. Hence, it was hypothesized that this PAS2 residue forms a key distinction between the phyA and phyB phytochrome species.

  9. Novel PTCH1 mutations in patients with keratocystic odontogenic tumors screened for nevoid basal cell carcinoma (NBCC syndrome.

    Directory of Open Access Journals (Sweden)

    Lorenza Pastorino

    Full Text Available Keratocystic odontogenic tumors (KCOTs are cystic tumors that arise sporadically or associated with nevoid basal cell carcinoma syndrome (NBCCS. NBCCS is a rare autosomal dominantly inherited disease mainly characterized by multiple basal cell carcinomas, KCOTs of the jaws and a variety of other tumors. PTCH1 mutation can be found both in sporadic or NBCCS associated KCOTs. The aim of the current study was to assess whether a combined clinical and bio-molecular approach could be suitable for the detection of NBCCS among patients with a diagnosis of keratocystic odontogenic tumors (KCOTs. The authors collected keratocystic odontogenic tumors recorded in the database of the Pathology Department of the University of Modena and Reggio Emilia during the period 1991-2011. Through interviews and examinations, family pedigrees were drawn for all patients affected by these odontogenic lesions. We found out that 18 of the 70 patients with KCOTs and/or multiple basal cell carcinomas actually met the clinical criteria for the diagnosis of NBCCS. A wide inter- and intra-familial phenotypic variability was evident in the families. Ameloblastomas (AMLs were reported in two probands that are also carriers of the PCTH1 germline mutations. Nine germline mutations in the PTCH1 gene, 5 of them novel, were evident in 14 tested probands. The clinical evaluation of the keratocystic odontogenic tumors can be used as screening for the detection of families at risk of NBCCS. Keratocystic odontogenic lesions are uncommon, and their discovery deserves the search for associated cutaneous basal cell carcinomas and other benign and malignant tumors related to NBCCS.

  10. Optimization of a genotyping screening based on hydrolysis probes to detect the main mutations related to Leber hereditary optic neuropathy (LHON).

    Science.gov (United States)

    Martins, Fábio Tadeu Arrojo; Miranda, Paulo Maurício do Amor Divino; Fernandes, Marcela Scabello Amaral; Maciel-Guerra, Andréa Trevas; Sartorato, Edi Lúcia

    2017-01-01

    Leber hereditary optic neuropathy (LHON) is a mitochondrial inherited disease characterized by bilateral vision problems, such as reduced visual acuity, dyschromatopsia, and central or centrocecal scotoma. Of these cases, 95% are caused by three mutations in mitochondrial DNA (mtDNA): m.G11778A, followed by m.T14484C and m.G3460A. The remaining 5% of cases of LHON are caused by rare mutations also present in mtDNA. Although conventional molecular tools for molecular screening of LHON are becoming popular, in most cases these tools are still expensive and time-consuming and are difficult to reproduce. Therefore, to meet the need for more accurate, faster, and cheaper techniques for molecular screening, as well as make it more accessible, we used the high-throughput method TaqMan(®) OpenArray(™) Genotyping platform for developing a customized high-throughput assay for the three main mutations related to LHON. The assay was performed for 87 individuals diagnosed with LHON or acquired optic neuropathy of unknown origin. The three main mutations were screened using the customized assay with the TaqMan(®) OpenArray(™) Genotyping platform, and all reactions were performed in triplicate. The positive and negative results were revalidated with restriction fragment length polymorphism PCR (RFLP-PCR) and Sanger sequencing. The main mutations related to LHON were detected in 34 patients with genotyping reactions, of which 27 cases had the m.G11778A mutation, and seven had the m.T14484C mutation. The TaqMan(®) OpenArray(™) Genotyping platform was shown to be an effective tool for molecular screening of the most common mutations related to LHON without presenting false positive or negative results for the analyzed mutations. In addition, this tool can be considered a cheaper, faster, and more accurate alternative for molecular screening of LHON mutations than PCR and Sanger sequencing, as 94 genotyping reactions can be performed within 6 h and specific Taq

  11. Optimization of a genotyping screening based on hydrolysis probes to detect the main mutations related to Leber hereditary optic neuropathy (LHON)

    Science.gov (United States)

    Miranda, Paulo Maurício do Amor Divino; Fernandes, Marcela Scabello Amaral; Maciel-Guerra, Andréa Trevas; Sartorato, Edi Lúcia

    2017-01-01

    Purpose Leber hereditary optic neuropathy (LHON) is a mitochondrial inherited disease characterized by bilateral vision problems, such as reduced visual acuity, dyschromatopsia, and central or centrocecal scotoma. Of these cases, 95% are caused by three mutations in mitochondrial DNA (mtDNA): m.G11778A, followed by m.T14484C and m.G3460A. The remaining 5% of cases of LHON are caused by rare mutations also present in mtDNA. Although conventional molecular tools for molecular screening of LHON are becoming popular, in most cases these tools are still expensive and time-consuming and are difficult to reproduce. Therefore, to meet the need for more accurate, faster, and cheaper techniques for molecular screening, as well as make it more accessible, we used the high-throughput method TaqMan® OpenArray™ Genotyping platform for developing a customized high-throughput assay for the three main mutations related to LHON. Methods The assay was performed for 87 individuals diagnosed with LHON or acquired optic neuropathy of unknown origin. The three main mutations were screened using the customized assay with the TaqMan® OpenArray™ Genotyping platform, and all reactions were performed in triplicate. The positive and negative results were revalidated with restriction fragment length polymorphism PCR (RFLP-PCR) and Sanger sequencing. Results The main mutations related to LHON were detected in 34 patients with genotyping reactions, of which 27 cases had the m.G11778A mutation, and seven had the m.T14484C mutation. Conclusions The TaqMan® OpenArray™ Genotyping platform was shown to be an effective tool for molecular screening of the most common mutations related to LHON without presenting false positive or negative results for the analyzed mutations. In addition, this tool can be considered a cheaper, faster, and more accurate alternative for molecular screening of LHON mutations than PCR and Sanger sequencing, as 94 genotyping reactions can be performed within 6 h

  12. DNA/RNA-based formulations for treatment of breast cancer

    DEFF Research Database (Denmark)

    Xie, Zhaolu; Zeng, Xianghui

    2017-01-01

    INTRODUCTION: To develop a successful formulation for the gene therapy of breast cancer, an effective therapeutic nucleic acid and a proper delivery system are essential. Increased understanding of breast cancer, and developments in biotechnology, material science and nanotechnology have provided...... a major impetus in the development of effective formulations for the gene therapy of breast cancer. Areas covered: We discuss DNA/RNA-based formulations that can inhibit the growth of breast cancer cells and control the progress of breast cancer. Targets for the gene therapy of breast cancer, DNA....../RNA-based therapeutics and delivery systems are summarized. And examples of successful DNA/RNA-based formulations for breast cancer gene therapy are reviewed. Expert opinion: Several challenges remain in developing effective DNA/RNA-based formulations for treatment of breast cancer. Firstly, most of the currently...

  13. NCG 4.0: the network of cancer genes in the era of massive mutational screenings of cancer genomes.

    Science.gov (United States)

    An, Omer; Pendino, Vera; D'Antonio, Matteo; Ratti, Emanuele; Gentilini, Marco; Ciccarelli, Francesca D

    2014-01-01

    NCG 4.0 is the latest update of the Network of Cancer Genes, a web-based repository of systems-level properties of cancer genes. In its current version, the database collects information on 537 known (i.e. experimentally supported) and 1463 candidate (i.e. inferred using statistical methods) cancer genes. Candidate cancer genes derive from the manual revision of 67 original publications describing the mutational screening of 3460 human exomes and genomes in 23 different cancer types. For all 2000 cancer genes, duplicability, evolutionary origin, expression, functional annotation, interaction network with other human proteins and with microRNAs are reported. In addition to providing a substantial update of cancer-related information, NCG 4.0 also introduces two new features. The first is the annotation of possible false-positive cancer drivers, defined as candidate cancer genes inferred from large-scale screenings whose association with cancer is likely to be spurious. The second is the description of the systems-level properties of 64 human microRNAs that are causally involved in cancer progression (oncomiRs). Owing to the manual revision of all information, NCG 4.0 constitutes a complete and reliable resource on human coding and non-coding genes whose deregulation drives cancer onset and/or progression. NCG 4.0 can also be downloaded as a free application for Android smart phones. Database URL: http://bio.ieo.eu/ncg/.

  14. The different faces of the p. A53T alpha-synuclein mutation: A screening of Greek patients with parkinsonism and/or dementia.

    Science.gov (United States)

    Breza, Marianthi; Koutsis, Georgios; Karadima, Georgia; Potagas, Constantin; Kartanou, Chrisoula; Papageorgiou, Sokratis G; Paraskevas, George P; Kapaki, Elisabeth; Stefanis, Leonidas; Panas, Marios

    2017-12-09

    The p. A53T mutation in the alpha-synuclein (SNCA) gene is a rare cause of autosomal dominant Parkinson's disease (PD). Although generally rare, it is particularly common in the Greek population due to a founder effect. A53T-positive PD patients often develop dementia during disease course and may very rarely present with dementia. We screened for the p. A53T SNCA mutation a total of 347 cases of Greek origin with parkinsonism and/or dementia, collected over 15 years at the Neurogenetics Unit, Eginition Hospital, University of Athens. Cases were classified into: "pure parkinsonism", "pure dementia" and "parkinsonism plus dementia". In total, 4 p. A53T SNCA mutation carriers were identified. All had autosomal dominant family history and early onset. Screening of the "pure parkinsonism" category revealed 2 cases with typical PD. The other two mutation carriers were identified in the "parkinsonism plus dementia" category. One had a diagnosis of PD dementia and the other of behavioral variant frontotemporal dementia. Screening of patients with "pure dementia" failed to identify any further A53T-positive cases. Our results confirm that the p. A53T SNCA mutation is relatively common in Greek patients with PD or PD plus dementia, particularly in cases with early onset and/or autosomal dominant family history. Copyright © 2017 Elsevier B.V. All rights reserved.

  15. Screening of R122H and N29I mutations in the PRSS1 gene and N34S mutation in the SPINK1 gene in Mexican pediatric patients with acute and recurrent pancreatitis.

    Science.gov (United States)

    Sánchez-Ramírez, Carmen A; Flores-Martínez, Silvia E; García-Zapién, Alejandra G; Montero-Cruz, Sergio A; Larrosa-Haro, Alfredo; Sánchez-Corona, José

    2012-07-01

    The study's objective was to assess the association between the PRSS1 R122H and N29I and the SPINK1 N34S mutations and acute pancreatitis (AP) and recurrent pancreatitis in Mexican pediatric patients. The N34S and R122H mutations were detected using polymerase chain reaction-restriction fragment length polymorphism, and the N29I mutation was detected using allele-specific polymerase chain reaction in 92 pancreatitis patients (58 AP and 34 recurrent pancreatitis patients) and 144 controls. We found 1 mutated allele in 4 (4.3%) of 92 pancreatitis patients and none in the controls. All 4 patients bearing mutations had AP, with a frequency of 6.8% (4/58). Three (5.2%) of 58 patients were heterozygous for the N34S mutation, and 1 (1.7%) of 58 patients was heterozygous for the N29I mutation. The comparison between the AP and control groups revealed both a significant number of patients carrying any mutations in the screened genes (P = 0.008) and bearing the N34S mutation (P = 0.023). Moreover, we found that the N34S G allele increased the risk of developing AP (odds ratio, 10.3; confidence interval, 1.1-248.8). Patients bearing the N34S G allele exhibited a 10-fold increased risk of developing AP compared with controls, suggesting that the SPINK1 N34S mutation represents an etiologic risk factor for AP in our Mexican pediatric patients.

  16. Distribution of CFTR mutations in Eastern Hungarians: relevance to genetic testing and to the introduction of newborn screening for cystic fibrosis.

    Science.gov (United States)

    Ivady, Gergely; Madar, Laszlo; Nagy, Bela; Gonczi, Ferenc; Ajzner, Eva; Dzsudzsak, Erika; Dvořáková, Lenka; Gombos, Eva; Kappelmayer, Janos; Macek, Milan; Balogh, Istvan

    2011-05-01

    The aim of this study was characterization of an updated distribution of CFTR mutations in a representative cohort of 40 CF patients with the classical form of the disease drawn from Eastern Hungary. Due to the homogeneity of the Hungarian population our data are generally applicable to other regions of the country, including the sizeable diaspora. We utilized the recommended "cascade" CFTR mutation screening approach, initially using a commercial assay, followed by examination of the common "Slavic" deletion CFTRdele2,3(21kb). Subsequently, the entire CFTR coding region of the CFTR gene was sequenced in patients with yet unidentified mutations. The Elucigene CF29(Tm) v2 assay detected 81.25% of all CF causing mutations. An addition of the CFTRdele2,3(21kb) increased the mutation detection rate to 86.25%. DNA sequencing enabled us to identify mutations on 79/80 CF alleles. Mutations [CFTRdele2,3(21kb), p.Gln685ThrfsX4 (2184insA) were found at an unusually high frequency, each comprising 5.00% of all CF alleles. We have identified common CF causing mutations in the Hungarian population with the most common mutations (p.Phe508del, p.Asn1303Lys, CFTRdele2,3(21kb), 2184insA, p.Gly542X, and p.Leu101X), comprising over 93.75% of all CF alleles. Obtained data are applicable to the improvement of DNA diagnostics in Hungary and beyond, and are the necessary prerequisite for the introduction of a nationwide "two tier" CF newborn screening program. Copyright © 2011 European Cystic Fibrosis Society. Published by Elsevier B.V. All rights reserved.

  17. Mutation screening and genotype phenotype correlation of α-crystallin, γ-crystallin and GJA8 gene in congenital cataract

    Science.gov (United States)

    Kumar, Manoj; Agarwal, Tushar; Khokhar, Sudarshan; Kumar, Manoj; Kaur, Punit; Roy, Tara Sankar

    2011-01-01

    Purpose To screen α-crystallin (CRYAB), γ-crystallin (CRYGC and CRYGD), and Connexin 50 (Cx-50 or GJA8) genes in congenital cataract patients and controls. Methods Thirty clinically diagnosed congenital cataract cases below 3 years of age from northern India, presenting at Dr. R. P. Centre for Ophthalmic Sciences (AIIMS, New Delhi, India) were enrolled in this study. Genomic DNA was extracted from peripheral blood, all coding and exon/intron regions were amplified using PCR and direct sequencing was performed to detect any nucleotide variation. ProtScale and Discovery Studio programs were used for insilico and structural analysis of non-synonymous mutations. Results DNA sequencing analysis of CRYAB, CRYGC, CRYGD, and GJA8 showed a total of six variations of which two were novel (CRYGC:p.R48H and GJA8:p.L281C) and four have been previously reported (CRYAB: rs11603779T>G, GJA8: p.L268L, CRYGD: p.R95R, and c.T564C). Both the novel changes, in CRYGC and GJA8 were found in 16.6% of the patients. Previously reported nucleotide alterations (CRYGD:p.R95R and c.T564C) were found in 90% of the patients. Insilico and structural analysis data suggested that two novel non-synonymous mutations altered the stability and solvent accessibility of γC-crystallin and Cx-50 proteins which may lead to lens opacification. Conclusions We observed two novel nonsynonymous variations and four reported variations in CRYAB, CRYGC, CRYGD, and GJA8. The p.R48H variation in γC-crystallin may disrupt the normal structure of lens and can cause cataract. Cx50 is responsible for joining the lens cells into a functional syncytium and a mutation (p.L281C) in GJA8 may lead to lens opacification resulting in cataract formation. This study further expands the mutation spectrum of congenital cataract and help understanding how mutant proteins lead to opacification of lens. PMID:21423869

  18. Screening and Mutation Breeding of the Celloulonmonas flavigena CR-14 and Optimization of Its Fermentation Conditions

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    YANG Ji-ye

    2016-05-01

    Full Text Available The Celloulonmonas flavigena CR-14 was screened from the rumen of cattle, which had the ability of producting cellulose and was stored in our laboratory. In order to improve the cellulose activity of the CR-14, it was treated with nitrite, ultraviolet(UV and complex mutagenesis. Initial selection identified 6 cellulase-producing strains, as demonstrated by their ability to turn Congo red plates yellow. These 6 strains were then rescreened by determining the cellulase activity of broth. Finally, through nitrite and UV mutagenesis,Y-UA-18 strain with the high cellulose yield and stable characteristics, which enzymatic activity was 10.57 U, 1.67 times of starting strain CR-14, was screened out. In order to provide a theoretical reference to the industrial production of the strain Y-UA-18, we conducted the single factor test and orthogonal test to the fermentation medium components and the fermentation conditions. The results showed that the best medium of strain Y-UA-18 enzyme production was as followed:straw powder 1.0%, nitrogen source 0.6%, MgSO4 0.1%, KH2PO4 0.1%, NaCl 0.08%. The optimum fermentation conditions were:initial pH 7.0, incubation temperature 30 ℃, fermentation time 3 d, the effect of ventilation for strain Y-UA-18 enzyme production was not obvious, but under the condition of anaerobic, the enzyme activity of fermentation liquor was reduced, 0.1% TW-80 had no effect for enzyme production.

  19. [Screening of the gene mutation in D-loop region of mitochondrial DNA in oral squamous cell carcinoma].

    Science.gov (United States)

    Sun, Yang; Yuan, Rong-tao; Chen, Wan-tao; Bu, Ling-xue; Jia, Mu-yun

    2013-05-01

    To investigate the gene mutation in D-loop region of mitochondrial DNA (mtDNA) in oral squamous cell carcinoma (OSCC) tissue and to explore the role of the gene mutation in D-loop region in the OSCC tumorigenesis. mtDNA was obtained from cancer, paracancerous and normal mucosa tissues of thirty patients with OSCC. The D-loop regions of mtDNA were amplified with PCR, sequencing and then analyzed by Chromas software and BLAST to identify the mutation site. Mutation in the D-loop region was found in eight cases, with the mutation rate of 27%. There were nine mutations totally, including one point mutation, two base deletions, three insertion mutations, three heterozygous mutations. In these mutations, base deletions were different from each other and heterozygous mutations had no same mutation form, while the three insertion mutations were same, the insertion of base C. One case had T/A heterozygous mutation and base C insertion at the same time. There were mutations in mtDNA D-loop in OSCC, but the relationship between occurrence of OSCC and mutation of mtDNA needs further study.

  20. High resolution melting curve analysis targeting the HBB gene mutational hot-spot offers a reliable screening approach for all common as well as most of the rare beta-globin gene mutations in Bangladesh.

    Science.gov (United States)

    Islam, Md Tarikul; Sarkar, Suprovath Kumar; Sultana, Nusrat; Begum, Mst Noorjahan; Bhuyan, Golam Sarower; Talukder, Shezote; Muraduzzaman, A K M; Alauddin, Md; Islam, Mohammad Sazzadul; Biswas, Pritha Promita; Biswas, Aparna; Qadri, Syeda Kashfi; Shirin, Tahmina; Banu, Bilquis; Sadya, Salma; Hussain, Manzoor; Sarwardi, Golam; Khan, Waqar Ahmed; Mannan, Mohammad Abdul; Shekhar, Hossain Uddin; Chowdhury, Emran Kabir; Sajib, Abu Ashfaqur; Akhteruzzaman, Sharif; Qadri, Syed Saleheen; Qadri, Firdausi; Mannoor, Kaiissar

    2018-01-02

    Bangladesh lies in the global thalassemia belt, which has a defined mutational hot-spot in the beta-globin gene. The high carrier frequencies of beta-thalassemia trait and hemoglobin E-trait in Bangladesh necessitate a reliable DNA-based carrier screening approach that could supplement the use of hematological and electrophoretic indices to overcome the barriers of carrier screening. With this view in mind, the study aimed to establish a high resolution melting (HRM) curve-based rapid and reliable mutation screening method targeting the mutational hot-spot of South Asian and Southeast Asian countries that encompasses exon-1 (c.1 - c.92), intron-1 (c.92 + 1 - c.92 + 130) and a portion of exon-2 (c.93 - c.217) of the HBB gene which harbors more than 95% of mutant alleles responsible for beta-thalassemia in Bangladesh. Our HRM approach could successfully differentiate ten beta-globin gene mutations, namely c.79G > A, c.92 + 5G > C, c.126_129delCTTT, c.27_28insG, c.46delT, c.47G > A, c.92G > C, c.92 + 130G > C, c.126delC and c.135delC in heterozygous states from the wild type alleles, implying the significance of the approach for carrier screening as the first three of these mutations account for ~85% of total mutant alleles in Bangladesh. Moreover, different combinations of compound heterozygous mutations were found to generate melt curves that were distinct from the wild type alleles and from one another. Based on the findings, sixteen reference samples were run in parallel to 41 unknown specimens to perform direct genotyping of the beta-thalassemia specimens using HRM. The HRM-based genotyping of the unknown specimens showed 100% consistency with the sequencing result. Targeting the mutational hot-spot, the HRM approach could be successfully applied for screening of beta-thalassemia carriers in Bangladesh as well as in other countries of South Asia and Southeast Asia. The approach could be a useful supplement of hematological and

  1. Identification of fragile X pre-mutation carriers in the Chinese obstetric population using a robust FMR1 polymerase chain reaction assay: implications for screening and prenatal diagnosis.

    Science.gov (United States)

    Cheng, Y Ky; Lin, C Sw; Kwok, Y Ky; Chan, Y M; Lau, T K; Leung, T Y; Choy, K W

    2017-04-01

    There is significant morbidity associated with fragile X syndrome. Unfortunately, most maternal carriers are clinically silent during their reproductive years. Because of this, many experts have put forward the notion of preconception or prenatal fragile X carrier screening for females. This study aimed to determine the prevalence of fragile X syndrome pre-mutation and asymptomatic full-mutation carriers in a Chinese pregnant population, and the distribution of cytosine-guanine-guanine (CGG) repeat numbers using a robust fragile X mental retardation 1 (FMR1) polymerase chain reaction assay. This was a cross-sectional survey in prospectively recruited pregnant women from a university hospital in Hong Kong. Chinese pregnant women without a family history of fragile X syndrome were recruited between April 2013 and May 2015. A specific FMR1 polymerase chain reaction assay was performed on peripheral blood to determine the CGG repeat number of the FMR1 gene. Prenatal counselling was offered to full-mutation and pre-mutation carriers. In 2650 Chinese pregnant women, two individuals with pre-mutation alleles (0.08%, one in 1325) and one asymptomatic woman with full-mutation (0.04%, one in 2650) alleles were identified. The overall prevalence of pre-mutation and full-mutation alleles was 0.11% (1 in 883). Furthermore, 30 (1.1%) individuals with intermediate alleles were detected. In the 2617 women with normal CGG repeats, the most common CGG repeat allele was 30. The overall prevalence of pre-mutation and asymptomatic full-mutation carriers in the Chinese pregnant population was one in 883, detected by a new FMR1 polymerase chain reaction assay.

  2. Molecular basis of non-syndromic hypospadias: systematic mutation screening and genome-wide copy-number analysis of 62 patients.

    Science.gov (United States)

    Kon, M; Suzuki, E; Dung, V C; Hasegawa, Y; Mitsui, T; Muroya, K; Ueoka, K; Igarashi, N; Nagasaki, K; Oto, Y; Hamajima, T; Yoshino, K; Igarashi, M; Kato-Fukui, Y; Nakabayashi, K; Hayashi, K; Hata, K; Matsubara, Y; Moriya, K; Ogata, T; Nonomura, K; Fukami, M

    2015-03-01

    What percentage of cases with non-syndromic hypospadias can be ascribed to mutations in known causative/candidate/susceptibility genes or submicroscopic copy-number variations (CNVs) in the genome? Monogenic and digenic mutations in known causative genes and cryptic CNVs account for >10% of cases with non-syndromic hypospadias. While known susceptibility polymorphisms appear to play a minor role in the development of this condition, further studies are required to validate this observation. Fifteen causative, three candidate, and 14 susceptible genes, and a few submicroscopic CNVs have been implicated in non-syndromic hypospadias. Systematic mutation screening and genome-wide copy-number analysis of 62 patients. The study group consisted of 57 Japanese and five Vietnamese patients with non-syndromic hypospadias. Systematic mutation screening was performed for 25 known causative/candidate/susceptibility genes using a next-generation sequencer. Functional consequences of nucleotide alterations were assessed by in silico assays. The frequencies of polymorphisms in the patient group were compared with those in the male general population. CNVs were analyzed by array-based comparative genomic hybridization and characterized by fluorescence in situ hybridization. Seven of 62 patients with anterior or posterior hypospadias carried putative pathogenic mutations, such as hemizygous mutations in AR, a heterozygous mutation in BNC2, and homozygous mutations in SRD5A2 and HSD3B2. Two of the seven patients had mutations in multiple genes. We did not find any rare polymorphisms that were abundant specifically in the patient group. One patient carried mosaic dicentric Y chromosome. The patient group consisted solely of Japanese and Vietnamese individuals and clinical and hormonal information of the patients remained rather fragmentary. In addition, mutation analysis focused on protein-altering substitutions. Our data provide evidence that pathogenic mutations can underlie both

  3. Evolutionary patterns of RNA-based duplication in non-mammalian chordates.

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    Ming Chen

    Full Text Available The role of RNA-based duplication, or retroposition, in the evolution of new gene functions in mammals, plants, and Drosophila has been widely reported. However, little is known about RNA-based duplication in non-mammalian chordates. In this study, we screened ten non-mammalian chordate genomes for retrocopies and investigated their evolutionary patterns. We identified numerous retrocopies in these species. Examination of the age distribution of these retrocopies revealed no burst of young retrocopies in ancient chordate species. Upon comparing these non-mammalian chordate species to the mammalian species, we observed that a larger fraction of the non-mammalian retrocopies was under strong evolutionary constraints than mammalian retrocopies are, as evidenced by signals of purifying selection and expression profiles. For the Western clawed frog, Medaka, and Sea squirt, many retrogenes have evolved gonad and brain expression patterns, similar to what was observed in human. Testing of retrogene movement in the Medaka genome, where the nascent sex chrosomes have been well assembled, did not reveal any significant gene movement. Taken together, our analyses demonstrate that RNA-based duplication generates many functional genes and can make a significant contribution to the evolution of non-mammalian genomes.

  4. Which screening strategy should be offered to women with BRCA1 or BRCA2 mutations? A simulation of comparative cost-effectiveness.

    Science.gov (United States)

    de Bock, G H; Vermeulen, K M; Jansen, L; Oosterwijk, J C; Siesling, S; Dorrius, M D; Feenstra, T; Houssami, N; Greuter, M J W

    2013-04-30

    There is no consensus on the most effective strategy (mammography or magnetic resonance imaging (MRI)) for screening women with BRCA1 or BRCA2 mutations. The effectiveness and cost-effectiveness of the Dutch, UK and US screening strategies, which involve mammography and MRI at different ages and intervals were evaluated in high-risk women with BRCA1 or BRCA2 mutations. Into a validated simulation screening model, outcomes and cost parameters were integrated from published and cancer registry data. Main outcomes were life-years gained and incremental cost-effectiveness ratios. The simulation was situated in the Netherlands as well as in the United Kingdom, comparing the Dutch, UK and US strategies with the population screening as a reference. A discount rate of 3% was applied to both costs and health benefits. In terms of life-years gained, the strategies from least to most cost-effective were the UK, Dutch and US screening strategy, respectively. However, the differences were small. Applying the US strategy in the Netherlands, the costs were €43 800 and 68 800 for an additional life-year gained for BRCA1 and BRCA2, respectively. At a threshold of €20 000 per life-year gained, implementing the US strategy in the Netherlands has a very low probability of being cost-effective. Stepping back to the less-effective UK strategy would save relatively little in costs and results in life-years lost. When implementing the screening strategies in the United Kingdom, the Dutch, as well as the US screening strategy have a high probability of being cost-effective. From a cost-effectiveness perspective, the Dutch screening strategy is preferred for screening high-risk women in the Netherlands as well as in the United Kingdom.

  5. Dual NRASQ61R and BRAFV600E mutation-specific immunohistochemistry completes molecular screening in melanoma samples in a routine practice.

    Science.gov (United States)

    Uguen, Arnaud; Guéguen, Paul; Legoupil, Delphine; Bouvier, Stéphanie; Costa, Sebastian; Duigou, Sandrine; Lemasson, Gilles; Ledé, Françoise; Sassolas, Bruno; Talagas, Matthieu; Férec, Claude; Le Maréchal, Cédric; De Braekeleer, Marc; Marcorelles, Pascale

    2015-11-01

    NRAS and BRAF mutational status has become mandatory to treat patients with metastatic melanomas. Mutation-specific immunohistochemistry (IHC) can help analyze challenging tumor samples. We report our experience integrating NRASQ61R (SP174) and BRAFV600E (VE1) IHC in routine practice in a cancer molecular genetic platform. All samples screened for BRAF and NRAS mutations during the year 2014 were analyzed by IHC and pyrosequencing, with an independent analysis of the 2 methods. Cases with first-line discordant results benefited from a complementary second-round IHC and next-generation sequencing (NGS) with a final interpretation taking into account the results of pyrosequencing, IHC, NGS, and quantification of the tumor cells. We analyzed 111 consecutive formalin-fixed and paraffin-embedded melanoma samples from 101 patients. Twenty-two and 11 samples were concordant for BRAFV600E and NRASQ61R mutations, respectively. Second-round analyses of 9 discordant and 1 molecularly inconclusive samples allowed conclusion in 4 further mutated samples (2 BRAFV600E and 2 NRASQ61R). A sample remained NRASQ61R IHC negative but NRASQ61R mutated with molecular methods. Overall, BRAFV600 and NRASQ61 mutation frequencies were 31.7% and 30.7%, respectively. When compared to molecular results, the sensitivity and specificity of IHC were 100% for BRAFV600E IHC and 92.3% and 98.9% for NRASQ61R IHC, respectively. IHC interpretation required a more stringent cutoff for BRAFV600E IHC than NRASQ61R to minimize false results. We conclude that NRASQ61R and BRAFV600E IHC coupled with NGS allow detection of mutations in melanoma challenging samples. Copyright © 2015 Elsevier Inc. All rights reserved.

  6. Screening mutations of OTOF gene in Chinese patients with auditory neuropathy, including a familial case of temperature-sensitive auditory neuropathy

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    Benedict-Alderfer Cindy

    2010-05-01

    Full Text Available Abstract Background Mutations in OTOF gene, encoding otoferlin, cause DFNB9 deafness and non-syndromic auditory neuropathy (AN. The aim of this study is to identify OTOF mutations in Chinese patients with non-syndromic auditory neuropathy. Methods 73 unrelated Chinese Han patients with AN, including one case of temperature sensitive non-syndromic auditory neuropathy (TS-NSRAN and 92 ethnicity-matched controls with normal hearing were screened. Forty-five pairs of PCR primers were designed to amplify all of the exons and their flanking regions of the OTOF gene. The PCR products were sequenced and analyzed for mutation identification. Results Five novel possibly pathogenic variants (c.1740delC, c.2975_2978delAG, c.1194T>A, c.1780G>A, c.4819C > T were identified in the group of 73 AN patients, in which two novel mutant alleles (c.2975_2978delAG + c.4819C > T were identified in one Chinese TS-NSRAN case. Besides, 10 non-pathogenic variants of the OTOF gene were found in AN patients and controls. Conclusions Screening revealed that mutations in the OTOF gene account for AN in 4 of 73(5.5% sporadic AN patients, which shows a lower genetic load of that gene in contrast to the previous studies based on other populations. Notably, we found two novel mutant alleles related to temperature sensitive non-syndromic auditory neuropathy. This mutation screening study further confirms that the OTOF gene contributes to ANs and to TS-NSRAN.

  7. Screening for MYO15A Gene Mutations in Autosomal Recessive Nonsyndromic, GJB2 Negative Iranian Deaf Population

    Science.gov (United States)

    Fattahi, Zohreh; Shearer, A. Eliot; Babanejad, Mojgan; Bazazzadegan, Niloofar; Almadani, Seyed Navid; Nikzat, Nooshin; Jalalvand, Khadijeh; Arzhangi, Sanaz; Esteghamat, Fatemehsadat; Abtahi, Rezvan; Azadeh, Batool; Smith, Richard J.H.; Kahrizi, Kimia; Najmabadi, Hossein

    2013-01-01

    MYO15A is located at the DFNB3 locus on chromosome 17p11.2, and encodes myosin-XV, an unconventional myosin critical for the formation of stereocilia in hair cells of cochlea. Recessive mutations in this gene lead to profound autosomal recessive nonsyndromic hearing loss (ARNSHL) in humans and the shaker2 (sh2) phenotype in mice. Here, we performed a study on 140 Iranian families in order to determine mutations causing ARNSHL. The families, who were negative for mutations in GJB2, were subjected to linkage analysis. Eight of these families showed linkage to the DFNB3 locus, suggesting a MYO15A mutation frequency of 5.71% in our cohort of Iranian population. Subsequent sequencing of the MYO15A gene led to identification of 7 previously unreported mutations, including 4 missense mutations, 1 nonsense mutation, and 2 deletions in different regions of the myosin-XV protein. PMID:22736430

  8. Mutation screen and association studies for the fatty acid amide hydrolase (FAAH gene and early onset and adult obesity

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    Rief Winfried

    2010-01-01

    Full Text Available Abstract Background The orexigenic effects of cannabinoids are limited by activation of the endocannabinoid degrading enzyme fatty acid amide hydrolase (FAAH. The aim of this study was to analyse whether FAAH alleles are associated with early and late onset obesity. Methods We initially assessed association of five single nucleotide polymorphisms (SNPs in FAAH with early onset extreme obesity in up to 521 German obese children and both parents. SNPs with nominal p-values ≤ 0.1 were subsequently analysed in 235 independent German obesity families. SNPs associated with childhood obesity (p-values ≤ 0.05 were further analysed in 8,491 adult individuals of a population-based cohort (KORA for association with adult obesity. One SNP was further analysed in 985 German obese adults and 588 normal and underweight controls. In parallel, we screened the FAAH coding region for novel sequence variants in 92 extremely obese children using single-stranded-conformation-polymorphism-analysis and denaturing HPLC and assessed the implication of the identified new variants for childhood obesity. Results The trio analysis revealed some evidence for an association of three SNPs in FAAH (rs324420 rs324419 and rs873978 with childhood obesity (two-sided p-values between 0.06 and 0.10. Although analyses of these variants in 235 independent obesity families did not result in statistically significant effects (two-sided p-values between 0.14 and 0.75, the combined analysis of all 603 obesity families supported the idea of an association of two SNPs in FAAH (rs324420 and rs2295632 with early onset extreme obesity (p-values between 0.02 and 0.03. No association was, however, found between these variants and adult obesity. The mutation screen revealed four novel variants, which were not associated with early onset obesity (p > 0.05. Conclusions As we observed some evidence for an association of the FAAH variants rs2295632 rs324420 with early onset but not adult obesity

  9. [Audiologic and molecular screening for hearing loss by 35delG mutation in connexin 26 gene and congenital cytomegalovirus infection].

    Science.gov (United States)

    Streitenberger, Edgardo Raúl; Suárez, Ariel Ignacio; Masciovecchio, María Verónica; Laurnagaray, Diana; Alda, Ernesto

    2011-12-01

    Hearing loss may be attributed to genetic and environmental factors. Mutations in the gene of the CX26 protein (connexin 26), are responsible for 30-80% of all cases of non-syndromic profound hearing loss. The 35delG is the most frequent variant in the caucasian population. As to environmental factors, the cytomegalovirus (CMV) is the main cause of congenital infection. To determine the prevalence of congenital CMV infection and the frequency of the 35delG mutation in newborns. To identify those at risk of suffering hearing loss in order to do an audiologic follow-up of detected cases. One thousand and twenty samples of dry blood spots corresponding to newborns were tested using conventional and real time PCR. Audiologic screening was performed to all newborns before hospital discharge. Fifteen out of 1020 subjects were heterozygous for the mutation. No homozygous patients were found. Six out of the samples tested positive for CMV (confirmed by a urine sample), out of which only one newborn was symptomatic. The auditory brainstem response was recorded in all these children. Hearing loss was found in three children with congenital CMV infection and two with 35delG mutation. The frecuency of 35delG mutation carriers in our population was 1.3% and the CMV congenital infection prevalence was 0.6%. Audiologic monitoring of these two populations allowed detection of hearing loss of late onset.

  10. Inactivating Mutation screening of Exon 6 and Exon 10E of FSHR gene in women with Polycystic Ovarian Syndrome in Vellore population

    Science.gov (United States)

    Sekar, Nishu; Sapre, Madhura; Kale, Vaikhari; Prabhu, Yogamaya D.; Renu, Kaviyarasi; Ramgir, Shalaka S.; Abilash, V. G.

    2017-11-01

    Polycystic Ovarian syndrome (PCOS) is a major cause of infertility in females of reproducing age and is typified by oligo-anovulation, hyperandrogenism, hirsutism and polycystic ovaries. FSHR gene located on chromosome 2 p21 is responsible for the normal follicular development and any deletion or mutation in the gene affects the interaction of FSH with its receptor. Thus, it becomes the candidate gene for PCOS study. Inactivating mutation in FSHR gene limits the receptor’s function by creating a complete block, changing the receptor-ligand complex or the basic hormone signal transduction.To screen the inactivating mutations in Exon 6 and Exon 10E of FSHR gene in women diagnosed with PCOS.PCR-RFLP analysis indicated that there were no inactivating mutations found in Exon 6 and Exon 10E. Variations in hormone levels were seen amongst the PCOS patients. There were no inactivating mutations found in FSHR gene of the women diagnosed with PCOS according to the Rotterdam criteria in Vellore population.

  11. Clinicians' attitudes toward general screening of the Ashkenazi-Jewish population for prevalent founder BRCA1/2 and LRRK2 mutations.

    Science.gov (United States)

    Shkedi-Rafid, S; Ofer-Bialer, G; Meiner, V; Calderon-Margalit, R

    2013-01-01

    Advances in genomics may eventually lead to genetic susceptibility screening of the general population, regardless of a personal or familial history of the disease in question. Yet, little is known about clinicians' attitudes toward such programs. We explored attitudes of family practitioners, medical geneticists and genetic counselors toward genetic screening of the general Ashkenazi-Jewish population for the common founder mutations in BRCA1/2 and LRRK2 genes (which increase the risk of hereditary breast/ovarian cancers and Parkinson's disease, respectively). Participants (n = 204) completed a specially designed questionnaire, distributed by e-mail, regular mail or in-person. Slightly more than half (52%) were in favor of BRCA screening, while the vast majority (86%) opposed to LRRK2 screening. About two-thirds (68%) of the respondents supported pre-test genetic counseling. Attitudes were largely independent of professional background and sociodemographic characteristics, though a correlation was found with personal interest in genetic self-testing for the above genes. Adverse psychological impact and discrimination in insurance and employment were the major concerns cited by respondents with regard to screening programs. Our findings suggest that the availability of measures for prevention and/or treatment is a major factor in the attitudes of healthcare providers toward population screening for late-onset conditions. Copyright © 2013 S. Karger AG, Basel.

  12. Mutation screening of the MECP2 gene in a large cohort of 613 fragile-X negative patients with mental retardation.

    Science.gov (United States)

    Lesca, Gaëtan; Bernard, Virginie; Bozon, Muriel; Touraine, Renaud; Gérard, Daniel; Edery, Patrick; Calender, Alain

    2007-01-01

    Mental retardation affects 2 to 3% of the population and is marked by significant etiological heterogeneity, including genetic and non genetic causes. FRAXA (FMR1) trinucleotide expansion is widely searched in routine screening, but found in only about 2% of the patients tested. Mutations of the MECP2 (methyl-CpG-binding protein) gene mainly cause Rett syndrome but were also shown to be involved in mental retardation. This study aimed to estimate the frequency of MECP2 gene mutations in a large group of mentally retarded patients without FRAXA expansion. Screening by heteroduplex analysis and SSCP followed by DNA sequencing of shifted bands were performed on 613 patients, including 442 males and 171 females. Eleven sequence variants were found, including nine polymorphisms. The two others may be pathogenetic. The first one, the double nucleotide substitution c.1162_1163delinsTA leading to a premature stop codon (p.Pro388X) was found in a female patient with random X-inactivation, presenting with borderline mental impairment without any features of Rett syndrome. The second one, the c.679C>G substitution, changing a glutamine to a glutamate in the transcriptional repression functional domain (p.Gln227Glu), was found in a female patient with a moderately biased X-chromosome inactivation profile and presenting with mild intellectual delay and minor psychotic features. The low mutation rate suggests that a large-scale routine screening for MECP2 in mentally retarded subjects is not cost-effective in clinical practice. Screening may be improved by a pre-selection based on clinical features that remain to be established.

  13. Sanger Sequencing for BRCA1 c.68_69del, BRCA1 c.5266dup and BRCA2 c.5946del Mutation Screen on Pap Smear Cytology Samples

    Directory of Open Access Journals (Sweden)

    Sin Hang Lee

    2016-02-01

    Full Text Available Three sets of polymerase chain reaction (PCR primers were designed for heminested PCR amplification of the target DNA fragments in the human genome which include the site of BRCA1 c.68_69del, BRCA1 c.5266dup and BRCA2 c.5946del respectively, to prepare the templates for direct Sanger sequencing screen of these three founder mutations. With a robust PCR mixture, crude proteinase K digestate of the fixed cervicovaginal cells in the liquid-based Papanicolaou (Pap cytology specimens can be used as the sample for target DNA amplification without pre-PCR DNA extraction, purification and quantitation. The post-PCR products can be used directly as the sequencing templates without further purification or quantitation. By simplifying the frontend procedures for template preparation, the cost for screening these three founder mutations can be reduced to about US $200 per test when performed in conjunction with human papillomavirus (HPV assays now routinely ordered for cervical cancer prevention. With this projected price structure, selective patients in a high-risk population can be tested and each provided with a set of DNA sequencing electropherograms to document the absence or presence of these founder mutations in her genome to help assess inherited susceptibility to breast and ovarian cancer in this era of precision molecular personalized medicine.

  14. Frequency of EGFR mutations in non-small cell lung cancer patients: screening data from West Siberia.

    Science.gov (United States)

    Gervas, Polina; Ivanova, Anna; Vasiliev, Nikolay; Ananina, Olga; Zharkova, Olga; Rogovieva, Olga; Verzhbitskaya, Natalia; Didichuk, Ivan; Cheremisina, Olga; Popova, Natalia; Goldberg, Victor; Cherdyntsev, Evgeny; Choynzonov, Evgeny; Cherdyntseva, Nadezda

    2015-01-01

    Incorporation of molecular analysis of the epidermal growth factor receptor (EGFR) gene into routine clinical practice has shown great promise to provide personalized therapy of the non-small cell lung cancer (NSCLC) in the developed world. However, the genetic testing of EGFR mutations has not yet become routine clinical practice in territories remote from the central regions of Russia. Therefore, we aimed to study the frequency of major types of activating mutations of the EGFR gene in NSCLC patients residing in West Siberia. We examined EGFR mutations in exons 19 and 21 in 147 NSCLC patients (excluding squamous cell lung carcinomas) by real time polymerase chain reaction. EGFR mutations were detected in 28 of the 147 (19%) patients. There were 19 (13%) cases with mutations in exon 19 and 9 cases (6%) in exon 21. Mutations were more frequently observed in women (42%, p=0.000) than in men (1%). A significantly higher incidence of EGFR mutations was observed in bronchioloalveolar carcinomas (28%, p=0.019) and in adenocarcinomas (21%, p=0.024) than in large cell carcinomas, mixed adenocarcinomas, and NOS (4%). The EGFR mutation rate was much higher in never-smokers than in smokers: 38% vs. 3% (p=0.000). The frequency of EGFR mutations in the Kemerovo and Tomsk regions was 19%. The incorporation of molecular analysis of the EGFR gene into routine clinical practice will allow clinicians to provide personalised therapy, resulting in a significant increase in survival rates and improvement in life quality of advanced NSCLC patients.

  15. Screening for MYO15A Gene Mutations in Autosomal Recessive Nonsyndromic, GJB2 Negative Iranian Deaf Population

    OpenAIRE

    Fattahi, Zohreh; Shearer, A Eliot; Babanejad, Mojgan; Bazazzadegan, Niloofar; Almadani, Seyed Navid; Nikzat, Nooshin; Jalalvand, Khadijeh; Arzhangi, Sanaz; Esteghamat, Fatemehsadat; Abtahi, Rezvan; Azadeh, Batool; Smith, Richard J. H.; Kahrizi, Kimia; Najmabadi, Hossein

    2012-01-01

    MYO15A is located at the DFNB3 locus on chromosome 17p11.2, and encodes myosin-XV, an unconventional myosin critical for the formation of stereocilia in hair cells of cochlea. Recessive mutations in this gene lead to profound autosomal recessive nonsyndromic hearing loss (ARNSHL) in humans and the shaker2 (sh2) phenotype in mice. Here, we performed a study on 140 Iranian families in order to determine mutations causing ARNSHL. The families, who were negative for mutations in GJB2, were subjec...

  16. Screening for Fabry disease in patients undergoing dialysis for chronic renal failure in Turkey: identification of new case with novel mutation.

    Science.gov (United States)

    Okur, Ilyas; Ezgu, Fatih; Biberoglu, Gursel; Tumer, Leyla; Erten, Yasemin; Isitman, Muzeyyen; Eminoglu, Fatma Tuba; Hasanoglu, Alev

    2013-09-15

    Chronic renal failure (CRF) is a serious complication of Fabry disease (FD). The aims of the present study were to determine the prevalence of unrecognized FD in Turkish hemodialysis population and to investigate the molecular background. Primarily, α-galactosidase A (α-Gal A) activity was investigated on DBS in 1136 patients of both sexes who underwent dialysis for CRF in Turkey. The disease was confirmed by analyzing enzyme activity in leukocyte and GLA gene sequencing in all patients in whom α-Gal A level was 40% of normal or less. Mean age of the patients (44.5% female, 52.5% male) was 56.46±15.85 years. Enzyme activity was found low with DBS method in 12 patients (four males, eight females). Two men, but no women, were diagnosed with FD by enzymatic and molecular analysis. In consequence of genetic analysis of a case, a new mutation [hemizygote c.638C>T (p.P214S) missense mutation in exon 5] was identified, which was not described in literature. Family screening of cases identified six additional cases. As a result of this initial screening study performed on hemodialysis patients for the first time with DBS method in Turkey, the prevalence of FD was detected as 0.17%. Although the prevalence seems to be low, screening studies are of great importance for detecting hidden cases as well as for identifying other effected family members. Copyright © 2013 Elsevier B.V. All rights reserved.

  17. A genetic screen for modifiers of Drosophila decapentaplegic signaling identifies mutations in punt, Mothers against dpp and the BMP-7 homologue, 60A.

    Science.gov (United States)

    Chen, Y; Riese, M J; Killinger, M A; Hoffmann, F M

    1998-05-01

    decapentaplegic (dpp) is a Transforming Growth Factor beta (TGF-beta)-related growth factor that controls multiple developmental processes in Drosophila. To identify components involved in dpp signaling, we carried out a genetic screen for dominant enhancer mutations of a hypomorphic allele of thick veins (tkv), a type I receptor for dpp. We recovered new alleles of tkv, punt, Mothers against dpp (Mad) and Medea (Med), all of which are known to mediate dpp signaling. We also recovered mutations in the 60A gene which encodes another TGF-beta-related factor in Drosophila. DNA sequence analysis established that all three 60A alleles were nonsense mutations in the prodomain of the 60A polypeptide. These mutations in 60A caused defects in midgut morphogenesis and fat body differentiation. We present evidence that when dpp signaling is compromised, lowering the level of 60A impairs several dpp-dependent developmental processes examined, including the patterning of the visceral mesoderm, the embryonic ectoderm and the imaginal discs. These results provide the first in vivo evidence for the involvement of 60A in the dpp pathway. We propose that 60A activity is required to maintain optimal signaling capacity of the dpp pathway, possibly by forming biologically active heterodimers with Dpp proteins.

  18. Screening LGI1 in a cohort of 26 lateral temporal lobe epilepsy patients with auditory aura from Turkey detects a novel de novo mutation.

    Science.gov (United States)

    Kesim, Yesim F; Uzun, Gunes Altiokka; Yucesan, Emrah; Tuncer, Feyza N; Ozdemir, Ozkan; Bebek, Nerses; Ozbek, Ugur; Iseri, Sibel A Ugur; Baykan, Betul

    2016-02-01

    Autosomal dominant lateral temporal lobe epilepsy (ADLTE) is an autosomal dominant epileptic syndrome characterized by focal seizures with auditory or aphasic symptoms. The same phenotype is also observed in a sporadic form of lateral temporal lobe epilepsy (LTLE), namely idiopathic partial epilepsy with auditory features (IPEAF). Heterozygous mutations in LGI1 account for up to 50% of ADLTE families and only rarely observed in IPEAF cases. In this study, we analysed a cohort of 26 individuals with LTLE diagnosed according to the following criteria: focal epilepsy with auditory aura and absence of cerebral lesions on brain MRI. All patients underwent clinical, neuroradiological and electroencephalography examinations and afterwards they were screened for mutations in LGI1 gene. The single LGI1 mutation identified in this study is a novel missense variant (NM_005097.2: c.1013T>C; p.Phe338Ser) observed de novo in a sporadic patient. This is the first study involving clinical analysis of a LTLE cohort from Turkey and genetic contribution of LGI1 to ADLTE phenotype. Identification of rare LGI1 gene mutations in sporadic cases supports diagnosis as ADTLE and draws attention to potential familial clustering of ADTLE in suggestive generations, which is especially important for genetic counselling. Copyright © 2015 Elsevier B.V. All rights reserved.

  19. Urine screening for patients with developmental disabilities detected a patient with creatine transporter deficiency due to a novel missense mutation in SLC6A8.

    Science.gov (United States)

    Kato, Hidekazu; Miyake, Fuyu; Shimbo, Hiroko; Ohya, Makoto; Sugawara, Hidenori; Aida, Noriko; Anzai, Rie; Takagi, Mariko; Okuda, Mitsuko; Takano, Kyoko; Wada, Takahito; Iai, Mizue; Yamashita, Sumimasa; Osaka, Hitoshi

    2014-08-01

    Creatine transporter deficiency (CTD) is an example of X-linked intellectual disability syndromes, caused by mutations in SLC6A8 on Xq28. Although this is the second most frequent genetic cause of intellectual disabilities in Europe or America after Fragile X syndrome, information on the morbidity of this disease is limited in Japan. Using the HPLC screening method we have established recently, we examined samples of urine of 105 patients (73 males and 32 females) with developmental disabilities at our medical center. And we have found a family with three ID boys with a novel missense mutation in SLC6A8. This is the second report of a Japanese family case of CTD. A systematic diagnostic system of this syndrome should be established in Japan to enable us to estimate its frequency and treatment. Copyright © 2013 The Japanese Society of Child Neurology. Published by Elsevier B.V. All rights reserved.

  20. Screening for ATM Mutations in an African-American Population to Identify a Predictor of Breast Cancer Susceptibility

    National Research Council Canada - National Science Library

    Rosenstein, Barry S

    2006-01-01

    ... ATM haplotype compared to African-American women without breast cancer. An additional objective is to determine the functional impact upon the protein encoded by the ATM gene for each mutation identified. Specific Aims...

  1. [Mutation screening and prenatal diagnosis of methylmalonic academia in a Chinese pedigree by Ion Torrent semiconductor sequencing].

    Science.gov (United States)

    Li, Li; Ma, Dingyuan; Sun, Yun; Zhang, Jingjing; Wang, Yuguo; Jiang, Tao; Xu, Zhengfeng

    2016-04-01

    To identify pathogenic mutations in a Chinese pedigree affected with methylmalonic academia for genetic counseling and prenatal diagnosis. Molecular analysis of the MUT, MMACHC, MMAA and MMAB genes was performed for the proband with methylmalonic academia by Ion Torrent semiconductor sequencing. Candidate mutations were validated by Sanger sequencing. The couple was offered prenatal diagnosis via analyzing of the fetal DNA through amniocentesis. The proband was found to be compound heterozygous for c.609G>A (p.Trp203X) and c.658-660del AAG (p.Lys220del) mutations, which were inherited respectively from each of his parents. Prenatal diagnosis showed that the fetus has inherited two wild-type parental alleles. The targeted Ion Torrent PGM sequencing has detected pathogenic mutations in the Chinese pedigree affected with methylmalonic academia, which has provided molecular evidence for clinical diagnosis, genetic counseling and prenatal diagnosis for the family.

  2. Targeted prostate cancer screening in BRCA1 and BRCA2 mutation carriers: Results from the initial screening round of the IMPACT study

    NARCIS (Netherlands)

    E. Bancroft (Elizabeth); E. Page (Elizabeth); E. Castro (Elena); H. Lilja (Hans); A.J. Vickers (Andrew); D. Sjoberg (Daniel); M. Assel (Matthias); C.S. Foster; G. Mitchell (Gillian); K. Drew (Kate); L. Maehle; K. Axcrona; D.G. Evans (Gareth); B. Bulman; D. Eccles (Diana); D. McBride (Donna); C.J. van Asperen (Christi); H. Vasen (Hans); L.A.L.M. Kiemeney (Bart); J. Ringelberg (Janneke); C. Cybulski (Cezary); D. Wokolorczyk (Dominika); C.G. Selkirk (Christina); P.J. Hulick (Peter); S.E. Bojesen (Stig); A.-B. Skytte (Anne-Bine); J. Lam (Jimmy); L. Taylor (Lorne); R.A. Oldenburg (Rogier); R.G.H.M. Cremers; G. Verhaegh (Gerald); W.A. van Zelst-Stams; J.C. Oosterwijk (Jan); I. Blanco (Ignacio); M. Salinas; J. Cook (Jackie); K. Rosario (Karyna); S.S. Buys (Saundra); T. Conner (Tom); M.G.E.M. Ausems (Margreet); K.-R. Ong (Kai-Ren); J. Hoffman (Jonathan); S.M. Domchek (Susan); J. Powers (Jacquelyn); P.J. Teixeira; S. Maia (Sofia); W.D. Foulkes (William); N. Taherian (Nassim); M.W.G. Ruijs (Marielle); A.T.H.-V. den Enden (Apollonia T. Helderman-Van); L. Izatt (Louise); R. Davidson (Rosemarie); M.A. Adank (Muriel); L.J. Walker (Lisa); R.K. Schmutzler (Rita); K. Tucker (Kathryn); J. Kirk (Judy); S.V. Hodgson (Shirley); M. Harris (Marion); F. Douglas (Fiona); G.J. Lindeman; J. Zgajnar (Janez); M. Tischkowitz (Marc); V. Clowes; R. Susman (Rachel); T. Ramon Y Cajal; N. Patcher (Nicholas); N. Gadea (Neus); A. Spigelman; T.A.M. van Os (Theo); A. Liljegren (Annelie); L. Side (Lucy); C. Brewer (Carole); A.F. Brady (Angela); A. Donaldson (Alan); V. Stefansdottir (Vigdis); E. Friedman (Eitan); R. Chen-Shtoyerman (Rakefet); D. Amor (David); L. Copakova (Lucia); J. Barwell (Julian); V.N. Giri (Veda); A.C. Murthy (Adeline C.); N. Nicolai (Nicola); S.-H. Teo; T. Greenhalgh (Trisha); S. Strom; A. Henderson (Alex); J. McGrath (John); D. Gallagher (David); N.K. Aaronson (Neil); A. Ardern-Jones (Audrey); C.H. Bangma (Chris); D. Dearnaley (David); A. Costello (Anthony); J. Eyfjord; J. Rothwell (Jeanette); A. Falconer; H. Grönberg (Henrik); F. Hamdy (Freddie); O.T. Johannson (Oskar); V. Khoo; Z. Kote-Jarai; J. Lubinski (Jan); K. Axcrona; J. Melia; E.T. McKinley (Enid); A. Mitra (Anita); C. Moynihan; G. Rennert (Gad); M. Suri (Mohnish); P. Wilson; S.R. Killick; S.M. Moss (Sue); R. Eeles (Rosalind)

    2014-01-01

    textabstractBackground Men with germline breast cancer 1, early onset (BRCA1) or breast cancer 2, early onset (BRCA2) gene mutations have a higher risk of developing prostate cancer (PCa) than noncarriers. IMPACT (Identification of Men with a genetic predisposition to ProstAte Cancer: Targeted

  3. 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.

  4. High-Throughput Genetic Screening of 51 Pediatric Cataract Genes Identifies Causative Mutations in Inherited Pediatric Cataract in South Eastern Australia

    Directory of Open Access Journals (Sweden)

    Shari Javadiyan

    2017-10-01

    Full Text Available Pediatric cataract is a leading cause of childhood blindness. This study aimed to determine the genetic cause of pediatric cataract in Australian families by screening known disease-associated genes using massively parallel sequencing technology. We sequenced 51 previously reported pediatric cataract genes in 33 affected individuals with a family history (cases with previously known or published mutations were excluded using the Ion Torrent Personal Genome Machine. Variants were prioritized for validation if they were predicted to alter the protein sequence and were absent or rare with minor allele frequency 60% of familial pediatric cataract in Australia, indicating that still more causative genes remain to be identified.

  5. Estimation of Wilson's disease incidence and carrier frequency in the Korean population by screening ATP7B major mutations in newborn filter papers using the SYBR green intercalator method based on the amplification refractory mutation system.

    Science.gov (United States)

    Kim, Gu-Hwan; Yang, Jeong Yoon; Park, Jung-Young; Lee, Jin Joo; Kim, Ju Hyun; Yoo, Han-Wook

    2008-09-01

    Wilson's disease (WD), an autosomal recessive disorder of copper transport, is one of the most common inherited metabolic disorders in Korea. Despite its frequency, the incidence and carrier frequency of WD has not yet been estimated in the Korean population. We therefore screened for four major missense mutations (p.Arg778Leu, p.Ala874Val, p.Leu1083Phe, and p.Asn1270Ser) of the ATP7B gene in 476 newborn filter papers by real-time multiplex PCR and melting curve analysis using the SYBR Green intercalator method based on the amplification refractory mutation system test. Newborn filter papers with abnormal melting curves were subjected to subsequent sequence analysis. Three mutated alleles, one p.Arg778Leu and two p.Ala874Val, were detected among the 476 newborn filter papers (952 alleles). The carrier frequency and incidence of WD in the Korean population were determined as 1 in 88.2 and 30,778, respectively, by reversely calculating based on the Hardy-Weinberg law.

  6. Screening for mutations in 17β-hydroxysteroid dehydrogenase and androgen receptor in women presenting with partially virilised 46,XY disorders of sex development.

    Science.gov (United States)

    Phelan, Niamh; Williams, Emma L; Cardamone, Stefanie; Lee, Marilyn; Creighton, Sarah M; Rumsby, Gill; Conway, Gerard S

    2015-06-01

    The precise diagnosis of partially virilised women with 46,XY disorders of sex development (DSD) is often obscure. In practice, this group often comes under the poorly defined, clinically based label of partial androgen insensitivity syndrome (PAIS). In a previous study, we found that 5α-reductase 2 (SRD5A2) mutations occurred in 43% of women in this subgroup. We expand this work to include biochemical and genetic screening for 17β-hydroxysteroid dehydrogenase (HSD17B3) and androgen receptor (AR) mutations. Analysis of serum androgens (androstenedione and testosterone) and genetic analyses for HSD17B3 and AR were performed in 42 women from 36 pedigrees with partially virilised 46,XY DSD in whom SRD5A2 deficiency had been excluded by urine steroid profiling. Out of 36 unrelated women, 14 (38%) were found to have HSD17B3 mutations and one (2.7%) to have an AR defect. Six novel pathogenic HSD17B3 mutations were identified: three splice site mutations and three missense changes. Seven patients with HSD17B3 deficiency tested before gonadectomy had basal testosterone/androstenedione (T/A) ratio XY female DSD population and is often associated with lesser degrees of virilisation compared with those with 5α-reductase deficiency. This diagnosis should be considered for individuals labelled as PAIS, particularly, but not exclusively, those who present with virilisation at puberty or primary amenorrhoea. Before gondadectomy, T/A ratio is useful to aid diagnosis, but after gonadectomy sequencing of HSD17B3 must be performed to confirm the diagnosis. © 2015 European Society of Endocrinology.

  7. High Resolution Melting (HRM) analysis for mutation screening of RGSL1,RGS16 and RGS8 in breast cancer

    DEFF Research Database (Denmark)

    Wiechec, Emilia; Wiuf, Carsten Henrik; Overgaard, Jens

    2011-01-01

    Abstract BACKGROUND: Identification of specific mutation targets in cancer may lead to discovery of the genes modulating cancer susceptibility and/or prognosis. The RGSL1, RGS16, and RGS8 genes within the 1q25.3 region belong to the novel family of regulators of G protein signaling (RGS) genes...... coding exons of RGSL1, RGS16, and RGS8 in tumors from 200 breast cancer patients. All sequence variants detected by HRM resulted in abnormal shape of the melting curves. The identified mutations and known single nucleotide polymorphisms (SNP) were subsequently confirmed by sequencing, and distribution...... cancer patients. In addition, a total of seven known SNPs were identified in this study. Genotype distributions were not significantly different between breast cancer patients and controls. CONCLUSIONS AND IMPACT: Identification of novel mutations within RGSL1 provides a new insight...

  8. Clinical Application of Screening for GJB2 Mutations before Cochlear Implantation in a Heterogeneous Population with High Rate of Autosomal Recessive Nonsyndromic Hearing Loss

    Directory of Open Access Journals (Sweden)

    Masoud Motasaddi Zarandy

    2011-01-01

    Full Text Available Clinical application of mutation screening and its effect on the outcome of cochlear implantation is widely debated. We investigated the effect of mutations in GJB2 gene on the outcome of cochlear implantation in a population with a high rate of consanguineous marriage and autosomal recessive nonsyndromic hearing loss. Two hundred and one children with profound prelingual sensorineural hearing loss were included. Forty-six patients had 35delG in GJB2. Speech awareness thresholds (SATs and speech recognition thresholds (SRTs improved following implantation, but there was no difference in performance between patients with GJB2-related deafness versus control (all >0.10. Both groups had produced their first comprehensible words within the same period of time following implantation (2.27 months in GJB2-related deaf versus 2.62 months in controls, =0.22. Although our findings demonstrate the need to uncover unidentified genetic causes of hereditary deafness, they do not support the current policy for genetic screening before cochlear implantation, nor prove a prognostic value.

  9. Glucose-6-Phosphate Dehydrogenase (G6PD) deficiency in Greek newborns: the Mediterranean C563T mutation screening.

    Science.gov (United States)

    Molou, Elina; Schulpis, Kleopatra H; Thodi, Georgia; Georgiou, Vassiliki; Dotsikas, Yannis; Papadopoulos, Konstantinos; Biti, Sofia; Loukas, Yannis L

    2014-04-01

    Glucose-6-Phosphate Dehydrogenase (G6PD) gene is located at the X-chromosome at Xq28 and the disease is recessively inherited predominantly in males. More than 400 variants have been proposed based on clinical and enzymatic studies. The aim of the current study was to identify C563T mutation in G6PD-deficient newborns and to correlate the enzyme residual activity with the presence of the mutation. Some 1189 full-term neonates aged 3-5 days old were tested for G6PD activity in dried blood spots from Guthrie cards using a commercial kit. DNA extraction from Guthrie cards and mutation identification among the deficient samples were performed with current techniques. A total of 92 (7.7%) newborns were G6PD-deficient. In 46 (50%), the mutation C563T was identified. The residual activity in C563T hemizygote males (n = 28) was statistically significantly lower (1.23 ± 0.93 U/g Hb) than that in non-C563T G6PD-deficient males (n = 25) (4.01 ± 1.20 U/g Hb, p G6PD deficiency and severe neonatal jaundice. G6PD activity showed statistically significant correlation with total bilirubin blood levels.

  10. Mutation-Guided Unbiased Modeling of the Fat Sensor GPR119 for High-Yield Agonist Screening

    DEFF Research Database (Denmark)

    Norn, Christoffer; Pedersen, Maria Hauge; Engelstoft, Maja S.

    2015-01-01

    multiple X-ray receptor structures in combination with a fully flexible ligand docking protocol to det. the binding conformation of AR231453, a small-mol. agonist, in the GPR119 receptor. Resulting models converge to one conformation that explains the majority of data from mutation studies...

  11. Efficient molecular screening of Lynch syndrome by specific 3' promoter methylation of the MLH1 or BRAF mutation in colorectal cancer with high-frequency microsatellite instability.

    Science.gov (United States)

    Nakagawa, Hitoshi; Nagasaka, Takeshi; Cullings, Harry M; Notohara, Kenji; Hoshijima, Naoko; Young, Joanne; Lynch, Henry T; Tanaka, Noriaki; Matsubara, Nagahide

    2009-06-01

    It is sometimes difficult to diagnose Lynch syndrome by the simple but strict clinical criteria, or even by the definitive genetic testing for causative germline mutation of mismatch repair genes. Thus, some practical and efficient screening strategy to select highly possible Lynch syndrome patients is exceedingly desirable. We performed a comprehensive study to evaluate the methylation status of whole MLH1 promoter region by direct bisulfite sequencing of the entire MLH1 promoter regions on Lynch and non-Lynch colorectal cancers (CRCs). Then, we established a convenient assay to detect methylation in key CpG islands responsible for the silencing of MLH1 expression. We studied the methylation status of MLH1 as well as the CpG island methylator phenotype (CIMP) and immunohistochemical analysis of mismatch repair proteins on 16 cases of Lynch CRC and 19 cases of sporadic CRCs with high-frequency microsatellite instability (MSI-H). Sensitivity to detect Lynch syndrome by MLH1 (CCAAT) methylation was 88% and the specificity was 84%. Positive likelihood ratio (PLR) was 5.5 and negative likelihood ratio (NLR) was 0.15. Sensitivity by mutational analysis of BRAF was 100%, specificity was 84%, PLR was 6.3 and NLR was zero. By CIMP analysis; sensitivity was 88%, specificity was 79%, PLR was 4.2, and NLR was 0.16. BRAF mutation or MLH1 methylation analysis combined with MSI testing could be a good alternative to screen Lynch syndrome patients in a cost effective manner. Although the assay for CIMP status also showed acceptable sensitivity and specificity, it may not be practical because of its rather complicated assay.

  12. In-silico identification of inhibitors against mutated BCR-ABL protein of chronic myeloid leukemia: a virtual screening and molecular dynamics simulation study.

    Science.gov (United States)

    Kumar, Himansu; Raj, Utkarsh; Gupta, Saurabh; Varadwaj, Pritish Kumar

    2016-10-01

    Aberrant and proliferative expression of the oncogene BCR-ABL in the bone marrow cells had been proven as the prime cause of chronic myeloid leukemia (CML). It has been established that tyrosine kinase domain of BCR-ABL protein is a potential therapeutic target for the treatment of CML. Imatinib is considered as a first-generation drug that can inhibit the enzymatic action by inhibiting the ATP binding with BCR-ABL protein. Later on, insensitivity of CML cells towards Imatinib has been observed may be due to mutation in tyrosine kinase domain of the ABL receptor. Subsequently, some other second-generation drugs have also been reported viz. Baustinib, Nilotinib, Dasatinib, Ponatinib, Bafetinib, etc., which can able to combat against mutated domain of ABL tyrosine kinase protein. By taking into account of bioavailability and resistance developed, there is an utmost need to find some more inhibitors for the mutated ABL tyrosine kinase protein. For virtual screening, a data-set has been generated by collecting the all available drug like natural compounds from ZINC and Drug Bank databases. Comparative docking analysis was also carried out on the active site of ABL tyrosine kinase receptor with reported reference inhibitors. Molecular dynamics simulation of the best screened interacting complex was done for 50 ns to validate the stability of the system. These selected inhibitors were further validated and analyzed through pharmacokinetics properties and series of ADMET parameters by in silico methods. Considering the above said parameters proposed molecules are concluded as potential leads for drug designing pipeline against CML.

  13. An RNA-based signature enables high specificity detection of circulating tumor cells in hepatocellular carcinoma.

    Science.gov (United States)

    Kalinich, Mark; Bhan, Irun; Kwan, Tanya T; Miyamoto, David T; Javaid, Sarah; LiCausi, Joseph A; Milner, John D; Hong, Xin; Goyal, Lipika; Sil, Srinjoy; Choz, Melissa; Ho, Uyen; Kapur, Ravi; Muzikansky, Alona; Zhang, Huidan; Weitz, David A; Sequist, Lecia V; Ryan, David P; Chung, Raymond T; Zhu, Andrew X; Isselbacher, Kurt J; Ting, David T; Toner, Mehmet; Maheswaran, Shyamala; Haber, Daniel A

    2017-01-31

    Circulating tumor cells (CTCs) are shed into the bloodstream by invasive cancers, but the difficulty inherent in identifying these rare cells by microscopy has precluded their routine use in monitoring or screening for cancer. We recently described a high-throughput microfluidic CTC-iChip, which efficiently depletes hematopoietic cells from blood specimens and enriches for CTCs with well-preserved RNA. Application of RNA-based digital PCR to detect CTC-derived signatures may thus enable highly accurate tissue lineage-based cancer detection in blood specimens. As proof of principle, we examined hepatocellular carcinoma (HCC), a cancer that is derived from liver cells bearing a unique gene expression profile. After identifying a digital signature of 10 liver-specific transcripts, we used a cross-validated logistic regression model to identify the presence of HCC-derived CTCs in nine of 16 (56%) untreated patients with HCC versus one of 31 (3%) patients with nonmalignant liver disease at risk for developing HCC (P digital CTC scoring was not correlated with the standard HCC serum protein marker alpha fetoprotein (P = 0.57). Modeling the sequential use of these two orthogonal markers for liver cancer screening in patients with high-risk cirrhosis generates positive and negative predictive values of 80% and 86%, respectively. Thus, digital RNA quantitation constitutes a sensitive and specific CTC readout, enabling high-throughput clinical applications, such as noninvasive screening for HCC in populations where viral hepatitis and cirrhosis are prevalent.

  14. Prevalence and mutations of β-thalassemia trait and abnormal hemoglobins in premarital screening in Çanakkale province, Turkey

    Directory of Open Access Journals (Sweden)

    Uludağ A

    2016-06-01

    Full Text Available The prevalence of β-thalassemia (β-thal carriers in Turkey varies according to region but in general it is 2.0%. Çanakkale is a city in the Aegean region of Turkey but no study about β-thal frequency in Çanakkale has been published to date. In this study, we aimed to investigate the frequency of β-thal mutations in this province. A total of 4452 couples (8904 individuals applied for premarital thalassemia scans at the Çanakkale State Health Directorate Laboratory between January 2008 and June 2012 and scanning was done with high performance liquid chromatography (HPLC. Of 125 β-thal carriers seen at the Medical Genetics Clinic, Çanakkale Onsekiz Mart University, Çanakkale, Turkey, for genetic counseling, 46 participated in the study. The remaining 79 patients could not be reached. The prevalence for β-thal carriers in Çanakkale was identified as 1.4% (125/8904. One couple were both β-thal carriers. β-Globin gene analysis of 46 carriers found the total frequency of the three most common mutations was 45.6%. These mutations were found to be HBB: c.93-21G>A [IVS-I-110 (G>A], 26.08% (12/46; HBB: c.17_ 18delCT [codon 5 (‒CT], 10.85% (5/46; HBB: c.20delA [codon 6 (‒A] 8.69% (4/46. This is the first report on the frequency and mutation profiles of β-thal for Çanakkale. The incidence of β-thal carriers in Çanakkale is below the average for Turkey. The most frequently observed mutation profile and rate of β-thal in our region is different from the other regions of Turkey.

  15. Screening for germline phosphatase and tensin homolog-mutations in suspected Cowden syndrome and Cowden syndrome-like families among uterine cancer patients.

    Science.gov (United States)

    Tzortzatos, Gerasimos; Aravidis, Christos; Lindblom, Annika; Mints, Miriam; Tham, Emma

    2015-04-01

    Cowden syndrome (CS) is an autosomal dominant disorder characterized by multiple hamartomas in the breast, thyroid and endometrium, with a prevalence of 1 per 250,000. Females with CS have a 21-28% lifetime risk of developing uterine cancer. Germline mutations in the phosphatase and tensin homolog (PTEN) gene, a tumor suppressor gene, are responsible for 30-80% of CS cases. PTEN is a nine-exon gene, located on chromosome 10q23.3, which encodes the 403 amino acid PTEN protein. It negatively regulates the phosphoinositide 3-kinase/protein kinase B/mammalian target of rapamycin pathway, affecting various cellular processes and signaling pathways. The present study examined whether PTEN mutations are present in CS-like families with uterine cancer (UC). UC patients underwent surgery at Karolinska University Hospital, Stockholm, Sweden (2008-2012). Pedigrees were analyzed and 54 unrelated CS-like families were identified. CS-like families were defined as having at least one occurrence of uterine cancer and one of breast cancer, as well as at least one additional Cowden-associated tumor (uterine, breast, thyroid, colon or kidney cancer) in the same individual or in first-degree relatives. Genomic DNA was amplified using polymerase chain reaction, and DNA sequencing analysis of all nine exons of the PTEN gene was conducted. No germline PTEN mutations or polymorphisms were identified. Germline PTEN mutations are rare in CS-like families with uterine cancer, therefore, genetic screening must be restricted to patients that meet the strict National Comprehensive Cancer Network criteria. Gynecologists must be aware of the CS criteria and identify potential cases of CS in females where uterine cancer is the sentinel cancer.

  16. Excited State Dynamics of DNA and RNA bases

    Science.gov (United States)

    Hudock, Hanneli; Levine, Benjamin; Martinez, Todd

    2007-03-01

    Recent ultrafast spectroscopic experiments have reported excited state lifetimes for DNA and RNA bases and assigned these lifetimes to various electronic states. We have used theoretical and simulation methods to describe the excited state dynamics of these bases in an effort to provide a mechanistic explanation for the observed lifetimes. Our simulations are based on ab initio molecular dynamics, where the electronic and nuclear Schrodinger equations are solved simultaneously. The results are further verified by comparison to high-level ab initio electronic structure methods, including dynamic electron correlation effects through multireference perturbation theory, at important points along the dynamical pathways. Our results provide an explanation of the photochemical mechanism leading to nonradiative decay of the electronic excited states and some suggestions as to the origin of the different lifetimes. Comparisons between pyrimidines illustrate how chemical differences impact excited state dynamics and may play a role in explaining the propensity for dimer formation in thymine.

  17. Screening for BRCA1 and BRCA2 mutations in breast cancer patients from Mexico: the public health perspective.

    Science.gov (United States)

    Narod, Steven A

    2009-01-01

    Genetic testing for mutations in BRCA1 and BRCA2 has potentially important public health implications. Through judicious testing of women believed to be at high risk for early-onset breast cancer and for ovarian cancer, it is possible to identify highly-predisposed women prior to the development of cancer. Current preventive options include preventive mastectomy, preventive oophorectomy, tamoxifen and oral contraceptives. The ability to offer genetic testing in Mexico on a widespread level is enhanced if the common founder mutations in the two genes can be discovered or if the cost of genetic sequencing is reduced. It is important that a genetic testing service be a multi-disciplinary effort with co-ordinated follow-up.

  18. Systematic screening for mutations in the promoter and the coding region of the 5-HT{sub 1A} gene

    Energy Technology Data Exchange (ETDEWEB)

    Erdmann, J.; Shimron-Abarbanell, D.; Cichon, S. [Univ. of Bonn (Germany)] [and others

    1995-10-09

    In the present study we sought to identify genetic variation in the 5-HT{sub 1A} receptor gene which through alteration of protein function or level of expression might contribute to the genetic predisposition to neuropsychiatric diseases. Genomic DNA samples from 159 unrelated subjects (including 45 schizophrenic, 46 bipolar affective, and 43 patients with Tourette`s syndrome, as well as 25 healthy controls) were investigated by single-strand conformation analysis. Overlapping PCR (polymerase chain reaction) fragments covered the whole coding sequence as well as the 5{prime} untranslated region of the 5-HT{sub 1A} gene. The region upstream to the coding sequence we investigated contains a functional promoter. We found two rare nucleotide sequence variants. Both mutations are located in the coding region of the gene: a coding mutation (A{yields}G) in nucleotide position 82 which leads to an amino acid exchange (Ile{yields}Val) in position 28 of the receptor protein and a silent mutation (C{yields}T) in nucleotide position 549. The occurrence of the Ile-28-Val substitution was studied in an extended sample of patients (n = 352) and controls (n = 210) but was found in similar frequencies in all groups. Thus, this mutation is unlikely to play a significant role in the genetic predisposition to the diseases investigated. In conclusion, our study does not provide evidence that the 5-HT{sub 1A} gene plays either a major or a minor role in the genetic predisposition to schizophrenia, bipolar affective disorder, or Tourette`s syndrome. 29 refs., 4 figs., 1 tab.

  19. Genomic sequencing in cystic fibrosis newborn screening: what works best, two-tier predefined CFTR mutation panels or second-tier CFTR panel followed by third-tier sequencing?

    Science.gov (United States)

    Currier, Robert J; Sciortino, Stan; Liu, Ruiling; Bishop, Tracey; Alikhani Koupaei, Rasoul; Feuchtbaum, Lisa

    2017-10-01

    PurposeThe purpose of this study was to model the performance of several known two-tier, predefined mutation panels and three-tier algorithms for cystic fibrosis (CF) screening utilizing the ethnically diverse California population.MethodsThe cystic fibrosis transmembrane conductance regulator (CFTR) mutations identified among the 317 CF cases in California screened between 12 August 2008 and 18 December 2012 were used to compare the expected CF detection rates for several two- and three-tier screening approaches, including the current California approach, which consists of a population-specific 40-mutation panel followed by third-tier sequencing when indicated.ResultsThe data show that the strategy of using third-tier sequencing improves CF detection following an initial elevated immunoreactive trypsinogen and detection of only one mutation on a second-tier panel.ConclusionIn a diverse population, the use of a second-tier panel followed by third-tier CFTR gene sequencing provides a better detection rate for CF, compared with the use of a second-tier approach alone, and is an effective way to minimize the referrals of CF carriers for sweat testing. Restricting screening to a second-tier testing to predefined mutation panels, even broad ones, results in some missed CF cases and demonstrates the limited utility of this approach in states that have diverse multiethnic populations.

  20. The Y42H mutation in medium-chain acyl-CoA dehydrogenase, which is prevalent in babies identified by MS/MS-based newborn screening, is temperature sensitive

    DEFF Research Database (Denmark)

    O'Reilly, Linda; Bross, Peter; Corydon, Thomas J

    2004-01-01

    variant. To distinguish between effects of temperature on folding/assembly and the stability of the native enzyme, the thermal stability of the variant proteins was studied after expression and purification by dye affinity chromatography. This showed that, compared with the wild-type enzyme......Medium-chain acyl-CoA dehydrogenase (MCAD) is a homotetrameric flavoprotein which catalyses the initial step of the beta-oxidation of medium-chain fatty acids. Mutations in MCAD may cause disease in humans. A Y42H mutation is frequently found in babies identified by newborn screening with MS....../MS, yet there are no reports of patients presenting clinically with this mutation. As a basis for judging its potential consequences we have examined the protein phenotype of the Y42H mutation and the common disease-associated K304E mutation. Our studies of the intracellular biogenesis of the variant...

  1. Genetic Panel Screening of Nearly 100 Mutations Reveals New Insights into the Breed Distribution of Risk Variants for Canine Hereditary Disorders

    Science.gov (United States)

    Donner, Jonas; Möller, Fredrik; Kyöstilä, Kaisa; Sankari, Satu; Hytönen, Marjo; Giger, Urs; Lohi, Hannes

    2016-01-01

    Background The growing number of identified genetic disease risk variants across dog breeds challenges the current state-of-the-art of population screening, veterinary molecular diagnostics, and genetic counseling. Multiplex screening of such variants is now technologically feasible, but its practical potential as a supportive tool for canine breeding, disease diagnostics, pet care, and genetics research is still unexplored. Results To demonstrate the utility of comprehensive genetic panel screening, we tested nearly 7000 dogs representing around 230 breeds for 93 disease-associated variants using a custom-designed genotyping microarray (the MyDogDNA® panel test). In addition to known breed disease-associated mutations, we discovered 15 risk variants in a total of 34 breeds in which their presence was previously undocumented. We followed up on seven of these genetic findings to demonstrate their clinical relevance. We report additional breeds harboring variants causing factor VII deficiency, hyperuricosuria, lens luxation, von Willebrand’s disease, multifocal retinopathy, multidrug resistance, and rod-cone dysplasia. Moreover, we provide plausible molecular explanations for chondrodysplasia in the Chinook, cerebellar ataxia in the Norrbottenspitz, and familiar nephropathy in the Welsh Springer Spaniel. Conclusions These practical examples illustrate how genetic panel screening represents a comprehensive, efficient and powerful diagnostic and research discovery tool with a range of applications in veterinary care, disease research, and breeding. We conclude that several known disease alleles are more widespread across different breeds than previously recognized. However, careful follow up studies of any unexpected discoveries are essential to establish genotype-phenotype correlations, as is readiness to provide genetic counseling on their implications for the dog and its breed. PMID:27525650

  2. Genetic Panel Screening of Nearly 100 Mutations Reveals New Insights into the Breed Distribution of Risk Variants for Canine Hereditary Disorders.

    Directory of Open Access Journals (Sweden)

    Jonas Donner

    Full Text Available The growing number of identified genetic disease risk variants across dog breeds challenges the current state-of-the-art of population screening, veterinary molecular diagnostics, and genetic counseling. Multiplex screening of such variants is now technologically feasible, but its practical potential as a supportive tool for canine breeding, disease diagnostics, pet care, and genetics research is still unexplored.To demonstrate the utility of comprehensive genetic panel screening, we tested nearly 7000 dogs representing around 230 breeds for 93 disease-associated variants using a custom-designed genotyping microarray (the MyDogDNA® panel test. In addition to known breed disease-associated mutations, we discovered 15 risk variants in a total of 34 breeds in which their presence was previously undocumented. We followed up on seven of these genetic findings to demonstrate their clinical relevance. We report additional breeds harboring variants causing factor VII deficiency, hyperuricosuria, lens luxation, von Willebrand's disease, multifocal retinopathy, multidrug resistance, and rod-cone dysplasia. Moreover, we provide plausible molecular explanations for chondrodysplasia in the Chinook, cerebellar ataxia in the Norrbottenspitz, and familiar nephropathy in the Welsh Springer Spaniel.These practical examples illustrate how genetic panel screening represents a comprehensive, efficient and powerful diagnostic and research discovery tool with a range of applications in veterinary care, disease research, and breeding. We conclude that several known disease alleles are more widespread across different breeds than previously recognized. However, careful follow up studies of any unexpected discoveries are essential to establish genotype-phenotype correlations, as is readiness to provide genetic counseling on their implications for the dog and its breed.

  3. Mutational screening of GLI3, SHH, preZRS, and ZRS in 102 Chinese children with nonsyndromic polydactyly.

    Science.gov (United States)

    Xiang, Ying; Jiang, Limin; Wang, Bo; Xu, Yunlan; Cai, Haiqing; Fu, Qihua

    2017-05-01

    Polydactyly is a group of congenital limb malformations that show high degree of phenotypic variability and genetic heterogeneity. In the present study, four genomic regions (exons of GLI3, SHH, and noncoding sequences of preZRS and ZRS) involved in hedgehog (Hh) signaling pathway were sequenced for 102 unrelated Chinese children with nonsyndromic polydactyly. Two GLI3 variants (c.2844 G > G/A; c.1486C > C/T) and four preZRS variants (chr7:156585336 A>G; chr7:156585421 C>A; chr7: 156585247 G>C; chr7:156585420 A > C) were observed in 2(2.0%) and 6(5.9%) patients, respectively. These variants are not over-represented in the Chinese healthy population. All the 8 cases showed preaxial polydactyly in hands. Additionally, no specific patterns of malformation predicted mutations in other candidate genes or sequences. This is the first report of the assessment of the frequency of GLI3/SHH/preZRS/ZRS in Chinese patients to show any higher possibility of mutations or variants for the 4 genes or sequences in China. Developmental Dynamics 246:392-402, 2017. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

  4. BPS: a database of RNA base-pair structures.

    Science.gov (United States)

    Xin, Yurong; Olson, Wilma K

    2009-01-01

    The BPS (http://bps.rutgers.edu) is a database of RNA base-pair structures, higher-order base interactions and isosteric pairs (base pairs with similar shape). The main functions of the BPS are to find and annotate the structural and chemical features of the Watson-Crick and non-Watson-Crick (noncanonical) base pairs in high-resolution RNA structures, and to provide a user-friendly interface to browse and search for the base pairs. The current database contains 91,265 bp and 3386 higher-order base interactions from 426 RNA crystal structures and 61,819 bp that fall into one of 17 different isosteric classes. The base-pair data can be accessed by searches of base-pair patterns, structure identifiers (IDs) and structural types. The BPS also includes an Atlas with representative images of the various base pairs, higher-order base interactions and isosteric pairs and links to statistical information about these groups of structures.

  5. A thermostable messenger RNA based vaccine against rabies.

    Science.gov (United States)

    Stitz, Lothar; Vogel, Annette; Schnee, Margit; Voss, Daniel; Rauch, Susanne; Mutzke, Thorsten; Ketterer, Thomas; Kramps, Thomas; Petsch, Benjamin

    2017-12-01

    Although effective rabies virus vaccines have been existing for decades, each year, rabies virus infections still cause around 50.000 fatalities worldwide. Most of these cases occur in developing countries, where these vaccines are not available. The reasons for this are the prohibitive high costs of cell culture or egg grown rabies virus vaccines and the lack of a functional cold chain in many regions in which rabies virus is endemic. Here, we describe the excellent temperature resistance of a non-replicating mRNA based rabies virus vaccine encoding the rabies virus glycoprotein (RABV-G). Prolonged storage of the vaccine from -80°C to up to +70°C for several months did not impact the protective capacity of the mRNA vaccine. Efficacy after storage was demonstrated by the induction of rabies specific virus neutralizing antibodies and protection in mice against lethal rabies infection. Moreover, storing the vaccine at oscillating temperatures between +4° and +56°C for 20 cycles in order to simulate interruptions of the cold chain during vaccine transport, did not affect the vaccine's immunogenicity and protective characteristics, indicating that maintenance of a cold chain is not essential for this vaccine.

  6. MicroRNA-based biotechnology for plant improvement.

    Science.gov (United States)

    Zhang, Baohong; Wang, Qinglian

    2015-01-01

    MicroRNAs (miRNAs) are an extensive class of newly discovered endogenous small RNAs, which negatively regulate gene expression at the post-transcription levels. As the application of next-generation deep sequencing and advanced bioinformatics, the miRNA-related study has been expended to non-model plant species and the number of identified miRNAs has dramatically increased in the past years. miRNAs play a critical role in almost all biological and metabolic processes, and provide a unique strategy for plant improvement. Here, we first briefly review the discovery, history, and biogenesis of miRNAs, then focus more on the application of miRNAs on plant breeding and the future directions. Increased plant biomass through controlling plant development and phase change has been one achievement for miRNA-based biotechnology; plant tolerance to abiotic and biotic stress was also significantly enhanced by regulating the expression of an individual miRNA. Both endogenous and artificial miRNAs may serve as important tools for plant improvement. © 2014 Wiley Periodicals, Inc.

  7. A thermostable messenger RNA based vaccine against rabies.

    Directory of Open Access Journals (Sweden)

    Lothar Stitz

    2017-12-01

    Full Text Available Although effective rabies virus vaccines have been existing for decades, each year, rabies virus infections still cause around 50.000 fatalities worldwide. Most of these cases occur in developing countries, where these vaccines are not available. The reasons for this are the prohibitive high costs of cell culture or egg grown rabies virus vaccines and the lack of a functional cold chain in many regions in which rabies virus is endemic. Here, we describe the excellent temperature resistance of a non-replicating mRNA based rabies virus vaccine encoding the rabies virus glycoprotein (RABV-G. Prolonged storage of the vaccine from -80°C to up to +70°C for several months did not impact the protective capacity of the mRNA vaccine. Efficacy after storage was demonstrated by the induction of rabies specific virus neutralizing antibodies and protection in mice against lethal rabies infection. Moreover, storing the vaccine at oscillating temperatures between +4° and +56°C for 20 cycles in order to simulate interruptions of the cold chain during vaccine transport, did not affect the vaccine's immunogenicity and protective characteristics, indicating that maintenance of a cold chain is not essential for this vaccine.

  8. A thermostable messenger RNA based vaccine against rabies

    Science.gov (United States)

    Stitz, Lothar; Vogel, Annette; Schnee, Margit; Voss, Daniel; Rauch, Susanne; Mutzke, Thorsten; Ketterer, Thomas; Kramps, Thomas

    2017-01-01

    Although effective rabies virus vaccines have been existing for decades, each year, rabies virus infections still cause around 50.000 fatalities worldwide. Most of these cases occur in developing countries, where these vaccines are not available. The reasons for this are the prohibitive high costs of cell culture or egg grown rabies virus vaccines and the lack of a functional cold chain in many regions in which rabies virus is endemic. Here, we describe the excellent temperature resistance of a non-replicating mRNA based rabies virus vaccine encoding the rabies virus glycoprotein (RABV-G). Prolonged storage of the vaccine from -80°C to up to +70°C for several months did not impact the protective capacity of the mRNA vaccine. Efficacy after storage was demonstrated by the induction of rabies specific virus neutralizing antibodies and protection in mice against lethal rabies infection. Moreover, storing the vaccine at oscillating temperatures between +4° and +56°C for 20 cycles in order to simulate interruptions of the cold chain during vaccine transport, did not affect the vaccine’s immunogenicity and protective characteristics, indicating that maintenance of a cold chain is not essential for this vaccine. PMID:29216187

  9. Identification of canine saliva using mRNA-based assay.

    Science.gov (United States)

    Nakanishi, Hiroaki; Ohmori, Takeshi; Hara, Masaaki; Yoneyama, Katsumi; Takada, Aya; Saito, Kazuyuki

    2017-01-01

    A dog saliva analysis in addition to a bite-mark analysis may be important for evidence when a crime involves a dog bite. In this study, the utility of detecting canine saliva-specific mRNAs to identify canine saliva was evaluated. Canine saliva swabs (n = 20), urine swabs (n = 20), body surface swabs (n = 20), whole blood samples (n = 10), human saliva (n = 20), human skin surface swabs (n = 20), and human whole blood (n = 20) were tested. The saliva-specific genes encoding statherin (STATH), carbonic anhydrase VI (CA-VI), and dog allergens (Canf1 and Canf2) were analyzed as candidate genes. Moreover, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as confirmation of canine mRNA extraction. STATH, CA-VI, Canf1, Canf2, and GAPDH mRNAs were detected in 19/20, 1/20, 11/20, 4/20, and 20/20 saliva samples, respectively. The STATH, CA-VI, Canf1, Canf2, and GAPDH mRNAs did not exhibit cross-reactivity with samples of human origin. This mRNA-based assay was also able to detect canine saliva in mock forensic samples. The results of this study indicated that the detection of STATH mRNA is useful for the identification of canine saliva, and GAPDH is a suitable marker for canine mRNA extraction.

  10. To Screen Inactivation Mutation of Exon 1 of FSHR Gene in Polycystic Ovarian Syndrome: A South Indian Cohort Study

    Science.gov (United States)

    Sekar, Nishu; Yeole, Samiksha; Pradeep, Rashmi; Prabhu, Yogamaya D.; Renu, Kaviyarasi; Ramgir, Shalaka S.; Abilash, V. G.

    2017-11-01

    Polycystic ovary syndrome is an endocrine disorder. Irregular menstrual cycle, acne, facial hair and elevated androgen levels are the most common signs for PCOS. PCOS has an estimated prevalence of 4-12% among reproductive age women, thus making it a forerunner in female infertility. FSHR plays an important role in FSH signaling pathway making it an important gene for PCOS. In this study, we aim to focus on any association between the FSHR gene and PCOS. Our study was to evaluate any polymorphism of exon 1 of FSHR gene associated with PCOS.PCR-RFLP technique was performed on the PCOS samples. Hormonal changes were found in the patients. Exon 1 inactivation mutation of FSHR gene was not observed in the patient sample. A study of this association needs to be done using large sample size.

  11. p53 transactivation and the impact of mutations, cofactors and small molecules using a simplified yeast-based screening system.

    Directory of Open Access Journals (Sweden)

    Virginia Andreotti

    Full Text Available BACKGROUND: The p53 tumor suppressor, which is altered in most cancers, is a sequence-specific transcription factor that is able to modulate the expression of many target genes and influence a variety of cellular pathways. Inactivation of the p53 pathway in cancer frequently occurs through the expression of mutant p53 protein. In tumors that retain wild type p53, the pathway can be altered by upstream modulators, particularly the p53 negative regulators MDM2 and MDM4. METHODOLOGY/PRINCIPAL FINDINGS: Given the many factors that might influence p53 function, including expression levels, mutations, cofactor proteins and small molecules, we expanded our previously described yeast-based system to provide the opportunity for efficient investigation of their individual and combined impacts in a miniaturized format. The system integrates i variable expression of p53 proteins under the finely tunable GAL1,10 promoter, ii single copy, chromosomally located p53-responsive and control luminescence reporters, iii enhanced chemical uptake using modified ABC-transporters, iv small-volume formats for treatment and dual-luciferase assays, and v opportunities to co-express p53 with other cofactor proteins. This robust system can distinguish different levels of expression of WT and mutant p53 as well as interactions with MDM2 or 53BP1. CONCLUSIONS/SIGNIFICANCE: We found that the small molecules Nutlin and RITA could both relieve the MDM2-dependent inhibition of WT p53 transactivation function, while only RITA could impact p53/53BP1 functional interactions. PRIMA-1 was ineffective in modifying the transactivation capacity of WT p53 and missense p53 mutations. This dual-luciferase assay can, therefore, provide a high-throughput assessment tool for investigating a matrix of factors that can influence the p53 network, including the effectiveness of newly developed small molecules, on WT and tumor-associated p53 mutants as well as interacting proteins.

  12. Screening of DFNB3 in Iranian families with autosomal recessive non-syndromic hearing loss reveals a novel pathogenic mutation in the MyTh4 domain of the MYO15A gene in a linked family

    Directory of Open Access Journals (Sweden)

    Somayeh Reiisi

    2016-07-01

    Full Text Available Objective(s: Non-syndromic sensorineural hearing loss (NSHL is a common disorder affecting approximately 1 in 500 newborns. This type of hearing loss is extremely heterogeneous and includes over 100 loci. Mutations in the GJB2 gene have been implicated in about half of autosomal recessive NSHL (ARNSHL cases, making this the most common cause of ARNSHL. For the latter form of deafness, most frequent genes proposed include GJB2, SLC26A4, MYO15A, OTOF, and CDH23 worldwide. Materials and Methods: the aim of the present study was to determine the role of MYO15A gene mutations in Iranian families. Thirty Iranian families with over three deaf children and negative for GJB2 using genetic linkage analysis (GLA, followed by mutation screening by DNA sequencing were enrolled. Results: One family (3.33% showed linkage to DFNB3 and a novel mutation was identified in the MYO15A gene (c.6442T>A as the disease-causing mutation. Mutation co-segregated with hearing loss in the family but was not present in the 100 ethnicity-matched controls. Conclusion: Our results confirmed that the hearing loss of the linked Iranian family was caused by a novel missense mutation in the MYO15A gene. This mutation is the first to be reported in the world and affects the first MyTH4 domain of the protein.

  13. Mutation screening of MIR146A/B and BRCA1/2 3'-UTRs in the GENESIS study.

    Science.gov (United States)

    Garcia, Amandine I; Buisson, Monique; Damiola, Francesca; Tessereau, Chloé; Barjhoux, Laure; Verny-Pierre, Carole; Sornin, Valérie; Dondon, Marie-Gabrielle; Eon-Marchais, Séverine; Caron, Olivier; Gautier-Villars, Marion; Coupier, Isabelle; Buecher, Bruno; Vennin, Philippe; Belotti, Muriel; Lortholary, Alain; Gesta, Paul; Dugast, Catherine; Noguès, Catherine; Fricker, Jean-Pierre; Faivre, Laurence; Stoppa-Lyonnet, Dominique; Andrieu, Nadine; Sinilnikova, Olga M; Mazoyer, Sylvie

    2016-08-01

    Although a wide number of breast cancer susceptibility alleles associated with various levels of risk have been identified to date, about 50% of the heritability is still missing. Although the major BRCA1 and BRCA2 genes are being extensively screened for truncating and missense variants in breast and/or ovarian cancer families, potential regulatory variants affecting their expression remain largely unexplored. In an attempt to identify such variants, we focused our attention on gene regulation mediated by microRNAs (miRs). We screened two genes, MIR146A and MIR146B, producing miR-146a and miR-146b-5p, respectively, that regulate BRCA1, and the 3'- untranslated regions (3'-UTRs) of BRCA1 and BRCA2 in the GENESIS French national case/control study (BRCA1- and BRCA2-negative breast cancer cases with at least one sister with breast cancer and matched controls). We identified one rare variant in MIR146A, four in MIR146B, five in BRCA1 3'-UTR and one in BRCA2 3'-UTR in 716 index cases and 619 controls. Among these 11 rare variants, 7 were identified each in 1 index case. None of the three relevant MIR146A/MIR146B variants affected the pre-miR sequences. The potential causality of the four relevant BRCA1/BRCA2 3'-UTRs variants was evaluated with luciferase reporter assays and co-segregation studies, as well as with bioinformatics analyses to predict miRs-binding sites, RNA secondary structures and RNA accessibility. This is the first study to report the screening of miR genes and of BRCA2 3'-UTR in a large series of familial breast cancer cases. None of the variant identified in this study gave convincing evidence of potential pathogenicity.

  14. Results of molecular genetic screening of mutations in the NLRP3, TNFRSF1A, and MVK genes in patients with autoinflammatory diseases and systemic juvenile arthritis

    Directory of Open Access Journals (Sweden)

    S. O. Salugina

    2017-01-01

    Full Text Available Autoinflammatory diseases (AIDs are being intensively studied. Molecular genetic testing of patients is of great importance for the diagnosis of AIDs since the basis for its development is pathological mutations that cause innate (antigen-nonspecific immunity system disorders and the development of inflammation. This also applies to patients with systemic juvenile arthritis (SJA that has been recently assigned to a group of AIDs due to the great similarity of symptoms. In this connection, the assumption that monogenic AIDs mask SJA in a number of patients was well founded. More than 25 genes, mutations in which lead to AIDs, are known; the NLRP3, TNFRSF1A, and MVK genes are most common and well investigated. These genes cause major monogenic AIDs, such as cryopyrin-associated periodic syndromes (CAPS, TNF receptor-associated periodic syndrome (TRAPS, and hyperimmunoglobulinemia D/deficit mevalonate kinase syndrome (HIDS.Objective: to identify patients with monogenic AIDs among those with fever, arthralgias, and other manifestations of systemic inflammatory response, including among those with SJA, through molecular genetic testing.Patients and methods. In 2012–2016, molecular genetic testing for mutations in the NLRP3, MVK, and TNFRSF1A genes was carried out within the framework of screening in 184 patients (94 women and 90 men. The investigation enrolled 117 patients with suspected AIDs (Group 1 and 67 patients with SJA (Group 2. The selection criteria were periodic or persistent fever, clinical manifestations of systemic inflammatory response (skin rashes, arthralgias/arthritis, lymphadenopathy, hepatolienal syndrome, serositis, etc., acute-phase markers when excluding infectious, oncohematologic, and autoimmune causes. SJA was diagnosed based on the ILAR criteria (2001. The patients' age ranged from 6 months to 60 years (mean age, 9.0 years [5; 15], disease duration, 2 months to 54 years (mean duration, 3.0 [1.0; 8.5]. To

  15. In Silico Screening of Mutated K-Ras Inhibitors from Malaysian Typhonium flagelliforme for Non-Small Cell Lung Cancer

    Directory of Open Access Journals (Sweden)

    Ayesha Fatima

    2014-01-01

    Full Text Available K-ras is an oncogenic GTPase responsible for at least 15–25% of all non-small cell lung cancer cases worldwide. Lung cancer of both types is increasing with an alarming rate due to smoking habits in Malaysia among men and women. Natural products always offer alternate treatment therapies that are safe and effective. Typhonium flagelliforme or Keladi Tikus is a local plant known to possess anticancer properties. The whole extract is considered more potent than individual constituents. Since K-ras is the key protein in lung cancer, our aim was to identify the constituents of the plant that could target the mutated K-ras. Using docking strategies, reported potentially active compounds of Typhonium flagelliforme were docked into the allosteric surface pockets and switch regions of the K-ras protein to identify possible inhibitors. The selected ligands were found to have a high binding affinity for the switch II and the interphase region of the ras-SOS binding surface.

  16. In Silico Screening, Alanine Mutation, and DFT Approaches for Identification of NS2B/NS3 Protease Inhibitors

    Directory of Open Access Journals (Sweden)

    R. Balajee

    2016-01-01

    Full Text Available To identify the ligand that binds to a target protein with high affinity is a nontrivial task in computer-assisted approaches. Antiviral drugs have been identified for NS2B/NS3 protease enzyme on the mechanism to cleave the viral protein using the computational tools. The consequence of the molecular docking, free energy calculations, and simulation protocols explores the better ligand. It provides in-depth structural insights with the catalytic triad of His51, Asp75, Ser135, and Gly133. The MD simulation was employed here to predict the stability of the complex. The alanine mutation has been performed and its stability was monitored by using the molecular dynamics simulation. The minimal RMSD value suggests that the derived complexes are close to equilibrium. The DFT outcome reveals that the HOMO-LUMO gap of Ligand19 is 2.86 kcal/mol. Among the considered ligands, Ligand19 shows the lowest gap and it is suggested that the HOMO of Ligand19 may transfer the electrons to the LUMO in the active regions. The calculated binding energy of Ligand19 using the DFT method is in good agreement with the docking studies. The pharmacological activity of ligand was performed and satisfies Lipinski rule of 5. Moreover, the computational results are compared with the available IC50 values of experimental results.

  17. Screening in asymptomatic SDHx mutation carriers: added value of {sup 18}F-FDG PET/CT at initial diagnosis and 1-year follow-up

    Energy Technology Data Exchange (ETDEWEB)

    Lepoutre-Lussey, C.; Deandreis, D.; Berdelou, A.; Nascimento, C.; Lumbroso, J.; Schlumberger, M.; Baudin, E.; Leboulleux, S. [Gustave Roussy Institut, Universite Paris-Sud, Department of Nuclear Medicine and Endocrine Oncology, Villejuif (France); Caramella, C.; Bidault, F.; Deschamps, F. [Gustave Roussy Institut, Department of Radiology, Villejuif (France); Al Ghuzlan, A. [Gustave Roussy Institut, Department of Medical Biology and Pathology, Villejuif (France); Hartl, D.; Dumont, F. [Gustave Roussy Institut, Department of Surgery, Villejuif (France); Borget, I. [Gustave Roussy Institut, Department of Biostatistic and Epidemiology, Villejuif (France); Paris-Sud University, Villejuif (France); Gimenez-Roqueplo, A.P. [Assistance Publique-Hopitaux de Paris, Hopital Europeen Georges Pompidou, Department of Genetics, Paris (France); Paris Descartes University, Faculty of Medicine, Paris (France); Guillaud Bataille, M. [Gustave Roussy Institut, Department of Genetics, Villejuif (France)

    2015-05-01

    Specific recommendations on screening modalities for paraganglioma (PGL) and phaeochromocytoma (PCC) in asymptomatic SDHx mutation carriers (relatives) are still lacking. We evaluated the added value of {sup 18}F-FDG PET/CT in comparison with morphological imaging at initial diagnosis and 1 year of follow-up in this population. The study included 30 consecutive relatives with a proven SDHx mutation who were investigated by {sup 18}F-FDG PET/CT, gadolinium-enhanced magnetic resonance angiography of the head and neck, thoracic/abdominal/pelvic (TAP) contrast-enhanced CT and/or TAP MRI. {sup 123}I-MIBG scintigraphy was performed in 20 subjects and somatostatin receptor scintigraphy (SRS) in 20 subjects. The gold standard was based on pathology or a composite endpoint as defined by any other positive imaging method and persistent tumour on follow-up. Images were considered as false-positive when the lesions were not detected by another imaging method or not confirmed at 1 year. At initial work-up, an imaging abnormality was found in eight subjects (27 %). The final diagnosis was true-positive in five subjects (two with abdominal PGL, one with PCC and two with neck PGL) and false-positives in the other three subjects (detected with {sup 18}F-FDG PET/CT in two and TAP MRI in one). At 1 year, an imaging abnormality was found in three subjects of which one was an 8-mm carotid body PGL in a patient with SDHD mutation and two were considered false-positive. The tumour detection rate was 100 % for {sup 18}F-FDG PET/CT and conventional imaging, 80 % for SRS and 60 % for {sup 123}I-MIBG scintigraphy. Overall, disease was detected in 4 % of the subjects at the 1-year follow-up. {sup 18}F-FDG PET/CT demonstrated excellent sensitivity but intermediate specificity justifying combined modality imaging in these patients. Given the slow progression of the disease, if {sup 18}F-FDG PET/CT and MRI are normal at baseline, the second imaging work-up should be delayed and an examination

  18. mRNA-based skin identification for forensic applications.

    Science.gov (United States)

    Visser, Mijke; Zubakov, Dmitry; Ballantyne, Kaye N; Kayser, Manfred

    2011-03-01

    Although the identification of human skin cells is of important relevance in many forensic cases, there is currently no reliable method available. Here, we present a highly specific and sensitive messenger RNA (mRNA) approach for skin identification, meeting the key requirements in forensic analyses. We examined 11 candidate genes with skin-specific expression, as ascertained from expression databases and the literature, as well as five candidate reference genes ascertained from previous studies, in skin samples and in other forensically relevant tissues. We identified mRNA transcripts from three genes CDSN, LOR and KRT9, showing strong over-expression in skin samples relative to samples from forensic body fluids, making them suitable markers for skin identification. Out of the candidate reference genes tested, only ACTB showed similarly high expression in skin and body-fluid samples, providing a suitable reference marker for quantitative real-time PCR (qPCR) analysis of skin. Analyses of palmar and thumbprint skin samples indicate that our qPCR approach for the three skin-targeted mRNA markers, as well as the reference mRNA marker ACTB, is highly sensitive, allowing successful detection of minute amounts of skin material including full, half and quarter thumbprints, albeit with decreased success in decreasing print material. Furthermore, thumbprints stored for 6.5 months provided similar results relative to freshly analysed samples implying reasonable time-wise stability of the three skin-targeted mRNAs as well as the ACTB reference mRNA. Our study represents the first attempt towards reliable mRNA-based skin identification in forensic applications with particular relevance for future trace/touched object analyses in forensic case work. Although the approach for skin identification introduced here can be informative when applied on its own, we recommend for increased reliability the integration of (one or more of) the skin-targeted mRNA markers presented here

  19. Triple-modality screening trial for familial breast cancer underlines the importance of magnetic resonance imaging and questions the role of mammography and ultrasound regardless of patient mutation status, age, and breast density.

    Science.gov (United States)

    Riedl, Christopher C; Luft, Nikolaus; Bernhart, Clemens; Weber, Michael; Bernathova, Maria; Tea, Muy-Kheng M; Rudas, Margaretha; Singer, Christian F; Helbich, Thomas H

    2015-04-01

    To evaluate the breast cancer screening efficacy of mammography, ultrasound, and magnetic resonance imaging (MRI) in a high-risk population and in various population subgroups. In a single-center, prospective, nonrandomized comparison study, BRCA mutation carriers and women with a high familial risk (> 20% lifetime risk) for breast cancer were offered screening with mammography, ultrasound, and MRI every 12 months. Diagnostic performance was compared between individual modalities and their combinations. Further comparisons were based on subpopulations dichotomized by screening rounds, mutation status, age, and breast density. There were 559 women with 1,365 complete imaging rounds included in this study. The sensitivity of MRI (90.0%) was significantly higher (P cancers, 18 (45.0%) were detected by MRI alone. Two cancers were found by mammography alone (a ductal carcinoma in situ [DCIS] with microinvasion and a DCIS with cancers were detected by ultrasound alone. Similarly, of 14 DCISs, all were detected by MRI, whereas mammography and ultrasound each detected five DCISs (35.7%). Age, mutation status, and breast density had no influence on the sensitivity of MRI and did not affect the superiority of MRI over mammography and ultrasound. MRI allows early detection of familial breast cancer regardless of patient age, breast density, or risk status. The added value of mammography is limited, and there is no added value of ultrasound in women undergoing MRI for screening. © 2015 by American Society of Clinical Oncology.

  20. Impact of Molecular Screening for Point Mutations and Rearrangements in Routine Air-Dried Fine-Needle Aspiration Samples of Thyroid Nodules

    DEFF Research Database (Denmark)

    Eszlinger, Markus; Krogdahl, Annelise; Münz, Sina

    2014-01-01

    formalin-fixed paraffin-embedded tissue (156 follicular adenomas [FAs], 32 FTCs, 44 papillary thyroid carcinomas [PTCs], 9 follicular variant PTCs, and 69 goiters). PAX8/PPARG and RET/PTC rearrangements were detected by qPCR, BRAF and RAS mutations by high-resolution melting PCR and by pyrosequencing....... Results: Forty-seven mutations were detected in the FNAs: 22 BRAF, 13 NRAS, and 3 HRAS mutations, 8 PAX8/PPARG, and one RET/PTC-rearrangement. While the presence of a BRAF and RET/PTC mutation was associated with cancer in 100% of samples each, the presence of a RAS and PAX8/PPARG mutation was associated...

  1. Prospective mutation screening of three common deafness genes in a large Taiwanese Cohort with idiopathic bilateral sensorineural hearing impairment reveals a difference in the results between families from hospitals and those from rehabilitation facilities.

    Science.gov (United States)

    Wu, Chen-Chi; Chen, Pei-Jer; Chiu, Yu-Hsun; Lu, Ying-Chang; Wu, Ming-Chueh; Hsu, Chuan-Jen

    2008-01-01

    Accurate epidemiological data on common deafness genes are essential to improve the efficiency and to reduce the cost of molecular diagnosis. They may depend on several factors, including a clear delineation of the source of patients being studied. In the present study, we hypothesize that patients with idiopathic sensorineural hearing loss recruited from different sources might reveal discrepancies in the epidemiological results of genetic screening, because patients from different sources might demonstrate distinct clinical or audiologic features and thus result in biased selection of subjects. To elucidate the relative importance of common deafness genes in Taiwanese and to verify our hypothesis, we conducted a prospective project screening mutations in GJB2, SLC26A4 and mitochondrial 12S rRNA gene in a total of 420 Taiwanese families with idiopathic bilateral sensorineural hearing loss, of which 325 families were recruited from hospitals and 95 from hearing rehabilitation facilities. Allele frequencies of common mutations in these three genes and distributions of the corresponding genotypes were then compared between the two groups. The allele frequencies of mutations in SLC26A4, GJB2 and mitochondrial 12S rRNA in the probands of the 420 families were 14.4, 21.7 and 3.8%, respectively. The allele frequency of SLC26A4 mutations in the hospital group was significantly higher than that in the rehabilitation facility group (16.2 vs. 8.4%, chi(2)-test, p < 0.05), whereas no difference in the frequencies of GJB2 mutations and mitochondrial 12S rRNA mutations was found between the two groups. Distributions of probands classified by SLC26A4 genotypes were also different between the two groups (chi(2)-test, p < 0.05). Accordingly, a discrepancy in the genetic screening results might exist between different sources of idiopathic hearing-impaired patients. Further analysis of audiological results and construction of a logistic regression model showed that different

  2. Can pretreatment screening for dhps and dhfr point mutations in Plasmodium falciparum infections be used to predict sulfadoxine-pyrimethamine treatment failure?

    Science.gov (United States)

    Omar, S A; Adagu, I S; Warhurst, D C

    2001-01-01

    This study examines the relationship between malaria treatment failure after sulfadoxine-pyrimethamine (S-P) chemotherapy and presence of mutations in the Plasmodium falciparum dihydropteroate synthase (dhps) and dihydrofolate reductase (dhfr) genes (associated with resistance in vitro to S and P) before treatment. In Kenya, 38 malaria patients in a holoendemic area, and 21 in an epidemic area, participated in the trial in 1997-98. In the 2 areas, drug failure occurred in 76% and 75% of cases where any mutation in dhfr was seen (positive predictive values 76% and 75%: P = 0.003 and 0.008) and an identical association was seen with dhfr Asn-108. In the holoendemic area all occurrences of > or = 2 mutations in dhfr predicted drug failure. Only 3 instances were seen in the epidemic focus, but treatment failed in all. Only in the epidemic focus, 7 (88%) of 8 occurrences of > or = 1 mutations in dhps, and all occurrences of the Gly-437 allele of dhps, predicted failure. Association between mutations in dhps and mutations in dhfr was noted in the combined sites, irrespective of outcome. Although this makes the relationship of combined dhfr and dhps mutations to failure more difficult to interpret, it nevertheless supports S-P selection acting on both genes. In the holoendemic site, treatment success increased with age. In this location, acquired immunity may mask the impact of mutations in dhps, since sulfadoxine is a less effective treatment than pyrimethamine.

  3. Molecular Screening of "MECP2" Gene in a Cohort of Lebanese Patients Suspected with Rett Syndrome: Report on a Mild Case with a Novel Indel Mutation

    Science.gov (United States)

    Corbani, S.; Chouery, E.; Fayyad, J.; Fawaz, A.; El Tourjuman, O.; Badens, C.; Lacoste, C.; Delague, V.; Megarbane, A.

    2012-01-01

    Background: Rett syndrome (RTT), an X-linked, dominant, neurodevelopment disorder represents 10% of female subjects with profound intellectual disability. Mutations in the "MECP2" gene are responsible for up to 95% of the classical RTT cases, and nearly 500 different mutations distributed throughout the gene have been reported. Methods:…

  4. Screening of three common mtDNA mutations among subjects with autosomal recessive non-syndromic hearing loss in Sistan va Baluchestan province, Iran

    Directory of Open Access Journals (Sweden)

    Fartemeh Azadegan-Dehkordi

    2011-10-01

    Full Text Available Background: Non-syndromic hearing loss may be induced by mutations in both nuclear and mitochondrial genes. Mutations in mtDNA are present in less than 1% of the children with pre-lingual deafness but are more prevalent later. Most of the molecular defects responsible for mitochondrial disorder, associated with hearing loss may be induced by mutations in the 12SrRNA and tRNA genes. This aim of this study was to investigate the frequency of three common mtDNA mutations including A1555G, A3243G and A7445G in a cohort of autosomal recessive non-syndromic hearing loss (ARNSHL subjects in Sistan va Baluchestan province. Material and Methods: In this descriptive- experimental based study, a total of 110. ARNSHL subjects from Sistan va Baluchestan province were investigated for three common mtDNA mutations using PCR-RFLP procedure. The possible mutations were confirmed by direct sequencing.Results: None of the A1555G and A7445G mutations were detected in this study. However, we found one sample to carry A3243G mutation (0.9%. Moreover abolishing a MTTL1 restriction site close to A3243G mutation revealed a G3316A allelic variant in 0.9% of patients studied.Conclusion: This study showed that mtDNA mutations are responsible for less than 1% of pre-lingual ARNSHL associated subjects. The present study will improve the genetic counseling of hearing impaired patients in Sistan va Baluchestan province, Iran

  5. Screening for mutations related to atovaquone/proguanil resistance in treatment failures and other imported isolates of Plasmodium falciparum in Europe

    DEFF Research Database (Denmark)

    Wichmann, Ole; Muehlberger, Nikolai; Jelinek, Tomas

    2004-01-01

    BACKGROUND: Two single-point mutations of the Plasmodium falciparum cytochrome b gene (Tyr268Asn and Tyr268Ser) were recently reported in cases of atovaquone/proguanil (Malarone) treatment failure. However, little is known about the prevalence of codon-268 mutations and their quantitative...... association with treatment failure. METHODS: We set out to assess the prevalence of codon-268 mutations in P. falciparum isolates imported into Europe and to quantify their association with atovaquone/proguanil treatment failure. Isolates of P. falciparum collected by the European Network on Imported...... failure but in 0 of 53 successfully treated patients. CONCLUSIONS: Tyr268Ser seems to be a sufficient, but not a necessary, cause for atovaquone/proguanil treatment failure. The prevalence of both codon-268 mutations is currently unlikely to be >1% in the European patient pool....

  6. Co segregation of the m.1555A>G mutation in the MT-RNR1 gene and mutations in MT-ATP6 gene in a family with dilated mitochondrial cardiomyopathy and hearing loss: A whole mitochondrial genome screening.

    Science.gov (United States)

    Alila-Fersi, Olfa; Chamkha, Imen; Majdoub, Imen; Gargouri, Lamia; Mkaouar-Rebai, Emna; Tabebi, Mouna; Tlili, Abdelaziz; Keskes, Leila; Mahfoudh, Abdelmajid; Fakhfakh, Faiza

    2017-02-26

    Mitochondrial disease refers to a heterogeneous group of disorders resulting in defective cellular energy production due to dysfunction of the mitochondrial respiratory chain, which is responsible for the generation of most cellular energy. Because cardiac muscles are one of the high energy demanding tissues, mitochondrial cardiomyopathies is one of the most frequent mitochondria disorders. Mitochondrial cardiomyopathy has been associated with several point mutations of mtDNA in both genes encoded mitochondrial proteins and mitochondrial tRNA and rRNA. We reported here the first description of mutations in MT-ATP6 gene in two patients with clinical features of dilated mitochondrial cardiomyopathy. The mutational analysis of the whole mitochondrial DNA revealed the presence of m.1555A>G mutation in MT-RNR1 gene associated to the m.8527A>G (p.M>V) and the m.8392C>T (p.136P>S) variations in the mitochondrial MT-ATP6 gene in patient1 and his family members with variable phenotype including hearing impairment. The second patient with isolated mitochondrial cardiomyopathy presented the m.8605C>T (p.27P>S) mutation in the MT-ATP6 gene. The three mutations p.M1V, p.P27S and p.P136S detected in MT-ATP6 affected well conserved residues of the mitochondrial protein ATPase 6. In addition, the substitution of proline residue at position 27 and 136 effect hydrophobicity and structure flexibility conformation of the protein. Copyright © 2017 Elsevier Inc. All rights reserved.

  7. Secondary variants in individuals undergoing exome sequencing: screening of 572 individuals identifies high-penetrance mutations in cancer-susceptibility genes.

    Science.gov (United States)

    Johnston, Jennifer J; Rubinstein, Wendy S; Facio, Flavia M; Ng, David; Singh, Larry N; Teer, Jamie K; Mullikin, James C; Biesecker, Leslie G

    2012-07-13

    Genome- and exome-sequencing costs are continuing to fall, and many individuals are undergoing these assessments as research participants and patients. The issue of secondary (so-called incidental) findings in exome analysis is controversial, and data are needed on methods of detection and their frequency. We piloted secondary variant detection by analyzing exomes for mutations in cancer-susceptibility syndromes in subjects ascertained for atherosclerosis phenotypes. We performed exome sequencing on 572 ClinSeq participants, and in 37 genes, we interpreted variants that cause high-penetrance cancer syndromes by using an algorithm that filtered results on the basis of mutation type, quality, and frequency and that filtered mutation-database entries on the basis of defined categories of causation. We identified 454 sequence variants that differed from the human reference. Exclusions were made on the basis of sequence quality (26 variants) and high frequency in the cohort (77 variants) or dbSNP (17 variants), leaving 334 variants of potential clinical importance. These were further filtered on the basis of curation of literature reports. Seven participants, four of whom were of Ashkenazi Jewish descent and three of whom did not meet family-history-based referral criteria, had deleterious BRCA1 or BRCA2 mutations. One participant had a deleterious SDHC mutation, which causes paragangliomas. Exome sequencing, coupled with multidisciplinary interpretation, detected clinically important mutations in cancer-susceptibility genes; four of such mutations were in individuals without a significant family history of disease. We conclude that secondary variants of high clinical importance will be detected at an appreciable frequency in exomes, and we suggest that priority be given to the development of more efficient modes of interpretation with trials in larger patient groups. Copyright © 2012 The American Society of Human Genetics. Published by Elsevier Inc. All rights

  8. Screening for germline BRCA1, BRCA2, TP53 and CHEK2 mutations in families at-risk for hereditary breast cancer identified in a population-based study from Southern Brazil

    Directory of Open Access Journals (Sweden)

    Edenir Inêz Palmero

    2016-01-01

    Full Text Available Abstract In Brazil, breast cancer is a public health care problem due to its high incidence and mortality rates. In this study, we investigated the prevalence of hereditary breast cancer syndromes (HBCS in a population-based cohort in Brazils southernmost capital, Porto Alegre. All participants answered a questionnaire about family history (FH of breast, ovarian and colorectal cancer and those with a positive FH were invited for genetic cancer risk assessment (GCRA. If pedigree analysis was suggestive of HBCS, genetic testing of the BRCA1, BRCA2, TP53, and CHEK2 genes was offered. Of 902 women submitted to GCRA, 214 had pedigrees suggestive of HBCS. Fifty of them underwent genetic testing: 18 and 40 for BRCA1/BRCA2 and TP53 mutation screening, respectively, and 7 for CHEK2 1100delC testing. A deleterious BRCA2 mutation was identified in one of the HBOC probands and the CHEK2 1100delC mutation occurred in one of the HBCC families. No deleterious germline alterations were identified in BRCA1 or TP53. Although strict inclusion criteria and a comprehensive testing approach were used, the suspected genetic risk in these families remains unexplained. Further studies in a larger cohort are necessary to better understand the genetic component of hereditary breast cancer in Southern Brazil.

  9. Primary erythermalgia as a sodium channelopathy: screening for SCN9A mutations: exclusion of a causal role of SCN10A and SCN11A.

    NARCIS (Netherlands)

    Drenth, J.P.H.; Morsche, R.H.M. te; Mansour, S.; Mortimer, P.S.

    2008-01-01

    OBJECTIVES: To elucidate the rate of missense mutations in the SCN9A gene (which encodes sodium channel Na(v)1.7) (OMIM 603415) among patients with primary erythermalgia and to examine the possibility that other sodium channels can cause the disease. DESIGN: Case series. SETTING: Department of

  10. Pan-cancer screen for mutations in non-coding elements with conservation and cancer specificity reveals correlations with expression and survival

    DEFF Research Database (Denmark)

    Hornshøj, Henrik; Nielsen, Morten Muhlig; Sinnott-Armstrong, Nicholas A

    2018-01-01

    characterize the driver potential of the identified elements and shortlist candidates, we identified elements where presence of mutations correlated significantly with expression levels (e.g., TERT and CDH10) and survival (e.g., CDH9 and CDH10) in an independent set of 505 TCGA whole-genome samples...

  11. Human fructose-1,6-bisphosphatase gene (FBP1): Exon-intron organization, localization to chromosome bands 9q22.2-q22.3, and mutation screening in subjects with fructose-1,6-bisphosphatase deficiency

    Energy Technology Data Exchange (ETDEWEB)

    El-Maghrabi, M.R.; Jiang, W. [State Univ. of New York, Stony Brook, NY (United States)] [and others

    1995-06-10

    Fructose-1,6-bisphosphatase (EC 3.1.3.11) is a key regulatory enzyme of gluconeogenesis that catalyzes the hydrolysis of fructose-1,6-bisphosphate to generate fructose-6-phosphate and inorganic phosphate. Deficiency of fructose-1,6-bisphosphatase is associated with fasting hypoglycemia and metabolic acidosis because of impaired gluconeogenesis. We have cloned and characterized the human liver fructose-1,6-bisphosphatase gene (FBP1). FBP1, localized to chromosome bands 9q22.2-q22.3 by fluorescence in situ hybridization, consists of seven exons that span > 31 kb, and the six introns are in the same position as in the rat gene. FBP1 was screened for mutations in two subjects with fructose-1,6-bisphosphatase deficiency. Four nucleotide substitutions were identified, two of which were silent mutations in the codons for Ala-216 (GCT {yields} GCC) and Gly-319 (GGG {yields} GGA). The other substitutions were in intron 3, a C {yields} T substitution 7 nucleotides downstream from the splice donor site, and in the promoter region, an A {yields} T substitution 188 nucleotides upstream from the start of transcription. These nucleotide substitutions were also found in normal unaffected subjects and thus are not the cause of fructose-1,6-bisphosphatase deficiency in the two subjects studied. The molecular basis of hepatic fructose-1,6-bisphosphatase deficiency in these subjects remains undetermined but could result from unidentified mutations in the promoter that decrease expression or from mutations in another gene that indirectly lead to decreased fructose-1,6-bisphosphatase activity. 18 refs., 3 figs., 3 tabs.

  12. Exploiting Nanotechnology for the Development of MicroRNA-Based Cancer Therapeutics.

    Science.gov (United States)

    Tyagi, Nikhil; Arora, Sumit; Deshmukh, Sachin K; Singh, Seema; Marimuthu, Saravanakumar; Singh, Ajay P

    2016-01-01

    MicroRNAs (miRNAs/miRs) represent a novel class of small non-coding RNAs that post-transcriptionally regulate gene expression by base pairing with complementary sequences in the 3' untranslated region (UTR) of target mRNAs. Functional studies suggest that miRNAs control almost every biological process, and their aberrant expression leads to a disease state, such as cancer. Differential expression of miRNAs in cancerous versus normal cells have generated enormous interest for the development of miRNA-based cancer cell-targeted therapeutics. Depending on the miRNA function and expression in cancer, two types of miRNA-based therapeutic strategies can be utilized that either restore or inhibit miRNA function through exogenous delivery of miRNAs mimics or inhibitors (anti-miRs). However, hydrophilic nature of miRNA mimics/anti-miRs, sensitivity to nuclease degradation in serum, poor penetration and reduced uptake by the tumor cells are chief hurdles in accomplishing their efficient in vivo delivery. To overcome these barriers, several nanotechnology-based systems are being developed and tested for delivery efficacy. This review summarizes the importance of miRNAs-based therapeutics in cancer, associated translational challenges and novel nanotechnology-assisted delivery systems that hold potential for next-generation miRNA-based cancer therapeutics.

  13. Associations between HIV-RNA-based indicators and virological and clinical outcomes

    DEFF Research Database (Denmark)

    Laut, Kamilla G.; Shepherd, Leah C.; Pedersen, Court

    2016-01-01

    Objectives: To evaluate and compare the performance of six HIV-RNA-based quality of care indicators for predicting short-term and long-term outcomes. Design: Multinational cohort study. Methods: We included EuroSIDA patients on antiretroviral therapy (ART) with at least three viral load measureme......Objectives: To evaluate and compare the performance of six HIV-RNA-based quality of care indicators for predicting short-term and long-term outcomes. Design: Multinational cohort study. Methods: We included EuroSIDA patients on antiretroviral therapy (ART) with at least three viral load...... measurements after baseline (the latest of 01/01/2001 or entry into EuroSIDA). Using multivariate Poisson regression, we modelled the association between short-term (resistance, triple-class failure) and long-term (all-cause mortality, any AIDS/non-AIDS clinical event) outcomes and the indicators: viraemia...... of care indicators were small and no indicator performed consistently better than current viral load. Given the simplicity in assessing and interpreting this indicator, we propose to use current viral load when HIV-RNA-based indicators are used to evaluate the efficacy of ART programs....

  14. MicroRNA-Based Classification of Hepatocellular Carcinoma and Oncogenic Role of miR-517a

    Science.gov (United States)

    TOFFANIN, SARA; HOSHIDA, YUJIN; LACHENMAYER, ANJA; VILLANUEVA, AUGUSTO; CABELLOS, LAIA; MINGUEZ, BEATRIZ; SAVIC, RADOSLAV; WARD, STEPHEN C.; THUNG, SWAN; CHIANG, DEREK Y.; ALSINET, CLARA; TOVAR, VICTORIA; ROAYAIE, SASAN; SCHWARTZ, MYRON; BRUIX, JORDI; WAXMAN, SAMUEL; FRIEDMAN, SCOTT L.; GOLUB, TODD; MAZZAFERRO, VINCENZO; LLOVET, JOSEP M.

    2013-01-01

    BACKGROUND & AIMS Hepatocellular carcinoma (HCC) is a heterogeneous tumor that develops via activation of multiple pathways and molecular alterations. It has been a challenge to identify molecular classes of HCC and design treatment strategies for each specific subtype. MicroRNAs (miRNAs) are involved in HCC pathogenesis and their expression profiles have been used to classify cancers. We analyzed miRNA expression in human HCC samples to identify molecular subclasses and oncogenic miRNAs. METHODS We performed miRNA profiling of 89 HCC samples using a ligation-mediated amplification method. Subclasses were identified by unsupervised clustering analysis. We identified molecular features specific for each subclass using expression pattern (Affymetrix U133 2.0), DNA change (Affymetrix STY Mapping Array), mutation (CTNNB1), and immunohistochemical (phosphor[p]-Akt, p-IGF-IR, p-S6, p-EGFR, β-catenin) analyses. The roles of selected miRNAs were investigated in cell lines and in an orthotopic model of HCC. RESULTS We identified 3 main clusters of HCCs: the Wnt (32 of 89; 36%), interferon-related (29 of 89; 33%), and proliferation (28 of 89; 31%) subclasses. A subset of patients with tumors in the proliferation subclass (8 of 89; 9%) overexpressed a family of poorly characterized miRNAs from chr19q13.42. Expression of miR-517a and miR-520c (from ch19q13.42) increased proliferation, migration, and invasion of HCC cells in vitro. MiR-517a promoted tumorigenesis and metastatic dissemination in vivo. CONCLUSIONS We propose miRNA-based classification of 3 subclasses of HCC. Among the proliferation class, miR-517a is an oncogenic miRNA that promotes tumor progression. There is rationale for developing therapies that miRNA 517 for patients with HCC. PMID:21324318

  15. Mutation screening of MIR146A/B and BRCA1/2 3′-UTRs in the GENESIS study

    OpenAIRE

    Garcia, Amandine I; Buisson, Monique; Damiola, Francesca; Tessereau, Chloé; Barjhoux, Laure; Verny-Pierre, Carole; Sornin, Valérie; Dondon, Marie-Gabrielle; Eon-Marchais, Séverine; Caron, Olivier; Gautier-Villars, Marion; Coupier, Isabelle; Buecher, Bruno; Vennin, Philippe; Belotti, Muriel

    2016-01-01

    Although a wide number of breast cancer susceptibility alleles associated with various levels of risk have been identified to date, about 50% of the heritability is still missing. Although the major BRCA1 and BRCA2 genes are being extensively screened for truncating and missense variants in breast and/or ovarian cancer families, potential regulatory variants affecting their expression remain largely unexplored. In an attempt to identify such variants, we focused our attention on gene regulati...

  16. ALS2 mutations

    Science.gov (United States)

    Schneider, Susanne A.; Carr, Lucinda; Deuschl, Guenther; Hopfner, Franziska; Stamelou, Maria; Wood, Nicholas W.; Bhatia, Kailash P.

    2014-01-01

    Objective: To determine the genetic etiology in 2 consanguineous families who presented a novel phenotype of autosomal recessive juvenile amyotrophic lateral sclerosis associated with generalized dystonia. Methods: A combination of homozygosity mapping and whole-exome sequencing in the first family and Sanger sequencing of candidate genes in the second family were used. Results: Both families were found to have homozygous loss-of-function mutations in the amyotrophic lateral sclerosis 2 (juvenile) (ALS2) gene. Conclusions: We report generalized dystonia and cerebellar signs in association with ALS2-related disease. We suggest that the ALS2 gene should be screened for mutations in patients who present with a similar phenotype. PMID:24562058

  17. Screening for microsatellite instability identifies frequent 3'-untranslated region mutation of the RB1-inducible coiled-coil 1 gene in colon tumors.

    Directory of Open Access Journals (Sweden)

    Bogdan C Paun

    Full Text Available BACKGROUND: Coding region microsatellite instability (MSI results in loss of gene products and promotion of microsatellite-unstable (MSI-H carcinogenesis. Recent studies have indicated that MSI within 3'-untranslated regions (3'UTRs may post-transcriptionally dysregulate gene products. Within this context, we conducted a broad mutational survey of 42 short 3'UTR microsatellites (MSs in 45 MSI-H colorectal tumors and their corresponding normal colonic mucosae. METHODOLOGY/PRINCIPAL FINDINGS: In order to estimate the overall susceptibility of MSs to MSI in MSI-H tumors, the observed MSI frequency of each MS was correlated with its length, interspecies sequence conservation level, and distance from some genetic elements (i.e., stop codon, polyA signal, and microRNA binding sites. All MSs were stable in normal colonic mucosae. The MSI frequency at each MS in MSI-H tumors was independent of sequence conservation level and distance from other genetic elements. In contrast, MS length correlated significantly with MSI frequency in MSI-H tumors (r=0.86, p=7.2x10(-13. 3'UTR MSs demonstrated MSI frequencies in MSI-H tumors higher than the 99% upper limit predicted by MS length for RB1-inducible coiled-coil 1(RB1CC1, mutation frequency 68.4%, NUAK family SNF1-like kinase 1(NUAK1, 31.0%, and Rtf1, Paf1/RNA polymerase II complex component, homolog (RTF1, 25.0%. An in silico prediction of RNA structure alterations was conducted for these MSI events to gauge their likelihood of affecting post-transcriptional regulation. RB1CC1 mutant was predicted to lose a microRNA-accessible loop structure at a putative binding site for the tumor-suppressive microRNA, miR-138. In contrast, the predicted 3'UTR structural change was minimal for NUAK1- and RTF1 mutants. Notably, real-time quantitative RT-PCR analysis revealed significant RB1CC1 mRNA overexpression vs. normal colonic mucosae in MSI-H cancers manifesting RB1CC1 3'UTR MSI (9.0-fold; p = 3.6x10(-4. CONCLUSIONS: This

  18. RNA-based Assay Demonstrated Enterococcus faecalis Metabolic Activity after Chemomechanical Procedures.

    Science.gov (United States)

    Pinheiro, Ericka T; Candeiro, George T; Teixeira, Sílvia R; Shin, Regina C; Prado, Laís C; Gavini, Giulio; Mayer, Márcia P A

    2015-09-01

    Because ribosomal RNA (rRNA) indicates metabolic cell activity, this study aimed to evaluate the sensitivity of rRNA-based quantitative polymerase chain reaction (RT-qPCR) for the identification of active Enterococcus faecalis in root canals samples compared with a method based on ribosomal DNA (rDNA) (rRNA genes). Samples were taken from 18 teeth with persistent/secondary intraradicular infection before (S1) and after (S2) chemomechanical preparation. RNA and DNA were extracted, and complementary DNA was synthesized from RNA using RT-PCR. Complementary DNA and genomic DNA were subjected to quantitative polymerase chain reaction with primers complementary for E. faecalis 16S rRNA sequence. E. faecalis was detected in 77.8% and 72.2% of S1 samples using rRNA- and rDNA-based assays, respectively. In contrast, E. faecalis was detected in only 33.3% of S2 samples using rDNA as the template compared with 61.1% using the rRNA-based method. The median concentration of rRNA copies of E. faecalis was significantly higher than rDNA copies, indicating a higher sensitivity for the method targeting rRNA in both S1 (P faecalis persisted in root canals after chemomechanical preparation. The RT-qPCR assay provides a sensitive method for the identification of active E. faecalis from endodontic samples. Furthermore, the rRNA-based assay indicated that E. faecalis viable cells persisted in treated root canals, suggesting that it may be a useful tool for monitoring microbial load during endodontic treatment. Copyright © 2015 American Association of Endodontists. Published by Elsevier Inc. All rights reserved.

  19. A de novo splice site mutation in EHMT1 resulting in Kleefstra syndrome with pharmacogenomics screening and behavior therapy for regressive behaviors.

    Science.gov (United States)

    Mitra, Amit Kumar; Dodge, Jessica; Van Ness, Jody; Sokeye, Israel; Van Ness, Brian

    2017-03-01

    Kleefstra syndrome (KS) is a rare autosomal dominant developmental disability, caused by microdeletions or intragenic mutations within the epigenetic regulator gene EHMT1 (euchromatic histone lysine N-methyltransferase 1). In addition to common features of autism, young adult regressive behaviors have been reported. However, the genetic downstream effects of the reported deletions or mutations on KS phenotype have not yet been completely explored. While genetic backgrounds affecting drug metabolism can have a profound effect on therapeutic interventions, pharmacogenomic variations are seldom considered in directing psychotropic therapies. In this report, we used next-generation sequencing (exome sequencing and high-throughput RNA sequencing) in a patient and his parents to identify causative genetic variants followed by pharmacogenomics-guided clinical decision-making for making positive changes toward his treatment strategies. The patient had an early autism diagnosis and showed significant regressive behaviors and physical aberrations at age 23. Exome sequencing identified a novel, de novo splice site variant NM_024757.4: c.2750-1G>T in EHMT1, a candidate gene for Kleefstra syndrome, in the patient that results in exon skipping and downstream frameshift and termination. Gene expression results from the patient showed, when compared to his parents, there was a significant decreased expression of several reported gene variants associated with autism risk. Further, using a pharmacogenomics genotyping panel, we discovered that the patient had the CYP2D6 nonfunctioning variant genotype *4/*4 that results in very low metabolic activity on a number of psychotropic drugs, including fluvoxamine which he was prescribed. As reported here, a change in psychotropic drugs and intense behavior therapies resulted in a significant reversal of the regressive behaviors and physical aberrations. These results demonstrate an individualized approach that integrated genetic information

  20. Mutation screening of MIR146A/B and BRCA1/2 3′-UTRs in the GENESIS study

    Science.gov (United States)

    Garcia, Amandine I; Buisson, Monique; Damiola, Francesca; Tessereau, Chloé; Barjhoux, Laure; Verny-Pierre, Carole; Sornin, Valérie; Dondon, Marie-Gabrielle; Eon-Marchais, Séverine; Caron, Olivier; Gautier-Villars, Marion; Coupier, Isabelle; Buecher, Bruno; Vennin, Philippe; Belotti, Muriel; Lortholary, Alain; Gesta, Paul; Dugast, Catherine; Noguès, Catherine; Fricker, Jean-Pierre; Faivre, Laurence; Stoppa-Lyonnet, Dominique; Andrieu, Nadine; Sinilnikova, Olga M; Mazoyer, Sylvie

    2016-01-01

    Although a wide number of breast cancer susceptibility alleles associated with various levels of risk have been identified to date, about 50% of the heritability is still missing. Although the major BRCA1 and BRCA2 genes are being extensively screened for truncating and missense variants in breast and/or ovarian cancer families, potential regulatory variants affecting their expression remain largely unexplored. In an attempt to identify such variants, we focused our attention on gene regulation mediated by microRNAs (miRs). We screened two genes, MIR146A and MIR146B, producing miR-146a and miR-146b-5p, respectively, that regulate BRCA1, and the 3′- untranslated regions (3′-UTRs) of BRCA1 and BRCA2 in the GENESIS French national case/control study (BRCA1- and BRCA2-negative breast cancer cases with at least one sister with breast cancer and matched controls). We identified one rare variant in MIR146A, four in MIR146B, five in BRCA1 3′-UTR and one in BRCA2 3′-UTR in 716 index cases and 619 controls. Among these 11 rare variants, 7 were identified each in 1 index case. None of the three relevant MIR146A/MIR146B variants affected the pre-miR sequences. The potential causality of the four relevant BRCA1/BRCA2 3′-UTRs variants was evaluated with luciferase reporter assays and co-segregation studies, as well as with bioinformatics analyses to predict miRs-binding sites, RNA secondary structures and RNA accessibility. This is the first study to report the screening of miR genes and of BRCA2 3′-UTR in a large series of familial breast cancer cases. None of the variant identified in this study gave convincing evidence of potential pathogenicity. PMID:26785832

  1. Unlocking the 'microbial black box' using RNA-based stable isotope probing technologies.

    Science.gov (United States)

    Whiteley, Andrew S; Manefield, Mike; Lueders, Tillmann

    2006-02-01

    Microbial ecologists have long sought to associate the transformation of compounds in the environment with the microbial clades responsible. The development of stable isotope probing (SIP) has made this possible in many ecological and biotechnological contexts. RNA-based SIP technologies represent a significant leap forward for culture-independent 'functional phylogeny' analyses, where specific consumption of a given compound carrying a (13)C signature can be associated with the small subunit ribosomal RNA molecules of the microbes that consume it. Recent advances have led to the unequivocal identification of microorganisms responsible for contaminant degradation in engineered systems, and to applications enhancing our understanding of carbon flow in terrestrial ecosystems.

  2. MicroRNA-based Therapy in Animal Models of Selected Gastrointestinal Cancers

    Directory of Open Access Journals (Sweden)

    Jana Merhautova

    2016-09-01

    Full Text Available Gastrointestinal cancer accounts for the 20 most frequent cancer diseases worldwide and there is a constant urge to bring new therapeutics with new mechanism of action into the clinical practice. Quantity of in vitro and in vivo evidences indicate, that exogenous change in pathologically imbalanced microRNAs (miRNAs is capable of transforming the cancer cell phenotype. This review analyzed preclinical miRNA-based therapy attempts in animal models of gastric, pancreatic, gallbladder, and colorectal cancer. From more than 400 original articles, 26 was found to assess the effect of miRNA mimics, precursors, expression vectors, or inhibitors administered locally or systemically being an approach with relatively high translational potential. We have focused on mapping available information on animal model used (animal strain, cell line, xenograft method, pharmacological aspects (oligonucleotide chemistry, delivery system, posology, route of administration and toxicology assessments. We also summarize findings in the field pharmacokinetics and toxicity of miRNA-based therapy.□

  3. Hydration sites of unpaired RNA bases: a statistical analysis of the PDB structures

    Directory of Open Access Journals (Sweden)

    Carugo Oliviero

    2011-10-01

    Full Text Available Abstract Background Hydration is crucial for RNA structure and function. X-ray crystallography is the most commonly used method to determine RNA structures and hydration and, therefore, statistical surveys are based on crystallographic results, the number of which is quickly increasing. Results A statistical analysis of the water molecule distribution in high-resolution X-ray structures of unpaired RNA nucleotides showed that: different bases have the same penchant to be surrounded by water molecules; clusters of water molecules indicate possible hydration sites, which, in some cases, match those of the major and minor grooves of RNA and DNA double helices; complex hydrogen bond networks characterize the solvation of the nucleotides, resulting in a significant rigidity of the base and its surrounding water molecules. Interestingly, the hydration sites around unpaired RNA bases do not match, in general, the positions that are occupied by the second nucleotide when the base-pair is formed. Conclusions The hydration sites around unpaired RNA bases were found. They do not replicate the atom positions of complementary bases in the Watson-Crick pairs.

  4. [The use of ultra deep sequencing technique in the screening program on HIV-1 drug resistance mutation among ART-naїve patients in Hunan province].

    Science.gov (United States)

    He, Jianmei; Zou, Xiaobai; Chen, Xi; Zheng, Jun

    2014-10-01

    To determine the prevalence rates of nucleotide reverse-transcriptase inhibitor (NRTI) and nonnucleoside reverse transcriptase inhibitor (NNRTI)TDRs among HIV-1 ART-naїve patients in Hunan province using the ultra deep sequencing (UDS) technique. ART-naїve subjects diagnosed in Hunan between 2010 and 2011 were evaluated by both UDS technique and Sanger sequencing techniques, to the 1% variant level. Mutations were scored using the Stanford HIVdb algorithm to infer the status on drug resistance. UDS method was performed on 90 ART-naїve subjects that seeking service of care, in Hunan. In total, 42.2% (38/90) of the subjects showed major NRTI or nonnucleoside reverse transcriptase inhibitor NNRTI TDRs by UDS technique, at a HIV variant frequency level of ≥1%, 15.6% (14/90) showed NRTI TDR, 16.7% (15/90) showed a major NNRTI TDR and 10% (9/90) were both resistant to NRTI and NNRTI when variants were analyzed by Stanford HIVdb. ART-naїve subjects from Hunan province, which had been predominately infected by subtype AE, would frequently possess HIV variants with NRTI/NNRTI TDRs that would affect the use of first line ART in the region, identified by the UDS technique. Further studies were needed to describe the prevalence of TDRs and to gather related information.

  5. "Hypothesis for the Modern RNA World": A pervasive Non-coding RNA-Based Genetic Regulation is a Prerequisite for the Emergence of Multicellular Complexity

    Science.gov (United States)

    Lozada-Chávez, Irma; Stadler, Peter F.; Prohaska, Sonja J.

    2011-12-01

    The transitions to multicellularity mark the most pivotal and distinctive events in life's history on Earth. Although several transitions to "simple" multicellularity (SM) have been recorded in both bacterial and eukaryotic clades, transitions to complex multicellularity (CM) have only happened a few times in eukaryotes. A large number of cell types (associated with large body size), increased energy consumption per gene expressed, and an increment of non-protein-coding DNA positively correlate with CM. These three factors can indeed be understood as the causes and consequences of the regulation of gene expression. Here, we discuss how a vast expansion of non-protein-coding RNA (ncRNAs) regulators rather than large numbers of novel protein regulators can easily contribute to the emergence of CM. We also propose that the evolutionary advantage of RNA-based gene regulation derives from the robustness of the RNA structure that makes it easy to combine genetic drift with functional exploration. We describe a model which aims to explain how the evolutionary dynamic of ncRNAs becomes dominated by the accessibility of advantageous mutations to innovate regulation in complex multicellular organisms. The information and models discussed here outline the hypothesis that pervasive ncRNA-based regulatory systems, only capable of being expanded and explored in higher eukaryotes, are prerequisite to complex multicellularity. Thereby, regulatory RNA molecules in Eukarya have allowed intensification of morphological complexity by stabilizing critical phenotypes and controlling developmental precision. Although the origin of RNA on early Earth is still controversial, it is becoming clear that once RNA emerged into a protocellular system, its relevance within the evolution of biological systems has been greater than we previously thought.

  6. Effects of Immediate Telephone Follow-Up with Providers on Sweat Chloride Test Timing after Cystic Fibrosis Newborn Screening Identifies a Single Mutation

    Science.gov (United States)

    La Pean, Alison; Farrell, Michael H.; Eskra, Kerry L.; Farrell, Philip M.

    2012-01-01

    Objectives To assess whether reporting “possible cystic fibrosis (CF)” newborn screening (NBS) results via fax plus simultaneous telephone contact with primary care providers (PCPs), versus fax alone, influenced three outcomes: getting a sweat chloride test, age at sweat chloride test, and sweat-testing before 8 weeks old. Study Design Retrospective cohort comparison of infants born in Wisconsin whose PCPs received telephone intervention (n=301), versus recent historical controls whose PCP did not (n=355). Intervention data were collected during a longitudinal research and quality improvement effort; de-identified comparison data were constructed from auxiliary NBS tracking information. Parametric and nonparametric statistical analyses tested for group differences. Results Most infants (92%) with “possible CF” NBS results whose PCPs lacked telephone intervention ultimately underwent sweat-testing, underlining efficacy for fax-only reporting. Telephone intervention was significantly associated with improvements in infants undergoing sweat-testing at both ≤6 and sweat-testing. The effect of telephone intervention was greater for PCPs whose patients underwent sweat-testing at community-affiliated medical centers versus academic medical centers (p=0.008). Conclusion Reporting “possible CF” NBS results via fax plus simultaneous telephone follow-up with PCPs increases the number of infants who have sweat chloride tests before 8 weeks of age, when affected infants are more likely to receive full benefits of early diagnosis and treatment. PMID:23102590

  7. Outcome and toxicities associated to chemotherapy in children with acute lymphoblastic leukemia and Gilbert syndrome. Usefulness of UGT1A1 mutational screening.

    Science.gov (United States)

    Berrueco, R; Alonso-Saladrigues, A; Martorell-Sampol, L; Català-Temprano, A; Ruiz-Llobet, A; Toll, T; Torrebadell, M; Naudó, M; Camós, M; Rives, S

    2015-07-01

    Acute lymphoblastic leukemia (ALL) is the most frequent cancer in childhood. Although intensive chemotherapy has improved survival in those patients, important side effects, including hyperbilirubinemia, are frequent. Gilbert syndrome (GS) is a frequent condition that causes a reduction in glucuronidation and intermittent hyperbilirubinemia episodes. This could provoke a greater exposure to some cytotoxic agents used in ALL, increasing the risk of toxicity. On the other hand, unexplained hyperbilirubinemia could lead to unnecessary modifications or even treatment withdrawals, which could increase the risk of relapse, but data regarding this in ALL pediatric population are scarce. Retrospective study to analyze toxicity, outcome and treatment modifications related to GS in children diagnosed with ALL. A total of 23 of 159 patients were diagnosed with GS. They had statistically higher hyperbilirubinemias during all treatment phases (P < 0.0001) and a slower methotrexate clearance when it was administered during a 24-hr infusion at high doses (patients with GS: 74 hr ± 19 vs. patients without GS: 64 hr ± 8; P <  .002). However, no relevant toxicity or delays in treatment were found in them. Finally, changes in treatment due to hyperbilirubinemia were only done in 5 patients with GS. Differences in outcome were not found in patients with GS. Universal screening for GS appears to be not necessary in pediatric patients with ALL. However, when hyperbilirubinemia is observed, it must be rule out in order to avoid unnecessary changes in treatment. © 2015 Wiley Periodicals, Inc.

  8. Bacterial Small RNA-based Negative Regulation: Hfq and Its Accomplices*

    Science.gov (United States)

    De Lay, Nicholas; Schu, Daniel J.; Gottesman, Susan

    2013-01-01

    A large group of bacterial small regulatory RNAs (sRNAs) use the Hfq chaperone to mediate pairing with and regulation of mRNAs. Recent findings help to clarify how Hfq acts and highlight the role of the endonuclease RNase E and its associated proteins (the degradosome) in negative regulation by these sRNAs. sRNAs frequently uncouple transcription and translation by blocking ribosome access to the mRNA, allowing other proteins access to the mRNA. As more examples of sRNA-mediated regulation are studied, more variations on how Hfq, RNase E, and other proteins collaborate to bring about sRNA-based regulation are being found. PMID:23362267

  9. Bacterial small RNA-based negative regulation: Hfq and its accomplices.

    Science.gov (United States)

    De Lay, Nicholas; Schu, Daniel J; Gottesman, Susan

    2013-03-22

    A large group of bacterial small regulatory RNAs (sRNAs) use the Hfq chaperone to mediate pairing with and regulation of mRNAs. Recent findings help to clarify how Hfq acts and highlight the role of the endonuclease RNase E and its associated proteins (the degradosome) in negative regulation by these sRNAs. sRNAs frequently uncouple transcription and translation by blocking ribosome access to the mRNA, allowing other proteins access to the mRNA. As more examples of sRNA-mediated regulation are studied, more variations on how Hfq, RNase E, and other proteins collaborate to bring about sRNA-based regulation are being found.

  10. 13C-detection in RNA bases: revealing structure-chemical shift relationships.

    Science.gov (United States)

    Farès, Christophe; Amata, Irene; Carlomagno, Teresa

    2007-12-26

    The chemical shifts of the unprotonated carbons in the proton-deficient nucleobases of RNA are rarely reported, despite the valuable information that they contain about base-pairing and base-stacking. We have developed 13C-detected 2D-experiments to identify the unprotonated 13C in the RNA bases and have assigned all the base nuclei of uniformly 13C,15N-labeled HIV-2 TAR-RNA. The 13C chemical shift distributions revealed perturbations correlated with the base-pairing and base-stacking properties of all four base-types. From this work, we conclude that the information contained in the chemical shift perturbations within the base rings can provide valuable restraint information for solving RNA structures, especially in conformational averaged regions, where NOE-based information is not available.

  11. Characterization and mapping of the human rhodopsin kinase gene and screening of the gene for mutations in patients with retinitis pigmentosa

    Energy Technology Data Exchange (ETDEWEB)

    Khani, S.C.; Lin, D.; Magovcevic, I. [Harvard Medical School, Boston, MA (United States)] [and others

    1994-09-01

    Rhodopsin kinase (RK) is a cytosolic enzyme in rod photoreceptors that initiates the deactivation of the phototransductions cascade by phosphorylating photoactivated rhodopsin. Although the cDNA sequence of bovine RK has been determined previously, no human cDNA or genomic sequence has thus far been available for genetic studies. In order to investigate the possible role of this candidate gene in retinitis pigmentosa (RP) and allied diseases, we have isolated and characterized human cDNA and genomic clones derived from the RK locus. The coding sequence of the human gene is 1692 nucleotides in length and is split into seven exons. The human and the bovine sequence show 84% identity at the nucleotide level and 92% identity at the amino acid level. Thus far, the intronic sequences flanking each exon except for one have been determined. We have also mapped the human RK gene to chromosome 13q34 using fluorescence in situ hybridization. To our knowledge, no RP gene has as yet been linked to this region. However, since the substrate for RK (rhodopsin) and other members of the phototransduction cascade have been implicated in the pathogenesis of RP, it is conceivable that defects in RK can also cause some forms of this disease. We are evaluating this possibility by screening DNA from 173 patients with autosomal recessive RP and 190 patients with autosomal dominant RP. So far, we have found 11 patients with variant bands. In one patient with autosomal dominant RP we discovered the missense change Ser536Leu. Cosegregation studies and further sequencing of the variant bands are currently underway.

  12. Minisequencing mitochondrial DNA pathogenic mutations

    Directory of Open Access Journals (Sweden)

    Carracedo Ángel

    2008-04-01

    Full Text Available Abstract Background There are a number of well-known mutations responsible of common mitochondrial DNA (mtDNA diseases. In order to overcome technical problems related to the analysis of complete mtDNA genomes, a variety of different techniques have been proposed that allow the screening of coding region pathogenic mutations. Methods We here propose a minisequencing assay for the analysis of mtDNA mutations. In a single reaction, we interrogate a total of 25 pathogenic mutations distributed all around the whole mtDNA genome in a sample of patients suspected for mtDNA disease. Results We have detected 11 causal homoplasmic mutations in patients suspected for Leber disease, which were further confirmed by standard automatic sequencing. Mutations m.11778G>A and m.14484T>C occur at higher frequency than expected by change in the Galician (northwest Spain patients carrying haplogroup J lineages (Fisher's Exact test, P-value Conclusion We here developed a minisequencing genotyping method for the screening of the most common pathogenic mtDNA mutations which is simple, fast, and low-cost. The technique is robust and reproducible and can easily be implemented in standard clinical laboratories.

  13. Depression Screening

    Science.gov (United States)

    ... Depression Screening Substance Abuse Screening Alcohol Use Screening Depression Screening (PHQ-9) - Instructions The following questions are ... this tool, there is also text-only version . Depression Screening - Manual Instructions The following questions are a ...

  14. Molecular screening in galactosemia

    Energy Technology Data Exchange (ETDEWEB)

    Elsas, L.J.; Singh, R.; Fernhoff, P.M. [Emory Univ., Atlanta, GA (United States)] [and others

    1994-09-01

    Classical galactosemia (G/G) is caused by the absence of galactose-1-phosphate uridyl transferase (GALT) activity while the Duarte allele produces partial impairment and a specific biochemical phenotype. Cloning and sequencing of the human GALT gene has enabled the identification of prevalent mutations for both Classical and Duarte alleles. The G allele is caused by a Q188R codon mutation in exon 6 in 70% of a Caucasian population while the D allele is caused by an N134D codon mutation in exon 10. Since the Q188R sequence creates a new Hpa II site and the N314D sequence creates a new Sin I site, it is relatively easy to screen for both mutations by multiplex PCR and restriction digest. Here we describe a method for detection of new mutations producing impaired GALT. Patient DNAs are subjected to SSCP (single strand conformational polymorphism) analysis of their 11 GALT exons. Direct sequencing of the exons targeted by SSCP has revealed many codon changes: IVSC 956 (a splice acceptor site loss), S135L, V151A, E203K, A320T, and Y323D. Two of these codon changes, V151A and S135L, have been confirmed as mutations by finding impaired GALT activity in a yeast expression system. We conclude that molecular screening of GALT DNA will clarify the structural biology of GALT and the pathophysiology of galactosemia.

  15. Causative mutations in FKBP10

    African Journals Online (AJOL)

    Prior to the current project, contact was made with medical colleagues in other centres in SA, and they were offered molecular genetic screening for mutations in their patients with OI-3 by the. Division of Human Genetics at UCT. The clinicians caring for affected persons with OI-3 and their families submitted blood or saliva.

  16. identification of a novel mutation

    Indian Academy of Sciences (India)

    in children and it mostly affects the liver, muscle and heart. (Koshy et al. 2006). GSDs can be differentiated ... novel mutation; Azeri Turkish sequencing. Journal of Genetics, DOI 10.1007/s12041-016-0734-y, Vol. ... patients were screened by their physicians at a children's hospital in Tabriz between February 2011 and July ...

  17. Mutations found in the Danish population causing Hereditary Hemorrhagic Telangiectasia

    DEFF Research Database (Denmark)

    Tørring, Pernille M; Brusgaard, Klaus

    2011-01-01

    been performing genetic screening of patients and relatives with HHT. The molecular genetic screening serves dual purposes, a) as part of the clinical management as genotype/phenotype correlations exists, b) to identify asymptomatic family members. Materials and Methods Inclusion of patient’s who were....... Results In 61 families we found mutations in either ENG (N=35) or ACVLR1 (N=26). In ENG a total of 22 different mutations were found 16 was unreported. In ACVLR1 24 different mutations were found 13 was unreported. The mutations were mainly of a familial character all though in ENG a single mutation...... is present in 11 families and 2 mutations are represented in 2 families. Likewise in ACVLR1 2 mutations was found in 2 different families. I ENG 1 and in ACVLR1 3 families had major deletions found by MLPA. No mutations were found in MADH4. Conclusion The majority of mutations found during clinical genetic...

  18. Nanomedicine Meets microRNA: Current Advances in RNA-Based Nanotherapies for Atherosclerosis.

    Science.gov (United States)

    Gadde, Suresh; Rayner, Katey J

    2016-09-01

    Cardiovascular disease (CVD) accounts for almost half of all deaths worldwide and has now surpassed infectious disease as the leading cause of death and disability in developing countries. At present, therapies such as low-density lipoprotein-lowering statins and antihypertensive drugs have begun to bend the morality curve for coronary artery disease (CAD); yet, as we come to appreciate the more complex pathophysiological processes in the vessel wall, there is an opportunity to fine-tune therapies to more directly target mechanisms that drive CAD. MicroRNAs (miRNAs) have been identified that control vascular cell homeostasis,(1-3) lipoprotein metabolism,(4-9) and inflammatory cell function.(10) Despite the importance of these miRNAs in driving atherosclerosis and vascular dysfunction, therapeutic modulation of miRNAs in a cell- and context-specific manner has been a challenge. In this review, we summarize the emergence of miRNA-based therapies as an approach to treat CAD by specifically targeting the pathways leading to the disease. We focus on the latest development of nanoparticles (NPs) as a means to specifically target the vessel wall and what the future of these nanomedicines may hold for the treatment of CAD. © 2016 American Heart Association, Inc.

  19. RNA-Based TWIST1 Inhibition via Dendrimer Complex to Reduce Breast Cancer Cell Metastasis

    Directory of Open Access Journals (Sweden)

    James Finlay

    2015-01-01

    Full Text Available Breast cancer is the leading cause of cancer-related deaths among women in the United States, and survival rates are lower for patients with metastases and/or triple-negative breast cancer (TNBC; ER, PR, and Her2 negative. Understanding the mechanisms of cancer metastasis is therefore crucial to identify new therapeutic targets and develop novel treatments to improve patient outcomes. A potential target is the TWIST1 transcription factor, which is often overexpressed in aggressive breast cancers and is a master regulator of cellular migration through epithelial-mesenchymal transition (EMT. Here, we demonstrate an siRNA-based TWIST1 silencing approach with delivery using a modified poly(amidoamine (PAMAM dendrimer. Our results demonstrate that SUM1315 TNBC cells efficiently take up PAMAM-siRNA complexes, leading to significant knockdown of TWIST1 and EMT-related target genes. Knockdown lasts up to one week after transfection and leads to a reduction in migration and invasion, as determined by wound healing and transwell assays. Furthermore, we demonstrate that PAMAM dendrimers can deliver siRNA to xenograft orthotopic tumors and siRNA remains in the tumor for at least four hours after treatment. These results suggest that further development of dendrimer-based delivery of siRNA for TWIST1 silencing may lead to a valuable adjunctive therapy for patients with TNBC.

  20. Synthetic Pre-miRNA-Based shRNA as Potent RNAi Triggers

    Directory of Open Access Journals (Sweden)

    Kazuya Terasawa

    2011-01-01

    Full Text Available RNA interference (RNAi is a powerful tool for studying gene function owing to the ease with which it can selectively silence genes of interest, and it has also attracted attention because of its potential for therapeutic applications. Chemically synthesized small interfering RNAs (siRNAs and DNA vector-based short hairpin RNAs (shRNAs are now widely used as RNAi triggers. In contrast to expressed shRNAs, the use of synthetic shRNAs is limited. Here we designed shRNAs modeled on a precursor microRNA (pre-miRNA and evaluated their biological activity. We demonstrated that chemically synthetic pre-miRNA-based shRNAs have more potent RNAi activity than their corresponding siRNAs and found that their antisense strands are more efficiently incorporated into the RNA-induced silencing complex. Although greater off-target effects and interferon responses were induced by shRNAs than by their corresponding siRNAs, these effects could be overcome by simply using a lower concentration or by optimizing and chemically modifying shRNAs similar to synthetic siRNAs. These are challenges for the future.

  1. Tracking fungal community responses to maize plants by DNA- and RNA-based pyrosequencing.

    Directory of Open Access Journals (Sweden)

    Eiko E Kuramae

    Full Text Available We assessed soil fungal diversity and community structure at two sampling times (t1 = 47 days and t2 = 104 days of plant age in pots associated with four maize cultivars, including two genetically modified (GM cultivars by high-throughput pyrosequencing of the 18S rRNA gene using DNA and RNA templates. We detected no significant differences in soil fungal diversity and community structure associated with different plant cultivars. However, DNA-based analyses yielded lower fungal OTU richness as compared to RNA-based analyses. Clear differences in fungal community structure were also observed in relation to sampling time and the nucleic acid pool targeted (DNA versus RNA. The most abundant soil fungi, as recovered by DNA-based methods, did not necessary represent the most "active" fungi (as recovered via RNA. Interestingly, RNA-derived community compositions at t1 were highly similar to DNA-derived communities at t2, based on presence/absence measures of OTUs. We recovered large proportions of fungal sequences belonging to arbuscular mycorrhizal fungi and Basidiomycota, especially at the RNA level, suggesting that these important and potentially beneficial fungi are not affected by the plant cultivars nor by GM traits (Bt toxin production. Our results suggest that even though DNA- and RNA-derived soil fungal communities can be very different at a given time, RNA composition may have a predictive power of fungal community development through time.

  2. RNA-Based Assessment of Diversity and Composition of Active Archaeal Communities in the German Bight

    Directory of Open Access Journals (Sweden)

    Bernd Wemheuer

    2012-01-01

    Full Text Available Archaea play an important role in various biogeochemical cycles. They are known extremophiles inhabiting environments such as thermal springs or hydrothermal vents. Recent studies have revealed a significant abundance of Archaea in moderate environments, for example, temperate sea water. Nevertheless, the composition and ecosystem function of these marine archaeal communities is largely unknown. To assess diversity and composition of active archaeal communities in the German Bight, seven marine water samples were taken and studied by RNA-based analysis of ribosomal 16S rRNA. For this purpose, total RNA was extracted from the samples and converted to cDNA. Archaeal community structures were investigated by pyrosequencing-based analysis of 16S rRNA amplicons generated from cDNA. To our knowledge, this is the first study combining next-generation sequencing and metatranscriptomics to study archaeal communities in marine habitats. The pyrosequencing-derived dataset comprised 62,045 archaeal 16S rRNA sequences. We identified Halobacteria as the predominant archaeal group across all samples with increased abundance in algal blooms. Thermoplasmatales (Euryarchaeota and the Marine Group I (Thaumarchaeota were identified in minor abundances. It is indicated that archaeal community patterns were influenced by environmental conditions.

  3. Viral evasion of a bacterial suicide system by RNA-based molecular mimicry enables infectious altruism.

    Directory of Open Access Journals (Sweden)

    Tim R Blower

    Full Text Available Abortive infection, during which an infected bacterial cell commits altruistic suicide to destroy the replicating bacteriophage and protect the clonal population, can be mediated by toxin-antitoxin systems such as the Type III protein-RNA toxin-antitoxin system, ToxIN. A flagellum-dependent bacteriophage of the Myoviridae, ΦTE, evolved rare mutants that "escaped" ToxIN-mediated abortive infection within Pectobacterium atrosepticum. Wild-type ΦTE encoded a short sequence similar to the repetitive nucleotide sequence of the RNA antitoxin, ToxI, from ToxIN. The ΦTE escape mutants had expanded the number of these "pseudo-ToxI" genetic repeats and, in one case, an escape phage had "hijacked" ToxI from the plasmid-borne toxIN locus, through recombination. Expression of the pseudo-ToxI repeats during ΦTE infection allowed the phage to replicate, unaffected by ToxIN, through RNA-based molecular mimicry. This is the first example of a non-coding RNA encoded by a phage that evolves by selective expansion and recombination to enable viral suppression of a defensive bacterial suicide system. Furthermore, the ΦTE escape phages had evolved enhanced capacity to transduce replicons expressing ToxIN, demonstrating virus-mediated horizontal transfer of genetic altruism.

  4. Analysis of the contributions of ring current and electric field effects to the chemical shifts of RNA bases.

    Science.gov (United States)

    Sahakyan, Aleksandr B; Vendruscolo, Michele

    2013-02-21

    Ring current and electric field effects can considerably influence NMR chemical shifts in biomolecules. Understanding such effects is particularly important for the development of accurate mappings between chemical shifts and the structures of nucleic acids. In this work, we first analyzed the Pople and the Haigh-Mallion models in terms of their ability to describe nitrogen base conjugated ring effects. We then created a database (DiBaseRNA) of three-dimensional arrangements of RNA base pairs from X-ray structures, calculated the corresponding chemical shifts via a hybrid density functional theory approach and used the results to parametrize the ring current and electric field effects in RNA bases. Next, we studied the coupling of the electric field and ring current effects for different inter-ring arrangements found in RNA bases using linear model fitting, with joint electric field and ring current, as well as only electric field and only ring current approximations. Taken together, our results provide a characterization of the interdependence of ring current and electric field geometric factors, which is shown to be especially important for the chemical shifts of non-hydrogen atoms in RNA bases.

  5. Screen-based identification and validation of four novel ion channels as regulators of renal ciliogenesis

    NARCIS (Netherlands)

    Slaats, Gisela G|info:eu-repo/dai/nl/413981452; Wheway, Gabrielle; Foletto, Veronica; Szymanska, Katarzyna; van Balkom, Bas W M|info:eu-repo/dai/nl/256594783; Logister, Ive; Den Ouden, Krista; Keijzer-Veen, Mandy G|info:eu-repo/dai/nl/292371950; Lilien, Marc R|info:eu-repo/dai/nl/271538899; Knoers, Nine V|info:eu-repo/dai/nl/298974460; Johnson, Colin A; Giles, Rachel H|info:eu-repo/dai/nl/173658725

    2015-01-01

    To investigate the contribution of ion channels to ciliogenesis we carried out an siRNA-based reverse genetics screen of all ion channels in the mouse genome in murine inner medullary collecting duct kidney cells. This screen revealed four candidate ion channel genes: Kcnq1, Kcnj10, Kcnf1 and Clcn4.

  6. Comparison of uncommon EGFR exon 21 L858R compound mutations with single mutation.

    Science.gov (United States)

    Peng, Liang; Song, Zhigang; Jiao, Shunchang

    2015-01-01

    Non-small-cell lung cancer with epidermal growth factor receptor (EGFR) mutation is sensitive to EGFR tyrosine kinase inhibitors (TKIs). But little is known about the response to EGFR TKIs and the prognostic role of compound mutations. This study compared the uncommon EGFR exon 21 L858R compound mutations with single mutation to characterize EGFR compound mutations and investigated their response to EGFR TKI treatment. We retrospectively screened 799 non-small-cell lung cancer patients from August 1, 2009 to June 1, 2012 by EGFR mutation testing. EGFR mutations were detected in 443 patients, with 22 (4.97%) compound mutations. Subsequently, six patients with EGFR exon 21 L858R compound mutations and 18 paired patients with single L858R mutation were well characterized. Finally, we also analyzed the EGFR TKI treatment response and patients' outcomes of compound or single L858R mutations. There was no differential treatment effect on the disease control rate and objective response rate between the L858R compound mutations and single mutation groups. No significant difference in overall survival or progression-free survival of these two groups was found by log-rank test. In conclusion, we demonstrated that no significant difference was detected in the response to EGFR TKIs and patients' outcomes in the compound and single mutation groups.

  7. Discordant diagnoses obtained by different approaches in antithrombin mutation analysis

    DEFF Research Database (Denmark)

    Feddersen, Søren; Nybo, Mads

    2014-01-01

    with a negative DHPLC mutation screening, discordant results were found in ten patients (62.5%) when using direct sequencing: Eight had the Basel mutation (c.218C>T), while two had the Cambridge II mutation (c.1246G>T). For seven of the ten patients this meant an altered clinical risk-assessment for future...

  8. Screening of three Mediterranean phenylketonuria mutations in ...

    Indian Academy of Sciences (India)

    Laboratory of Biochemistry, Department of Paediatrics, Rabta Hospital, 1007 Jebbari, Tunis, Tunisia; Laboratory of Biochemistry and Molecular Biology, Children's Hospital, 1007 Jebbari, Tunis, Tunisia; Research Unit UR04/SP03, Pasteur Institute, 1002 Tunis-Belvedere, Tunis, Tunisia; Department of Paediatrics, Rabta ...

  9. A miRNA-based signature detected in primary melanoma tissue predicts development of brain metastasis

    Science.gov (United States)

    Hanniford, Doug; Zhong, Judy; Koetz, Lisa; Gaziel-Sovran, Avital; Lackaye, Daniel J.; Shang, Shulian; Pavlick, Anna; Shapiro, Richard; Berman, Russell; Darvishian, Farbod; Shao, Yongzhao; Osman, Iman; Hernando, Eva

    2015-01-01

    Purpose Brain metastasis is the major cause of mortality among melanoma patients. A molecular prognostic test that can reliably stratify patients at initial melanoma diagnosis by risk of developing brain metastasis may inform the clinical management of these patients. Experimental Design We performed a retrospective, cohort-based study analyzing genome-wide and targeted microRNA expression profiling of primary melanoma tumors of three patient cohorts (n= 92, n= 119, n= 45) with extensive clinical follow up. We used Cox regression analysis to establish a microRNA-based signature that improves the ability of the current clinicopathologic staging system to predict the development of brain metastasis. Results Our analyses identified a 4-microRNA (miR-150–5p, miR-15b-5p, miR-16–5p, and miR-374b-3p) prognostic signature that, in combination with stage, distinguished primary melanomas that metastasized to the brain from non-recurrent and non-brain-metastatic primary tumors (training cohort: C-index=81.4%, validation cohort: C-index=67.4%, independent cohort: C-index=76.9%). Corresponding Kaplan-Meier curves of high- vs. low-risk patients displayed a clear separation in brain-metastasis-free and overall survival (training: pmelanomas metastatic to brain, correlated with presence of CD45+ tumor infiltrating lymphocytes. Conclusions A prognostic assay based on the described miRNA expression signature combined with the currently used staging criteria may improve accuracy of primary melanoma patient prognoses and aid clinical management of patients, including selection for adjuvant treatment or clinical trials of adjuvant therapies. PMID:26089374

  10. Medium-chain acyl-CoA dehydrogenase (MCAD) mutations identified by MS/MS-based prospective screening of newborns differ from those observed in patients with clinical symptoms

    DEFF Research Database (Denmark)

    Andresen, B S; Dobrowolski, S F; O'Reilly, L

    2001-01-01

    Medium-chain acyl-CoA dehydrogenase (MCAD) deficiency is the most frequently diagnosed mitochondrial beta-oxidation defect, and it is potentially fatal. Eighty percent of patients are homozygous for a common mutation, 985A-->G, and a further 18% have this mutation in only one disease allele...

  11. Live Cell Imaging of Endogenous mRNA Using RNA-Based Fluorescence "Turn-On" Probe.

    Science.gov (United States)

    Ong, Wei Qiang; Citron, Y Rose; Sekine, Sayaka; Huang, Bo

    2017-01-20

    Messenger RNA (mRNA) plays a critical role in cellular growth and development. However, there have been limited methods available to visualize endogenous mRNA in living cells with ease. We have designed RNA-based fluorescence "turn-on" probes that target mRNA by fusing an unstable form of Spinach with target-complementary sequences. These probes have been demonstrated to be selective, stable, and capable of targeting various mRNAs for live E. coli imaging.

  12. Small-interfering RNA (siRNA)-based functional micro- and nanostructures for efficient and selective gene silencing.

    Science.gov (United States)

    Lee, Soo Hyeon; Chung, Bong Hyun; Park, Tae Gwan; Nam, Yoon Sung; Mok, Hyejung

    2012-07-17

    Because of RNA's ability to encode structure and functional information, researchers have fabricated diverse geometric structures from this polymer at the micro- and nanoscale. With their tunable structures, rigidity, and biocompatibility, novel two-dimensional and three-dimensional RNA structures can serve as a fundamental platform for biomedical applications, including engineered tissues, biosensors, and drug delivery vehicles. The discovery of the potential of small-interfering RNA (siRNA) has underscored the applications of RNA-based micro- and nanostructures in medicine. Small-interfering RNA (siRNA), synthetic double-stranded RNA consisting of approximately 21 base pairs, suppresses problematic target genes in a sequence-specific manner via inherent RNA interference (RNAi) processing. As a result, siRNA offers a potential strategy for treatment of many human diseases. However, due to inefficient delivery to cells and off-target effects, the clinical application of therapeutic siRNA has been very challenging. To address these issues, researchers have studied a variety of nanocarrier systems for siRNA delivery. In this Account, we describe several strategies for efficient siRNA delivery and selective gene silencing. We took advantage of facile chemical conjugation and complementary hybridization to design novel siRNA-based micro- and nanostructures. Using chemical crosslinkers and hydrophobic/hydrophilic polymers at the end of siRNA, we produced various RNA-based structures, including siRNA block copolymers, micelles, linear siRNA homopolymers, and microhydrogels. Because of their increased charge density and flexibility compared with conventional siRNA, these micro- and nanostructures can form polyelectrolyte complexes with poorly charged and biocompatible cationic carriers that are both more condensed and more homogenous than the complexes formed in other carrier systems. In addition, the fabricated siRNA-based structures are linked by cleavable disulfide

  13. APC mutation spectrum of Norwegian familial adenomatous polyposis families: high ratio of novel mutations.

    Science.gov (United States)

    Andresen, Per Arne; Heimdal, Ketil; Aaberg, Kristin; Eklo, Katrine; Eklo, Kristin; Ariansen, Sarah; Silye, Alexandra; Fausa, Olav; Aabakken, Lars; Aretz, Stefan; Eide, Tor J; Gedde-Dahl, Tobias

    2009-10-01

    Familial adenomatous polyposis (FAP) is an autosomal dominantly inherited disease caused by mutations in the adenomatous polyposis coli (APC) gene. Massive formation of colorectal adenomas, of which some will inevitably develop into adenocarcinomas, is the hallmark of the disease. Characterization of causative APC mutations allows presymptomatic diagnosis, close follow-up and prophylactic intervention in families. To date more than 900 different germline mutations have been characterized worldwide demonstrating allelic heterogeneity. The germline mutation spectrum of APC identified in 69 apparently unrelated Norwegian FAP families are presented and discussed with reference to clinical phenotype and novel mutation rate. Different methods have been used over the years. However, all mutations were confirmed detectable by an implemented denaturing high-performance liquid chromatography screening approach. Multiplex ligation-dependent probe amplification analysis was employed for potential gross rearrangements. Fifty-three distinctive mutations were detected, of which 22 have been detected in Norway exclusively. Except for two major deletion mutations encompassing the entire APC, all mutations resulted in premature truncation of translation caused by non-sense (31%) or change in reading frame (69%). A high ratio of novel APC mutations continues to contribute to APC mutation heterogeneity causing FAP. This is the first comprehensive report of APC germline mutation spectrum in Norway.

  14. Auditory screening concurrent deafness predisposing genes screening in 10,043 neonates in Gansu province, China.

    Science.gov (United States)

    Zhang, Zhewen; Ding, Wenjuan; Liu, Xiaowen; Xu, Baicheng; Du, Wan; Nan, Shuling; Guo, Yufen

    2012-07-01

    The aim of our study was to evaluate the effectiveness of combining newborn hearing screening with screening for genetic mutations associated with deafness. Ten thousand forty-three newborn babies, born between December 2009 and April 2011 in Gansu province, China, were screened for hearing loss using the otoacoustic emissions test or automatic auditory brainstem response test and genetic mutations associated with deafness using a standard protocol. In the hearing screening, the referral rate for hearing loss in the first-step screening was 14.4% (1409/9786), decreasing significantly to 3.8% (362/9506) upon retesting. After the second-step screening, a total of 537 newborns were lost to follow-up. The genetic screening found that about 2.29% (230/10,043) individuals carried one or more recessive risk alleles or the mitochondrial mutation. Among them, 18 babies had the pathogenic mitochondrial DNA mutation, 92 babies were SLC26A4 heterozygote carriers, one case with both SLC26A4 and 12S rRNA 1555A>G mutation, 117 babies were GJB2 heterozygote carriers, and two babies were GJB2 homozygote carriers. However, 83.5% (192/230) neonates passed the conventional hearing screening among these carriers. It might be effective to complement the conventional hearing screening with gene screening for the purpose of early diagnosis and discovery of the late-onset hearing loss. Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.

  15. Three new BLM gene mutations associated with Bloom syndrome.

    Science.gov (United States)

    Amor-Guéret, Mounira; Dubois-d'Enghien, Catherine; Laugé, Anthony; Onclercq-Delic, Rosine; Barakat, Abdelhamid; Chadli, Elbekkay; Bousfiha, Ahmed Aziz; Benjelloun, Meriem; Flori, Elisabeth; Doray, Bérénice; Laugel, Vincent; Lourenço, Maria Teresa; Gonçalves, Rui; Sousa, Silvia; Couturier, Jérôme; Stoppa-Lyonnet, Dominique

    2008-06-01

    Bloom's syndrome (BS) is a rare autosomal recessive disease predisposing patients to all types of cancers affecting the general population. BS cells display a high level of genetic instability, including a 10-fold increase in the rate of sister chromatid exchanges, currently the only objective criterion for BS diagnosis. We have developed a method for screening the BLM gene for mutations based on direct genomic DNA sequencing. A questionnaire based on clinical information, cytogenetic features, and family history was addressed to physicians prescribing BS genetic screening, with the aim of confirming or guiding diagnosis. We report here four BLM gene mutations, three of which have not been described before. Three of the mutations are frameshift mutations, and the fourth is a nonsense mutation. All these mutations introduce a stop codon, and may therefore be considered to have deleterious biological effect. This approach should make it possible to identify new mutations and to correlate them with clinical information.

  16. Spontaneous Mutation Rate of Measles Virus: Direct Estimation Based on Mutations Conferring Monoclonal Antibody Resistance

    Science.gov (United States)

    Schrag, Stephanie J.; Rota, Paul A.; Bellini, William J.

    1999-01-01

    High mutation rates typical of RNA viruses often generate a unique viral population structure consisting of a large number of genetic microvariants. In the case of viral pathogens, this can result in rapid evolution of antiviral resistance or vaccine-escape mutants. We determined a direct estimate of the mutation rate of measles virus, the next likely target for global elimination following poliovirus. In a laboratory tissue culture system, we used the fluctuation test method of estimating mutation rate, which involves screening a large number of independent populations initiated by a small number of viruses each for the presence or absence of a particular single point mutation. The mutation we focused on, which can be screened for phenotypically, confers resistance to a monoclonal antibody (MAb 80-III-B2). The entire H gene of a subset of mutants was sequenced to verify that the resistance phenotype was associated with single point mutations. The epitope conferring MAb resistance was further characterized by Western blot analysis. Based on this approach, measles virus was estimated to have a mutation rate of 9 × 10−5 per base per replication and a genomic mutation rate of 1.43 per replication. The mutation rates we estimated for measles virus are comparable to recent in vitro estimates for both poliovirus and vesicular stomatitis virus. In the field, however, measles virus shows marked genetic stability. We briefly discuss the evolutionary implications of these results. PMID:9847306

  17. Small RNA-based silencing strategies for transposons in the process of invading Drosophila species

    Science.gov (United States)

    Rozhkov, Nikolay V.; Aravin, Alexei A.; Zelentsova, Elena S.; Schostak, Natalia G.; Sachidanandam, Ravi; McCombie, W. Richard; Hannon, Gregory J.; Evgen'ev, Michael B.

    2010-01-01

    Colonization of a host by an active transposon can increase mutation rates or cause sterility, a phenotype termed hybrid dysgenesis. As an example, intercrosses of certain Drosophila virilis strains can produce dysgenic progeny. The Penelope element is present only in a subset of laboratory strains and has been implicated as a causative agent of the dysgenic phenotype. We have also introduced Penelope into Drosophila melanogaster, which are otherwise naive to the element. We have taken advantage of these natural and experimentally induced colonization processes to probe the evolution of small RNA pathways in response to transposon challenge. In both species, Penelope was predominantly targeted by endo-small-interfering RNAs (siRNAs) rather than by piwi-interacting RNAs (piRNAs). Although we do observe correlations between Penelope transcription and dysgenesis, we could not correlate differences in maternally deposited Penelope piRNAs with the sterility of progeny. Instead, we found that strains that produced dysgenic progeny differed in their production of piRNAs from clusters in subtelomeric regions, possibly indicating that changes in the overall piRNA repertoire underlie dysgenesis. Considered together, our data reveal unexpected plasticity in small RNA pathways in germ cells, both in the character of their responses to invading transposons and in the piRNA clusters that define their ability to respond to mobile elements. PMID:20581131

  18. HFE mutations in the elderly.

    Science.gov (United States)

    Willis, Gavin; Wimperis, Jennie Z; Smith, Katy; Fellows, Ian W; Jennings, Barbara A

    2003-01-01

    Most individuals diagnosed with hereditary hemochromatosis have mutations in both copies of the HFE gene, with such mutations being common in populations of north European origin. The number of individuals currently diagnosed and treated for hemochromatosis is small relative to the number carrying two HFE mutations. Studies searching for undiagnosed hemochromatosis cases among disease cohorts have generally failed to find the number of cases that would be expected if disease were the commonest outcome for individuals with two C282Y HFE mutations. Our aim was to test the hypothesis that individuals with two HFE mutations would be under-represented in an elderly population because many would have died from disease caused by hemochromatosis before they reached old age. This is a cross-sectional study of elderly patients referred for full blood counts at the Norfolk and Norwich University Hospital. We screened blood samples from 1,000 elderly men (aged 85 and over) and women (aged 89 and over) for the C282Y, H63D, and S65C mutations of the HFE gene. We also analyzed any recent laboratory data relevant to signs of hemochromatosis. None of the ten possible genotypes was significantly under- or over-represented compared to the expected frequency calculated from the Hardy-Weinberg equation. Four C282Y homozygotes were found. There were few significant differences in the laboratory findings between the genotypes. Our data suggest that most people with HFE mutations survive to old age and do not suffer from signs of iron overload and hemochromatosis.

  19. p53 mutations in urinary bladder cancer

    OpenAIRE

    Berggren, P; Steineck, G; Adolfsson, J; Hansson, J; Jansson, O; Larsson, P; Sandstedt, B; Wijkstr?m, H; Hemminki, K

    2001-01-01

    We have screened for mutations in exons 5?8 of the p53 gene in a series consisting of 189 patients with urinary bladder neoplasms. 82 (44%) neoplasms were lowly malignant (Ta, G1?G2a) and 106 (56%) were highly malignant (G2b?G4 or ?T1). Only one mutation was in a lowly malignant urinary bladder neoplasm, in total we found p53 mutations in 26 (14%) of the 189 patients. 30% of the samples had loss of heterozygosity (LOH) for one or both of the p53 exogenic (CA)n repeat and the p53 intragenic (A...

  20. Spectrum of small mutations in the dystrophin coding region.

    Science.gov (United States)

    Prior, T W; Bartolo, C; Pearl, D K; Papp, A C; Snyder, P J; Sedra, M S; Burghes, A H; Mendell, J R

    1995-07-01

    Duchenne and Becker muscular dystrophies (DMD and BMD) are caused by defects in the dystrophin gene. About two-thirds of the affected patients have large deletions or duplications, which occur in the 5' and central portion of the gene. The nondeletion/duplication cases are most likely the result of smaller mutations that cannot be identified by current diagnostic screening strategies. We screened approximately 80% of the dystrophin coding sequence for small mutations in 158 patients without deletions or duplications and identified 29 mutations. The study indicates that many of the DMD and the majority of the BMD small mutations lie in noncoding regions of the gene. All of the mutations identified were unique to single patients, and most of the mutations resulted in protein truncation. We did not find a clustering of small mutations similar to the deletion distribution but found > 40% of the small mutations 3' of exon 55. The extent of protein truncation caused by the 3' mutations did not determine the phenotype, since even the exon 76 nonsense mutation resulted in the severe DMD phenotype. Our study confirms that the dystrophin gene is subject to a high rate of mutation in CpG sequences. As a consequence of not finding any hotspots or prevalent small mutations, we conclude that it is presently not possible to perform direct carrier and prenatal diagnostics for many families without deletions or duplications.

  1. Diversity of ARSACS mutations in French-Canadians.

    Science.gov (United States)

    Thiffault, I; Dicaire, M J; Tetreault, M; Huang, K N; Demers-Lamarche, J; Bernard, G; Duquette, A; Larivière, R; Gehring, K; Montpetit, A; McPherson, P S; Richter, A; Montermini, L; Mercier, J; Mitchell, G A; Dupré, N; Prévost, C; Bouchard, J P; Mathieu, J; Brais, B

    2013-01-01

    The growing number of spastic ataxia of Charlevoix-Saguenay (SACS) gene mutations reported worldwide has broadened the clinical phenotype of autosomal recessive spastic ataxia of Charlevoix-Saguenay (ARSACS). The identification of Quebec ARSACS cases without two known SACS mutation led to the development of a multi-modal genomic strategy to uncover mutations in this large gene and explore phenotype variability. Search for SACS mutations by combining various methods on 20 cases with a classical French-Canadian ARSACS phenotype without two mutations and a group of 104 sporadic or recessive spastic ataxia cases of unknown cause. Western blot on lymphoblast protein from cases with different genotypes was probed to establish if they still expressed sacsin. A total of 12 mutations, including 7 novels, were uncovered in Quebec ARSACS cases. The screening of 104 spastic ataxia cases of unknown cause for 98 SACS mutations did not uncover carriers of two mutations. Compounds heterozygotes for one missense SACS mutation were found to minimally express sacsin. The large number of SACS mutations present even in Quebec suggests that the size of the gene alone may explain the great genotypic diversity. This study does not support an expanding ARSACS phenotype in the French-Canadian population. Most mutations lead to loss of function, though phenotypic variability in other populations may reflect partial loss of function with preservation of some sacsin expression. Our results also highlight the challenge of SACS mutation screening and the necessity to develop new generation sequencing methods to ensure low cost complete gene sequencing.

  2. Tay-Sachs disease preconception screening in Australia: self-knowledge of being an Ashkenazi Jew predicts carrier state better than does ancestral origin, although there is an increased risk for c.1421 + 1G > C mutation in individuals with South African heritage.

    Science.gov (United States)

    Lew, Raelia; Burnett, Leslie; Proos, Anné

    2011-12-01

    The Australasian Community Genetics Program provided a preconception screening for Tay-Sachs disease (TSD) to 4,105 Jewish high school students in Sydney and Melbourne over the 12-year period 1995-2007. By correlating the frequencies of mutant HEXA, MIM *606869 (gene map locus 15q23-q24) alleles with subjects' nominated ethnicity (Ashkenazi/Sephardi/Mixed) and grandparental birthplaces, we established that Ashkenazi ethnicity is a better predictor of TSD carrier status than grandparental ancestral origins. Screening self-identified Ashkenazi subjects detected 95% of TSD carriers (carrier frequency 1:25). Having mixed Ashkenazi and non-Ashkenazi heritage reduced the carrier frequency (1:97). South African heritage conveyed a fourfold risk of c.1421 + 1G > C mutation compared with other AJ subjects (odds ratio (OR), 4.19; 95% confidence interval (CI), 1.83-9.62, p = 0.001), but this was the only specific case of ancestral origin improving diagnostic sensitivity over that based on determining Ashkenazi ethnicity. Carriers of c.1278insTATC mutations were more likely to have heritage from Western Europe (OR, 1.65 (95% CI, 1.04-2.60), p = 0.032) and South Eastern Europe (OR, 1.77 (95% CI, 1.14-2.73), p = 0.010). However, heritage from specific European countries investigated did not significantly alter the overall odds of TSD carrier status.

  3. One third of Danish hypertrophic cardiomyopathy patients with MYH7 mutations have mutations [corrected] in MYH7 rod region.

    Science.gov (United States)

    Hougs, Lotte; Havndrup, Ole; Bundgaard, Henning; Køber, Lars; Vuust, Jens; Larsen, Lars Allan; Christiansen, Michael; Andersen, Paal Skytt

    2005-02-01

    Familial hypertrophic cardiomyopathy (FHC) is, in most cases, a disease of the sarcomere, caused by a mutation in one of 10 known sarcomere disease genes. More than 266 mutations have been identified since 1989. The FHC disease gene first characterized MYH7, encodes the cardiac beta-myosin heavy chain, and contains more than 115 of these mutations. However, in most studies, only the region encoding the globular head and the hinge region of the mature cardiac beta-myosin heavy chain have been investigated. Furthermore, most studies carries out screening for mutations in the most prevalent disease genes, and discontinues screening when an apparent disease-associated mutation has been identified. The aim of the present study was to screen for mutations in the rod region of the MYH7 gene in all probands of the cohort, regardless of the known genetic status of the proband. Three disease-causing mutations were identified in the rod region in four probands using capillary electrophoresis single-strand conformation polymorphism as a screening method. All mutations were novel: N1327K, R1712W, and E1753K. Two of the probands had already been shown to carry other FHC-associated mutations. In conclusion, we show that in the Danish cohort we find one third of all MYH7 mutations in the rod-encoding region and we find that two of the patients carrying these mutations also carry mutations in other FHC disease genes stressing the need for a complete screening of all known disease genes in FHC-patients.

  4. A comparative study of mutation screening of sarcomeric genes ( MYBPC3 , MYH7 , TNNT2 ) using single gene approach versus targeted gene panel next generation sequencing in a cohort of HCM patients in Egypt

    National Research Council Canada - National Science Library

    Heba Sh Kassem; Roddy Walsh; Paul J Barton; Besra S Abdelghany; Remon S Azer; Rachel Buchan; Shibu John; Ahmed Elguindy; Sarah Moharem-ElGamal; Hala M Badran; Hoda Shehata; Stuart A Cook; Magdi H Yacoub

    2017-01-01

    ..., genetic testing for HCM has focused on mutations in myosin heavy chain 7 (MYH7), myosin-binding protein C3 (MYBPC3) and cardiac troponin T (TNNT2), which account for between 35% and 60% of cases [5]. Traditionally such studies have been performed by single gene analysis, utilising either polymerase chain reaction (PCR) or denaturing high perfor...

  5. TILLING to detect induced mutations in soybean.

    Science.gov (United States)

    Cooper, Jennifer L; Till, Bradley J; Laport, Robert G; Darlow, Margaret C; Kleffner, Justin M; Jamai, Aziz; El-Mellouki, Tarik; Liu, Shiming; Ritchie, Rae; Nielsen, Niels; Bilyeu, Kristin D; Meksem, Khalid; Comai, Luca; Henikoff, Steven

    2008-01-24

    Soybean (Glycine max L. Merr.) is an important nitrogen-fixing crop that provides much of the world's protein and oil. However, the available tools for investigation of soybean gene function are limited. Nevertheless, chemical mutagenesis can be applied to soybean followed by screening for mutations in a target of interest using a strategy known as Targeting Induced Local Lesions IN Genomes (TILLING). We have applied TILLING to four mutagenized soybean populations, three of which were treated with ethyl methanesulfonate (EMS) and one with N-nitroso-N-methylurea (NMU). We screened seven targets in each population and discovered a total of 116 induced mutations. The NMU-treated population and one EMS mutagenized population had similar mutation density (approximately 1/140 kb), while another EMS population had a mutation density of approximately 1/250 kb. The remaining population had a mutation density of approximately 1/550 kb. Because of soybean's polyploid history, PCR amplification of multiple targets could impede mutation discovery. Indeed, one set of primers tested in this study amplified more than a single target and produced low quality data. To address this problem, we removed an extraneous target by pretreating genomic DNA with a restriction enzyme. Digestion of the template eliminated amplification of the extraneous target and allowed the identification of four additional mutant alleles compared to untreated template. The development of four independent populations with considerable mutation density, together with an additional method for screening closely related targets, indicates that soybean is a suitable organism for high-throughput mutation discovery even with its extensively duplicated genome.

  6. TILLING to detect induced mutations in soybean

    Directory of Open Access Journals (Sweden)

    Nielsen Niels

    2008-01-01

    Full Text Available Abstract Background Soybean (Glycine max L. Merr. is an important nitrogen-fixing crop that provides much of the world's protein and oil. However, the available tools for investigation of soybean gene function are limited. Nevertheless, chemical mutagenesis can be applied to soybean followed by screening for mutations in a target of interest using a strategy known as Targeting Induced Local Lesions IN Genomes (TILLING. We have applied TILLING to four mutagenized soybean populations, three of which were treated with ethyl methanesulfonate (EMS and one with N-nitroso-N-methylurea (NMU. Results We screened seven targets in each population and discovered a total of 116 induced mutations. The NMU-treated population and one EMS mutagenized population had similar mutation density (~1/140 kb, while another EMS population had a mutation density of ~1/250 kb. The remaining population had a mutation density of ~1/550 kb. Because of soybean's polyploid history, PCR amplification of multiple targets could impede mutation discovery. Indeed, one set of primers tested in this study amplified more than a single target and produced low quality data. To address this problem, we removed an extraneous target by pretreating genomic DNA with a restriction enzyme. Digestion of the template eliminated amplification of the extraneous target and allowed the identification of four additional mutant alleles compared to untreated template. Conclusion The development of four independent populations with considerable mutation density, together with an additional method for screening closely related targets, indicates that soybean is a suitable organism for high-throughput mutation discovery even with its extensively duplicated genome.

  7. Toxicology screen

    Science.gov (United States)

    ... this page: //medlineplus.gov/ency/article/003578.htm Toxicology screen To use the sharing features on this page, please enable JavaScript. A toxicology screen refers to various tests that determine the ...

  8. Carrier Screening

    Science.gov (United States)

    ... pregnant are offered carrier screening for cystic fibrosis, hemoglobinopathies , and spinal muscular atrophy . You can have screening ... caused by a change in genes or chromosomes. Hemoglobinopathies: Any inherited disorder caused by changes in the ...

  9. Airport Screening

    Science.gov (United States)

    Health Physics Society Specialists in Radiation Safety Airport Screening Fact Sheet Adopted: May 2011 Photo courtesy of Dan Paluska/Flickr Denver Airport Security Screening Introduction With air travel regaining popularity and ...

  10. MRSA Screening

    Science.gov (United States)

    ... Links Patient Resources For Health Professionals Subscribe Search MRSA Screening Send Us Your Feedback Choose Topic At ... Related Content Related Images View Sources Formal Name Methicillin-resistant Staphylococcus aureus Screening This article was last reviewed on February ...

  11. Recurrent BRCA1 and BRCA2 mutations in Mexican women with breast cancer.

    Science.gov (United States)

    Torres-Mejía, Gabriela; Royer, Robert; Llacuachaqui, Marcia; Akbari, Mohammad R; Giuliano, Anna R; Martínez-Matsushita, Louis; Angeles-Llerenas, Angélica; Ortega-Olvera, Carolina; Ziv, Elad; Lazcano-Ponce, Eduardo; Phelan, Catherine M; Narod, Steven A

    2015-03-01

    Germline mutations in the BRCA1 and BRCA2 genes confer an estimated 58% to 80% lifetime risk of breast cancer. In general, screening is done for cancer patients if a relative has been diagnosed with breast or ovarian cancer. There are few data on the prevalence of mutations in these genes in Mexican women with breast cancer and this hampers efforts to develop screening policies in Mexico. We screened 810 unselected women with breast cancer from three cities in Mexico (Mexico City, Veracruz, and Monterrey) for mutations in BRCA1 and BRCA2, including a panel of 26 previously reported mutations. Thirty-five mutations were identified in 34 women (4.3% of total) including 20 BRCA1 mutations and 15 BRCA2 mutations. Twenty-two of the 35 mutations were recurrent mutations (62.8%). Only five of the 34 mutation carriers had a first-degree relative with breast cancer (three with BRCA1 and two with BRCA2 mutations). These results support the rationale for a strategy of screening for recurrent mutations in all women with breast cancer in Mexico, as opposed to restricting screening to those with a sister or mother with breast or ovarian cancer. These results will impact cancer genetic testing in Mexico and the identification of at-risk individuals who will benefit from increased surveillance. Cancer Epidemiol Biomarkers Prev; 24(3); 498-505. ©2014 AACR. ©2014 American Association for Cancer Research.

  12. Cancer Screening

    OpenAIRE

    Krishna Prasad

    2004-01-01

    Cancer screening is a means to detect cancer early with the goal of decreasing morbidity and mortality. At present, there is a reasonable consensus regarding screening for breast, cervical and colorectal cances and the role of screening is under trial in case of cancers of the lung,  ovaries and prostate. On the other hand, good screening tests are not available for some of the commonest cancers in India like the oral, pharyngeal, esophageal and stomach cancers.

  13. Targeting miRNA-based medicines to cystic fibrosis airway epithelial cells using nanotechnology.

    Science.gov (United States)

    McKiernan, Paul J; Cunningham, Orla; Greene, Catherine M; Cryan, Sally-Ann

    2013-01-01

    Cystic fibrosis (CF) is an inherited disorder characterized by chronic airway inflammation. microRNAs (miRNAs) are endogenous small RNAs which act on messenger (m) RNA at a post transcriptional level, and there is a growing understanding that altered expression of miRNA is involved in the CF phenotype. Modulation of miRNA by replacement using miRNA mimics (premiRs) presents a new therapeutic paradigm for CF, but effective and safe methods of delivery to the CF epithelium are limiting clinical translation. Herein, polymeric nanoparticles are investigated for delivery of miRNA mimics into CF airway epithelial cells, using miR-126 as a proof-of-concept premiR cargo to determine efficiency. Two polymers, polyethyleneimine (PEI) and chitosan, were used to prepare miRNA nanomedicines, characterized for their size, surface (zeta) potential, and RNA complexation efficiency, and screened for delivery and cytotoxicity in CFBE41o- (human F508del cystic fibrosis transmembrane conductance regulator bronchial epithelial) cells using a novel high content analysis method. RNA extraction was carried out 24 hours post transfection, and miR-126 and TOM1 (target of Myb1) expression (a validated miR-126 target) was assessed. Manufacture was optimized to produce small nanoparticles that effectively complexed miRNA. Using high content analysis, PEI-based nanoparticles were more effective than chitosan-based nanoparticles in facilitating uptake of miRNA into CFBE41o- cells and this was confirmed in miR-126 assays. PEI-premiR-126 nanoparticles at low nitrogen/phosphate (N/P) ratios resulted in significant knockdown of TOM1 in CFBE41o- cells, with the most significant reduction of 66% in TOM1 expression elicited at an N/P ratio of 1:1 while chitosan-based miR-126 nanomedicines failed to facilitate statistically significant knockdown of TOM1 and both nanoparticles appeared relatively nontoxic. miRNA nanomedicine uptake can be qualitatively and quantitatively assessed rapidly by high content

  14. Isolated congenital hepatic fibrosis associated with TMEM67 mutations

    DEFF Research Database (Denmark)

    Vogel, Ida; Ott, Peter; Lildballe, Dorte

    2017-01-01

    We report an otherwise healthy 32-year-old man with portal hypertension, variceal bleeding, and congenital hepatic fibrosis with ductal plate malformation. Genetic screening identified two TMEM67 mutations. Biallelic TMEM67 mutations are known to cause Joubert/Meckel syndrome or nephronopthisis...

  15. Clinicopathological characteristics and mutation profiling in primary cutaneous melanoma.

    Science.gov (United States)

    Yaman, Banu; Akalin, Taner; Kandiloğlu, Gülşen

    2015-05-01

    The incidence of mutations in malignant melanoma varies remarkably according to the subtype of melanoma, and this in itself is affected by racial and geographical factors. Studies screening melanoma case series for different types of mutations are relatively rare. The authors analyzed the frequency of various somatic point mutations of 10 genes in 106 primary cutaneous melanoma cases. The mutations (BRAF, NRAS, KIT, CDKN2A, KRAS, HRAS, PIK3CA, STK11, GNAQ, CTNNB1) were evaluated with real-time PCR-based PCR-Array through allele-specific amplification, and the results were correlated with various clinicopathological characteristics. Mutations were found in 64.2% of the melanomas overall. BRAF (42.5%), NRAS (15.1%), and CDKN2A (13.2%) were the 3 most common mutations. BRAF and NRAS mutations were more frequent in nodular and superficial spreading melanomas (P < 0.001). Associations with BRAF mutation were as follows: male gender [odds ratio (OR) = 2.4], younger age (OR = 2.7), superficial spreading (OR = 15.6) and nodular melanoma (OR = 9.5), trunk localization (OR = 6.3), and intermittent sun exposure (OR = 4.6). A considerable percentage of V600K (44.4%) mutations were found among the BRAF mutations, whereas KIT mutations (3.8%) were less frequent. Multiple mutations were detected in 13.2% of the melanomas. The most common co-occurrences were in the BRAF, NRAS, and CDKN2A genes. The authors analyzed 10 somatic mutations in the main subtypes of primary cutaneous melanomas from the western region of Turkey. Mutations were found in 64.2% of the melanomas overall. The most common mutations were in the BRAF and NRAS genes. In addition to other less common mutations, a notable number of multiple mutations were encountered. The multiplicity and concurrence of mutations in this study may provide further study areas for personalized targeted therapy.

  16. Antifolate drug resistance: Novel mutations and haplotype ...

    Indian Academy of Sciences (India)

    N P Sarmah

    2017-09-27

    Sep 27, 2017 ... Malaria is a major public health concern in Northeast India with a preponderance of drug-resistant strains. Until recently ... India, were screened for malaria, and of these, 75 were found to be positive for Plasmodium falciparum. Samples ... Double mutation in dhfr gene causes intermediate resistance, while.

  17. Analysis of mutations causing familial hypercholesterolaemia in ...

    African Journals Online (AJOL)

    the LDLR (coding region, promoter and intron/exon boundaries), APOB (part of exon 26) and PCSK9 genes (exon 7), using high-resolution melting. Results. Eight LDLR mutations were identified, ... screened by four overlapping PCR fragments and exon 10 (228 bp) by two overlapping fragments. Amplification of most of the ...

  18. Development and validation of a microRNA based diagnostic assay for primary tumor site classification of liver core biopsies

    DEFF Research Database (Denmark)

    Perell, Katharina; Vincent, Martin; Vainer, Ben

    2015-01-01

    negatively affect the accuracy and usability of molecular classifiers. We have developed and validated a microRNA-based classifier, which predicts the primary tumor site of liver biopsies, containing a limited number of tumor cells. Concurrently we explored the influence of surrounding normal tissue...... for normal liver tissue contamination. Performance was estimated by cross-validation, followed by independent validation on 55 liver core biopsies with a tumor content as low as 10%. A microRNA classifier developed, using the statistical contamination model, showed an overall classification accuracy of 74...... on classification. MicroRNA profiling was performed using quantitative Real-Time PCR on formalin-fixed paraffin-embedded samples. 278 primary tumors and liver metastases, representing nine primary tumor classes, as well as normal liver samples were used as a training set. A statistical model was applied to adjust...

  19. Spliceosomal small nuclear RNAs of Tetrahymena thermophila and some possible snRNA-snRNA base-pairing interactions

    DEFF Research Database (Denmark)

    Orum, H; Nielsen, Henrik; Engberg, J

    1991-01-01

    organisms. Furthermore, secondary structures closely similar to phylogenetically proven models can be inferred from the T. thermophila data. Analysis of the snRNA sequences identifies three potential snRNA-snRNA base-pairing interactions, all of which are consistent with available phylogenetic data. Two......We have identified and characterized the full set of spliceosomal small nuclear RNAs (snRNAs; U1, U2, U4, U5 and U6) from the ciliated protozoan Tetrahymena thermophila. With the exception of U4 snRNA, the sizes of the T. thermophila snRNAs are closely similar to their metazoan homologues. The T....... thermophila snRNAs all have unique 5' ends, which start with an adenine residue. In contrast, with the exception of U6, their 3' ends show some size heterogeneity. The primary sequences of the T. thermophila snRNAs contain the sequence motifs shown, or proposed, to be of functional importance in other...

  20. The P3 domain of eukaryotic RNases P/MRP: making a protein-rich RNA-based enzyme.

    Science.gov (United States)

    Perederina, Anna; Krasilnikov, Andrey S

    2010-01-01

    Nuclear Ribonuclease (RNase) P is a universal essential RNA-based enzyme made of a catalytic RNA component and a protein part; eukaryotic RNase P is closely related to a universal eukaryotic ribonucleoprotein RNase MRP. The protein part of the eukaryotic RNases P/MRP is dramatically more complex than that in bacterial and archaeal RNases P. The increase in the complexity of the protein part in eukaryotic RNases P/MRP was accompanied by the appearance of a novel structural element in the RNA component: an essential and phylogenetically conserved helix-loop-helix P3 RNA domain. The crystal structure of the P3 RNA domain in a complex with protein components Pop6 and Pop7 has been recently solved. Here we discuss the most salient structural features of the P3 domain as well as its possible role in the evolutionary transition to the protein-rich eukaryotic RNases P/MRP.

  1. Double screening

    Energy Technology Data Exchange (ETDEWEB)

    Gratia, Pierre [Department of Physics, University of Chicago,South Ellis Avenue, Chicago, IL 60637 (United States); Hu, Wayne [Department of Astronomy and Astrophysics, University of Chicago,South Ellis Avenue, Chicago, IL 60637 (United States); Enrico Fermi Institute and Kavli Institute for Cosmological Physics, University of Chicago,South Ellis Avenue, Chicago, IL 60637 (United States); Joyce, Austin [Enrico Fermi Institute and Kavli Institute for Cosmological Physics, University of Chicago,South Ellis Avenue, Chicago, IL 60637 (United States); Ribeiro, Raquel H. [School of Physics and Astronomy, Queen Mary University of London,Mile End Road, London, E1 4NS (United Kingdom)

    2016-06-15

    Attempts to modify gravity in the infrared typically require a screening mechanism to ensure consistency with local tests of gravity. These screening mechanisms fit into three broad classes; we investigate theories which are capable of exhibiting more than one type of screening. Specifically, we focus on a simple model which exhibits both Vainshtein and kinetic screening. We point out that due to the two characteristic length scales in the problem, the type of screening that dominates depends on the mass of the sourcing object, allowing for different phenomenology at different scales. We consider embedding this double screening phenomenology in a broader cosmological scenario and show that the simplest examples that exhibit double screening are radiatively stable.

  2. Rastreamento da mutação mitocondrial A1555G em pacientes com deficiência auditiva sensorioneural Screening of the mitochondrial A1555G mutation in patients with sensorineural hearing loss

    Directory of Open Access Journals (Sweden)

    Luciano Pereira Maniglia

    2008-10-01

    Full Text Available A mutação mitocondrial A1555G é a principal alteração associada à surdez ocasionada pelo uso de aminoglicosídeos. OBJETIVO: Investigar a prevalência da mutação A1555G em pacientes com deficiência auditiva sensorioneural com e sem uso de antibióticos aminoglicosídeos. MATERIAL E MÉTODO: Estudo em amostras de 27 pacientes com surdez, como casos, e em 100 neonatos, com audição normal, como grupo controle. O DNA foi extraído de leucócitos de amostras de sangue e "primers" específicos foram utilizados para amplificar o gene do citocromo b e a região que abrange a mutação A1555G do DNA mitocondrial, usando as técnicas da Reação em Cadeia da Polimerase e do Polimorfismo no Comprimento de Fragmentos de Restrição. DESENHO CIENTÍFICO: Estudo de casos em corte transversal. RESULTADOS: A região do gene do citocromo b foi amplificada, sendo confirmada a presença do DNA mitocondrial em todas as 127 amostras do estudo. A mutação A1555G não foi identificada nos 27 pacientes com deficiência auditiva e no grupo controle (100 neonatos. CONCLUSÕES: Os resultados são concordantes com estudos que relatam que a mutação A1555G não é prevalente nas Américas. Há interesse na determinação da real prevalência dessa mutação e na investigação de outras mutações que possam ocasionar deficiência auditiva associada ou não ao uso de aminoglicosídeos na população brasileira.The A1555G mitochondrial mutation is the main alteration associated with aminoglycoside-induced deafness. AIM: to investigate the prevalence of the A1555G mutation in patients sensorineural hearing loss patients with and without aminoglycosides antibiotic use. MATERIAL AND METHOD: a study of 27 cases with deafness as the sample, and 100 neonates with normal hearing as the control group. DNA was extracted from blood leukocyte samples, and specific oligonucleotide primers were designed to amplify the cytochrome b gene and the region which encloses the A1555

  3. A comparative study of mutation screening of sarcomeric genes (MYBPC3, MYH7, TNNT2 using single gene approach versus targeted gene panel next generation sequencing in a cohort of HCM patients in Egypt

    Directory of Open Access Journals (Sweden)

    Heba Sh. Kassem

    2017-10-01

    Conclusion: NGS provides a useful and rapid tool to allow panoramic screening of several genes simultaneously with a high sensitivity rate amongst genes of known etiologic role allowing high throughput analysis of HCM patients and relevant control series in a less characterised population.

  4. Two novel mutations in ILDR1 gene cause autosomal recessive ...

    Indian Academy of Sciences (India)

    In a recent screening programme on hearing loss (HL), we examined 17 common autosomal recessive nonsyndromic hearing loss (ARNSHL) genes in every consanguineous Ira- nian family with ARNSHL that was referred to our centre. We first screened GJB2 mutations and then utilized a panel of three to four short ...

  5. Systematic screening for mutations in the 5{prime}-regulatory region of the human dopamine D{sub 1} receptor (DRD1) gene in patients with schizophrenia and bipolar affective disorder

    Energy Technology Data Exchange (ETDEWEB)

    Cichon, S.; Noethen, M.M.; Stoeber, G. [Univ. of Bonn (Germany)] [and others

    1996-07-26

    A possible dysregulation of dopaminergic neurotransmission has been implicated in a variety of neuropsychiatric diseases. In the present study we systematically searched for the presence of mutations in the 5{prime}-flanking region of the dopamine D{sub 1} receptor (DRD1) gene. This region has previously been shown to contain a functional promoter. We investigated 119 unrelated individuals (including 36 schizophrenic patients, 38 bipolar affective patients, and 45 healthy controls) using single-strand conformation analysis (SSCA). Eleven overlapping PCR fragments covered 2,189 bp of DNA sequence. We identified six single base substitutions: -2218T/C, -2102C/A, -2030T/C, -1992G/A, -1251G/C, and -800T/C. None of the mutations was found to be located in regions which have important influence on the level of transcriptional activity. Allele frequencies were similar in patients and controls, indicating that genetic variation in the 5{prime}-regulatory region of the DRD1 gene is unlikely to play a frequent, major role in the genetic predisposition to either schizophrenia or bipolar affective disorder. 31 refs., 3 tabs.

  6. Multiple gene mutations, not the type of mutation, are the modifier of left ventricle hypertrophy in patients with hypertrophic cardiomyopathy.

    Science.gov (United States)

    Zou, Yubao; Wang, Jizheng; Liu, Xuan; Wang, Yilu; Chen, Yi; Sun, Kai; Gao, Shuo; Zhang, Channa; Wang, Zhimin; Zhang, Yin; Feng, Xinxing; Song, Ying; Wu, Yajie; Zhang, Hongju; Jia, Lei; Wang, Hu; Wang, Dong; Yan, Chaowu; Lu, Minjie; Zhou, Xianliang; Song, Lei; Hui, Rutai

    2013-06-01

    Genotype-phenotype correlation of hypertrophic cardiomyopathy (HCM) has been challenging because of the genetic and clinical heterogeneity. To determine the mutation profile of Chinese patients with HCM and to correlate genotypes with phenotypes, we performed a systematic mutation screening of the eight most commonly mutated genes encoding sarcomere proteins in 200 unrelated Chinese adult patients using direct DNA sequencing. A total of 98 mutations were identified in 102 mutation carriers. The frequency of mutations in MYH7, MYBPC3, TNNT2 and TNNI3 was 26.0, 18.0, 4.0 and 3.5 % respectively. Among the 200 genotyped HCM patients, 83 harbored a single mutation, and 19 (9.5 %) harbored multiple mutations. The number of mutations was positively correlated with the maximum wall thickness. We found that neither particular gene nor specific mutation was correlated to clinical phenotype. In summary, the frequency of multiple mutations was greater in Chinese HCM patients than in the Caucasian population. Multiple mutations in sarcomere protein may be a risk factor for left ventricular wall thickness.

  7. Developmental Screening

    Science.gov (United States)

    ... bye,” and pointing to something interesting are all developmental milestones, or things most children can do by a ... screening are ways to look for your child’s developmental milestones. Developmental Monitoring Developmental Screening WHO: You — parents, grandparents, ...

  8. Mapping Mutations on Phylogenies

    DEFF Research Database (Denmark)

    Nielsen, Rasmus

    2005-01-01

    This chapter provides a short review of recent methodologies developed for mapping mutations on phylogenies. Mapping of mutations, or character changes in general, using the maximum parsimony principle has been one of the most powerful tools in phylogenetics, and it has been used in a variety...... uncertainty in the mapping. Recently developed probabilistic methods can incorporate statistical uncertainty in the character mappings. In these methods, focus is on a probability distribution of mutational mappings instead of a single estimate of the mutational mapping....

  9. [Prenatal screening and diagnosis of genetic deafness by microarray].

    Science.gov (United States)

    Sun, Lian-hua; Li, Lei; Wang, Xiao-wen; Zhu, Ya-zhong; Chai, Yong-chuan; Li, Xiao-hua; Wu, Hao; Yang, Tao

    2012-12-01

    To evaluate a microarray-based mutation screening method for genetic deafness and its application in prenatal diagnosis. Mutation screening of common deafness genes was performed in pregnant women and volunteers spouses. Nine common mutations in four major deafness genes, GJB2, GJB3, SLC26A4 and mitochondrial 12S rRNA, were detected simultaneously by a microarray-based method. Genetic counseling was given based on their testing results. 5.11% of pregnant women carried at least one mutation. Among them, seven carried mutation in the mitochondria 12S rRNA gene and were offered aminoglycoside-induced ototoxicity warning. For other mutation carriers of GJB2 or SLC26A4 genes, additional mutation screening was performed in their husbands by direct sequencing. A total of 20 couples were at risk of giving birth to children with genetic deafness. Of five couples who selected to undergo prenatal diagnostic testing of the fetus, four were diagnosed as wild type or heterozygous for the tested genes and one as p.V37I/c.235delC compound heterozygous for GJB2. DNA microarray is a quick, easy and reliable method to screen mutations in genetic deafness genes. Application of this method in prenatal screening and diagnosis might effectively reduce the occurrence of genetic deafness.

  10. Detection of Sialic Acid-Utilising Bacteria in a Caecal Community Batch Culture Using RNA-Based Stable Isotope Probing

    Directory of Open Access Journals (Sweden)

    Wayne Young

    2015-03-01

    Full Text Available Sialic acids are monosaccharides typically found on cell surfaces and attached to soluble proteins, or as essential components of ganglioside structures that play a critical role in brain development and neural transmission. Human milk also contains sialic acid conjugated to oligosaccharides, glycolipids, and glycoproteins. These nutrients can reach the large bowel where they may be metabolised by the microbiota. However, little is known about the members of the microbiota involved in this function. To identify intestinal bacteria that utilise sialic acid within a complex intestinal community, we cultured the caecal microbiota from piglets in the presence of 13C-labelled sialic acid. Using RNA-based stable isotope probing, we identified bacteria that consumed 13C-sialic acid by fractionating total RNA in isopycnic buoyant density gradients followed by 16S rRNA gene analysis. Addition of sialic acid caused significant microbial community changes. A relative rise in Prevotella and Lactobacillus species was accompanied by a corresponding reduction in the genera Escherichia/Shigella, Ruminococcus and Eubacterium. Inspection of isotopically labelled RNA sequences suggests that the labelled sialic acid was consumed by a wide range of bacteria. However, species affiliated with the genus Prevotella were clearly identified as the most prolific users, as solely their RNA showed significantly higher relative shares among the most labelled RNA species. Given the relevance of sialic acid in nutrition, this study contributes to a better understanding of their microbial transformation in the intestinal tract with potential implications for human health.

  11. Confirming the RNAi-mediated mechanism of action of siRNA-based cancer therapeutics in mice.

    Science.gov (United States)

    Judge, Adam D; Robbins, Marjorie; Tavakoli, Iran; Levi, Jasna; Hu, Lina; Fronda, Anna; Ambegia, Ellen; McClintock, Kevin; MacLachlan, Ian

    2009-03-01

    siRNAs that specifically silence the expression of cancer-related genes offer a therapeutic approach in oncology. However, it remains critical to determine the true mechanism of their therapeutic effects. Here, we describe the preclinical development of chemically modified siRNA targeting the essential cell-cycle proteins polo-like kinase 1 (PLK1) and kinesin spindle protein (KSP) in mice. siRNA formulated in stable nucleic acid lipid particles (SNALP) displayed potent antitumor efficacy in both hepatic and subcutaneous tumor models. This was correlated with target gene silencing following a single intravenous administration that was sufficient to cause extensive mitotic disruption and tumor cell apoptosis. Our siRNA formulations induced no measurable immune response, minimizing the potential for nonspecific effects. Additionally, RNAi-specific mRNA cleavage products were found in tumor cells, and their presence correlated with the duration of target mRNA silencing. Histological biomarkers confirmed that RNAi-mediated gene silencing effectively inhibited the target's biological activity. This report supports an RNAi-mediated mechanism of action for siRNA antitumor effects, suggesting a new methodology for targeting other key genes in cancer development with siRNA-based therapeutics.

  12. Mutational landscape of yeast mutator strains.

    Science.gov (United States)

    Serero, Alexandre; Jubin, Claire; Loeillet, Sophie; Legoix-Né, Patricia; Nicolas, Alain G

    2014-02-04

    The acquisition of mutations is relevant to every aspect of genetics, including cancer and evolution of species on Darwinian selection. Genome variations arise from rare stochastic imperfections of cellular metabolism and deficiencies in maintenance genes. Here, we established the genome-wide spectrum of mutations that accumulate in a WT and in nine Saccharomyces cerevisiae mutator strains deficient for distinct genome maintenance processes: pol32Δ and rad27Δ (replication), msh2Δ (mismatch repair), tsa1Δ (oxidative stress), mre11Δ (recombination), mec1Δ tel1Δ (DNA damage/S-phase checkpoints), pif1Δ (maintenance of mitochondrial genome and telomere length), cac1Δ cac3Δ (nucleosome deposition), and clb5Δ (cell cycle progression). This study reveals the diversity, complexity, and ultimate unique nature of each mutational spectrum, composed of punctual mutations, chromosomal structural variations, and/or aneuploidies. The mutations produced in clb5Δ/CCNB1, mec1Δ/ATR, tel1Δ/ATM, and rad27Δ/FEN1 strains extensively reshape the genome, following a trajectory dependent on previous events. It comprises the transmission of unstable genomes that lead to colony mosaicisms. This comprehensive analytical approach of mutator defects provides a model to understand how genome variations might accumulate during clonal evolution of somatic cell populations, including tumor cells.

  13. Comparison of viable cell counts and fluorescence in situ hybridization using specific rRNA-based probes for the quantification of human fecal bacteria

    NARCIS (Netherlands)

    Harmsen, HJM; Gibson, GR; Elfferich, P; Raangs, GC; Wildeboer-Veloo, ACM; Argaiz, A; Roberfroid, MB; Welling, GW

    2000-01-01

    Conventional cultivation and fluorescence in situ hybridization (FISH) using 16S rRNA-based probes were compared for the enumeration of human colonic bacteria. Groups of common intestinal anaerobic bacteria were enumerated in slurries prepared From fecal samples of three healthy volunteers. To

  14. 16S rRNA-based bacterial diversity in the organic-rich sediments underlying oxygen-deficient waters of the eastern Arabian Sea

    Digital Repository Service at National Institute of Oceanography (India)

    Divya, B.; Parvathi, A.; LokaBharathi, P.A.; Nair, S.

    =UTF-8 Author version: World J. Microbiol. Biotechnol., vol.27; 2011; 2821-2833 16S rRNA-based bacterial diversity in the organic-rich sediments underlying oxygen deficient waters of eastern Arabian Sea Baby Divya 1 , Ammini Parvathi 2 , P. A...

  15. Linking phylogenetic identities of bacteria to starch fermentation in an in vitro model of the large intestine by RNA-based stable isotope probing

    NARCIS (Netherlands)

    Kovatcheva-Datchary, P.P.; Egert, M.G.G.; Maathuis, A.; Rajilic-Stojanovic, M.; Graaf, de A.A.; Smidt, H.; Vos, de W.M.; Venema, K.

    2009-01-01

    Summary Carbohydrates, including starches, are an important energy source for humans, and are known for their interactions with the microbiota in the digestive tract. Largely, those interactions are thought to promote human health. Using 16S ribosomal RNA (rRNA)-based stable isotope probing (SIP),

  16. Distinct summer and winter bacterial communities in the active layer of Svalbard permafrost revealed by DNA- and RNA-based analyses

    DEFF Research Database (Denmark)

    Schostag, Morten; Stibal, Marek; Jacobsen, Carsten S.

    2015-01-01

    was significantly correlated with pH and dissolved organic carbon, while the potentially active (RNA-based) community structure was not significantly correlated with any of the measured soil parameters. A large fraction of the 16S rRNA transcripts was assigned to nitrogen-fixing bacteria (up to 24% in June...

  17. Founder mutations characterise the mutation panorama in 200 Swedish index cases referred for Long QT syndrome genetic testing.

    Science.gov (United States)

    Stattin, Eva-Lena; Boström, Ida Maria; Winbo, Annika; Cederquist, Kristina; Jonasson, Jenni; Jonsson, Björn-Anders; Diamant, Ulla-Britt; Jensen, Steen M; Rydberg, Annika; Norberg, Anna

    2012-10-25

    Long QT syndrome (LQTS) is an inherited arrhythmic disorder characterised by prolongation of the QT interval on ECG, presence of syncope and sudden death. The symptoms in LQTS patients are highly variable, and genotype influences the clinical course. This study aims to report the spectrum of LQTS mutations in a Swedish cohort. Between March 2006 and October 2009, two hundred, unrelated index cases were referred to the Department of Clinical Genetics, Umeå University Hospital, Sweden, for LQTS genetic testing. We scanned five of the LQTS-susceptibility genes (KCNQ1, KCNH2, SCN5A, KCNE1, and KCNE2) for mutations by DHPLC and/or sequencing. We applied MLPA to detect large deletions or duplications in the KCNQ1, KCNH2, SCN5A, KCNE1, and KCNE2 genes. Furthermore, the gene RYR2 was screened in 36 selected LQTS genotype-negative patients to detect cases with the clinically overlapping disease catecholaminergic polymorphic ventricular tachycardia (CPVT). In total, a disease-causing mutation was identified in 103 of the 200 (52%) index cases. Of these, altered exon copy numbers in the KCNH2 gene accounted for 2% of the mutations, whereas a RYR2 mutation accounted for 3% of the mutations. The genotype-positive cases stemmed from 64 distinct mutations, of which 28% were novel to this cohort. The majority of the distinct mutations were found in a single case (80%), whereas 20% of the mutations were observed more than once. Two founder mutations, KCNQ1 p.Y111C and KCNQ1 p.R518*, accounted for 25% of the genotype-positive index cases. Genetic cascade screening of 481 relatives to the 103 index cases with an identified mutation revealed 41% mutation carriers who were at risk of cardiac events such as syncope or sudden unexpected death. In this cohort of Swedish index cases with suspected LQTS, a disease-causing mutation was identified in 52% of the referred patients. Copy number variations explained 2% of the mutations and 3 of 36 selected cases (8%) harboured a mutation in the

  18. Founder mutations characterise the mutation panorama in 200 Swedish index cases referred for Long QT syndrome genetic testing

    Directory of Open Access Journals (Sweden)

    Stattin Eva-Lena

    2012-10-01

    Full Text Available Abstract Background Long QT syndrome (LQTS is an inherited arrhythmic disorder characterised by prolongation of the QT interval on ECG, presence of syncope and sudden death. The symptoms in LQTS patients are highly variable, and genotype influences the clinical course. This study aims to report the spectrum of LQTS mutations in a Swedish cohort. Methods Between March 2006 and October 2009, two hundred, unrelated index cases were referred to the Department of Clinical Genetics, Umeå University Hospital, Sweden, for LQTS genetic testing. We scanned five of the LQTS-susceptibility genes (KCNQ1, KCNH2, SCN5A, KCNE1, and KCNE2 for mutations by DHPLC and/or sequencing. We applied MLPA to detect large deletions or duplications in the KCNQ1, KCNH2, SCN5A, KCNE1, and KCNE2 genes. Furthermore, the gene RYR2 was screened in 36 selected LQTS genotype-negative patients to detect cases with the clinically overlapping disease catecholaminergic polymorphic ventricular tachycardia (CPVT. Results In total, a disease-causing mutation was identified in 103 of the 200 (52% index cases. Of these, altered exon copy numbers in the KCNH2 gene accounted for 2% of the mutations, whereas a RYR2 mutation accounted for 3% of the mutations. The genotype-positive cases stemmed from 64 distinct mutations, of which 28% were novel to this cohort. The majority of the distinct mutations were found in a single case (80%, whereas 20% of the mutations were observed more than once. Two founder mutations, KCNQ1 p.Y111C and KCNQ1 p.R518*, accounted for 25% of the genotype-positive index cases. Genetic cascade screening of 481 relatives to the 103 index cases with an identified mutation revealed 41% mutation carriers who were at risk of cardiac events such as syncope or sudden unexpected death. Conclusion In this cohort of Swedish index cases with suspected LQTS, a disease-causing mutation was identified in 52% of the referred patients. Copy number variations explained 2% of the

  19. Mutation analysis of ATP7B gene in Turkish Wilson disease patients: identification of five novel mutations.

    Science.gov (United States)

    Simsek Papur, Ozlenen; Akman, Sezin Asik; Cakmur, Raif; Terzioglu, Orhan

    2013-04-01

    Wilson disease is an autosomal recessive disorder of copper metabolism caused by mutations in the ATP7B gene that encodes a P-type copper transporting ATPase. The aim of this study was to screen and detect mutations of the ATP7B gene in unrelated Turkish Wilson disease patients (n = 46) and control group (n = 52). Mutations were screened and detected by DNA sequencing. 30 out of 46 patients had mutations. 24 different Wilson disease related mutations were identified in those patients. The distribution of mutations in ATP7B gene was as follow: 17 missense, 3 nonsense, 1 silent, 3 frameshift (1 insertion, 2 deletion). None of them were not found in the control group. Five out of 24 mutations were found to be novel. Four of them were missense (c.2363C > T, c.3106G > A, c.3451C > T, c.3733C > A). The last one was deletion (c.3111delC). 10 single nucleotide polymorphisms (SNPs) given in the literature were found in both control and patients groups. Moreover one new polymorphism in exon 18 (c.3727G > A) not reported previously was discovered in both groups. It was striking that most of the mutations were found in exons 8, 12-14. This is the first study covering Turkish Wilson disease patients and control groups for mutation screening in all the coding regions of ATP7B gene by DNA sequencing method and adding five new mutations and one polymorphism into the HUGO Wilson disease mutation database. Copyright © 2013 Elsevier Masson SAS. All rights reserved.

  20. UV Signature Mutations

    Science.gov (United States)

    2014-01-01

    Sequencing complete tumor genomes and exomes has sparked the cancer field's interest in mutation signatures for identifying the tumor's carcinogen. This review and meta-analysis discusses signatures and their proper use. We first distinguish between a mutagen's canonical mutations – deviations from a random distribution of base changes to create a pattern typical of that mutagen – and the subset of signature mutations, which are unique to that mutagen and permit inference backward from mutations to mutagen. To verify UV signature mutations, we assembled literature datasets on cells exposed to UVC, UVB, UVA, or solar simulator light (SSL) and tested canonical UV mutation features as criteria for clustering datasets. A confirmed UV signature was: ≥60% of mutations are C→T at a dipyrimidine site, with ≥5% CC→TT. Other canonical features such as a bias for mutations on the non-transcribed strand or at the 3' pyrimidine had limited application. The most robust classifier combined these features with criteria for the rarity of non-UV canonical mutations. In addition, several signatures proposed for specific UV wavelengths were limited to specific genes or species; non-signature mutations induced by UV may cause melanoma BRAF mutations; and the mutagen for sunlight-related skin neoplasms may vary between continents. PMID:25354245

  1. Comparison of DNA-, PMA-, and RNA-based 16S rRNA Illumina sequencing for detection of live bacteria in water.

    Science.gov (United States)

    Li, Ru; Tun, Hein Min; Jahan, Musarrat; Zhang, Zhengxiao; Kumar, Ayush; Fernando, Dilantha; Farenhorst, Annemieke; Khafipour, Ehsan

    2017-07-18

    The limitation of 16S rRNA gene sequencing (DNA-based) for microbial community analyses in water is the inability to differentiate live (dormant cells as well as growing or non-growing metabolically active cells) and dead cells, which can lead to false positive results in the absence of live microbes. Propidium-monoazide (PMA) has been used to selectively remove DNA from dead cells during downstream sequencing process. In comparison, 16S rRNA sequencing (RNA-based) can target live microbial cells in water as both dormant and metabolically active cells produce rRNA. The objective of this study was to compare the efficiency and sensitivity of DNA-based, PMA-based and RNA-based 16S rRNA Illumina sequencing methodologies for live bacteria detection in water samples experimentally spiked with different combination of bacteria (2 gram-negative and 2 gram-positive/acid fast species either all live, all dead, or combinations of live and dead species) or obtained from different sources (First Nation community drinking water; city of Winnipeg tap water; water from Red River, Manitoba, Canada). The RNA-based method, while was superior for detection of live bacterial cells still identified a number of 16S rRNA targets in samples spiked with dead cells. In environmental water samples, the DNA- and PMA-based approaches perhaps overestimated the richness of microbial community compared to RNA-based method. Our results suggest that the RNA-based sequencing was superior to DNA- and PMA-based methods in detecting live bacterial cells in water.

  2. Clinical presentation and mutations in Danish patients with Wilson disease

    DEFF Research Database (Denmark)

    Møller, Lisbeth Birk; Horn, Nina; Jeppesen, Tina Dysgaard

    2011-01-01

    consistently indicative of WND, with the exception of the 24-h urine-Cu test, which is always outside the normal range. Mutations were identified in 100% of the screened ATP7B alleles (70 unrelated), including five novel mutations: p.1021K; p.G1158V; p.L1304F; IVS20-2A>G; Ex5_6del. In all, 70% of mutations...... were found in exons 8, 14, 17, 18, and 20. The most frequent mutation, p.H1069Q, comprised 18%. We propose a new and simple model that correlates genotype and age of onset. By assuming that the milder of two mutations is 'functionally dominant' and determines the age of onset, we classified 25....../27 mutations as either severe (age of onset 20 years), and correctly predicted the age of onset in 37/39 patients. This method should be tested in other Wilson populations....

  3. The Mutations Associated with Dilated Cardiomyopathy

    Directory of Open Access Journals (Sweden)

    Ruti Parvari

    2012-01-01

    Full Text Available Cardiomyopathy is an important cause of heart failure and a major indication for heart transplantation in children and adults. This paper describes the state of the genetic knowledge of dilated cardiomyopathy (DCM. The identification of the causing mutation is important since presymptomatic interventions of DCM have proven value in preventing morbidity and mortality. Additionally, as in general in genetic studies, the identification of the mutated genes has a direct clinical impact for the families and population involved. Identifying causative mutations immediately amplifies the possibilities for disease prevention through carrier screening and prenatal testing. This often lifts a burden of social isolation from affected families, since healthy family members can be assured of having healthy children. Identification of the mutated genes holds the potential to lead to the understanding of disease etiology, pathophysiology, and therefore potential therapy. This paper presents the genetic variations, or disease-causing mutations, contributing to the pathogenesis of hereditary DCM, and tries to relate these to the functions of the mutated genes.

  4. TOX3 mutations in breast cancer.

    Directory of Open Access Journals (Sweden)

    James Owain Jones

    Full Text Available TOX3 maps to 16q12, a region commonly lost in breast cancers and recently implicated in the risk of developing breast cancer. However, not much is known of the role of TOX3 itself in breast cancer biology. This is the first study to determine the importance of TOX3 mutations in breast cancers. We screened TOX3 for mutations in 133 breast tumours and identified four mutations (three missense, one in-frame deletion of 30 base pairs in six primary tumours, corresponding to an overall mutation frequency of 4.5%. One potentially deleterious missense mutation in exon 3 (Leu129Phe was identified in one tumour (genomic DNA and cDNA. Whilst copy number changes of 16q12 are common in breast cancer, our data show that mutations of TOX3 are present at low frequency in tumours. Our results support that TOX3 should be further investigated to elucidate its role in breast cancer biology.

  5. Streptococcal screen

    Science.gov (United States)

    ... Considerations This test screens for the group A streptococcus bacteria only. It ... MJ, eds. Mandell, Douglas and Bennett's Principles and Practice of Infectious Diseases . 8th ed. Philadelphia, PA: Elsevier Saunders; 2015:chap ...

  6. Hypertension screening

    Science.gov (United States)

    Foulke, J. M.

    1975-01-01

    An attempt was made to measure the response to an announcement of hypertension screening at the Goddard Space Center, to compare the results to those of previous statistics. Education and patient awareness of the problem were stressed.

  7. Targeted Screening of Fabry Disease in Male Hemodialysis Patients in Brazil Highlights Importance of Family Screening.

    Science.gov (United States)

    Silva, Cassiano Augusto Braga; Barreto, Fellype Carvalho; Dos Reis, Marlene Antonia; Moura Junior, José Andrade; Cruz, Constança Margarida Sampaio

    2016-01-01

    Fabry disease (FD) is a lysosomal storage disorder caused by enzyme α galactosidase A (α-Gal A) deficiency due to mutations in the galactosidase alpha (GLA) gene. It leads to damage several organs, such as the kidneys, due to progressive accumulation of glycosphingolipids. To estimate the prevalence of FD among male hemodialysis (HD) patients in a northern state of Brazil. Screening was performed using a dried blood spot on filter paper to identify patients with low α-Gal A enzyme activity (≤2.2 µmol/l/h). Those with low enzyme activity underwent genetic analysis of the GLA gene. Family screening was conducted in the index cases. 2,583 male HD patients (age: 52 (18-91 years)) were screened. The α-Gal A assay identified 72 males (2.78%) with low enzyme activity. Genotyping identified 3 patients with GLA mutations: W204X, A368T, both previously reported; and C52F, a novel missense mutation. Only the patient with W204X mutation had classic FD. The prevalence rate was 0.12%. Family screening of the index cases identified 23 family members with the same mutations. The prevalence of FD amongst male HD patients found in the Northern of Brazil was low (0.12%). However, family screening of the 3 index cases identified family members at an early stage of the disease, which may benefit from earlier treatment. © 2016 S. Karger AG, Basel.

  8. A novel miRNA-based predictive model for biochemical failure following post-prostatectomy salvage radiation therapy.

    Directory of Open Access Journals (Sweden)

    Erica Hlavin Bell

    Full Text Available To develop a microRNA (miRNA-based predictive model for prostate cancer patients of 1 time to biochemical recurrence after radical prostatectomy and 2 biochemical recurrence after salvage radiation therapy following documented biochemical disease progression post-radical prostatectomy.Forty three patients who had undergone salvage radiation therapy following biochemical failure after radical prostatectomy with greater than 4 years of follow-up data were identified. Formalin-fixed, paraffin-embedded tissue blocks were collected for all patients and total RNA was isolated from 1mm cores enriched for tumor (>70%. Eight hundred miRNAs were analyzed simultaneously using the nCounter human miRNA v2 assay (NanoString Technologies; Seattle, WA. Univariate and multivariate Cox proportion hazards regression models as well as receiver operating characteristics were used to identify statistically significant miRNAs that were predictive of biochemical recurrence.Eighty eight miRNAs were identified to be significantly (p36 months. Nine miRNAs were identified to be significantly (p<0.05 associated by multivariate analysis with biochemical failure after salvage radiation therapy. A new predictive model for biochemical recurrence after salvage radiation therapy was developed; this model consisted of miR-4516 and miR-601 together with, Gleason score, and lymph node status. The area under the ROC curve (AUC was improved to 0.83 compared to that of 0.66 for Gleason score and lymph node status alone.miRNA signatures can distinguish patients who fail soon after radical prostatectomy versus late failures, giving insight into which patients may need adjuvant therapy. Notably, two novel miRNAs (miR-4516 and miR-601 were identified that significantly improve prediction of biochemical failure post-salvage radiation therapy compared to clinico-histopathological factors, supporting the use of miRNAs within clinically used predictive models. Both findings warrant further

  9. Long Distance Metabolic Regulation through Adipose-Derived Circulating Exosomal miRNAs: A Trail for RNA-Based Therapies?

    Directory of Open Access Journals (Sweden)

    Farah Fatima

    2017-08-01

    Full Text Available The contribution of non-coding RNAs, such as microRNAs (miRNAs in regulating physiological and pathological states has been intensively elucidated during last 15 years. The discovery of circulating miRNAs (cir-miRNAs in variety of body fluids, is, however a recent focus of interest in understanding pathophysiological states of their originating cells/organs. Yet another stimulating debate that takes miRNAs to the next level is their presence in exosomes, and this is truly interesting area of research. Exosomes are cell-derived extracellular vesicles, and are naturally equipped biological vehicles that not only enable functional transfer of miRNAs between cells (horizontal transfer but also foster inter-organ communication, presumably guided by organ specific receptors—decorated on their surface. However, understandings on inter-organ communication elicited by tissue specific exosomal-miRNA fingerprints remain elusive. Recently, Thomou et al., has discovered that adipose tissue contributes a large fraction of adipose specific exosomal-miRNA fingerprints in blood circulation. Experimental evidence emphasize adipose tissue as major depot of cir-miRNAs that sail through blood flow and reach to distal organs—primarily in the liver, where they regulate gene expression of host tissue and elicit metabolic control. This appears to be a genetic form of adipokines (endocrine factors secreted from adipose tissue. We review such offshore metabolic insults, and make an effort to address few important missing links between miRNAs processing and their incorporation into exosomes. We provide potential perspectives on how this knowledge could be steered towards RNA-based therapeutics for monitoring complex metabolic diseases and beyond.

  10. Optimization of Critical Hairpin Features Allows miRNA-based Gene Knockdown Upon Single-copy Transduction

    Directory of Open Access Journals (Sweden)

    Renier Myburgh

    2014-01-01

    Full Text Available Gene knockdown using micro RNA (miRNA-based vector constructs is likely to become a prominent gene therapy approach. It was the aim of this study to improve the efficiency of gene knockdown through optimizing the structure of miRNA mimics. Knockdown of two target genes was analyzed: CCR5 and green fluorescent protein. We describe here a novel and optimized miRNA mimic design called mirGE comprising a lower stem length of 13 base pairs (bp, positioning of the targeting strand on the 5′ side of the miRNA, together with nucleotide mismatches in upper stem positions 1 and 12 placed on the passenger strand. Our mirGE proved superior to miR-30 in four aspects: yield of targeting strand incorporation into RNA-induced silencing complex (RISC; incorporation into RISC of correct targeting strand; precision of cleavage by Drosha; and ratio of targeting strand over passenger strand. A triple mirGE hairpin cassette targeting CCR5 was constructed. It allowed CCR5 knockdown with an efficiency of over 90% upon single-copy transduction. Importantly, single-copy expression of this construct rendered transduced target cells, including primary human macrophages, resistant to infection with a CCR5-tropic strain of HIV. Our results provide new insights for a better knockdown efficiency of constructs containing miRNA. Our results also provide the proof-of-principle that cells can be rendered HIV resistant through single-copy vector transduction, rendering this approach more compatible with clinical applications.

  11. Long Distance Metabolic Regulation through Adipose-Derived Circulating Exosomal miRNAs: A Trail for RNA-Based Therapies?

    Science.gov (United States)

    Fatima, Farah; Nawaz, Muhammad

    2017-01-01

    The contribution of non-coding RNAs, such as microRNAs (miRNAs) in regulating physiological and pathological states has been intensively elucidated during last 15 years. The discovery of circulating miRNAs (cir-miRNAs) in variety of body fluids, is, however a recent focus of interest in understanding pathophysiological states of their originating cells/organs. Yet another stimulating debate that takes miRNAs to the next level is their presence in exosomes, and this is truly interesting area of research. Exosomes are cell-derived extracellular vesicles, and are naturally equipped biological vehicles that not only enable functional transfer of miRNAs between cells (horizontal transfer) but also foster inter-organ communication, presumably guided by organ specific receptors—decorated on their surface. However, understandings on inter-organ communication elicited by tissue specific exosomal-miRNA fingerprints remain elusive. Recently, Thomou et al., has discovered that adipose tissue contributes a large fraction of adipose specific exosomal-miRNA fingerprints in blood circulation. Experimental evidence emphasize adipose tissue as major depot of cir-miRNAs that sail through blood flow and reach to distal organs—primarily in the liver, where they regulate gene expression of host tissue and elicit metabolic control. This appears to be a genetic form of adipokines (endocrine factors secreted from adipose tissue). We review such offshore metabolic insults, and make an effort to address few important missing links between miRNAs processing and their incorporation into exosomes. We provide potential perspectives on how this knowledge could be steered towards RNA-based therapeutics for monitoring complex metabolic diseases and beyond. PMID:28824444

  12. RNA-based molecular survey of biodiversity of limestone tombstone microbiota in response to atmospheric sulphur pollution.

    Science.gov (United States)

    Villa, F; Vasanthakumar, A; Mitchell, R; Cappitelli, F

    2015-01-01

    Outdoor stoneworks sustain biofilm formation and are constantly at risk of deterioration by micro-organisms. In this study, the biofilm microflora of historic limestone tombstones located in a highly polluted urban environment (Cambridge, MA) and in a less polluted location (Lexington, MA) were compared using comprehensive RNA-based molecular analyses of 16S rRNA gene sequences as well as sequences of genes for different pathways of sulphur metabolism (soxB, apsA, dsrA). The metabolically active micro-organisms detected by denaturing gradient gel electrophoresis analysis of 16S rRNA fragments were predominantly represented by cyanobacteria (belonging to the family Nostocaceae and to the genus Chroococcidiopsis) in both polluted and unpolluted environments. The investigation of soxB, apsA, dsrA transcripts reflected the abundance and the diversity of sulphur-oxidizing and sulphate-reducing bacteria in the Cambridge samples in comparison with the Lexington samples. The investigation revealed that in addition to phototrophic sulphur bacteria belonging to the genera Thiocapsa, Halochromatium, Allochromatium, Thiococcus and Thermochromatium, other sulphate-oxidizing prokaryotes (e.g. the genus Thiobacillus) as well as sequences of Deltaproteobacteria from the genus Desulfovibrio occurred at the polluted urban site. The interactions between the main functional groups retrieved from the limestone tombstones were discussed. The biofilm microflora inhabiting historic limestones are a multi-component open ecosystem sensitively reacting to all environmental factors including air pollutants. Little is known about specific target groups that are active in the biofilm and their physiological functions. For the first time, transcripts involved in important energy-yielding processes were investigated to reveal the metabolic capabilities of the microflora in response to atmospheric sulphur pollution. This work provides novel and important information about the ecology of limestone

  13. Oncogene mutation profiling reveals poor prognosis associated with FGFR1/3 mutation in liposarcoma.

    Science.gov (United States)

    Li, Chengfang; Shen, Yaoyuan; Ren, Yan; Liu, Wei; Li, Man; Liang, Weihua; Liu, Chunxia; Li, Feng

    2016-09-01

    Liposarcoma (LPS) is one of the most prevalent soft tissue sarcomas. LPS shows a poor response to radiation and chemotherapy. The causes of death in patients with LPS include locally recurrent and metastatic disease. We sought to examine novel gene mutations and pathways in primary and matched recurrent LPSs to identify potential therapeutic targets. We conducted a high-throughput analysis of 238 known mutations in 19 oncogenes using Sequenom MassARRAY technology. Nucleic acids were extracted from 19 primary and recurrent LPS samples, encompassing 9 dedifferentiated LPSs (DDLPS), 9 myxoid/round cell LPSs, and 1 pleomorphic LPS. Mutation screening revealed missense mutations in 21.1% (4/19) of the LPS specimens, including 4 different genes (FGFR1, FGFR3, PIK3CA, and KIT). Based on histologic subtypes, 22.2% DDLPS (2/9) and 22.2% myxoid cell LPS (2/9) contained gene mutations. Specifically, 3 (23.1%) of 13 primary tumors harbored mutations. Furthermore, although gene mutations were identified in 1 (11.1%) of 9 recurrent LPS samples, the difference between the primary and the recurrence was not statistically significant. Analysis of patient survival data indicated that patients harboring FGFR1/3 mutations experienced reduced overall survival (P<.05). Despite the limited number of samples, our findings provide the first evidence of FGFR1/3 mutations in DDLPS, which were associated with poor clinical outcomes. The FGFR pathway may play an important role in the development and progression of DDLPS and warrants further investigation; moreover, PIK3CA mutation is a common event (11.1%) in myxoid cell LPS. Copyright © 2016 Elsevier Inc. All rights reserved.

  14. A high frequency of distinct ATM gene mutations in ataxia-telangiectasia

    Energy Technology Data Exchange (ETDEWEB)

    Wright, J.; Teraoka, S.; Concannon, P. [Univ. of Washington School of Medicine, Seattle, WA (United States)] [and others

    1996-10-01

    The clinical features of the autosomal recessive disorder ataxia-telangiectasia (AT) include a progressive cerebellar ataxia, hypersensitivity to ionizing radiation, and an increased susceptibility to malignancies. Epidemiological studies have suggested that AT heterozygotes may also be at increased risk for malignancy, possibly as a consequence of radiation exposure. A gene mutated in AT patients (ATM) has recently been isolated, making mutation screening in both patients and the general population possible. Because of the relatively large size of the ATM gene, the design of screening programs will depend on the types and distribution of mutations in the general population. In this report, we describe 30 mutations identified in a panel of unrelated AT patients and controls. Twenty-five of the 30 were distinct, and most patients were compound heterozygotes. The most frequently detected mutation was found in three different families and had previously been reported in five others. This corresponds to a frequency of 8% of all reported ATM mutations. Twenty-two of the alterations observed would be predicted to lead to protein truncation at sites scattered throughout the molecule. Two fibroblast cell lines, which displayed normal responses to ionizing radiation, also proved to be heterozygous for truncation mutations of ATM. These observations suggest that the carrier frequency of ATM mutations may be sufficiently high to make population screening practical. However, such screening may need to be done prospectively, that is, by searching for new mutations rather than by screening for just those already identified in AT families. 33 refs., 1 fig., 1 tab.

  15. OBSCN Mutations Associated with Dilated Cardiomyopathy and Haploinsufficiency.

    Directory of Open Access Journals (Sweden)

    Steven Marston

    Full Text Available Studies of the functional consequences of DCM-causing mutations have been limited to a few cases where patients with known mutations had heart transplants. To increase the number of potential tissue samples for direct investigation we performed whole exon sequencing of explanted heart muscle samples from 30 patients that had a diagnosis of familial dilated cardiomyopathy and screened for potentially disease-causing mutations in 58 HCM or DCM-related genes.We identified 5 potentially disease-causing OBSCN mutations in 4 samples; one sample had two OBSCN mutations and one mutation was judged to be not disease-related. Also identified were 6 truncating mutations in TTN, 3 mutations in MYH7, 2 in DSP and one each in TNNC1, TNNI3, MYOM1, VCL, GLA, PLB, TCAP, PKP2 and LAMA4. The mean level of obscurin mRNA was significantly greater and more variable in healthy donor samples than the DCM samples but did not correlate with OBSCN mutations. A single obscurin protein band was observed in human heart myofibrils with apparent mass 960 ± 60 kDa. The three samples with OBSCN mutations had significantly lower levels of obscurin immunoreactive material than DCM samples without OBSCN mutations (45±7, 48±3, and 72±6% of control level.Obscurin levels in DCM controls, donor heart and myectomy samples were the same.OBSCN mutations may result in the development of a DCM phenotype via haploinsufficiency. Mutations in the obscurin gene should be considered as a significant causal factor of DCM, alone or in concert with other mutations.

  16. Mutations in the sarcomere gene MYH7 in Ebstein anomaly.

    Science.gov (United States)

    Postma, Alex V; van Engelen, Klaartje; van de Meerakker, Judith; Rahman, Thahira; Probst, Susanne; Baars, Marieke J H; Bauer, Ulrike; Pickardt, Thomas; Sperling, Silke R; Berger, Felix; Moorman, Antoon F M; Mulder, Barbara J M; Thierfelder, Ludwig; Keavney, Bernard; Goodship, Judith; Klaassen, Sabine

    2011-02-01

    Ebstein anomaly is a rare congenital heart malformation characterized by adherence of the septal and posterior leaflets of the tricuspid valve to the underlying myocardium. An association between Ebstein anomaly with left ventricular noncompaction (LVNC) and mutations in MYH7 encoding β-myosin heavy chain has been shown; in this report, we have screened for MYH7 mutations in a cohort of probands with Ebstein anomaly in a large population-based study. Mutational analysis in a cohort of 141 unrelated probands with Ebstein anomaly was performed by next-generation sequencing and direct DNA sequencing of MYH7. Heterozygous mutations were identified in 8 of 141 samples (6%). Seven distinct mutations were found; 5 were novel and 2 were known to cause hypertrophic cardiomyopathy. All mutations except for 1 3-bp deletion were missense mutations; 1 was a de novo change. Mutation-positive probands and family members showed various congenital heart malformations as well as LVNC. Among 8 mutation-positive probands, 6 had LVNC, whereas among 133 mutation-negative probands, none had LVNC. The frequency of MYH7 mutations was significantly different between probands with and without LVNC accompanying Ebstein anomaly (PMYH7 mutation in the pedigrees of 3 of the probands, 1 of which also included another individual with Ebstein anomaly. Ebstein anomaly is a congenital heart malformation that is associated with mutations in MYH7. MYH7 mutations are predominantly found in Ebstein anomaly associated with LVNC and may warrant genetic testing and family evaluation in this subset of patients.

  17. Mutation distributions and clinical correlations of PIK3CA gene mutations in breast cancer.

    Science.gov (United States)

    Dirican, Ebubekir; Akkiprik, Mustafa; Özer, Ayşe

    2016-06-01

    Breast cancer (BCa) is the most common cancer and the second cause of death among women. Phosphoinositide 3-kinase (PI3K) signaling pathway has a crucial role in the cellular processes such as cell survival, growth, division, and motility. Moreover, oncogenic mutations in the PI3K pathway generally involve the activation phosphatidylinositol-4,5-bisphosphate 3-kinase-catalytic subunit alpha (PIK3CA) mutation which has been identified in numerous BCa subtypes. In this review, correlations between PIK3CA mutations and their clinicopathological parameters on BCa will be described. It is reported that PIK3CA mutations which have been localized mostly on exon 9 and 20 hot spots are detected 25-40 % in BCa. This relatively high frequency can offer an advantage for choosing the best treatment options for BCa. PIK3CA mutations may be used as biomarkers and have been major focus of drug development in cancer with the first clinical trials of PI3K pathway inhibitors currently in progress. Screening of PIK3CA gene mutations might be useful genetic tests for targeted therapeutics or diagnosis. Increasing data about PIK3CA mutations and its clinical correlations with BCa will help to introduce new clinical applications in the near future.

  18. HCC screening; HCC-Screening

    Energy Technology Data Exchange (ETDEWEB)

    Albrecht, T. [Charite-Unversitaetsmedizin,Freie Universitaet und Humboldt-Universitaet zu Berlin, Klinik und Hochschulambulanz fuer Radiologie und Nuklearmedizin,Campus Benjamin Franklin, Berlin (Germany)

    2008-01-15

    Hepatocellular carcinoma (HCC) is one of the most frequently diagnosed tumour diseases throughout the world. In the vast majority of cases those affected are high-risk patients with chronic viral hepatitis and/or liver cirrhosis, which means there is a clearly identifiable target group for HCC screening. With resection, transplantation, and interventional procedures for local ablation, following early diagnosis curative treatment options are available with which 5-year survival rates of over 60% can be reached. Such early diagnosis is a reality only in a minority of patients, however, and in the majority of cases the disease is already in an advanced stage at diagnosis. One of the objects of HCC screening is diagnosis in an early stage when curative treatment is still possible. Precisely this is achieved by screening, so that the proportion of patients treated with curative intent is decisively higher. There is not yet any clear evidence as to whether this leads to a lowering of the mortality of HCC. As lower mortality is the decisive indicator of success for a screening programme the benefit of HCC screening has so far been neither documented nor refuted. Nonetheless, in large regions of the world it is the practice for high-risk patients to undergo HCC screening in the form of twice-yearly ultrasound examination and determination of AFP. (orig.) [German] Das hepatozellulaere Karzinom (HCC) ist eine der weltweit haeufigsten Tumorerkrankungen. Es tritt in der grossen Mehrzahl der Faelle bei Hochrisikopatienten mit chronischer Virushepatitis bzw. Leberzirrhose auf, woraus sich eine klar identifizierbare Zielgruppe fuer das HCC-Screening ergibt. Mit der Resektion, der Transplantation und interventionellen lokal ablativen Verfahren stehen bei rechtzeitiger Diagnosestellung kurative Therapieoptionen zur Verfuegung, die 5-Jahres-Ueberlebensraten von >60% erreichen. Diese rechtzeitige Diagnosestellung erfolgt jedoch nur bei einer Minderzahl der Patienten, waehrend die

  19. 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

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

    Science.gov (United States)

    Tomar, Swati; Sethi, Raman; Sundar, Gangadhara; Quah, Thuan Chong; Quah, Boon Long; Lai, Poh San

    2017-01-01

    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 asymptomatic

  1. Filaggrin Mutation in Korean Patients with Atopic Dermatitis.

    Science.gov (United States)

    On, Hye Rang; Lee, Sang Eun; Kim, Song Ee; Hong, Won Jin; Kim, Hyun Jung; Nomura, Toshifumi; Suzuki, Shotaro; Shimizu, Hiroshi; Kim, Soo Chan

    2017-03-01

    Atopic dermatitis (AD) is a chronic, relapsing eczematous inflammatory skin disease. Mutations in the filaggrin gene (FLG) are major predisposing factors for AD. Ethnic differences exist between Asian and European populations in the frequency and spectrum of FLG mutations. Moreover, a distinct set of FLG mutations has been reported in Asian populations. The aim of this study was to examine the spectrum of FLG mutations in Koreans with AD. We also investigated the association of FLG mutations and clinical features of AD and compared the Korean FLG landscape with that of other East Asian countries. Seventy Korean patients with AD were enrolled in this study. Fourteen FLG mutations previously detected in Korean, Japanese, and Chinese patients were screened by genotyping. Four FLG null mutations (3321delA, K4022X, S3296X, and S2889X) were identified in eleven patients (15.7%). The most commonly detected mutations in Korean patients with AD were 3321delA (n=6, 9.1%) and K4022X (n=3, 4.5%). FLG mutations were significantly associated with elevated IgE (≥200 KIU/L and/or MAST-CLA >3+, p=0.005), palmar hyperlinearity (pKoreans and revealed an association between FLG mutations and AD phenotype.

  2. Identification of new mutations in familial amyotrophic lateral sclerosis

    Energy Technology Data Exchange (ETDEWEB)

    Siddique, T.; Deng, H.X.; Hentati, A. [Northwestern Univ. Medical School, Chicago, IL (United States)] [and others

    1994-09-01

    Amyotrophic lateral sclerosis (ALS) is a lethal neurodegenerative disease due to motor neuron death in the cortex, brain stem and spinal cord. Ten percent of ALS cases are familial (FALS). Previously a subset of FALS families have been mapped to chromosome 21 and mutations in the Cu,Zn superoxide dismutase gene have been identified in those families. Nineteen different mutations at 16 distinct codons have been documented, of which 12 different mutations were identified in our 29 FALS families. These mutations account for about twenty percent of all FALS families screened. The mutations identified in our FALS families are A4V, A4T, G37R, G41D, H43R, G85R, G93A, E100G, L106V, I113T, L144F, and V148G. Mutation A4V is the most frequent one which occurred in 14 out of our 29 FALS families. In further screening of our FALS families, two new mutations, V14M and L84V, have been identified. Thus a total of 21 different mutations at 18 distinct codon sites have been identified in SOD1.

  3. 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.

  4. Absence of ras-gene hot-spot mutations in canine fibrosarcomas and melanomas.

    Science.gov (United States)

    Murua Escobar, Hugo; Günther, Kathrin; Richter, Andreas; Soller, Jan T; Winkler, Susanne; Nolte, Ingo; Bullerdiek, Jörn

    2004-01-01

    Point mutations within ras proto-oncogenes, particularly within the mutational hot-spot codons 12, 13 and 61, are frequently detected in human malignancies and in different types of experimentally-induced tumours in animals. So far little is known about ras mutations in naturally occurring canine fibrosarcomas or K-ras mutations in canine melanomas. To elucidate whether ras mutations exist in these naturally occurring tumours in dogs, in the present study we screened 13 canine fibrosarcomas, 2 feline fibrosarcomas and 11 canine melanomas for point mutations, particularly within the mutational hot-spots, making this the first study to investigate a large number of canine fibrosarcomas. None of the samples showed a K- or N-ras hot spot mutation. Thus, our data strongly suggest that ras mutations at the hot-spot loci are very rare and do not play a major role in the pathogenesis of the spontaneously occurring canine tumours investigated.

  5. Use of denaturing gradient gel electrophoresis in screening ...

    African Journals Online (AJOL)

    G. Christopoulos

    2012-08-24

    Aug 24, 2012 ... mutation system (ARMS), the synthetic oligonucleotide hybridization, the restriction enzyme digestion of the product or the use of denaturant high performance liquid chromatog- raphy (DHPLC) has greatly facilitated the screening for known mutations [3–6]. Furthermore, the use of PCR amplifi- cation and ...

  6. High frequency of additional gene mutations in acute myeloid leukemia with MLL partial tandem duplication: DNMT3A mutation is associated with poor prognosis.

    Science.gov (United States)

    Kao, Hsiao-Wen; Liang, D Cherng; Kuo, Ming-Chung; Wu, Jin-Hou; Dunn, Po; Wang, Po-Nan; Lin, Tung-Liang; Shih, Yu-Shu; Liang, Sung-Tzu; Lin, Tung-Huei; Lai, Chen-Yu; Lin, Chun-Hui; Shih, Lee-Yung

    2015-10-20

    The mutational profiles of acute myeloid leukemia (AML) with partial tandem duplication of mixed-lineage leukemia gene (MLL-PTD) have not been comprehensively studied. We studied 19 gene mutations for 98 patients with MLL-PTD AML to determine the mutation frequency and clinical correlations. MLL-PTD was screened by reverse-transcriptase PCR and confirmed by real-time quantitative PCR. The mutational analyses were performed with PCR-based assays followed by direct sequencing. Gene mutations of signaling pathways occurred in 63.3% of patients, with FLT3-ITD (44.9%) and FLT3-TKD (13.3%) being the most frequent. 66% of patients had gene mutations involving epigenetic regulation, and DNMT3A (32.7%), IDH2 (18.4%), TET2 (18.4%), and IDH1 (10.2%) mutations were most common. Genes of transcription pathways and tumor suppressors accounted for 23.5% and 10.2% of patients. RUNX1 mutation occurred in 23.5% of patients, while none had NPM1 or double CEBPA mutation. 90.8% of MLL-PTD AML patients had at least one additional gene mutation. Of 55 MLL-PTD AML patients who received standard chemotherapy, age older than 50 years and DNMT3A mutation were associated with inferior outcome. In conclusion, gene mutations involving DNA methylation and activated signaling pathway were common co-existed gene mutations. DNMT3A mutation was a poor prognostic factor in MLL-PTD AML.

  7. 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.

  8. Mitochondrial mutations in cancer.

    Science.gov (United States)

    Brandon, M; Baldi, P; Wallace, D C

    2006-08-07

    The metabolism of solid tumors is associated with high lactate production while growing in oxygen (aerobic glycolysis) suggesting that tumors may have defects in mitochondrial function. The mitochondria produce cellular energy by oxidative phosphorylation (OXPHOS), generate reactive oxygen species (ROS) as a by-product, and regulate apoptosis via the mitochondrial permeability transition pore (mtPTP). The mitochondria are assembled from both nuclear DNA (nDNA) and mitochondrial DNA (mtDNA) genes. The mtDNA codes for 37 genes essential of OXPHOS, is present in thousands of copies per cell, and has a very high mutations rate. In humans, severe mtDNA mutations result in multisystem disease, while some functional population-specific polymorphisms appear to have permitted humans to adapt to new environments. Mutations in the nDNA-encoded mitochondrial genes for fumarate hydratase and succinate dehydrogenase have been linked to uterine leiomyomas and paragangliomas, and cancer cells have been shown to induce hexokinase II which harnesses OXPHOS adenosine triphosphate (ATP) production to drive glycolysis. Germline mtDNA mutations at nucleotides 10398 and 16189 have been associated with breast cancer and endometrial cancer. Tumor mtDNA somatic mutations range from severe insertion-deletion and chain termination mutations to mild missense mutations. Surprisingly, of the 190 tumor-specific somatic mtDNA mutations reported, 72% are also mtDNA sequence variants found in the general population. These include 52% of the tumor somatic mRNA missense mutations, 83% of the tRNA mutations, 38% of the rRNA mutations, and 85% of the control region mutations. Some associations might reflect mtDNA sequencing errors, but analysis of several of the tumor-specific somatic missense mutations with population counterparts appear legitimate. Therefore, mtDNA mutations in tumors may fall into two main classes: (1) severe mutations that inhibit OXPHOS, increase ROS production and promote tumor

  9. International distribution and age estimation of the Portuguese BRCA2 c.156_157insAlu founder mutation

    NARCIS (Netherlands)

    Peixoto, Ana; Santos, Catarina; Pinheiro, Manuela; Pinto, Pedro; Soares, Maria Jose; Rocha, Patricia; Gusmao, Leonor; Amorim, Antonio; van der Hout, Annemarie; Gerdes, Anne-Marie; Thomassen, Mads; Kruse, Torben A.; Cruger, Dorthe; Sunde, Lone; Bignon, Yves-Jean; Uhrhammer, Nancy; Cornil, Lucie; Rouleau, Etienne; Lidereau, Rosette; Yannoukakos, Drakoulis; Pertesi, Maroulio; Narod, Steven; Royer, Robert; Costa, Mauricio M.; Lazaro, Conxi; Feliubadalo, Lidia; Grana, Begona; Blanco, Ignacio; de la Hoya, Miguel; Caldes, Trinidad; Maillet, Philippe; Benais-Pont, Gaelle; Pardo, Bruno; Laitman, Yael; Friedman, Eitan; Velasco, Eladio A.; Duran, Mercedes; Miramar, Maria-Dolores; Rodriguez Valle, Ana; Calvo, Maria-Teresa; Vega, Ana; Blanco, Ana; Diez, Orland; Gutierrez-Enriquez, Sara; Balmana, Judith; Ramon y Cajal, Teresa; Alonso, Carmen; Baiget, Montserrat; Foulkes, William; Tischkowitz, Marc; Kyle, Rachel; Sabbaghian, Nelly; Ashton-Prolla, Patricia; Ewald, Ingrid P.; Rajkumar, Thangarajan; Mota-Vieira, Luisa; Giannini, Giuseppe; Gulino, Alberto; Achatz, Maria I.; Carraro, Dirce M.; de Paillerets, Brigitte Bressac; Remenieras, Audrey; Benson, Cindy; Casadei, Silvia; King, Mary-Claire; Teugels, Erik; Teixeira, Manuel R.

    The c.156_157insAlu BRCA2 mutation has so far only been reported in hereditary breast/ovarian cancer (HBOC) families of Portuguese origin. Since this mutation is not detectable using the commonly used screening methodologies and must be specifically sought, we screened for this rearrangement in a

  10. Testicular Cancer Screening

    Science.gov (United States)

    ... Professional Testicular Cancer Treatment Testicular Cancer Screening Testicular Cancer Screening (PDQ®)–Patient Version What is screening? Go to ... testicles, and need to be followed closely. Testicular Cancer Screening Key Points Tests are used to screen for ...

  11. Alcohol Use Screening

    Science.gov (United States)

    ... Depression Screening Substance Abuse Screening Alcohol Use Screening Alcohol Use Screening (AUDIT-C) - Instructions The following questions ... this tool, there is also text-only version . Alcohol Use Screening (AUDIT-C) - Manual Instructions The following ...

  12. An RNA-based analysis of changes in biodiversity indices in response to Sus scrofa domesticus decomposition.

    Science.gov (United States)

    Bergmann, R C; Ralebitso-Senior, T K; Thompson, T J U

    2014-08-01

    Despite emergent research initiatives, significant knowledge gaps remain of soil microbiology-associated cadaver decomposition. Nevertheless, preliminary studies have shown that the vast diversity and complex interactions of soil microbial communities have great potential for forensic applications such as clandestine grave location and postmortem interval estimation. This study investigated changes in soil bacterial communities during pig (Sus scrofa domesticus) leg decomposition. 16S rRNA, instead of the usually applied 16S rDNA marker, was used to compare the metabolically active bacteria. Total bacterial RNA was extracted from soil samples of three different layers on day 3, 28 and 77 after the shallow burial of a pig leg. The V3 region of the 16S rRNA was amplified, analysed by RT-PCR DGGE, and compared with control soil bacterial community profiles. Statistically significant differences in soil bacterial biodiversity were observed. For the control, bacterial diversity (H') and species richness (S) of the three layers averaged 2.48±0.14 (H') and 18.8±2.5 (S), respectively, while for the test soil increases (p=0.027) were recorded between day 3 (H'=2.71±0.02; S=21.3±2.0) and 28 (H'=3.46±0.32; S=60.3±16.9), particularly in the middle (10-20 cm) and bottom (20-30 cm) soil layers. Between day 28 and 77 the diversity and richness then decreased on average for all three layers (H'=3.43±0.20; S=60.0±17.3) but remained higher than on day 3. Thus, responses in soil bacterial profiles and activity to carcass decomposition, detected and characterised by RNA-based DGGE, could be used together with RNA sequencing data, changes in physico-chemical variables (carbon, nitrogen, phosphorus, temperature, redox potential, water activity and pH) and conventional macroecology markers (e.g. insects and vegetation), to develop a suite of analytical protocols for different forensic scenarios. Copyright © 2014. Published by Elsevier Ireland Ltd.

  13. Determination of Resistant Starch Assimilating Bacteria in Fecal Samples of Mice by In vitro RNA-Based Stable Isotope Probing

    Directory of Open Access Journals (Sweden)

    Elena Herrmann

    2017-07-01

    Full Text Available The impact of the intestinal microbiota on human health is becoming increasingly appreciated in recent years. In consequence, and fueled by major technological advances, the composition of the intestinal microbiota in health and disease has been intensively studied by high throughput sequencing approaches. Observations linking dysbiosis of the intestinal microbiota with a number of serious medical conditions including chronic inflammatory disorders and allergic diseases suggest that restoration of the composition and activity of the intestinal microbiota may be a treatment option at least for some of these diseases. One possibility to shape the intestinal microbiota is the administration of prebiotic carbohydrates such as resistant starch (RS. In the present study, we aim at establishing RNA-based stable isotope probing (RNA-SIP to identify bacterial populations that are involved in the assimilation of RS using anaerobic in vitro fermentation of murine fecal material with stable [U13C] isotope-labeled potato starch. Total RNA from these incubations was extracted, processed by gradient ultracentrifugation and fractionated by density. 16S rRNA gene sequences were amplified from reverse transcribed RNA of high and low density fractions suspected to contain labeled and unlabeled RNA, respectively. Phylogenetic analysis of the obtained sequences revealed a distinct subset of the intestinal microbiota involved in starch metabolism. The results suggest Bacteroidetes, in particular genera affiliated with Prevotellaceae, as well as members of the Ruminococcacea family to be primary assimilators of resistant starch due to a significantly higher relative abundance in higher density fractions in RNA samples isolated after 2 h of incubation. Using high performance liquid chromatography coupled to isotope ratio mass spectrometry (HPLC-IRMS analysis, some stable isotope label was recovered from acetate, propionate and butyrate. Here, we demonstrate the

  14. Challenging a dogma: co-mutations exist in MAPK pathway genes in colorectal cancer.

    Science.gov (United States)

    Grellety, Thomas; Gros, Audrey; Pedeutour, Florence; Merlio, Jean-Philippe; Duranton-Tanneur, Valerie; Italiano, Antoine; Soubeyran, Isabelle

    2016-10-01

    Sequencing of genes encoding mitogen-activated protein kinase (MAPK) pathway proteins in colorectal cancer (CRC) has established as dogma that of the genes in a pathway only a single one is ever mutated. We searched for cases with a mutation in more than one MAPK pathway gene (co-mutations). Tumor tissue samples of all patients presenting with CRC, and referred between 01/01/2008 and 01/06/2015 to three French cancer centers for determination of mutation status of RAS/RAF+/-PIK3CA, were retrospectively screened for co-mutations using Sanger sequencing or next-generation sequencing. We found that of 1791 colorectal patients with mutations in the MAPK pathway, 20 had a co-mutation, 8 of KRAS/NRAS, and some even with a third mutation. More than half of the mutations were in codons 12 and 13. We also found 3 cases with a co-mutation of NRAS/BRAF and 9 with a co-mutation of KRAS/BRAF. In 2 patients with a co-mutation of KRAS/NRAS, the co-mutation existed in the primary as well as in a metastasis, which suggests that co-mutations occur early during carcinogenesis and are maintained when a tumor disseminates. We conclude that co-mutations exist in the MAPK genes but with low frequency and as yet with unknown outcome implications.

  15. Secondary and tertiary structure modeling reveals effects of novel mutations in polycystic liver disease genes PRKCSH and SEC63.

    NARCIS (Netherlands)

    Waanders, E.; Venselaar, H.; Morsche, R.H.M. te; Koning, D.B. de; Kamath, P.S.; Torres, V.E.; Somlo, S.; Drenth, J.P.H.

    2010-01-01

    Polycystic liver disease (PCLD) is characterized by intralobular bile duct cysts in the liver. It is caused by mutations in PRKCSH, encoding hepatocystin, and SEC63, encoding Sec63p. The main goals of this study were to screen for novel mutations and to analyze mutations for effects on protein

  16. Spectrum of rhodopsin mutations in Korean patients with retinitis pigmentosa

    Science.gov (United States)

    Kim, Kwang Joong; Kim, Cinoo; Bok, Jeong; Kim, Kyung-Seon; Lee, Eun-Ju; Park, Sung Pyo; Chung, Hum; Han, Bok-Ghee; Kim, Hyung-Lae; Kimm, Kuchan; Yu, Hyeong Gon

    2011-01-01

    Purpose To determine the spectrum and frequency of rhodopsin gene (RHO) mutations in Korean patients with retinitis pigmentosa (RP) and to characterize genotype–phenotype correlations in patients with mutations. Methods The RHO mutations were screened by direct sequencing, and mutation prevalence was measured in patients and controls. The impact of missense mutations to RP was predicted by segregation analysis, peptide sequence alignment, and in silico analysis. The severity of disease in patients with the missense mutations was compared by visual acuity, electroretinography, optical coherence tomography, and kinetic visual field testing. Results Five heterozygous mutations were identified in six of 302 probands with RP, including a novel mutation (c.893C>A, p.A298D) and four known mutations (c.50C>T, p.T17M; c.533A>G, p.Y178C; c.888G>T, p.K296N; and c.1040C>T, p.P347L). The allele frequency of missense mutations was measured in 114 ethnically matched controls. p.A298D, newly identified in a sporadic patient, had never been found in controls and was predicted to be pathogenic. Among the patients with the missense mutations, we observed the most severe phenotype in patients with p.P347L, less severe phenotypes in patients with p.Y178C or p.A298D, and a relatively moderate phenotype in a patient with p.T17M. Conclusions The results reveal the spectrum of RHO mutations in Korean RP patients and clinical features that vary according to mutations. Our findings will be useful for understanding these genetic spectra and the genotype–phenotype correlations and will therefore help with predicting disease prognosis and facilitating the development of gene therapy. PMID:21677794

  17. Revertant mutation releases confined lethal mutation, opening Pandora's box: a novel genetic pathogenesis.

    Directory of Open Access Journals (Sweden)

    Yasushi Ogawa

    2014-05-01

    Full Text Available When two mutations, one dominant pathogenic and the other "confining" nonsense, coexist in the same allele, theoretically, reversion of the latter may elicit a disease, like the opening of Pandora's box. However, cases of this hypothetical pathogenic mechanism have never been reported. We describe a lethal form of keratitis-ichthyosis-deafness (KID syndrome caused by the reversion of the GJB2 nonsense mutation p.Tyr136X that would otherwise have confined the effect of another dominant lethal mutation, p.Gly45Glu, in the same allele. The patient's mother had the identical misssense mutation which was confined by the nonsense mutation. The biological relationship between the parents and the child was confirmed by genotyping of 15 short tandem repeat loci. Haplotype analysis using 40 SNPs spanning the >39 kbp region surrounding the GJB2 gene and an extended SNP microarray analysis spanning 83,483 SNPs throughout chromosome 13 in the family showed that an allelic recombination event involving the maternal allele carrying the mutations generated the pathogenic allele unique to the patient, although the possibility of coincidental accumulation of spontaneous point mutations cannot be completely excluded. Previous reports and our mutation screening support that p.Gly45Glu is in complete linkage disequilibrium with p.Tyr136X in the Japanese population. Estimated from statisitics in the literature, there may be approximately 11,000 p.Gly45Glu carriers in the Japanese population who have this second-site confining mutation, which acts as natural genetic protection from the lethal disease. The reversion-triggered onset of the disesase shown in this study is a previously unreported genetic pathogenesis based on Mendelian inheritance.

  18. Hearing Screening

    Science.gov (United States)

    Johnson-Curiskis, Nanette

    2012-01-01

    Hearing levels are threatened by modern life--headsets for music, rock concerts, traffic noises, etc. It is crucial we know our hearing levels so that we can draw attention to potential problems. This exercise requires that students receive a hearing screening for their benefit as well as for making the connection of hearing to listening.

  19. Screening Tests

    Science.gov (United States)

    ... gov/publications/AssessingAlcohol/index.htm .) This issue of Alcohol Research & Health highlights some of the most popular screening ... tolerance to more than two drinks (the T question) = 2 points. The Alcohol Use Disorders Identification Test (AUDIT) can detect alcohol ...

  20. Immunohistochemical Detections of EGFR Mutations in NSCLC

    Directory of Open Access Journals (Sweden)

    Chang LIU

    2014-09-01

    Full Text Available In recent years, it has been well known that non-small cell lung cancer (NSCLC patients with mutations of epidermal growth factor receptor (EGFR response better to EGFR-tyrosine kinase inhibitor treatment. Although DNA-based assays (e.g. DNA sequencing are the most frequently used and a relatively reliable method to detect EGFR mutations, they are complex, time-consuming and relatively expensive for routine use in clinical laboratories, besides they require high quality tumor samples. In contrast, the immunohistochemistry (IHC methods make up fully for the above shortcomings and can serve as screening tests for EGFR mutations. However, there are many factors that can influence the results of IHC methods, such as different staining procedures, different antigen retrieval solutions and different sets of criteria, etc. Thus the IHC methods for detecting EGFR mutations have not been widely used in clinic and only in the research stage. This article reviews the use of IHC methods by different researchers and further discusses how to make the IHC methods work best for the detection of EGFR mutations.

  1. Expanding CEP290 mutational spectrum in ciliopathies.

    Science.gov (United States)

    Travaglini, Lorena; Brancati, Francesco; Attie-Bitach, Tania; Audollent, Sophie; Bertini, Enrico; Kaplan, Josseline; Perrault, Isabelle; Iannicelli, Miriam; Mancuso, Brunella; Rigoli, Luciana; Rozet, Jean-Michel; Swistun, Dominika; Tolentino, Jerlyn; Dallapiccola, Bruno; Gleeson, Joseph G; Valente, Enza Maria; Zankl, A; Leventer, R; Grattan-Smith, P; Janecke, A; D'Hooghe, M; Sznajer, Y; Van Coster, R; Demerleir, L; Dias, K; Moco, C; Moreira, A; Kim, C Ae; Maegawa, G; Petkovic, D; Abdel-Salam, G M H; Abdel-Aleem, A; Zaki, M S; Marti, I; Quijano-Roy, S; Sigaudy, S; de Lonlay, P; Romano, S; Touraine, R; Koenig, M; Lagier-Tourenne, C; Messer, J; Collignon, P; Wolf, N; Philippi, H; Kitsiou Tzeli, S; Halldorsson, S; Johannsdottir, J; Ludvigsson, P; Phadke, S R; Udani, V; Stuart, B; Magee, A; Lev, D; Michelson, M; Ben-Zeev, B; Fischetto, R; Benedicenti, F; Stanzial, F; Borgatti, R; Accorsi, P; Battaglia, S; Fazzi, E; Giordano, L; Pinelli, L; Boccone, L; Bigoni, S; Ferlini, A; Donati, M A; Caridi, G; Divizia, M T; Faravelli, F; Ghiggeri, G; Pessagno, A; Briguglio, M; Briuglia, S; Salpietro, C D; Tortorella, G; Adami, A; Castorina, P; Lalatta, F; Marra, G; Riva, D; Scelsa, B; Spaccini, L; Uziel, G; Del Giudice, E; Laverda, A M; Ludwig, K; Permunian, A; Suppiej, A; Signorini, S; Uggetti, C; Battini, R; Di Giacomo, M; Cilio, M R; Di Sabato, M L; Leuzzi, V; Parisi, P; Pollazzon, M; Silengo, M; De Vescovi, R; Greco, D; Romano, C; Cazzagon, M; Simonati, A; Al-Tawari, A A; Bastaki, L; Mégarbané, A; Sabolic Avramovska, V; de Jong, M M; Stromme, P; Koul, R; Rajab, A; Azam, M; Barbot, C; Martorell Sampol, L; Rodriguez, B; Pascual-Castroviejo, I; Teber, S; Anlar, B; Comu, S; Karaca, E; Kayserili, H; Yüksel, A; Akcakus, M; Al Gazali, L; Sztriha, L; Nicholl, D; Woods, C G; Bennett, C; Hurst, J; Sheridan, E; Barnicoat, A; Hennekam, R; Lees, M; Blair, E; Bernes, S; Sanchez, H; Clark, A E; DeMarco, E; Donahue, C; Sherr, E; Hahn, J; Sanger, T D; Gallager, T E; Dobyns, W B; Daugherty, C; Krishnamoorthy, K S; Sarco, D; Walsh, C A; McKanna, T; Milisa, J; Chung, W K; De Vivo, D C; Raynes, H; Schubert, R; Seward, A; Brooks, D G; Goldstein, A; Caldwell, J; Finsecke, E; Maria, B L; Holden, K; Cruse, R P; Swoboda, K J; Viskochil, D

    2009-10-01

    Ciliopathies are an expanding group of rare conditions characterized by multiorgan involvement, that are caused by mutations in genes encoding for proteins of the primary cilium or its apparatus. Among these genes, CEP290 bears an intriguing allelic spectrum, being commonly mutated in Joubert syndrome and related disorders (JSRD), Meckel syndrome (MKS), Senior-Loken syndrome and isolated Leber congenital amaurosis (LCA). Although these conditions are recessively inherited, in a subset of patients only one CEP290 mutation could be detected. To assess whether genomic rearrangements involving the CEP290 gene could represent a possible mutational mechanism in these cases, exon dosage analysis on genomic DNA was performed in two groups of CEP290 heterozygous patients, including five JSRD/MKS cases and four LCA, respectively. In one JSRD patient, we identified a large heterozygous deletion encompassing CEP290 C-terminus that resulted in marked reduction of mRNA expression. No copy number alterations were identified in the remaining probands. The present work expands the CEP290 genotypic spectrum to include multiexon deletions. Although this mechanism does not appear to be frequent, screening for genomic rearrangements should be considered in patients in whom a single CEP290 mutated allele was identified.

  2. BRCA2 Mutations in 154 Finnish Male Breast Cancer Patients

    Directory of Open Access Journals (Sweden)

    Kirsi Syrjäkoski

    2004-09-01

    Full Text Available The etiology and pathogenesis of male breast cancer (MBC are poorly known. This is due to the fact that the disease is rare, and large-scale genetic epidemiologic studies have been difficult to carry out. Here, we studied the frequency of eight recurrent Finnish BRCA2 founder mutations in a large cohort of 154 MBC patients (65% diagnosed in Finland from 1967 to 1996. Founder mutations were detected in 10 patients (6.5%, eight of whom carried the 9346(-2 A>G mutation. Two novel mutations (4075 delGT and 5808 del5 were discovered in a screening of the entire BRCA2 coding region in 34 samples. However, these mutations were not found in the rest of the 120 patients studied. Patients with positive family history of breast and/or ovarian cancer were often BRCA2 mutation carriers (44%, whereas those with no family history showed a low frequency of involvement (3.6%; P < .0001. Finally, we found only one Finnish MBC patient with 999 dell, the most common founder mutation in Finnish female breast cancer (FBC patients, and one that explains most of the hereditary FBC and MBC cases in Iceland. The variation in BRCA2 mutation spectrum between Finnish MBC patients and FBC patients in Finland and breast cancer patients in Iceland suggests that modifying genetic and environmental factors may significantly influence the penetrance of MBC and FBC in individuals carrying germline BRCA2 mutations in some populations.

  3. Mutation analysis of the gene involved in adrenoleukodystrophy

    Energy Technology Data Exchange (ETDEWEB)

    Oost, B.A. van; Ligtenberg, M.J.L. [Univ. Hospital Nijmegen (Netherlands); Kemp, S.; Bolhuis, P.A. [Academic Medical Center, Amsterdam (Netherlands)

    1994-09-01

    A gene responsible for the X-linked genetic disorder adrenoleukodystrophy (ALD) that is characterized by demyelination of the nervous system and adrenocortical insufficiency has been identified by positional cloning. The gene encodes an ATP-binding transporter which is located in the peroxisomal membrane. Deficiency of the gene leads to accumulation of unsaturated very long chain fatty acids due to impaired peroxisomal {beta}-oxidation. A systematic analysis of the open reading frame of the ALD gene unraveled the mutations in 28 different families using reverse transcriptase-PCR followed by direct sequencing. No entire gene deletions or drastic promoter mutations have been detected. Only in one family did the mutation involved multiple exons. The remaining mutations were subtle alterations leading to missense (about 50%) or nonsense mutations, frameshifts or splice acceptor site defects. In one patient a single codon was missing. Mutations affecting a single amino acid were concentrated in the region between the third and fourth putative membrane spanning fragments and in the ATP-binding domain. This overview of mutations aids in the determination of structural and functional important regions and facilitates the screening for mutations in other ALD patients. The detection of mutations in virtually all ALD families tested indicates that the isolated gene is the only gene responsible for ALD located in Xq28.

  4. Discovery Genetics - The History and Future of Spontaneous Mutation Research.

    Science.gov (United States)

    Davisson, Muriel T; Bergstrom, David E; Reinholdt, Laura G; Donahue, Leah Rae

    2012-06-01

    Historically, spontaneous mutations in mice have served as valuable models of heritable human diseases, contributing substantially to our understanding of both disease mechanisms and basic biological pathways. While advances in molecular technologies have improved our ability to create mouse models of human disease through targeted mutagenesis and transgenesis, spontaneous mutations continue to provide valuable research tools for discovery of novel genes and functions. In addition, the genetic defects caused by spontaneous mutations are molecularly similar to mutations in the human genome and, therefore often produce phenotypes that more closely resemble those characteristic of human disease than do genetically engineered mutations. Due to the rarity with which spontaneous mutations arise and the animal intensive nature of their genetic analysis, large-scale spontaneous mutation analysis has traditionally been limited to large mammalian genetics institutes. More recently, ENU mutagenesis and new screening methods have increased the rate of mutant strain discovery, and high-throughput DNA sequencing has enabled rapid identification of the underlying genes and their causative mutations. Here, we discuss the continued value of spontaneous mutations for biomedical research.

  5. 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...

  6. Mutations of the BRAF gene in human cancer

    OpenAIRE

    Davies, H.; Bignell, G.R.; Cox, C.; Stephens, P.; Edkins, S.; Clegg, S.; Teague, J.; Woffendin, H.; Garnett, M.J.; Bottomley, W.; Davis, N.; Dicks, E.; Ewing, R.; Floyd, Y.; Gray, K.

    2002-01-01

    Cancers arise owing to the accumulation of mutations in critical genes that alter normal programmes of cell proliferation, differentiation and death. As the first stage of a systematic genome-wide screen for these genes, we have prioritized for analysis signalling pathways in which at least one gene is mutated in human cancer. The RAS RAF MEK ERK MAP kinase pathway mediates cellular responses to growth signals. RAS is mutated to an oncogenic form in about 15% of human cancer. The three RAF ge...

  7. The Mutational Landscape of Circulating Tumor Cells in Multiple Myeloma

    Directory of Open Access Journals (Sweden)

    Yuji Mishima

    2017-04-01

    Full Text Available The development of sensitive and non-invasive “liquid biopsies” presents new opportunities for longitudinal monitoring of tumor dissemination and clonal evolution. The number of circulating tumor cells (CTCs is prognostic in multiple myeloma (MM, but there is little information on their genetic features. Here, we have analyzed the genomic landscape of CTCs from 29 MM patients, including eight cases with matched/paired bone marrow (BM tumor cells. Our results show that 100% of clonal mutations in patient BM were detected in CTCs and that 99% of clonal mutations in CTCs were present in BM MM. These include typical driver mutations in MM such as in KRAS, NRAS, or BRAF. These data suggest that BM and CTC samples have similar clonal structures, as discordances between the two were restricted to subclonal mutations. Accordingly, our results pave the way for potentially less invasive mutation screening of MM patients through characterization of CTCs.

  8. RAS gene hot-spot mutations in canine neoplasias.

    Science.gov (United States)

    Richter, A; Murua Escobar, H; Günther, K; Soller, J T; Winkler, S; Nolte, I; Bullerdiek, J

    2005-01-01

    Point mutations in the cellular homologues HRAS, KRAS2, and NRAS of the viral Harvey and Kirsten rat sarcoma virus oncogenes are commonly involved in the onset of malignancies in humans and other species such as dog, mouse, and rat. Most often, three particular hot-spot codons are affected, with one amino acid exchange being sufficient for the induction of tumor growth. While RAS genes have been shown to play an important role in canine tumors such as non-small lung cell carcinomas, data about RAS mutations in canine fibrosarcomas as well as KRAS2 mutations in canine melanomas is sparse. To increase the number of tumors examined, we recently screened 13 canine fibrosarcomas and 11 canine melanomas for point mutations, particularly within the mutational hot spots. The results were compared to the already existing data from other studies about these tumors in dogs.

  9. Prevalence of deleterious ATM germline mutations in gastric cancer patients.

    Science.gov (United States)

    Huang, Dong-Sheng; Tao, Hou-Quan; He, Xu-Jun; Long, Ming; Yu, Sheng; Xia, Ying-Jie; Wei, Zhang; Xiong, Zikai; Jones, Sian; He, Yiping; Yan, Hai; Wang, Xiaoyue

    2015-12-01

    Besides CDH1, few hereditary gastric cancer predisposition genes have been previously reported. In this study, we discovered two germline ATM mutations (p.Y1203fs and p.N1223S) in a Chinese family with a history of gastric cancer by screening 83 cancer susceptibility genes. Using a published exome sequencing dataset, we found deleterious germline mutations of ATM in 2.7% of 335 gastric cancer patients of different ethnic origins. The frequency of deleterious ATM mutations in gastric cancer patients is significantly higher than that in general population (p=0.0000435), suggesting an association of ATM mutations with gastric cancer predisposition. We also observed biallelic inactivation of ATM in tumors of two gastric cancer patients. Further evaluation of ATM mutations in hereditary gastric cancer will facilitate genetic testing and risk assessment.

  10. Clinical Grade "SNaPshot" Genetic Mutation Profiling in Multiple Myeloma.

    Science.gov (United States)

    O'Donnell, Elizabeth; Mahindra, Anuj; Yee, Andrew J; Nardi, Valentina; Birrer, Nicole; Horick, Nora; Borger, Darrell; Finkelstein, Dianne; Iafrate, John A; Raje, Noopur

    2015-01-01

    Whole genome sequencing studies have identified several oncogenic mutations in multiple myeloma (MM). As MM progresses, it evolves genetically underscoring the need to have tools for rapid detection of targetable mutations to optimize individualized treatment. Massachusetts General Hospital (MGH) has developed a Clinical Laboratory Improvement Amendments (CLIA)-approved, high-throughput, genotyping platform to determine the mutation status of a panel of known oncogenes. Sequence analysis using SNaPshot on DNA extracted from bone marrow and extramedullary plasmacytomas is feasible and leads to the detection of potentially druggable mutations. Screening MM patients for somatic mutations in oncogenes may provide novel targets leading to additional therapies for this patient population.

  11. Autosomal dominant hereditary spastic paraplegia: Novel mutations in the REEP1 gene (SPG31

    Directory of Open Access Journals (Sweden)

    Epplen Joerg T

    2008-07-01

    Full Text Available Abstract Background Mutations in the SPG4 gene (spastin and in the SPG3A gene (atlastin account for the majority of 'pure' autosomal dominant form of hereditary spastic paraplegia (HSP. Recently, mutations in the REEP1 gene were identified to cause autosomal dominant HSP type SPG31. The purpose of this study was to determine the prevalence of REEP1 mutations in a cohort of 162 unrelated Caucasian index patients with 'pure' HSP and a positive family history (at least two persons per family presented symptoms. Methods 162 patients were screened for mutations by, both, DHPLC and direct sequencing. Results Ten mutations were identified in the REEP1 gene, these included eight novel mutations comprising small insertions/deletions causing frame shifts and subsequently premature stop codons, one nonsense mutation and one splice site mutation as well as two missense mutations. Both missense mutations and the splice site mutation were not identified in 170 control subjects. Conclusion In our HSP cohort we found pathogenic mutations in 4.3% of cases with autosomal dominant inheritance. Our results confirm the previously observed mutation range of 3% to 6.5%, respectively, and they widen the spectrum of REEP1 mutations.

  12. Minkowski Polynomials and Mutations

    Directory of Open Access Journals (Sweden)

    Mohammad Akhtar

    2012-12-01

    Full Text Available Given a Laurent polynomial f, one can form the period of f: this is a function of one complex variable that plays an important role in mirror symmetry for Fano manifolds. Mutations are a particular class of birational transformations acting on Laurent polynomials in two variables; they preserve the period and are closely connected with cluster algebras. We propose a higher-dimensional analog of mutation acting on Laurent polynomials f in n variables. In particular we give a combinatorial description of mutation acting on the Newton polytope P of f, and use this to establish many basic facts about mutations. Mutations can be understood combinatorially in terms of Minkowski rearrangements of slices of P, or in terms of piecewise-linear transformations acting on the dual polytope P* (much like cluster transformations. Mutations map Fano polytopes to Fano polytopes, preserve the Ehrhart series of the dual polytope, and preserve the period of f. Finally we use our results to show that Minkowski polynomials, which are a family of Laurent polynomials that give mirror partners to many three-dimensional Fano manifolds, are connected by a sequence of mutations if and only if they have the same period.

  13. Is high pressure liquid chromatography an effective screening tool for characterization of molecular defects in hemoglobinopathies?

    Science.gov (United States)

    Moorchung, Nikhil; Phillip, Joseph; Sarkar, Ravi Shankar; Prasad, Rupesh; Dutta, Vibha

    2013-01-01

    Hemoglobinopathies constitute entities that are generated by either abnormal hemoglobin or thalassemias. high pressure liquid chromatography (HPLC) is one of the best methods for screening and detection of various hemoglobinopathies but it has intrinsic interpretive problems. The study was designed to evaluate the different mutations seen in cases of hemoglobinopathies and compare the same with screening tests. 68 patients of hemoglobinopathies were screened by HPLC. Mutation studies in the beta globin gene was performed using the polymerase chain reaction (PCR)-based allele-specific Amplification Refractory Mutation System (ARMS). Molecular analysis for the sickle cell mutation was done by standard methods. The IVS 1/5 mutation was the commonest mutation seen and it was seen in 26 (38.23%) of the cases. This was followed by the IVS 1/1, codon 41/42, codon 8/9, del 22 mutation, codon 15 mutation and the -619 bp deletion. No mutation was seen in eight cases. There was a 100% concordance between the sickle cell trait as diagnosed by HPLC and genetic testing. Our study underlies the importance of molecular testing in all cases of hemoglobinopathies. Although HPLC is a useful screening tool, molecular testing is very useful in accurately diagnosing the mutations. Molecular testing is especially applicable in cases with an abnormal hemoglobin (HbD, HbE and HbS) because there may be a concomitant inheritance of a beta thalassemia mutation. Molecular testing is the gold standard when it comes to the diagnosis of hemoglobinopathies.

  14. ATM Mutations and the Development of Severe Radiation-Induced Morbidity Following Radiotherapy for Breast Cancer

    National Research Council Canada - National Science Library

    Rosenstein, Barry

    2003-01-01

    ... of this project are to (1) screen 50 breast cancer patients for a ATM mutations who developed radiation-induced grade 3/4 late subcutaneous tissue morbidity as defined by the RTOG/EORTC scoring scheme, (2...

  15. Mutations in TERT promoter and FGFR3 and telomere length in bladder cancer

    National Research Council Canada - National Science Library

    Hosen, Ismail; Rachakonda, P. Sivaramakrishna; Heidenreich, Barbara; de Verdier, Petra J; Ryk, Charlotta; Steineck, Gunnar; Hemminki, Kari; Kumar, Rajiv

    2015-01-01

    .... In this study, we screened DNA from 327 urothelial bladder carcinomas from well‐documented patients, with different stages and grades and known TERT promoter mutational status, for FGFR3 alterations and measured relative telomere length (RTL...

  16. BRCA1 and BRCA2 mutations in breast cancer patients from Venezuela

    National Research Council Canada - National Science Library

    Lara, Karlena; Consigliere, Nigmet; Pérez, Jorge; Porco, Antonietta

    2012-01-01

    A sample of 58 familial breast cancer patients from Venezuela were screened for germline mutations in the coding sequences and exon-intron boundaries of BRCA1 (MIM no. 113705) and BRCA2 (MIM no. 600185...

  17. Hereditary Diffuse Gastric Cancer Syndrome: CDH1 Mutations and Beyond.

    Science.gov (United States)

    Hansford, Samantha; Kaurah, Pardeep; Li-Chang, Hector; Woo, Michelle; Senz, Janine; Pinheiro, Hugo; Schrader, Kasmintan A; Schaeffer, David F; Shumansky, Karey; Zogopoulos, George; Santos, Teresa Almeida; Claro, Isabel; Carvalho, Joana; Nielsen, Cydney; Padilla, Sarah; Lum, Amy; Talhouk, Aline; Baker-Lange, Katie; Richardson, Sue; Lewis, Ivy; Lindor, Noralane M; Pennell, Erin; MacMillan, Andree; Fernandez, Bridget; Keller, Gisella; Lynch, Henry; Shah, Sohrab P; Guilford, Parry; Gallinger, Steven; Corso, Giovanni; Roviello, Franco; Caldas, Carlos; Oliveira, Carla; Pharoah, Paul D P; Huntsman, David G

    2015-04-01

    E-cadherin (CDH1) is a cancer predisposition gene mutated in families meeting clinically defined hereditary diffuse gastric cancer (HDGC). Reliable estimates of cancer risk and spectrum in germline mutation carriers are essential for management. For families without CDH1 mutations, genetic-based risk stratification has not been possible, resulting in limited clinical options. To derive accurate estimates of gastric and breast cancer risks in CDH1 mutation carriers and determine if germline mutations in other genes are associated with HDGC. Testing for CDH1 germline mutations was performed on 183 index cases meeting clinical criteria for HDGC. Penetrance was derived from 75 mutation-positive families from within this and other cohorts, comprising 3858 probands (353 with gastric cancer and 89 with breast cancer). Germline DNA from 144 HDGC probands lacking CDH1 mutations was screened using multiplexed targeted sequencing for 55 cancer-associated genes. Accurate estimates of gastric and breast cancer risks in CDH1 mutation carriers and the relative contribution of other cancer predisposition genes in familial gastric cancers. Thirty-one distinct pathogenic CDH1 mutations (14 novel) were identified in 34 of 183 index cases (19%). By the age of 80 years, the cumulative incidence of gastric cancer was 70% (95% CI, 59%-80%) for males and 56% (95% CI, 44%-69%) for females, and the risk of breast cancer for females was 42% (95% CI, 23%-68%). In CDH1 mutation-negative index cases, candidate mutations were identified in 16 of 144 probands (11%), including mutations within genes of high and moderate penetrance: CTNNA1, BRCA2, STK11, SDHB, PRSS1, ATM, MSR1, and PALB2. This is the largest reported series of CDH1 mutation carriers, providing more precise estimates of age-associated risks of gastric and breast cancer that will improve counseling of unaffected carriers. In HDGC families lacking CDH1 mutations, testing of CTNNA1 and other tumor suppressor genes should be considered

  18. Mapping EGFR1 mutations in patients with lung adenocarcinoma.

    Science.gov (United States)

    Vlastos, Fotis; Zinszner, Julie; Hussenet, Thomas; du Manoir, Stanislas; Vordonis, Leonidas; Nikolakopoulou, Sofia; Hardavella, Georgia; Lacomme, Stéfanie; Vignaud, Jean Michel; Martinet, Nadine

    2010-12-01

    Unselected lung cancer patients seem unable to gain in terms of survival from treatment with epidermal growth factor receptor (EGFR) inhibitors. Screening for specific molecular targets involves detection of EGFR1 mutations. The aim of our study was to develop a simple set of tests to detect mutations at the tyrosine kinase domain of the EGFR1 gene while avoiding expensive DNA sequencing to select patients eligible for treatment. DNA samples from 85 adenocarcinoma patients were analyzed. The cohort consisted of 65 female (40 nonsmokers and 25 smokers) and 20 male patients [15 smokers and 5 diagnosed with bronchioloalveolar carcinomas (BAC)]. Different restriction enzymes were identified that recognize mutations at the EGFR1's tyrosine kinase domain. Biocomputing and polymerase chain reaction were used to develop molecular screening tools. Eight mutations were found in 7 patients, of which 5 were female nonsmokers (14.3%), 1 was a male nonsmoker, and 1 a male smoker. Among the mutations that were discovered, 5 (71%) were found at exon 19 and 3 (29%) at exon 20. At exon 19, 4 were deletions found in nonsmoker women, whereas the fifth was a deletion-insertion found in a nonsmoker male patient with BAC. At exon 20, 3 mutations were identified in 2 patients: a duplication (in a nonsmoker woman) and 2 substitutions (in a smoker male with BAC). No mutations were found at exons 18 and 21. Gene copy number was increased in 6 patients (4 female and 2 male) with the highest being found in a smoking female patient diagnosed with BAC. Mapping of EGFR1 mutations by alternative methods should be used in the screening of patients with non-small cell lung cancer who are candidates for EGFR inhibitor treatment. Patients with an increased EGFR1 copy number could benefit from the monoclonal antibody therapy.

  19. The first PTPN1 1 mutations in hotspot exons reported in Moroccan children with Noonan syndrome and comparison of mutation rate to previous studies.

    Science.gov (United States)

    El Bouchikhi, Ihssane; Samri, Imane; Iraqui Houssaini, Mohammed; Trhanint, Saaid; Bouguenouch, Laila; Sayel, Hanane; Hida, Moustapha; Atmani, Samir; Ouldim, Karim

    2015-01-01

    Noonan syndrome is an autosomal dominant disorder with an incidence of 1/1000-2500. It results from protein-tyrosine phosphatase, nonreceptor type 11 (PTPN11) mutations in roughly 50% of cases. Mutational screening of PTPN11 has been carried out in different populations. Thus, the aim of this study was to screen, for the first time, PTPN11 mutations in a series of Moroccan Noonan syndrome patients. We used bidirectional sequencing of exons 3 and 8, considered as PTPN11 mutation hot spots, and then compared the rate of mutational events of these exons between different populations using chi-square and Fisher's exact tests. We detected 3 heterozygous mutations (Asp6lGly, Tyr63Cys, and Asn308Ser) in 4 individuals of 16 sporadic patients (25%). The rate of mutation in our cohort did not differ from that of other populations. However, we found significant differences in the mutation rate of exon 8 between one Japanese cohort and some populations, which requires more investigations to be explained. The present study allowed identification of mutations clustered in exons 3 and 8 of the PTPN11 gene in a Moroccan Noonan syndrome cohort and enabled us to give appropriate genetic counseling to the mutation-positive patients.

  20. Defining a mutational panel and predicting the prevalence of cystic fibrosis in oman.

    Science.gov (United States)

    Fass, Uwe W; Al-Salmani, Majid; Bendahhou, Said; Shivalingam, Ganji; Norrish, Catherine; Hebal, Kallesh; Clark, Fiona; Heming, Thomas; Al-Khusaiby, Saleh

    2014-08-01

    Cystic fibrosis transmembrane conductance regulator (CFTR) mutations form distinct mutational panels in different populations and subgroups. The frequency of cystic fibrosis (CF) mutations and prevalence are unknown in Oman. This study aimed to elucidate the mutational panel and prevalence of CF for the North Al Batinah (NAB) region in Oman and to estimate the national prevalence of CF based on the carrier screening of unrelated volunteers. The study included retrospective and prospective analyses of CF cases in the NAB region for 1998-2012. Genetic analysis of disease-causing mutations was conducted by screening of the entire coding sequence and exon-intron borders. The obtained mutational panel was used for the carrier screening of 408 alleles of unrelated and unaffected Omani individuals. S549R and F508del were the major mutations, accounting for 89% of mutations in the patient population. Two private mutations, c.1733-1734delTA and c.1175T>G, were identified in the patient cohort. Two carriers, one for F508del and another for S549R, were identified by screening of the volunteer cohort, resulting in a predicted prevalence for Oman of 1 in 8,264. The estimated carrier frequency of CF in Oman was 1 in 94. The carrier frequency in the NAB region was 3.9 times higher. The mutational panel for the NAB region and the high proportion of S549R mutations emphasises the need for specific screening for CF in Oman. The different distribution of allele frequencies suggests a spatial clustering of CF in the NAB region.

  1. Mutations in GABRB3

    DEFF Research Database (Denmark)

    Møller, Rikke S; Wuttke, Thomas V; Helbig, Ingo

    2017-01-01

    OBJECTIVE: To examine the role of mutations in GABRB3 encoding the β3 subunit of the GABAA receptor in individual patients with epilepsy with regard to causality, the spectrum of genetic variants, their pathophysiology, and associated phenotypes. METHODS: We performed massive parallel sequencing...... of GABRB3 in 416 patients with a range of epileptic encephalopathies and childhood-onset epilepsies and recruited additional patients with epilepsy with GABRB3 mutations from other research and diagnostic programs. RESULTS: We identified 22 patients with heterozygous mutations in GABRB3, including 3...... probands from multiplex families. The phenotypic spectrum of the mutation carriers ranged from simple febrile seizures, genetic epilepsies with febrile seizures plus, and epilepsy with myoclonic-atonic seizures to West syndrome and other types of severe, early-onset epileptic encephalopathies...

  2. Mutational founder effect in recessive dystrophic epidermolysis bullosa families from Southern Tunisia.

    Science.gov (United States)

    Ben Brick, Ahlem Sabrine; Laroussi, Nadia; Mesrati, Hela; Kefi, Rym; Bchetnia, Mbarka; Lasram, Khaled; Ben Halim, Nizar; Romdhane, Lilia; Ouragini, Houyem; Marrakchi, Salaheddine; Boubaker, Mohamed Samir; Meddeb Cherif, Mounira; Castiglia, Daniele; Hovnanian, Alain; Abdelhak, Sonia; Turki, Hamida

    2014-05-01

    Dystrophic epidermolysis bullosa (DEB) is a group of heritable bullous skin disorders caused by mutations in the COL7A1 gene. One of the most severe forms of DEB is the severe generalized [recessive dystrophic epidermolysis bullosa (RDEB-SG)] subtype, which is inherited in an autosomal recessive manner. This subtype is most often due to COL7A1 mutations resulting in a premature termination codon on both alleles. We report here, the molecular investigation of 15 patients belonging to 14 nuclear families from the city of Sfax in Southern Tunisia, with clinical features of RDEB-SG complicated by squamous cell carcinoma in 3 patients. We identified two novel mutations, p.Val769LeufsX1 and p.Ala2297SerfsX91, in addition to one previously reported mutation (p.Arg2063Trp). The p.Val769LeufsX1 mutation was shared by 11 families and haplotype analysis indicated that it is a founder mutation. The p.Ala2297SerfsX91 mutation was a private mutation found in only one family. Together with the previously described recurrent mutations in Tunisia, screening for the founder p.Val769LeufsX1 mutation should provide a rapid molecular diagnosis tool for mutation screening in RDEB patients from Southern Tunisia and possibly from other Mediterranean populations sharing the same genetic background.

  3. EGR2 mutations define a new clinically aggressive subgroup of chronic lymphocytic leukemia.

    Science.gov (United States)

    Young, E; Noerenberg, D; Mansouri, L; Ljungström, V; Frick, M; Sutton, L-A; Blakemore, S J; Galan-Sousa, J; Plevova, K; Baliakas, P; Rossi, D; Clifford, R; Roos-Weil, D; Navrkalova, V; Dörken, B; Schmitt, C A; Smedby, K E; Juliusson, G; Giacopelli, B; Blachly, J S; Belessi, C; Panagiotidis, P; Chiorazzi, N; Davi, F; Langerak, A W; Oscier, D; Schuh, A; Gaidano, G; Ghia, P; Xu, W; Fan, L; Bernard, O A; Nguyen-Khac, F; Rassenti, L; Li, J; Kipps, T J; Stamatopoulos, K; Pospisilova, S; Zenz, T; Oakes, C C; Strefford, J C; Rosenquist, R; Damm, F

    2017-07-01

    Recurrent mutations within EGR2 were recently reported in advanced-stage chronic lymphocytic leukemia (CLL) patients and associated with a worse outcome. To study their prognostic impact, 2403 CLL patients were examined for mutations in the EGR2 hotspot region including a screening (n=1283) and two validation cohorts (UK CLL4 trial patients, n=366; CLL Research Consortium (CRC) patients, n=490). Targeted deep-sequencing of 27 known/postulated CLL driver genes was also performed in 38 EGR2-mutated patients to assess concurrent mutations. EGR2 mutations were detected in 91/2403 (3.8%) investigated cases, and associated with younger age at diagnosis, advanced clinical stage, high CD38 expression and unmutated IGHV genes. EGR2-mutated patients frequently carried ATM lesions (42%), TP53 aberrations (18%) and NOTCH1/FBXW7 mutations (16%). EGR2 mutations independently predicted shorter time-to-first-treatment (TTFT) and overall survival (OS) in the screening cohort; they were confirmed associated with reduced TTFT and OS in the CRC cohort and independently predicted short OS from randomization in the UK CLL4 cohort. A particularly dismal outcome was observed among EGR2-mutated patients who also carried TP53 aberrations. In summary, EGR2 mutations were independently associated with an unfavorable prognosis, comparable to CLL patients carrying TP53 aberrations, suggesting that EGR2-mutated patients represent a new patient subgroup with very poor outcome.

  4. PRRT2 gene mutations

    Science.gov (United States)

    Gardiner, Alice R.; Bhatia, Kailash P.; Stamelou, Maria; Dale, Russell C.; Kurian, Manju A.; Schneider, Susanne A.; Wali, G.M.; Counihan, Tim; Schapira, Anthony H.; Spacey, Sian D.; Valente, Enza-Maria; Silveira-Moriyama, Laura; Teive, Hélio A.G.; Raskin, Salmo; Sander, Josemir W.; Lees, Andrew; Warner, Tom; Kullmann, Dimitri M.; Wood, Nicholas W.; Hanna, Michael

    2012-01-01

    ABSTRACT Objective: The proline-rich transmembrane protein (PRRT2) gene was recently identified using exome sequencing as the cause of autosomal dominant paroxysmal kinesigenic dyskinesia (PKD) with or without infantile convulsions (IC) (PKD/IC syndrome). Episodic neurologic disorders, such as epilepsy, migraine, and paroxysmal movement disorders, often coexist and are thought to have a shared channel-related etiology. To investigate further the frequency, spectrum, and phenotype of PRRT2 mutations, we analyzed this gene in 3 large series of episodic neurologic disorders with PKD/IC, episodic ataxia (EA), and hemiplegic migraine (HM). Methods: The PRRT2 gene was sequenced in 58 family probands/sporadic individuals with PKD/IC, 182 with EA, 128 with HM, and 475 UK and 96 Asian controls. Results: PRRT2 genetic mutations were identified in 28 out of 58 individuals with PKD/IC (48%), 1/182 individuals with EA, and 1/128 individuals with HM. A number of loss-of-function and coding missense mutations were identified; the most common mutation found was the p.R217Pfs*8 insertion. Males were more frequently affected than females (ratio 52:32). There was a high proportion of PRRT2 mutations found in families and sporadic cases with PKD associated with migraine or HM (10 out of 28). One family had EA with HM and another large family had typical HM alone. Conclusions: This work expands the phenotype of mutations in the PRRT2 gene to include the frequent occurrence of migraine and HM with PKD/IC, and the association of mutations with EA and HM and with familial HM alone. We have also extended the PRRT2 mutation type and frequency in PKD and other episodic neurologic disorders. PMID:23077024

  5. Mutations in Lettuce Improvement

    OpenAIRE

    Mou, Beiquan

    2012-01-01

    Lettuce is a major vegetable in western countries. Mutations generated genetic variations and played an important role in the domestication of the crop. Many traits derived from natural and induced mutations, such as dwarfing, early flowering, male sterility, and chlorophyll deficiency, are useful in physiological and genetic studies. Mutants were also used to develop new lettuce products including miniature and herbicide-tolerant cultivars. Mutant analysis was critical in lettuce genomic stu...

  6. Mental Health Screening Center

    Science.gov (United States)

    ... Releases & Announcements Public Service Announcements Partnering with DBSA Mental Health Screening Center These online screening tools are not ... you have any concerns, see your doctor or mental health professional. Depression Screening for Adult Depression Screening for ...

  7. Breast cancer screening

    Science.gov (United States)

    Mammogram - breast cancer screening; Breast exam - breast cancer screening; MRI - breast cancer screening ... is performed to screen women to detect early breast cancer when it is more likely to be cured. ...

  8. Expanding the clinical phenotype of patients with a ZDHHC9 mutation

    OpenAIRE

    Masurel-Paulet, A.; Kalscheuer, V.; Lebrun, N.; Hu, H.; Levy, F.; Thauvin-Robinet, C.; Darmency-Stamboul, V.; El Chehadeh, S.; Thevenon, J.; Chancenotte, S.; Ruffier-Bourdet, M.; Bonnet, M.; Pinoit, J.; Huet, F.; Desportes, V.

    2014-01-01

    In 2007, 250 families with X-linked intellectual disability (XLID) were screened for mutations in genes on the X-chromosome, and in 4 of these families, mutations in the ZDHHC9 gene were identified. The ID was either isolated or associated with a marfanoid habitus. ZDHHC9 encodes a palmitoyl transferase that catalyzes the posttranslational modification of NRAS and HRAS. Since this first description, no additional patient with a ZDHHC9 mutation has been reported in the literature. Here, we des...

  9. The Potential Contribution of BRCA Mutations to Early Onset and Familial Breast Cancer in Uzbekistan

    Directory of Open Access Journals (Sweden)

    Abdulla Abdikhakimov

    2016-12-01

    Conclusion: This preliminary evidence suggests a potential contribution of BRCA1 5382insC mutation to breast cancer development in Uzbek population. Taking into account a high disease penetrance in carriers of BRCA1 mutation, it seems reasonable to recommend inclusion of the 5382insC mutation test in future research on the development of screening programs for breast cancer prevention in Uzbekistan.

  10. Co-Inheritance of Sickle Cell Trait and Thalassemia Mutations in South Central Iran

    OpenAIRE

    Mohammadi-Anaie, M; Saleh-gohari, N

    2012-01-01

    Background: We aimed to determine the incidence of co-inheritance as well as interaction of sickle cell trait (SCT) and ?thal/?thal mutations in south and south central of Iran. Method: We employed a PCR and restriction fragment length polymorphism techniques to confirm diagnosis of sickle cell trait. All subjects were screened for any ?/? ?thalassemia mutations using a gap-polymerase chain reaction and amplification refractory mutations system. Results: Our results showed combination of sick...

  11. Mutations in the vesicular trafficking protein annexin A11 are associated with amyotrophic lateral sclerosis

    NARCIS (Netherlands)

    Smith, Bradley N.; Topp, Simon D.; Fallini, Claudia; Shibata, Hideki; Chen, Han-Jou; Troakes, Claire; King, Andrew; Ticozzi, Nicola; Kenna, Kevin P.; Soragia-Gkazi, Athina; Miller, Jack W.; Sato, Akane; Dias, Diana Marques; Jeon, Maryangel; Vance, Caroline; Wong, Chun Hao; de Majo, Martina; Kattuah, Wejdan; Mitchell, Jacqueline C.; Scotter, Emma L.; Parkin, Nicholas W.; Sapp, Peter C.; Nolan, Matthew; Nestor, Peter J.; Simpson, Michael; Weale, Michael; Lek, Monkel; Baas, Frank; Vianney de Jong, J. M.; ten Asbroek, Anneloor L. M. A.; Redondo, Alberto Garcia; Esteban-Pérez, Jesús; Tiloca, Cinzia; Verde, Federico; Duga, Stefano; Leigh, Nigel; Pall, Hardev; Morrison, Karen E.; Al-Chalabi, Ammar; Shaw, Pamela J.; Kirby, Janine; Turner, Martin R.; Talbot, Kevin; Hardiman, Orla; Glass, Jonathan D.; de Belleroche, Jacqueline; Maki, Masatoshi; Moss, Stephen E.; Miller, Christopher; Gellera, Cinzia; Ratti, Antonia; Al-Sarraj, Safa; Brown, Robert H.; Silani, Vincenzo; Landers, John E.; Shaw, Christopher E.

    2017-01-01

    Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disorder. We screened 751 familial ALS patient whole-exome sequences and identified six mutations including p.D40G in the ANXA11 gene in 13 individuals. The p.D40G mutation was absent from 70,000 control whole-exome sequences. This

  12. high prevalence of the cys282tyr hfe mutation facilitates an improved ...

    African Journals Online (AJOL)

    screened for two common haemochromatosis (HFE) gene mutations. The local frequencies of mutations C282Y and. H63D were determined and the DNA results correlated with biochemical parameters. Setting. Patients were referred from private practitioners, health workers and pathologists for a molecular diagnosis of.

  13. Gly118Asp is a SCA14 founder mutation in the Dutch ataxia population

    NARCIS (Netherlands)

    Verbeek, DS; van de Warrenburg, BPC; Hennekam, FAM; Dooijes, D; Ippel, PF; Verschuuren-Bemelmans, CC; Kremer, HPH; Sinke, RJ

    Missense mutations in the PRKCG gene have recently been identified in spinocerebellar ataxia 14 (SCA14) patients; these include the Gly118Asp mutation that we found in a large Dutch autosomal dominant cerebellar ataxia (ADCA) family. We subsequently screened the current Dutch ataxia cohort

  14. Fanconi Anemia Gene Mutations Are Not Involved in Sporadic Wilms Tumor

    NARCIS (Netherlands)

    Adank, Muriel A.; Segers, Heidi; van Mil, Saskia E.; van Helsdingen, Yvette M.; Ameziane, Najim; van den Ouweland, Ans M. W.; Wagner, Anja; Meijers-Heijboer, Hanne; Kool, Marcel; de Kraker, Jan; Waisfisz, Quinten; van den Heuvel-Eibrink, Marry M.

    2010-01-01

    Bi-allelic germline mutations of the Fancom anemia (FA) genes, PALB2/FANCN and BRCA2/FANCD1, have been reported in a few Wilms tumor (WT) patients with an atypical FA phenotype Therefore, we screened a random cohort of 47 Dutch WT cases for germline mutations in these two FA-genes by DNA sequencing

  15. De novo mutations in the genome organizer CTCF cause intellectual disability

    DEFF Research Database (Denmark)

    Gregor, Anne; Oti, Martin; Kouwenhoven, Evelyn N

    2013-01-01

    An increasing number of genes involved in chromatin structure and epigenetic regulation has been implicated in a variety of developmental disorders, often including intellectual disability. By trio exome sequencing and subsequent mutational screening we now identified two de novo frameshift mutat...

  16. Correlation between clinical features and MECP2 gene mutations in patients with Rett syndrome

    Directory of Open Access Journals (Sweden)

    Hisham Megahed

    2015-03-01

    Conclusions: Mutation screening for MECP2 is a fast and reliable method to diagnose patients clinically suspected to suffer from Rett syndrome or female patients with atypical Rett syndrome features, mental retardation, developmental delay and other neurological abnormalities who do not fit any specific diagnosis. Also, patients with MECP2 mutation presented with a more severe phenotype.

  17. REEP1 Mutation Spectrum and Genotype/Phenotype Correlation in Hereditary Spastic Paraplegia Type 31

    Science.gov (United States)

    Beetz, Christian; Schule, Rebecca; Deconinck, Tine; Tran-Viet, Khanh-Nhat; Zhu, Hui; Kremer, Berry P. H.; Frints, Suzanna G. M.; van Zelst-Stams, Wendy A. G.; Byrne, Paula; Otto, Susanne; Nygren, Anders O. H.; Baets, Jonathan; Smets, Katrien; Ceulemans, Berten; Dan, Bernard; Nagan, Narasimhan; Kassubek, Jan; Klimpe, Sven; Klopstock, Thomas; Stolze, Henning; Smeets, Hubert J. M.; Schrander-Stumpel, Constance T. R. M.; Hutchinson, Michael; van de Warrenburg, Bart P.; Braastad, Corey; Deufel, Thomas; Pericak-Vance, Margaret; Schols, Ludger; de Jonghe, Peter; Zuchner, Stephan

    2008-01-01

    Mutations in the receptor expression enhancing protein 1 (REEP1) have recently been reported to cause autosomal dominant hereditary spastic paraplegia (HSP) type SPG31. In a large collaborative effort, we screened a sample of 535 unrelated HSP patients for "REEP1" mutations and copy number variations. We identified 13 novel and 2 known "REEP1"…

  18. Simultaneous mutation detection of three homoeologous genes in wheat by High Resolution Melting analysis and Mutation Surveyor®

    Directory of Open Access Journals (Sweden)

    Vincent Kate

    2009-12-01

    Full Text Available Abstract Background TILLING (Targeting Induced Local Lesions IN Genomes is a powerful tool for reverse genetics, combining traditional chemical mutagenesis with high-throughput PCR-based mutation detection to discover induced mutations that alter protein function. The most popular mutation detection method for TILLING is a mismatch cleavage assay using the endonuclease CelI. For this method, locus-specific PCR is essential. Most wheat genes are present as three similar sequences with high homology in exons and low homology in introns. Locus-specific primers can usually be designed in introns. However, it is sometimes difficult to design locus-specific PCR primers in a conserved region with high homology among the three homoeologous genes, or in a gene lacking introns, or if information on introns is not available. Here we describe a mutation detection method which combines High Resolution Melting (HRM analysis of mixed PCR amplicons containing three homoeologous gene fragments and sequence analysis using Mutation Surveyor® software, aimed at simultaneous detection of mutations in three homoeologous genes. Results We demonstrate that High Resolution Melting (HRM analysis can be used in mutation scans in mixed PCR amplicons containing three homoeologous gene fragments. Combining HRM scanning with sequence analysis using Mutation Surveyor® is sensitive enough to detect a single nucleotide mutation in the heterozygous state in a mixed PCR amplicon containing three homoeoloci. The method was tested and validated in an EMS (ethylmethane sulfonate-treated wheat TILLING population, screening mutations in the carboxyl terminal domain of the Starch Synthase II (SSII gene. Selected identified mutations of interest can be further analysed by cloning to confirm the mutation and determine the genomic origin of the mutation. Conclusion Polyploidy is common in plants. Conserved regions of a gene often represent functional domains and have high sequence

  19. Screening of BRCA1 sequence variants within exon 11 by heteroduplex analysis

    Directory of Open Access Journals (Sweden)

    Lucian Negura

    2013-03-01

    Full Text Available Germ-line mutations of either BRCA1 or BRCA2 represents the major hereditary risk to breast and ovariancancer. Screening for mutations in these genes is now standard practice in molecular diagnosis, opening the way tooncogenetic counselling and follow-up. Because mutations in both BRCA1 and BRCA2 are distributed throughout theloci, accepted clinical protocols involve screening their entire coding regions. Systematic Sanger sequencing is time andmoney consuming. Therefore, a lot of pre-screening techniques evolved over time in order to identify anomalousamplicons prior to sequencing. Because BRCA mutations are always heterozygous, heteroduplex analysis proved to be asuitable pre-screening step. We previously implemented mismatch specific endonuclease heteroduplex analysis forBRCA1 exon7. Here we show the utility of the same method for mutations and SNPs found in BRCA1 exon 11

  20. Relations between DNA- and RNA-based molecular methods for cyanobacteria and microcystin concentration at Maumee Bay State Park Lakeside Beach, Oregon, Ohio, 2012

    Science.gov (United States)

    Stelzer, Erin A.; Loftin, Keith A.; Struffolino, Pamela

    2013-01-01

    Water samples were collected from Maumee Bay State Park Lakeside Beach, Oregon, Ohio, during the 2012 recreational season and analyzed for selected cyanobacteria gene sequences by DNA-based quantitative polymerase chain reaction (qPCR) and RNA-based quantitative reverse-transcription polymerase chain reaction (qRT-PCR). Results from the four DNA assays (for quantifying total cyanobacteria, total Microcystis, and Microcystis and Planktothrix strains that possess the microcystin synthetase E (mcyE) gene) and two RNA assays (for quantifying Microcystis and Planktothrix genera that are expressing the microcystin synthetase E (mcyE) gene) were compared to microcystin concentration results determined by an enzyme-linked immunosorbent assay (ELISA). Concentrations of the target in replicate analyses were log10 transformed. The average value of differences in log10 concentrations for the replicates that had at least one detection were found to range from 0.05 to >0.37 copy per 100 milliliters (copy/100 mL) for DNA-based methods and from >0.04 to >0.17 copy/100 mL for RNA-based methods. RNA has a shorter half-life than DNA; consequently, a 24-hour holding-time study was done to determine the effects of holding time on RNA concentrations. Holding-time comparisons for the RNA-based Microcystis toxin mcyE assay showed reductions in the number of copies per 100 milliliters over 24 hours. The log difference between time 2 hours and time 24 hours was >0.37 copy/100 mL, which was higher than the analytical variability (log difference of >0.17 copy/100 mL). Spearman’s correlation analysis indicated that microcystin toxin concentrations were moderately to highly related to DNA-based assay results for total cyanobacteria (rho=0.69), total Microcystis (rho=0.74), and Microcystis strains that possess the mcyE gene (rho=0.81). Microcystin toxin concentrations were strongly related with RNA-based assay results for Microcystis mcyE gene expression (rho=0.95). Correlation analysis could

  1. Driver Gene Mutations in Stools of Colorectal Carcinoma Patients Detected by Targeted Next-Generation Sequencing.

    Science.gov (United States)

    Armengol, Gemma; Sarhadi, Virinder K; Ghanbari, Reza; Doghaei-Moghaddam, Masoud; Ansari, Reza; Sotoudeh, Masoud; Puolakkainen, Pauli; Kokkola, Arto; Malekzadeh, Reza; Knuutila, Sakari

    2016-07-01

    Detection of driver gene mutations in stool DNA represents a promising noninvasive approach for screening colorectal cancer (CRC). Amplicon-based next-generation sequencing (NGS) is a good option to study mutations in many cancer genes simultaneously and from a low amount of DNA. Our aim was to assess the feasibility of identifying mutations in 22 cancer driver genes with Ion Torrent technology in stool DNA from a series of 65 CRC patients. The assay was successful in 80% of stool DNA samples. NGS results showed 83 mutations in cancer driver genes, 29 hotspot and 54 novel mutations. One to five genes were mutated in 75% of cases. TP53, KRAS, FBXW7, and SMAD4 were the top mutated genes, consistent with previous studies. Of samples with mutations, 54% presented concomitant mutations in different genes. Phosphatidylinositol 3-kinase/mitogen-activated protein kinase pathway genes were mutated in 70% of samples, with 58% having alterations in KRAS, NRAS, or BRAF. Because mutations in these genes can compromise the efficacy of epidermal growth factor receptor blockade in CRC patients, identifying mutations that confer resistance to some targeted treatments may be useful to guide therapeutic decisions. In conclusion, the data presented herein show that NGS procedures on stool DNA represent a promising tool to detect genetic mutations that could be used in the future for diagnosis, monitoring, or treating CRC. Copyright © 2016 American Society for Investigative Pathology and the Association for Molecular Pathology. Published by Elsevier Inc. All rights reserved.

  2. Beta-thalassaemia mutations in northern India (Delhi).

    Science.gov (United States)

    Madan, N; Sharma, S; Rusia, U; Sen, S; Sood, S K

    1998-03-01

    The present study was undertaken to define beta-thalassaemia mutations prevalent in northern India (Delhi). Forty six children of beta-thalassaemia major and their families were investigated. DNA was extracted from leucocytes and screened for mutations prevalent in the Indian population. These mutations included 619bp deletion, IVS 1-1 (G-T), IVS 1-5 (G-C), frameshift mutations FS 8/9 (+G), FS 41/42 (-CTTT), Codon 16(-C), Codon 15 (G-A), codon 30 (G-C), IVS 1-110 (G-A) and -88 (C-T). 619 bp deletion mutation was detected directly by amplification of DNA by PCR followed by agarose gel electrophoresis. Other mutations were studied by DNA amplification and dot blot hybridization using synthetic normal and mutant oligonucleotide probes labelled at 5' end with gamma-32 P-ATP. Five mutations accounted for all the chromosomes in 46 patients. 619 bp deletion mutation was found to be the commonest mutation (34.8%) followed by IVS 1-5 (G-C) in 22.8 per cent, IVS 1-1 (G-T) in 19.6 per cent, FS 8/9 (+G) in 13 per cent and FS 41/42 (-CTTT) in 9.8 per cent. Nineteen (41.3%) patients were homozygous and 27 (58.7%) double heterozygous for different beta-thalassaemia mutations. This observation of limited number of mutations is significant and will be useful in planning strategies for prenatal diagnosis of beta-thalassaemia in northern India.

  3. Changes in screening behaviors and attitudes toward screening from pre-test genetic counseling to post-disclosure in Lynch syndrome families

    Science.gov (United States)

    Burton-Chase, Allison M.; Hovick, Shelly R.; Peterson, Susan K.; Marani, Salma K.; Vernon, Sally W.; Amos, Christopher I.; Frazier, Marsha L.; Lynch, Patrick M.; Gritz, Ellen R.

    2013-01-01

    Purpose This study examined colonoscopy adherence and attitudes towards colorectal cancer (CRC) screening in individuals who underwent Lynch syndrome genetic counseling and testing. Methods We evaluated changes in colonoscopy adherence and CRC screening attitudes in 78 cancer-unaffected relatives of Lynch syndrome mutation carriers before pre-test genetic counseling (baseline) and at 6 and 12 months post-disclosure of test results (52 mutation-negative, 26 mutation-positive). Results While both groups were similar at baseline, at 12 months post-disclosure, a greater number of mutation-positive individuals had had a colonoscopy compared with mutation-negative individuals. From baseline to 12 months post-disclosure, the mutation-positive group demonstrated an increase in mean scores on measures of colonoscopy commitment, self-efficacy, and perceived benefits of CRC screening, and a decrease in mean scores for perceived barriers to CRC screening. Mean scores on colonoscopy commitment decreased from baseline to 6 months in the mutation-negative group. Conclusion Adherence to risk-appropriate guidelines for CRC surveillance improved after genetic counseling and testing for Lynch syndrome. Mutation-positive individuals reported increasingly positive attitudes toward CRC screening after receiving genetic test results, potentially reinforcing longer term colonoscopy adherence. PMID:23414081

  4. Screening for fragile X syndrome.

    Science.gov (United States)

    Murray, J; Cuckle, H; Taylor, G; Hewison, J

    1997-01-01

    BACKGROUND AND AIM OF REVIEW. In 1991, the gene responsible for fragile X syndrome, a common cause of learning disability, was discovered. As a result, diagnosis of the disorder has improved and its molecular genetics are now understood. This report seems to provide the information needed to decide whether to use DNA testing to screen for the disorder. HOW THE RESEARCH WAS CONDUCTED. A literature search of electronic reference databases of published and 'grey' literature was undertaken together with hand searching of the most recent publications. RESEARCH FINDINGS. NATURAL HISTORY. Physical characteristics of fragile X syndrome include facial atypia, joint laxity and, in boys, macro-orchidism. Most affected males have moderate-to-severe learning disabilities with IQs under 50 whereas most females have borderline IQs of 70-85. Behavioural problems are similar to those seen with autism and attention-deficit disorders. Although fragile X syndrome is not curable there are a number of medical, educational, psychological and social interventions that can improve the symptoms. About 6% of those with learning disabilities tested in institutions have fragile X syndrome. Population prevalence figures are 1 in 4000 in males and 1 in 8000 in females. GENETICS. The disorder is caused by a mutation in a gene on the X chromosome which includes a trinucleotide repeat sequence. The mutation is characterized by hyper-expansion of the repeat sequence leading to down-regulation of the gene. In males an allele with repeat size in excess of 200, termed a full mutation (FM), is always associated with the affected phenotype, whereas in females only half are affected. Individuals with alleles having repeat size in the range 55-199 are unaffected but in females the sequence is heritably unstable so that it is at high risk of expansion to an FM in her offspring. This allele is known as a pre-mutation (PM) to contrast it with the FM found in the affected individual. No spontaneous expansions

  5. SRSF2-p95 hotspot mutation is highly associated with advanced forms of mastocytosis and mutations in epigenetic regulator genes.

    Science.gov (United States)

    Hanssens, Katia; Brenet, Fabienne; Agopian, Julie; Georgin-Lavialle, Sophie; Damaj, Gandhi; Cabaret, Laure; Chandesris, Maria Olivia; de Sepulveda, Paulo; Hermine, Olivier; Dubreuil, Patrice; Soucie, Erinn

    2014-05-01

    Mastocytosis is a rare and chronic disease with phenotypes ranging from indolent to severe. Prognosis for this disease is variable and very few biomarkers to predict disease evolution or outcome are currently known. We have performed comprehensive screening in our large cohort of mastocytosis patients for mutations previously found in other myeloid diseases and that could serve as prognostic indicators. KIT, SRSF2-P95 and TET2 mutations were by far the most frequent, detected in 81%, 24% and 21% of patients, respectively. Where TET2 and SRSF2-P95 mutation both correlated with advanced disease phenotypes, SRSF2-P95 hotspot mutation was found almost exclusively in patients diagnosed with associated clonal hematologic non-mast cell disease. Statistically, TET2 and SRSF2-P95 mutations were highly associated, suggesting a mechanistic link between these two factors. Finally, analysis of both clonal and sorted cell populations from patients confirms the presence of these mutations in the mast cell component of the disease, suggests an ontological mutation hierarchy and provides evidence for the expansion of multiple clones. This highlights the prognostic potential of such approaches, if applied systematically, for delineating the roles of specific mutations in predisposing and/or driving distinct disease phenotypes.

  6. Improved detection of p53 point mutations by dideoxyfingerprinting (ddF).

    Science.gov (United States)

    Martincic, D; Whitlock, J A

    1996-11-07

    Two screening techniques for identifying point mutations (single-strand conformational polymorphism (SSCP) and dideoxyfingerprinting (ddF)) were compared to sequencing to determine their efficiency in detecting mutations in exons 5-8 of the p53 tumor suppressor gene. Twelve human glioblastoma cell lines were studied by each of the three methods. Ten mutations were identified by sequencing; of these, 10/10 were detected by ddF, while SSCP detected 6/10 true mutations and falsely identified two presumed mutations not confirmed by sequencing. We examined the impact of parameters which influence DNA conformation (gel temperature, gel composition, and PCR product size) on the ability of SSCP and ddF to detect mutations. The sensitivity of SSCP varied with both gel temperature and the size of the PCR product; in contrast, ddF was not influenced by either gel temperature or product length (up to 460 nucleotides). We conclude that the increased sensitivity of ddF, together with its greater ease of application due to the lack of need for optimization, provides significant advantages over SSCP in screening DNA sequences for the presence of point mutations. Our results also suggest that the incidence of p53 mutations may be underestimated in studies of human cancers which utilize SSCP as the method of mutational screening.

  7. [Value of immunohistochemical staining with mutation-specific antibodies in detecting EGFR mutations: a meta-analysis].

    Science.gov (United States)

    Ma, Qing; Wang, Jing; Zhong, Diansheng; Ning, Chao; Liu, Chang; Xiao, Ping

    2014-06-20

    It has been proven that epidermal growth factor receptor (EGFR) mutation is the most important predictive factor for determining the effect of EGFR tyrosine kinase inhibitors (TKIs) applied to non-small cell lung cancer (NSCLC) patients. The patients with EGFR mutations response better to TKIs. To detect EGFR mutation has been particularly essential to select first-line treatment for lung cancer patients. To research and analyze the sensitivity and specificity of immunohistochemistry (IHC) using mutation specific antibodies in detecting EGFR mutations compared with DNA sequencing, and further evaluate the accuracy and clinical application value of IHC. All required articles in Pubmed database were searched. The deadline of retrieval was March 26, 2013. Then further screening the articles based on the inclusion and exclusion criteria. Meta analysis of diagnostic test was applied to analyze the sensitivity and specificity of IHC compared with DNA sequencing for the detection of EGFR mutations. Ten articles were included in the meta analysis, there were 1,679 samples in L858R group and 1,041 samples in E746-A750del group. The DOR were 225.17 (95%CI: 55.67-910.69) and 267.16 (95%CI: 132.45-538.88) respectively; the AUC of SROC were 0.948,4 (SEAUC=0.014,4) and 0.981,3 (SEAUC=0.009,9) respectively; the Q values were 0.888,3 (SEQ*=0.019,2) and 0.939,7 (SEQ*=0.019,1) respectively. The specificity and sensitivity of IHC method using these two mutation-specific antibodies were relatively high. As a screening method for EGFR mutations, the IHC with mutation specific antibodies is of clinical value.

  8. Identification of novel mutations in congenital afibrinogenemia patients and molecular modeling of missense mutations in Pakistani population.

    Science.gov (United States)

    Naz, Arshi; Biswas, Arijit; Khan, Tehmina Nafees; Goodeve, Anne; Ahmed, Nisar; Saqlain, Nazish; Ahmed, Shariq; Ujjan, Ikram Din; Shamsi, Tahir S; Oldenburg, Johannes

    2017-01-01

    Congenital afibrinogenemia (OMIM #202400) is a rare coagulation disorder that was first described in 1920. It is transmitted as an autosomal recessive trait that is characterized by absent levels of fibrinogen (factor I) in plasma. Consanguinity in Pakistan and its neighboring countries has resulted in a higher number of cases of congenital fibrinogen deficiency in their respective populations. This study focused on the detection of mutations in fibrinogen genes using DNA sequencing and molecular modeling of missense mutations in all three genes [Fibrinogen gene alpha (FGA), beta (FGB) and gamma (FGG)] in Pakistani patients. This descriptive and cross sectional study was conducted in Karachi and Lahore and fully complied with the Declaration of Helsinki. Patients with fibrinogen deficiency were screened for mutations in the Fibrinogen alpha (FGA), beta (FGB) and gamma (FGG) genes by direct sequencing. Molecular modeling was performed to predict the putative structure functional impact of the missense mutations identified in this study. Ten patients had mutations in FGA followed by three mutations in FGB and three mutations in FGG, respectively. Twelve of these mutations were novel. The missense mutations were predicted to result in a loss of stability because they break ordered regions and cause clashes in the hydrophobic core of the protein. Congenital afibrinogenemia is a rapidly growing problem in regions where consanguinity is frequently practiced. This study illustrates that mutations in FGA are relatively more common in Pakistani patients and molecular modeling of the missense mutations has shown damaging protein structures which has profounding effect on phenotypic bleeding manifestations in these patients.

  9. Mutation scanning of peach floral genes

    Directory of Open Access Journals (Sweden)

    Wilde H Dayton

    2011-05-01

    Full Text Available Abstract Background Mutation scanning technology has been used to develop crop species with improved traits. Modifications that improve screening throughput and sensitivity would facilitate the targeted mutation breeding of crops. Technical innovations for high-resolution melting (HRM analysis are enabling the clinic-based screening for human disease gene polymorphism. We examined the application of two HRM modifications, COLD-PCR and QMC-PCR, to the mutation scanning of genes in peach, Prunus persica. The targeted genes were the putative floral regulators PpAGAMOUS and PpTERMINAL FLOWER I. Results HRM analysis of PpAG and PpTFL1 coding regions in 36 peach cultivars found one polymorphic site in each gene. PpTFL1 and PpAG SNPs were used to examine approaches to increase HRM throughput. Cultivars with SNPs could be reliably detected in pools of twelve genotypes. COLD-PCR was found to increase the sensitivity of HRM analysis of pooled samples, but worked best with small amplicons. Examination of QMC-PCR demonstrated that primary PCR products for further analysis could be produced from variable levels of genomic DNA. Conclusions Natural SNPs in exons of target peach genes were discovered by HRM analysis of cultivars from a southeastern US breeding program. For detecting natural or induced SNPs in larger populations, HRM efficiency can be improved by increasing sample pooling and template production through approaches such as COLD-PCR and QMC-PCR. Technical advances developed to improve clinical diagnostics can play a role in the targeted mutation breeding of crops.

  10. Screening for calreticulin mutations in a cohort of patients suspected ...

    African Journals Online (AJOL)

    [4] The N domain is important for proper folding of CALR and enables its interaction with numerous other protein molecules such as α-integrin. The P domain comprises specific amino acid sequence repeats permitting binding of high-affinity calcium ions. (Ca2+) and lectin-like chaperoning.[5] The C domain contains a KDEL.

  11. A comparative study of mutation screening of sarcomeric genes ...

    African Journals Online (AJOL)

    However there have been limited direct comparisons between NGS and more established technologies to assess the sensitivity and false negative rates of this new approach. The scope of the present manuscript is to compare variants detected in MYBPC3, MYH7 and TNNT2 genes using the stepwise dHPLC/ Sanger ...

  12. RESEARCH ARTICLE Mutational screening of PKD2 gene in north ...

    Indian Academy of Sciences (India)

    2017-01-12

    Jan 12, 2017 ... genetically compatible kidney donor in future and the risk assessment studies. The spectrum of genetic variants identified in this study includes one/two nucleotide deletion responsible for truncated protein formation and single nucleotide substitution causing missense and silent variation at protein level.

  13. Detecting clusters of mutations.

    Directory of Open Access Journals (Sweden)

    Tong Zhou

    Full Text Available Positive selection for protein function can lead to multiple mutations within a small stretch of DNA, i.e., to a cluster of mutations. Recently, Wagner proposed a method to detect such mutation clusters. His method, however, did not take into account that residues with high solvent accessibility are inherently more variable than residues with low solvent accessibility. Here, we propose a new algorithm to detect clustered evolution. Our algorithm controls for different substitution probabilities at buried and exposed sites in the tertiary protein structure, and uses random permutations to calculate accurate P values for inferred clusters. We apply the algorithm to genomes of bacteria, fly, and mammals, and find several clusters of mutations in functionally important regions of proteins. Surprisingly, clustered evolution is a relatively rare phenomenon. Only between 2% and 10% of the genes we analyze contain a statistically significant mutation cluster. We also find that not controlling for solvent accessibility leads to an excess of clusters in terminal and solvent-exposed regions of proteins. Our algorithm provides a novel method to identify functionally relevant divergence between groups of species. Moreover, it could also be useful to detect artifacts in automatically assembled genomes.

  14. Identification of a novel CLRN1 gene mutation in Usher syndrome type 3: two case reports.

    Science.gov (United States)

    Yoshimura, Hidekane; Oshikawa, Chie; Nakayama, Jun; Moteki, Hideaki; Usami, Shin-Ichi

    2015-05-01

    This study examines the CLRN1 gene mutation analysis in Japanese patients who were diagnosed with Usher syndrome type 3 (USH3) on the basis of clinical findings. Genetic analysis using massively parallel DNA sequencing (MPS) was conducted to search for 9 causative USH genes in 2 USH3 patients. We identified the novel pathogenic mutation in the CLRN1 gene in 2 patients. The missense mutation was confirmed by functional prediction software and segregation analysis. Both patients were diagnosed as having USH3 caused by the CLRN1 gene mutation. This is the first report of USH3 with a CLRN1 gene mutation in Asian populations. Validating the presence of clinical findings is imperative for properly differentiating among USH subtypes. In addition, mutation screening using MPS enables the identification of causative mutations in USH. The clinical diagnosis of this phenotypically variable disease can then be confirmed. © The Author(s) 2015.

  15. Lack of TERT Promoter Mutations in Human B-Cell Non-Hodgkin Lymphoma

    Directory of Open Access Journals (Sweden)

    Gary Lam

    2016-10-01

    Full Text Available Non-Hodgkin lymphomas (NHL are a heterogeneous group of immune cell neoplasms that comprise molecularly distinct lymphoma subtypes. Recent work has identified high frequency promoter point mutations in the telomerase reverse transcriptase (TERT gene of different cancer types, including melanoma, glioma, liver and bladder cancer. TERT promoter mutations appear to correlate with increased TERT expression and telomerase activity in these cancers. In contrast, breast, pancreatic, and prostate cancer rarely demonstrate mutations in this region of the gene. TERT promoter mutation prevalence in NHL has not been thoroughly tested thus far. We screened 105 B-cell lymphoid malignancies encompassing nine NHL subtypes and acute lymphoblastic leukemia, for TERT promoter mutations. Our results suggest that TERT promoter mutations are rare or absent in most NHL. Thus, the classical TERT promoter mutations may not play a major oncogenic role in TERT expression and telomerase activation in NHL.

  16. DMSO increases mutation-scanning detection sensitivity in clinical samples using high resolution melting

    Science.gov (United States)

    Song, Chen; Castellanos-Rizaldos, Elena; Bejar, Rafael; Ebert, Benjamin L.; Makrigiorgos, G. Mike

    2016-01-01

    BACKGROUND Mutation scanning provides the simplest, lowest cost method for identifying DNA variations on single PCR amplicons, and it may be performed prior to sequencing to avoid screening of non-informative wild type samples. High resolution melting (HRM) is the most commonly used method for mutation scanning. However, by using PCR-HRM mutations below ≈ 3–10% that can still be clinically significant may often be missed. Therefore, enhancing HRM detection sensitivity is important for mutation scanning and its clinical application. METHODS We used serial dilution of TP53 exon 8 mutation containing cell lines to demonstrate the improvement in detection sensitivity for conventional-PCR-HRM in the presence of DMSO. We also conducted full-COLD-PCR to further enrich low-level mutations prior to HRM±DMSO and employed droplet-digital PCR to derive the optimal conditions for mutation enrichment. Both conventional-PCR-HRM and full-COLD-PCR-HRM ±DMSO were used for mutation scanning in TP53 exon 8 in cancer samples containing known mutations and in myelodysplastic syndrome samples with unknown mutations. Mutations in other genes were also examined. RESULTS The detection sensitivity of PCR-HRM-scanning increases 2–5-fold in the presence of DMSO, depending also on mutation type and sequence context, and can typically detect mutation abundance of about 1%. When mutation enrichment is applied during amplification using full-COLD-PCR and followed by HRM in the presence of DMSO, mutations with 0.2–0.3% mutation abundance in TP53 exon 8 can be detected. CONCLUSIONS DMSO improves HRM mutation scanning sensitivity. When full-COLD-PCR is employed, followed by DMSO-HRM, the overall improvement is about 20-fold as compared to conventional PCR-HRM. PMID:26432802

  17. Frequent mutations of genes encoding ubiquitin-mediated proteolysis pathway components in clear cell renal cell carcinoma

    DEFF Research Database (Denmark)

    Guo, Guangwu; Gui, Yaoting; Gao, Shengjie

    2012-01-01

    We sequenced whole exomes of ten clear cell renal cell carcinomas (ccRCCs) and performed a screen of similar to 1,100 genes in 88 additional ccRCCs, from which we discovered 12 previously unidentified genes mutated at elevated frequencies in ccRCC. Notably, we detected frequent mutations in the u......We sequenced whole exomes of ten clear cell renal cell carcinomas (ccRCCs) and performed a screen of similar to 1,100 genes in 88 additional ccRCCs, from which we discovered 12 previously unidentified genes mutated at elevated frequencies in ccRCC. Notably, we detected frequent mutations...

  18. Correlation of RET somatic mutations with clinicopathological features in sporadic medullary thyroid carcinomas

    Science.gov (United States)

    Moura, M M; Cavaco, B M; Pinto, A E; Domingues, R; Santos, J R; Cid, M O; Bugalho, M J; Leite, V

    2009-01-01

    Screening of REarranged during Transfection (RET) gene mutations has been carried out in different series of sporadic medullary thyroid carcinomas (MTC). RET-positive tumours seem to be associated to a worse clinical outcome. However, the correlation between the type of RET mutation and the patients' clinicopathological data has not been evaluated yet. We analysed RET exons 5, 8, 10–16 in fifty-one sporadic MTC, and found somatic mutations in thirty-three (64.7%) tumours. Among the RET-positive cases, exon 16 was the most frequently affected (60.6%). Two novel somatic mutations (Cys630Gly, c.1881del18) were identified. MTC patients were divided into three groups: group 1, with mutations in RET exons 15 and 16; group 2, with other RET mutations; group 3, having no RET mutations. Group 1 had higher prevalence (P=0.0051) and number of lymph node metastases (P=0.0017), and presented more often multifocal tumours (P=0.037) and persistent disease at last control (P=0.0242) than group 2. Detectable serum calcitonin levels at last screening (P=0.0119) and stage IV disease (P=0.0145) were more frequent in group 1, than in the other groups. Our results suggest that, among the sporadic MTC, cases with RET mutations in exons 15 and 16 are associated with the worst prognosis. Cases with other RET mutations have the most indolent course, and those with no RET mutations have an intermediate risk. PMID:19401695

  19. MUTATIONS IN CALMODULIN GENES

    DEFF Research Database (Denmark)

    2013-01-01

    The present invention relates to an isolated polynucleotide encoding at least a part of calmodulin and an isolated polypeptide comprising at least a part of a calmodulin protein, wherein the polynucleotide and the polypeptide comprise at least one mutation associated with a cardiac disorder...... the binding of calmodulin to ryanodine receptor 2 and use of such compound in a treatment of an individual having a cardiac disorder. The invention further provides a kit that can be used to detect specific mutations in calmodulin encoding genes....

  20. Are There Mutator Polymerases?

    Directory of Open Access Journals (Sweden)

    Miguel Garcia-Diaz

    2003-01-01

    Full Text Available DNA polymerases are involved in different cellular events, including genome replication and DNA repair. In the last few years, a large number of novel DNA polymerases have been discovered, and the biochemical analysis of their properties has revealed a long list of intriguing features. Some of these polymerases have a very low fidelity and have been suggested to play mutator roles in different processes, like translesion synthesis or somatic hypermutation. The current view of these processes is reviewed, and the current understanding of DNA polymerases and their role as mutator enzymes is discussed.

  1. Effects of intronic mutations in the LDLR gene on pre-mRNA splicing: Comparison of wet-lab and bioinformatics analyses

    NARCIS (Netherlands)

    Holla, Øystein L.; Nakken, Sigve; Mattingsdal, Morten; Ranheim, Trine; Berge, Knut Erik; Defesche, Joep C.; Leren, Trond P.

    2009-01-01

    Screening for mutations in the low density lipoprotein receptor (LDLR) gene has identified more than 1000 mutations as the cause of familial hypercholesterolemia (FH). In addition, numerous intronic mutations with uncertain effects on pre-mRNA splicing have also been identified. In this study, we

  2. Factor V Leiden Mutation and PT 20210 Mutation Test

    Science.gov (United States)

    ... Patient Resources For Health Professionals Subscribe Search Factor V Leiden Mutation and PT 20210 Mutation Send Us ... As Activated Protein C Resistance APC Resistance Factor V R506Q PT G20210A Factor II 20210 Factor II ...

  3. BCR-ABL kinase domain mutations, including 2 novel mutations in imatinib resistant Malaysian chronic myeloid leukemia patients-Frequency and clinical outcome.

    Science.gov (United States)

    Elias, Marjanu Hikmah; Baba, Abdul Aziz; Azlan, Husin; Rosline, Hassan; Sim, Goh Ai; Padmini, Menon; Fadilah, S Abdul Wahid; Ankathil, Ravindran

    2014-04-01

    Discovery of imatinib mesylate (IM) as the targeted BCR-ABL protein tyrosine kinase inhibitor (TKI) has resulted in its use as the frontline therapy for chronic myeloid leukemia (CML) across the world. Although high response rates are observed in CML patients who receive IM treatment, a significant number of patients develop resistance to IM. Resistance to IM in patients has been associated with a heterogeneous array of mechanisms of which point mutations within the ABL tyrosine kinase domain (TKD) are the frequently documented. The types and frequencies of mutations reported in different population studies have shown wide variability. We screened 125 Malaysian CML patients on IM therapy who showed either TKI refractory or resistance to IM to investigate the frequency and pattern of BCR-ABL kinase domain mutations among Malaysian CML patients undergoing IM therapy and to determine the clinical significance. Mutational screening using denaturing high performance liquid chromatography (dHPLC) followed by DNA sequencing was performed on 125 IM resistant Malaysian CML patients. Mutations were detected in 28 patients (22.4%). Fifteen different types of mutations (T315I, E255K, G250E, M351T, F359C, G251E, Y253H, V289F, E355G, N368S, L387M, H369R, A397P, E355A, D276G), including 2 novel mutations were identified, with T315I as the predominant type of mutation. The data generated from clinical and molecular parameters studied were correlated with the survival of CML patients. Patients with Y253H, M351T and E355G TKD mutations showed poorer prognosis compared to those without mutation. Interestingly, when the prognostic impact of the observed mutations was compared inter-individually, E355G and Y253H mutations were associated with more adverse prognosis and shorter survival (P=0.025 and 0.005 respectively) than T315I mutation. Results suggest that apart from those mutations occurring in the three crucial regions (catalytic domain, P-loop and activation-loop), other rare

  4. An update of Gaucher mutations distribution in the Ashkenazi Jewish population: prevalence and country of origin of the mutation R496H.

    Science.gov (United States)

    Bronstein, Silvia; Karpati, Mazal; Peleg, Leah

    2014-11-01

    Gaucher disease is the most prevalent inherited disorder among Ashkenazi Jews (carrier frequency of about 6%) and six mutations account for about 96% of their mutant alleles. Two mutations, N370S and R496H, have been reported only in mildly affected or asymptomatic patients. Due to the rarity of R496H, it was recommended that it be excluded from screening programs. To verify the frequency and trace the origin of Gaucher mutations in screened individuals whose Ashkenazi ethnicity was confirmed by the birthplace of their grandparents. We conducted a retrospective analysis of the screened results for the period 2006-2011. Mutations were identified by restriction analysis, Tag-It detection system, Pronto diagnostic kit and Nanogen technology (NanoChip 400). The heterozygote frequency of eight mutations was estimated in a cohort of 16,910 alleles. Two mutations, N370S and R496H, were the most frequent in our population. However, while the occurrence of N370S carriers was similar to other reports (1:19.4), that of R496H carriers was considerably elevated (1:207). Examination of the screened individuals' ethnicity showed a significant difference in the distribution pattern of the country of origin between the carriers of these two mutations. The origin pattern differences between the two groups of heterozygotes might reflect a separate geographic region of introduction for various mutations. As a result, secondary subgroups could be formed within the Ashkenazi population. This might clarify the dissimilarities in the occurrence of R496H mutation reported by various centers.

  5. CF Mutation Panel

    Science.gov (United States)

    ... Testing Leptin Levetiracetam Lipase Lipid Profile Lipoprotein (a) Lithium Liver Panel Lp-PLA2 Lupus Anticoagulant Testing Luteinizing ... L. et. al. (2011 September 29). Kids in America Newborn Screening for Cystic Fibrosis. Medscape Today News ...

  6. [Screening of common deaf genes in pregnant women and prevention of deafness at birth].

    Science.gov (United States)

    Shao, Minjie; Liu, Ping; Zhao, Nan; Zhong, Su; Zhao, Yangyu; Wei, Yuan

    2015-06-01

    To determine the carrier rate for common mutations causing deafness among pregnant women in order to prevent births of deaf children. For 893 pregnant women, 2 mL peripheral venous blood was taken and DNA was extracted. A deafness DNA microarray screening was applied to such samples, and DNA sequencing was applied to husbands of women with positive screening results. A total of 40 carriers were detected, with the overall mutation rate being 4.48%. Among such carriers, GJB2 235delC was the most common heterozygous mutation (18 cases) and the mutation rate was 2.02%. GJB2 299A-T heterozygous mutation was detected in 7 cases with a mutation rate of 0.78%. IVS7-2A to G heterozygous mutation was detected in 9 cases with a mutation rate of 1.02%. There were 2 cases carrying GJB3 heterozygous mutation and 2 cases of mitochondrial 12S rRNA heterozygous mutation, with a mutation rate of 0.22%. IVS7-2A>G with GJB3 538C>T double heterozygous mutation was detected in 1 case, and IVS7-2A>G with GJB2 299A-T double heterozygous mutation was detected in another case, with the mutation rate of each being 0.11%. DNA sequencing has failed to find presence of mutations in the same gene in the husbands. The results of neonatal hearing follow-up were all normal. Applications of the deaf genes screening in pregnant women may play prove to be valuable for the early detection for neonatal deafness.

  7. Msx1 Mutations

    Science.gov (United States)

    Wang, Y.; Kong, H.; Mues, G.; D’Souza, R.

    2011-01-01

    Mutations in the transcription factors PAX9 and MSX1 cause selective tooth agenesis in humans. In tooth bud mesenchyme of mice, both proteins are required for the expression of Bmp4, which is the key signaling factor for progression to the next step of tooth development. We have previously shown that Pax9 can transactivate a 2.4-kb Bmp4 promoter construct, and that most tooth-agenesis-causing PAX9 mutations impair DNA binding and Bmp4 promoter activation. We also found that Msx1 by itself represses transcription from this proximal Bmp4 promoter, and that, in combination with Pax9, it acts as a potentiator of Pax9-induced Bmp4 transactivation. This synergism of Msx1 with Pax9 is significant, because it is currently the only documented mechanism for Msx1-mediated activation of Bmp4. In this study, we investigated whether the 5 known tooth-agenesis-causing MSX1 missense mutations disrupt this Pax9-potentiation effect, or if they lead to deficiencies in protein stability, protein-protein interactions, nuclear translocation, and DNA-binding. We found that none of the studied molecular mechanisms yielded a satisfactory explanation for the pathogenic effects of the Msx1 mutations, calling for an entirely different approach to the investigation of this step of odontogenesis on the molecular level. PMID:21297014

  8. Mitochondrial mutations in cancer

    National Research Council Canada - National Science Library

    Brandon, M; Baldi, P; Wallace, D C

    2006-01-01

    ...). The mitochondria are assembled from both nuclear DNA (nDNA) and mitochondrial DNA (mtDNA) genes. The mtDNA codes for 37 genes essential of OXPHOS, is present in thousands of copies per cell, and has a very high mutations rate...

  9. Mutation analysis and evaluation of the cardiac localization of TMEM43 in arrhythmogenic right ventricular cardiomyopathy

    DEFF Research Database (Denmark)

    Christensen, A H; Andersen, C B; Tybjærg-Hansen, A

    2011-01-01

    Christensen AH, Andersen CB, Tybjærg-Hansen A, Haunso S, Svendsen JH. Mutation analysis and evaluation of the cardiac localization of TMEM43 in arrhythmogenic right ventricular cardiomyopathy. A single report has associated mutations in TMEM43 (LUMA) with a distinctive form of arrhythmogenic right...... ventricular cardiomyopathy (ARVC). We aimed at performing mutational analysis of the gene and characterizing the associated immunohistochemical features. Sixty-five unrelated patients (55 fulfilling Task Force criteria and 10 borderline cases) were screened for mutations in TMEM43. Immunohistochemistry...

  10. Mutations in galactosemia

    Energy Technology Data Exchange (ETDEWEB)

    Reichardt, J.K.V. [Univ. of Southern California School of Medicine, Los Angeles, CA (United States)

    1995-10-01

    This Letter raises four issues concerning two papers on galactosemia published in the March 1995 of the Journal. First, table 2 in the paper by Elsas et al. incorrectly attributes seven galactose-l-phosphate uridyl transferase (GALT) mutations (S135L, L195P, K285N, N314D, R333W, R333G, and K334R). The table also fails to mention that others have reported the same two findings attributed to {open_quotes}Leslie et al.; Elsas et al. and in press{close_quotes} and {open_quotes}Leslie et al.; Elsas et al.{close_quotes} The first finding on the prevalence of the Q188R galactosemia mutation in the G/G Caucasian population has also been described by Ng et al., and the second finding on the correlation of the N314D GALT mutation with the Duarte variant was reported by Lin et al. Second, Elsas et al. suggest that the E203K and N314D mutations may {open_quotes}produce intra-allelic complementation when in cis{close_quotes}. This speculation is supported by the activity data of individual III-2 but is inconsistent with the activities of three other individuals I-1, II-1, and III-1 of the same pedigree. The GALT activity measured in these three individuals suggests a dominant negative effect of E203K in E203K-N314D chromosomes, since they all have less than normal activity. Thus, the preponderance of the data in this paper is at odds with the authors speculation. It is worth recalling that Lin et al. also identified four N314D GALT mutations on 95 galactosemic chromosomes examined. A similar situation also appears to be the case in proband III-1 (with genotype E203K-N314D/IVSC) in the Elsas et al. paper. 9 refs.

  11. Pediatric MDS: GATA screen the germline.

    Science.gov (United States)

    Stieglitz, Elliot; Loh, Mignon L

    2016-03-17

    In this issue of Blood, Wlodarski and colleagues demonstrate that as many as 72% of adolescents diagnosed with myelodysplastic syndrome (MDS) and monosomy 7 harbor germline mutations in GATA2. Although pediatric MDS is a very rare diagnosis, occurring in 0.8 to 4 cases per million, Wlodarski et al screened >600 cases of primary or secondary MDS in children and adolescents who were enrolled in the European Working Group on MDS consortium over a period of 15 years. The overall frequency of germline GATA2 mutations in children with primary MDS was 7%, and 15% in those presenting with advanced disease. Notably, mutations in GATA2 were absent in patients with therapy-related MDS or acquired aplastic anemia.

  12. A Novel c.554+5C>T Mutation in the DUOXA2 Gene Combined with p.R885Q Mutation in the DUOX2 Gene Causing Congenital Hypothyroidism.

    Science.gov (United States)

    Zheng, Xiao; Ma, Shao Gang; Qiu, Ya Li; Guo, Man Li; Shao, Xiao Juan

    2016-06-05

    The coexistence of mutations in the dual oxidase maturation factor 2 (DUOXA2) and dual oxidase 2 (DUOX2) genes is rarely identified in congenital hypothyroidism (CH). This study reports a boy with CH due to a novel splice-site mutation in the DUOXA2 gene and a missense mutation in the DUOX2 gene. A four-year-old boy was diagnosed with CH at neonatal screening and was enrolled in this study. The DUOXA2, DUOX2, thyroid peroxidase (TPO), and thyrotropin receptor (TSHR) genes were considered for genetic defects screening. Genomic DNA was extracted from peripheral blood leukocytes, and Sanger sequencing was used to screen the mutations in the exon fragments. Family members of the patient and the controls were also enrolled and evaluated. The boy harbored compound heterozygous mutations including a novel splice-site mutation c.554+5C>T in the maternal DUOXA2 allele and c.2654G>A (p.R885Q) in the paternal DUOX2 allele. The germline mutations from his parents were consistent with an autosomal recessive inheritance pattern. No mutations in the TPO and TSHR genes were detected. A novel splice-site mutation c.554+5C>T in the DUOXA2 gene and a mutation p.R885Q in the DUOX2 gene were identified in a 4-year-old patient with goitrous CH.

  13. RBC Antibody Screen

    Science.gov (United States)

    ... products and services. Advertising & Sponsorship: Policy | Opportunities RBC Antibody Screen Share this page: Was this page helpful? ... Indirect Coombs Test; Indirect Anti-human Globulin Test; Antibody Screen Formal name: Red Blood Cell Antibody Screen ...

  14. Screen time and children

    Science.gov (United States)

    ... page: //medlineplus.gov/ency/patientinstructions/000355.htm Screen time and children To use the sharing features on ... videos is considered unhealthy screen time. Current Screen Time Guidelines Children under age 2 should have no ...

  15. Prenatal Genetic Screening Tests

    Science.gov (United States)

    ... Education & Events Advocacy For Patients About ACOG Prenatal Genetic Screening Tests Home For Patients Search FAQs Prenatal ... Screening Tests FAQ165, July 2017 PDF Format Prenatal Genetic Screening Tests Pregnancy What is prenatal genetic testing? ...

  16. Endometrial Cancer Screening

    Science.gov (United States)

    ... Treatment Endometrial Cancer Prevention Endometrial Cancer Screening Research Endometrial Cancer Screening (PDQ®)–Patient Version What is screening? ... These are called diagnostic tests . General Information About Endometrial Cancer Key Points Endometrial cancer is a disease ...

  17. Stomach (Gastric) Cancer Screening

    Science.gov (United States)

    ... Stomach Cancer Prevention Stomach Cancer Screening Research Stomach (Gastric) Cancer Screening (PDQ®)–Patient Version What is screening? Go ... are called diagnostic tests . General Information About Stomach (Gastric) Cancer Key Points Stomach cancer is a disease in ...

  18. Normosmic congenital hypogonadotropic hypogonadism due to TAC3/TACR3 mutations: characterization of neuroendocrine phenotypes and novel mutations.

    Directory of Open Access Journals (Sweden)

    Bruno Francou

    Full Text Available CONTEXT: TAC3/TACR3 mutations have been reported in normosmic congenital hypogonadotropic hypogonadism (nCHH (OMIM #146110. In the absence of animal models, studies of human neuroendocrine phenotypes associated with neurokinin B and NK3R receptor dysfunction can help to decipher the pathophysiology of this signaling pathway. OBJECTIVE: To evaluate the prevalence of TAC3/TACR3 mutations, characterize novel TACR3 mutations and to analyze neuroendocrine profiles in nCHH caused by deleterious TAC3/TACR3 biallelic mutations. RESULTS: From a cohort of 352 CHH, we selected 173 nCHH patients and identified nine patients carrying TAC3 or TACR3 variants (5.2%. We describe here 7 of these TACR3 variants (1 frameshift and 2 nonsense deleterious mutations and 4 missense variants found in 5 subjects. Modeling and functional studies of the latter demonstrated the deleterious consequence of one missense mutation (Tyr267Asn probably caused by the misfolding of the mutated NK3R protein. We found a statistically significant (p<0.0001 higher mean FSH/LH ratio in 11 nCHH patients with TAC3/TACR3 biallelic mutations than in 47 nCHH patients with either biallelic mutations in KISS1R, GNRHR, or with no identified mutations and than in 50 Kallmann patients with mutations in KAL1, FGFR1 or PROK2/PROKR2. Three patients with TAC3/TACR3 biallelic mutations had an apulsatile LH profile but low-frequency alpha-subunit pulses. Pulsatile GnRH administration increased alpha-subunit pulsatile frequency and reduced the FSH/LH ratio. CONCLUSION: The gonadotropin axis dysfunction associated with nCHH due to TAC3/TACR3 mutations is related to a low GnRH pulsatile frequency leading to a low frequency of alpha-subunit pulses and to an elevated FSH/LH ratio. This ratio might be useful for pre-screening nCHH patients for TAC3/TACR3 mutations.

  19. Establishment and Validation of RNA-Based Predictive Models for Understanding Survival of Vibrio parahaemolyticus in Oysters Stored at Low Temperatures

    Science.gov (United States)

    Liao, Chao; Zhao, Yong

    2017-01-01

    ABSTRACT This study developed RNA-based predictive models describing the survival of Vibrio parahaemolyticus in Eastern oysters (Crassostrea virginica) during storage at 0, 4, and 10°C. Postharvested oysters were inoculated with a cocktail of five V. parahaemolyticus strains and were then stored at 0, 4, and 10°C for 21 or 11 days. A real-time reverse transcription-PCR (RT-PCR) assay targeting expression of the tlh gene was used to evaluate the number of surviving V. parahaemolyticus cells, which was then used to establish primary molecular models (MMs). Before construction of the MMs, consistent expression levels of the tlh gene at 0, 4, and 10°C were confirmed, and this gene was used to monitor the survival of the total V. parahaemolyticus cells. In addition, the tdh and trh genes were used for monitoring the survival of virulent V. parahaemolyticus. Traditional models (TMs) were built based on data collected using a plate counting method. From the MMs, V. parahaemolyticus populations had decreased 0.493, 0.362, and 0.238 log10 CFU/g by the end of storage at 0, 4, and 10°C, respectively. Rates of reduction of V. parahaemolyticus shown in the TMs were 2.109, 1.579, and 0.894 log10 CFU/g for storage at 0, 4, and 10°C, respectively. Bacterial inactivation rates (IRs) estimated with the TMs (−0.245, −0.152, and −0.121 log10 CFU/day, respectively) were higher than those estimated with the MMs (−0.134, −0.0887, and −0.0732 log10 CFU/day, respectively) for storage at 0, 4, and 10°C. Higher viable V. parahaemolyticus numbers were predicted using the MMs than using the TMs. On the basis of this study, RNA-based predictive MMs are the more accurate and reliable models and can prevent false-negative results compared to TMs. IMPORTANCE One important method for validating postharvest techniques and for monitoring the behavior of V. parahaemolyticus is to establish predictive models. Unfortunately, previous predictive models established based on plate

  20. A collaborative European exercise on mRNA-based body fluid/skin typing and interpretation of DNA and RNA results

    DEFF Research Database (Denmark)

    van den Berge, M; Carracedo, A; Gomes, I

    2014-01-01

    The European Forensic Genetics Network of Excellence (EUROFORGEN-NoE) undertook a collaborative project on mRNA-based body fluid/skin typing and the interpretation of the resulting RNA and DNA data. Although both body fluids and skin are composed of a variety of cell types with different functions......, the participating laboratories were asked to integrate the DNA and RNA results and associate donor and cell type where possible. A large variation for the integrated interpretations of the DNA and RNA data was obtained including correct interpretations. We infer that with expertise in analysing RNA profiles, clear...... that ranged from reference PCR products, cDNAs of indicated or unnamed cell type source(s), to challenging mock casework stains. From this specimen set, information on the overall sensitivity and specificity of the various markers was obtained. In addition, the reliability of a scoring system for inference...

  1. 2-Nitropropane-induced liver DNA and RNA base modifications: differences between Sprague-Dawley rats and New Zealand white rabbits.

    Science.gov (United States)

    Fiala, E S; Nie, G; Sodum, R; Conaway, C C; Sohn, O S

    1993-10-15

    2-Nitropropane (2-NP), a hepatocarcinogen in male Sprague-Dawley rats but not, under the same conditions, in male New Zealand White rabbits, induces characteristic base modifications in rat liver DNA and RNA including increases in 8-oxoguanine and the formation of 8-aminoguanine. We compared the levels of these modifications in the two animal species at 6, 18 and 42 h after a single i.p. treatment with 1.12 mmol/kg 2-NP. Significantly less nucleic acid base modifications were found to be produced in rabbit liver than in rat liver. Thus, the relative resistance of the rabbit to the hepatocarcinogenicity of 2-NP correlates with decreased levels of 2-NP-induced liver DNA and RNA base damage.

  2. Mutation profile of BBS genes in Iranian patients with Bardet-Biedl syndrome: genetic characterization and report of nine novel mutations in five BBS genes.

    Science.gov (United States)

    Fattahi, Zohreh; Rostami, Parvin; Najmabadi, Amin; Mohseni, Marzieh; Kahrizi, Kimia; Akbari, Mohammad Reza; Kariminejad, Ariana; Najmabadi, Hossein

    2014-07-01

    Bardet-Biedl syndrome (BBS) is a rare ciliopathy disorder that is clinically and genetically heterogeneous with 18 known genes. This study was performed to characterize responsible genes and mutation spectrum in a cohort of 14 Iranian families with BBS. Sanger sequencing of the most commonly mutated genes (BBS1, BBS2 and BBS10) accounting for ∼50% of BBS patients determined mutations only in BBS2, including three novel mutations. Next, three of the remaining patients were subjected to whole exome sequencing with 96% at 20 × depth of coverage that revealed novel BBS4 mutation. Observation of no mutation in the other patients represents the possible presence of novel genes. Screening of the remaining patients for six other genes (BBS3, BBS4, BBS6, BBS7, BBS9 and BBS12) revealed five novel mutations. This result represents another indication for the genetic heterogeneity of BBS and extends the mutational spectrum of the disease by introducing nine novel mutations in five BBS genes. In conclusion, although BBS1 and BBS10 are among the most commonly mutated genes in other populations like Caucasian, these two seem not to have an important role in Iranian patients. This suggests that a different strategy in molecular genetics diagnostic approaches in Middle Eastern countries such as Iran should be considered.

  3. Genetic Etiology of Parkinson Disease Associated with Mutations in the SNCA, PARK2, PINK1, PARK7, and LRRK2 Genes: A Mutation Update

    Science.gov (United States)

    Nuytemans, Karen; Theuns, Jessie; Cruts, Marc; Van Broeckhoven, Christine

    2010-01-01

    To date, molecular genetic analyses have identified over 500 distinct DNA variants in five disease genes associated with familial Parkinson disease; α-synuclein (SNCA), parkin (PARK2), PTEN-induced putative kinase 1 (PINK1), DJ-1 (PARK7), and Leucine-rich repeat kinase 2 (LRRK2). These genetic variants include ∼82% simple mutations and ∼18% copy number variations. Some mutation subtypes are likely underestimated because only few studies reported extensive mutation analyses of all five genes, by both exonic sequencing and dosage analyses. Here we present an update of all mutations published to date in the literature, systematically organized in a novel mutation database (http://www.molgen.ua.ac.be/PDmutDB). In addition, we address the biological relevance of putative pathogenic mutations. This review emphasizes the need for comprehensive genetic screening of Parkinson patients followed by an insightful study of the functional relevance of observed genetic variants. Moreover, while capturing existing data from the literature it became apparent that several of the five Parkinson genes were also contributing to the genetic etiology of other Lewy Body Diseases and Parkinson-plus syndromes, indicating that mutation screening is recommendable in these patient groups. Hum Mutat 31:763–780, 2010. © 2010 Wiley-Liss, Inc. PMID:20506312

  4. 220 patients with autosomal dominant spastic paraplegia do not display mutations in the SLC33A1-gene (SPG42)

    OpenAIRE

    Bauer, Peter; Schlipf, Nina; Beetz, Christian; Schüle, Rebecca; Stevanin, Giovanni; Erichsen, Anne Kjersti; Forlani, Sylvie; Zaros, Cécile; Karle, Kathrin; Klebe, Stephan; Klimpe, Sven; Durr, Alexandra; Otto, Susanne; Tallaksen, Chantal; Riess, Olaf

    2010-01-01

    Abstract The most frequent causes of autosomal dominant HSP (ADHSP) are mutations in the SPAST-gene (SPG4 locus). However, roughly 60% of the patients are negative for SPAST-mutations despite their family history is compatible with autosomal dominant inheritance. A mutation in the gene for an acetyl-CoA transporter (SLC33A1) has recently been reported in one Chinese family to cause ADHSP type SPG42. Here we screened 220 independent SPAST-mutation-negative ADHSP samples for mutation...

  5. Fabry disease mimicking hypertrophic cardiomyopathy: genetic screening needed for establishing the diagnosis in women

    DEFF Research Database (Denmark)

    Havndrup, Ole; Christiansen, Michael; Stoevring, Birgitte

    2010-01-01

    tested for. METHODS AND RESULTS: We evaluated 90 consecutively recruited HCM probands and their relatives. Probands without sarcomere-gene mutations were tested for alpha-galactosidase A gene (GLA) mutations. Of the 90 families, 31 (34%) had sarcomere gene mutations and were therefore excluded...... mutation (p.G271S). She had one affected daughter but her two other children were unaffected. The affected daughter had three children, of whom two were also affected--a boy aged 8 and a daughter aged 10 years. CONCLUSION: This is the first report of systematic mutation screening of GLA in HCM patients...... without sarcomere gene mutations. GLA mutations were found in 3/90 (3%) of HCM families and in 2/20 (10%) of females without sarcomere-gene mutations. None of the probands presented other indices of Fabry disease. This, in combination with putative reversibility of cardiac changes by enzyme replacement...

  6. LRRK2 exon 41 mutations in sporadic Parkinson disease in Europeans.

    Science.gov (United States)

    Lesage, Suzanne; Janin, Sabine; Lohmann, Ebba; Leutenegger, Anne-Louise; Leclere, Laurence; Viallet, François; Pollak, Pierre; Durif, Franck; Thobois, Stéphane; Layet, Valérie; Vidailhet, Marie; Agid, Yves; Dürr, Alexandra; Brice, Alexis; Bonnet, Anne-Marie; Borg, Michel; Broussolle, Emmanuel; Damier, Philippe; Destée, Alain; Martinez, Maria; Penet, Christiane; Rasco, Olivier; Tison, François; Tranchan, Christine; Vérin, Marc

    2007-03-01

    Mutations in leucine-rich repeat kinase 2 gene (LRRK2), particularly the G2019S mutation in exon 41, have been detected in familial and sporadic Parkinson disease (PD) cases. To assess the frequency of LRRK2 exon 41 mutations in a series of sporadic PD cases from Europe and to determine the clinical features of LRRK2 mutation carriers. We analyzed European cases of sporadic PD for the presence of LRRK2 exon 41 mutations. These mutations were screened by denaturing high-performance liquid chromatography, and abnormal chromatograph traces were investigated by direct sequencing to determine the exact nature of the variants. Early-onset sporadic PD cases were also screened for parkin mutations. The haplotypes associated with the G2019S mutation were determined. The clinical characteristics of patients carrying LRRK2 mutations were detailed. French Network for the Study of Parkinson Disease Genetics. Patients Three hundred twenty patients with apparently sporadic PD from Europe. Results of genetic analyses. We found the G2019S mutation in 6 patients and identified 2 new variants (Y2006H and T2031S) in 1 patient each. Their clinical features were similar to those of typical PD. All G2019S mutation carriers shared a common haplotype. The G2019S mutation is almost as frequent in sporadic cases (1.9%) as in previously reported familial cases (2.9%) in Europe and occurs in the same common founder. We identified 2 novel variants. Although the phenotype of LRRK2 mutation carriers closely resembles that of typical PD, the age at onset was younger (29 years in 1 patient) than previously described, and 3 patients were improved by deep brain stimulation.

  7. Analytical Performances of Human Immunodeficiency Virus Type 1 RNA-Based Amplix® Real-Time PCR Platform for HIV-1 RNA Quantification

    Directory of Open Access Journals (Sweden)

    Christian Diamant Mossoro-Kpinde

    2016-01-01

    Full Text Available Objectives. We evaluated the performances of Amplix real-time PCR platform developed by Biosynex (Strasbourg, France, combining automated station extraction (Amplix station 16 Dx and real-time PCR (Amplix NG, for quantifying plasma HIV-1 RNA by lyophilized HIV-1 RNA-based Amplix reagents targeting gag and LTR, using samples from HIV-1-infected adults from Central African Republic. Results. Amplix real-time PCR assay showed low limit of detection (28 copies/mL, across wide dynamic range (1.4–10 log copies/mL, 100% sensitivity and 99% specificity, high reproducibility, and accuracy with mean bias < 5%. The assay showed excellent correlations and concordance of 95.3% with the reference HIV-1 RNA load assay (Roche, with mean absolute bias of +0.097 log copies/mL by Bland-Altman analysis. The assay was able to detect and quantify the most prevalent HIV-1 subtype strains and the majority of non-B subtypes, CRFs of HIV-1 group M, and HIV-1 groups N and O circulating in Central Africa. The Amplix assay showed 100% sensitivity and 99.6% specificity to diagnose virological failure in clinical samples from antiretroviral drug-experienced patients. Conclusions. The HIV-1 RNA-based Amplix real-time PCR platform constitutes sensitive and reliable system for clinical monitoring of HIV-1 RNA load in HIV-1-infected children and adults, particularly adapted to intermediate laboratory facilities in sub-Saharan Africa.

  8. Identification of pathways controlling DNA damage induced mutation in Saccharomyces cerevisiae.

    Science.gov (United States)

    Lis, Ewa T; O'Neill, Bryan M; Gil-Lamaignere, Cristina; Chin, Jodie K; Romesberg, Floyd E

    2008-05-03

    Mutation in response to most types of DNA damage is thought to be mediated by the error-prone sub-branch of post-replication repair and the associated translesion synthesis polymerases. To further understand the mutagenic response to DNA damage, we screened a collection of 4848 haploid gene deletion strains of Saccharomyces cerevisiae for decreased damage-induced mutation of the CAN1 gene. Through extensive quantitative validation of the strains identified by the screen, we identified ten genes, which included error-prone post-replication repair genes known to be involved in induced mutation, as well as two additional genes, FYV6 and RNR4. We demonstrate that FYV6 and RNR4 are epistatic with respect to induced mutation, and that they function, at least partially, independently of post-replication repair. This pathway of induced mutation appears to be mediated by an increase in dNTP levels that facilitates lesion bypass by the replicative polymerase Pol delta, and it is as important as error-prone post-replication repair in the case of UV- and MMS-induced mutation, but solely responsible for EMS-induced mutation. We show that Rnr4/Pol delta-induced mutation is efficiently inhibited by hydroxyurea, a small molecule inhibitor of ribonucleotide reductase, suggesting that if similar pathways exist in human cells, intervention in some forms of mutation may be possible.

  9. Clinical and biological characteristics of cervical neoplasias with FGFR3 mutation

    Directory of Open Access Journals (Sweden)

    Thiery Jean

    2005-05-01

    Full Text Available Abstract Background We have previously reported activating mutations of the gene coding for the fibroblast growth factor receptor 3 (FGFR3 in invasive cervical carcinoma. To further analyze the role of FGFR3 in cervical tumor progression, we extended our study to screen a total of 75 invasive tumors and 80 cervical intraepithelial neoplasias (40 low-grade and 40 high-grade lesions. Results Using single strand conformation polymorphism (SSCP followed by DNA sequencing, we found FGFR3 mutation (S249C in all cases in 5% of invasive cervical carcinomas and no mutation in intraepithelial lesions. These results suggest that, unlike in bladder carcinoma, FGFR3 mutation does not or rarely occur in non invasive lesions. Compared to patients with wildtype FGFR3 tumor, patients with S249C FGFR3 mutated tumors were older (mean age 64 vs. 49.4 years, P = 0.02, and were more likely to be associated with a non-16/18 HPV type in their tumor. Gene expression analysis demonstrated that FGFR3 mutated tumors were associated with higher FGFR3b mRNA expression levels compared to wildtype FGFR3 tumors. Supervised analysis of Affymetrix expression data identified a significant number of genes specifically differentially expressed in tumors with respect to FGFR3 mutation status. Conclusion This study suggest that tumors with FGFR3 mutation appear to have distinctive clinical and biological characteristics that may help in defining a population of patients for FGFR3 mutation screening.

  10. Mutation detection in autosomal dominant Hirschsprung disease: SSCP analysis of the RET proto-oncogene

    Energy Technology Data Exchange (ETDEWEB)

    Angrist, M.; Bolk, S.; Chakravarti, A. [Case Western Reserve Univ., Cleveland, OH (United States)

    1994-09-01

    Hirschsprung disease (HSCR), or congenital aganglionic megacolon, is the most common cause of congenital bowel obstruction, with an incidence of 1 in 5000. Recently, linkage of an incompletely penetrant, dominant form of HSCR to the pericentromeric region of chromosome 10 was reported, followed by identification of mutations in the RET proto-oncogene in HSCR patients. RET mutations have also been reported in both sporadic and familial forms of three neuroendrocrine tumor syndromes. Unlike the clustered RET mutations observed in these syndromes, the 18 reported HSCR mutations are distributed throughout the extracellular and tryosine kinase domains of RET. In an effort to determine the frequency of RET mutations in HSCR and correlate genotype with phenotype, we have begun to screen for mutations among 80 HSCR probands representing a wide range of phenotypes and pedigree structures. Non-isotopic single strand conformation of polymorphism (SSCP) analysis was carried out using the Pharmacia PhastSystem{trademark}. Initial screening of exons 2 through 6 detected variants in 11 patients not seen in 24 controls. One additional band shift in exon 6 has been observed in both patients and controls. Preliminary sequence analysis has revealed two putative familial mutations in exon 2: a single base pair deletion (49Pro del C 296) and a point mutation that leads to a conservative amino acid substitution (93Gly{r_arrow}Ser). These results suggest that HSCR may be associated with a range of alterations in the coding sequence of the RET extracellular domain. Additional mutations will be described.

  11. KRAS mutational concordance between primary and metastatic colorectal adenocarcinoma

    Science.gov (United States)

    PALIOGIANNIS, PANAGIOTIS; COSSU, ANTONIO; TANDA, FRANCESCO; PALMIERI, GIUSEPPE; PALOMBA, GRAZIA

    2014-01-01

    KRAS mutation analysis is commonly performed on tissue samples obtained from primary colorectal cancers (CRCs). The metastatic lesions of CRC are usually considered as qualitatively similar or even identical to the primary tumors. The aim of this study was to evaluate the spectrum and distribution of KRAS mutations in a large collection of CRCs, while also evaluating the concordance of primary and metastatic lesions among available paired specimens from the same patients. A total of 729 patients with histologically confirmed advanced CRC at the University Hospital and Local Health Unit (Sassari, Italy) were included. Clinical and pathological features were obtained from medical records and/or pathology reports. Formalin-fixed, paraffin-embedded tissue samples were used for mutation analysis. Genomic DNA was isolated using a standard protocol; the coding sequence and splice junctions of exons 2 and 3 in the KRAS gene were screened by direct automated sequencing. Overall, 219 (30%) KRAS mutations were found; 208 (30.1%) were identified in the 690 primary tumors and 11 (28.2%) in the 39 metastatic tissue samples. Among the 31 (4.3%) patients who had paired samples of primary CRC and synchronous or asynchronous metastases, 28 (90.3%) showed consistent mutation patterns between the primary tumors and metastatic lesions. In one case, an additive mutation (Q61L) was found in the metastatic tissue, while two other discrepant cases exhibited a different mutation distribution; Q61H in the primitive lesion and G13V in the metastatic lesion in one case, and a mutated primary tumor (Q61L) and wild-type metastasis in another case. The results of this study confirm that a high concordance exists between the results of KRAS mutation analysis performed in primitive and metastatic CRCs; independent subclones may be generated in a limited amount of patients. PMID:25202344

  12. Mutations of PIK3CA in gastric adenocarcinoma

    Directory of Open Access Journals (Sweden)

    So Samuel

    2005-03-01

    Full Text Available Abstract Background Activation of the phosphatidylinositol 3-kinase (PI3K through mutational inactivation of PTEN tumour suppressor gene is common in diverse cancer types, but rarely reported in gastric cancer. Recently, mutations in PIK3CA, which encodes the p110α catalytic subunit of PI3K, have been identified in various human cancers, including 3 of 12 gastric cancers. Eighty percent of these reported mutations clustered within 2 regions involving the helical and kinase domains. In vitro study on one of the "hot-spot" mutants has demonstrated it as an activating mutation. Methods Based on these data, we initiated PIK3CA mutation screening in 94 human gastric cancers by direct sequencing of the gene regions in which 80% of all the known PIK3CA mutations were found. We also examined PIK3CA expression level by extracting data from the previous large-scale gene expression profiling study. Using Significance Analysis of Microarrays (SAM, we further searched for genes that show correlating expression with PIK3CA. Results We have identified PIK3CA mutations in 4 cases (4.3%, all involving the previously reported hotspots. Among these 4 cases, 3 tumours demonstrated microsatellite instability and 2 tumours harboured concurrent KRAS mutation. Data extracted from microarray studies showed an increased expression of PIK3CA in gastric cancers when compared with the non-neoplastic gastric mucosae (p PIK3CA. Conclusion Our data suggested that activation of the PI3K signalling pathway in gastric cancer may be achieved through up-regulation or mutation of PIK3CA, in which the latter may be a consequence of mismatch repair deficiency.

  13. A dominant mutation in hexokinase 1 (HK1) causes retinitis pigmentosa.

    Science.gov (United States)

    Sullivan, Lori S; Koboldt, Daniel C; Bowne, Sara J; Lang, Steven; Blanton, Susan H; Cadena, Elizabeth; Avery, Cheryl E; Lewis, Richard A; Webb-Jones, Kaylie; Wheaton, Dianna H; Birch, David G; Coussa, Razck; Ren, Huanan; Lopez, Irma; Chakarova, Christina; Koenekoop, Robert K; Garcia, Charles A; Fulton, Robert S; Wilson, Richard K; Weinstock, George M; Daiger, Stephen P

    2014-09-04

    To identify the cause of retinitis pigmentosa (RP) in UTAD003, a large, six-generation Louisiana family with autosomal dominant retinitis pigmentosa (adRP). A series of strategies, including candidate gene screening, linkage exclusion, genome-wide linkage mapping, and whole-exome next-generation sequencing, was used to identify a mutation in a novel disease gene on chromosome 10q22.1. Probands from an additional 404 retinal degeneration families were subsequently screened for mutations in this gene. Exome sequencing in UTAD003 led to identification of a single, novel coding variant (c.2539G>A, p.Glu847Lys) in hexokinase 1 (HK1) present in all affected individuals and absent from normal controls. One affected family member carries two copies of the mutation and has an unusually severe form of disease, consistent with homozygosity for this mutation. Screening of additional adRP probands identified four other families (American, Canadian, and Sicilian) with the same mutation and a similar range of phenotypes. The families share a rare 450-kilobase haplotype containing the mutation, suggesting a founder mutation among otherwise unrelated families. We identified an HK1 mutation in five adRP families. Hexokinase 1 catalyzes phosphorylation of glucose to glucose-6-phosphate. HK1 is expressed in retina, with two abundant isoforms expressed at similar levels. The Glu847Lys mutation is located at a highly conserved position in the protein, outside the catalytic domains. We hypothesize that the effect of this mutation is limited to the retina, as no systemic abnormalities in glycolysis were detected. Prevalence of the HK1 mutation in our cohort of RP families is 1%. Copyright 2014 The Association for Research in Vision and Ophthalmology, Inc.

  14. Potassium channel gene mutations rarely cause atrial fibrillation

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    Nam Edwin G

    2006-08-01

    Full Text Available Abstract Background Mutations in several potassium channel subunits have been associated with rare forms of atrial fibrillation. In order to explore the role of potassium channels in inherited typical forms of the arrhythmia, we have screened a cohort of patients from a referral clinic for mutations in the channel subunit genes implicated in the arrhythmia. We sought to determine if mutations in KCNJ2 and KCNE1-5 are a common cause of atrial fibrillation. Methods Serial patients with lone atrial fibrillation or atrial fibrillation with hypertension were enrolled between June 1, 2001 and January 6, 2005. Each patient underwent a standardized interview and physical examination. An electrocardiogram, echocardiogram and blood sample for genetic analysis were also obtained. Patients with a family history of AF were screened for mutations in KCNJ2 and KCNE1-5 using automated sequencing. Results 96 patients with familial atrial fibrillation were enrolled. Eighty-three patients had lone atrial fibrillation and 13 had atrial fibrillation and hypertension. Patients had a mean age of 56 years at enrollment and 46 years at onset of atrial fibrillation. Eighty-one percent of patients had paroxysmal atrial fibrillation at enrollment. Unlike patients with an activating mutation in KCNQ1, the patients had a normal QTc interval with a mean of 412 ± 42 ms. Echocardiography revealed a normal mean ejection fraction of 62.0 ± 7.2 % and mean left atrial dimension of 39.9 ± 7.0 mm. A number of common polymorphisms in KCNJ2 and KCNE1-5 were identified, but no mutations were detected. Conclusion Mutations in KCNJ2 and KCNE1-5 rarely cause typical atrial fibrillation in a referral clinic population.

  15. Prenatal screening and genetics

    DEFF Research Database (Denmark)

    Alderson, P; Aro, A R; Dragonas, T

    2001-01-01

    Although the term 'genetic screening' has been used for decades, this paper discusses how, in its most precise meaning, genetic screening has not yet been widely introduced. 'Prenatal screening' is often confused with 'genetic screening'. As we show, these terms have different meanings, and we...... examine definitions of the relevant concepts in order to illustrate this point. The concepts are i) prenatal, ii) genetic screening, iii) screening, scanning and testing, iv) maternal and foetal tests, v) test techniques and vi) genetic conditions. So far, prenatal screening has little connection...... with precisely defined genetics. There are benefits but also disadvantages in overstating current links between them in the term genetic screening. Policy making and professional and public understandings about screening could be clarified if the distinct meanings of prenatal screening and genetic screening were...

  16. Kin Selection - Mutation Balance

    DEFF Research Database (Denmark)

    Dyken, J. David Van; Linksvayer, Timothy Arnold; Wade, Michael J.

    2011-01-01

    Abstract Social conflict, in the form of intraspecific selfish "cheating" has been observed in a number of natural systems. However, a formal, evolutionary genetic theory of social cheating that provides an explanatory, predictive framework for these observations is lacking. Here we derive the kin...... selection-mutation balance, which provides an evolutionary null hypothesis for the statics and dynamics of cheating. When social interactions have linear fitness effects and Hamilton´s rule is satisfied, selection is never strong enough to eliminate recurrent cheater mutants from a population, but cheater...... lineages are transient and do not invade. Instead, cheating lineages are eliminated by kin selection but are constantly reintroduced by mutation, maintaining a stable equilibrium frequency of cheaters. The presence of cheaters at equilibrium creates a "cheater load" that selects for mechanisms of cheater...

  17. Peripheral lung adenocarcinomas with KRAS mutations are more likely to invade visceral pleura.

    Science.gov (United States)

    Raparia, Kirtee; Villa, Celina; Raj, Rishi; Cagle, Philip T

    2015-02-01

    Kirsten-RAS (KRAS) mutations play an important role in the carcinogenesis of a subset of lung adenocarcinomas and are associated with poorer prognosis. To investigate the relationship of KRAS mutation status to the histologic subtype of adenocarcinoma according to the recent classification, patient demographics, tumor size, predominant histologic subtype, nodal status, and visceral pleural invasion, in an attempt to uncover the reason for the worse prognosis associated with KRAS mutation. A total of 187 consecutive resected lung adenocarcinomas from our institution from 2008 to 2011 that were diagnosed according to the new International Association for the Study of Lung Cancer/American Thoracic Society/European Respiratory Society classification and screened for KRAS mutations were included in the study. A total of 32% of the adenocarcinomas harbored the KRAS mutation. The median age in the KRAS mutation group was 69 years (range, 43-86 years), and male to female ratio was 1:2.3. The proportion of heavy smokers was significantly higher in tumors with KRAS mutation compared with wild type (83% versus 62%; P = .01). A total of 27% of tumors with KRAS mutation had pleural invasion versus 11% of tumors without KRAS mutation (P = .009). A total of 59 tumor samples were positive for KRAS mutation (25 for G12C, 14 for G12A, 8 for G12V, 7 for G12D, 3 for G12S, and 1 for G12T), and only 3 tumors harbored codon 13 mutations (G13C). Two tumors had double mutations. KRAS mutations are more common in heavy smokers, and lung adenocarcinomas with KRAS mutation are more likely to invade the visceral pleura. Increased frequency of visceral pleural invasion may explain in part the worse prognosis associated with KRAS mutations.

  18. Adverse events in families with hypertrophic or dilated cardiomyopathy and mutations in the MYBPC3 gene

    Directory of Open Access Journals (Sweden)

    Lehrke Stephanie

    2008-10-01

    Full Text Available Abstract Background Mutations in MYBPC3 encoding myosin binding protein C belong to the most frequent causes of hypertrophic cardiomyopathy (HCM and may also lead to dilated cardiomyopathy (DCM. MYBPC3 mutations initially were considered to cause a benign form of HCM. The aim of this study was to examine the clinical outcome of patients and their relatives with 18 different MYBPC3 mutations. Methods 87 patients with HCM and 71 patients with DCM were screened for MYBPC3 mutations by denaturing gradient gel electrophoresis and sequencing. Close relatives of mutation carriers were genotyped for the respective mutation. Relatives with mutation were then evaluated by echocardiography and magnetic resonance imaging. A detailed family history regarding adverse clinical events was recorded. Results In 16 HCM (18.4% and two DCM (2.8% index patients a mutation was detected. Seven mutations were novel. Mutation carriers exhibited no additional mutations in genes MYH7, TNNT2, TNNI3, ACTC and TPM1. Including relatives of twelve families, a total number of 42 mutation carriers was identified of which eleven (26.2% had at least one adverse event. Considering the twelve families and six single patients with mutations, 45 individuals with cardiomyopathy and nine with borderline phenotype were identified. Among the 45 patients, 23 (51.1% suffered from an adverse event. In eleven patients of seven families an unexplained sudden death was reported at the age between 13 and 67 years. Stroke or a transient ischemic attack occurred in six patients of five families. At least one adverse event occurred in eleven of twelve families. Conclusion MYBPC3 mutations can be associated with cardiac events such as progressive heart failure, stroke and sudden death even at younger age. Therefore, patients with MYBPC3 mutations require thorough clinical risk assessment.

  19. Identification of two point mutations and a one base deletion in exon 19 of the dystrophin gene by heteroduplex formation.

    Science.gov (United States)

    Prior, T W; Papp, A C; Snyder, P J; Burghes, A H; Sedra, M S; Western, L M; Bartello, C; Mendell, J R

    1993-03-01

    Two thirds of the Duchenne muscular dystrophy population have either gene deletions or duplications. The nondeletion/duplication cases are most likely the result of point mutations or small deletions and duplications that cannot be easily identified by current strategies. The major obstacle in identifying small mutations is due to the large size of the dystrophin gene. We selectively screened 5 DMD exons containing CpG dinucleotides in 110 DMD patients without detectable deletions or duplications. Nonsenses mutations are frequently due to a C- to -T transition within a CG dinucleotide pair. To screen for the nonsense mutations, we used the heteroduplex method. Utilizing this approach, we identified 2 different nonsense mutations and a single base deletion all occurring in exon 19. This is the first report of a clustering of small mutations in the dystrophin gene.

  20. Investigation of CYP21A2 mutations in Turkish patients with 21-hydroxylase deficiency and a novel founder mutation.

    Science.gov (United States)

    Toraman, Bayram; Ökten, Ayşenur; Kalay, Ersan; Karagüzel, Gülay; Dinçer, Tuba; Açıkgöz, Emel Gül; Karagüzel, Ahmet

    2013-01-15

    Congenital adrenal hyperplasia (CAH) is a group of autosomal recessively inherited disorders characterized by impaired production of adrenal steroids. Approximately 95% of all CAH are caused by mutations of the CYP21A2 that encodes 21-hydroxylase. In this study, mutation analyses of CYP21A2 were performed in 48 CAH patients from 45 Turkish families with the clinical diagnosis of 21-hydroxylase deficiency (21OHD). While in 39 (86.7%) of 21OHD patients, disease causing CYP21A2 mutations were identified in both alleles, in two 21OHD patients CYP21A2 mutations were identified only in one allele. In four patients, mutation was not detected at all. In total, seventeen known and one novel, disease causing CYP21A2 mutations were observed. Among identified mutations, previously described c.293-13C/A>G, large rearrangements and p.Q319X mutations were the most common mutations accounting for 33.3%, 14.4% and 12.2% of all evaluated chromosomes, respectively. In six families (13.3%) a novel founder mutation, c.2T>C (p.M1?), inactivating the translation initiation codon was found. This mutation is not present in pseudogene CYP21A1P and causes the classical form of the disease in six patients. In addition, depending on the nature of the rearrangements CYP21A1P/CYP21A2 chimeras were further classified as CH(c/d), and CH-1(c) was shown to be the most prominent chimera in our study group. In conclusion, with this study we identified a novel founder CYP21A2 mutation and suggest a further classification for CYP21A1P/CYP21A2 chimeras depending on the combination of junction site position and whether it is occurred as a result of deletion or conversion. Absence of disease causing mutation of CYP21A2 in ten of screened ninety chromosomes suggests the contribution of regulatory elements in occurrences of CAH due to the 21OHD. Copyright © 2012 Elsevier B.V. All rights reserved.

  1. Septin mutations in human cancers

    Directory of Open Access Journals (Sweden)

    Elias T Spiliotis

    2016-11-01

    Full Text Available Septins are GTP-binding proteins that are evolutionarily and structurally related to the RAS oncogenes. Septin expression levels are altered in many cancers and new advances point to how abnormal septin expression may contribute to the progression of cancer. In contrast to the RAS GTPases, which are frequently mutated and actively promote tumorigenesis, little is known about the occurrence and role of septin mutations in human cancers. Here, we review septin missense mutations that are currently in the Catalog of Somatic Mutations in Cancer (COSMIC database. The majority of septin mutations occur in tumors of the large intestine, skin, endometrium and stomach. Over 25% of the annotated mutations in SEPT2, SEPT4 and SEPT9 belong to large intestine tumors. From all septins, SEPT9 and SEPT14 exhibit the highest mutation frequencies in skin, stomach and large intestine cancers. While septin mutations occur with frequencies lower than 3%, recurring mutations in several invariant and highly conserved amino acids are found across different septin paralogs and tumor types. Interestingly, a significant number of these mutations occur in the GTP-binding pocket and septin dimerization interfaces. Future studies may determine how these somatic mutations affect septin structure and function, whether they contribute to the progression of specific cancers and if they could serve as tumor-specific biomarkers.

  2. The first USH2A mutation analysis of Japanese autosomal recessive retinitis pigmentosa patients: a totally different mutation profile with the lack of frequent mutations found in Caucasian patients.

    Science.gov (United States)

    Zhao, Yang; Hosono, Katsuhiro; Suto, Kimiko; Ishigami, Chie; Arai, Yuuki; Hikoya, Akiko; Hirami, Yasuhiko; Ohtsubo, Masafumi; Ueno, Shinji; Terasaki, Hiroko; Sato, Miho; Nakanishi, Hiroshi; Endo, Shiori; Mizuta, Kunihiro; Mineta, Hiroyuki; Kondo, Mineo; Takahashi, Masayo; Minoshima, Shinsei; Hotta, Yoshihiro

    2014-09-01

    Retinitis pigmentosa (RP) is a highly heterogeneous genetic disease. The USH2A gene, which accounts for approximately 74-90% of Usher syndrome type 2 (USH2) cases, is also one of the major autosomal recessive RP (arRP) causative genes among Caucasian populations. To identify disease-causing USH2A gene mutations in Japanese RP patients, all 73 exons were screened for mutations by direct sequencing. In total, 100 unrelated Japanese RP patients with no systemic manifestations were identified, excluding families with obvious autosomal dominant inheritance. Of these 100 patients, 82 were included in this present study after 18 RP patients with very likely pathogenic EYS (eyes shut homolog) mutations were excluded. The mutation analysis of the USH2A revealed five very likely pathogenic mutations in four patients. A patient had only one very likely pathogenic mutation and the others had two of them. Caucasian frequent mutations p.C759F in arRP and p.E767fs in USH2 were not found. All the four patients exhibited typical clinical features of RP. The observed prevalence of USH2A gene mutations was approximately 4% among Japanese arRP patients, and the profile of the USH2A gene mutations differed largely between Japanese patients and previously reported Caucasian populations.

  3. 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.

  4. High frequency of RPL22 mutations in microsatellite-unstable colorectal and endometrial tumors.

    Science.gov (United States)

    Ferreira, Ana M; Tuominen, Iina; van Dijk-Bos, Krista; Sanjabi, Bahram; van der Sluis, Tineke; van der Zee, Ate G; Hollema, Harry; Zazula, Monika; Sijmons, Rolf H; Aaltonen, Lauri A; Westers, Helga; Hofstra, Robert M W

    2014-12-01

    Ribosomal Protein L22 (RPL22) encodes a protein that is a component of the 60S subunit of the ribosome. Variants in this gene have recently been linked to cancer development. Mutations in an A8 repeat in exon 2 were found in a recent study in 52% of microsatellite-unstable endometrial tumors. These tumors are particularly prone to mutations in repeats due to mismatch repair deficiency. We screened this coding repeat in our collection of microsatellite-unstable endometrial tumors (EC) and colorectal tumors (CRC). We found 50% mutation frequency for EC and 77% mutation frequency for CRC. These results confirm the previous study on the involvement of RPL22 in EC and, more importantly, reports for the first time such high mutation frequency in this gene in colorectal cancer. Furthermore, considering the high mutation frequency found, our data point toward an important role for RPL22 in microsatellite instability carcinogenesis. © 2014 WILEY PERIODICALS, INC.

  5. ELOVL5 mutations cause spinocerebellar ataxia 38.

    Science.gov (United States)

    Di Gregorio, Eleonora; Borroni, Barbara; Giorgio, Elisa; Lacerenza, Daniela; Ferrero, Marta; Lo Buono, Nicola; Ragusa, Neftj; Mancini, Cecilia; Gaussen, Marion; Calcia, Alessandro; Mitro, Nico; Hoxha, Eriola; Mura, Isabella; Coviello, Domenico A; Moon, Young-Ah; Tesson, Christelle; Vaula, Giovanna; Couarch, Philippe; Orsi, Laura; Duregon, Eleonora; Papotti, Mauro Giulio; Deleuze, Jean-François; Imbert, Jean; Costanzi, Chiara; Padovani, Alessandro; Giunti, Paola; Maillet-Vioud, Marcel; Durr, Alexandra; Brice, Alexis; Tempia, Filippo; Funaro, Ada; Boccone, Loredana; Caruso, Donatella; Stevanin, Giovanni; Brusco, Alfredo

    2014-08-07

    Spinocerebellar ataxias (SCAs) are a heterogeneous group of autosomal-dominant neurodegenerative disorders involving the cerebellum and 23 different genes. We mapped SCA38 to a 56 Mb region on chromosome 6p in a SCA-affected Italian family by whole-genome linkage analysis. Targeted resequencing identified a single missense mutation (c.689G>T [p.Gly230Val]) in ELOVL5. Mutation screening of 456 independent SCA-affected individuals identified the same mutation in two further unrelated Italian families. Haplotyping showed that at least two of the three families shared a common ancestor. One further missense variant (c.214C>G [p.Leu72Val]) was found in a French family. Both missense changes affect conserved amino acids, are predicted to be damaging by multiple bioinformatics tools, and were not identified in ethnically matched controls or within variant databases. ELOVL5 encodes an elongase involved in the synthesis of polyunsaturated fatty acids of the ω3 and ω6 series. Arachidonic acid and docosahexaenoic acid, two final products of the enzyme, were reduced in the serum of affected individuals. Immunohistochemistry on control mice and human brain demonstrated high levels in Purkinje cells. In transfection experiments, subcellular localization of altered ELOVL5 showed a perinuclear distribution with a signal increase in the Golgi compartment, whereas the wild-type showed a widespread signal in the endoplasmic reticulum. SCA38 and SCA34 are examples of SCAs due to mutations in elongase-encoding genes, emphasizing the importance of fatty-acid metabolism in neurological diseases. Copyright © 2014 The American Society of Human Genetics. Published by Elsevier Inc. All rights reserved.

  6. PDCD10 gene mutations in multiple cerebral cavernous malformations.

    Science.gov (United States)

    Cigoli, Maria Sole; Avemaria, Francesca; De Benedetti, Stefano; Gesu, Giovanni P; Accorsi, Lucio Giordano; Parmigiani, Stefano; Corona, Maria Franca; Capra, Valeria; Mosca, Andrea; Giovannini, Simona; Notturno, Francesca; Ciccocioppo, Fausta; Volpi, Lilia; Estienne, Margherita; De Michele, Giuseppe; Antenora, Antonella; Bilo, Leda; Tavoni, Antonietta; Zamponi, Nelia; Alfei, Enrico; Baranello, Giovanni; Riva, Daria; Penco, Silvana

    2014-01-01

    Cerebral cavernous malformations (CCMs) are vascular abnormalities that may cause seizures, intracerebral haemorrhages, and focal neurological deficits. Familial form shows an autosomal dominant pattern of inheritance with incomplete penetrance and variable clinical expression. Three genes have been identified causing familial CCM: KRIT1/CCM1, MGC4607/CCM2, and PDCD10/CCM3. Aim of this study is to report additional PDCD10/CCM3 families poorly described so far which account for 10-15% of hereditary cerebral cavernous malformations. Our group investigated 87 consecutive Italian affected individuals (i.e. positive Magnetic Resonance Imaging) with multiple/familial CCM through direct sequencing and Multiplex Ligation-Dependent Probe Amplification (MLPA) analysis. We identified mutations in over 97.7% of cases, and PDCD10/CCM3 accounts for 13.1%. PDCD10/CCM3 molecular screening revealed four already known mutations and four novel ones. The mutated patients show an earlier onset of clinical manifestations as compared to CCM1/CCM2 mutated patients. The study of further families carrying mutations in PDCD10/CCM3 may help define a possible correlation between genotype and phenotype; an accurate clinical follow up of the subjects would help define more precisely whether mutations in PDCD10/CCM3 lead to a characteristic phenotype.

  7. PDCD10 gene mutations in multiple cerebral cavernous malformations.

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    Maria Sole Cigoli

    Full Text Available Cerebral cavernous malformations (CCMs are vascular abnormalities that may cause seizures, intracerebral haemorrhages, and focal neurological deficits. Familial form shows an autosomal dominant pattern of inheritance with incomplete penetrance and variable clinical expression. Three genes have been identified causing familial CCM: KRIT1/CCM1, MGC4607/CCM2, and PDCD10/CCM3. Aim of this study is to report additional PDCD10/CCM3 families poorly described so far which account for 10-15% of hereditary cerebral cavernous malformations. Our group investigated 87 consecutive Italian affected individuals (i.e. positive Magnetic Resonance Imaging with multiple/familial CCM through direct sequencing and Multiplex Ligation-Dependent Probe Amplification (MLPA analysis. We identified mutations in over 97.7% of cases, and PDCD10/CCM3 accounts for 13.1%. PDCD10/CCM3 molecular screening revealed four already known mutations and four novel ones. The mutated patients show an earlier onset of clinical manifestations as compared to CCM1/CCM2 mutated patients. The study of further families carrying mutations in PDCD10/CCM3 may help define a possible correlation between genotype and phenotype; an accurate clinical follow up of the subjects would help define more precisely whether mutations in PDCD10/CCM3 lead to a characteristic phenotype.

  8. GJA12 mutations in children with recessive hypomyelinating leukoencephalopathy.

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    Bugiani, M; Al Shahwan, S; Lamantea, E; Bizzi, A; Bakhsh, E; Moroni, I; Balestrini, M R; Uziel, G; Zeviani, M

    2006-07-25

    Pelizaeus-Merzbacher-like disease (PMLD) is an inherited hypomyelinating leukoencephalopathy with onset in early infancy. Like Pelizaeus-Merzbacher disease (PMD), PMLD is characterized clinically by nystagmus, cerebellar ataxia, and spasticity, due to a permanent lack of myelin deposition in the brain. Mutations in the GJA12 gene, encoding connexin 47 (Cx47), were recently reported in five children with autosomal recessive PMLD. To evaluate the impact of mutations in the GJA12 gene in, and define the clinical and neuroimaging features of, autosomal recessive PMLD. The authors screened for GJA12 mutations in 10 additional PMLD families originating from Italy, Pakistan, and Saudi Arabia. Three novel homozygous GJA12 mutations were identified in 12 mutant cases distributed in 3 of 10 families. The mutations segregated with the disease according to an autosomal recessive trait and included one missense (G236S) and two nonsense (L281fs285X and P131fs144X) changes. The identification of homozygous mutations predicting the synthesis of aberrant and truncated polypeptides, and their tight segregation with the disease in very large families, clearly demonstrate that the loss of Cx47 function is the cause of the disease. The phenotype of GJA12-related Pelizaeus-Merzbacher-like disease is fairly homogeneous and similar to that of Pelizaeus-Merzbacher disease. However, slower progression of symptoms, greater preservation of cognitive functions, and partial myelination of corticospinal tracts at MRI were distinctive features, which could help in the differential diagnosis.

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

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    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.

  10. Mutations in genes involved in nonsense mediated decay ameliorate the phenotype of sel-12 mutants with amber stop mutations in Caenorhabditis elegans

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    Aubert Sylvie

    2009-03-01

    Full Text Available Abstract Background Presenilin proteins are part of a complex of proteins that can cleave many type I transmembrane proteins, including Notch Receptors and the Amyloid Precursor Protein, in the middle of the transmembrane domain. Dominant mutations in the human presenilin genes PS1 and PS2 lead to Familial Alzheimer's disease. Mutations in the Caenorhabditis elegans sel-12 presenilin gene cause a highly penetrant egg-laying defect due to reduction of signalling through the lin-12/Notch receptor. Mutations in six spr genes (for suppressor of presenilin are known to strongly suppress sel-12. Mutations in most strong spr genes suppress sel-12 by de-repressing the transcription of the largely functionally equivalent hop-1 presenilin gene. However, how mutations in the spr-2 gene suppress sel-12 is unknown. Results We show that spr-2 mutations increase the levels of sel-12 transcripts with Premature translation Termination Codons (PTCs in embryos and L1 larvae. mRNA transcripts from sel-12 alleles with PTCs undergo degradation by a process known as Nonsense Mediated Decay (NMD. However, spr-2 mutations do not appear to affect NMD. Mutations in the smg genes, which are required for NMD, can restore sel-12(PTC transcript levels and ameliorate the phenotype of sel-12 mutants with amber PTCs. However, the phenotypic suppression of sel-12 by smg genes is nowhere near as strong as the effect of previously characterized spr mutations including spr-2. Consistent with this, we have identified only two mutations in smg genes among the more than 100 spr mutations recovered in genetic screens. Conclusion spr-2 mutations do not suppress sel-12 by affecting NMD of sel-12(PTC transcripts and appear to have a novel mechanism of suppression. The fact that mutations in smg genes can ameliorate the phenotype of sel-12 alleles with amber PTCs suggests that some read-through of sel-12(amber alleles occurs in smg backgrounds.

  11. Mutations in genes involved in nonsense mediated decay ameliorate the phenotype of sel-12 mutants with amber stop mutations in Caenorhabditis elegans.

    Science.gov (United States)

    Gontijo, Alisson M; Aubert, Sylvie; Roelens, Ingele; Lakowski, Bernard

    2009-03-20

    Presenilin proteins are part of a complex of proteins that can cleave many type I transmembrane proteins, including Notch Receptors and the Amyloid Precursor Protein, in the middle of the transmembrane domain. Dominant mutations in the human presenilin genes PS1 and PS2 lead to Familial Alzheimer's disease. Mutations in the Caenorhabditis elegans sel-12 presenilin gene cause a highly penetrant egg-laying defect due to reduction of signalling through the lin-12/Notch receptor. Mutations in six spr genes (for suppressor of presenilin) are known to strongly suppress sel-12. Mutations in most strong spr genes suppress sel-12 by de-repressing the transcription of the largely functionally equivalent hop-1 presenilin gene. However, how mutations in the spr-2 gene suppress sel-12 is unknown. We show that spr-2 mutations increase the levels of sel-12 transcripts with Premature translation Termination Codons (PTCs) in embryos and L1 larvae. mRNA transcripts from sel-12 alleles with PTCs undergo degradation by a process known as Nonsense Mediated Decay (NMD). However, spr-2 mutations do not appear to affect NMD. Mutations in the smg genes, which are required for NMD, can restore sel-12(PTC) transcript levels and ameliorate the phenotype of sel-12 mutants with amber PTCs. However, the phenotypic suppression of sel-12 by smg genes is nowhere near as strong as the effect of previously characterized spr mutations including spr-2. Consistent with this, we have identified only two mutations in smg genes among the more than 100 spr mutations recovered in genetic screens. spr-2 mutations do not suppress sel-12 by affecting NMD of sel-12(PTC) transcripts and appear to have a novel mechanism of suppression. The fact that mutations in smg genes can ameliorate the phenotype of sel-12 alleles with amber PTCs suggests that some read-through of sel-12(amber) alleles occurs in smg backgrounds.

  12. 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.

  13. Dealing with the unexpected: consumer responses to direct-access BRCA mutation testing.<