Simulating Phase Variation: A Practical Approach to Teaching Mutation and Diversity

Science.gov (United States)

Wanford, Joe; Aidley, Jack; Bayliss, Chris; Ketley, Julian; Goodwin, Mark

2018-01-01

Mutation, diversity, natural selection and the biology of human pathogens (including antibiotic resistance) are key features of the biosciences curriculum at A Level and undergraduate study. Few resources exist to allow students to engage with these topics in an interactive manner. This paper describes an interactive, online simulation of mutation…

Diversity-Guided Evolutionary Algorithms

DEFF Research Database (Denmark)

Ursem, Rasmus Kjær

2002-01-01

Population diversity is undoubtably a key issue in the performance of evolutionary algorithms. A common hypothesis is that high diversity is important to avoid premature convergence and to escape local optima. Various diversity measures have been used to analyze algorithms, but so far few...... algorithms have used a measure to guide the search. The diversity-guided evolutionary algorithm (DGEA) uses the wellknown distance-to-average-point measure to alternate between phases of exploration (mutation) and phases of exploitation (recombination and selection). The DGEA showed remarkable results...

Global Rebalancing of Cellular Resources by Pleiotropic Point Mutations Illustrates a Multi-scale Mechanism of Adaptive Evolution

DEFF Research Database (Denmark)

Utrilla, José; O'Brien, Edward J.; Chen, Ke

2016-01-01

Pleiotropic regulatory mutations affect diverse cellular processes, posing a challenge to our understanding of genotype-phenotype relationships across multiple biological scales. Adaptive laboratory evolution (ALE) allows for such mutations to be found and characterized in the context of clear se...

Change of point mutations in Helicobacter pylori rRNA associated with clarithromycin resistance in Italy.

Science.gov (United States)

De Francesco, Vincenzo; Zullo, Angelo; Giorgio, Floriana; Saracino, Ilaria; Zaccaro, Cristina; Hassan, Cesare; Ierardi, Enzo; Di Leo, Alfredo; Fiorini, Giulia; Castelli, Valentina; Lo Re, Giovanna; Vaira, Dino

2014-03-01

Primary clarithromycin resistance is the main factor affecting the efficacy of Helicobacter pylori therapy. This study aimed: (i) to assess the concordance between phenotypic (culture) and genotypic (real-time PCR) tests in resistant strains; (ii) to search, in the case of disagreement between the methods, for point mutations other than those reported as the most frequent in Europe; and (iii) to compare the MICs associated with the single point mutations. In order to perform real-time PCR, we retrieved biopsies from patients in whom H. pylori infection was successful diagnosed by bacterial culture and clarithromycin resistance was assessed using the Etest. Only patients who had never been previously treated, and with H. pylori strains that were either resistant exclusively to clarithromycin or without any resistance, were included. Biopsies from 82 infected patients were analysed, including 42 strains that were clarithromycin resistant and 40 that were clarithromycin susceptible on culture. On genotypic analysis, at least one of the three most frequently reported point mutations (A2142C, A2142G and A2143G) was detected in only 23 cases (54.8%), with a concordance between the two methods of 0.67. Novel point mutations (A2115G, G2141A and A2144T) were detected in a further 14 out of 19 discordant cases, increasing the resistance detection rate of PCR to 88% (Presistance; and (iii) none of the tested point mutations is associated with significantly higher MIC values than the others.

Efficient Generation of Orthologous Point Mutations in Pigs via CRISPR-assisted ssODN-mediated Homology-directed Repair

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Kankan Wang

2016-01-01

Full Text Available Precise genome editing in livestock is of great value for the fundamental investigation of disease modeling. However, genetically modified pigs carrying subtle point mutations were still seldom reported despite the rapid development of programmable endonucleases. Here, we attempt to investigate single-stranded oligonucleotides (ssODN mediated knockin by introducing two orthologous pathogenic mutations, p.E693G for Alzheimer's disease and p.G2019S for Parkinson's disease, into porcine APP and LRRK2 loci, respectively. Desirable homology-directed repair (HDR efficiency was achieved in porcine fetal fibroblasts (PFFs by optimizing the dosage and length of ssODN templates. Interestingly, incomplete HDR alleles harboring partial point mutations were observed in single-cell colonies, which indicate the complex mechanism of ssODN-mediated HDR. The effect of mutation-to-cut distance on incorporation rate was further analyzed by deep sequencing. We demonstrated that a mutation-to-cut distance of 11 bp resulted in a remarkable difference in HDR efficiency between two point mutations. Finally, we successfully obtained one cloned piglet harboring the orthologous p.C313Y mutation at the MSTN locus via somatic cell nuclear transfer (SCNT. Our proof-of-concept study demonstrated efficient ssODN-mediated incorporation of pathogenic point mutations in porcine somatic cells, thus facilitating further development of disease modeling and genetic breeding in pigs.

Comparison of poliovirus recombinants: accumulation of point mutations provides further advantages.

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Savolainen-Kopra, Carita; Samoilovich, Elena; Kahelin, Heidi; Hiekka, Anna-Kaisa; Hovi, Tapani; Roivainen, Merja

2009-08-01

The roles of recombination and accumulation of point mutations in the origin of new poliovirus (PV) characteristics have been hypothesized, but it is not known which are essential to evolution. We studied phenotypic differences between recombinant PV strains isolated from successive stool specimens of an oral PV vaccine recipient. The studied strains included three PV2/PV1 recombinants with increasing numbers of mutations in the VP1 gene, two of the three with an amino acid change I-->T in the DE-loop of VP1, their putative PV1 parent and strains Sabin 1 and 2. Growth of these viruses was examined in three cell lines: colorectal adenocarcinoma, neuroblastoma and HeLa. The main observation was a higher growth rate between 4 and 6 h post-infection of the two recombinants with the I-->T substitution. All recombinants grew at a higher rate than parental strains in the exponential phase of the replication cycle. In a temperature sensitivity test, the I-->T-substituted recombinants replicated equally well at an elevated temperature. Complete genome sequencing of the three recombinants revealed 12 (3), 19 (3) and 27 (3) nucleotide (amino acid) differences from Sabin. Mutations were located in regions defining attenuation, temperature sensitivity, antigenicity and the cis-acting replicating element. The recombination site was in the 5' end of 3D. In a competition assay, the most mutated recombinant beat parental Sabin in all three cell lines, strongly suggesting that this virus has an advantage. Two independent intertypic recombinants, PV3/PV1 and PV3/PV2, also showed similar growth advantages, but they also contained several point mutations. Thus, our data defend the hypothesis that accumulation of certain advantageous mutations plays a key role in gaining increased fitness.

c-Ha-ras BamHI RFLP in human urothelial tumors and point mutations in hot codons

International Nuclear Information System (INIS)

Weismanova, E; Skovraga, M.; Kaluz, S.

1993-01-01

High-molecular weights DNAs from 30 bladder and renal cell carcinomas (RCC) were isolated and the c-Ha-ras the c-Ha-ras gene BamHI RFLP was examined. Amplification of c-Ha-ras with normal localization with regard to the size of alleles was found only in the case. One of the normally localized c-Ha-ras allele termed RCC c-H-ras of a length of about 6.6 kbp was cloned and an oncogene-activating point mutation was identified using two restriction enzymes. After comparison of CfrI and Cfr10I cleavage maps of RCC c-Ha-ras to complete nucleotide sequences of EJ/T24 c-Ha-ras oncogene and its normal counterpart, a point mutation was identified within codon 11 or 12. The use of CfrI and Cfr10I is of value for clinical practice in identification of point mutations in c-Ha-ras PCR product in neoplasia accompanied by somatic mutation of c-Ha-ras. The correlation among c-Ha-ras allele, amplification/loss, presence of point mutation and progression of neoplasia is discussed. (author)

Adenylosuccinate lyase (ADSL) and infantile autism: Absence of previously reported point mutation

Energy Technology Data Exchange (ETDEWEB)

Fon, E.A.; Sarrazin, J.; Rouleau, G.A. [Montreal General Hospital (Canada)] [and others

1995-12-18

Autism is a heterogeneous neuropsychiatric syndrome of unknown etiology. There is evidence that a deficiency in the enzyme adenylosuccinate lyase (ADSL), essential for de novo purine biosynthesis, could be involved in the pathogenesis of certain cases. A point mutation in the ADSL gene, resulting in a predicted serine-to-proline substitution and conferring structural instability to the mutant enzyme, has been reported previously in 3 affected siblings. In order to determine the prevalence of the mutation, we PCR-amplified the exon spanning the site of this mutation from the genomic DNA of patients fulfilling DSM-III-R criteria for autistic disorder. None of the 119 patients tested were found to have this mutation. Furthermore, on preliminary screening using single-strand conformation polymorphism (SSCP), no novel mutations were detected in the coding sequence of four ADSL exons, spanning approximately 50% of the cDNA. In light of these findings, it appears that mutations in the ADSL gene represent a distinctly uncommon cause of autism. 12 refs., 2 figs.

Pigmentary retinopathy associated with the mitochondrial DNA 3243 point mutation.

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Sue, C M; Mitchell, P; Crimmins, D S; Moshegov, C; Byrne, E; Morris, J G

1997-10-01

Fourteen patients from four unrelated families were studied to determine the prevalence of retinal pigmentary abnormalities associated with the MELAS A to G 3243 point mutation. Neurologic and ophthalmic examinations, retinal photography, pattern shift visual evoked potentials, and electroretinography were performed in all patients. Eight of the 14 patients had retinal pigmentary abnormalities characterized by symmetric areas of depigmentation involving predominantly the posterior pole and midperipheral retina. None of the patients had optic atrophy and only one patient with pigmentary retinal abnormalities had impaired visual acuity. None of the diabetic subjects (n = 6) had signs of diabetic retinopathy. Fluorescein angiography demonstrated mottled hyper- and hypofluorescent areas indicating multiple window defects in the retinal pigmentary epithelium. Visual evoked potentials showed delayed P100 responses in four of the eight patients with retinal pigmentary abnormalities. We conclude that there is a high prevalence of retinal pigmentary abnormalities in patients with MELAS A to G 3243 point mutation. These abnormalities are usually asymptomatic and best detected by retinal photography.

Human Prolactin Point Mutations and Their Projected Effect on Vasoinhibin Generation and Vasoinhibin-Related Diseases

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Jakob Triebel

2017-11-01

Full Text Available BackgroundA dysregulation of the generation of vasoinhibin hormones by proteolytic cleavage of prolactin (PRL has been brought into context with diabetic retinopathy, retinopathy of prematurity, preeclampsia, pregnancy-induced hypertension, and peripartum cardiomyopathy. Factors governing vasoinhibin generation are incompletely characterized, and the composition of vasoinhibin isoforms in human tissues or compartments, such as the circulation, is unknown. The aim of this study was to determine the possible contribution of PRL point mutations to the generation of vasoinhibins as well as to project their role in vasoinhibin-related diseases.MethodsProlactin sequences, point mutations, and substrate specificity information about the PRL cleaving enzymes cathepsin D, matrix metalloproteinases 8 and 13, and bone-morphogenetic protein 1 were retrieved from public databases. The consequences of point mutations in regard to their possible effect on vasoinhibin levels were projected on the basis of a score indicating the suitability of a particular sequence for enzymatic cleavage that result in vasoinhibin generation. The relative abundance and type of vasoinhibin isoforms were estimated by comparing the relative cleavage efficiency of vasoinhibin-generating enzymes.ResultsSix point mutations leading to amino acid substitutions in vasoinhibin-generating cleavage sites were found and projected to either facilitate or inhibit vasoinhibin generation. Four mutations affecting vasoinhibin generation in cancer tissues were found. The most likely composition of the relative abundance of vasoinhibin isoforms is projected to be 15 > 17.2 > 16.8 > 17.7 > 18 kDa vasoinhibin.ConclusionProlactin point mutations are likely to influence vasoinhibin levels by affecting the proteolysis efficiency of vasoinhibin-generating enzymes and should be monitored in patients with vasoinhibin-related diseases. Attempts to characterize vasoinhibin-related diseases

RUNX1/AML1 point mutations take part in the pathogenesis of radiation-and therapy-related myeloid neoplasms

International Nuclear Information System (INIS)

Harada, Yuka; Kimura, Akiro; Harada, Hironori

2012-01-01

High frequency of myelodysplastic syndrome (MDS) has been reported in Hiroshima A-bomb exposed survivors, in resident around Semipalatinsk Nuclear Laboratory and in exposed people by Chernobyl Nuclear Power Station Accident. MDS/acute myeloid leukemia (AML) is thought to be caused by mutation of runt-related transcription factor 1 (RUNX1) gene after a long time post exposure to relatively low dose radiation. In this study, participation of RUNX1/AML1 point mutations was examined in pathogenesis of the title neoplasms experienced in authors' facility. Subjects were 18/417 cases in whom myeloproliferative neoplasms (MPN) had switched to MDS or AML in the follow-up period of 1-25 years, and 11/124 cases in whom t-MN (therapy-related myeloid neoplasms) had developed during the remission of acute promyelocytic leukemia (APL) in the 1-9.7 years follow up. Point mutations were analyzed by PCR-single strand conformation polymorphism (PCR-SSCP) followed by base sequencing. In the former cases above, RUNX1 point mutation was found in 5/18 cases and in the latter, 4/11. When patients with persistent decrease of blood cells post therapy of APL were followed up for mutation, their RUNX1 point mutation was detected before they were diagnosed to be morbid of MDS/AML. The point mutation was thus a biomarker of myelo-hematogenic cancer, and was thought useful for early diagnosis of MDS and AML. (T.T.)

Investigating the Impact of Asp181 Point Mutations on Interactions between PTP1B and Phosphotyrosine Substrate

Science.gov (United States)

Liu, Mengyuan; Wang, Lushan; Sun, Xun; Zhao, Xian

2014-05-01

Protein tyrosine phosphatase 1B (PTP1B) is a key negative regulator of insulin and leptin signaling, which suggests that it is an attractive therapeutic target in type II diabetes and obesity. The aim of this research is to explore residues which interact with phosphotyrosine substrate can be affected by D181 point mutations and lead to increased substrate binding. To achieve this goal, molecular dynamics simulations were performed on wild type (WT) and two mutated PTP1B/substrate complexes. The cross-correlation and principal component analyses show that point mutations can affect the motions of some residues in the active site of PTP1B. Moreover, the hydrogen bond and energy decomposition analyses indicate that apart from residue 181, point mutations have influence on the interactions of substrate with several residues in the active site of PTP1B.

An efficient method for the prediction of deleterious multiple-point mutations in the secondary structure of RNAs using suboptimal folding solutions

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Barash Danny

2008-04-01

Full Text Available Abstract Background RNAmute is an interactive Java application which, given an RNA sequence, calculates the secondary structure of all single point mutations and organizes them into categories according to their similarity to the predicted structure of the wild type. The secondary structure predictions are performed using the Vienna RNA package. A more efficient implementation of RNAmute is needed, however, to extend from the case of single point mutations to the general case of multiple point mutations, which may often be desired for computational predictions alongside mutagenesis experiments. But analyzing multiple point mutations, a process that requires traversing all possible mutations, becomes highly expensive since the running time is O(nm for a sequence of length n with m-point mutations. Using Vienna's RNAsubopt, we present a method that selects only those mutations, based on stability considerations, which are likely to be conformational rearranging. The approach is best examined using the dot plot representation for RNA secondary structure. Results Using RNAsubopt, the suboptimal solutions for a given wild-type sequence are calculated once. Then, specific mutations are selected that are most likely to cause a conformational rearrangement. For an RNA sequence of about 100 nts and 3-point mutations (n = 100, m = 3, for example, the proposed method reduces the running time from several hours or even days to several minutes, thus enabling the practical application of RNAmute to the analysis of multiple-point mutations. Conclusion A highly efficient addition to RNAmute that is as user friendly as the original application but that facilitates the practical analysis of multiple-point mutations is presented. Such an extension can now be exploited prior to site-directed mutagenesis experiments by virologists, for example, who investigate the change of function in an RNA virus via mutations that disrupt important motifs in its secondary

De novo point mutations in patients diagnosed with ataxic cerebral palsy.

Science.gov (United States)

Parolin Schnekenberg, Ricardo; Perkins, Emma M; Miller, Jack W; Davies, Wayne I L; D'Adamo, Maria Cristina; Pessia, Mauro; Fawcett, Katherine A; Sims, David; Gillard, Elodie; Hudspith, Karl; Skehel, Paul; Williams, Jonathan; O'Regan, Mary; Jayawant, Sandeep; Jefferson, Rosalind; Hughes, Sarah; Lustenberger, Andrea; Ragoussis, Jiannis; Jackson, Mandy; Tucker, Stephen J; Németh, Andrea H

2015-07-01

Cerebral palsy is a sporadic disorder with multiple likely aetiologies, but frequently considered to be caused by birth asphyxia. Genetic investigations are rarely performed in patients with cerebral palsy and there is little proven evidence of genetic causes. As part of a large project investigating children with ataxia, we identified four patients in our cohort with a diagnosis of ataxic cerebral palsy. They were investigated using either targeted next generation sequencing or trio-based exome sequencing and were found to have mutations in three different genes, KCNC3, ITPR1 and SPTBN2. All the mutations were de novo and associated with increased paternal age. The mutations were shown to be pathogenic using a combination of bioinformatics analysis and in vitro model systems. This work is the first to report that the ataxic subtype of cerebral palsy can be caused by de novo dominant point mutations, which explains the sporadic nature of these cases. We conclude that at least some subtypes of cerebral palsy may be caused by de novo genetic mutations and patients with a clinical diagnosis of cerebral palsy should be genetically investigated before causation is ascribed to perinatal asphyxia or other aetiologies. © The Author (2015). Published by Oxford University Press on behalf of the Guarantors of Brain.

Evaluation of point mutations in dystrophin gene in Iranian Duchenne and Becker muscular dystrophy patients: introducing three novel variants.

Science.gov (United States)

Haghshenas, Maryam; Akbari, Mohammad Taghi; Karizi, Shohreh Zare; Deilamani, Faravareh Khordadpoor; Nafissi, Shahriar; Salehi, Zivar

2016-06-01

Duchenne and Becker muscular dystrophies (DMD and BMD) are X-linked neuromuscular diseases characterized by progressive muscular weakness and degeneration of skeletal muscles. Approximately two-thirds of the patients have large deletions or duplications in the dystrophin gene and the remaining one-third have point mutations. This study was performed to evaluate point mutations in Iranian DMD/BMD male patients. A total of 29 DNA samples from patients who did not show any large deletion/duplication mutations following multiplex polymerase chain reaction (PCR) and multiplex ligation-dependent probe amplification (MLPA) screening were sequenced for detection of point mutations in exons 50-79. Also exon 44 was sequenced in one sample in which a false positive deletion was detected by MLPA method. Cycle sequencing revealed four nonsense, one frameshift and two splice site mutations as well as two missense variants.

Identification of the mutation causing progressive retinal atrophy in Old Danish Pointing Dog.

Science.gov (United States)

Karlskov-Mortensen, P; Proschowsky, H F; Gao, F; Fredholm, M

2018-04-06

Progressive retinal atrophy (PRA) is a common cause of blindness in many dog breeds. It is most often inherited as a simple Mendelian trait, but great genetic heterogeneity has been demonstrated both within and between breeds. In many breeds the genetic cause of the disease is not known, and until now, the Old Danish Pointing Dog (ODP) has been one of those breeds. ODP is one of the oldest dog breeds in Europe. Seventy years ago the breed almost vanished, but today a population still exists, primarily in Denmark but with some dogs in Germany and Sweden. PRA has been diagnosed in ODP since the late 1990s. It resembles late onset PRA in other dog breeds, and it is inherited as an autosomal recessive trait. In the present study, we performed whole-genome sequencing and identified a single base insertion (c.3149_3150insC) in exon 1 of C17H2orf71. This is the same mutation previously found to cause PRA in Gordon Setters and Irish Setters, and it was later found in Tibetan Terrier, Standard Poodle and the Polski Owczarek Nizinny. The presence of the mutation in such a diverse range of breeds indicates an origin preceding creation of modern dog breeds. Hence, we screened 262 dogs from 44 different breeds plus four crossbred dogs, and can subsequently add Miniature Poodle and another polish sheepdog, the Polski Owczarek Podhalanski, to the list of affected breeds. © 2018 Stichting International Foundation for Animal Genetics.

Molecular alterations underlying the spontaneous and γ-ray-induced point mutations at the white locus of Drosophila Melanogaster

International Nuclear Information System (INIS)

Aleksandrova, M.V.; Lapidus, I.L.; Aleksandrov, I.D.; Karpovskij, A.L.

1996-01-01

The white locus in D.Melanogaster was selected as a target gene for the study of the mutational spectra of spontaneously arising and radiation-induced gene mutations in a whole organism. Analysis of 6 spontaneous and 73 γ-ray-induced white mutations by a combination of cytological, genetic and molecular techniques revealed that on the chromosomal and genetic levels all spontaneous mutations showed themselves to be point mutants. The share of such mutants among all heritable radiation-induced gene mutations is about 40%, whereas the rest ones are due to exchange breaks (8%) as well as multilocus, single-locus or partial-locus (intragenic) deletions (52%). The DNAs from 4 spontaneous and 17 γ-ray-induced point mutants were analysed by Southern blot-hybridization. The three spontaneous and 7 radiation mutants showed an altered DNA sequence at the left (distal) half of the white gene due to insertion or DNA rearrangement. The rest (58%) of the radiation-induced point mutations did not indicate any alternations in this part of the gene as detected by this technique and probes employed. 15 refs., 3 figs., 1 tab

Specific regulation of point-mutated K-ras-immortalized cell proliferation by a photodynamic antisense strategy.

Science.gov (United States)

Higuchi, Maiko; Yamayoshi, Asako; Kato, Kiyoko; Kobori, Akio; Wake, Norio; Murakami, Akira

2010-02-01

It has been reported that point mutations in genes are responsible for various cancers, and the selective regulation of gene expression is an important factor in developing new types of anticancer drugs. To develop effective drugs for the regulation of point-mutated genes, we focused on photoreactive antisense oligonucleotides. Previously, we reported that photoreactive oligonucleotides containing 2'-O-psoralenylmethoxyethyl adenosine (2'-Ps-eom) showed drastic photoreactivity in a strictly sequence-specific manner. Here, we demonstrated the specific gene regulatory effects of 2'-Ps-eom on [(12)Val]K-ras mutant (GGT --> GTT). Photo-cross-linking between target mRNAs and 2'-Ps-eom was sequence-specific, and the effect was UVA irradiation-dependent. Furthermore, 2'-Ps-eom was able to inhibit K-ras-immortalized cell proliferation (K12V) but not Vco cells that have the wild-type K-ras gene. These results suggest that the 2'-Ps-eom will be a powerful nucleic acid drug to inhibit the expression of disease-causing point mutation genes, and has great therapeutic potential in the treatment of cancer.

The effect of point mutations on structure and mechanical properties of collagen-like fibril: A molecular dynamics study

International Nuclear Information System (INIS)

Marlowe, Ashley E.; Singh, Abhishek; Yingling, Yaroslava G.

2012-01-01

Understanding sequence dependent mechanical and structural properties of collagen fibrils is important for the development of artificial biomaterials for medical and nanotechnological applications. Moreover, point mutations are behind many collagen associated diseases, including Osteogenesis Imperfecta (OI). We conducted a combination of classical and steered atomistic molecular dynamics simulations to examine the effect of point mutations on structure and mechanical properties of short collagen fibrils which include mutations of glycine to alanine, aspartic acid, cysteine, and serine or mutations of hydroxyproline to arginine, asparagine, glutamine, and lysine. We found that all mutations disrupt structure and reduce strength of the collagen fibrils, which may affect the hierarchical packing of the fibrils. The glycine mutations were more detrimental to mechanical strength of the fibrils (WT > Ala > Ser > Cys > Asp) than that of hydroxyproline (WT > Arg > Gln > Asn > Lys). The clinical outcome for glycine mutations agrees well with the trend in reduction of fibril's tensile strength predicted by our simulations. Overall, our results suggest that the reduction in mechanical properties of collagen fibrils may be used to predict the clinical outcome of mutations. Highlights: ► All mutations disrupt structure and bonding pattern and reduce strength of the collagen fibrils. ► Gly based mutations are worst to mechanical integrity of fibrils than that of Hyp. ► Lys and Arg mutations most dramatically destabilize collagen fibril properties. ► Clinical outcome of mutations may be related to the reduced mechanical properties of fibrils.

Identification of a mutation in the CHAT gene of Old Danish Pointing Dogs affected with congenital myasthenic syndrome

DEFF Research Database (Denmark)

Proschowsky, Helle Friis; Flagstad, Annette; Cirera, Susanna

2007-01-01

The presence of a recessive inherited muscle disease in Old Danish Pointing Dogs has been well known for years. Comparisons of this disease with myasthenic diseases of other dog breeds and humans have pointed toward a defect in the synthesis of the neurotransmitter acetylcholine possibly due...... to decreased activity of the enzyme choline acetyltransferase. We sequenced exons 5-18 of the gene encoding choline acetyltransferase (CHAT) in 2 affected and 2 unaffected dogs and identified a G to A missense mutation in exon 6. The mutation causes a valine to methionine substitution and segregates...... in agreement with the inheritance of the disease. The mutation was not detected in 50 dogs representing 25 other dog breeds. A DNA test has been developed and is now available to the breeders of Old Danish Pointing Dogs....

Diversity and frequency of kdr mutations within Anopheles sinensis populations from Guangxi, China.

Science.gov (United States)

Yang, Chan; Feng, Xiangyang; Huang, Zushi; Li, Mei; Qiu, Xinghui

2016-08-15

Anopheles sinensis is a major vector of malaria in China and its control is under great threat as the development of insecticide resistance. Voltage-gated sodium channel (VGSC) is the target of several classes of insecticides. Genetic mutations of VGSC have been documented to confer knockdown resistance (kdr) to dichlorodiphenyltrichloroethane (DDT) and pyrethroids in mosquitoes. To control this vector efficiently, it is important to know the resistance-associated genetic mutations, their distribution frequencies and genealogical relations. Three hundreds and thirteen (313) adults of An. sinensis collected from nine locations across Guangxi Zhuang Autonomous Region were used. The partial sequence of the An. sinensis voltage gated sodium channel gene (AS-VGSC) containing codon 1014 was sequenced. PHASE2.1 was used to construct the haplotypes of each individual, and the accuracy of haplotypes was further confirmed by clone sequencing. The genealogical relations of kdr mutations in AS-VGSC was analysed using TCS 2.1 and Network 5.0. Sixteen AS-VGSC haplotypes including seven haplotypes carrying non-synonymous mutations at codon 1014, and fifty-five AS-VGSC genotypes were identified from 313 mosquitoes collected from nine geographical locations across Guangxi. The number of haplotypes in each of the nine populations ranged from 5 to 13. The frequency of haplotypes carrying kdr mutations ranged from 2.7 to 80.0 % within the nine populations, of which 1014C was unexpectedly high in the northeast of Guangxi. Genealogical analysis suggested multiple origins of kdr mutations in An. sinensis. Diverse haplotypes of AS-VGSC are distributed in Guangxi. The presence of haplotypes carrying mutations at codon 1014 indicates a risk of pyrethroid and DDT resistance. The kdr mutations show differential distribution geographically, with high frequencies occurred in the northeast of Guangxi. Genealogical analysis suggests multiple origins of kdr mutations in An. sinensis populations

High mutation rates limit evolutionary adaptation in Escherichia coli.

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Kathleen Sprouffske

2018-04-01

Full Text Available Mutation is fundamental to evolution, because it generates the genetic variation on which selection can act. In nature, genetic changes often increase the mutation rate in systems that range from viruses and bacteria to human tumors. Such an increase promotes the accumulation of frequent deleterious or neutral alleles, but it can also increase the chances that a population acquires rare beneficial alleles. Here, we study how up to 100-fold increases in Escherichia coli's genomic mutation rate affect adaptive evolution. To do so, we evolved multiple replicate populations of asexual E. coli strains engineered to have four different mutation rates for 3000 generations in the laboratory. We measured the ability of evolved populations to grow in their original environment and in more than 90 novel chemical environments. In addition, we subjected the populations to whole genome population sequencing. Although populations with higher mutation rates accumulated greater genetic diversity, this diversity conveyed benefits only for modestly increased mutation rates, where populations adapted faster and also thrived better than their ancestors in some novel environments. In contrast, some populations at the highest mutation rates showed reduced adaptation during evolution, and failed to thrive in all of the 90 alternative environments. In addition, they experienced a dramatic decrease in mutation rate. Our work demonstrates that the mutation rate changes the global balance between deleterious and beneficial mutational effects on fitness. In contrast to most theoretical models, our experiments suggest that this tipping point already occurs at the modest mutation rates that are found in the wild.

High mutation rates limit evolutionary adaptation in Escherichia coli

Science.gov (United States)

Wagner, Andreas

2018-01-01

Mutation is fundamental to evolution, because it generates the genetic variation on which selection can act. In nature, genetic changes often increase the mutation rate in systems that range from viruses and bacteria to human tumors. Such an increase promotes the accumulation of frequent deleterious or neutral alleles, but it can also increase the chances that a population acquires rare beneficial alleles. Here, we study how up to 100-fold increases in Escherichia coli’s genomic mutation rate affect adaptive evolution. To do so, we evolved multiple replicate populations of asexual E. coli strains engineered to have four different mutation rates for 3000 generations in the laboratory. We measured the ability of evolved populations to grow in their original environment and in more than 90 novel chemical environments. In addition, we subjected the populations to whole genome population sequencing. Although populations with higher mutation rates accumulated greater genetic diversity, this diversity conveyed benefits only for modestly increased mutation rates, where populations adapted faster and also thrived better than their ancestors in some novel environments. In contrast, some populations at the highest mutation rates showed reduced adaptation during evolution, and failed to thrive in all of the 90 alternative environments. In addition, they experienced a dramatic decrease in mutation rate. Our work demonstrates that the mutation rate changes the global balance between deleterious and beneficial mutational effects on fitness. In contrast to most theoretical models, our experiments suggest that this tipping point already occurs at the modest mutation rates that are found in the wild. PMID:29702649

Genetic interaction analysis of point mutations enables interrogation of gene function at a residue-level resolution

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Braberg, Hannes; Moehle, Erica A.; Shales, Michael; Guthrie, Christine; Krogan, Nevan J.

2014-01-01

We have achieved a residue-level resolution of genetic interaction mapping – a technique that measures how the function of one gene is affected by the alteration of a second gene – by analyzing point mutations. Here, we describe how to interpret point mutant genetic interactions, and outline key applications for the approach, including interrogation of protein interaction interfaces and active sites, and examination of post-translational modifications. Genetic interaction analysis has proven effective for characterizing cellular processes; however, to date, systematic high-throughput genetic interaction screens have relied on gene deletions or knockdowns, which limits the resolution of gene function analysis and poses problems for multifunctional genes. Our point mutant approach addresses these issues, and further provides a tool for in vivo structure-function analysis that complements traditional biophysical methods. We also discuss the potential for genetic interaction mapping of point mutations in human cells and its application to personalized medicine. PMID:24842270

Quantitative PCR high-resolution melting (qPCR-HRM) curve analysis, a new approach to simultaneously screen point mutations and large rearrangements: application to MLH1 germline mutations in Lynch syndrome.

Science.gov (United States)

Rouleau, Etienne; Lefol, Cédrick; Bourdon, Violaine; Coulet, Florence; Noguchi, Tetsuro; Soubrier, Florent; Bièche, Ivan; Olschwang, Sylviane; Sobol, Hagay; Lidereau, Rosette

2009-06-01

Several techniques have been developed to screen mismatch repair (MMR) genes for deleterious mutations. Until now, two different techniques were required to screen for both point mutations and large rearrangements. For the first time, we propose a new approach, called "quantitative PCR (qPCR) high-resolution melting (HRM) curve analysis (qPCR-HRM)," which combines qPCR and HRM to obtain a rapid and cost-effective method suitable for testing a large series of samples. We designed PCR amplicons to scan the MLH1 gene using qPCR HRM. Seventy-six patients were fully scanned in replicate, including 14 wild-type patients and 62 patients with known mutations (57 point mutations and five rearrangements). To validate the detected mutations, we used sequencing and/or hybridization on a dedicated MLH1 array-comparative genomic hybridization (array-CGH). All point mutations and rearrangements detected by denaturing high-performance liquid chromatography (dHPLC)+multiplex ligation-dependent probe amplification (MLPA) were successfully detected by qPCR HRM. Three large rearrangements were characterized with the dedicated MLH1 array-CGH. One variant was detected with qPCR HRM in a wild-type patient and was located within the reverse primer. One variant was not detected with qPCR HRM or with dHPLC due to its proximity to a T-stretch. With qPCR HRM, prescreening for point mutations and large rearrangements are performed in one tube and in one step with a single machine, without the need for any automated sequencer in the prescreening process. In replicate, its reagent cost, sensitivity, and specificity are comparable to those of dHPLC+MLPA techniques. However, qPCR HRM outperformed the other techniques in terms of its rapidity and amount of data provided.

Ultra-deep sequencing reveals high prevalence and broad structural diversity of hepatitis B surface antigen mutations in a global population.

Science.gov (United States)

Gencay, Mikael; Hübner, Kirsten; Gohl, Peter; Seffner, Anja; Weizenegger, Michael; Neofytos, Dionysios; Batrla, Richard; Woeste, Andreas; Kim, Hyon-Suk; Westergaard, Gaston; Reinsch, Christine; Brill, Eva; Thu Thuy, Pham Thi; Hoang, Bui Huu; Sonderup, Mark; Spearman, C Wendy; Pabinger, Stephan; Gautier, Jérémie; Brancaccio, Giuseppina; Fasano, Massimo; Santantonio, Teresa; Gaeta, Giovanni B; Nauck, Markus; Kaminski, Wolfgang E

2017-01-01

The diversity of the hepatitis B surface antigen (HBsAg) has a significant impact on the performance of diagnostic screening tests and the clinical outcome of hepatitis B infection. Neutralizing or diagnostic antibodies against the HBsAg are directed towards its highly conserved major hydrophilic region (MHR), in particular towards its "a" determinant subdomain. Here, we explored, on a global scale, the genetic diversity of the HBsAg MHR in a large, multi-ethnic cohort of randomly selected subjects with HBV infection from four continents. A total of 1553 HBsAg positive blood samples of subjects originating from 20 different countries across Africa, America, Asia and central Europe were characterized for amino acid variation in the MHR. Using highly sensitive ultra-deep sequencing, we found 72.8% of the successfully sequenced subjects (n = 1391) demonstrated amino acid sequence variation in the HBsAg MHR. This indicates that the global variation frequency in the HBsAg MHR is threefold higher than previously reported. The majority of the amino acid mutations were found in the HBV genotypes B (28.9%) and C (25.4%). Collectively, we identified 345 distinct amino acid mutations in the MHR. Among these, we report 62 previously unknown mutations, which extends the worldwide pool of currently known HBsAg MHR mutations by 22%. Importantly, topological analysis identified the "a" determinant upstream flanking region as the structurally most diverse subdomain of the HBsAg MHR. The highest prevalence of "a" determinant region mutations was observed in subjects from Asia, followed by the African, American and European cohorts, respectively. Finally, we found that more than half (59.3%) of all HBV subjects investigated carried multiple MHR mutations. Together, this worldwide ultra-deep sequencing based genotyping study reveals that the global prevalence and structural complexity of variation in the hepatitis B surface antigen have, to date, been significantly underappreciated.

Effect of point mutations on Herbaspirillum seropedicae NifA activity

International Nuclear Information System (INIS)

Aquino, B.; Stefanello, A.A.; Oliveira, M.A.S.; Pedrosa, F.O.; Souza, E.M.; Monteiro, R.A.; Chubatsu, L.S.

2015-01-01

NifA is the transcriptional activator of the nif genes in Proteobacteria. It is usually regulated by nitrogen and oxygen, allowing biological nitrogen fixation to occur under appropriate conditions. NifA proteins have a typical three-domain structure, including a regulatory N-terminal GAF domain, which is involved in control by fixed nitrogen and not strictly required for activity, a catalytic AAA+ central domain, which catalyzes open complex formation, and a C-terminal domain involved in DNA-binding. In Herbaspirillum seropedicae, a β-proteobacterium capable of colonizing Graminae of agricultural importance, NifA regulation by ammonium involves its N-terminal GAF domain and the signal transduction protein GlnK. When the GAF domain is removed, the protein can still activate nif genes transcription; however, ammonium regulation is lost. In this work, we generated eight constructs resulting in point mutations in H. seropedicae NifA and analyzed their effect on nifH transcription in Escherichia coli and H. seropedicae. Mutations K22V, T160E, M161V, L172R, and A215D resulted in inactive proteins. Mutations Q216I and S220I produced partially active proteins with activity control similar to wild-type NifA. However, mutation G25E, located in the GAF domain, resulted in an active protein that did not require GlnK for activity and was partially sensitive to ammonium. This suggested that G25E may affect the negative interaction between the N-terminal GAF domain and the catalytic central domain under high ammonium concentrations, thus rendering the protein constitutively active, or that G25E could lead to a conformational change comparable with that when GlnK interacts with the GAF domain

Effect of point mutations on Herbaspirillum seropedicae NifA activity

Directory of Open Access Journals (Sweden)

B. Aquino

2015-08-01

Full Text Available NifA is the transcriptional activator of the nif genes in Proteobacteria. It is usually regulated by nitrogen and oxygen, allowing biological nitrogen fixation to occur under appropriate conditions. NifA proteins have a typical three-domain structure, including a regulatory N-terminal GAF domain, which is involved in control by fixed nitrogen and not strictly required for activity, a catalytic AAA+ central domain, which catalyzes open complex formation, and a C-terminal domain involved in DNA-binding. In Herbaspirillum seropedicae, a β-proteobacterium capable of colonizing Graminae of agricultural importance, NifA regulation by ammonium involves its N-terminal GAF domain and the signal transduction protein GlnK. When the GAF domain is removed, the protein can still activate nif genes transcription; however, ammonium regulation is lost. In this work, we generated eight constructs resulting in point mutations in H. seropedicae NifA and analyzed their effect on nifH transcription in Escherichia coli and H. seropedicae. Mutations K22V, T160E, M161V, L172R, and A215D resulted in inactive proteins. Mutations Q216I and S220I produced partially active proteins with activity control similar to wild-type NifA. However, mutation G25E, located in the GAF domain, resulted in an active protein that did not require GlnK for activity and was partially sensitive to ammonium. This suggested that G25E may affect the negative interaction between the N-terminal GAF domain and the catalytic central domain under high ammonium concentrations, thus rendering the protein constitutively active, or that G25E could lead to a conformational change comparable with that when GlnK interacts with the GAF domain.

Effect of point mutations on Herbaspirillum seropedicae NifA activity.

Science.gov (United States)

Aquino, B; Stefanello, A A; Oliveira, M A S; Pedrosa, F O; Souza, E M; Monteiro, R A; Chubatsu, L S

2015-08-01

NifA is the transcriptional activator of the nif genes in Proteobacteria. It is usually regulated by nitrogen and oxygen, allowing biological nitrogen fixation to occur under appropriate conditions. NifA proteins have a typical three-domain structure, including a regulatory N-terminal GAF domain, which is involved in control by fixed nitrogen and not strictly required for activity, a catalytic AAA+ central domain, which catalyzes open complex formation, and a C-terminal domain involved in DNA-binding. In Herbaspirillum seropedicae, a β-proteobacterium capable of colonizing Graminae of agricultural importance, NifA regulation by ammonium involves its N-terminal GAF domain and the signal transduction protein GlnK. When the GAF domain is removed, the protein can still activate nif genes transcription; however, ammonium regulation is lost. In this work, we generated eight constructs resulting in point mutations in H. seropedicae NifA and analyzed their effect on nifH transcription in Escherichia coli and H. seropedicae. Mutations K22V, T160E, M161V, L172R, and A215D resulted in inactive proteins. Mutations Q216I and S220I produced partially active proteins with activity control similar to wild-type NifA. However, mutation G25E, located in the GAF domain, resulted in an active protein that did not require GlnK for activity and was partially sensitive to ammonium. This suggested that G25E may affect the negative interaction between the N-terminal GAF domain and the catalytic central domain under high ammonium concentrations, thus rendering the protein constitutively active, or that G25E could lead to a conformational change comparable with that when GlnK interacts with the GAF domain.

Effect of point mutations on Herbaspirillum seropedicae NifA activity

Energy Technology Data Exchange (ETDEWEB)

Aquino, B.; Stefanello, A.A.; Oliveira, M.A.S.; Pedrosa, F.O.; Souza, E.M.; Monteiro, R.A.; Chubatsu, L.S. [Departamento de Bioquímica e Biologia Molecular, Universidade Federal do Paraná, Curitiba, PR (Brazil)

2015-07-10

NifA is the transcriptional activator of the nif genes in Proteobacteria. It is usually regulated by nitrogen and oxygen, allowing biological nitrogen fixation to occur under appropriate conditions. NifA proteins have a typical three-domain structure, including a regulatory N-terminal GAF domain, which is involved in control by fixed nitrogen and not strictly required for activity, a catalytic AAA+ central domain, which catalyzes open complex formation, and a C-terminal domain involved in DNA-binding. In Herbaspirillum seropedicae, a β-proteobacterium capable of colonizing Graminae of agricultural importance, NifA regulation by ammonium involves its N-terminal GAF domain and the signal transduction protein GlnK. When the GAF domain is removed, the protein can still activate nif genes transcription; however, ammonium regulation is lost. In this work, we generated eight constructs resulting in point mutations in H. seropedicae NifA and analyzed their effect on nifH transcription in Escherichia coli and H. seropedicae. Mutations K22V, T160E, M161V, L172R, and A215D resulted in inactive proteins. Mutations Q216I and S220I produced partially active proteins with activity control similar to wild-type NifA. However, mutation G25E, located in the GAF domain, resulted in an active protein that did not require GlnK for activity and was partially sensitive to ammonium. This suggested that G25E may affect the negative interaction between the N-terminal GAF domain and the catalytic central domain under high ammonium concentrations, thus rendering the protein constitutively active, or that G25E could lead to a conformational change comparable with that when GlnK interacts with the GAF domain.

Optimized knock-in of point mutations in zebrafish using CRISPR/Cas9.

Science.gov (United States)

Prykhozhij, Sergey V; Fuller, Charlotte; Steele, Shelby L; Veinotte, Chansey J; Razaghi, Babak; Robitaille, Johane M; McMaster, Christopher R; Shlien, Adam; Malkin, David; Berman, Jason N

2018-06-14

We have optimized point mutation knock-ins into zebrafish genomic sites using clustered regularly interspaced palindromic repeats (CRISPR)/Cas9 reagents and single-stranded oligodeoxynucleotides. The efficiency of knock-ins was assessed by a novel application of allele-specific polymerase chain reaction and confirmed by high-throughput sequencing. Anti-sense asymmetric oligo design was found to be the most successful optimization strategy. However, cut site proximity to the mutation and phosphorothioate oligo modifications also greatly improved knock-in efficiency. A previously unrecognized risk of off-target trans knock-ins was identified that we obviated through the development of a workflow for correct knock-in detection. Together these strategies greatly facilitate the study of human genetic diseases in zebrafish, with additional applicability to enhance CRISPR-based approaches in other animal model systems.

Dew inspired breathing-based detection of genetic point mutation visualized by naked eye

Science.gov (United States)

Xie, Liping; Wang, Tongzhou; Huang, Tianqi; Hou, Wei; Huang, Guoliang; Du, Yanan

2014-09-01

A novel label-free method based on breathing-induced vapor condensation was developed for detection of genetic point mutation. The dew-inspired detection was realized by integration of target-induced DNA ligation with rolling circle amplification (RCA). The vapor condensation induced by breathing transduced the RCA-amplified variances in DNA contents into visible contrast. The image could be recorded by a cell phone for further or even remote analysis. This green assay offers a naked-eye-reading method potentially applied for point-of-care liver cancer diagnosis in resource-limited regions.

Association of a novel point mutation in MSH2 gene with familial multiple primary cancers

Directory of Open Access Journals (Sweden)

Hai Hu

2017-10-01

Full Text Available Abstract Background Multiple primary cancers (MPC have been identified as two or more cancers without any subordinate relationship that occur either simultaneously or metachronously in the same or different organs of an individual. Lynch syndrome is an autosomal dominant genetic disorder that increases the risk of many types of cancers. Lynch syndrome patients who suffer more than two cancers can also be considered as MPC; patients of this kind provide unique resources to learn how genetic mutation causes MPC in different tissues. Methods We performed a whole genome sequencing on blood cells and two tumor samples of a Lynch syndrome patient who was diagnosed with five primary cancers. The mutational landscape of the tumors, including somatic point mutations and copy number alternations, was characterized. We also compared Lynch syndrome with sporadic cancers and proposed a model to illustrate the mutational process by which Lynch syndrome progresses to MPC. Results We revealed a novel pathologic mutation on the MSH2 gene (G504 splicing that associates with Lynch syndrome. Systematical comparison of the mutation landscape revealed that multiple cancers in the proband were evolutionarily independent. Integrative analysis showed that truncating mutations of DNA mismatch repair (MMR genes were significantly enriched in the patient. A mutation progress model that included germline mutations of MMR genes, double hits of MMR system, mutations in tissue-specific driver genes, and rapid accumulation of additional passenger mutations was proposed to illustrate how MPC occurs in Lynch syndrome patients. Conclusion Our findings demonstrate that both germline and somatic alterations are driving forces of carcinogenesis, which may resolve the carcinogenic theory of Lynch syndrome.

The Number of Point Mutations in Induced Pluripotent Stem Cells and Nuclear Transfer Embryonic Stem Cells Depends on the Method and Somatic Cell Type Used for Their Generation.

Science.gov (United States)

Araki, Ryoko; Mizutani, Eiji; Hoki, Yuko; Sunayama, Misato; Wakayama, Sayaka; Nagatomo, Hiroaki; Kasama, Yasuji; Nakamura, Miki; Wakayama, Teruhiko; Abe, Masumi

2017-05-01

Induced pluripotent stem cells hold great promise for regenerative medicine but point mutations have been identified in these cells and have raised serious concerns about their safe use. We generated nuclear transfer embryonic stem cells (ntESCs) from both mouse embryonic fibroblasts (MEFs) and tail-tip fibroblasts (TTFs) and by whole genome sequencing found fewer mutations compared with iPSCs generated by retroviral gene transduction. Furthermore, TTF-derived ntESCs showed only a very small number of point mutations, approximately 80% less than the number observed in iPSCs generated using retrovirus. Base substitution profile analysis confirmed this greatly reduced number of point mutations. The point mutations in iPSCs are therefore not a Yamanaka factor-specific phenomenon but are intrinsic to genome reprogramming. Moreover, the dramatic reduction in point mutations in ntESCs suggests that most are not essential for genome reprogramming. Our results suggest that it is feasible to reduce the point mutation frequency in iPSCs by optimizing various genome reprogramming conditions. We conducted whole genome sequencing of ntES cells derived from MEFs or TTFs. We thereby succeeded in establishing TTF-derived ntES cell lines with far fewer point mutations. Base substitution profile analysis of these clones also indicated a reduced point mutation frequency, moving from a transversion-predominance to a transition-predominance. Stem Cells 2017;35:1189-1196. © 2017 AlphaMed Press.

Preliminary studies on DNA retardation by MutS applied to the detection of point mutations in clinical samples

International Nuclear Information System (INIS)

Stanislawska-Sachadyn, Anna; Paszko, Zygmunt; Kluska, Anna; Skasko, Elzibieta; Sromek, Maria; Balabas, Aneta; Janiec-Jankowska, Aneta; Wisniewska, Alicja; Kur, Jozef; Sachadyn, Pawel

2005-01-01

MutS ability to bind DNA mismatches was applied to the detection of point mutations in PCR products. MutS recognized mismatches from single up to five nucleotides and retarded the electrophoretic migration of mismatched DNA. The electrophoretic detection of insertions/deletions above three nucleotides is also possible without MutS, thanks to the DNA mobility shift caused by the presence of large insertion/deletion loops in the heteroduplex DNA. Thus, the method enables the search for a broad range of mutations: from single up to several nucleotides. The mobility shift assays were carried out in polyacrylamide gels stained with SYBR-Gold. One assay required 50-200 ng of PCR product and 1-3 μg of Thermus thermophilus his 6 -MutS protein. The advantages of this approach are: the small amounts of DNA required for the examination, simple and fast staining, no demand for PCR product purification, no labelling and radioisotopes required. The method was tested in the detection of cancer predisposing mutations in RET, hMSH2, hMLH1, BRCA1, BRCA2 and NBS1 genes. The approach appears to be promising in screening for unknown point mutations

Efficient Knock-in of a Point Mutation in Porcine Fibroblasts Using the CRISPR/Cas9-GMNN Fusion Gene.

Science.gov (United States)

Gerlach, Max; Kraft, Theresia; Brenner, Bernhard; Petersen, Björn; Niemann, Heiner; Montag, Judith

2018-06-13

During CRISPR/Cas9 mediated genome editing, site-specific double strand breaks are introduced and repaired either unspecific by non-homologous end joining (NHEJ) or sequence dependent by homology directed repair (HDR). Whereas NHEJ-based generation of gene knock-out is widely performed, the HDR-based knock-in of specific mutations remains a bottleneck. Especially in primary cell lines that are essential for the generation of cell culture and animal models of inherited human diseases, knock-in efficacy is insufficient and needs significant improvement. Here, we tested two different approaches to increase the knock-in frequency of a specific point mutation into the MYH7 -gene in porcine fetal fibroblasts. We added a small molecule inhibitor of NHEJ, SCR7 (5,6-bis((E)-benzylideneamino)-2-mercaptopyrimidin-4-ol), during genome editing and screened cell cultures for the point mutation. However, this approach did not yield increased knock-in rates. In an alternative approach, we fused humanized Cas9 (hCas9) to the N-terminal peptide of the Geminin gene ( GMNN ). The fusion protein is degraded in NHEJ-dominated cell cycle phases, which should increase HDR-rates. Using hCas9- GMNN and point mutation-specific real time PCR screening, we found a two-fold increase in genome edited cell cultures. This increase of HDR by hCas9- GMNN provides a promising way to enrich specific knock-in in porcine fibroblast cultures for somatic cloning approaches.

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

Science.gov (United States)

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

2015-12-15

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

Identification of a rare point mutation at C-terminus of merozoite surface antigen-1 gene of Plasmodium falciparum in eastern Indian isolates.

Science.gov (United States)

Raj, Dipak Kumar; Das, Bibhu Ranjan; Dash, A P; Supakar, Prakash C

2004-01-01

Merozoite surface antigen-1 (MSA-1) of Plasmodium falciparum is highly immunogenic in human. Several studies suggest that MSA-1 protein is an effective target for a protective immune response. Attempt has been made to find new point mutations by analyzing 244 bp [codon 1655(R) to 1735 (I)] relatively conserved C-terminus region of MSA-1 gene in 125 isolates. This region contains two EGF like domains, which are involved in generating protective immune response in human. Point mutations in this region are very much important in view of vaccine development. Searching of mutational hot spots in MSA-1 protein by sequencing method in a representative number of isolates is quite critical and expensive. Therefore, in this study slot blot and PCR-SSCP method have been used to find out new mutations in the individual isolates showing alterations in the mobility of DNA fragment. Sequencing of the altered bands from the SSCP gel shows a rare non-synonymous point mutation in 7 (5.6%) of the 125 isolates at amino acid position 1704 of MSA-1 gene where isoleucine is replaced by valine.

Two α1-Globin Gene Point Mutations Causing Severe Hb H Disease.

Science.gov (United States)

Jiang, Hua; Huang, Lv-Yin; Zhen, Li; Jiang, Fan; Li, Dong-Zhi

Hb H disease is generally a moderate form of α-thalassemia (α-thal) that rarely requires regular blood transfusions. In this study, two Chinese families with members carrying transfusion-dependent Hb H disease were investigated for rare mutations on the α-globin genes (HBA1, HBA2). In one family, Hb Zürich-Albisrieden [α59(E8)Gly→Arg; HBA1: c.178G>C] in combination with the Southeast Asian (- - SEA ) deletion was the defect responsible for the severe phenotype. In another family, a novel hemoglobin (Hb) variant named Hb Sichuan (HBA1: c.393_394insT), causes α-thal and a severe phenotype when associated with the - - SEA deletion. As these two HBA1 mutations can present as continuous blood transfusion-dependent α-thal, it is important to take this point into account for detecting the carriers, especially in couples in which one partner is already a known α 0 -thal carrier.

Familial isolated primary hyperparathyroidism/hyperparathyroidism-jaw tumour syndrome caused by germline gross deletion or point mutations of CDC73 gene in Chinese.

Science.gov (United States)

Kong, Jing; Wang, Ou; Nie, Min; Shi, Jie; Hu, Yingying; Jiang, Yan; Li, Mei; Xia, Weibo; Meng, Xunwu; Xing, Xiaoping

2014-08-01

Hyperparathyroidism-jaw tumour syndrome (HPT-JT) and familial isolated primary hyperparathyroidism (FIHP) are two subtypes of familial primary hyperparathyroidism, which are rarely reported in Chinese population. Here, we reported three FIHP families and one HPT-JT family with long-term follow-up and genetic analysis. A total of 22 patients, from four FIHP/HPT-JT families of Chinese descent, were recruited and genomic DNA was extracted from their peripheral blood lymphocytes. Direct sequencing for MEN1, CDC73, CASR gene was conducted. Reverse transcription PCR (RT-PCR) and quantitative real-time PCR (qRT-PCR) were used to study the effect of splice site mutations and gross deletion mutations. Immunohistochemistry was performed to analyse parafibromin expression in parathyroid tumours. Genotype-phenotype correlations were assessed through clinical characteristics and long-term follow-up data. Genetic analysis revealed four CDC73 germline mutations that were responsible for the four kindreds, including two novel point mutation (c.157 G>T and IVS3+1 G>A), one recurrent point mutation (c.664 C>T) and one deletion mutation (c.307+?_513-?del exons 4, 5, 6). RT-PCR confirmed that IVS3+1 G>A generated an aberrant transcript with exon3 deletion. Immunohistochemical analysis demonstrated reduced nuclear parafibromin expression in tumours supporting the pathogenic effects of these mutations. This study supplies information on mutations and phenotypes of HPT-JT/FIHP syndrome in Chinese. Screening for gross deletion and point mutations of the CDC73 gene is necessary in susceptible subjects. © 2014 John Wiley & Sons Ltd.

Discovery of Point Mutations in the Voltage-Gated Sodium Channel from African Aedes aegypti Populations: Potential Phylogenetic Reasons for Gene Introgression

Science.gov (United States)

Muranami, Yuto; Kawashima, Emiko; Osei, Joseph H. N.; Sakyi, Kojo Yirenkyi; Dadzie, Samuel; de Souza, Dziedzom K.; Appawu, Maxwell; Ohta, Nobuo; Minakawa, Noboru

2016-01-01

Background Yellow fever is endemic in some countries in Africa, and Aedes aegpyti is one of the most important vectors implicated in the outbreak. The mapping of the nation-wide distribution and the detection of insecticide resistance of vector mosquitoes will provide the beneficial information for forecasting of dengue and yellow fever outbreaks and effective control measures. Methodology/Principal Findings High resistance to DDT was observed in all mosquito colonies collected in Ghana. The resistance and the possible existence of resistance or tolerance to permethrin were suspected in some colonies. High frequencies of point mutations at the voltage-gated sodium channel (F1534C) and one heterozygote of the other mutation (V1016I) were detected, and this is the first detection on the African continent. The frequency of F1534C allele and the ratio of F1534C homozygotes in Ae. aegypti aegypti (Aaa) were significantly higher than those in Ae. aegypti formosus (Aaf). We could detect the two types of introns between exon 20 and 21, and the F1534C mutations were strongly linked with one type of intron, which was commonly found in South East Asian and South and Central American countries, suggesting the possibility that this mutation was introduced from other continents or convergently selected after the introgression of Aaa genes from the above area. Conclusions/Significance The worldwide eradication programs in 1940s and 1950s might have caused high selection pressure on the mosquito populations and expanded the distribution of insecticide-resistant Ae. aegypti populations. Selection of the F1534C point mutation could be hypothesized to have taken place during this period. The selection of the resistant population of Ae. aegypti with the point mutation of F1534C, and the worldwide transportation of vector mosquitoes in accordance with human activity such as trading of used tires, might result in the widespread distribution of F1534C point mutation in tropical countries

Serial MRI in early Creutzfeldt-Jacob disease with a point mutation of prion protein at codon 180

International Nuclear Information System (INIS)

Ishida, S.; Sugino, M.; Shinoda, K.; Ohsawa, N.; Koizumi, N.; Ohta, T.; Kitamoto, T.; Tateishi, J.

1995-01-01

We report a 66-year-old woman with histologically diagnosed Creutzfeld-Jacob disease (CJD), followed with MRI from an early clinical stage. MRI demonstrated expansion of the high cortical signal on T2-weighted images, which differs from previous MRI reports of CJD. This patient followed an atypical clinical course: 16 months had passed before she developed akinetic mutism, and periodic sharp waves had not been detected on EEG after 2 years in spite of her akinetic mutism. Brain biopsy showed primary spongiform changes in the grey matter, and a point mutation of the prion protein gene at codon 180 was discovered using polymerase chain reaction direct sequencing and Tth 111 I cutting. This is the first case with the point mutation of the codon 180 variant with an atypical clinical course and characteristic MRI findings. (orig.)

Protein truncation test: analysis of two novel point mutations at the carboxy-terminus of the human dystrophin gene associated with mental retardation.

Science.gov (United States)

Tuffery, S; Lenk, U; Roberts, R G; Coubes, C; Demaille, J; Claustres, M

1995-01-01

Approximately one-third of the mutations responsible for Duchenne muscular dytrophy (DMD) do not involve gross rearrangements of the dystrophin gene. Methods for intensive mutation screening have recently been applied to this immense gene, which resulted in the identification of a number of point mutations in DMD patients, mostly translation-terminating mutations. A number of data raised the possibility that the C-terminal region of dystrophin might be involved in some cases of mental retardation associated with DMD. Using single-strand conformation analysis of products amplified by polymerase chain reaction (PCR-SSCA) to screen the terminal domains of the dystrophin gene (exons 60-79) of 20 unrelated patients with DMD or BMD, we detected two novel point mutations in two mentally retarded DMD patients: a 1-bp deletion in exon 70 (10334delC) and a 5' splice donor site alteration in intron 69 (10294 + 1G-->T). Both mutations should result in a premature translation termination of dystrophin. The possible effects on the reading frame were analyzed by the study of reverse transcripts amplified from peripheral blood lymphocytes mRNA and by the protein truncation test.

One adenosine deaminase allele in a patient with severe combined immunodeficiency contains a point mutation abolishing enzyme activity.

OpenAIRE

Valerio, D; Dekker, B M; Duyvesteyn, M G; van der Voorn, L; Berkvens, T M; van Ormondt, H; van der Eb, A J

1986-01-01

We have cloned and sequenced an adenosine deaminase (ADA) gene from a patient with severe combined immunodeficiency (SCID) caused by inherited ADA deficiency. Two point mutations were found, resulting in amino acid substitutions at positions 80 (Lys to Arg) and 304 (Leu to Arg) of the protein. Hybridization experiments with synthetic oligonucleotide probes showed that the determined mutations are present in both DNA and RNA from the ADA-SCID patient. In addition, wild-type sequences could be ...

Identification of point mutations in clinical Staphylococcus aureus strains that produce small-colony variants auxotrophic for menadione.

Science.gov (United States)

Dean, Melissa A; Olsen, Randall J; Long, S Wesley; Rosato, Adriana E; Musser, James M

2014-04-01

Staphylococcus aureus small-colony variants (SCVs) are implicated in chronic and relapsing infections that are difficult to diagnose and treat. Despite many years of study, the underlying molecular mechanisms and virulence effect of the small-colony phenotype remain incompletely understood. We sequenced the genomes of five S. aureus SCV strains recovered from human patients and discovered previously unidentified nonsynonymous point mutations in three genes encoding proteins in the menadione biosynthesis pathway. Analysis of genetic revertants and complementation with wild-type alleles confirmed that these mutations caused the SCV phenotype and decreased virulence for mice.

The identification of point mutations in Duchenne muscular dystrophy patients by using reverse-transcription PCR and the protein truncation test

Energy Technology Data Exchange (ETDEWEB)

Gardner, R.J.; Bobrow, M.; Roberts, R.G. [St. Thomas`s Hospitals, London (United Kingdom)

1995-08-01

The protein truncation test (PTT) is a mutation-detection method that monitors the integrity of the open reading frame (ORF). More than 60% of cases of Duchenne muscular dystrophy (DMD) result from gross frameshifting deletions in the dystrophin gene that are detectable by multiplex PCR system. It has become apparent that virtually all of the remaining DMD mutations also disrupt the translational reading frame, making the PTT a logical next step toward a comprehensive strategy for the identification of all DMD mutations. We report here a pilot study involving 22 patients and describe the mutations characterized. These constitute 12 point mutations or small insertions/deletions and 4 gross rearrangements. We also have a remaining five patients in whom there does not appear to be mutation in the ORF. We believe that reverse-transcription-PCR/PTT is an efficient method by which to screen for small mutations in DMD patients with no deletion. 29 refs., 2 figs., 3 tabs.

Somatic point mutations in mtDNA control region are influenced by genetic background and associated with healthy aging: a GEHA study

DEFF Research Database (Denmark)

Rose, Giuseppina; Romeo, Giuseppe; Dato, Serena

2010-01-01

and of mortality risk in the elderly. Our study provides new evidence on the relevance of mtDNA somatic mutations in aging and longevity and confirms that the occurrence of specific point mutations in the mtDNA control region may represent a strategy for the age-related remodelling of organismal functions....

A novel germ-line point mutation in RET exon 8 (Gly(533)Cys) in a large kindred with familial medullary thyroid carcinoma

OpenAIRE

Silva, Adriana Madeira Alvares da [UNIFESP; Maciel, Rui Monteiro de Barros [UNIFESP; Dias-da-Silva, Magnus Régios [UNIFESP; Toledo, Silvia Regina Caminada de [UNIFESP; De Carvalho, Marcos B.; Cerutti, Janete Maria [UNIFESP

2003-01-01

Familial medullary thyroid carcinoma is related to germ-line mutations in the RET oncogene, mainly in cysteine codon 10 or 11, whereas noncysteine mutations in codons 13 - 15 are rare. We now report a new missense point mutation in exon 8 of the RET gene (1597G-->T) corresponding to a Gly(533)Cys substitution in the cystein-rich domain of RET protein in 76 patients from a 6-generation Brazilian family with 229 subjects, with ascendants from Spain. It is likely that the mutation causes familia...

Point mutation in the MITF gene causing Waardenburg syndrome type II in a three-generation Indian family.

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Lalwani, A K; Attaie, A; Randolph, F T; Deshmukh, D; Wang, C; Mhatre, A; Wilcox, E

1998-12-04

Waardenburg syndrome (WS) is an autosomal-dominant neural crest cell disorder phenotypically characterized by hearing impairment and disturbance of pigmentation. A presence of dystopia canthorum is indicative of WS type 1, caused by loss of function mutation in the PAX3 gene. In contrast, type 2 WS (WS2) is characterized by normally placed medial canthi and is genetically heterogeneous; mutations in MITF (microphthalmia associated transcription factor) associated with WS2 have been identified in some but not all affected families. Here, we report on a three-generation Indian family with a point mutation in the MITF gene causing WS2. This mutation, initially reported in a Northern European family, creates a stop codon in exon 7 and is predicted to result in a truncated protein lacking the HLH-Zip or Zip structure necessary for normal interaction with its target DNA motif. Comparison of the phenotype between the two families demonstrates a significant difference in pigmentary disturbance of the eye. This family, with the first documented case of two unrelated WS2 families harboring identical mutations, provides additional evidence for the importance of genetic background on the clinical phenotype.

Abiotic stress leads to somatic and heritable changes in homologous recombination frequency, point mutation frequency and microsatellite stability in Arabidopsis plants

International Nuclear Information System (INIS)

Yao Youli; Kovalchuk, Igor

2011-01-01

In earlier studies, we showed that abiotic stresses, such as ionizing radiation, heavy metals, temperature and water, trigger an increase in homologous recombination frequency (HRF). We also demonstrated that many of these stresses led to inheritance of high-frequency homologous recombination, HRF. Although an increase in recombination frequency is an important indicator of genome rearrangements, it only represents a minor portion of possible stress-induced mutations. Here, we analyzed the influence of heat, cold, drought, flood and UVC abiotic stresses on two major types of mutations in the genome, point mutations and small deletions/insertions. We used two transgenic lines of Arabidopsis thaliana, one allowing an analysis of reversions in a stop codon-containing inactivated β-glucuronidase transgene and another one allowing an analysis of repeat stability in a microsatellite-interrupted β-glucuronidase transgene. The transgenic Arabidopsis line carrying the β-glucuronidase-based homologous recombination substrate was used as a positive control. We showed that the majority of stresses increased the frequency of point mutations, homologous recombination and microsatellite instability in somatic cells, with the frequency of homologous recombination being affected the most. The analysis of transgenerational changes showed an increase in HRF to be the most prominent effect observed in progeny. Significant changes in recombination frequency were observed upon exposure to all types of stress except drought, whereas changes in microsatellite instability were observed upon exposure to UVC, heat and cold. The frequency of point mutations in the progeny of stress-exposed plants was the least affected; an increase in mutation frequency was observed only in the progeny of plants exposed to UVC. We thus conclude that transgenerational changes in genome stability in response to stress primarily involve an increase in recombination frequency.

Mutation rate heterogeneity and the generation of allele diversity at the human minisatellite MS205 (D16S309).

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May, C A; Jeffreys, A J; Armour, J A

1996-11-01

Many tandemly repeated minisatellite loci display extreme levels of length variation as a consequence of high rates of spontaneous germline mutation altering repeat copy number. Direct screening for new allele lengths by small-pool PCR has shown that instability at the human minisatellite locus MS205 (D16S309) is largely germline specific and usually results in the gain or loss of just a few repeat units. Structural analysis of the order of variant repeats has shown that these events occur preferentially at one end of the tandem array and can result in complex rearrangements including the inter-allelic transfer of repeat units. In contrast, putative mutants recovered from somatic DNA occur at a substantially lower rate and are simple and non-polar in nature. Germline mutation rates vary considerably between alleles, consistent with regulation occurring in cis. Although examination of DNA sequence polymorphisms immediately flanking the minisatellite reveals no definitive associations with germline mutation rate variation, differences in rate may be paralleled by changes in mutation spectrum. These findings help to explain the diversity of MS205 allele structures in modern humans and suggest a common mutation pathway with some other minisatellites.

Terbinafine Resistance of Trichophyton Clinical Isolates Caused by Specific Point Mutations in the Squalene Epoxidase Gene.

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Yamada, Tsuyoshi; Maeda, Mari; Alshahni, Mohamed Mahdi; Tanaka, Reiko; Yaguchi, Takashi; Bontems, Olympia; Salamin, Karine; Fratti, Marina; Monod, Michel

2017-07-01

Terbinafine is one of the allylamine antifungal agents whose target is squalene epoxidase (SQLE). This agent has been extensively used in the therapy of dermatophyte infections. The incidence of patients with tinea pedis or unguium tolerant to terbinafine treatment prompted us to screen the terbinafine resistance of all Trichophyton clinical isolates from the laboratory of the Centre Hospitalier Universitaire Vaudois collected over a 3-year period and to identify their mechanism of resistance. Among 2,056 tested isolates, 17 (≈1%) showed reduced terbinafine susceptibility, and all of these were found to harbor SQLE gene alleles with different single point mutations, leading to single amino acid substitutions at one of four positions (Leu 393 , Phe 397 , Phe 415 , and His 440 ) of the SQLE protein. Point mutations leading to the corresponding amino acid substitutions were introduced into the endogenous SQLE gene of a terbinafine-sensitive Arthroderma vanbreuseghemii (formerly Trichophyton mentagrophytes ) strain. All of the generated A. vanbreuseghemii transformants expressing mutated SQLE proteins exhibited obvious terbinafine-resistant phenotypes compared to the phenotypes of the parent strain and of transformants expressing wild-type SQLE proteins. Nearly identical phenotypes were also observed in A. vanbreuseghemii transformants expressing mutant forms of Trichophyton rubrum SQLE proteins. Considering that the genome size of dermatophytes is about 22 Mb, the frequency of terbinafine-resistant clinical isolates was strikingly high. Increased exposure to antifungal drugs could favor the generation of resistant strains. Copyright © 2017 American Society for Microbiology.

Differential evolution enhanced with multiobjective sorting-based mutation operators.

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Wang, Jiahai; Liao, Jianjun; Zhou, Ying; Cai, Yiqiao

2014-12-01

Differential evolution (DE) is a simple and powerful population-based evolutionary algorithm. The salient feature of DE lies in its mutation mechanism. Generally, the parents in the mutation operator of DE are randomly selected from the population. Hence, all vectors are equally likely to be selected as parents without selective pressure at all. Additionally, the diversity information is always ignored. In order to fully exploit the fitness and diversity information of the population, this paper presents a DE framework with multiobjective sorting-based mutation operator. In the proposed mutation operator, individuals in the current population are firstly sorted according to their fitness and diversity contribution by nondominated sorting. Then parents in the mutation operators are proportionally selected according to their rankings based on fitness and diversity, thus, the promising individuals with better fitness and diversity have more opportunity to be selected as parents. Since fitness and diversity information is simultaneously considered for parent selection, a good balance between exploration and exploitation can be achieved. The proposed operator is applied to original DE algorithms, as well as several advanced DE variants. Experimental results on 48 benchmark functions and 12 real-world application problems show that the proposed operator is an effective approach to enhance the performance of most DE algorithms studied.

Mitochondrial DNA mutation load in a family with the m.8344A>G point mutation and lipomas

DEFF Research Database (Denmark)

Jeppesen, Tina Dysgaard; Al-Hashimi, Noor; Duno, Morten

2017-01-01

Studies have shown that difference in mtDNA mutation load among tissues is a result of postnatal modification. We present five family members with the m.8344A>G with variable phenotypes but uniform intrapersonal distribution of mutation load, indicating that there is no postnatal modification of mt......DNA mutation load in this genotype....

The point mutation process in proteins

Science.gov (United States)

Schwartz, R. M.; Dayhoff, M. O.

1978-01-01

An optimized scoring matrix for residue-by-residue comparisons of distantly related protein sequences has been developed. The scoring matrix is based on observed exchanges and mutabilities of amino acids in 1572 closely related sequences derived from a cross-section of protein groups. Very few superimposed or parallel mutations are included in the data. The scoring matrix is most useful for demonstrating the relatedness of proteins between 65 and 85% different.

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

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

2015-01-01

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

Novel Point Mutations and A8027G Polymorphism in Mitochondrial-DNA-Encoded Cytochrome c Oxidase II Gene in Mexican Patients with Probable Alzheimer Disease

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Verónica Loera-Castañeda

2014-01-01

Full Text Available Mitochondrial dysfunction has been thought to contribute to Alzheimer disease (AD pathogenesis through the accumulation of mitochondrial DNA mutations and net production of reactive oxygen species (ROS. Mitochondrial cytochrome c-oxidase plays a key role in the regulation of aerobic production of energy and is composed of 13 subunits. The 3 largest subunits (I, II, and III forming the catalytic core are encoded by mitochondrial DNA. The aim of this work was to look for mutations in mitochondrial cytochrome c-oxidase gene II (MTCO II in blood samples from probable AD Mexican patients. MTCO II gene was sequenced in 33 patients with diagnosis of probable AD. Four patients (12% harbored the A8027G polymorphism and three of them were early onset (EO AD cases with familial history of the disease. In addition, other four patients with EOAD had only one of the following point mutations: A8003C, T8082C, C8201T, or G7603A. Neither of the point mutations found in this work has been described previously for AD patients, and the A8027G polymorphism has been described previously; however, it hasn’t been related to AD. We will need further investigation to demonstrate the role of the point mutations of mitochondrial DNA in the pathogenesis of AD.

A protein-targeting strategy used to develop a selective inhibitor of the E17K point mutation in the PH domain of Akt1

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Deyle, Kaycie M.; Farrow, Blake; Qiao Hee, Ying; Work, Jeremy; Wong, Michelle; Lai, Bert; Umeda, Aiko; Millward, Steven W.; Nag, Arundhati; Das, Samir; Heath, James R.

2015-05-01

Ligands that can bind selectively to proteins with single amino-acid point mutations offer the potential to detect or treat an abnormal protein in the presence of the wild type (WT). However, it is difficult to develop a selective ligand if the point mutation is not associated with an addressable location, such as a binding pocket. Here we report an all-chemical synthetic epitope-targeting strategy that we used to discover a 5-mer peptide with selectivity for the E17K-transforming point mutation in the pleckstrin homology domain of the Akt1 oncoprotein. A fragment of Akt1 that contained the E17K mutation and an I19[propargylglycine] substitution was synthesized to form an addressable synthetic epitope. Azide-presenting peptides that clicked covalently onto this alkyne-presenting epitope were selected from a library using in situ screening. One peptide exhibits a 10:1 in vitro selectivity for the oncoprotein relative to the WT, with a similar selectivity in cells. This 5-mer peptide was expanded into a larger ligand that selectively blocks the E17K Akt1 interaction with its PIP3 (phosphatidylinositol (3,4,5)-trisphosphate) substrate.

Mitochondrial DNA triplication and punctual mutations in patients with mitochondrial neuromuscular disorders

Energy Technology Data Exchange (ETDEWEB)

Mkaouar-Rebai, Emna, E-mail: emna.mkaouar@gmail.com [Département des Sciences de la Vie, Faculté des Sciences de Sfax, Université de Sfax (Tunisia); Felhi, Rahma; Tabebi, Mouna [Laboratoire de Génétique Moléculaire Humaine, Faculté de Médecine de Sfax, Université de Sfax (Tunisia); Alila-Fersi, Olfa; Chamkha, Imen [Département des Sciences de la Vie, Faculté des Sciences de Sfax, Université de Sfax (Tunisia); Maalej, Marwa; Ammar, Marwa [Laboratoire de Génétique Moléculaire Humaine, Faculté de Médecine de Sfax, Université de Sfax (Tunisia); Kammoun, Fatma [Service de pédiatrie, C.H.U. Hedi Chaker de Sfax (Tunisia); Keskes, Leila [Laboratoire de Génétique Moléculaire Humaine, Faculté de Médecine de Sfax, Université de Sfax (Tunisia); Hachicha, Mongia [Service de pédiatrie, C.H.U. Hedi Chaker de Sfax (Tunisia); Fakhfakh, Faiza, E-mail: faiza.fakhfakh02@gmail.com [Département des Sciences de la Vie, Faculté des Sciences de Sfax, Université de Sfax (Tunisia)

2016-04-29

Mitochondrial diseases are a heterogeneous group of disorders caused by the impairment of the mitochondrial oxidative phosphorylation system which have been associated with various mutations of the mitochondrial DNA (mtDNA) and nuclear gene mutations. The clinical phenotypes are very diverse and the spectrum is still expanding. As brain and muscle are highly dependent on OXPHOS, consequently, neurological disorders and myopathy are common features of mtDNA mutations. Mutations in mtDNA can be classified into three categories: large-scale rearrangements, point mutations in tRNA or rRNA genes and point mutations in protein coding genes. In the present report, we screened mitochondrial genes of complex I, III, IV and V in 2 patients with mitochondrial neuromuscular disorders. The results showed the presence the pathogenic heteroplasmic m.9157G>A variation (A211T) in the MT-ATP6 gene in the first patient. We also reported the first case of triplication of 9 bp in the mitochondrial NC7 region in Africa and Tunisia, in association with the novel m.14924T>C in the MT-CYB gene in the second patient with mitochondrial neuromuscular disorder. - Highlights: • We reported 2 patients with mitochondrial neuromuscular disorders. • The heteroplasmic MT-ATP6 9157G>A variation was reported. • A triplication of 9 bp in the mitochondrial NC7 region was detected. • The m.14924T>C transition (S60P) in the MT-CYB gene was found.

A novel natural killer cell line (KHYG-1) from a patient with aggressive natural killer cell leukemia carrying a p53 point mutation.

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Yagita, M; Huang, C L; Umehara, H; Matsuo, Y; Tabata, R; Miyake, M; Konaka, Y; Takatsuki, K

2000-05-01

We present the establishment of a natural killer (NK) leukemia cell line, designated KHYG-1, from the blood of a patient with aggressive NK leukemia, which both possessed the same p53 point mutation. The immunophenotype of the primary leukemia cells was CD2+, surface CD3-, cytoplasmic CD3epsilon+, CD7+, CD8alphaalpha+, CD16+, CD56+, CD57+ and HLA-DR+. A new cell line (KHYG-1) was established by culturing peripheral leukemia cells with 100 units of recombinant interleukin (IL)-2. The KHYG-1 cells showed LGL morphology with a large nucleus, coarse chromatin, conspicuous nucleoli, and abundant basophilic cytoplasm with many azurophilic granules. The immunophenotype of KHYG-1 cells was CD1-, CD2+, surface CD3-, cytoplasmic CD3epsilon+, CD7+, CD8alphaalpha+, CD16-, CD25-, CD33+, CD34-, CD56+, CD57-, CD122+, CD132+, and TdT-. Southern blot analysis of these cells revealed a normal germline configuration for the beta, delta, and gamma chains of the T cell receptor and the immunoglobulin heavy-chain genes. Moreover, the KHYG-1 cells displayed NK cell activity and IL-2-dependent proliferation in vitro, suggesting that they are of NK cell origin. Epstein-Barr virus (EBV) DNA was not detected in KHYG-1 cells by Southern blot analysis with a terminal repeat probe from an EBV genome. A point mutation in exon 7 of the p53 gene was detected in the KHYG-1 cells by PCR/SSCP analysis, and direct sequencing revealed the conversion of C to T at nucleotide 877 in codon 248. The primary leukemia cells also carried the same point mutation. Although the precise role of the p53 point mutation in leukemogenesis remains to be clarified, the establishment of an NK leukemia cell line with a p53 point mutation could be valuable in the study of leukemogenesis.

A new point mutation in the iron-sulfur subunit of succinate dehydrogenase confers resistance to boscalid in Sclerotinia sclerotiorum.

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Wang, Yong; Duan, Yabing; Wang, Jianxin; Zhou, Mingguo

2015-09-01

Research has established that mutations in highly conserved amino acids of the succinate dehydrogenase (SDH) complex in various fungi confer SDH inhibitor (SDHI) resistance. For Sclerotinia sclerotiorum (Lib.) de Bary, a necrotrophic fungus with a broad host range and a worldwide distribution, boscalid resistance has been attributed to the mutation H132R in the highly conserved SdhD subunit protein of the SDH complex. In our previous study, however, only one point mutation, A11V in SdhB (GCA to GTA change in SdhB), was detected in S. sclerotiorum boscalid-resistant (BR) mutants. In the current study, replacement of the SdhB gene in a boscalid-sensitive (BS) S. sclerotiorum strain with the mutant SdhB gene conferred resistance. Compared with wild-type strains, BR and GSM (SdhB gene in the wild-type strain replaced by the mutant SdhB gene) mutants were more sensitive to osmotic stress, lacked the ability to produce sclerotia and exhibited lower expression of the pac1 gene. Importantly, the point mutation was not located in the highly conserved sequence of the iron-sulfur subunit of SDH. These results suggest that resistance based on non-conserved vs. conserved protein domains differs in mechanism. In addition to increasing our understanding of boscalid resistance in S. sclerotiorum, the new information will be useful for the development of alternative antifungal drugs. © 2014 BSPP AND JOHN WILEY & SONS LTD.

Multiple contacts with diversion at the point of arrest.

Science.gov (United States)

Riordan, Sharon; Wix, Stuart; Haque, M Sayeed; Humphreys, Martin

2003-04-01

A diversion at the point of arrest (DAPA) scheme was set up in five police stations in South Birmingham in 1992. In a study of all referrals made over a four-year period a sub group of multiple contact individuals was identified. During that time four hundred and ninety-two contacts were recorded in total, of which 130 were made by 58 individuals. The latter group was generally no different from the single contact group but did have a tendency to be younger. This research highlights the need for a re-evaluation of service provision and associated education of police officers and relevant mental health care professionals.

Hereditary neuropathy with liability to pressure palsy (HNPP): report of a family with a new point mutation in PMP22 gene.

Science.gov (United States)

Fusco, Carlo; Spagnoli, Carlotta; Salerno, Grazia Gabriella; Pavlidis, Elena; Frattini, Daniele; Pisani, Francesco

2017-10-27

Hereditary neuropathy with liability to pressure palsy (HNPP) is an autosomal dominant disorder most commonly presenting with acute-onset, non-painful focal sensory and motor mononeuropathy. Approximately 80% of patients carry a 1.5 Mb deletion of chromosome 17p11.2 involving the peripheral myelin protein 22 gene (PMP22), the same duplicated in Charcot-Marie-Tooth 1A patients. In a small proportion of patients the disease is caused by PMP22 point mutations. We report on a familial case harbouring a new point mutation in the PMP22 gene. The proband is a 4-years-old girl with acute onset of focal numbness and weakness in her right hand. Electroneurography demonstrated transient sensory and motor radial nerves involvement. In her father, reporting chronic symptoms (cramps and exercise-induced myalgia), we uncovered mild atrophy and areflexia on clinical examination and a mixed (predominantly demyelinating) polyneuropathy with sensory-motor involvement on electrophysiological study. Both carried a nucleotidic substitution c.178 + 2 T > C on intron 3 of the PMP22 gene, involving the splicing donor site, not reported on databases but predicted to be likely pathogenic. We described a previously unreported point mutation in PMP22 gene, which led to the development of a HNPP phenotype in a child and her father. In children evaluated for a sensory and motor transient episode, HNPP disorder due to PMP22 mutations should be suspected. Clinical and electrophysiological studies should be extended to all family members even in the absence of previous episodes suggestive for HNPP.

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.

Evolution of Salmonella enterica virulence via point mutations in the fimbrial adhesin.

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Dagmara I Kisiela

Full Text Available Whereas the majority of pathogenic Salmonella serovars are capable of infecting many different animal species, typically producing a self-limited gastroenteritis, serovars with narrow host-specificity exhibit increased virulence and their infections frequently result in fatal systemic diseases. In our study, a genetic and functional analysis of the mannose-specific type 1 fimbrial adhesin FimH from a variety of serovars of Salmonella enterica revealed that specific mutant variants of FimH are common in host-adapted (systemically invasive serovars. We have found that while the low-binding shear-dependent phenotype of the adhesin is preserved in broad host-range (usually systemically non-invasive Salmonella, the majority of host-adapted serovars express FimH variants with one of two alternative phenotypes: a significantly increased binding to mannose (as in S. Typhi, S. Paratyphi C, S. Dublin and some isolates of S. Choleraesuis, or complete loss of the mannose-binding activity (as in S. Paratyphi B, S. Choleraesuis and S. Gallinarum. The functional diversification of FimH in host-adapted Salmonella results from recently acquired structural mutations. Many of the mutations are of a convergent nature indicative of strong positive selection. The high-binding phenotype of FimH that leads to increased bacterial adhesiveness to and invasiveness of epithelial cells and macrophages usually precedes acquisition of the non-binding phenotype. Collectively these observations suggest that activation or inactivation of mannose-specific adhesive properties in different systemically invasive serovars of Salmonella reflects their dynamic trajectories of adaptation to a life style in specific hosts. In conclusion, our study demonstrates that point mutations are the target of positive selection and, in addition to horizontal gene transfer and genome degradation events, can contribute to the differential pathoadaptive evolution of Salmonella.

Somatic point mutation calling in low cellularity tumors.

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Karin S Kassahn

Full Text Available Somatic mutation calling from next-generation sequencing data remains a challenge due to the difficulties of distinguishing true somatic events from artifacts arising from PCR, sequencing errors or mis-mapping. Tumor cellularity or purity, sub-clonality and copy number changes also confound the identification of true somatic events against a background of germline variants. We have developed a heuristic strategy and software (http://www.qcmg.org/bioinformatics/qsnp/ for somatic mutation calling in samples with low tumor content and we show the superior sensitivity and precision of our approach using a previously sequenced cell line, a series of tumor/normal admixtures, and 3,253 putative somatic SNVs verified on an orthogonal platform.

Co-occurrence of point mutations in the voltage-gated sodium channel of pyrethroid-resistant Aedes aegypti populations in Myanmar.

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Kawada, Hitoshi; Oo, Sai Zaw Min; Thaung, Sein; Kawashima, Emiko; Maung, Yan Naung Maung; Thu, Hlaing Myat; Thant, Kyaw Zin; Minakawa, Noboru

2014-01-01

Single amino acid substitutions in the voltage-gated sodium channel associated with pyrethroid resistance constitute one of the main causative factors of knockdown resistance in insects. The kdr gene has been observed in several mosquito species; however, point mutations in the para gene of Aedes aegypti populations in Myanmar have not been fully characterized. The aim of the present study was to determine the types and frequencies of mutations in the para gene of Aedes aegypti collected from used tires in Yangon City, Myanmar. We determined high pyrethroid resistance in Aedes aegypti larvae at all collection sites in Yangon City, by using a simplified knockdown bioassay. We showed that V1016G and S989P mutations were widely distributed, with high frequencies (84.4% and 78.8%, respectively). By contrast, we were unable to detect I1011M (or I1011V) or L1014F mutations. F1534C mutations were also widely distributed, but with a lower frequency than the V1016G mutation (21.2%). High percentage of co-occurrence of the homozygous V1016G/S989P mutations was detected (65.7%). Additionally, co-occurrence of homozygous V1016G/F1534C mutations (2.9%) and homozygous V1016G/F1534C/S989P mutations (0.98%) were detected in the present study. Pyrethroid insecticides were first used for malaria control in 1992, and have since been constantly used in Myanmar. This intensive use may explain the strong selection pressure toward Aedes aegypti, because this mosquito is generally a domestic and endophagic species with a preference for indoor breeding. Extensive use of DDT for malaria control before the use of this chemical was banned may also explain the development of pyrethroid resistance in Aedes aegypti.

Co-occurrence of Point Mutations in the Voltage-Gated Sodium Channel of Pyrethroid-Resistant Aedes aegypti Populations in Myanmar

Science.gov (United States)

Kawada, Hitoshi; Oo, Sai Zaw Min; Thaung, Sein; Kawashima, Emiko; Maung, Yan Naung Maung; Thu, Hlaing Myat; Thant, Kyaw Zin; Minakawa, Noboru

2014-01-01

Background Single amino acid substitutions in the voltage-gated sodium channel associated with pyrethroid resistance constitute one of the main causative factors of knockdown resistance in insects. The kdr gene has been observed in several mosquito species; however, point mutations in the para gene of Aedes aegypti populations in Myanmar have not been fully characterized. The aim of the present study was to determine the types and frequencies of mutations in the para gene of Aedes aegypti collected from used tires in Yangon City, Myanmar. Methodology/Principal Findings We determined high pyrethroid resistance in Aedes aegypti larvae at all collection sites in Yangon City, by using a simplified knockdown bioassay. We showed that V1016G and S989P mutations were widely distributed, with high frequencies (84.4% and 78.8%, respectively). By contrast, we were unable to detect I1011M (or I1011V) or L1014F mutations. F1534C mutations were also widely distributed, but with a lower frequency than the V1016G mutation (21.2%). High percentage of co-occurrence of the homozygous V1016G/S989P mutations was detected (65.7%). Additionally, co-occurrence of homozygous V1016G/F1534C mutations (2.9%) and homozygous V1016G/F1534C/S989P mutations (0.98%) were detected in the present study. Conclusions/Significance Pyrethroid insecticides were first used for malaria control in 1992, and have since been constantly used in Myanmar. This intensive use may explain the strong selection pressure toward Aedes aegypti, because this mosquito is generally a domestic and endophagic species with a preference for indoor breeding. Extensive use of DDT for malaria control before the use of this chemical was banned may also explain the development of pyrethroid resistance in Aedes aegypti. PMID:25077956

HIV populations are large and accumulate high genetic diversity in a nonlinear fashion.

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Maldarelli, Frank; Kearney, Mary; Palmer, Sarah; Stephens, Robert; Mican, JoAnn; Polis, Michael A; Davey, Richard T; Kovacs, Joseph; Shao, Wei; Rock-Kress, Diane; Metcalf, Julia A; Rehm, Catherine; Greer, Sarah E; Lucey, Daniel L; Danley, Kristen; Alter, Harvey; Mellors, John W; Coffin, John M

2013-09-01

HIV infection is characterized by rapid and error-prone viral replication resulting in genetically diverse virus populations. The rate of accumulation of diversity and the mechanisms involved are under intense study to provide useful information to understand immune evasion and the development of drug resistance. To characterize the development of viral diversity after infection, we carried out an in-depth analysis of single genome sequences of HIV pro-pol to assess diversity and divergence and to estimate replicating population sizes in a group of treatment-naive HIV-infected individuals sampled at single (n = 22) or multiple, longitudinal (n = 11) time points. Analysis of single genome sequences revealed nonlinear accumulation of sequence diversity during the course of infection. Diversity accumulated in recently infected individuals at rates 30-fold higher than in patients with chronic infection. Accumulation of synonymous changes accounted for most of the diversity during chronic infection. Accumulation of diversity resulted in population shifts, but the rates of change were low relative to estimated replication cycle times, consistent with relatively large population sizes. Analysis of changes in allele frequencies revealed effective population sizes that are substantially higher than previous estimates of approximately 1,000 infectious particles/infected individual. Taken together, these observations indicate that HIV populations are large, diverse, and slow to change in chronic infection and that the emergence of new mutations, including drug resistance mutations, is governed by both selection forces and drift.

A high proportion of ADA point mutations associated with a specific alanine-to-valine substitution.

OpenAIRE

Markert, M L; Norby-Slycord, C; Ward, F E

1989-01-01

In 15%-20% of children with severe combined immunodeficiency (SCID), the underlying defect is adenosine deaminase (ADA) deficiency. The overall goal of our research has been to identify the precise molecular defects in patients with ADA-deficient SCID. In this study, we focused on a patient whom we found to have normal sized ADA mRNA by Northern analysis and an intact ADA structural gene by Southern analysis. By cloning and sequencing this patient's ADA cDNA, we found a C-to-T point mutation ...

Diversity of Pol IV function is defined by mutations at the maize rmr7 locus.

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Jennifer L Stonaker

2009-11-01

Full Text Available Mutations affecting the heritable maintenance of epigenetic states in maize identify multiple small RNA biogenesis factors including NRPD1, the largest subunit of the presumed maize Pol IV holoenzyme. Here we show that mutations defining the required to maintain repression7 locus identify a second RNA polymerase subunit related to Arabidopsis NRPD2a, the sole second largest subunit shared between Arabidopsis Pol IV and Pol V. A phylogenetic analysis shows that, in contrast to representative eudicots, grasses have retained duplicate loci capable of producing functional NRPD2-like proteins, which is indicative of increased RNA polymerase diversity in grasses relative to eudicots. Together with comparisons of rmr7 mutant plant phenotypes and their effects on the maintenance of epigenetic states with parallel analyses of NRPD1 defects, our results imply that maize utilizes multiple functional NRPD2-like proteins. Despite the observation that RMR7/NRPD2, like NRPD1, is required for the accumulation of most siRNAs, our data indicate that different Pol IV isoforms play distinct roles in the maintenance of meiotically-heritable epigenetic information in the grasses.

Mutations in THAP1/DYT6 reveal that diverse dystonia genes disrupt similar neuronal pathways and functions.

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Zuchra Zakirova

2018-01-01

Full Text Available Dystonia is characterized by involuntary muscle contractions. Its many forms are genetically, phenotypically and etiologically diverse and it is unknown whether their pathogenesis converges on shared pathways. Mutations in THAP1 [THAP (Thanatos-associated protein domain containing, apoptosis associated protein 1], a ubiquitously expressed transcription factor with DNA binding and protein-interaction domains, cause dystonia, DYT6. There is a unique, neuronal 50-kDa Thap1-like immunoreactive species, and Thap1 levels are auto-regulated on the mRNA level. However, THAP1 downstream targets in neurons, and the mechanism via which it causes dystonia are largely unknown. We used RNA-Seq to assay the in vivo effect of a heterozygote Thap1 C54Y or ΔExon2 allele on the gene transcription signatures in neonatal mouse striatum and cerebellum. Enriched pathways and gene ontology terms include eIF2α Signaling, Mitochondrial Dysfunction, Neuron Projection Development, Axonal Guidance Signaling, and Synaptic LongTerm Depression, which are dysregulated in a genotype and tissue-dependent manner. Electrophysiological and neurite outgrowth assays were consistent with those enrichments, and the plasticity defects were partially corrected by salubrinal. Notably, several of these pathways were recently implicated in other forms of inherited dystonia, including DYT1. We conclude that dysfunction of these pathways may represent a point of convergence in the pathophysiology of several forms of inherited dystonia.

PIK3CA mutations frequently coexist with RAS and BRAF mutations in patients with advanced cancers.

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Filip Janku

Full Text Available Oncogenic mutations of PIK3CA, RAS (KRAS, NRAS, and BRAF have been identified in various malignancies, and activate the PI3K/AKT/mTOR and RAS/RAF/MEK pathways, respectively. Both pathways are critical drivers of tumorigenesis.Tumor tissues from 504 patients with diverse cancers referred to the Clinical Center for Targeted Therapy at MD Anderson Cancer Center starting in October 2008 were analyzed for PIK3CA, RAS (KRAS, NRAS, and BRAF mutations using polymerase chain reaction-based DNA sequencing.PIK3CA mutations were found in 54 (11% of 504 patients tested; KRAS in 69 (19% of 367; NRAS in 19 (8% of 225; and BRAF in 31 (9% of 361 patients. PIK3CA mutations were most frequent in squamous cervical (5/14, 36%, uterine (7/28, 25%, breast (6/29, 21%, and colorectal cancers (18/105, 17%; KRAS in pancreatic (5/9, 56%, colorectal (49/97, 51%, and uterine cancers (3/20, 15%; NRAS in melanoma (12/40, 30%, and uterine cancer (2/11, 18%; BRAF in melanoma (23/52, 44%, and colorectal cancer (5/88, 6%. Regardless of histology, KRAS mutations were found in 38% of patients with PIK3CA mutations compared to 16% of patients with wild-type (wtPIK3CA (p = 0.001. In total, RAS (KRAS, NRAS or BRAF mutations were found in 47% of patients with PIK3CA mutations vs. 24% of patients wtPIK3CA (p = 0.001. PIK3CA mutations were found in 28% of patients with KRAS mutations compared to 10% with wtKRAS (p = 0.001 and in 20% of patients with RAS (KRAS, NRAS or BRAF mutations compared to 8% with wtRAS (KRAS, NRAS or wtBRAF (p = 0.001.PIK3CA, RAS (KRAS, NRAS, and BRAF mutations are frequent in diverse tumors. In a wide variety of tumors, PIK3CA mutations coexist with RAS (KRAS, NRAS and BRAF mutations.

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

Energy Technology Data Exchange (ETDEWEB)

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

1994-03-01

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

Bystander effect-induced mutagenicity in HPRT locus of CHO cells following BNCT neutron irradiation: Characteristics of point mutations by sequence analysis

Energy Technology Data Exchange (ETDEWEB)

Kinashi, Yuko [Research Reactor Institute, Kyoto University, Kumatori-cho, Sennan-gun, Osaka (Japan)], E-mail: kinashi@rri.kyoto-u.ac.jp; Suzuki, Minoru; Masunaga, Shinichiro; Ono, Koji [Research Reactor Institute, Kyoto University, Kumatori-cho, Sennan-gun, Osaka (Japan)

2009-07-15

To investigate bystander mutagenic effects induced by alpha particles during boron neutron capture therapy (BNCT), we mixed cells that were electroporated with borocaptate sodium (BSH), which led to the accumulation of {sup 10}B inside the cells, with cells that did not contain the boron compound. BSH-containing cells were irradiated with {alpha} particles produced by the {sup 10}B(n,{alpha}){sup 7}Li reaction, whereas cells without boron were only affected by the {sup 1}H(n,{gamma}){sup 2}H and {sup 14}N(n,{rho}){sup 14}C reactions. The frequency of mutations induced in the hypoxanthine-guanine phosphoribosyltransferase (HPRT) locus was examined in Chinese hamster ovary (CHO) cells irradiated with neutrons (Kyoto University Research Reactor: 5 MW). Neutron irradiation of 1:1 mixtures of cells with and without BSH resulted in a survival fraction of 0.1, and the cells that did not contain BSH made up 99.4% of the surviving cell population. Using multiplex polymerase chain reactions (PCRs), molecular structural analysis indicated that most of the mutations induced by the bystander effect were point mutations and that the frequencies of total and partial deletions induced by the bystander effect were lower than those resulting from the {alpha} particles produced by the {sup 10}B(n,{alpha}){sup 7}Li reaction or the neutron beam from the {sup 1}H(n,{gamma}){sup 2}H and {sup 14}N(n,{rho}){sup 14}C reactions. The types of point mutations induced by the BNCT bystander effect were analyzed by cloning and sequencing methods. These mutations were comprised of 65.5% base substitutions, 27.5% deletions, and 7.0% insertions. Sequence analysis of base substitutions showed that transversions and transitions occurred in 64.7% and 35.3% of cases, respectively. G:C{yields}T:A transversion induced by 8-oxo-guanine in DNA occurred in 5.9% of base substitution mutants in the BNCT bystander group. The characteristic mutations seen in this group, induced by BNCT {alpha} particles

[Clinical characteristics of human recombination activating gene 1 mutations in 8 immunodeficiency patients with diverse phenotypes].

Science.gov (United States)

Yu, G; Wang, W J; Liu, D R; Tao, Z F; Hui, X Y; Hou, J; Sun, J Q; Wang, X C

2018-03-02

3 cases, increased serum IgE levels in 5 cases. RAG1 homozygous mutations were detected in 5 cases and RAG1 compound heterozygous mutations in 3 cases. Two novel mutations and six previously reported mutations were identified. Three cases were successfully treated with hematopoietic stem cell transplantation. Four cases died due to infections, and the 13 year-old patient was still under follow-up in the outpatient clinic. Conclusions: Different RAG1 gene mutations can lead to diverse clinical presentations and immune phenotypes. Clinicians should pay attention to the family history of infant death with severe infection. In that situation, immunological evaluation and gene detection should be performed as early as possible.

The m.3291T>C mt-tRNALeu(UUR) mutation is definitely pathogenic and causes multisystem mitochondrial disease

Science.gov (United States)

Yarham, John W.; Blakely, Emma L.; Alston, Charlotte L.; Roberts, Mark E.; Ealing, John; Pal, Piyali; Turnbull, Douglass M.; McFarland, Robert; Taylor, Robert W.

2013-01-01

Mitochondrial tRNA point mutations are important causes of human disease, and have been associated with a diverse range of clinical phenotypes. Definitively proving the pathogenicity of any given mt-tRNA mutation requires combined molecular, genetic and functional studies. Subsequent evaluation of the mutation using a pathogenicity scoring system is often very helpful in concluding whether or not the mutation is causing disease. Despite several independent reports linking the m.3291T>C mutation to disease in humans, albeit in association with several different phenotypes, its pathogenicity remains controversial. A lack of conclusive functional evidence and an over-emphasis on the poor evolutionary conservation of the affected nucleotide have contributed to this controversy. Here we describe an adult patient who presented with deafness and lipomas and evidence of mitochondrial abnormalities in his muscle biopsy, who harbours the m.3291T > C mutation, providing conclusive evidence of pathogenicity through analysis of mutation segregation with cytochrome c oxidase (COX) deficiency in single muscle fibres, underlining the importance of performing functional studies when assessing pathogenicity. PMID:23273904

Highly sensitive chemiluminescent point mutation detection by circular strand-displacement amplification reaction.

Science.gov (United States)

Shi, Chao; Ge, Yujie; Gu, Hongxi; Ma, Cuiping

2011-08-15

Single nucleotide polymorphism (SNP) genotyping is attracting extensive attentions owing to its direct connections with human diseases including cancers. Here, we have developed a highly sensitive chemiluminescence biosensor based on circular strand-displacement amplification and the separation by magnetic beads reducing the background signal for point mutation detection at room temperature. This method took advantage of both the T4 DNA ligase recognizing single-base mismatch with high selectivity and the strand-displacement reaction of polymerase to perform signal amplification. The detection limit of this method was 1.3 × 10(-16)M, which showed better sensitivity than that of most of those reported detection methods of SNP. Additionally, the magnetic beads as carrier of immobility was not only to reduce the background signal, but also may have potential apply in high through-put screening of SNP detection in human genome. Copyright © 2011 Elsevier B.V. All rights reserved.

Somatic mosaicism of a point mutation in the dystrophin gene in a patient presenting with an asymmetrical muscle weakness and contractures

NARCIS (Netherlands)

Helderman-van den Enden, A. T. J. M.; Ginjaar, H. B.; Kneppers, A. L. J.; Bakker, E.; Breuning, M. H.; de Visser, M.

2003-01-01

We describe a patient with somatic mosaicism of a point mutation in the dystrophin gene causing benign muscular dystrophy with an unusual asymmetrical distribution of muscle weakness and contractures. To our knowledge this is the first patient with asymmetrical weakness and contractures in an

Selection-Mutation Dynamics of Signaling Games

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Josef Hofbauer

2015-01-01

Full Text Available We study the structure of the rest points of signaling games and their dynamic behavior under selection-mutation dynamics by taking the case of three signals as our canonical example. Many rest points of the replicator dynamics of signaling games are not isolated and, therefore, not robust under perturbations. However, some of them attract open sets of initial conditions. We prove the existence of certain rest points of the selection-mutation dynamics close to Nash equilibria of the signaling game and show that all but the perturbed rest points close to strict Nash equilibria are dynamically unstable. This is an important result for the evolution of signaling behavior, since it shows that the second-order forces that are governed by mutation can increase the chances of successful signaling.

Real-time resolution of point mutations that cause phenovariance in mice

Science.gov (United States)

Wang, Tao; Zhan, Xiaowei; Bu, Chun-Hui; Lyon, Stephen; Pratt, David; Hildebrand, Sara; Choi, Jin Huk; Zhang, Zhao; Zeng, Ming; Wang, Kuan-wen; Turer, Emre; Chen, Zhe; Zhang, Duanwu; Yue, Tao; Wang, Ying; Shi, Hexin; Wang, Jianhui; Sun, Lei; SoRelle, Jeff; McAlpine, William; Hutchins, Noelle; Zhan, Xiaoming; Fina, Maggy; Gobert, Rochelle; Quan, Jiexia; Kreutzer, McKensie; Arnett, Stephanie; Hawkins, Kimberly; Leach, Ashley; Tate, Christopher; Daniel, Chad; Reyna, Carlos; Prince, Lauren; Davis, Sheila; Purrington, Joel; Bearden, Rick; Weatherly, Jennifer; White, Danielle; Russell, Jamie; Sun, Qihua; Tang, Miao; Li, Xiaohong; Scott, Lindsay; Moresco, Eva Marie Y.; McInerney, Gerald M.; Karlsson Hedestam, Gunilla B.; Xie, Yang; Beutler, Bruce

2015-01-01

With the wide availability of massively parallel sequencing technologies, genetic mapping has become the rate limiting step in mammalian forward genetics. Here we introduce a method for real-time identification of N-ethyl-N-nitrosourea-induced mutations that cause phenotypes in mice. All mutations are identified by whole exome G1 progenitor sequencing and their zygosity is established in G2/G3 mice before phenotypic assessment. Quantitative and qualitative traits, including lethal effects, in single or multiple combined pedigrees are then analyzed with Linkage Analyzer, a software program that detects significant linkage between individual mutations and aberrant phenotypic scores and presents processed data as Manhattan plots. As multiple alleles of genes are acquired through mutagenesis, pooled “superpedigrees” are created to analyze the effects. Our method is distinguished from conventional forward genetic methods because it permits (1) unbiased declaration of mappable phenotypes, including those that are incompletely penetrant (2), automated identification of causative mutations concurrent with phenotypic screening, without the need to outcross mutant mice to another strain and backcross them, and (3) exclusion of genes not involved in phenotypes of interest. We validated our approach and Linkage Analyzer for the identification of 47 mutations in 45 previously known genes causative for adaptive immune phenotypes; our analysis also implicated 474 genes not previously associated with immune function. The method described here permits forward genetic analysis in mice, limited only by the rates of mutant production and screening. PMID:25605905

Fetal-juvenile origins of point mutations in the adult human tracheal-bronchial epithelium: Absence of detectable effects of age, gender or smoking status

Energy Technology Data Exchange (ETDEWEB)

Sudo, Hiroko [Massachusetts Institute of Technology, Department of Biological Engineering, 21 Ames St., 16-743 Cambridge, MA 02139 (United States); Toray Industries, Inc., New Frontiers Research Laboratories 10-1, Tebiro 6-chome, Kamakura, Kanagawa 248-8555 (Japan); Li-Sucholeiki, Xiao-Cheng [Massachusetts Institute of Technology, Department of Biological Engineering, 21 Ames St., 16-743 Cambridge, MA 02139 (United States); Agencourt Bioscience Corp., 500 Cummings Center, Suite 2450, Beverly, MA 01915 (United States); Marcelino, Luisa A. [Massachusetts Institute of Technology, Department of Biological Engineering, 21 Ames St., 16-743 Cambridge, MA 02139 (United States); Biomedical Engineering Department, Northwestern University, 633 Clark Street, Evanston, IL 60208 (United States); Gruhl, Amanda N. [Massachusetts Institute of Technology, Department of Biological Engineering, 21 Ames St., 16-743 Cambridge, MA 02139 (United States); Herrero-Jimenez, Pablo [Massachusetts Institute of Technology, Department of Biological Engineering, 21 Ames St., 16-743 Cambridge, MA 02139 (United States); SLC Ontario, 690 Dorval Drive, Suite 200, Oakville, Ontario L6K 3W7 Canada (Canada); Zarbl, Helmut [UMDNJ-Robert Wood Johnson Medical School, Environmental and Occupational Health Sciences Institute, 170 Freylinghuysen Road, Room 426, Piscataway, NJ 08854 (United States); Willey, James C. [Medical College of Ohio, 3120 Glendale Avenue, Room 12, Toledo, OH 43614 (United States); Furth, Emma E. [University of Pennsylvania Medical Center, Department of Pathology, 3400 Spruce Street, 6 Founders Building, Philadelphia, PA 19104 (United States); Morgenthaler, Stephan [Institute of Applied Mathematics, Swiss Federal Institute of Technology (EPFL), SB/IMA, 1015 Lausanne (Switzerland)] (and others)

2008-11-10

Allele-specific mismatch amplification mutation assays (MAMA) of anatomically distinct sectors of the upper bronchial tracts of nine nonsmokers revealed many numerically dispersed clusters of the point mutations C742T, G746T, G747T of the TP53 gene, G35T of the KRAS gene and G508A of the HPRT1 gene. Assays of these five mutations in six smokers have yielded quantitatively similar results. One hundred and eighty four micro-anatomical sectors of 0.5-6 x 10{sup 6} tracheal-bronchial epithelial cells represented en toto the equivalent of approximately 1.7 human smokers' bronchial trees to the fifth bifurcation. Statistically significant mutant copy numbers above the 95% upper confidence limits of historical background controls were found in 198 of 425 sector assays. No significant differences (P = 0.1) for negative sector fractions, mutant fractions, distributions of mutant cluster size or anatomical positions were observed for smoking status, gender or age (38-76 year). Based on the modal cluster size of mitochondrial point mutants, the size of the adult bronchial epithelial maintenance turnover unit was estimated to be about 32 cells. When data from all 15 lungs were combined the log 2 of nuclear mutant cluster size plotted against log 2 of the number of clusters of a given cluster size displayed a slope of {approx}1.1 over a range of cluster sizes from {approx}2{sup 6} to 2{sup 15} mutant copies. A parsimonious interpretation of these nuclear and previously reported data for lung epithelial mitochondrial point mutant clusters is that they arose from mutations in stem cells at a high but constant rate per stem cell doubling during at least ten stem cell doublings of the later fetal-juvenile period. The upper and lower decile range of summed point mutant fractions among lungs was about 7.5-fold, suggesting an important source of stratification in the population with regard to risk of tumor initiation.

Mutational Context and Diverse Clonal Development in Early and Late Bladder Cancer

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Iver Nordentoft

2014-06-01

Full Text Available Bladder cancer (or urothelial cell carcinoma [UCC] is characterized by field disease (malignant alterations in surrounding mucosa and frequent recurrences. Whole-genome, exome, and transcriptome sequencing of 38 tumors, including four metachronous tumor pairs and 20 superficial tumors, identified an APOBEC mutational signature in one-third. This was biased toward the sense strand, correlated with mean expression level, and clustered near breakpoints. A > G mutations were up to eight times more frequent on the sense strand (p < 0.002 in [ACG]AT contexts. The patient-specific APOBEC signature was negatively correlated to repair-gene expression and was not related to clinicopathological parameters. Mutations in gene families and single genes were related to tumor stage, and expression of chromatin modifiers correlated with survival. Evolutionary and subclonal analyses of early/late tumor pairs showed a unitary origin, and discrete tumor clones contained mutated cancer genes. The ancestral clones contained Pik3ca/Kdm6a mutations and may reflect the field-disease mutations shared among later tumors.

In silico reversal of repeat-induced point mutation (RIP identifies the origins of repeat families and uncovers obscured duplicated genes

Directory of Open Access Journals (Sweden)

Hane James K

2010-11-01

Full Text Available Abstract Background Repeat-induced point mutation (RIP is a fungal genome defence mechanism guarding against transposon invasion. RIP mutates the sequence of repeated DNA and over time renders the affected regions unrecognisable by similarity search tools such as BLAST. Results DeRIP is a new software tool developed to predict the original sequence of a RIP-mutated region prior to the occurrence of RIP. In this study, we apply deRIP to the genome of the wheat pathogen Stagonospora nodorum SN15 and predict the origin of several previously uncharacterised classes of repetitive DNA. Conclusions Five new classes of transposon repeats and four classes of endogenous gene repeats were identified after deRIP. The deRIP process is a new tool for fungal genomics that facilitates the identification and understanding of the role and origin of fungal repetitive DNA. DeRIP is open-source and is available as part of the RIPCAL suite at http://www.sourceforge.net/projects/ripcal.

Molecular analysis of point mutations in a barley genome exposed to MNU and gamma rays

Energy Technology Data Exchange (ETDEWEB)

Kurowska, Marzena, E-mail: mkurowsk@us.edu.pl [Department of Genetics, Faculty of Biology and Environmental Protection, University of Silesia, Jagiellonska 28, 40-032 Katowice (Poland); Labocha-Pawlowska, Anna; Gnizda, Dominika; Maluszynski, Miroslaw; Szarejko, Iwona [Department of Genetics, Faculty of Biology and Environmental Protection, University of Silesia, Jagiellonska 28, 40-032 Katowice (Poland)

2012-10-15

We present studies aimed at determining the types and frequencies of mutations induced in the barley genome after treatment with chemical (N-methyl-N-nitrosourea, MNU) and physical (gamma rays) mutagens. We created M{sub 2} populations of a doubled haploid line and used them for the analysis of mutations in targeted DNA sequences and over an entire barley genome using TILLING (Targeting Induced Local Lesions in Genomes) and AFLP (Amplified Fragment Length Polymorphism) technique, respectively. Based on the TILLING analysis of the total DNA sequence of 4,537,117 bp in the MNU population, the average mutation density was estimated as 1/504 kb. Only one nucleotide change was found after an analysis of 3,207,444 bp derived from the highest dose of gamma rays applied. MNU was clearly a more efficient mutagen than gamma rays in inducing point mutations in barley. The majority (63.6%) of the MNU-induced nucleotide changes were transitions, with a similar number of G > A and C > T substitutions. The similar share of G > A and C > T transitions indicates a lack of bias in the repair of O{sup 6}-methylguanine lesions between DNA strands. There was, however, a strong specificity of the nucleotide surrounding the O{sup 6}-meG at the -1 position. Purines formed 81% of nucleotides observed at the -1 site. Scanning the barley genome with AFLP markers revealed ca. a three times higher level of AFLP polymorphism in MNU-treated as compared to the gamma-irradiated population. In order to check whether AFLP markers can really scan the whole barley genome for mutagen-induced polymorphism, 114 different AFLP products, were cloned and sequenced. 94% of bands were heterogenic, with some bands containing up to 8 different amplicons. The polymorphic AFLP products were characterised in terms of their similarity to the records deposited in a GenBank database. The types of sequences present in the polymorphic bands reflected the organisation of the barley genome.

Intrahost Diversity of Feline Coronavirus: A Consensus between the Circulating Virulent/Avirulent Strains and the Internal Mutation Hypotheses?

Directory of Open Access Journals (Sweden)

Aline S. Hora

2013-01-01

Full Text Available To evaluate the most controversial issue concerning current feline coronavirus (FCoV virology, the coexisting hypotheses of the intrahost and interhost origins of feline infectious peritonitis virus (FIPV in regard to the pathogenesis of feline infectious peritonitis (FIP, this study aimed to assess the molecular diversity of the membrane gene FCoVs in 190 samples from 10 cats with signs of FIP and in 5 faecal samples from cats without signs of FIP. All samples from the non-FIP cats and 25.26% of the samples from the FIP cats were positive for the FCoV membrane (M gene. Mutations in this gene consisted of SNP changes randomly scattered among the sequences; few mutations resulted in amino acid changes. No geographic pattern was observed. Of the cats without FIP that harboured FECoV, the amino acid sequence identities for the M gene were 100% among cats (Cats 1–3 from the same cattery, and the overall sequence identity for the M gene was ≥91%. In one cat, two different lineages of FCoV, one enteric and one systemic, were found that segregated apart in the M gene tree. In conclusion, the in vivo mutation transition hypothesis and the circulating high virulent-low virulent FCoV hypothesis have been found to be plausible according to the results obtained from sequencing the M gene.

A point mutation of valine-311 to methionine in Bacillus subtilis protoporphyrinogen oxidase does not greatly increase resistance to the diphenyl ether herbicide oxyfluorfen.

Science.gov (United States)

Jeong, Eunjoo; Houn, Thavrak; Kuk, Yongin; Kim, Eun-Seon; Chandru, Hema Kumar; Baik, Myunggi; Back, Kyoungwhan; Guh, Ja-Ock; Han, Oksoo

2003-10-01

In an effort to asses the effect of Val311Met point mutation of Bacillus subtilis protoporphyrinogen oxidase on the resistance to diphenyl ether herbicides, a Val311Met point mutant of B. subtilis protoporphyrinogen oxidase was prepared, heterologously expressed in Escherichia coli, and the purified recombinant Val311Met mutant protoporphyrinogen oxidase was kinetically characterized. The mutant protoporphyrinogen oxidase showed very similar kinetic patterns to wild type protoporphyrinogen oxidase, with slightly decreased activity dependent on pH and the concentrations of NaCl, Tween 20, and imidazole. When oxyfluorfen was used as a competitive inhibitor, the Val311Met mutant protoporphyrinogen oxidase showed an increased inhibition constant about 1.5 times that of wild type protoporphyrinogen oxidase. The marginal increase of the inhibition constant indicates that the Val311Met point mutation in B. subtilis protoporphyrinogen oxidase may not be an important determinant in the mechanism that protects protoporphyrinogen oxidase against diphenyl ether herbicides.

EGFR and KRAS mutation coexistence in lung adenocarcinomas

Directory of Open Access Journals (Sweden)

Vitor Manuel Leitão de Sousa

2015-04-01

Full Text Available Lung cancer is one of the most common causes of cancer deaths. The development of EGFR targeted therapies, including monoclonal antibodies and tyrosine kinase inhibitors have generated an interest in the molecular characterization of these tumours. KRAS mutations are associated with resistance to EGFR TKIs. EGFR and KRAS mutations have been considered as mutually exclusive. This paper presents three bronchial-pulmonary carcinomas, two adenocarcinomas and one pleomorphic sarcomatoid carcinoma, harboring EGFR and KRAS mutations. Case 1 corresponded to an adenocarcinoma with EGFR exon 21 mutation (L858R and KRAS codon 12 point mutation (G12V; case 2, a  mucinous adenocarcinoma expressed coexistence of EGFR exon 21 mutation (L858R and KRAS codon 12 point mutation (G12V; and case 3 a sarcomatoid carcinoma with EGFR exon 19 deletion – del 9bp and KRAS codon 12 point mutation (G12C - cysteine. Based on our experience and on the literature, we conclude that EGFR and KRAS mutations can indeed coexist in the same bronchial-pulmonary carcinoma, either in the same histological type or in different patterns. The biological implications of this coexistence are still poorly understood mainly because these cases are not frequent or currently searched. It is therefore necessary to study larger series of cases with the two mutations to better understand the biological, clinical and therapeutic implications.

Mutations in the Norrie disease gene.

Science.gov (United States)

Schuback, D E; Chen, Z Y; Craig, I W; Breakefield, X O; Sims, K B

1995-01-01

We report our experience to date in mutation identification in the Norrie disease (ND) gene. We carried out mutational analysis in 26 kindreds in an attempt to identify regions presumed critical to protein function and potentially correlated with generation of the disease phenotype. All coding exons, as well as noncoding regions of exons 1 and 2, 636 nucleotides in the noncoding region of exon 3, and 197 nucleotides of 5' flanking sequence, were analyzed for single-strand conformation polymorphisms (SSCP) by polymerase chain reaction (PCR) amplification of genomic DNA. DNA fragments that showed altered SSCP band mobilities were sequenced to locate the specific mutations. In addition to three previously described submicroscopic deletions encompassing the entire ND gene, we have now identified 6 intragenic deletions, 8 missense (seven point mutations, one 9-bp deletion), 6 nonsense (three point mutations, three single bp deletions/frameshift) and one 10-bp insertion, creating an expanded repeat in the 5' noncoding region of exon 1. Thus, mutations have been identified in a total of 24 of 26 (92%) of the kindreds we have studied to date. With the exception of two different mutations, each found in two apparently unrelated kindreds, these mutations are unique and expand the genotype database. Localization of the majority of point mutations at or near cysteine residues, potentially critical in protein tertiary structure, supports a previous protein model for norrin as member of a cystine knot growth factor family (Meitinger et al., 1993). Genotype-phenotype correlations were not evident with the limited clinical data available, except in the cases of larger submicroscopic deletions associated with a more severe neurologic syndrome.(ABSTRACT TRUNCATED AT 250 WORDS)

Personalized medicine and human genetic diversity.

Science.gov (United States)

Lu, Yi-Fan; Goldstein, David B; Angrist, Misha; Cavalleri, Gianpiero

2014-07-24

Human genetic diversity has long been studied both to understand how genetic variation influences risk of disease and infer aspects of human evolutionary history. In this article, we review historical and contemporary views of human genetic diversity, the rare and common mutations implicated in human disease susceptibility, and the relevance of genetic diversity to personalized medicine. First, we describe the development of thought about diversity through the 20th century and through more modern studies including genome-wide association studies (GWAS) and next-generation sequencing. We introduce several examples, such as sickle cell anemia and Tay-Sachs disease that are caused by rare mutations and are more frequent in certain geographical populations, and common treatment responses that are caused by common variants, such as hepatitis C infection. We conclude with comments about the continued relevance of human genetic diversity in medical genetics and personalized medicine more generally. Copyright © 2014 Cold Spring Harbor Laboratory Press; all rights reserved.

Comprehensive mutational profiling of core binding factor acute myeloid leukemia.

Science.gov (United States)

Duployez, Nicolas; Marceau-Renaut, Alice; Boissel, Nicolas; Petit, Arnaud; Bucci, Maxime; Geffroy, Sandrine; Lapillonne, Hélène; Renneville, Aline; Ragu, Christine; Figeac, Martin; Celli-Lebras, Karine; Lacombe, Catherine; Micol, Jean-Baptiste; Abdel-Wahab, Omar; Cornillet, Pascale; Ifrah, Norbert; Dombret, Hervé; Leverger, Guy; Jourdan, Eric; Preudhomme, Claude

2016-05-19

Acute myeloid leukemia (AML) with t(8;21) or inv(16) have been recognized as unique entities within AML and are usually reported together as core binding factor AML (CBF-AML). However, there is considerable clinical and biological heterogeneity within this group of diseases, and relapse incidence reaches up to 40%. Moreover, translocations involving CBFs are not sufficient to induce AML on its own and the full spectrum of mutations coexisting with CBF translocations has not been elucidated. To address these issues, we performed extensive mutational analysis by high-throughput sequencing in 215 patients with CBF-AML enrolled in the Phase 3 Trial of Systematic Versus Response-adapted Timed-Sequential Induction in Patients With Core Binding Factor Acute Myeloid Leukemia and Treating Patients with Childhood Acute Myeloid Leukemia with Interleukin-2 trials (age, 1-60 years). Mutations in genes activating tyrosine kinase signaling (including KIT, N/KRAS, and FLT3) were frequent in both subtypes of CBF-AML. In contrast, mutations in genes that regulate chromatin conformation or encode members of the cohesin complex were observed with high frequencies in t(8;21) AML (42% and 18%, respectively), whereas they were nearly absent in inv(16) AML. High KIT mutant allele ratios defined a group of t(8;21) AML patients with poor prognosis, whereas high N/KRAS mutant allele ratios were associated with the lack of KIT or FLT3 mutations and a favorable outcome. In addition, mutations in epigenetic modifying or cohesin genes were associated with a poor prognosis in patients with tyrosine kinase pathway mutations, suggesting synergic cooperation between these events. These data suggest that diverse cooperating mutations may influence CBF-AML pathophysiology as well as clinical behavior and point to potential unique pathogenesis of t(8;21) vs inv(16) AML. © 2016 by The American Society of Hematology.

Diversity of [beta]-globin mutations in Israeli ethnic groups reflects recent historic events

Energy Technology Data Exchange (ETDEWEB)

Filon, D.; Oron, V.; Krichevski, S.; Shaag, A.; Goldfarb, A.; Aker, M.; Rachmilewitz, E.A.; Rund, D.; Oppenheim, A. (Hebrew Univ. Hadassah-Medical School, Jerusalem (Israel)) (and others)

1994-05-01

The authors characterized nearly 500 [beta]-thalassemia genes from the Israeli population representing a variety of ethnic subgroups. They found 28 different mutations in the [beta]-globin gene, including three mutations ([beta][sup S], [beta][sup C], and [beta][sup O-Arab]) causing hemoglobinopathies. Marked genetic heterogeneity was observed in both the Arab (20 mutations) and Jewish (17 mutations) populations. On the other hand, two ethnic isolates - Druze and Samaritans - had a single mutation each. Fifteen of the [beta]-thalassemia alleles are Mediterranean in type, 5 originated in Kurdistan, 2 are of Indian origin, and 2 sporadic alleles came from Europe. Only one mutant allele-nonsense codon 37-appears to be indigenous to Israel. While human habitation in Israel dates back to early prehistory, the present-day spectrum of [beta]-globin mutations can be largely explained by migration events that occurred in the past millennium. 26 refs., 2 figs., 3 tabs.

Electroclinical presentation and genotype-phenotype relationships in patients with Unverricht-Lundborg disease carrying compound heterozygous CSTB point and indel mutations.

Science.gov (United States)

Canafoglia, Laura; Gennaro, Elena; Capovilla, Giuseppe; Gobbi, Giuseppe; Boni, Antonella; Beccaria, Francesca; Viri, Maurizio; Michelucci, Roberto; Agazzi, Pamela; Assereto, Stefania; Coviello, Domenico A; Di Stefano, Maria; Rossi Sebastiano, Davide; Franceschetti, Silvana; Zara, Federico

2012-12-01

Unverricht-Lundborg disease (EPM1A) is frequently due to an unstable expansion of a dodecamer repeat in the CSTB gene, whereas other types of mutations are rare. EPM1A due to homozygous expansion has a rather stereotyped presentation with prominent action myoclonus. We describe eight patients with five different compound heterozygous CSTB point or indel mutations in order to highlight their particular phenotypical presentations and evaluate their genotype-phenotype relationships. We screened CSTB mutations by means of Southern blotting and the sequencing of the genomic DNA of each proband. CSTB messenger RNA (mRNA) aberrations were characterized by sequencing the complementary DNA (cDNA) of lymphoblastoid cells, and assessing the protein concentrations in the lymphoblasts. The patient evaluations included the use of a simplified myoclonus severity rating scale, multiple neurophysiologic tests, and electroencephalography (EEG)-polygraphic recordings. To highlight the particular clinical features and disease time-course in compound heterozygous patients, we compared some of their characteristics with those observed in a series of 40 patients carrying the common homozygous expansion mutation observed at the C. Besta Foundation, Milan, Italy. The eight compound heterozygous patients belong to six EPM1A families (out of 52; 11.5%) diagnosed at the Laboratory of Genetics of the Galliera Hospitals in Genoa, Italy. They segregated five different heterozygous point or indel mutations in association with the common dodecamer expansion. Four patients from three families had previously reported CSTB mutations (c.67-1G>C and c.168+1_18del); one had a novel nonsense mutation at the first exon (c.133C>T) leading to a premature stop codon predicting a short peptide; the other three patients from two families had a complex novel indel mutation involving the donor splice site of intron 2 (c.168+2_169+21delinsAA) and leading to an aberrant transcript with a partially retained intron

Rapid mutation of Spirulina platensis by a new mutagenesis system of atmospheric and room temperature plasmas (ARTP and generation of a mutant library with diverse phenotypes.

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Mingyue Fang

Full Text Available In this paper, we aimed to improve the carbohydrate productivity of Spirulina platensis by generating mutants with increased carbohydrate content and growth rate. ARTP was used as a new mutagenesis tool to generate a mutant library of S. platensis with diverse phenotypes. Protocol for rapid mutation of S. platensis by 60 s treatment with helium driven ARTP and high throughput screening method of the mutants using the 96-well microplate and microplate reader was established. A mutant library of 62 mutants was then constructed and ideal mutants were selected out. The characteristics of the mutants after the mutagenesis inclined to be stable after around 9(th subculture, where the total mutation frequency and positive mutation frequency in terms of specific growth rate reached 45% and 25%, respectively. The mutants in mutant library showed diverse phenotypes in terms of cell growth rate, carbohydrate content and flocculation intensity. The positive mutation frequency in terms of cellular carbohydrate content with the increase by more than 20% percent than the wild strain was 32.3%. Compared with the wild strain, the representative mutants 3-A10 and 3-B2 showed 40.3% and 78.0% increase in carbohydrate content, respectively, while the mutant 4-B3 showed 10.5% increase in specific growth rate. The carbohydrate contents of the representative mutants were stable during different subcultures, indicating high genetic stability. ARTP was demonstrated to be an effective and non-GMO mutagenesis tool to generate the mutant library for multicellular microalgae.

Mu Opioid Receptor Gene: New Point Mutations in Opioid Addicts

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Amin Dinarvand

2014-02-01

Full Text Available Introduction: Association between single-nucleotide polymorphisms (SNPs in mu opioid receptor gene and drug addiction has been shown in various studies. Here, we have evaluated the existence of polymorphisms in exon 3 of this gene in Iranian population and investigated the possible association between these mutations and opioid addiction.  Methods: 79 opioid-dependent subjects (55 males, 24 females and 134 non-addict or control individuals (74 males, 60 females participated in the study. Genomic DNA was extracted from volunteers’ peripheral blood and exon 3 of the mu opioid receptor gene was amplified by polymerase chain reaction (PCR whose products were then sequenced.  Results: Three different heterozygote polymorphisms were observed in 3 male individuals: 759T>C and 877G>A mutations were found in 2 control volunteers and 1043G>C substitution was observed in an opioid-addicted subject. Association between genotype and opioid addiction for each mutation was not statistically significant.  Discussion: It seems that the sample size used in our study is not enough to confirm or reject any association between 759T>C, 877G>A and 1043G>C substitutions in exon 3 of the mu opioid receptor gene and opioid addiction susceptibility in Iranian population.

Intrachromosomal amplification, locus deletion and point mutation in the aquaglyceroporin AQP1 gene in antimony resistant Leishmania (Viannia guyanensis.

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Rubens Monte-Neto

2015-02-01

Full Text Available Antimony resistance complicates the treatment of infections caused by the parasite Leishmania.Using next generation sequencing, we sequenced the genome of four independent Leishmania guyanensis antimony-resistant (SbR mutants and found different chromosomal alterations including aneuploidy, intrachromosomal gene amplification and gene deletion. A segment covering 30 genes on chromosome 19 was amplified intrachromosomally in three of the four mutants. The gene coding for the multidrug resistance associated protein A involved in antimony resistance was also amplified in the four mutants, most likely through chromosomal translocation. All mutants also displayed a reduced accumulation of antimony mainly due to genomic alterations at the level of the subtelomeric region of chromosome 31 harboring the gene coding for the aquaglyceroporin 1 (LgAQP1. Resistance involved the loss of LgAQP1 through subtelomeric deletions in three mutants. Interestingly, the fourth mutant harbored a single G133D point mutation in LgAQP1 whose role in resistance was functionality confirmed through drug sensitivity and antimony accumulation assays. In contrast to the Leishmania subspecies that resort to extrachromosomal amplification, the Viannia strains studied here used intrachromosomal amplification and locus deletion.This is the first report of a naturally occurred point mutation in AQP1 in antimony resistant parasites.

RAC1 Missense Mutations in Developmental Disorders with Diverse Phenotypes.

Science.gov (United States)

Reijnders, Margot R F; Ansor, Nurhuda M; Kousi, Maria; Yue, Wyatt W; Tan, Perciliz L; Clarkson, Katie; Clayton-Smith, Jill; Corning, Ken; Jones, Julie R; Lam, Wayne W K; Mancini, Grazia M S; Marcelis, Carlo; Mohammed, Shehla; Pfundt, Rolph; Roifman, Maian; Cohn, Ronald; Chitayat, David; Millard, Tom H; Katsanis, Nicholas; Brunner, Han G; Banka, Siddharth

2017-09-07

RAC1 is a widely studied Rho GTPase, a class of molecules that modulate numerous cellular functions essential for normal development. RAC1 is highly conserved across species and is under strict mutational constraint. We report seven individuals with distinct de novo missense RAC1 mutations and varying degrees of developmental delay, brain malformations, and additional phenotypes. Four individuals, each harboring one of c.53G>A (p.Cys18Tyr), c.116A>G (p.Asn39Ser), c.218C>T (p.Pro73Leu), and c.470G>A (p.Cys157Tyr) variants, were microcephalic, with head circumferences between -2.5 to -5 SD. In contrast, two individuals with c.151G>A (p.Val51Met) and c.151G>C (p.Val51Leu) alleles were macrocephalic with head circumferences of +4.16 and +4.5 SD. One individual harboring a c.190T>G (p.Tyr64Asp) allele had head circumference in the normal range. Collectively, we observed an extraordinary spread of ∼10 SD of head circumferences orchestrated by distinct mutations in the same gene. In silico modeling, mouse fibroblasts spreading assays, and in vivo overexpression assays using zebrafish as a surrogate model demonstrated that the p.Cys18Tyr and p.Asn39Ser RAC1 variants function as dominant-negative alleles and result in microcephaly, reduced neuronal proliferation, and cerebellar abnormalities in vivo. Conversely, the p.Tyr64Asp substitution is constitutively active. The remaining mutations are probably weakly dominant negative or their effects are context dependent. These findings highlight the importance of RAC1 in neuronal development. Along with TRIO and HACE1, a sub-category of rare developmental disorders is emerging with RAC1 as the central player. We show that ultra-rare disorders caused by private, non-recurrent missense mutations that result in varying phenotypes are challenging to dissect, but can be delineated through focused international collaboration. Copyright © 2017 American Society of Human Genetics. Published by Elsevier Inc. All rights reserved.

Experimental estimation of mutation rates in a wheat population with a gene genealogy approach.

Science.gov (United States)

Raquin, Anne-Laure; Depaulis, Frantz; Lambert, Amaury; Galic, Nathalie; Brabant, Philippe; Goldringer, Isabelle

2008-08-01

Microsatellite markers are extensively used to evaluate genetic diversity in natural or experimental evolving populations. Their high degree of polymorphism reflects their high mutation rates. Estimates of the mutation rates are therefore necessary when characterizing diversity in populations. As a complement to the classical experimental designs, we propose to use experimental populations, where the initial state is entirely known and some intermediate states have been thoroughly surveyed, thus providing a short timescale estimation together with a large number of cumulated meioses. In this article, we derived four original gene genealogy-based methods to assess mutation rates with limited bias due to relevant model assumptions incorporating the initial state, the number of new alleles, and the genetic effective population size. We studied the evolution of genetic diversity at 21 microsatellite markers, after 15 generations in an experimental wheat population. Compared to the parents, 23 new alleles were found in generation 15 at 9 of the 21 loci studied. We provide evidence that they arose by mutation. Corresponding estimates of the mutation rates ranged from 0 to 4.97 x 10(-3) per generation (i.e., year). Sequences of several alleles revealed that length polymorphism was only due to variation in the core of the microsatellite. Among different microsatellite characteristics, both the motif repeat number and an independent estimation of the Nei diversity were correlated with the novel diversity. Despite a reduced genetic effective size, global diversity at microsatellite markers increased in this population, suggesting that microsatellite diversity should be used with caution as an indicator in biodiversity conservation issues.

Distinct pattern of p53 mutations in bladder cancer

DEFF Research Database (Denmark)

Spruck, C H; Rideout, W M; Olumi, A F

1993-01-01

A distinct mutational spectrum for the p53 tumor suppressor gene in bladder carcinomas was established in patients with known exposures to cigarette smoke. Single-strand conformational polymorphism analysis of exons 5 through 8 of the p53 gene showed inactivating mutations in 16 of 40 (40%) bladder...... tumors from smokers and 13 of 40 (33%) tumors from lifetime nonsmokers. Overall, 13 of the 50 (26%) total point mutations discovered in this and previous work were G:C-->C:G transversions, a relatively rare mutational type in human tumors. In six tumors, identical AGA (Arg)-->ACA (Thr) point mutations...... double mutations, four of which were tandem mutations on the same allele. No double mutations were found in tumors from nonsmoking patients. None of the mutations in smokers were G:C-->T:A transversions, which would be anticipated for exposure to the suspected cigarette smoke carcinogen 4-aminobiphenyl...

Mutation breeding in malting barley

Energy Technology Data Exchange (ETDEWEB)

Hiraki, Makoto; Sanada, Matsuyoshi

1984-03-01

The released varieties of malting barley through mutation breeding is more than ten in number, including foreign varieties. In Japan four varieties has been released so far. We started mutation breeding in 1956 together with cross breeding that we employed before. Until now, Gamma 4, Amagi Nijo 1 and Fuji Nijo 2 have been produced from the direct use of induced mutations and Nirasaki Nijo 8 from the indirect use of them. Mutation breeding has been used mainly in the partial improvement of agronomic characteristics since the selection for malting quality was very complicated. As the variety bred by induced mutation is usually equivalent to the original variety in malting quality, both this new variety and the original one could be cultivated in the same area without any problem on later malt production. Particularly when one farmer cultivates barley in an extensive acreage, he can harvest at the best time according to the different maturing time of each variety. From these points of view, mutation breeding is an efficient tool in malting barley breeding. Mutagens we have used so far are X-rays, ..gamma..-rays, neutron and chemicals such as dES. From our experience in selection, the low dose of radiation and chemical mutagens are more effective in selection of point mutation than the high dose of radiation which tends to produce many abnormal but few practical mutants. (author).

Rapid point-of-care testing for epidermal growth factor receptor gene mutations in patients with lung cancer using cell-free DNA from cytology specimen supernatants.

Science.gov (United States)

Asaka, Shiho; Yoshizawa, Akihiko; Saito, Kazusa; Kobayashi, Yukihiro; Yamamoto, Hiroshi; Negishi, Tatsuya; Nakata, Rie; Matsuda, Kazuyuki; Yamaguchi, Akemi; Honda, Takayuki

2018-06-01

Epidermal growth factor receptor (EGFR) mutations are associated with responses to EGFR tyrosine kinase inhibitors (EGFR-TKIs) in non-small-cell lung cancer (NSCLC). Our previous study revealed a rapid point-of-care system for detecting EGFR mutations. This system analyzes cell pellets from cytology specimens using droplet-polymerase chain reaction (d-PCR), and has a reaction time of 10 min. The present study aimed to validate the performance of the EGFR d-PCR assay using cell-free DNA (cfDNA) from supernatants obtained from cytology specimens. Assay results from cfDNA supernatant analyses were compared with those from cell pellets for 90 patients who were clinically diagnosed with, or suspected of having, lung cancer (80 bronchial lavage fluid samples, nine pleural effusion samples and one spinal fluid sample). EGFR mutations were identified in 12 and 15 cases using cfDNA supernatants and cell pellets, respectively. The concordance rates between cfDNA-supernatant and cell‑pellet assay results were 96.7% [kappa coefficient (K)=0.87], 98.9% (K=0.94), 98.9% (K=0.79) and 98.9% (K=0.79) for total EGFR mutations, L858R, E746_A750del and T790M, respectively. All 15 patients with EGFR mutation-positive results, as determined by EGFR d-PCR assay using cfDNA supernatants or cell pellets, also displayed positive results by conventional EGFR assays using tumor tissue or cytology specimens. Notably, EGFR mutations were even detected in five cfDNA supernatants for which the cytological diagnoses of the corresponding cell pellets were 'suspicious for malignancy', 'atypical' or 'negative for malignancy.' In conclusion, this rapid point-of-care system may be considered a promising novel screening method that may enable patients with NSCLC to receive EGFR-TKI therapy more rapidly, whilst also reserving cell pellets for additional morphological and molecular analyses.

Roles of Mitochondrial DNA Mutations in Stem Cell Ageing

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Tianhong Su

2018-03-01

Full Text Available Mitochondrial DNA (mtDNA mutations accumulate in somatic stem cells during ageing and cause mitochondrial dysfunction. In this review, we summarize the studies that link mtDNA mutations to stem cell ageing. We discuss the age-related behaviours of the somatic mtDNA mutations in stem cell populations and how they potentially contribute to stem cell ageing by altering mitochondrial properties in humans and in mtDNA-mutator mice. We also draw attention to the diverse fates of the mtDNA mutations with different origins during ageing, with potential selective pressures on the germline inherited but not the somatic mtDNA mutations.

New parkin mutations and atypical phenotypes in families with autosomal recessive parkinsonism.

NARCIS (Netherlands)

Rawal, N.; Periquet, M.; Lohmann, E.; Lucking, C.B.; Teive, H.; Ambrosio, G.; Raskin, S.; Lincoln, S.; Hattori, N.; Guimaraes, J.; Horstink, M.W.I.M.; Santos Bele, W. Dos; Brousolle, E.; Destee, A.; Mizuno, Y.; Farrer, M.; Deleuze, J.F.; Michele, G. de; Agid, Y.; Durr, A.; Brice, A.

2003-01-01

The frequency of parkin mutations was evaluated in 30 families of highly diverse geographic origin with early-onset autosomal recessive parkinsonism. Twelve different mutations, six of which were new, were found in 10 families from Europe and Brazil. Patients with parkin mutations had significantly

Single point mutations distributed in 10 soluble and membrane regions of the Nicotiana plumbaginifolia plasma membrane PMA2 H+-ATPase activate the enzyme and modify the structure of the C-terminal region.

Science.gov (United States)

Morsomme, P; Dambly, S; Maudoux, O; Boutry, M

1998-12-25

The Nicotiana plumbaginifolia pma2 (plasma membrane H+-ATPase) gene is capable of functionally replacing the H+-ATPase genes of the yeast Saccharomyces cerevisiae, provided that the external pH is kept above 5.0. Single point mutations within the pma2 gene were previously identified that improved H+-ATPase activity and allowed yeast growth at pH 4.0. The aim of the present study was to identify most of the PMA2 positions, the mutation of which would lead to improved growth and to determine whether all these mutations result in similar enzymatic and structural modifications. We selected additional mutants in total 42 distinct point mutations localized in 30 codons. They were distributed in 10 soluble and membrane regions of the enzyme. Most mutant PMA2 H+-ATPases were characterized by a higher specific activity, lower inhibition by ADP, and lower stimulation by lysophosphatidylcholine than wild-type PMA2. The mutants thus seem to be constitutively activated. Partial tryptic digestion and immunodetection showed that the PMA2 mutants had a conformational change making the C-terminal region more accessible. These data therefore support the hypothesis that point mutations in various H+-ATPase parts displace the inhibitory C-terminal region, resulting in enzyme activation. The high density of mutations within the first half of the C-terminal region suggests that this part is involved in the interaction between the inhibitory C-terminal region and the rest of the enzyme.

Galactosemia caused by a point mutation that activates cryptic donor splice site in the galactose-1-phosphate uridyltransferase gene

Energy Technology Data Exchange (ETDEWEB)

Wadelius, C.; Lagerkvist, A. (Univ. Hospital, Uppsala (Sweden) Uppsala Univ. (Sweden)); Molin, A.K.; Larsson, A. (Univ. Hospital, Uppsala (Sweden)); Von Doebeln, U. (Karolinska Institute, Stockholm (Sweden))

1993-08-01

Galactosemia affects 1/84,000 in Sweden and is manifested in infancy when the child is exposed to galactose in the diet. If untreated there is a risk of severe early symptoms and, even with a lactose-free diet, late symptoms such as mental retardation and ovarial dysfunction may develop. In classical galactosemia, galactose-1-phosphate uridyltransferase (GALT) (EC 2.7.7.12) is defective and the normal cDNA sequence of this enzyme has been characterized. Recently eight mutations leading to galactosemia were published. Heparinized venous blood was drawn from a patient with classical galactosemia. In the cDNA from the patient examined, an insertion of 54 bp was found at position 1087. Amplification of the relevant genomic region of the patient's DNA was performed. Exon-intron boundaries and intronic sequences thus determined revealed that the 54-bp insertion was located immediately downstream of exon 10. It was further found that the patient was heterozygous for a point mutation, changing a C to a T (in 5 of 9 clones) at the second base in the intron downstream of the insertion. This alteration creates a sequence which, as well as the ordinary splice site, differs in only two positions from the consensus sequence. It was found that the mutation occurred in only one of the 20 alleles from galactosemic patients and in none of the 200 alleles from normal controls. The mutation is inherited from the mother, who also was found to express the 54-bp-long insertion at the mRNA level. Sequences from the 5[prime] end of the coding region were determined after genomic amplification, revealing a sequence identical to that reported. The mutation on the paternal allele has not been identified. 9 refs., 1 fig.

Aphid thermal tolerance is governed by a point mutation in bacterial symbionts.

Directory of Open Access Journals (Sweden)

Helen E Dunbar

2007-05-01

Full Text Available Symbiosis is a ubiquitous phenomenon generating biological complexity, affecting adaptation, and expanding ecological capabilities. However, symbionts, which can be subject to genetic limitations such as clonality and genomic degradation, also impose constraints on hosts. A model of obligate symbiosis is that between aphids and the bacterium Buchnera aphidicola, which supplies essential nutrients. We report a mutation in Buchnera of the aphid Acyrthosiphon pisum that recurs in laboratory lines and occurs in field populations. This single nucleotide deletion affects a homopolymeric run within the heat-shock transcriptional promoter for ibpA, encoding a small heat-shock protein. This Buchnera mutation virtually eliminates the transcriptional response of ibpA to heat stress and lowers its expression even at cool or moderate temperatures. Furthermore, this symbiont mutation dramatically affects host fitness in a manner dependent on thermal environment. Following a short heat exposure as juveniles, aphids bearing short-allele symbionts produced few or no progeny and contained almost no Buchnera, in contrast to aphids bearing symbionts without the deletion. Conversely, under constant cool conditions, aphids containing symbionts with the short allele reproduced earlier and maintained higher reproductive rates. The short allele has appreciable frequencies in field populations (up to 20%, further supporting the view that lowering of ibpA expression improves host fitness under some conditions. This recurring Buchnera mutation governs thermal tolerance of aphid hosts. Other cases in which symbiont microevolution has a major effect on host ecological tolerance are likely to be widespread because of the high mutation rates of symbiotic bacteria and their crucial roles in host metabolism and development.

Loss and recovery of genetic diversity in adapting populations of HIV.

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Pleuni S Pennings

2014-01-01

Full Text Available The evolution of drug resistance in HIV occurs by the fixation of specific, well-known, drug-resistance mutations, but the underlying population genetic processes are not well understood. By analyzing within-patient longitudinal sequence data, we make four observations that shed a light on the underlying processes and allow us to infer the short-term effective population size of the viral population in a patient. Our first observation is that the evolution of drug resistance usually occurs by the fixation of one drug-resistance mutation at a time, as opposed to several changes simultaneously. Second, we find that these fixation events are accompanied by a reduction in genetic diversity in the region surrounding the fixed drug-resistance mutation, due to the hitchhiking effect. Third, we observe that the fixation of drug-resistance mutations involves both hard and soft selective sweeps. In a hard sweep, a resistance mutation arises in a single viral particle and drives all linked mutations with it when it spreads in the viral population, which dramatically reduces genetic diversity. On the other hand, in a soft sweep, a resistance mutation occurs multiple times on different genetic backgrounds, and the reduction of diversity is weak. Using the frequency of occurrence of hard and soft sweeps we estimate the effective population size of HIV to be 1.5 x 10(5 (95% confidence interval [0.8 x 10(5,4.8 x 10(5]. This number is much lower than the actual number of infected cells, but much larger than previous population size estimates based on synonymous diversity. We propose several explanations for the observed discrepancies. Finally, our fourth observation is that genetic diversity at non-synonymous sites recovers to its pre-fixation value within 18 months, whereas diversity at synonymous sites remains depressed after this time period. These results improve our understanding of HIV evolution and have potential implications for treatment strategies.

50 Detecting adenosine triphosphatase 6 point mutations that may ...

African Journals Online (AJOL)

mutations at codons for the key residues Lys 260, Leu263, Gln266, Ser769 .... agarose gel and visualized under UV transillumination after treatment with ..... Li, W., Mo, W., Shen, D., Sun, L., Wang, J., Lu, S., Gitschier, J.M. & Zhou, B. (2005) Yeast ... Nagamune, K., Beatty, W.L., & Sibley, D. (2007) Artemisinin induces Calcium ...

Rare beneficial mutations can halt Muller's ratchet

Science.gov (United States)

Balick, Daniel; Goyal, Sidhartha; Jerison, Elizabeth; Neher, Richard; Shraiman, Boris; Desai, Michael

2012-02-01

In viral, bacterial, and other asexual populations, the vast majority of non-neutral mutations are deleterious. This motivates the application of models without beneficial mutations. Here we show that the presence of surprisingly few compensatory mutations halts fitness decay in these models. Production of deleterious mutations is balanced by purifying selection, stabilizing the fitness distribution. However, stochastic vanishing of fitness classes can lead to slow fitness decay (i.e. Muller's ratchet). For weakly deleterious mutations, production overwhelms purification, rapidly decreasing population fitness. We show that when beneficial mutations are introduced, a stable steady state emerges in the form of a dynamic mutation-selection balance. We argue this state is generic for all mutation rates and population sizes, and is reached as an end state as genomes become saturated by either beneficial or deleterious mutations. Assuming all mutations have the same magnitude selective effect, we calculate the fraction of beneficial mutations necessary to maintain the dynamic balance. This may explain the unexpected maintenance of asexual genomes, as in mitochondria, in the presence of selection. This will affect in the statistics of genetic diversity in these populations.

Natural Selection Reduced Diversity on Human Y Chromosomes

Science.gov (United States)

Wilson Sayres, Melissa A.; Lohmueller, Kirk E.; Nielsen, Rasmus

2014-01-01

The human Y chromosome exhibits surprisingly low levels of genetic diversity. This could result from neutral processes if the effective population size of males is reduced relative to females due to a higher variance in the number of offspring from males than from females. Alternatively, selection acting on new mutations, and affecting linked neutral sites, could reduce variability on the Y chromosome. Here, using genome-wide analyses of X, Y, autosomal and mitochondrial DNA, in combination with extensive population genetic simulations, we show that low observed Y chromosome variability is not consistent with a purely neutral model. Instead, we show that models of purifying selection are consistent with observed Y diversity. Further, the number of sites estimated to be under purifying selection greatly exceeds the number of Y-linked coding sites, suggesting the importance of the highly repetitive ampliconic regions. While we show that purifying selection removing deleterious mutations can explain the low diversity on the Y chromosome, we cannot exclude the possibility that positive selection acting on beneficial mutations could have also reduced diversity in linked neutral regions, and may have contributed to lowering human Y chromosome diversity. Because the functional significance of the ampliconic regions is poorly understood, our findings should motivate future research in this area. PMID:24415951

Sequence diversity and copy number variation of Mutator-like transposases in wheat

Directory of Open Access Journals (Sweden)

Nobuaki Asakura

2008-01-01

Full Text Available Partial transposase-coding sequences of Mutator-like elements (MULEs were isolated from a wild einkorn wheat, Triticum urartu, by degenerate PCR. The isolated sequences were classified into a MuDR or Class I clade and divided into two distinct subclasses (subclass I and subclass II. The average pair-wise identity between members of both subclasses was 58.8% at the nucleotide sequence level. Sequence diversity of subclass I was larger than that of subclass II. DNA gel blot analysis showed that subclass I was present as low copy number elements in the genomes of all Triticum and Aegilops accessions surveyed, while subclass II was present as high copy number elements. These two subclasses seemed uncapable of recognizing each other for transposition. The number of copies of subclass II elements was much higher in Aegilops with the S, Sl and D genomes and polyploid Triticum species than in diploid Triticum with the A genome, indicating that active transposition occurred in S, Sl and D genomes before polyploidization. DNA gel blot analysis of six species selected from three subfamilies of Poaceae demonstrated that only the tribe Triticeae possessed both subclasses. These results suggest that the differentiation of these two subclasses occurred before or immediately after the establishment of the tribe Triticeae.

A new mitochondrial point mutation in the transfer RNA(Lys) gene associated with progressive external ophthalmoplegia with impaired respiratory regulation.

Science.gov (United States)

Wolf, Joachim; Obermaier-Kusser, Bert; Jacobs, Martina; Milles, Cornelia; Mörl, Mario; von Pein, Harald D; Grau, Armin J; Bauer, Matthias F

2012-05-15

We report a novel heteroplasmic point mutation G8299A in the gene for mitochondrial tRNA(Lys) in a patient with progressive external ophthalmoplegia complicated by recurrent respiratory insufficiency. Biochemical analysis of respiratory chain complexes in muscle homogenate showed a combined complex I and IV deficiency. The transition does not represent a known neutral polymorphism and affects a position in the tRNA acceptor stem which is conserved in primates, leading to a destabilization of this functionally important domain. In vitro analysis of an essential maturation step of the tRNA transcript indicates the probable pathogenicity of this mutation. We hypothesize that there is a causal relationship between the novel G8299A transition and progressive external ophthalmoplegia with recurrent respiratory failure due to a depressed respiratory drive. Copyright © 2012 Elsevier B.V. All rights reserved.

Multiplex Detection and Genotyping of Point Mutations Involved in Charcot-Marie-Tooth Disease Using a Hairpin Microarray-Based Assay

Directory of Open Access Journals (Sweden)

Yasser Baaj

2009-01-01

Full Text Available We previously developed a highly specific method for detecting SNPs with a microarray-based system using stem-loop probes. In this paper we demonstrate that coupling a multiplexing procedure with our microarray method is possible for the simultaneous detection and genotyping of four point mutations, in three different genes, involved in Charcot-Marie-Tooth disease. DNA from healthy individuals and patients was amplified, labeled with Cy3 by multiplex PCR; and hybridized to microarrays. Spot signal intensities were 18 to 74 times greater for perfect matches than for mismatched target sequences differing by a single nucleotide (discrimination ratio for “homozygous” DNA from healthy individuals. “Heterozygous” mutant DNA samples gave signal intensity ratios close to 1 at the positions of the mutations as expected. Genotyping by this method was therefore reliable. This system now combines the principle of highly specific genotyping based on stem-loop structure probes with the advantages of multiplex analysis.

Identification of weak points prone for mutation in ferredoxin of Trichomonas vaginalis

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Wiwanitkit V

2008-01-01

Full Text Available Trichomonas vaginalis , the causative agent for human trichomoniasis, is a problematic sexually transmitted disease mainly in women. At present, metronidazole-resistant trichomoniasis is an infrequent but challenging problem with no universally successful treatment. Genetic mutation is believed to be an important factor leading to increasing drug resistance. Understanding the mutation status will help to design accurate strategies of therapy against mutant strains of T. vaginalis . The author performed a bioinformatic analysis to determine positions that tend to comply peptide motifs in the amino acid sequence of ferridoxin of T. vaginalis . Based on this study, the weak linkages in the studied protein can be identified and can be useful information for prediction of possible new mutations that can lead to drug resistance. In addition, the results from this study can be good information for further research on the diagnosis for mutants and new effective drug development.

Repair-resistant mutation in Neurospora

International Nuclear Information System (INIS)

Stadler, D.; Macleod, H.; Loo, M.

1987-01-01

Chronic UV treatment produces severalfold fewer mutations in Neurospora conidia than does the same total dose of acute UV. Experiments were designed to determine the conditions required for chronic UV mutagenesis. Measurement of the coincidence frequency for two independent mutations revealed the existence of a subset of cells which are mutable by chronic UV. Analysis of forward mutation at the mtr locus showed that the genetic alterations produced by chronic UV were virtually all point mutants, even though the assay system could detect alterations or deletions extending into neighboring genes. A significant fraction of the mutants produced by acute UV were multigenic deletions. The size of the dose-rate effect (acute UV mutation frequency divided by chronic UV mutation frequency) was compared for several different mutation assay systems. Forward mutations (recessive lethals and mtr) gave values ranging from four to nine. For events which were restricted to specific molecular sites (specific reversions and nonsense suppressor mutations), there was a wider range of dose-rate ratios. This suggests that chronic UV mutation may be restricted to certain molecular sequences or configurations

Assessment of genetic diversity and variation of acer mono max seedlings after spaceflight

International Nuclear Information System (INIS)

Long, C.; Li, Y.; Zhang, Y.; Yang, M.

2015-01-01

Genetic diversity and variation of Acer Mono Maxim seedlings sampled from space-mutated (sm) populations were compared to seedlings from parallel control (ck) ones using molecular markers. RAMP analysis showed that the percentage of polymorphic band, Shannon diversity index and Nei gene diversity index of the space-mutated populations were higher than those of the control ones, which indicated that genetic variation increased after spaceflight in populations of Acer Mono Maxim. By using un-weighted pair group method with arithmetic mean (UPGMA) method, three space-mutated repeats (populations) were clustered together, and control groups clustered separately, which further indicated that there was difference between the space-mutated ones and the control ones, which may be caused by space mutation. Further analysis of genomic inconsistency between the root and leaf samples from the same tree showed that a total variation rate of 6.3% and 1.7% were obtained in ten space-mutated individuals by using RAMP and SSR markers, respectively, however, the variation rate was zero in control ones. It provided that space mutation may be caused the individual variation of Acer Mono Maxim. (author)

A point mutation of human p53, which was not detected as a mutation by a yeast functional assay, led to apoptosis but not p21Waf1/Cip1/Sdi1 expression in response to ionizing radiation in a human osteosarcoma cell line, Saos-2

International Nuclear Information System (INIS)

Okaichi, Kumio; Wang Lihong; Sasaki, Ji-ichiro; Saya, Hideyuki; Tada, Mitsuhiro; Okumura, Yutaka

1999-01-01

Purpose: The 123A point mutation of p53 showed increased radiosensitivity, whereas other mutations (143A, 175H, and 273H) were not affected. To determine the reason for increased radiosensitivity of the 123A mutation, the response of the transformant of 123A mutation to ionizing radiation (IR) was examined and compared to those of transformants with the wild type p53 or other point mutations (143A, 175H, and 273H). Methods and Materials: Stable transformants with a mutant or wild type p53 made by introducing cDNA into the human osteosarcoma cell line, Saos-2, which lacks an endogenous p53 were used. The transcriptional activity of mutant p53 was examined using a yeast functional assay. The transformants were examined for the accumulation of p53, the induction of p21 Waf1/Cip1/Sdi1 (hereafter referred to as p21), and the other response of p53-responsive genes (MDM2, Bax, and Bcl-2) by Western blotting. Apoptosis was analyzed by detection of DNA fragmentation. Results: The 123A point mutation of p53 was detected as a wild type in the yeast functional assay. The 123A mutant accumulated p53 in response to IR. The 123A mutant did not induce p21, but normally responded to MDM2, Bax, and Bcl-2. The 123A mutant entered apoptosis earlier than the wild type p53 transformant, and induced Fas at earlier in response to IR. Conclusion: The 123A mutant led to apoptosis, but not p21 expression in response to IR. The occurrence of apoptosis, but not induction of p21, corresponded to the radiosensitivity in the transformant. The early occurrence of apoptosis in 123A transformants may depend on the early induction of Fas

The role of viral population diversity in adaptation of bovine coronavirus to new host environments.

Directory of Open Access Journals (Sweden)

Monica K Borucki

Full Text Available The high mutation rate of RNA viruses enables a diverse genetic population of viral genotypes to exist within a single infected host. In-host genetic diversity could better position the virus population to respond and adapt to a diverse array of selective pressures such as host-switching events. Multiple new coronaviruses, including SARS, have been identified in human samples just within the last ten years, demonstrating the potential of coronaviruses as emergent human pathogens. Deep sequencing was used to characterize genomic changes in coronavirus quasispecies during simulated host-switching. Three bovine nasal samples infected with bovine coronavirus were used to infect human and bovine macrophage and lung cell lines. The virus reproduced relatively well in macrophages, but the lung cell lines were not infected efficiently enough to allow passage of non lab-adapted samples. Approximately 12 kb of the genome was amplified before and after passage and sequenced at average coverages of nearly 950×(454 sequencing and 38,000×(Illumina. The consensus sequence of many of the passaged samples had a 12 nucleotide insert in the consensus sequence of the spike gene, and multiple point mutations were associated with the presence of the insert. Deep sequencing revealed that the insert was present but very rare in the unpassaged samples and could quickly shift to dominate the population when placed in a different environment. The insert coded for three arginine residues, occurred in a region associated with fusion entry into host cells, and may allow infection of new cell types via heparin sulfate binding. Analysis of the deep sequencing data indicated that two distinct genotypes circulated at different frequency levels in each sample, and support the hypothesis that the mutations present in passaged strains were "selected" from a pre-existing pool rather than through de novo mutation and subsequent population fixation.

IFITM5 mutations and osteogenesis imperfecta.

Science.gov (United States)

Hanagata, Nobutaka

2016-03-01

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

Effects of Point Mutations in the Major Capsid Protein of Beet Western Yellows Virus on Capsid Formation, Virus Accumulation, and Aphid Transmission

Science.gov (United States)

Brault, V.; Bergdoll, M.; Mutterer, J.; Prasad, V.; Pfeffer, S.; Erdinger, M.; Richards, K. E.; Ziegler-Graff, V.

2003-01-01

Point mutations were introduced into the major capsid protein (P3) of cloned infectious cDNA of the polerovirus beet western yellows virus (BWYV) by manipulation of cloned infectious cDNA. Seven mutations targeted sites on the S domain predicted to lie on the capsid surface. An eighth mutation eliminated two arginine residues in the R domain, which is thought to extend into the capsid interior. The effects of the mutations on virus capsid formation, virus accumulation in protoplasts and plants, and aphid transmission were tested. All of the mutants replicated in protoplasts. The S-domain mutant W166R failed to protect viral RNA from RNase attack, suggesting that this particular mutation interfered with stable capsid formation. The R-domain mutant R7A/R8A protected ∼90% of the viral RNA strand from RNase, suggesting that lower positive-charge density in the mutant capsid interior interfered with stable packaging of the complete strand into virions. Neither of these mutants systemically infected plants. The six remaining mutants properly packaged viral RNA and could invade Nicotiana clevelandii systemically following agroinfection. Mutant Q121E/N122D was poorly transmitted by aphids, implicating one or both targeted residues in virus-vector interactions. Successful transmission of mutant D172N was accompanied either by reversion to the wild type or by appearance of a second-site mutation, N137D. This finding indicates that D172 is also important for transmission but that the D172N transmission defect can be compensated for by a “reverse” substitution at another site. The results have been used to evaluate possible structural models for the BWYV capsid. PMID:12584348

Plant mutation reports. Vol. 1, No. 3, December 2007

International Nuclear Information System (INIS)

2007-12-01

This issue of the Plant Mutation Reports emphasises the recently concluded IAEA-RCA project on Mutant Multi-location Trial and Mutational Enhancement of genetic Diversity (RAS/5/040) which has developed 33 mutant varieties and dozens of promising mutant breeding lines with high yield potantial and/or improved agronomic characters, resistance and end user quality traits

Simultaneous DNA and RNA mapping of somatic mitochondrial mutations across diverse human cancers

DEFF Research Database (Denmark)

Stewart, James B.; Alaei-Mahabadi, Babak; Radhakrishnan, Sabarinathan

2015-01-01

of evidence from both genomic and transcriptomic sequencing. We find that there is selective pressure against deleterious coding mutations, supporting that functional mitochondria are required in tumor cells, and also observe a strong mutational strand bias, compatible with endogenous replication...

Epilepsy caused by CDKL5 mutations.

Science.gov (United States)

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

2011-01-01

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

RGE of fission neutrons under the recessive mutation induction in Drosophila Melanogaster

International Nuclear Information System (INIS)

Aleksandrov, I.D.; Aleksandrova, M.V.; Lapidus, I.L.; Korablinova, S.V.; )

2001-01-01

The RCR-analysis of 81 γ- and neutron-induced vg recessive mutations in ripe sperm of Drosophila melanogaster males of combined with complementation assay with the vg[nw83b27] deletion mutation is used to detect precisely the RGE values of neutrons (0.85 MeV) under the chromosome and point mutation induction. The results obtained show that all genetic end-points increase linearly with γ-ray and neutron dose. Thereby, the efficacy of neutrons is found to be twice (and more) as large as that of γ-rays under the all macro- and micro-aberration mutation induction. Unlike that, the RGE of neutrons are more than twice as low as that of γ-rays under the gene/point mutation induction [ru

Photodynamic antisense regulation of mRNA having a point mutation with psoralen-conjugated oligonucleotide.

Science.gov (United States)

Higuchi, Maiko; Yamayoshi, Asako; Kobori, Akio; Murakami, Akira

2008-01-01

Nucleic acid-based drugs, such as antisense oligonucleotide, ribozyme, and small interfering RNA, are specific compounds that inhibit gene expression at the post-transcriptional level. To develop more effective nucleic acid-based drugs, we focused on photo-reactive antisense oligonucleotides. We have optimized the structure of psoralen-conjugated oligonucleotide to improve their sequence selectivity and photo-crosslinking efficiency. Previously, we reported that photo reactive oligonucleotides containing 2'-O-psoralenyl-methoxyethyl adenosine (2'-Ps-eom) showed drastic photo-reactivity with a strictly sequence specific manner in vitro. In this report, we evaluated the binding ability toward intracellular target mRNA. The 2'-Ps-eom selectively photo-cross-linked to the target mRNA extracted from cells. The 2'-Ps-eom also cross-linked to target mRNA in cells. Furthermore, 2'-Ps-eom did not cross-link to mRNA having a mismatch base. These results suggest that 2'-Ps-eom is a powerful antisense molecule to inhibit the expression of mRNA having a point mutation.

The SHOX region and its mutations.

Science.gov (United States)

Capone, L; Iughetti, L; Sabatini, S; Bacciaglia, A; Forabosco, A

2010-06-01

The short stature homeobox-containing (SHOX) gene lies in the pseudoautosomal region 1 (PAR1) that comprises 2.6 Mb of the short-arm tips of both the X and Y chromosomes. It is known that its heterozygous mutations cause Leri-Weill dyschondrosteosis (LWD) (OMIM #127300), while its homozygous mutations cause a severe form of dwarfism known as Langer mesomelic dysplasia (LMD) (OMIM #249700). The analysis of 238 LWD patients between 1998 and 2007 by multiple authors shows a prevalence of deletions (46.4%) compared to point mutations (21.2%). On the whole, deletions and point mutations account for about 67% of LWD patients. SHOX is located within a 1000 kb desert region without genes. The comparative genomic analysis of this region between genomes of different vertebrates has led to the identification of evolutionarily conserved non-coding DNA elements (CNE). Further functional studies have shown that one of these CNE downstream of the SHOX gene is necessary for the expression of SHOX; this is considered to be typical "enhancer" activity. Including the enhancer, the overall mutation of the SHOX region in LWD patients does not hold in 100% of cases. Various authors have demonstrated the existence of other CNE both downstream and upstream of SHOX regions. The resulting conclusion is that it is necessary to reanalyze all LWD/LMD patients without SHOX mutations for the presence of mutations in the 5'- and 3'-flanking SHOX regions.

In vivo somatic mutation systems in the mouse

International Nuclear Information System (INIS)

Russell, L.B.

1979-01-01

In an effort to meet the need for a fast and cheap in vivo prescreen for inherited mammalian point mutations, a somatic forward-mutation method, originally developed in an x-ray experiment, has more recently been tested in work with chemical mutagens. The method makes use of coat-color mutations because the gene product is usually locally expressed, mosaics can be detected with minimal effort, and opportunities for making comparison with induction of germinal point mutations are greatest.--Following treatment of embryos that are heterozygous at specific coat-color loci, various induced genetic changes can result in expression of the recessive (RS) in clones derived from mutant melanocyte precursor cells. However, other events, such as decrease in the number of precursor cells, or disturbed differentiation, can also result in spots, which with careful classification can usually be distinguished from RS's on the basis of their location and color. When this is done, the relative RS frequencies for a series of compounds at least roughly parallel the relative spermatogonial mutation rates. The fact that easily measurable (though low) RS rates are obtained with compounds that have yielded negative results in spermatogonial tests is not surprising in view of the fact that RS's can be caused by several mechanisms besides point mutation.--In spite of the parallelism observed in one laboratory, the usefulness of the in vivo somatic mutation method as a prescreen could come to be doubted because of major discrepancies between results of similar experiments at different laboratories. However, It appears probable that at least some of these discrepancies are due to failure to discriminate between spots that probably resulted from melanocyte insufficiency and spots that resulted from expression of the recessive.--Reverse somatic mutation systems can potentially avoid some of the pitfalls of forward mutation systems. Such system are still in developmental stages

Skin pH, Atopic Dermatitis, and Filaggrin Mutations

DEFF Research Database (Denmark)

Bandier, Josefine; Johansen, Jeanne Duus; Petersen, Lars Jelstrup

2014-01-01

mutations may influence skin pH. OBJECTIVE: We aimed to determine the epidermal pH in different groups stratified by filaggrin mutations and atopic dermatitis. Further, we investigated the changes in pH according to severity of mutational status among patients with dermatitis, irrespective of skin condition....... METHODS: pH was measured with a multiprobe system pH probe (PH 905), and the study population was composed of 67 individuals, who had all been genotyped for 3 filaggrin mutations (R501X, 2282del4, R2447X). RESULTS: We found no clear pattern in relation to filaggrin mutation carrier status. Individuals...... with wild-type filaggrin displayed both the most acidic and most alkaline values independent of concomitant skin disease; however, no statistical differences between the groups were found. CONCLUSIONS: The lack of significant diversity in skin pH in relation to filaggrin mutation carrier status suggests...

Exonization of an Intronic LINE-1 Element Causing Becker Muscular Dystrophy as a Novel Mutational Mechanism in Dystrophin Gene.

Science.gov (United States)

Gonçalves, Ana; Oliveira, Jorge; Coelho, Teresa; Taipa, Ricardo; Melo-Pires, Manuel; Sousa, Mário; Santos, Rosário

2017-10-03

A broad mutational spectrum in the dystrophin ( DMD ) gene, from large deletions/duplications to point mutations, causes Duchenne/Becker muscular dystrophy (D/BMD). Comprehensive genotyping is particularly relevant considering the mutation-centered therapies for dystrophinopathies. We report the genetic characterization of a patient with disease onset at age 13 years, elevated creatine kinase levels and reduced dystrophin labeling, where multiplex-ligation probe amplification (MLPA) and genomic sequencing failed to detect pathogenic variants. Bioinformatic, transcriptomic (real time PCR, RT-PCR), and genomic approaches (Southern blot, long-range PCR, and single molecule real-time sequencing) were used to characterize the mutation. An aberrant transcript was identified, containing a 103-nucleotide insertion between exons 51 and 52, with no similarity with the DMD gene. This corresponded to the partial exonization of a long interspersed nuclear element (LINE-1), disrupting the open reading frame. Further characterization identified a complete LINE-1 (~6 kb with typical hallmarks) deeply inserted in intron 51. Haplotyping and segregation analysis demonstrated that the mutation had a de novo origin. Besides underscoring the importance of mRNA studies in genetically unsolved cases, this is the first report of a disease-causing fully intronic LINE-1 element in DMD , adding to the diversity of mutational events that give rise to D/BMD.

Exonization of an Intronic LINE-1 Element Causing Becker Muscular Dystrophy as a Novel Mutational Mechanism in Dystrophin Gene

Science.gov (United States)

Gonçalves, Ana; Coelho, Teresa; Melo-Pires, Manuel; Sousa, Mário

2017-01-01

A broad mutational spectrum in the dystrophin (DMD) gene, from large deletions/duplications to point mutations, causes Duchenne/Becker muscular dystrophy (D/BMD). Comprehensive genotyping is particularly relevant considering the mutation-centered therapies for dystrophinopathies. We report the genetic characterization of a patient with disease onset at age 13 years, elevated creatine kinase levels and reduced dystrophin labeling, where multiplex-ligation probe amplification (MLPA) and genomic sequencing failed to detect pathogenic variants. Bioinformatic, transcriptomic (real time PCR, RT-PCR), and genomic approaches (Southern blot, long-range PCR, and single molecule real-time sequencing) were used to characterize the mutation. An aberrant transcript was identified, containing a 103-nucleotide insertion between exons 51 and 52, with no similarity with the DMD gene. This corresponded to the partial exonization of a long interspersed nuclear element (LINE-1), disrupting the open reading frame. Further characterization identified a complete LINE-1 (~6 kb with typical hallmarks) deeply inserted in intron 51. Haplotyping and segregation analysis demonstrated that the mutation had a de novo origin. Besides underscoring the importance of mRNA studies in genetically unsolved cases, this is the first report of a disease-causing fully intronic LINE-1 element in DMD, adding to the diversity of mutational events that give rise to D/BMD. PMID:28972564

A Substantial Fraction of Barley (Hordeum vulgare L. Low Phytic Acid Mutations Have Little or No Effect on Yield across Diverse Production Environments

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Victor Raboy

2015-04-01

Full Text Available The potential benefits of the low phytic acid (lpa seed trait for human and animal nutrition, and for phosphorus management in non-ruminant animal production, are well documented. However, in many cases the lpa trait is associated with impaired seed or plant performance, resulting in reduced yield. This has given rise to the perception that the lpa trait is tightly correlated with reduced yield in diverse crop species. Here we report a powerful test of this correlation. We measured grain yield in lines homozygous for each of six barley (Hordeum vulgare L. lpa mutations that greatly differ in their seed phytic acid levels. Performance comparisons were between sibling wild-type and mutant lines obtained following backcrossing, and across two years in five Idaho (USA locations that greatly differ in crop yield potential. We found that one lpa mutation (Hvlpa1-1 had no detectable effect on yield and a second (Hvlpa4-1 resulted in yield losses of only 3.5%, across all locations. When comparing yields in three relatively non-stressful production environments, at least three lpa mutations (Hvlpa1-1, Hvlpa3-1, and Hvlpa4-1 typically had yields similar to or within 5% of the wild-type sibling isoline. Therefore in the case of barley, lpa mutations can be readily identified that when simply incorporated into a cultivar result in adequately performing lines, even with no additional breeding for performance within the lpa line. In conclusion, while some barley lpa mutations do impact field performance, a substantial fraction appears to have little or no effect on yield.

Induction of different types of mutations in yeast Saccharomyces serevisiae by γ-radiation

International Nuclear Information System (INIS)

Lyubimova, K.A.; Shvaneva, N.V.; Koltovaya, N.A.

2005-01-01

Several tester systems were used to study a wide spectrum of genetic changes induced by γ-radiation in the yeast Saccharomyces cerevisiae. The tester systems allow one to identify a loss of chromosomes, recombination (crossing over) and point mutations (frame shifts and base-pair substitutions.) Large genome changes were induced by γ-rays more efficiently than the point mutations. The dose dependence of the point mutations frequency was linear. Spontaneous and induced mutation rates per base pair corresponded with the known literature data for the same tester systems. Our finding shows that the used tester systems are not specific. They are useful for further study of mutations induced by ionizing radiation with various physical characteristics

Single-Point Mutation with a Rotamer Library Toolkit: Toward Protein Engineering.

Science.gov (United States)

Pottel, Joshua; Moitessier, Nicolas

2015-12-28

Protein engineers have long been hard at work to harness biocatalysts as a natural source of regio-, stereo-, and chemoselectivity in order to carry out chemistry (reactions and/or substrates) not previously achieved with these enzymes. The extreme labor demands and exponential number of mutation combinations have induced computational advances in this domain. The first step in our virtual approach is to predict the correct conformations upon mutation of residues (i.e., rebuilding side chains). For this purpose, we opted for a combination of molecular mechanics and statistical data. In this work, we have developed automated computational tools to extract protein structural information and created conformational libraries for each amino acid dependent on a variable number of parameters (e.g., resolution, flexibility, secondary structure). We have also developed the necessary tool to apply the mutation and optimize the conformation accordingly. For side-chain conformation prediction, we obtained overall average root-mean-square deviations (RMSDs) of 0.91 and 1.01 Å for the 18 flexible natural amino acids within two distinct sets of over 3000 and 1500 side-chain residues, respectively. The commonly used dihedral angle differences were also evaluated and performed worse than the state of the art. These two metrics are also compared. Furthermore, we generated a family-specific library for kinases that produced an average 2% lower RMSD upon side-chain reconstruction and a residue-specific library that yielded a 17% improvement. Ultimately, since our protein engineering outlook involves using our docking software, Fitted/Impacts, we applied our mutation protocol to a benchmarked data set for self- and cross-docking. Our side-chain reconstruction does not hinder our docking software, demonstrating differences in pose prediction accuracy of approximately 2% (RMSD cutoff metric) for a set of over 200 protein/ligand structures. Similarly, when docking to a set of over 100

Mutation induction by heavy ions

Science.gov (United States)

Kiefer, J.; Stoll, U.; Schneider, E.

1994-10-01

Mutation induction by heavy ions is compared in yeast and mammalian cells. Since mutants can only be recovered in survivors the influence of inactivation cross sections has to be taken into account. It is shown that both the size of the sensitive cellular site as well as track structure play an important role. Another parameter which influences the probability of mutation induction is repair: Contrary to naive assumptions primary radiation damage does not directly lead to mutations but requires modification to reconstitute the genetic machinery so that mutants can survive. The molecular structure of mutations was analyzed after exposure to deuterons by amplification with the aid of polymerase chain reaction. The results-although preliminary-demonstrate that even with densely ionizing particles a large fraction does not carry big deletions which suggests that point mutations may also be induced by heavy ions.

Site-Selective Ribosylation of Fluorescent Nucleobase Analogs Using Purine-Nucleoside Phosphorylase as a Catalyst: Effects of Point Mutations

Directory of Open Access Journals (Sweden)

Alicja Stachelska-Wierzchowska

2015-12-01

Full Text Available Enzymatic ribosylation of fluorescent 8-azapurine derivatives, like 8-azaguanine and 2,6-diamino-8-azapurine, with purine-nucleoside phosphorylase (PNP as a catalyst, leads to N9, N8, and N7-ribosides. The final proportion of the products may be modulated by point mutations in the enzyme active site. As an example, ribosylation of the latter substrate by wild-type calf PNP gives N7- and N8-ribosides, while the N243D mutant directs the ribosyl substitution at N9- and N7-positions. The same mutant allows synthesis of the fluorescent N7-β-d-ribosyl-8-azaguanine. The mutated form of the E. coli PNP, D204N, can be utilized to obtain non-typical ribosides of 8-azaadenine and 2,6-diamino-8-azapurine as well. The N7- and N8-ribosides of the 8-azapurines can be analytically useful, as illustrated by N7-β-d-ribosyl-2,6-diamino-8-azapurine, which is a good fluorogenic substrate for mammalian forms of PNP, including human blood PNP, while the N8-riboside is selective to the E. coli enzyme.

Mutations du gene de la filamine et syndromes malformatifs | Koffi ...

African Journals Online (AJOL)

Filamin is a cytoskeletal protein that occurs in the control of cytoskeleton structure and activity, the modulation of cell shape and migration as well as in the maintaining of cell shape. Mutations in the genes FLNA and FLNB provoke diverse malformative diseases in human. Mutations in the gene FLNA cause four X-Linked ...

Catalase-Negative Staphylococcus lugdunensis Strain with a Novel Point Mutation in the Catalase Gene Isolated from a Patient with Chronic Suppurative Otitis Media

OpenAIRE

Lu, Yong; Wang, Yiping; Ling, Buzhi; Ke, Xianfu; Ying, Jianfei; Yu, Yanhong; He, Mingyang; Li, Xiangyang

2013-01-01

This report describes the results of the sequence analysis of a methicillin-susceptible strain of catalase-negative Staphylococcus lugdunensis. Molecular characterization of the deduced sequence revealed a novel point mutation in the catalase gene. To our knowledge, this is the first report of a catalase-negative S. lugdunensis strain, although catalase-negative isolates of Staphylococcus aureus and Staphylococcus epidermidis have been previously reported.

A neutral theory for interpreting correlations between species and genetic diversity in communities.

Science.gov (United States)

Laroche, Fabien; Jarne, Philippe; Lamy, Thomas; David, Patrice; Massol, Francois

2015-01-01

Spatial patterns of biological diversity have been extensively studied in ecology and population genetics, because they reflect the forces acting on biodiversity. A growing number of studies have found that genetic (within-species) and species diversity can be correlated in space (the so-called species-gene diversity correlation [SGDC]), which suggests that they are controlled by nonindependent processes. Positive SGDCs are generally assumed to arise from parallel responses of genetic and species diversity to variation in site size and connectivity. However, this argument implicitly assumes a neutral model that has yet to be developed. Here, we build such a model to predict SGDC in a metacommunity. We describe how SGDC emerges from competition within sites and variation in connectivity and carrying capacity among sites. We then introduce the formerly ignored mutation process, which affects genetic but not species diversity. When mutation rate is low, our model confirms that variation in the number of migrants among sites creates positive SGDCs. However, when considering high mutation rates, interactions between mutation, migration, and competition can produce negative SGDCs. Neutral processes thus do not always contribute positively to SGDCs. Our approach provides empirical guidelines for interpreting these novel patterns in natura with respect to evolutionary and ecological forces shaping metacommunities.

Structural stability of human protein tyrosine phosphatase ρ catalytic domain: effect of point mutations.

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Alessandra Pasquo

Full Text Available Protein tyrosine phosphatase ρ (PTPρ belongs to the classical receptor type IIB family of protein tyrosine phosphatase, the most frequently mutated tyrosine phosphatase in human cancer. There are evidences to suggest that PTPρ may act as a tumor suppressor gene and dysregulation of Tyr phosphorylation can be observed in diverse diseases, such as diabetes, immune deficiencies and cancer. PTPρ variants in the catalytic domain have been identified in cancer tissues. These natural variants are nonsynonymous single nucleotide polymorphisms, variations of a single nucleotide occurring in the coding region and leading to amino acid substitutions. In this study we investigated the effect of amino acid substitution on the structural stability and on the activity of the membrane-proximal catalytic domain of PTPρ. We expressed and purified as soluble recombinant proteins some of the mutants of the membrane-proximal catalytic domain of PTPρ identified in colorectal cancer and in the single nucleotide polymorphisms database. The mutants show a decreased thermal and thermodynamic stability and decreased activation energy relative to phosphatase activity, when compared to wild- type. All the variants show three-state equilibrium unfolding transitions similar to that of the wild- type, with the accumulation of a folding intermediate populated at ~4.0 M urea.

Efficient algorithms for probing the RNA mutation landscape.

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Jérôme Waldispühl

Full Text Available The diversity and importance of the role played by RNAs in the regulation and development of the cell are now well-known and well-documented. This broad range of functions is achieved through specific structures that have been (presumably optimized through evolution. State-of-the-art methods, such as McCaskill's algorithm, use a statistical mechanics framework based on the computation of the partition function over the canonical ensemble of all possible secondary structures on a given sequence. Although secondary structure predictions from thermodynamics-based algorithms are not as accurate as methods employing comparative genomics, the former methods are the only available tools to investigate novel RNAs, such as the many RNAs of unknown function recently reported by the ENCODE consortium. In this paper, we generalize the McCaskill partition function algorithm to sum over the grand canonical ensemble of all secondary structures of all mutants of the given sequence. Specifically, our new program, RNAmutants, simultaneously computes for each integer k the minimum free energy structure MFE(k and the partition function Z(k over all secondary structures of all k-point mutants, even allowing the user to specify certain positions required not to mutate and certain positions required to base-pair or remain unpaired. This technically important extension allows us to study the resilience of an RNA molecule to pointwise mutations. By computing the mutation profile of a sequence, a novel graphical representation of the mutational tendency of nucleotide positions, we analyze the deleterious nature of mutating specific nucleotide positions or groups of positions. We have successfully applied RNAmutants to investigate deleterious mutations (mutations that radically modify the secondary structure in the Hepatitis C virus cis-acting replication element and to evaluate the evolutionary pressure applied on different regions of the HIV trans-activation response

Loss of mutL homolog-1 (MLH1) expression promotes acquisition of oncogenic and inhibitor-resistant point mutations in tyrosine kinases.

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Springuel, Lorraine; Losdyck, Elisabeth; Saussoy, Pascale; Turcq, Béatrice; Mahon, François-Xavier; Knoops, Laurent; Renauld, Jean-Christophe

2016-12-01

Genomic instability drives cancer progression by promoting genetic abnormalities that allow for the multi-step clonal selection of cells with growth advantages. We previously reported that the IL-9-dependent TS1 cell line sequentially acquired activating substitutions in JAK1 and JAK3 upon successive selections for growth factor independent and JAK inhibitor-resistant cells, suggestive of a defect in mutation avoidance mechanisms. In the first part of this paper, we discovered that the gene encoding mutL homolog-1 (MLH1), a key component of the DNA mismatch repair system, is silenced by promoter methylation in TS1 cells. By means of stable ectopic expression and RNA interference methods, we showed that the high frequencies of growth factor-independent and inhibitor-resistant cells with activating JAK mutations can be attributed to the absence of MLH1 expression. In the second part of this paper, we confirm the clinical relevance of our findings by showing that chronic myeloid leukemia relapses upon ABL-targeted therapy correlated with a lower expression of MLH1 messenger RNA. Interestingly, the mutational profile observed in our TS1 model, characterized by a strong predominance of T:A>C:G transitions, was identical to the one described in the literature for primitive cells derived from chronic myeloid leukemia patients. Taken together, our observations demonstrate for the first time a causal relationship between MLH1-deficiency and incidence of oncogenic point mutations in tyrosine kinases driving cell transformation and acquired resistance to kinase-targeted cancer therapies.

Lethal mutagenesis: targeting the mutator phenotype in cancer.

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Fox, Edward J; Loeb, Lawrence A

2010-10-01

The evolution of cancer and RNA viruses share many similarities. Both exploit high levels of genotypic diversity to enable extensive phenotypic plasticity and thereby facilitate rapid adaptation. In order to accumulate large numbers of mutations, we have proposed that cancers express a mutator phenotype. Similar to cancer cells, many viral populations, by replicating their genomes with low fidelity, carry a substantial mutational load. As high levels of mutation are potentially deleterious, the viral mutation frequency is thresholded at a level below which viral populations equilibrate in a traditional mutation-selection balance, and above which the population is no longer viable, i.e., the population undergoes an error catastrophe. Because their mutation frequencies are fine-tuned just below this error threshold, viral populations are susceptible to further increases in mutational load and, recently this phenomenon has been exploited therapeutically by a concept that has been termed lethal mutagenesis. Here we review the application of lethal mutagenesis to the treatment of HIV and discuss how lethal mutagenesis may represent a novel therapeutic approach for the treatment of solid cancers. Copyright © 2010 Elsevier Ltd. All rights reserved.

Point mutations in acetylcholinesterase 1 associated with chlorpyrifos resistance in the brown planthopper, Nilaparvata lugens Stål.

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Zhang, Y; Yang, B; Li, J; Liu, M; Liu, Z

2017-08-01

Insecticide resistance frequently results from target-site insensitivity, such as point mutations in acetylcholinesterases (AChEs) for resistance to organophosphates and carbamates. From a field-originated population of Nilaparvata lugens, a major rice pest, a resistant population (R9) was obtained by nine-generation continuous selection with chlorpyrifos. From the same field population, a relatively susceptible population (S9) was also constructed through rearing without any insecticides. Compared to the susceptible strain, Sus [medium lethal dose (LC 50 ) = 0.012 mg/l], R9 had a resistance ratio (RR) of 253.08-fold, whereas the RR of S9 was only 2.25-fold. Piperonyl butoxide and triphenyl phosphate synergized chlorpyrifos in R9 less than three-fold, indicating other important mechanisms for high resistance. The target-site insensitivity was supported by the key property differences of crude AChEs between R9 and S9. Compared to S9, three mutations (G119S, F331C and I332L) were detected in NlAChE1 from individuals of the R9 and field populations, but no mutation was detected in NlAChE2. G119S and F331C could decreased insecticide sensitivities in recombinant NlAChE1, whereas I332L took effect through increasing the influence of F331C on target insensitivity. F331C might be deleterious because of its influence on the catalytic efficiency of NlAChE1, whereas I332L would decrease these adverse effects and maintain the normal functions of AChEs. © 2017 The Royal Entomological Society.

Mutation induction by ion beams in plants

International Nuclear Information System (INIS)

Tanaka, Atsushi

2001-01-01

The effect of ion beams such as C, He, and Ne ions was investigated on the mutation induction in plants with the expectation that ion beams of high linear energy transfer (LET) can frequently produce large DNA alternation such as inversion, translocation and large deletion rather than point mutation. Mutation frequency was investigated using Arabidopsis visible phenotype loci and was 8 to 33 fold higher for 220 MeV carbon ions than for electrons. Mutation spectrum was investigated on the flower color of chrysanthemum cv to find that flower mutants induced by ion beams show complex and stripe types rather than single color. Polymerase chain reaction analysis was performed to investigate DNA alteration of mutations. In conclusion, the characteristics of ion beams for the mutation induction are 1) high frequency, 2) broad mutation spectrum, and 3) novel mutants. (S. Ohno)

Mutation induction by ion beams in plants

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Tanaka, Atsushi [Japan Atomic Energy Research Inst., Takasaki, Gunma (Japan). Takasaki Radiation Chemistry Research Establishment

2001-03-01

The effect of ion beams such as C, He, and Ne ions was investigated on the mutation induction in plants with the expectation that ion beams of high linear energy transfer (LET) can frequently produce large DNA alternation such as inversion, translocation and large deletion rather than point mutation. Mutation frequency was investigated using Arabidopsis visible phenotype loci and was 8 to 33 fold higher for 220 MeV carbon ions than for electrons. Mutation spectrum was investigated on the flower color of chrysanthemum cv to find that flower mutants induced by ion beams show complex and stripe types rather than single color. Polymerase chain reaction analysis was performed to investigate DNA alteration of mutations. In conclusion, the characteristics of ion beams for the mutation induction are 1) high frequency, 2) broad mutation spectrum, and 3) novel mutants. (S. Ohno)

Mutations of the KRAS oncogene in endometrial hyperplasia and carcinoma.

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Wiesława Niklińska

2009-05-01

Full Text Available The aim of this study was to examine the prevalence and clinicopathological significance of KRAS point mutation in endometrial hyperplasia and carcinoma. We analysed KRAS in 11 cases of complex atypical hyperplasia and in 49 endometrial carcinomas using polymerase chain reaction associated with restriction fragment length polymorphism (PCR-RFPL. Point mutations at codon 12 of KRAS oncogene were identified in 7 of 49 (14,3% tumor specimens and in 2 of 11 (18,2% hyperplasias. No correlation was found between KRAS gene mutation and age at onset, histology, grade of differentiation and clinical stage. We conclude that KRAS mutation is a relatively common event in endometrial carcinogenesis, but with no prognostic value.

Drug resistance-related mutations in multidrug-resistant Mycobacterium tuberculosis isolates from diverse geographical regions

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Senia Rosales-Klintz

2012-01-01

Conclusion: This study confirms that there are significant geographical differences in the distribution of resistance-related mutations and suggests that an increased understanding of such differences in the specific distribution of resistance conferring mutations is crucial for development of new, generally applicable, molecular tools for rapid diagnosis of drug-resistant TB. The fact that a narrower distribution of mutations in high MDR-TB prevalence settings was seen suggests that much of the problems in these settings can be a result of an ongoing transmission of certain MDR-TB strains.

Epidermal Growth Factor Receptor Activating Mutations in Squamous Histology of Lung Cancer Patients of Southern Bulgaria

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Genova Silvia N.

2015-12-01

Full Text Available There is only limited data on the prevalence of epidermal growth factor receptor (EGFR activating mutations in squamous cell carcinomas and adenosquamous carcinomas of the lung in patients of the Southern Bulgarian region and the efficacy of EGFR tyrosine kinase inhibitors. AIM: Previous reports for Bulgarian population showed high incidence of EGFR mutations in the squamous cell carcinomas, so we set the goal to investigate their frequency in Southern Bulgaria, after precise immunohistochemical verification of lung cancers. MATERIALS AND METHODS: Two hundred and thirty-six lung carcinomas were included in this prospective study. All biopsies were initially analysed with p63, TTF1, Napsin A, CK7, CK34βE12, synaptophysin, CK20 and CDX2. Two hundred and twenty-five non-small cell lung carcinomas were studied with real-time PCR technology to assess the status of the EGFR gene. RESULTS: We detected 132 adenocarcinomas (58.7%, 89 squamous cell carcinomas (39.2%, 4 adenosquamous carcinomas (1.8%, 9 large cell neuroendocrine carcinomas (3.8% and 2 metastatic colorectal adenocarcinomas (0.8%. Activating mutations in the EGF receptor had 3 out of 89 squamous cell carcinomas (3.37%. We have established mutations in L858R, deletion in exon 19 and rare mutation in S7681. One out of four adenosquamous carcinomas had a point mutation in the L858R (25%. CONCLUSIONS: The frequency of EGFR mutations we found in lung squamous cell carcinomas in a Southern Bulgarian region is lower than that in European countries. Ethnic diversity in the region does not play role of an independent predictive factor in terms of mutation frequency.

Different mutations of the human c-mpl gene indicate distinct haematopoietic diseases.

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He, Xin; Chen, Zhigang; Jiang, Yangyan; Qiu, Xi; Zhao, Xiaoying

2013-01-25

The human c-mpl gene (MPL) plays an important role in the development of megakaryocytes and platelets as well as the self-renewal of haematopoietic stem cells. However, numerous MPL mutations have been identified in haematopoietic diseases. These mutations alter the normal regulatory mechanisms and lead to autonomous activation or signalling deficiencies. In this review, we summarise 59 different MPL mutations and classify these mutations into four different groups according to the associated diseases and mutation rates. Using this classification, we clearly distinguish four diverse types of MPL mutations and obtain a deep understand of their clinical significance. This will prove to be useful for both disease diagnosis and the design of individual therapy regimens based on the type of MPL mutations.

Parkinson's disease-related LRRK2 G2019S mutation results from independent mutational events in humans.

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Lesage, Suzanne; Patin, Etienne; Condroyer, Christel; Leutenegger, Anne-Louise; Lohmann, Ebba; Giladi, Nir; Bar-Shira, Anat; Belarbi, Soraya; Hecham, Nassima; Pollak, Pierre; Ouvrard-Hernandez, Anne-Marie; Bardien, Soraya; Carr, Jonathan; Benhassine, Traki; Tomiyama, Hiroyuki; Pirkevi, Caroline; Hamadouche, Tarik; Cazeneuve, Cécile; Basak, A Nazli; Hattori, Nobutaka; Dürr, Alexandra; Tazir, Meriem; Orr-Urtreger, Avi; Quintana-Murci, Lluis; Brice, Alexis

2010-05-15

Mutations in the leucine-rich-repeat kinase 2 (LRRK2) gene have been identified in families with autosomal dominant Parkinson's disease (PD) and in sporadic cases; the G2019S mutation is the single most frequent. Intriguingly, the frequency of this mutation in PD patients varies greatly among ethnic groups and geographic origins: it is present at <0.1% in East Asia, approximately 2% in European-descent patients and can reach frequencies of up to 15-40% in PD Ashkenazi Jews and North African Arabs. To ascertain the evolutionary dynamics of the G2019S mutation in different populations, we genotyped 74 markers spanning a 16 Mb genomic region around G2019S, in 191 individuals carrying the mutation from 126 families of different origins. Sixty-seven families were of North-African Arab origin, 18 were of North/Western European descent, 37 were of Jewish origin, mostly from Eastern Europe, one was from Japan, one from Turkey and two were of mixed origins. We found the G2019S mutation on three different haplotypes. Network analyses of the three carrier haplotypes showed that G2019S arose independently at least twice in humans. In addition, the population distribution of the intra-allelic diversity of the most widespread carrier haplotype, together with estimations of the age of G2019S determined by two different methods, suggests that one of the founding G2019S mutational events occurred in the Near East at least 4000 years ago.

Point mutations in the post-M2 region of human alpha-ENaC regulate cation selectivity.

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Ji, H L; Parker, S; Langloh, A L; Fuller, C M; Benos, D J

2001-07-01

We tested the hypothesis that an arginine-rich region immediately following the second transmembrane domain may constitute part of the inner mouth of the epithelial Na+ channel (ENaC) pore and, hence, influence conduction and/or selectivity properties of the channel by expressing double point mutants in Xenopus oocytes. Double point mutations of arginines in this post-M2 region of the human alpha-ENaC (alpha-hENaC) led to a decrease and increase in the macroscopic conductance of alphaR586E,R587Ebetagamma- and alphaR589E,R591Ebetagamma-hENaC, respectively, but had no effect on the single-channel conductance of either double point mutant. However, the apparent equilibrium dissociation constant for Na+ was decreased for both alphaR586E,R587Ebetagamma- and alphaR589E,R591Ebetagamma-hENaC, and the maximum amiloride-sensitive Na+ current was decreased for alphaR586E,R587Ebetagamma-hENaC and increased for alphaR589E,R591Ebetagamma-hENaC. The relative permeabilities of Li+ and K+ vs. Na+ were increased 11.25- to 27.57-fold for alphaR586E,R587Ebetagamma-hENaC compared with wild type. The relative ion permeability of these double mutants and wild-type ENaC was inversely related to the crystal diameter of the permeant ions. Thus the region of positive charge is important for the ion permeation properties of the channel and may form part of the pore itself.

Achievements and trends of using induced mutations in crop improvement

International Nuclear Information System (INIS)

Nichterlein, K.; Maluszynski, M.; ); Bohlmann, H.; Nielen, S.; )

2000-01-01

Mutation techniques have been employed for the genetic improvement of crops and ornamentals leading to the official release of more than 2200 improved varieties. Some of them have made a major impact on crop productivity and achieved great economic success. Induced mutations play an important role in plant genome research to understand the function of genes aiming to improve food security and diversity. (author)

Expanded Mutational Spectrum in Cohen Syndrome, Tissue Expression, and Transcript Variants of COH1

NARCIS (Netherlands)

Seifert, Wenke; Holder-Espinasse, Muriel; Kuehnisch, Jirko; Kahrizi, Kimia; Tzschach, Andreas; Garshasbi, Masoud; Najmabadi, Hossein; Kuss, Andreas Walter; Kress, Wolfram; Laureys, Genevieve; Loeys, Bart; Brilstra, Eva; Mancini, Grazia M. S.; Dollfus, Helene; Dahan, Karin; Apse, Kira; Hennies, Hans Christian; Horn, Denise

Cohen syndrome is characterised by mental retardation, postnatal microcephaly, facial dysmorphism, pigmentary retinopathy, myopia, and intermittent neutropenia. Mutations in COH1 (VPS13B) have been found in patients with Cohen syndrome from diverse ethnic origins. We have carried out mutation

Introduction of a point mutation into an HLA class I single-chain trimer induces enhancement of CTL priming and antitumor immunity

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Masanori Matsui

2014-01-01

Full Text Available We previously discovered one particular HLA-A*02:01 mutant that enhanced peptide-specific cytotoxic T lymphocyte (CTL recognition in vitro compared to wild-type HLA-A*02:01. This mutant contains a single amino acid substitution from histidine to leucine at position 74 (H74L that is located in the peptide-binding groove. To investigate the effect of the H74L mutation on the in vivo CTL priming, we took advantage of the technology of the HLA class I single-chain trimer (SCT in which three components involving a peptide, β2 microglobulin and the HLA class I heavy chain are joined together via flexible linkers. We generated recombinant adenovirus expressing SCT comprised influenza A matrix protein (FMP-derived peptide, β2 microglobulin and the H74L heavy chain. HLA-A*02:01 transgenic mice were immunized with the adenovirus, and the induction of peptide-specific CTLs and antitumor immunity was investigated. It was clearly shown that the H74L mutation enabled the HLA-A*02:01 SCT molecule to dramatically enhance both in vivo priming of FMP-specific CTLs and protection against a lethal challenge of tumor cells expressing FMP. These data present the first evidence that a simple point mutation in the HLA class I heavy chain of SCT is beneficial for improving CTL-based immunotherapy and prophylaxis to control tumors.

Heart tissue of harlequin (hq)/Big Blue mice has elevated reactive oxygen species without significant impact on the frequency and nature of point mutations in nuclear DNA

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Crabbe, Rory A. [Department of Biology, University of Western Ontario, London, Ontario, N6A 5B7 (Canada); Hill, Kathleen A., E-mail: khill22@uwo.ca [Department of Biology, University of Western Ontario, London, Ontario, N6A 5B7 (Canada)

2010-09-10

Age is a major risk factor for heart disease, and cardiac aging is characterized by elevated mitochondrial reactive oxygen species (ROS) with compromised mitochondrial and nuclear DNA integrity. To assess links between increased ROS levels and mutations, we examined in situ levels of ROS and cII mutation frequency, pattern and spectrum in the heart of harlequin (hq)/Big Blue mice. The hq mouse is a model of premature aging with mitochondrial dysfunction and increased risk of oxidative stress-induced heart disease with the means for in vivo mutation detection. The hq mutation produces a significant downregulation in the X-linked apoptosis-inducing factor gene (Aif) impairing both the antioxidant and oxidative phosphorylation functions of AIF. Brain and skin of hq disease mice have elevated frequencies of point mutations in nuclear DNA and histopathology characterized by cell loss. Reports of associated elevations in ROS in brain and skin have mixed results. Herein, heart in situ ROS levels were elevated in hq disease compared to AIF-proficient mice (p < 0.0001) yet, mutation frequency and pattern were similar in hq disease, hq carrier and AIF-proficient mice. Heart cII mutations were also assessed 15 days following an acute exposure to an exogenous ROS inducer (10 mg paraquat/kg). Acute paraquat exposure with a short mutant manifestation period was insufficient to elevate mutation frequency or alter mutation pattern in the post-mitotic heart tissue of AIF-proficient mice. Paraquat induction of ROS requires mitochondrial complex I and thus is likely compromised in hq mice. Results of this preliminary survey and the context of recent literature suggest that determining causal links between AIF deficiency and the premature aging phenotypes of specific tissues is better addressed with assay of mitochondrial ROS and large-scale changes in mitochondrial DNA in specific cell types.

Heart tissue of harlequin (hq)/Big Blue mice has elevated reactive oxygen species without significant impact on the frequency and nature of point mutations in nuclear DNA

International Nuclear Information System (INIS)

Crabbe, Rory A.; Hill, Kathleen A.

2010-01-01

Age is a major risk factor for heart disease, and cardiac aging is characterized by elevated mitochondrial reactive oxygen species (ROS) with compromised mitochondrial and nuclear DNA integrity. To assess links between increased ROS levels and mutations, we examined in situ levels of ROS and cII mutation frequency, pattern and spectrum in the heart of harlequin (hq)/Big Blue mice. The hq mouse is a model of premature aging with mitochondrial dysfunction and increased risk of oxidative stress-induced heart disease with the means for in vivo mutation detection. The hq mutation produces a significant downregulation in the X-linked apoptosis-inducing factor gene (Aif) impairing both the antioxidant and oxidative phosphorylation functions of AIF. Brain and skin of hq disease mice have elevated frequencies of point mutations in nuclear DNA and histopathology characterized by cell loss. Reports of associated elevations in ROS in brain and skin have mixed results. Herein, heart in situ ROS levels were elevated in hq disease compared to AIF-proficient mice (p < 0.0001) yet, mutation frequency and pattern were similar in hq disease, hq carrier and AIF-proficient mice. Heart cII mutations were also assessed 15 days following an acute exposure to an exogenous ROS inducer (10 mg paraquat/kg). Acute paraquat exposure with a short mutant manifestation period was insufficient to elevate mutation frequency or alter mutation pattern in the post-mitotic heart tissue of AIF-proficient mice. Paraquat induction of ROS requires mitochondrial complex I and thus is likely compromised in hq mice. Results of this preliminary survey and the context of recent literature suggest that determining causal links between AIF deficiency and the premature aging phenotypes of specific tissues is better addressed with assay of mitochondrial ROS and large-scale changes in mitochondrial DNA in specific cell types.

Characterization of carbon ion-induced mutations in Arabidopsis thaliana

International Nuclear Information System (INIS)

Shikazono, N.; Suzuki, C.; Kitamura, S.; Watanabe, H.; Tano, S.; Tanaka, A.

2003-01-01

Full text: Irradiation of Arabidopsis thaliana by carbon ions was carried out to investigate the mutational effect of ion particles in higher plants. The averaged mutation rate of carbon ions was 2.0 X 10 -6 / Gy, which was 18-fold higher than that of electrons. PCR analysis of the carbon ion-induced mutants showed that, out of 28 mutant alleles, 14 had point-like mutations within the gene, while 14 contained large structural alterations. In the case of 12 electron-induced mutants, 9 had point-like mutations within the gene, while 3 contained large structural alterations. These results suggest that carbon ions are more likely to induce large structural alterations compared with electrons. Further sequence analysis revealed that most of the point-like mutations induced by carbon ions were short deletions. In the case of rearrangements, DNA strand breaks were found to be rejoined using, if present, short homologous sequences for both types of radiation. After carbon ion-irradiation, small deletions were frequently observed around the breakpoints, whereas duplications of terminal sequence were found after electron-irradiation. These results suggest that non-homologous end joining (NHEJ) pathway operates after plant cells are exposed to both ion particles and electrons but that different mode of rejoining deals with the broken ends produced by each radiation. From the present results, it seems reasonable to assume that carbon ions could predominantly induce null mutations in Arabidopsis. The fact that the molecular nature of carbon ion-induced mutation was different from that of electrons and that the molecular mechanisms of cells to induce mutations appeared to be also different implicates that ion particle is not only valuable as a new mutagen but also useful as a new tool to study repair mechanisms of certain types of DNA damage

Gypsy Phenylketonuria: A point mutation of the phenylalanine hydroxylase gene in Gypsy families from Slovakia

Energy Technology Data Exchange (ETDEWEB)

Kalanin, J. [Institute for Clinical and Experical Medicine, Praha (Czechoslovakia); Takarada, Y. [Toyobo Research Center, Shiga (Japan); Kagawa, S.; Yamashita, K.; Ohtsuka, N.; Matsuoka, A. [Hyogo College of Medicine, Nishinomiya (Japan)

1994-01-15

A direct mutational analysis of the phenylalanine hydroxylase gene (PAH) in Gypsy families with phenylketonuria (PKU) has not yet been presented. However, they obviously represent a group at high risk for this inherited disease. The authors analyzed the PAH loci of 65 Gypsies originating from Eastern Slovakia by a combination of PCR amplification, direct sequencing and ASO hybridization. These studies uncovered 10 {open_quotes}classical PKU{close_quotes} patients to be homozygous for a R252W (CGG-TGG) transition, and 29 heterozygous carriers of this mutation. Fifteen control Caucasoid PKU patients from the Czech and Slovak Republics were selected. In this group they detected R252W mutation in two subjects (6.67% of all mutant alleles). Both were compound heterozygous for two different mutations. Previous haplotype studies of Welsh Gypsies with PKU were uninformative in the determination of heterozygosity. ASO hybridization served effectively for the consequent analyses in Gypsy PKU-related families and to identify the carriers among the unrelated subjects. 19 refs., 2 figs.

A Constant Rate of Spontaneous Mutation in DNA-Based Microbes

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Drake, John W.

1991-08-01

In terms of evolution and fitness, the most significant spontaneous mutation rate is likely to be that for the entire genome (or its nonfrivolous fraction). Information is now available to calculate this rate for several DNA-based haploid microbes, including bacteriophages with single- or double-stranded DNA, a bacterium, a yeast, and a filamentous fungus. Their genome sizes vary by ≈6500-fold. Their average mutation rates per base pair vary by ≈16,000-fold, whereas their mutation rates per genome vary by only ≈2.5-fold, apparently randomly, around a mean value of 0.0033 per DNA replication. The average mutation rate per base pair is inversely proportional to genome size. Therefore, a nearly invariant microbial mutation rate appears to have evolved. Because this rate is uniform in such diverse organisms, it is likely to be determined by deep general forces, perhaps by a balance between the usually deleterious effects of mutation and the physiological costs of further reducing mutation rates.

Petroleum pollution and mutation in mangroves

International Nuclear Information System (INIS)

Klekowski, E.J. Jr.; Corredor, J.E.; Morell, J.M.; Del Castillo, C.A.

1994-01-01

Chlorophyll-deficiency has often been used as a sensitive genetic end-point in plant mutation research. The frequency of trees heterozygous for nuclear chlorophyll-deficient mutations was determined for mangrove populations growing along the southwest coast of Puerto Rico. The frequency of heterozygotes was strongly correlated with the concentration of polycyclic aromatic hydrocarbons in the underlying sediment and with both acute and chronic petroleum pollution. Although epidemiological studies can seldom prove causation, a strong correlation is certainly compatible with a cause-effect relationship. Our results suggest that the biota of oil-polluted habitats may be experiencing increased mutation. (Author)

Progranulin mutations as risk factors for Alzheimer disease.

Science.gov (United States)

Perry, David C; Lehmann, Manja; Yokoyama, Jennifer S; Karydas, Anna; Lee, Jason Jiyong; Coppola, Giovanni; Grinberg, Lea T; Geschwind, Dan; Seeley, William W; Miller, Bruce L; Rosen, Howard; Rabinovici, Gil

2013-06-01

Mutations in the progranulin gene are known to cause diverse clinical syndromes, all attributed to frontotemporal lobar degeneration. We describe 2 patients with progranulin gene mutations and evidence of Alzheimer disease (AD) pathology. We also conducted a literature review. This study focused on case reports of 2 unrelated patients with progranulin mutations at the University of California, San Francisco, Memory and Aging Center. One patient presented at age 65 years with a clinical syndrome suggestive of AD and showed evidence of amyloid aggregation on positron emission tomography. Another patient presented at age 54 years with logopenic progressive aphasia and, at autopsy, showed both frontotemporal lobar degeneration with TDP-43 inclusions and AD. In addition to autosomal-dominant frontotemporal lobar degeneration, mutations in the progranulin gene may be a risk factor for AD clinical phenotypes and neuropathology.

Dynamic Harmony Search with Polynomial Mutation Algorithm for Valve-Point Economic Load Dispatch

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M. Karthikeyan

2015-01-01

mutation (DHSPM algorithm to solve ORPD problem. In DHSPM algorithm the key parameters of HS algorithm like harmony memory considering rate (HMCR and pitch adjusting rate (PAR are changed dynamically and there is no need to predefine these parameters. Additionally polynomial mutation is inserted in the updating step of HS algorithm to favor exploration and exploitation of the search space. The DHSPM algorithm is tested with three power system cases consisting of 3, 13, and 40 thermal units. The computational results show that the DHSPM algorithm is more effective in finding better solutions than other computational intelligence based methods.

Experimental Determination and Prediction of the Fitness Effects of Random Point Mutations in the Biosynthetic Enzyme HisA

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Lundin, Erik; Tang, Po-Cheng; Guy, Lionel; Näsvall, Joakim; Andersson, Dan I

2018-01-01

Abstract The distribution of fitness effects of mutations is a factor of fundamental importance in evolutionary biology. We determined the distribution of fitness effects of 510 mutants that each carried between 1 and 10 mutations (synonymous and nonsynonymous) in the hisA gene, encoding an essential enzyme in the l-histidine biosynthesis pathway of Salmonella enterica. For the full set of mutants, the distribution was bimodal with many apparently neutral mutations and many lethal mutations. For a subset of 81 single, nonsynonymous mutants most mutations appeared neutral at high expression levels, whereas at low expression levels only a few mutations were neutral. Furthermore, we examined how the magnitude of the observed fitness effects was correlated to several measures of biophysical properties and phylogenetic conservation.We conclude that for HisA: (i) The effect of mutations can be masked by high expression levels, such that mutations that are deleterious to the function of the protein can still be neutral with regard to organism fitness if the protein is expressed at a sufficiently high level; (ii) the shape of the fitness distribution is dependent on the extent to which the protein is rate-limiting for growth; (iii) negative epistatic interactions, on an average, amplified the combined effect of nonsynonymous mutations; and (iv) no single sequence-based predictor could confidently predict the fitness effects of mutations in HisA, but a combination of multiple predictors could predict the effect with a SD of 0.04 resulting in 80% of the mutations predicted within 12% of their observed selection coefficients. PMID:29294020

Mosquito bottlenecks alter viral mutant swarm in a tissue and time-dependent manner with contraction and expansion of variant positions and diversity.

Science.gov (United States)

Patterson, Edward I; Khanipov, Kamil; Rojas, Mark M; Kautz, Tiffany F; Rockx-Brouwer, Dedeke; Golovko, Georgiy; Albayrak, Levent; Fofanov, Yuriy; Forrester, Naomi L

2018-01-01

Viral diversity is theorized to play a significant role during virus infections, particularly for arthropod-borne viruses (arboviruses) that must infect both vertebrate and invertebrate hosts. To determine how viral diversity influences mosquito infection and dissemination Culex taeniopus mosquitoes were infected with the Venezuelan equine encephalitis virus endemic strain 68U201. Bodies and legs/wings of the mosquitoes were collected individually and subjected to multi-parallel sequencing. Virus sequence diversity was calculated for each tissue. Greater diversity was seen in mosquitoes with successful dissemination versus those with no dissemination. Diversity across time revealed that bottlenecks influence diversity following dissemination to the legs/wings, but levels of diversity are restored by Day 12 post-dissemination. Specific minority variants were repeatedly identified across the mosquito cohort, some in nearly every tissue and time point, suggesting that certain variants are important in mosquito infection and dissemination. This study demonstrates that the interaction between the mosquito and the virus results in changes in diversity and the mutational spectrum and may be essential for successful transition of the bottlenecks associated with arbovirus infection.

Identification of seven novel mutations including the first two genomic rearrangements in SLC26A3 mutated in congenital chloride diarrhea.

Science.gov (United States)

Höglund, P; Sormaala, M; Haila, S; Socha, J; Rajaram, U; Scheurlen, W; Sinaasappel, M; de Jonge, H; Holmberg, C; Yoshikawa, H; Kere, J

2001-09-01

Congenital chloride diarrhea (CLD) is an autosomal recessive disorder characterized by defective intestinal electrolyte absorption, resulting in voluminous osmotic diarrhea with high chloride content. A variety of mutations in the solute carrier family 26, member 3 gene (SLC26A3, previously known as CLD or DRA) are responsible for the disease. Since the identification of the SLC26A3 gene and the determination of its genomic structure, altogether three founder and 17 private mutations have been characterized within miscellaneous ethnic groups. We screened for mutations in seven unrelated families with CLD. The diagnoses were confirmed by fecal chloride measurements. The combined PCR-SSCP and sequencing analyses revealed altogether seven novel mutations including two missense mutations (S206P, D468V), two splicing defects (IVS12-1G>C, IVS13-2delA), one nonsense mutation (Q436X), one insertion/deletion mutation (2104-2105delGGins29-bp), and an intragenic deletion of SLC26A3 exons 7 and 8. Two previously identified mutations were also found. This is the first report of rearrangement mutations in SLC26A3. Molecular features predisposing SLC26A3 for the two rearrangements may include repetitive elements and palindromic-like sequences. The increasingly wide diversity of SLC26A3 mutations suggests that mutations in the SLC26A3 gene may not be rare events. Copyright 2001 Wiley-Liss, Inc.

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.

Mandibulofacial Dysostosis with Microcephaly: Mutation and Database Update

DEFF Research Database (Denmark)

Gregersen, Pernille Axel

2016-01-01

, we review the molecular basis of MFDM in the 69 individuals described to date, and report mutations in 38 new individuals, bringing the total number of reported individuals to 107 individuals from 94 kindreds. Pathogenic EFTUD2 variants comprise 76 distinct mutations and 7 microdeletions. Among point...... mutations, missense substitutions are infrequent (14/76; 18%) relative to stopgain (29/76; 38%), and splicing (33/76; 43%) mutations. Where known, mutation origin was de novo in 48/64 individuals (75%), dominantly-inherited in 12/64 (19%), and due to proven germline mosaicism in 4/64 (6%). Highly penetrant......-reported anomalies, include vestibular and ossicular malformations, reduced mouth opening, atrophy of cerebral white matter, structural brain malformations, and epibulbar dermoid. All reported EFTUD2 mutations can be found in the EFTUD2 mutation database (http://databases.lovd.nl/shared/genes/EFTUD2). This article...

Novel Mutations in the PC Gene in Patients with Type B Pyruvate Carboxylase Deficiency

DEFF Research Database (Denmark)

Ostergaard, Elsebet; Duno, Morten; Møller, Lisbeth Birk

2013-01-01

We have investigated seven patients with the type B form of pyruvate carboxylase (PC) deficiency. Mutation analysis revealed eight mutations, all novel. In a patient with exon skipping on cDNA analysis, we identified a homozygous mutation located in a potential branch point sequence, the first...... possible branch point mutation in PC. Two patients were homozygous for missense mutations (with normal protein amounts on western blot analysis), and two patients were homozygous for nonsense mutations. In addition, a duplication of one base pair was found in a patient who also harboured a splice site...... mutation. Another splice site mutation led to the activation of a cryptic splice site, shown by cDNA analysis.All patients reported until now with at least one missense mutation have had the milder type A form of PC deficiency. We thus report for the first time two patients with homozygous missense...

ASSOCIATIONS BETWEEN GENETIC DIVERSITY AND ANTHROPOGENIC DISTURBANCE IN MIDWESTERN STREAM-DWELLING MINNOWS

Science.gov (United States)

Anthropogenic disturbances may leave imprints on patterns of intraspecific genetic diversity through their effects on population size, adaptation, migration, and mutation. We examined patterns of genetic diversity for a stream-dwelling minnow (the central stoneroller, Campostoma...

Identification of a point mutation in growth factor repeat C of the low density lipoprotein-receptor gene in a patient with homozygous familial hypercholesterolemia

International Nuclear Information System (INIS)

Soutar, A.K.; Knight, B.L.; Patel, D.D.

1989-01-01

The coding region of the low density lipoprotein (LDL)-receptor gene from a patient (MM) with homozygous familial hypercholesterolemia (FH) has been sequenced from six overlapping 500-base-pair amplified fragments of the cDNA from cultured skin fibroblasts. Two separate single nucleotide base changes from the normal sequence were detected. The first involved substitution of guanine for adenine in the third position of the codon for amino acid residue Cys-27 and did not affect the protein sequence. The second mutation was substitution of thymine for cytosine in the DNA for the codon for amino acid residue 664, changing the codon from CCG (proline) to CTG (leucine) and introducing a new site for the restriction enzyme PstI. MM is a true homozygote with two identical genes, and the mutation cosegregated with clinically diagnosed FH in his family in which first cousin marriages occurred frequently. LDL receptors in MM's skin fibroblasts bind less LDL than normal and with reduced affinity. Thus this naturally occurring single point mutation affects both intracellular transport of the protein and ligand binding and occurs in growth factor-like repeat C, a region that has not previously been found to influence LDL binding

Two Tales of Prokaryotic Genomic Diversity: Escherichia coli and Halophiles

Directory of Open Access Journals (Sweden)

Lejla Pašić

2014-01-01

Full Text Available Prokaryotes are generally characterized by vast genomic diversity that has been shaped by mutations, horizontal gene transfer, bacteriocins and phage predation. Enormous genetic diversity has developed as a result of stresses imposed in harsh environments and the ability of microorganisms to adapt. Two examples of prokaryotic diversity are presented: on intraspecies level, exemplified by Escherichia coli, and the diversity of the hypersaline environment, with the discussion of food-related health issues and biotechnological potential.

Invasion Dynamics and Genotypic Diversity of Cogongrass (Imperata cylindrica) at the Point of Introduction in the Southeastern United States

Science.gov (United States)

Ludovic J. A. Capo-chichi; Wilson H. Faircloth; A. G. Williamson; Michael G. Patterson; James H. Miller; Edzard van Santen

2008-01-01

Nine sites of cogongrass were included in a study of genotypic dimity and spread dynamics at the point of introduction and its adjacent areas in the southern United States. Clones evaluated with two primer pairs yielded a total of 137 amplified fragment length polymorphism (AFLP) hi of which 102 (74.4%) were polymorphic. Genetic diversity was measured as the percentage...

Genetic Divergence and Chemotype Diversity in the Fusarium Head Blight Pathogen Fusarium poae.

Science.gov (United States)

Vanheule, Adriaan; De Boevre, Marthe; Moretti, Antonio; Scauflaire, Jonathan; Munaut, Françoise; De Saeger, Sarah; Bekaert, Boris; Haesaert, Geert; Waalwijk, Cees; van der Lee, Theo; Audenaert, Kris

2017-08-23

Fusarium head blight is a disease caused by a complex of Fusarium species. F. poae is omnipresent throughout Europe in spite of its low virulence. In this study, we assessed a geographically diverse collection of F. poae isolates for its genetic diversity using AFLP (Amplified Fragment Length Polymorphism). Furthermore, studying the mating type locus and chromosomal insertions, we identified hallmarks of both sexual recombination and clonal spread of successful genotypes in the population. Despite the large genetic variation found, all F. poae isolates possess the nivalenol chemotype based on Tri7 sequence analysis. Nevertheless, Tri gene clusters showed two layers of genetic variability. Firstly, the Tri1 locus was highly variable with mostly synonymous mutations and mutations in introns pointing to a strong purifying selection pressure. Secondly, in a subset of isolates, the main trichothecene gene cluster was invaded by a transposable element between Tri5 and Tri6 . To investigate the impact of these variations on the phenotypic chemotype, mycotoxin production was assessed on artificial medium. Complex blends of type A and type B trichothecenes were produced but neither genetic variability in the Tri genes nor variability in the genome or geography accounted for the divergence in trichothecene production. In view of its complex chemotype, it will be of utmost interest to uncover the role of trichothecenes in virulence, spread and survival of F. poae .

Investigation of FANCA mutations in Greek patients.

Science.gov (United States)

Selenti, Nikoletta; Sofocleous, Christalena; Kattamis, Antonis; Kolialexi, Aggeliki; Kitsiou, Sophia; Fryssira, Elena; Polychronopoulou, Sophia; Kanavakis, Emmanouel; Mavrou, Ariadni

2013-08-01

Fanconi anemia (FA) is a rare genetic disease characterized by considerable heterogeneity. Fifteen subtypes are currently recognised and deletions of the Fanconi anemia complementation group A (FANCA) gene account for more than 65% of FA cases. We report on the results from a cohort of 166 patients referred to the Department of Medical Genetics of Athens University for genetic investigation after the clinical suspicion of FA. For clastogen-induced chromosome damage, cultures were set up with the addition of mitomycin C (MMC) and diepoxybutane (DEB), respectively. Following a positive cytogenetic result, molecular analysis was performed to allow identification of causative mutations in the FANCA gene. A total of 13/166 patients were diagnosed with FA and 8/13 belonged to the FA-A subtype. A novel point mutation was identified in exon 26 of FANCA gene. In our study 62% of FA patients were classified in the FA-A subtype and a point mutation in exon 26 was noted for the first time.

Predicting Binding Free Energy Change Caused by Point Mutations with Knowledge-Modified MM/PBSA Method.

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Marharyta Petukh

2015-07-01

Full Text Available A new methodology termed Single Amino Acid Mutation based change in Binding free Energy (SAAMBE was developed to predict the changes of the binding free energy caused by mutations. The method utilizes 3D structures of the corresponding protein-protein complexes and takes advantage of both approaches: sequence- and structure-based methods. The method has two components: a MM/PBSA-based component, and an additional set of statistical terms delivered from statistical investigation of physico-chemical properties of protein complexes. While the approach is rigid body approach and does not explicitly consider plausible conformational changes caused by the binding, the effect of conformational changes, including changes away from binding interface, on electrostatics are mimicked with amino acid specific dielectric constants. This provides significant improvement of SAAMBE predictions as indicated by better match against experimentally determined binding free energy changes over 1300 mutations in 43 proteins. The final benchmarking resulted in a very good agreement with experimental data (correlation coefficient 0.624 while the algorithm being fast enough to allow for large-scale calculations (the average time is less than a minute per mutation.

Performance evaluation of FSO system using wavelength and time diversity over malaga turbulence channel with pointing errors

Science.gov (United States)

Balaji, K. A.; Prabu, K.

2018-03-01

There is an immense demand for high bandwidth and high data rate systems, which is fulfilled by wireless optical communication or free space optics (FSO). Hence FSO gained a pivotal role in research which has a added advantage of both cost-effective and licence free huge bandwidth. Unfortunately the optical signal in free space suffers from irradiance and phase fluctuations due to atmospheric turbulence and pointing errors which deteriorates the signal and degrades the performance of communication system over longer distance which is undesirable. In this paper, we have considered polarization shift keying (POLSK) system applied with wavelength and time diversity technique over Malaga(M)distribution to mitigate turbulence induced fading. We derived closed form mathematical expressions for estimating the systems outage probability and average bit error rate (BER). Ultimately from the results we can infer that wavelength and time diversity schemes enhances these systems performance.

Analysis of point mutations in an ultraviolet-irradiated shuttle vector plasmid propagated in cells from Japanese xeroderma pigmentosum patients in complementation groups A and F

International Nuclear Information System (INIS)

Yagi, T.; Tatsumi-Miyajima, J.; Sato, M.; Kraemer, K.H.; Takebe, H.

1991-01-01

To assess the contribution to mutagenesis by human DNA repair defects, a UV-treated shuttle vector plasmid, pZ189, was passed through fibroblasts derived from Japanese xeroderma pigmentosum (XP) patients in two different DNA repair complementation groups (A and F). Patients with XP have clinical and cellular UV hypersensitivity, increased frequency of skin cancer, and defects in DNA repair. The XP DNA repair defects represented by complementation groups A (XP-A) and F (XP-F) are more common in Japan than in Europe or the United States. In comparison to results with DNA repair-proficient human cells (W138-VA13), UV-treated pZ189 passed through the XP-A [XP2OS(SV)] or XP-F [XP2YO(SV)] cells showed fewer surviving plasmids (XP-A less than XP-F) and a higher frequency of mutated plasmids (XP-A greater than XP-F). Base sequence analysis of more than 200 mutated plasmids showed the major type of base substitution mutation to be the G:C----A:T transition with all three cell lines. The XP-A and XP-F cells revealed a higher frequency of G:C----A:T transitions and a lower frequency of transversions among plasmids with single or tandem mutations and a lower frequency of plasmids with multiple point mutations compared to the normal line. The spectrum of mutations in pZ189 with the XP-A cells was similar to that with the XP-F cells. Seventy-six to 91% of the single base substitution mutations occurred at G:C base pairs in which the 5'-neighboring base of the cytosine was thymine or cytosine. These studies indicate that the DNA repair defects in Japanese XP patients in complementation groups A and F result in different frequencies of plasmid survival and mutagenesis but in similar types of mutagenic abnormalities despite marked differences in clinical features

Human aging and somatic point mutations in mtDNA: a comparative study of generational differences (grandparents and grandchildren

Directory of Open Access Journals (Sweden)

Anderson Nonato do Rosário Marinho

2011-01-01

Full Text Available The accumulation of somatic mutations in mtDNA is correlated with aging. In this work, we sought to identify somatic mutations in the HVS-1 region (D-loop of mtDNA that might be associated with aging. For this, we compared 31 grandmothers (mean age: 63 ± 2.3 years and their 62 grandchildren (mean age: 15 ± 4.1 years, the offspring of their daughters. Direct DNA sequencing showed that mutations absent in the grandchildren were detected in a presumably homoplasmic state in three grandmothers and in a heteroplasmic state in an additional 13 grandmothers; no mutations were detected in the remaining 15 grandmothers. However, cloning followed by DNA sequencing in 12 grandmothers confirmed homoplasia in only one of the three mutations previously considered to be homoplasmic and did not confirm heteroplasmy in three out of nine grandmothers found to be heteroplasmic by direct sequencing. Thus, of 12 grandmothers in whom mtDNA was analyzed by cloning, eight were heteroplasmic for mutations not detected in their grandchildren. In this study, the use of genetically related subjects allowed us to demonstrate the occurrence of age-related (> 60 years old mutations (homoplasia and heteroplasmy. It is possible that both of these situations (homoplasia and heteroplasmy were a long-term consequence of mitochondrial oxidative phosphorylation that can lead to the accumulation of mtDNA mutations throughout life.

The degree of attenuation of tick-borne encephalitis virus depends on the cumulative effects of point mutations.

Science.gov (United States)

Gritsun, T S; Desai, A; Gould, E A

2001-07-01

An infectious clone (pGGVs) of the tick-borne encephalitis complex virus Vasilchenko (Vs) was constructed previously. Virus recovered from pGGVs produced slightly smaller plaques than the Vs parental virus. Sequence analysis demonstrated five nucleotide differences between the original Vs virus and pGGVs; four of these mutations resulted in amino acid substitutions, while the fifth mutation was located in the 3' untranslated region (3'UTR). Two mutations were located in conserved regions and three mutations were located in variable regions of the virus genome. Reverse substitutions from the conserved regions of the genome, R(496)-->H in the envelope (E) gene and C(10884)-->T in the 3'UTR, were introduced both separately and together into the infectious clone and their biological effect on virus phenotype was evaluated. The engineered viruses with R(496) in the E protein produced plaques of smaller size than viruses with H(496) at this position. This mutation also affected the growth and neuroinvasiveness of the virus. In contrast, the consequence of a T(10884)-->C substitution within the 3'UTR was noticeable only in cytotoxicity and neuroinvasiveness tests. However, all virus mutants engineered by modification of the infectious clone, including one with two wild-type mutations, H(496) and T(10884), showed reduced neuroinvasiveness in comparison with the Vs parental virus. Therefore, although the H(496)-->R and T(10884)-->C substitutions clearly reduce virus virulence, the other mutations within the variable regions of the capsid (I(45)-->F) and the NS5 (T(2688)-->A and M(3385)-->I) genes also contribute to the process of attenuation. In terms of developing flavivirus vaccines, the impact of accumulating apparently minor mutations should be assessed in detail.

Plant Breeding by Using Radiation Mutation

International Nuclear Information System (INIS)

Kang, Si Yong; Kim, Dong Sub; Lee, Geung Joo

2007-06-01

A mutation breeding is to use physical or chemical mutagens to induce mutagenesis, followed by individual selections with favorable traits. The mutation breeding has many advantages over other breeding methods, which include the usefulness for improving one or two inferior characteristics, applications to broad species with different reproductive systems or to diverse plant materials, native or plant introduction with narrow genetic background, time and cost-effectiveness, and valuable mutant resources for genomic researches. Recent applications of the radiation breeding techniques to developments of flowering plants or food crops with improved functional constituents heightened the public's interests in agriculture and in our genetic resources and seed industries. The goals of this project, therefore, include achieving advances in domestic seed industries and agricultural productivities by developing and using new radiation mutants with favored traits, protecting an intellectual property right of domestic seeds or germplasm, and sharing the valuable mutants and mutated gene information for the genomic and biotech researches that eventually leads to economic benefits

Plant Breeding by Using Radiation Mutation

Energy Technology Data Exchange (ETDEWEB)

Kang, Si Yong; Kim, Dong Sub; Lee, Geung Joo (and others)

2007-06-15

A mutation breeding is to use physical or chemical mutagens to induce mutagenesis, followed by individual selections with favorable traits. The mutation breeding has many advantages over other breeding methods, which include the usefulness for improving one or two inferior characteristics, applications to broad species with different reproductive systems or to diverse plant materials, native or plant introduction with narrow genetic background, time and cost-effectiveness, and valuable mutant resources for genomic researches. Recent applications of the radiation breeding techniques to developments of flowering plants or food crops with improved functional constituents heightened the public's interests in agriculture and in our genetic resources and seed industries. The goals of this project, therefore, include achieving advances in domestic seed industries and agricultural productivities by developing and using new radiation mutants with favored traits, protecting an intellectual property right of domestic seeds or germplasm, and sharing the valuable mutants and mutated gene information for the genomic and biotech researches that eventually leads to economic benefits.

A novel OPA1 mutation in a Chinese family with autosomal dominant optic atrophy

Energy Technology Data Exchange (ETDEWEB)

Zhang, Juanjuan; Yuan, Yimin; Lin, Bing; Feng, Hao; Li, Yan [School of Ophthalmology and Optometry, Wenzhou Medical College, Wenzhou 325027, Zhejiang (China); Dai, Xianning; Zhou, Huihui [Attardi Institute of Mitochondrial Biomedicine and Zhejiang Provincial Key Laboratory of Medical Genetics, School of Life Sciences, Wenzhou Medical College, Wenzhou 325035, Zhejiang (China); Dong, Xujie [School of Ophthalmology and Optometry, Wenzhou Medical College, Wenzhou 325027, Zhejiang (China); Liu, Xiao-Ling, E-mail: lxl@mail.eye.ac.cn [School of Ophthalmology and Optometry, Wenzhou Medical College, Wenzhou 325027, Zhejiang (China); Guan, Min-Xin, E-mail: min-xin.guan@cchmc.org [Attardi Institute of Mitochondrial Biomedicine and Zhejiang Provincial Key Laboratory of Medical Genetics, School of Life Sciences, Wenzhou Medical College, Wenzhou 325035, Zhejiang (China); Institute of Genetics, Zhejiang University, Hangzhou, Zhejiang 310012 (China); Division of Human Genetics, Cincinnati Children' s Hospital Medical Center, OH 45229 (United States)

2012-03-23

Highlights: Black-Right-Pointing-Pointer We report the characterization of a four-generation large Chinese family with ADOA. Black-Right-Pointing-Pointer We find a new heterozygous mutation c.C1198G in OPA1 gene which may be a novel pathogenic mutation in this pedigree. Black-Right-Pointing-Pointer We do not find any mitochondrial DNA mutations associated with optic atrophy. Black-Right-Pointing-Pointer Other factors may also contribute to the phenotypic variability of ADOA in this pedigree. -- Abstract: A large four-generation Chinese family with autosomal dominant optic atrophy (ADOA) was investigated in the present study. Eight of the family members were affected in this pedigree. The affected family members exhibited early-onset and progressive visual impairment, resulting in mild to profound loss of visual acuity. The average age-at-onset was 15.9 years. A new heterozygous mutation c.C1198G was identified by sequence analysis of the 12th exon of the OPA1 gene. This mutation resulted in a proline to alanine substitution at codon 400, which was located in an evolutionarily conserved region. This missense mutation in the GTPase domain was supposed to result in a loss of function for the encoded protein and act through a dominant negative effect. No other mutations associated with optic atrophy were found in our present study. The c.C1198G heterozygous mutation in the OPA1 gene may be a novel key pathogenic mutation in this pedigree with ADOA. Furthermore, additional nuclear modifier genes, environmental factors, and psychological factors may also contribute to the phenotypic variability of ADOA in this pedigree.

Identification of novel mutations in Chinese Hans with autosomal dominant polycystic kidney disease

Directory of Open Access Journals (Sweden)

Yu Chaowen

2011-12-01

Full Text Available Abstract Background Autosomal dominant polycystic kidney disease (ADPKD is the most common inherited renal disease with an incidence of 1 in 400 to 1000. The disease is genetically heterogeneous, with two genes identified: PKD1 (16p13.3 and PKD2 (4q21. Molecular diagnosis of the disease in at-risk individuals is complicated due to the structural complexity of PKD1 gene and the high diversity of the mutations. This study is the first systematic ADPKD mutation analysis of both PKD1 and PKD2 genes in Chinese patients using denaturing high-performance liquid chromatography (DHPLC. Methods Both PKD1 and PKD2 genes were mutation screened in each proband from 65 families using DHPLC followed by DNA sequencing. Novel variations found in the probands were checked in their family members available and 100 unrelated normal controls. Then the pathogenic potential of the variations of unknown significance was examined by evolutionary comparison, effects of amino acid substitutions on protein structure, and effects of splice site alterations using online mutation prediction resources. Results A total of 92 variations were identified, including 27 reported previously. Definitely pathogenic mutations (ten frameshift, ten nonsense, two splicing defects and one duplication were identified in 28 families, and probably pathogenic mutations were found in an additional six families, giving a total detection level of 52.3% (34/65. About 69% (20/29 of the mutations are first reported with a recurrent mutation rate of 31%. Conclusions Mutation study of PKD1 and PKD2 genes in Chinese Hans with ADPKD may contribute to a better understanding of the genetic diversity between different ethnic groups and enrich the mutation database. Besides, evaluating the pathogenic potential of novel variations should also facilitate the clinical diagnosis and genetic counseling of the disease.

Extraordinary Genetic Diversity in a Wood Decay Mushroom.

Science.gov (United States)

Baranova, Maria A; Logacheva, Maria D; Penin, Aleksey A; Seplyarskiy, Vladimir B; Safonova, Yana Y; Naumenko, Sergey A; Klepikova, Anna V; Gerasimov, Evgeny S; Bazykin, Georgii A; James, Timothy Y; Kondrashov, Alexey S

2015-10-01

Populations of different species vary in the amounts of genetic diversity they possess. Nucleotide diversity π, the fraction of nucleotides that are different between two randomly chosen genotypes, has been known to range in eukaryotes between 0.0001 in Lynx lynx and 0.16 in Caenorhabditis brenneri. Here, we report the results of a comparative analysis of 24 haploid genotypes (12 from the United States and 12 from European Russia) of a split-gill fungus Schizophyllum commune. The diversity at synonymous sites is 0.20 in the American population of S. commune and 0.13 in the Russian population. This exceptionally high level of nucleotide diversity also leads to extreme amino acid diversity of protein-coding genes. Using whole-genome resequencing of 2 parental and 17 offspring haploid genotypes, we estimate that the mutation rate in S. commune is high, at 2.0 × 10(-8) (95% CI: 1.1 × 10(-8) to 4.1 × 10(-8)) per nucleotide per generation. Therefore, the high diversity of S. commune is primarily determined by its elevated mutation rate, although high effective population size likely also plays a role. Small genome size, ease of cultivation and completion of the life cycle in the laboratory, free-living haploid life stages and exceptionally high variability of S. commune make it a promising model organism for population, quantitative, and evolutionary genetics. © The Author 2015. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.

Mutation Pattern of Paired Immunoglobulin Heavy and Light Variable Domains in Chronic Lymphocytic Leukemia B Cells

Science.gov (United States)

Ghiotto, Fabio; Marcatili, Paolo; Tenca, Claudya; Calevo, Maria Grazia; Yan, Xiao-Jie; Albesiano, Emilia; Bagnara, Davide; Colombo, Monica; Cutrona, Giovanna; Chu, Charles C; Morabito, Fortunato; Bruno, Silvia; Ferrarini, Manlio; Tramontano, Anna; Fais, Franco; Chiorazzi, Nicholas

2011-01-01

B-cell chronic lymphocytic leukemia (CLL) patients display leukemic clones bearing either germline or somatically mutated immunoglobulin heavy variable (IGHV ) genes. Most information on CLL immunoglobulins (Igs), such as the definition of stereotyped B-cell receptors (BCRs), was derived from germline unmutated Igs. In particular, detailed studies on the distribution and nature of mutations in paired heavy- and light-chain domains of CLL clones bearing mutated Igs are lacking. To address the somatic hyper-mutation dynamics of CLL Igs, we analyzed the mutation pattern of paired IGHV–diversity-joining (IGHV-D-J ) and immunoglobulin kappa/lambda variable-joining (IGK/LV-J ) rearrangements of 193 leukemic clones that displayed ≥2% mutations in at least one of the two immunoglobulin variable (IGV ) genes (IGHV and/or IGK/LV ). The relationship between the mutation frequency in IGHV and IGK/LV complementarity determining regions (CDRs) and framework regions (FRs) was evaluated by correlation analysis. Replacement (R) mutation frequency within IGK/LV chain CDRs correlated significantly with mutation frequency of paired IGHV CDRs in λ but not κ isotype CLL clones. CDRs of IGKV-J rearrangements displayed a lower percentage of R mutations than IGHVs. The frequency/pattern of mutations in kappa CLL Igs differed also from that in κ-expressing normal B cells described in the literature. Instead, the mutation frequency within the FRs of IGHV and either IGKV or IGLV was correlated. Notably, the amount of diversity introduced by replaced amino acids was comparable between IGHVs and IGKVs. The data indicate a different mutation pattern between κ and λ isotype CLL clones and suggest an antigenic selection that, in κ samples, operates against CDR variation. PMID:21785810

Mutation Pattern of Paired Immunoglobulin Heavy and Light Variable Domains in Chronic Lymphocytic Leukemia B Cells

KAUST Repository

Ghiotto, Fabio; Marcatili, Paolo

2011-01-01

B-cell chronic lymphocytic leukemia (CLL) patients display leukemic clones bearing either germline or somatically mutated immunoglobulin heavy variable (IGHV ) genes. Most information on CLL immunoglobulins (Igs), such as the definition of stereotyped B-cell receptors (BCRs), was derived from germline unmutated Igs. In particular, detailed studies on the distribution and nature of mutations in paired heavy- and light-chain domains of CLL clones bearing mutated Igs are lacking. To address the somatic hyper-mutation dynamics of CLL Igs, we analyzed the mutation pattern of paired IGHV-diversity-joining (IGHV-D-J ) and immunoglobulin kappa/lambda variable-joining (IGK/LV-J ) rearrangements of 193 leukemic clones that displayed ≥ 2% mutations in at least one of the two immunoglobulin variable (IGV ) genes (IGHV and/or IGK/LV ). The relationship between the mutation frequency in IGHV and IGK/LV complementarity determining regions (CDRs) and framework regions (FRs) was evaluated by correlation analysis. Replacement (R) mutation frequency within IGK/LV chain CDRs correlated significantly with mutation frequency of paired IGHV CDRs in λ but not κ isotype CLL clones. CDRs of IGKV-J rearrangements displayed a lower percentage of R mutations than IGHVs. The frequency/pattern of mutations in kappa CLL Igs differed also from that in κ-expressing normal B cells described in the literature. Instead, the mutation frequency within the FRs of IGHV and either IGKV or IGLV was correlated. Notably, the amount of diversity introduced by replaced amino acids was comparable between IGHVs and IGKVs. The data indicate a different mutation pattern between κ and λ isotype CLL clones and suggest an antigenic selection that, in κ samples, operates against CDR variation.

Deleterious mutation accumulation in organelle genomes.

Science.gov (United States)

Lynch, M; Blanchard, J L

1998-01-01

It is well established on theoretical grounds that the accumulation of mildly deleterious mutations in nonrecombining genomes is a major extinction risk in obligately asexual populations. Sexual populations can also incur mutational deterioration in genomic regions that experience little or no recombination, i.e., autosomal regions near centromeres, Y chromosomes, and organelle genomes. Our results suggest, for a wide array of genes (transfer RNAs, ribosomal RNAs, and proteins) in a diverse collection of species (animals, plants, and fungi), an almost universal increase in the fixation probabilities of mildly deleterious mutations arising in mitochondrial and chloroplast genomes relative to those arising in the recombining nuclear genome. This enhanced width of the selective sieve in organelle genomes does not appear to be a consequence of relaxed selection, but can be explained by the decline in the efficiency of selection that results from the reduction of effective population size induced by uniparental inheritance. Because of the very low mutation rates of organelle genomes (on the order of 10(-4) per genome per year), the reduction in fitness resulting from mutation accumulation in such genomes is a very long-term process, not likely to imperil many species on time scales of less than a million years, but perhaps playing some role in phylogenetic lineage sorting on time scales of 10 to 100 million years.

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.

Genetic mutations associated with status epilepticus.

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Bhatnagar, M; Shorvon, S

2015-08-01

This paper reports the results of a preliminary search of the literature aimed at identifying the genetic mutations reported to be strongly associated with status epilepticus. Genetic mutations were selected for inclusion if status epilepticus was specifically mentioned as a consequence of the mutation in standard genetic databases or in a case report or review article. Mutations in 122 genes were identified. The genetic mutations identified were found in only rare conditions (sometimes vanishingly rare) and mostly in infants and young children with multiple other handicaps. Most of the genetic mutations can be subdivided into those associated with cortical dysplasias, inborn errors of metabolism, mitochondrial disease, or epileptic encephalopathies and childhood syndromes. There are no identified 'pure status epilepticus genes'. The range of genes underpinning status epilepticus differs in many ways from the range of genes underpinning epilepsy, which suggests that the processes underpinning status epilepticus differ from those underpinning epilepsy. It has been frequently postulated that status epilepticus is the result of a failure of 'seizure termination mechanisms', but the wide variety of genes affecting very diverse biochemical pathways identified in this survey makes any unitary cause unlikely. The genetic influences in status epilepticus are likely to involve a wide range of mechanisms, some related to development, some to cerebral energy production, some to diverse altered biochemical pathways, some to transmitter and membrane function, and some to defects in networks or systems. The fact that many of the identified genes are involved with cerebral development suggests that status epilepticus might often be a system or network phenomenon. To date, there are very few genes identified which are associated with adult-onset status epilepticus (except in those with preexisting neurological damage), and this is disappointing as the cause of many adult

Highly diverse, massive organic data as explored by a composite QSPR strategy: an advanced study of boiling point.

Science.gov (United States)

Ivanova, A A; Ivanov, A A; Oliferenko, A A; Palyulin, V A; Zefirov, N S

2005-06-01

An improved strategy of quantitative structure-property relationship (QSPR) studies of diverse and inhomogeneous organic datasets has been proposed. A molecular connectivity term was successively corrected for different structural features encoded in fragmental descriptors. The so-called solvation index 1chis (a weighted Randic index) was used as a "leading" variable and standardized molecular fragments were employed as "corrective" class-specific variables. Performance of the new approach was illustrated by modelling a dataset of experimental normal boiling points of 833 organic compounds belonging to 20 structural classes. Firstly, separate QSPR models were derived for each class and for eight groups of structurally similar classes. Finally, a general model formed by combining all the classes together was derived (r2=0.957, s=12.9degreesC). The strategy outlined can find application in QSPR analyses of massive, highly diverse databases of organic compounds.

Understanding the lid movements of LolA in Escherichia coli using molecular dynamics simulation and in silico point mutation.

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Murahari, Priyadarshini; Anishetty, Sharmila; Pennathur, Gautam

2013-12-01

The Lol system in Escherichia coli is involved in localization of lipoproteins and hence is essential for growth of the organism. LolA is a periplasmic chaperone that binds to outer-membrane specific lipoproteins and transports them from inner membrane to outer membrane through LolB. The hydrophobic lipid-binding cavity of LolA consists of α-helices which act as a lid in regulating the transfer of lipoproteins from LolA to LolB. The current study aims to investigate the structural changes observed in LolA during the transition from open to closed conformation in the absence of lipoprotein. Molecular dynamics (MD) simulations were carried out for two LolA crystal structures; LolA(R43L), and in silico mutated MsL43R for a simulation time of 50 ns in water environment. We have performed an in silico point mutation of leucine to arginine in MsL43R to evaluate the importance of arginine to induce structural changes and impact the stability of protein structure. A complete dynamic analysis of open to closed conformation reveals the existence of two distinct levels; closing of lid and closing of entrance of hydrophobic cavity. Our analysis reveals that the structural flexibility of LolA is an important factor for its role as a periplasmic chaperone. Copyright © 2013 Elsevier Ltd. All rights reserved.

Genomic analysis of globally diverse Mycobacterium tuberculosis strains provides insights into emergence and spread of multidrug resistance

Science.gov (United States)

Manson, Abigail L.; Cohen, Keira A.; Abeel, Thomas; Desjardins, Christopher A.; Armstrong, Derek T.; Barry, Clifton E.; Brand, Jeannette; Chapman, Sinéad B.; Cho, Sang-Nae; Gabrielian, Andrei; Gomez, James; Jodals, Andreea M.; Joloba, Moses; Jureen, Pontus; Lee, Jong Seok; Malinga, Lesibana; Maiga, Mamoudou; Nordenberg, Dale; Noroc, Ecaterina; Romancenco, Elena; Salazar, Alex; Ssengooba, Willy; Velayati, A. A.; Winglee, Kathryn; Zalutskaya, Aksana; Via, Laura E.; Cassell, Gail H.; Dorman, Susan E.; Ellner, Jerrold; Farnia, Parissa; Galagan, James E.; Rosenthal, Alex; Crudu, Valeriu; Homorodean, Daniela; Hsueh, Po-Ren; Narayanan, Sujatha; Pym, Alexander S.; Skrahina, Alena; Swaminathan, Soumya; Van der Walt, Martie; Alland, David; Bishai, William R.; Cohen, Ted; Hoffner, Sven; Birren, Bruce W.; Earl, Ashlee M.

2017-01-01

Multidrug-resistant tuberculosis (MDR-TB), caused by drug resistant strains of Mycobacterium tuberculosis, is an increasingly serious problem worldwide. In this study, we examined a dataset of 5,310 M. tuberculosis whole genome sequences from five continents. Despite great diversity with respect to geographic point of isolation, genetic background and drug resistance, patterns of drug resistance emergence were conserved globally. We have identified harbinger mutations that often precede MDR. In particular, the katG S315T mutation, conferring resistance to isoniazid, overwhelmingly arose before rifampicin resistance across all lineages, geographic regions, and time periods. Molecular diagnostics that include markers for rifampicin resistance alone will be insufficient to identify pre-MDR strains. Incorporating knowledge of pre-MDR polymorphisms, particularly katG S315, into molecular diagnostics will enable targeted treatment of patients with pre-MDR-TB to prevent further development of MDR-TB. PMID:28092681

Thyroglobulin Gene Mutation with Cold Nodule on Thyroid Scintigraphy

Directory of Open Access Journals (Sweden)

Toshio Kahara

2012-01-01

Full Text Available Thyroglobulin gene mutation is a rare cause of congenital hypothyroidism, but thyroglobulin gene mutations are thought to be associated with thyroid cancer development. A 21-year-old Japanese man treated with levothyroxine for congenital hypothyroidism had an enlarged thyroid gland with undetectable serum thyroglobulin despite elevated serum TSH level. The patient was diagnosed with thyroglobulin gene mutation, with compound heterozygosity for Gly304Cys missense mutation and Arg432X nonsense mutation. Ultrasonography showed a hypovascular large tumor in the left lobe that appeared as a cold nodule on thyroid scintigraphy. He underwent total thyroidectomy, but pathological study did not reveal findings of thyroid carcinoma, but rather a hyperplastic nodule with hemorrhage. Strong cytoplasmic thyroglobulin immunostaining was observed, but sodium iodide symporter immunostaining was hardly detected in the hyperplastic nodule. The clinical characteristics of patients with thyroglobulin gene mutations are diverse, and some patients are diagnosed by chance on examination of goiter in adults. The presence of thyroid tumors that appear as cold nodules on thyroid scintigraphy should consider the potential for thyroid carcinoma, if the patient has relatively low serum thyroglobulin concentration in relation to the degree of TSH without thyroglobulin autoantibody.

A new nonsense mutation in the NF1 gene with neurofibromatosis-Noonan syndrome phenotype.

Science.gov (United States)

Yimenicioğlu, Sevgi; Yakut, Ayten; Karaer, Kadri; Zenker, Martin; Ekici, Arzu; Carman, Kürşat Bora

2012-12-01

Neurofibromatosis-Noonan syndrome is a rare autosomal dominant disorder which combines neurofibromatosis type 1 (NF1) features with Noonan syndrome. NF1 gene mutations are reported in the majority of these patients. Sequence analysis of the established genes for Noonan syndrome revealed no mutation; a heterozygous NF1 point mutation c.7549C>T in exon 51, creating a premature stop codon (p.R2517X), had been demonstrated. Neurofibromatosis-Noonan syndrome recently has been considered a subtype of NF1 and caused by different NF1 mutations. We report the case of a 14-year-old boy with neurofibromatosis type 1 with Noonan-like features, who complained of headache with triventricular hydrocephaly and a heterozygous NF1 point mutation c.7549C>T in exon 51.

CDH23 mutation and phenotype heterogeneity: a profile of 107 diverse families with Usher syndrome and nonsyndromic deafness.

Science.gov (United States)

Astuto, L M; Bork, J M; Weston, M D; Askew, J W; Fields, R R; Orten, D J; Ohliger, S J; Riazuddin, S; Morell, R J; Khan, S; Riazuddin, S; Kremer, H; van Hauwe, P; Moller, C G; Cremers, C W R J; Ayuso, C; Heckenlively, J R; Rohrschneider, K; Spandau, U; Greenberg, J; Ramesar, R; Reardon, W; Bitoun, P; Millan, J; Legge, R; Friedman, T B; Kimberling, W J

2002-08-01

Usher syndrome type I is characterized by congenital hearing loss, retinitis pigmentosa (RP), and variable vestibular areflexia. Usher syndrome type ID, one of seven Usher syndrome type I genetic localizations, have been mapped to a chromosomal interval that overlaps with a nonsyndromic-deafness localization, DFNB12. Mutations in CDH23, a gene that encodes a putative cell-adhesion protein with multiple cadherin-like domains, are responsible for both Usher syndrome and DFNB12 nonsyndromic deafness. Specific CDH23 mutational defects have been identified that differentiate these two phenotypes. Only missense mutations of CDH23 have been observed in families with nonsyndromic deafness, whereas nonsense, frameshift, splice-site, and missense mutations have been identified in families with Usher syndrome. In the present study, a panel of 69 probands with Usher syndrome and 38 probands with recessive nonsyndromic deafness were screened for the presence of mutations in the entire coding region of CDH23, by heteroduplex, single-strand conformation polymorphism, and direct sequence analyses. A total of 36 different CDH23 mutations were detected in 45 families; 33 of these mutations were novel, including 18 missense, 3 nonsense, 5 splicing defects, 5 microdeletions, and 2 insertions. A total of seven mutations were common to more than one family. Numerous exonic and intronic polymorphisms also were detected. Results of ophthalmologic examinations of the patients with nonsyndromic deafness have found asymptomatic RP-like manifestations, indicating that missense mutations may have a subtle effect in the retina. Furthermore, patients with mutations in CDH23 display a wide range of hearing loss and RP phenotypes, differing in severity, age at onset, type, and the presence or absence of vestibular areflexia.

CDH23 Mutation and Phenotype Heterogeneity: A Profile of 107 Diverse Families with Usher Syndrome and Nonsyndromic Deafness

Science.gov (United States)

Astuto, L. M.; Bork, J. M.; Weston, M. D.; Askew, J. W.; Fields, R. R.; Orten, D. J.; Ohliger, S. J.; Riazuddin, S.; Morell, R. J.; Khan, S.; Riazuddin, S.; Kremer, H.; van Hauwe, P.; Moller, C. G.; Cremers, C. W. R. J.; Ayuso, C.; Heckenlively, J. R.; Rohrschneider, K.; Spandau, U.; Greenberg, J.; Ramesar, R.; Reardon, W.; Bitoun, P.; Millan, J.; Legge, R.; Friedman, T. B.; Kimberling, W. J.

2002-01-01

Usher syndrome type I is characterized by congenital hearing loss, retinitis pigmentosa (RP), and variable vestibular areflexia. Usher syndrome type ID, one of seven Usher syndrome type I genetic localizations, have been mapped to a chromosomal interval that overlaps with a nonsyndromic-deafness localization, DFNB12. Mutations in CDH23, a gene that encodes a putative cell-adhesion protein with multiple cadherin-like domains, are responsible for both Usher syndrome and DFNB12 nonsyndromic deafness. Specific CDH23 mutational defects have been identified that differentiate these two phenotypes. Only missense mutations of CDH23 have been observed in families with nonsyndromic deafness, whereas nonsense, frameshift, splice-site, and missense mutations have been identified in families with Usher syndrome. In the present study, a panel of 69 probands with Usher syndrome and 38 probands with recessive nonsyndromic deafness were screened for the presence of mutations in the entire coding region of CDH23, by heteroduplex, single-strand conformation polymorphism, and direct sequence analyses. A total of 36 different CDH23 mutations were detected in 45 families; 33 of these mutations were novel, including 18 missense, 3 nonsense, 5 splicing defects, 5 microdeletions, and 2 insertions. A total of seven mutations were common to more than one family. Numerous exonic and intronic polymorphisms also were detected. Results of ophthalmologic examinations of the patients with nonsyndromic deafness have found asymptomatic RP–like manifestations, indicating that missense mutations may have a subtle effect in the retina. Furthermore, patients with mutations in CDH23 display a wide range of hearing loss and RP phenotypes, differing in severity, age at onset, type, and the presence or absence of vestibular areflexia. PMID:12075507

Three novel PHEX gene mutations in four Chinese families with X-linked dominant hypophosphatemic rickets

Energy Technology Data Exchange (ETDEWEB)

Kang, Qing-lin [Department of Orthopedic Surgery, Shanghai Jiao Tong University Affiliated Sixth People' s Hospital, Shanghai 200233 (China); Xu, Jia [Department of Orthopedic Surgery, Shanghai Jiao Tong University Affiliated Sixth People' s Hospital, Shanghai 200233 (China); Metabolic Bone Disease and Genetic Research Unit, Department of Osteoporosis and Bone Diseases, Shanghai Jiao Tong University Affiliated Sixth People' s Hospital, Shanghai 200233 (China); Medical College of Soochow University, Suzhou, Jiangsu province 215000 (China); Zhang, Zeng [Department of Orthopedic Surgery, Shanghai Jiao Tong University Affiliated Sixth People' s Hospital, Shanghai 200233 (China); Metabolic Bone Disease and Genetic Research Unit, Department of Osteoporosis and Bone Diseases, Shanghai Jiao Tong University Affiliated Sixth People' s Hospital, Shanghai 200233 (China); He, Jin-wei [Metabolic Bone Disease and Genetic Research Unit, Department of Osteoporosis and Bone Diseases, Shanghai Jiao Tong University Affiliated Sixth People' s Hospital, Shanghai 200233 (China); Lu, Lian-song [Department of Orthopedic Surgery, Shanghai Jiao Tong University Affiliated Sixth People' s Hospital, Shanghai 200233 (China); Medical College of Soochow University, Suzhou, Jiangsu province 215000 (China); Fu, Wen-zhen [Metabolic Bone Disease and Genetic Research Unit, Department of Osteoporosis and Bone Diseases, Shanghai Jiao Tong University Affiliated Sixth People' s Hospital, Shanghai 200233 (China); Zhang, Zhen-lin, E-mail: zzl2002@medmail.com.cn [Metabolic Bone Disease and Genetic Research Unit, Department of Osteoporosis and Bone Diseases, Shanghai Jiao Tong University Affiliated Sixth People' s Hospital, Shanghai 200233 (China)

2012-07-13

Highlights: Black-Right-Pointing-Pointer In our study, all of the patients were of Han Chinese ethnicity, which were rarely reported. Black-Right-Pointing-Pointer We identified three novel PHEX gene mutations in four unrelated families with XLH. Black-Right-Pointing-Pointer We found that the relationship between the phenotype and genotype of the PHEX gene was not invariant. Black-Right-Pointing-Pointer We found that two PHEX gene sites, p.534 and p.731, were conserved. -- Abstract: Background: X-linked hypophosphatemia (XLH), the most common form of inherited rickets, is a dominant disorder that is characterized by renal phosphate wasting with hypophosphatemia, abnormal bone mineralization, short stature, and rachitic manifestations. The related gene with inactivating mutations associated with XLH has been identified as PHEX, which is a phosphate-regulating gene with homologies to endopeptidases on the X chromosome. In this study, a variety of PHEX mutations were identified in four Chinese families with XLH. Methods: We investigated four unrelated Chinese families who exhibited typical features of XLH by using PCR to analyze mutations that were then sequenced. The laboratory and radiological investigations were conducted simultaneously. Results: Three novel mutations were found in these four families: one frameshift mutation, c.2033dupT in exon 20, resulting in p.T679H; one nonsense mutation, c.1294A > T in exon 11, resulting in p.K432X; and one missense mutation, c.2192T > C in exon 22, resulting in p.F731S. Conclusions: We found that the PHEX gene mutations were responsible for XLH in these Chinese families. Our findings are useful for understanding the genetic basis of Chinese patients with XLH.

Decrease in specific micronutrient intake in colorectal cancer patients with tumors presenting Ki-ras mutation

OpenAIRE

JORDI SALAS; NURIA LASO; SERGI MAS; M. JOSE LAFUENTE; XAVIER CASTERAD; MANUEL TRIAS; ANTONIO BALLESTA; RAFAEL MOLINA; CARLOS ASCASO; SHICHUN ZHENG; JOHN K. WIENCKE; AMALIA LAFUENTE

2004-01-01

Decrease in specific micronutrient intake in colorectal cancer patients with tumors presenting Ki-ras mutation BACKGROUND: The diversity of the Mediterranean diet and the heterogeneity of acquired genetic alterations in colorectal cancer (CRC) led us to examine the possible association between dietary factors and mutations, such as Ki-ras mutations, in genes implicated in the pathogenesis of these neoplasms. PATIENTS AND METHODS: The study was based on 246 cases and 296 controls. For th...

Hybrid Capture-Based Comprehensive Genomic Profiling Identifies Lung Cancer Patients with Well-Characterized Sensitizing Epidermal Growth Factor Receptor Point Mutations That Were Not Detected by Standard of Care Testing.

Science.gov (United States)

Suh, James H; Schrock, Alexa B; Johnson, Adrienne; Lipson, Doron; Gay, Laurie M; Ramkissoon, Shakti; Vergilio, Jo-Anne; Elvin, Julia A; Shakir, Abdur; Ruehlman, Peter; Reckamp, Karen L; Ou, Sai-Hong Ignatius; Ross, Jeffrey S; Stephens, Philip J; Miller, Vincent A; Ali, Siraj M

2018-03-14

In our recent study, of cases positive for epidermal growth factor receptor ( EGFR ) exon 19 deletions using comprehensive genomic profiling (CGP), 17/77 (22%) patients with prior standard of care (SOC) EGFR testing results available were previously negative for exon 19 deletion. Our aim was to compare the detection rates of CGP versus SOC testing for well-characterized sensitizing EGFR point mutations (pm) in our 6,832-patient cohort. DNA was extracted from 40 microns of formalin-fixed paraffin-embedded sections from 6,832 consecutive cases of non-small cell lung cancer (NSCLC) of various histologies (2012-2015). CGP was performed using a hybrid capture, adaptor ligation-based next-generation sequencing assay to a mean coverage depth of 576×. Genomic alterations (pm, small indels, copy number changes and rearrangements) involving EGFR were recorded for each case and compared with prior testing results if available. Overall, there were 482 instances of EGFR exon 21 L858R (359) and L861Q (20), exon 18 G719X (73) and exon 20 S768I (30) pm, of which 103 unique cases had prior EGFR testing results that were available for review. Of these 103 cases, CGP identified 22 patients (21%) with sensitizing EGFR pm that were not detected by SOC testing, including 9/75 (12%) patients with L858R, 4/7 (57%) patients with L861Q, 8/20 (40%) patients with G719X, and 4/7 (57%) patients with S768I pm (some patients had multiple EGFR pm). In cases with available clinical data, benefit from small molecule inhibitor therapy was observed. CGP, even when applied to low tumor purity clinical-grade specimens, can detect well-known EGFR pm in NSCLC patients that would otherwise not be detected by SOC testing. Taken together with EGFR exon 19 deletions, over 20% of patients who are positive for EGFR -activating mutations using CGP are previously negative by SOC EGFR mutation testing, suggesting that thousands of such patients per year in the U.S. alone could experience improved clinical

Ras mutations are rare in solitary cold and toxic thyroid nodules.

Science.gov (United States)

Krohn, K; Reske, A; Ackermann, F; Müller, A; Paschke, R

2001-08-01

Activation of ras proto-oncogenes as a result of point mutations is detectable in a significant percentage of most types of tumour. Similar to neoplasms of other organs, mutations of all three ras genes can be found in thyroid tumours. H-, K- and N-ras mutations have been detected in up to 20% of follicular adenomas and adenomatous nodules which were not functionally characterized. This raises the question as to whether ras mutations are specific for hypofunctional nodules and TSH receptor mutations for hyperfunctioning nodules. To investigate ras and TSH receptor mutations with respect to functional differentiation we studied 41 scintigraphically cold nodules and 47 toxic thyroid nodules. To address the likelihood of a somatic mutation we also studied the clonal origin of these tumours. Genomic DNA was extracted from nodular and surrounding tissue. Mutational hot spots in exons 1 and 2 of the H- and K-ras gene were PCR amplified and sequenced using big dye terminator chemistry. Denaturing gradient gel electrophoresis (DGGE) was used to verify sequencing results for the H-ras gene and to analyse the N-ras gene because its greater sensitivity in detecting somatic mutations. Clonality of nodular thyroid tissue was evaluated using X-Chromosome inactivation based on PCR amplification of the human androgen receptor locus. Monoclonal origin was detectable in 14 of 23 informative samples from cold thyroid nodules. In toxic thyroid nodules the frequency of clonal tissue was 20 in 30 informative cases. Only one point mutation could be found in the N-ras gene codon 61 (Gly to Arg) in a cold adenomatous nodule which was monoclonal. In toxic thyroid nodules no ras mutation was detectable. Our study suggests that ras mutations are rare in solitary cold and toxic thyroid nodules and that the frequent monoclonal origin of these tumours implies somatic mutations in genes other than H-, K- and N-ras.

The functional importance of disease-associated mutation

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Klein Teri E

2002-09-01

Full Text Available Abstract Background For many years, scientists believed that point mutations in genes are the genetic switches for somatic and inherited diseases such as cystic fibrosis, phenylketonuria and cancer. Some of these mutations likely alter a protein's function in a manner that is deleterious, and they should occur in functionally important regions of the protein products of genes. Here we show that disease-associated mutations occur in regions of genes that are conserved, and can identify likely disease-causing mutations. Results To show this, we have determined conservation patterns for 6185 non-synonymous and heritable disease-associated mutations in 231 genes. We define a parameter, the conservation ratio, as the ratio of average negative entropy of analyzable positions with reported mutations to that of every analyzable position in the gene sequence. We found that 84.0% of the 231 genes have conservation ratios less than one. 139 genes had eleven or more analyzable mutations and 88.0% of those had conservation ratios less than one. Conclusions These results indicate that phylogenetic information is a powerful tool for the study of disease-associated mutations. Our alignments and analysis has been made available as part of the database at http://cancer.stanford.edu/mut-paper/. Within this dataset, each position is annotated with the analysis, so the most likely disease-causing mutations can be identified.

Point mutations in EBV gH that abrogate or differentially affect B cell and epithelial cell fusion

International Nuclear Information System (INIS)

Wu Liguo; Hutt-Fletcher, Lindsey M.

2007-01-01

Cell fusion mediated by Epstein-Barr virus requires three conserved glycoproteins, gB and gHgL, but activation is cell type specific. B cell fusion requires interaction between MHC class II and a fourth virus glycoprotein, gp42, which complexes non-covalently with gHgL. Epithelial cell fusion requires interaction between gHgL and a novel epithelial cell coreceptor and is blocked by excess gp42. We show here that gp42 interacts directly with gH and that point mutations in the region of gH recognized by an antibody that differentially inhibits epithelial and B cell fusion significantly impact both the core fusion machinery and cell-specific events. Substitution of alanine for glycine at residue 594 completely abrogates fusion with either B cells or epithelial cells. Substitution of alanine for glutamic acid at residue 595 reduces fusion with epithelial cells, greatly enhances fusion with B cells and allows low levels of B cell fusion even in the absence of gL

Exploration of Structural and Functional Variations Owing to Point Mutations in α-NAGA.

Science.gov (United States)

Meshach Paul, D; Rajasekaran, R

2018-03-01

Schindler disease is a lysosomal storage disorder caused due to deficiency or defective activity of alpha-N-acetylgalactosaminidase (α-NAGA). Mutations in gene encoding α-NAGA cause wide range of diseases, characterized with mild to severe clinical features. Molecular effects of these mutations are yet to be explored in detail. Therefore, this study was focused on four missense mutations of α-NAGA namely, S160C, E325K, R329Q and R329W. Native and mutant structures of α-NAGA were analysed to determine geometrical deviations such as the contours of root mean square deviation, root mean square fluctuation, percentage of residues in allowed regions of Ramachandran plot and solvent accessible surface area, using conformational sampling technique. Additionally, global energy-minimized structures of native and mutants were further analysed to compute their intra-molecular interactions, hydrogen bond dilution and distribution of secondary structure. In addition, docking studies were also performed to determine variations in binding energies between native and mutants. The deleterious effects of mutants were evident due to variations in their active site residues pertaining to spatial conformation and flexibility, comparatively. Hence, variations exhibited by mutants, namely S160C, E325K, R329Q and R329W to that of native, consequently, lead to the detrimental effects causing Schindler disease. This study computationally explains the underlying reasons for the pathogenesis of the disease, thereby aiding future researchers in drug development and disease management.

High prevalence of BRCA1 founder mutations in Greek breast/ovarian families.

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Konstantopoulou, I; Tsitlaidou, M; Fostira, F; Pertesi, M; Stavropoulou, A-V; Triantafyllidou, O; Tsotra, E; Tsiftsoglou, A P; Tsionou, C; Droufakou, S; Dimitrakakis, C; Fountzilas, G; Yannoukakos, D

2014-01-01

We have screened 473 breast/ovarian cancer patients with family history, aiming to define the prevalence and enrich the spectrum of BRCA1/2 pathogenic mutations occurring in the Greek population. An overall mutation prevalence of 32% was observed. Six BRCA1 recurrent/founder mutations dominate the observed spectrum (58.5% of all mutations found). These include three mutations in exon 20 and three large genomic deletions. Of the 44 different deleterious mutations found in both genes, 16 are novel and reported here for the first time. Correlation with available histopathology data showed that 80% of BRCA1 carriers presented a triple-negative breast cancer phenotype while 82% of BRCA2 carriers had oestrogen receptor positive tumours. This study provides a comprehensive view of the frequency, type and distribution of BRCA1/2 mutations in the Greek population as well as an insight of the screening strategy of choice for patients of Greek origin. We conclude that the Greek population has a diverse mutation spectrum influenced by strong founder effects. © 2013 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Ultra-deep sequencing of mouse mitochondrial DNA: mutational patterns and their origins.

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Adam Ameur

2011-03-01

Full Text Available Somatic mutations of mtDNA are implicated in the aging process, but there is no universally accepted method for their accurate quantification. We have used ultra-deep sequencing to study genome-wide mtDNA mutation load in the liver of normally- and prematurely-aging mice. Mice that are homozygous for an allele expressing a proof-reading-deficient mtDNA polymerase (mtDNA mutator mice have 10-times-higher point mutation loads than their wildtype siblings. In addition, the mtDNA mutator mice have increased levels of a truncated linear mtDNA molecule, resulting in decreased sequence coverage in the deleted region. In contrast, circular mtDNA molecules with large deletions occur at extremely low frequencies in mtDNA mutator mice and can therefore not drive the premature aging phenotype. Sequence analysis shows that the main proportion of the mutation load in heterozygous mtDNA mutator mice and their wildtype siblings is inherited from their heterozygous mothers consistent with germline transmission. We found no increase in levels of point mutations or deletions in wildtype C57Bl/6N mice with increasing age, thus questioning the causative role of these changes in aging. In addition, there was no increased frequency of transversion mutations with time in any of the studied genotypes, arguing against oxidative damage as a major cause of mtDNA mutations. Our results from studies of mice thus indicate that most somatic mtDNA mutations occur as replication errors during development and do not result from damage accumulation in adult life.

Building diversity in REU programs through MIMSUP at the Shannon Point Marine Center

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Bingham, B. L.; Sulkin, S.

2011-12-01

The road to a career in the ocean sciences can be long and challenging, particularly for students from racial/ethnic groups underrepresented in the field. For the past 21 years, faculty and staff at the Shannon Point Marine Center, Western Washington University have annually administered the NSF-funded Multicultural Initiative in the Marine Sciences: Undergraduate Participation (MIMSUP) program. The goal of MIMSUP is to increase diversity in the ocean sciences by moving students though their undergraduate programs into advanced education and leadership positions in the field. Helping students find positions in REU and other focused research programs is an important step along this path. Primary obstacles for the students include 1) a lack of knowledge about opportunities available to them, 2) a lack of experience preparing quality applications and 3) a lack of confidence in their ability to compete for positions. Focused mentoring, with an emphasis on skills development is important in helping outstanding, though inexperienced, students find and excel in REU programs.

Prevalence of common MEFV mutations and carrier frequencies in a ...

Indian Academy of Sciences (India)

Christian Oberkanins, oberkanins@viennalab.com. 1967; Pras et al. ... affected populations with spreading very diverse mutational patterns, especially ..... Arabs, mainly living in Syria, Lebanon, Palestine, Jordan and Iraq, and North .... Mediterranean fever suspected patients and gender correlation: a retrospective study.

Environmental stresses can alleviate the average deleterious effect of mutations

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Leibler Stanislas

2003-05-01

Full Text Available Abstract Background Fundamental questions in evolutionary genetics, including the possible advantage of sexual reproduction, depend critically on the effects of deleterious mutations on fitness. Limited existing experimental evidence suggests that, on average, such effects tend to be aggravated under environmental stresses, consistent with the perception that stress diminishes the organism's ability to tolerate deleterious mutations. Here, we ask whether there are also stresses with the opposite influence, under which the organism becomes more tolerant to mutations. Results We developed a technique, based on bioluminescence, which allows accurate automated measurements of bacterial growth rates at very low cell densities. Using this system, we measured growth rates of Escherichia coli mutants under a diverse set of environmental stresses. In contrast to the perception that stress always reduces the organism's ability to tolerate mutations, our measurements identified stresses that do the opposite – that is, despite decreasing wild-type growth, they alleviate, on average, the effect of deleterious mutations. Conclusions Our results show a qualitative difference between various environmental stresses ranging from alleviation to aggravation of the average effect of mutations. We further show how the existence of stresses that are biased towards alleviation of the effects of mutations may imply the existence of average epistatic interactions between mutations. The results thus offer a connection between the two main factors controlling the effects of deleterious mutations: environmental conditions and epistatic interactions.

The role of human demographic history in determining the distribution and frequency of transferase-deficient galactosaemia mutations.

LENUS (Irish Health Repository)

Flanagan, J M

2010-02-01

Classical or transferase-deficient galactosaemia is an inherited metabolic disorder caused by mutation in the human Galactose-1-phosphate uridyl transferase (GALT) gene. Of some 170 causative mutations reported, fewer than 10% are observed in more than one geographic region or ethnic group. To better understand the population history of the common GALT mutations, we have established a haplotyping system for the GALT locus incorporating eight single nucleotide polymorphisms and three short tandem repeat markers. We analysed haplotypes associated with the three most frequent GALT gene mutations, Q188R, K285N and Duarte-2 (D2), and estimated their age. Haplotype diversity, in conjunction with measures of genetic diversity and of linkage disequilibrium, indicated that Q188R and K285N are European mutations. The Q188R mutation arose in central Europe within the last 20 000 years, with its observed east-west cline of increasing relative allele frequency possibly being due to population expansion during the re-colonization of Europe by Homo sapiens in the Mesolithic age. K285N was found to be a younger mutation that originated in Eastern Europe and is probably more geographically restricted as it arose after all major European population expansions. The D2 variant was found to be an ancient mutation that originated before the expansion of Homo sapiens out of Africa.

How much do we know about spontaneous human mutation rates

Energy Technology Data Exchange (ETDEWEB)

Crow, J.F. (Univ. of Wisconsin, Madison, WI (United States))

1993-01-01

The much larger number of cell divisions between zygote and sperm than between zygote and egg, the increased age of fathers of children with new dominant mutations, and the greater evolution rate of pseudogenes on the Y chromosome than of those on autosomes all point to a much higher mutation rate in human males than in females, as first pointed out by Haldane in his classical study of X-linked hemophilia. The age of the father is the main factor determining the human spontaneous mutation rate, and probably the total mutation rate. The total mutation rate in Drosophila males of genes causing minor reduction in viability is at least 0.4 per sperm and may be considerably higher. The great mutation load implied by a rate of [approx] 1 per zygote can be greatly ameliorated by quasi-transition selection. Corresponding data are not available for the human population. The evolution rate of pseudogenes in primates suggests some 10[sup 2] new mutations per zygote. Presumably the overwhelming majority of these are neutral, but even the approximate fraction is not known. Statistical evidence in Drosophilia shows that mutations with minor effects cause about the same heterozygous impairment of fitness as those that are lethal when homozygous. The magnitude of heterozygous effect is such that almost all mutant genes are eliminated as heterozygotes before ever becoming homozygous. Although quantitative data in the human species are lacking, anecdotal information supports the conclusion that partial dominance is the rule here as well. This suggests that if the human mutation rate were increased or decreased, the effects would be spread over a period of 50-100 generations. 31 refs., 3 figs., 2 tabs.

Seeking new mutation clues from Bacillus licheniformis amylase by molecular dynamics simulations

Science.gov (United States)

Lu, Tao

2009-07-01

Amylase is one of the most important industrial enzymes in the world. Researchers have been searching for a highly thermal stable mutant for many years, but most focus on point mutations of one or few nitrogenous bases. According to this molecular dynamic simulation of amylase from Bacillus licheniformis (BLA), the deletion of some nitrogenous bases would be more efficacious than point mutations. The simulation reveals strong fluctuation of the BLA structure at optimum temperature. The fluctuation of the outer domains of BLA is stronger than that of the core domain. Molecular simulation provides a clue to design thermal stable amylases through deletion mutations in the outer domain.

A point mutation in the polymerase protein PB2 allows a reassortant H9N2 influenza isolate of wild-bird origin to replicate in human cells.

Science.gov (United States)

Hussein, Islam T.M.; Ma, Eric J.; Meixell, Brandt; Hill, Nichola J.; Lindberg, Mark S.; Albrecht , Randy A.; Bahl, Justin; Runstadler, Jonathan A.

2016-01-01

H9N2 influenza A viruses are on the list of potentially pandemic subtypes. Therefore, it is important to understand how genomic reassortment and genetic polymorphisms affect phenotypes of H9N2 viruses circulating in the wild bird reservoir. A comparative genetic analysis of North American H9N2 isolates of wild bird origin identified a naturally occurring reassortant virus containing gene segments derived from both North American and Eurasian lineage ancestors. The PB2 segment of this virus encodes 10 amino acid changes that distinguish it from other H9 strains circulating in North America. G590S, one of the 10 amino acid substitutions observed, was present in ~ 12% of H9 viruses worldwide. This mutation combined with R591 has been reported as a marker of pathogenicity for human pandemic 2009 H1N1 viruses. Screening by polymerase reporter assay of all the natural polymorphisms at these two positions identified G590/K591 and S590/K591 as the most active, with the highest polymerase activity recorded for the SK polymorphism. Rescued viruses containing these two polymorphic combinations replicated more efficiently in MDCK cells and they were the only ones tested that were capable of establishing productive infection in NHBE cells. A global analysis of all PB2 sequences identified the K591 signature in six viral HA/NA subtypes isolated from several hosts in seven geographic locations. Interestingly, introducing the K591 mutation into the PB2 of a human-adapted H3N2 virus did not affect its polymerase activity. Our findings demonstrate that a single point mutation in the PB2 of a low pathogenic H9N2 isolate could have a significant effect on viral phenotype and increase its propensity to infect mammals. However, this effect is not universal, warranting caution in interpreting point mutations without considering protein sequence context.

wKinMut: An integrated tool for the analysis and interpretation of mutations in human protein kinases

DEFF Research Database (Denmark)

Gonzalez-Izarzugaza, Jose Maria; Vazquez, Miguel; del Pozo, Angela

2013-01-01

mutation remains a considerable challenge. Results The wKinMut web-server offers direct prediction of the potential pathogenicity of the mutations from a number of methods, including our recently developed prediction method based on the combination of information from a range of diverse sources, including...

Variable mutation rates as an adaptive strategy in replicator populations.

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Michael Stich

2010-06-01

Full Text Available For evolving populations of replicators, there is much evidence that the effect of mutations on fitness depends on the degree of adaptation to the selective pressures at play. In optimized populations, most mutations have deleterious effects, such that low mutation rates are favoured. In contrast to this, in populations thriving in changing environments a larger fraction of mutations have beneficial effects, providing the diversity necessary to adapt to new conditions. What is more, non-adapted populations occasionally benefit from an increase in the mutation rate. Therefore, there is no optimal universal value of the mutation rate and species attempt to adjust it to their momentary adaptive needs. In this work we have used stationary populations of RNA molecules evolving in silico to investigate the relationship between the degree of adaptation of an optimized population and the value of the mutation rate promoting maximal adaptation in a short time to a new selective pressure. Our results show that this value can significantly differ from the optimal value at mutation-selection equilibrium, being strongly influenced by the structure of the population when the adaptive process begins. In the short-term, highly optimized populations containing little variability respond better to environmental changes upon an increase of the mutation rate, whereas populations with a lower degree of optimization but higher variability benefit from reducing the mutation rate to adapt rapidly. These findings show a good agreement with the behaviour exhibited by actual organisms that replicate their genomes under broadly different mutation rates.

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.

Mutations of alpha-galactosidase A gene in two unusual cases of Fabry disease

NARCIS (Netherlands)

Beyer, EM; Kopishinskaya, SV; Van Amstel, JKP; Tsvetkova, [No Value

1999-01-01

The mutation analysis of alpha-galactosidase A gene was carried out in two families with Fabry disease described by us earlier. In the family P. a new point mutation E341K (a G to A transition at position 10999 of the gene) was identified. The mutation causes a Glu341Lys substitution in

HIV-1 diversity and drug-resistant mutations in infected individuals in Changchun, China.

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Ming Yan

Full Text Available OBJECTIVES: Human immunodeficiency virus type 1 (HIV-1 infection has been detected in all provinces of China. Although epidemiological and phylogenetic studies have been conducted in many regions, such analyses are lacking from Jilin province in northeastern China. METHOD: Epidemiological and phylogenetic analyses, as well as detection of drug-resistant mutations, were conducted on 57 HIV-1 infected patients from Changchun city identified and confirmed through annual surveillance by local Centers for Disease Control in Jilin province of northeastern China in 2012. RESULTS: Sexual contact was determined to be the major pathway for HIV-1 transmission in Jilin, where hetero- and homosexual activities contributed almost equally. Phylogenetic analyses detected multiple subtypes of HIV-1 including subtype G circulating in Jilin, with multiple origins for each of them. Both subtype B and CRF01_AE were dominant, and evidence of subtype B transmitting between different high-risk groups was observed. Mutations in the viral protease at position 71 indicated the presence of a selective pressure. Several drug-resistant mutations were detected, although they were predicted with low-level resistance to antiviral treatments. CONCLUSIONS: Information from this study fills the gap in knowledge of HIV-1 transmission in Changchun city, Jilin province, China. By revealing the origin and evolutionary status of local HIV-1 strains, this work contributes to ongoing efforts in the control and prevention of AIDS.

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

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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

A genomic point mutation in the extracellular domain of the thyrotropin receptor in patients with Graves` ophthalmopathy

Energy Technology Data Exchange (ETDEWEB)

Bahn, R.S.; Dutton, C.M.; Heufelder, A.E.; Sarkar, G. [Mayo Clinic/Foundation, Rochester, MN (United States)]|[Ludwig-Maximilians-Universitat, Munich (Germany)

1994-02-01

Orbital and pretibial fibroblasts are targets of autoimmune attack in Graves` ophthalmopathy (GO) and pretibial dermopathy (PTD). The fibroblast autoantigen involved in these peripheral manifestations of Graves` disease and the reason for the association of GO and PTD with hyperthyroidism are unknown. RNA encoding the full-length extracellular domain of the TSH receptor has been demonstrated in orbital and dermal fibroblasts from patients with GO and normal subjects, suggesting a possible antigenic link between fibroblasts and thyrocytes. RNA was isolated from cultured orbital, pretibial, and abdominal fibroblasts obtained from patients with severe GO (n = 22) and normal subjects (n = 5). RNA was reverse transcribed, and the resulting cDNA was amplified by the polymerase chain reaction, using primers spanning overlapping regions of the entire extracellular domain of the TSH receptor. Nucleotide sequence analysis showed an A for C substitution in the first position of codon 52 in 2 of the patients, both of whom had GO, PTD, and acropachy. Genomic DNA isolated from the 2 affected patients, and not from an additional 12 normal subjects, revealed the codon 52 mutation by direct sequencing and AciI restriction enzyme digestions. In conclusion, the authors have demonstrated the presence of a genomic point mutation, leading to a threonine for proline amino acid shift in the predicted peptide, in the extracellular domain of the TSH receptor in two patients with severe GO, PTD, acropachy, and high thyroid-stimulating immunoglobulin levels. RNA encoding this mutant product was demonstrated in the fibroblasts of these patients. They suggest that the TSH receptor may be an important fibroblast autoantigen in GO and PTD, and that this mutant form of the receptor may have unique immunogenic properties. 28 refs., 3 figs., 2 tabs.

Parkinson disease: α-synuclein mutational screening and new clinical insight into the p.E46K mutation.

Science.gov (United States)

Pimentel, Márcia M G; Rodrigues, Fabíola C; Leite, Marco Antônio A; Campos Júnior, Mário; Rosso, Ana Lucia; Nicaretta, Denise H; Pereira, João S; Silva, Delson José; Della Coletta, Marcus V; Vasconcellos, Luiz Felipe R; Abreu, Gabriella M; Dos Santos, Jussara M; Santos-Rebouças, Cíntia B

2015-06-01

Amongst Parkinson's disease-causing genetic factors, missense mutations and genomic multiplications in the gene encoding α-synuclein are well established causes of the disease, although genetic data in populations with a high degree of admixture, such as the Brazilian one, are still scarce. In this study, we conducted a molecular screening of α-synuclein point mutations and copy number variation in the largest cohort of Brazilian patients with Parkinson's disease (n = 549) and also in twelve Portuguese and one Bolivian immigrants. Genomic DNA was isolated from peripheral blood leukocytes or saliva, and the mutational screening was performed by quantitative and qualitative real-time PCR. The only alteration identified was the p.E46K mutation in a 60-year-old man, born in Bolivia, with a familial history of autosomal dominant Parkinson's disease. This is the second family ever reported, in which this rare pathogenic mutation is segregating. The same mutation was firstly described ten years ago in a Spanish family with a neurodegenerative syndrome combining parkinsonism, dementia and visual hallucinations. The clinical condition of our proband reveals a less aggressive phenotype than previously described and reinforces that marked phenotypic heterogeneity is common among patients with Parkinson's disease, even among those carriers sharing the same mutation. Our findings add new insight into the preexisting information about α-synuclein p.E46K, improving our understanding about the endophenotypes associated to this mutation and corroborate that missense alterations and multiplications in α-synuclein are uncommon among Brazilian patients with Parkinson's disease. Copyright © 2015 Elsevier Ltd. All rights reserved.

Elucidating the Interdependence of Drug Resistance from Combinations of Mutations.

Science.gov (United States)

Ragland, Debra A; Whitfield, Troy W; Lee, Sook-Kyung; Swanstrom, Ronald; Zeldovich, Konstantin B; Kurt-Yilmaz, Nese; Schiffer, Celia A

2017-11-14

HIV-1 protease is responsible for the cleavage of 12 nonhomologous sites within the Gag and Gag-Pro-Pol polyproteins in the viral genome. Under the selective pressure of protease inhibition, the virus evolves mutations within (primary) and outside of (secondary) the active site, allowing the protease to process substrates while simultaneously countering inhibition. The primary protease mutations impede inhibitor binding directly, while the secondary mutations are considered accessory mutations that compensate for a loss in fitness. However, the role of secondary mutations in conferring drug resistance remains a largely unresolved topic. We have shown previously that mutations distal to the active site are able to perturb binding of darunavir (DRV) via the protein's internal hydrogen-bonding network. In this study, we show that mutations distal to the active site, regardless of context, can play an interdependent role in drug resistance. Applying eigenvalue decomposition to collections of hydrogen bonding and van der Waals interactions from a series of molecular dynamics simulations of 15 diverse HIV-1 protease variants, we identify sites in the protease where amino acid substitutions lead to perturbations in nonbonded interactions with DRV and/or the hydrogen-bonding network of the protease itself. While primary mutations are known to drive resistance in HIV-1 protease, these findings delineate the significant contributions of accessory mutations to resistance. Identifying the variable positions in the protease that have the greatest impact on drug resistance may aid in future structure-based design of inhibitors.

Genetic diversity estimates point to immediate efforts for conserving the endangered Tibetan sheep of India.

Science.gov (United States)

Sharma, Rekha; Kumar, Brijesh; Arora, Reena; Ahlawat, Sonika; Mishra, A K; Tantia, M S

2016-06-01

Tibetan is a valuable Himalayan sheep breed classified as endangered. Knowledge of the level and distribution of genetic diversity in Tibetan sheep is important for designing conservation strategies for their sustainable survival and to preserve their evolutionary potential. Thus, for the first time, genetic variability in the Tibetan population was accessed with twenty five inter-simple sequence repeat markers. All the microsatellites were polymorphic and a total of 148 alleles were detected across these loci. The observed number of alleles across all the loci was more than the effective number of alleles and ranged from 3 (BM6506) to 11 (BM6526) with 5.920 ± 0.387 mean number of alleles per locus. The average observed heterozygosity was less than the expected heterozygosity. The observed and expected heterozygosity values ranged from 0.150 (BM1314) to 0.9 (OarCP20) with an overall mean of 0.473 ± 0.044 and from 0.329 (BM8125) to 0.885 (BM6526) with an overall mean 0.672 ± 0.030, respectively. The lower heterozygosity pointed towards diminished genetic diversity in the population. Thirteen microsatellite loci exhibited significant (P scientific management of the population so as to increase the population hand in hand with retaining the founder alleles to the maximum possible extent.

Molecular analysis of formaldehyde-induced mutations in human lymphoblasts and E. coli

International Nuclear Information System (INIS)

Crosby, R.M.; Richardson, K.K.; Craft, T.R.; Benforado, K.B.; Liber, H.L.; Skopek, T.R.

1988-01-01

The molecular nature of formaldehyde (HCHO)-induced mutations was studied in both human lymphoblasts and E. coli. Thirty HPRT - human lymphoblast colonies induced by eight repetitive 150 μM HCHO treatments were characterized by Southern blot analysis. Fourteen of these mutants (47%) had visible deletions of some or all of the X-linked HPRT bands, indicating that HCHO can induce large losses of DNA in human lymphoblasts. In E. coli., DNA alterations induced by HCHO were characterized with use of the xanthine guanine phosphoribosyl transferase (gpt) gene as the genetic target. Exposure of E. coli to 4 mM HCHO for 1 hr induced large insertions (41%), large deletions (18%), and point mutations (41%). Dideoxy DNA sequencing revealed that most of the point mutations were transversions at GC base pairs. In contrast, exposure of E. coli to 40 mM HCHO for 1 hr produced 92% point mutations, 62% of which were transitions at a single AT base pair in the gene. Therefore, HCHO is capable of producing different genetic alterations in E. coli at different concentrations, suggesting fundamental differences in the mutagenic mechanisms operating at the two concentrations used. Naked pSV2gpt plasmid DNA was exposed to 3.3 or 10 mM HCHO and transformed into E. coli. Most of the resulting mutations were frameshifts, again suggesting a different mutagenic mechanism

FLT3 mutation incidence and timing of origin in a population case series of pediatric leukemia

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Chang Jeffrey

2010-09-01

Full Text Available Abstract Background Mutations in FLT3 result in activated tyrosine kinase activity, cell growth stimulation, and a poor prognosis among various subtypes of leukemia. The causes and timing of the mutations are not currently known. We evaluated the prevalence and timing of origin of FLT3 mutations in a population series of childhood leukemia patients from Northern California. Methods We screened and sequenced FLT3 mutations (point mutations and internal tandem duplications, ITDs among 517 childhood leukemia patients, and assessed whether these mutations occurred before or after birth using sensitive "backtracking" methods. Results We determined a mutation prevalence of 9 of 73 acute myeloid leukemias (AMLs, 12% and 9 of 441 acute lymphocytic leukemias (ALLs, 2%. Among AMLs, FLT3 mutations were more common in older patients, and among ALLs, FLT3 mutations were more common in patients with high hyperdiploidy (3.7% than those without this cytogenetic feature (1.4%. Five FLT3 ITDs, one deletion mutation, and 3 point mutations were assessed for their presence in neonatal Guthrie spots using sensitive real-time PCR techniques, and no patients were found to harbor FLT3 mutations at birth. Conclusions FLT3 mutations were not common in our population-based patient series in California, and patients who harbor FLT3 mutations most likely acquire them after they are born.

Both point mutations and low expression levels of the nicotinic acetylcholine receptor β1 subunit are associated with imidacloprid resistance in an Aphis gossypii (Glover) population from a Bt cotton field in China.

Science.gov (United States)

Chen, Xuewei; Li, Fen; Chen, Anqi; Ma, Kangsheng; Liang, Pingzhuo; Liu, Ying; Song, Dunlun; Gao, Xiwu

2017-09-01

Aphis gossypii Glover is a destructive pest of numerous crops throughout the world. Although the expansion of Bt cotton cultivation has helped to control some insect pests, the damage from cotton aphids has not been mitigated. The evolution of aphid resistance to imidacloprid has made its chemical control more difficult since its introduction in 1991. Field populations of A. gossypii that were collected from different transgenic (Bt) cotton planting areas of China in 2014 developed different levels of resistance to imidacloprid. The IMI_R strain has developed high resistance to imidacloprid with the resistance ratio >1200-fold. Compared with the susceptible IMI_S strain, the IMI_R strain also developed a high level cross resistance to sulfoxaflor and acetamiprid. The limited synergism with either PBO or DEF suggests that resistance may be due to the site mutation of molecular target rather than to enhanced detoxification. Three target-site mutations within the nicotinic acetylcholine receptor (nAChR) β1 subunit were detected in the IMI_R strain. The R81T mutation has been reported to be responsible for imidacloprid resistance in A. gossypii and M. persicae. Both V62I and K264E were first detected in A. gossypii. These point mutations are also present in field populations, suggesting that they play a role in the resistance to imidacloprid. Furthermore, the expression level of transcripts encoding β1 subunit was decreased significantly in the IMI_R strain compared with the IMI_S strain, suggesting that both point mutations and the down-regulation of nAChR β1 subunit expression may be involved in the resistance mechanism for imidacloprid in A. gossypii. These results should be useful for the management of imidacloprid-resistant cotton aphids in Bt cotton fields in China. Copyright © 2016 Elsevier B.V. All rights reserved.

No evidence for association of autism with rare heterozygous point mutations in Contactin-Associated Protein-Like 2 (CNTNAP2, or in Other Contactin-Associated Proteins or Contactins.

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John D Murdoch

2015-01-01

Full Text Available Contactins and Contactin-Associated Proteins, and Contactin-Associated Protein-Like 2 (CNTNAP2 in particular, have been widely cited as autism risk genes based on findings from homozygosity mapping, molecular cytogenetics, copy number variation analyses, and both common and rare single nucleotide association studies. However, data specifically with regard to the contribution of heterozygous single nucleotide variants (SNVs have been inconsistent. In an effort to clarify the role of rare point mutations in CNTNAP2 and related gene families, we have conducted targeted next-generation sequencing and evaluated existing sequence data in cohorts totaling 2704 cases and 2747 controls. We find no evidence for statistically significant association of rare heterozygous mutations in any of the CNTN or CNTNAP genes, including CNTNAP2, placing marked limits on the scale of their plausible contribution to risk.

Molecular Epidemiology of P. vivax in Iran: High Diversity and Complex Sub-Structure Using Neutral Markers, but No Evidence of Y976F Mutation at pvmdr1.

Science.gov (United States)

Hamedi, Yaghoob; Sharifi-Sarasiabi, Khojasteh; Dehghan, Farzaneh; Safari, Reza; To, Sheren; Handayuni, Irene; Trimarsanto, Hidayat; Price, Ric N; Auburn, Sarah

2016-01-01

Malaria remains endemic at low levels in the south-eastern provinces of Iran bordering Afghanistan and Pakistan, with the majority of cases attributable to P. vivax. The national guidelines recommend chloroquine (CQ) as blood-stage treatment for uncomplicated P. vivax, but the large influx of imported cases enhances the risk of introducing CQ resistance (CQR). The genetic diversity at pvmdr1, a putative modulator of CQR, and across nine putatively neutral short tandem repeat (STR) markers were assessed in P. vivax clinical isolates collected between April 2007 and January 2013 in Hormozgan Province, south-eastern Iran. One hundred blood samples were collected from patients with microscopy-confirmed P. vivax enrolled at one of five district clinics. In total 73 (73%) were autochthonous cases, 23 (23%) imported cases from Afghanistan or Pakistan, and 4 (4%) with unknown origin. 97% (97/100) isolates carried the F1076L mutation, but none carried the Y976F mutation. STR genotyping was successful in 71 (71%) isolates, including 57(57%) autochthonous and 11 (11%) imported cases. Analysis of population structure revealed 2 major sub-populations, K1 and K2, with further sub-structure within K2. The K1 sub-population had markedly lower diversity than K2 (HE = 0.06 vs HE = 0.82) suggesting that the sub-populations were sustained by distinct reservoirs with differing transmission dynamics, possibly reflecting local versus imported/introduced populations. No notable separation was observed between the local and imported cases although the sample size was limited. The contrasting low versus high diversity in the two sub-populations (K1 and K2) infers that a combination of local transmission and cross-border malaria from higher transmission regions shape the genetic make-up of the P. vivax population in south-eastern Iran. There was no molecular evidence of CQR amongst the local or imported cases, but ongoing clinical surveillance is warranted.

Molecular Epidemiology of P. vivax in Iran: High Diversity and Complex Sub-Structure Using Neutral Markers, but No Evidence of Y976F Mutation at pvmdr1.

Directory of Open Access Journals (Sweden)

Yaghoob Hamedi

Full Text Available Malaria remains endemic at low levels in the south-eastern provinces of Iran bordering Afghanistan and Pakistan, with the majority of cases attributable to P. vivax. The national guidelines recommend chloroquine (CQ as blood-stage treatment for uncomplicated P. vivax, but the large influx of imported cases enhances the risk of introducing CQ resistance (CQR.The genetic diversity at pvmdr1, a putative modulator of CQR, and across nine putatively neutral short tandem repeat (STR markers were assessed in P. vivax clinical isolates collected between April 2007 and January 2013 in Hormozgan Province, south-eastern Iran. One hundred blood samples were collected from patients with microscopy-confirmed P. vivax enrolled at one of five district clinics. In total 73 (73% were autochthonous cases, 23 (23% imported cases from Afghanistan or Pakistan, and 4 (4% with unknown origin. 97% (97/100 isolates carried the F1076L mutation, but none carried the Y976F mutation. STR genotyping was successful in 71 (71% isolates, including 57(57% autochthonous and 11 (11% imported cases. Analysis of population structure revealed 2 major sub-populations, K1 and K2, with further sub-structure within K2. The K1 sub-population had markedly lower diversity than K2 (HE = 0.06 vs HE = 0.82 suggesting that the sub-populations were sustained by distinct reservoirs with differing transmission dynamics, possibly reflecting local versus imported/introduced populations. No notable separation was observed between the local and imported cases although the sample size was limited.The contrasting low versus high diversity in the two sub-populations (K1 and K2 infers that a combination of local transmission and cross-border malaria from higher transmission regions shape the genetic make-up of the P. vivax population in south-eastern Iran. There was no molecular evidence of CQR amongst the local or imported cases, but ongoing clinical surveillance is warranted.

Multi-center analysis of glucocerebrosidase mutations in Parkinson disease

Science.gov (United States)

Sidransky, Ellen; Nalls, Michael A.; Aasly, Jan O.; Aharon-Peretz, Judith; Annesi, Grazia; Barbosa, Egberto Reis; Bar-Shira, Anat; Berg, Daniela; Bras, Jose; Brice, Alexis; Chen, Chiung-Mei; Clark, Lorraine N.; Condroyer, Christel; De Marco, Elvira Valeria; Dürr, Alexandra; Eblan, Michael J.; Fahn, Stanley; Farrer, Matthew; Fung, Hon-Chung; Gan-Or, Ziv; Gasser, Thomas; Gershoni-Baruch, Ruth; Giladi, Nir; Griffith, Alida; Gurevich, Tanya; Januario, Cristina; Kropp, Peter; Lang, Anthony E.; Lee-Chen, Guey-Jen; Lesage, Suzanne; Marder, Karen; Mata, Ignacio F.; Mirelman, Anat; Mitsui, Jun; Mizuta, Ikuko; Nicoletti, Giuseppe; Oliveira, Catarina; Ottman, Ruth; Orr-Urtreger, Avi; Pereira, Lygia V.; Quattrone, Aldo; Rogaeva, Ekaterina; Rolfs, Arndt; Rosenbaum, Hanna; Rozenberg, Roberto; Samii, Ali; Samaddar, Ted; Schulte, Claudia; Sharma, Manu; Singleton, Andrew; Spitz, Mariana; Tan, Eng-King; Tayebi, Nahid; Toda, Tatsushi; Troiano, André; Tsuji, Shoji; Wittstock, Matthias; Wolfsberg, Tyra G.; Wu, Yih-Ru; Zabetian, Cyrus P.; Zhao, Yi; Ziegler, Shira G.

2010-01-01

Background Recent studies indicate an increased frequency of mutations in the gene for Gaucher disease, glucocerebrosidase (GBA), among patients with Parkinson disease. An international collaborative study was conducted to ascertain the frequency of GBA mutations in ethnically diverse patients with Parkinson disease. Methods Sixteen centers participated, including five from the Americas, six from Europe, two from Israel and three from Asia. Each received a standard DNA panel to compare genotyping results. Genotypes and phenotypic data from patients and controls were analyzed using multivariate logistic regression models and the Mantel Haenszel procedure to estimate odds ratios (ORs) across studies. The sample included 5691 patients (780 Ashkenazi Jews) and 4898 controls (387 Ashkenazi Jews). Results All 16 centers could detect GBA mutations, L444P and N370S, and the two were found in 15.3% of Ashkenazi patients with Parkinson disease (ORs = 4.95 for L444P and 5.62 for N370S), and in 3.2% of non-Ashkenazi patients (ORs = 9.68 for L444P and 3.30 for N370S). GBA was sequenced in 1642 non-Ashkenazi subjects, yielding a frequency of 6.9% for all mutations, demonstrate that limited mutation screens miss half the mutant alleles. The presence of any GBA mutation was associated with an OR of 5.43 across studies. Clinically, although phenotypes varied, subjects with a GBA mutation presented earlier, and were more likely to have affected relatives and atypical manifestations. Conclusion Data collected from sixteen centers demonstrate that there is a strong association between GBA mutations and Parkinson disease. PMID:19846850

Development and inter-laboratory validation of unlabeled probe melting curve analysis for detection of JAK2 V617F mutation in polycythemia vera.

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Wu, Zhiyuan; Yuan, Hong; Zhang, Xinju; Liu, Weiwei; Xu, Jinhua; Zhang, Wei; Guan, Ming

2011-01-01

JAK2 V617F, a somatic point mutation that leads to constitutive JAK2 phosphorylation and kinase activation, has been incorporated into the WHO classification and diagnostic criteria of myeloid neoplasms. Although various approaches such as restriction fragment length polymorphism, amplification refractory mutation system and real-time PCR have been developed for its detection, a generic rapid closed-tube method, which can be utilized on routine genetic testing instruments with stability and cost-efficiency, has not been described. Asymmetric PCR for detection of JAK2 V617F with a 3'-blocked unlabeled probe, saturate dye and subsequent melting curve analysis was performed on a Rotor-Gene® Q real-time cycler to establish the methodology. We compared this method to the existing amplification refractory mutation systems and direct sequencing. Hereafter, the broad applicability of this unlabeled probe melting method was also validated on three diverse real-time systems (Roche LightCycler® 480, Applied Biosystems ABI® 7500 and Eppendorf Mastercycler® ep realplex) in two different laboratories. The unlabeled probe melting analysis could genotype JAK2 V617F mutation explicitly with a 3% mutation load detecting sensitivity. At level of 5% mutation load, the intra- and inter-assay CVs of probe-DNA heteroduplex (mutation/wild type) covered 3.14%/3.55% and 1.72%/1.29% respectively. The method could equally discriminate mutant from wild type samples on the other three real-time instruments. With a high detecting sensitivity, unlabeled probe melting curve analysis is more applicable to disclose JAK2 V617F mutation than conventional methodologies. Verified with the favorable inter- and intra-assay reproducibility, unlabeled probe melting analysis provided a generic mutation detecting alternative for real-time instruments.

Development and inter-laboratory validation of unlabeled probe melting curve analysis for detection of JAK2 V617F mutation in polycythemia vera.

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Zhiyuan Wu

Full Text Available BACKGROUND: JAK2 V617F, a somatic point mutation that leads to constitutive JAK2 phosphorylation and kinase activation, has been incorporated into the WHO classification and diagnostic criteria of myeloid neoplasms. Although various approaches such as restriction fragment length polymorphism, amplification refractory mutation system and real-time PCR have been developed for its detection, a generic rapid closed-tube method, which can be utilized on routine genetic testing instruments with stability and cost-efficiency, has not been described. METHODOLOGY/PRINCIPAL FINDINGS: Asymmetric PCR for detection of JAK2 V617F with a 3'-blocked unlabeled probe, saturate dye and subsequent melting curve analysis was performed on a Rotor-Gene® Q real-time cycler to establish the methodology. We compared this method to the existing amplification refractory mutation systems and direct sequencing. Hereafter, the broad applicability of this unlabeled probe melting method was also validated on three diverse real-time systems (Roche LightCycler® 480, Applied Biosystems ABI® 7500 and Eppendorf Mastercycler® ep realplex in two different laboratories. The unlabeled probe melting analysis could genotype JAK2 V617F mutation explicitly with a 3% mutation load detecting sensitivity. At level of 5% mutation load, the intra- and inter-assay CVs of probe-DNA heteroduplex (mutation/wild type covered 3.14%/3.55% and 1.72%/1.29% respectively. The method could equally discriminate mutant from wild type samples on the other three real-time instruments. CONCLUSIONS: With a high detecting sensitivity, unlabeled probe melting curve analysis is more applicable to disclose JAK2 V617F mutation than conventional methodologies. Verified with the favorable inter- and intra-assay reproducibility, unlabeled probe melting analysis provided a generic mutation detecting alternative for real-time instruments.

Screening for mutations in the androgen receptor gene (AR) causing infertility in Syrian men using real-time PCR

International Nuclear Information System (INIS)

Madania, A.; Ghouri, I.; Abou-Alshamat, Gh.; Issa, M.; Al-Halabi, M.

2012-01-01

14 known point mutations in the androgen receptor gene (AR) causing male infertility were screened by real time PCR and by DNA sequencing, in order to identify point mutations in the AR gene causing infertility in azoospermic men. We screened 110 Syrian patients suffering from non-obstructive azoospermia with no chromosomal aberrations or AZF micro deletions. We discovered a new AR mutation, del 57Leu, described for the first time as a possible cause of male infertility. Furthermore, we found two patients with the Ala474Val mutation and one patient bearing the Pro390Ser mutation. Our results indicate that these mutations are significant markers for idiopathic male infertility in the Syrian society and in Mediterranean populations in general. (author)

Mutational spectrum of Duchenne muscular dystrophy in Spain: Study of 284 cases.

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Vieitez, I; Gallano, P; González-Quereda, L; Borrego, S; Marcos, I; Millán, J M; Jairo, T; Prior, C; Molano, J; Trujillo-Tiebas, M J; Gallego-Merlo, J; García-Barcina, M; Fenollar, M; Navarro, C

Duchenne muscular dystrophy (DMD) is a severe X-linked recessive neuromuscular disease that affects one in 3500 live-born males. The total absence of dystrophin observed in DMD patients is generally caused by mutations that disrupt the reading frame of the DMD gene, and about 80% of cases harbour deletions or duplications of one or more exons. We reviewed 284 cases of males with a genetic diagnosis of DMD between 2007 and 2014. These patients were selected from 8 Spanish reference hospitals representing most areas of Spain. Multiplex PCR, MLPA, and sequencing were performed to identify mutations. Most of these DMD patients present large deletions (46.1%) or large duplications (19.7%) in the dystrophin gene. The remaining 34.2% correspond to point mutations, and half of these correspond to nonsense mutations. In this study we identified 23 new mutations in DMD: 7 large deletions and 16 point mutations. The algorithm for genetic diagnosis applied by the participating centres is the most appropriate for genotyping patients with DMD. The genetic specificity of different therapies currently being developed emphasises the importance of identifying the mutation appearing in each patient; 38.7% of the cases in this series are eligible to participate in current clinical trials. Copyright © 2016 Sociedad Española de Neurología. Publicado por Elsevier España, S.L.U. All rights reserved.

Genomic Profiling on an Unselected Solid Tumor Population Reveals a Highly Mutated Wnt/β-Catenin Pathway Associated with Oncogenic EGFR Mutations

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Jingrui Jiang

2018-04-01

Full Text Available Oncogenic epidermal growth factor receptors (EGFRs can recruit key effectors in diverse cellular processes to propagate oncogenic signals. Targeted and combinational therapeutic strategies have been successfully applied for treating EGFR-driven cancers. However, a main challenge in EGFR therapies is drug resistance due to mutations, oncogenic shift, alternative signaling, and other potential mechanisms. To further understand the genetic alterations associated with oncogenic EGFRs and to provide further insight into optimal and personalized therapeutic strategies, we applied a proprietary comprehensive next-generation sequencing (NGS-based assay of 435 genes to systematically study the genomic profiles of 1565 unselected solid cancer patient samples. We found that activating EGFR mutations were predominantly detected in lung cancer, particularly in non-small cell lung cancer (NSCLC. The mutational landscape of EGFR-driven tumors covered most key signaling pathways and biological processes. Strikingly, the Wnt/β-catenin pathway was highly mutated (48 variants detected in 46% of the EGFR-driven tumors, and its variant number topped that in the TP53/apoptosis and PI3K-AKT-mTOR pathways. Furthermore, an analysis of mutation distribution revealed a differential association pattern of gene mutations between EGFR exon 19del and EGFR L858R. Our results confirm the aggressive nature of the oncogenic EGFR-driven tumors and reassure that a combinational strategy should have advantages over an EGFR-targeted monotherapy and holds great promise for overcoming drug resistance.

Accurate Measurement of the Effects of All Amino-Acid Mutations on Influenza Hemagglutinin.

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Doud, Michael B; Bloom, Jesse D

2016-06-03

Influenza genes evolve mostly via point mutations, and so knowing the effect of every amino-acid mutation provides information about evolutionary paths available to the virus. We and others have combined high-throughput mutagenesis with deep sequencing to estimate the effects of large numbers of mutations to influenza genes. However, these measurements have suffered from substantial experimental noise due to a variety of technical problems, the most prominent of which is bottlenecking during the generation of mutant viruses from plasmids. Here we describe advances that ameliorate these problems, enabling us to measure with greatly improved accuracy and reproducibility the effects of all amino-acid mutations to an H1 influenza hemagglutinin on viral replication in cell culture. The largest improvements come from using a helper virus to reduce bottlenecks when generating viruses from plasmids. Our measurements confirm at much higher resolution the results of previous studies suggesting that antigenic sites on the globular head of hemagglutinin are highly tolerant of mutations. We also show that other regions of hemagglutinin-including the stalk epitopes targeted by broadly neutralizing antibodies-have a much lower inherent capacity to tolerate point mutations. The ability to accurately measure the effects of all influenza mutations should enhance efforts to understand and predict viral evolution.

Accurate Measurement of the Effects of All Amino-Acid Mutations on Influenza Hemagglutinin

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Michael B. Doud

2016-06-01

Full Text Available Influenza genes evolve mostly via point mutations, and so knowing the effect of every amino-acid mutation provides information about evolutionary paths available to the virus. We and others have combined high-throughput mutagenesis with deep sequencing to estimate the effects of large numbers of mutations to influenza genes. However, these measurements have suffered from substantial experimental noise due to a variety of technical problems, the most prominent of which is bottlenecking during the generation of mutant viruses from plasmids. Here we describe advances that ameliorate these problems, enabling us to measure with greatly improved accuracy and reproducibility the effects of all amino-acid mutations to an H1 influenza hemagglutinin on viral replication in cell culture. The largest improvements come from using a helper virus to reduce bottlenecks when generating viruses from plasmids. Our measurements confirm at much higher resolution the results of previous studies suggesting that antigenic sites on the globular head of hemagglutinin are highly tolerant of mutations. We also show that other regions of hemagglutinin—including the stalk epitopes targeted by broadly neutralizing antibodies—have a much lower inherent capacity to tolerate point mutations. The ability to accurately measure the effects of all influenza mutations should enhance efforts to understand and predict viral evolution.

Nucleotide diversity and gene expression of Catalase and Glutathione peroxidase in irradiated Scots pine (Pinus sylvestris L.) from the Chernobyl exclusion zone

International Nuclear Information System (INIS)

Vornam, Barbara; Arkhipov, Andrey; Finkeldey, Reiner

2012-01-01

In the Chernobyl exclusion zone forest trees have to tolerate and to adapt to ionizing radiation, therefore the molecular basis of their adaptive responses is of the utmost interest. Based on SNP analysis and real time PCR nucleotide diversity and expression profiles of gene fragments of catalase (Cat) and glutathione peroxidase (GPx), which are known as radical scavenging genes, were analysed in the needles of irradiated pine trees of the Chernobyl exclusion zone. In acutely and chronically irradiated trees (50 years old) planted before the accident a higher nucleotide diversity of Cat and more somatic mutations were found compared to their control. Chronically irradiated trees (20 years old) planted after the accident showed a similar nucleotide diversity of Cat compared to their control and in both collectives one somatic mutation was found. The nucleotide diversity of GPx was higher in all analysed trees compared to Cat. No somatic mutation events were found in GPx. For both gene fragments, no association between the received dose in a tree and the nucleotide diversity and mutation events was detected. The expression profiles of Cat and GPx in acutely and chronically and in chronically irradiated trees were similar. Compared to their corresponding control collectives, Cat was up-regulated and GPx slightly down-regulated.

Population Size vs. Mutation Strength for the (1+λ) EA on OneMax

DEFF Research Database (Denmark)

Gießen, Christian; Witt, Carsten

2015-01-01

The (1+1) EA with mutation probability c/n, where c>0 is an arbitrary constant, is studied for the classical OneMax function. Its expected optimization time is analyzed exactly (up to lower order terms) as a function of c and λ. It turns out that 1/n is the only optimal mutation probability if λ......=o(ln n ln ln n/ln ln ln n), which is the cut-off point for linear mnspeed-up. However, if λ is above this cut-off point then the standard mutation probability 1/n is no longer the only optimal choice. Instead, the expected number of generations is (up to lower order terms) independent of c...

Diversity of Poissonian populations.

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Eliazar, Iddo I; Sokolov, Igor M

2010-01-01

Populations represented by collections of points scattered randomly on the real line are ubiquitous in science and engineering. The statistical modeling of such populations leads naturally to Poissonian populations-Poisson processes on the real line with a distinguished maximal point. Poissonian populations are infinite objects underlying key issues in statistical physics, probability theory, and random fractals. Due to their infiniteness, measuring the diversity of Poissonian populations depends on the lower-bound cut-off applied. This research characterizes the classes of Poissonian populations whose diversities are invariant with respect to the cut-off level applied and establishes an elemental connection between these classes and extreme-value theory. The measures of diversity considered are variance and dispersion, Simpson's index and inverse participation ratio, Shannon's entropy and Rényi's entropy, and Gini's index.

Suppression of different classes of somatic mutations in Arabidopsis by vir gene-expressing Agrobacterium strains.

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Shah, Jasmine M; Ramakrishnan, Anantha Maharasi; Singh, Amit Kumar; Ramachandran, Subalakshmi; Unniyampurath, Unnikrishnan; Jayshankar, Ajitha; Balasundaram, Nithya; Dhanapal, Shanmuhapreya; Hyde, Geoff; Baskar, Ramamurthy

2015-08-26

Agrobacterium infection, which is widely used to generate transgenic plants, is often accompanied by T-DNA-linked mutations and transpositions in flowering plants. It is not known if Agrobacterium infection also affects the rates of point mutations, somatic homologous recombinations (SHR) and frame-shift mutations (FSM). We examined the effects of Agrobacterium infection on five types of somatic mutations using a set of mutation detector lines of Arabidopsis thaliana. To verify the effect of secreted factors, we exposed the plants to different Agrobacterium strains, including wild type (Ach5), its derivatives lacking vir genes, oncogenes or T-DNA, and the heat-killed form for 48 h post-infection; also, for a smaller set of strains, we examined the rates of three types of mutations at multiple time-points. The mutation detector lines carried a non-functional β-glucuronidase gene (GUS) and a reversion of mutated GUS to its functional form resulted in blue spots. Based on the number of blue spots visible in plants grown for a further two weeks, we estimated the mutation frequencies. For plants co-cultivated for 48 h with Agrobacterium, if the strain contained vir genes, then the rates of transversions, SHRs and FSMs (measured 2 weeks later) were lower than those of uninfected controls. In contrast, co-cultivation for 48 h with any of the Agrobacterium strains raised the transposition rates above control levels. The multiple time-point study showed that in seedlings co-cultivated with wild type Ach5, the reduced rates of transversions and SHRs after 48 h co-cultivation represent an apparent suppression of an earlier short-lived increase in mutation rates (peaking for plants co-cultivated for 3 h). An increase after 3 h co-cultivation was also seen for rates of transversions (but not SHR) in seedlings exposed to the strain lacking vir genes, oncogenes and T-DNA. However, the mutation rates in plants co-cultivated for longer times with this strain subsequently

Ferredoxin Gene Mutation in Iranian Trichomonas Vaginalis Isolates

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Soudabeh Heidari

2013-09-01

Full Text Available Background: Trichomonas vaginalis causes trichomoniasis and metronidazole is its chosen drug for treatment. Ferredoxin has role in electron transport and carbohydrate metabolism and the conversion of an inactive form of metronidazole (CO to its active form (CPR. Ferredoxin gene mutations reduce gene expression and increase its resistance to metronidazole. In this study, the frequency of ferredoxin gene mutations in clinical isolates of T.vaginalis in Tehran has been studied.Methods: Forty six clinical T. vaginalis isolates of vaginal secretions and urine sediment were collected from Tehran Province since 2011 till 2012. DNA was extracted and ferredoxin gene was amplified by PCR technique. The ferredoxin gene PCR products were sequenced to determine gene mutations.Results: In four isolates (8.69% point mutation at nucleotide position -239 (the translation start codon of the ferredoxin gene were detected in which adenosine were converted to thymine.Conclusion: Mutation at nucleotide -239 ferredoxin gene reduces translational regulatory protein’s binding affinity which concludes reduction of ferredoxin expression. For this reduction, decrease in activity and decrease in metronidazole drug delivery into the cells occur. Mutations in these four isolates may lead to resistance of them to metronidazole.

Characterization of phospholipase C gamma enzymes with gain-of-function mutations.

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Everett, Katy L; Bunney, Tom D; Yoon, Youngdae; Rodrigues-Lima, Fernando; Harris, Richard; Driscoll, Paul C; Abe, Koichiro; Fuchs, Helmut; de Angelis, Martin Hrabé; Yu, Philipp; Cho, Wohnwa; Katan, Matilda

2009-08-21

Phospholipase C gamma isozymes (PLC gamma 1 and PLC gamma 2) have a crucial role in the regulation of a variety of cellular functions. Both enzymes have also been implicated in signaling events underlying aberrant cellular responses. Using N-ethyl-N-nitrosourea (ENU) mutagenesis, we have recently identified single point mutations in murine PLC gamma 2 that lead to spontaneous inflammation and autoimmunity. Here we describe further, mechanistic characterization of two gain-of-function mutations, D993G and Y495C, designated as ALI5 and ALI14. The residue Asp-993, mutated in ALI5, is a conserved residue in the catalytic domain of PLC enzymes. Analysis of PLC gamma 1 and PLC gamma 2 with point mutations of this residue showed that removal of the negative charge enhanced PLC activity in response to EGF stimulation or activation by Rac. Measurements of PLC activity in vitro and analysis of membrane binding have suggested that ALI5-type mutations facilitate membrane interactions without compromising substrate binding and hydrolysis. The residue mutated in ALI14 (Tyr-495) is within the spPH domain. Replacement of this residue had no effect on folding of the domain and enhanced Rac activation of PLC gamma 2 without increasing Rac binding. Importantly, the activation of the ALI14-PLC gamma 2 and corresponding PLC gamma 1 variants was enhanced in response to EGF stimulation and bypassed the requirement for phosphorylation of critical tyrosine residues. ALI5- and ALI14-type mutations affected basal activity only slightly; however, their combination resulted in a constitutively active PLC. Based on these data, we suggest that each mutation could compromise auto-inhibition in the inactive PLC, facilitating the activation process; in addition, ALI5-type mutations could enhance membrane interaction in the activated state.

Metabolic engineering of an ATP-neutral Embden-Meyerhof-Parnas pathway in Corynebacterium glutamicum: growth restoration by an adaptive point mutation in NADH dehydrogenase.

Science.gov (United States)

Komati Reddy, Gajendar; Lindner, Steffen N; Wendisch, Volker F

2015-03-01

Corynebacterium glutamicum uses the Embden-Meyerhof-Parnas pathway of glycolysis and gains 2 mol of ATP per mol of glucose by substrate-level phosphorylation (SLP). To engineer glycolysis without net ATP formation by SLP, endogenous phosphorylating NAD-dependent glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was replaced by nonphosphorylating NADP-dependent glyceraldehyde-3-phosphate dehydrogenase (GapN) from Clostridium acetobutylicum, which irreversibly converts glyceraldehyde-3-phosphate (GAP) to 3-phosphoglycerate (3-PG) without generating ATP. As shown recently (S. Takeno, R. Murata, R. Kobayashi, S. Mitsuhashi, and M. Ikeda, Appl Environ Microbiol 76:7154-7160, 2010, http://dx.doi.org/10.1128/AEM.01464-10), this ATP-neutral, NADPH-generating glycolytic pathway did not allow for the growth of Corynebacterium glutamicum with glucose as the sole carbon source unless hitherto unknown suppressor mutations occurred; however, these mutations were not disclosed. In the present study, a suppressor mutation was identified, and it was shown that heterologous expression of udhA encoding soluble transhydrogenase from Escherichia coli partly restored growth, suggesting that growth was inhibited by NADPH accumulation. Moreover, genome sequence analysis of second-site suppressor mutants that were able to grow faster with glucose revealed a single point mutation in the gene of non-proton-pumping NADH:ubiquinone oxidoreductase (NDH-II) leading to the amino acid change D213G, which was shared by these suppressor mutants. Since related NDH-II enzymes accepting NADPH as the substrate possess asparagine or glutamine residues at this position, D213G, D213N, and D213Q variants of C. glutamicum NDH-II were constructed and were shown to oxidize NADPH in addition to NADH. Taking these findings together, ATP-neutral glycolysis by the replacement of endogenous NAD-dependent GAPDH with NADP-dependent GapN became possible via oxidation of NADPH formed in this pathway by mutant NADPH

Death and population dynamics affect mutation rate estimates and evolvability under stress in bacteria.

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Frenoy, Antoine; Bonhoeffer, Sebastian

2018-05-01

The stress-induced mutagenesis hypothesis postulates that in response to stress, bacteria increase their genome-wide mutation rate, in turn increasing the chances that a descendant is able to better withstand the stress. This has implications for antibiotic treatment: exposure to subinhibitory doses of antibiotics has been reported to increase bacterial mutation rates and thus probably the rate at which resistance mutations appear and lead to treatment failure. More generally, the hypothesis posits that stress increases evolvability (the ability of a population to generate adaptive genetic diversity) and thus accelerates evolution. Measuring mutation rates under stress, however, is problematic, because existing methods assume there is no death. Yet subinhibitory stress levels may induce a substantial death rate. Death events need to be compensated by extra replication to reach a given population size, thus providing more opportunities to acquire mutations. We show that ignoring death leads to a systematic overestimation of mutation rates under stress. We developed a system based on plasmid segregation that allows us to measure death and division rates simultaneously in bacterial populations. Using this system, we found that a substantial death rate occurs at the tested subinhibitory concentrations previously reported to increase mutation rate. Taking this death rate into account lowers and sometimes removes the signal for stress-induced mutagenesis. Moreover, even when antibiotics increase mutation rate, we show that subinhibitory treatments do not increase genetic diversity and evolvability, again because of effects of the antibiotics on population dynamics. We conclude that antibiotic-induced mutagenesis is overestimated because of death and that understanding evolvability under stress requires accounting for the effects of stress on population dynamics as much as on mutation rate. Our goal here is dual: we show that population dynamics and, in particular, the

Integrated analysis of whole genome and transcriptome sequencing reveals diverse transcriptomic aberrations driven by somatic genomic changes in liver cancers.

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Yuichi Shiraishi

Full Text Available Recent studies applying high-throughput sequencing technologies have identified several recurrently mutated genes and pathways in multiple cancer genomes. However, transcriptional consequences from these genomic alterations in cancer genome remain unclear. In this study, we performed integrated and comparative analyses of whole genomes and transcriptomes of 22 hepatitis B virus (HBV-related hepatocellular carcinomas (HCCs and their matched controls. Comparison of whole genome sequence (WGS and RNA-Seq revealed much evidence that various types of genomic mutations triggered diverse transcriptional changes. Not only splice-site mutations, but also silent mutations in coding regions, deep intronic mutations and structural changes caused splicing aberrations. HBV integrations generated diverse patterns of virus-human fusion transcripts depending on affected gene, such as TERT, CDK15, FN1 and MLL4. Structural variations could drive over-expression of genes such as WNT ligands, with/without creating gene fusions. Furthermore, by taking account of genomic mutations causing transcriptional aberrations, we could improve the sensitivity of deleterious mutation detection in known cancer driver genes (TP53, AXIN1, ARID2, RPS6KA3, and identified recurrent disruptions in putative cancer driver genes such as HNF4A, CPS1, TSC1 and THRAP3 in HCCs. These findings indicate genomic alterations in cancer genome have diverse transcriptomic effects, and integrated analysis of WGS and RNA-Seq can facilitate the interpretation of a large number of genomic alterations detected in cancer genome.

Amelogenesis imperfecta caused by N-terminal enamelin point mutations in mice and men is driven by endoplasmic reticulum stress.

Science.gov (United States)

Brookes, Steven J; Barron, Martin J; Smith, Claire E L; Poulter, James A; Mighell, Alan J; Inglehearn, Chris F; Brown, Catriona J; Rodd, Helen; Kirkham, Jennifer; Dixon, Michael J

2017-05-15

'Amelogenesis imperfecta' (AI) describes a group of inherited diseases of dental enamel that have major clinical impact. Here, we identify the aetiology driving AI in mice carrying a p.S55I mutation in enamelin; one of the most commonly mutated proteins underlying AI in humans. Our data indicate that the mutation inhibits the ameloblast secretory pathway leading to ER stress and an activated unfolded protein response (UPR). Initially, with the support of the UPR acting in pro-survival mode, Enamp.S55I heterozygous mice secreted structurally normal enamel. However, enamel secreted thereafter was structurally abnormal; presumably due to the UPR modulating ameloblast behaviour and function in an attempt to relieve ER stress. Homozygous mutant mice failed to produce enamel. We also identified a novel heterozygous ENAMp.L31R mutation causing AI in humans. We hypothesize that ER stress is the aetiological factor in this case of human AI as it shared the characteristic phenotype described above for the Enamp.S55I mouse. We previously demonstrated that AI in mice carrying the Amelxp.Y64H mutation is a proteinopathy. The current data indicate that AI in Enamp.S55I mice is also a proteinopathy, and based on comparative phenotypic analysis, we suggest that human AI resulting from the ENAMp.L31R mutation is another proteinopathic disease. Identifying a common aetiology for AI resulting from mutations in two different genes opens the way for developing pharmaceutical interventions designed to relieve ER stress or modulate the UPR during enamel development to ameliorate the clinical phenotype. © The Author 2017. Published by Oxford University Press.

Presence of two alternative kdr-like mutations, L1014F and L1014S, and a novel mutation, V1010L, in the voltage gated Na+ channel of Anopheles culicifacies from Orissa, India

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Bhatt Rajendra M

2010-05-01

Full Text Available Abstract Background Knockdown resistance in insects resulting from mutation(s in the voltage gated Na+ channel (VGSC is one of the mechanisms of resistance against DDT and pyrethroids. Recently a point mutation leading to Leu-to-Phe substitution in the VGSC at residue 1014, a most common kdr mutation in insects, was reported in Anopheles culicifacies-a major malaria vector in the Indian subcontinent. This study reports the presence of two additional amino acid substitutions in the VGSC of an An. culicifacies population from Malkangiri district of Orissa, India. Methods Anopheles culicifacies sensu lato (s.l. samples, collected from a population of Malkangiri district of Orissa (India, were sequenced for part of the second transmembrane segment of VGSC and analyzed for the presence of non-synonymous mutations. A new primer introduced restriction analysis-PCR (PIRA-PCR was developed for the detection of the new mutation L1014S. The An. culicifacies population was genotyped for the presence of L1014F substitution by an amplification refractory mutation system (ARMS and for L1014S substitutions by using a new PIRA-PCR developed in this study. The results were validated through DNA sequencing. Results DNA sequencing of An. culicifacies individuals collected from district Malkangiri revealed the presence of three amino acid substitutions in the IIS6 transmembrane segments of VGSC, each one resulting from a single point mutation. Two alternative point mutations, 3042A>T transversion or 3041T>C transition, were found at residue L1014 leading to Leu (TTA-to-Phe (TTT or -Ser (TCA changes, respectively. A third and novel substitution, Val (GTG-to-Leu (TTG or CTG, was identified at residue V1010 resulting from either of the two transversions–3028G>T or 3028G>C. The L1014S substitution co-existed with V1010L in all the samples analyzed irrespective of the type of point mutation associated with the latter. The PIRA-PCR strategy developed for the

Next-generation sequencing reveals the mutational landscape of clinically diagnosed Usher syndrome: copy number variations, phenocopies, a predominant target for translational read-through, and PEX26 mutated in Heimler syndrome.

Science.gov (United States)

Neuhaus, Christine; Eisenberger, Tobias; Decker, Christian; Nagl, Sandra; Blank, Cornelia; Pfister, Markus; Kennerknecht, Ingo; Müller-Hofstede, Cornelie; Charbel Issa, Peter; Heller, Raoul; Beck, Bodo; Rüther, Klaus; Mitter, Diana; Rohrschneider, Klaus; Steinhauer, Ute; Korbmacher, Heike M; Huhle, Dagmar; Elsayed, Solaf M; Taha, Hesham M; Baig, Shahid M; Stöhr, Heidi; Preising, Markus; Markus, Susanne; Moeller, Fabian; Lorenz, Birgit; Nagel-Wolfrum, Kerstin; Khan, Arif O; Bolz, Hanno J

2017-09-01

Combined retinal degeneration and sensorineural hearing impairment is mostly due to autosomal recessive Usher syndrome (USH1: congenital deafness, early retinitis pigmentosa (RP); USH2: progressive hearing impairment, RP). Sanger sequencing and NGS of 112 genes (Usher syndrome, nonsyndromic deafness, overlapping conditions), MLPA, and array-CGH were conducted in 138 patients clinically diagnosed with Usher syndrome. A molecular diagnosis was achieved in 97% of both USH1 and USH2 patients, with biallelic mutations in 97% (USH1) and 90% (USH2), respectively. Quantitative readout reliably detected CNVs (confirmed by MLPA or array-CGH), qualifying targeted NGS as one tool for detecting point mutations and CNVs. CNVs accounted for 10% of identified USH2A alleles, often in trans to seemingly monoallelic point mutations. We demonstrate PTC124-induced read-through of the common p.Trp3955* nonsense mutation (13% of detected USH2A alleles), a potential therapy target. Usher gene mutations were found in most patients with atypical Usher syndrome, but the diagnosis was adjusted in case of double homozygosity for mutations in OTOA and NR2E3 , genes implicated in isolated deafness and RP. Two patients with additional enamel dysplasia had biallelic PEX26 mutations, for the first time linking this gene to Heimler syndrome. Targeted NGS not restricted to Usher genes proved beneficial in uncovering conditions mimicking Usher syndrome.

Evaluator competencies in the context of diversity training: The practitioners' point of view.

Science.gov (United States)

Froncek, Benjamin; Mazziotta, Agostino; Piper, Verena; Rohmann, Anette

2018-04-01

Evaluator competencies have been discussed since the beginnings of program evaluation literature. More recently, the Essential Competencies for Program Evaluators (Ghere et al., 2006; Stevahn, King, Ghere & Minnema, 2005a) have proven to be a useful taxonomy for learning and improving evaluation practice. Evaluation is critical to diversity training activities, and diversity training providers face the challenge of conducting evaluations of their training programs. We explored what competencies are viewed as instrumental to conducting useful evaluations in this specific field of evaluation practice. In an online survey, N = 172 diversity training providers were interviewed via an open answer format about their perceptions of evaluator competencies, with n = 95 diversity training providers contributing statements. The Essential Competencies for Program Evaluators were used to conduct a deductive qualitative content analysis of the statements. While systematic inquiry, reflective practice, and interpersonal competence were well represented, situational analysis and project management were not. Implications are discussed for evaluation capacity building among diversity training providers and for negotiating evaluation projects with evaluation professionals. Copyright © 2018 Elsevier Ltd. All rights reserved.

Common Β- Thalassaemia Mutations in

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P Azarfam

2005-01-01

Full Text Available Introduction: β –Thalassaemia was first explained by Thomas Cooly as Cooly’s anaemia in 1925. The β- thalassaemias are hereditary autosomal disorders with decreased or absent β-globin chain synthesis. The most common genetic defects in β-thalassaemias are caused by point mutations, micro deletions or insertions within the β-globin gene. Material and Methods: In this research , 142 blood samples (64 from childrens hospital of Tabriz , 15 samples from Shahid Gazi hospital of Tabriz , 18 from Urumia and 45 samples from Aliasghar hospital of Ardebil were taken from thalassaemic patients (who were previously diagnosed .Then 117 non-familial samples were selected . The DNA of the lymphocytes of blood samples was extracted by boiling and Proteinase K- SDS procedure, and mutations were detected by ARMS-PCR methods. Results: From the results obtained, eleven most common mutations,most of which were Mediterranean mutations were detected as follows; IVS-I-110(G-A, IVS-I-1(G-A ،IVS-I-5(G-C ,Frameshift Codon 44 (-C,( codon5(-CT,IVS-1-6(T-C, IVS-I-25(-25bp del ,Frameshift 8.9 (+G ,IVS-II-1(G-A ,Codon 39(C-T, Codon 30(G-C the mutations of the samples were defined. The results showed that Frameshift 8.9 (+G, IVS-I-110 (G-A ,IVS-II-I(G-A, IVS-I-5(G-C, IVS-I-1(G-A , Frameshift Codon 44(-C , codon5(-CT , IVS-1-6(T-C , IVS-I-25(-25bp del with a frequency of 29.9%, 25.47%,17.83%, 7.00%, 6.36% , 6.63% , 3.8% , 2.5% , 0.63% represented the most common mutations in North - west Iran. No mutations in Codon 39(C-T and Codon 30(G-C were detected. Cunclusion: The frequency of the same mutations in patients from North - West of Iran seems to be different as compared to other regions like Turkey, Pakistan, Lebanon and Fars province of Iran. The pattern of mutations in this region is more or less the same as in the Mediterranean region, but different from South west Asia and East Asia.

A Point Mutation in Suppressor of Cytokine Signalling 2 (Socs2 Increases the Susceptibility to Inflammation of the Mammary Gland while Associated with Higher Body Weight and Size and Higher Milk Production in a Sheep Model.

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Rachel Rupp

2015-12-01

Full Text Available Mastitis is an infectious disease mainly caused by bacteria invading the mammary gland. Genetic control of susceptibility to mastitis has been widely evidenced in dairy ruminants, but the genetic basis and underlying mechanisms are still largely unknown. We describe the discovery, fine mapping and functional characterization of a genetic variant associated with elevated milk leukocytes count, or SCC, as a proxy for mastitis. After implementing genome-wide association studies, we identified a major QTL associated with SCC on ovine chromosome 3. Fine mapping of the region, using full sequencing with 12X coverage in three animals, provided one strong candidate SNP that mapped to the coding sequence of a highly conserved gene, suppressor of cytokine signalling 2 (Socs2. The frequency of the SNP associated with increased SCC was 21.7% and the Socs2 genotype explained 12% of the variance of the trait. The point mutation induces the p.R96C substitution in the SH2 functional domain of SOCS2 i.e. the binding site of the protein to various ligands, as well-established for the growth hormone receptor GHR. Using surface plasmon resonance we showed that the p.R96C point mutation completely abrogates SOCS2 binding affinity for the phosphopeptide of GHR. Additionally, the size, weight and milk production in p.R96C homozygote sheep, were significantly increased by 24%, 18%, and 4.4%, respectively, when compared to wild type sheep, supporting the view that the point mutation causes a loss of SOCS2 functional activity. Altogether these results provide strong evidence for a causal mutation controlling SCC in sheep and highlight the major role of SOCS2 as a tradeoff between the host's inflammatory response to mammary infections, and body growth and milk production, which are all mediated by the JAK/STAT signaling pathway.

Amino acid substitutions in random mutagenesis libraries: lessons from analyzing 3000 mutations.

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Zhao, Jing; Frauenkron-Machedjou, Victorine Josiane; Kardashliev, Tsvetan; Ruff, Anna Joëlle; Zhu, Leilei; Bocola, Marco; Schwaneberg, Ulrich

2017-04-01

The quality of amino acid substitution patterns in random mutagenesis libraries is decisive for the success in directed evolution campaigns. In this manuscript, we provide a detailed analysis of the amino acid substitutions by analyzing 3000 mutations of three random mutagenesis libraries (1000 mutations each; epPCR with a low-mutation and a high-mutation frequency and SeSaM-Tv P/P) employing lipase A from Bacillus subtilis (bsla). A comparison of the obtained numbers of beneficial variants in the mentioned three random mutagenesis libraries with a site saturation mutagenesis (SSM) (covering the natural diversity at each amino acid position of BSLA) concludes the diversity analysis. Seventy-six percent of the SeSaM-Tv P/P-generated substitutions yield chemically different amino acid substitutions compared to 64% (epPCR-low) and 69% (epPCR-high). Unique substitutions from one amino acid to others are termed distinct amino acid substitutions. In the SeSaM-Tv P/P library, 35% of all theoretical distinct amino acid substitutions were found in the 1000 mutation library compared to 25% (epPCR-low) and 26% (epPCR-high). Thirty-six percent of distinct amino acid substitutions found in SeSaM-Tv P/P were unobtainable by epPCR-low. Comparison with the SSM library showed that epPCR-low covers 15%, epPCR-high 18%, and SeSaM-Tv P/P 21% of obtainable beneficial amino acid positions. In essence, this study provides first insights on the quality of epPCR and SeSaM-Tv P/P libraries in terms of amino acid substitutions, their chemical differences, and the number of obtainable beneficial amino acid positions.

Characteristics, causes and evolutionary consequences of male-biased mutation.

Science.gov (United States)

Ellegren, Hans

2007-01-07

Mutation has traditionally been considered a random process, but this paradigm is challenged by recent evidence of divergence rate heterogeneity in different genomic regions. One facet of mutation rate variation is the propensity for genetic change to correlate with the number of germ cell divisions, reflecting the replication-dependent origin of many mutations. Haldane was the first to connect this association of replication and mutation to the difference in the number of cell divisions in oogenesis (low) and spermatogenesis (usually high), and the resulting sex difference in the rate of mutation. The concept of male-biased mutation has been thoroughly analysed in recent years using an evolutionary approach, in which sequence divergence of autosomes and/or sex chromosomes are compared to allow inference about the relative contribution of mothers and fathers in the accumulation of mutations. For instance, assuming that a neutral sequence is analysed, that rate heterogeneity owing to other factors is cancelled out by the investigation of many loci and that the effect of ancestral polymorphism is properly taken into account, the male-to-female mutation rate ratio, alpham, can be solved from the observed difference in rate of X and Y chromosome divergence. The male mutation bias is positively correlated with the relative excess of cell divisions in the male compared to the female germ line, as evidenced by a generation time effect: in mammals, alpham is estimated at approximately 4-6 in primates, approximately 3 in carnivores and approximately 2 in small rodents. Another life-history correlate is sexual selection: when there is intense sperm competition among males, increased sperm production will be associated with a larger number of mitotic cell divisions in spermatogenesis and hence an increase in alpham. Male-biased mutation has implications for important aspects of evolutionary biology such as mate choice in relation to mutation load, sexual selection and the

Mutation Spectrum of GNE Myopathy in the Indian Sub-Continent.

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Bhattacharya, Sudha; Khadilkar, Satish V; Nalini, Atchayaram; Ganapathy, Aparna; Mannan, Ashraf U; Majumder, Partha P; Bhattacharya, Alok

GNE myopathy is an adult onset recessive genetic disorder that affects distal muscles sparing the quadriceps. GNE gene mutations have been identified in GNE myopathy patients all over the world. Homozygosity is a common feature in GNE myopathy patients worldwide. The major objective of this study was to investigate the mutation spectrum of GNE myopathy in India in relation to the population diversity in the country. We have collated GNE mutation data of Indian GNE myopathy patients from published literature and from recently identified patients. We also used data of people of Indian subcontinent from 1000 genomes database, South Asian Genome database and Strand Life Science database to determine frequency of GNE mutations in the general population. A total of 67 GNE myopathy patients were studied, of whom 21% were homozygous for GNE variants, while the rest were compound heterozygous. Thirty-five different mutations in the GNE gene were recorded, of which 5 have not been reported earlier. The most frequent mutation was p.Val727Met (65%) found mainly in the heterozygous form. Another mutation, p.Ile618Thr was also common (16%) but was found mainly in patients from Rajasthan, while p.Val727Met was more widely distributed. The latter was also seen at a high frequency in general population of Indian subcontinent in all the databases. It was also present in Thailand but was absent in general population elsewhere in the world. p.Val727Met is likely to be a founder mutation of Indian subcontinent.

Screening for mutations in two exons of FANCG gene in Pakistani population.

Science.gov (United States)

Aymun, Ujala; Iram, Saima; Aftab, Iram; Khaliq, Saba; Nadir, Ali; Nisar, Ahmed; Mohsin, Shahida

2017-06-01

Fanconi anemia is a rare autosomal recessive disorder of genetic instability. It is both molecularly and clinically, a heterogeneous disorder. Its incidence is 1 in 129,000 births and relatively high in some ethnic groups. Sixteen genes have been identified among them mutations in FANCG gene are most common after FANCA and FANCC gene mutations. To study mutations in exon 3 and 4 of FANCG gene in Pakistani population. Thirty five patients with positive Diepoxybutane test were included in the study. DNA was extracted and amplified for exons 3 and 4. Thereafter Sequencing was done and analyzed for the presence of mutations. No mutation was detected in exon 3 whereas a carrier of known mutation c.307+1 G>T was found in exon 4 of the FANCG gene. Absence of any mutation in exon 3 and only one heterozygous mutation in exon 4 of FANCG gene points to a different spectrum of FA gene pool in Pakistan that needs extensive research in this area.

Activating mutations in FGFR3 and HRAS reveal a shared genetic origin for congenital disorders and testicular tumors

DEFF Research Database (Denmark)

Goriely, Anne; Hansen, Ruth M S; Taylor, Indira B

2009-01-01

Genes mutated in congenital malformation syndromes are frequently implicated in oncogenesis, but the causative germline and somatic mutations occur in separate cells at different times of an organism's life. Here we unify these processes to a single cellular event for mutations arising in male germ...... cells that show a paternal age effect. Screening of 30 spermatocytic seminomas for oncogenic mutations in 17 genes identified 2 mutations in FGFR3 (both 1948A>G, encoding K650E, which causes thanatophoric dysplasia in the germline) and 5 mutations in HRAS. Massively parallel sequencing of sperm DNA...... a common 'selfish' pathway supporting proliferation in the testis, leading to diverse phenotypes in the next generation including fetal lethality, congenital syndromes and cancer predisposition....

Isolated growth hormone deficiency in two siblings because of paternal mosaicism for a mutation in the GH1 gene.

Science.gov (United States)

Tsubahara, Mayuko; Hayashi, Yoshitaka; Niijima, Shin-ichi; Yamamoto, Michiyo; Kamijo, Takashi; Murata, Yoshiharu; Haruna, Hidenori; Okumura, Akihisa; Shimizu, Toshiaki

2012-03-01

  Mutations in the GH1 gene have been identified in patients with isolated growth hormone deficiency (IGHD). Mutations causing aberrant splicing of exon 3 of GH1 that have been identified in IGHD are inherited in an autosomal dominant manner, whereas other mutations in GH1 that have been identified in IGHD are inherited in an autosomal recessive manner.   Two siblings born from nonconsanguineous healthy parents exhibited IGHD. To elucidate the cause, GH1 in all family members was analysed.   Two novel mutations in GH1, a point mutation in intron 3 and a 16-bp deletion in exon 3, were identified by sequence analyses. The intronic mutation was present in both siblings and was predicted to cause aberrant splicing. The deletion was present in one of the siblings as well as the mother with normal stature and was predicted to cause rapid degradation of mRNA through nonsense-mediated mRNA decay. The point mutation was not identified in the parents' peripheral blood DNA; however, it was detected in the DNA extracted from the father's sperms. As a trace of the mutant allele was detected in the peripheral blood of the father using PCR-RFLP, the mutation is likely to have occurred de novo at an early developmental stage before differentiation of somatic cells and germline cells.   This is the first report of mosaicism for a mutation in GH1 in a family with IGHD. It is clear that the intronic mutation plays a dominant role in the pathogenesis of IGHD in this family, as one of the siblings who had only the point mutation was affected. On the other hand, the other sibling was a compound heterozygote for the point mutation and the 16-bp deletion and it may be arguable whether IGHD in this patient should be regarded as autosomal dominant or recessive. © 2012 Blackwell Publishing Ltd.

Board diversity in family firms

OpenAIRE

Menozzi, Anna; Fraquelli, Giovanni; Novara, Jolanda de

2015-01-01

The paper deals with diversity as a key factor to improve the board of directors’ decision process in family firms. The empirical literature about board diversity points at the positive impact of diversity on board functioning and firm performance. The paper uses a statistical diversity index to capture the heterogeneity of board of directors and put it in relation with firm performance, as measured by firm profitability. The empirical analysis is based on a newly collected panel of 327 famil...

In silico analysis of conformational changes induced by mutation of aromatic binding residues: consequences for drug binding in the hERG K+ channel.

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Kirsten Knape

Full Text Available Pharmacological inhibition of cardiac hERG K(+ channels is associated with increased risk of lethal arrhythmias. Many drugs reduce hERG current by directly binding to the channel, thereby blocking ion conduction. Mutation of two aromatic residues (F656 and Y652 substantially decreases the potency of numerous structurally diverse compounds. Nevertheless, some drugs are only weakly affected by mutation Y652A. In this study we utilize molecular dynamics simulations and docking studies to analyze the different effects of mutation Y652A on a selected number of hERG blockers. MD simulations reveal conformational changes in the binding site induced by mutation Y652A. Loss of π-π-stacking between the two aromatic residues induces a conformational change of the F656 side chain from a cavity facing to cavity lining orientation. Docking studies and MD simulations qualitatively reproduce the diverse experimentally observed modulatory effects of mutation Y652A and provide a new structural interpretation for the sensitivity differences.

Recurrent SETBP1 mutations in atypical chronic myeloid leukemia

Science.gov (United States)

Piazza, Rocco; Valletta, Simona; Winkelmann, Nils; Redaelli, Sara; Spinelli, Roberta; Pirola, Alessandra; Antolini, Laura; Mologni, Luca; Donadoni, Carla; Papaemmanuil, Elli; Schnittger, Susanne; Kim, Dong-Wook; Boultwood, Jacqueline; Rossi, Fabio; Gaipa, Giuseppe; De Martini, Greta P; di Celle, Paola Francia; Jang, Hyun Gyung; Fantin, Valeria; Bignell, Graham R; Magistroni, Vera; Haferlach, Torsten; Pogliani, Enrico Maria; Campbell, Peter J; Chase, Andrew J; Tapper, William J; Cross, Nicholas C P; Gambacorti-Passerini, Carlo

2013-01-01

Atypical chronic myeloid leukemia (aCML) shares clinical and laboratory features with CML, but it lacks the BCR-ABL1 fusion. We performed exome sequencing of eight aCMLs and identified somatic alterations of SETBP1 (encoding a p.Gly870Ser alteration) in two cases. Targeted resequencing of 70 aCMLs, 574 diverse hematological malignancies and 344 cancer cell lines identified SETBP1 mutations in 24 cases, including 17 of 70 aCMLs (24.3%; 95% confidence interval (CI) = 16–35%). Most mutations (92%) were located between codons 858 and 871 and were identical to changes seen in individuals with Schinzel-Giedion syndrome. Individuals with mutations had higher white blood cell counts (P = 0.008) and worse prognosis (P = 0.01). The p.Gly870Ser alteration abrogated a site for ubiquitination, and cells exogenously expressing this mutant exhibited higher amounts of SETBP1 and SET protein, lower PP2A activity and higher proliferation rates relative to those expressing the wild-type protein. In summary, mutated SETBP1 represents a newly discovered oncogene present in aCML and closely related diseases. PMID:23222956

Bibliography. Examples of literature related to the use of induced mutations in cross-breeding

International Nuclear Information System (INIS)

Micke, A.

1976-01-01

The bibliography contains about 400 references arranged alphabetically under the following 20 headings: Genetic analysis of mutants; Mutant gene combination and interaction; Pleiotropy versus linkage; Genetic background; Heterosis and overdominance; Mutations in heterozygous plants such as vegetatively propagated plants; Mutations in hybrids of self-pollinators; Distant hybridization; Increasing recombination; Alteration in the reproductive system; Alteration of photoperiodic response; Self and cross-incompatibility; Male or female sterility; Adaptability of mutants and mutant hybrids; Mutation induction in cross pollinators; Dwarfing mutant genes in cross-breeding; Protein mutants in cross-breeding; Disease resistant mutants in cross-breeding; Practical cross-breeding programmes using mutants; Spontaneous versus induced genetic diversity

Mutation choice to eliminate buried free cysteines in protein therapeutics.

Science.gov (United States)

Xia, Xue; Longo, Liam M; Blaber, Michael

2015-02-01

Buried free-cysteine (Cys) residues can contribute to an irreversible unfolding pathway that promotes protein aggregation, increases immunogenic potential, and significantly reduces protein functional half-life. Consequently, mutation of buried free-Cys residues can result in significant improvement in the storage, reconstitution, and pharmacokinetic properties of protein-based therapeutics. Mutational design to eliminate buried free-Cys residues typically follows one of two common heuristics: either substitution by Ser (polar and isosteric), or substitution by Ala or Val (hydrophobic); however, a detailed structural and thermodynamic understanding of Cys mutations is lacking. We report a comprehensive structure and stability study of Ala, Ser, Thr, and Val mutations at each of the three buried free-Cys positions (Cys16, Cys83, and Cys117) in fibroblast growth factor-1. Mutation was almost universally destabilizing, indicating a general optimization for the wild-type Cys, including van der Waals and H-bond interactions. Structural response to Cys mutation characteristically involved changes to maintain, or effectively substitute, local H-bond interactions-by either structural collapse to accommodate the smaller oxygen radius of Ser/Thr, or conversely, expansion to enable inclusion of novel H-bonding solvent. Despite the diverse structural effects, the least destabilizing average substitution at each position was Ala, and not isosteric Ser. © 2014 Wiley Periodicals, Inc. and the American Pharmacists Association.

Genotyping of K-ras codons 12 and 13 mutations in colorectal cancer by capillary electrophoresis.

Science.gov (United States)

Chen, Yen-Ling; Chang, Ya-Sian; Chang, Jan-Gowth; Wu, Shou-Mei

2009-06-26

Point mutations of the K-ras gene located in codons 12 and 13 cause poor responses to the anti-epidermal growth factor receptor (anti-EGFR) therapy of colorectal cancer (CRC) patients. Besides, mutations of K-ras gene have also been proven to play an important role in human tumor progression. We established a simple and effective capillary electrophoresis (CE) method for simultaneous point mutation detection in codons 12 and 13 of K-ras gene. We combined one universal fluorescence-based nonhuman-sequence primer and two fragment-oriented primers in one tube, and performed this two-in-one polymerase chain reaction (PCR). PCR fragments included wild type and seven point mutations at codons 12 and 13 of K-ras gene. The amplicons were analyzed by single-strand conformation polymorphism (SSCP)-CE method. The CE analysis was performed by using a 1x Tris-borate-EDTA (TBE) buffer containing 1.5% (w/v) hydroxyethylcellulose (HEC) (MW 250,000) under reverse polarity with 15 degrees C and 30 degrees C. Ninety colorectal cancer patients were blindly genotyped using this developed method. The results showed good agreement with those of DNA sequencing method. The SSCP-CE was feasible for mutation screening of K-ras gene in populations.

Analysis of the sex-determining region of the Y chromosome (SRY) in sex reversed patients: point-mutation in SRY causing sex-reversion in a 46,XY female

DEFF Research Database (Denmark)

Müller, Jørn; Schwartz, M; Skakkebaek, N E

1992-01-01

The first and essential step in normal sexual differentiation takes place during the 5th-6th week of gestation. The testis determining factor (TDF) directs the undifferentiated gonad into a testis, which secretes hormones responsible for normal male development. A new candidate for TDF has recently...... been reported, and it has been called the sex determining region of the Y (SRY). The hypothesis has been supported by the finding of XX individuals with SRY, and two females with 46,XY karyotype and a mutation in SRY. However, XX males without SRY has been reported, and the role of SRY still has...... to be determined. We have tested three human females with 46,XY karyotype and gonadal dysgenesis and two 46,XX males for the presence of SRY using the polymerase chain reaction and subsequent DNA sequencing. Both 46,XX males contained SRY, whereas one of the 46,XY females had suffered a point mutation in SRY...

A novel mitochondrial mutation m.8989G>C associated with neuropathy, ataxia, retinitis pigmentosa - the NARP syndrome

DEFF Research Database (Denmark)

Duno, Morten; Wibrand, Flemming; Baggesen, Kirsten

2013-01-01

mitochondrial point mutation, m.8989G>C, in a patient presenting with neuropathy, ataxia and retinitis pigmentosa constituting the classical NARP phenotype. This mutation alters the amino acid right next to canonical NARP mutation. We suggest that classic NARP syndrome relates to a defined dysfunction of p...

Mutation spectrum of the rhodopsin gene among patients with autosomal dominant retinitis pigmentosa

International Nuclear Information System (INIS)

Dryja, T.P.; Han, L.B.; Cowley, G.S.; McGee, T.L.; Berson, E.L.

1991-01-01

The authors searched for point mutations in every exon of the rhodopsin gene in 150 patients from separate families with autosomal dominant retinitis pigmentosa. Including the 4 mutations the authors reported previously, they found a total of 17 different mutations that correlate with the disease. Each of these mutations is a single-base substitution corresponding to a single amino acid substitution. Based on current models for the structure of rhodopsin, 3 of the 17 mutant amino acids are normally located on the cytoplasmic side of the protein, 6 in transmembrane domains, and 8 on the intradiscal side. Forty-three of the 150 patients (29%) carry 1 of these mutations, and no patient has more than 1 mutation. In every family with a mutation so far analyzed, the mutation cosegregates with the disease. They found one instance of a mutation in an affected patient that was absent in both unaffected parents (i.e., a new germ-line mutation), indicating that some isolate cases of retinitis pigmentosa carry a mutation of the rhodopsin gene

A point mutation in the DNA-binding domain of HPV-2 E2 protein increases its DNA-binding capacity and reverses its transcriptional regulatory activity on the viral early promoter

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Gao Chen

2012-02-01

Full Text Available Abstract Background The human papillomavirus (HPV E2 protein is a multifunctional DNA-binding protein. The transcriptional activity of HPV E2 is mediated by binding to its specific binding sites in the upstream regulatory region of the HPV genomes. Previously we reported a HPV-2 variant from a verrucae vulgaris patient with huge extensive clustered cutaneous, which have five point mutations in its E2 ORF, L118S, S235P, Y287H, S293R and A338V. Under the control of HPV-2 LCR, co-expression of the mutated HPV E2 induced an increased activity on the viral early promoter. In the present study, a series of mammalian expression plasmids encoding E2 proteins with one to five amino acid (aa substitutions for these mutations were constructed and transfected into HeLa, C33A and SiHa cells. Results CAT expression assays indicated that the enhanced promoter activity was due to the co-expressions of the E2 constructs containing A338V mutation within the DNA-binding domain. Western blots analysis demonstrated that the transiently transfected E2 expressing plasmids, regardless of prototype or the A338V mutant, were continuously expressed in the cells. To study the effect of E2 mutations on its DNA-binding activity, a serial of recombinant E2 proteins with various lengths were expressed and purified. Electrophoresis mobility shift assays (EMSA showed that the binding affinity of E2 protein with A338V mutation to both an artificial probe with two E2 binding sites or HPV-2 and HPV-16 promoter-proximal LCR sequences were significantly stronger than that of the HPV-2 prototype E2. Furthermore, co-expression of the construct containing A338V mutant exhibited increased activities on heterologous HPV-16 early promoter P97 than that of prototype E2. Conclusions These results suggest that the mutation from Ala to Val at aa 338 is critical for E2 DNA-binding and its transcriptional regulation.

A novel mutation of PAX3 in a Chinese family with Waardenburg syndrome.

Science.gov (United States)

Qin, Wei; Shu, Anli; Qian, Xueqing; Gao, Jianjun; Xing, Qinghe; Zhang, Juan; Zheng, Yonglan; Li, Xingwang; Li, Sheng; Feng, Guoyin; He, Lin

2006-08-28

The molecular characterization of 34 members of a Chinese family, with 22 members in four generations, affected with Waardenburg syndrome (WS1). A detailed family history and clinical data were collected. A genome-wide scan by two-point linkage analysis using more than 400 microsatellite markers in combination with haplotype analysis was performed. Mutation screening was carried out in the candidate gene by sequencing of amplified products. A maximum two-point lod score of 6.53 at theta = 0.00 was obtained with marker D2S2248. Haplotype analysis placed the WS1 locus to a 45.74 cM region between D2S117 and D2S206, in close proximity to the PAX3 gene on chromosome 2q35. Mutation screening in PAX3 identified a 701T > C mutation which converted a highly conserved Leu to Pro. This nucleotide alteration was neither seen in unaffected members of the family nor found in 50 unrelated control subjects. The present study identified a novel 701T > C mutation in PAX3. The mutation observed in this family highlights the phenotypic heterogeneity of the disorder.

Induction of mutation and recombination following UV irradiation during meiosis in Saccharomyces cerevisiae

Energy Technology Data Exchange (ETDEWEB)

Kelly, S.L.; Parry, J.M. (University Coll. of Swansea (UK). Dept. of Genetics)

1983-03-01

Irradiation of yeast cultures with ultraviolet light at discrete stages during meiosis produces cyclic variations in sensitivity, i.e. cells are more sensitive to the lethal effects of UV light prior to entry into the meiotic DNA synthesis, and this corresponds to a peak of induction of point mutation. Cells become more resistant to both induced point mutation and lethality as they enter meiotic DNA synthesis, but become more sensitive again during spore formation. The induced level of intragenic recombination rises during the period of commitment ot recombination to a level indistinguishable from the full meiotic level of spontaneous intragenic recombination. Induced reciprocal recombination remains above the spontaneous level up to the point of commitment to sporulation.

Racial Diversity within the Marine Corps

National Research Council Canada - National Science Library

Alexander, Clinton D

2008-01-01

.... Diversity is an increasingly important topic in the civilian world as well as in the military. Experts in organizational behavior tend to agree that diversity fosters multiple points of view in problem solving and decreases group think...

Dynamic of Mutational Events in Variable Number Tandem Repeats of Escherichia coli O157:H7

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A. V. Bustamante

2013-01-01

Full Text Available VNTRs regions have been successfully used for bacterial subtyping; however, the hypervariability in VNTR loci is problematic when trying to predict the relationships among isolates. Since few studies have examined the mutation rate of these markers, our aim was to estimate mutation rates of VNTRs specific for verotoxigenic E. coli O157:H7. The knowledge of VNTR mutational rates and the factors affecting them would make MLVA more effective for epidemiological or microbial forensic investigations. For this purpose, we analyzed nine loci performing parallel, serial passage experiments (PSPEs on 9 O157:H7 strains. The combined 9 PSPE population rates for the 8 mutating loci ranged from 4.4 × 10−05 to 1.8 × 10−03 mutations/generation, and the combined 8-loci mutation rate was of 2.5 × 10−03 mutations/generation. Mutations involved complete repeat units, with only one point mutation detected. A similar proportion between single and multiple repeat changes was detected. Of the 56 repeat mutations, 59% were insertions and 41% were deletions, and 72% of the mutation events corresponded to O157-10 locus. For alleles with up to 13 UR, a constant and low mutation rate was observed; meanwhile longer alleles were associated with higher and variable mutation rates. Our results are useful to interpret data from microevolution and population epidemiology studies and particularly point out that the inclusion or not of O157-10 locus or, alternatively, a differential weighting data according to the mutation rates of loci must be evaluated in relation with the objectives of the proposed study.

Common mutations of hepatitis B virus and their clinical significance

Directory of Open Access Journals (Sweden)

HU Airong

2016-06-01

Full Text Available Hepatitis B virus (HBV tends to mutate easily due to its special structure and life cycle. Mutation changes the biological behavior of HBV and its sensitivity to antiviral drugs and even affects therapeutic effect and accelerate disease progression. The point mutations are commonly see in the pre-S/S open reading frame (ORF, which may be associated with immune escape and occult HBV infection. The G1896A mutation is often observed in the pre-C/C-ORF and is associated with the development of HBeAg-negative chronic hepatitis B (CHB, hepatocellular carcinoma (HCC, and severe chronic hepatitis (liver failure. The mutations in P-ORF mainly occur in the reverse transcriptase (RT domain and are closely related to the resistance to nucleos(tide analogues. The A1762T and G1764A mutations occur in the basal core promoter (BCP, which overlaps with X-ORF, and may be associated with HBeAg-negative CHB, HCC, and severe chronic hepatitis (liver failure. Clarification of the association between these mutations and diseases helps to develop tailor-made diagnostic and therapeutic regimens for patients with HBV infection.

Evolutionary constraints and the neutral theory. [mutation-caused nucleotide substitutions in DNA

Science.gov (United States)

Jukes, T. H.; Kimura, M.

1984-01-01

The neutral theory of molecular evolution postulates that nucleotide substitutions inherently take place in DNA as a result of point mutations followed by random genetic drift. In the absence of selective constraints, the substitution rate reaches the maximum value set by the mutation rate. The rate in globin pseudogenes is about 5 x 10 to the -9th substitutions per site per year in mammals. Rates slower than this indicate the presence of constraints imposed by negative (natural) selection, which rejects and discards deleterious mutations.

Molecular analysis of carbon ion-induced mutations in Arabidopsis thaliana

International Nuclear Information System (INIS)

Shikazono, Naoya; Tanaka, Atsushi; Watanabe, Hiroshi; Tano, Shigemitsu; Yokota, Yukihiko

1998-01-01

In order to elucidate the characteristics of the mutations induced by ion particles at the molecular level in plants, mutated loci in carbon ion-induced mutants of Arabidopsis were investigated by PCR and Southern blot analyses. In the present study, two lines of gl1 mutant and two lines of tt4 mutant were isolated after carbon ion-irradiation. Out of four mutants, one had a deletion, other two contained rearrangements, and one had a point-like mutation. From the present result, it was suggested that ion particles induced different kinds of alterations of the DNA and therefore they could produce various types of mutant alleles in plants. (author)

EGFR Mutations in Surgically Resected Fresh Specimens from 697 Consecutive Chinese Patients with Non-Small Cell Lung Cancer and Their Relationships with Clinical Features

Directory of Open Access Journals (Sweden)

Yuanyang Lai

2013-12-01

Full Text Available We aimed to reveal the true status of epidermal growth factor receptor (EGFR mutations in Chinese patients with non-small cell lung cancer (NSCLC after lung resections. EGFR mutations of surgically resected fresh tumor samples from 697 Chinese NSCLC patients were analyzed by Amplification Refractory Mutation System (ARMS. Correlations between EGFR mutation hotspots and clinical features were also explored. Of the 697 NSCLC patients, 235 (33.7% patients had tyrosine kinase inhibitor (TKIs sensitive EGFR mutations in 41 (14.5% of the 282 squamous carcinomas, 155 (52.9% of the 293 adenocarcinomas, 34 (39.5% of the 86 adenosquamous carcinomas, one (9.1% of the 11 large-cell carcinomas, 2 (11.1% of the 18 sarcomatoid carcinomas, and 2 (28.6% of the 7 mucoepidermoid carcinomas. TKIs sensitive EGFR mutations were more frequently found in female patients (p < 0.001, non-smokers (p = 0.047 and adenocarcinomas (p < 0.001. The rates of exon 19 deletion mutation (19-del, exon 21 L858R point mutation (L858R, exon 21 L861Q point mutation (L861Q, exon 18 G719X point mutations (G719X, including G719C, G719S, G719A were 43.4%, 48.1%, 1.7% and 6.8%, respectively. Exon 20 T790M point mutation (T790M was detected in 3 squamous carcinomas and 3 adenocarcinomas and exon 20 insertion mutation (20-ins was detected in 2 patients with adenocarcinoma. Our results show the rates of EGFR mutations are higher in all types of NSCLC in Chinese patients. 19-del and L858R are two of the more frequent mutations. EGFR mutation detection should be performed as a routine postoperative examination in Chinese NSCLC patients.

Mutations of the SRY-responsive enhancer of SOX9 are uncommon in XY gonadal dysgenesis.

Science.gov (United States)

Georg, I; Bagheri-Fam, S; Knower, K C; Wieacker, P; Scherer, Gerd; Harley, V R

2010-01-01

During mouse sex determination, SRY upregulates the core testis-specific enhancer of Sox9, TESCO. Mutations in human SRY are found in one third of cases with XY pure gonadal dysgenesis (XY GD; Swyer syndrome), while two thirds remain unexplained. Heterozygous SOX9 mutations can cause XY GD in association with the skeletal malformation syndrome campomelic dysplasia. We hypothesized that human TESCO mutations could cause isolated XY GD. Sixty-six XY GD cases with an intact SRY were analyzed for TESCO point mutations or deletions. No mutations were identified. We conclude that TESCO mutations are not a common cause of XY GD. Copyright © 2010 S. Karger AG, Basel.

Genetic polymorphisms and mutation rates of 27 Y-chromosomal STRs in a Han population from Guangdong Province, Southern China.

Science.gov (United States)

Wang, Ying; Zhang, Yong-Ji; Zhang, Chu-chu; Li, Ran; Yang, Yang; Ou, Xue-Ling; Tong, Da-yue; Sun, Hong-Yu

2016-03-01

In this study, we collected blood samples from 1033 father-son pairs of a Han population from Guangdong Province, Southern China, of which 1007 fathers were unrelated male individuals. All together, 2040 male individuals were analyzed at 27 Y-chromosomal short tandem repeats (Y-STRs) with Yfiler(®) Plus system. A total of 1003 different haplotypes were observed among 1007 unrelated fathers, with the overall haplotype diversity (HD) 0.999992 and discrimination capacity (DC) 0.996. The gene diversity (GD) values for the 27 Y-STR loci ranged from 0.4400 at DYS438 to 0.9597 at DYS385a/b. 11 off-ladder alleles and 25 copy number variants were detected in 1007 males. Population relationships were analyzed by comparison with 19 other worldwide populations. With 27,920 allele transfers in 1033 father-son pairs, 124 mutation events occurred, of which 118 were one-step mutations and 6 were two-step mutations. Eleven father-son pairs were found to have mutations at two loci, while one pair at three loci. The estimated locus-specific mutation rates varied from 0 to 1.74×10(-2), with an average estimated mutation rate 4.4×10(-3) (95%CI: 3.7×10(-3) to 5.3×10(-3)). Mutations were most frequently observed at three rapidly mutating Y-STRs (RM Y-STRs), DYS576, DYS518 and DYS627. However, at DYS570, DYS449 and DYF387S1 loci, which were also described as RM Y-STRs, the mutation rates in Guangdong Han population were not as high as estimated in other populations. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

Novel mutations of the nucleophosmin (NPM-1) gene in Egyptian patients with acute myeloid leukemia: A pilot study

International Nuclear Information System (INIS)

Neemat Kassem, N.; Abel Hamid, A.; Tarek Attia, T.; Mahmoud, S.; Moemen, E.; Baathallah, Sh.; Safwat, E.; Khalaf, M.; Shaker, O.

2011-01-01

Mutations of the nucleophosmin (NPM-1) gene have been reported in 50-60% of acute myeloid leukemia (AML) patients with normal karyotype. This work was designed to study the prevalence and nature of NPM1 gene mutations in a group of Egyptian patients with AML to get an idea about the profile of NPM1 gene mutations in our society. In 45 previously untreated patients with de novo AML, peripheral blood and/or bone marrow samples from all patients were subjected to microscopic morphologic examination, cytochemical analysis, immuno phenotyping and karyotyping. Patients with normal cytogenetic results were selected for molecular analysis of NPM1 exon 12 by PCR amplification followed by DNA sequencing of the amplified product. Twenty-one patients (46.7%) had abnormal karyotype: six cases with ;(15;17), five cases with (8;21), five cases had trisomy 8, two cases carrying inv(3) and three cases had monosomy 7. The remaining 24 patients (53.3%) had normal karyotype. These patients were then subjected to molecular analysis. Out of these 24 patients with normal karyotype, mutant NPM-1 was detected in 11 patients (45.8%) by DNA sequencing; 2 cases showed type A mutation, 2 cases were harboring [ins 1015-4019 (CACG)], with point mutation [1006C→G], while the remaining 7 cases showed heterozygous deletion of nt A [del 1178 (A)]. Conclusion: Two novel NPM1 gene mutations were detected among our study population of AML patients identified as: the insertion CACG associated with point mutation, deletion of one base, or associated with point mutation. NPM1 gene mutations may become a new tool for monitoring minimal residual disease in AML with normal karyotype. Whether these previously unreported NPM-1 mutations will confer the same better outcome as previously reported mutations is currently unknown and warrants a larger study.

DNA sequence analysis of X-ray induced Adh null mutations in Drosophila melanogaster

International Nuclear Information System (INIS)

Mahmoud, J.; Fossett, N.G.; Arbour-Reily, P.; McDaniel, M.; Tucker, A.; Chang, S.H.; Lee, W.R.

1991-01-01

The mutational spectrum for 28 X-ray induced mutations and 2 spontaneous mutations, previously determined by genetic and cytogenetic methods, consisted of 20 multilocus deficiencies (19 induced and 1 spontaneous) and 10 intragenic mutations (9 induced and 1 spontaneous). One of the X-ray induced intragenic mutations was lost, and another was determined to be a recombinant with the allele used in the recovery scheme. The DNA sequence of two X-ray induced intragenic mutations has been published. This paper reports the results of DNA sequence analysis of the remaining intragenic mutations and a summary of the X-ray induced mutational spectrum. The combination of DNA sequence analysis with genetic complementation analysis shows a continuous distribution in size of deletions rather than two different types of mutations consisting of deletions and 'point mutations'. Sequencing is shown to be essential for detecting intragenic deletions. Of particular importance for future studies is the observation that all of the intragenic deletions consist of a direct repeat adjacent to the breakpoint with one of the repeats deleted

Population structure and genetic diversity of Indian Major Carp, Labeo rohita (Hamilton, 1822) from three phylo-geographically isolated riverine ecosystems of India as revealed by mtDNA cytochrome b region sequences.

Science.gov (United States)

Behera, Bijay Kumar; Baisvar, Vishwamitra Singh; Kunal, Swaraj Priyaranjan; Meena, Dharmendra Kumar; Panda, Debarata; Pakrashi, Sudip; Paria, Prasenjit; Das, Pronob; Bhakta, Dibakar; Debnath, Dipesh; Roy, Suvra; Suresh, V R; Jena, J K

2018-03-01

The population structure and genetic diversity of Rohu (Labeo rohita Hamilton, 1822) was studied by analysis of the partial sequences of mitochondrial DNA cytochrome b region. We examined 133 samples collected from six locations in three geographically isolated rivers of India. Analysis of 11 haplotypes showed low haplotype diversity (0.00150), nucleotide diversity (π) (0.02884) and low heterogeneity value (0.00374). Analysis of molecular variance (AMOVA) revealed the genetic diversity of L. rohita within population is very high than between the populations. The Fst scores (-0.07479 to 0.07022) were the indication of low genetic structure of L. rohita populations of three rivers of India. Conspicuously, Farakka-Bharuch population pair Fst score of 0.0000, although the sampling sites are from different rivers. The phylogenetic reconstruction of unique haplotypes revealed sharing of a single central haplotype (Hap_1) by all the six populations with a point mutations ranging from 1-25 nucleotides.

Molecular Dynamics and Bioactivity of a Novel Mutated Human ...

African Journals Online (AJOL)

Keywords: Parathyroid hormone, Mutation prediction, Molecular dynamics, RANKL/OPG, UAMS-32P cell. Tropical .... PTH1R were used as MD simulation starting points. A full-atom ... Values of RMSD, Rg, and potential energy evaluation ...

Presymptomatic generalized brain atrophy in frontotemporal dementia caused by CHMP2B mutation

DEFF Research Database (Denmark)

Rohrer, Jonathan D; Ahsan, R Laila; Isaacs, Adrian M

2009-01-01

mutation carriers with a control group of 7 mutation-negative family members. Volumetric MRI brain scans were performed on all subjects at two time points, and rates of volume change were compared between the two groups. RESULTS: We demonstrate that generalized atrophy occurs presymptomatically in CHMP2B...... gene mutation carriers. CONCLUSIONS: This finding suggests that mutations in CHMP2B have widespread effects throughout the brain, leading to a neuro-anatomical signature distinct from other diseases in the frontotemporal lobar degeneration spectrum........ There are no detailed studies of brain imaging in CHMP2B mutation-associated FTD. This study aimed to investigate whether there were early or presymptomatic changes in this group of patients. METHODS: Subjects comprised 16 members of a Danish family with CHMP2B mutation-associated FTD. Nine subjects were presymptomatic...

Mutation induction in repair-deficient strains of Drosophila

International Nuclear Information System (INIS)

Wuergler, F.E.; Graf, U.

1980-01-01

Experimental evidence indicates a polygenic control of mutagenesis in Drosophila melanogaster. In oocytes chromosome aberrations detected as half-translocations or dominant lethals depend on a repair system which in a number of genetically nonrelated strains shows different repair capacities. Sister chromatid exchanges are easily studied as ring chromosome losses. They develop through a genotype controlled mechanism from, premutational lesions. Stocks with particular pairs of third chromosomes were discovered in which increased sensitivity of larvae to the toxic effects of a monofunctional alkylating agent correlates with high frequencies of x-ray induced SCE's. Sex-linked mutagen-sensitive mutants could be shown to control mutation fixation: pronounced maternal effects were found when sperm carrying particular types of premutational lesions were introduced into different types of mutant oocytes. The mutant mus(1)101D1 was found to be unable to process lesions induced by the crosslinking agent nitrogen mustard into point mutations. Alkylation damage leads to increased point mutation frequencies in the excision repair deficient mutant mei-9L1, but to reduced frequencies in the post-replication repair deficient mutant mei-41D5. It became clear that the study of maternal effects on mutagenized sperm represents an efficient tool to analyze the gentic control of mutagenesis in the eukaryotic genome of Drosophila melanogaster

Mutanalyst, an online tool for assessing the mutational spectrum of epPCR libraries with poor sampling

DEFF Research Database (Denmark)

Ferla, Matteo

2016-01-01

of mutations per sequence it does so by fitting to a Poisson distribution, which is more robust than calculating the average in light of the small sampling size.Conclusion: As a result of the added measures to keep into account of small sample size the user can better assess whether the library is satisfactory...... of mutations of a specific nucleobase to another-is calculated enabling the user to make more informed predictions on library diversity and coverage. However, the calculations of the mutational spectrum are severely affected by the limited sample sizes.Results: Here an online program, called Mutanalyst...

Performance of mitochondrial DNA mutations detecting early stage cancer

International Nuclear Information System (INIS)

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

2008-01-01

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

Mutation screening of the PCDH15 gene in Spanish patients with Usher syndrome type I.

Science.gov (United States)

Jaijo, Teresa; Oshima, Aki; Aller, Elena; Carney, Carol; Usami, Shin-ichi; Millán, José M; Kimberling, William J

2012-01-01

PCDH15 codes for protocadherin-15, a cell-cell adhesion protein essential in the morphogenesis and cohesion of stereocilia bundles and in the function or preservation of photoreceptor cells. Mutations in the PCDH15 gene are responsible for Usher syndrome type I (USH1F) and non-syndromic hearing loss (DFNB23). The purpose of this work was to perform PCDH15 mutation screening to identify the genetic cause of the disease in a cohort of Spanish patients with Usher syndrome type I and establish phenotype-genotype correlation. Mutation analysis of PCDH15 included additional exons recently identified and was performed by direct sequencing. The screening was performed in 19 probands with USH already screened for mutations in the most prevalent USH1 genes, myosin VIIA (MYO7A) and cadherin-23 (CDH23), and for copy number variants in PCDH15. Seven different point mutations, five novel, were detected. Including the large PCDH15 rearrangements previously reported in our cohort of patients, a total of seven of 19 patients (36.8%) were carriers of at least one pathogenic allele. Thirteen out of the 38 screened alleles carried pathogenic PCDH15 variants (34.2%). Five out of the seven point mutations reported in the present study are novel, supporting the idea that most PCDH15 mutations are private. Furthermore, no mutational hotspots have been identified. In most patients, detected mutations led to a truncated protein, reinforcing the hypothesis that severe mutations cause the Usher I phenotype and that missense variants are mainly responsible for non-syndromic hearing impairment.

Association of disease severity with skin microbiome and filaggrin gene mutations in adult atopic dermatitis

DEFF Research Database (Denmark)

Clausen, Maja Lisa; Agner, Tove; Lilje, Berit

2018-01-01

IMPORTANCE Skin microbiome correlates with disease severity for lesional and nonlesional skin, indicating a global influence of atopic dermatitis (AD). A relation between skin microbiome and filaggrin gene (FLG) mutations proposes a possible association between skin microbiome and host genetics....... OBJECTIVES To assess skin and nasal microbiome diversity and composition in patients with AD and compare with healthy controls, and to investigate the microbiome in relation to disease severity and FLG mutations in patients with AD. DESIGN, SETTING, AND PARTICIPANTS An observational case-control study of 45...... analyses of the microbiome were analyzed using R statistical software (version 3.3.1, R Foundation Inc). MAIN OUTCOMES AND MEASURES Skin microbiomeswere investigated using next-generation sequencing targeting 16S ribosomal RNA. RESULTS Microbiome alpha diversity was lower in patients with AD compared...

Comparison of the efficacy of icotinib in patients with non-small-cell lung cancer according to the type of epidermal growth factor receptor mutation.

Science.gov (United States)

Xue, Zhang Xiao; Wen, Wang Xiu; Zhuang, Yu; Hua, Zang Jian; Xia, Yang Ni

2016-09-01

Icotinib hydrochloride is a novel epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor (TKI) with preclinical and clinical activity in non-small-cell lung cancer (NSCLC). Exon 19 deletion and L858R point mutation are the most commonly encountered EGFR mutations in NSCLC, and they predict improved clinical outcomes following treatment with icotinib. The objective of this study was to evaluate the differential clinical efficacy of icotinib in patients with exon 19 deletion or L858R point mutation of the EGFR gene. A total of 104 patients with advanced NSCLC, who harbored exon 19 deletion or L858R point mutation of EGFR and were treated with icotinib, were enrolled in this study. The tumor response and progression-free survival were evaluated. There were no significant differences between patients with EGFR exon 19 deletion and those with L858R point mutation who received treatment with icotinib.

Significance of Coexisting Mutations on Determination of the Degree of Isoniazid Resistance in Mycobacterium tuberculosis Strains.

Science.gov (United States)

Karunaratne, Galbokka Hewage Roshanthi Eranga; Wijesundera, Sandhya Sulochana; Vidanagama, Dhammika; Adikaram, Chamila Priyangani; Perera, Jennifer

2018-04-23

The emergence and spread of drug-resistant tuberculosis (TB) pose a threat to TB control in Sri Lanka. Isoniazid (INH) is a key element of the first-line anti-TB treatment regimen. Resistance to INH is mainly associated with point mutations in katG, inhA, and ahpC genes. The objective of this study was to determine mutations of these three genes in INH-resistant Mycobacterium tuberculosis (MTb) strains in Sri Lanka. Complete nucleotide sequence of the three genes was amplified by polymerase chain reaction and subjected to DNA sequencing. Point mutations in the katG gene were identified in 93% isolates, of which the majority (78.6%) were at codon 315. Mutations at codons 212 and 293 of the katG gene have not been reported previously. Novel mutations were recognized in the promoter region of the inhA gene (C deletion at -34), fabG1 gene (codon 27), and ahpC gene (codon 39). Single S315T mutation in the katG gene led to a high level of resistance, while a low level of resistance with high frequency (41%) was observed when katG codon 315 coexisted with the mutation at codon 463. Since most of the observed mutations of all three genes coexisted with the katG315 mutation, screening of katG315 mutations will be a useful marker for molecular detection of INH resistance of MTb in Sri Lanka.

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...... mutations and one de novo missense mutation in CTCF in individuals with intellectual disability, microcephaly, and growth retardation. Furthermore, an individual with a larger deletion including CTCF was identified. CTCF (CCCTC-binding factor) is one of the most important chromatin organizers in vertebrates...... and is involved in various chromatin regulation processes such as higher order of chromatin organization, enhancer function, and maintenance of three-dimensional chromatin structure. Transcriptome analyses in all three individuals with point mutations revealed deregulation of genes involved in signal transduction...

Current study on ionizing radiation-induced mitochondial DNA damage and mutations

International Nuclear Information System (INIS)

Zhou Xin; Wang Zhenhua; Zhang Hong

2012-01-01

Current advance in ionizing radiation-induced mitochondrial DNA damage and mutations is reviewed, in addition with the essential differences between mtDNA and nDNA damage and mutations. To extent the knowledge about radiation induced mitochondrial alterations, the researchers in Institute of Modern Physics, Chinese Academy of Sciences developed some technics such as real-time PCR, long-PCR for accurate quantification of radiation induced damage and mutations, and in-depth investigation about the functional changes of mitochondria based on mtDNA damage and mutations were also carried out. In conclusion, the important role of mitochondrial study in radiation biology is underlined, and further study on mitochondrial study associated with late effect and metabolism changes in radiation biology is pointed out. (authors)

Genotype-Phenotype Correlation of Maternally Inherited Disorders due to Mutations in Mitochondrial DNA

Directory of Open Access Journals (Sweden)

Peterus Thajeb

2006-09-01

Full Text Available Mitochondrial disorders are heterogeneous systemic ailments that are most often caused by maternal inheritance of a variety of mutations of the mitochondrial (mt DNA. Paternal inheritance and somatic mutation are rare. The disorders are well recognized not only for the genotypic heterogeneity, but also the phenotypic variation among the affected members of a single family. The genotype-phenotype correlation of the diversity of the syndromic and non-syndromic features of mitochondrial disorders are discussed. Some aspects of the molecular mechanisms of this heterogeneity, and the histopathologic findings are highlighted.

A single point-mutation within the melanophilin gene causes the lavender plumage colour dilution phenotype in the chicken

Directory of Open Access Journals (Sweden)

Tixier-Boichard Michèle

2008-01-01

Full Text Available Abstract Background The lavender phenotype in the chicken causes the dilution of both black (eumelanin and red/brown (phaeomelanin pigments. Defects in three genes involved in intracellular melanosomal transport, previously described in mammals, give rise to similar diluted pigmentation phenotypes as those seen in lavender chickens. Results We have used a candidate-gene approach based on an expectation of homology with mammals to isolate a gene involved in pigmentation in chicken. Comparative sequence analysis of candidate genes in the chicken identified a strong association between a mutation in the MLPH gene and the diluted pigmentation phenotype. This mutation results in the amino acid change R35W, at a site also associated with similar phenotypes in mice, humans and cats. Conclusion This is the first time that an avian species with a mutation in the MLPH gene has been reported.

Nucleotide diversity maps reveal variation in diversity among wheat genomes and chromosomes

Directory of Open Access Journals (Sweden)

McGuire Patrick E

2010-12-01

chromosomal regions. The net effect of these factors in T. aestivum is large variation in diversity among genomes and chromosomes, which impacts the development of SNP markers and their practical utility. Accumulation of new mutations in older polyploid species, such as wild emmer, results in increased diversity and its more uniform distribution across the genome.

FireProt: Energy- and Evolution-Based Computational Design of Thermostable Multiple-Point Mutants.

Science.gov (United States)

Bednar, David; Beerens, Koen; Sebestova, Eva; Bendl, Jaroslav; Khare, Sagar; Chaloupkova, Radka; Prokop, Zbynek; Brezovsky, Jan; Baker, David; Damborsky, Jiri

2015-11-01

There is great interest in increasing proteins' stability to enhance their utility as biocatalysts, therapeutics, diagnostics and nanomaterials. Directed evolution is a powerful, but experimentally strenuous approach. Computational methods offer attractive alternatives. However, due to the limited reliability of predictions and potentially antagonistic effects of substitutions, only single-point mutations are usually predicted in silico, experimentally verified and then recombined in multiple-point mutants. Thus, substantial screening is still required. Here we present FireProt, a robust computational strategy for predicting highly stable multiple-point mutants that combines energy- and evolution-based approaches with smart filtering to identify additive stabilizing mutations. FireProt's reliability and applicability was demonstrated by validating its predictions against 656 mutations from the ProTherm database. We demonstrate that thermostability of the model enzymes haloalkane dehalogenase DhaA and γ-hexachlorocyclohexane dehydrochlorinase LinA can be substantially increased (ΔTm = 24°C and 21°C) by constructing and characterizing only a handful of multiple-point mutants. FireProt can be applied to any protein for which a tertiary structure and homologous sequences are available, and will facilitate the rapid development of robust proteins for biomedical and biotechnological applications.

FireProt: Energy- and Evolution-Based Computational Design of Thermostable Multiple-Point Mutants.

Directory of Open Access Journals (Sweden)

David Bednar

2015-11-01

Full Text Available There is great interest in increasing proteins' stability to enhance their utility as biocatalysts, therapeutics, diagnostics and nanomaterials. Directed evolution is a powerful, but experimentally strenuous approach. Computational methods offer attractive alternatives. However, due to the limited reliability of predictions and potentially antagonistic effects of substitutions, only single-point mutations are usually predicted in silico, experimentally verified and then recombined in multiple-point mutants. Thus, substantial screening is still required. Here we present FireProt, a robust computational strategy for predicting highly stable multiple-point mutants that combines energy- and evolution-based approaches with smart filtering to identify additive stabilizing mutations. FireProt's reliability and applicability was demonstrated by validating its predictions against 656 mutations from the ProTherm database. We demonstrate that thermostability of the model enzymes haloalkane dehalogenase DhaA and γ-hexachlorocyclohexane dehydrochlorinase LinA can be substantially increased (ΔTm = 24°C and 21°C by constructing and characterizing only a handful of multiple-point mutants. FireProt can be applied to any protein for which a tertiary structure and homologous sequences are available, and will facilitate the rapid development of robust proteins for biomedical and biotechnological applications.

Brain white matter 1 H MRS in Leber optic neuropathy mutation carriers

DEFF Research Database (Denmark)

Ostojic, Jelena; Jancic, Jasna; Kozic, Dusko

2009-01-01

OBJECTIVE: This study was conducted in order to test the hypothesis that proton MR spectroscopic (1H MRS) profile of Leber's hereditary optic neuropathy (LHON) mutation carriers group (including both symptomatic and asymptomatic) differs from group of healthy individuals and to determine metabolite...... or ratio that contributes most to differentiation. PATIENTS AND METHODS: We performed single voxel 1H MRS in normal appearing white matter of eighteen LHON mtDNA mutation carriers bearing one of three LHON mtDNA point mutations and in fifty control subjects. RESULTS: ANOVA showed significant difference...

Fidelity and Mutational Spectrum of Pfu DNA Polymerase on a Human Mitochondrial DNA Sequence

Science.gov (United States)

André, Paulo; Kim, Andrea; Khrapko, Konstantin; Thilly, William G.

1997-01-01

The study of rare genetic changes in human tissues requires specialized techniques. Point mutations at fractions at or below 10−6 must be observed to discover even the most prominent features of the point mutational spectrum. PCR permits the increase in number of mutant copies but does so at the expense of creating many additional mutations or “PCR noise”. Thus, each DNA sequence studied must be characterized with regard to the DNA polymerase and conditions used to avoid interpreting a PCR-generated mutation as one arising in human tissue. The thermostable DNA polymerase derived from Pyrococcus furiosus designated Pfu has the highest fidelity of any DNA thermostable polymerase studied to date, and this property recommends it for analyses of tissue mutational spectra. Here, we apply constant denaturant capillary electrophoresis (CDCE) to separate and isolate the products of DNA amplification. This new strategy permitted direct enumeration and identification of point mutations created by Pfu DNA polymerase in a 96-bp low melting domain of a human mitochondrial sequence despite the very low mutant fractions generated in the PCR process. This sequence, containing part of the tRNA glycine and NADH dehydrogenase subunit 3 genes, is the target of our studies of mitochondrial mutagenesis in human cells and tissues. Incorrectly synthesized sequences were separated from the wild type as mutant/wild-type heteroduplexes by sequential enrichment on CDCE. An artificially constructed mutant was used as an internal standard to permit calculation of the mutant fraction. Our study found that the average error rate (mutations per base pair duplication) of Pfu was 6.5 × 10−7, and five of its more frequent mutations (hot spots) consisted of three transversions (GC → TA, AT → TA, and AT → CG), one transition (AT → GC), and one 1-bp deletion (in an AAAAAA sequence). To achieve an even higher sensitivity, the amount of Pfu-induced mutants must be

Fidelity and mutational spectrum of Pfu DNA polymerase on a human mitochondrial DNA sequence.

Science.gov (United States)

André, P; Kim, A; Khrapko, K; Thilly, W G

1997-08-01

The study of rare genetic changes in human tissues requires specialized techniques. Point mutations at fractions at or below 10(-6) must be observed to discover even the most prominent features of the point mutational spectrum. PCR permits the increase in number of mutant copies but does so at the expense of creating many additional mutations or "PCR noise". Thus, each DNA sequence studied must be characterized with regard to the DNA polymerase and conditions used to avoid interpreting a PCR-generated mutation as one arising in human tissue. The thermostable DNA polymerase derived from Pyrococcus furiosus designated Pfu has the highest fidelity of any DNA thermostable polymerase studied to date, and this property recommends it for analyses of tissue mutational spectra. Here, we apply constant denaturant capillary electrophoresis (CDCE) to separate and isolate the products of DNA amplification. This new strategy permitted direct enumeration and identification of point mutations created by Pfu DNA polymerase in a 96-bp low melting domain of a human mitochondrial sequence despite the very low mutant fractions generated in the PCR process. This sequence, containing part of the tRNA glycine and NADH dehydrogenase subunit 3 genes, is the target of our studies of mitochondrial mutagenesis in human cells and tissues. Incorrectly synthesized sequences were separated from the wild type as mutant/wild-type heteroduplexes by sequential enrichment on CDCE. An artificially constructed mutant was used as an internal standard to permit calculation of the mutant fraction. Our study found that the average error rate (mutations per base pair duplication) of Pfu was 6.5 x 10(-7), and five of its more frequent mutations (hot spots) consisted of three transversions (GC-->TA, AT-->TA, and AT-->CG), one transition (AT-->GC), and one 1-bp deletion (in an AAAAAA sequence). To achieve an even higher sensitivity, the amount of Pfu-induced mutants must be reduced.

Potential use of gradient denaturing gel electrophoresis in obtaining mutational spectra from human cells

International Nuclear Information System (INIS)

Thilly, W.G.

1985-01-01

A method is described to isolate mutations in DNA in human cells. When a double-stranded DNA migrates through an electric field on an electrophoretic gel, it is compact hydrodynamic structure relative to the same material in a melted form. Normally the solution in electrophoretic gels is uniform, but a way has been devised to set up a stable gradient of increasing solute concentration in the direction of DNA motion. Thus, as a double-stranded DNA molecule is drawn by the electric field into higher and higher concentrations of urea/formamide, it will eventually reach a point at which the concentration is high enough to melt the lower-melting-point region. The melting results in an essentially immobile structure within the gel so that the position at which the DNA molecule stops on the gradient gel is determined by its melting point, which is uniquely determined by its nucleotide sequence. A single base pair substitution within a low melting point sequence of some 100 base pairs changed the expected melting point by 0.4 0 C and resulted in about a 2-cm displacement under appropriate denaturing gel conditions. This expectation leads to the idea that if a mixture of DNA sequences derived from point mutations within the same restriction fragment were permitted to anneal with a complementary wild-type sequence, the melting point of each type of heteroduplex would differ depending on the kind and position of each mutation

U2AF1 mutations alter splice site recognition in hematological malignancies.

Science.gov (United States)

Ilagan, Janine O; Ramakrishnan, Aravind; Hayes, Brian; Murphy, Michele E; Zebari, Ahmad S; Bradley, Philip; Bradley, Robert K

2015-01-01

Whole-exome sequencing studies have identified common mutations affecting genes encoding components of the RNA splicing machinery in hematological malignancies. Here, we sought to determine how mutations affecting the 3' splice site recognition factor U2AF1 alter its normal role in RNA splicing. We find that U2AF1 mutations influence the similarity of splicing programs in leukemias, but do not give rise to widespread splicing failure. U2AF1 mutations cause differential splicing of hundreds of genes, affecting biological pathways such as DNA methylation (DNMT3B), X chromosome inactivation (H2AFY), the DNA damage response (ATR, FANCA), and apoptosis (CASP8). We show that U2AF1 mutations alter the preferred 3' splice site motif in patients, in cell culture, and in vitro. Mutations affecting the first and second zinc fingers give rise to different alterations in splice site preference and largely distinct downstream splicing programs. These allele-specific effects are consistent with a computationally predicted model of U2AF1 in complex with RNA. Our findings suggest that U2AF1 mutations contribute to pathogenesis by causing quantitative changes in splicing that affect diverse cellular pathways, and give insight into the normal function of U2AF1's zinc finger domains. © 2015 Ilagan et al.; Published by Cold Spring Harbor Laboratory Press.

RDEL: Restart Differential Evolution algorithm with Local Search Mutation for global numerical optimization

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Ali Wagdy Mohamed

2014-11-01

Full Text Available In this paper, a novel version of Differential Evolution (DE algorithm based on a couple of local search mutation and a restart mechanism for solving global numerical optimization problems over continuous space is presented. The proposed algorithm is named as Restart Differential Evolution algorithm with Local Search Mutation (RDEL. In RDEL, inspired by Particle Swarm Optimization (PSO, a novel local mutation rule based on the position of the best and the worst individuals among the entire population of a particular generation is introduced. The novel local mutation scheme is joined with the basic mutation rule through a linear decreasing function. The proposed local mutation scheme is proven to enhance local search tendency of the basic DE and speed up the convergence. Furthermore, a restart mechanism based on random mutation scheme and a modified Breeder Genetic Algorithm (BGA mutation scheme is combined to avoid stagnation and/or premature convergence. Additionally, an exponent increased crossover probability rule and a uniform scaling factors of DE are introduced to promote the diversity of the population and to improve the search process, respectively. The performance of RDEL is investigated and compared with basic differential evolution, and state-of-the-art parameter adaptive differential evolution variants. It is discovered that the proposed modifications significantly improve the performance of DE in terms of quality of solution, efficiency and robustness.

Osteogenesis imperfecta type 3 in South Africa: Causative mutations in FKBP10

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Alvera Vorster

2017-05-01

Full Text Available Background. A relatively high frequency of autosomal recessively inherited osteogenesis imperfecta (OI type 3 (OI-3 is present in the indigenous black southern African population. Affected persons may be severely handicapped as a result of frequent fractures, progressive deformity of the tubular bones and spinal malalignment. Objective. To delineate the molecular basis for the condition. Methods. Molecular investigations were performed on 91 affected persons from seven diverse ethnolinguistic groups in this population. Results. Following polymerase chain reaction amplification and direct cycle sequencing, FKBP10 mutations were identified in 45.1% (41/91 OI-3-affected persons. The homozygous FKBP10 c.831dupC frameshift mutation was confirmed in 35 affected individuals in the study cohort. Haplotype analysis suggests that this mutation is identical among these OI-3-affected persons by descent, thereby confirming that they had a common ancestor. Compound heterozygosity of this founder mutation was observed, in combination with three different deleterious FKBP10 mutations, in six additional persons in the cohort. Four of these individuals had the c.831delC mutation. Conclusion. The burden of the disorder, both in frequency and severity, warrants the establishment of a dedicated service for molecular diagnostic confirmation and genetic management of persons and families with OI in southern Africa.

Mutations in the human adenosine deaminase gene that affect protein structure and RNA splicing

International Nuclear Information System (INIS)

Akeson, A.L.; Wiginton, D.A.; States, C.J.; Perme, C.M.; Dusing, M.R.; Hutton, J.J.

1987-01-01

Adenosine deaminase deficiency is one cause of the genetic disease severe combined immunodeficiency. To identify mutations responsible for ADA deficiency, the authors synthesized cDNAs to ADA mRNAs from two cell lines, GM2756 and GM2825A, derived from ADA-deficient immunodeficient patients. Sequence analysis of GM2756 cDNA clones revealed a different point mutation in each allele that causes amino acid changes of alanine to valine and arginine to histidine. One allele of GM2825A also has a point mutation that causes an alanine to valine substitution. The other allele of GM2825A was found to produce an mRNA in which exon 4 had been spliced out but had no other detrimental mutations. S1 nuclease mapping of GM2825A mRNA showed equal abundance of the full-length ADA mRNA and the ADA mRNA that was missing exon 4. Several of the ADA cDNA clones extended 5' of the major initiation start site, indicating multiple start sites for ADA transcription. The point mutations in GM2756 and GM2825A and the absence of exon 4 in GM2825A appear to be directly responsible for the ADA deficiency. Comparison of a number of normal and mutant ADA cDNA sequences showed a number of changes in the third base of codons. These change do not affect the amino acid sequence. Analyses of ADA cDNAs from different cell lines detected aberrant RNA species that either included intron 7 or excluded exon 7. Their presence is a result of aberrant splicing of pre-mRNAs and is not related to mutations that cause ADA deficiency

VNTR diversity in Yersinia pestis isolates from an animal challenge study reveals the potential for in vitro mutations during laboratory cultivation

Science.gov (United States)

Vogler, Amy J.; Nottingham, Roxanne; Busch, Joseph D.; Sahl, Jason W.; Shuey, Megan M.; Foster, Jeffrey T.; Schupp, James M.; Smith, Susan; Rocke, Tonie E.; Klein, Paul; Wagner, David M.

2016-01-01

Underlying mutation rates and other evolutionary forces shape the population structure of bacteria in nature. Although easily overlooked, similar forces are at work in the laboratory and may influence observed mutations. Here, we investigated tissue samples and Yersinia pestis isolates from a rodent laboratory challenge with strain CO92 using whole genome sequencing and multi-locus variable-number tandem repeat (VNTR) analysis (MLVA). We identified six VNTR mutations that were found to have occurred in vitro during laboratory cultivation rather than in vivo during the rodent challenge. In contrast, no single nucleotide polymorphism (SNP) mutations were observed, either in vivo or in vitro. These results were consistent with previously published mutation rates and the calculated number of Y. pestis generations that occurred during the in vitro versus the in vivo portions of the experiment. When genotyping disease outbreaks, the potential for in vitro mutations should be considered, particularly when highly variable genetic markers such as VNTRs are used.

Plant Mutation Reports, Vol. 2, No. 3, April 2011

Energy Technology Data Exchange (ETDEWEB)

NONE

2011-04-15

Enhancing crop yields, improving food quality and value in an environmentally friendly manner, and sustaining crop biodiversity continue to be key goals for improving agricultural production. Mutation induction techniques are undergoing a renaissance in crop improvement because of advancements in modern efficiency enhancing biotechnologies - irreplaceable tools in the tool box of the breeder. In the context of climate change and variability, mutation induction is a proven way to generate diversity in existing crop varieties, to widen the extent of adaptability and enhance productivity of crop biomass. We are encouraged by the contributions from our Member States to this journal. In many countries, we see that a broad variety of plant species and target traits are addressed using mutation induction. In this issue, the technical papers highlight studies on induced mutagenesis using either physical or chemical mutagens in a range of food and industrial crops. Dr Tulmann Neto gives an extensive review of 40 years of induced mutations in plant breeding in Brazil. A number of mutant derived varieties ranging from cereals, legumes, fruits and spices were developed together with commercial breeding companies and have significant economic value in Latin America. We trust that these reports will reignite a drive in experienced users of induced mutants in plant breeding and stir an interest in younger scientists

Plant Mutation Reports, Vol. 2, No. 3, April 2011

International Nuclear Information System (INIS)

2011-04-01

Enhancing crop yields, improving food quality and value in an environmentally friendly manner, and sustaining crop biodiversity continue to be key goals for improving agricultural production. Mutation induction techniques are undergoing a renaissance in crop improvement because of advancements in modern efficiency enhancing biotechnologies - irreplaceable tools in the tool box of the breeder. In the context of climate change and variability, mutation induction is a proven way to generate diversity in existing crop varieties, to widen the extent of adaptability and enhance productivity of crop biomass. We are encouraged by the contributions from our Member States to this journal. In many countries, we see that a broad variety of plant species and target traits are addressed using mutation induction. In this issue, the technical papers highlight studies on induced mutagenesis using either physical or chemical mutagens in a range of food and industrial crops. Dr Tulmann Neto gives an extensive review of 40 years of induced mutations in plant breeding in Brazil. A number of mutant derived varieties ranging from cereals, legumes, fruits and spices were developed together with commercial breeding companies and have significant economic value in Latin America. We trust that these reports will reignite a drive in experienced users of induced mutants in plant breeding and stir an interest in younger scientists

Hereditary Persistence of Fetal Hemoglobin Caused by Single Nucleotide Promoter Mutations in Sickle Cell Trait and Hb SC Disease.

Science.gov (United States)

Akinbami, Anthony O; Campbell, Andrew D; Han, Zeqiu J; Luo, Hong-Yuan; Chui, David H K; Steinberg, Martin H

2016-01-01

Hereditary persistence of fetal hemoglobin (HPFH) can be caused by point mutations in the γ-globin gene promoters. We report three rare cases: a child compound heterozygous for Hb S (HBB: c.20A > T) and HPFH with a novel point mutation in the (A)γ-globin gene promoter who had 42.0% Hb S, 17.0% Hb A and 38.0% Hb F; a man with Hb SC (HBB: c.19G > A) disease and a point mutation in the (G)γ-globin gene promoter who had 54.0% Hb S, 18.0% Hb C and 25.0% Hb F; a child heterozygous for Hb S and HPFH due to mutations in both the (A)γ- and (G)γ-globin gene promoters in cis [(G)γ(A)γ(β(+)) HPFH], with 67.0% Hb A, 6.5% Hb S and 25.0% Hb F.

Case Report Identification of a novel SLC45A2 mutation in albinism by targeted next-generation sequencing.

Science.gov (United States)

Xue, J J; Xue, J F; Xue, H Q; Guo, Y Y; Liu, Y; Ouyang, N

2016-09-19

Albinism is a diverse group of hypopigmentary disorders caused by multiple-genetic defects. The genetic diagnosis of patients affected with albinism by Sanger sequencing is often complex, expensive, and time-consuming. In this study, we performed targeted next-generation sequencing to screen for 16 genes in a patient with albinism, and identified 21 genetic variants, including 19 known single nucleotide polymorphisms, one novel missense mutation (c.1456 G>A), and one disease-causing mutation (c.478 G>C). The novel mutation was not observed in 100 controls, and was predicted to be a damaging mutation by SIFT and Polyphen. Thus, we identified a novel mutation in SLC45A2 in a Chinese family, expanding the mutational spectrum of albinism. Our results also demonstrate that targeted next-generation sequencing is an effective genetic test for albinism.

Retinal phenotype-genotype correlation of pediatric patients expressing mutations in the Norrie disease gene.

Science.gov (United States)

Wu, Wei-Chi; Drenser, Kimberly; Trese, Michael; Capone, Antonio; Dailey, Wendy

2007-02-01

To correlate the ophthalmic findings of patients with pediatric vitreoretinopathies with mutations occurring in the Norrie disease gene (NDP). One hundred nine subjects with diverse pediatric vitreoretinopathies and 54 control subjects were enrolled in the study. Diagnoses were based on retinal findings at each patient's first examination. Samples of DNA from each patient underwent polymerase chain reaction amplification and direct sequencing of the NDP gene. Eleven male patients expressing mutations in the NDP gene were identified in the test group, whereas the controls demonstrated wild-type NDP. All patients diagnosed as having Norrie disease had mutations in the NDP gene. Four of the patients with Norrie disease had mutations involving a cysteine residue in the cysteine-knot motif. Four patients diagnosed as having familial exudative vitreoretinopathy were found to have noncysteine mutations. One patient with retinopathy of prematurity had a 14-base deletion in the 5' untranslated region (exon 1), and 1 patient with bilateral persistent fetal vasculature syndrome expressed a noncysteine mutation in the second exon. Mutations disrupting the cysteine-knot motif corresponded to severe retinal dysgenesis, whereas patients with noncysteine mutations had varying degrees of avascular peripheral retina, extraretinal vasculature, and subretinal exudate. Patients exhibiting severe retinal dysgenesis should be suspected of carrying a mutation that disrupts the cysteine-knot motif in the NDP gene.

The induction of mutation and recombination following UV irradiation during meiosis in Saccharomyces cerevisiae

International Nuclear Information System (INIS)

Kelly, S.L.; Parry, J.M.

1983-01-01

Irradiation of yeast cultures with ultraviolet light at discrete stages during meiosis produces cyclic variations in sensitivity, i.e. cells are more sensitive to the lethal effects of UV light prior to entry into the meiotic DNA synthesis, and this corresponds to a peak of induction of point mutation. Cells become more resistant to both induced point mutation and lethality as they enter meiotic DNA synthesis, but become more sensitive again during spore formation. The induced level of intragenic recombination rises during the period of commitment ot recombination to a level indistinguishable from the full meiotic level of spontaneous intragenic recombination. Induced reciprocal recombination remains above the spontaneous level up to the point of commitment to sporulation. (orig.)

The induction of mutation and recombination following UV irradiation during meiosis in Saccharomyces cerevisiae.

Science.gov (United States)

Kelly, S L; Parry, J M

1983-03-01

Irradiation of yeast cultures with ultraviolet light at discrete stages during meiosis produces cyclic variations in sensitivity, i.e. cells are more sensitive to the lethal effects of UV light prior to entry into the meiotic DNA synthesis, and this corresponds to a peak of induction of point mutation. Cells become more resistant to both induced point mutation and lethality as they enter meiotic DNA synthesis, but become more sensitive again during spore formation. The induced level of intragenic recombination rises during the period of commitment to recombination to a level indistinguishable from the full meiotic level of spontaneous intragenic recombination. Induced reciprocal recombination remains above the spontaneous level up to the point of commitment to sporulation.

Gender and Diversity in European Media

DEFF Research Database (Denmark)

Flyger, Tabitta

2013-01-01

The chapter contains the representation of findings from the Eurosphere project related to gender and diversity in the European media. The point of departure is the question of how articulations of diversity in the media draw on and contribute to country-specific discourses and whether...

Concurrent mutation in exons 1 and 2 of the K-ras oncogene in colorectal cancer

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Fiorella Guadagni

2012-01-01

Full Text Available The K-ras gene is frequently mutated in colorectal cancer and has been associated with tumor initiation and progression; approximately 90% of the activating mutations are found in codons 12 and 13 of exon 1 and just under 5% in codon 61 located in exon 2. These mutations determine single aminoacidic substitutions in the GTPase pocket leading to a block of the GTP hydrolytic activity of the K-ras p21 protein, and therefore to its constitutive activation. Point mutations in sites of the K-ras gene, other than codons 12, 13 and 61, and other types of genetic alterations, may occur in a minority of cases, such as in the less frequent cases of double mutations in the K-ras gene. However, all mutations in this gene, even those which occur in non-canonical sites or double mutations, are relevant oncogenic alterations in colorectal cancer and may underlie K-ras pathway hyperactivation. In the present study, we report the case of a patient with colorectal cancer presenting a concurrent point mutation in exons 1 and 2 of the K-ras gene, a GGT to TGT substitution (Glycine to Cysteine at codon 12, and a GAC to AAC substitution (Aspartic Acid to Asparagine at codon 57. In addition, we found in the same patient’s sample a silent polymorphism at codon 11 (Ala11Ala of exon 1. (Folia Histochemica et Cytobiologica 2011; Vol. 49, No. 4, pp. 729–733

Mutation induction by ion beams in arabidopsis

International Nuclear Information System (INIS)

Tanaka, Atsushi

1999-01-01

An investigation was made on characteristics of ion beams for the biological effects and the induction of mutation using Arabidopsis plant as a model plant for the molecular genetics. Here, the characteristics of mutation at the molecular level as well as new mutants induced by ion beams were described. The ast and sep1 were obtained from the offspring of 1488 carbon ion-irradiated seeds respectively. The uvi1-uvi4 mutants were also induced from 1280 M 1 lines. Thus, ion beams can induce not only known mutants such as tt, gl and hy but also novel mutants with high frequency. Even in the tt phenotype, two new mutant loci other than known loci were found. In chrysanthemum, several kinds of single, complex or stripped flower-color mutants that have been never induced by γirradiation, indicating that ion beams could produce a variety of mutants with the same phenotype. In conclusion, ion beams for the mutation induction are characterized by 1) to induce mutants with high frequency, 2) to show broad mutation spectrum and 3) to produce novel mutants. For these reasons, chemical mutagens such as EMS and low LET ionizing radiation such as X-rays and γ-rays will predominantly induce many but small modifications or DNA damages on the DNA strands. As the result, several point mutations will be produced on the genome. On the contrary, ion beams as a high LET ionizing radiation will not cause so many but large and irreparable DNA damage locally, resulting in production of limited number of null mutation. (M.N.)

Mutation induction by ion beams in arabidopsis

Energy Technology Data Exchange (ETDEWEB)

Tanaka, Atsushi [Japan Atomic Energy Research Inst., Takasaki, Gunma (Japan). Takasaki Radiation Chemistry Research Establishment

1999-07-01

An investigation was made on characteristics of ion beams for the biological effects and the induction of mutation using Arabidopsis plant as a model plant for the molecular genetics. Here, the characteristics of mutation at the molecular level as well as new mutants induced by ion beams were described. The ast and sep1 were obtained from the offspring of 1488 carbon ion-irradiated seeds respectively. The uvi1-uvi4 mutants were also induced from 1280 M{sub 1} lines. Thus, ion beams can induce not only known mutants such as tt, gl and hy but also novel mutants with high frequency. Even in the tt phenotype, two new mutant loci other than known loci were found. In chrysanthemum, several kinds of single, complex or stripped flower-color mutants that have been never induced by {gamma}irradiation, indicating that ion beams could produce a variety of mutants with the same phenotype. In conclusion, ion beams for the mutation induction are characterized by 1) to induce mutants with high frequency, 2) to show broad mutation spectrum and 3) to produce novel mutants. For these reasons, chemical mutagens such as EMS and low LET ionizing radiation such as X-rays and {gamma}-rays will predominantly induce many but small modifications or DNA damages on the DNA strands. As the result, several point mutations will be produced on the genome. On the contrary, ion beams as a high LET ionizing radiation will not cause so many but large and irreparable DNA damage locally, resulting in production of limited number of null mutation. (M.N.)

A novel mutation of the fibrillin gene causing Ectopia lentis

Energy Technology Data Exchange (ETDEWEB)

Loennqvist, L.; Kainulainen, K.; Puhakka, L.; Peltonen, L. (National Public Health Institute, Helsinki (Finland)); Child, A. (St. George' s Hospital Medical School, London (United Kingdom)); Peltonen, L. (Duncan Guthrie Institute, Glasgow, Scotland (United Kingdom))

1994-02-01

Ectopia lentis (EL), a dominantly inherited connective tissue disorder, has been genetically linked to the fibrillin gene on chromosome 15 (FBN1) in earlier studies. Here, the authors report the first EL mutation in the FBN1 gene confirming that EL is caused by mutations of this gene. So far, several mutations in the FBN1 gene have been reported in patients with Marfan syndrome (MFS). EL and MFS are clinically related but distinct conditions with typical manifestations in the ocular and skeletal systems, the fundamental difference between them being the absence of cardiovascular involvement in EL. They report a point mutation, cosegregating with the disease in the described family, that displays EL over four generations. The mutation changes a conserved glutamic acid residue in an EGF-like motif, which is the major structural component of the fibrillin and is repeated throughout the polypeptide. In vitro mutagenetic studies have demonstrated the necessity of an analogous glutamic acid residue for calcium binding in an EGF-like repeat of human factor IX. This provides a possible explanation for the role of this mutation in the disease pathogenesis. 32 refs., 2 figs., 1 tab.

Novel mutations of CYP3A4 in Chinese.

Science.gov (United States)

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

2001-03-01

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

Mutated genes as research tool

International Nuclear Information System (INIS)

1981-01-01

mutations, it was pointed out that analogous genetical structures exist in all living organisms, the more closely related, the more similar. This is reflected in strikingly similar biochemical pathways, leading from the primary gene message to the ultimate compound or trait. Induced mutations are a unique tool for analysing these gene-controlled pathways, thus leading also to a better understanding of natural evolution

Point mutations in the transmembrane region of the clic1 ion channel selectively modify its biophysical properties.

Directory of Open Access Journals (Sweden)

Stefania Averaimo

Full Text Available Chloride intracellular Channel 1 (CLIC1 is a metamorphic protein that changes from a soluble cytoplasmic protein into a transmembrane protein. Once inserted into membranes, CLIC1 multimerises and is able to form chloride selective ion channels. Whilst CLIC1 behaves as an ion channel both in cells and in artificial lipid bilayers, its structure in the soluble form has led to some uncertainty as to whether it really is an ion channel protein. CLIC1 has a single putative transmembrane region that contains only two charged residues: arginine 29 (Arg29 and lysine 37 (Lys37. As charged residues are likely to have a key role in ion channel function, we hypothesized that mutating them to neutral alanine to generate K37A and R29A CLIC1 would alter the electrophysiological characteristics of CLIC1. By using three different electrophysiological approaches: i single channel Tip-Dip in artificial bilayers using soluble recombinant CLIC1, ii cell-attached and iii whole-cell patch clamp recordings in transiently transfected HEK cells, we determined that the K37A mutation altered the single-channel conductance while the R29A mutation affected the single-channel open probability in response to variation in membrane potential. Our results show that mutation of the two charged amino acids (K37 and R29 in the putative transmembrane region of CLIC1 alters the biophysical properties of the ion channel in both artificial bilayers and cells. Hence these charged residues are directly involved in regulating its ion channel activity. This strongly suggests that, despite its unusual structure, CLIC1 itself is able to form a chloride ion channel.

UPF1 silenced cellular model systems for screening of read-through agents active on β039 thalassemia point mutation.

Science.gov (United States)

Salvatori, Francesca; Pappadà, Mariangela; Breveglieri, Giulia; D'Aversa, Elisabetta; Finotti, Alessia; Lampronti, Ilaria; Gambari, Roberto; Borgatti, Monica

2018-05-15

Nonsense mutations promote premature translational termination, introducing stop codons within the coding region of mRNAs and causing inherited diseases, including thalassemia. For instance, in β 0 39 thalassemia the CAG (glutamine) codon is mutated to the UAG stop codon, leading to premature translation termination and to mRNA destabilization through the well described NMD (nonsense-mediated mRNA decay). In order to develop an approach facilitating translation and, therefore, protection from NMD, ribosomal read-through molecules, such as aminoglycoside antibiotics, have been tested on mRNAs carrying premature stop codons. These findings have introduced new hopes for the development of a pharmacological approach to the β 0 39 thalassemia therapy. While several strategies, designed to enhance translational read-through, have been reported to inhibit NMD efficiency concomitantly, experimental tools for systematic analysis of mammalian NMD inhibition by translational read-through are lacking. We developed a human cellular model of the β 0 39 thalassemia mutation with UPF-1 suppressed and showing a partial NMD suppression. This novel cellular model could be used for the screening of molecules exhibiting preferential read-through activity allowing a great rescue of the mutated transcripts.

A lack of Birbeck granules in Langerhans cells is associated with a naturally occurring point mutation in the human Langerin gene.

Science.gov (United States)

Verdijk, Pauline; Dijkman, Remco; Plasmeijer, Elsemieke I; Mulder, Aat A; Zoutman, Willem H; Mieke Mommaas, A; Tensen, Cornelis P

2005-04-01

A heterozygous mutation in the Langerin gene corresponding to position 837 in the Langerin mRNA was identified in a person deficient in Birbeck granules (BG). This mutation results in an amino acid replacement of tryptophan by arginine at position 264 in the carbohydrate recognition domain of the Langerine protein. Expression of mutated Langerin in human fibroblasts induces tubular-like structures that are negative for BG-specific antibodies and do not resemble the characteristic structural features of BG.

VNTR diversity in Yersinia pestis isolates from an animal challenge study reveals the potential for in vitro mutations during laboratory cultivation.

Science.gov (United States)

Vogler, Amy J; Nottingham, Roxanne; Busch, Joseph D; Sahl, Jason W; Shuey, Megan M; Foster, Jeffrey T; Schupp, James M; Smith, Susan R; Rocke, Tonie E; Keim, Paul; Wagner, David M

2016-11-01

Underlying mutation rates and other evolutionary forces shape the population structure of bacteria in nature. Although easily overlooked, similar forces are at work in the laboratory and may influence observed mutations. Here, we investigated tissue samples and Yersinia pestis isolates from a rodent laboratory challenge with strain CO92 using whole genome sequencing and multi-locus variable-number tandem repeat (VNTR) analysis (MLVA). We identified six VNTR mutations that were found to have occurred in vitro during laboratory cultivation rather than in vivo during the rodent challenge. In contrast, no single nucleotide polymorphism (SNP) mutations were observed, either in vivo or in vitro. These results were consistent with previously published mutation rates and the calculated number of Y. pestis generations that occurred during the in vitro versus the in vivo portions of the experiment. When genotyping disease outbreaks, the potential for in vitro mutations should be considered, particularly when highly variable genetic markers such as VNTRs are used. Copyright © 2016 Elsevier B.V. All rights reserved.

De novo mutations in HCN1 cause early infantile epileptic encephalopathy.

Science.gov (United States)

Nava, Caroline; Dalle, Carine; Rastetter, Agnès; Striano, Pasquale; de Kovel, Carolien G F; Nabbout, Rima; Cancès, Claude; Ville, Dorothée; Brilstra, Eva H; Gobbi, Giuseppe; Raffo, Emmanuel; Bouteiller, Delphine; Marie, Yannick; Trouillard, Oriane; Robbiano, Angela; Keren, Boris; Agher, Dahbia; Roze, Emmanuel; Lesage, Suzanne; Nicolas, Aude; Brice, Alexis; Baulac, Michel; Vogt, Cornelia; El Hajj, Nady; Schneider, Eberhard; Suls, Arvid; Weckhuysen, Sarah; Gormley, Padhraig; Lehesjoki, Anna-Elina; De Jonghe, Peter; Helbig, Ingo; Baulac, Stéphanie; Zara, Federico; Koeleman, Bobby P C; Haaf, Thomas; LeGuern, Eric; Depienne, Christel

2014-06-01

Hyperpolarization-activated, cyclic nucleotide-gated (HCN) channels contribute to cationic Ih current in neurons and regulate the excitability of neuronal networks. Studies in rat models have shown that the Hcn1 gene has a key role in epilepsy, but clinical evidence implicating HCN1 mutations in human epilepsy is lacking. We carried out exome sequencing for parent-offspring trios with fever-sensitive, intractable epileptic encephalopathy, leading to the discovery of two de novo missense HCN1 mutations. Screening of follow-up cohorts comprising 157 cases in total identified 4 additional amino acid substitutions. Patch-clamp recordings of Ih currents in cells expressing wild-type or mutant human HCN1 channels showed that the mutations had striking but divergent effects on homomeric channels. Individuals with mutations had clinical features resembling those of Dravet syndrome with progression toward atypical absences, intellectual disability and autistic traits. These findings provide clear evidence that de novo HCN1 point mutations cause a recognizable early-onset epileptic encephalopathy in humans.

The influence of recombination on human genetic diversity.

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Chris C A Spencer

2006-09-01

Full Text Available In humans, the rate of recombination, as measured on the megabase scale, is positively associated with the level of genetic variation, as measured at the genic scale. Despite considerable debate, it is not clear whether these factors are causally linked or, if they are, whether this is driven by the repeated action of adaptive evolution or molecular processes such as double-strand break formation and mismatch repair. We introduce three innovations to the analysis of recombination and diversity: fine-scale genetic maps estimated from genotype experiments that identify recombination hotspots at the kilobase scale, analysis of an entire human chromosome, and the use of wavelet techniques to identify correlations acting at different scales. We show that recombination influences genetic diversity only at the level of recombination hotspots. Hotspots are also associated with local increases in GC content and the relative frequency of GC-increasing mutations but have no effect on substitution rates. Broad-scale association between recombination and diversity is explained through covariance of both factors with base composition. To our knowledge, these results are the first evidence of a direct and local influence of recombination hotspots on genetic variation and the fate of individual mutations. However, that hotspots have no influence on substitution rates suggests that they are too ephemeral on an evolutionary time scale to have a strong influence on broader scale patterns of base composition and long-term molecular evolution.

Allelic heterogeneity and genetic modifier loci contribute to clinical variation in males with X-linked retinitis pigmentosa due to RPGR mutations.

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Abigail T Fahim

Full Text Available Mutations in RPGR account for over 70% of X-linked retinitis pigmentosa (XlRP, characterized by retinal degeneration and eventual blindness. The clinical consequences of RPGR mutations are highly varied, even among individuals with the same mutation: males demonstrate a wide range of clinical severity, and female carriers may or may not be affected. This study describes the phenotypic diversity in a cohort of 98 affected males from 56 families with RPGR mutations, and demonstrates the contribution of genetic factors (i.e., allelic heterogeneity and genetic modifiers to this diversity. Patients were categorized as grade 1 (mild, 2 (moderate or 3 (severe according to specific clinical criteria. Patient DNAs were genotyped for coding SNPs in 4 candidate modifier genes with products known to interact with RPGR protein: RPGRIP1, RPGRIP1L, CEP290, and IQCB1. Family-based association testing was performed using PLINK. A wide range of clinical severity was observed both between and within families. Patients with mutations in exons 1-14 were more severely affected than those with ORF15 mutations, and patients with predicted null alleles were more severely affected than those predicted to make RPGR protein. Two SNPs showed association with severe disease: the minor allele (N of I393N in IQCB1 (p = 0.044 and the common allele (R of R744Q in RPGRIP1L (p = 0.049. These data demonstrate that allelic heterogeneity contributes to phenotypic diversity in XlRP and suggest that this may depend on the presence or absence of RPGR protein. In addition, common variants in 2 proteins known to interact with RPGR are associated with severe disease in this cohort.

Restriction digest screening facilitates efficient detection of site-directed mutations introduced by CRISPR in C. albicans UME6.

Science.gov (United States)

Evans, Ben A; Smith, Olivia L; Pickerill, Ethan S; York, Mary K; Buenconsejo, Kristen J P; Chambers, Antonio E; Bernstein, Douglas A

2018-01-01

Introduction of point mutations to a gene of interest is a powerful tool when determining protein function. CRISPR-mediated genome editing allows for more efficient transfer of a desired mutation into a wide range of model organisms. Traditionally, PCR amplification and DNA sequencing is used to determine if isolates contain the intended mutation. However, mutation efficiency is highly variable, potentially making sequencing costly and time consuming. To more efficiently screen for correct transformants, we have identified restriction enzymes sites that encode for two identical amino acids or one or two stop codons. We used CRISPR to introduce these restriction sites directly upstream of the Candida albicans UME6 Zn 2+ -binding domain, a known regulator of C. albicans filamentation. While repair templates coding for different restriction sites were not equally successful at introducing mutations, restriction digest screening enabled us to rapidly identify isolates with the intended mutation in a cost-efficient manner. In addition, mutated isolates have clear defects in filamentation and virulence compared to wild type C. albicans . Our data suggest restriction digestion screening efficiently identifies point mutations introduced by CRISPR and streamlines the process of identifying residues important for a phenotype of interest.

Efficient detection of factor IX mutations by denaturing high-performance liquid chromatography in Taiwanese hemophilia B patients, and the identification of two novel mutations

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Pei-Chin Lin

2014-04-01

Full Text Available Hemophilia B (HB is an X-linked recessive disorder characterized by mutations in the clotting factor IX (FIX gene that result in FIX deficiency. Previous studies have shown a wide variation of FIX gene mutations in HB. Although the quality of life in HB has greatly improved mainly because of prophylactic replacement therapy with FIX concentrates, there exists a significant burden on affected families and the medical care system. Accurate detection of FIX gene mutations is critical for genetic counseling and disease prevention in HB. In this study, we used denaturing high-performance liquid chromatography (DHPLC, which has proved to be a highly informative and practical means of detecting mutations, for the molecular diagnosis of our patients with HB. Ten Taiwanese families affected by HB were enrolled. We used the DHPLC technique followed by direct sequencing of suspected segments to detect FIX gene mutations. In all, 11 FIX gene mutations (8 point mutations, 2 small deletions/insertions, and 1 large deletion, including two novel mutations (exon6 c.687–695, del 9 mer and c.460–461, ins T were found. According to the HB pedigrees, 25% and 75% of our patients were defined as familial and sporadic HB cases, respectively. We show that DHPLC is a highly sensitive and cost-effective method for FIX gene analysis and can be used as a convenient system for disease prevention.

A novel point mutation (G[sup [minus]1] to T) in a 5[prime] splice donor site of intron 13 of the dystrophin gene results in exon skipping and is responsible for Becker Muscular Dystrophy

Energy Technology Data Exchange (ETDEWEB)

Hagiwara, Yoko; Nishio, Hisahide; Kitoh, Yoshihiko; Takeshima, Yasuhiro; Narita, Naoko; Wada, Hiroko; Yokoyama, Mitsuhiro; Nakamura, Hajime; Matsuo, Masafumi (Kobe Univ. School of Medicine (Japan))

1994-01-01

The mutations in one-third of Duchenne and Becker muscular dystrophy patients remain unknown, as they do not involve gross rearrangements of the dystrophin gene. The authors now report a defect in the splicing of precursor mRNA (pre-mRNA), resulting from a maternally inherited mutation of the dystrophin gene in a patient with Becker muscular dystrophy. This defect results from a G-to-T transversion at the terminal nucleotide of exon 13, within the 5[prime] splice site of intron 13, and causes complete skipping of exon 13 during processing of dystrophin pre-mRNA. The predicted polypeptide encoded by the aberrant mRNA is a truncated dystrophin lacking 40 amino acids from the amino-proximal end of the rod domain. This is the first report of an intraexon point mutation that completely inactivates a 5[prime] splice donor site in dystrophin pre-mRNA. Analysis of the genomic context of the G[sup [minus]1]-to-T mutation at the 5[prime] splice site supports the exon-definition model of pre-mRNA splicing and contributes to the understanding of splice-site selection. 48 refs., 5 figs.

Phenotypic spectrum associated with mutations of the mitochondrial polymerase gamma gene.

Science.gov (United States)

Horvath, Rita; Hudson, Gavin; Ferrari, Gianfrancesco; Fütterer, Nancy; Ahola, Sofia; Lamantea, Eleonora; Prokisch, Holger; Lochmüller, Hanns; McFarland, Robert; Ramesh, V; Klopstock, Thomas; Freisinger, Peter; Salvi, Fabrizio; Mayr, Johannes A; Santer, Rene; Tesarova, Marketa; Zeman, Jiri; Udd, Bjarne; Taylor, Robert W; Turnbull, Douglass; Hanna, Michael; Fialho, Doreen; Suomalainen, Anu; Zeviani, Massimo; Chinnery, Patrick F

2006-07-01

Mutations in the gene coding for the catalytic subunit of the mitochondrial DNA (mtDNA) polymerase gamma (POLG1) have recently been described in patients with diverse clinical presentations, revealing a complex relationship between genotype and phenotype in patients and their families. POLG1 was sequenced in patients from different European diagnostic and research centres to define the phenotypic spectrum and advance understanding of the recurrence risks. Mutations were identified in 38 cases, with the majority being sporadic compound heterozygotes. Eighty-nine DNA sequence changes were identified, including 2 predicted to alter a splice site, 1 predicted to cause a premature stop codon and 13 predicted to cause novel amino acid substitutions. The majority of children had a mutation in the linker region, often 1399G-->A (A467T), and a mutation affecting the polymerase domain. Others had mutations throughout the gene, and 11 had 3 or more substitutions. The clinical presentation ranged from the neonatal period to late adult life, with an overlapping phenotypic spectrum from severe encephalopathy and liver failure to late-onset external ophthalmoplegia, ataxia, myopathy and isolated muscle pain or epilepsy. There was a strong gender bias in children, with evidence of an environmental interaction with sodium valproate. POLG1 mutations cause an overlapping clinical spectrum of disease with both dominant and recessive modes of inheritance. 1399G-->A (A467T) is common in children, but complete POLG1 sequencing is required to identify multiple mutations that can have complex implications for genetic counselling.

Exact Identification of a Quantum Change Point

Science.gov (United States)

Sentís, Gael; Calsamiglia, John; Muñoz-Tapia, Ramon

2017-10-01

The detection of change points is a pivotal task in statistical analysis. In the quantum realm, it is a new primitive where one aims at identifying the point where a source that supposedly prepares a sequence of particles in identical quantum states starts preparing a mutated one. We obtain the optimal procedure to identify the change point with certainty—naturally at the price of having a certain probability of getting an inconclusive answer. We obtain the analytical form of the optimal probability of successful identification for any length of the particle sequence. We show that the conditional success probabilities of identifying each possible change point show an unexpected oscillatory behavior. We also discuss local (online) protocols and compare them with the optimal procedure.

Molecular characterization and diversity of a novel non-autonomous mutator-like transposon family in brassica

International Nuclear Information System (INIS)

Nouroz, F.

2015-01-01

Transposable elements (TEs) are capable of mobilizing from one genomic location to other, with changes in their copy numbers. Mutator-like elements (MULEs) are DNA transposons characterized by 9 bp target site duplications (TSDs), with high variability in sequence and length, and include non-conserved terminal inverted repeats (TIRs). We identified and characterized a family of Mutator-like elements designated as Shahroz. The structural and molecular analyses revealed that family had a small number of mostly defective non-autonomous MULEs and has shown limited activity in the evolutionary history of the Brassica A-genome. The Shahroz elements range in size from 2734 to 3160 bp including 76 bp imperfect TIRs and 9 bp variable TSDs. The individual copies have shown high homology (52-99%) in their entire lengths. The study revealed that the elements are less in numbers but active in Brassica rapa genomes and PCR amplification revealed their specificity and amplification in A-genome containing diploid and polyploids Brassica. The phylogenetic analysis of Brassica MULEs with other plant Mutator elements revealed that no correlation exists between Brassica MULEs and other elements suggesting a separate line of evolution. Analyzing the regions flanking the insertions revealed that the insertions have showed a preference for AT rich regions. The detailed study of these insertions revealed that although less in number and small sizes, they have played a role in Brassica genome evolution by their mobilization. (author)

Impact of mutations in Toll-like receptor pathway genes on esophageal carcinogenesis.

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Daffolyn Rachael Fels Elliott

2017-05-01

Full Text Available Esophageal adenocarcinoma (EAC develops in an inflammatory microenvironment with reduced microbial diversity, but mechanisms for these influences remain poorly characterized. We hypothesized that mutations targeting the Toll-like receptor (TLR pathway could disrupt innate immune signaling and promote a microenvironment that favors tumorigenesis. Through interrogating whole genome sequencing data from 171 EAC patients, we showed that non-synonymous mutations collectively affect the TLR pathway in 25/171 (14.6%, PathScan p = 8.7x10-5 tumors. TLR mutant cases were associated with more proximal tumors and metastatic disease, indicating possible clinical significance of these mutations. Only rare mutations were identified in adjacent Barrett's esophagus samples. We validated our findings in an external EAC dataset with non-synonymous TLR pathway mutations in 33/149 (22.1%, PathScan p = 0.05 tumors, and in other solid tumor types exposed to microbiomes in the COSMIC database (10,318 samples, including uterine endometrioid carcinoma (188/320, 58.8%, cutaneous melanoma (377/988, 38.2%, colorectal adenocarcinoma (402/1519, 26.5%, and stomach adenocarcinoma (151/579, 26.1%. TLR4 was the most frequently mutated gene with eleven mutations in 10/171 (5.8% of EAC tumors. The TLR4 mutants E439G, S570I, F703C and R787H were confirmed to have impaired reactivity to bacterial lipopolysaccharide with marked reductions in signaling by luciferase reporter assays. Overall, our findings show that TLR pathway genes are recurrently mutated in EAC, and TLR4 mutations have decreased responsiveness to bacterial lipopolysaccharide and may play a role in disease pathogenesis in a subset of patients.

Preaxial polydactyly/triphalangeal thumb is associated with changed transcription factor-binding affinity in a family with a novel point mutation in the long-range cis-regulatory element ZRS

DEFF Research Database (Denmark)

Farooq, Muhammad; Troelsen, Jesper T; Boyd, Mette

2010-01-01

A cis-regulatory sequence also known as zone of polarizing activity (ZPA) regulatory sequence (ZRS) located in intron 5 of LMBR1 is essential for expression of sonic hedgehog (SHH) in the developing posterior limb bud mesenchyme. Even though many point mutations causing preaxial duplication defects...... demonstrated a marked difference between wild-type and the mutant probe, which uniquely bound one or several transcription factors extracted from Caco-2 cells. This finding supports a model in which ectopic anterior SHH expression in the developing limb results from abnormal binding of one or more...

Diverse functional consequences of mutations in the Na+/K+-ATPase alpha2-subunit causing familial hemiplegic migraine type 2.

NARCIS (Netherlands)

Tavraz, N.N.; Friedrich, T.; Durr, K.L.; Koenderink, J.B.; Bamberg, E.; Freilinger, T.; Dichgans, M.

2008-01-01

Mutations in ATP1A2, the gene coding for the Na(+)/K(+)-ATPase alpha(2)-subunit, are associated with both familial hemiplegic migraine and sporadic cases of hemiplegic migraine. In this study, we examined the functional properties of 11 ATP1A2 mutations associated with familial or sporadic

Human immunodeficiency virus type-1 (HIV-1) genetic diversity and ...

African Journals Online (AJOL)

The presence of human immunodeficiency virus (HIV) type-1 diversity has an impact on vaccine efficacy and drug resistance. It is important to know the circulating genetic variants and associated drug-resistance mutations in the context of scale up of antiretroviral therapy (ART) in Nigeria. The objective of this study was to ...

Insights into the binding specificity of wild type and mutated wheat germ agglutinin towards Neu5Acα(2-3)Gal: a study by in silico mutations and molecular dynamics simulations.

Science.gov (United States)

Parasuraman, Ponnusamy; Murugan, Veeramani; Selvin, Jeyasigamani F A; Gromiha, M Michael; Fukui, Kazuhiko; Veluraja, Kasinadar

2014-08-01

Wheat germ agglutinin (WGA) is a plant lectin, which specifically recognizes the sugars NeuNAc and GlcNAc. Mutated WGA with enhanced binding specificity can be used as biomarkers for cancer. In silico mutations are performed at the active site of WGA to enhance the binding specificity towards sialylglycans, and molecular dynamics simulations of 20 ns are carried out for wild type and mutated WGAs (WGA1, WGA2, and WGA3) in complex with sialylgalactose to examine the change in binding specificity. MD simulations reveal the change in binding specificity of wild type and mutated WGAs towards sialylgalactose and bound conformational flexibility of sialylgalactose. The mutated polar amino acid residues Asn114 (S114N), Lys118 (G118K), and Arg118 (G118R) make direct and water mediated hydrogen bonds and hydrophobic interactions with sialylgalactose. An analysis of possible hydrogen bonds, hydrophobic interactions, total pair wise interaction energy between active site residues and sialylgalactose and MM-PBSA free energy calculation reveals the plausible binding modes and the role of water in stabilizing different binding modes. An interesting observation is that the binding specificity of mutated WGAs (cyborg lectin) towards sialylgalactose is found to be higher in double point mutation (WGA3). One of the substituted residues Arg118 plays a crucial role in sugar binding. Based on the interactions and energy calculations, it is concluded that the order of binding specificity of WGAs towards sialylgalactose is WGA3 > WGA1 > WGA2 > WGA. On comparing with the wild type, double point mutated WGA (WGA3) exhibits increased specificity towards sialylgalactose, and thus, it can be effectively used in targeted drug delivery and as biological cell marker in cancer therapeutics. Copyright © 2014 John Wiley & Sons, Ltd.

A missense mutation in the agouti signaling protein gene (ASIP) is associated with the no light points coat phenotype in donkeys.

Science.gov (United States)

Abitbol, Marie; Legrand, Romain; Tiret, Laurent

2015-04-08

Seven donkey breeds are recognized by the French studbook and are characterized by a black, bay or grey coat colour including light cream-to-white points (LP). Occasionally, Normand bay donkeys give birth to dark foals that lack LP and display the no light points (NLP) pattern. This pattern is more frequent and officially recognized in American miniature donkeys. The LP (or pangare) phenotype resembles that of the light bellied agouti pattern in mouse, while the NLP pattern resembles that of the mammalian recessive black phenotype; both phenotypes are associated with the agouti signaling protein gene (ASIP). We used a panel of 127 donkeys to identify a recessive missense c.349 T > C variant in ASIP that was shown to be in complete association with the NLP phenotype. This variant results in a cysteine to arginine substitution at position 117 in the ASIP protein. This cysteine is highly-conserved among vertebrate ASIP proteins and was previously shown by mutagenesis experiments to lie within a functional site. Altogether, our results strongly support that the identified mutation is causative of the NLP phenotype. Thus, we propose to name the c.[349 T > C] allele in donkeys, the a(nlp) allele, which enlarges the panel of coat colour alleles in donkeys and ASIP recessive loss-of-function alleles in animals.

Functional Assessment of Residues in the Amino- and Carboxyl-Termini of Crustacean Hyperglycemic Hormone (CHH in the Mud Crab Scylla olivacea Using Point-Mutated Peptides.

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Chun-Jing Liu

Full Text Available To assess functional importance of the residues in the amino- and carboxyl-termini of crustacean hyperglycemic hormone in the mud crab Scylla olivacea (Sco-CHH, both wild-type and point-mutated CHH peptides were produced with an amidated C-terminal end. Spectral analyses of circular dichroism, chromatographic retention time, and mass spectrometric analysis of the recombinant peptides indicate that they were close in conformation to native CHH and were produced with the intended substitutions. The recombinant peptides were subsequently used for an in vivo hyperglycemic assay. Two mutants (R13A and I69A rSco-CHH completely lacked hyperglycemic activity, with temporal profiles similar to that of vehicle control. Temporal profiles of hyperglycemic responses elicited by 4 mutants (I2A, F3A, D12A, and D60A Sco-CHH were different from that elicited by wild-type Sco-CHH; I2A was unique in that it exhibited significantly higher hyperglycemic activity, whereas the remaining 3 mutants showed lower activity. Four mutants (D4A, Q51A, E54A, and V72A rSco-CHH elicited hyperglycemic responses with temporal profiles similar to those evoked by wild-type Sco-CHH. In contrast, the glycine-extended version of V72A rSco-CHH (V72A rSco-CHH-Gly completely lost hyperglycemic activity. By comparing our study with previous ones of ion-transport peptide (ITP and molt-inhibiting hormone (MIH using deleted or point-mutated mutants, detail discussion is made regarding functionally important residues that are shared by both CHH and ITP (members of Group I of the CHH family, and those that discriminate CHH from ITP, and Group-I from Group-II peptides. Conclusions summarized in the present study provide insights into understanding of how functional diversification occurred within a peptide family of multifunctional members.

Targeted next-generation sequencing reveals novel USH2A mutations associated with diverse disease phenotypes: implications for clinical and molecular diagnosis.

Science.gov (United States)

Chen, Xue; Sheng, Xunlun; Liu, Xiaoxing; Li, Huiping; Liu, Yani; Rong, Weining; Ha, Shaoping; Liu, Wenzhou; Kang, Xiaoli; Zhao, Kanxing; Zhao, Chen

2014-01-01

USH2A mutations have been implicated in the disease etiology of several inherited diseases, including Usher syndrome type 2 (USH2), nonsyndromic retinitis pigmentosa (RP), and nonsyndromic deafness. The complex genetic and phenotypic spectrums relevant to USH2A defects make it difficult to manage patients with such mutations. In the present study, we aim to determine the genetic etiology and to characterize the correlated clinical phenotypes for three Chinese pedigrees with nonsyndromic RP, one with RP sine pigmento (RPSP), and one with USH2. Family histories and clinical details for all included patients were reviewed. Ophthalmic examinations included best corrected visual acuities, visual field measurements, funduscopy, and electroretinography. Targeted next-generation sequencing (NGS) was applied using two sequence capture arrays to reveal the disease causative mutations for each family. Genotype-phenotype correlations were also annotated. Seven USH2A mutations, including four missense substitutions (p.P2762A, p.G3320C, p.R3719H, and p.G4763R), two splice site variants (c.8223+1G>A and c.8559-2T>C), and a nonsense mutation (p.Y3745*), were identified as disease causative in the five investigated families, of which three reported to have consanguineous marriage. Among all seven mutations, six were novel, and one was recurrent. Two homozygous missense mutations (p.P2762A and p.G3320C) were found in one individual family suggesting a potential double hit effect. Significant phenotypic divergences were revealed among the five families. Three families of the five families were affected with early, moderated, or late onset RP, one with RPSP, and the other one with USH2. Our study expands the genotypic and phenotypic variability relevant to USH2A mutations, which would help with a clear insight into the complex genetic and phenotypic spectrums relevant to USH2A defects, and is complementary for a better management of patients with such mutations. We have also

Identification of mutated driver pathways in cancer using a multi-objective optimization model.

Science.gov (United States)

Zheng, Chun-Hou; Yang, Wu; Chong, Yan-Wen; Xia, Jun-Feng

2016-05-01

New-generation high-throughput technologies, including next-generation sequencing technology, have been extensively applied to solve biological problems. As a result, large cancer genomics projects such as the Cancer Genome Atlas (TCGA) and the International Cancer Genome Consortium are producing large amount of rich and diverse data in multiple cancer types. The identification of mutated driver genes and driver pathways from these data is a significant challenge. Genome aberrations in cancer cells can be divided into two types: random 'passenger mutation' and functional 'driver mutation'. In this paper, we introduced a Multi-objective Optimization model based on a Genetic Algorithm (MOGA) to solve the maximum weight submatrix problem, which can be employed to identify driver genes and driver pathways promoting cancer proliferation. The maximum weight submatrix problem defined to find mutated driver pathways is based on two specific properties, i.e., high coverage and high exclusivity. The multi-objective optimization model can adjust the trade-off between high coverage and high exclusivity. We proposed an integrative model by combining gene expression data and mutation data to improve the performance of the MOGA algorithm in a biological context. Copyright © 2016 Elsevier Ltd. All rights reserved.

A mutation of the fission yeast EB1 overcomes negative regulation by phosphorylation and stabilizes microtubules

Energy Technology Data Exchange (ETDEWEB)

Iimori, Makoto; Ozaki, Kanako [Graduate School of Biostudies, Kyoto University, Kitashirakawa-Oiwake cho, Sakyo ku, Kyoto, 606-8502 (Japan); Chikashige, Yuji [Kobe Advanced ICT Research Center, National Institute of Information and Communications Technology, Kobe, 651-2492 (Japan); Habu, Toshiyuki [Graduate School of Biostudies, Kyoto University, Kitashirakawa-Oiwake cho, Sakyo ku, Kyoto, 606-8502 (Japan); Radiation Biology Center, Kyoto University, Yoshida-Konoe cho, Sakyo ku, Kyoto, 606-8501 (Japan); Hiraoka, Yasushi [Kobe Advanced ICT Research Center, National Institute of Information and Communications Technology, Kobe, 651-2492 (Japan); Graduate School of Frontier Biosciences, Osaka University, 1-3 Yamadaoka, Suita, 565-0871 (Japan); Maki, Takahisa; Hayashi, Ikuko [Graduate School of Nanobioscience, Yokohama City University, Tsurumi, Yokohama, 230-0045 (Japan); Obuse, Chikashi [Graduate School of Life Science, Hokkaido University, Sapporo 001-0021 (Japan); Matsumoto, Tomohiro, E-mail: tmatsumo@house.rbc.kyoto-u.ac.jp [Graduate School of Biostudies, Kyoto University, Kitashirakawa-Oiwake cho, Sakyo ku, Kyoto, 606-8502 (Japan); Radiation Biology Center, Kyoto University, Yoshida-Konoe cho, Sakyo ku, Kyoto, 606-8501 (Japan)

2012-02-01

Mal3 is a fission yeast homolog of EB1, a plus-end tracking protein (+ TIP). We have generated a mutation (89R) replacing glutamine with arginine in the calponin homology (CH) domain of Mal3. Analysis of the 89R mutant in vitro has revealed that the mutation confers a higher affinity to microtubules and enhances the intrinsic activity to promote the microtubule-assembly. The mutant Mal3 is no longer a + TIP, but binds strongly the microtubule lattice. Live cell imaging has revealed that while the wild type Mal3 proteins dissociate from the tip of the growing microtubules before the onset of shrinkage, the mutant Mal3 proteins persist on microtubules and reduces a rate of shrinkage after a longer pausing period. Consequently, the mutant Mal3 proteins cause abnormal elongation of microtubules composing the spindle and aster. Mal3 is phosphorylated at a cluster of serine/threonine residues in the linker connecting the CH and EB1-like C-terminal motif domains. The phosphorylation occurs in a microtubule-dependent manner and reduces the affinity of Mal3 to microtubules. We propose that because the 89R mutation is resistant to the effect of phosphorylation, it can associate persistently with microtubules and confers a stronger stability of microtubules likely by reinforcing the cylindrical structure. -- Highlights: Black-Right-Pointing-Pointer We characterize a mutation (mal3-89R) in fission yeast homolog of EB1. Black-Right-Pointing-Pointer The mutation enhances the activity to assemble microtubules. Black-Right-Pointing-Pointer Mal3 is phosphorylated in a microtubule-dependent manner. Black-Right-Pointing-Pointer The phosphorylation negatively regulates the Mal3 activity.

Augmented Nonlinear Controller for Maximum Power-Point Tracking with Artificial Neural Network in Grid-Connected Photovoltaic Systems

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Suliang Ma

2016-11-01

Full Text Available Photovoltaic (PV systems have non-linear characteristics that generate maximum power at one particular operating point. Environmental factors such as irradiance and temperature variations greatly affect the maximum power point (MPP. Diverse offline and online techniques have been introduced for tracking the MPP. Here, to track the MPP, an augmented-state feedback linearized (AFL non-linear controller combined with an artificial neural network (ANN is proposed. This approach linearizes the non-linear characteristics in PV systems and DC/DC converters, for tracking and optimizing the PV system operation. It also reduces the dependency of the designed controller on linearized models, to provide global stability. A complete model of the PV system is simulated. The existing maximum power-point tracking (MPPT and DC/DC boost-converter controller techniques are compared with the proposed ANN method. Two case studies, which simulate realistic circumstances, are presented to demonstrate the effectiveness and superiority of the proposed method. The AFL with ANN controller can provide good dynamic operation, faster convergence speed, and fewer operating-point oscillations around the MPP. It also tracks the global maxima under different conditions, especially irradiance-mutating situations, more effectively than the conventional methods. Detailed mathematical models and a control approach for a three-phase grid-connected intelligent hybrid system are proposed using MATLAB/Simulink.

Excess of mutational jackpot events in expanding populations revealed by spatial Luria–Delbrück experiments

Science.gov (United States)

Fusco, Diana; Gralka, Matti; Kayser, Jona; Anderson, Alex; Hallatschek, Oskar

2016-01-01

The genetic diversity of growing cellular populations, such as biofilms, solid tumours or developing embryos, is thought to be dominated by rare, exceptionally large mutant clones. Yet, the emergence of these mutational jackpot events is only understood in well-mixed populations, where they stem from mutations that arise during the first few cell divisions. To study jackpot events in spatially structured populations, we track mutant clones in microbial populations using fluorescence microscopy and population sequencing. High-frequency mutations are found to be massively enriched in microbial colonies compared with well-shaken liquid cultures, as a result of late-occurring mutations surfing at the edge of range expansions. Thus, jackpot events can be generated not only when mutations arise early but also when they occur at favourable locations, which exacerbates their role in adaptation and disease. In particular, because spatial competition with the wild type keeps most mutant clones in a quiescent state, strong selection pressures that kill the wild type promote drug resistance. PMID:27694797

Excess of mutational jackpot events in expanding populations revealed by spatial Luria-Delbrück experiments.

Science.gov (United States)

Fusco, Diana; Gralka, Matti; Kayser, Jona; Anderson, Alex; Hallatschek, Oskar

2016-10-03

The genetic diversity of growing cellular populations, such as biofilms, solid tumours or developing embryos, is thought to be dominated by rare, exceptionally large mutant clones. Yet, the emergence of these mutational jackpot events is only understood in well-mixed populations, where they stem from mutations that arise during the first few cell divisions. To study jackpot events in spatially structured populations, we track mutant clones in microbial populations using fluorescence microscopy and population sequencing. High-frequency mutations are found to be massively enriched in microbial colonies compared with well-shaken liquid cultures, as a result of late-occurring mutations surfing at the edge of range expansions. Thus, jackpot events can be generated not only when mutations arise early but also when they occur at favourable locations, which exacerbates their role in adaptation and disease. In particular, because spatial competition with the wild type keeps most mutant clones in a quiescent state, strong selection pressures that kill the wild type promote drug resistance.

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.

Iron Overload Leading to Torsades de Pointes in β-Thalassemia and Long QT Syndrome

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Refaat, Marwan M; El Hage, Lea; Steffensen, Annette Buur

2016-01-01

The authors present a unique case of torsades de pointes in a β-thalassemia patient with early iron overload in the absence of any structural abnormalities as seen in hemochromatosis. Genetic testing showed a novel KCNQ1 gene mutation 1591C>T [Gln531Ter(X)]. Testing of the gene mutation in Xenopus...

Mutation of mtDNA ND1 Gene in 20 Type 2 Diabetes Mellitus Patients of Gorontalonese and Javanese Ethnicity

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AMIEN RAMADHAN ISHAK

2014-12-01

Full Text Available Mitochondrial gene mutation plays a role in the development of type two diabetes mellitus (T2DM. A point mutation in the mitochondrial gene Nicotinamide adenine dinucleotide dehydrogenase 1 (mtDNA ND1 gene mainly reported as the most common mutation related to T2DM. However, several studies have identified another SNP (single-nucleotide polymorphisms in the RNA region of mtDNA from patients from specific ethnic populations in Indonesia. Building on those findings, this study aimed to use PCR and DNA sequencing technology to identify nucleotides in RNA and ND1 fragment from 20 Gorontalonese and 20 Javanese T2DM patients, that may trigger T2DM expression. The results showed successful amplification of RNA along 294 bp for all samples. From these samples, we found two types of point mutation in Javanese patients in the G3316A and T3200C points of the rRNA and ND1 gene. In samples taken from Gorontalonese patients, no mutation were found in the RNA or ND1 region. We conclude that T2DM was triggered differently in our two populations. While genetic mutation is implicated for the 20 Javanese patients, T2DM pathogenesis in the Gorontalonese patients must be traced to other genetic, environmental, or behavioral factors.

Mutation in LIM2 Is Responsible for Autosomal Recessive Congenital Cataracts.

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Bushra Irum

Full Text Available To identify the molecular basis of non-syndromic autosomal recessive congenital cataracts (arCC in a consanguineous family.All family members participating in the study received a comprehensive ophthalmic examination to determine their ocular phenotype and contributed a blood sample, from which genomic DNA was extracted. Available medical records and interviews with the family were used to compile the medical history of the family. The symptomatic history of the individuals exhibiting cataracts was confirmed by slit-lamp biomicroscopy. A genome-wide linkage analysis was performed to localize the disease interval. The candidate gene, LIM2 (lens intrinsic membrane protein 2, was sequenced bi-directionally to identify the disease-causing mutation. The physical changes caused by the mutation were analyzed in silico through homology modeling, mutation and bioinformatic algorithms, and evolutionary conservation databases. The physiological importance of LIM2 to ocular development was assessed in vivo by real-time expression analysis of Lim2 in a mouse model.Ophthalmic examination confirmed the diagnosis of nuclear cataracts in the affected members of the family; the inheritance pattern and cataract development in early infancy indicated arCC. Genome-wide linkage analysis localized the critical interval to chromosome 19q with a two-point logarithm of odds (LOD score of 3.25. Bidirectional sequencing identified a novel missense mutation, c.233G>A (p.G78D in LIM2. This mutation segregated with the disease phenotype and was absent in 192 ethnically matched control chromosomes. In silico analysis predicted lower hydropathicity and hydrophobicity but higher polarity of the mutant LIM2-encoded protein (MP19 compared to the wild-type. Moreover, these analyses predicted that the mutation would disrupt the secondary structure of a transmembrane domain of MP19. The expression of Lim2, which was detected in the mouse lens as early as embryonic day 15 (E15

rpoB gene mutations among Mycobacterium tuberculosis isolates from extrapulmonary sites.

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Khosravi, Azar Dokht; Meghdadi, Hossein; Ghadiri, Ata A; Alami, Ameneh; Sina, Amir Hossein; Mirsaeidi, Mehdi

2018-03-01

The aim of this study was to analyze mutations occurring in the rpoB gene of Mycobacterium tuberculosis (MTB) isolates from clinical samples of extrapulmonary tuberculosis (EPTB). Seventy formalin-fixed, paraffin-embedded samples and fresh tissue samples from confirmed EPTB cases were analyzed. Nested PCR based on the rpoB gene was performed on the extracted DNAs, combined with cloning and subsequent sequencing. Sixty-seven (95.7%) samples were positive for nester PCR. Sequence analysis of the 81 bp region of the rpoB gene demonstrated mutations in 41 (61.2%) of 67 sequenced samples. Several point mutations including deletion mutations at codons 510, 512, 513 and 515, with 45% and 51% of the mutations in codons 512 and 513 respectively were seen, along with 26% replacement mutations at codons 509, 513, 514, 518, 520, 524 and 531. The most common alteration was Gln → His, at codon 513, presented in 30 (75.6%) isolates. This study demonstrated sequence alterations in codon 513 of the 81 bp region of the rpoB gene as the most common mutation occurred in 75.6% of molecularly confirmed rifampin-resistant strains. In addition, simultaneous mutation at codons 512 and 513 was demonstrated in 34.3% of the isolates. © 2018 APMIS. Published by John Wiley & Sons Ltd.

Rare mutations predisposing to familial adenomatous polyposis in Greek FAP patients

International Nuclear Information System (INIS)

Mihalatos, Markos; Fountzilas, George; Agnantis, Niki J; Nasioulas, Georgios; Apessos, Angela; Dauwerse, Hans; Velissariou, Voula; Psychias, Aristidis; Koliopanos, Alexander; Petropoulos, Konstantinos; Triantafillidis, John K; Danielidis, Ioannis

2005-01-01

Familial Adenomatous Polyposis (FAP) is caused by germline mutations in the APC (Adenomatous Polyposis Coli) gene. The vast majority of APC mutations are point mutations or small insertions / deletions which lead to truncated protein products. Splicing mutations or gross genomic rearrangements are less common inactivating events of the APC gene. In the current study genomic DNA or RNA from ten unrelated FAP suspected patients was examined for germline mutations in the APC gene. Family history and phenotype were used in order to select the patients. Methods used for testing were dHPLC (denaturing High Performance Liquid Chromatography), sequencing, MLPA (Multiplex Ligation – dependent Probe Amplification), Karyotyping, FISH (Fluorescence In Situ Hybridization) and RT-PCR (Reverse Transcription – Polymerase Chain Reaction). A 250 Kbp deletion in the APC gene starting from intron 5 and extending beyond exon 15 was identified in one patient. A substitution of the +5 conserved nucleotide at the splice donor site of intron 9 in the APC gene was shown to produce frameshift and inefficient exon skipping in a second patient. Four frameshift mutations (1577insT, 1973delAG, 3180delAAAA, 3212delA) and a nonsense mutation (C1690T) were identified in the rest of the patients. Screening for APC mutations in FAP patients should include testing for splicing defects and gross genomic alterations

ENU-induced phenovariance in mice: inferences from 587 mutations

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Arnold Carrie N

2012-10-01

Full Text Available Abstract Background We present a compendium of N-ethyl-N-nitrosourea (ENU-induced mouse mutations, identified in our laboratory over a period of 10 years either on the basis of phenotype or whole genome and/or whole exome sequencing, and archived in the Mutagenetix database. Our purpose is threefold: 1 to formally describe many point mutations, including those that were not previously disclosed in peer-reviewed publications; 2 to assess the characteristics of these mutations; and 3 to estimate the likelihood that a missense mutation induced by ENU will create a detectable phenotype. Findings In the context of an ENU mutagenesis program for C57BL/6J mice, a total of 185 phenotypes were tracked to mutations in 129 genes. In addition, 402 incidental mutations were identified and predicted to affect 390 genes. As previously reported, ENU shows strand asymmetry in its induction of mutations, particularly favoring T to A rather than A to T in the sense strand of coding regions and splice junctions. Some amino acid substitutions are far more likely to be damaging than others, and some are far more likely to be observed. Indeed, from among a total of 494 non-synonymous coding mutations, ENU was observed to create only 114 of the 182 possible amino acid substitutions that single base changes can achieve. Based on differences in overt null allele frequencies observed in phenotypic vs. non-phenotypic mutation sets, we infer that ENU-induced missense mutations create detectable phenotype only about 1 in 4.7 times. While the remaining mutations may not be functionally neutral, they are, on average, beneath the limits of detection of the phenotypic assays we applied. Conclusions Collectively, these mutations add to our understanding of the chemical specificity of ENU, the types of amino acid substitutions it creates, and its efficiency in causing phenovariance. Our data support the validity of computational algorithms for the prediction of damage caused by

Simulation Study for Transfer of Antibiotic Resistance via Mutator Subpopulation

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Philipsen, Kirsten Riber; Christiansen, Lasse Engbo; Aarestrup, Frank Møller

Evolution of antibiotic resistance in bacterial populations is an increasing problem having fatal consequences for treatment of diseases. Therefore it is very important to understand this evolution. Traditionally evolution is considered to happen by single point mutations, where each mutant must...

Design of thermostable rhamnogalacturonan lyase mutants from Bacillus licheniformis by combination of targeted single point mutations

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da Silva, Ines Isabel Cardoso Rodrigues; Jers, Carsten; Otten, Harm

2014-01-01

Rhamnogalacturonan I lyases (RGI lyases) (EC 4.2.2.-) catalyze cleavage of α-1,4 bonds between rhamnose and galacturonic acid in the backbone of pectins by β-elimination. In the present study, targeted improvement of the thermostability of a PL family 11 RGI lyase from Bacillus licheniformis (DSM......, were obtained due to additive stabilizing effects of single amino acid mutations (E434L, G55V, and G326E) compared to the wild type. The crystal structure of the B. licheniformis wild-type RGI lyase was also determined; the structural analysis corroborated that especially mutation of charged amino...

The Trojan Female Technique for pest control: a candidate mitochondrial mutation confers low male fertility across diverse nuclear backgrounds in Drosophila melanogaster.

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Dowling, Damian K; Tompkins, Daniel M; Gemmell, Neil J

2015-10-01

Pest species represent a major ongoing threat to global biodiversity. Effective management approaches are required that regulate pest numbers, while minimizing collateral damage to nontarget species. The Trojan Female Technique (TFT) was recently proposed as a prospective approach to biological pest control. The TFT draws on the evolutionary hypothesis that maternally inherited mitochondrial genomes are prone to the accumulation of male, but not female, harming mutations. These mutations could be harnessed to provide trans-generational fertility-based control of pest species. A candidate TFT mutation was recently described in the fruit fly, Drosophila melanogaster, which confers male-only sterility in the specific isogenic nuclear background in which it is maintained. However, applicability of the TFT relies on mitochondrial mutations whose male-sterilizing effects are general across nuclear genomic contexts. We test this assumption, expressing the candidate TFT-mutation bearing haplotype alongside a range of nuclear backgrounds and comparing its fertility in males, relative to that of control haplotypes. We document consistently lower fertility for males harbouring the TFT mutation, in both competitive and noncompetitive mating contexts, across all nuclear backgrounds screened. This indicates that TFT mutations conferring reduced male fertility can segregate within populations and could be harnessed to facilitate this novel form of pest control.

Geographic structuring of the Plasmodium falciparum sarco(endoplasmic reticulum Ca2+ ATPase (PfSERCA gene diversity.

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Ronan Jambou

Full Text Available Artemisinin, a thapsigargin-like sesquiterpene has been shown to inhibit the Plasmodium falciparum sarco/endoplasmic reticulum calcium-ATPase PfSERCA. To collect baseline pfserca sequence information before field deployment of Artemisinin-based Combination therapies that may select mutant parasites, we conducted a sequence analysis of 100 isolates from multiple sites in Africa, Asia and South America. Coding sequence diversity was large, with 29 mutated codons, including 32 SNPs (average of one SNP/115 bp, of which 19 were novel mutations. Most SNP detected in this study were clustered within a region in the cytosolic head of the protein. The PfSERCA functional domains were very well conserved, with non synonymous mutations located outside the functional domains, except for the S769N mutation associated in French Guiana with elevated IC(50 for artemether. The S769N mutation is located close to the hinge of the headpiece, which in other species modulates calcium affinity and in consequence efficacy of inhibitors, possibly linking calcium homeostasis to drug resistance. Genetic diversity was highest in Senegal, Brazil and French Guiana, and few mutations were identified in Asia. Population genetic analysis was conducted for a partial fragment of the gene encompassing nucleotide coordinates 87-2862 (unambiguous sequence available for 96 isolates. This supported a geographic clustering, with a separation between Old and New World samples and one dominant ancestral haplotype. Genetic drift alone cannot explain the observed polymorphism, suggesting that other evolutionary mechanisms are operating. One possible contributor could be the frequency of haemoglobinopathies that are associated with calcium dysregulation in the erythrocyte.

A molecular dynamics investigation on the crizotinib resistance mechanism of C1156Y mutation in ALK

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Sun, Hui-Yong [Shandong University of Technology, Zibo 255049 (China); Ji, Feng-Qin, E-mail: fengqinji@mail.hzau.edu.cn [National Key Laboratory of Crop Genetic Improvement, College of Life Science and Technology, Huazhong Agricultural University, Wuhan 430070 (China); Center for Bioinformatics, Huazhong Agricultural University, Wuhan 430070 (China)

2012-06-29

Highlights: Black-Right-Pointing-Pointer The study revealed the detailed resistance mechanism of the non-active mutation C1156Y in ALK. Black-Right-Pointing-Pointer C1156Y leads to crizotinib displacement and conformational changes in the binding cavity. Black-Right-Pointing-Pointer The conformations cause a decline in the vdW and electrostatic energy between crizotinib and ALK. -- Abstract: Crizotinib is an anaplastic lymphoma kinase (ALK) inhibitor that has recently been approved in the US for the treatment of non-small cell lung carcinoma (NSCLC). Despite its outstanding safety and efficacy, several resistant mutations against crizotinib have been detected in the treatment of NSCLC. However, in contrast to the widely accepted mechanism of steric hindrance by mutations at the active site, the mechanism by which the C1156Y non-active site mutation confers resistance against crizotinib remains unclear. In the present study, the resistance mechanism of C1156Y in ALK was investigated using molecular dynamics simulations. The results suggest that despite the non-active site mutation, C1156Y causes the dislocation of crizotinib as well as the indirect conformational changes in the binding cavity, which results in a marked decrease in the van der Waals and electrostatic interactions between crizotinib and ALK. The obtained results provide a detailed explanation of the resistance caused by C1156Y and may give a vital clue for the design of drugs to combat crizotinib resistance.

Clinical and Molecular Characteristics in 100 Chinese Pediatric Patients with m.3243A>G Mutation in Mitochondrial DNA

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Chang-Yu Xia

2016-01-01

Conclusions: Our study showed that half of Chinese pediatric patients with m.3243A>G mutation presented seizures, short stature, abnormal MRI/CT changes, elevated plasma lactate, vomiting, and headache. Pediatric patients with these recurrent symptoms should be considered for screening m.3243A>G mutation. Clinical manifestations and laboratory abnormalities should be carefully monitored in patients with this point mutation.

Mutation Spectrum and Phenotypic Features in Noonan Syndrome with PTPN11 Mutations: Definition of Two Novel Mutations.

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Atik, Tahir; Aykut, Ayca; Hazan, Filiz; Onay, Huseyin; Goksen, Damla; Darcan, Sukran; Tukun, Ajlan; Ozkinay, Ferda

2016-06-01

To evaluate the spectrum of PTPN11 gene mutations in Noonan syndrome patients and to study the genotype-phenotype associations. In this study, twenty Noonan syndrome patients with PTPN11 mutations were included. The patients underwent a detailed clinical and physical evaluation. To identify inherited cases, parents of all mutation positive patients were analyzed. Thirteen different PTPN11 mutations, two of them being novel, were detected in the study group. These mutations included eleven missense mutations: p.G60A, p.D61N, p.Y62D, p.Y63C, p.E69Q, p.Q79R, p.Y279C,p.N308D, p.N308S, p.M504V, p.Q510R and two novel missense mutations: p.I56V and p.I282M. The frequency of cardiac abnormalities and short stature were found to be 80 % and 80 %, respectively. Mental retardation was not observed in patients having exon 8 mutations. No significant correlations were detected between other phenotypic features and genotypes. By identifying genotype-phenotype correlations, this study provides information on phenotypes observed in NS patients with different PTPN11 mutations.

Ectopic expression of AID in a non-B cell line triggers A:T and G:C point mutations in non-replicating episomal vectors.

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Tihana Jovanic

Full Text Available Somatic hypermutation (SHM of immunoglobulin genes is currently viewed as a two step process initiated by the deamination of deoxycytidine (C to deoxyuridine (U, catalysed by the activation induced deaminase (AID. Phase 1 mutations arise from DNA replication across the uracil residue or the abasic site, generated by the uracil-DNA glycosylase, yielding transitions or transversions at G:C pairs. Phase 2 mutations result from the recognition of the U:G mismatch by the Msh2/Msh6 complex (MutS Homologue, followed by the excision of the mismatched nucleotide and the repair, by the low fidelity DNA polymerase eta, of the gap generated by the exonuclease I. These mutations are mainly focused at A:T pairs. Whereas in activated B cells both G:C and A:T pairs are equally targeted, ectopic expression of AID was shown to trigger only G:C mutations on a stably integrated reporter gene. Here we show that when using non-replicative episomal vectors containing a GFP gene, inactivated by the introduction of stop codons at various positions, a high level of EGFP positive cells was obtained after transient expression in Jurkat cells constitutively expressing AID. We show that mutations at G:C and A:T pairs are produced. EGFP positive cells are obtained in the absence of vector replication demonstrating that the mutations are dependent only on the mismatch repair (MMR pathway. This implies that the generation of phase 1 mutations is not a prerequisite for the expression of phase 2 mutations.

IDH1/2 mutations target a key hallmark of cancer by deregulating cellular metabolism in glioma.

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Zhang, Chunzhi; Moore, Lynette M; Li, Xia; Yung, W K Alfred; Zhang, Wei

2013-09-01

Isocitrate dehydrogenase (IDH) enzymes have recently become a focal point for research aimed at understanding the biology of glioma. IDH1 and IDH2 are mutated in 50%-80% of astrocytomas, oligodendrogliomas, oligoastrocytomas, and secondary glioblastomas but are seldom mutated in primary glioblastomas. Gliomas with IDH1/2 mutations always harbor other molecular aberrations, such as TP53 mutation or 1p/19q loss. IDH1 and IDH2 mutations may serve as prognostic factors because patients with an IDH-mutated glioma survive significantly longer than those with an IDH-wild-type tumor. However, the molecular pathogenic role of IDH1/2 mutations in the development of gliomas is unclear. The production of 2-hydroxyglutarate and enhanced NADP+ levels in tumor cells with mutant IDH1/2 suggest mechanisms through which these mutations contribute to tumorigenesis. Elucidating the pathogenesis of IDH mutations will improve understanding of the molecular mechanisms of gliomagenesis and may lead to development of a new molecular classification system and novel therapies.

Subclinical hyperthyroidism due to a thyrotropin receptor (TSHR) gene mutation (S505R).

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Pohlenz, Joachim; Pfarr, Nicole; Krüger, Silvia; Hesse, Volker

2006-12-01

To identify the molecular defect by which non-autoimmune subclinical hyperthyroidism was caused in a 6-mo-old infant who presented with weight loss. Congenital non-autoimmune hyperthyroidism is caused by activating germline mutations in the thyrotropin receptor (TSHR) gene. Therefore, the TSHR gene was sequenced directly from the patient's genomic DNA. Molecular analysis revealed a heterozygous point mutation (S505R) in the TSHR gene as the underlying defect. A constitutively activating mutation in the TSHR gene has to be considered not only in patients with severe congenital non-autoimmune hyperthyroidism, but also in children with subclinical non-autoimmune hyperthyroidism.

Functional analysis of a point mutation in the ryanodine receptor of Plutella xylostella (L.) associated with resistance to chlorantraniliprole.

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Guo, Lei; Wang, Yi; Zhou, Xuguo; Li, Zhenyu; Liu, Shangzhong; Pei, Liang; Gao, Xiwu

2014-07-01

The diamondback moth, Plutella xylostella (L.) has developed extremely high resistance to chlorantraniliprole and other diamide insecticides in the field. A glycine to glutamic acid substitution (G4946E) in the P. xylostella ryanodine receptor (PxRyR) has been found in two resistant populations collected in Thailand and Philippines and was considered associated with the diamide insecticides resistance but no experimental evidence was provided. The present study aimed to clarify the function of the reported mutation in chlorantraniliprole resistance in P. xylostella. We identified the same mutation (G4946E) in PxRyR from four field collected chlorantraniliprole resistant populations of Plutella xylostella in China. Most importantly, we found that the frequency of the G4946E mutation is significantly correlated to the chlorantraniliprole resistance ratios in P. xylostella (R(2)  = 0.82, P = 0.0003). Ligand binding assays showed that the binding affinities of the PxRyR to the chlorantraniliprole in three field resistant populations were 2.41-, 2.54- and 2.60-times lower than that in the susceptible one. For the first time we experimentally proved that the G4946E mutation in PxRyR confers resistance to chlorantraniliprole in Plutella xylostella. These findings pave the way for the complete understanding of the mechanisms of diamide insecticides resistance in insects. © 2013 Society of Chemical Industry.

Nurse Scheduling by Cooperative GA with Effective Mutation Operator

Science.gov (United States)

Ohki, Makoto

In this paper, we propose an effective mutation operators for Cooperative Genetic Algorithm (CGA) to be applied to a practical Nurse Scheduling Problem (NSP). The nurse scheduling is a very difficult task, because NSP is a complex combinatorial optimizing problem for which many requirements must be considered. In real hospitals, the schedule changes frequently. The changes of the shift schedule yields various problems, for example, a fall in the nursing level. We describe a technique of the reoptimization of the nurse schedule in response to a change. The conventional CGA is superior in ability for local search by means of its crossover operator, but often stagnates at the unfavorable situation because it is inferior to ability for global search. When the optimization stagnates for long generation cycle, a searching point, population in this case, would be caught in a wide local minimum area. To escape such local minimum area, small change in a population should be required. Based on such consideration, we propose a mutation operator activated depending on the optimization speed. When the optimization stagnates, in other words, when the optimization speed decreases, the mutation yields small changes in the population. Then the population is able to escape from a local minimum area by means of the mutation. However, this mutation operator requires two well-defined parameters. This means that user have to consider the value of these parameters carefully. To solve this problem, we propose a periodic mutation operator which has only one parameter to define itself. This simplified mutation operator is effective over a wide range of the parameter value.

Combined exposure to X-irradiation followed by N-ethyl-N-nitrosourea treatment alters the frequency and spectrum of Ikaros point mutations in murine T-cell lymphoma

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Kakinuma, Shizuko, E-mail: skakinum@nirs.go.jp [Radiobiology for Children' s Health Research Program, Research Center for Radiation Protection, National Institute of Radiological Sciences, 4-9-1 Anagawa, Inage-ku, Chiba 263-8555 (Japan); Nishimura, Mayumi; Amasaki, Yoshiko; Takada, Mayumi; Yamauchi, Kazumi; Sudo, Satomi; Shang, Yi [Radiobiology for Children' s Health Research Program, Research Center for Radiation Protection, National Institute of Radiological Sciences, 4-9-1 Anagawa, Inage-ku, Chiba 263-8555 (Japan); Doi, Kazutaka; Yoshinaga, Shinji [Regulatory Sciences Research Program, Research Center for Radiation Protection, National Institute of Radiological Sciences, 4-9-1 Anagawa, Inage-ku, Chiba 263-8555 (Japan); Shimada, Yoshiya [Radiobiology for Children' s Health Research Program, Research Center for Radiation Protection, National Institute of Radiological Sciences, 4-9-1 Anagawa, Inage-ku, Chiba 263-8555 (Japan)

2012-09-01

Ionizing radiation is a well-known carcinogen, but its potency may be influenced by other environmental carcinogens, which is of practical importance in the assessment of risk. Data are scarce, however, on the combined effect of radiation with other environmental carcinogens and the underlying mechanisms involved. We studied the mode and mechanism of the carcinogenic effect of radiation in combination with N-ethyl-N-nitrosourea (ENU) using doses approximately equal to the corresponding thresholds. B6C3F1 mice exposed to fractionated X-irradiation (Kaplan's method) followed by ENU developed T-cell lymphomas in a dose-dependent manner. Radiation doses above an apparent threshold acted synergistically with ENU to promote lymphoma development, whereas radiation doses below that threshold antagonized lymphoma development. Ikaros, which regulates the commitment and differentiation of lymphoid lineage cells, is a critical tumor suppressor gene frequently altered in both human and mouse lymphomas and shows distinct mutation spectra between X-ray- and ENU-induced lymphomas. In the synergistically induced lymphomas, we observed a low frequency of LOH and an inordinate increase of Ikaros base substitutions characteristic of ENU-indcued point mutations, G:C to A:T at non-CpG, A:T to G:C, G:C to T:A and A:T to T:A. This suggests that radiation doses above an apparent threshold activate the ENU mutagenic pathway. This is the first report on the carcinogenic mechanism elicited by combined exposure to carcinogens below and above threshold doses based on the mutation spectrum of the causative gene. These findings constitute a basis for assessing human cancer risk following exposure to multiple carcinogens.

The FH mutation database: an online database of fumarate hydratase mutations involved in the MCUL (HLRCC tumor syndrome and congenital fumarase deficiency

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Tomlinson Ian PM

2008-03-01

Full Text Available Abstract Background Fumarate hydratase (HGNC approved gene symbol – FH, also known as fumarase, is an enzyme of the tricarboxylic acid (TCA cycle, involved in fundamental cellular energy production. First described by Zinn et al in 1986, deficiency of FH results in early onset, severe encephalopathy. In 2002, the Multiple Leiomyoma Consortium identified heterozygous germline mutations of FH in patients with multiple cutaneous and uterine leiomyomas, (MCUL: OMIM 150800. In some families renal cell cancer also forms a component of the complex and as such has been described as hereditary leiomyomatosis and renal cell cancer (HLRCC: OMIM 605839. The identification of FH as a tumor suppressor was an unexpected finding and following the identification of subunits of succinate dehydrogenase in 2000 and 2001, was only the second description of the involvement of an enzyme of intermediary metabolism in tumorigenesis. Description The FH mutation database is a part of the TCA cycle gene mutation database (formerly the succinate dehydrogenase gene mutation database and is based on the Leiden Open (source Variation Database (LOVD system. The variants included in the database were derived from the published literature and annotated to conform to current mutation nomenclature. The FH database applies HGVS nomenclature guidelines, and will assist researchers in applying these guidelines when directly submitting new sequence variants online. Since the first molecular characterization of an FH mutation by Bourgeron et al in 1994, a series of reports of both FH deficiency patients and patients with MCUL/HLRRC have described 107 variants, of which 93 are thought to be pathogenic. The most common type of mutation is missense (57%, followed by frameshifts & nonsense (27%, and diverse deletions, insertions and duplications. Here we introduce an online database detailing all reported FH sequence variants. Conclusion The FH mutation database strives to systematically

Targeted next-generation sequencing reveals novel USH2A mutations associated with diverse disease phenotypes: implications for clinical and molecular diagnosis.

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Xue Chen

Full Text Available USH2A mutations have been implicated in the disease etiology of several inherited diseases, including Usher syndrome type 2 (USH2, nonsyndromic retinitis pigmentosa (RP, and nonsyndromic deafness. The complex genetic and phenotypic spectrums relevant to USH2A defects make it difficult to manage patients with such mutations. In the present study, we aim to determine the genetic etiology and to characterize the correlated clinical phenotypes for three Chinese pedigrees with nonsyndromic RP, one with RP sine pigmento (RPSP, and one with USH2. Family histories and clinical details for all included patients were reviewed. Ophthalmic examinations included best corrected visual acuities, visual field measurements, funduscopy, and electroretinography. Targeted next-generation sequencing (NGS was applied using two sequence capture arrays to reveal the disease causative mutations for each family. Genotype-phenotype correlations were also annotated. Seven USH2A mutations, including four missense substitutions (p.P2762A, p.G3320C, p.R3719H, and p.G4763R, two splice site variants (c.8223+1G>A and c.8559-2T>C, and a nonsense mutation (p.Y3745*, were identified as disease causative in the five investigated families, of which three reported to have consanguineous marriage. Among all seven mutations, six were novel, and one was recurrent. Two homozygous missense mutations (p.P2762A and p.G3320C were found in one individual family suggesting a potential double hit effect. Significant phenotypic divergences were revealed among the five families. Three families of the five families were affected with early, moderated, or late onset RP, one with RPSP, and the other one with USH2. Our study expands the genotypic and phenotypic variability relevant to USH2A mutations, which would help with a clear insight into the complex genetic and phenotypic spectrums relevant to USH2A defects, and is complementary for a better management of patients with such mutations. We have

High Frequency of AML1/RUNX1 Point Mutations in Radiation-Associated Myelodysplastic Syndrome Around Semipalatinsk Nuclear Test Site

OpenAIRE

Dinara, ZHARLYGANOVA; Hironori, HARADA; Yuka, HARADA; Sergey, SHINKAREV; Zhaxybay, ZHUMADILOV; Aigul, ZHUNUSOVA; Naylya J., TCHAIZHUNUSOVA; Kazbek N., APSALIKOV; Vadim, KEMAIKIN; Kassym, ZHUMADILOV; Noriyuki, KAWANO; Akiro, KIMURA; Masaharu, HOSHI; Department of Radiation Biophysics, Research Institute for Radiation Biology and Medicine, Hiroshima University; Department of Hematology and Oncology, Research Institute for Radiation Biology and Medicine, Hiroshima University

2008-01-01

It is known that bone marrow is a sensitive organ to ionizing radiation, and many patients with acute myeloid leukemia (AML) or myelodysplastic syndrome (MDS) have been diagnosed in radiation-treated cases and atomic bomb survivors in Hiroshima and Nagasaki. The AML1/RUNX1 gene has been known to be frequently mutated in MDS/AML patients among atomic bomb survivors and radiation therapy-related MDS/AML patients. In this study, we investigated the AML1 mutations in radiation-exposed patients wi...

Prognostic Value of RUNX1 Mutations in AML: A Meta-Analysis

Science.gov (United States)

Jalili, Mahdi; Yaghmaie, Marjan; Ahmadvand, Mohammad; Alimoghaddam, Kamran; Mousavi, Seyed Asadollah; Vaezi, Mohammad; Ghavamzadeh, Ardeshir

2018-02-26

The RUNX1 (AML1) gene is a relatively infrequent mutational target in cases of acute myeloid leukemia (AML). Previous work indicated that RUNX1 mutations can have pathological and prognostic implications. To evaluate prognostic value, we conducted a meta-analysis of 4 previous published works with data for survival according to RUNX1 mutation status. Pooled hazard ratios for overall survival and disease-free survival were 1.55 (95% confidence interval (CI) = 1.11–2.15; p-value = 0.01) and 1.76 (95% CI = 1.24–2.52; p-value = 0.002), respectively, for cases positive for RUNX1 mutations. This evidence supports clinical implications of RUNX1 mutations in the development and progression of AML cases and points to the possibility of a distinct category within the newer WHO classification. Though it must be kept in mind that the present work was based on data extracted from observational studies, the findings suggest that the RUNX1 status can contribute to risk-stratification and decision-making in management of AML. Creative Commons Attribution License

Sentinel and other mutational effects in offspring of cancer survivors

International Nuclear Information System (INIS)

Mulvihill, J.J.

1990-01-01

To date, no agent has been documented to cause germ cell mutation in human beings, with the possible exception of radiation causing abnormal meiotic chromosomes in testes. For studies in humans, mutation epidemiologists prefer the cohort approach, starting with an exposed population and looking for mutations that may be expressed in offspring as variants in health, chromosomes, proteins, or nucleic acids. Currently patients with cancer are the cohort exposed to the largest doses of potential mutagens, i.e., radiotherapy and drugs. In 12 large studies with over 825 patients and 1573 pregnancies, 46 (4%) of 1240 liveborns had a major birth defect, a rate comparable to that in the general population. One of these was a classic sentinel phenotype, i.e., a new sporadic case of a dominant mendelian syndrome. In collaboration with 5 U.S. cancer registries, we interviewed a retrospective cohort of 2383 patients diagnosed with cancer under age 20 years, from 1945 through 1975. Records were sought to verify major genetic disease, defined as a cytogenetic or single gene disorder or 1 of 15 isolated birth defects. In 2308 offspring of survivors, 5 had a chromosomal syndrome, 11 had a single gene disorder, and 62 had at least one major malformation. Among 4722 offspring of sibling controls, the respective numbers were 7, 12, and 127, nonsignificant differences. 7% of the parents of the offspring with possibly new mutations received potentially mutagenic therapy, compared with 12% of parents of normal children. Since pregnancy in or by cancer survivors is still a rare event, future efforts to document germ cell mutation may be best studied through international cooperation coupled with diverse laboratory measures of mutation

Functional assessment of compound mutations in the KCNQ1 and KCNH2 genes associated with long QT syndrome

DEFF Research Database (Denmark)

Grunnet, Morten; Behr, Elijah Raphael; Calloe, Kirstine

2005-01-01

BACKGROUND: Long QT syndrome (LQTS) is a cardiovascular disorder characterized by prolonged QTc time, syncope, or sudden death caused by torsades de pointes and ventricular fibrillation. We investigated the clinical and electrophysiologic phenotype of individual mutations and the compound mutations...

MPL mutation profile in JAK2 mutation-negative patients with myeloproliferative disorders.

Science.gov (United States)

Ma, Wanlong; Zhang, Xi; Wang, Xiuqiang; Zhang, Zhong; Yeh, Chen-Hsiung; Uyeji, Jennifer; Albitar, Maher

2011-03-01

Mutations in the thrombopoietin receptor gene (myeloproliferative leukemia, MPL) have been reported in patients with JAK2 V617F-negative chronic myeloproliferative disorders (MPDs). We evaluated the prevalence of MPL mutations relative to JAK2 mutations in patients with suspected MPDs. A total of 2790 patient samples submitted for JAK2 mutation analysis were tested using real-time polymerase chain reaction and bidirectional sequencing of plasma RNA. JAK2 V617F-negative samples were tested for JAK2 exons 12 to 14 mutations, and those with negative results were then tested for mutations in MPL exons 10 and 11. Of the 2790 patients, 529 (18.96%) had V617F, 12 (0.43%) had small insertions or deletions in exon 12, and 7 (0.25%) had other JAK2 mutations in exons 12 to 14. Of the 2242 JAK2 mutation-negative patients, 68 (3.03%) had MPL mutations. W515L was the predominant MPL mutation (n=46; 68%), and 10 (15%) patients had other W515 variants. The remaining MPL mutations (n=12, 17%) were detected at other locations in exons 10 and 11 and included 3 insertion/deletion mutations. The S505N mutation, associated with familial MPD, was detected in 3 patients. Overall, for every 100 V617F mutations in patients with suspected MPDs, there were 12.9 MPL mutations, 2.3 JAK2 exon 12 mutations, and 1.3 JAK2 exons 13 to 14 mutations. These findings suggest that MPL mutation screening should be performed before JAK2 exons 12 to 14 testing in JAK2 V617F-negative patients with suspected MPDs.

Discovery and prioritization of somatic mutations in diffuse large B-cell lymphoma (DLBCL) by whole-exome sequencing

Science.gov (United States)

Lohr, Jens G.; Stojanov, Petar; Lawrence, Michael S.; Auclair, Daniel; Chapuy, Bjoern; Sougnez, Carrie; Cruz-Gordillo, Peter; Knoechel, Birgit; Asmann, Yan W.; Slager, Susan L.; Novak, Anne J.; Dogan, Ahmet; Ansell, Stephen M.; Zou, Lihua; Gould, Joshua; Saksena, Gordon; Stransky, Nicolas; Rangel-Escareño, Claudia; Fernandez-Lopez, Juan Carlos; Hidalgo-Miranda, Alfredo; Melendez-Zajgla, Jorge; Hernández-Lemus, Enrique; Schwarz-Cruz y Celis, Angela; Imaz-Rosshandler, Ivan; Ojesina, Akinyemi I.; Jung, Joonil; Pedamallu, Chandra S.; Lander, Eric S.; Habermann, Thomas M.; Cerhan, James R.; Shipp, Margaret A.; Getz, Gad; Golub, Todd R.

2012-01-01

To gain insight into the genomic basis of diffuse large B-cell lymphoma (DLBCL), we performed massively parallel whole-exome sequencing of 55 primary tumor samples from patients with DLBCL and matched normal tissue. We identified recurrent mutations in genes that are well known to be functionally relevant in DLBCL, including MYD88, CARD11, EZH2, and CREBBP. We also identified somatic mutations in genes for which a functional role in DLBCL has not been previously suspected. These genes include MEF2B, MLL2, BTG1, GNA13, ACTB, P2RY8, PCLO, and TNFRSF14. Further, we show that BCL2 mutations commonly occur in patients with BCL2/IgH rearrangements as a result of somatic hypermutation normally occurring at the IgH locus. The BCL2 point mutations are primarily synonymous, and likely caused by activation-induced cytidine deaminase–mediated somatic hypermutation, as shown by comprehensive analysis of enrichment of mutations in WRCY target motifs. Those nonsynonymous mutations that are observed tend to be found outside of the functionally important BH domains of the protein, suggesting that strong negative selection against BCL2 loss-of-function mutations is at play. Last, by using an algorithm designed to identify likely functionally relevant but infrequent mutations, we identify KRAS, BRAF, and NOTCH1 as likely drivers of DLBCL pathogenesis in some patients. Our data provide an unbiased view of the landscape of mutations in DLBCL, and this in turn may point toward new therapeutic strategies for the disease. PMID:22343534

Molecular analysis on germline mutation caused by low-dose irradiation

International Nuclear Information System (INIS)

Uchiyama, R.; Fujikawa, K.; Nishimura, M.; Adzuma, H.; Shimada, Y.; Yamauchi, M.

2003-01-01

Full text: Genetic heterogeneity and a low frequency of germline mutation at single-copy gene loci have limited the direct measurement of germline mutation in human populations. Two conflicting results have been reported for the effect of ionizing radiation on germline mutation in human populations. A study conducted on the first-generation progeny of the survivors of the atomic bombs at Hiroshima and Nagasaki found no significant increase in germline mutations. On the other hand, a significant increase in germline mutation was reported among the human population in the Belarus area after the Chernobyl accident in 1986. We investigated the germline mutation at the molecular level using experimental mouse strains with different genetic backgrounds to assess the risk of ionizing radiation on human populations. The C3H male parents were exposed to X ray (0, 0.3, 1, and 3Gy) and mated with unexposed C57BL females after two weeks interval, so as to detect the germline mutation occurred at the spermatid stage. Genomic DNA samples were prepared from the both parents and F1s, and the genomic DNA sequences were compared between parents and offspring at the specific genomic gene loci, such as adenine phosphoribosyl transferase (aprt) gene and cytidine triphosphate synthetase (ctps) gene, using the automated DNA sequencer. Also hypervariable Pc-1 (Ms6-hm) minisatellite repeat locus was analyzed by using Southern blot hybridization technique. Our preliminary results indicated that the changes of the restriction DNA fragment length in offspring did not reflect the occurrence of the mutation, such as point mutation, insertion, and deletion, in the genomic gene loci including the intervening sequence (intron)

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

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Eun Hyun Ahn

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

A Mutation in the Dmp1 Gene Alters Phosphate Responsiveness in Mice

Science.gov (United States)

Gerard-O'Riley, Rita L.; Acton, Dena; McQueen, Amie K.; Strobel, Isabel E.; Witcher, Phillip C.; Feng, Jian Q.; Econs, Michael J.

2017-01-01

Mutations in the dentin matrix protein 1 (DMP1) gene cause autosomal recessive hypophosphatemic rickets (ARHR). Hypophosphatemia in ARHR results from increased circulating levels of the phosphaturic hormone, fibroblast growth factor 23 (FGF23). Similarly, elevated FGF23, caused by mutations in the PHEX gene, is responsible for the hypophosphatemia in X-linked hypophosphatemic rickets (XLH). Previously, we demonstrated that a Phex mutation in mice creates a lower set point for extracellular phosphate, where an increment in phosphorus further stimulates Fgf23 production to maintain low serum phosphorus levels. To test the presence of the similar set point defect in ARHR, we generated 4- and 12-week-old Dmp1/Galnt3 double knockout mice and controls, including Dmp1 knockout mice (a murine model of ARHR), Galnt3 knockout mice (a murine model of familial tumoral calcinosis), and phenotypically normal double heterozygous mice. Galnt3 knockout mice had increased proteolytic cleavage of Fgf23, leading to low circulating intact Fgf23 levels with consequent hyperphosphatemia. In contrast, Dmp1 knockout mice had little Fgf23 cleavage and increased femoral Fgf23 expression, resulting in hypophosphatemia and low femoral bone mineral density (BMD). However, introduction of the Galnt3 null allele to Dmp1 knockout mice resulted in a significant increase in serum phosphorus and normalization of BMD. This increased serum phosphorus was accompanied by markedly elevated Fgf23 expression and circulating Fgf23 levels, an attempt to reduce serum phosphorus in the face of improving phosphorus levels. These data indicate that a Dmp1 mutation creates a lower set point for extracellular phosphate and maintains it through the regulation of Fgf23 cleavage and expression. PMID:28005411

Viral Diversity Threshold for Adaptive Immunity in Prokaryotes

Science.gov (United States)

Weinberger, Ariel D.; Wolf, Yuri I.; Lobkovsky, Alexander E.; Gilmore, Michael S.; Koonin, Eugene V.

2012-01-01

ABSTRACT Bacteria and archaea face continual onslaughts of rapidly diversifying viruses and plasmids. Many prokaryotes maintain adaptive immune systems known as clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated genes (Cas). CRISPR-Cas systems are genomic sensors that serially acquire viral and plasmid DNA fragments (spacers) that are utilized to target and cleave matching viral and plasmid DNA in subsequent genomic invasions, offering critical immunological memory. Only 50% of sequenced bacteria possess CRISPR-Cas immunity, in contrast to over 90% of sequenced archaea. To probe why half of bacteria lack CRISPR-Cas immunity, we combined comparative genomics and mathematical modeling. Analysis of hundreds of diverse prokaryotic genomes shows that CRISPR-Cas systems are substantially more prevalent in thermophiles than in mesophiles. With sequenced bacteria disproportionately mesophilic and sequenced archaea mostly thermophilic, the presence of CRISPR-Cas appears to depend more on environmental temperature than on bacterial-archaeal taxonomy. Mutation rates are typically severalfold higher in mesophilic prokaryotes than in thermophilic prokaryotes. To quantitatively test whether accelerated viral mutation leads microbes to lose CRISPR-Cas systems, we developed a stochastic model of virus-CRISPR coevolution. The model competes CRISPR-Cas-positive (CRISPR-Cas+) prokaryotes against CRISPR-Cas-negative (CRISPR-Cas−) prokaryotes, continually weighing the antiviral benefits conferred by CRISPR-Cas immunity against its fitness costs. Tracking this cost-benefit analysis across parameter space reveals viral mutation rate thresholds beyond which CRISPR-Cas cannot provide sufficient immunity and is purged from host populations. These results offer a simple, testable viral diversity hypothesis to explain why mesophilic bacteria disproportionately lack CRISPR-Cas immunity. More generally, fundamental limits on the adaptability of biological

DiversePathsJ: diverse shortest paths for bioimage analysis.

Science.gov (United States)

Uhlmann, Virginie; Haubold, Carsten; Hamprecht, Fred A; Unser, Michael

2018-02-01

We introduce a formulation for the general task of finding diverse shortest paths between two end-points. Our approach is not linked to a specific biological problem and can be applied to a large variety of images thanks to its generic implementation as a user-friendly ImageJ/Fiji plugin. It relies on the introduction of additional layers in a Viterbi path graph, which requires slight modifications to the standard Viterbi algorithm rules. This layered graph construction allows for the specification of various constraints imposing diversity between solutions. The software allows obtaining a collection of diverse shortest paths under some user-defined constraints through a convenient and user-friendly interface. It can be used alone or be integrated into larger image analysis pipelines. http://bigwww.epfl.ch/algorithms/diversepathsj. michael.unser@epfl.ch or fred.hamprecht@iwr.uni-heidelberg.de. Supplementary data are available at Bioinformatics online. © The Author(s) 2017. Published by Oxford University Press.

Human impacts on genetic diversity in forest ecosystems

Energy Technology Data Exchange (ETDEWEB)

Ledig, F T [Inst. of Forest Genetics, Southwest Forest and Range Experiment Station, USDA Forest Service, Berkeley (US)

1992-01-01

Humans have converted forest to agricultural and urban uses, exploited species, fragmented wildlands, changed the demographic structure of forests, altered habitat, degraded the environment with atmospheric and soil pollutants, introduced exotic pests and competitors, and domesticated favored species. None of these activities is new; perhaps with the exception of atmospheric pollution, they date back to prehistory. All have impacted genetic diversity by their influence on the evolutionary processes of extinction, selection, drift, gene flow, and mutation, sometimes increasing diversity, as int he case of domestication, but often reducing it. Even in the absence of changes in diversity, mating systems were altered, changing the genetic structure of populations. Demographic changes influenced selection by increasing the incidence of disease. Introduction of exotic diseases, insects, mammalian herbivores, and competing vegetation has had the best-documented effects on genetic diversity, reducing both species diversity and intraspecific diversity. Deforestation has operated on a vast scale to reduce diversity by direct elimination of locally-adapted populations. Atmospheric pollution and global warming will be a major threat in the near future, particularly because forests are fragmented and migration is impeded. Past impacts can be estimated with reference to expert knowledge, but hard data are often laching. Baselines are needed to quantify future impacts and provide an early warning of problems. Genetic inventories of indicator species can provide the baselines against which to measure changes in diversity. (author) (44 refs.).

Bacteriophage Resistance Mechanisms in the Fish Pathogen Flavobacterium psychrophilum: Linking Genomic Mutations to Changes in Bacterial Virulence Factors

DEFF Research Database (Denmark)

Castillo, Daniel; Christiansen, Rói Hammershaimb; Dalsgaard, Inger

2015-01-01

-resistant strains had all obtained unique insertions and/or deletions and point mutations distributed among intergenic and genic regions. Mutations in genes related to cell surface properties, gliding motility, and biosynthesis of lipopolysaccharides and cell wall were found. The observed links between phage...

Ancient genes establish stress-induced mutation as a hallmark of cancer.

Science.gov (United States)

Cisneros, Luis; Bussey, Kimberly J; Orr, Adam J; Miočević, Milica; Lineweaver, Charles H; Davies, Paul

2017-01-01

stress that evolved among prokaryotes was co-opted to maintain diversity in the germline and immune system, while the original phenotype is restored in cancer. Reversion to a stress-induced mutational response is a hallmark of cancer that allows for effectively searching "protected" genome space where genes causally implicated in cancer are located and underlies the high adaptive potential and concomitant therapeutic resistance that is characteristic of cancer.

Sequence of the intron/exon junctions of the coding region of the human androgen receptor gene and identification of a point mutation in a family with complete androgen insensitivity

International Nuclear Information System (INIS)

Lubahn, D.B.; Simental, J.A.; Higgs, H.N.; Wilson, E.M.; French, F.S.; Brown, T.R.; Migeon, C.J.

1989-01-01

Androgens act through a receptor protein (AR) to mediate sex differentiation and development of the male phenotype. The authors have isolated the eight exons in the amino acid coding region of the AR gene from a human X chromosome library. Nucleotide sequences of the AR gene intron/exon boundaries were determined for use in designing synthetic oligonucleotide primers to bracket coding exons for amplification by the polymerase chain reaction. Genomic DNA was amplified from 46, XY phenotypic female siblings with complete androgen insensitivity syndrome. AR binding affinity for dihydrotestosterone in the affected siblings was lower than in normal males, but the binding capacity was normal. Sequence analysis of amplified exons demonstrated within the AR steroid-binding domain (exon G) a single guanine to adenine mutation, resulting in replacement of valine with methionine at amino acid residue 866. As expected, the carrier mother had both normal and mutant AR genes. Thus, a single point mutation in the steroid-binding domain of the AR gene correlated with the expression of an AR protein ineffective in stimulating male sexual development

The TREAT-NMD DMD Global Database: Analysis of More than 7,000 Duchenne Muscular Dystrophy Mutations

Science.gov (United States)

Bladen, Catherine L; Salgado, David; Monges, Soledad; Foncuberta, Maria E; Kekou, Kyriaki; Kosma, Konstantina; Dawkins, Hugh; Lamont, Leanne; Roy, Anna J; Chamova, Teodora; Guergueltcheva, Velina; Chan, Sophelia; Korngut, Lawrence; Campbell, Craig; Dai, Yi; Wang, Jen; Barišić, Nina; Brabec, Petr; Lahdetie, Jaana; Walter, Maggie C; Schreiber-Katz, Olivia; Karcagi, Veronika; Garami, Marta; Viswanathan, Venkatarman; Bayat, Farhad; Buccella, Filippo; Kimura, En; Koeks, Zaïda; van den Bergen, Janneke C; Rodrigues, Miriam; Roxburgh, Richard; Lusakowska, Anna; Kostera-Pruszczyk, Anna; Zimowski, Janusz; Santos, Rosário; Neagu, Elena; Artemieva, Svetlana; Rasic, Vedrana Milic; Vojinovic, Dina; Posada, Manuel; Bloetzer, Clemens; Jeannet, Pierre-Yves; Joncourt, Franziska; Díaz-Manera, Jordi; Gallardo, Eduard; Karaduman, A Ayşe; Topaloğlu, Haluk; El Sherif, Rasha; Stringer, Angela; Shatillo, Andriy V; Martin, Ann S; Peay, Holly L; Bellgard, Matthew I; Kirschner, Jan; Flanigan, Kevin M; Straub, Volker; Bushby, Kate; Verschuuren, Jan; Aartsma-Rus, Annemieke; Béroud, Christophe; Lochmüller, Hanns

2015-01-01

Analyzing the type and frequency of patient-specific mutations that give rise to Duchenne muscular dystrophy (DMD) is an invaluable tool for diagnostics, basic scientific research, trial planning, and improved clinical care. Locus-specific databases allow for the collection, organization, storage, and analysis of genetic variants of disease. Here, we describe the development and analysis of the TREAT-NMD DMD Global database (http://umd.be/TREAT_DMD/). We analyzed genetic data for 7,149 DMD mutations held within the database. A total of 5,682 large mutations were observed (80% of total mutations), of which 4,894 (86%) were deletions (1 exon or larger) and 784 (14%) were duplications (1 exon or larger). There were 1,445 small mutations (smaller than 1 exon, 20% of all mutations), of which 358 (25%) were small deletions and 132 (9%) small insertions and 199 (14%) affected the splice sites. Point mutations totalled 756 (52% of small mutations) with 726 (50%) nonsense mutations and 30 (2%) missense mutations. Finally, 22 (0.3%) mid-intronic mutations were observed. In addition, mutations were identified within the database that would potentially benefit from novel genetic therapies for DMD including stop codon read-through therapies (10% of total mutations) and exon skipping therapy (80% of deletions and 55% of total mutations). PMID:25604253

A novel heterozygous SOX2 mutation causing anophthalmia/microphthalmia with genital anomalies.

Science.gov (United States)

Pedace, Lucia; Castori, Marco; Binni, Francesco; Pingi, Alberto; Grammatico, Barbara; Scommegna, Salvatore; Majore, Silvia; Grammatico, Paola

2009-01-01

Anophthalmia/microphthalmia is a rare developmental craniofacial defect, which recognizes a wide range of causes, including chromosomal abnormalities, single-gene mutations as well as environmental factors. Heterozygous mutations in the SOX2 gene are the most common monogenic form of anophthalmia/microphthalmia, as they are reported in up to 10-15% cases. Here, we describe a sporadic patient showing bilateral anophthalmia/microphthalmia and micropenis caused by a novel mutation (c.59_60insGG) in the SOX2 gene. Morphological and endocrinological evaluations excluded any anomaly of the hypothalamus-pituitary axis. Our finding supports the hypothesis that SOX2 is particularly prone to slipped-strand mispairing, which results in a high frequency of point deletions/insertions.

A novel splicing mutation in the V2 vasopressin receptor

DEFF Research Database (Denmark)

Kamperis, Konstantinos; Siggaard, C; Herlin, Troels

2000-01-01

as clinical investigations comprising a fluid deprivation test and a 1-deamino-8-D-arginine-vasopressin (dDAVP) infusion test in the study subject and his mother. We found a highly unusual, novel, de novo 1447A-->C point mutation (gDNA), involving the invariable splice acceptor of the second intron...... of the gene in both the affected male (hemizygous) and his mother (heterozygous). This mutation is likely to cause aberrant splicing of the terminal intron of the gene, leading to a non-functional AVP receptor. The clinical studies were consistent with such a hypothesis, as the affected subject had a severe...