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Mutation Detection (mutation + detection)
Terms modified by Mutation Detection Selected AbstractsA Sensitive Fluorescence Anisotropy Method for Point Mutation Detection by Using Core,Shell Fluorescent Nanoparticles and High-Fidelity DNA LigaseCHEMISTRY - A EUROPEAN JOURNAL, Issue 27 2007Ting Deng Dr. Abstract The present study reports a proof-of-principle for a sensitive genotyping assay approach that can detect single nucleotide polymorphisms (SNPs) based on fluorescence anisotropy measurements through a core,shell fluorescent nanoparticles assembly and ligase reaction. By incorporating the core,shell fluorescent nanoparticles into fluorescence anisotropy measurements, this assay provided a convenient and sensitive detection assay that enabled straightforward single-base discrimination without the need of complicated operational steps. The assay was implemented via two steps: first, the hybridization reaction that allowed two nanoparticle-tagged probes to hybridize with the target DNA strand and the ligase reaction that generated the ligation between perfectly matched probes while no ligation occurred between mismatched ones were implemented synchronously in the same solution. Then, a thermal treatment at a relatively high temperature discriminated the ligation of probes. When the reaction mixture was heated to denature the duplex formed, the fluorescence anisotropy value of the perfect-match solution does not revert to the initial value, while that of the mismatch again comes back as the assembled fluorescent nanoparticles dispart. The present approach has been demonstrated with the discrimination of a single base mutation in codon 12 of a K-ras oncogene that is of significant value for colorectal cancers diagnosis, and the wild type and mutant type were successfully scored. Due to its ease of operation and high sensitivity, it was expected that the proposed detection approach might hold great promise in practical clinical diagnosis. [source] A parylene-based dual channel micro-electrophoresis system for rapid mutation detection via heteroduplex analysis,ELECTROPHORESIS, Issue 18 2008Sertan Sukas Abstract A new dual channel micro-electrophoresis system for rapid mutation detection based on heteroduplex analysis was designed and implemented. Mutation detection was successfully achieved in a total separation length of 250,,m in less than 3,min for a 590,bp DNA sample harboring a 3,bp mutation causing an amino acid change. Parylene-C was used as the structural material for fabricating the micro-channels as it provides conformal deposition, transparency, biocompatibility, and low background fluorescence without any surface treatment. A new dual channel architecture was derived from the traditional cross-channel layout by forming two identical channels with independent sample loading and waste reservoirs. The control of injected sample volume was accomplished by a new u-turn injection technique with pull-back method. The use of heteroduplex analysis as a mutation detection method on a cross-linked polyacrylamide medium provided accurate mutation detection in an extremely short length and time. The presence of two channels on the microchip offers the opportunity of comparing the sample to be tested with a desired control sample rapidly, which is very critical for the accuracy and reliability of the mutation analyses, especially for clinical and research purposes. [source] Quantification of sequence exchange events between PMS2 and PMS2CL provides a basis for improved mutation scanning of lynch syndrome patients,HUMAN MUTATION, Issue 5 2010Heleen M. van der Klift Abstract Heterozygous mutations in PMS2 are involved in Lynch syndrome, whereas biallelic mutations are found in Constitutional mismatch repair-deficiency syndrome patients. Mutation detection is complicated by the occurrence of sequence exchange events between the duplicated regions of PMS2 and PMS2CL. We investigated the frequency of such events with a nonspecific polymerase chain reaction (PCR) strategy, coamplifying both PMS2 and PMS2CL sequences. This allowed us to score ratios between gene and pseudogene-specific nucleotides at 29 PSV sites from exon 11 to the end of the gene. We found sequence transfer at all investigated PSVs from intron 12 to the 3, end of the gene in 4 to 52% of DNA samples. Overall, sequence exchange between PMS2 and PMS2CL was observed in 69% (83/120) of individuals. We demonstrate that mutation scanning with PMS2 -specific PCR primers and MLPA probes, designed on PSVs, in the 3, duplicated region is unreliable, and present an RNA-based mutation detection strategy to improve reliability. Using this strategy, we found 19 different putative pathogenic PMS2 mutations. Four of these (21%) are lying in the region with frequent sequence transfer and are missed or called incorrectly as homozygous with several PSV-based mutation detection methods. Hum Mutat 31:578,587, 2010. © 2010 Wiley-Liss, Inc. [source] Screening of 25 Italian patients with Niemann-Pick a reveals fourteen new mutations, one common and thirteen private, in SMPD1,,HUMAN MUTATION, Issue 1 2004V. Ricci Abstract Niemann-Pick disease (NPD) results from the deficiency of lysosomal acid sphingomyelinase (SMPD1). To date, out of more than 70-disease associated alleles only a few of them have a significant frequency in various ethnic groups. In contrast, the remainder of the mutations are rare or private. In this paper we report the molecular characterization of an Italian series consisting of twenty-five NPD patients with the severe neurodegenerative A phenotype. Mutation detection identified a total of nineteen different mutations, including 14 novel mutations and five previously reported lesions. The known p.P189fs and the novel p.T542fs were the most frequent mutations accounting for 34% and 18% of the alleles, respectively. Screening the alleles for the three common polymorphisms revealed the variant c.1516G>A (exon 6) and the repeat in exon 1, but not the variant c.965C>T (exon 2). In absence of frequent mutations, the prognostic value of genotyping is limited. However, new genotype/phenotype correlations were observed for this disorder that could in the future facilitate genetic counseling and guide selection of patients for therapy. © 2004 Wiley-Liss, Inc. [source] Large Genomic Mutations within the ATM Gene Detected by MLPA, Including a Duplication of 41 kb from Exon 4 to 20ANNALS OF HUMAN GENETICS, Issue 1 2008Simona Cavalieri Summary Mutation detection remains problematic for large genes, primarily because PCR-based methodology fails to detect heterozygous deletions and any duplication. In the ATM gene only a handful of multi-exon deletions have been described to date, and this type of mutation has been considered rare. To address this issue we tested a new MLPA (Multiplex Ligation Probe Amplification) kit that covers 33 of the 66 ATM exons, using for controls two previously characterized genomic deletions in addition to three A-T patients, taken from a survey of nine, who had missing four mutations unidentified after conventional mutation screening. We identified for the first time: 1) a ,41 kb genomic duplication spanning exons 4,20 (c.-30_2816dup41kb)(a.k.a., ATM dup 41 kb); 2) a novel genomic deletion including exon 31, and 3) in hemizygosis a point mutation in the non-deleted exon 31. In this study we extended mutation detection to nine new Italian A-T patients, using a combined approach of haplotype analysis, DHPLC and MLPA. Overall we achieved a mutation detection rate of >97%, and can now define a spectrum of ATM mutations based on twenty-one consecutive Italian families with A-T. [source] Four novel mutations in the Tyrosine Hydroxylase gene in patients with infantile parkinsonismANNALS OF HUMAN GENETICS, Issue 1 2000R. J. M. SWAANS Mutation detection in the Tyrosine Hydroxylase gene (TH) was performed in patients from two families. DNA sequencing revealed the presence of four novel missense mutations (exon 9 and 14 in family A, exon 8 and 9 in family B); the mutations were confirmed with restriction enzyme analysis, and did not occur in control alleles. Three mutations are in the catalytic domain of the enzyme and one may disturb tetramerization. At the moment, all patients are in the fourth decade of life. For more than 30 years they have been able to live a normal life with low-dose l -DOPA medication. [source] Cover Picture: Electrophoresis 10'2010ELECTROPHORESIS, Issue 10 2010Article first published online: 18 MAY 2010 Issue no. 10 is a regular issue comprising 19 manuscripts distributed over four distinct parts. Part I is on microfluidics and miniaturized systems and has 5 articles; Part II is on nucleic acids with 4 articles on restriction endonuclease fingerprinting, mutation detection and DNA separation and detection; Part III has 7 articles on monolithic stationary phases for CEC, single walled carbon nanohorns as pseudo-stationary phases for CEC and EKC, MEEKC, cyclodextrin-modified gold nanoparticles for enantioseparations by CEC, use of divalent dipeptides as counter ions in CE and capillary coating for CE of proteins; and Part IV has 3 articles on proteomics methodologies. Featured articles include: Microfluidic preparative free-flow isoelectric focusing in a triangular channel: System development and characterization ((10.1002/elps.200900577)) Separation and recovery of nucleic acids with improved biological activity by acid-degradable polyacrylamide gel electrophoresis ((10.1002/elps.200900783)) Evaluation of the performance of single-walled carbon nanohorns in capillary electrophoresis ((10.1002/elps.200900628)) The inter- and intra-operator variability in manual spot segmentation and its effect on spot quantitation in two dimensional electrophoresis analysis. ((10.1002/elps.200900674)) [source] A new approach to long QT syndrome mutation detection by Sequenom MassARRAY® systemELECTROPHORESIS, Issue 10 2010Catarina Allegue Abstract Congenital long QT syndrome is an inherited cardiac disorder characterized by a prolonged QT interval and polymorphic ventricular arrhythmias that could result in recurrent syncope, seizures or sudden death as the most dramatic event. Until now QT interval mutations have been described in 12 genes, where the majority of mutations reside in three genes KCNQ1, KCNH2, and SCN5A. Diagnosis and prognosis are directly related with the gene and mutation involved. We have developed a diagnostic approach for long QT syndrome and Brugada syndrome based on published mutations and Sequenom MassArray® system. Three diagnostic tests have been developed, oriented to each of the three most prevalent genes in the long QT syndrome. A total of 433 mutations are analyzed in 38 multiplex reactions, allowing their detection in about 48,h. Tests were validated on 502 samples from individuals with different clinical conditions and family history. The average call rates obtained for each of the tests were 93, 83, and 73% in KCNQ1, KCNH2, and SCNA, respectively. Sequenom MassARRAY mutation detection is a reliable, highly flexible, and cost-efficient alternative to conventional methods for genetic testing in long QT syndrome and Brugada syndrome, facilitating flexible upgrades of the version of the test presented here with the inclusion of new mutations. [source] A parylene-based dual channel micro-electrophoresis system for rapid mutation detection via heteroduplex analysis,ELECTROPHORESIS, Issue 18 2008Sertan Sukas Abstract A new dual channel micro-electrophoresis system for rapid mutation detection based on heteroduplex analysis was designed and implemented. Mutation detection was successfully achieved in a total separation length of 250,,m in less than 3,min for a 590,bp DNA sample harboring a 3,bp mutation causing an amino acid change. Parylene-C was used as the structural material for fabricating the micro-channels as it provides conformal deposition, transparency, biocompatibility, and low background fluorescence without any surface treatment. A new dual channel architecture was derived from the traditional cross-channel layout by forming two identical channels with independent sample loading and waste reservoirs. The control of injected sample volume was accomplished by a new u-turn injection technique with pull-back method. The use of heteroduplex analysis as a mutation detection method on a cross-linked polyacrylamide medium provided accurate mutation detection in an extremely short length and time. The presence of two channels on the microchip offers the opportunity of comparing the sample to be tested with a desired control sample rapidly, which is very critical for the accuracy and reliability of the mutation analyses, especially for clinical and research purposes. [source] An optimized microchip electrophoresis system for mutation detection by tandem SSCP and heteroduplex analysis for p53,gene exons,5,9ELECTROPHORESIS, Issue 19 2006Christa N. Hestekin Abstract With the complete sequencing of the human genome, there is a growing need for rapid, highly sensitive genetic mutation detection methods suitable for clinical implementation. DNA-based diagnostics such as single-strand conformational polymorphism (SSCP) and heteroduplex analysis (HA) are commonly used in research laboratories to screen for mutations, but the slab gel electrophoresis (SGE) format is ill-suited for routine clinical use. The translation of these assays from SGE to microfluidic chips offers significant speed, cost, and sensitivity advantages; however, numerous parameters must be optimized to provide highly sensitive mutation detection. Here we present a methodical study of system parameters including polymer matrix, wall coating, analysis temperature, and electric field strengths on the effectiveness of mutation detection by tandem SSCP/HA for DNA samples from exons,5,9 of the p53 gene. The effects of polymer matrix concentration and average molar mass were studied for linear polyacrylamide (LPA) solutions. We determined that a matrix of 8%,w/v 600,kDa LPA provides the most reliable SSCP/HA mutation detection on chips. The inclusion of a small amount of the dynamic wall-coating polymer poly- N -hydroxyethylacrylamide in the matrix substantially improves the resolution of SSCP conformers and extends the coating lifetime. We investigated electrophoresis temperatures between 17 and 35°C and found that the lowest temperature accessible on our chip electrophoresis system gives the best condition for high sensitivity of the tandem SSCP/HA method, especially for the SSCP conformers. Finally, the use of electrical fields between 350 and 450,V/cm provided rapid separations (<10,min) with well-resolved DNA peaks for both SSCP and HA. [source] Analysis of Genetically Complex EpilepsiesEPILEPSIA, Issue 2005Ruth Ottman Summary:, During the last decade, great progress has been made in the discovery of genes that influence risk for epilepsy. However, these gene discoveries have been in epilepsies with Mendelian modes of inheritance, which comprise only a tiny fraction of all epilepsy. Most people with epilepsy have no affected relatives, suggesting that the great majority of all epilepsies are genetically complex: multiple genes contribute to their etiology, none of which has a major effect on disease risk. Gene discovery in the genetically complex epilepsies is a formidable task. It is unclear which epilepsy phenotypes are most advantageous to study, and chromosomal localization and mutation detection are much more difficult than in Mendelian epilepsies. Association studies are very promising for the identification of complex epilepsy genes, but we are still in the earliest stages of their application in the epilepsies. Future studies should employ very large sample sizes to ensure adequate statistical power, clinical phenotyping methods of the highest quality, designs and analytic techniques that control for population stratification, and state-of-the-art molecular methods. Collaborative studies are essential to achieve these goals. [source] Point of care mutation detectionHUMAN MUTATION, Issue 2 2010Mats Nilsson No abstract is available for this article. [source] Masking selected sequence variation by incorporating mismatches into melting analysis probes,HUMAN MUTATION, Issue 3 2006Rebecca L. Margraf Abstract Hybridization probe melting analysis can be complicated by the presence of sequence variation (benign polymorphisms or other mutations) near the targeted mutation. We investigated the use of "masking" probes to differentiate alleles with similar probe melting temperatures. Selected sequence variation was masked by incorporating mismatches (deletion, unmatched nucleotide, or universal base) into hybridization probes at the polymorphic location. Such masking probes create a probe/target mismatch with all possible alleles at the selected polymorphic location. Any allele with additional variation at another site is identified by a lower probe melting temperature than alleles that vary only at the masked position. This "masking technique" was applied to RET protooncogene and HPA6 mutation detection using unlabeled hybridization probes, a saturating dsDNA dye, and high-resolution melting analysis. Masking probes clearly distinguished all targeted mutations from polymorphisms when at least 1 base pair (bp) separated the mutation from the masked variation. We were able to mask polymorphisms immediately adjacent to mutations, except in certain cases, such as those involving single-base deletion probes when both adjacent positions had the same polymorphic nucleotides. The masking probes can also localize mutations to specific codons or nucleotide positions. Masking probes can simplify melting analysis of complex regions and eliminate the need for sequencing. Hum Mutat 27(3), 269,278, 2006. © 2006 Wiley-Liss, Inc. [source] Eight novel MSH6 germline mutations in patients with familial and nonfamilial colorectal cancer selected by loss of protein expression in tumor tissue,,HUMAN MUTATION, Issue 3 2004Jens Plaschke Abstract Germline mutations in mismatch repair (MMR) genes, predominantly in MLH1 and MSH2, are responsible for hereditary nonpolyposis colorectal cancer (HNPCC), a cancer-susceptibility syndrome with high penetrance. In addition, MSH6 mutations have been reported to account for about 10% of all germline mismatch repair (MMR) gene mutations in HNPCC patients, and have been associated with a later age of onset of the disease compared to MLH1 and MSH2 mutations. Here, we report eight novel germline mutations in MSH6. The patients were selected by having developed tumors with loss of MSH6 protein expression. All tumors showed high-level microsatellite instability (MSI-H). Seven mutations resulted in premature stop codons, comprised of two nonsense mutations (c.426G>A [p.W142X], c.2105C>A [p.S702X]), two insertions (c.2611_2614dupATTA [p.I872fsX10], c.3324dupT [p.I1109fsX3]) and three deletions (c.1190_1191delAT [p.Y397fsX3], c.1632_1635delAAAA [p.E544fsX26], c.3513_3514delTA [p.1171fsX5]). In addition, an amino acid substitution of an arginine residue (c.2314C>T [p.R772W]) conserved throughout a wide variety of mutS homologs has been found in a patient not fulfilling the Bethesda criteria for HNPCC. Our results emphasize the suitability of IHC as a pre-selection tool for MSH6 mutation analysis and the high frequency of germline mutation detection in patients with MSH6 -deficient tumors. In addition, our findings point towards a broad variability regarding penetrance associated with MSH6 germline mutations. © 2004 Wiley-Liss, Inc. [source] H intragenic polymorphisms and haplotype analysis in the ornithine transcarbamylase (OTC) gene and their relevance for tracking the inheritance of OTC deficiency,,HUMAN MUTATION, Issue 5 2002Consuelo Climent Abstract The "private" nature of most mutations causing ornithine transcarbamylase (OTC) deficiency makes mutation identification in the patients difficult. Further, the PCR-amplification technology generally used for the genetic diagnosis of the deficiency misses large deletions in carrier females. Intragenic OTC polymorphisms may allow detection of these deletions and may represent an alternative to mutation detection for prenatal diagnosis and carrier identification in families with a history of inherited OTC deficiency. A new highly informative polymorphism (allele frequencies, 0.66/0.34) in intron 3 of the OTC gene (IVS3-39_40insT) is reported here, and allelic frequencies of 16 additional intragenic OTC polymorphisms are determined in 133-35 (average per polymorphism, 72) unrelated chromosomes. In addition to the novel polymorphism, only three of the studied polymorphisms (Lys46Arg, allelic frequency 0.68/0.32; IVS3-8A>T, 0.34/0.66; Gln270Arg, 0.97/0.03) are confirmed to be informative. These provide, together with another reported polymorphism (IVS4-7A>G; reported allelic frequency 0.71/0.29; Plante and Tuchman, 1998), a set of highly valuable markers of the OTC gene. Nevertheless, the combined informativity of the studied polymorphisms is limited by their distribution in only four haplotypes with one of them predominating (65% of the sampled chromosomes). Although this haplotype composition may be restricted to the Iberian peninsula (the origin of the samples), more informative polymorphisms are required to increase the diagnostic potential and, particularly, to identify large deletions affecting OTC gene exons 5-10, where only one polymorphism of weak diagnostic value is known. © 2002 Wiley-Liss, Inc. [source] Analysis of the glucocerebrosidase gene and mutation profile in 144 Italian gaucher patients,,HUMAN MUTATION, Issue 3 2002Mirella Filocamo Abstract Gaucher disease (GD), the most prevalent lysosomal storage disease characterized by a remarkable degree of clinical variability, results from deleterious mutations in the glucocerebrosidase gene (GBA). In this paper we report the molecular characterization of 144 unrelated Italian GD patients with the three types of the disease. The allelic frequencies of Italians are reported and the mutation profile is analyzed. Besides the common N370S, L444P, RecNciI, G202R, IVS2+1G>A, D409H, F213I mutations, the different molecular strategies, used for the mutation detection, identified the rare N107L, R131C, R170C, R170P, N188S, S196P, R285C, R285H, W312C, D399N, A446P, IVS10-1G>A, Rec,55, total gene deletion, as well as 12 mutant alleles that were exclusively present in the Italian population until now: the previously reported R353G, N370S+S488P mosaicism, IVS8(-11delC)-14T>A), Rec I, Y418C, and the seven novel alleles D127X, P159T, V214X, T231R, L354X, H451R, and G202R+M361I. The wide phenotypic differences observed within the genotypic groups as well as between siblings implicate a significant contribution of other modifying genetic and/or non-genetic factors and claim a comprehensive valuation of the patient including clinical., biochemical and molecular investigations for prognosis, appropriate interventive therapy and reliable genetic counseling. © 2002 Wiley-Liss, Inc. [source] Denaturing capillary electrophoresis for automated detection of L858R mutation in exon 21 of the epidermal growth factor receptor gene in prediction of the outcome of lung cancer therapyJOURNAL OF SEPARATION SCIENCE, JSS, Issue 15 2010Lucie Benesova Abstract The presence of activating mutations within the tyrosine kinase domain of the epidermal growth factor receptor gene has been attributed to a positive response to biological therapy of lung cancer by small-molecular tyrosine kinase inhibitors, gefitinib and erlotinib. Among the two most significant mutation types are deletions in exon 19 and a single point substitution in exon 21 (termed L858R). The exon 19 deletions can readily be examined by fragment analysis, due to the characteristic length difference between the normal and mutated PCR product. Analysis of the L858R point mutation, however, presents a greater challenge. The current paper is aimed at developing a sensitive, yet simple, low-cost mutation detection assay directed at the L858R mutation using a method based on CE of heteroduplexes under partial denaturing conditions. We perform optimization of separation conditions on different commercial instruments including ones equipped with 8, 16 and 96 capillaries. We present normalized migration reproducibility in the range from 1 (8 and 16) to 5% (96) RSD. A reliable distinction of the R836R silent polymorphism from a potential presence of the L858R mutation is also demonstrated. In its implementation, the presented assay is just another application running on a conventional CE platform without the need of dedicated instrumentation. [source] Prenatal diagnosis of carnitine palmitoyltransferase 2 deficiency in chorionic villi: a novel approachPRENATAL DIAGNOSIS, Issue 11 2003Bernadette Chadefaux Vekemans Abstract Carnitine palmitoyltransferase 2 (CPT2) deficiency, the most common autosomal recessive inherited disease of the mitochondrial long-chain fatty acid (LCFA) ,-oxidation, may result in three distinct clinical phenotypes, namely, a mild adult muscular form, a severe infantile hepatocardiomuscular disease, and a neonatal form, which includes dysmorphic features in addition to hepatocardiomuscular symptoms. Both the latter forms are life-threatening diseases, and prenatal diagnosis (PND) can be offered to couples at a one-fourth risk of having an affected child. PND of CPT2 deficiency hitherto relied mostly on mutation detection from fresh chorionic villi (10 weeks' gestation), since CPT2 activity could be assayed on cultured amniocytes only (16,17 weeks' gestation). We devised a CPT2 activity assay from 10 mg of chorionic villi sampling (CVS). Combining this enzymatic assay to haplotype study using polymorphic markers linked to the CPT2 gene, we were able to carry out within 2 days, CPT2 deficiency PND, in two unrelated families, using a CVS performed at the 11th week of gestation. Copyright © 2003 John Wiley & Sons, Ltd. [source] Prenatal diagnosis of 21-hydroxylase deficiency caused by gene conversion and rearrangements: pitfalls and molecular diagnostic solutionsPRENATAL DIAGNOSIS, Issue 13 2002Rong Mao Abstract Objectives The present paper reports the prenatal diagnosis of congenital adrenal hyperplasia (CAH) in two cases of 21-hydroxylase deficiency. DNA diagnostic errors can be caused by the presence of the highly homologous 21-hydroxylase pseudogene, CYP21P, adjacent to the functional gene, CYP21. The present paper details how complex gene conversions and rearrangements between the CYP21 and CYP21P pose unique complications for prenatal diagnosis. Methods Analysis of eight common mutations in the 21-hydroxylase gene as well as deletion of the entire gene is accomplished using polymerase chin reaction (PCR) followed by amplified created restriction site (ACRS) or allele-specific oligohybridization (ASO) and Southern blot followed by hybridization to a CYP21-specific probe. Linkage analysis was performed using microsatellite markers flanking the CYP21 gene. Results The direct mutation detection assay indicated a complicated gene conversion and rearrangement in the probands of both families. Interpretation of these rearrangements made it difficult to determine whether or not the fetuses would be affected with CAH. Linkage studies revealed that each fetus had inherited both parental disease chromosomes and was therefore predicted to be affected with CAH. Conclusion As observed in the two reported cases, direct DNA analysis may provide limited information due to gene conversion or rearrangement between the CYP21 and CYP21P genes. These cases suggest that direct mutation detection should be supported by linkage analysis, whenever possible, to provide more comprehensive information for the family. Copyright © 2002 John Wiley & Sons, Ltd. [source] High-resolution Melting Facilitates Mutation Screening of PYGM in Patients with McArdle DiseaseANNALS OF HUMAN GENETICS, Issue 3 2009Morten Duno Summary Mutations in PYGM, encoding the muscle-specific glycogen phosphorylase (myophosphorylase), are responsible for McArdle disease. Among Caucasians, a large proportion of patients are homozygous for the R50X mutation, but other mutations can affect all the 20 exons of PYGM, making mutation detection laborious. We have developed a high-resolution melting (HRM) assay for mutation detection in PYGM. Twelve McArdle patients were investigated, in whom pre-screening had ruled out homozygosity or compound heterozygosity for the two common G205S and R50X mutations. In total, we identified 16 different variations. Thirteen of these are pathogenic, and three were classified as polymorphisms. Nine variations had not previously been described. One of the novel mutations, c.2430C > T, was initially predicted to result in a silent G810G change, but cDNA analysis demonstrated that the mutation led to abnormal mRNA processing. The HRM protocol reduced the need for direct sequencing by approximately 85%, and is a good approach to search for new mutations in PYGM. [source] Large Genomic Mutations within the ATM Gene Detected by MLPA, Including a Duplication of 41 kb from Exon 4 to 20ANNALS OF HUMAN GENETICS, Issue 1 2008Simona Cavalieri Summary Mutation detection remains problematic for large genes, primarily because PCR-based methodology fails to detect heterozygous deletions and any duplication. In the ATM gene only a handful of multi-exon deletions have been described to date, and this type of mutation has been considered rare. To address this issue we tested a new MLPA (Multiplex Ligation Probe Amplification) kit that covers 33 of the 66 ATM exons, using for controls two previously characterized genomic deletions in addition to three A-T patients, taken from a survey of nine, who had missing four mutations unidentified after conventional mutation screening. We identified for the first time: 1) a ,41 kb genomic duplication spanning exons 4,20 (c.-30_2816dup41kb)(a.k.a., ATM dup 41 kb); 2) a novel genomic deletion including exon 31, and 3) in hemizygosis a point mutation in the non-deleted exon 31. In this study we extended mutation detection to nine new Italian A-T patients, using a combined approach of haplotype analysis, DHPLC and MLPA. Overall we achieved a mutation detection rate of >97%, and can now define a spectrum of ATM mutations based on twenty-one consecutive Italian families with A-T. [source] Leukocyte cDNA Analysis of NSD1 Derived from Confirmed Sotos Syndrome PatientsANNALS OF HUMAN GENETICS, Issue 6 2007M. Duno Summary Background: Haploinsufficiency of the NSD1 gene leads to Sotos syndrome (Sos), which is characterised by excessive growth, especially during childhood, distinct craniofacial features and variable degree of mental impairment. A wide spectrum of NSD1 mutations have been described in Sos patients, ranging from more than 100 different single nucleotide changes, to partial gene deletions, and to microdeletions of various sizes comprising the entire NSD1 locus. Objective: To investigate the NSD1 cDNA sequence in genetically confirmed Sos patients harbouring truncating and missense mutations. Method: Total RNA was isolated from a 250 ,l standard EDTA blood sample from nine genetically verified Sos patients, and subsequent reverse-transcribed into cDNA followed by PCR and direct sequencing of specific NSD1 cDNA sequences. Results: All nine mutations, including missense, nonsense and whole exon deletions, previously identified in genomic DNA, could confidently be detected in cDNA. Several NSD1 transcript splice variants were detected. Conclusion: Despite the fact that Sos is caused by haploinsufficiency, NSD1 transcripts containing nonsense and frame shift mutations can be detected in leukocyte-derived cDNA. The possibility therefore exists that certain NSD1 mutations are expressed and contribute to the phenotypic variability of Sos. NSD1 cDNA analysis is likely to enhance mutation detection in Sos patients. [source] An audit of genetic testing in diagnosis of inherited retinal disorders: a prerequisite for gene-specific interventionCLINICAL & EXPERIMENTAL OPHTHALMOLOGY, Issue 7 2009Monika Pradhan MS MRCOphth Abstract Background:, There has been an exponential increase in the number of genes implicated in inherited retinal disease over the last decade, but the genetic and phenotypic heterogeneity limited mutation detection. The high cost of sequencing and long turn around times meant that gene testing was not a viable option, particularly in New Zealand. Recently, advancements including development of micro-array-based mutation analysis and non-for-profit laboratories have resulted in affordable and time-efficient testing. This has enabled genetic diagnostics to become an integral component of the work-up for inherited retinal disease. Methods:, Genetic testing for inherited retinal disorders was initiated via the Ocular Genetic Clinic in Auckland 2 years ago. A retrospective audit of genetic testing over this period was carried out. The results of these tests and outcomes are discussed. Results:, Thirty-five probands have undergone genetic testing for retinal disorders. This has included X-Linked Retinoschisis, Leber Congenital Amaurosis, Retinitis Pigmentosa, Albinism, Achromatopsia, Usher syndrome, Stargardt disease and Mitochondrial disease. Of these, 54% of tests (19/35) showed a rare variant or pathogenic mutation. Three couples have proceeded to investigate the options of prenatal diagnosis and/or pre-implantation genetic diagnosis. Conclusion:, The introduction of genetic testing, largely via disease arrays, has been highly successful at clarifying disease genotype in our cohort. It is now a timely and cost-effective investigation that should be elemental to the assessment of inherited retinal disease. Genetic testing in an opportune fashion permits genetic counselling, enables families to make reproductive choices and might allow the possibility of gene therapy interventions. [source] | ||||||||||||||||