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At least 613 records · Page 34Linked to original sources
PubMed2005

A complete screen for mutations of the rhodopsin gene in a panel of Chinese patients with autosomal dominant retinitis pigmentosa.

OBJECTIVE: To evaluate the prevalence of rhodopsin (RHO) mutations and the genotype-phenotype relationships in Chinese patients with autosomal dominant retinitis pigmentosa (ADRP) by conformation sensitive gel electrophoresis (CSGE) and direct DNA sequencing. METHODS: We have screened the five coding exons and splice sites of RHO gene in 27 probands who had no relativity from Chinese ADRP families and 100 normal controls to identify disease-associated mutations, using CSGE and direct DNA sequencing. Family members of some probands with disease-associated mutations were also genotyped to determine whether the RHO mutations segregated with retinitis pigmentosa (RP) in their families. RESULTS: Two RHO mutations, Pro347Leu and Pro327 (1-bp del), were identified separately in two families, thus the frequency of RHO mutations among this set of Chinese ADRP families is about 7.4% (2/27). Pro347Leu mutation was found in one ADRP proband as well as three her children who also had RP. She had relatively early onset at about 17 years. The only one child without this mutation had no symptom or sign of RP at age of 34. Pro327 (1-bp del) was identified in a late-onset ADRP patient, who appeared night blindness around 30 years old and in her fifties electroretinogram (ERG) has been flat in both scotopic and photopic phases. Family analysis showed that this mutation also existed in her younger daughter and her elder sister, both of them also had RP. Three other family members were genotypically and phenotypically normal. Neither of the two mutations was detected in 100 normal controls. CONCLUSIONS: The frequency of RHO mutations in Chinese patients was lower than that in Europe and North America. The phenotype of the patients with Pro347Leu corresponded to type 1 ADRP, with severe rod degeneration and some cone preservation later, while the phenotype of the patients carrying Pro327 (1-bp del) corresponded to type 2 ADRP, with a concomitant loss of rod and cone visual function. CSGE was found to be a sensitive, simple, and practical method for the screening of a large number of samples under highly reproducible conditions, and could be utilized in routine molecular diagnostic laboratories.

Asian People↗
PubMed2004

Haplotype analysis of BRCA1 gene reveals a new gene rearrangement: characterization of a 19.9 KBP deletion.

Germ-line mutations in the BRCA1 gene cause hereditary predisposition to breast and ovarian cancer. BRCA1 and BRCA2 mutations account for about 40% of high-risk families. Mutation-screening methods generally focus on genomic DNA and are usually PCR based; they enable the detection of sequence alterations such as point mutations and small deletions and insertions. However, they do not allow the detection of partial or entire exon(s) loss, because the presence of the homologous allele results in a positive PCR signal, giving rise to a false-negative result. Identification of unusual haplotypes in patient samples by an expectation maximization algorithm has recently been suggested as a method for identifying hemizygous regions caused by large intragenic deletions. Using a similar approach, we identified a novel BRCA1 genomic rearrangement in a breast/ovarian cancer family negative at the first mutation screening; we detected a deletion encompassing exons 14-19, probably due to replication slippage between Alu sequences.

Alu Elements↗
PubMed1998

Novel trabecular meshwork inducible glucocorticoid response mutation in an eight-generation juvenile-onset primary open-angle glaucoma pedigree.

OBJECTIVE: This study aimed to update a large kindred with juvenile-onset primary open-angle glaucoma (POAG) first described in 1940 and to identify the underlying genetic cause of the disease. DESIGN: Molecular genetic study of a single kindred, including clinical examination, retrospective review of clinical and family history records, linkage analysis, and mutation screening. PARTICIPANTS: The retrospective review included 957 members of a single large family. The linkage study included 40 members of 1 branch of the family in which juvenile-onset POAG is segregating in an autosomal-dominant pattern. Mutation screening included 15 at-risk family members with juvenile-onset POAG, probands of 40 families with adult-onset POAG, probands of 11 additional unrelated juvenile-onset POAG families, and 43 unrelated normal control subjects. INTERVENTION: Clinical and family history records were obtained, ophthalmologic examinations were performed, and blood samples were drawn for use in genotyping. MAIN OUTCOME MEASURES: Allele sizes of microsatellite repeat genetic markers from the vicinity of the GLC1A glaucoma gene on chromosome 1q were assigned based on size fractionation of DNA fragments generated by polymerase chain reaction (PCR). Linkage was established by the method of lod scores. Mutations were identified by determination of the DNA sequence of PCR products amplified from the trabecular meshwork inducible glucocorticoid response (TIGR) gene. Glaucoma status for purposes of linkage and mutation analysis was based on a combination of ophthalmologic examination, clinical records, family history, and previously published information. For some individuals reported in the pedigree, but not included in the genotyping studies, less information was available as presented in the text and tables. RESULTS: Autosomal-dominant POAG was confirmed or reported for 78 members of an 8-generation family. Linkage analysis showed significant evidence for linkage of juvenile-onset POAG in one branch of the family to D1S452 (maximum lod score of 6.42 at a recombination fraction of 0.00) and other markers in the vicinity of the GLC1A gene on chromosome 1q. Screening of the TIGR gene identified a mutation that results in substitution of asparagine for isoleucine at codon 477 near the carboxyterminal end of the protein. CONCLUSIONS: The authors' findings strongly suggest that the juvenile-onset POAG locus in this family is the GLC1A locus and that the underlying cause of the disease is the IIe477Asn TIGR mutation that cosegregates with juvenile-onset POAG in one branch of this large family. Lack of samples from deceased individuals prevented the authors from determining whether reported adult-onset cases in this family could also be attributed to the IIe477Asn TIGR mutation. Absence of the IIe477Asn TIGR mutation from other juvenile- and adult-onset POAG families implies that this TIGR mutation is not a common cause of glaucoma.

Adolescent↗
PubMed2000

Histological and genetic analysis and risk assessment for chromosomal aberration after ICSI for patients presenting with CBAVD.

Intracytoplasmic sperm injection (ICSI) has opened a new field in the treatment of male infertility, leading to a debate concerning its genetic safety. In this study we present an analysis of 11 patients presenting congenital bilateral absence of the vas deferens (CBAVD). In all 11 cases, genetic counselling, histological analysis of testicular biopsies, cystic fibrosis transmembrane conductance regulator (CFTR) mutation screenings of both partners and spermatozoa three-colour fluorescent in-situ hybridization (FISH) analysis were performed. A total of 31 CFTR mutations were screened and mutations were found in eight out of 11 cases, with DeltaF508 being the most common mutation found. Histological analyses showed that seven out of 11 patients had normal tubule/membrane/interstitium (TMI) and Johnsen scores, while the remaining four patients had mild impairment of testicular parenchyma. The average aneuploidy rate was 6.8 +/- 3.9% compared with two control subjects with 4.4 and 5.4% aneuploidy rates respectively, using FISH analysis. After ICSI, the fertilization and pregnancy rates were 66.2 and 22.7% respectively. Thus, in our case of CBAVD, the risk of chromosomal aberration following ICSI, in the absence of a CFTR mutation in the male patient and/or in his partner, was not higher than in normal fertile men. Furthermore, the pregnancy success rate following ICSI of these CBAVD patients was comparable to the general ICSI population, even when histological analysis showed limited spermatogenesis.

Adult↗
PubMed1999

A proven de novo germline mutation in HNPCC.

Hereditary non-polyposis colon cancer (HNPCC) is a heterogeneous group of tumour predisposition syndromes caused by germline mutations in at least four different mismatch repair genes. HNPCC patients are prone to the development of carcinomas of the intestinal tract and other specific sites. Identification of presumptive HNPCC patients is primarily based on a positive family history of colorectal cancer in at least two generations. In the course of mutation screening of the MLH1 and MSH2 genes in patients manifesting a carcinoma of the HNPCC tumour spectrum before the age of 45 years, we identified a germline MSH2 344delA frameshift mutation in a male proband. This index patient, at the age of 25 years, initially developed a large rectal adenoma that was removed by polypectomy. Ten years later he was operated on for an invasive right sided colon carcinoma in the caecum (International Union Against Cancer (UICC) stage III). The mother and father, aged 61 and 66 years, respectively, were healthy and had no family history of colorectal cancer. Subsequent molecular analyses excluded the germinal MSH2 344delA alteration identified in their son and at the same time paternity was confirmed with a set of informative polymorphic markers. Thus, the genetic alteration identified in our patient definitely represented a de novo germline mutation in one of the major HNPCC genes. This case report of a patient with colorectal cancer at a relatively young age with no family history is intended to encourage mutation screening of the MSH2 and MLH1 genes in similar cases to find out whether this group of patients contains an increased proportion of de novo mutations in mismatch repair genes.

Adaptor Proteins, Signal Transducing↗
PubMed2005

In vitro mutation and selection of doubled-haploid Brassica napus lines with improved resistance to Sclerotinia sclerotiorum.

This paper describes a new protocol to develop doubled-haploid (DH) Brassica napus lines with improved resistance to Sclerotinia sclerotiorum. In this protocol, haploid seedlings derived from microspore cultures of B. napus were used to produce haploid calli for in vitro mutation-selection. For routine screening, mutation was induced by EMS (ethylmethane sulfonate) or occurred spontaneously, and screening for resistant mutants occurred on media with added oxalic acid (OA) as a selection agent. In tests with selected lines, the optimal concentration of EMS for mutation was determined to be 0.15%, and the optimal concentration of OA for in vitro screening was 3 mmol/l (half lethal dose was 3.1 mmol/l) for the first cycle of screening. There was an accumulated effect of OA toxicity on calli over two cycles of screening, but the growth and capacity of the surviving calli for regenerating seedlings were not affected by OA. Of the 54 DH lines produced from the in vitro mutation-selection, two DH lines of resistant mutants, named M083 and M004, were selected following seedling and glasshouse tests. The resistance of M083 and M004 to S. sclerotiorum following tests with both mycelial inoculum and OA was greater than that of their donor lines and the resistant control Zhongyou 821. In both glasshouse and field disease nurseries, disease indices on M083 and M004 were less than 50% of those of the control. The time required for M083 and M004 to mature was 14 days and 10 days shorter, respectively, than that of their donor lines. Furthermore, M083 had more pods per inflorescence, a greater 1,000 seed weight and higher yield than its donor line. Random amplified polymorphic DNA characterisation showed that M083 had DNA band patterns that differed from its donor line.

Ascomycota↗
PubMed2006

A novel missense RP1 mutation in retinitis pigmentosa.

AIMS: More than 20 mutations associated with retinitis pigmentosa (RP) have been identified in the retinitis pigmentosa 1 (RP1) gene, all of them leading to the production of a truncated protein without 50-70% of the C-terminal of the RP1 protein. RP1 was recently found to be a microtubule-associated protein (MAP) and responsible for the organisation of the photoreceptor outer segment. The N-terminal doublecortin (DCX) domain of RP1 is essential for its function. But how the C-terminal of the protein affects its function is still not known. This study aims to get a better understanding of the RP1 gene by mutation screening on RP patients. METHODS: Peripheral blood was taken from 72 RP patients. Together with 101 RP patients and 190 control subjects previously reported, mutation screening was performed by polymerase chain reaction (PCR) and direct sequencing. Statistical analysis was performed using SPSS. RESULTS: Two novel missense sequence changes, D984G and C727W, and one novel variant, 6492T>G, at the 3' untranslated region were found. They were not found in 190 control subjects. D984G causes RP. It creates two possible N-myristoylation sites according to PROSITE. C727W does not segregate with RP in the family. It abolishes an N-myristoylation site. R872H, a previously reported polymorphism, was predominantly present in control subjects (P=0.001). CONCLUSIONS: Our results suggest that disruption of the C-terminal of RP1 may be associated with the development of RP, and the possible involvement of the RP1 polypeptide downstream of its DCX domain in normal RP1 function.

Adult↗
PubMed1995

The DNA and steroid binding domains of the glucocorticoid receptor are not altered in mononuclear cells of treated CLL patients.

The aim of this study was to investigate whether mutations in the glucocorticoid receptor could account for the increasing unresponsiveness of patients with chronic lymphatic leukemia (CLL) to combination chemotherapy. The receptor was tested immunocytochemically, in steroid binding assays, and by a mutation screening (denaturing gradient gel electrophoresis) of the receptor-cDNA. The receptor concentration, as measured by staining and steroid binding test, varied considerably but showed no clear correlation to clinical response. Using a highly sensitive mutation screening assay of the DNA- and the steroid-binding region, none of the treated patients revealed any mutation, suggesting that the glucocorticoid receptor in the CLL patients tested is not altered in these domains. In one individual who had not been treated before analysis a silent mutation was found in one receptor allele. The results suggest that mechanisms other than altered ligand or DNA binding of the receptor may be responsible for the lack of response to chemotherapy. This conclusion is discussed in relation to the mechanism of corticoid resistance in mouse and human lymphoma cells in culture.

Animals↗
PubMed2001

Screening for mutations in the steroidogenic acute regulatory protein and steroidogenic factor-1 genes, and in CYP11A and dosage-sensitive sex reversal-adrenal hypoplasia gene on the X chromosome, gene-1 (DAX-1), in hyperandrogenic hirsute women.

Abstract Abnormalities in adrenal and/or ovarian steroidogenesis are found in most patients with hirsutism. The rate-limiting step in the synthesis of steroids in the ovary and the adrenal is the conversion of cholesterol into pregnenolone by cholesterol side-chain cleavage enzyme (P450scc), encoded by the gene CYP11A, after cholesterol is introduced into the mitochondria by the steroidogenic acute regulatory protein (StAR). DAX-1 is a repressor of StAR gene expression, and steroidogenic factor-1 (SF-1) is a regulator of CYP11A, DAX-1, and StAR gene. Mutations in any of these factors resulting in gain of function, or loss of repression, of StAR or P450scc might contribute to the steroidogenic abnormalities present in hirsute patients. In the present study we have screened, using heteroduplex analysis, the genes encoding StAR and SF-1 as well as DAX-1 and CYP11A for mutations in genomic DNA from 19 women presenting with hirsutism and increased serum androgen levels. When variants were found, analysis was extended to a larger group of hyperandrogenic patients and nonaffected women. Two variants were identified in the SF-1 gene. A G-->C change in exon 6, resulting in an Arg(365)Pro mutation, was found in 1 of 45 patients, but not in controls. Also, a Gly(146)Ala missense mutation, resulting from a G-->C change in exon 4, was found in 2 of 48 patients and in 2 of 50 nonaffected individuals. We identified a C-->T base pair change at position -33 of the StAR gene. Three of 48 patients and 3 of 43 controls presented this variant. No mutations were found in coding regions of the StAR gene. Analysis of CYP11A-coding regions identified a G-->A change in exon 3, resulting in a Val(179)Ile missense mutation. This mutation was found in 1 of 29 patients studied and was not present in 50 controls. Finally, analysis of DAX-1 showed no variant in any of the women studied. In conclusion, mutations in StAR, SF-1, CYP11A, and DAX-1 are seldom found in hirsute patients and do not explain the steroidogenic abnormalities found in these women.

Adrenal Glands↗
PubMed1996

Population-specific screening by mutation analysis for diseases frequent in Ashkenazi Jews.

We describe a partially automated DNA mutation assay for detecting the most frequent mutations in the alpha-subunit of beta-hexosaminidase A, the acid beta-glucosidase and the cystic fibrosis transmembrane conductance regulator genes for the Ashkenazi Jewish population. The assay detects carriers for Tay-Sachs disease, Gaucher disease, and cystic fibrosis with sensitivities of at least 92%, 96%, and 97%, respectively. Among 1,364 young adults of Ashkenazic ancestry in the Dor Yeshurim community who were tested, 52 were Tay-Sachs carriers, 110 were Gaucher carriers, and 62 were cystic fibrosis carriers. Ten individuals were carriers for two diseases, and three unsuspected cases were diagnosed with Gaucher disease based on mutation test results. In addition to Tay-Sachs mutation data, results for hexosaminidase A activity were also available. All of 1,254 samples normal by enzyme quantitation were also negative for the three alpha-subunit mutations tested, and all of 43 samples with 'inconclusive' enzyme results were negative by DNA. Only 52 of 67 samples positive by enzyme assay were also positive for one of the three mutations tested for Tay-Sachs disease. The data suggest a high degree of false positivity inherent in enzyme identification of carriers. There are no correlative methods to assess the sensitivity of Gaucher and CF carrier testing. The results show that population screening can be carried out efficiently by DNA analysis, with the accrual of carrier information for three separate diseases conducted as a single test. Furthermore, the DNA method for Tay-Sachs screening appears to exceed the specificity of hexosaminidase A enzyme testing.

Adult↗
PubMed1995

Screening for mutations in haemophilia A patients by multiplex PCR-SSCP, Southern blotting and RNA analysis: the detection of a genetic abnormality in the factor VIII gene in 30 out of 35 patients.

The molecular characterization of mutations in haemophilia A patients in this study was carried out by PCR-SSCP, Southern blotting, and reverse transcribed-PCR. A multiplex PCR in which four to eight exons were co-amplified was developed to reduce the time needed for screening the coding region of the factor VIII gene. PCR-SSCP was used to screen for small molecular defects, and reverse transcriptase PCR combined with Southern blotting was used to screen DNA for the inversions that occur frequently in intron 22 of the factor VIII gene. A group of 35 haemophilia A patients was analysed by these methods and 31 mutations were detected. In one patient two mutations were identified. The cases of mild and moderate haemophilia A showed changes in single nucleotides which predicted amino acid changes. The patients affected by severe haemophilia A showed two types of mutations. First, deletions or insertions that result in a frameshift in the coding DNA sequence were observed. Second, inversions were found which result in a disruption of the gene. With the screening strategies used we succeeded in elucidating an abnormality in the factor VIII gene in 30/35 haemophilia A patients.

Base Sequence↗
PubMed1998

Enzymatic mutation detection. Procedure for screening and mapping of mutations by immobilised endonuclease VII.

Endonuclease VII (endo VII) binds to non-pairing nucleotides in DNA. This served as the basis for the development of a mutation detection assay involving immobilised endo VII and heteroduplex DNAs made by hybridisation of mutant and wild type DNA. The use of microtiter plates allows screening of large numbers of samples. Localisation of mutations in positive samples can be done in the same assay in a second optional step.

DNA↗
PubMed2004

Evaluation of a DHPLC-based assay for rapid detection of RET germline mutations in Italian patients with medullary thyroid carcinoma.

Causative gain-of-function mutations of the RET tyrosine-kinase receptor gene have been reported in more than 95% of inherited cases of medullary thyroid carcinoma (MTC; OMIM# 155240). Most RET activating mutations are clustered in mutational "hot spots" in exons 10, 11, 13, 14, 15 and 16 and are usually detected by single-strand conformation polymorphism (SSCP) followed by direct sequencing. To improve sensitivity, time and costs of mutational screening we have developed a denaturing high performance chromatography (DHPLC) protocol, based on the detection of heteroduplex molecules by ion-pair reverse-phase liquid chromatography under partially denaturing conditions. The mutational screening of RET exons 10, 11, 13-16 was performed in a total of 111 subjects, including 45 MTC patients and 49 relatives with known RET mutations and 17 individuals, being at risk of hereditary MTC and carrying unknown RET alleles. Heteroduplex peaks with a distinct and reproducible DHPLC elution profile allowed the detection of both rare and common RET mutations. Overall, the DHPLC-based methodology showed a high level of sensitivity and accuracy, nearing 100%. Furthermore, our protocol showed the ability to identify: 1) all the mutated codons of RET located in the "hot spots" domain; 2) the different point mutations occurring in the same codon of RET gene; 3) less frequent or rare mutations; 4) polymorphisms. As such, it can be proposed as a relatively simple and highly accurate method for a rapid genetic testing for members of MTC families.

Carcinoma, Medullary↗
PubMed2003

Screening for mutations in the RYR1 gene in families with malignant hyperthermia.

Malignant hyperthermia (MH) is a potentially lethal pharmacogenetic predisposition associated with a susceptibility to volatile anesthetics and depolarizing muscle relaxants that lead to a fulminant anesthetic crisis with hyperthermia, skeletal muscle rigidity, respiratory and metabolic acidosis, and muscle rhabdomyolysis. Malignant hyperthermia crises are caused by an abnormal regulation of the calcium release mechanism, which reflects the consequences of disturbed skeletal muscle calcium homeostasis. We screened 64 individuals of 27 unrelated families for the most frequently described mutations associated with MH in the genes RYR1 and CACNL1A3. We identified only one family with the Arg614Cys mutation but with a discordant segregating pattern to the in vitro contracture test (IVCT). To elucidate which other mechanism could lead to susceptibility in the members of this family, we tested it for further MH susceptibility loci. The same haplotype was shown to segregate with the individuals carrying the Arg614Cys mutation in chromosome 19; however, the other susceptible and equivocal individuals do not share this haplotype. Markers for the susceptible locus in chromosome regions 17q, 7q, 3q, and 5p did not segregate with the IVCT phenotype in the susceptible individuals, suggesting that the positivity of the IVCT could be attributable to other ambient factors.

Calcium↗
PubMed2002

Nijmegen breakage syndrome: clinical characteristics and mutation analysis in eight unrelated Russian families.

OBJECTIVE: The purpose of the study was to ascertain patients with Nijmegen breakage syndrome (NBS) in the Russian population and characterize the clinical phenotype and molecular genotype of these patients. STUDY DESIGN: Eight unrelated Russian patients with possible diagnoses of NBS were identified. Family histories were collected and clinical and laboratory analyses were carried out. Mutation screening of the NBS1 gene was carried out to confirm the diagnosis in 7 cases. RESULTS: All patients had the key diagnostic features of NBS. One patient had acute myeloblastic leukemia (AML). Two patients had bone marrow aplasia, not previously described as a feature of NBS. Mutation screening of the NBS1 gene revealed that 6 patients were homozygous for the 657del5 mutation, whereas a seventh patient was a compound heterozygote, having the 657del5 mutation and an additional novel mutation, 681delT. CONCLUSIONS: Molecular analyses confirmed the diagnosis of NBS in 7 of the patients. The surprising finding of bone marrow aplasia or AML in 3 of 7 patients raises the possibility of a connection between NBS and another DNA damage disorder, Fanconi anemia.

Chromosome Fragility↗
PubMed2003

Rapid detection of beta-globin gene mutations and polymorphisms by temporal temperature gradient gel electrophoresis.

BACKGROUND: Inherited hemoglobin disorders represent the most common Mendelian disease worldwide. Prevention programs based on molecular diagnosis of heterozygous carriers and/or patients require the use of reliable mutation scanning methods in at-risk populations. METHODS: We developed a rapid and highly specific mutation-screening test based on temporal temperature gradient gel electrophoresis (TTGE). We analyzed 889 beta-thalassemia genes from homozygous beta-thalassemia patients and unrelated individuals with heterozygous beta-thalassemia. Previously reported common mutations were screened by reverse dot blots using allele-specific probes. The rare mutations were analyzed by TTGE. RESULTS: We found common mutations in 753 beta-thalassemia genes. TTGE analysis in the rest of the genes showed the presence of mutations in different regions of the beta-globin gene in 134 of them, and these mutations were characterized by DNA sequencing. In the two genes in which mutations were not identified, large deletions spanning beta-globin gene were suspected. CONCLUSIONS: Compared with other approaches for comprehensive mutation screening, the reported method is rapid, highly sensitive, cost-effective, and suitable for high-throughput screening of a large number of samples.

Electrophoresis, Polyacrylamide Gel↗
PubMed2005

Comparative genomics and gene expression analysis identifies BBS9, a new Bardet-Biedl syndrome gene.

Bardet-Biedl syndrome (BBS) is an autosomal recessive, genetically heterogeneous, pleiotropic human disorder characterized by obesity, retinopathy, polydactyly, renal and cardiac malformations, learning disabilities, and hypogenitalism. Eight BBS genes representing all known mapped loci have been identified. Mutation analysis of the known BBS genes in BBS patients indicate that additional BBS genes exist and/or that unidentified mutations exist in the known genes. To identify new BBS genes, we performed homozygosity mapping of small, consanguineous BBS pedigrees, using moderately dense SNP arrays. A bioinformatics approach combining comparative genomic analysis and gene expression studies of a BBS-knockout mouse model was used to prioritize BBS candidate genes within the newly identified loci for mutation screening. By use of this strategy, parathyroid hormone-responsive gene B1 (B1) was found to be a novel BBS gene (BBS9), supported by the identification of homozygous mutations in BBS patients. The identification of BBS9 illustrates the power of using a combination of comparative genomic analysis, gene expression studies, and homozygosity mapping with SNP arrays in small, consanguineous families for the identification of rare autosomal recessive disorders. We also demonstrate that small, consanguineous families are useful in identifying intragenic deletions. This type of mutation is likely to be underreported because of the difficulty of deletion detection in the heterozygous state by the mutation screening methods that are used in many studies.

Alleles↗
PubMed2000

Rapid detection of deletion, insertion, and substitution mutations via heteroduplex analysis using capillary- and microchip-based electrophoresis.

In this report, we explore the potential of capillary and microchip electrophoresis for heteroduplex analysis- (HDA) based mutation detection. Fluorescent dye-labeled primers (6-FAM-tagged) were used to amplify the DNA fragments ranging from 130 to 400 bp. The effects of DNA fragment length, matrix additives, pH, and salt were evaluated for capillary electrophoresis- (CE) and/or microchip electrophoresis-based HDA, using six heterozygous mutations, 185delAG, E1250X (3867GT), R1443G (4446CG), 5382insC, 5677insA in BRCA1, and 6174delT in BRCA2. For this system, the effective fragment size for CE-based HDA was found in the range of 200-300 bp, however, the effective range was 150-260 bp for microchip-based HDA. Sensitivity studies show CE-based HDA could detect a mutated DNA present at only 1%-10% of the total DNA. Discrimination between wild-type and deletion or insertion mutations in BRCA1 and BRCA2 with CE-based HDA could be achieved in <8 min, while the substitution mutations required 14 min of analysis time. For each mutation region, 15 samples were run to confirm the accuracy and reproducibility of the method. Using the method described, two previously reported mutations, E1038G (3232AG, missense) and 4427 C/T (4427CT, polymorphism), were detected in the tested samples and confirmed by DNA sequencing. Translation of the CE-based methodology to the microchip format allowed the analysis time for each mutation to be decreased to 130 sec. Based on the results obtained with this model system, it is possible that CE-based HDA methodologies can be developed and used effectively in genetic testing. The fast separation time and automated operation afforded with CE instrumentation provide a powerful system for screening mutations that include small deletions, insertions, and point mutations. Translation to the microchip platform, especially to a multichannel microchip system, would allow for screening mutations with high throughput.

BRCA2 Protein↗