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Direct Sequencing Method (direct + sequencing_method)
Selected AbstractsA Screening test for the prediction of Dravet syndrome before one year of ageEPILEPSIA, Issue 4 2008Junri Hattori Summary Purpose: Our aim was to develop a screening test to predict Dravet syndrome before the first birthday based on the clinical characteristics of infants and the SCN1A mutation analysis. Methods: Ninety-six patients who experienced febrile seizures before the age of one were enrolled. The patients were divided into two groups,the Dravet syndrome group (n = 46) and the non-Dravet syndrome group (n = 50). We compared the clinical characteristics before one year of age of the two groups. We analyzed all coding exons of the SCN1A gene by the direct sequencing method. Scores from 0 to 3 were assigned to each risk factor based on the odds ratio and p-value. Results: An age of onset of febrile seizure , 7 months, a total number of seizures , 5, and prolonged seizures lasting more than 10 min. were regarded as significant risk factors for Dravet syndrome. Other factors highly predictive of this syndrome were hemiconvulsions, partial seizures, myoclonic seizures, and hot water,induced seizures. A total clinical score of six or above was the cutoff value indicating a high risk of Dravet syndrome. SCN1A missense and truncated mutations were detected significantly more often in the Dravet syndrome group than in the non-Dravet syndrome group. Discussion: This simple screening test was designed to be used by general pediatricians. It could help to predict Dravet syndrome before one year of age. If the sum of the clinical risk score is , 6, then the performance of an SCN1A mutation analysis is recommended. [source] The effects of RANTES/CCR5 promoter polymorphisms on HIV disease progression in HIV-infected KoreansINTERNATIONAL JOURNAL OF IMMUNOGENETICS, Issue 2 2008D. H. Jang Summary Recent studies have reported that two single nucleotide polymorphisms (SNPs) in the RANTES gene promoter region, ,403G/A and ,28C/G, are associated with a slower rate of decline in CD4+ T-cell number, whereas genetic polymorphisms within the CCR5 promoter are linked to acceleration of AIDS progression. In this study, we investigated the distribution of SNPs in the RANTES and CCR5 promoters and the association between these SNPs and HIV-1 disease progression in HIV-infected Koreans. Twenty-seven long-term non-progressors (LTNPs), 29 AIDS patients and 39 HIV-uninfected persons were enrolled in this study. SNPs for the RANTES and CCR5 promoters were determined by polymerase chain reaction,restriction fragment length polymorphism (PCR-RFLP) and a direct sequencing method. In the analysis of RANTES promoter polymorphisms, the genotypic and allelic frequencies of the RANTES ,28G mutation were significantly lower in HIV-infected patients than in HIV-uninfected persons (P = 0.005 and P = 0.001, respectively). The genotypic frequencies of RANTES ,28G and ,403A mutations did not differ significantly between LTNPs and AIDS patients. The frequencies of three CCR5 promoter polymorphisms, designated 59029 G/A, 59353T/C, and 59402G/A, did not differ significantly between HIV-uninfected and HIV-infected patients. However, the allelic frequency of CCR559353C was significantly higher in AIDS patients than in LTNPs (P = 0.003). These results suggest that RANTES-28G and CCR5 59353C mutations might be associated with HIV infection or pathogenesis in the Korean population. [source] Mutational Analysis and Functional Correlation With Phenotype in German Patients With Childhood-Type HypophosphatasiaJOURNAL OF BONE AND MINERAL RESEARCH, Issue 12 2001Hideo Orimo Abstract The tissue-nonspecific alkaline phosphatase (TNSALP) gene from five German family members with childhood-type hypophosphatasia (HOPS) was analyzed using the polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP)-direct sequencing method. Four novel missense mutations (T51M, R54S, L258P, and R374H) and two that had been described previously (A160T and R206W) were detected in the respective patients. Mutation A160T was detected in 3 distinct patients, and a polymorphism V505A that had been described previously was detected in the same allele as L258P mutation in 1 patient and in 2 fathers whose V505A alleles were not transmitted to the probands. No other mutations were found in 2 patients. Transient expression of the mutant proteins in COS-1 cells showed that the four novel mutations and R206W were severe alleles, whereas A160T was a moderate allele. Analysis of its enzymatic activity and genetic transmission patterns confirmed that V505A was a polymorphism. Immunoprecipitation of the transiently expressed proteins showed that levels of the 80-kDa mature form of the enzyme were diminished or absent with the severe alleles; instead, levels of high-molecular mass disulfide-linked aggregates were increased. These results suggest that in compound heterozygotes, the combination of severe and moderate alleles may combine to cause the mild phenotype seen in childhood-type HOPS. [source] Nijmegen breakage syndrome gene (NBS1) alterations and its protein (nibrin) expression in human ovarian tumoursANNALS OF HUMAN GENETICS, Issue 5-6 2002J. PLISIECKA-HA We looked for NBS1 gene (602667) alterations and changes in nibrin expression in 162 human gynaecological tumours, mostly ovarian. Exons 6,8 and 10 of the NBS1 gene were evaluated by the SSCP and direct sequencing method. Nibrin expression was detected immunohistochemically with the use of the p95NBS1 (Ab-1) antibody. The 657del5 mutation (Slavic mutation) was found in two of 117 carcinomas studied (1.7%) , in both cases it was present in the germline; one of these tumours showed loss of heterozygosity (LOH) for the 657del5 mutation and loss of nibrin expression. We have found three types of novel germline intron variants: (1) two concomitant transitions (G to A) at bases 14009 and 14256; (2) C to T transition at base 13998; (3) G to C transversion at base 20035. Among the carcinomas studied, the intron variants were associated with a clear cell histological type (p = 0.004). Our results may suggest that NBS1 gene alterations contribute to the development of rare ovarian carcinomas. LOH for 657del5 in tumour tissue may support the hypothesis that the NBS1 gene functions as a tumour suppressor. [source] | ||||||||||