2-(trimethylsilyl)ethanethiol in nucleoside chemistry. A short route for preparing thionucleosides and their methyl disulfides.
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Disulfides↗
Biomedical subjects
S Chambert
Publications and source records attributed to S Chambert.
Point mutations in the dystrophin gene: evidence for frequent use of cryptic splice sites as a result of splicing defects.
Ten different mutations have been identified in patients with Becker (n = 1) or Duchenne (n = 9) muscular dystrophy using reverse transcription of total RNA, polymerase chain reaction amplification of the whole coding region of the gene and protein truncation test (PTT) analysis. Seven mutations had not been reported previously, and these consist in three nonsense mutations (Q2522X, E2726X, R3381X), three frameshifting deletions (3686-3687delGT, 5126delA, 5759delC), and four splicing defects of which the effects on the muscle dystrophin mRNA transcripts have been analyzed. In one case, a 3' splice-site mutation (IVS74-2A-->G) resulted in a complex pattern of exon skipping involving exons of the C-terminal domain. In the three other cases, nucleotide substitutions in splice donor (IVS26+2T-->A, IVS65+1G-->A) or acceptor (IVS8-15A-->G) recognition sequences led to the use of cryptic splice sites, with consequent insertions of intronic sequences in the processed mRNA. Up to 34% (70/203) of the point mutations reported to date in the dystrophin database (http://www.dmd.nl) affect splice sites of the dystrophin gene. However, altered mRNA splicing has been confirmed experimentally in only 23% of cases (16/70). Combined with PTT, the transcript analysis protocol defined in this study permits direct determination of the impact of intronic variations on the structure of dystrophin mRNA and of the resulting consequences on the translational reading frame. We present evidence for a frequent use of cryptic splice sites as a result of splicing defects.
Base Sequence↗
Mutation analysis of the dystrophin gene in Southern French DMD or BMD families: from Southern blot to protein truncation test.
Data from 6 years of experience in molecular diagnosis of Duchenne (DMD) and Becker (BMD) muscular dystrophy in Southern France are reported. DMD and BMD patients have been extensively analyzed for deletions and for point mutations in the dystrophin gene. By scanning the whole coding sequence as reverse-transcribed from lymphocytes or muscular RNA by the protein truncation test, we have reached a minimum of an 86% detection rate for point mutations responsible for DMD; these mutations consist of nonsense, frameshifting, and splicing mutations. Four of 12 small alterations identified in our sample are novel and described in this study. We also present an improved protocol for the automated detection of fluorescently labeled duplex polymerase chain reactions of six known intragenic microsatellites (Dys II, TG 15, STRs 44, 45, 49, and 50). Accurate sizing of the alleles at each locus was performed, and we elucidated the sequence of several repeat units. Allele frequencies at each of the six microsatellite loci and at one restriction fragment length polymorphism site (intron 16/TaqI) were defined in a sample of normal, DMD, and BMD X chromosomes from Southern France. The determination of the grandparental origin of either deletions or point mutations revealed differences depending on the type of the mutation, with most of the deletions occurring in oogenesis and most of the point mutations occurring in spermatogenesis.
Blotting, Southern↗
[Genotypic diagnosis of Duchenne and Becker muscular dystrophies].
Duchenne (DMD) and Becker (BMD) are allelic forms of a X-linked neuromuscular disorder. Both are caused by mutations arising in the gene encoding dystrophin, a cytoskeletal protein. Two-thirds of DMD/BMD patients have large deletions localised in two hot spots, and the remaining cases are presumed to be caused by point mutations. Since Duchenne muscular dystrophy is a serious disorder for which at present there is no effective treatment, much emphasis has been given to prevention. This involves the ascertainment of women likely to have an affected son, and the provision of genetic counselling and prenatal diagnosis for such women. Accurate carrier detection and genetic counselling depend upon identifying the mutation itself in the proband. Large deletions are easily identified using multiplex polymerase chain reaction (PCR) whereas detection of point mutations is restricted to a few number of specialized laboratories. Hence carrier and prenatal diagnosis in 40% of families rely heavily on indirect approaches which presents major drawbacks and are not applicable in sporadic cases of DMD or BMD. We developped a strategy for searching small alterations in the dystrophin gene. As most of the non-deletion mutations cause a premature termination of translation, we have used the Protein Truncation Test to scan specifically the dystrophin transcripts isolated from muscle biopsies. This approach allowed to detect the disease-causing mutations in more than 90% of the patients who have been investigated; his efficiency is thus significantly higher than DNA-based strategies. The identification of the mutation in non deleted sporadic cases allows the at-risks females to benefit from an accurate diagnosis. Also, the characterization of the molecular defects provides a better understanding of the molecular pathology of the dystrophin gene.
Dystrophin↗
[RNA isolation and purification methods].
Recent advances in human, bacterial and viral genome projects and the development of quantitative real-time reverse transcription-polymerase chain reaction methods offer the possibility of analysing a large number of gene transcripts. These molecular developments represent an important advancein the field of genetics, cancer, virology, bacteriology and hematology. A limiting step remains the isolation of high quality mRNA purified from biological samples. This review describes the different methods used to isolate mRNA from biological samples and to verify RNA integrity and gives precise details about RNA storage conditions.
Blotting, Northern↗