Detection of a transcript abnormality in mRNA of the SLC12A3 gene extracted from urinary sediment cells of a patient with Gitelman's syndrome.

To date, many mutations, including intronic nucleotide changes, in the SLC12A3 gene encoding the thiazide-sensitive sodium-chloride cotransporter (NCCT) have been reported in Gitelman's syndrome (GS) patients. However, it has not been clarified whether intronic nucleotide changes affect mRNA content. Since mRNA analysis is possible only after obtaining renal biopsy specimens, no studies have been conducted to identify transcript abnormalities in GS. In the study reported here, we investigated such transcript abnormalities for the first time by using mRNA expressed in a patient's urinary sediment cells. Direct sequencing analysis of leukocyte DNA disclosed one known missense mutation (R399C) and one known nucleotide change of the splicing acceptor site of intron 13 (1670-1 g > t). mRNA extracted from the urinary sediment cells was analyzed by RT-PCR to determine the pathogenic role of the intron mutation. A fragment encompassing exon 13 to 15 was amplified as two products, one consisting of all three exons and the other lacking only exon 14 in its entirety. Our investigation was the first to demonstrate exon 14 skipping in an NCCT transcript in renal cells. This methodology thus constitutes a potential noninvasive analytical tool for every inherited kidney disease.

C-terminal truncated dystrophin identified in skeletal muscle of an asymptomatic boy with a novel nonsense mutation of the dystrophin gene.

Mutations that cause premature stop codons in the dystrophin gene lead to a complete loss of dystrophin from skeletal muscle, resulting in severe Duchenne muscular dystrophy. Here, a C-terminally truncated dystrophin resulting from a novel nonsense mutation is shown for the first time to be localized to the muscle plasma membrane. An asymptomatic 8-y-old boy was examined for dystrophin in skeletal muscle because of high serum creatine kinase activity. Remarkably, no dystrophin labeling was seen with an MAb against the C-terminal domain, suggesting the presence of an early stop codon in the dystrophin gene. Labeling with an antibody specific to the N-terminal domain, however, revealed weak, patchy, and discontinuous staining, suggesting limited production of a truncated form of the protein. Molecular analysis revealed a novel nonsense mutation (Q3625X) as a result of a single nucleotide change in the patient's genomic DNA (C10873T), leaving 1.6% of dystrophin gene product unsynthesized at the C terminus. Dystrophin mRNA analysis did not show rescue of the nonsense mutation as a result of exon-skipping by an alternative splicing mechanism. This is the first report of an asymptomatic dystrophinopathy with a nonsense mutation in the dystrophin gene.

A novel cryptic exon in intron 3 of the dystrophin gene was incorporated into dystrophin mRNA with a single nucleotide deletion in exon 5.

The dystrophin gene, which is mutated in Duchenne muscular dystrophy, is the largest human gene. A full spectrum of the gene transcripts has not been fully elucidated yet, although two cryptic exons have so far been identified in the 5' region of the dystrophin gene. Here, a novel dystrophin mRNA containing a 62-nucleotide insertion between exons 3 and 4 was identified in lymphocytes from a Japanese Duchenne muscular dystrophy patient with a single nucleotide deletion in exon 5. The inserted 62-nucleotide sequence was found to be homologous to part of intron 3 and it was revealed that the insertion possessed branch point and both acceptor and donor splice site consensus sequences perfectly. Therefore, the 62-bp insertion sequence was considered to be a novel exon and was designated as exon 3a. However, this insertion was not present in the patient's muscle and 12 different normal tissues that were screened. The physiological role of the novel cryptic exon remains to be clarified.