Direct detection of exon deletions/duplications in female carriers of and male patients with Duchenne/Becker muscular dystrophy.

Duchenne and Becker muscular dystrophies (DMD/ BMD) are X-linked allelic neuromuscular disorders that have a prevalence of 1 in 3500 live-born males. The genetic defect is the result of mutations of the dystrophin gene, which encodes a 427-kDa rod-shaped cytoskeletal protein. The locus is very unstable: one-third of all DMD/ BMD cases represent new mutations without a family history of the disease. Approximately 50–70% of DMD/BMD cases are the result of macrodeletions, and partial gene duplications have been reported in 6% of patients. Both macrodeletions and macroduplications are preferentially clustered in two areas, the aminoterminal (exons 3–7) and the central (exons 44–55) regions. The remaining cases are presumably attributable to point mutations or small insertions/deletions scattered along the entire gene. PCR detection of macrodeletions is very useful in the analysis of affected males, but it provides no information about the carrier status of at-risk women. Carrier status within families is usually assessed by haplotype analysis, fluorescence in situ hybridization, amplification of ectopic transcripts, dosage analysis on Southern blots, or separation of quantitative PCR products by gel electrophoresis. Semiquantitative methods, based on the separation of fluorescently labeled amplified exons by gel or capillary electrophoresis, are also available. Here we report a quantitative PCR method, followed by separation by capillary gel electrophoresis of the fluorescently labeled amplified exons of hot spot regions of the dystrophin gene, which allowed us to detect 99% patients (affected males and female carriers) with macrodeletions and 89% with macroduplications, and to identify small insertions or deletions in those regions.