Automated sequence screening of the entire dystrophin cDNA in Duchenne dystrophy: point mutation detection

This is the first report of direct sequencing of the complete 11 kb coding sequence of the dystrophin gene affording high sensitivity for all types of mutations of both coding sequence and splicing. Direct automated capillary gel sequence analysis of dystrophin reverse-transcribed polymerase chain reaction (RT-PCR) products was carried out in 15 Duchenne muscular dystrophy (DMD) patient muscle biopsies (170,000 bp sequenced). We identified mutations in 67% of patients tested (10/15); including premature stop codons (n = 5) and small deletions/duplications (n = 5). Mutation-negative patients (n = 5) were also negative for promoter mutations. All were tested for the possibility of transcription abnormalities using quantitative multiplex fluorescence polymerase chain studies (QMF-PCR), however, equal ratios of mRNA transcripts were identified at the 5'and 3' regions, with mild reduction in overall quantity, suggesting that transcription abnormalities were less likely. We suggested that such patients might have a problem with the 3.5 kb 3' UTR, polyA site or undetected stop codons. It is also possible that splicing defects could result in addition of intron sequence which could lead to preferential amplification of low level residual normal transcript skipping.