Duchenne and Becker muscular dystrophies (DMD and BMD) are allelic X-linked disorders arising from mutations in the (2.4 Mb) dystrophin gene at Xp21. We have applied the reverse transcriptase-polymerase chain reaction (RT-PCR) to identify a larger than normal dystrophin mRNA from a male with Duchenne muscular dystrophy and his younger affected brother. The increased size of the dystrophin mRNA was due to a splice site mutation at the exon 26:intron 26 junction where a T to G substitution prevented normal RNA processing. A cryptic splice site, downstream of the mutation, was activated during processing, resulting in the inclusion of 117 bases of intron 26. This insertion introduced an in-frame stop codon into the mature dystrophin mRNA. An allele specific test was developed to identify the mutation and was applied to this family. Interestingly, the mother of the two affected boys did not carry the mutation, as determined by allele-specific amplification and direct DNA sequence analysis, indicating gonadal mosaicism. Her eldest daughter, designated as a carrier based upon conventional testing and haplotype analysis, also did not carry the family mutation. Initial haplotyping of the family appeared to be straightforward with gonadal mosaicism becoming evident only after allele-specific analysis. The application of linked markers to identify the disease allele for conventional genetic counselling would have been erroneous in this family and highlights the diagnostic power of precise identification of the disease causing mutation. (C) 1994 Wiley-Liss, Inc.