Detection of mutations in the dystrophin gene via automated DHPLC screening and direct sequencing

Using denaturing high performance liquid chromatography (DHPLC) screening and direct sequencing, 86 PCR amplicons of genomic DNA from the dystrophin gene were screened for mutations in eight patients diagnosed with DMD who had tested negative for large DNA rearragements. Mutations likely to be disease-causative were found in six of the eight patients. All 86 amplicons from the two patients in whom no likely disease-causative mutations were found were completely sequenced and only polymorphisms were found.We have shown that it is now feasible for clinical laboratories to begin testing for both point mutations and large deletions/duplications in the dystrophin gene. The detection rate will rise from 65% to greater than 92% with only a moderate increase in cost.Dystrophinopathies are X-linked recessive diseases caused by primary dystrophin deficiency, and include: Duchenne Muscular Dystrophy (DMD), Becker Muscular Dystrophy (BMD), manifesting DMD/BMD carrier females, X-linked dilated cardiomyopathy, isolated quadriceps myopathy, muscle cramps with myoglobinuria and asymptomatic elevation of muscle enzymes [1].The DMD gene spans 2.4 million base pairs of genomic DNA on the X chromosome and its 14 kb transcript encodes a full-length protein (dystrophin) of 427 kiloDaltons. Dystrophin is a sarcolemmal protein that through its interaction with many other proteins participates in the linkage of the extracellular matrix to the cytoplasmic cytoskeleton [2-4]. Mutations in this gene result in DMD, BMD or other dystrophinopathy. A major consequence of the dystrophin gene's large genomic size is a high rate of mutation; close to 30% of cases prove to be spontaneous mutations[5]. Approximately 60% of mutations causing DMD are deletions of large segments of the gene usually including one or more exons [6-8]. Approximately 5% of mutations are duplications of large segments of the gene[8]. Large deletions and duplications are detected using multiplexed PCR primers to amplify a subse