A quantitative polymerase chain reaction (PCR) assay completely discriminates between Duchenne and Becker muscular dystrophy deletion carriers and normal females

Duchenne/Becker muscular dystrophy (DMD/BMD) is a severe X-linked myopathy. In 65% of the patients, the mutations responsible for the disease are macrodeletions in the dystrophin-encoding gene that can be identified with multiplex polymerase chain reaction (PCR) technology. We developed a method for quantitative PCR analysis of deletion carriers involving the use of phosphorimager-based scanning of radioactive-labelled PCR products. We calculated the ratios between the areas of two peaks, one corresponding to the deleted segments to be analysed and the other taken as a reference. In carriers, these ratios (R value) were always about half those obtained in normal females. The final diagnostic result, the diagnostic index (DI), is the ratio of the R values between the propositus and a normal subject. We also assessed the variability of each step of the procedure and the overall variability of the DI value, thus obtaining cut-off values that completely discriminated BMD/DMD deletion carriers from normal females. We were also able to classify, as either «carrier» or «normal», several females whose status was not identified with linkage analysis.