A Competitive Allele-specific Oligomers Polymerase Chain Reaction Assay for the cis Double Mutation in AMPD1 That Is...

Background: Myo-adenylate deaminase deficiency (mADD) is the most common enzyme deficiency restricted to skeletal muscle, with a frequency of 1-2% in frozen muscle biopsies and complaints of easy fatigue and muscle cramping on exertion. A double C > T transition at coding bases 34 in exon 2 and 143 in exon 3 is the main cause of mADD. A 1-day assay using allele-specific oligomers and no isotope would be valuable for single cases. Methods and Results: Downstream primers with penultimate mismatch and 3' terminus matching the mutant or the normal base in exons 2 and 3 are used with a common upstream primer for each exon, to give amplimers of 150 bp for exon 2 and 200 bp for exon 3. A short common primer further downstream in exon 3 provides a competing 300-bp apmlimer whose product contribution is readily controlled by adjusting the annealing temperature. The entire procdure could be done in 1 day: DNA isolation, polymerase chain reaction (PCR), electrophoresis in agarose gel with ethidium bromide, and visualization by ultraviolet light. Deficient individuals have bands only with the mutant primers, normal persons have bands only with the normal primers, and heterozygotes (carriers) show bands with both primer sets. The empty slots show the 300-bp competing band, proving the PCR amplified the correct template. Allele-specific oligomers PCR results were verfied by dot blots and by restriction endonuclease analysis of exon 2. Conclusions: A simple and reliable allele-specific PCR assay using DNA from blood (or muscle) is now available for the diagnosis of individual cases of mADD caused by the common double-mtant AMPD1 gene, including the rare instances arising from homologous recombination between the two mutations.