Reverse dot-blot hybridization as an improved tool for the molecular diagnosis of point mutations in congenital adrenal hyperplasia caused by 21-hydroxylase deficiency

Background: More than 90% of cases of congenital adrenal hyperplasia (CAH) are caused by mutations of the CYP21 gene that result in deficiencies of th e enzyme 21-hydroxylase. Allele-specific PCR, allele-specific oligonucleoti de hybridization, and Southern blot analysis are the most common methods to detect point mutations and deletions in the CYP21 gene. Methods and Results: This report is the first application of the reverse do t-blot (RDB) assay for diagnosis of the nine most common point mutations in the CYP21 gene associated with CAH (P30L, g.659A>G or g.659C>G, I172N, 123 6N-V237E-M239K, V281L, g.1767-1768insT, Q318X, R356W, P453S). Normal and mu tant oligonucleotides spanning these nine mutation sites were spotted onto a nylon membrane. DNA was extracted from dried blood spots, and exons. enco mpassing mutations from samples to be tested were amplified and labeled wit h biotin-dUTP by PCR. These exons then were hybridized to membrane strips. Signal detection was achieved by chemiluminescence. Thirty clinically confi rmed cases that were identified by the Texas Newborn Screening Program were tested. All mutations were subsequently confirmed by automated DNA sequenc ing. Conclusion: The RDB method has the advantages of being accurate and cost-ef fective for the molecular diagnosis of CYP21 point mutations in CAH. It per mits simultaneous detection of a panel of point mutations with only one hyb ridization per sample and could be automated to study many samples.