DETECTION OF STEROID 21-HYDROXYLASE ALLELES USING GENE-SPECIFIC PCR AND A MULTIPLEXED LIGATION DETECTION REACTION

Steroid 21-hydroxylase deficiency is the most common cause of congenit al adrenal hyperplasia, an inherited inability to synthesize cortisol that occurs in 1 in 10,000-15,000 births. Affected females are born wi th ambiguous genitalia, a condition that can be ameliorated by adminis tering dexamethasone to the mother for most of gestation, Prenatal dia gnosis is required for accurate treatment of affected females as well as for genetic counseling purposes. Approximately 95% of mutations cau sing this disorder result from recombinations between the gene encodin g the 21-hydroxylase enzyme (CYP21) and a linked, highly homologous ps eudogene (CYP21P), Approximately 20% of these mutations are gene delet ions, and the remainder are gene conversions that transfer any of nine deleterious mutations from the CYP21P pseudogene to CYP21. We describ e a methodology for genetic diagnosis of 21-hydroxylase deficiency tha t utilizes gene-specific PCR amplification in conjunction with thermos table DNA ligase to discriminate single nucleotide variations in a mul tiplexed ligation detection assay, The assay has been designed to be u sed with either fluorescent or radioactive detection of ligation produ cts by electrophoresis on denaturing acrylamide gels and is readily ad aptable for use in other disease systems. (C) 1995 Academic Press, Inc .