The most common cause of congenital adrenal hyperplasia is steroid 21-hydroxylase deficiency. The molecular genetics of this disease are such that genotyping is a potentially useful tool in its diagnosis. An assay was developed using real-time, quantitative PCR to detect deletions of the steroid 21-hydroxylase gene (CYP21A2). This assay was able to detect heterozygous gene deletions with an ␣ error rate of less than 5%, with a power greater than 95%. When combined with allele-specific PCR,

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... yping for the nine most common mutations can be completed within hours of blood sampling. This technique was used to study subjects with 21-hydroxylase deficiency in North Florida. Twenty-eight subjects with congenital adrenal hyperplasia, seven first-degree relatives and thirteen normal subjects, were characterized. Of 96 chromosomes, 69 abnor-mal alleles were identified. Among unrelated abnormal alleles, the frequency of specific mutations was 28% for a gene deletion, 24% for the intron 2 splice mutation, 10% for ile172asn, 8% each for val281leu and the exon 6 cluster, and 6% for gln318x mutations. These frequencies, as well as the genotype/phenotype correlation, were similar to those found in comparable populations. The utility of genotyping in the diagnosis of 21-hydroxylase deficiency is increased by the rapidity of the analysis. With quantitative PCR, the need for more expensive and time consuming Southern blot analysis is reduced and limited to the clarification of certain genotypes. Faster results will allow for more timely initiation of appropriate therapy and limit the exposure of potentially unnecessary therapy. (J Clin Endocrinol Metab 87: 735-741, 2002)