Identification of Molecular Defects Causing Congenital Adrenal Hyperplasia by Cloning and Differential Hybridization of Polymerase Chain Reaction-Amplified 21-Hydroxylase (CYP21) Genes

摘要

ABSTRACTCongenital adrenal hyperplasia (CAH), one of the most common autosomal recessive disorders, is caused primarily by defects in the gene encoding steroid 21-hydroxylase,CYP21B. The molecular diagnosis of CAH, important for prenatal diagnosis, carrier detection, and a better understanding of the various clinical CAH forms, is complicated by the close proximity of a highly similar pseudogene,CYP21A, containing (and probably donating, by gene conversion-like events) most of the defects underlying CAH. In this study, we describe an efficient strategy to identify molecular defects causing CAH: polymerase chain reaction-amplifiedCYP21loci are cloned and hybridized to a set of oligonucleotides, allowing rapid and allele-specific identification of all knownCYP21Bmutations relevant to 21-hydroxylase function. Possible new mutations can be identified by subsequent nucleic acid sequencing provided they reside within the clonedCYP21Bfragment (from the TATA box to the 8thof the 10CYP21Bgene exons). Using this method, theCYP21Bgene mutations of a heterozygous carrier and 25 CAH patients have been identified by oligonucleotide hybridization. All disease haplotypes seem to have been generated by recombinational events involving theCYP21Apseudogene. In 5 individuals, these data were subsequently verified by nucleic acid sequencing. The procedure can be used for diagnostic applications and may facilitate identification of newCYP21Bdefects.