The investigations described will elucidate the relationship between clinical phenotype and molecular genotype in congenital adrenal hyperplasia due to 21-hydroxylase deficiency (21-OHD) . Patients with each of the three phenotypic forms i.e., salt-wasting, simple virilizing, and nonclassic 21-OHD will be categorized by detailed endocrinologic studies, and DNA samples will be obtained from patients and family members.
In Specific Aim 1, specific alleles land genotype combinations will be identified and correlated with each of the above-named groups. Presence or absence of 8 functionally important mutations in the CYP21 gene encoding the active 21-hydroxylase will be analyzed by allele-specific oligonucleotide hybridizations after amplification of genomic DNA in the polymerase chain reaction; gene copy number will be assessed by Southern blot. Segregation of mutations found in individual patients will be determined by studying parents. De novo mutations may be identified in this manner. Novel mutations in CYP21 may be sought by direct sequencing of amplified DNA in patients in whom none of the typical gene conversion mutations are found. An attempt will be made to relate in vivo measures of 21-hydroxylase activity, such as 17-hydroxyprogesterone and aldosterone levels, to the degree of enzyme dysfunction as predicted from previously published in vitro expression studies.
In Specific Aim 2, dietary sodium deprivation will be instituted to identify patients who recover from salt-wasting. The ratio of plasma renin activity to aldosterone will be used as an index of the adrenal's efficiency in producing aldosterone in patients of pre- and post-pubertal ages. Parallel in vitro studies will focus on characterization of genes encoding other P450 enzymes capable of performing steroid 21-hydroxylation in the adrenal.
Specific Aim 3 calls for molecular genetic screening of a newborn population for the mutation found in approximately 80% of nonclassic haplotypes bearing HLA-B14;DR1. This screening will both confirm the high frequency of this disorder, and allow for study of the evolution of clinical manifestations of nonclassic 21-OHD in the young child.
Specific Aim 4 will test the usefulness of allele-- specific hybridization in prenatal diagnosis where the CYP21 mutation is known. Safety and efficacy of dexamethasone prenatal treatment of pregnancies at risk for classic 21-OHD will be assessed through careful evaluation of complications of pregnancy and early childhood growth and development of offspring.
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