GENE MUTATION IN 21-HYDROXYLASE DEFICIENCY

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All forms of 21-hydroxylase deficiency, salt-losing, simple virilizing, and late-onset, share a common gene locus (CYP21B) in the class III region of the major histocompatibility complex on chromosome 6 in tandem duplication with a pseudogene (CYP21A). Gene deletions, conversions, and point mutations have been described in patients with 21-hydroxylase deficiency. This heterogeneity of clinical phenotypes and mutational events suggests that there is a wide variation in the expression of 21-hydroxylase activity. To evaluate our hypothesis that greater disturbance of gene expression leads to greater decrease in 21-hydroxylase activity manifested as more severe phenotypic disease, we propose to correlate ,the frequency of alterations in the structural portion of the 21-hydroxylase gene, detected through genomic DNA amplification utilizing polymerase chain reaction and dot-blot analysis, with the clinical phenotype, clinical severity and 17-hydroxyprogesterone response to synthetic ACTH, in our population of affected families Since defects may occur in the regulatory portion of the gene, we will examine the regulatory portion if there are no apparent nucleotide sequence alterations in the structural portion of the gene. To confirm that detected sequence alterations affect enzyme activity, expression of the altered sequence in pKCRH-2 transfected into COS-7 monkey kidney cells with determination of conversion of 17-hydroxy[14C]progesterone into 14C-lldeoxycortisol will be performed. Results of expressed enzyme activity will be correlated with phenotype and genotype to determine if phenotype predicts genotype.