Congenital adrenal hyperplasia (CAH) due to 21-hydrorylase (21-0H) deficiency is one of the most common inherited metabolic disorders. Marked deficiency of the adrenocortical cytochrome P450 21-0H results in the salt-losing form of CAH and is manifested by glucocorticoid and mineralocorticoid deficiencies and androgen excess. There is a life-long dependence on hormonal replacement therapy, however some patients recover from mineralocorticoid dependence, perhaps due to another P45U capable of 21-0H activity. Less marked 21-0H deficiency results in non-salt-losing CAH. Two HLA-linked 21-0H genes, the CYP21A pseudogene and the CYP21B active gene, lie in tandem duplication with the genes encoding the fourth components of complement, C4A and C4B. This duplicated region has undergone frequent unequal crossing-over events resulting in deletions, duplications and apparent conversions of the C4 and CYP21 genes. Deletions or conversions of the CYP21B gene account for up to half of the mutations causing salt-losing CAH. The remainder of CAH-linked mutations have not been as extensively characterized. Patients with salt-losing CAH have a high frequency of C4B genes which are present but unexpressed.
The specific aims of this FIRST Award are to: 1.) Determine sites of unequal crossing-over within recombinant (hybrid) CYP21 and C4 genes in order to identify sequences which may predispose to recombination. Regions within the CYP21 genes containing these sites will be localized by analysis of unique restriction fragments for the CYP21A and CYP21B genes, and by hybridization with allele-specific oligonucleotides for the C4A and C4B genes. Specific sites of crossing-over within these regions will then be determined by sequence analysis of enzymatically amplified DNA segments. 2.) Determine CYP21B mutations in patients with salt-losing and non-salt-losing CAH. CYP21B mutations which are characteristic of CYP21A will be identified with mutation- specific oligonucleotide probes. As yet undescribed mutations will be detected by DNA sequence analysis of cloned or enzymatically amplified mutant CYP21B genes. We will also analyze neighboring loci for CAH-linked restriction fragment length polymorphisms. 3.) Determine the identity of a human genomic DNA clone which is homologous to but different from the CYP21 genes. 4.) Determine the DNA sequence of the C4B/CYP21B intragenic region to assess for the presence of an element capable of controlling the expression of both genes.
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