A form of congenital adrenal hyperplasia (CAH) is associated with accumulation of steroid metabolites indicating impaired 17alpha-hydroxylase and 21-hydroxylase activities. However, sequencing of CYP17 and CYP21 genes does not reveal mutations, suggesting the involvement of a co-factor interacting with both enzymes. Affected females present with ambiguous genitalia at delivery, but circulating androgens are low and virilisation does not progress, a paradox yet to be explained. Here we analyzed three affected children from two unrelated families. Both girls presented with ambiguous genitalia at birth; pregnancy with the affected boy led to maternal virilisation resolving after delivery. Urinary steroid analysis (GC/MS) revealed the disease-specific increase in pregnenolone, progesterone, 17-hydroxyprogesterone and corticosterone metabolites and concurrently low androgen metabolites. We sequenced the gene encoding P450 oxidoreductase (OR), which catalyzes electron transfer from NADPH to P450c17 and P450c21, in both families. We found four mutations encoding for single amino acid changes Y178D, A284P, R454H, and C566Y. All patients were compound heterozygotes while parents harbored a mutation in one allele only. By contrast, no mutations were found in a normal control cohort (n=100). All identified mutations are located in close proximity to flavoprotein or NADPH binding sites of OR. Recombinant wild-type and mutant OR proteins were bacterially expressed, purified and assayed for cytochrome c reductase activity, revealing total (Y178D) and partial disruption of enzymatic activity (A284P, R454H, and C566Y with 82%, 77% and 14% of wild-type catalytic efficiency Vmax/Km), thereby confirming the inactivating nature of the mutations. Thus we revealed that OR deficiency explains this variant of CAH. In addition, we propose that these patients paradigmatically indicate an alternative pathway in human androgen synthesis, present in fetal life only, explaining the concurrent presence of antenatal androgen excess and postnatal androgen deficiency. OR deficiency further illustrates the importance of intracellular redox potential in the control of steroidogenesis.
22 - 24 Mar 2004
British Endocrine Societies