Philipp Lachenmann: DELPHI Rationale


Schoeller Junkmann Preis 2004

Dr. Wiebke Arlt

The Medical School, University of Birmingham, United Kingdom

Congenital adrenal hyperplasia with apparent combined P450c17 and P450c21 deficiency is caused by mutations in P450 oxidoreductase

A variant of congenital adrenal hyperplasia (CAH) first described in 1985 (N Engl J Med 1985, 313:1182-1191) is associated with accumulation of steroids indicating impaired 17?-hydroxylase and 21-hydroxylase activities. However, sequencing of CYP17 and CYP21 genes does not reveal mutations. Affected females present with ambiguous genitalia at delivery, but circulating androgens are low and virilization 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 virilization 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 hypothesized that a co-factor interacting with both P450c17 and P450c21 may be the underlying cause and therefore sequenced the gene encoding P450 oxidoreductase (OR), which transfers electrons from NADPH to both steroidogenic enzymes. 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 (A284P, R454H, C566Y) disruption of enzymatic activity, as compared to wild-type catalytic efficiency (Vmax/Km), thereby confirming the inactivating nature of the mutations.

Thus we revealed that P450 oxidoreductase deficiency (ORD) explains this variant of CAH. ORD further illustrates the importance of intracellular redox potential in the control of steroidogenesis. In addition, we propose that ORD paradigmatically indicates the existence of an alternative pathway in human androgen synthesis, which is supported by the results of steroid analysis in the three affected children. This alternative androgen pathway is present in fetal life only and explains the concurrent presence of antenatal androgen excess and postnatal androgen deficiency. It involves conversion of 5?-pregnanediolone to androsterone and has been previously described in the fetal gonad of the tammar wallaby (Endocrinology 2003, 144:575-580). Future research will need to clarify the role of this novel androgen pathway in human fetal development.



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