Congenital adrenal hyperplasia caused by mutant P450 oxidoreductase and human androgen synthesis: analytical study

Summary

Background

Congenital adrenal hyperplasia with apparent combined P450C17 and P450C21 deficiency is associated with accumulation of steroid metabolites, indicating impaired activity of 17α-hydroxylase and 21-hydroxylase. However, no mutations have been reported in the CYP17 and CYP21 genes, which encode these P450 enzymes. Affected girls are born with ambiguous genitalia, but their circulating androgens are low, and virilisation does not progress. We aimed to investigate the underlying molecular basis of congenital adrenal hyperplasia with apparent combined P450C17 and P450C21 deficiency in affected children.

Methods

We did sequence analysis of the human gene encoding P450 oxidoreductase, an enzyme that is important in electron transfer from NADPH to P450C17 and P450C21. We studied two unrelated families with a total of three affected children and 100 healthy controls. Wild-type and mutant P450 oxidoreductase proteins were bacterially expressed, purified, and assayed for cytochrome c reductase activity.

Findings

We identified four mutations encoding single aminoacid changes in P450 oxidoreductase. All patients were compound heterozygotes, whereas their parents and an unaffected sibling harboured a mutation in only one allele. By contrast, no mutations were noted in the controls. Bacterial expression of recombinant mutant proteins revealed deficient or reduced enzyme activity.

Interpretation

Molecular pathogenesis of this form of congenital adrenal hyperplasia is caused by mutations in the gene encoding P450 oxidoreductase. Deficiency of this enzyme could suggest an alternative pathway in human androgen synthesis, present only in fetal life, which explains the combination of antenatal androgen excess and postnatal androgen deficiency.

Published online June 14,2004 http://image.thelancet.com/extras/04art4419web.pdf

Introduction

Apparent combined P450C17 and P450C21 deficiency is a rare variant of congenital adrenal hyperplasia, which was first reported in 1985.¹ The most striking phenotypic feature is that affected girls are born with ambiguous genitalia, indicating intrauterine androgen excess. After birth, however, virilisation does not progress and amounts of circulating androgens are low or normal. Conversely, affected boys are sometimes born undermasculinised. Boys and girls can also present with bone malformations, in some cases resembling the pattern seen in patients with Antley-Bixler congenital malformation syndrome.²

During the past 20 years, several case reports of this variant of congenital adrenal hyperplasia have been published.³ Findings of biochemical investigations of urinary steroid excretion in affected patients have shown accumulation of steroid metabolites, indicating impaired C17 and C21 hydroxylation. In turn, these results suggest concurrent partial deficiencies of two steroidogenic enzymes, P450C17 and P450C21 (figure 1). However, sequencing of the genes encoding these enzymes has not show any mutations,2, 4 which accords with the idea of a defect in a cofactor that interacts with both enzymes.⁵

P450C21 has a role in glucocorticoid biosynthesis and P450C17 catalyses the biosynthesis of dehydroepiandrosterone and androstenedione—precursors of the active androgens testosterone and 5α-dihydrotestosterone(figure 1). In keeping with partial impairment of enzymatic activities, affected patients can show reduced excretion of glucocorticoid metabolites, and concentrations of androgen metabolites are generally low.³ To date, a plausible hypothesis unifying the contradictory findings of antenatal androgen excess and postnatal androgen deficiency has been absent. Apparent combined P450C17 and P450C21 deficiency has, therefore, remained an enigmatic disease of steroidogenesis.

We aimed to investigate the underlying molecular basis of congenital adrenal hyperplasia with apparent combined P450C17 and P450C21 deficiency in affected children.