Molecular basis of congenital adrenal hyperplasia due to 3β-hydroxysteroid dehydrogenase deficiency

Congenital adrenal hyperplasia is the most frequent cause of adrenal insufficiency and ambiguous genitalia in newborn children. In contrast to congenital adrenal hyperplasia due to 21-hydroxylase and 11β-hydroxylase deficiencies, which impair steroid formation in the adrenal cortex, exclusively, classical 3β-hydroxysteroid dehydrogenase (3β-HSD) deficiency affects steroid biosynthesis in the gonads as well as in the adrenals. The structures of the highly homologous type I and II 3β-HSD genes have been analyzed in three male pseudohermaphrodite 3β-HSD deficient patients from unrelated families in order to elucidate the molecular basis of classical 3β-HSD deficiency from patients exhibiting various degrees of severity of salt losing. The nucleotide sequence of DNA fragments generated by selective polymerase chain reaction amplification that span the four exons, the exon-intron boundaries, as well as the 5′-flanking region of each of the two 3β-HSD genes have been determined in the three male patients. The five point mutations characterized were all detected in the type II 3β-HSD gene, which is the gene predominantly expressed in the adrenals and gonads, while no mutation was detected in the type I 3β-HSD gene, predominantly expressed in the placenta and peripheral tissues. The two male patients suffering from severe salt-losing 3β-HSD deficiency are compound heterozygotes, one bearing the frame-shift mutation 186/insC/187 and the missense mutation Y253N, while the other bears the nonsense mutation W171X and the missense mutation E142K. The influence of the detected missense mutations on enzymatic activity was assessed by in vitro expression analysis of mutant recombinant enzymes generated by site-directed mutagenesis in heterologous mammalian cells. Recombinant mutant type II 3β-HSD enzymes carrying Y253N or E142K substitutions exhibit no detectable activity. On the other hand, the nonsalt-losing patient is homozygous for the missense mutation A245P. This mutation decreases 3β-HSD activity by approximately 90%. The present findings, describing the first missense mutations in the human type II 3β-HSD gene, provide unique information on the structure-activity relationships of the 3β-HSD superfamily. Moreover, the present findings provide a molecular explanation for the enzymatic heterogeneity responsible for the severe salt-losing form to the clinically inapparent salt-wasting form of classical 3β-HSD deficiency. The impairment of steroid formation in both the adrenal and gonadal tissues of patients suffering from classical 3β-HSD deficiency thus results from mutation(s) in the type II 3β-HSD gene, causing various levels of impairment of enzymatic activity and, consequently, varying clinical seventy of the disease. On the other hand, the finding of a normal type I 3β-HSD gene provides the basis for the well recognized intact peripheral intracrine steroidogenesis in these patients.