MOLECULAR-BASIS OF CONGENITAL ADRENAL-HYPERPLASIA DUE TO 3-BETA-HYDROXYSTEROID DEHYDROGENASE-DEFICIENCY

Congenital adrenal hyperplasia is the most frequent cause of adrenal i nsufficiency and ambiguous genitalia in newborn children. In contrast to congenital adrenal hyperplasia due to 21-hydroxylase and 11beta-hyd roxylase deficiencies, which impair steroid formation in the adrenal c ortex, exclusively, classical 3beta-hydroxysteroid dehydrogenase (3bet a-HSD) deficiency affects steroid biosynthesis in the gonads as well a s in the adrenals. The structures of the highly homologous type I and II 3beta-HSD genes have been analyzed in three male pseudohermaphrodit e 3beta-HSD deficient patients from unrelated families in order to elu cidate the molecular basis of classical 3beta-HSD deficiency from pati ents exhibiting various degrees of severity of salt losing. The nucleo tide sequence of DNA fragments generated by selective polymerase chain reaction amplification that span the four exons, the exon-intron boun daries, as well as the 5'-flanking region of each of the two 3beta-HSD genes have been determined in the three male patients. The five point mutations characterized were all detected in the type II 3beta-HSD ge ne, which is the gene predominantly expressed in the adrenals and gona ds, while no mutation was detected in the type I 3beta-HSD gene, predo minantly expressed in the placenta and peripheral tissues. The two mal e patients suffering from severe salt-losing 3beta-HSD deficiency are compound heterozygotes, one bearing the frame-shift mutation 186/insC/ 187 and the missense mutation Y253N, while the other bears the nonsens e 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 s ite-directed mutagenesis in heterologous mammalian cells. Recombinant mutant type II 3beta-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 3beta-HSD activity by approximately 90%. The present finding s, describing the first missense mutations in the human type II 3beta- HSD gene, provide unique information on the structure-activity relatio nships of the 3beta-HSD superfamily. Moreover, the present findings pr ovide a molecular explanation for the enzymatic heterogeneity responsi ble for the severe salt-losing form to the clinically inapparent salt- wasting form of classical 3beta-HSD deficiency. The impairment of ster oid formation in both the adrenal and gonadal tissues of patients suff ering from classical 3beta-HSD deficiency thus results from mutation(s ) in the type II 3beta-HSD gene, causing various levels of impairment of enzymatic activity and, consequently, varying clinical severity of the disease. On the other hand, the finding of a normal type I 3beta-H SD gene provides the basis for the weil recognized intact peripheral i ntracrine steroidogenesis in these patients.