NONSALT-LOSING MALE PSEUDOHERMAPHRODITISM DUE TO THE NOVEL HOMOZYGOUSN100S MUTATION IN THE TYPE-II 3-BETA-HYDROXYSTEROID DEHYDROGENASE GENE

Recently, the structure of two genes encoding isoenzymes responsible f or 3 beta-hydroxysteroid dehydrogenase/Delta(5)-Delta(4)-isomerase (3 beta HSD) activity in the human was elucidated. This activity is an es sential step in the biosynthesis of all classes of steroid hormones. I n the classic severe form of 3 beta HSD deficiency, patients present w ith adrenal insufficiency, various degrees of salt loss, and incomplet e masculinization in males. Here we report the characterization of the molecular basis of congenital adrenal hyperplasia due to 3 beta HSD d eficiency in a male pseudohermaphrodite born from consanguineous paren ts and having no clinical, salt loss. To analyze the structure of the type I and II 3 beta HSD genes of the patient, DNA fragments, generate d by polymerase chain reaction amplification of the four exons and the exon-intron boundaries of these genes, were directly sequenced. The p atient carried a homozygous missense mutation converting Asn(100) to S er in exon 3 of his type II 3 beta HSD gene. His parents were heterozy gous for the same point mutation. The absence of clinical salt loss as sociated with a male pseudohermaphroditism suggested that 3 beta HSD a ctivity was impaired to different levels in the testes and adrenal. To elucidate whether this N100S missense mutation affected preferentiall y a steroidogenic pathway, enzymatic activity was analyzed by in vitro analysis of mutant recombinant enzyme generated by site-directed muta genesis after its transient expression in COS-1 cells. Using homogenat es from transferred cells:, the N100S 3 beta HSD enzyme showed a K-m v alue for pregnenolone of 25 +/- 3 mu mol/L compared with 3.5 +/- 0.2 m u mol/L for the normal human type II 3 beta HSD enzyme. Similar result s were obtained using dehydroepiandrosterone as substrate. In addition to decreasing apparent affinity, the N100S mutation decreased the rel ative specific activity (V-max), leading to a relative specificity (re lative V-max/K-m) 2.7% and 11% that of normal type II 3 beta HSD using pregnenolone or dehydroepiandrosterone as substrate, respectively. Mo reover, the mutant N100S protein had an apparent decreased affinity fo r NAD(+), with a K-m value of 650 +/- 66 mu mol/L, compared with 20 +/ - 2 mu mol/L for normal type II 3 beta HSD. Except for the hypothetica l effect of local factors. these Findings suggest that a very weak res idual activity DE the normal type II 3 beta HSD enzyme could prevent s alt loss. but it was insufficient for normal male sex differentiation N100S is the first mutation to significantly affect both the affinity and specific activity of the type II 3 beta HSD enzyme for steroid sub strates and for its cofactor, thus providing unique information about the structure-activity relationships of the 3 beta HSD family. Conserv ation of Asn(100) in ail 3 beta HSD enzymes characterized so Far in ma mmalian as well as bacterial and viral members of the 3 beta HSD super family supports the crucial importance of this amino acid in the funct ion of the enzyme.