A novel A10E homozygous mutation in the HSD3B2 gene causing severe salt-wasting 3beta-hydroxysteroid dehydrogenase deficiency in 46,XX and 46,XY French-Canadians: evaluation of gonadal function after puberty.

Severe 3beta-hydroxysteroid dehydrogenase (3betaHSD) deficiency is a rare form of congenital adrenal hyperplasia resulting from mutations in the HSD3B2 gene that impair steroidogenesis in both the adrenals and gonads and cause salt-wasting in both sexes and incomplete masculinization of the external genitalia in genetic males. About two thirds of the reported patients are 46,XY. We describe two French-Canadian patients from two families without a known relationship who presented with severe salt-wasting 3betaHSD deficiency in infancy. Although the diagnosis was considered clinically, plasma steroid profiles were confusing. We have thus directly sequenced DNA fragments generated by PCR amplification of the four exons, exon-intron boundaries, and the 5'-flanking regions of the HSD3B2 gene. Sequencing of exon II revealed the presence of a C to A transversion in both alleles of these two cases, thus converting codon 10 (GCA), which codes for Ala, into GAA, encoding Glu. This Ala is highly conserved in the vertebrate 3betaHSD gene family and is located in the putative NAD-binding domain of the enzyme. The mutant type II 3betaHSD enzyme carrying an A10E substitution exhibited no detectable activity in intact transfected Ad293 cells. Both homozygous patients share the same haplotype, spanning approximately 3.3 centimorgans surrounding the HSD3B2 locus, which is consistent with a founder effect for this missense mutation. The 46,XY patient presented with ambiguous genitalia at birth and underwent normal masculinization at puberty, but was azoospermic at 18.5 yr of age. The 46,XX patient presented progressive breast development, menarche, and evidence of progesterone secretion. The only previously reported cases with pubertal follow-up revealed paternity in one male and hypogonadism in one female. These findings demonstrate the complex relationships between the genotype and the gonadal phenotype in severe 3betaHSD deficiency and the difficulty in predicting fertility.

A new insight into the molecular basis of 3beta-hydroxysteroid dehydrogenase deficiency.

Classical 3beta-hydroxysteroid dehydrogenase/delta5-delta4 isomerase (3beta-HSD) deficiency is a rare form of congenital adrenal hyperplasia that impairs steroidogenesis in both the adrenals and gonads resulting from mutations in the HSD3B2 gene, causing varying degrees of salt-loss in both sexes and incomplete masculinization of the external genitalia in genetic males. To date a total of 34 mutations (including 5 frameshift, 4 nonsense, 1 in-frame deletion, 1 splicing and 23 missense mutations) have been identified in the HSD3B2 gene. Results from functional charaterization studies of the mutant proteins agrees with the prediction that no functional type II 3beta-HSD isoenzyme is expressed in the adrenals and gonads of the patients with the severe salt-losing form, whereas the nonsalt-losing form causes an incomplete loss in enzymatic activity, thereby leaving sufficient enzymatic activity to prevent salt loss. Recent studies have highlighted the fact that various mutations appear to have a drastic effect upon the stability of the protein, therefore providing molecular evidence of a new mechanism involved in classical 3beta-HSD deficiency. Finally, the functional characterization of the missense mutations known to be involved in this autosomal recessive disorder provides valuable information concerning the structure-function relationships of the 3beta-HSD enzyme superfamily.

New insight into the molecular basis of 3beta-hydroxysteroid dehydrogenase deficiency: identification of eight mutations in the HSD3B2 gene eleven patients from seven new families and comparison of the functional properties of twenty-five mutant enzymes.

Classical 3beta-hydroxysteroid dehydrogenase/delta5-delta4 isomerase (3betaHSD) deficiency is a form of congenital adrenal hyperplasia that impairs steroidogenesis in both the adrenals and gonads resulting from mutations in the HSD3B2 gene and causing various degrees of salt-wasting in both sexes and incomplete masculinization of the external genitalia in genetic males. To identify the molecular lesion(s) in the HSD3B2 gene in the 11 patients from the seven new families suffering from classical 3betaHSD deficiency, the complete nucleotide sequence of the whole coding region and exon-intron splicing boundaries of this gene was determined by direct sequencing. Five of these families were referred to Morel's molecular diagnostics laboratory in France, whereas the two other families were investigated by Peter's group in Germany. Functional characterization studies were performed by Simard's group in Canada. Following transient expression in 293 cells of each of the mutant recombinant proteins generated by site-directed mutagenesis, the effect of the 25 mutations on enzyme activity was assessed by incubating intact cells in culture with 10 nM [14C]-DHEA as substrate. The stability of the mutant proteins has been investigated using a combination of Northern and Western blot analyses, as well as an in vitro transcription/translation assay using rabbit reticulocyte lysates. The present report describes the identification of 8 mutations, in seven new families with individuals suffering from classical 3betaHSD deficiency, thus increasing the number of known HSD3B2 mutations involved in this autosomal recessive disorder to 31 (1 splicing, 1 in-frame deletion, 3 nonsense, 4 frameshift and 22 missense mutations). In addition to the mutations reported here in these new families, we have also investigated for the first time the functional significance of previously reported missense mutations and or sequence variants namely, A82T, A167V, L173R, L205P, S213G and K216E, P222H, T259M, and T259R, which have not previously been functionally characterized. Furthermore, their effects have been compared with those of the 10 previously reported mutant enzymes to provide a more consistent and comprehensive study. The present results are in accordance with the prediction that no functional 3betaHSD type 2 isoenzyme is expressed in the adrenals and gonads of the patients suffering from a severe salt-wasting form of CAH due to classical 3betaHSD deficiency. Whereas the nonsalt-losing form also results from missense mutation(s) in the HSD3B2 gene, which cause an incomplete loss in enzyme activity, thus leaving sufficient enzymatic activity to prevent salt wasting. The functional data described in the present study concerning the sequence variants A167V, S213G, K216E and L236S, which were detected with premature pubarche or hyperandrogenic adolescent girls suspected to be affected from nonclassical 3betaHSD deficiency, coupled with the previous studies reporting that no mutations were found in both HSD3B1 and/or HSD3B2 genes in such patients strongly support the conclusion that this disorder does not result from a mutant 3betaHSD isoenzyme. The present study provides biochemical evidence supporting the involvement of a new molecular mechanism in classical 3betaHSD deficiency involving protein instability and further illustrates the complexity of the genotype-phenotype relationships of this disease, in addition to providing further valuable information concerning the structure-function relationships of the 3betaHSD superfamily.