New Insight into the Molecular Basis of 3β-Hydroxysteroid Dehydrogenase Deficiency: Identification of Eight Mutations in the HSD3B2 Gene in Eleven Patients from Seven New Families and Comparison of the Functional Properties of Twenty-Five Mutant Enzymes1

Abstract

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 ...

Two novel HSD3B2 missense mutations with diverse residual enzymatic activities for Δ5‐steroids

“…The mutations, p.Y190C and p.S218P, are located between the two substrate‐binding domains. In the region, including the two amino acid residues of the present study, six amino acid residues (Y190, L205, S213, S218, P222 and L236) have been reported to have missense mutations that affect the enzymatic activity without impairing the stability of the product. These finding suggest that the region has an essential role in the enzyme's activity.…”

“…The enzyme has a key role in steroid biosynthesis, converting Δ5‐steroids to Δ4‐steroids in adrenal glands and gonads. Patients with type II 3β‐HSDD show impaired synthesis of glucocorticoids and mineralcorticoids in adrenal glands with an excess of Δ5‐steroids, resulting in adrenal insufficiency with or without salt wasting.…”

Regulation of human 3-beta-hydroxysteroid dehydrogenase type-2 (3βHSD2) by molecular chaperones and the mitochondrial environment affects steroidogenesis

“…In contrast to exported poly- 11 peptides (e.g., insulin, growth hormone, digestive enzymes, etc.) 12 that are first synthesized and then stored in secretory vesicles, 13 steroidogenic cells store very little steroid hormone, necessitating 14 a rapid steroidogenic response (e.g., adrenal secretion of cortisol in 15 the classic 'fight or flight' response) that requires de novo steroid 16 synthesis.…”

“…Estradiol 56 induces increased expression of oxytocin in placental tissues 57 (5-times greater than oxytocin from the posterior pituitary) and 58 of oxytocin receptors in the myometrium in a concentration- 59 dependent manner [8]. 60 3bHSD gene mutations, which induce amino acid changes 61 throughout the protein sequence, are not restricted to any 62 ethnicity or geographic location, but are responsible for congenital 63 adrenal hyperplasia (CAH) [9][10][11][12][13][14][15][16][17][18][19][20]. Some 3bHSD mutations alter 64 its activity [9,11,14,21,22].…”

“…60 3bHSD gene mutations, which induce amino acid changes 61 throughout the protein sequence, are not restricted to any 62 ethnicity or geographic location, but are responsible for congenital 63 adrenal hyperplasia (CAH) [9][10][11][12][13][14][15][16][17][18][19][20]. Some 3bHSD mutations alter 64 its activity [9,11,14,21,22]. While, the exact mechanism of action of 65 3bHSD remains unknown [23], changes in 3bHSD activity can 66 have a wide range of effects given the central role of 3bHSD in 67 steroidogenesis.…”