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New Insight into the Molecular Basis of 3ß-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

Medical Research Council Group in Molecular Endocrinology, Centre Hospitalier Université Laval Research Center and Laval University (A.-M.M., M.L.R, M.D., J.S.), Québec, Canada G1V 4G2; Laboratoire de Biochimie Endocrinienne (V.T., F.M., M.G.F., Y.M.), Institut National de la Sante et de la Recherche Medicale U329, Université de Lyon and Hôpital Debrousse, 69322 Lyon Cedex 05, France; Service d’Explorations Fonctionnelles Endocriniennes (S.C., M.C.R.-D.), Hôpital Armand Trousseau, Paris, France; Service d’Endocrinologie Pédiatrique (J.-L.C.), Hôpital Saint-Vincent de Paul, Paris, France; Department of Paediatrics (W.G.S., M.P.), Division of Paediatric Endocrinology, Christian Albrechts-University of Kiel, Germany D-23946

Address correspondence and requests for reprints to: Dr. Jacques Simard, Laboratory of Hereditary Cancers, Centre Hospitalier Université Laval Research Center, 2705 Laurier Boulevard, Québec City, Québec, Canada, G1V 4G2. E-mail: jacques.simard{at}crchul.ulaval.ca

Classical 3ß-hydroxysteroid dehydrogenase/⁵-⁴ isomerase (3ßHSD) 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 3ßHSD 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 3ßHSD 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 3ßHSD 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 3ßHSD 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 3ßHSD 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 3ßHSD isoenzyme. The present study provides biochemical evidence supporting the involvement of a new molecular mechanism in classical 3ßHSD 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 3ßHSD superfamily.

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