Commentary - New insight into the molecular basis of 3 beta-hydroxysteroiddehydrogenase deficiency: Identification of fight mutations in the HSD3B2 gene in eleven patients from seven new families and comparison of the functional properties of twenty-five mutant enzymes

Classical 3 beta-hydroxysteroid dehydrogenase/Delta(5)-Delta(4) isomerase ( 3 beta HSD) deficiency is a form of congenital adrenal hyperplasia that imp airs steroidogenesis in both the adrenals and gonads resulting from mutatio ns in the HSD3B2 gene and causing various degrees of salt-wasting in both s exes and incomplete masculinization of the external genitalia in genetic ma les. To identify the molecular lesion(s) in the HSD3B2 gene in the II patie nts from the seven new families suffering from classical 3 beta HSD deficie ncy, the complete nucleotide sequence of the whole coding region and exon-i ntron splicing boundaries of this gene was determined by direct sequencing. Five of these families were referred to Morel's molecular diagnostics labo ratory in France, whereas the two other families were investigated by Peter 's group in Germany. Functional characterization studies mere performed by Simard's group in Canada. Following transient expression in 293 cells of ea ch of the mutant recombinant proteins generated by site-directed mutagenesi s, the effect of the 25 mutations on enzyme activity was assessed by incuba ting intact cells in culture with 10 nM [C-14]-DHEA as substrate. The stabi lity of the mutant proteins has been investigated using a combination of No rthern and Western blot analyses, as well as an in vitro transcription/tran slation assay using rabbit reticulocyte lysates. The present report describ es the identification of 8 mutations, in seven new families with individual s suffering from classical 3 beta HSD deficiency, thus increasing the numbe r of known HSD3B2 mutations involved in this autosomal recessive disorder t o 31 (1 splicing, 1 in-frame deletion, 3 nonsense, 4 frameshift and 22 miss ense mutations). In addition to the mutations reported here in these new fa milies, we have also investigated for the first time the functional signifi cance of previously reported missense mutations and or sequence variants na mely, A82T, A167V, L173R, L205P, S213G and K216E, P222H, T259M, and T259R, which have not previously been functionally characterized Furthermore, thei r effects have been compared with those of the 10 previously reported mutan t enzymes to provide a more consistent and comprehensive study. The present results are in accordance with the prediction that no functional 3 beta HS D 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 3 beta HS D deficiency. Whereas the nonsalt-losing form also results fl om missense m utation(s) in the HSD3B2 gene, which cause an incomplete loss in enzyme act ivity, 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 3 beta HSD deficiency, con pled with the previous studies repo rting that no mutations were found in both HSD3B1 and/or HSD3B2 genes in su ch patients strongly support the conclusion that this disorder does not res ult from a mutant 3 beta HSD isoenzyme. The present study provides biochemi cal evidence supporting the involvement of a new molecular mechanism in cla ssical 3 beta HSD deficiency involving protein instability and further illu strates the complexity of the genotype-phenotype relationships of this dise ase, in addition to providing further valuable information concerning the s tructure-function relationships of the 3 beta HSD superfamily.