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A Novel Nonstop Mutation in the Stop Codon and a Novel Missense Mutation in the Type II 3ß-Hydroxysteroid Dehydrogenase (3ß-HSD) Gene Causing, Respectively, Nonclassic and Classic 3ß-HSD Deficiency Congenital Adrenal Hyperplasia

Department of Pediatrics (S.P.,W.W., Y.T.C., G.C.), College of Medicine, University of Illinois, Chicago, Illinois 60612; Department of Pediatrics (B.R.), School of Medicine, University of Chicago, Chicago, Illinois 60637; New York University (R.D.), New York, New York 10016; Department of Pediatrics (S.E.M.), St. Louis University, St. Louis, Missouri 63104; Department of Reproductive and Developmental Sciences (Clinical Biochemistry Section) (A.F.H., K.J.S., J.I.M.), University of Edinburgh, EH3 9YW Edinburgh, Scotland, United Kingdom

Address all correspondence and requests for reprints to: Dr. Songya Pang, Department of Pediatrics (M/C 856), University of Illinois, 840 South Wood Street, Chicago, Illinois 60612.

Abstract

We investigated two novel point mutations in the human type II 3ß-hydroxysteroid dehydrogenase (3ß-HSD) gene causing a mild and a severe form of 3ß-HSD deficiency congenital adrenal hyperplasia. The first is a nonstop mutation in the normal stop codon 373 of the gene in exon IV [TGA (Stop) TGC (Cys) = Stop373C) identified from one allele of a female child with premature pubarche whose second allele had an E142K mutation. The Stop373C mutation predictably results in an open reading frame and a mutant-type (MT) II 3ß-HSD protein containing 467 amino acid residues, compared with the 372 amino acid residues of wild-type (WT) protein. The second is a homozygous missense mutation in codon 222 [CCA (Pro) ACT (Thr) = P222T] in the gene identified from a female neonate with salt-wasting disorder. The pcDNA vectors containing the constructs of WT II 3ß-HSD cDNA, WT cDNA with the open reading frame (WT cDNA⁺), MT Stop373C with the open reading frame (Stop373C⁺) and MT P222T cDNA were transfected in COS-I and 293T cells and expressed a similar amount of 3ß-HSD mRNA. The enzyme activity in intact cells using pregnenolone and dehydroepiandrosterone as substrate in the medium (1 µmol/liter) was identical between the WT cDNA and the WT cDNA⁺, but was decreased to 27% of the WT enzymes at 6 h by MT Stop373C⁺ enzyme, and was undetectable by P222T enzyme. In the homogenates of the cells, both MT Stop373C⁺ and P222T enzyme activities and enzymes were undetectable despite clear detection of WT enzyme activities and WT enzymes. LH response to an LHRH analog stimulation in the pubertal female with the Stop373C/E142K genotypes and in a pubertal female with compound 273/318 frameshift genotypes were comparable to and higher than control females, respectively. In conclusion, a structurally lengthy MT II 3ß-HSD enzyme due to a nonstop mutation was relatively detrimental in intact cells causing the nonclassic phenotype of 3ß-HSD deficiency. A missense P222T mutation was seriously detrimental, causing the classic phenotype of 3ß-HSD deficiency. The undetectable Stop373C and P222T enzymes on Western blottings, together with the respective in vivo and in vitro data, suggest that a relative instability of Stop373C enzyme and a profound instability of the P222T enzyme are likely the detrimental molecular mechanisms. The increased LH in the female with the frameshift genotype and the appropriate LH response in the female with the nonstop genotype correlated with predictably severe and mild ovarian type II 3ß-HSD deficiency, respectively.

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