We investigated two novel point mutations in the human type II 3beta-hydroxysteroid
dehydrogenase (3beta-HSD) gene causing a mild and a severe form of
3beta-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 3beta-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 3beta-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 3beta-HSD
mRNA. The
enzyme activity in intact cells using
pregnenolone and
dehydroepiandrosterone as substrate in the medium (1 micromol/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 3beta-HSD
enzyme due to a nonstop mutation was relatively detrimental in intact cells causing the nonclassic phenotype of 3beta-HSD deficiency. A missense P222T mutation was seriously detrimental, causing the classic phenotype of 3beta-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 3beta-HSD deficiency, respectively.