MOLECULAR-BASIS OF CONGENITAL ADRENAL-HYPERPLASIA IN 2 SIBLINGS WITH CLASSICAL NONSALT-LOSING 3-BETA-HYDROXYSTEROID DEHYDROGENASE-DEFICIENCY

We report mutations of the type II 3 beta-hydroxysteroid dehydrogenase (3 beta HSD) gene in two siblings, male and female, with congenital a drenal hyperplasia caused by classical nonsalt-losing 3 beta HSD defic iency. During childhood, the male sibling, born with ambiguous genital ia, and the female sibling, born with normal genitalia, both manifeste d symptoms of mild androgen excess; both apparently had normal tons gl omerulosa function. Gonadal dynamic study at puberty showed the presen ce of partial gonadal 3 beta HSD deficiency in both siblings despite t heir spontaneous pubertal maturation. The 5'-region as well as exons I -II, III, and IV and portions of the adjacent introns of the type II 3 beta HSD gene were amplified by polymerase chain reaction and sequenc ed. In both siblings and their mother, an identical single nucleotide substitution mutation in intron III, six bases up-stream from exon IV, was identified in one allele. This mutation, G to A at nucleotide 665 1, may create a new splicing junction and affect the normal splicing o f the messenger ribonucleic acid. In the other allele of both siblings , a missense mutation from GGG (Gly) to AGG (Arg) at codon 129 (G129R) in exon IV was found. We assessed the effect of the G129R missense mu tation on enzymatic activity by in vitro analysis of the mutant recomb inant enzyme generated by site-directed mutagenesis after its transien t expression in COS-1 cells. Using homogenates from transfected cells, the G129R 3 beta HSD enzyme showed a K-m value for pregnenolone of 10 +/- 2 mu mol/L compared with 1.00 +/- 0.03 mu mol/L for the wild-type type II 3 beta HSD enzyme. When dehydroepiandrosterone was used as su bstrate, the K, value for G129R 3 beta HSD was 14 +/- 2 mu mol/L compa red with 2.1 +/- 0.2 mu mol/L for the wild-type type II 3 beta HSD enz yme. In addition to an apparent decrease in affinity, the G129R mutati on caused a marked decrease in the apparent relative specific activity , thus leading to apparent relative specific efficiencies (relative sp ecific activity/K-m) of 2.0% and 4.7% that of the normal type II 3 bet a HSD using pregnenolone or dehydroepiandrosterone as substrate, respe ctively. It appears likely that this low level of activity is sufficie nt to prevent salt loss, but it is also possible that part of the enzy matic activity comes from the putative remaining percentage of correct ly spliced n6651 allele in these patients.