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We investigated adrenal steroidogenic enzymes, their activity and mRNA expression, and in vitro biosynthesis of an enzyme in rabbits with congenital adrenal hyperplasia (CAH; weight: CAH, 19 +/- 5 mg/adrenal; normal, 2.7 +/- 1.0 mg/adrenal). Serum pregnenolone (delta 5-P) levels in CAH newborn rabbits (12-36 h) were normal (mean/range, 438/51-2191 ng/dl), but corticosterone levels were low [0.05 +/- 0.05 microgram/dl; P less than 0.001 vs. normal (0.66 +/- 0.57)]. Serum Na+ levels in CAH newborn rabbits were in the normal range (143 +/- 30 meq/liter), but K+ levels were elevated [7 +/- 1.1 meq/liter; P less than 0.05 vs. normal (5.9 +/- 0.6 meq/liter)]. Minced normal adrenal tissue incubated with [3H] cholesterol (30-100 pmol/flask) and ACTH (100 mU/flask) produced [3H]delta 5-P (newborn, 21 and 45 fmol/100 mg; adult, 3 and 5 fmol/100 mg) and [3H]corticosterone (newborn, 23 fmol/100 mg; adult, 11.3 fmol/100 mg), but CAH adrenals produced no product (less than 1.3 fmol/100 mg). Adrenal mitochondria from normal newborn rabbits produced delta 5-P (4.4-7 nmol/mg protein), but CAH adrenals did not, while CAH adrenal mitochondria demonstrated over 4 times greater 11 beta-hydroxylase activity. A Western blot of adrenal homogenate from normal newborn rabbits revealed a cholesterol side-chain cleavage cytochrome P450 (P450scc)-immunoreactive species (mol wt, 53 x 10(3), but this species was absent in CAH adrenals; CAH adrenals had a normal adrenodoxin and intensified 17 alpha-hydroxylase cytochrome P450 (P450(17)alpha) band compared to normal adrenals. In vitro translation of RNA in a cell-free rabbit reticulocyte lysate system containing [35S] methionine yielded a precursor P450scc protein (mol wt, 58.5 x 10(3)) with normal adrenal RNA, but not with CAH adrenal RNA. P450scc mRNA was detected in all normal adrenals, but was not detected in all CAH adrenals. 21-Hydroxylase cytochrome P450 mRNA expression was detected at a similar level in both normal and CAH adrenals. We conclude that CAH in the rabbit is caused by inherited absent P450scc gene expression. The clinical, pathological, and biochemical manifestations of P450scc deficiency in the rabbit are nearly identical to the human disorder. Increased 11 beta-hydroxylase activity and increased P450(17)alpha on Western blot of CAH adrenals indicate altered gene expression of other steroidogenic enzymes due to CAH. Further molecular analysis of the P450scc gene in this animal CAH model will facilitate understanding of P450scc deficiency CAH.

Conversion of cholesterol to pregnenolone is mediated by the cholesterol side-chain cleavage (SCC) enzyme, P450scc. Deficient SCC activity causes congenital lipoid adrenal hyperplasia (also known as 20,22 desmolase deficiency), a potentially lethal defect in the synthesis of all steroid hormones. To probe for possible genetic defects causing this disease we synthesized four oligodeoxyribonucleotides containing 63 to 72 bases corresponding to portions of the bovine complementary DNA (cDNA) sequence for P450scc. The bovine oligonucleotides were labeled and used directly to probe Southern blots of normal human genomic DNA, revealing a pattern indicating there is a single P450scc gene in the human genome. Hybridization to Northern blots of normal human and bovine adrenal messenger RNA indicates that P450scc messenger RNA is about 2.0 kilobases long in both species. Hybridizations of the oligonucleotides to genomic DNA from three unrelated patients with SCC deficiency did not detect a deletion in the human P450scc gene. The bovine sequence oligonucleotides were then used to isolate a human P450scc cDNA clone. The isolated P450scc cDNA fragment contains 818 bases encoding 239 amino acids of the protein, the translation termination signal, and 98 bases of the 3' untranslated region. The sequence of this carboxy-terminal half of the human P450scc protein is 72% homologous with the bovine sequence and contains an additional amino acid not found in bovine P450scc; the human and bovine nucleotide sequences are 81% homologous. Repetition of the genomic DNA blotting studies with the cDNA probe gave the same results obtained with the bovine-sequence oligonucleotide probes, confirming that SCC deficiency is not due to a deletion in the regions of the P450scc hybridizing with the probes. Long, chemically synthesized heterologous sequence oligonucleotides containing unknown numbers of base mismatches with human sequences may thus be used to study human genes so that access to a cDNA is not necessary for such studies.