Varied clinical presentations of seven patients with mutations in CYP11A1 encoding the cholesterol side-chain cleavage enzyme, P450scc

Abstract

Context: The cholesterol side-chain cleavage enzyme P450scc, encoded by CYP11A1, converts cholesterol to pregnenolone to initiate steroidogenesis. P450scc deficiency can disrupt adrenal and gonadal steroidogenesis, resembling congenital lipoid adrenal hyperplasia clinically and hormonally; only 12 such patients have been reported previously.

Objective: We sought to expand clinical and genetic experience with P450scc deficiency.

Patients and methods: We sequenced candidate genes in 7 children with adrenal insufficiency who lacked disordered sexual development. P450scc missense mutations were recreated in the F2 vector, which expresses the fusion protein P450scc-Ferredoxin Reductase-Ferredoxin. COS-1 cells were transfected, production of pregnenolone was assayed, and apparent kinetic parameters were calculated. Previously described P450scc mutants were assayed in parallel.

Results: Four of five Bedouin children in one kindred were compound heterozygotes for mutations c.694C>T (Arg232Stop) and c.644T>C (Phe215Ser). Single-nucleotide polymorphism analysis confirmed segregation of these mutations. The fifth kindred member and another Bedouin patient presented in infancy and were homozygous for Arg232Stop. A patient from Fiji presenting in infancy was homozygous for c.358T>C (Arg120Stop). All mutations are novel. As assayed in the F2 fusion protein, P450scc Phe215Ser retained 2.5% of wild-type activity; previously described mutants Leu141Trp and Ala269Val had 2.6% and 12% of wild-type activity, respectively, and Val415Glu and c.835delA lacked detectable activity.

Conclusions: Although P450scc is required to produce placental progesterone required to maintain pregnancy, severe mutations in P450scc are compatible with term gestation; milder P450scc mutations may present later without disordered sexual development. Enlarged adrenals usually distinguish steroidogenic acute regulatory protein deficiency from P450scc deficiency, but only DNA sequencing is definitive.

Figure 1.

Family pedigree and haplotype analysis for patients 1 to 5. Data are shown for 6 SNPs: two within CYP11A1 (rs2959014 and rs1484213) and two on each side of the gene. These data show the chromosomal regions inherited from each parent. The mutation Arg232Stop is on the B chromosome indicated by dark gray (the B alleles), and the mutation Phe215Ser is on the A chromosome indicated by light gray (the A alleles). DNA was not available from individuals in generations I and II, but the A and B annotations indicate the probable segregation of the chromosomes bearing the mutations. Individuals III.1 and III.2 are shown as consanguineous because III.1 is reportedly related to individual I.4, but the relationship is not certain. All available individuals in generations III and IV were screened for the mutations; heterozygosity for Arg232Stop is designated by symbols filled on the right, and Phe215Ser is designated by symbols filled on the left. The NCBI genomic and protein reference sequences are NG_007973 and NP_000772 respectively.

Figure 2.

Activities of the mutants. A, Western blot of mitochondrial proteins extracted from transfected COS-1 cells, probed with anti-P450scc antiserum. B, Michaelis-Menten analysis. P450scc fusion constructs for WT-F2, Leu141Trp-F2, Phe215Ser-F2, Ala269Val-F2, Val415Glu, and c.835delA were transfected into COS-1 cells for 24 hours and incubated with 0.3μM, 1μM, 3μM, and 5μM 22R-hydroxycholesterol for 24 hours. Pregnenolone production in the culture media was measured by ELISA. The data are shown as mean ± SD. Analyses were not done for Val415Glu and 835delA, because their activities were below the sensitivity of the assay.

Figure 3.

Model of human P450scc structure. The model is based on the crystal structure of human P450scc complexed with 22R-hydroxycholesterol (blue) and adrenodoxin (green); the heme is shown in red, and the boundary for the putative mitochondrial membrane is shown by the gray horizontal line. Residues Leu141, Phe215, Ala269, and Val415 are depicted as packed sphere images: white, carbon; red, carboxyl groups; blue, amine groups. The figure was created using Molsoft ICM MolBrowser (Molsoft LLC, La Jolla, California).