Background: CYP11A1 encodes the P450 side chain cleavage enzyme (P450scc) responsible for initiating steroidogenesis and classically gives rise to disordered sex development plus adrenal and gonadal insufficiency. The rs6161 variant in exon 5 of CYP11A1 (c.940G>A; p.E314K) has previously been considered as benign. When next generation sequencing was performed in patients with primary adrenal insufficiency of unknown etiology the rs6161 variant was found as a heterozygous change in 15 subjects (minor allele frequency 0.0625 (our cohort) vs 0.0024 (ExAC database)). In 13 patients the c.940G>A variant occurred in trans with a second clearly disruptive heterozygous change, in the remaining two patients the only other novel variant was a synonymous substitution (c.990G>A (p.T330=), c.1173C>T (p.S391=)). Given the enrichment of the c.940A allele and rarity of the two synonymous variants c.990A and c.1173T (MAF 0 and 3×106 respectively), we suspected they were pathogenic and therefore investigated their effects on mRNA processing and/or protein function.
Results: An in vitro splicing assay demonstrated aberrant splicing for all three variants, causing exon 5 skipping in most RNA transcripts for variants c.940A and c.990A and complete exon 7 skipping for c.1173T. In each instance when the exon is skipped a frameshift and premature translation-termination codon will occur. We corroborated the findings for variants c.940A and c.990A in vivo in patient samples. Mutant p.314K P450scc enzyme activity did not differ from WT in a validated functional assay in E. coli. However, when expressed in a eukaryotic cell line the mutant protein is truncated to 30-35 kDa, and the half-life is much shorter than for WT.
Conclusion: This comprehensive analysis illustrates how multiple mechanisms might contribute to loss-of-function and highlights the fact that in silico prediction tools are inadequate. Therefore functional analyses need to be undertaken on all rare variants to determine pathogenicity and correctly assign clinical significance.