Endocrine Abstracts (2017) 50 OC4.1 | DOI: 10.1530/endoabs.50.OC4.1

Predicted benign and silent SNPs in CYP11A1 cause primary adrenal insufficiency through missplicing

Avinaash Maharaj1, Federica Buonocore2, Eirini Meimaridou1, Gerard Ruiz-Babot1, Leonardo Guasti1, Hwei-Ming Peng3, Cameron Capper3, Rathi Prasad1, Elizabeth Crowne4, Timothy Cheetham5, Caroline Brain2, Jenifer Suntharalingham2, Niccolo Striglioni2, Bilgin Yuksel6, Fatih Gurbuz7, Richard Auchus3, Helen Spoudeas2, Tulay Guran7, Stephanie Johnson8,9, Dallas Fowler9,10, Emma Duncan8,9, Louise Conwell8,9, Delphine Drui11, Bertrand Cariou12, Juan Pedro Lopez Siguero13, Mark Harris8,9, Malcolm Donaldson14, John Achermann2 & Lou Metherell1


1Centre for Endocrinology, William Harvey Research Institute, Queen Mary, University of London, London, UK; 2Genetics and Genomic Medicine, UCL Great Ormond Street Institute of Child Health, University College London, London, UK; 3Division of Metabolism, Endocrinology, and Diabetes, Department of Internal Medicine, and Department of Pharmacology, University of Michigan, Ann Arbor, MI, USA; 4Department Paediatric Endocrinology and Diabetes, Bristol Royal Hospital for Children, Bristol, UK; 5Institute of Genetic Medicine, Newcastle University, Newcastle, UK; 6Cukurova University, Department Pediatric Endocrinology and Diabetes, Adana, Turkey; 7Marmara University, Department Pediatric Endocrinology and Diabetes, Istanbul, Turkey; 8Lady Cilento Children’s Hospital, Brisbane, Australia; 9University of Queensland, Brisbane, Australia; 10Lady Cilento Children’s Hospital; 11CHU de Nantes, l’institut du thorax, Department of Endocrinology, Nantes, France; 12INSERM UMR 1087, CNRS UMR 6291, Université de Nantes, l’institut du thorax, Nantes, France; 13Pediatric Endocrinology Unit Children’s Hospital, Malaga, Spain; 14Section of Child Health, Glasgow University School of Medicine, Glasgow, UK.


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.

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