Aberrant Splicing Is the Pathogenicity Mechanism of the p.Glu314Lys Variant in CYP11A1 Gene

Abstract

Context: The cholesterol side chain cleavage enzyme (CYP11A1) catalyzes the conversion of cholesterol to pregnenolone, the first rate-limiting step of steroidogenesis. CYP11A1 mutations are associated with primary adrenal insufficiency (PAI) as well as disorders of sex development (DSD) in 46,XY patients.Objective: To define the pathogenicity mechanism for the p.Glu314Lys variant, previously reported, and found in four additional patients with CYP11A1 deficiency.Subjects and Methods: DNA of four patients presenting with delayed PAI and/or 46,XY DSD were studied by Sanger or Massively Parallel sequencing. Three CYP11A1 mutations were characterized in vitro and in silico, and one by mRNA analysis on testicular tissue.Results: All patients were compound heterozygous for the previously described p.Glu314Lys variant. In silico studies predicted this mutation as benign with no effect on splicing but mRNA analysis found that it led to incomplete exon 5 skipping. This mechanism was confirmed by minigene experiment. The protein carrying this mutation without exon skipping should conserve almost normal activity, according to in vitro studies. Two other mutations found in trans, the p.Arg120Gln and p.Arg465Trp, had similar activity compared to negative control, consistent with the in silico studies.Conclusions: We provide biological proof that the p. Glu314Lys variant is pathogenic due to its impact on splicing and seems responsible for the moderate phenotype of the four patients reported herein. The present study highlights the importance of considering the potential effect of a missense variant on splicing when it is not predicted to be disease causing.

Long-term outcome of partial P450 side-chain cleavage enzyme deficiency in three brothers: the importance of early diagnosis

“…The partial nature of P450scc deficiency in this family is consistent with most of the published cases with similar genetic findings (19,24,25,26).…”

“…To date, fewer than 40 patients with P450scc deficiency have been reported. Classic deficiency presents with severe earlyonset adrenal insufficiency in the neonatal period and female external genitalia in 46,XY individuals (11,13,14,15,16,17,18,19). Partial defects result in late-onset adrenal insufficiency or glucocorticoid insufficiency alone, associated with either normal genitalia or variable degrees of underandrogenisation (11,15,19,20,21,22,23,24,25).…”

“…Recently, Maharaj and colleagues reported that the missense mutation rs6161, c.940G>A, p.Glu314Lys, previously predicted to be a benign variant, results in PAI in the context of compound heterozygosity when combined with variations harboring disruptive changes (26). In all, 28 cases of P450scc deficiency related to the presence of the rs6161, c.940G>A, p.Glu314Lys variant have been described, 19 by Maharaj et al (26) and 9 from other centres (19,24,25).…”

Primary adrenal insufficiency: New genetic causes and their long‐term consequences

“…Genetic analysis has revealed that many European individuals and families of European ancestry with this condition are compound (double) heterozygous for a c.940G > A variant (rs6161) on one allele of CYP11A1 and a severely disruptive change on the other allele (Figure A) . Although the c.940G>A variant is predicted to cause a benign protein change (p.E314K), detailed molecular studies have shown that it generates a novel splice site so that missplicing occurs . This variant is carried by approximately 1:140 people of European descent, but is likely to cause adrenal insufficiency only when inherited with a very rare disruptive change on the other allele.…”

“…64 Although the c.940G>A variant is predicted to cause a benign protein change (p.E314K), detailed molecular studies have shown that it generates a novel splice site so that missplicing occurs. 64,65 This variant is carried by approximately 1:140 people of European descent, but is likely to cause adrenal insufficiency only when inherited with a very rare disruptive change on the other allele. Partial CYP11A1 insufficiency can also occur in other populations, such as due to the p.R451W variant common in central Turkey.…”