Cytochrome P450 side-chain cleavage enzyme (CYP11A1) catalyses the first and rate-limiting step of steroidogenesis, facilitating conversion of cholesterol to pregnenolone. Cholesterol, transported by steroidogenic acute regulatory protein (StAR) into the inner mitochondrial membrane, is converted by CYP11A1 into 22R-hydroxycholesterol. Subsequently, CYP11A1 converts 22R-hydroxycholesterol by 20alpha-hydroxylation and cleavage of the C20C22 bond into pregnenolone. All patients with CYP11A1 deficiency described so far presented with combined adrenal insufficiency (AI) and 46,XY disorder of sex development (DSD).
Herein, we describe two siblings (46,XY) presenting with late-onset isolated AI with normal male genital development. The older sibling had first symptoms suggestive of AI at the age of 2 years with the diagnosis finally established at the age of 4.1 years. His younger brother was diagnosed with adrenal insufficiency at the age of 2.5 years. A maternal uncle died at the age of 9 years during an episode of febrile seizures.
Mutation analysis of the DAX1 and StAR genes was performed but did not reveal disease-causing mutations. Finally, we detected a novel homozygous CYP11A1 mutation (c.1351C>T; p.Arg451Trp) in both siblings. Segregation analysis confirmed that both parents were heterozygous carriers.
Functional in vitro analysis was performed in COS7 cells co-transfected with wild-type or mutant CYP11A1, with or without wild-type StAR and adrenodoxin. Transfected cells were incubated with either cholesterol or 22R-hydroxycholesterol and pregnenolone production was measured by liquid chromatography/tandem mass spectrometry (LCMS/MS). This demonstrated partial inactivation of CYP11A1 activity by the p.Ar451Trp mutation consistent with results of in silico analysis.
For the first time, mutant CYP11A1 enzyme activity has been assessed in vitro employing the natural substrate cholesterol and the intermediate product 22R-hydroxycholesterol using LCMS/MS. Importantly, we present a novel entity of isolated AI caused by a partially inactivating CYP11A1 mutation, which should be considered as differential diagnosis in patients with unexplained isolated adrenal insufficiency.