ESPE Abstracts

Backgrounds and Aims: Primary adrenal insufficiency (PAI) is due to impaired production of steroid hormones by the adrenal cortex. Among PAI of genetic origin, most cases have congenital adrenal hyperplasia (CAH), due to 21-hydroxylase or less frequently 11β-hydroxylase deficiency (11OHD), but 5% have no clear genetic support. Adrenal steroidogenesis pathway comprises three P450 cytochrome-based mitochondrial oxidative steps (CYP11A1, CYP11B1 and CYP11B2). Their enzymatic properties rely on the electron transferring partnership with the iron-sulfur protein adrenodoxin (FDX1). Loss of FDX1 function related damage is expected to be multiple, and reported in vivo inactivation models were all sought to be lethal. We present for the first time two unrelated consanguineous families with apparent 11OHD and bone rickets due to a FDX1 mutation.

Patients and Methods: Two patients, each from unrelated consanguineous families, presented with the same phenotype of apparent 11OHD associated with bone rickets resistant to vitamin D. No mutation was identified in CYP11B1 or in the vitamin D hydroxylation related gene (CYP27B1). We hypothesized an anomaly in a redox partner protein of these two cytochromes and identified the NM_004109.4 (FDX1): c.346C>T missense (p.H116Y) variation. RT-qPCR and western-blotting were performed on cultured fibroblasts from patients, carrier and healthy control. Functional in vitro study was performed on transiently transfected non-steroidogenic COS-1 cells. The mutation was introduced by site-directed mutagenesis. Mutated or wild-type adrenodoxin, adrenodoxin reductase, and P450 cytochromes were encoded by pCMV and pSV40 plasmids. Cells were incubated with specific steroid precursors of steroidogenesis cytochromes, and products were measured by tandem mass spectrometry in order to evaluate residual enzymatic kinetics.

Results and Discussion: The NM_004109.4 (FDX1): c.346C>T variation was absent from all population-wide genome databases. Histidine 116 is located at the close junction between iron-sulfur cluster and redox partner interaction domain. A lowered but non-null (13.5%) expression of p.H116Y adrenodoxin was found in patients culture fibroblasts compared to carrier and healthy controls. In vitro enzymatic study revealed that CYP11B1 and CYP11B2 activities were drastically decreased when co-expressed with p.H116Y adrenodoxin compared to wild-type form (P <0.001), and that CYP11A1 conserved 62% of residual activity when co-expressed with p.H116Y adrenodoxin (P <0.001). Our results are highly supportive for the existence of a maintained partially active form of mutated adrenodoxin with cytochrome-specific metabolic impact, in concordance with observed phenotypes. To sum up, we report a first FDX1 mutation in humans causing an atypical form of congenital adrenal hyperplasia by apparent 11OHD associated with bone rickets.