Endocrine Abstracts

JOINT467

Several different human diseases are caused by mutations in the NADPH:cytochrome P450 oxidoreductase (POR) that may severely impair its electron transfer ability. As the energy provider to microsomal cytochrome P450 (CYP) enzymes, effects of mutations in POR reach far beyond steroid biosynthesis. Human variants in POR manifest with a very broad phenotype affecting steroid and drug metabolism. POR is composed of a NADPH-binding domain and FAD and FMN-binding domains, linked by a hinge region. This hinge region allows the protein to undergo the conformational changes needed for the electron transfer from the electron donor to the final acceptor, either a CYP, another protein partner, or a small molecule. Variations in different regions of the protein contribute to the heterogeneous effects of POR variants in multiple pathologies. We report two POR variants, Val334Ile and Val472Leu, located in the FAD-binding domain and identified, respectively, in patients with primary adrenal insufficiency and familial glucocorticoid deficiency. Both variants were found to have been reported in population genetics databases, with a higher allele frequency in individuals with South Asian ancestry. The variants were expressed in bacterial systems and partially purified. CYP electron transfer activity was assessed using standard fluorescent or radiolabelled substrates, and small molecule reduction was assessed spectrophotometrically following cytochrome c reduction. Both variants resulted in a decrease in activity of drug metabolizing CYPs 2C9, 2C19, and 3A4, more pronounced for the Val334Ile variant, which maintained only approximately 20% of CYP2C9 and CYP2C19 activity and 70% of CYP3A4 activity. Moreover, preliminary data suggests a pronounced effect for the Val334Ile POR variant in the steroidogenic activity of CYPs 17A1 and 21A2, with approximately 35% of activity compared to wild-type POR. Cytochrome c reduction by POR was also impaired more severely by the Val334Ile variant. In silico predictions of stability and conservation highlighted the Val334 amino acid residue as more conserved and predicted a more deleterious effect of Val334Ile compared to Val472Leu, in accordance with the experimental data.