Abstract
A broad spectrum of human diseases are caused by mutations in the NADPH cytochrome P450 oxidoreductase (POR)1. POR transfers electrons from NADPH to several microsomal cytochrome P450 proteins (CYPs). A number of POR mutations and polymorphisms have been characterized from patients and genome sequencing databases.
We performed a database search of 1000 genome sequences to identify potentially disease causing variants in POR. Our aim was to check if POR variations from nonclinical samples can be disruptive. Y607C variant (rs72557954, NM_000941.2:c. 1820A>G) has been reported in population studies and prevalent in south Asians (2), but was predicted to be likely pathogenic.
We analysed the ability of POR wild type (WT) and Y607C variant to reduce ferricyanide, MTT, cytochrome c and drug /steroid metabolizing CYP450. POR WT and Y607C and CYP19A1/CYP3A4 were produced as recombinant proteins. The effect of mutation on cofactor (FAD/FMN) binding and activities under varying substrate and cofactor conditions were measured.
We found varied effects of Y607C mutation on activities with different substrates. As compared to WT, Y607C variant showed 66% cytochrome c and 91 % ferricyanide reduction activity but had only 13 % MTT reduction activity. Y607C did not affect POR flavin content but NADPH binding was severely affected. With varying NADPH, Y607C showed 95% decrease in supporting CYP19A1 and CYP3A4 activities. This mutation was later identified in patients with POR deficiency.
Identification of severe effects on both drug and steroid metabolizing CYP450s indicates that likely pathogenic mutations may be found in apparently normal (non-clinical) population. Their combination may lead to severe impact on both steroid and drug metabolism by modification of redox partners activities. Variations in POR need to be evaluated individually. Advanced identification of disease causing variants in POR will help in understanding the POR deficiency in patients if the same mutations are later identified.
References
1 Pandey AV, Fluck CE. NADPH P450 oxidoreductase: structure, function, and pathology of diseases. Pharmacol Ther. (2013) 138:229-54.
2 Burkhard FZ, Parween S, Udhane SS, Fluck CE, Pandey AV. P450 Oxidoreductase deficiency: Analysis of mutations and polymorphisms. J Steroid Biochem Mol Biol. (2017) 165:38-50.
Funding: Swiss National Science Foundation (31003A_134926).
