Analysis of novel variants in the Cytochrome P450 Oxidoreductase gene found in the Genome Aggregation Database applying Bioinformatics Tools and Functional assays

Abstract

<b>Abstract ID 17329</b> <b>Poster Board 584</b> Cytochrome P450 oxidoreductase (POR) is the obligatory redox partner of steroid and drug-metabolizing cytochrome P450s located in the endoplasmic reticulum. Mutations in POR cause a broad range of disorders like congenital adrenal hyperplasia and alter the metabolism of clinically relevant drugs. In this study, we analyzed around 400 missense variants of the POR gene indexed in the Genome Aggregation Database (gnomAD) using eleven different<i> in silico</i> prediction tools. We identified 64 novel variants that were predicted to be disease-causing in most of the tools. We performed further population analysis of these variants and selected two of them for functional studies as proof of concept. We expressed the human POR wild type and the R268W and L577P variants in bacteria. Subsequently, we performed Kinetics assays of POR activities using model substrates. At last, POR (WT or variants) was mixed with purified cytochrome P450 proteins, and the activities of several cytochrome P450 proteins were assayed. In the kinetics studies, the rates of the reactions with the single point mutations R268W and L577P were considerably lower than the reactions with WT POR. Furthermore, we observed a decrease in the enzymatic activities of the main drug metabolizing enzymes CYP3A4, CYP3A5, CYP2C9, and CYP2C19 in a range between 35 to 85 % with variants of POR compared to WT. These results validated our method of curating a big amount of data from the wide genome projects. To be highlighted, we provided an updated and curated reference source for the diagnosis of POR deficiency. The identification and characterization of POR variants is a useful approach to studying the correlation between structural and functional changes in the POR variants and a valuable tool for clinical diagnosis and genetic counseling. Supported by grants to Amit V Pandey from the Swiss National Science Foundation, Burgergemeinde Bern and Novartis Foundation for Medical Biological Research. Maria Natalia Rojas Velazquez is supported by a Swiss Government Excellence Scholarship (ESKAS).