IMPE Abstracts

Introduction: Cytochrome P450 oxidoreductase (POR) acts as a redox partner of all microsomal cytochrome P450s which metabolize drugs and steroid hormones. Mutations in POR cause congenital adrenal hyperplasia (CAH) and/or disorders of sexual development (DSD) due to defective biosynthesis of steroid hormones in the adrenal and gonads, and affect the hepatic metabolism of drugs. The variant V631I was previously identified only in healthy heterozygous carriers. Functional studies performed by our group predicted this variant to significantly reduce the dual activity of CYP17A1 by about 50 %. In 2022, this variant was reported as a compound heterozygous in an XY patient with POR deficiency.

Aims: Expand the characterization of POR variant V631I found in a patient with PORD.

Methods: We analyzed the ability of POR wild-type (WT) and V631I to reduce resazurin, MTT, cytochrome c, and activity towards the drug and steroid metabolizing cytochrome P450. POR WT and Val631Ile were expressed and produced as recombinant proteins in bacteria (E. coli C41(DE3) and combined with recombinant P450 proteins and small molecule substrates for enzyme assays using UV-Vis and fluorescence spectroscopy and analysis of steroids.

Results: We found significant effects of Val631I mutation on activities with different substrates. As compared to WT, Val631I showed between 20 to 50 % of the WT activity in cytochrome C, MTT, and Resazurin reduction. Activities of CYP3A4, CYP3A5, CYP2C9, and CYP2C19 supported by POR-V631I were inhibited in a range of 15-40%. The 21, hydroxylase showed about 85% of activity. However, 17,20 lyase activity of CYP17A1 was only 45% compared to WT POR.

Discussion: The V631 residue is located in the NADPH binding region of POR. Computational analysis predicted the instability of POR V631I which was confirmed with the functional assays. Therefore, an adverse effect on multiple P450 enzyme activities due to V631I mutation in POR was predicted and then confirmed by experiments with purified recombinant proteins.

Conclusion: The fact that this variant was sorted by our group as a loss of function variant years before an actual case was reported reaffirms the importance of employing big genome data analysis to search and predict disease causing mutation and we showed that this could represent a helpful.