Biochemical and Functional Characterization of a Novel Mutation in the NADPH Cytochrome P450 Oxidoreductase

Abstract

BackgroundCytochrome P450 oxidoreductase (POR) deficiency (PORD) is a form of congenital adrenal hyperplasia (CAH) and results in loss of steroid and drug metabolizing cytochrome P450 activities. Mutations in POR cause mild to severe forms of CAH with/without bone malformation. We report a novel POR Arg550Trp mutation identified in a 46, XX patient with signs of CYP19A1 deficiency. Child and mother had signs of virilization. Mother had elevated T on the fifth day that returned to normal in the fourth month. The daughter was born with fused labioscrotal folds. Ultrasound revealed the presence of uterus and ovaries. Sequencing of the CYP19A1 gene did not reveal any defects, and further analysis revealed compound heterozygous mutations c.70_71delTC/p.Leu25PhefsTer93 and c.1648C>T/p.Arg550Trp in POR. At eight years the adrenal function of the patient is normal except for slightly elevated 17‐OH‐prog.MethodsDNA was analyzed with a targeted Disorders of Sexual Development NGS panel (DSDSeq.V1, 111 genes) on a NextSeq (Illumina) platform. We analyzed the ability of POR wild‐type (WT) and Arg550Trp to reduce ferricyanide, MTT, cytochrome c and activity towards the drug and steroid metabolizing cytochromes P450. The POR WT and Arg550Trp variants were expressed and produced as recombinant proteins in bacteria and combined with recombinant P450 proteins and small molecule substrates for enzyme assays.ResultsWe found severe effects of Arg550Trp mutation on activities with different substrates. Arg550Trp showed 41% of the WT activity in cytochrome c and only 7.7% activity towards the reduction of MTT. A 2.75 fold increase in Michaelis constant (Km) was observed in ferricyanide reduction assays compared to WT POR. Severe effect on NADPH binding by R550W mutation was observed using both cytochrome c and MTT as substrates with variable NADPH concentration. Assays of CYP19A1 activity showed loss of >90% activities using androstenedione as substrate.ConclusionThe mutation Arg550Trp is located in the NADPH binding region of POR. Computational analysis predicted instability in the NADPH binding region of POR by R550W mutation, Computationally predicted adverse effect on aromatase activity as well as a reduction in binding of NADPH was confirmed by experiments using recombinant proteins. These results suggest a pathological effect of POR R550W and a diagnosis of PORD in the patient with p.Arg550Trp/p.Leu25PhefsTer93 in POR.Support or Funding InformationSupported by grants to Amit V Pandey from the Swiss National Science Foundation, and Novartis Foundation for Medical Biological Research and to Mónica Fernández‐Cancio and Sara Benito from Instituto de Salud Carlos III, Madrid, Spain.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.