ESPE Abstracts (2018) 89 P-P2-343

Clinical, Biochemical, Structural and Functional Characterization of a Novel P450 Oxidoreductase Mutation Causing Virilization in a 46,XX Patient

Núria Camatsa, Sara Benito-Sanzb, Shaheena Parweenc, Juan-Pedro López-Siguerod, Mónica Fernández-Cancioa, Christa E Flückc, Sameer S Udhanec, Norio Kagawae, Laura Audía & Amit V Pandeyc

aVall d’Hebron Research Institute (VHIR), Hospital Universitario Vall d’Hebron, Centre for Biomedical Research on Rare Diseases (CIBERER), ISCIII, Barcelona, Spain; bINGEMM, Hospital Universitario La Paz, Centre for Biomedical Research on Rare Diseases (CIBERER), ISCIII, Madrid, Spain; cPediatric Endocrinology, Diabetology and Metabolism, University Children’s Hospital, Bern, and Department of Clinical Research, University of Bern, Bern, Switzerland; dPediatric Endocrinology Unit, Hospital Carlos Haya, Málaga, Spain; eNagoya University, Nagoya, Japan

Background: Cytochrome P450 oxidoreductase (POR) deficiency (PORD) is a form of congenital adrenal hyperplasia (CAH) and results in steroid-production loss from cytochrome P450 proteins. Mutations in POR cause mild to severe forms of CAH with/without bone malformation symptoms resembling Antley-Bixler syndrome. We report a novel POR Arg550Trp mutation identified in a 46,XX patient with signs of aromatase (ARO) deficiency. Child (first pregnancy) and mother presented signs of virilization from 6th month. Mother had elevated T at 5th day that returned to normal at 4th month. Daughter was born with fused labioscrotal folds (genital tubercle 1.5 cm with urethral opening, Prader 3). Ultrasound revealed presence of uterus and ovaries. Sequencing of CYP19A1 gene did not reveal any defects and candidate-gene screening revealed compound heterozygous novel mutations c.70_71delTC/p.Leu25PhefsTer93 and c.1648C>T/p.Arg550Trp in POR. At 8y adrenal function is normal except for slightly elevated 17-OH-progesterone.

Methods: 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 cytochrome P450. POR WT and Arg550Trp 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. The CYP19A1 was produced in E.coli JM109 cells with chaperons GroEL and GroES for folding and purified by metal chelate column chromatography.

Results: We 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 reduction of MTT. A 2.75 fold increase in Michaelis constant (Km) was observed in ferricyanide reduction assays compared to WT POR. Further ongoing assays with ARO activity (D4A to E1) and change of NADPH in assays will provide detailed information.

Conclusion: Arg550Trp is located in the NADPH binding region of POR. Binding of NADPH is crucial for electron transfer in POR and supply of redox equivalents to partner proteins and small molecules. Computational analysis predicted instability in the NADPH binding region of POR, which may affect ARO activity to a higher degree than other partner enzymes because ARO requires 6 molecules of NADPH per reaction cycle compared to 2 molecules for other cytochrome P450 partners of POR. Therefore, an adverse effect on ARO activity due to Arg550W mutation in POR is predicted. This mutation combined with p.Leu25PhefsTer93 explains the patient phenotype of PORD.