Endocrine Abstracts

JOINT388

P450 oxidoreductase (POR) is an essential enzyme in steroidogenesis. It transfers the electrons from NADPH to microsomal cytochrome P450 (CYP) proteins that drive the synthesis of glucocorticoids, mineralocorticoids, and sex hormones in the adrenal cortex. Patients with POR deficiency could present with metabolic disorders, including impaired sexual development, skeletal anomalies characteristic of the Antley-Bixler phenotype, and congenital adrenal hyperplasia. Over 200 deleterious mutations in POR have been discovered, with a few of them being characterized using purified protein systems. To gain deeper insights into mutated POR activities within the context of living cells, the development of personalized human cell models is crucial. Using CRISPR/Cas9 system we performed the POR knockout in the NCI – H295R (adrenal carcinoma cells) and HEK 293T (human embryonic kidney cells). Resulting cells were subcloned to obtain monogenic cultures representing unified genotype. To evaluate the status of POR, we employed Sanger sequencing of POR gene, Western blot analysis, and the assays on POR function. For HEK 293T cells, the CYP17A1 and CYP21A2 activity were measured using thin layer chromatography. For NCI – H295R cells, the full steroid profile was obtained using LC–MS/MS. Adrenal – derived POR knockout cells were characterized by the significant inhibition of aldosterone, cortisol, and testosterone pathways indicating reduced activities of CYP21A2 and CYP17A1 – two key metabolic partners of POR in steroid biosynthesis. The Progesterone levels were significantly higher in POR knockout adrenal cells and 17OH-Progesterone levels were comparable to controls. In HEK293T cells, loss of POR showed 3.6% of wild-type activity towards CYP21A2 and 0% activity towards CYP17A1. The generated cell lines resemble the steroid abnormalities found in patients with POR deficiency. Additionally, they can be utilized to study patient-specific POR mutations by artificially inducing their expression, thereby supporting the development of personalized disease models. By reflecting individual genetic profiles, these cell models offer valuable insights into the complex metabolic processes in the adrenal glands while also serving as a screening platform to characterize the impact of the POR mutations on steroidogenesis and drug response. Such models hold significant potential for improving both the diagnosis and treatment of POR deficiency.