Complete POR ablation in human adrenal cells reveals steroidogenic rerouting and variant-dependent loss of cytochrome P450 support

This study provides a novel approach to understanding the effects of POR mutations on steroid hormone synthesis using engineered human cell models.

Summary and findings

This study engineered complete biallelic POR knockout in human adrenal-derived NCI-H295R and HEK293T cells to investigate the effects on steroidogenesis. The findings indicated a blockade in canonical steroidogenesis with upstream accumulation of pregnenolone and progesterone, and depletion of downstream glucocorticoids and mineralocorticoids. The study also evaluated the functional complementation of clinically relevant POR variants.

Abstract

Abstract Cytochrome P450 oxidoreductase (POR) is the obligate electron donor for microsomal cytochrome P450 enzymes. Recessive mutations in the POR gene cause POR deficiency (PORD), a severe metabolic disorder characterized by skeletal malformations, ambiguous genitalia, and adrenal insufficiency. Because global POR knockout is embryonically lethal in mammalian models, the mechanistic study of PORD has historically been restricted to reconstituted biochemical assays or non-steroidogenic cellular backgrounds. Here, we engineered complete biallelic POR knockout in human adrenal-derived NCI-H295R and human embryonic kidney HEK293T cells using CRISPR/Cas9. Transcriptomic and mass spectrometric steroid profiling of the adrenal clones revealed a blockade in canonical steroidogenesis, characterized by upstream accumulation of pregnenolone and progesterone, and severe depletion of downstream glucocorticoids and mineralocorticoids. Strikingly, knockout cells maintained low-level synthesis of dehydroepiandrosterone (DHEA) despite the complete absence of canonical CYP17A1 catalysis, supporting the possibility of alternative, CYP17A1-independent steroidogenic routes. Furthermore, using the HEK293T platform, we evaluated the functional complementation of clinically relevant variants (A287P, R457H, P228L, and delP399_E401). Notably, the highly prevalent P228L variant exhibited selective preservation of CYP17A1 activity while severely impairing CYP19A1 aromatase function. A direct comparison between episomal overexpression and endogenous CRISPR prime editing of P228L highlighted critical differences in enzyme efficiency under native regulatory control. Exploratory imaging further suggested altered redox-associated Fe(II) handling in POR-deficient or P228L-edited cells. These engineered human cell models provide a highly tractable platform for interrogating mutant-specific pharmacogenomics and developing targeted interventions.

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