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Description
Context: 17α-hydroxylase/17,20-lyase deficiency (17-OHD) is a rare form of congenital adrenal hyperplasia characterized by hypertension, hypokalemia, and sexual infantilism. Its spectrum ranges from complete deficiency to partial forms with spontaneous puberty and fertility, yet the molecular basis of this heterogeneity remains unclear.
Objective: To elucidate the pathophysiology of 17-OHD by integrating phenotyping with functional and structural characterization of seven CYP17A1 variants: T131P, L209P, G303D, L361P, I296T, R340H, and R449H.
Methods: Clinical and hormonal data were obtained from seven probands, including one with the novel T131P mutation. Variants were generated by site-directed mutagenesis in pcDNA3.1 vectors and expressed in HEK-293T cells. Enzymatic activities of 17α-hydroxylase and 17,20-lyase were measured using 14C-progesterone and 3H-17OH-pregnenolone substrates and normalized to protein expression by Western blot. In silico structural modeling localized mutations to catalytic or regulatory domains.
Results: Western blot showed mutant proteins were expressed at levels comparable to wild-type, indicating preserved stability. L209P and G303D showed near-complete loss of hydroxylase activity (<9% of WT), consistent with severe phenotypes. The T131P variant retained ~7% activity, sufficient to permit spontaneous menarche. R449H retained lyase activity, consistent with a history of fertility. Structural modeling linked activity loss to disruptions in the F-helix (L209P), I-helix (G303D), or redox partner-binding surfaces (R449H), rather than gross protein misfolding.
Conclusion: These variants cause disease through specific catalytic or regulatory defects rather than protein instability. Residual activity showed a non-linear relationship with clinical severity, with <10% supporting secondary sexual development. Structural analysis may help predict clinical outcomes and fertility potential in 17-OHD.
Funding: This research was supported by CANCER RESEARCH SWITZERLAND grant number KFS-5557-02-2022 to A.V.P. J.Y. is partially funded by a SWISS GOVERNMENT EXCELLENCE SCHOLARSHIP (ESKAS) grant number 2022.0470
