Functional and structural characterization of POR splicing variants reveals pathogenic mechanisms in PORD

Background Pathogenic POR variants cause P450 oxidoreductase deficiency, a rare steroidogenesis disorder. Missense changes are well characterized, but the clinical and molecular consequences of splicing defects remain unclear. Methods We identified a novel homozygous splice variant (c.1249-2A>C) in a patient with disorders of sex development and Antley-Bixler syndrome -like skeletal malformations. By reviewing 12 published cases and performing minigene assays on five variants (c.731 + 1G>A, c.732-2A>T, c.947 + 1G>A, c.948-30G>A, c.1249-2A>C), we characterized their splicing outcomes. Structural consequences were predicted using AlphaFold; nonsense-mediated mRNA degradation was assessed for c.1249-2A>C using cycloheximide block. Results c.731 + 1G>A and c.947 + 1G>A caused intron retention with premature termination, deleting FAD/NADPH-binding domains. c.732-2A>T and c.1249-2A>C skipped exons 8 and 12, which altered FAD-binding site conformation. c.1249-2A>C mRNA reduction was not rescued by cycloheximide, arguing against NMD and suggesting nuclear retention or intranuclear decay. We classified two variants as pathogenic (c.731 + 1G>A, c.947 + 1G>A), two as likely pathogenic (c.732-2A>T and the novel c.1249-2A>C), and one as likely benign (c.948-30G>A). Conclusion Our findings establish that POR splicing variants, whether causing exon skipping or intron retention, disrupt essential domains and produce severe disease. Minigene-based functional testing enables precise variant classification and sharpens genotype-phenotype correlations, supporting improved diagnosis and informed genetic counseling.