A long first intron of PROS1 modulates protein S expression across tissues in mice and humans

Introns can modulate gene expression through enhancer- or silencer-like elements as well as intron-mediated enhancement. Protein S (PS), encoded by PROS1, is an anticoagulant cofactor for activated protein C and tissue factor pathway inhibitor. PROS1 spans ∼100 kb and harbors an unusually long first intron (∼46 kb). We asked whether intron 1 contributes to PS regulation. Reporter assays in HepG2 cells identified discrete intron 1 regions that enhanced or repressed PROS1 promoter activity. CRISPR/Cas9-mediated deletion of most of intron 1 in HepG2 cells reduced endogenous PS mRNA and secreted PS. Mice carrying large intron 1 deletions were viable but showed markedly reduced PS antigen in plasma and platelets compared with wild-type mice. Tissue profiling revealed variable decreases in PS mRNA across organs, indicating tissue-dependent regulation. In a patient with congenital PS deficiency who was initially negative by conventional coding-region sequencing, long-read sequencing uncovered rare intron 1 variants that reduced reporter activity. These data show that regulatory sequences within PROS1 intron 1 are necessary to maintain PS expression in vivo and suggest that noncoding intronic variants may contribute to congenital PS deficiency. Our findings support the incorporation of PROS1 intron 1 and other noncoding PROS1 regions into genetic testing for PS deficiency.