Abstract 3137: Partial alternative splicing in carriers of <i>DPYD</i> rs75017182 explains modest association with severe 5-FU toxicity to 5-FU

Abstract Dihydropyrimidine dehydrogenase (DPD, encoded by the DPYD gene) is the rate-limiting enzyme in the catabolism of the widely used chemotherapeutic drug 5-fluorouracil (5-FU). Genetic variations in DPYD are predictive biomarkers of 5-FU toxicity. One such biomarker, rs75017182 (the causal allele linked to the “HapB3” haplotype), has been suggested to promote alternative splicing of DPYD, leading to expression of an inactive proteoform. However, carriers of rs75017182 exhibit only moderately increased risk of 5-FU toxicity and modestly decreased ex vivo DPD activity compared to carriers of another well-studied splice variant, rs3918290 (*2A). We hypothesized that, unlike *2A, the rs75017182 variant does not cause obligate alternative splicing but instead promotes partial alternative splicing. To test this, we generated dual-fluorescent minigene reporter constructs containing either the G (wildtype) or C (variant) allele. In our minigene system, alternative splicing was elevated in cells transfected with C-containing vectors via flow cytometry, though levels were lower than those observed in a parallel *2A model. To investigate contributions of other variants in LD with rs75017182, we expanded the reporter to include additional relevant genomic regions. The analyses were complemented by the study of in vivo splicing in cells engineered to harbor various combinations of variants and in lymphoblastoid lines carrying specific genotype combinations. Overall, our findings demonstrate that rs75017182-C promotes only partial alternative splicing, eliciting a modest decrease in DPD activity and explaining its comparatively weak association with severe 5-FU toxicity. Additional variants in LD have a minimal impact on splicing and 5-FU sensitivity. Citation Format: Lauryn Allyn Hahn, Ryan Schwartz, Hannah Marie Krause, Lulu Jiang, Brianna Bembenek, Kelly Bouchonville, Steven M. Offer. Partial alternative splicing in carriers of DPYD rs75017182 explains modest association with severe 5-FU toxicity to 5-FU [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 3137.