3008-LB: Variant-to-Function analysis of the C2CD4A/B T2D GWAS Signal in Skeletal Muscle Fibers

Introduction and Objective: Genome-wide association studies (GWAS) and mechanistic analyses of insulin resistance (IR) have largely focused on the fasting state, leaving fed-state mechanisms poorly understood. The C2CD4A/B locus harbors a type 2 diabetes (T2D) GWAS signal that colocalizes with an insulin fold change (IFC) signal, an index of fed-state IR. We previously identified a chromatin accessibility QTL (caQTL) at this locus in skeletal muscle, where the T2D risk allele is associated with reduced accessibility at a putative enhancer; the caQTL colocalizes with the T2D and IFC signals. Here, we functionally characterize this variant to elucidate the mechanism underlying the GWAS association. Methods: Luciferase assays were performed in LHCN-M2 human myotubes using constructs centered on the risk or non-risk allele; data were analyzed using a mixed-effects model. Candidate regulatory factors at the caQTL were identified by FIMO motif scan, and an interaction QTL analysis was performed in type 1 fibers. We applied an enhancer-gene linking deep learning model and nominated VPS13C as the effector gene. siRNA-mediated VPS13C knockdown in LHCN-M2 myotubes was assessed by insulin-stimulated glucose uptake (ISGU) assays. Results: Concordant with the caQTL, constructs carrying the risk allele showed reduced reporter activity relative to the non-risk allele (p = 0.004). The interaction QTL revealed that the effect of genotype on chromatin accessibility is modulated by MEF2D expression (βgi = −0.181, p = 0.003), suggesting that the risk allele disrupts MEF2D-dependent enhancer activation in a dose-dependent manner. ISGU assays showed decreased glucose uptake of VPS13C knockdown relative to controls. Conclusion: We provide functional evidence that the C2CD4A/B T2D risk variant reduces enhancer activity through a MEF2D-dependent mechanism in skeletal muscle, and that VPS13C plays a role in ISGU. Collectively, this work advances our understanding of the context-specific mechanisms linking the C2CD4A/B locus to T2D and fed-state IR. Disclosure L. Epelle: None. A. Tovar: None. A.K. Huber: None. A. Varshney: None. P. Orchard: None. S. Parker: Research Support; Current; Pfizer Inc. Consultant; Ended; Novo Nordisk.