Multiancestry GWAS And Functional Genomics Analysis Of HNF1A Locus To Define Novel Pathogenic Mechanism For Ischemic Stroke

Background: Ischemic stroke (IS) disproportionately affects populations of African descent due to genetic predispositions. A multiancestry Genome-wide Association Study (GWAS) identified non-coding SNPs in HNF1A locus highly associated with IS, exclusively to African ancestry. Analysis of this ancestry-specific genomic architecture can reveal novel pathogenic mechanism for IS. Methods: The IS-associated SNPs in HNF1A locus were analysed. Electrophoretic Mobility Shift Assay (EMSA) identified functional SNPs (fSNP) that allele-specifically bind to nuclear proteins from human vascular endothelial cells (ECs). Proteomics identified transcription factors (TFs) binding to fSNPs. siRNA knockdown was conducted to determine the function of TFs on HNF1A expression. The effects of stroke-like condition hypoxia on TFs and HNF1A were studied in various vascular cells. Results: GWAS-revealed non-coding tag SNP rs55931441 in HNF1A locus was associated with IS risk exclusively in African population. The frequency of risk allele A of tag SNP was 3.8% in African but absent in other races, supporting the causative role of this locus in determining the genetic risk of IS. EMSA revealed no nuclear protein binding to rs55931441, suggesting it is unlikely an fSNP with TF-mediated regulation on target genes. We expanded the screening to 7 haplotype SNPs in linkage disequilibrium with R 2 >0.8. Among them, rs144534697 presented with allele-imbalanced nuclear protein binding. Proteomics revealed ILF3, a vascular function-related TF, binds preferentially to non-risk allele C. Functional studies showed that ILF3 regulates HNF1A as a repressor. Less binding of ILF3 to risk allele A results in a loss of repression to HNF1A and augments its function as vascular pathogenic factor. Furthermore, vascular stressor hypoxia downregulated ILF3 and upregulated HNF1A in vascular ECs, smooth muscle cells and fibroblasts. Conclusion: Our functional genomics analysis of disease-associated SNPs identified by multiancestry GWAS offers a novel mechanistic approach for diseases with ancestry prominence. The newly identified ILF3-HNF1A pathway may serve as a general mechanistic explanation for the pathogenesis of IS, given their response to vascular stressor hypoxia.