Abstract 74: The rs1173771 Haplotype Associated with Blood Pressure Influences Chromatin Interactions Involving the Natriuretic Peptide Receptor 3 Gene Promoter

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Determining the mechanisms of action for human blood pressure (BP) variants in the noncoding regions of genome, particularly large haplotypes containing multiple variants, is both critical and challenging. Human BP-associated rs1173771 locus includes a haplotype containing 11 single nucleotide polymorphisms (SNPs) spanning 17.4 kbp. The Linkage Disequilibrium (LD) region is approximately 126 kbp from the transcription start site of the closest protein-coding gene NPR3 (natriuretic peptide receptor 3). We used genome editing and region-capture Micro-C to examine the effect of the rs1173771 haplotype on the expression of neighboring genes and the chromatin mechanisms involved. The BP-elevating and -lowering haplotypes of rs1173771 locus were precisely reconstituted in human induced pluripotent stem cells (iPSCs) using an efficient, two-step genome editing technique. We then differentiated the edited iPSCs to endothelial (iEC) and vascular smooth muscle cells (iVSMC). The BP-elevating haplotype significantly (p ≤ 0.05) decreased NPR3 expression in iEC and iVSMC. Using region capture Micro-C targeting the rs1173771 haplotype region and the promoter regions of neighboring genes, we found in both iECs and iVSMCs, the BP-elevating rs1173771 haplotype increased chromatin contacts between the rs1173771 haplotype region and NPR3 promoter region, compared to the BP-lowering haplotype. Statistical comparison of chromatin interaction using HiCCompare showed the interactions of a genomic segment overlapping the 5’ end of the rs1173771 haplotype region with the NPR3 promoter region and a region just upstream of the promoter were 10 and 6 fold stronger (p.adj ≤ 0.001) in iECs with homozygous BP-elevating haplotype compared to BP-lowering haplotype. Similarly, in iVSMCs, the interaction of a genomic segment overlapping the rs1173771 haplotype region with a region just upstream of the NPR3 promoter was 4 fold stronger (p.adj = 0.02) for homozygous BP-elevating haplotype compared to BP-lowering haplotype. These results suggest that the BP-elevating rs1173771 haplotype interacts with the NPR3 promoter more strongly to suppress NPR3 expression, possibly through the recruitment of inhibitory protein complexes.

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