Physiological role and mechanisms of action for a long noncoding haplotype region [Hi-C]

Direct targeting of noncoding genomic regions harboring common sequence variants associated with human traits through in vivo animal model studies and precise genome editing in human cells is essential for closing the critical gap between genetic discoveries and physiological understanding. However, such investigation has been impractical for most of these variants as they have small effect sizes and are in haplotypes containing multiple single nucleotide polymorphisms (SNPs) spanning thousands of base pairs. We developed an integrated approach to address this challenge, combining an efficient two-step technique to precisely edit large haplotypes in human induced pluripotent stem cells and orthologous region deletion in phenotypically permissive animal models. As proof of principle, we applied this approach to examine a blood pressure associated locus with a noncoding haplotype containing 11 SNPs spanning 17.4 kbp. We found a robust blood pressure effect of nearly 10 mmHg and identified the physiological and molecular mechanisms involved. Overall design: Human iPSC cells with homozygous BP-elevating or BP-lowering rs1173771 haplotypes were differentiated into endothelial (iEC) and vascular smooth muscle (iVSMC) cells. Region capture Micro-C with Dovetail Micro-C kit and Dovetail targeted enrichment panels plus customer designed probe panel targeting human chr5: 32,443,700-33,470,100 (hg38) to compare the chromatin conformation of the rs1173771 region.