Blood pressure-associated non-coding single nucleotide polymorphism rs217727 modulates gene expression in human endothelial cells

Abstract

Majority of Blood Pressure (BP)-associated single nucleotide polymorphisms (SNPs) are in intronic or intergenic regions. Allele G of the noncoding SNP rs217727 is associated with decreased systolic, diastolic and pulse pressure. MRPL23 is the closest protein-coding gene to rs217727, and the transcriptional start site of MRPL23 is 48.3kb away from rs217727. rs217727 is an expression quantitative trait locus for MRPL23 and TNNT3 (76.1kb away). We performed a series of studies to experimentally identify genes modulated by rs217727 and to begin to understand the mechanism. We differentiated a human induced pluripotent stem cell (iPSC) line to endothelial cells (iECs). The iECs were purified using VE-cadherin sorting. Chromatin conformation analysis using Micro-C identified several genes including MRPL23, IGF2 (141.6kb away) and TNNT3 as sharing a chromatin loop with rs217727. We used CRISPR-Cas9 to generate isogenic iPSC lines with either homozygous low BP (G) or high BP allele (A) of rs217727. The screening of several hundred clones led to the establishment of three and four clones with low BP and high BP genotypes. The edited iPSC lines were subjected to six rounds of differentiation to iECs for targeted real-time PCR analysis of genes near rs217727, including the genes mentioned above. We found significantly (p<0.05) decreased expression of TNNT3 (0.44±0.04 fold), LSP1 (142.6kb away; 0.12±0.02 fold) and IGF2 (0.78±0.06 fold) and slightly increased expression of CD81 (380.5kb away; 1.09±0.03 fold) in iECs with the high BP allele (n≥43) compared to the low BP allele (n≥34) of rs217727 (p<0.05). TNNT3 encodes troponin T3 fast skeletal type, LSP1 encodes lymphocyte specific protein 1 and IGF2 encodes insulin-like growth factor 2. The closest gene MRPL23, CTSD (231.8kb away), and MRPS6, a gene on a different chromosome, were not significantly differentially expressed. In summary, the results indicate that rs217727 influences the expression of several local genes in an allele-specific manner. The results provide new insights into the function of a BP-associated noncoding SNP. The work is supported by NHLBI P01 HL149620. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.