Analysis of the combined effect of rs699 and rs5051 on angiotensinogen expression and hypertension

Background Hypertension(HTN)involves genetic variability in the renin-angiotensin system and influences antihypertensive response.We previously reported that angiotensinogen(AGT)messenger RNA(mRNA)is endogenously bound by miR-122-5p and rs699 A>G decreases reporter mRNA in the microRNA functional-assay PASSPORT-seq.The AGT promoter variant rs5051 C>T is in linkage disequilibrium(LD)with rs699 A>G and increases AGT transcription.The independent effect of these variants is understudied due to their LD therefore we aimed to test the hypothesis that increased AGT by rs5051 C>T counterbalances AGT decreased by rs699 A>G,and when these variants occur independently,it translates to HTN-related phenotypes.Methods We used in silico,in vitro,in vivo,and retrospective models to test this hypothesis.Results In silico,rs699 A>G is predicted to increase miR-122-5p binding affinity by 3%.Mir-eCLIP results show rs699 is 40–45 nucleotides from the strongest microRNA-binding site in the AGT mRNA.Unexpectedly,rs699 A>G increases AGT mRNA in an AGT-plasmid-cDNA HepG2 expression model.Genotype-Tissue Expression(GTEx)and UK Biobank analyses demonstrate liver AGT expression and HTN phenotypes are not different when rs699 A>G occurs independently from rs5051 C>T.However,GTEx and the in vitro experiments suggest rs699 A>G confers cell-type-specific effects on AGT mRNA abundance,and suggest paracrine renal renin-angiotensin-system perturbations could mediate the rs699 A>G associations with HTN.Conclusions We found that rs5051 C>T and rs699 A>G significantly associate with systolic blood pressure in Black participants in the UK Biobank,demonstrating a fourfold larger effect than in White participants.Further studies are warranted to determine if altered antihypertensive response in Black individuals might be due to rs5051 C>T or rs699 A>G.Studies like this will help clinicians move beyond the use of race as a surrogate for genotype.