Analysis of the Combined Effect of rs699 and rs5051 on Angiotensinogen Expression and Hypertension

Hypertension (HTN) involves genetic variability in the renin-angiotensin system and characterizing this variability will help advance precision antihypertensive treatments. We previously reported that angiotensinogen (AGT) mRNA is endogenously bound by mir-122-5p and that rs699 A>G significantly decreases reporter mRNA in the functional mirSNP assay PASSPORT-seq. The AGT promoter variant rs5051 C>T is in linkage disequilibrium (LD) with rs699 A>G and increases AGT transcription. We hypothesized that the increased AGT by rs5051 C>T counterbalances AGT decrease by rs699 A>G, and when these variants occur independently, would translate to HTN-related phenotypes. The independent effect of each of these variants is understudied due to their LD, therefore, we used in silico, in vitro, in vivo, and retrospective clinical and biobank analyses to assess HTN and AGT expression phenotypes where rs699 A>G occurs independently from rs5051 C>T. In silico, rs699 A>G is predicted to increase mir-122-5p binding strength by 3%. Mir-eCLIP assay results show that rs699 is 40-45 nucleotides from the strongest microRNA binding site in the AGT mRNA. Unexpectedly, rs699 A>G increases AGT mRNA in a plasmid cDNA HepG2 expression model. GTEx and UK Biobank analyses demonstrate that liver AGT expression and HTN phenotypes were not different when rs699 A>G occurs independently from rs5051 C>T, allowing us to reject the original hypothesis. However, both GTEx and our in vitro experiments suggest rs699 A>G confers cell-type specific effects on AGT mRNA abundance. 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 4-fold larger effect than in White participants. Further studies are warranted to determine if the 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.