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

Abstract

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.

Keywords: angiotensinogen; genetic and genomic medicine; hypertension; rs699.

Figure 1

Schematic showing the RNAduplex predicted increase in binding strength with the rs699 A > G variant. The variant position is highlighted in yellow. Nucleotide bonds that are predicted to arise due to the variant are shown in red. GC pairs are shown with double lines, AU pairs are shown with single lines, and GU pairs are shown with single lines with open dots. The variant nomenclature “rs699 A > G” is complemented to the positive strand, whereas the nucleotides shown are for the AGT mRNA structure, which is transcribed from the negative DNA strand. A, adenine; AGT, angiotensinogen; C, cytosine; deltaG, kcal/mol change in free energy; G, guanine; hsa, homo sapien; miR, microRNA; mRNA, messenger RNA; U, uracil.

Figure 2

UCSC Genome Browser view of our mir‐eCLIP data in BedGraph format, showing the proximity of rs699 to the most prominent microRNA‐binding peak in the AGT mRNA. AGT, angiotensinogen; kb, kilobase; max, maximum; mRNA, messenger RNA; UTR, untranslated region.

Figure 3

GTEx web‐portal data. (Left) Bulk mRNA expression data in log transformed units. (Middle) eQTLs for rs699. (Right) Violin plots showing the more granular eQTL data for rs699 for Liver, and tissues with the most extreme eQTLs or potential relevance to the renin‐angiotensin system. AGT, angiotensinogen; eQTL, expression quantitative trait loci; mRNA, messenger RNA; NES, normalized expression slope; TPM, transcripts per million.

Figure 4

Liver AGT expression across the genotype groups shown with the hypothesized effect on AGT expression. (Left column; A–C) Log transformed TPM AGT reads in liver n = 208, brain n = 209, and colon n = 318. (Right column; D–F) Log transformed TPM AGT reads in kidney n = 76, coronary artery n = 213, and tibial artery n = 583. AGT, angiotensinogen; mRNA, messenger RNA; TPM, transcripts per million.

Figure 5

(A–C) qPCR results for three different cell lines. (A) AGT expression in HepG2 liver cells, (B) AGT expression in HT29 colon cells, and (C) AGT expression in HEK293 kidney cells. Biorep, biological replicate; Ct, cycle threshold; NA, not assessed; qPCR, quantitative polymerase chain reaction; reps, replicates; WT, wild type.

Figure 6

Indiana University Advanced Precision Genomics cohort results for (A) total fills per year and (B) total maximum concomitant meds at any given time.

Figure 7

Blood pressure phenotype regression estimates (corrected for covariates and normalized to standard deviation) with (A) genotype groups according to our ordinal hypothesis, (B) binned genotype groups that test only groups with unbalanced rs699 G and rs5051 T, and (C) rs5051 genotype group. Systolic blood pressure boxplots are shown for (D) genotype groups according to our hypothesis, (E) binned genotype groups that test only groups with unbalanced rs699 G and rs5051 T, and (F) rs5051 genotype group. Boxplots display median (black bar), 25th–75th percentile (box), and mean values are labeled over the box. HTN, hypertension.

Figure 8

Analysis of (A) rs5051 C > T association with systolic blood pressure in Black participants in the UK Biobank data, and (B) allele haplotype frequencies for rs699 and rs5051 in the 1000 Genomes Project for those with African ancestry. Boxplot displays median (black bar), 25th–75th percentile (box), and mean values are labeled over the box. LD, linkage disequilibrium.