Discovery of rare variants associated with blood pressure regulation through meta-analysis of 1.3 million individuals

Abstract

Genetic studies of blood pressure (BP) to date have mainly analyzed common variants (minor allele frequency > 0.05). In a meta-analysis of up to ~1.3 million participants, we discovered 106 new BP-associated genomic regions and 87 rare (minor allele frequency ≤ 0.01) variant BP associations (P < 5 × 10^-8), of which 32 were in new BP-associated loci and 55 were independent BP-associated single-nucleotide variants within known BP-associated regions. Average effects of rare variants (44% coding) were ~8 times larger than common variant effects and indicate potential candidate causal genes at new and known loci (for example, GATA5 and PLCB3). BP-associated variants (including rare and common) were enriched in regions of active chromatin in fetal tissues, potentially linking fetal development with BP regulation in later life. Multivariable Mendelian randomization suggested possible inverse effects of elevated systolic and diastolic BP on large artery stroke. Our study demonstrates the utility of rare-variant analyses for identifying candidate genes and the results highlight potential therapeutic targets.

Extended Data Fig. 1. Power estimation for stage 2 meta-analyses

Power calculations were performed assuming that, for any given variant, there were 1,318,884 individuals for EAWAS PA analyses, 1,164,961 participants for EAWAS EA analyses, and 670,472 participants for RV-GWAS analyses. Calculations were performed in R (https://genome.sph.umich.edu/wiki/Power_Calculations:_Quantitative_Traits).

Extended Data Fig. 2. Expression of genes implicated by the rare SNVs in GTEx v7 tissues

We used FUMA GWAS to perform these analyses. We included genes closest to the identified rare variants from the EAWAS and the RV-GWAS.

Extended Data Fig. 3. Tissue enrichment of rare variant gene expression levels in GTEx v7

We used FUMA GWAS to perform these analyses. We included genes closest to the identified rare variants from the EAWAS and the RV-GWAS.

Figure 1. Study design for single variant discovery.

a, Exome array-wide association study (EAWAS) of SBP, DBP, PP and HTN. In Stage 1, we performed two fixed effect meta-analyses for each of the blood pressure (BP) phenotypes SBP, DBP, PP and HTN: one meta-analysis including 810,865 individuals of European (EUR) ancestry and a second pan-ancestry (PA) meta-analysis including 870,217 individuals of EUR, South Asians (SAS), East Asians (EAS), African Ancestry (AA), Hispanics (HIS) and Native Americans (NAm) (Supplementary Tables 23 and 24; Methods). Summary association statistics for SNVs with P < 5 × 10^-8 in Stage 1 that were outside of previously reported BP loci (Methods, Supplementary Tables 1 and 25) were requested in independent studies (up to 448,667 participants; Supplementary Table 24). In Stage 2, we performed both a EUR and a PA meta-analyses for each trait of Stage 1 results and summary statistics from the additional studies. Only SNVs that were associated with a BP trait at P < 5 × 10^-8 in the combined Stage 2 EUR or PA meta-analyses and had concordant directions of effect across studies (P _{heterogeneity} > 1 × 10^-4; Methods) were considered significant. Further details are provided in the Methods and Supplementary Information. b, Rare variant GWAS (RV-GWAS) of SBP, DBP and PP. For SNVs outside of the previously reported BP loci (Methods, Supplementary Tables 1 and 6) with P < 1 × 10^-7 in Stage 1, summary association statistics were requested from MVP (up to 225,112 participants; Supplementary Table 24). In Stage 2, we performed meta-analyses of Stage 1 and MVP for SBP, DBP and PP in EUR. SNVs that were associated with a BP trait at P < 5 × 10^-8 in the combined Stage 2 EUR with concordant directions of effect across UKBB and MVP (P _{heterogeneity} >1 × 10^-4; Methods) were considered significant. Justification of the significance thresholds used and further information on the statistical methods are detailed in the Methods and Supplementary Information. *Total number of participants analyzed within each study that provided single variant association summaries following the data request—EAWAS EUR: Million Veterans Program (MVP: 225,113), deCODE (127,478) and GENOA (1,505); EAWAS PA: Million Veterans Program (MVP: 225,113 EUR; 63,490 AA; 22,802 HIS; 2,695 Nam; 4,792 EAS), deCODE (127,478 participants from Iceland) and GENOA (1,505 EUR; 792 AA); RV-GWAS EUR: Million Veterans Program (MVP: 225,112 EUR).

Figure 2. New BP associations.

a, Fuji plot of the genome-wide significant BP-associated SNVs from the Stage 2 EAWAS and Stage 2 rare variant GWAS. The first four circles (from inside-out) and the last circle (locus annotation) summarize pleiotropic effects, while circles 5 to 8 summarize the genome-wide significant associations. Every dot or square represents a BP-associated locus, and large dots represent novel BP-associated loci, while small dots represent loci containing novel variants identified in this study, which are in linkage disequilibrium with a variant reported by Evangelou et al. and/or Giri et al.. All loci are independent of each other, but due to the scale of the plot, dots for loci in close proximity overlap. *Loci with rare variant associations. b, Venn diagram showing the overlap of the 107 new BP loci across the analyzed BP traits. c, Functional annotation from VEP of all the identified rare variants in known and novel regions. d, Plots of minor allele frequency against effect estimate on the transformed scale for the BP-associated SNVs. Blue squares are new BP-associated SNVs, black dots represent SNVs at known loci, and red dots are newly identified distinct BP-associated SNVs at known loci. Effect estimates and SEs for the novel loci are taken from the Stage 2 EUR analyses (up to 1,164,961 participants), while for the known are from the Stage 1 analyses (up to 810,865 participants). Results are from the EAWAS where available and the GWAS (up to 670,472 participants) if the known variants were not on the exome array (data from Supplementary Tables 1, 3, 7, 8, and 25 were used).

Figure 3. Annotation of BP loci.

a, BP associations shared with eQTL from GTEx through multi-trait colocalization analyses. Expressed gene and the colocalized SNV are provided on the y-axis. BP trait and eQTL tissues are provided on the x-axis. The color indicates whether the candidate SNV increases BP and gene expression (brown), decreases BP and gene expression (orange), or has the inverse effects on BP and gene expression (blue). b, Enrichment of BP-associated SNVs in DNase I hypersensitivity hot spots (active chromatin). The top plot is for SBP, middle is for DBP, and bottom represents PP. Height of the bar indicates the fold enrichment in the listed tissues, with error bars representing the 95% confidence intervals. The colors represent the enrichment P-value.

Figure 4. Phenome-wide associations of the new BP loci.

a, Modified Fuji plot of the genome-wide significant associated SNVs from the Stage 2 EAWAS and Stage 2 rare variant GWAS (novel loci only). Each dot resents a novel locus where a conditionally independent variant or a variant in LD with the conditionally independent variant has been previously associated with one or more traits unrelated to blood pressure, and each circle represents different trait category (Supplementary Table 20). Locus annotation is plotted in the outer circle, and * sign denotes loci where the conditionally independent signal maps to a gene which is different to the one closest to the sentinel variant. b, Bar chart showing the distribution of traits (x-axis) and number of distinct BP-associated variants per trait (y-axis) that the SNVs in a are associated with. c, Bar chart of the number of traits included in b (y-axis) by trait category (x-axis). The color coding for a and b is relative to c.

Figure 5. Causal association of BP with stroke and coronary artery disease.

Mendelian randomization analyses of the effect of blood pressure on stroke and coronary artery disease. a, Univariable analyses. b, Multivariable analyses (Methods). Analyses were performed using summary association statistics (Methods). The causal estimates are on the odds ratio (OR) scale (the square in the plot). The whiskers on the plots are the 95% confidence intervals for these ORs. Results on the standard deviation scale are provided in Supplementary Table 22. The genetic variants for the estimation of the causal effects in this plot are sets of SNVs after removing the confounding SNVs and invalid instrumental variant. OR, odds ratio (P-value from the inverse variance weighted two sample Mendelian randomization method). n, number of disease cases.