Identifying multiple causative genes at a single GWAS locus

Abstract

Genome-wide association studies (GWAS) are useful for nominating candidate genes, but typically are unable to establish disease causality or differentiate between the effects of variants in linkage disequilibrium (LD). Additionally, some GWAS loci might contain multiple causative variants or genes that contribute to the overall disease susceptibility at a single locus. However, the majority of current GWAS lack the statistical power to test whether multiple causative genes underlie the same locus, prompting us to adopt an alternative approach to testing multiple GWAS genes empirically. We used gene targeting in a disease-susceptible rat model of genetic hypertension to test all six genes at the Agtrap-Plod1 locus (Agtrap, Mthfr, Clcn6, Nppa, Nppb, and Plod1) for blood pressure (BP) and renal phenotypes. This revealed that the majority of genes at this locus (five out of six) can impact hypertension by modifying BP and renal phenotypes. Mutations of Nppa, Plod1, and Mthfr increased disease susceptibility, whereas Agtrap and Clcn6 mutations decreased hypertension risk. Reanalysis of the human AGTRAP-PLOD1 locus also implied that disease-associated haplotype blocks with polygenic effects were not only possible, but rather were highly plausible. Combined, these data demonstrate for the first time that multiple modifiers of hypertension can cosegregate at a single GWAS locus.

Figure 1.

Mean arterial pressure (MAP) (A), systolic blood pressure (SBP) (B), and diastolic blood pressure (DBP) (C) measured by radiotelemetry in conscious WT and mutant SS rat strains on 4% NaCl diet (n = 8–25 per strain). Data are presented as mean BP ± SEM. (*) P < 0.05, (**) P < 0.01, and (***) P < 0.001 vs. WT as determined by one-way ANOVA followed by a Holm-Sidak post-hoc test. (D) Rats were placed in metabolic cages overnight to acclimate, followed by a 24-h urine collection. Data are presented as mean protein excretion ± SEM (n = 8–25 rats per strain). (*) P < 0.05 and (***) P < 0.001 vs. WT as determined by one-way ANOVA followed by a Holm-Sidak post-hoc test. (E) BP and renal damage are linked with multiple AGTRAP-PLOD1 locus genes in human association studies and mutant SS rat strains. For human, genes were considered linked to a phenotype if an associated SNP or SNPs in LD (r² > 0.6) with an associated SNP caused nonsynonymous mutations or were significantly associated with expression of a gene. Blue lines indicate decreased BP and/or proteinuria associated with a gene in the rat. Red lines indicate increased BP and/or proteinuria associated with a gene in the rat.

Figure 2.

(A) Proportions of functional consequences of all SNPs in LD (r² > 0.6) with the SNPs associated with BP and renal phenotypes at the AGTRAP-PLOD1 locus (see Supplemental Table 1). (B) Genes linked with lead SNPs of haplotypes associated with BP and renal phenotypes at the AGTRAP-PLOD1 locus. A lead SNP was considered linked to a gene if the lead SNP or SNPs in LD (r² > 0.6) with a lead SNP caused nonsynonymous mutations or were significantly associated with expression of a gene. (C) An example of a BP-associated nSNP in NPPA (rs5063) that is in LD (r² > 0.6) with an nSNP in MTHFR (rs2274976). Both are also in LD with fSNPs correlated with the expression of MTHFR, CLCN6, and NPPB. Note that a total of two nSNPs (boxed), two eSNPs (blue), and five fSNPs (green) are in LD with rs5063. (D) An example of an fSNP (rs12121543) that is in LD with two SNPs (rs1801131 and rs484609) associated with BP and renal phenotypes. An overview of the region between genes MTHFR and CLCN6 shows that rs12121543 falls in a transcriptional active region (vertical black bar overlapping histone modification, DNase hypersensitivity, and ChIP-seq peaks). rs12121543 is predicted to change a conserved nucleotide in a consensus STAT1-binding site and is associated with significant differences in MTHFR and CLCN6 expression compared with the major allele. (A,C,D) nSNP, nonsynonymous SNP; eSNP, expression SNP; and fSNP, functional SNP.