Abstract 50: Characterization of the Gwas-nominated Agtrap-plod1 Locus Using Zfn-mutated Rats
Background: Genome-wide association studies (GWAS) are frequently used to nominate candidate genes for complex diseases, but are largely unable to identify the causative variant(s). One example: the AGTRAP-PLOD1 locus contains 6 genes in close proximity that are all associated with blood pressure (BP), making it difficult to delineate specific allele(s) that underlie human hypertension at this locus. Here we present a novel rat model that was developed to rapidly dissect GWAS loci using high-throughput gene mutation on a single hypertensive background.
METHODS: Agtrap, Mthfr, Clcn6, Nppa, Nppb, and Plod1 were individually mutated in the Dahl SS rat by zinc finger nuclease (ZFN) injections. ZFN-mutated and wild type (WT) control SS rats (n=8-25 per group) were assessed for BP and renal damage after 10 days on a 4% NaCl diet. Temporal gene expression during development of hypertension was assessed by qRT-PCR and confirmed by RNA-seq.
Results: Expression of all AGTRAP-PLOD1 transcripts changed significantly in response to elevated BP, indicating possible roles of the entire AGTRAP-PLOD1 locus in salt-sensitive hypertension. However, compared with WT, only NPPA mutation further increased mean BP (Δ+27mmHg, P<0.001) and heart weight (1.62±0.03 vs. 1.13±0.03g, P<0.001) in response to 4% NaCl diet. In contrast, CLCN6 mutation significantly decreased diastolic BP (Δ-22mmHg, P<0.001) but had no effect on systolic BP compared to WT. Following salt challenge, proteinuria was elevated by mutation of PLOD1 (147±12 mg/day, P<0.001) and MTHFR (132±23 mg/day, P<0.05) compared with WT littermates (91±5 mg/day), whereas AGTRAP mutation decreased protein excretion (54±9 mg/day, P<0.05). Analysis of haplotype blocks that are associated with gene expression and human hypertension confirmed that NPPA, CLCN6, and MTHFR are the primary mediators of BP changes linked to the AGTRAP-PLOD1 locus.
Conclusions: Combined with human GWAS, our data show for the first time that NPPA and CLCN6 are divergent mediators of BP at the AGTRAP-PLOD1 locus, while MTHFR mutation directly increases susceptibility to end-stage renal disease. These novel mechanistic data provide rationale for developing haplotype-specific therapies for treating hypertension.
- © 2012 by American Heart Association, Inc.