WNK1 and its role in hypertension

Abstract

WNK1 is a serine-threonine protein kinase involved in blood pressure (BP) control and electrolyte homeostasis. Intronic deletions in the WNK1 gene result in over-expression and lead to pseudohypoaldosteronism type II, a disorder characterized by hypertension and hyperkalemia. We have previously reported association between WNK1 polymorphisms and BP (associated SNPs map to promoter and intron 1). This PhD investigated whether the associated variants in WNK1 affect gene expression and also explored the role of low frequency haplotypes and copy number variation (CNV) in WNK1 with blood pressure phenotypes.

Bioinformatic analysis was performed to identify conserved regions across species for intron 1 sequence and to test for the presence of any transcriptional factor binding sites (TFBS). Analysis revealed five conserved regions in intron 1 and many putative TFBS, two of these regions, Int1.8 and Int1.9 were tested for functionality using a dual-luciferase assay. Analyses revealed the Int1.8 region to have a 1.33 fold increase (p = 0.002) and Int1.9 a 0.73 fold decrease (p = 0.04) in luciferase expression in HEK293 cells, and results were consistent in the MDCK cells. Bioinformatic analysis performed on BP associated SNPs (rs1468326, rs3088353, rs2107612 and rs765250) revealed a number of TFBS present. One SNP, rs1468326 was tested for functionality using a dual luciferase assay. Analysis revealed allele A to have a 2.5 fold increase in relative luciferase activity compared to allele C (p = 4.8 x 10-11) in HEK293 cells, results were consistent in MDCK cells. To determine if there were proteins binding to rs1468326 sequence an electrophoretic mobility shift assay (EMSA) was performed, followed by LC-MS/MS for protein identification. The data revealed a protein binding to both alleles in HEK293 and MDCK cells but protein identification was not successful due to high non-specific binding. An EMSA experiment for rs3088353 was also performed and an unknown protein was found to bind to both alleles in HEK293 and MDCK cells.

Real-time quantitative PCR (Q-PCR) analysis was performed on mRNA from sixty human kidney tissues to test if 4 BP associated SNPs correlate with WNK1 expression. No significant difference in expression was observed for any of the SNPs tested.

Finally 24 tSNPs were genotyped in 3 case/control resources to try and validate the association of low frequency haplotypes in WNK1 with decreased risk of hypertension. Analyses revealed no association between haplotypes of WNK1 and hypertension. The role of WNK1 CNV and hypertension was also explored using Q-PCR analysis and access to large collaboration studies. No association with CNV in WNK1 was found with hypertension in either WTCCC or GBPG study.

In conclusion, the functional studies performed on WNK1 variants suggest 2 SNPs are potentially functional, but further work will be required to identify the binding proteins. Genetic studies found no evidence for CNV or confirmation of low frequency haplotypes in WNK1 associated with BP phenotypes. The data in this thesis and larger GWAS recently published suggest common variants in WNK1 are not associated with BP, but the data thus far does not rule out the possibility of rare variants affecting BP at this locus.