We found multiple WNK1
tSNPs and haplotypes to be significantly associated with BP, EH and urinary potassium excretion. The strong prior functional and genetic evidence for the role of WNK1
in BP regulation 
together with replication in additional populations, provides further support for the role of WNK1
in BP regulation in both hypertensives and the general population. Although environmental effects, such as diet and drug treatment will confound the reported associations and may lead to inaccurate estimates of effect size, our data supports observations that WNK1
regulates BP and K+
excretion in vivo
, however, the association with K+
homoeostasis remains to be confirmed 
In the BRIGHT study resource, the strongest tSNP association was seen between rs765250 and SBP. This association was replicated in additional populations, suggesting that the original association is unlikely to be a false positive. This association, however, was not replicated in all cohorts tested. Failure to replicate the effect of rs765250 on EH and BP in every population could be due to genetic heterogeneity across populations, small effect sizes or low power. Although the associations did not reach statistical significance in some of the individual cohorts, for the majority of populations, the direction of the effect was consistent with that seen in BRIGHT, with overlapping 95% confidence intervals. Encouragingly, in the combined meta-analyses, the evidence for association with SBP increased. Notably, this same SNP has previously been associated with ambulatory SBP and DBP in the families from the GRAPHIC study (min p
, and more recently, with DBP gradient (p
0.02) in children from the Avon Longitudinal Study of Parent and Children Study 
, lending further support to the reported findings.
In the single tSNP analyses, our primary associations with BP variation and EH were observed with variants located in the WNK1
promoter regions and intron 1. In contrast to this, the tSNPS associated with urinary K+
excretion span the entire length of the gene. However, there is some overlap between those tSNPs associated with BP and variation in urinary K+
. In particular, the variant rs765250, located in intron 1, which demonstrated the strongest evidence for association with EH and BP variation, is also associated with decreased in urinary K+
excretion. These novel genetic data correlate well with what is known about WNK1
function, especially in relation to the primary phenotypes that characterise PHA2 - hyperkalemia and hypertension 
. That is, we would expect true functional variants (or those in LD with these polymorphisms) to be associated with both BP and altered potassium excretion; this is what we observe.
Although there is some overlap between those tSNPs associated with BP and urinary potassium excretion (eg. 3/7 BP SNPs are also associated with variation in urinary potassium), not all BP associated variants were associated with urinary potassium and vice-versa. Furthermore, haplotypes associated with increased BP were not also associated with decreased urine potassium excretion. This discrepancy may represent complex interactions between WNK1 polymorphisms and may also reflect the complexity of WNK1 regulation and its role in electrolyte homeostasis.
There are two major isoforms of WNK1
is ubiquitously expressed, but Ks-WNK1
has so far only been found to be expressed in the kidney 
. Both L-WNK1
interact with each other to regulate common downstream targets involved in electrolyte homeostasis and BP regulation, via both kinase dependent and independent mechanisms [(e.g., sodium chloride co-transporter (SLC12A3
), epithelial sodium channel (ENaC) and the renal outer medullary potassium channel (KCNJ1
)]. These isoforms are under the control of alternative promoters – one located 5′ of the gene for L-WNK1
, and the other in intron 4, controlling expression of Ks-WNK1 
. Furthermore, both isoforms undergo tissue specific splicing and further variation is achieved by the use of two polyadenylation sites 
. These data imply that there are multiple functional sites along the gene through which genetic variation could effect WNK1
expression and function. Furthermore, it has been observed in some PHA2 patients carrying the WNK1
deletion mutations, that the development of hyperkalemia may be separate from hypertension, and often precedes the development of high BP in these patients i.e., there may be no clear “cause and effect” relationship between the two phenotypes 
. Therefore, it is possible that different WNK1
polymorphisms, either singly or in combination, could contribute to the two different phenotypes. This could account for some of our observations and will need to be explored with further studies.
We found multiple tSNPs spanning the entire length of the gene and several haplotypes to be associated with the traits of interest, suggesting there may be multiple causal variants across the WNK1
locus. Even though we used a comprehensive tSNP set that captured all known common WNK1
variation in HapMap, HapMap does not contain a complete catalogue of all genetic variation, thus fine mapping will be required to identify the true causal variants. Interestingly, all common HapMap SNPs in strong LD (r2
>0.8) with rs765250 and the other BP associated tSNPs, map to the L-WNK1
promoter, the Ks-WNK1
promoter located in intron 4 
and regions in intron 1 that span the sites of the PHA2 deletions (Figure S2
), thus highlighting a few potential regions for targeted re-sequencing.
The most striking observations from our analyses were the identification of low frequency haplotypes with large BP lowering effects and their increased prevalence in the control population. Loss of WNK1
function is deleterious, as demonstrated by homozygous knockout mice which are embryonic lethal 
. On the other hand, heterozygous knockout mice have low blood pressure, and this is associated with decreased WNK1
expression at the mRNA and protein level. Therefore, we can hypothesise that loss of function/expression mutations in WNK1
would be selected against and be rare in the general population. More subtle mutations, however, that lead to decreased WNK1
expression or function may be ‘protective’ against hypertension, and preserved at low frequencies in the general population. However, the effects of genetic drift should not be underestimated in terms of allowing slightly deleterious alleles to persist in human populations 
. This may explain some of our observations. The ability of common tSNPs/haplotypes to capture rare functional variants has previously been demonstrated for the angiotensinogen (AGT) gene 
. It is possible that our tSNP analysis set is capturing rare loss-of-function mutations that may be embedded in these low frequency haplotype backgrounds, thus highlighting the need to re-sequence individuals carrying these low frequency haplotypes.
Our data suggest that multiple common WNK1
variants, with relatively weak effects, and multiple rare variants with large effects may be associated with blood pressure variation, and this should be explored further. Our findings are consistent with recent studies by Ji et al (2008) 
and Tobin et al (2008) 
. Both groups have performed a systematic analysis of the effect of variants in genes involved in renal salt handling on blood pressure and the development of hypertension. Many of which are regulated by WNK1
. Ji et al (2008) have identified rare variants in these genes that are associated with significantly lower blood pressure and protect from the development of hypertension in members of the Framingham Heart Study (FHS) 
. In addition, Tobin et. al. (2008) have reported associations between common variants in these genes and blood pressure in families from the general population 
. The findings suggest that both common and rare variants in genes responsible for some Mendelian disorders of hypertension and hypotension may also affect blood pressure variation in the general population. Our data lends further support to these observations.
The importance of rare variants to quantitative trait variability and susceptibility to disease has now been demonstrated for a number of other important phenotypes; including high density lipoprotein cholesterol (HDL-C), low density lipoprotein cholesterol (LDL-C) plasma triglycerides and body weight 
. We believe there is now compelling evidence to initiate further studies to identify functional WNK1
variants that have a significant impact on BP variation, electrolyte homeostasis and risk of EH in the general population; thus taking an important step forward in our understanding of the pathogenesis of human essential hypertension.