Plasma potassium is a moderately heritable phenotype, but no robust associations between common single nucleotide polymorphisms (SNPs) and plasma potassium have previously been described. Genetic influences on renal potassium handling could be important in the aetiology of hypertension. We have tested whether common genetic variation in the gene encoding the beta subunit of the epithelial sodium channel (SCNN1B) affects plasma potassium and blood pressure level in a study of 1425 members of 248

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... families ascertained on a proband with hypertension. We characterised family members for blood pressure using ambulatory monitoring, measured plasma potassium in venous blood samples, and genotyped four SNPs which spanned the SCNN1B gene. We found highly significant association between genotype at the SCNN1B rs889299 SNP situated in intron 4 of the gene and plasma potassium. Homozygotes for the rarer T allele had on average a 0.15mM lower plasma potassium than homozygotes for the common C allele, with an intermediate value for heterozygotes (trend p=0.0003). Genotype at rs889299 accounted for approximately 1% of the total variability in plasma potassium, or around 3% of the total heritable fraction. There was no association between genotype at any SCNN1B SNP and blood pressure considered as a quantitative trait, or with hypertension affection status. We have shown a modest sized but highly significant effect of common genetic variation in the SCNN1B gene on plasma potassium. Interaction between the rs889299 SNP and functional SNPs in other genes influencing aldosterone-responsive distal tubular electrolyte transport may be important in the aetiology of essential hypertension. BACKGROUND The epithelial sodium channel (ENaC) plays a key role in the maintenance of blood pressure and electrolyte homeostasis in the distal nephron. ENaC consists of three subunits , and , which combine in a 1:1:1 ratio to form the functional channel. (Jasti et al. 2007) Activating mutations in the and subunits of ENaC (encoded by the SCNN1B and SCNN1G genes respectively) give rise to Liddle's syndrome, an autosomal dominant condition involving amiloride-sensitive hypertension, hypokalemia and metabolic alkalosis. (Hansson et al. 1995; Shimkets et al. 1994) Conversely, loss of function mutations in the , or subunits of ENaC lead to recessive pseudohypoaldosteronism type I, a childhood condition characterised by severe salt wasting and hyperkalemia. (Chang et al. 1996) A number of groups have investigated the role of common polymorphisms in the genes encoding the ENaC subunits as potential susceptibility alleles influencing the risk of essential hypertension to a moderate degree. However, no common single nucleotide polymorphism in any subunit of ENaC has been consistently identified as contributing to blood pressure variation in human populations thus far. (Pratt 2005) Both plasma potassium and 24-hour urinary potassium excretion show significant heritabilities estimated at between 30-60%, suggesting that genetic influences on potassium handling may be identifiable. (Hunter et al. 2002; Whitfield and Martin 1984, 1985) However, no studies so far have shown robust evidence for association between common genetic variants and plasma levels of potassium. Although there is no simple correlation between plasma potassium and blood pressure (in the absence of secondary causes of hypertension which cause hypo-or hyperkalemia), a diet that is high in potassium reduces levels of blood pressure. (Appel et al. 1997) The mechanism whereby increased dietary potassium reduces blood pressure is unknown, since total body content of potassium is not different in patients with essential hypertension and Page 3 of 20 RESULTS Characteristics of the participants are summarised in Table 1 . 52% of the sample were female and 36% were classified as hypertensive. As expected given the selection of families through a hypertensive proband, ambulatory BPs tended to be higher than would be expected in a nonselected population. Quantitative daytime recordings of ambulatory BP were available in 958 people, with on-treatment recordings available in a further 224. Due to participants electing to switch off the monitor at night, fewer night recordings were available: quantitative night recordings were available in 770 people with on-treatment recordings available in a further 162. The median family size was 5 people, 60% of families comprising between 4 and 6 genotyped and phenotyped members. 71% of families were 2-generation and 29% were 3-generation. Plasma potassium was approximately Normally distributed, so was analysed without transformation. All blood pressure phenotypes departed significantly from Normal, and were therefore log-transformed prior to analysis. Blood pressure variables were adjusted by regression for the significant covariates age, sex, smoking and physical exercise habit. Plasma potassium was adjusted for the following significant covariates: age ( =0.005; p<0.001); sex ( =0.120; female K + lower than male K + ; p<0.001); treatment with diuretics ( =-0.272; p<0.001); treatment with ACE inhibitors ( =0.083; p=0.008); and current smoking ( =0.052; p=0.029), which, as previously described by others, was associated with a higher plasma potassium level. (Hawthorne et al. 1974; Wannamethee et al. 1997) Age, sex and smoking explained 5.38% of the variability in plasma K + , and drug therapy explained a further 5.93%, the majority of that effect being due to diuretics. Thus, the covariates together explained only around 11% of the variability in plasma potassium. Plasma potassium was not significantly correlated with blood pressure, hypertension status, or body mass index. Plasma potassium after adjustment for covariates was significantly heritable (h 2 =35.4%; p<10 -6 ) in these families. Page 8 of 20 levels of aldosterone excretion in healthy subjects. (Imrie et al. 2006) There is therefore a strong rationale to test the hypothesis that genetic interaction between SCNN1B and CYP11B1/B2 SNPs influence the risk of hypertension. However, studies far larger than any conducted to date would be required to achieve reliable results. Previous studies that have reported genetic associations with plasma potassium have focused on the C825T SNP in the G-protein beta-3 subunit (GNB3) gene, but the results are contradictory and study sizes relatively small. A Japanese study of 211 subjects showed significantly higher plasma potassium in carriers of the 825T allele, but a more recent study of 144 European subjects showed significant association in the opposite direction. (Ishikawa et al. 2000; Martin et al. 2005) The present study includes data on genotype and plasma potassium for 1332 individuals and is much larger than these previous studies. Our result accounts for around 3% of the heritability of plasma potassium. Larger effects may therefore remain to be discovered. Genomewide SNP association studies coupled with intensive study of other genes in the signalling pathway regulating sodium reabsorption in the distal nephron (for example, WNK4, NEDD4) are complementary approaches which may both be required to identify such effects. This study has certain limitations. No urine specimens suitable for the quantification of sodium and potassium excretion were available; such data would have enabled us to account to some extent for variability in dietary intake of these electrolytes and potentially increased the power of the study. Further studies incorporating these urinary phenotypes would be of interest in developing our findings. The SCNN1B gene is large (~80Kb) and in a region of relatively low linkage disequilibrium, and so the four SNPs we typed did not capture all common genetic variation present. Associations of larger magnitude between other SCNN1B SNPs and plasma potassium may therefore remain to be discovered in future fine-mapping studies. Finally, as Genetic variants in the epithelial sodium channel in relation to aldosterone and potassium excretion and risk for hypertension. Hypertension 34: 631-637, 1999. Appel LJ, Moore TJ, Obarzanek E, Vollmer WM, Svetkey LP, Sacks FM, Bray GA, Vogt TM, Cutler JA, Windhauser MM, Lin PH and Karanja N. A clinical trial of the effects of dietary patterns on blood pressure. DASH Collaborative Research Group. N Engl J Med 336: 1117-1124, 1997. Baker EH, Dong YB, Sagnella GA, Rothwell M, Onipinla AK, Markandu ND, Cappuccio FP, Cook DG, Persu A, Corvol P, Jeunemaitre X, Carter ND and MacGregor GA. Association of hypertension with T594M mutation in beta subunit of epithelial sodium channels in black people resident in London. Shimkets RA, Nelson-Williams C, Rossier BC and Lifton RP. Mutations in subunits of the epithelial sodium channel cause salt wasting with hyperkalaemic acidosis, pseudohypoaldosteronism type 1. Nat Genet 12: 248-253, 1996. Cleland JG, Dargie HJ, Robertson I, Robertson JI and East BW. Total body electrolyte composition in patients with heart failure: a comparison with normal subjects and patients with untreated hypertension.