Journal of Human Hypertension (2014) 28, 504–509 & 2014 Macmillan Publishers Limited All rights reserved 0950-9240/14 www.nature.com/jhh

ORIGINAL ARTICLE Tag polymorphisms of solute carrier family 12 member 3 modify the risk of hypertension in northeastern Han Chinese

Y-L Wang1,YQi2, J-N Bai1, Z-M Qi1, J-R Li1, H-Y Zhao3, Y-F Wang3, C-Z Lu3, Y Xiao3, N Jia4,5, B Wang3 and W-Q Niu5,6

Converging evidence suggests that the gene encoding solute carrier family 12 member 3 (SLC12A3) is a logical candidate involved in the underlying cause of hypertension. We therefore selected four tag polymorphisms (rs2304483, rs5804, rs8063291 and rs6499857) from SLC12A3 gene to investigate their individual and interactive associations with hypertension in northeastern Han Chinese. There were 1009 hypertensive patients and 756 normotensive controls. Data were analyzed by Haplo.Stats and multifactor dimensionality reduction (MDR) softwares, and risk estimates were expressed as odds ratio (OR) and 95% confidence interval (95% CI). Overall, there were significant differences in the genotype (P ¼ 0.002) and allele (P ¼ 0.002) distributions of rs5804 between patients and controls. Compared with the most common haplotype A-C-T-G, haplotype G-C-T-G that was overrepresented in controls (Po0.001) reduced the crude and adjusted risk of hypertension by 36% (OR 0.64; 95% CI 0.50–0.81; Po0.001) and 39% (OR 0.61; 95% CI 0.48–0.79; Po0.001), respectively. Further interaction analyses identified an overall best MDR model including rs5804 and body mass index (P ¼ 0.001), which was validated by logistic regression analysis. Taken together, our findings demonstrate a predominant role played by SLC12A3 gene rs5804 in determining hypertension risk among northeastern Han Chinese. Moreover, the interaction of this polymorphism with obesity can enhance risk prediction.

Journal of Human Hypertension (2014) 28, 504–509; doi:10.1038/jhh.2013.134; published online 16 January 2014 Keywords: sodium-chloride symporter; solute carrier family 12 member 3; polymorphism; case–control study

INTRODUCTION homogeneous. Given the ubiquitous nature of epistasis in human Sodium-chloride symporter is a renal thiazide-sensitive cotran- genetics, it is a high priority to examine the interaction of multiple sporter that not only has a role in electrolyte homeostasis by genetic variants from hypertension-susceptibility or reabsorbing sodium and chloride ions from the tubular fluid into pathways. Bearing these in mind, we sought to investigate the the cells of the distal convoluted tubule of the nephron, but also associations of four tag polymorphisms (rs2304483, rs5804, serves as the target of thiazide diuretics that are generally rs8063291 and rs6499857) in SLC12A3 gene, both individually prescribed to treat high in clinical practice.1,2 In and interactively, with the risk of having hypertension in humans, sodium-chloride symporter is encoded by solute carrier northeastern Han Chinese. family 12 member 3 gene (SLC12A3, 16q13), and its loss-of-function are described as causing Gitelman’s syndrome, an autosomic recessive disease characterized by salt MATERIALS AND METHODS wasting and low blood pressure, hypokalemic metabolic alkalosis, Study population hypomagnesemia and hypocalciuria.3,4 It is therefore reasonable This is a hospital-based case–control study. All study subjects were local to hypothesize that SLC12A3 gene might be a logical candidate residents of Han descent from Qiqihar city, Heilongjiang province in the involved in the underlying cause of hypertension. northeast of China. The Ethics Committee of Qiqihar Medical University The gene encoding SLC12A3 consists of 26 exons, and spans approved this study, and signed informed consent was obtained from each 5 subject. This study was conducted according to the guidelines of the about 4.8 kbp. The genomic sequence of SLC12A3 gene is highly Declaration of Helsinki. polymorphic, and so far over a hundred mutations in this gene have been reported. Although epidemiologic studies on SLC12A3– hypertension associations have been undertaken extensively Diagnosis across various ethnic groups, the results are inconsistent and All study subjects were divided into the hypertensive patient group and inconclusive. Although candidate gene approach may not replace normotensive control group according to their clinical examinations and laboratory indices by experienced physicians. genome-wide scan strategy in unveiling the genetic under- Diagnosis of hypertension was based on a mean systolic blood pressure pinnings of complex disease, it is an important alternative, (SBP; X140 mm Hg) and/or diastolic blood pressure (DBPX90 mm Hg) especially when the genes or polymorphisms selected are on two occasions, and/or the current usage of antihypertensive drug biologically plausible and the population studied is genetically treatment. Blood pressure was measured using a calibrated mercury

1The Third Division of Cardiology, Department of Internal Medicine, The Second Affiliated Hospital of Qiqihar Medical University, Heilongjiang Province, China; 2Department of Epidemiology, Beijing An Zhen Hospital, Capital Medical University, Beijing Institute of Heart, Lung and Blood Vessel Diseases, Beijing, China; 3Department of Physiology, Qiqihar Medical University, Heilongjiang, China; 4Department of Hypertension, Ruijin Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai, China; 5Shanghai Institute of Hypertension, Ruijin Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai, China and 6State Key Laboratory of Medical Genomics, Ruijin Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai, China. Correspondence: Associate Professor Y-L Wang, The Third Division of Cardiology, Department of Internal Medicine, The Second Affiliated Hospital of Qiqihar Medical University, Zhonghua West Road 41, Jianhua District, Qiqihar 161006, Heilongjiang Province, China or Associate Professor W-Q Niu, State Key Laboratory of Medical Genomics, Ruijin Hospital, School of Medicine, Shanghai Jiao Tong University, Ruijin Second Road 197, Shanghai 200025, China. E-mail: [email protected] or [email protected] Received 7 August 2013; revised 17 October 2013; accepted 28 October 2013; published online 16 January 2014 SLC12A3 tag polymorphisms and hypertension Y-L Wang et al 505 sphygmomanometer with appropriate adult cuff size by certified and phosphorylated at the 50 end. The multiplex ligation reaction was examiners. Hypertensive patients who exhibited clinical manifest of carried out in a reaction volume of 10 ml containing 2 ml of PCR product, secondary hypertension and renal-relevant diseases were removed. To 1 ml10Â Taq DNA ligase buffer, 1 mM of each discriminating probe, 5 U Taq correct the impact of antihypertensive drugs, SBP and DBP were imputed DNA ligase and the ligation parameters were 30 cycles of 94 1C for 30 s and by adding 15 mm Hg and 10 mm Hg, respectively, as suggested by Tobin 56 1C for 3 min. After reaction, 1 ml LDR reaction product was mixed with et al.,6 and this imputation method was also adopted by Newton-Cheh 1 ml ROX passive reference and 1 ml loading buffer, and then denatured at et al.7 in a genome-wide association study of blood pressure. Unless 95 1C for 3 min, chilled rapidly in ice water. The fluorescent products of otherwise indicated, adjusted SBP and DBP were analyzed. LDR were differentiated using ABI 3730XL sequencer (Applied Biosystems, Foster City, CA, USA). Sample size To test the accuracy of PCR-LDR method, 96 samples were randomly selected as internal references, and were re-genotyped for four examined In total, 1009 sporadic patients constituted the hypertensive patient group polymorphisms. There was no difference in duplicate results. and they were aged 64.48 (s.d.: 8.53) years with 54.31% male gender. The rest 756 subjects who had normal blood pressure formed the age- and gender-matched normotensive control group. Statistical analysis Data were analyzed by STATA software (version 11.0) for Windows Demographic and clinical measurement (StataCorp LP, College Station, TX, USA). Study power was estimated by the Power and Sample Size Calculations (PS) software (version 3.0.7, Nashville, Age and gender, as well as body weight and height, were recorded before TN, USA). enrollment. Body mass index (BMI) was computed as weight in kilograms Continuous and categorical variables were compared between hyper- divided by height in meters squared. tensive patients and normotensive controls by unpaired t-test or Mann– Venous blood samples were extracted from all subjects after an Whitney U-test and w2 test, respectively. Hardy–Weinberg equilibrium overnight fasting, and the upper sera were isolated simultaneously after was tested by a Pearson goodness-of-fit test. The genotype and allele centrifugation. Plasma triglyceride, total cholesterol, high-density lipo- distributions of four tag polymorphisms examined between the two cholesterol, blood urea nitrogen, creatinine and urea acid were groups were compared by w2 test. Linear regression analyses were determined enzymatically using available kits and auto-analyzers. High- conducted to investigate the association of four examined polymorphisms sensitivity C-reactive protein was measured using a particle-enhanced with adjusted SBP and DBP as continuous traits after adjusting for age, immunoturbidimetric method. gender and BMI. For linear regression model, the genotype effect was assumed to be additive, that is, scores of 0, 1 and 2 were assigned for Selection of tag polymorphisms genotype wild homozygotes, heterozygotes and mutant homozygotes of Tag polymorphisms of SLC12A3 gene were selected first by detecting each , respectively. haplotype blocks from the HapMap database using the Haploview Haplotypes were constructed based on SLC12A3 gene four tag software (version 4.2, Cambridge, MA, USA), and a total of four blocks polymorphisms, and their frequencies were estimated by haplo.em were partitioned. Then, under the criteria of r2X0.8 and minor allele program. This program computes the maximum likelihood estimates of frequency of at least 0.05, one tag polymorphism that has been described haplotype probabilities using the progressive insertion algorithm, which in hypertension association studies8–12 was selected from each haplotype progressively inserts batches of loci into haplotypes of growing lengths. In block. Therefore, four tag polymorphisms of SLC12A3 gene including this study, only haplotypes with frequency X1% in all subjects were rs2304483, rs5804, rs8063291 and rs6499857 were genotyped in all study analyzed. Simulated P-values are calculated based on 1000 replicates. Risk subjects. Except for a synonymous polymorphism rs5804 in the 22nd exon, estimates were expressed as odds ratio (OR) and 95% confidence interval the other three polymorphisms were all intronic. (95% CI) before and after adjusting for age, gender and BMI. Moreover, the haplo.score program is used to model an individual’s phenotype as a function of each inferred haplotype, weighted by their estimated Genotyping probability, to account for haplotype ambiguity. This program is based on Genomic DNA was isolated from peripheral blood leukocytes, and was score statistics, which provide both global tests and haplotype-specific stored at À 40 1C until batch genotyping. The genotypes of four tag tests.14 The haplo.em and haplo.score programs are implemented in polymorphisms were determined by the PCR-ligase detection reaction Haplo.Stats software (version 1.4.0, Rochester, MN, USA) operated in the (LDR) method as previously described.13 The primers for PCR amplification R language (version 2.14, available at the website http://www.r-project.org). and the probes for LDR are available on request. To identify and characterize the interactions of multiple polymorphisms For each polymorphism, two specific probes were synthesized to in SLC12A3 gene, an open-source data-mining multifactor dimensionality discriminate specific bases, and additionally one common probe was reduction (MDR) approach (version 3.0, available at the website synthesized and labeled at the 3’ end with 6-carboxy-fluorescein (FAM) www.epistasis.org) was adopted.15,16 The aim of this approach is to con-

Table 1. The baseline characteristics of all study subjects

Characteristics Patients (n ¼ 1009) Controls (n ¼ 756) P-valuea

Age (years) 64.48±8.53 64.23±10.13 0.751 Gender (males) 54.31% 53.84% 0.843 BMI (kg m–2) 27.89±6.29 23.18±3.77 o0.001 Antihypertensive treatment 14.17% 0% o0.001 Adjusted SBP (mm Hg) 147.31±16.52 109.76±17.97 o0.001 Adjusted DBP (mm Hg) 89.09±15.92 71.37±11.43 o0.001 Fasting glucose (mmol l–1) 6.14±2.15 5.33±1.12 o0.001 TG (mmol l–1) 1.90±1.04 1.77±0.95 0.024 TC (mmol l–1) 4.59±1.18 4.59±0.91 0.995 HDL-C (mmol l–1) 1.12±0.32 1.24±0.34 o0.001 BUN (mmol l–1) 5.92±3.88 5.79±4.35 0.557 Creatinine (mmol l–1) 87.47±36.79 81.80±25.13 0.002 Uric acid (mmol l–1) 329.12±100.31 333.98±95.54 0.374 hsCRP (mmol l–1) 12.37±41.42 2.21±3.71 0.001 Abbreviations: BMI, body mass index; BUN, blood urea nitrogen; DBP, diastolic blood pressure; HDL-C, high-density lipoprotein cholesterol; hsCRP, high-sensitivity C-reactive protein; SBP, systolic blood pressure; TC, total cholesterol; TG, triglyceride. aP-values were computed by unpaired t-test or Mann–Whitney U-test for quantitative variables and by w2 test for qualitative variables.

& 2014 Macmillan Publishers Limited Journal of Human Hypertension (2014) 504 – 509 SLC12A3 tag polymorphisms and hypertension Y-L Wang et al 506 struct all possible combinations of four tag polymorphisms and categorical The distributions of age (P ¼ 0.751) and gender (P ¼ 0.843) did not indices to construct the overall best model. The accuracy of each MDR differed significantly between patients and controls. BMI (Po0.001), model is evaluated by a Bayes classifier in the context of 10-fold cross- blood pressures (both Po0.001), fasting glucose (Po0.001), validation. A single best model has the maximal testing accuracy and triglycerides (P ¼ 0.024), creatinine (P ¼ 0.002) and high-sensitivity cross-validation consistency simultaneously. Here, the cross-validation C-reactive protein (P 0.001) levels were significantly higher in consistency is a measure of the number of times of 10 divisions of the data o set that the best model is extracted. Statistical significance was evaluated patients than in controls, whereas the levels of high-density using a 1000-fold permutation test to compare observed testing accuracies lipoprotein cholesterol were significantly lowered (Po0.001). The with those expected under the null hypothesis of null association. rate of patients under antihypertensive treatment was 14.17%. Permutation testing corrects for multiple testing by repeating the entire analyses on 1000 data sets that are consistent with the null hypothesis. Finally, to validate the effectiveness of MDR approach, a classical logistic Single-locus analysis regression analysis was performed to check the derived overall best model. The genotype distributions and allele frequencies of four tag polymorphisms in SLC12A3 gene between hypertensive patients RESULTS and normotensive controls are compared in Table 2. Hardy– Baseline characteristics Weinberg equilibrium was satisfied in both patients and controls for all polymorphisms examined (P40.05). Overall, there were Table 1 shows the comparisons of demographic and clinical vari- significant differences in the genotype (P ¼ 0.002) and allele ables between hypertensive patients and normotensive controls. (P ¼ 0.002) distributions of rs5804 between the two groups. This significance was preserved after the Bonferroni correction (Bonferroni significance threshold P ¼ 0.05 divided by the total Table 2. Genotype distributions and allele frequencies of four tag number of examined polymorphisms (n ¼ 4): P ¼ 0.0125). The polymorphisms in SLC12A3 gene between hypertensive patients and power to reject the null hypothesis of no differences in mutant normotensive controls genotype and allele frequencies of rs5804 between the two

a 2 groups was 93.1% and 84.7%, respectively. No significance was Polymorphism Patients Controls w P- observed for the other three polymorphisms. (n ¼ 1009) (n ¼ 756) valueb Linear regression of four tag polymorphisms in SLC12A3 gene rs2304483 on adjusted SBP and DBP is shown in Table 3. After adjusting for GG 361 (32.32%) 254 (33.60%) age, gender and BMI, rs5804 was associated with a significant GA 546 (48.88%) 374 (49.47%) 1.13 0.569 increase of 1.77 mm Hg in adjusted SBP (P ¼ 0.037), and with an AA 210 (18.80%) 128 (16.93%) increase of 0.88 mm Hg in adjusted DBP (P ¼ 0.104) under an A allele 43.24% 41.67% 0.91 0.339 additive effect. Similarly, there was still no significance for the other three polymorphisms. rs5804 CC 615 (55.06%) 453 (59.92%) CT 418 (37.42%) 275 (36.38%) 12.98 0.002 TT 84 (7.52%) 28 (3.70%) T allele 26.23% 21.89% 9.18 0.002

rs8063291 Table 3. Linear regression of four tag polymorphisms in SLC12A3 gene TT 654 (58.55%) 434 (57.41%) on adjusted systolic and diastolic blood pressures TC 406 (36.35%) 275 (36.38%) 1.11 0.574 CC 57 (5.55%) 47 (6.22%) C allele 23.28% 24.40% 0.63 0.426 Polymorphisms Adjusted SBP Adjusted DBP

rs6499857 Coef. b P-valuea Coef. b P-valuea GG 708 (63.38%) 480 (63.49%) GC 366 (32.77%) 236 (31.22%) 2.45 0.294 rs2304483 1.54 0.05 0.088 0.67 0.04 0.186 CC 43 (3.85%) 40 (5.29%) rs5804 1.77 0.07 0.037 0.88 0.05 0.104 C allele 20.23% 20.90% 0.25 0.620 rs8063291 0.77 0.03 0.458 0.14 0.01 0.822 rs6499857 0.64 0.02 0.527 0.26 0.02 0.671 aGenotypes of four tag polymorphisms were expressed as count (percentage). bP-values were computed by using w2 test based on the Abbreviations: b, standardized coefficient; Coef., unstandardized 3 Â 2 contingency tables for genotype comparisons and on the 2 Â 2 coefficient; DBP, diastolic blood pressure; SBP, systolic blood pressure. contingency tables for allele comparisons. aP-values were adjusted for age, gender and body mass index.

Table 4. Frequencies of derived haplotypes (X1% in total subjects) from four tag polymorphisms in SLC12A3 gene between hypertensive patients and normotensive controls, and their risk prediction for hypertension

a b Haplotype Patients Controls PSim OR; 95% CI;P-value OR; 95% CI;P-value

A-C-T-G 42.13 41.59 0.431 Reference group Reference group G-T-T-G 25.37 21.67 0.006 1.11; 0.94–1.32; 0.221 1.09; 0.91–1.31; 0.325 G-C-C-C 17.01 18.16 0.194 0.93; 0.77–1.12; 0.443 0.89; 0.73–1.08; 0.229 G-C-T-G 5.84 9.82 o0.001 0.64; 0.50–0.81; o0.001 0.61; 0.48–0.79; o0.001 G-C-C-G 5.61 5.95 0.663 0.89; 0.66–1.19; 0.435 0.90; 0.66–1.22; 0.492 G-C-T-C 2.75 2.52 0.982 1.05; 0.69–1.61; 0.810 1.00; 0.64–1.56; 0.994 Abbreviations: OR, odds ratio; 95% CI, 95% confidence interval. aAlleles incorporated in a haplotype were in order of rs2304483, rs5804, rs8063291 and rs6499857 in SLC12A3 gene. bAdjustment for age, gender and body mass index.

Journal of Human Hypertension (2014) 504 – 509 & 2014 Macmillan Publishers Limited SLC12A3 tag polymorphisms and hypertension Y-L Wang et al 507 Haplotype analysis in association with the derived haplotypes as a whole in both The frequencies of derived haplotypes (X1%) from four tag patients and controls. polymorphisms in SLC12A3 gene are summarized and compared between hypertensive patients and normotensive controls in Interaction analysis Table 4. The cumulative variance explained by these haplotypes To explore the potential interactions of four tag polymorphisms in reached 98.71% and 99.71% for patients and controls, respec- SLC12A3 gene and three confounding factors (age, gender and tively. Haplotype A-C-T-G was the most common haplotype and BMI), a promising data-mining analytical approach MDR was used its frequency was comparable between patients and controls (Table 6). Here, age was categorized by 50 years for males and (P ¼ 0.431), and this haplotype therefore was assigned as the Sim 55 years for females, and BMI was categorized by 25, (25, 30) reference group in risk estimates. Haplotype G-C-T-G that was p and 430 kg m–2. overrepresented in controls (P 0.001) reduced the crude and Simo Each best model is evaluated by testing accuracy, cross- adjusted risk of hypertension by 36% (OR 0.64; 95% CI 0.50–0.81; validation consistency and significance level as determined by P 0.001) and 39% (OR 0.61; 95% CI 0.48–0.79; P 0.001), o o permutation test. The overall best MDR model included rs5804 respectively. The adjusted factors included age, gender and and BMI. This model had the maximal testing accuracy of 0.6331 BMI. This significance also survived the Bonferroni correction with a cross-validation consistency of 10 out of 10, and was (significance threshold P ¼ 0.05 divided by the total number of significant at P ¼ 0.001, indicating that a model this good or better comparisons (n ¼ 6): P ¼ 0.0083). No significance was observed for was observed 1 out of 1000 permutations and thus unlikely the other haplotypes derived. hinged on the null hypothesis of null association. Further a classical logistic analysis was used to regress the product of rs5804 and BMI, age and gender. Consistently, the interaction of rs5804 Haplotype–phenotype analysis and BMI was found to be significantly associated with 1.23-fold The omnibus associations of all derived haplotypes with demo- increased risk of having hypertension under the additive model, graphic indices and clinical biomarkers are presented in Table 5. reinforcing the robustness of the MDR approach. There was no statistical significance for all indices and biomarkers

DISCUSSION Table 5. Global testing of omnibus haplotypes with anthropometric The most noteworthy finding of this study was a predominant role indexes and clinical biomarkers in both hypertensive patients and played by SLC12A3 gene rs5804 polymorphism in determining normotensive controls hypertension risk in northeastern Han Chinese. Moreover, the Characteristics Patients Controls interaction of this polymorphism with obesity can enhance risk prediction. To the best of our knowledge, this study is the first to report potential interactions of SLC12A3 genetic polymorphisms Global- Global Global- Global and environmental factors in Chinese. These findings not only stat PSim stat PSim deepen our understandings that complex genetic–environmental Age 7.29 0.774 7.20 0.408 interactions might account for disease risk, but also yield strong Gender 11.17 0.429 10.95 0.141 evidence in favor of the logical candidacy of SLC12A3 gene in the BMI 12.07 0.358 9.83 0.198 pathogenesis of hypertension. Adjusted SBP 7.90 0.722 9.45 0.222 Literature, being abundant with studies associating SLC12A3 Adjusted DBP 8.05 0.708 7.77 0.353 genetic mutations with the incidence of Gitelman’s and Bartter’s Triglyceride 5.20 0.921 6.27 0.508 syndromes,3,16–18 paves the way to examine the predictive value TC 5.78 0.887 11.56 0.116 of these mutations on the risk for hypertension. Evidence HDL-C 10.42 0.493 10.30 0.174 is converging supporting that SLC12A3 genetic mutations can Glucose 7.60 0.748 1.94 0.963 BUN 5.37 0.864 4.06 0.773 reduce the capability of sodium-chloride cotransporter to Creatinine 4.75 0.907 11.72 0.109 reabsorb in the distal renal tubules where the cotransporter is 3,17 Uric acid 8.78 0.553 7.67 0.363 specifically expressed. In the past decade, there is an increasing hsCRP 7.87 0.548 3.86 0.695 awareness of the contributory role of SLC12A3 gene in the pathogenesis of hypertension. For example, homozygous carriers Abbreviations: BMI, body mass index; BUN, blood urea nitrogen; DBP, of 904Gln allele (Arg904Gln, rs11643718) had an increased risk diastolic blood pressure; HDL-C, high-density lipoprotein cholesterol; of essential hypertension in southern Swedes3 and in young hsCRP, high-sensitivity C-reactive protein; SBP, systolic blood pressure; 18 TC, total cholesterol. Japanese women. However, a reverse tendency was identified as 904Gln allele was overrepresented in controls of Kazak ancestry,

Table 6. Summary of MDR analysis

Best combination of each model Testing accuracy Cross-validation consistency P-value

rs5804 0.5960 7 0.0099 rs5804, BMI 0.6331 10 0.0010a rs5804, BMI, rs2304483 0.6128 10 0.0057 rs5804, BMI, rs2304483, rs8063291 0.6127 8 0.0058 rs5804, BMI, rs2304483, rs8063291, age 0.6200 10 0.0034 rs5804, BMI, rs2304483, rs8063291, age, rs6499857 0.6227 9 0.0533 rs5804, BMI, rs2304483, rs8063291, age, rs6499857, gender 0.6245 10 0.0230 Abbreviations: BMI, body mass index; MDR, multifactor dimensionality reduction. aThe overall best MDR model.

& 2014 Macmillan Publishers Limited Journal of Human Hypertension (2014) 504 – 509 SLC12A3 tag polymorphisms and hypertension Y-L Wang et al 508 and was comparable between hypertensive patients and of multiple genetic and environmental interactions to predict normotensive controls of Uyghur ancestry from the Northwest high-risk individuals. of China.19 One possible explanation for these inconsistent findings is genetic heterogeneity across ethnicities,20 which can be somewhat avoided when homogeneous populations are used.21 In fact, our study population is characterized by genetic What is known about this topic K Sodium-chloride symporter is a renal thiazide-sensitive cotransporter homogeneity and geographic stability, and they are probably 1,2 that has a role in electrolyte homeostasis. more uniform in their environmental exposures, including habitual 22 K The solute carrier family 12 member 3 (SLC12A3) gene, encoding dietary intake of high salt and high fat, and the lower rate sodium-chloride symporter, might be a logical candidate involved in the of hypertension recognition and treatment, rendering this underlying cause of hypertension.3,4 population more appropriate to better understand genetic K The genomic sequence of SLC12A3 gene is highly polymorphic, and so predisposition to hypertension risk. far over a hundred mutations in this gene have been reported. To capture the sequence variability of SLC12A3 gene, we resorted to the HapMap database to select four tag polymorph- What this study adds isms, and found only a synonymous polymorphism rs5804 was K SLC12A3 gene rs5804 polymorphism was independently associated with independently associated with significant risk of hypertension, the significant risk of developing hypertension in single-locus analysis. K Haplotype G-C-T-G that possessed the rs5804-C allele was associated even after the stringent Bonferroni correction. The exact function with reduced risk of hypertension, even after adjusting for confounding of this synonymous polymorphism is unclear at present, and it is factor. speculated that if involved, this polymorphism might be in linkage K Our interaction analyses identified an overall best MDR model including disequilibrium with another functional locus in SLC12A3 gene that rs5804 and BMI, suggesting the potential genetic–environmental can affect the final bioavailability of sodium-chloride symporter. interactions in the pathogenesis of hypertension. Considering the fact that the evolutionary history of linkage disequilibrium patterns will vary significantly in different ethnic populations,23 there is a need to construct a database of CONFLICT OF INTEREST hypertension-susceptibility genes or polymorphisms in each The authors declare no conflict of interest. ethnic group. Another important finding of this study was that environmental factors might modify the risk of SLC12A3 gene for hypertension. ACKNOWLEDGEMENTS Hypertension is a multifactorial disorder with up to 50% of This study received grants from the National Natural Science Foundation of China blood pressure variation currently attributed to an individual’s (grant no. 81000109 and 30900808), the Department of Education Science and genetic makeup.24,25 Genetic factors may predispose individuals Technology Research Project in Heilongjiang Province (grant no. 12521621), the to develop a disease; however, its incidence largely depends on Beijing New Star Program (grant no. Z111107054511072) and the Shanghai Rising the exposure to a certain environment. To yield more information, Star Program (grant no. 11QA1405500). we adopted a promising data-mining approach MDR, which is nonparametric and model free in design, and has been REFERENCES successfully applied to detect and characterize high-order gene- to-gene and gene-to-environment interactions in studies with 1 Eladari D, Hubner CA. Novel mechanisms for NaCl reabsorption in the collecting 26,27 duct. Curr Opin Nephrol Hypertens 2011; 20(5): 506–511. relatively small samples. By using this approach, we detected a 2 Karadsheh F, Weir MR. Thiazide and thiazide-like diuretics: an opportunity to potential interaction of rs5804 with BMI in northeastern reduce blood pressure in patients with advanced kidney disease. Curr Hypertens Han Chinese, which was further validated by logistic analysis. Rep 2012; 14(5): 416–420. The interpretation of this observation might be due to the harmful 3 Melander O, Orho-Melander M, Bengtsson K, Lindblad U, Rastam L, Groop L et al. impact of high-salt and high-fat diet, which are more prevalent in Genetic variants of thiazide-sensitive NaCl-cotransporter in Gitelman’s syndrome northeastern Chinese. As documented in animal studies, changes and primary hypertension. Hypertension 2000; 36(3): 389–394. in NaCl and K( þ ) intake can alter the expression and phos- 4 Fava C, Montagnana M, Rosberg L, Burri P, Almgren P, Jonsson A et al. Subjects phorylation of SLC12A3 gene,28 suggesting the potential heterozygous for genetic loss of function of the thiazide-sensitive cotransporter interaction of this gene with high-salt diet. On the basis of these have reduced blood pressure. Hum Mol Genet 2008; 17(3): 413–418. 5 Simon DB, Nelson-Williams C, Bia MJ, Ellison D, Karet FE, Molina AM et al. observations, it is reasonable to believe that disregarding Gitelman’s variant of Bartter’s syndrome, inherited hypokalaemic alkalosis, is environmental factors could lead to the failure in identifying caused by mutations in the thiazide-sensitive Na-Cl cotransporter. Nat Genet 1996; genetic determinants responsible for disease risk. 12(1): 24–30. Several limitations of this study merit consideration. First, the 6 Tobin MD, Sheehan NA, Scurrah KJ, Burton PR. Adjusting for treatment effects retrospective design of this study has inherent drawbacks and in studies of quantitative traits: antihypertensive therapy and systolic blood precludes causal inferences.29 Second, this study of 1765 subjects pressure. Stat Med 2005; 24(19): 2911–2935. may be underpowered to demonstrate small risk effects. However, 7 Newton-Cheh C, Johnson T, Gateva V, Tobin MD, Bochud M, Coin L et al. a sample size calculation ensured us with 484% statistical power Genome-wide association study identifies eight loci associated with blood to detect significance. Third, only four tag polymorphisms were pressure. Nat Genet 2009; 41(6): 666–676. 8 Kokubo Y, Kamide K, Inamoto N, Tanaka C, Banno M, Takiuchi S et al. Identification examined, and it is highly encouraged to incorporate other of 108 SNPs in TSC, WNK1, and WNK4 and their association with hypertension in a polymorphisms, especially the low-penetrance ones in SLC12A3 Japanese general population. J Hum Genet 2004; 49(9): 507–515. gene or other validated hypertension candidacy genes, such as 9 Keszei AP, Tisler A, Backx PH, Andrulis IL, Bull SB, Logan AG. Molecular variants of 30 the apelin-AGTRL1 pathway genes. Fourth, the fact that our the thiazide-sensitive Na þ -Cl- cotransporter in hypertensive families. J Hypertens study subjects were of northeastern Han Chinese descent limited 2007; 25(10): 2074–2081. the generalizability of our findings, calling for further confirmation 10 Chen LY, Zhao WH, Tian W, Guo J, Jiang F, Jin LJ et al. STK39 is an independent risk in other ethnic populations. factor for male hypertension in Han Chinese. Int J Cardiol 2012; 154(2): 122–127. Taken together, our findings demonstrate a predominant role 11 Kardia SL, Greene MT, Boerwinkle E, Turner ST, Kullo IJ. Investigating the complex played by the SLC12A3 gene rs5804 polymorphism in determining genetic architecture of ankle-brachial index, a measure of peripheral arterial disease, in non-Hispanic whites. BMC Med Genomics 2008; 1:16. hypertension risk among northeastern Han Chinese. Moreover, the 12 Syren ML, Borsa Ghiringhelli N, Bettinelli A, Colussi G, Vargas-Poussou R, interaction of this polymorphism with obesity can enhance risk Tammaro F et al. The mutation c.1196_1202dup7bp (p.Ser402X) in the SLC12A3 prediction. As hypertension is a multifactorial complex trait, more gene clusters in Italian Gitelman syndrome patients and reflects the presence of a emphases should be placed on the detection and characterization common ancestor. Nephrol Dial Transplant 2011; 26(2): 557–561.

Journal of Human Hypertension (2014) 504 – 509 & 2014 Macmillan Publishers Limited SLC12A3 tag polymorphisms and hypertension Y-L Wang et al 509 13 Niu W, Zhang Y, Ji K, Gu M, Gao P, Zhu D. Confirmation of top polymorphisms in 23 Niu W, Qi Y, Wu Z, Liu Y, Zhu D, Jin W. A meta-analysis of receptor for advanced hypertension genome wide association study among Han Chinese. Clin Chim Acta glycation end products gene: four well-evaluated polymorphisms with diabetes 2010; 411(19-20): 1491–1495. mellitus. Mol Endocrinol 2012; 358(1): 9–17. 14 Schaid DJ, Rowland CM, Tines DE, Jacobson RM, Poland GA. Score tests for 24 Niu W, Qi Y, Qian Y, Gao P, Zhu D. The relationship between apolipoprotein E association between traits and haplotypes when linkage phase is ambiguous. epsilon2/epsilon3/epsilon4 polymorphisms and hypertension: a meta-analysis of Am J Hum Genet 2002; 70(2): 425–434. six studies comprising 1812 cases and 1762 controls. Hypertens Res 2009; 32(12): 15 Pattin KA, White BC, Barney N, Gui J, Nelson HH, Kelsey KT et al. A computationally 1060–1066. efficient hypothesis testing method for epistasis analysis using multifactor 25 Marteau JB, Zaiou M, Siest G, Visvikis-Siest S. Genetic determinants of blood dimensionality reduction. Genet Epidemiol 2009; 33(1): 87–94. pressure regulation. J Hypertens 2005; 23(12): 2127–2143. 16 Hahn LW, Ritchie MD, Moore JH. Multifactor dimensionality reduction software for 26 Qi Y, Niu W, Zhu T, Zhou W, Qiu C. Synergistic effect of the genetic detecting gene-gene and gene-environment interactions. Bioinformatics 2003; polymorphisms of the renin-angiotensin-aldosterone system on high-altitude 19(3): 376–382. pulmonary edema: a study from Qinghai-Tibet altitude. Eur J Epidemiol 2008; 17 Nakhoul F, Nakhoul N, Dorman E, Berger L, Skorecki K, Magen D. Gitelman’s 23(2): 143–152. syndrome: a pathophysiological and clinical update. Endocrine 2012; 41(1): 53–57. 27 Niu W, Qi Y, Hou S, Zhai X, Zhou W, Qiu C. Haplotype-based association of the 18 Matsuo A, Katsuya T, Ishikawa K, Sugimoto K, Iwashima Y, Yamamoto K et al. renin-angiotensin-aldosterone system genes polymorphisms with essential G2736A polymorphism of thiazide-sensitive Na-Cl cotransporter gene predisposes hypertension among Han Chinese: the Fangshan study. J Hypertens 2009; 27(7): to hypertension in young women. J Hypertens 2004; 22(11): 2123–2127. 1384–1391. 19 Wang XF, Lin RY, Wang SZ, Zhang LP, Qian J, Lu DR et al. Association study of 28 Vallon V, Schroth J, Lang F, Kuhl D, Uchida S. Expression and phosphorylation variants in two ion-channel genes (TSC and CLCNKB) and hypertension in two of the Na þ -Cl- cotransporter NCC in vivo is regulated by dietary ethnic groups in Northwest China. Clin Chim Acta 2008; 388(1-2): 95–98. salt, potassium, and SGK1. Am J Physiol Renal Physiol 2009; 297(3): 20 Kato N. Ethnic differences in genetic predisposition to hypertension. Hypertens Res F704–F712. 2012; 35(6): 574–581. 29 Gu M, Dong X, Zhang X, Wang X, Qi Y, Yu J et al. Strong association between two 21 Pineda-Krch M, Lehtila K. Costs and benefits of genetic heterogeneity within polymorphisms on 15q25.1 and lung cancer risk: a meta-analysis. PLoS One 2012; organisms. J Evol Biol 2004; 17(6): 1167–1177. 7(6): e37970. 22 Niu W, Qi Y. Association of alpha-adducin and G-protein beta3 genetic 30 Niu W, Wu S, Zhang Y, Li W, Ji K, Gao P et al. Validation of genetic association in polymorphisms with hypertension: a meta-analysis of Chinese populations. PLoS apelin-AGTRL1 system with hypertension in a larger Han Chinese population. One 2011; 6(2): e17052. J Hypertens 2010; 28(9): 1854–1861.

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