Research ArticleNovel Association of WNK4 Gene Ala589SerPolymorphism in Essential Hypertension and Type 2Diabetes Mellitus in Malaysia

Nooshin Ghodsian1 Patimah Ismail1 Salma Ahmadloo1 Farzad Heidari1

Polin Haghvirdizadeh1 Sima Ataollahi Eshkoor2 and Ali Etemad1

1Genetic Research Group Department of Biomedical Science Faculty of Medicine and Health SciencesUniversiti Putra Malaysia Serdang Selangor Malaysia2Iranian Research Center on Aging University of Social Welfare and Rehabilitation Science Tehran Iran

Correspondence should be addressed to Patimah Ismail patimahismailgmailcom

Received 24 December 2015 Revised 12 February 2016 Accepted 22 February 2016

Academic Editor Yajing Wang

Copyright copy 2016 Nooshin Ghodsian et al This is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work is properlycited

With-no-lysine (K) Kinase-4 (WNK4) consisted of unique serine and threonine protein kinases genetically associated with anautosomal dominant form of hypertension Argumentative consequences have lately arisen on the association of specific singlenucleotide polymorphisms ofWNK4 gene and essential hypertension (EHT)The aim of this studywas to determine the associationof Ala589Ser polymorphism of WNK4 gene with essential hypertensive patients in Malaysia WNK4 gene polymorphism wasspecified utilizingmutagenically separated polymerase chain reaction (PCR) and restriction fragment length polymorphism (RFLP)method in 320 subjects including 163 cases and 157 controls Close relation between Ala589Ser polymorphism and elevated systolicand diastolic blood pressure (SBP and DBP) was recognized Sociodemographic factors including body mass index (BMI) age thelevel of fasting blood sugar (FBS) low density lipoprotein (LDL) and triglyceride (TG) in the cases and healthy subjects exhibitedstrong differences (119901 lt 005) The distribution of allele frequency and genotype ofWNK4 gene Ala589Ser polymorphism showedsignificant differences (119901 lt 005) between EHT subjects with or without type 2 diabetes mellitus (T2DM) and normotensivesubjects statistically The WNK4 gene variation influences significantly blood pressure increase Ala589Ser probably has effectson the enzymic activity leading to enhanced predisposition to the disorder

1 Introduction

Essential hypertension as a significant health risk factorleading to renal and cardiovascular disorders is a commondisorder developed by unhealthy lifestyle habits and heritableelements The genetic influence or heritability estimation onblood pressure (BP) variation displays remarkable range (30to 50) according to Timberlake et al [1] Genetic variationscan considerably affect EHT genesis which significantlyexhibits risk factor for progressive renal damage strokeischemic heart disease and peripheral vascular disease [2]The specified genes are desired objectives for genetic physio-logical and functional research on EHT however these raremutations do not restate BP variation in general population[3]

Previous prospective and case control studies have shownthat hypertension progression is an independent predictorof T2DM [4] Several possible factors are likely to becauses of the association between T2DM and EHT Manyreports have emphasized that patients with chronic diseasessuch as hypertension have diabetes [5] The relationshipbetween hypertension and diabetes also was obtained byother research groups [6]

The serine-threonine kinase with-no-lysine (K) 4 gene(MIM 601844) is a hot locus for blood pressure that is on17th chromosome [7] In addition to the rare variants spec-ification in WNK4 that is reliable for the pseudohypoaldos-teronism type II common single nucleotide polymorphisms(SNPs) in these genes have established relationship with

Hindawi Publishing CorporationJournal of Diabetes ResearchVolume 2016 Article ID 8219543 6 pageshttpdxdoiorg10115520168219543

2 Journal of Diabetes Research

BP variation and susceptibility to hypertension in generalpopulation in children and adults [8 9] The substitute ofa nonpolar residue alanine by a polar residue serine canbe produced by the Ala589Ser polymorphism as a missensemutation According to Wilson and colleagues mutations inWNK4 can cause some autosomal dominant Mendelian traitcontributing to hypertension [10] Recent studies also illus-trate thatWNK4 influences dominantly the distal convolutedtubule located in the kidneys as significant areas balancingreabsorption of water and salt leading to blood pressuredetermination [10ndash12] According to Sun et al [13] theWNK4 gene seems to influence considerably pathogenesisof essential hypertension Ala589Ser polymorphism (as amissense mutation) might also make alterations to enzymersquosfunctions leading to increased susceptibility to the disorderHowever monogenic disorders are generally resulting fromuncommon polymorphism in a gene coding sequence usualdisorders seem to be produced by genetic polymorphismin gene regulatory factors changing the locus expressionalprofile [14 15]

Considering a strong relation between hypertension anddiabetes in this study our major target aimed at hypertensionas well as T2DM to specify role of the gene underlying thedisease tomodify the prognostication of those at risk and alsoadminister further antihypertensive treatments The currentresearch principal objective was to explore the relation ofWNK4 gene Ala589Ser polymorphism between EHT andT2DM inMalaysian subjects Several screening tests resultedin the recognition of human-specific variant Ala589Ser inWNK4 exon 8This polymorphism (Ala589Ser) was targetedto be conducted onMalaysian population through evolution-ary genetic analysis and to explore its relation with BP andFBS development as well

2 Methods

21 Study Subjects Ethical approval was acquired fromSeremban hospital as well as Universiti Putra Malaysia withreference number [UPMFPSKPADST7-MJETIKAPerF01-JSB-Mac] All participants were asked to fill in informedconsent questionnaires In the current study 320 Malaysianparticipants were interviewed The blood pressure was mea-sured on the right arm (three times) and averaged after resting(5min intervals) by digital Sphygmomanometer on the basisof inclusion criteria [SBP above 140mmHg and DBP above90mmHg (for patients)] Participantsrsquo height and weightwere taken to measure BMI utilizing the formula [weight(kg)height (m2)] All participants had biochemical analyzingtest using plasma electrolytes

22 Genomic DNA Extraction In the study the buccal andblood cells were collected from cases (hypertensive patients)and controls respectively The blood was kept in ethylenediamine tetra acetic acid (EDTA) tube and stored at 4∘C fora maximum of three days before utilizing The DNA wasapplied for amplification after extracting it from buccal andblood cell samples using Qiagen kit (Germany) then it wasstored at minus20∘C for later usage DNA was qualified right

after all primers were optimized by PCRmethod By utilizingthe Nanodrop in two optical density (OD) wavelengths(260 nm and 280 nm) the extracted DNA concentration wasexamined

23 Biochemical Analysis In this section peripheral venousblood samples were collected after keeping an overnightfast by control subjects Moreover lipid profiles [includingTG LDL total cholesterol (TCH) and high-density protein(HDL)] were determined We also measured FBS with stan-dard laboratory techniques however the diabetic glucoselevels were taken from the medical history It was noticeablethat we had referred to the hospital patientsrsquo documents toassess biochemical information for cases

24 Determination of WNK4 Gene Ala589Ser Polymorphism241 Polymerase Chain Reaction (PCR) Genomic DNAwas amplified by multiplex-PCR reactions The examinedpolymorphism of WNK4 (Ala589Ser) was identified usingPCR based RFLP PCRs were carried out with forward primer51015840-TGGAAACCCATTTTCCCCTGG-31015840 and reverse primer51015840-AGGTGGTGAGGCCTAGAAAGT-31015840 at the specific tem-peratures Each reaction was composed of 6x master mix(which embodies DNA polymerase MgCl

2 dNTPs and

reaction buffers) 06 120583L relative primers (03 120583L forward and03 120583L reverse) and 1 120583L of genomic DNA and ultimatelydistilled water was added to a final volume of 25120583LTheDNAwas amplified using initial denaturation of 94∘C (5 minutes)followed by 40 cycles of 55∘C annealing temperature forWNK4 gene polymorphism (90 seconds) 72∘C extension (60seconds) and kept at 72∘C (10 minute) for final extensionAmplified PCR products were analyzed by gel electrophoresismethods with agarose

242 Restriction Fragment Length Polymorphism (RFLP)The enzyme AlwNI was used for restriction of the PCRproducts A total of 10 120583L PCR products were incubatedat 37∘C for at least 3 hours The restriction enzyme andcorresponding buffer (5120583L) were added to the final restric-tion volume After incubating the achieved digested PCRproduct proceeded for electrophoresis on 2 agarose gelat 100 bp DNA molecular weight markers The followingelectrophoresis was observed to determine the genotype ofWNK4 gene Ala589Ser polymorphism The size of the PCRproduct could be determined comparing with a 100 bp DNAladder which was used as a DNA marker Finally the gelwas washed with water for fewminutes before viewing underAlpha Imager

25 Statistical Analysis The statistical analysis in presentstudy was conducted by statistical package for the socialscience (SPSS version 22) Utilizing two-tailed Studentrsquos 119905-testand one-wayANOVA test all variables among the groups andthe groupmeans were being contrasted (119901 lt 005was viewedstatistically significant) The distribution of genotype withHardy-Weinberg expectations was calculated using a Chi-squared test and allelic frequencies were analyzed by gene-counting method In order to detect the effects of high risk

Journal of Diabetes Research 3

alleles odds ratios (OR) with 95 confidence intervals (CI)were checked as well

3 Results

31 Clinical Characteristics In the study 320 volunteersdivided into hypertensive (163) and normotensive (157) par-ticipated for screening Ala589Ser polymorphism in WNK4The clinical characteristicsmean and standard deviation (SD)were clearly indicated in Table 1 however the EHT meanage (5907 plusmn 1044) was noticeably higher in contrast to thecontrols (5251plusmn941) Considering age SBP DBP BMI FBSLDL and TG the subjectsrsquo clinical characteristics illustratedsignificant differences for most parameters between hyper-tensive and normotensive subjects (119901 lt 005) ExcludingHDL and TCH other blood pressure related characteristicsand the Ala589Ser polymorphism in WNK4 exon 8 werefound to be related with elevated risk for the disorder(Table 1) Table 1 also shows significant differences for T2DMbetween hypertensive and normotensive subjects (119901 lt 005)

32 Genotype and Allele Frequency Multiple PCRs were usedto determine the absence and presence genotype ofAla589Serof WNK4 gene in samples Before doing main RCR clarityof the bands was checked by optimization of the methodGene was set up separately with internal control to find outthe sharp band Each gel illustrated the optimization on onegene with four different samples PCR products were run on2 gel with four different samples 50 bp ladder was usedas a DNA marker After PCR product digestion by AlwNIrestriction enzyme the gene encompassingAla589Ser variantwas detected through RFLP method using 2 agarose gelelectrophoresis The enzyme digested the variant type allelesin two fragments of A589 that was divided into 180 and 112 bpwhereas wild type (S589) remained uncut Figure 1 shows theAla589Ser polymorphism amplification product of WNK4gene

Table 2 shows the WNK4 gene Ala589Ser polymor-phism distribution between hypertensive and normotensivesubjects Homozygote (GG) genotype calculated the highestfrequency where case and control were 95 (583) and 109(694) respectively while lower frequencies were observedin heterozygote (GT) genotypes in case 57 (350) andcontrol 48 (306) Interestingly mutant (TT) genotypedetermined the lowest frequency in cases which was 11 (67)but not in the controls Significant differences in genotypeswere detected between cases and controls

In Table 2 G allele was found to be higher in thecontrol subjects which was 266 (847) as compared topatientswhile T allele was higher inpatients Significantdifferences in genotypes and allele frequencies were observedbetween cases and controls with 119901 value of 0005 and 0015respectively (119901 lt 005) Significant values were specified byone-way ANOVA with post hoc test for genotypes TT versusGG (0001) and TT versus GT (0004) between hypertensiveand normotensive subjects however no significant value wasobtained for TG versus GG No significant difference wasachieved from the gender and racersquos genotype for WNK4

112 bp180 bp292bp

50bp

M GG TT GTGT GG TT

Figure 1 Detection of Ala589Ser polymorphism of WNK4 gene byPCR-RFLP utilizing 20 agarose gel electrophoresis PCR productswere digested by AlwNI Wild type was cut (into 180 and 112 bp)while variant type remained uncut Homozygous genotypes areindicated as GG genotypes heterozygous genotypes are determinedas GT genotypes and TT genotypes show mutant genotypes Mrepresented a 50 bp DNA ladder plus (Bioline)

gene Ala589Ser polymorphismwithin genotypes of cases andcontrols

33 Genotype-Clinical Characteristics Correlations To spec-ify genotype-clinical characteristics correlations clinicalcharacteristics of patients between genotypes of Ala589Serpolymorphism were contrasted (Table 3) One-way ANOVAwas applied to analyze the relation of clinical characteristicfactors within genotype groups in patients The WNK4 Tallele established no relation with the genotype of the clinicalcharacteristics of patients It must be noted that no differencewas identified in clinical disability in regard to WNK4Ala589Ser gene polymorphism

34 DNA Sequencing In order to confirm the genotypingresults random samples were used and repeated with thesame PCR conditions To receive a final confirmation ofthe nucleotide sequence purified PCR products were sentto Research Biolabs Malaysia The sequencing results werealigned with the gene sequence from the NCBI-GenBank bythe MEGA4 software [16]

4 Discussion

We targeted Ala589Ser polymorphism of WNK4 as a can-didate gene in hypertension to specify potential associationaffecting high blood pressure determination We specified ahuman-specific polymorphic Ala589Ser in WNK4 exon 8In spite of the fact that the relevant research on Ala589Serpolymorphism variation of WNK4 gene was rather basicclose relation of WNK4 gene between cases and controls wasrecognized well recently [13] Sun et al indicated close rela-tion between Ala589Ser polymorphism and both increasedSBP andDBP In addition to kidneysWNK4 gene expressionwas observed in several other organs as well [13] Kamide andcolleagues identified 3 novel missense mutations of WNK4gene in hypertensive patients among Japanese population[17] The studies by Cao et al (2010) on Kazakhs ethnicgroup in Xinjiang proposed that Ala589Ser polymorphism ofWNK4 gene exhibited relation with high blood pressure andthe T allele possibly produced risk factor for EHT [18] In this

4 Journal of Diabetes Research

Table 1 Baseline characteristics of the subjects plusmn

Parameter Hypertensive subjects Normotensive subjects 119901 valueGender (malefemale) (9074) (7383) NSAge (year) 5907 plusmn 1044 5251 plusmn 941 lt00001lowast

SBP (mmHg) 14911 plusmn 2100 12221 plusmn 1123 lt00001lowast

DBP (mmHg) 8602 plusmn 951 7618 plusmn 876 lt00001lowast

BMI chol (mmolL) 2709 plusmn 508 2480 plusmn 368 lt00001lowast

FBS chol (mmolL) 637 plusmn 115 488 plusmn 058 lt00001lowast

T-chol (mmolL) 471 plusmn 098 488 plusmn 131 NSLDL-chol (mmolL) 269 plusmn 097 321 plusmn 110 lt00001lowast

HDL-chol (mmolL) 126 plusmn 032 128 plusmn 052 NSTG (mmolL) 155 plusmn 052 122 plusmn 055 00001lowast

Diabetes (yesno) (8875) (19138) 0000Family history (yesno) (7389) (2155) 0000Smoking habits (yesno) (30133) (13144) 0008Alcohol drinking (yesno) (23140) (2155) 0000Data are mean plusmn SD unless otherwise specified lowastThe 119901 value of genotypes and alleles was calculated using 119905-test NS not significant

Table 2 Genotype and allelic frequency distribution betweenhypertensive and normotensive subjects

Hypertensive subjects Normotensivesubjects 119901 value

GenotypeGG 95 (583) 109 (694)

0005lowastTG 57 (350) 48 (306)TT 11 (67) 0 (00)

Allele frequencyG 247 (758) 266 (847) 0015lowastT 79 (242) 48 (153)

Post hoc test 119901 value (95 confidenceinterval)

TT versus TG 0004 015ndash076TT versus GG 0001 023ndash083TG versus GG 0193 minus019ndash04lowastSignificant value (119901 lt 005) was achieved through Chi-square test ascompared with controls Data were reported in number with percent inparentheses EHT in comparison with control subjects

study genotype and allele frequencies of Ala589Ser showedsignificant association with EHT (119901 = 0004 119901 = 0003)

Although some researchers as Lu in china and Lv inUyghur ethnicity did not observe significant associationof Ala589Ser for genotype and allelic frequency [19 20]research conducted by Erlich and colleagues on Ala589Serof WNK4 demonstrated that there was no association ingenotype or in allele frequencies with hypertension amongAfrican American population [21]

Guo et al (2014) also pooled five studies in their meta-analysis they managed to illustrate significant associationbetween Ala589Ser of WNK4 and hypertension using bothdominant genetic (OR = 5185 95 CI 107ndash319 and 119901 =003) and allele contrast models (OR = 5162 95 CI 111ndash238 and 119901 = 001) [22]

WNK4 is expressed mainly in collecting ducts and distalconvoluted tubules of the kidney and colon Ala589Ser poly-morphism of WNK4 is mapped in Homo sapiens chromo-somes 17 [23]The recent studies demonstrated that the equi-librium between potassium secretion (K+) and sodium chlo-ride (Na+Clminus) reabsorption was regulated through WNK4(both in vitro and in vivo) as a molecular switch bymodulating the effect of the paracellular chloride (Cl

2)

pathways the renal outer medullary potassium (ROMK)channel the epithelia sodium channel (ENaC) and theNa+Clminus cotransporter (NCC) [22 24ndash26] Expression ofNCCincrease in distal convoluted tubule (DCT) enhancement inparacellular Cl

2permeability of the distal tubule and the

decrease in ROMK channels surface expression might beinduced bymutations ofWNK4 [22 25 27 28]Hypertensiondevelopment is also affected by NCC overactivity causedby Na+ retention in the DCT Moreover the results of thiscase control study confirmed the association of WNK4 genevariation with the susceptibility of hypertension

The present study perhaps is the first effort to specifythe relation between WNK4 gene Ala589Ser polymorphismand EHT in Malaysia Significant association of Ala589Serpolymorphism WNK4 gene and hypertension has beenspecified in Malaysian genotype Close association betweenTT genotypesT allele of WNK4 gene and EHT has beendemonstrated in contrast to controls in Malaysia (119901 lt005) Statistically substantial differences have been observedbetween EHT and controls (119901 = 0000) which matchedChinese (119901 lt 0035) and African American population (119901 lt005) [10] The genotype distribution with Hardy-Weinbergexpectations was calculated using a Chi-squared test andallelic frequencies were analyzed by gene-counting methodOdds ratios with 95 confidence intervals were checked inorder to determine the influence of high risk alleles

Considering the fact that 88 individuals out of 163 hyper-tension cases were diagnosed with T2DM based on theirhospital records a strong relation was established betweenhypertension with T2DM and Ala589Ser (119901 lt 005) in the

Journal of Diabetes Research 5

Table 3 Genotype-clinical characteristics correlations in patients

Variable GG GT TT 119901 valueGender (malefemale) 52 (547)43 (453) 31 (544)26 (456) 7 (636)4 (364) NSAge (year) 5563 plusmn 1021 5583 plusmn 1073 6027 plusmn 1249 NSSBP (mmHg) 13497 plusmn 2150 13599 plusmn 2202 15273 plusmn 1169 NSDBP (mmHg) 8025 plusmn 981 8287 plusmn 1122 8291 plusmn 1096 0041BMI (cmkg) 2583 plusmn 493 2624 plusmn 392 2587 plusmn 410 NSFBS (mmole) 551 plusmn 109 587 plusmn 131 587 plusmn 101 NSTCH (mmole) 487 plusmn 126 464 plusmn 096 475 plusmn 072 NSLDL (mmole) 301 plusmn 113 282 plusmn 095 291 plusmn 094 NSHDL (mmole) 130 plusmn 046 121 plusmn 036 134 plusmn 035 NSTG (mmole) 139 plusmn 056 138 plusmn 056 134 plusmn 051 NSDiabetes (noyes) 46 (484)49 (516) 22 (386)35 (614) 7 (77)4 (44) NSFamily history (noyes) 42 (447)52 (553) 23 (404)34 (506) 8 (727)3 (273) NSSmoking habit (noyes) 77 (811)18 (189) 48 (842)9 (158) 8 (727)3 (273) 0045Alcohol consumption (noyes) 86 (905)9 (95) 44 (772)13 (228) 10 (909)1 (91) NSData are presented as mean plusmn SD 119901 value gt 005

Table 4 Genotypes and allele frequencies distribution of genepolymorphisms between two patient groups and control subjects

EHT119899 ()

EHT+T2DM119899 ()

Control119899 ()

Genotype andallele frequency

GG 46 (613) 49 (557) 109 (694)GT 22 (293) 35 (398) 48 (306)TT 7 (93) 4 (45) 0 (0)G 114 (76) 133 (756) 266 (847)T 36 (24) 43 (244) 48 (153)119901 value 0001lowast0017lowast 0004lowast0009lowast

Odds ratio (95CI) 571 (252ndash928) 0558

(0352ndash885)Post hoc testTT versus GT 0000 0018TT versus GG 0000 0004GT versus GG 0790 0083lowast119901 value lt 005

study To reveal a relation between T2DM and Ala589Serpolymorphism I made a careful analysis which was thenovelty of this study I divided the case group into EHT andEHT+T2DM Significant differences in genotypes and allelefrequencies were observed between EHTpatients and controlgroup with 119901 value of lt0001 and lt0017 respectively (119901 lt005) In hypertensive subjects with T2DM the significantrecord was shown for their genotypes and allele frequencieswhich were 0004 and 0009 respectively in comparison withcontrols (119901 lt 005) (Table 4)

Significant differences in age SBP DBP BMI FBS LDLand TG were found between hypertensive and normoten-sive subjects (119901 lt 005) although HDL and TCH levelsshowed no significant differences in hypertensive subjectsand controls (119901 gt 005) Significant differences of clinical

characteristics (BMI HDL LDL TG TCH SBP and DBP)were identified between healthy individuals and EHT asreported previously [13] Interactive influences were alsoobserved by Cao et al between WNK4 gene Ala589Serpolymorphism BMI and gender [18]

In summary we analyzed Ala589Ser polymorphism ofWNK4 gene The Ala589Ser polymorphism displayed sig-nificant association between genotypeallele frequency andhypertension this polymorphism has a potential effect on theclinical characteristics profile of the subjects According toSun et al different hormonesmight have effect on theWNK4gene expression in the kidneys Increasing susceptibility inessential hypertension may be significantly influenced by theAla589Ser polymorphism as a missense mutation [13]

The findings of the present research indicate that TTgenotypesT allele of WNK4 gene produce a close rela-tionship with EHT and T2DM The current research mustbe interpreted within its context restrictions The proof ofthe relationship between TG variant of WNK4 gene andEHT at the gene level has been provided only neverthelessthe function and mechanism of T variant have not beenaddressed Second in contrast to cases the control subjectsare relatively young but not age matched however morerelative studies with larger population concerning other can-didate gene polymorphisms or TG polymorphism ofWNK4gene in relation to cardiovascular disorder are suggested

Competing Interests

The authors declare that they have no competing interests

Authorsrsquo Contributions

Nooshin Ghodsian conceived the study and participatedin the experimental design data acquisition and analysisand interpretation of results and drafted the paper NooshinGhodsian Patimah Ismail Salma Ahmadloo Farzad Hei-dari Polin Haghvirdizadeh Sima Ataollahi Eshkoor and Ali

6 Journal of Diabetes Research

Etemad interpreted the results and critically reviewed thestudy for important intellectual content All authors approvedthe final version of the paper

Acknowledgments

The authors fully acknowledge the cooperation of the vol-unteers and all participants who generate their generoussupport They are also grateful to Mr Mohammad Akhlaghiwho edited this paper grammatically and in that regardimproved the paper significantly

References

[1] D S Timberlake D T OrsquoConnor and R J Parmer ldquoMoleculargenetics of essential hypertension recent results and emergingstrategiesrdquo Current Opinion in Nephrology and Hypertensionvol 10 no 1 pp 71ndash79 2001

[2] F Cambien and L Tiret ldquoGenetics of cardiovascular diseasesfrom single mutations to the whole genomerdquo Circulation vol116 no 15 pp 1714ndash1724 2007

[3] W Ji J N Foo B J OrsquoRoak et al ldquoRare independent mutationsin renal salt handling genes contribute to blood pressurevariationrdquo Nature Genetics vol 40 no 5 pp 592ndash599 2008

[4] D Conen P M Ridker S Mora J E Buring and R J GlynnldquoBlood pressure and risk of developing type 2 diabetes mellitustheWomenrsquos Health Studyrdquo European Heart Journal vol 28 no23 pp 2937ndash2943 2007

[5] C C Doak and L G Doak ldquoThe literacy challenge in diabeteseducation so that clients may understandrdquo Diabetes Care andEducation vol 14 no 2 pp 9ndash11 1993

[6] J R Banegas E Lopez-Garcıa A Graciani et al ldquoRelationshipbetween obesity hypertension and diabetes and health-relatedquality of life among the elderlyrdquo European Journal of Cardio-vascular Prevention amp Rehabilitation vol 14 no 3 pp 456ndash4622007

[7] D Levy A L DeStefano M G Larson et al ldquoEvidence for agene influencing blood pressure on chromosome 17 Genomescan linkage results for longitudinal blood pressure phenotypesin subjects from the Framingham heart studyrdquo Hypertensionvol 36 no 4 pp 477ndash483 2000

[8] Y Kokubo K Kamide N Inamoto et al ldquoIdentification of108 SNPs in TSC WNK1 and WNK4 and their associationwith hypertension in a Japanese general populationrdquo Journal ofHuman Genetics vol 49 no 9 pp 507ndash515 2004

[9] Y Osada R Miyauchi T Goda et al ldquoVariations in theWNK1gene modulates the effect of dietary intake of sodium andpotassium on blood pressure determinationrdquo Journal of HumanGenetics vol 54 no 8 pp 474ndash478 2009

[10] F H Wilson S Disse-Nicodeme K A Choate et al ldquoHumanhypertension caused by mutations in WNK kinasesrdquo Sciencevol 293 no 5532 pp 1107ndash1112 2001

[11] F Verıssimo and P Jordan ldquoWNK kinases a novel proteinkinase subfamily in multi-cellular organismsrdquo Oncogene vol20 no 39 pp 5562ndash5569 2001

[12] R P Lifton A G Gharavi and D S Geller ldquoMolecularmechanisms of human hypertensionrdquo Cell vol 104 no 4 pp545ndash556 2001

[13] Z-J Sun Y Li J-Y Lu et al ldquoAssociation of Ala589Serpolymorphism of WNK4 gene with essential hypertension in

a high-risk Chinese populationrdquo The Journal of PhysiologicalSciences vol 59 no 2 pp 81ndash86 2009

[14] T Pastinen and T J Hudson ldquoCis-acting regulatory variationin the human genomerdquo Science vol 306 no 5696 pp 647ndash6502004

[15] A Visel E M Rubin and L A Pennacchio ldquoGenomic viewsof distant-acting enhancersrdquoNature vol 461 no 7261 pp 199ndash205 2009

[16] S Kumar K Tamura I B Jakobsen and M Nei ldquoMEGA2molecular evolutionary genetics analysis softwarerdquo Bioinfor-matics vol 17 no 12 pp 1244ndash1245 2001

[17] K Kamide S Takiuchi C Tanaka et al ldquoThree novel mis-sense mutations of WNK4 a kinase mutated in inheritedhypertension in Japanese hypertensives implication of clinicalphenotypesrdquoAmerican Journal ofHypertension vol 17 no 5 pp446ndash449 2004

[18] F F Cao H Han F Wang et al ldquoStudy on the associationbetween genetic polymorphism on WNK4 genes and essentialhypertension among Kazakhs ethnic population in XinjiangrdquoZhonghua Liuxingbingxue Zazhi vol 31 no 4 pp 375ndash3782010

[19] M Lu X Wang F Wang et al ldquoWNK4 polymorphisms andessential hypertension in the Uyghur populationrdquo Clinical andExperimental Hypertension vol 31 no 2 pp 179ndash185 2009

[20] J Y LvThe research on the expression of WNK genes [PhD the-sis] China Medical University Beijing China 2003 (Chinese)

[21] P M Erlich J Cui I Chazaro et al ldquoGenetic variants ofWNK4 in whites and African Americans with hypertensionrdquoHypertension vol 41 no 6 pp 1191ndash1195 2003

[22] X-G Guo J Ding H Xu et al ldquoComprehensive assessmentof the association ofWNK4 polymorphisms with hypertensionevidence from a meta-analysisrdquo Scientific Reports vol 4 article6507 2014

[23] L L J Koppes J M Dekker H F J Hendriks L M Bouterand R J Heine ldquoModerate alcohol consumption lowers the riskof type 2 diabetes a meta-analysis of prospective observationalstudiesrdquo Diabetes Care vol 28 no 3 pp 719ndash725 2005

[24] S-S Yang K Yamauchi T Rai et al ldquoRegulation of apicallocalization of the thiazide-sensitive NaCl cotransporter byWNK4 in polarized epithelial cellsrdquo Biochemical and Biophysi-cal Research Communications vol 330 no 2 pp 410ndash414 2005

[25] K T Kahle F HWilson and R P Lifton ldquoRegulation of diverseion transport pathways by WNK4 kinase a novel molecularswitchrdquo Trends in Endocrinology amp Metabolism vol 16 no 3pp 98ndash103 2005

[26] A M Ring S X Cheng Q Leng et al ldquoWNK4 regulatesactivity of the epithelial Na+ channel in vitro and in vivordquoProceedings of the National Academy of Sciences of the UnitedStates of America vol 104 no 10 pp 4020ndash4024 2007

[27] P W Flatman ldquoCotransporters WNKs and hypertension anupdaterdquo Current Opinion in Nephrology and Hypertension vol17 no 2 pp 186ndash192 2008

[28] G Gamba ldquoRole ofWNK kinases in regulating tubular salt andpotassium transport and in the development of hypertensionrdquoAmerican Journal of Physiology-Renal Physiology vol 288 no 2pp F245ndashF252 2005

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2 Journal of Diabetes Research

BP variation and susceptibility to hypertension in generalpopulation in children and adults [8 9] The substitute ofa nonpolar residue alanine by a polar residue serine canbe produced by the Ala589Ser polymorphism as a missensemutation According to Wilson and colleagues mutations inWNK4 can cause some autosomal dominant Mendelian traitcontributing to hypertension [10] Recent studies also illus-trate thatWNK4 influences dominantly the distal convolutedtubule located in the kidneys as significant areas balancingreabsorption of water and salt leading to blood pressuredetermination [10ndash12] According to Sun et al [13] theWNK4 gene seems to influence considerably pathogenesisof essential hypertension Ala589Ser polymorphism (as amissense mutation) might also make alterations to enzymersquosfunctions leading to increased susceptibility to the disorderHowever monogenic disorders are generally resulting fromuncommon polymorphism in a gene coding sequence usualdisorders seem to be produced by genetic polymorphismin gene regulatory factors changing the locus expressionalprofile [14 15]

Considering a strong relation between hypertension anddiabetes in this study our major target aimed at hypertensionas well as T2DM to specify role of the gene underlying thedisease tomodify the prognostication of those at risk and alsoadminister further antihypertensive treatments The currentresearch principal objective was to explore the relation ofWNK4 gene Ala589Ser polymorphism between EHT andT2DM inMalaysian subjects Several screening tests resultedin the recognition of human-specific variant Ala589Ser inWNK4 exon 8This polymorphism (Ala589Ser) was targetedto be conducted onMalaysian population through evolution-ary genetic analysis and to explore its relation with BP andFBS development as well

2 Methods

21 Study Subjects Ethical approval was acquired fromSeremban hospital as well as Universiti Putra Malaysia withreference number [UPMFPSKPADST7-MJETIKAPerF01-JSB-Mac] All participants were asked to fill in informedconsent questionnaires In the current study 320 Malaysianparticipants were interviewed The blood pressure was mea-sured on the right arm (three times) and averaged after resting(5min intervals) by digital Sphygmomanometer on the basisof inclusion criteria [SBP above 140mmHg and DBP above90mmHg (for patients)] Participantsrsquo height and weightwere taken to measure BMI utilizing the formula [weight(kg)height (m2)] All participants had biochemical analyzingtest using plasma electrolytes

22 Genomic DNA Extraction In the study the buccal andblood cells were collected from cases (hypertensive patients)and controls respectively The blood was kept in ethylenediamine tetra acetic acid (EDTA) tube and stored at 4∘C fora maximum of three days before utilizing The DNA wasapplied for amplification after extracting it from buccal andblood cell samples using Qiagen kit (Germany) then it wasstored at minus20∘C for later usage DNA was qualified right

after all primers were optimized by PCRmethod By utilizingthe Nanodrop in two optical density (OD) wavelengths(260 nm and 280 nm) the extracted DNA concentration wasexamined

23 Biochemical Analysis In this section peripheral venousblood samples were collected after keeping an overnightfast by control subjects Moreover lipid profiles [includingTG LDL total cholesterol (TCH) and high-density protein(HDL)] were determined We also measured FBS with stan-dard laboratory techniques however the diabetic glucoselevels were taken from the medical history It was noticeablethat we had referred to the hospital patientsrsquo documents toassess biochemical information for cases

24 Determination of WNK4 Gene Ala589Ser Polymorphism241 Polymerase Chain Reaction (PCR) Genomic DNAwas amplified by multiplex-PCR reactions The examinedpolymorphism of WNK4 (Ala589Ser) was identified usingPCR based RFLP PCRs were carried out with forward primer51015840-TGGAAACCCATTTTCCCCTGG-31015840 and reverse primer51015840-AGGTGGTGAGGCCTAGAAAGT-31015840 at the specific tem-peratures Each reaction was composed of 6x master mix(which embodies DNA polymerase MgCl

2 dNTPs and

reaction buffers) 06 120583L relative primers (03 120583L forward and03 120583L reverse) and 1 120583L of genomic DNA and ultimatelydistilled water was added to a final volume of 25120583LTheDNAwas amplified using initial denaturation of 94∘C (5 minutes)followed by 40 cycles of 55∘C annealing temperature forWNK4 gene polymorphism (90 seconds) 72∘C extension (60seconds) and kept at 72∘C (10 minute) for final extensionAmplified PCR products were analyzed by gel electrophoresismethods with agarose

242 Restriction Fragment Length Polymorphism (RFLP)The enzyme AlwNI was used for restriction of the PCRproducts A total of 10 120583L PCR products were incubatedat 37∘C for at least 3 hours The restriction enzyme andcorresponding buffer (5120583L) were added to the final restric-tion volume After incubating the achieved digested PCRproduct proceeded for electrophoresis on 2 agarose gelat 100 bp DNA molecular weight markers The followingelectrophoresis was observed to determine the genotype ofWNK4 gene Ala589Ser polymorphism The size of the PCRproduct could be determined comparing with a 100 bp DNAladder which was used as a DNA marker Finally the gelwas washed with water for fewminutes before viewing underAlpha Imager

25 Statistical Analysis The statistical analysis in presentstudy was conducted by statistical package for the socialscience (SPSS version 22) Utilizing two-tailed Studentrsquos 119905-testand one-wayANOVA test all variables among the groups andthe groupmeans were being contrasted (119901 lt 005was viewedstatistically significant) The distribution of genotype withHardy-Weinberg expectations was calculated using a Chi-squared test and allelic frequencies were analyzed by gene-counting method In order to detect the effects of high risk

Journal of Diabetes Research 3

alleles odds ratios (OR) with 95 confidence intervals (CI)were checked as well

3 Results

31 Clinical Characteristics In the study 320 volunteersdivided into hypertensive (163) and normotensive (157) par-ticipated for screening Ala589Ser polymorphism in WNK4The clinical characteristicsmean and standard deviation (SD)were clearly indicated in Table 1 however the EHT meanage (5907 plusmn 1044) was noticeably higher in contrast to thecontrols (5251plusmn941) Considering age SBP DBP BMI FBSLDL and TG the subjectsrsquo clinical characteristics illustratedsignificant differences for most parameters between hyper-tensive and normotensive subjects (119901 lt 005) ExcludingHDL and TCH other blood pressure related characteristicsand the Ala589Ser polymorphism in WNK4 exon 8 werefound to be related with elevated risk for the disorder(Table 1) Table 1 also shows significant differences for T2DMbetween hypertensive and normotensive subjects (119901 lt 005)

32 Genotype and Allele Frequency Multiple PCRs were usedto determine the absence and presence genotype ofAla589Serof WNK4 gene in samples Before doing main RCR clarityof the bands was checked by optimization of the methodGene was set up separately with internal control to find outthe sharp band Each gel illustrated the optimization on onegene with four different samples PCR products were run on2 gel with four different samples 50 bp ladder was usedas a DNA marker After PCR product digestion by AlwNIrestriction enzyme the gene encompassingAla589Ser variantwas detected through RFLP method using 2 agarose gelelectrophoresis The enzyme digested the variant type allelesin two fragments of A589 that was divided into 180 and 112 bpwhereas wild type (S589) remained uncut Figure 1 shows theAla589Ser polymorphism amplification product of WNK4gene

Table 2 shows the WNK4 gene Ala589Ser polymor-phism distribution between hypertensive and normotensivesubjects Homozygote (GG) genotype calculated the highestfrequency where case and control were 95 (583) and 109(694) respectively while lower frequencies were observedin heterozygote (GT) genotypes in case 57 (350) andcontrol 48 (306) Interestingly mutant (TT) genotypedetermined the lowest frequency in cases which was 11 (67)but not in the controls Significant differences in genotypeswere detected between cases and controls

In Table 2 G allele was found to be higher in thecontrol subjects which was 266 (847) as compared topatientswhile T allele was higher inpatients Significantdifferences in genotypes and allele frequencies were observedbetween cases and controls with 119901 value of 0005 and 0015respectively (119901 lt 005) Significant values were specified byone-way ANOVA with post hoc test for genotypes TT versusGG (0001) and TT versus GT (0004) between hypertensiveand normotensive subjects however no significant value wasobtained for TG versus GG No significant difference wasachieved from the gender and racersquos genotype for WNK4

112 bp180 bp292bp

50bp

M GG TT GTGT GG TT

Figure 1 Detection of Ala589Ser polymorphism of WNK4 gene byPCR-RFLP utilizing 20 agarose gel electrophoresis PCR productswere digested by AlwNI Wild type was cut (into 180 and 112 bp)while variant type remained uncut Homozygous genotypes areindicated as GG genotypes heterozygous genotypes are determinedas GT genotypes and TT genotypes show mutant genotypes Mrepresented a 50 bp DNA ladder plus (Bioline)

gene Ala589Ser polymorphismwithin genotypes of cases andcontrols

33 Genotype-Clinical Characteristics Correlations To spec-ify genotype-clinical characteristics correlations clinicalcharacteristics of patients between genotypes of Ala589Serpolymorphism were contrasted (Table 3) One-way ANOVAwas applied to analyze the relation of clinical characteristicfactors within genotype groups in patients The WNK4 Tallele established no relation with the genotype of the clinicalcharacteristics of patients It must be noted that no differencewas identified in clinical disability in regard to WNK4Ala589Ser gene polymorphism

34 DNA Sequencing In order to confirm the genotypingresults random samples were used and repeated with thesame PCR conditions To receive a final confirmation ofthe nucleotide sequence purified PCR products were sentto Research Biolabs Malaysia The sequencing results werealigned with the gene sequence from the NCBI-GenBank bythe MEGA4 software [16]

4 Discussion

We targeted Ala589Ser polymorphism of WNK4 as a can-didate gene in hypertension to specify potential associationaffecting high blood pressure determination We specified ahuman-specific polymorphic Ala589Ser in WNK4 exon 8In spite of the fact that the relevant research on Ala589Serpolymorphism variation of WNK4 gene was rather basicclose relation of WNK4 gene between cases and controls wasrecognized well recently [13] Sun et al indicated close rela-tion between Ala589Ser polymorphism and both increasedSBP andDBP In addition to kidneysWNK4 gene expressionwas observed in several other organs as well [13] Kamide andcolleagues identified 3 novel missense mutations of WNK4gene in hypertensive patients among Japanese population[17] The studies by Cao et al (2010) on Kazakhs ethnicgroup in Xinjiang proposed that Ala589Ser polymorphism ofWNK4 gene exhibited relation with high blood pressure andthe T allele possibly produced risk factor for EHT [18] In this

4 Journal of Diabetes Research

Table 1 Baseline characteristics of the subjects plusmn

Parameter Hypertensive subjects Normotensive subjects 119901 valueGender (malefemale) (9074) (7383) NSAge (year) 5907 plusmn 1044 5251 plusmn 941 lt00001lowast

SBP (mmHg) 14911 plusmn 2100 12221 plusmn 1123 lt00001lowast

DBP (mmHg) 8602 plusmn 951 7618 plusmn 876 lt00001lowast

BMI chol (mmolL) 2709 plusmn 508 2480 plusmn 368 lt00001lowast

FBS chol (mmolL) 637 plusmn 115 488 plusmn 058 lt00001lowast

T-chol (mmolL) 471 plusmn 098 488 plusmn 131 NSLDL-chol (mmolL) 269 plusmn 097 321 plusmn 110 lt00001lowast

HDL-chol (mmolL) 126 plusmn 032 128 plusmn 052 NSTG (mmolL) 155 plusmn 052 122 plusmn 055 00001lowast

Diabetes (yesno) (8875) (19138) 0000Family history (yesno) (7389) (2155) 0000Smoking habits (yesno) (30133) (13144) 0008Alcohol drinking (yesno) (23140) (2155) 0000Data are mean plusmn SD unless otherwise specified lowastThe 119901 value of genotypes and alleles was calculated using 119905-test NS not significant

Table 2 Genotype and allelic frequency distribution betweenhypertensive and normotensive subjects

Hypertensive subjects Normotensivesubjects 119901 value

GenotypeGG 95 (583) 109 (694)

0005lowastTG 57 (350) 48 (306)TT 11 (67) 0 (00)

Allele frequencyG 247 (758) 266 (847) 0015lowastT 79 (242) 48 (153)

Post hoc test 119901 value (95 confidenceinterval)

TT versus TG 0004 015ndash076TT versus GG 0001 023ndash083TG versus GG 0193 minus019ndash04lowastSignificant value (119901 lt 005) was achieved through Chi-square test ascompared with controls Data were reported in number with percent inparentheses EHT in comparison with control subjects

study genotype and allele frequencies of Ala589Ser showedsignificant association with EHT (119901 = 0004 119901 = 0003)

Although some researchers as Lu in china and Lv inUyghur ethnicity did not observe significant associationof Ala589Ser for genotype and allelic frequency [19 20]research conducted by Erlich and colleagues on Ala589Serof WNK4 demonstrated that there was no association ingenotype or in allele frequencies with hypertension amongAfrican American population [21]

Guo et al (2014) also pooled five studies in their meta-analysis they managed to illustrate significant associationbetween Ala589Ser of WNK4 and hypertension using bothdominant genetic (OR = 5185 95 CI 107ndash319 and 119901 =003) and allele contrast models (OR = 5162 95 CI 111ndash238 and 119901 = 001) [22]

WNK4 is expressed mainly in collecting ducts and distalconvoluted tubules of the kidney and colon Ala589Ser poly-morphism of WNK4 is mapped in Homo sapiens chromo-somes 17 [23]The recent studies demonstrated that the equi-librium between potassium secretion (K+) and sodium chlo-ride (Na+Clminus) reabsorption was regulated through WNK4(both in vitro and in vivo) as a molecular switch bymodulating the effect of the paracellular chloride (Cl

2)

pathways the renal outer medullary potassium (ROMK)channel the epithelia sodium channel (ENaC) and theNa+Clminus cotransporter (NCC) [22 24ndash26] Expression ofNCCincrease in distal convoluted tubule (DCT) enhancement inparacellular Cl

2permeability of the distal tubule and the

decrease in ROMK channels surface expression might beinduced bymutations ofWNK4 [22 25 27 28]Hypertensiondevelopment is also affected by NCC overactivity causedby Na+ retention in the DCT Moreover the results of thiscase control study confirmed the association of WNK4 genevariation with the susceptibility of hypertension

The present study perhaps is the first effort to specifythe relation between WNK4 gene Ala589Ser polymorphismand EHT in Malaysia Significant association of Ala589Serpolymorphism WNK4 gene and hypertension has beenspecified in Malaysian genotype Close association betweenTT genotypesT allele of WNK4 gene and EHT has beendemonstrated in contrast to controls in Malaysia (119901 lt005) Statistically substantial differences have been observedbetween EHT and controls (119901 = 0000) which matchedChinese (119901 lt 0035) and African American population (119901 lt005) [10] The genotype distribution with Hardy-Weinbergexpectations was calculated using a Chi-squared test andallelic frequencies were analyzed by gene-counting methodOdds ratios with 95 confidence intervals were checked inorder to determine the influence of high risk alleles

Considering the fact that 88 individuals out of 163 hyper-tension cases were diagnosed with T2DM based on theirhospital records a strong relation was established betweenhypertension with T2DM and Ala589Ser (119901 lt 005) in the

Journal of Diabetes Research 5

Table 3 Genotype-clinical characteristics correlations in patients

Variable GG GT TT 119901 valueGender (malefemale) 52 (547)43 (453) 31 (544)26 (456) 7 (636)4 (364) NSAge (year) 5563 plusmn 1021 5583 plusmn 1073 6027 plusmn 1249 NSSBP (mmHg) 13497 plusmn 2150 13599 plusmn 2202 15273 plusmn 1169 NSDBP (mmHg) 8025 plusmn 981 8287 plusmn 1122 8291 plusmn 1096 0041BMI (cmkg) 2583 plusmn 493 2624 plusmn 392 2587 plusmn 410 NSFBS (mmole) 551 plusmn 109 587 plusmn 131 587 plusmn 101 NSTCH (mmole) 487 plusmn 126 464 plusmn 096 475 plusmn 072 NSLDL (mmole) 301 plusmn 113 282 plusmn 095 291 plusmn 094 NSHDL (mmole) 130 plusmn 046 121 plusmn 036 134 plusmn 035 NSTG (mmole) 139 plusmn 056 138 plusmn 056 134 plusmn 051 NSDiabetes (noyes) 46 (484)49 (516) 22 (386)35 (614) 7 (77)4 (44) NSFamily history (noyes) 42 (447)52 (553) 23 (404)34 (506) 8 (727)3 (273) NSSmoking habit (noyes) 77 (811)18 (189) 48 (842)9 (158) 8 (727)3 (273) 0045Alcohol consumption (noyes) 86 (905)9 (95) 44 (772)13 (228) 10 (909)1 (91) NSData are presented as mean plusmn SD 119901 value gt 005

Table 4 Genotypes and allele frequencies distribution of genepolymorphisms between two patient groups and control subjects

EHT119899 ()

EHT+T2DM119899 ()

Control119899 ()

Genotype andallele frequency

GG 46 (613) 49 (557) 109 (694)GT 22 (293) 35 (398) 48 (306)TT 7 (93) 4 (45) 0 (0)G 114 (76) 133 (756) 266 (847)T 36 (24) 43 (244) 48 (153)119901 value 0001lowast0017lowast 0004lowast0009lowast

Odds ratio (95CI) 571 (252ndash928) 0558

(0352ndash885)Post hoc testTT versus GT 0000 0018TT versus GG 0000 0004GT versus GG 0790 0083lowast119901 value lt 005

study To reveal a relation between T2DM and Ala589Serpolymorphism I made a careful analysis which was thenovelty of this study I divided the case group into EHT andEHT+T2DM Significant differences in genotypes and allelefrequencies were observed between EHTpatients and controlgroup with 119901 value of lt0001 and lt0017 respectively (119901 lt005) In hypertensive subjects with T2DM the significantrecord was shown for their genotypes and allele frequencieswhich were 0004 and 0009 respectively in comparison withcontrols (119901 lt 005) (Table 4)

Significant differences in age SBP DBP BMI FBS LDLand TG were found between hypertensive and normoten-sive subjects (119901 lt 005) although HDL and TCH levelsshowed no significant differences in hypertensive subjectsand controls (119901 gt 005) Significant differences of clinical

characteristics (BMI HDL LDL TG TCH SBP and DBP)were identified between healthy individuals and EHT asreported previously [13] Interactive influences were alsoobserved by Cao et al between WNK4 gene Ala589Serpolymorphism BMI and gender [18]

In summary we analyzed Ala589Ser polymorphism ofWNK4 gene The Ala589Ser polymorphism displayed sig-nificant association between genotypeallele frequency andhypertension this polymorphism has a potential effect on theclinical characteristics profile of the subjects According toSun et al different hormonesmight have effect on theWNK4gene expression in the kidneys Increasing susceptibility inessential hypertension may be significantly influenced by theAla589Ser polymorphism as a missense mutation [13]

The findings of the present research indicate that TTgenotypesT allele of WNK4 gene produce a close rela-tionship with EHT and T2DM The current research mustbe interpreted within its context restrictions The proof ofthe relationship between TG variant of WNK4 gene andEHT at the gene level has been provided only neverthelessthe function and mechanism of T variant have not beenaddressed Second in contrast to cases the control subjectsare relatively young but not age matched however morerelative studies with larger population concerning other can-didate gene polymorphisms or TG polymorphism ofWNK4gene in relation to cardiovascular disorder are suggested

Competing Interests

The authors declare that they have no competing interests

Authorsrsquo Contributions

Nooshin Ghodsian conceived the study and participatedin the experimental design data acquisition and analysisand interpretation of results and drafted the paper NooshinGhodsian Patimah Ismail Salma Ahmadloo Farzad Hei-dari Polin Haghvirdizadeh Sima Ataollahi Eshkoor and Ali

6 Journal of Diabetes Research

Etemad interpreted the results and critically reviewed thestudy for important intellectual content All authors approvedthe final version of the paper

Acknowledgments

The authors fully acknowledge the cooperation of the vol-unteers and all participants who generate their generoussupport They are also grateful to Mr Mohammad Akhlaghiwho edited this paper grammatically and in that regardimproved the paper significantly

References

[1] D S Timberlake D T OrsquoConnor and R J Parmer ldquoMoleculargenetics of essential hypertension recent results and emergingstrategiesrdquo Current Opinion in Nephrology and Hypertensionvol 10 no 1 pp 71ndash79 2001

[2] F Cambien and L Tiret ldquoGenetics of cardiovascular diseasesfrom single mutations to the whole genomerdquo Circulation vol116 no 15 pp 1714ndash1724 2007

[3] W Ji J N Foo B J OrsquoRoak et al ldquoRare independent mutationsin renal salt handling genes contribute to blood pressurevariationrdquo Nature Genetics vol 40 no 5 pp 592ndash599 2008

[4] D Conen P M Ridker S Mora J E Buring and R J GlynnldquoBlood pressure and risk of developing type 2 diabetes mellitustheWomenrsquos Health Studyrdquo European Heart Journal vol 28 no23 pp 2937ndash2943 2007

[5] C C Doak and L G Doak ldquoThe literacy challenge in diabeteseducation so that clients may understandrdquo Diabetes Care andEducation vol 14 no 2 pp 9ndash11 1993

[6] J R Banegas E Lopez-Garcıa A Graciani et al ldquoRelationshipbetween obesity hypertension and diabetes and health-relatedquality of life among the elderlyrdquo European Journal of Cardio-vascular Prevention amp Rehabilitation vol 14 no 3 pp 456ndash4622007

[7] D Levy A L DeStefano M G Larson et al ldquoEvidence for agene influencing blood pressure on chromosome 17 Genomescan linkage results for longitudinal blood pressure phenotypesin subjects from the Framingham heart studyrdquo Hypertensionvol 36 no 4 pp 477ndash483 2000

[8] Y Kokubo K Kamide N Inamoto et al ldquoIdentification of108 SNPs in TSC WNK1 and WNK4 and their associationwith hypertension in a Japanese general populationrdquo Journal ofHuman Genetics vol 49 no 9 pp 507ndash515 2004

[9] Y Osada R Miyauchi T Goda et al ldquoVariations in theWNK1gene modulates the effect of dietary intake of sodium andpotassium on blood pressure determinationrdquo Journal of HumanGenetics vol 54 no 8 pp 474ndash478 2009

[10] F H Wilson S Disse-Nicodeme K A Choate et al ldquoHumanhypertension caused by mutations in WNK kinasesrdquo Sciencevol 293 no 5532 pp 1107ndash1112 2001

[11] F Verıssimo and P Jordan ldquoWNK kinases a novel proteinkinase subfamily in multi-cellular organismsrdquo Oncogene vol20 no 39 pp 5562ndash5569 2001

[12] R P Lifton A G Gharavi and D S Geller ldquoMolecularmechanisms of human hypertensionrdquo Cell vol 104 no 4 pp545ndash556 2001

[13] Z-J Sun Y Li J-Y Lu et al ldquoAssociation of Ala589Serpolymorphism of WNK4 gene with essential hypertension in

a high-risk Chinese populationrdquo The Journal of PhysiologicalSciences vol 59 no 2 pp 81ndash86 2009

[14] T Pastinen and T J Hudson ldquoCis-acting regulatory variationin the human genomerdquo Science vol 306 no 5696 pp 647ndash6502004

[15] A Visel E M Rubin and L A Pennacchio ldquoGenomic viewsof distant-acting enhancersrdquoNature vol 461 no 7261 pp 199ndash205 2009

[16] S Kumar K Tamura I B Jakobsen and M Nei ldquoMEGA2molecular evolutionary genetics analysis softwarerdquo Bioinfor-matics vol 17 no 12 pp 1244ndash1245 2001

[17] K Kamide S Takiuchi C Tanaka et al ldquoThree novel mis-sense mutations of WNK4 a kinase mutated in inheritedhypertension in Japanese hypertensives implication of clinicalphenotypesrdquoAmerican Journal ofHypertension vol 17 no 5 pp446ndash449 2004

[18] F F Cao H Han F Wang et al ldquoStudy on the associationbetween genetic polymorphism on WNK4 genes and essentialhypertension among Kazakhs ethnic population in XinjiangrdquoZhonghua Liuxingbingxue Zazhi vol 31 no 4 pp 375ndash3782010

[19] M Lu X Wang F Wang et al ldquoWNK4 polymorphisms andessential hypertension in the Uyghur populationrdquo Clinical andExperimental Hypertension vol 31 no 2 pp 179ndash185 2009

[20] J Y LvThe research on the expression of WNK genes [PhD the-sis] China Medical University Beijing China 2003 (Chinese)

[21] P M Erlich J Cui I Chazaro et al ldquoGenetic variants ofWNK4 in whites and African Americans with hypertensionrdquoHypertension vol 41 no 6 pp 1191ndash1195 2003

[22] X-G Guo J Ding H Xu et al ldquoComprehensive assessmentof the association ofWNK4 polymorphisms with hypertensionevidence from a meta-analysisrdquo Scientific Reports vol 4 article6507 2014

[23] L L J Koppes J M Dekker H F J Hendriks L M Bouterand R J Heine ldquoModerate alcohol consumption lowers the riskof type 2 diabetes a meta-analysis of prospective observationalstudiesrdquo Diabetes Care vol 28 no 3 pp 719ndash725 2005

[24] S-S Yang K Yamauchi T Rai et al ldquoRegulation of apicallocalization of the thiazide-sensitive NaCl cotransporter byWNK4 in polarized epithelial cellsrdquo Biochemical and Biophysi-cal Research Communications vol 330 no 2 pp 410ndash414 2005

[25] K T Kahle F HWilson and R P Lifton ldquoRegulation of diverseion transport pathways by WNK4 kinase a novel molecularswitchrdquo Trends in Endocrinology amp Metabolism vol 16 no 3pp 98ndash103 2005

[26] A M Ring S X Cheng Q Leng et al ldquoWNK4 regulatesactivity of the epithelial Na+ channel in vitro and in vivordquoProceedings of the National Academy of Sciences of the UnitedStates of America vol 104 no 10 pp 4020ndash4024 2007

[27] P W Flatman ldquoCotransporters WNKs and hypertension anupdaterdquo Current Opinion in Nephrology and Hypertension vol17 no 2 pp 186ndash192 2008

[28] G Gamba ldquoRole ofWNK kinases in regulating tubular salt andpotassium transport and in the development of hypertensionrdquoAmerican Journal of Physiology-Renal Physiology vol 288 no 2pp F245ndashF252 2005

Submit your manuscripts athttpwwwhindawicom

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Behavioural Neurology

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Disease Markers

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OncologyJournal of

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Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

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Diabetes ResearchJournal of

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Research and TreatmentAIDS

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Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

Journal of Diabetes Research 3

alleles odds ratios (OR) with 95 confidence intervals (CI)were checked as well

3 Results

31 Clinical Characteristics In the study 320 volunteersdivided into hypertensive (163) and normotensive (157) par-ticipated for screening Ala589Ser polymorphism in WNK4The clinical characteristicsmean and standard deviation (SD)were clearly indicated in Table 1 however the EHT meanage (5907 plusmn 1044) was noticeably higher in contrast to thecontrols (5251plusmn941) Considering age SBP DBP BMI FBSLDL and TG the subjectsrsquo clinical characteristics illustratedsignificant differences for most parameters between hyper-tensive and normotensive subjects (119901 lt 005) ExcludingHDL and TCH other blood pressure related characteristicsand the Ala589Ser polymorphism in WNK4 exon 8 werefound to be related with elevated risk for the disorder(Table 1) Table 1 also shows significant differences for T2DMbetween hypertensive and normotensive subjects (119901 lt 005)

32 Genotype and Allele Frequency Multiple PCRs were usedto determine the absence and presence genotype ofAla589Serof WNK4 gene in samples Before doing main RCR clarityof the bands was checked by optimization of the methodGene was set up separately with internal control to find outthe sharp band Each gel illustrated the optimization on onegene with four different samples PCR products were run on2 gel with four different samples 50 bp ladder was usedas a DNA marker After PCR product digestion by AlwNIrestriction enzyme the gene encompassingAla589Ser variantwas detected through RFLP method using 2 agarose gelelectrophoresis The enzyme digested the variant type allelesin two fragments of A589 that was divided into 180 and 112 bpwhereas wild type (S589) remained uncut Figure 1 shows theAla589Ser polymorphism amplification product of WNK4gene

Table 2 shows the WNK4 gene Ala589Ser polymor-phism distribution between hypertensive and normotensivesubjects Homozygote (GG) genotype calculated the highestfrequency where case and control were 95 (583) and 109(694) respectively while lower frequencies were observedin heterozygote (GT) genotypes in case 57 (350) andcontrol 48 (306) Interestingly mutant (TT) genotypedetermined the lowest frequency in cases which was 11 (67)but not in the controls Significant differences in genotypeswere detected between cases and controls

In Table 2 G allele was found to be higher in thecontrol subjects which was 266 (847) as compared topatientswhile T allele was higher inpatients Significantdifferences in genotypes and allele frequencies were observedbetween cases and controls with 119901 value of 0005 and 0015respectively (119901 lt 005) Significant values were specified byone-way ANOVA with post hoc test for genotypes TT versusGG (0001) and TT versus GT (0004) between hypertensiveand normotensive subjects however no significant value wasobtained for TG versus GG No significant difference wasachieved from the gender and racersquos genotype for WNK4

112 bp180 bp292bp

50bp

M GG TT GTGT GG TT

Figure 1 Detection of Ala589Ser polymorphism of WNK4 gene byPCR-RFLP utilizing 20 agarose gel electrophoresis PCR productswere digested by AlwNI Wild type was cut (into 180 and 112 bp)while variant type remained uncut Homozygous genotypes areindicated as GG genotypes heterozygous genotypes are determinedas GT genotypes and TT genotypes show mutant genotypes Mrepresented a 50 bp DNA ladder plus (Bioline)

gene Ala589Ser polymorphismwithin genotypes of cases andcontrols

33 Genotype-Clinical Characteristics Correlations To spec-ify genotype-clinical characteristics correlations clinicalcharacteristics of patients between genotypes of Ala589Serpolymorphism were contrasted (Table 3) One-way ANOVAwas applied to analyze the relation of clinical characteristicfactors within genotype groups in patients The WNK4 Tallele established no relation with the genotype of the clinicalcharacteristics of patients It must be noted that no differencewas identified in clinical disability in regard to WNK4Ala589Ser gene polymorphism

34 DNA Sequencing In order to confirm the genotypingresults random samples were used and repeated with thesame PCR conditions To receive a final confirmation ofthe nucleotide sequence purified PCR products were sentto Research Biolabs Malaysia The sequencing results werealigned with the gene sequence from the NCBI-GenBank bythe MEGA4 software [16]

4 Discussion

We targeted Ala589Ser polymorphism of WNK4 as a can-didate gene in hypertension to specify potential associationaffecting high blood pressure determination We specified ahuman-specific polymorphic Ala589Ser in WNK4 exon 8In spite of the fact that the relevant research on Ala589Serpolymorphism variation of WNK4 gene was rather basicclose relation of WNK4 gene between cases and controls wasrecognized well recently [13] Sun et al indicated close rela-tion between Ala589Ser polymorphism and both increasedSBP andDBP In addition to kidneysWNK4 gene expressionwas observed in several other organs as well [13] Kamide andcolleagues identified 3 novel missense mutations of WNK4gene in hypertensive patients among Japanese population[17] The studies by Cao et al (2010) on Kazakhs ethnicgroup in Xinjiang proposed that Ala589Ser polymorphism ofWNK4 gene exhibited relation with high blood pressure andthe T allele possibly produced risk factor for EHT [18] In this

4 Journal of Diabetes Research

Table 1 Baseline characteristics of the subjects plusmn

Parameter Hypertensive subjects Normotensive subjects 119901 valueGender (malefemale) (9074) (7383) NSAge (year) 5907 plusmn 1044 5251 plusmn 941 lt00001lowast

SBP (mmHg) 14911 plusmn 2100 12221 plusmn 1123 lt00001lowast

DBP (mmHg) 8602 plusmn 951 7618 plusmn 876 lt00001lowast

BMI chol (mmolL) 2709 plusmn 508 2480 plusmn 368 lt00001lowast

FBS chol (mmolL) 637 plusmn 115 488 plusmn 058 lt00001lowast

T-chol (mmolL) 471 plusmn 098 488 plusmn 131 NSLDL-chol (mmolL) 269 plusmn 097 321 plusmn 110 lt00001lowast

HDL-chol (mmolL) 126 plusmn 032 128 plusmn 052 NSTG (mmolL) 155 plusmn 052 122 plusmn 055 00001lowast

Diabetes (yesno) (8875) (19138) 0000Family history (yesno) (7389) (2155) 0000Smoking habits (yesno) (30133) (13144) 0008Alcohol drinking (yesno) (23140) (2155) 0000Data are mean plusmn SD unless otherwise specified lowastThe 119901 value of genotypes and alleles was calculated using 119905-test NS not significant

Table 2 Genotype and allelic frequency distribution betweenhypertensive and normotensive subjects

Hypertensive subjects Normotensivesubjects 119901 value

GenotypeGG 95 (583) 109 (694)

0005lowastTG 57 (350) 48 (306)TT 11 (67) 0 (00)

Allele frequencyG 247 (758) 266 (847) 0015lowastT 79 (242) 48 (153)

Post hoc test 119901 value (95 confidenceinterval)

TT versus TG 0004 015ndash076TT versus GG 0001 023ndash083TG versus GG 0193 minus019ndash04lowastSignificant value (119901 lt 005) was achieved through Chi-square test ascompared with controls Data were reported in number with percent inparentheses EHT in comparison with control subjects

study genotype and allele frequencies of Ala589Ser showedsignificant association with EHT (119901 = 0004 119901 = 0003)

Although some researchers as Lu in china and Lv inUyghur ethnicity did not observe significant associationof Ala589Ser for genotype and allelic frequency [19 20]research conducted by Erlich and colleagues on Ala589Serof WNK4 demonstrated that there was no association ingenotype or in allele frequencies with hypertension amongAfrican American population [21]

Guo et al (2014) also pooled five studies in their meta-analysis they managed to illustrate significant associationbetween Ala589Ser of WNK4 and hypertension using bothdominant genetic (OR = 5185 95 CI 107ndash319 and 119901 =003) and allele contrast models (OR = 5162 95 CI 111ndash238 and 119901 = 001) [22]

WNK4 is expressed mainly in collecting ducts and distalconvoluted tubules of the kidney and colon Ala589Ser poly-morphism of WNK4 is mapped in Homo sapiens chromo-somes 17 [23]The recent studies demonstrated that the equi-librium between potassium secretion (K+) and sodium chlo-ride (Na+Clminus) reabsorption was regulated through WNK4(both in vitro and in vivo) as a molecular switch bymodulating the effect of the paracellular chloride (Cl

2)

pathways the renal outer medullary potassium (ROMK)channel the epithelia sodium channel (ENaC) and theNa+Clminus cotransporter (NCC) [22 24ndash26] Expression ofNCCincrease in distal convoluted tubule (DCT) enhancement inparacellular Cl

2permeability of the distal tubule and the

decrease in ROMK channels surface expression might beinduced bymutations ofWNK4 [22 25 27 28]Hypertensiondevelopment is also affected by NCC overactivity causedby Na+ retention in the DCT Moreover the results of thiscase control study confirmed the association of WNK4 genevariation with the susceptibility of hypertension

The present study perhaps is the first effort to specifythe relation between WNK4 gene Ala589Ser polymorphismand EHT in Malaysia Significant association of Ala589Serpolymorphism WNK4 gene and hypertension has beenspecified in Malaysian genotype Close association betweenTT genotypesT allele of WNK4 gene and EHT has beendemonstrated in contrast to controls in Malaysia (119901 lt005) Statistically substantial differences have been observedbetween EHT and controls (119901 = 0000) which matchedChinese (119901 lt 0035) and African American population (119901 lt005) [10] The genotype distribution with Hardy-Weinbergexpectations was calculated using a Chi-squared test andallelic frequencies were analyzed by gene-counting methodOdds ratios with 95 confidence intervals were checked inorder to determine the influence of high risk alleles

Considering the fact that 88 individuals out of 163 hyper-tension cases were diagnosed with T2DM based on theirhospital records a strong relation was established betweenhypertension with T2DM and Ala589Ser (119901 lt 005) in the

Journal of Diabetes Research 5

Table 3 Genotype-clinical characteristics correlations in patients

Variable GG GT TT 119901 valueGender (malefemale) 52 (547)43 (453) 31 (544)26 (456) 7 (636)4 (364) NSAge (year) 5563 plusmn 1021 5583 plusmn 1073 6027 plusmn 1249 NSSBP (mmHg) 13497 plusmn 2150 13599 plusmn 2202 15273 plusmn 1169 NSDBP (mmHg) 8025 plusmn 981 8287 plusmn 1122 8291 plusmn 1096 0041BMI (cmkg) 2583 plusmn 493 2624 plusmn 392 2587 plusmn 410 NSFBS (mmole) 551 plusmn 109 587 plusmn 131 587 plusmn 101 NSTCH (mmole) 487 plusmn 126 464 plusmn 096 475 plusmn 072 NSLDL (mmole) 301 plusmn 113 282 plusmn 095 291 plusmn 094 NSHDL (mmole) 130 plusmn 046 121 plusmn 036 134 plusmn 035 NSTG (mmole) 139 plusmn 056 138 plusmn 056 134 plusmn 051 NSDiabetes (noyes) 46 (484)49 (516) 22 (386)35 (614) 7 (77)4 (44) NSFamily history (noyes) 42 (447)52 (553) 23 (404)34 (506) 8 (727)3 (273) NSSmoking habit (noyes) 77 (811)18 (189) 48 (842)9 (158) 8 (727)3 (273) 0045Alcohol consumption (noyes) 86 (905)9 (95) 44 (772)13 (228) 10 (909)1 (91) NSData are presented as mean plusmn SD 119901 value gt 005

Table 4 Genotypes and allele frequencies distribution of genepolymorphisms between two patient groups and control subjects

EHT119899 ()

EHT+T2DM119899 ()

Control119899 ()

Genotype andallele frequency

GG 46 (613) 49 (557) 109 (694)GT 22 (293) 35 (398) 48 (306)TT 7 (93) 4 (45) 0 (0)G 114 (76) 133 (756) 266 (847)T 36 (24) 43 (244) 48 (153)119901 value 0001lowast0017lowast 0004lowast0009lowast

Odds ratio (95CI) 571 (252ndash928) 0558

(0352ndash885)Post hoc testTT versus GT 0000 0018TT versus GG 0000 0004GT versus GG 0790 0083lowast119901 value lt 005

study To reveal a relation between T2DM and Ala589Serpolymorphism I made a careful analysis which was thenovelty of this study I divided the case group into EHT andEHT+T2DM Significant differences in genotypes and allelefrequencies were observed between EHTpatients and controlgroup with 119901 value of lt0001 and lt0017 respectively (119901 lt005) In hypertensive subjects with T2DM the significantrecord was shown for their genotypes and allele frequencieswhich were 0004 and 0009 respectively in comparison withcontrols (119901 lt 005) (Table 4)

Significant differences in age SBP DBP BMI FBS LDLand TG were found between hypertensive and normoten-sive subjects (119901 lt 005) although HDL and TCH levelsshowed no significant differences in hypertensive subjectsand controls (119901 gt 005) Significant differences of clinical

characteristics (BMI HDL LDL TG TCH SBP and DBP)were identified between healthy individuals and EHT asreported previously [13] Interactive influences were alsoobserved by Cao et al between WNK4 gene Ala589Serpolymorphism BMI and gender [18]

In summary we analyzed Ala589Ser polymorphism ofWNK4 gene The Ala589Ser polymorphism displayed sig-nificant association between genotypeallele frequency andhypertension this polymorphism has a potential effect on theclinical characteristics profile of the subjects According toSun et al different hormonesmight have effect on theWNK4gene expression in the kidneys Increasing susceptibility inessential hypertension may be significantly influenced by theAla589Ser polymorphism as a missense mutation [13]

The findings of the present research indicate that TTgenotypesT allele of WNK4 gene produce a close rela-tionship with EHT and T2DM The current research mustbe interpreted within its context restrictions The proof ofthe relationship between TG variant of WNK4 gene andEHT at the gene level has been provided only neverthelessthe function and mechanism of T variant have not beenaddressed Second in contrast to cases the control subjectsare relatively young but not age matched however morerelative studies with larger population concerning other can-didate gene polymorphisms or TG polymorphism ofWNK4gene in relation to cardiovascular disorder are suggested

Competing Interests

The authors declare that they have no competing interests

Authorsrsquo Contributions

Nooshin Ghodsian conceived the study and participatedin the experimental design data acquisition and analysisand interpretation of results and drafted the paper NooshinGhodsian Patimah Ismail Salma Ahmadloo Farzad Hei-dari Polin Haghvirdizadeh Sima Ataollahi Eshkoor and Ali

6 Journal of Diabetes Research

Etemad interpreted the results and critically reviewed thestudy for important intellectual content All authors approvedthe final version of the paper

Acknowledgments

The authors fully acknowledge the cooperation of the vol-unteers and all participants who generate their generoussupport They are also grateful to Mr Mohammad Akhlaghiwho edited this paper grammatically and in that regardimproved the paper significantly

References

[1] D S Timberlake D T OrsquoConnor and R J Parmer ldquoMoleculargenetics of essential hypertension recent results and emergingstrategiesrdquo Current Opinion in Nephrology and Hypertensionvol 10 no 1 pp 71ndash79 2001

[2] F Cambien and L Tiret ldquoGenetics of cardiovascular diseasesfrom single mutations to the whole genomerdquo Circulation vol116 no 15 pp 1714ndash1724 2007

[3] W Ji J N Foo B J OrsquoRoak et al ldquoRare independent mutationsin renal salt handling genes contribute to blood pressurevariationrdquo Nature Genetics vol 40 no 5 pp 592ndash599 2008

[4] D Conen P M Ridker S Mora J E Buring and R J GlynnldquoBlood pressure and risk of developing type 2 diabetes mellitustheWomenrsquos Health Studyrdquo European Heart Journal vol 28 no23 pp 2937ndash2943 2007

[5] C C Doak and L G Doak ldquoThe literacy challenge in diabeteseducation so that clients may understandrdquo Diabetes Care andEducation vol 14 no 2 pp 9ndash11 1993

[6] J R Banegas E Lopez-Garcıa A Graciani et al ldquoRelationshipbetween obesity hypertension and diabetes and health-relatedquality of life among the elderlyrdquo European Journal of Cardio-vascular Prevention amp Rehabilitation vol 14 no 3 pp 456ndash4622007

[7] D Levy A L DeStefano M G Larson et al ldquoEvidence for agene influencing blood pressure on chromosome 17 Genomescan linkage results for longitudinal blood pressure phenotypesin subjects from the Framingham heart studyrdquo Hypertensionvol 36 no 4 pp 477ndash483 2000

[8] Y Kokubo K Kamide N Inamoto et al ldquoIdentification of108 SNPs in TSC WNK1 and WNK4 and their associationwith hypertension in a Japanese general populationrdquo Journal ofHuman Genetics vol 49 no 9 pp 507ndash515 2004

[9] Y Osada R Miyauchi T Goda et al ldquoVariations in theWNK1gene modulates the effect of dietary intake of sodium andpotassium on blood pressure determinationrdquo Journal of HumanGenetics vol 54 no 8 pp 474ndash478 2009

[10] F H Wilson S Disse-Nicodeme K A Choate et al ldquoHumanhypertension caused by mutations in WNK kinasesrdquo Sciencevol 293 no 5532 pp 1107ndash1112 2001

[11] F Verıssimo and P Jordan ldquoWNK kinases a novel proteinkinase subfamily in multi-cellular organismsrdquo Oncogene vol20 no 39 pp 5562ndash5569 2001

[12] R P Lifton A G Gharavi and D S Geller ldquoMolecularmechanisms of human hypertensionrdquo Cell vol 104 no 4 pp545ndash556 2001

[13] Z-J Sun Y Li J-Y Lu et al ldquoAssociation of Ala589Serpolymorphism of WNK4 gene with essential hypertension in

a high-risk Chinese populationrdquo The Journal of PhysiologicalSciences vol 59 no 2 pp 81ndash86 2009

[14] T Pastinen and T J Hudson ldquoCis-acting regulatory variationin the human genomerdquo Science vol 306 no 5696 pp 647ndash6502004

[15] A Visel E M Rubin and L A Pennacchio ldquoGenomic viewsof distant-acting enhancersrdquoNature vol 461 no 7261 pp 199ndash205 2009

[16] S Kumar K Tamura I B Jakobsen and M Nei ldquoMEGA2molecular evolutionary genetics analysis softwarerdquo Bioinfor-matics vol 17 no 12 pp 1244ndash1245 2001

[17] K Kamide S Takiuchi C Tanaka et al ldquoThree novel mis-sense mutations of WNK4 a kinase mutated in inheritedhypertension in Japanese hypertensives implication of clinicalphenotypesrdquoAmerican Journal ofHypertension vol 17 no 5 pp446ndash449 2004

[18] F F Cao H Han F Wang et al ldquoStudy on the associationbetween genetic polymorphism on WNK4 genes and essentialhypertension among Kazakhs ethnic population in XinjiangrdquoZhonghua Liuxingbingxue Zazhi vol 31 no 4 pp 375ndash3782010

[19] M Lu X Wang F Wang et al ldquoWNK4 polymorphisms andessential hypertension in the Uyghur populationrdquo Clinical andExperimental Hypertension vol 31 no 2 pp 179ndash185 2009

[20] J Y LvThe research on the expression of WNK genes [PhD the-sis] China Medical University Beijing China 2003 (Chinese)

[21] P M Erlich J Cui I Chazaro et al ldquoGenetic variants ofWNK4 in whites and African Americans with hypertensionrdquoHypertension vol 41 no 6 pp 1191ndash1195 2003

[22] X-G Guo J Ding H Xu et al ldquoComprehensive assessmentof the association ofWNK4 polymorphisms with hypertensionevidence from a meta-analysisrdquo Scientific Reports vol 4 article6507 2014

[23] L L J Koppes J M Dekker H F J Hendriks L M Bouterand R J Heine ldquoModerate alcohol consumption lowers the riskof type 2 diabetes a meta-analysis of prospective observationalstudiesrdquo Diabetes Care vol 28 no 3 pp 719ndash725 2005

[24] S-S Yang K Yamauchi T Rai et al ldquoRegulation of apicallocalization of the thiazide-sensitive NaCl cotransporter byWNK4 in polarized epithelial cellsrdquo Biochemical and Biophysi-cal Research Communications vol 330 no 2 pp 410ndash414 2005

[25] K T Kahle F HWilson and R P Lifton ldquoRegulation of diverseion transport pathways by WNK4 kinase a novel molecularswitchrdquo Trends in Endocrinology amp Metabolism vol 16 no 3pp 98ndash103 2005

[26] A M Ring S X Cheng Q Leng et al ldquoWNK4 regulatesactivity of the epithelial Na+ channel in vitro and in vivordquoProceedings of the National Academy of Sciences of the UnitedStates of America vol 104 no 10 pp 4020ndash4024 2007

[27] P W Flatman ldquoCotransporters WNKs and hypertension anupdaterdquo Current Opinion in Nephrology and Hypertension vol17 no 2 pp 186ndash192 2008

[28] G Gamba ldquoRole ofWNK kinases in regulating tubular salt andpotassium transport and in the development of hypertensionrdquoAmerican Journal of Physiology-Renal Physiology vol 288 no 2pp F245ndashF252 2005

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

4 Journal of Diabetes Research

Table 1 Baseline characteristics of the subjects plusmn

Parameter Hypertensive subjects Normotensive subjects 119901 valueGender (malefemale) (9074) (7383) NSAge (year) 5907 plusmn 1044 5251 plusmn 941 lt00001lowast

SBP (mmHg) 14911 plusmn 2100 12221 plusmn 1123 lt00001lowast

DBP (mmHg) 8602 plusmn 951 7618 plusmn 876 lt00001lowast

BMI chol (mmolL) 2709 plusmn 508 2480 plusmn 368 lt00001lowast

FBS chol (mmolL) 637 plusmn 115 488 plusmn 058 lt00001lowast

T-chol (mmolL) 471 plusmn 098 488 plusmn 131 NSLDL-chol (mmolL) 269 plusmn 097 321 plusmn 110 lt00001lowast

HDL-chol (mmolL) 126 plusmn 032 128 plusmn 052 NSTG (mmolL) 155 plusmn 052 122 plusmn 055 00001lowast

Diabetes (yesno) (8875) (19138) 0000Family history (yesno) (7389) (2155) 0000Smoking habits (yesno) (30133) (13144) 0008Alcohol drinking (yesno) (23140) (2155) 0000Data are mean plusmn SD unless otherwise specified lowastThe 119901 value of genotypes and alleles was calculated using 119905-test NS not significant

Table 2 Genotype and allelic frequency distribution betweenhypertensive and normotensive subjects

Hypertensive subjects Normotensivesubjects 119901 value

GenotypeGG 95 (583) 109 (694)

0005lowastTG 57 (350) 48 (306)TT 11 (67) 0 (00)

Allele frequencyG 247 (758) 266 (847) 0015lowastT 79 (242) 48 (153)

Post hoc test 119901 value (95 confidenceinterval)

TT versus TG 0004 015ndash076TT versus GG 0001 023ndash083TG versus GG 0193 minus019ndash04lowastSignificant value (119901 lt 005) was achieved through Chi-square test ascompared with controls Data were reported in number with percent inparentheses EHT in comparison with control subjects

study genotype and allele frequencies of Ala589Ser showedsignificant association with EHT (119901 = 0004 119901 = 0003)

Although some researchers as Lu in china and Lv inUyghur ethnicity did not observe significant associationof Ala589Ser for genotype and allelic frequency [19 20]research conducted by Erlich and colleagues on Ala589Serof WNK4 demonstrated that there was no association ingenotype or in allele frequencies with hypertension amongAfrican American population [21]

Guo et al (2014) also pooled five studies in their meta-analysis they managed to illustrate significant associationbetween Ala589Ser of WNK4 and hypertension using bothdominant genetic (OR = 5185 95 CI 107ndash319 and 119901 =003) and allele contrast models (OR = 5162 95 CI 111ndash238 and 119901 = 001) [22]

WNK4 is expressed mainly in collecting ducts and distalconvoluted tubules of the kidney and colon Ala589Ser poly-morphism of WNK4 is mapped in Homo sapiens chromo-somes 17 [23]The recent studies demonstrated that the equi-librium between potassium secretion (K+) and sodium chlo-ride (Na+Clminus) reabsorption was regulated through WNK4(both in vitro and in vivo) as a molecular switch bymodulating the effect of the paracellular chloride (Cl

2)

pathways the renal outer medullary potassium (ROMK)channel the epithelia sodium channel (ENaC) and theNa+Clminus cotransporter (NCC) [22 24ndash26] Expression ofNCCincrease in distal convoluted tubule (DCT) enhancement inparacellular Cl

2permeability of the distal tubule and the

decrease in ROMK channels surface expression might beinduced bymutations ofWNK4 [22 25 27 28]Hypertensiondevelopment is also affected by NCC overactivity causedby Na+ retention in the DCT Moreover the results of thiscase control study confirmed the association of WNK4 genevariation with the susceptibility of hypertension

The present study perhaps is the first effort to specifythe relation between WNK4 gene Ala589Ser polymorphismand EHT in Malaysia Significant association of Ala589Serpolymorphism WNK4 gene and hypertension has beenspecified in Malaysian genotype Close association betweenTT genotypesT allele of WNK4 gene and EHT has beendemonstrated in contrast to controls in Malaysia (119901 lt005) Statistically substantial differences have been observedbetween EHT and controls (119901 = 0000) which matchedChinese (119901 lt 0035) and African American population (119901 lt005) [10] The genotype distribution with Hardy-Weinbergexpectations was calculated using a Chi-squared test andallelic frequencies were analyzed by gene-counting methodOdds ratios with 95 confidence intervals were checked inorder to determine the influence of high risk alleles

Considering the fact that 88 individuals out of 163 hyper-tension cases were diagnosed with T2DM based on theirhospital records a strong relation was established betweenhypertension with T2DM and Ala589Ser (119901 lt 005) in the

Journal of Diabetes Research 5

Table 3 Genotype-clinical characteristics correlations in patients

Variable GG GT TT 119901 valueGender (malefemale) 52 (547)43 (453) 31 (544)26 (456) 7 (636)4 (364) NSAge (year) 5563 plusmn 1021 5583 plusmn 1073 6027 plusmn 1249 NSSBP (mmHg) 13497 plusmn 2150 13599 plusmn 2202 15273 plusmn 1169 NSDBP (mmHg) 8025 plusmn 981 8287 plusmn 1122 8291 plusmn 1096 0041BMI (cmkg) 2583 plusmn 493 2624 plusmn 392 2587 plusmn 410 NSFBS (mmole) 551 plusmn 109 587 plusmn 131 587 plusmn 101 NSTCH (mmole) 487 plusmn 126 464 plusmn 096 475 plusmn 072 NSLDL (mmole) 301 plusmn 113 282 plusmn 095 291 plusmn 094 NSHDL (mmole) 130 plusmn 046 121 plusmn 036 134 plusmn 035 NSTG (mmole) 139 plusmn 056 138 plusmn 056 134 plusmn 051 NSDiabetes (noyes) 46 (484)49 (516) 22 (386)35 (614) 7 (77)4 (44) NSFamily history (noyes) 42 (447)52 (553) 23 (404)34 (506) 8 (727)3 (273) NSSmoking habit (noyes) 77 (811)18 (189) 48 (842)9 (158) 8 (727)3 (273) 0045Alcohol consumption (noyes) 86 (905)9 (95) 44 (772)13 (228) 10 (909)1 (91) NSData are presented as mean plusmn SD 119901 value gt 005

Table 4 Genotypes and allele frequencies distribution of genepolymorphisms between two patient groups and control subjects

EHT119899 ()

EHT+T2DM119899 ()

Control119899 ()

Genotype andallele frequency

GG 46 (613) 49 (557) 109 (694)GT 22 (293) 35 (398) 48 (306)TT 7 (93) 4 (45) 0 (0)G 114 (76) 133 (756) 266 (847)T 36 (24) 43 (244) 48 (153)119901 value 0001lowast0017lowast 0004lowast0009lowast

Odds ratio (95CI) 571 (252ndash928) 0558

(0352ndash885)Post hoc testTT versus GT 0000 0018TT versus GG 0000 0004GT versus GG 0790 0083lowast119901 value lt 005

study To reveal a relation between T2DM and Ala589Serpolymorphism I made a careful analysis which was thenovelty of this study I divided the case group into EHT andEHT+T2DM Significant differences in genotypes and allelefrequencies were observed between EHTpatients and controlgroup with 119901 value of lt0001 and lt0017 respectively (119901 lt005) In hypertensive subjects with T2DM the significantrecord was shown for their genotypes and allele frequencieswhich were 0004 and 0009 respectively in comparison withcontrols (119901 lt 005) (Table 4)

Significant differences in age SBP DBP BMI FBS LDLand TG were found between hypertensive and normoten-sive subjects (119901 lt 005) although HDL and TCH levelsshowed no significant differences in hypertensive subjectsand controls (119901 gt 005) Significant differences of clinical

characteristics (BMI HDL LDL TG TCH SBP and DBP)were identified between healthy individuals and EHT asreported previously [13] Interactive influences were alsoobserved by Cao et al between WNK4 gene Ala589Serpolymorphism BMI and gender [18]

In summary we analyzed Ala589Ser polymorphism ofWNK4 gene The Ala589Ser polymorphism displayed sig-nificant association between genotypeallele frequency andhypertension this polymorphism has a potential effect on theclinical characteristics profile of the subjects According toSun et al different hormonesmight have effect on theWNK4gene expression in the kidneys Increasing susceptibility inessential hypertension may be significantly influenced by theAla589Ser polymorphism as a missense mutation [13]

The findings of the present research indicate that TTgenotypesT allele of WNK4 gene produce a close rela-tionship with EHT and T2DM The current research mustbe interpreted within its context restrictions The proof ofthe relationship between TG variant of WNK4 gene andEHT at the gene level has been provided only neverthelessthe function and mechanism of T variant have not beenaddressed Second in contrast to cases the control subjectsare relatively young but not age matched however morerelative studies with larger population concerning other can-didate gene polymorphisms or TG polymorphism ofWNK4gene in relation to cardiovascular disorder are suggested

Competing Interests

The authors declare that they have no competing interests

Authorsrsquo Contributions

Nooshin Ghodsian conceived the study and participatedin the experimental design data acquisition and analysisand interpretation of results and drafted the paper NooshinGhodsian Patimah Ismail Salma Ahmadloo Farzad Hei-dari Polin Haghvirdizadeh Sima Ataollahi Eshkoor and Ali

6 Journal of Diabetes Research

Etemad interpreted the results and critically reviewed thestudy for important intellectual content All authors approvedthe final version of the paper

Acknowledgments

The authors fully acknowledge the cooperation of the vol-unteers and all participants who generate their generoussupport They are also grateful to Mr Mohammad Akhlaghiwho edited this paper grammatically and in that regardimproved the paper significantly

References

[1] D S Timberlake D T OrsquoConnor and R J Parmer ldquoMoleculargenetics of essential hypertension recent results and emergingstrategiesrdquo Current Opinion in Nephrology and Hypertensionvol 10 no 1 pp 71ndash79 2001

[2] F Cambien and L Tiret ldquoGenetics of cardiovascular diseasesfrom single mutations to the whole genomerdquo Circulation vol116 no 15 pp 1714ndash1724 2007

[3] W Ji J N Foo B J OrsquoRoak et al ldquoRare independent mutationsin renal salt handling genes contribute to blood pressurevariationrdquo Nature Genetics vol 40 no 5 pp 592ndash599 2008

[4] D Conen P M Ridker S Mora J E Buring and R J GlynnldquoBlood pressure and risk of developing type 2 diabetes mellitustheWomenrsquos Health Studyrdquo European Heart Journal vol 28 no23 pp 2937ndash2943 2007

[5] C C Doak and L G Doak ldquoThe literacy challenge in diabeteseducation so that clients may understandrdquo Diabetes Care andEducation vol 14 no 2 pp 9ndash11 1993

[6] J R Banegas E Lopez-Garcıa A Graciani et al ldquoRelationshipbetween obesity hypertension and diabetes and health-relatedquality of life among the elderlyrdquo European Journal of Cardio-vascular Prevention amp Rehabilitation vol 14 no 3 pp 456ndash4622007

[7] D Levy A L DeStefano M G Larson et al ldquoEvidence for agene influencing blood pressure on chromosome 17 Genomescan linkage results for longitudinal blood pressure phenotypesin subjects from the Framingham heart studyrdquo Hypertensionvol 36 no 4 pp 477ndash483 2000

[8] Y Kokubo K Kamide N Inamoto et al ldquoIdentification of108 SNPs in TSC WNK1 and WNK4 and their associationwith hypertension in a Japanese general populationrdquo Journal ofHuman Genetics vol 49 no 9 pp 507ndash515 2004

[9] Y Osada R Miyauchi T Goda et al ldquoVariations in theWNK1gene modulates the effect of dietary intake of sodium andpotassium on blood pressure determinationrdquo Journal of HumanGenetics vol 54 no 8 pp 474ndash478 2009

[10] F H Wilson S Disse-Nicodeme K A Choate et al ldquoHumanhypertension caused by mutations in WNK kinasesrdquo Sciencevol 293 no 5532 pp 1107ndash1112 2001

[11] F Verıssimo and P Jordan ldquoWNK kinases a novel proteinkinase subfamily in multi-cellular organismsrdquo Oncogene vol20 no 39 pp 5562ndash5569 2001

[12] R P Lifton A G Gharavi and D S Geller ldquoMolecularmechanisms of human hypertensionrdquo Cell vol 104 no 4 pp545ndash556 2001

[13] Z-J Sun Y Li J-Y Lu et al ldquoAssociation of Ala589Serpolymorphism of WNK4 gene with essential hypertension in

a high-risk Chinese populationrdquo The Journal of PhysiologicalSciences vol 59 no 2 pp 81ndash86 2009

[14] T Pastinen and T J Hudson ldquoCis-acting regulatory variationin the human genomerdquo Science vol 306 no 5696 pp 647ndash6502004

[15] A Visel E M Rubin and L A Pennacchio ldquoGenomic viewsof distant-acting enhancersrdquoNature vol 461 no 7261 pp 199ndash205 2009

[16] S Kumar K Tamura I B Jakobsen and M Nei ldquoMEGA2molecular evolutionary genetics analysis softwarerdquo Bioinfor-matics vol 17 no 12 pp 1244ndash1245 2001

[17] K Kamide S Takiuchi C Tanaka et al ldquoThree novel mis-sense mutations of WNK4 a kinase mutated in inheritedhypertension in Japanese hypertensives implication of clinicalphenotypesrdquoAmerican Journal ofHypertension vol 17 no 5 pp446ndash449 2004

[18] F F Cao H Han F Wang et al ldquoStudy on the associationbetween genetic polymorphism on WNK4 genes and essentialhypertension among Kazakhs ethnic population in XinjiangrdquoZhonghua Liuxingbingxue Zazhi vol 31 no 4 pp 375ndash3782010

[19] M Lu X Wang F Wang et al ldquoWNK4 polymorphisms andessential hypertension in the Uyghur populationrdquo Clinical andExperimental Hypertension vol 31 no 2 pp 179ndash185 2009

[20] J Y LvThe research on the expression of WNK genes [PhD the-sis] China Medical University Beijing China 2003 (Chinese)

[21] P M Erlich J Cui I Chazaro et al ldquoGenetic variants ofWNK4 in whites and African Americans with hypertensionrdquoHypertension vol 41 no 6 pp 1191ndash1195 2003

[22] X-G Guo J Ding H Xu et al ldquoComprehensive assessmentof the association ofWNK4 polymorphisms with hypertensionevidence from a meta-analysisrdquo Scientific Reports vol 4 article6507 2014

[23] L L J Koppes J M Dekker H F J Hendriks L M Bouterand R J Heine ldquoModerate alcohol consumption lowers the riskof type 2 diabetes a meta-analysis of prospective observationalstudiesrdquo Diabetes Care vol 28 no 3 pp 719ndash725 2005

[24] S-S Yang K Yamauchi T Rai et al ldquoRegulation of apicallocalization of the thiazide-sensitive NaCl cotransporter byWNK4 in polarized epithelial cellsrdquo Biochemical and Biophysi-cal Research Communications vol 330 no 2 pp 410ndash414 2005

[25] K T Kahle F HWilson and R P Lifton ldquoRegulation of diverseion transport pathways by WNK4 kinase a novel molecularswitchrdquo Trends in Endocrinology amp Metabolism vol 16 no 3pp 98ndash103 2005

[26] A M Ring S X Cheng Q Leng et al ldquoWNK4 regulatesactivity of the epithelial Na+ channel in vitro and in vivordquoProceedings of the National Academy of Sciences of the UnitedStates of America vol 104 no 10 pp 4020ndash4024 2007

[27] P W Flatman ldquoCotransporters WNKs and hypertension anupdaterdquo Current Opinion in Nephrology and Hypertension vol17 no 2 pp 186ndash192 2008

[28] G Gamba ldquoRole ofWNK kinases in regulating tubular salt andpotassium transport and in the development of hypertensionrdquoAmerican Journal of Physiology-Renal Physiology vol 288 no 2pp F245ndashF252 2005

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

Journal of Diabetes Research 5

Table 3 Genotype-clinical characteristics correlations in patients

Variable GG GT TT 119901 valueGender (malefemale) 52 (547)43 (453) 31 (544)26 (456) 7 (636)4 (364) NSAge (year) 5563 plusmn 1021 5583 plusmn 1073 6027 plusmn 1249 NSSBP (mmHg) 13497 plusmn 2150 13599 plusmn 2202 15273 plusmn 1169 NSDBP (mmHg) 8025 plusmn 981 8287 plusmn 1122 8291 plusmn 1096 0041BMI (cmkg) 2583 plusmn 493 2624 plusmn 392 2587 plusmn 410 NSFBS (mmole) 551 plusmn 109 587 plusmn 131 587 plusmn 101 NSTCH (mmole) 487 plusmn 126 464 plusmn 096 475 plusmn 072 NSLDL (mmole) 301 plusmn 113 282 plusmn 095 291 plusmn 094 NSHDL (mmole) 130 plusmn 046 121 plusmn 036 134 plusmn 035 NSTG (mmole) 139 plusmn 056 138 plusmn 056 134 plusmn 051 NSDiabetes (noyes) 46 (484)49 (516) 22 (386)35 (614) 7 (77)4 (44) NSFamily history (noyes) 42 (447)52 (553) 23 (404)34 (506) 8 (727)3 (273) NSSmoking habit (noyes) 77 (811)18 (189) 48 (842)9 (158) 8 (727)3 (273) 0045Alcohol consumption (noyes) 86 (905)9 (95) 44 (772)13 (228) 10 (909)1 (91) NSData are presented as mean plusmn SD 119901 value gt 005

Table 4 Genotypes and allele frequencies distribution of genepolymorphisms between two patient groups and control subjects

EHT119899 ()

EHT+T2DM119899 ()

Control119899 ()

Genotype andallele frequency

GG 46 (613) 49 (557) 109 (694)GT 22 (293) 35 (398) 48 (306)TT 7 (93) 4 (45) 0 (0)G 114 (76) 133 (756) 266 (847)T 36 (24) 43 (244) 48 (153)119901 value 0001lowast0017lowast 0004lowast0009lowast

Odds ratio (95CI) 571 (252ndash928) 0558

(0352ndash885)Post hoc testTT versus GT 0000 0018TT versus GG 0000 0004GT versus GG 0790 0083lowast119901 value lt 005

study To reveal a relation between T2DM and Ala589Serpolymorphism I made a careful analysis which was thenovelty of this study I divided the case group into EHT andEHT+T2DM Significant differences in genotypes and allelefrequencies were observed between EHTpatients and controlgroup with 119901 value of lt0001 and lt0017 respectively (119901 lt005) In hypertensive subjects with T2DM the significantrecord was shown for their genotypes and allele frequencieswhich were 0004 and 0009 respectively in comparison withcontrols (119901 lt 005) (Table 4)

Significant differences in age SBP DBP BMI FBS LDLand TG were found between hypertensive and normoten-sive subjects (119901 lt 005) although HDL and TCH levelsshowed no significant differences in hypertensive subjectsand controls (119901 gt 005) Significant differences of clinical

characteristics (BMI HDL LDL TG TCH SBP and DBP)were identified between healthy individuals and EHT asreported previously [13] Interactive influences were alsoobserved by Cao et al between WNK4 gene Ala589Serpolymorphism BMI and gender [18]

In summary we analyzed Ala589Ser polymorphism ofWNK4 gene The Ala589Ser polymorphism displayed sig-nificant association between genotypeallele frequency andhypertension this polymorphism has a potential effect on theclinical characteristics profile of the subjects According toSun et al different hormonesmight have effect on theWNK4gene expression in the kidneys Increasing susceptibility inessential hypertension may be significantly influenced by theAla589Ser polymorphism as a missense mutation [13]

The findings of the present research indicate that TTgenotypesT allele of WNK4 gene produce a close rela-tionship with EHT and T2DM The current research mustbe interpreted within its context restrictions The proof ofthe relationship between TG variant of WNK4 gene andEHT at the gene level has been provided only neverthelessthe function and mechanism of T variant have not beenaddressed Second in contrast to cases the control subjectsare relatively young but not age matched however morerelative studies with larger population concerning other can-didate gene polymorphisms or TG polymorphism ofWNK4gene in relation to cardiovascular disorder are suggested

Competing Interests

The authors declare that they have no competing interests

Authorsrsquo Contributions

Nooshin Ghodsian conceived the study and participatedin the experimental design data acquisition and analysisand interpretation of results and drafted the paper NooshinGhodsian Patimah Ismail Salma Ahmadloo Farzad Hei-dari Polin Haghvirdizadeh Sima Ataollahi Eshkoor and Ali

6 Journal of Diabetes Research

Etemad interpreted the results and critically reviewed thestudy for important intellectual content All authors approvedthe final version of the paper

Acknowledgments

The authors fully acknowledge the cooperation of the vol-unteers and all participants who generate their generoussupport They are also grateful to Mr Mohammad Akhlaghiwho edited this paper grammatically and in that regardimproved the paper significantly

References

[1] D S Timberlake D T OrsquoConnor and R J Parmer ldquoMoleculargenetics of essential hypertension recent results and emergingstrategiesrdquo Current Opinion in Nephrology and Hypertensionvol 10 no 1 pp 71ndash79 2001

[2] F Cambien and L Tiret ldquoGenetics of cardiovascular diseasesfrom single mutations to the whole genomerdquo Circulation vol116 no 15 pp 1714ndash1724 2007

[3] W Ji J N Foo B J OrsquoRoak et al ldquoRare independent mutationsin renal salt handling genes contribute to blood pressurevariationrdquo Nature Genetics vol 40 no 5 pp 592ndash599 2008

[4] D Conen P M Ridker S Mora J E Buring and R J GlynnldquoBlood pressure and risk of developing type 2 diabetes mellitustheWomenrsquos Health Studyrdquo European Heart Journal vol 28 no23 pp 2937ndash2943 2007

[5] C C Doak and L G Doak ldquoThe literacy challenge in diabeteseducation so that clients may understandrdquo Diabetes Care andEducation vol 14 no 2 pp 9ndash11 1993

[6] J R Banegas E Lopez-Garcıa A Graciani et al ldquoRelationshipbetween obesity hypertension and diabetes and health-relatedquality of life among the elderlyrdquo European Journal of Cardio-vascular Prevention amp Rehabilitation vol 14 no 3 pp 456ndash4622007

[7] D Levy A L DeStefano M G Larson et al ldquoEvidence for agene influencing blood pressure on chromosome 17 Genomescan linkage results for longitudinal blood pressure phenotypesin subjects from the Framingham heart studyrdquo Hypertensionvol 36 no 4 pp 477ndash483 2000

[8] Y Kokubo K Kamide N Inamoto et al ldquoIdentification of108 SNPs in TSC WNK1 and WNK4 and their associationwith hypertension in a Japanese general populationrdquo Journal ofHuman Genetics vol 49 no 9 pp 507ndash515 2004

[9] Y Osada R Miyauchi T Goda et al ldquoVariations in theWNK1gene modulates the effect of dietary intake of sodium andpotassium on blood pressure determinationrdquo Journal of HumanGenetics vol 54 no 8 pp 474ndash478 2009

[10] F H Wilson S Disse-Nicodeme K A Choate et al ldquoHumanhypertension caused by mutations in WNK kinasesrdquo Sciencevol 293 no 5532 pp 1107ndash1112 2001

[11] F Verıssimo and P Jordan ldquoWNK kinases a novel proteinkinase subfamily in multi-cellular organismsrdquo Oncogene vol20 no 39 pp 5562ndash5569 2001

[12] R P Lifton A G Gharavi and D S Geller ldquoMolecularmechanisms of human hypertensionrdquo Cell vol 104 no 4 pp545ndash556 2001

[13] Z-J Sun Y Li J-Y Lu et al ldquoAssociation of Ala589Serpolymorphism of WNK4 gene with essential hypertension in

a high-risk Chinese populationrdquo The Journal of PhysiologicalSciences vol 59 no 2 pp 81ndash86 2009

[14] T Pastinen and T J Hudson ldquoCis-acting regulatory variationin the human genomerdquo Science vol 306 no 5696 pp 647ndash6502004

[15] A Visel E M Rubin and L A Pennacchio ldquoGenomic viewsof distant-acting enhancersrdquoNature vol 461 no 7261 pp 199ndash205 2009

[16] S Kumar K Tamura I B Jakobsen and M Nei ldquoMEGA2molecular evolutionary genetics analysis softwarerdquo Bioinfor-matics vol 17 no 12 pp 1244ndash1245 2001

[17] K Kamide S Takiuchi C Tanaka et al ldquoThree novel mis-sense mutations of WNK4 a kinase mutated in inheritedhypertension in Japanese hypertensives implication of clinicalphenotypesrdquoAmerican Journal ofHypertension vol 17 no 5 pp446ndash449 2004

[18] F F Cao H Han F Wang et al ldquoStudy on the associationbetween genetic polymorphism on WNK4 genes and essentialhypertension among Kazakhs ethnic population in XinjiangrdquoZhonghua Liuxingbingxue Zazhi vol 31 no 4 pp 375ndash3782010

[19] M Lu X Wang F Wang et al ldquoWNK4 polymorphisms andessential hypertension in the Uyghur populationrdquo Clinical andExperimental Hypertension vol 31 no 2 pp 179ndash185 2009

[20] J Y LvThe research on the expression of WNK genes [PhD the-sis] China Medical University Beijing China 2003 (Chinese)

[21] P M Erlich J Cui I Chazaro et al ldquoGenetic variants ofWNK4 in whites and African Americans with hypertensionrdquoHypertension vol 41 no 6 pp 1191ndash1195 2003

[22] X-G Guo J Ding H Xu et al ldquoComprehensive assessmentof the association ofWNK4 polymorphisms with hypertensionevidence from a meta-analysisrdquo Scientific Reports vol 4 article6507 2014

[23] L L J Koppes J M Dekker H F J Hendriks L M Bouterand R J Heine ldquoModerate alcohol consumption lowers the riskof type 2 diabetes a meta-analysis of prospective observationalstudiesrdquo Diabetes Care vol 28 no 3 pp 719ndash725 2005

[24] S-S Yang K Yamauchi T Rai et al ldquoRegulation of apicallocalization of the thiazide-sensitive NaCl cotransporter byWNK4 in polarized epithelial cellsrdquo Biochemical and Biophysi-cal Research Communications vol 330 no 2 pp 410ndash414 2005

[25] K T Kahle F HWilson and R P Lifton ldquoRegulation of diverseion transport pathways by WNK4 kinase a novel molecularswitchrdquo Trends in Endocrinology amp Metabolism vol 16 no 3pp 98ndash103 2005

[26] A M Ring S X Cheng Q Leng et al ldquoWNK4 regulatesactivity of the epithelial Na+ channel in vitro and in vivordquoProceedings of the National Academy of Sciences of the UnitedStates of America vol 104 no 10 pp 4020ndash4024 2007

[27] P W Flatman ldquoCotransporters WNKs and hypertension anupdaterdquo Current Opinion in Nephrology and Hypertension vol17 no 2 pp 186ndash192 2008

[28] G Gamba ldquoRole ofWNK kinases in regulating tubular salt andpotassium transport and in the development of hypertensionrdquoAmerican Journal of Physiology-Renal Physiology vol 288 no 2pp F245ndashF252 2005

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

6 Journal of Diabetes Research

Etemad interpreted the results and critically reviewed thestudy for important intellectual content All authors approvedthe final version of the paper

Acknowledgments

The authors fully acknowledge the cooperation of the vol-unteers and all participants who generate their generoussupport They are also grateful to Mr Mohammad Akhlaghiwho edited this paper grammatically and in that regardimproved the paper significantly

References

[1] D S Timberlake D T OrsquoConnor and R J Parmer ldquoMoleculargenetics of essential hypertension recent results and emergingstrategiesrdquo Current Opinion in Nephrology and Hypertensionvol 10 no 1 pp 71ndash79 2001

[2] F Cambien and L Tiret ldquoGenetics of cardiovascular diseasesfrom single mutations to the whole genomerdquo Circulation vol116 no 15 pp 1714ndash1724 2007

[3] W Ji J N Foo B J OrsquoRoak et al ldquoRare independent mutationsin renal salt handling genes contribute to blood pressurevariationrdquo Nature Genetics vol 40 no 5 pp 592ndash599 2008

[4] D Conen P M Ridker S Mora J E Buring and R J GlynnldquoBlood pressure and risk of developing type 2 diabetes mellitustheWomenrsquos Health Studyrdquo European Heart Journal vol 28 no23 pp 2937ndash2943 2007

[5] C C Doak and L G Doak ldquoThe literacy challenge in diabeteseducation so that clients may understandrdquo Diabetes Care andEducation vol 14 no 2 pp 9ndash11 1993

[6] J R Banegas E Lopez-Garcıa A Graciani et al ldquoRelationshipbetween obesity hypertension and diabetes and health-relatedquality of life among the elderlyrdquo European Journal of Cardio-vascular Prevention amp Rehabilitation vol 14 no 3 pp 456ndash4622007

[7] D Levy A L DeStefano M G Larson et al ldquoEvidence for agene influencing blood pressure on chromosome 17 Genomescan linkage results for longitudinal blood pressure phenotypesin subjects from the Framingham heart studyrdquo Hypertensionvol 36 no 4 pp 477ndash483 2000

[8] Y Kokubo K Kamide N Inamoto et al ldquoIdentification of108 SNPs in TSC WNK1 and WNK4 and their associationwith hypertension in a Japanese general populationrdquo Journal ofHuman Genetics vol 49 no 9 pp 507ndash515 2004

[9] Y Osada R Miyauchi T Goda et al ldquoVariations in theWNK1gene modulates the effect of dietary intake of sodium andpotassium on blood pressure determinationrdquo Journal of HumanGenetics vol 54 no 8 pp 474ndash478 2009

[10] F H Wilson S Disse-Nicodeme K A Choate et al ldquoHumanhypertension caused by mutations in WNK kinasesrdquo Sciencevol 293 no 5532 pp 1107ndash1112 2001

[11] F Verıssimo and P Jordan ldquoWNK kinases a novel proteinkinase subfamily in multi-cellular organismsrdquo Oncogene vol20 no 39 pp 5562ndash5569 2001

[12] R P Lifton A G Gharavi and D S Geller ldquoMolecularmechanisms of human hypertensionrdquo Cell vol 104 no 4 pp545ndash556 2001

[13] Z-J Sun Y Li J-Y Lu et al ldquoAssociation of Ala589Serpolymorphism of WNK4 gene with essential hypertension in

a high-risk Chinese populationrdquo The Journal of PhysiologicalSciences vol 59 no 2 pp 81ndash86 2009

[14] T Pastinen and T J Hudson ldquoCis-acting regulatory variationin the human genomerdquo Science vol 306 no 5696 pp 647ndash6502004

[15] A Visel E M Rubin and L A Pennacchio ldquoGenomic viewsof distant-acting enhancersrdquoNature vol 461 no 7261 pp 199ndash205 2009

[16] S Kumar K Tamura I B Jakobsen and M Nei ldquoMEGA2molecular evolutionary genetics analysis softwarerdquo Bioinfor-matics vol 17 no 12 pp 1244ndash1245 2001

[17] K Kamide S Takiuchi C Tanaka et al ldquoThree novel mis-sense mutations of WNK4 a kinase mutated in inheritedhypertension in Japanese hypertensives implication of clinicalphenotypesrdquoAmerican Journal ofHypertension vol 17 no 5 pp446ndash449 2004

[18] F F Cao H Han F Wang et al ldquoStudy on the associationbetween genetic polymorphism on WNK4 genes and essentialhypertension among Kazakhs ethnic population in XinjiangrdquoZhonghua Liuxingbingxue Zazhi vol 31 no 4 pp 375ndash3782010

[19] M Lu X Wang F Wang et al ldquoWNK4 polymorphisms andessential hypertension in the Uyghur populationrdquo Clinical andExperimental Hypertension vol 31 no 2 pp 179ndash185 2009

[20] J Y LvThe research on the expression of WNK genes [PhD the-sis] China Medical University Beijing China 2003 (Chinese)

[21] P M Erlich J Cui I Chazaro et al ldquoGenetic variants ofWNK4 in whites and African Americans with hypertensionrdquoHypertension vol 41 no 6 pp 1191ndash1195 2003

[22] X-G Guo J Ding H Xu et al ldquoComprehensive assessmentof the association ofWNK4 polymorphisms with hypertensionevidence from a meta-analysisrdquo Scientific Reports vol 4 article6507 2014

[23] L L J Koppes J M Dekker H F J Hendriks L M Bouterand R J Heine ldquoModerate alcohol consumption lowers the riskof type 2 diabetes a meta-analysis of prospective observationalstudiesrdquo Diabetes Care vol 28 no 3 pp 719ndash725 2005

[24] S-S Yang K Yamauchi T Rai et al ldquoRegulation of apicallocalization of the thiazide-sensitive NaCl cotransporter byWNK4 in polarized epithelial cellsrdquo Biochemical and Biophysi-cal Research Communications vol 330 no 2 pp 410ndash414 2005

[25] K T Kahle F HWilson and R P Lifton ldquoRegulation of diverseion transport pathways by WNK4 kinase a novel molecularswitchrdquo Trends in Endocrinology amp Metabolism vol 16 no 3pp 98ndash103 2005

[26] A M Ring S X Cheng Q Leng et al ldquoWNK4 regulatesactivity of the epithelial Na+ channel in vitro and in vivordquoProceedings of the National Academy of Sciences of the UnitedStates of America vol 104 no 10 pp 4020ndash4024 2007

[27] P W Flatman ldquoCotransporters WNKs and hypertension anupdaterdquo Current Opinion in Nephrology and Hypertension vol17 no 2 pp 186ndash192 2008

[28] G Gamba ldquoRole ofWNK kinases in regulating tubular salt andpotassium transport and in the development of hypertensionrdquoAmerican Journal of Physiology-Renal Physiology vol 288 no 2pp F245ndashF252 2005

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom