research article endothelial nitric oxide synthase...

Research ArticleEndothelial Nitric Oxide Synthase Gene Polymorphisms and theRisk of Hypertension in an Indian Population

Priyanka Shankarishan1 Prasanta Kumar Borah1

Giasuddin Ahmed2 and Jagadish Mahanta1

1 Regional Medical Research Centre NE Region ICMR PO Box 105 Dibrugarh Assam 786001 India2Department of Biotechnology Gauhati University Guwahati Assam 781014 India

Correspondence should be addressed to Jagadish Mahanta jmahantahotmailcom

Received 28 February 2014 Revised 2 July 2014 Accepted 16 July 2014 Published 6 August 2014

Academic Editor Kazuhiko Kotani

Copyright copy 2014 Priyanka Shankarishan 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

Genetic variants of eNOS gene play a significant role in the pathogenesis of hypertension Many environmental factors have alsobeen implicated in the aetiology of hypertension We carried out an age-matched case-control study among adults Hypertensionwas defined according to JNC-VII criteria and eNOS gene polymorphisms were determined by PCR and PCR followed by PCR-RFLP eNOS intron 4 aa genotype (adjusted OR 681 95 CI 229ndash2025) and eNOS 894TT genotype (adjusted OR 784 95 CI257ndash2396) were associated with the risk of hypertension Tobacco users (either smokingchewing or both) with eNOS intron 4 aagenotype (OR 1400 95 CI 120ndash16337) eNOS 894GG genotype (OR 556 95 CI 372ndash831) and eNOS T-786C CC genotype(OR 900 95 CI 114ndash7104) were at an increased risk of hypertension Similarly a significant gene-environment interaction wasobserved between individuals consuming alcohol with eNOS intron 4 aa genotype (OR 1200 95CI 120ndash14373) and eNOS 894GGgenotype (OR 195 95 CI 135ndash281) The present study identified few susceptible genotypes of the eNOS gene with the risk ofhypertension Moreover the interactive effects between the environmental factors and the risk of hypertension were dependent onthe eNOS genotypes

1 Introduction

Nitric oxide (NO) produced from L-arginine by endothelialnitric oxide synthase (eNOS) plays a significant role in theregulation of vascular tone and in the control of bloodpressure [1] Reduction in basal NO release may predisposeto hypertension thrombosis vasospasm and atherosclerosis[2] and inhibition of eNOS elevates blood pressure in healthyhumans [3] Studies have shown that disruption of eNOSgene leads to hypertension in mice [4] Furthermore whole-body NO production in patients with essential hypertensionis diminished under basal conditions [5] Because of theseimportant evidences of NO and eNOS involvement in theblood pressure regulation the eNOS gene has therefore beenstudied as a putative candidate gene for hypertension Anumber of eNOS gene polymorphisms have been identified

so far Among these eNOS gene intron 4 ab polymorphismeNOS gene exon 7 Glu298Asp variant (rs 1799983) andeNOS gene T786C polymorphism (rs 2070744) have beenmost studied for an association with hypertension Althoughthe role of eNOS gene polymorphisms in the incidence ofhypertension seems to vary in different populations onlyfew studies on these polymorphisms have been conducted inIndian population [6 7]

Moreover the complex interplay between the environ-mental factors and genes for the risk of hypertension isstill not clear and many dietary and lifestyle factors havebeen implicated in the aetiology of hypertension In earlierstudies both tobacco use (either smokingchewing or both)and alcohol consumption were associated with an increasedrisk of hypertension [8ndash10] We in our previous study [11]have illustrated a dose-response relation between the number

Hindawi Publishing CorporationBioMed Research InternationalVolume 2014 Article ID 793040 11 pageshttpdxdoiorg1011552014793040

2 BioMed Research International

of cigarettes smoked per day (1205942 for trend = 2607 119875 lt00001) and the amount of alcohol consumption per day (1205942for trend = 2426 119875 lt 00001) and the risk of hypertensionThe same study also calculated the population attributablerisk (PAR) and were found to be 703 (95CI 630ndash775) fortobacco use 453 (95 CI 371ndash534) for tobacco chewing315 (95 CI 213ndash409) for smoking and 336 (95 CI229ndash444) for alcohol consumption

This prompted us to conduct an investigation to study theassociation between the polymorphisms of the endothelialnitric oxide synthase gene and hypertension in the teagarden worker community of northeastern India where theprevalence of hypertension is high [12]

2 Materials and Methods

21 Study Area and Study Subjects The study area and thestudy population were identical with that described previ-ously [11]

211 Study Area Dibrugarh the largest tea exporting districtin India and has the largest land area of tea cultivation amongall the districts of Assam In view of the importance ofDibrugarh district in the tea industry the tea gardens in theDibrugarh district were selected to conduct the present study

212 Study Subjects The study is a case-control study and700 subjects (350 cases and 350 age matched controls) fromthe tea garden worker community in the age group 20ndash65years after obtaining signed informed consent were enrolledfrom ten randomly selected tea gardens of Dibrugarh districtAssam India

Definition of Hypertension for the Present Study The hyper-tension status of the subjects was defined by the criteriaformulated by the US Seventh Joint National Commit-tee on Detection Evaluation and Treatment of Hyperten-sion (JNC-VII) that is SBP ge 140mmHg andor DBP ge90mmHg and those on antihypertensive medication [13]Initial identification of the cases was based on the hos-pital records maintained in the tea garden hospital andsubsequently rechecked before enrolment For this threeblood pressure readings by mercury sphygmomanome-ter were taken at an interval of ten minutes and themean of the three readings was taken for categorizing thesubjects

Exclusion Criteria Adopted for the Present Study The exclu-sion criteria adopted during recruitment of the study subjectsincluded subjectrsquos previous history of cardiovascular diseasewomen receiving oral contraceptives or hormone replace-ment therapy pregnant women or lactating mothers andinability to give informed consent or comply with the studyprotocol and subjects above or below the desired age

Ethical Issue Study was approved by the Institutional EthicalCommittee (IEC) of Regional Medical Research Centre NERegion Indian Council of Medical Research Dibrugarh

India Selected subjects were briefed about the study protocoland informed and signed consent was obtained from each ofthe selected study subjects

Collection of Epidemiological Data A pretested peer-reviewedproforma containing questions pertaining to the demo-graphic profile socioeconomic status and educational statuswas used to interview the study subjects The anthropomet-ric measurements included height and weight Height wasmeasured to the nearest centimeter using a stadiometer andweight to the nearest kilogram using a weighing machineBody weight was measured in light clothing without shoesBody mass index (BMI) was calculated using the formulaweight in kgheight in meter squared and was categorizedas underweight (lt18 kgm2) normal (180ndash249 kgm2) over-weight (250ndash299 kgm2) or obese (ge30 kgm2) according tothe 2000 WHO criteria [14]

The study population had the habit of taking extra salt (asa side dish) which means the raw salt taken as such as a dishin addition to the salt that is used in the preparation of themeal by the side of their plate during lunch and dinner andeating it during the course of their meal either alone or witha chilli

In the study population tobacco is consumed mainly intwo forms smoked tobacco in the form of bidi cigaretteand smokeless tobacco chewing in the form of khaini (atobacco quid containing a mixture of tobacco and lime withor without betel nut) A tobacco user was defined as one whohas consumed tobacco in any form (cigarette bidi or chewedtobacco) in the past one month A tobacco chewer wasdefined as one who consumed tobacco orally and includedkhaini alone or along with betel quidwith or without limepan and pan-masala or gutka (containing chewable tobacco)A smoker was defined as one who has consumed smokedtobacco in the form of bidi cigarette or any other formsof tobacco smoking in the past one month A nonuser oftobacco was defined as one who has never consumed tobaccoin any form (cigarette bidi or chewed tobacco) Those whodid not consume tobacco in any form in the past one yearwere defined as past users of tobacco

Local brew (locally known as haria) prepared from riceand other herbal ingredients was the most common alcoholdrink We categorized the habit of alcohol consumption intothe following categoriesmdashnonuser past user occasional userand moderate user An alcohol user was defined as onewho consumed alcohol in any form in the past one monthA nonuser of alcohol was defined as one who has neverconsumed alcohol in any form Those who did not consumealcohol in any form in the past one year were defined as pastusers of alcohol An occasional user of alcohol was definedas one who took 1ndash5 drinks a week whereas a moderateuser of alcohol was defined as one who consumed ge2 drinksdaily

Biochemical Investigations The biochemical indicators in-cluded fasting blood sugar (FBS) and renal function tests(blood urea and serum creatinine) and lipid profile testsThe 12-hour fasting blood samples for the glucose assay werecollected in the morning into tubes containing NaF and

BioMed Research International 3

plasma glucose concentrations were determined using theglucose oxidase and peroxidase method [15] Blood for therenal function tests was collected in sterile empty vials Bloodurea was estimated by enzymatic method [16] while serumcreatinine was estimated by alkaline Jaffersquos Picrate method[17] whereas total cholesterol HDL cholesterol and serumtriglycerides were analyzed enzymatically with commerciallyavailable reagents For participants LDL cholesterol levelswere calculated from the Friedewald equation (LDL choles-terol = total cholesterol minusHDL cholesterol minus triglycerides5)All these laboratory parameters were analyzed by usingcommercially available Randox reagent kits and with helpof a fully automated biochemical analyzer (Model-FullyBiochemical System International Italy)

Measurement of Blood Pressure The intent and purpose ofthe blood pressure measurement was explained to the studysubjects in a reassuring manner and every effort was madeto put the subject at ease A standard mercury sphygmo-manometer was used to measure the blood pressure withthe subject in the sitting posture The blood pressure wasrecorded in the right arm using a cuff of appropriate sizewith the instrument at the level of the subjectrsquos heart Thefirst and fifth Korotkoff sounds were taken as the systolicblood pressure and diastolic blood pressure respectivelyThree readings were taken at an interval of 10 minutes andthe average of the three readings was taken to determine thehypertension status of the subject The hypertension status ofthe subjects was defined by the criteria formulated by the USSeventh Joint National Committee on Detection Evaluationand Treatment of Hypertension (JNC-VII) that is SBP ge140mmHg andor DBP ge 90 and those on antihypertensivemedication [13]

22 Genetic Analysis

221 eNOS Gene Intron 4 ab Polymorphism The eNOS geneintron 4 ab polymorphism was determined bypolymerasechain reactionusing forward primer 51015840 AGG CCC TAT GGTAGTGCCTTT31015840 and reverse primer 51015840 TCTCTTAGTGCTGTG GTC AC 31015840 to amplify the ldquoardquo and ldquobrdquo alleles whichwill result in the amplification of 393 bp and 420 bp productsrespectively Polymerase chain reaction (PCR) consisted of05mM of each primer (Sigma) 10mM Tris-HCL pH 90(Bangalore Genei) 02mM dNTPs (Bangalore genei) 1 U ofTaq DNA polymerase (Bangalore Genei) and 50ndash100 ng ofgenomic DNA following standardized protocol with slightmodifications [18] The PCR reactions are amplified usinga Thermal Cycler (Gene Amp PCR Systems 9700 Version308 Applied Biosystems) at the following conditions initialdenaturation at 940∘C for 5 minutes 32 cycles of denatura-tion at 940∘C for 1 minute annealing temperature at 570∘Cfor 1 minute and extension at 720∘C for 2 minutes followedby final extension at 720∘C for 7 minutes Amplificationof PCR products is confirmed by electrophoresis on 35agarose gel The eNOS intron 4 ab polymorphism results inthree genotypes namely aa homozygous (aa) bb homozy-gous (bb) and ab heterozygous (ab)

222 eNOS Gene Exon 7 Glu298Asp Variant (rs 1799983)To genotype the G894T polymorphism in the exon 7 weperformed a polymerase chain reaction (PCR) amplificationof 206 bp fragment encompassing the variant followed byrestriction fragment length polymorphism (RFLP) using therestriction endonucleaseMbo1 (Promega) Polymerase chainreaction consisted of 05mM of each primer (Sigma) 10mMTris-HCL pH 90 (Bangalore Genei) 02mM of dNTPs(Bangalore Genei) 24U of Taq DNA polymerase (BangaloreGenei) and 50ndash100 ng of genomic DNA The PCR primerswere 51015840 CAT GAGGCT CAG CCC CAGAAC 31015840 (sense) and51015840 AGT CAA TCC CTT TGG TGC TCA C 31015840 (antisense)The PCR amplification conditions consisted of 94∘C for 5minutes followed by 40 cycles of 94∘C for 30 seconds 66∘Cfor 30 seconds 72∘C for 30 seconds and then 72∘C for 8minutes The PCR amplicons were then incubated at 37∘Cfor 3 hours The 206 bp amplicon containing a thymine atnucleotide position 894 (corresponding to an aspartic acid atamino acid position 298) was cleaved into two fragments of119 bp and 87 bp in length by Mbo1 digestion but not for aguanine in this positionThe restriction digest products wereanalyzed by electrophoresis on a 25 agarose gel [19]

223 eNOS Gene T-786C Polymorphism (rs 2070744) Thepresence of the T rarr C conversion at nucleotide position 786in the 51015840-flanking region of the eNOS gene was determinedby PCR amplification with the primers 51015840-ATG CTC CCACCA GGG CAT CA-31015840 (sense) and 51015840-GTC CTT GAG TCTGAC ATT AGG G-31015840 (antisense) followed by restrictionfragment length polymorphism (RFLP) using the restrictionendonucleaseNg0AIV (Promega) Polymerase chain reactionconsisted of 05mM of each primer (Sigma) 10mM Tris-HCL pH90 (BangaloreGenei) 02mMof dNTPs (BangaloreGenei) 24U of Taq DNA polymerase (Bangalore Genei)and 50ndash100 ng of genomic DNA The PCR amplificationconditions consisted of 94∘C for 6 minutes followed by 40cycles of 94∘C for 30 seconds 65∘C for 30 seconds 72∘Cfor 1 minute and then 72∘C for 8 minutes The 236 bp PCRfragments were digested withNg0AIV restriction enzyme for4 hours at 37∘C The T-allele has no Ng0AIV cleavage sitewhereas the PCR product is cleaved into two fragments of203 bp and 33 bp in the presence of the C786 allele [20]

23 Statistical Analysis The software Statistical Package forSocial Sciences (SPSS) version 130 and Epi-Info 2002 (CDCAtlanta USA) are used in the analysis of the data generatedA 119875 value (two-sided) le 005 was considered as statisticallysignificant Results are presented as number and percentageormean plusmn SD Chi-square test was used to test the associationbetween categorized variables and independent 119905-test andANOVAwas used to comparemeans of continuous variablesThe crude (unadjusted) relationship between the exposurevariables and the risk of hypertension was examined inunivariate logistic regression analyses Multivariate logisticregression analysis was done to evaluate the simultaneouseffects of various exposure variables with adjustment for thepotential confounding effects of other factors The frequen-cies for all the possible haplotypes were calculated with the


cubic exact solutions for the estimation of pairwise haplotypefrequencies (httpwwwoegeorgsoftwarecubex) [21] Thisprogramme estimates the haplotype frequencies Lewontinrsquosstandardized disequilibrium coefficient (1198631015840) and squaredcorrelation coefficient (1199032) for pairwise linkage disequilib-rium between two loci

3 Results

The study was designed as an age-adjusted case-control studyand included a total of 700 study participants (350 cases and350 controls) in the age group 20ndash65 years of both sexesDistribution of sociodemographic and clinical characteristicsof the study population has been shown in Table 1

Genotype and allelic frequencies for the eNOS genepolymorphisms for eNOS gene intron 4 ab polymorphismand eNOS gene exon 7 Glu298Asp variant were in agreementwith the frequencies predicted by the Hardy-Weinberg equi-librium (1205942 = 112 df = 2 119875 gt 005 and 1205942 = 267 df = 2119875 gt 005 resp)

To test the association of the eNOS gene polymorphismswith hypertension genotypic odds ratios (ORs) were cal-culated (Table 2) In univariate logistic regression analysiseNOS intron 4 aa genotype was found to be associated withincreased risk of hypertension Inmultivariatemodel of logis-tic regression too the risk persisted even after adjustment forage sex extra salt intake smoking tobacco chewing andhabit of alcohol consumption (eNOS intron 4 aa genotype(OR 681 95CI 229ndash2025) and eNOS intron 4 ab genotype(OR 223 95 CI 155minus320))

In univariate logistic regression analysis eNOS 894TTgenotype was found to be a risk factor In multivariatemodel of logistic regression adjusted for age sex extrasalt intake smoking tobacco chewing and habit of alcoholconsumption the eNOS 894TT genotype (OR 784 95 CI257ndash2396) and eNOS 894GT genotype (OR 398 95 CI265ndash598) were at an increased risk of hypertension

Pairwise comparison of the three studied polymorphisms(eNOS intron 4ab eNOS exon 7 and eNOS T-786C) ofeNOS genedepicting LDmeasures is presented in Table 3Weobserved linkage disequilibrium between eNOS intron 4 andeNOS exon 7 pairs (1198631015840 = minus0144 1199032 = 0172 1205942 = 1204119875 lt 00001)

The associations of the possible haplotypes of the threepolymorphisms of the eNOS gene with the risk of hyperten-sion in the study population are presented in Table 4 Thehaplotypes aT aG and bT of the variants eNOS-4 and eNOS-7 aT of the variants eNOS intron 4 and eNOS T-786C andTT and TC of the variants eNOS exon 7 and eNOS T-786Cwere found to be significantly associated with the risk ofhypertension in the study population

We performed stratified analysis to study interaction oreffect modification according to eNOS gene polymorphism

The risk of hypertension was increased among tobaccousers (either smokingchewing or both) carrying eNOSintron 4 aa genotype (OR 1400 95 CI 120minus16337) incomparison with the subjects who had the habit of tobaccouse (either smokingchewing or both) belonging to eNOS

intron 4 ab genotype (OR 806 95 CI 441minus1473) andeNOS intron 4 bb genotype (OR 409 95 CI 274minus612)Similarly the risk of hypertension was higher among subjectswho had the habit of alcohol consumption carrying eNOSintron 4 aa genotype (OR 1200 95 CI 120ndash14373) incomparison with the subjects who had the habit of alcoholconsumption carrying eNOS intron 4 ab genotype (OR 15695 CI 091minus265) and eNOS intron 4 bb genotype (OR 20795 CI 141minus304) (Table 5)

Risk of hypertension was increased among subjects whohad the habit of tobacco use (either smokingchewing orboth) carrying eNOS 894GG genotype (OR 556 95 CI372minus831) in comparison with the subjects who had the habitof tobacco use (either smokingchewing or both) with eNOS894GT genotype (OR 386 95 CI 195minus767) and eNOS894TT genotype (OR 229 95 CI 027ndash1966) Similarlythe risk of hypertension was increased among subjects witheNOS 894GG genotype who consume alcohol (OR 195 95CI 135minus281) in comparison to those who consume alcoholwith eNOS 894GT genotype (OR 241 95CI 124minus469) andeNOS 894TTgenotype (OR 109 95 CI 013minus912) (Table 5)

The risk of hypertension was increased among subjectswho had the habit of tobacco use (either smokingchewing orboth) carrying eNOS T-786C CC genotype (OR 900 95 CI114ndash7104) in comparisonwith the subjects who had the habitof tobacco use (either smokingchewing or both) carryingeNOS T-786C TC genotype (OR 451 95 CI 269ndash756) andeNOS T-786C TT genotype (OR 584 95 CI 379minus898)(Table 5)

4 Discussion

We conducted an age-adjusted case-control study to explorethe association between the endothelial nitric oxide synthasegene polymorphisms and hypertension in the tea gardenpopulation of northeastern India in the age group 20ndash65 yearsof both sexes

In the present study the frequency of the a-allele and b-allele was found to be 020 and 080 respectively The ldquobrdquo-allele frequency observed in our study is similar to studiesconducted in Japanese and UK populations [22 23]

The eNOS intron 4 aa genotype in the present studyhas been associated with an increased risk of hypertensionin comparison with the eNOS intron 4 ab genotype andeNOS intron 4 bb genotype Although the eNOS intron 4 abpolymorphism is an intronic polymorphism it is reportedthat the eNOS intron 4 ab polymorphism modulate tran-scription influencing translation efficiency mRNA stabilityand enzyme levels [24] Further a meta-analysis of 35 geneticstudies also supported the association between eNOS intron4 ab polymorphism and hypertension [25] These findingshighlight the significance of the eNOS intron 4 ab polymor-phism in the development of hypertension

As reported from different parts of India (north Indian[6] and south Indian populations [26])the eNOS exon 7homozygous GG genotype (eNOS 894GG) (700) waspredominant followed by eNOS exon 7 heterozygous GT


Table 1 Distribution of sociodemographic and clinical characteristics of the study population

Variable Study subjects (119873 = 700)119875 value

Control (119899 = 350) Case (119899 = 350)Age (mean plusmn SD) 362 plusmn 123 364 plusmn 123 0806Systolic blood pressure (mmHg mean plusmn SD) 1163 plusmn 98 1536 plusmn 196 0000lowast

Diastolic blood pressure (mmHg mean plusmn SD) 747 plusmn 68 929 plusmn 99 0000lowast

SexMale 143 (409) 134 (383) 0536Female 207 (591) 216 (617)

BMIUnderweight 117 (334) 103 (294)

0005lowastNormal 228 (651) 224 (640)Overweight 4 (11) 15 (43)Obese 1 (03) 8 (23)

Alcohol intakeYes 155 (443) 213 (609) 0000lowast

Alcohol consumptionNonuser 195 (557) 137 (391)

0000lowastPast user 26 (74) 18 (51)1ndash5 drinks per week 75 (214) 103 (294)ge2 drinks daily 54 (154) 92 (263)

Habit of tobacco useTobacco user 126 (360) 262 (749) 0000lowast

Smoking habitYes 58 (166) 112 (320) 0000lowast

Frequency of smokingNonuser 292 (834) 238 (680)

0000lowastPast user 11 (31) 12 (34)Rare 11 (31) 20 (57)1ndash4 nosday 30 (86) 56 (160)5ndash10 nosday 4 (11) 14 (40)More than 10 nosday 2 (06) 10 (29)

Tobacco chewerYes 91 (260) 183 (523) 0000lowast

Blood glucose (mgdL mean plusmn SD) 964 plusmn 175 1049 plusmn 242 0000lowast

Blood urea (mgdL mean plusmn SD) 213 plusmn 65 237 plusmn 83 0000lowast

Serum creatinine (mgdL mean plusmn SD) 09 plusmn 05 09 plusmn 03 0000lowast

Serum sodium (mmolL mean plusmn SD) 1407 plusmn 75 1462 plusmn 85 0000lowast

Serum potassium (mmolL mean plusmn SD) 65 plusmn 21 55 plusmn 15 0000lowast

Serum cholesterol (mgdL mean plusmn SD) 1448 plusmn 162 1463 plusmn 389 0003lowast

Serum HDL cholesterol (mgdL mean plusmn SD) 419 plusmn 74 4038 plusmn 662 0000lowast

Serum triglycerides (mgdL mean plusmn SD) 1408 plusmn 389 1484 plusmn 367 0027lowast

Serum LDL (mgdL mean plusmn SD) 686 plusmn 166 739 plusmn 225 0059All values within parenthesis are percentagesSD standard deviationBMI body mass indexHDL high density lipoproteinLDL low density lipoproteinlowastStatistically significant (119875 value le 005)


Table 2 Endothelial nitric oxide synthase (eNOS) gene polymorphisms and the risk of hypertension in the study population

eNOS gene polymorphisms Control(119899 = 350)

Case(119899 = 350)

Crude OR(95 CI) 119875 value Adjusted

OR(95 CI) 119875 value

eNOS geneintron 4 abpolymorphism

bb 256 (732) 190 (543) 1000 mdash 1000 mdash

ab 89 (254) 142 (406) 215(155ndash297) 0002lowast 223

(155ndash320) 0000lowast

aa 5 (14) 18 (51) 485(177ndash1330) 0000lowast 681


b 601 (859) 522 (746) 1000 mdash mdash mdash

a 99 (141) 178 (254) 207(156ndash274) 0000lowast mdash mdash

(aa + ab)bb 229(167ndash315) 0000lowast 233


aa(ab + bb) 374(137ndash1019) 0010lowast 385


Homozygous for rare allele versus homozygous forcommon allele

485(177ndash1330) 0002lowast 681


Heterozygous for the common allele versus homozygousfor the common allele

215(155ndash297) 0000lowast 221


eNOS gene exon7 Glu298Asppolymorphism

GG 296 (846) 194 (554) 1000 mdash 1000 mdash

GT 50 (143) 133 (380) 406(280ndash589) 0000lowast 398


TT 4 (11) 23 (66) 877(299ndash2576) 0000lowast 784


G 642 (917) 521 (744) 1000 mdash mdash mdash

T 58 (83) 179 (256) 380(274ndash529) 0000lowast mdash mdash

(TT + GT)GG 441(308ndash631) 0000lowast 425


TT(GT + GG) 608(208ndash1778) 0001lowast 635



877(299ndash2576) 0000lowast 784


Heterozygous for the common alleleversus homozygous for the common allele

406(280ndash589) 0000lowast 388


eNOS geneT786Cpolymorphism

TT 214 (611) 200 (571) 1000 mdash 1000 mdash

TC 127 (363) 139 (398) 117(086ndash159) 0315 119

(085ndash166) 0324

CC 9 (26) 11 (31) 131(053ndash322) 0560 141

(052ndash381) 0497

T 555 (793) 539 (770) 1000 mdash mdash mdash

C 145 (207) 161 (230) 114(088ndash149) 0301 mdash mdash

(CC + TC)TT 118(087ndash160) 0282 121

(087ndash167) 0255

CC + (TC + TT) 123(050ndash301) 0651 125

(048ndash324) 0647


131(053ndash322) 0560 141

(052ndash381) 0497


117(086ndash159) 0315 119

(085ndash166) 0324

OR odds ratioCI confidence intervalAdjusted for age sex extra salt intake smoking tobacco chewing and habit of alcohol consumption and eNOS gene polymorphismslowastStatistically significant (119875 value le 005)


Table 3 Measures of linkage disequilibrium observed in a pairwise comparison of the two polymorphisms of the endothelial nitric oxidesynthase (eNOS) gene among the study population

Variant 1 Variant 2 1198631015840

1199032

1205942 Linkage disequilibrium

eNOS 4 eNOS 7 minus0144 00172 1204 In linkage disequilibriumeNOS 4 eNOS T786C 0125 00043 301 Not in linkage disequilibriumeNOS 7 eNOS T786C minus0309 00054 378 Not in linkage disequilibrium1198631015840 Lewontinrsquos standardized disequilibrium coefficient1199032 squared correlation coefficient for pairwise linkage disequilibrium between two loci1205942 Chi-square value

Table 4 Haplotype frequency distribution (case versus control) of the three polymorphisms of the endothelial nitric oxide synthase (eNOS)gene in the study population

Variants Haplotype Haplotype frequencyOR (95 CI) 119875 value

Case (119899 = 350) Control (119899 = 350)

eNOS 4 and eNOS 7

bG 05550 07912 033(023ndash046)

0000lowast

aT 00650 00112 608(197ndash2099)

0002lowast

aG 01850 01288 155(100ndash239)

0048lowast

bT 01950 00688 328(195ndash552)

0000lowast

eNOS 4 and eNOS T786C

bT 05775 06794 064(047ndash089)

0006lowast

bC 01725 01806 094(063ndash142)

0843

aT 01925 01106 189(121ndash296)

0004lowast

aC 00575 00294 217(096ndash502)

007


GT 05698 07268 050(036ndash069)

0000lowast

GC 01702 01932 086(057ndash128)

047

TT 02002 00632 373(219ndash638)

0000lowast

TC 00598 00168 366(138ndash1026)

0006lowast

OR odds ratioCI confidence intervallowastStatistically significant (119875 value le 005)

genotype (eNOS 894GT) (2614) and eNOS exon 7 homozy-gous TT genotype (eNOS 894TT) (386)

A low (386) frequency of the homozygous mutanteNOS 894TT genotype was observed The study revealed aldquoTrdquo allele frequency of 017 among the tea garden communitywhich is comparable to that observed among south Indian(013) [26] and north Indian populations (015) [6]

Present study demonstrated a significant associationbetween eNOS exon 7 894TT genotype and the risk ofhypertension These results indicate that eNOS gene exon 7Glu298Asp variant plays an important role in blood pressure

regulation and may be a risk factor of hypertension forthe tea garden community of Assam The production ofbasal nitric oxide is significantly decreased in hypertensivecases as compared to healthy controls [27] The eNOSgene exon 7 Glu298Asp variant causes a Glu298 changeto 298Asp which alters the structure of eNOS and affectsits activity by decreasing the production of nitric oxideand ultimately increasing blood pressure [28] Similarly asignificantly higher frequency of the T allele has been foundto be associated with hypertension [29] and higher bloodpressure levels [30] in Japanese subjects


Table 5 Stratified analysis to study the relation between endothelial nitric oxide synthase (eNOS) gene polymorphisms and the risk ofhypertension in the subgroups with selected habits in the study population

eNOS genepolymorphism Parameter Odds ratio

(95 CI) 119875 value

eNOS gene intron4 ab polymorphism

Tobacco use (eithersmokingchewingor both)

Subjects who have the habit of tobaccouse (either smokingchewing or both)

with eNOS4 aa genotype

1400(120ndash16337) 0035lowast


with eNOS4 ab genotype

806(441ndash1473) 0000lowast


with eNOS4 bb genotype409


Alcoholconsumption

Subjects who have the habit of alcoholconsumption with eNOS4 aa genotype

1200(120ndash14373) 0035lowast

Subjects who have the habit of alcoholconsumption with eNOS4 ab genotype

156(091ndash265) 0105

Subjects who have the habit of alcoholconsumption with eNOS4 bb genotype

207(141ndash304) 0000lowast

eNOS gene exon 7Glu298Asppolymorphism



with eNOS7 GG genotype



with eNOS7 GT genotype



with eNOS7 TT genotype229

(027ndash1966) 0451

Alcoholconsumption

Subjects who have the habit of alcoholconsumption with eNOS7 GG genotype


Subjects who have the habit of alcoholconsumption with eNOS7 GT genotype


Subjects who have the habit of alcoholconsumption with eNOS7 TT genotype

109(013ndash912) 0936

eNOS gene T786Cpolymorphism



with eNOS T786C TT genotype



with eNOS T786C TC genotype



with eNOS T786C CC genotype900


Alcoholconsumption

Subjects who have the habit of alcoholconsumption eNOS T786C TT genotype

207(047ndash306) 0547

Subjects who have the habit of alcoholconsumption with eNOS T786C TC

genotype

175(018ndash286) 0084

Subjects who have the habit of alcoholconsumption with eNOS T786C CC

genotype

292(041ndash2090) 0287

lowastStatistically significant (119875 value le 005)CI confidence interval


In the study no association was detected betweeneNOS gene T-786C polymorphism and hypertension as alsoreported elsewhere [31ndash33]

Individual gene polymorphisms may not be consistentand reliable risk markers for developing hypertension andhaplotypes can sometimes provide greater power than single-marker analyses for genetic disease associations Tanus-Santos and his team investigated the role eNOS haplotypes insusceptibility to cardiovascular diseases and reportedmarkedinterethnic differences in the distribution of eNOS genepolymorphisms haplotype frequency and the associationbetween the eNOS variants in Caucasians and African-Americans and in white and black Brazilians [34 35] Thepresent study considered the pairwise comparison of thethree polymorphisms of the eNOS gene (namely eNOSintron 4 ab eNOS exon 7 and eNOS T-786C) observed asignificant linkage disequilibrium between eNOS intron 4and eNOS exon 7 pairs

The haplotypes aT aG and bT of the variants eNOS-4and eNOS-7 aT of the variants eNOS intron 4 and eNOST-786C and TT and TC of the variants eNOS exon 7and eNOS T-786C were found to be significantly associatedwith the risk of hypertension in the study population Ourstudy does not reveal the mechanism for the association ofhypertension with specific eNOS haplotypes but is supportedby other studies that reported a major influence of the eNOShaplotypes on disease risks [36ndash39] The study conductedby Sandrim and his group (2006) [40] reported a protectiveeffect for the ldquoC-Glu-brdquo haplotype against hypertension andthat the ldquoC-Asp-brdquo haplotype increases the susceptibility tohypertension Moreover their results suggested that eNOShaplotypes were not associated with resistance to antihyper-tensive therapy Another study [41] from the same groupsuggested a protective role for eNOShaplotype ldquoC-Glu-brdquoagainst the development of hypertension and that the hap-lotype ldquoC-Asp-brdquo increases the susceptibility to hypertensionin patients with or without type 2 diabetes mellitus Ourfindings suggest a contribution of eNOS haplotypes to thedevelopment of hypertension that may be obscured whenspecific eNOS genotypes alone are considered

The complex interplay between genes and environmen-tal factors affecting blood pressure regulation is not wellunderstood It is the coexistence of adverse environmentalfactors on the background of genetic susceptibility thatdetermines the initiation and progression of hypertensionBecause these exposures are modifiable their interactionwith genetic susceptibility to hypertension is of substantialpublic health importance

The study also documented a significant gene-environ-ment interaction between eNOS intron 4 aa genotype and thehabit of alcohol consumption for the risk of hypertension inthe study population Mechanisms underlying the relation-ship between alcohol and blood pressure remain ambiguousthough several mechanisms have been proposed [42 43]Some suggested mechanisms include stimulation of the sym-pathetic nervous system inhibition of nitric oxide depletionof ions and increased intracellular calcium especially invascular smooth muscle

An interesting surprising finding of the present study wasa significant gene-environment interaction between eNOSexon 7 894GG genotype and behavioral risk factors liketobacco chewing and alcohol consumption for the risk ofhypertension The molecular effect of the eNOS exon 7Glu298Asp polymorphism on eNOS enzyme function is stillnot clear Lacolley et al [44] reported 894G allele to beassociated with an increased risk of hypertension in Cau-casians In the present study we observed a significant linkagedisequilibrium between eNOS intron 4 and eNOS 7 pair andfound the haplotype aG of the variants eNOS-4 and eNOS-7 to be significantly associated with the risk of hypertensionThis may be a possible explanation However further studiesto assess to gene-environment relationship between eNOS894GG genotype and the habit of alcohol consumption in thepathogenesis of hypertension are warranted

Some of the limitations faced during the study shouldalso be considered During assessment of demographicvariables we adopted a recall method that may introducesome bias in estimations of demographic variables A smallproportion (lt20) dropout due to nonavailability of consentfor blood sample collection occurred in the study

5 Conclusion

The present study detected the association of endothelialnitric oxide synthase gene polymorphisms with hypertensionand identified few susceptible genotypes of the endothelialnitric oxide synthase gene with the risk of hypertensionThe present results suggested that eNOS gene variants andtheir interactions with some environmental risk factors playan important role in the pathophysiology of hypertensionThe understanding of such mechanisms may help betterappreciate the molecular basis of hypertension

Conflict of Interests

The authors declare that they have no conflict of interestsregarding the publication of this paper

Acknowledgments

Financial support in the form of ICMR-SRF Fellowship wasobtained from the Indian Council of Medical ResearchAnsari Nagar New Delhi India

References

[1] P Vallance J Collier and S Moncada ldquoEffects of endothelium-derived nitric oxide on peripheral arteriolar tone in manrdquo TheLancet vol 2 no 8670 pp 997ndash1000 1989

[2] B S Oemar M R Tschudi N Godoy V Brovkovich TMalinski and T F Luscher ldquoReduced endothelial nitric oxidesynthase expression and production in human atherosclerosisrdquoCirculation vol 97 no 25 pp 2494ndash2498 1998

[3] W G Haynes J P Noon B R Walker and D J WebbldquoInhibition of nitric oxide synthesis increases blood pressurein healthy humansrdquo Journal of Hypertension vol 11 no 12 pp1375ndash1380 1993


[4] P L Huang Z Huang H Mashimo et al ldquoHypertension inmice lacking the gene for endothelial nitric oxide synthaserdquoNature vol 377 no 6546 pp 239ndash242 1995

[5] P Forte M Copland L M Smith E Milne J Sutherlandand N Benjamin ldquoBasal nitric oxide synthesis in essentialhypertensionrdquoThe Lancet vol 349 no 9055 pp 837ndash842 1997

[6] K Srivastava U K Biswas R Narang J J Varghese andN Das ldquoPrevalence of eNOS Glu298Asp polymorphism inhealthy volunteers froma region of northern IndiardquoCommunityGenetics vol 8 no 3 pp 180ndash183 2005

[7] A Nejatizadeh R Kumar T Stobdan et al ldquoEndothelial nitricoxide synthase gene haplotypes and circulating nitric oxidelevels significantly associate with risk of essential hypertensionrdquoFree Radical Biology and Medicine vol 44 no 11 pp 1912ndash19182008

[8] G Siasos D Tousoulis C Vlachopoulos et al ldquoShort-term treatment with L-arginine prevents the smoking-inducedimpairment of endothelial function and vascular elastic prop-erties in young individualsrdquo International Journal of Cardiologyvol 126 no 3 pp 394ndash399 2008

[9] P D Arkwright L J Beilin I Rouse B K Armstrong and RVandongen ldquoEffects of alcohol use and other aspects of lifestyleon blood pressure levels and prevalence of hypertension in aworking populationrdquo Circulation vol 66 no 1 pp 60ndash66 1982

[10] R Yin H Li J Wu et al ldquoEffects of alcohol consumptionand other lifestyle behaviors on blood pressure for the middle-aged and elderly in the Guangxi Hei Yi Zhuang and Hanpopulationsrdquo Alcohol vol 41 no 8 pp 541ndash550 2007

[11] P Shankarishan P K Borah P K Mohapatra G Ahmedand J Mahanta ldquoPopulation attributable risk estimates for riskfactors associated with hypertension in an Indian populationrdquoEuropean Journal of Preventive Cardiology vol 20 no 6 pp963ndash971 2012

[12] N C Hazarika D Biswas K Narain H C Kalita and JMahanta ldquoHypertension and its risk factors in tea gardenworkers of Assamrdquo National Medical Journal of India vol 15no 2 pp 63ndash68 2002

[13] A V Chobanian G L Bakris H R Black et al ldquoThe seventhreport of the joint national committee on prevention detectionevaluation and treatment of high blood pressure the JNC 7reportrdquo The Journal of the American Medical Association vol289 no 19 pp 2560ndash2572 2003

[14] WHOThe Problem of Overweight and Obesity 2000 PreventingandManaging the Global Epidemic vol 537 of Report Series 894Wt World Health Organization Geneva Switzerland 2000

[15] P Trinder ldquoDetermination of glucose in blood using glucoseoxidase with an alternative oxygen acceptotrdquo Annals of ClinicalBiochemistry vol 6 no 1 pp 24ndash27 1969

[16] J K Fawcett and J E Scott ldquoA rapid and precise method for thedetermination of ureardquo Journal of Clinical Pathology vol 13 pp156ndash159 1960

[17] J A Owen B Iggo F J SCANDRETT and C P Scandrett ldquoThedetermination of creatinine in plasma or serum and in urinea critical examinationrdquo Biochemical Journal vol 58 no 3 pp426ndash437 1954

[18] X LWang A S Sim R F Badenhop RMichaelMccredie andD E LWilcken ldquoA smoking-dependent risk of coronary arterydisease associatedwith a polymorphismof the endothelial nitricoxide synthase generdquo Nature Medicine vol 2 no 1 pp 41ndash451996

[19] Q Zhao S-Y Su S-F Chen B Li and D-F Gu ldquoAssociationstudy of the endothelial nitric oxide synthase gene polymor-phisms with essential hypertension in Northern Han ChineserdquoChinese Medical Journal vol 119 no 13 pp 1065ndash1071 2006

[20] M E Hyndman H G Parsons S Verma et al ldquoThe T-786rarrCmutation in endothelial nitric oxide synthase is associated withhypertensionrdquo Hypertension vol 39 no 4 pp 919ndash922 2002

[21] T R Gaunt S Rodrıguez and I N M Day ldquoCubic exactsolutions for the estimation of pairwise haplotype frequenciesImplications for linkage disequilibrium analyses and a web toolldquoCubeXrdquordquo BMC Bioinformatics vol 8 article 428 2007

[22] J Hwang C Tsai H Yeh et al ldquoThe 27-bp tandem repeatpolymorphism in intron 4 of the endothelial nitric oxidesynthase gene is not associated with coronary artery disease ina hospital-based Taiwanese populationrdquo Cardiology vol 97 no2 pp 67ndash72 2002

[23] F G R Fowkes A J Lee C M Hau A Cooke J M Connorand G D O Lowe ldquoMethylene tetrahydrofolate reductase(MTHFR) and nitric oxide synthase (ecNOS) genes and risksof peripheral arterial disease and coronary heart disease edin-burgh artery studyrdquo Atherosclerosis vol 150 no 1 pp 179ndash1852000

[24] T Tsukada K Yokoyama T Arai et al ldquoEvidence of associationof the ecNOS gene polymorphism with plasma NO metabolitelevels in humansrdquo Biochemical and Biophysical Research Com-munications vol 245 no 1 pp 190ndash193 1998

[25] E Zintzaras G Kitsios and I Stefanidis ldquoEndothelial NO syn-thase gene polymorphisms and hypertension a meta-analysisrdquoHypertension vol 48 no 4 pp 700ndash710 2006

[26] N S Nishevitha T Angeline and N Jeyaraj ldquoEndothelial nitricoxide synthase (eNOS) Glu298rarrAsp polymorphism (G894T)among South Indiansrdquo Indian Journal of Medical Research vol129 no 1 pp 68ndash71 2009

[27] J P Cooke and V J Dzau ldquoNitric oxide synthase role in thegenesis of vascular diseaserdquo Annual Review of Medicine vol 48pp 489ndash509 1997

[28] M Yoshimura H Yasue M Nakayama et al ldquoA missenseGlu298Asp variant in the endothelial nitric oxide synthase geneis associated with coronary spasm in the Japaneserdquo HumanGenetics vol 103 no 1 pp 65ndash69 1998

[29] Y Miyamoto Y Saito N Kajiyama et al ldquoEndothelial nitricoxide synthase gene is positively associated with essentialhypertensionrdquo Hypertension vol 32 no 1 pp 3ndash8 1998

[30] M Shoji S Tsutaya R Saito H Takamatu and M YasujimaldquoPositive association of endothelial nitric oxide synthase genepolymorphism with hypertension in northern Japanrdquo LifeSciences vol 66 no 26 pp 2557ndash2562 2000

[31] N Kajiyama Y Saito Y Miyamoto et al ldquoLack of associationbetween T-786 rarrC mutation in the 51015840-flanking region of theendothelial nitric oxide synthase gene and essential hyperten-sionrdquo Hypertension Research vol 23 no 6 pp 561ndash565 2000

[32] Y Tsujita S Baba R Yamauchi et al ldquoAssociation analysesbetween genetic polymorphisms of endothelial nitric oxidesynthase gene and hypertension in Japanese the Suita StudyrdquoJournal of Hypertension vol 19 no 11 pp 1941ndash1948 2001

[33] R Li D Lyn R Lapu-Bula et al ldquoRelation of endothelial nitricoxide synthase gene to plasma nitric oxide level endothelialfunction and blood pressure in African AmericansrdquoTheAmer-ican Journal of Hypertension vol 17 no 7 pp 560ndash567 2004

[34] J E Tanus -Santos M Desai and D A Flockhart ldquoEffects ofethnicity on the distribution of clinically relevant endothelial


nitric oxide variantsrdquo Pharmacogenetics vol 11 no 8 pp 719ndash725 2001

[35] A SMarroni I FMetzger D C Souza-Costa et al ldquoConsistentinterethnic differences in the distribution of clinically rele-vant endothelial nitric oxide synthase genetic polymorphismsrdquoNitric Oxide vol 12 no 3 pp 177ndash182 2005

[36] I F Metzger D C Souza-Costa A S Marroni et al ldquoEndothe-lial nitric oxide synthase gene haplotypes associated withcirculating concentrations of nitric oxide products in healthymenrdquo Pharmacogenet Genomics vol 15 no 8 pp 565ndash5702005

[37] I F Metzger J T C Sertorio and J E Tanus-Santos ldquoMod-ulation of nitric oxide formation by endothelial nitric oxidesynthase gene haplotypesrdquo Free Radical Biology and Medicinevol 43 no 6 pp 987ndash992 2007

[38] I F Metzger M H Ishizawa F Rios-Santos W A Carvalhoand J E Tanus-Santos ldquoEndothelial nitric oxide synthase genehaplotypes affect nitrite levels in black subjectsrdquo Pharmacoge-nomics Journal vol 11 no 6 pp 393ndash399 2011

[39] V C Sandrim RW C de Syllos H R K Lisboa G S Tres andJ E Tanus-Santos ldquoInfluence of eNOShaplotypes on the plasmanitric oxide products concentrations in hypertensive and type 2diabetes mellitus patientsrdquo Nitric Oxide vol 16 no 3 pp 348ndash355 2007

[40] V C Sandrim E B Coelho F Nobre G M Arado V LLanchote and J E Tanus-Santos ldquoSusceptible and protectiveeNOS haplotypes in hypertensive black and white subjectsrdquoAtherosclerosis vol 186 no 2 pp 428ndash432 2006

[41] V C Sandrim J C Yugar-Toledo Z Desta D A FlockhartH Moreno Jr and J E Tanus-Santos ldquoEndothelial nitric oxidesynthase haplotypes are related to blood pressure elevation butnot to resistance to antihypertensive drug therapyrdquo Journal ofHypertension vol 24 no 12 pp 2393ndash2397 2006

[42] D Randin P Vollenweider L Tappy E Jequier P Nicod andU Scherrer ldquoSuppression of alcohol-induced hypertension bydexamethasonerdquoTheNew England Journal of Medicine vol 332no 26 pp 1733ndash1737 1995

[43] Y Yamada Y Noborisaka M Ishizaki I Tsuritani R Hondaand S Yamada ldquoAlcohol consumption homeostasis modelassessment indices and blood pressure in middle-aged healthymenrdquo Journal of Human Hypertension vol 18 no 5 pp 343ndash350 2004

[44] P Lacolley S Gautier O Poirier B Pannier F Cambien andA Benetos ldquoNitric oxide synthase gene polymorphisms bloodpressure and aortic stiffness in normotensive and hypertensivesubjectsrdquo Journal of Hypertension vol 16 no 1 pp 31ndash35 1998

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of


Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology


Molecular Biology International

GenomicsInternational Journal of


The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014


BioinformaticsAdvances in

Marine BiologyJournal of



Signal TransductionJournal of


BioMed Research International

Evolutionary BiologyInternational Journal of



Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014


Genetics Research International


Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational



Enzyme Research



Microbiology


of cigarettes smoked per day (1205942 for trend = 2607 119875 lt00001) and the amount of alcohol consumption per day (1205942for trend = 2426 119875 lt 00001) and the risk of hypertensionThe same study also calculated the population attributablerisk (PAR) and were found to be 703 (95CI 630ndash775) fortobacco use 453 (95 CI 371ndash534) for tobacco chewing315 (95 CI 213ndash409) for smoking and 336 (95 CI229ndash444) for alcohol consumption

This prompted us to conduct an investigation to study theassociation between the polymorphisms of the endothelialnitric oxide synthase gene and hypertension in the teagarden worker community of northeastern India where theprevalence of hypertension is high [12]

2 Materials and Methods

21 Study Area and Study Subjects The study area and thestudy population were identical with that described previ-ously [11]

211 Study Area Dibrugarh the largest tea exporting districtin India and has the largest land area of tea cultivation amongall the districts of Assam In view of the importance ofDibrugarh district in the tea industry the tea gardens in theDibrugarh district were selected to conduct the present study

212 Study Subjects The study is a case-control study and700 subjects (350 cases and 350 age matched controls) fromthe tea garden worker community in the age group 20ndash65years after obtaining signed informed consent were enrolledfrom ten randomly selected tea gardens of Dibrugarh districtAssam India

Definition of Hypertension for the Present Study The hyper-tension status of the subjects was defined by the criteriaformulated by the US Seventh Joint National Commit-tee on Detection Evaluation and Treatment of Hyperten-sion (JNC-VII) that is SBP ge 140mmHg andor DBP ge90mmHg and those on antihypertensive medication [13]Initial identification of the cases was based on the hos-pital records maintained in the tea garden hospital andsubsequently rechecked before enrolment For this threeblood pressure readings by mercury sphygmomanome-ter were taken at an interval of ten minutes and themean of the three readings was taken for categorizing thesubjects

Exclusion Criteria Adopted for the Present Study The exclu-sion criteria adopted during recruitment of the study subjectsincluded subjectrsquos previous history of cardiovascular diseasewomen receiving oral contraceptives or hormone replace-ment therapy pregnant women or lactating mothers andinability to give informed consent or comply with the studyprotocol and subjects above or below the desired age

Ethical Issue Study was approved by the Institutional EthicalCommittee (IEC) of Regional Medical Research Centre NERegion Indian Council of Medical Research Dibrugarh

India Selected subjects were briefed about the study protocoland informed and signed consent was obtained from each ofthe selected study subjects

Collection of Epidemiological Data A pretested peer-reviewedproforma containing questions pertaining to the demo-graphic profile socioeconomic status and educational statuswas used to interview the study subjects The anthropomet-ric measurements included height and weight Height wasmeasured to the nearest centimeter using a stadiometer andweight to the nearest kilogram using a weighing machineBody weight was measured in light clothing without shoesBody mass index (BMI) was calculated using the formulaweight in kgheight in meter squared and was categorizedas underweight (lt18 kgm2) normal (180ndash249 kgm2) over-weight (250ndash299 kgm2) or obese (ge30 kgm2) according tothe 2000 WHO criteria [14]

The study population had the habit of taking extra salt (asa side dish) which means the raw salt taken as such as a dishin addition to the salt that is used in the preparation of themeal by the side of their plate during lunch and dinner andeating it during the course of their meal either alone or witha chilli

In the study population tobacco is consumed mainly intwo forms smoked tobacco in the form of bidi cigaretteand smokeless tobacco chewing in the form of khaini (atobacco quid containing a mixture of tobacco and lime withor without betel nut) A tobacco user was defined as one whohas consumed tobacco in any form (cigarette bidi or chewedtobacco) in the past one month A tobacco chewer wasdefined as one who consumed tobacco orally and includedkhaini alone or along with betel quidwith or without limepan and pan-masala or gutka (containing chewable tobacco)A smoker was defined as one who has consumed smokedtobacco in the form of bidi cigarette or any other formsof tobacco smoking in the past one month A nonuser oftobacco was defined as one who has never consumed tobaccoin any form (cigarette bidi or chewed tobacco) Those whodid not consume tobacco in any form in the past one yearwere defined as past users of tobacco

Local brew (locally known as haria) prepared from riceand other herbal ingredients was the most common alcoholdrink We categorized the habit of alcohol consumption intothe following categoriesmdashnonuser past user occasional userand moderate user An alcohol user was defined as onewho consumed alcohol in any form in the past one monthA nonuser of alcohol was defined as one who has neverconsumed alcohol in any form Those who did not consumealcohol in any form in the past one year were defined as pastusers of alcohol An occasional user of alcohol was definedas one who took 1ndash5 drinks a week whereas a moderateuser of alcohol was defined as one who consumed ge2 drinksdaily

Biochemical Investigations The biochemical indicators in-cluded fasting blood sugar (FBS) and renal function tests(blood urea and serum creatinine) and lipid profile testsThe 12-hour fasting blood samples for the glucose assay werecollected in the morning into tubes containing NaF and




22 Genetic Analysis







3 Results












4 Discussion










SexMale 143 (409) 134 (383) 0536Female 207 (591) 216 (617)























Case(119899 = 350)




bb 256 (732) 190 (543) 1000 mdash 1000 mdash

ab 89 (254) 142 (406) 215(155ndash297) 0002lowast 223


aa 5 (14) 18 (51) 485(177ndash1330) 0000lowast 681









485(177ndash1330) 0002lowast 681



215(155ndash297) 0000lowast 221



GG 296 (846) 194 (554) 1000 mdash 1000 mdash

GT 50 (143) 133 (380) 406(280ndash589) 0000lowast 398


TT 4 (11) 23 (66) 877(299ndash2576) 0000lowast 784









877(299ndash2576) 0000lowast 784



406(280ndash589) 0000lowast 388



TT 214 (611) 200 (571) 1000 mdash 1000 mdash

TC 127 (363) 139 (398) 117(086ndash159) 0315 119

(085ndash166) 0324

CC 9 (26) 11 (31) 131(053ndash322) 0560 141

(052ndash381) 0497



(CC + TC)TT 118(087ndash160) 0282 121

(087ndash167) 0255

CC + (TC + TT) 123(050ndash301) 0651 125

(048ndash324) 0647


131(053ndash322) 0560 141

(052ndash381) 0497


117(086ndash159) 0315 119

(085ndash166) 0324





1199032





Case (119899 = 350) Control (119899 = 350)

eNOS 4 and eNOS 7

bG 05550 07912 033(023ndash046)

0000lowast

aT 00650 00112 608(197ndash2099)

0002lowast

aG 01850 01288 155(100ndash239)

0048lowast

bT 01950 00688 328(195ndash552)

0000lowast


bT 05775 06794 064(047ndash089)

0006lowast

bC 01725 01806 094(063ndash142)

0843

aT 01925 01106 189(121ndash296)

0004lowast

aC 00575 00294 217(096ndash502)

007


GT 05698 07268 050(036ndash069)

0000lowast

GC 01702 01932 086(057ndash128)

047

TT 02002 00632 373(219ndash638)

0000lowast

TC 00598 00168 366(138ndash1026)

0006lowast









(95 CI) 119875 value





1400(120ndash16337) 0035lowast



806(441ndash1473) 0000lowast




Alcoholconsumption


1200(120ndash14373) 0035lowast


156(091ndash265) 0105













(027ndash1966) 0451

Alcoholconsumption






109(013ndash912) 0936












Alcoholconsumption


207(047ndash306) 0547


genotype

175(018ndash286) 0084


genotype

292(041ndash2090) 0287










5 Conclusion




Acknowledgments


References























































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology




22 Genetic Analysis







3 Results












4 Discussion










SexMale 143 (409) 134 (383) 0536Female 207 (591) 216 (617)























Case(119899 = 350)




bb 256 (732) 190 (543) 1000 mdash 1000 mdash

ab 89 (254) 142 (406) 215(155ndash297) 0002lowast 223


aa 5 (14) 18 (51) 485(177ndash1330) 0000lowast 681









485(177ndash1330) 0002lowast 681



215(155ndash297) 0000lowast 221



GG 296 (846) 194 (554) 1000 mdash 1000 mdash

GT 50 (143) 133 (380) 406(280ndash589) 0000lowast 398


TT 4 (11) 23 (66) 877(299ndash2576) 0000lowast 784









877(299ndash2576) 0000lowast 784



406(280ndash589) 0000lowast 388



TT 214 (611) 200 (571) 1000 mdash 1000 mdash

TC 127 (363) 139 (398) 117(086ndash159) 0315 119

(085ndash166) 0324

CC 9 (26) 11 (31) 131(053ndash322) 0560 141

(052ndash381) 0497



(CC + TC)TT 118(087ndash160) 0282 121

(087ndash167) 0255

CC + (TC + TT) 123(050ndash301) 0651 125

(048ndash324) 0647


131(053ndash322) 0560 141

(052ndash381) 0497


117(086ndash159) 0315 119

(085ndash166) 0324





1199032





Case (119899 = 350) Control (119899 = 350)

eNOS 4 and eNOS 7

bG 05550 07912 033(023ndash046)

0000lowast

aT 00650 00112 608(197ndash2099)

0002lowast

aG 01850 01288 155(100ndash239)

0048lowast

bT 01950 00688 328(195ndash552)

0000lowast


bT 05775 06794 064(047ndash089)

0006lowast

bC 01725 01806 094(063ndash142)

0843

aT 01925 01106 189(121ndash296)

0004lowast

aC 00575 00294 217(096ndash502)

007


GT 05698 07268 050(036ndash069)

0000lowast

GC 01702 01932 086(057ndash128)

047

TT 02002 00632 373(219ndash638)

0000lowast

TC 00598 00168 366(138ndash1026)

0006lowast









(95 CI) 119875 value





1400(120ndash16337) 0035lowast



806(441ndash1473) 0000lowast




Alcoholconsumption


1200(120ndash14373) 0035lowast


156(091ndash265) 0105













(027ndash1966) 0451

Alcoholconsumption






109(013ndash912) 0936












Alcoholconsumption


207(047ndash306) 0547


genotype

175(018ndash286) 0084


genotype

292(041ndash2090) 0287










5 Conclusion




Acknowledgments


References























































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology



3 Results












4 Discussion










SexMale 143 (409) 134 (383) 0536Female 207 (591) 216 (617)























Case(119899 = 350)




bb 256 (732) 190 (543) 1000 mdash 1000 mdash

ab 89 (254) 142 (406) 215(155ndash297) 0002lowast 223


aa 5 (14) 18 (51) 485(177ndash1330) 0000lowast 681









485(177ndash1330) 0002lowast 681



215(155ndash297) 0000lowast 221



GG 296 (846) 194 (554) 1000 mdash 1000 mdash

GT 50 (143) 133 (380) 406(280ndash589) 0000lowast 398


TT 4 (11) 23 (66) 877(299ndash2576) 0000lowast 784









877(299ndash2576) 0000lowast 784



406(280ndash589) 0000lowast 388



TT 214 (611) 200 (571) 1000 mdash 1000 mdash

TC 127 (363) 139 (398) 117(086ndash159) 0315 119

(085ndash166) 0324

CC 9 (26) 11 (31) 131(053ndash322) 0560 141

(052ndash381) 0497



(CC + TC)TT 118(087ndash160) 0282 121

(087ndash167) 0255

CC + (TC + TT) 123(050ndash301) 0651 125

(048ndash324) 0647


131(053ndash322) 0560 141

(052ndash381) 0497


117(086ndash159) 0315 119

(085ndash166) 0324





1199032





Case (119899 = 350) Control (119899 = 350)

eNOS 4 and eNOS 7

bG 05550 07912 033(023ndash046)

0000lowast

aT 00650 00112 608(197ndash2099)

0002lowast

aG 01850 01288 155(100ndash239)

0048lowast

bT 01950 00688 328(195ndash552)

0000lowast


bT 05775 06794 064(047ndash089)

0006lowast

bC 01725 01806 094(063ndash142)

0843

aT 01925 01106 189(121ndash296)

0004lowast

aC 00575 00294 217(096ndash502)

007


GT 05698 07268 050(036ndash069)

0000lowast

GC 01702 01932 086(057ndash128)

047

TT 02002 00632 373(219ndash638)

0000lowast

TC 00598 00168 366(138ndash1026)

0006lowast









(95 CI) 119875 value





1400(120ndash16337) 0035lowast



806(441ndash1473) 0000lowast




Alcoholconsumption


1200(120ndash14373) 0035lowast


156(091ndash265) 0105













(027ndash1966) 0451

Alcoholconsumption






109(013ndash912) 0936












Alcoholconsumption


207(047ndash306) 0547


genotype

175(018ndash286) 0084


genotype

292(041ndash2090) 0287










5 Conclusion




Acknowledgments


References























































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology






SexMale 143 (409) 134 (383) 0536Female 207 (591) 216 (617)























Case(119899 = 350)




bb 256 (732) 190 (543) 1000 mdash 1000 mdash

ab 89 (254) 142 (406) 215(155ndash297) 0002lowast 223


aa 5 (14) 18 (51) 485(177ndash1330) 0000lowast 681









485(177ndash1330) 0002lowast 681



215(155ndash297) 0000lowast 221



GG 296 (846) 194 (554) 1000 mdash 1000 mdash

GT 50 (143) 133 (380) 406(280ndash589) 0000lowast 398


TT 4 (11) 23 (66) 877(299ndash2576) 0000lowast 784









877(299ndash2576) 0000lowast 784



406(280ndash589) 0000lowast 388



TT 214 (611) 200 (571) 1000 mdash 1000 mdash

TC 127 (363) 139 (398) 117(086ndash159) 0315 119

(085ndash166) 0324

CC 9 (26) 11 (31) 131(053ndash322) 0560 141

(052ndash381) 0497



(CC + TC)TT 118(087ndash160) 0282 121

(087ndash167) 0255

CC + (TC + TT) 123(050ndash301) 0651 125

(048ndash324) 0647


131(053ndash322) 0560 141

(052ndash381) 0497


117(086ndash159) 0315 119

(085ndash166) 0324





1199032





Case (119899 = 350) Control (119899 = 350)

eNOS 4 and eNOS 7

bG 05550 07912 033(023ndash046)

0000lowast

aT 00650 00112 608(197ndash2099)

0002lowast

aG 01850 01288 155(100ndash239)

0048lowast

bT 01950 00688 328(195ndash552)

0000lowast


bT 05775 06794 064(047ndash089)

0006lowast

bC 01725 01806 094(063ndash142)

0843

aT 01925 01106 189(121ndash296)

0004lowast

aC 00575 00294 217(096ndash502)

007


GT 05698 07268 050(036ndash069)

0000lowast

GC 01702 01932 086(057ndash128)

047

TT 02002 00632 373(219ndash638)

0000lowast

TC 00598 00168 366(138ndash1026)

0006lowast









(95 CI) 119875 value





1400(120ndash16337) 0035lowast



806(441ndash1473) 0000lowast




Alcoholconsumption


1200(120ndash14373) 0035lowast


156(091ndash265) 0105













(027ndash1966) 0451

Alcoholconsumption






109(013ndash912) 0936












Alcoholconsumption


207(047ndash306) 0547


genotype

175(018ndash286) 0084


genotype

292(041ndash2090) 0287










5 Conclusion




Acknowledgments


References























































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology




Case(119899 = 350)




bb 256 (732) 190 (543) 1000 mdash 1000 mdash

ab 89 (254) 142 (406) 215(155ndash297) 0002lowast 223


aa 5 (14) 18 (51) 485(177ndash1330) 0000lowast 681









485(177ndash1330) 0002lowast 681



215(155ndash297) 0000lowast 221



GG 296 (846) 194 (554) 1000 mdash 1000 mdash

GT 50 (143) 133 (380) 406(280ndash589) 0000lowast 398


TT 4 (11) 23 (66) 877(299ndash2576) 0000lowast 784









877(299ndash2576) 0000lowast 784



406(280ndash589) 0000lowast 388



TT 214 (611) 200 (571) 1000 mdash 1000 mdash

TC 127 (363) 139 (398) 117(086ndash159) 0315 119

(085ndash166) 0324

CC 9 (26) 11 (31) 131(053ndash322) 0560 141

(052ndash381) 0497



(CC + TC)TT 118(087ndash160) 0282 121

(087ndash167) 0255

CC + (TC + TT) 123(050ndash301) 0651 125

(048ndash324) 0647


131(053ndash322) 0560 141

(052ndash381) 0497


117(086ndash159) 0315 119

(085ndash166) 0324





1199032





Case (119899 = 350) Control (119899 = 350)

eNOS 4 and eNOS 7

bG 05550 07912 033(023ndash046)

0000lowast

aT 00650 00112 608(197ndash2099)

0002lowast

aG 01850 01288 155(100ndash239)

0048lowast

bT 01950 00688 328(195ndash552)

0000lowast


bT 05775 06794 064(047ndash089)

0006lowast

bC 01725 01806 094(063ndash142)

0843

aT 01925 01106 189(121ndash296)

0004lowast

aC 00575 00294 217(096ndash502)

007


GT 05698 07268 050(036ndash069)

0000lowast

GC 01702 01932 086(057ndash128)

047

TT 02002 00632 373(219ndash638)

0000lowast

TC 00598 00168 366(138ndash1026)

0006lowast









(95 CI) 119875 value





1400(120ndash16337) 0035lowast



806(441ndash1473) 0000lowast




Alcoholconsumption


1200(120ndash14373) 0035lowast


156(091ndash265) 0105













(027ndash1966) 0451

Alcoholconsumption






109(013ndash912) 0936












Alcoholconsumption


207(047ndash306) 0547


genotype

175(018ndash286) 0084


genotype

292(041ndash2090) 0287










5 Conclusion




Acknowledgments


References























































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology




1199032





Case (119899 = 350) Control (119899 = 350)

eNOS 4 and eNOS 7

bG 05550 07912 033(023ndash046)

0000lowast

aT 00650 00112 608(197ndash2099)

0002lowast

aG 01850 01288 155(100ndash239)

0048lowast

bT 01950 00688 328(195ndash552)

0000lowast


bT 05775 06794 064(047ndash089)

0006lowast

bC 01725 01806 094(063ndash142)

0843

aT 01925 01106 189(121ndash296)

0004lowast

aC 00575 00294 217(096ndash502)

007


GT 05698 07268 050(036ndash069)

0000lowast

GC 01702 01932 086(057ndash128)

047

TT 02002 00632 373(219ndash638)

0000lowast

TC 00598 00168 366(138ndash1026)

0006lowast









(95 CI) 119875 value





1400(120ndash16337) 0035lowast



806(441ndash1473) 0000lowast




Alcoholconsumption


1200(120ndash14373) 0035lowast


156(091ndash265) 0105













(027ndash1966) 0451

Alcoholconsumption






109(013ndash912) 0936












Alcoholconsumption


207(047ndash306) 0547


genotype

175(018ndash286) 0084


genotype

292(041ndash2090) 0287










5 Conclusion




Acknowledgments


References























































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology




(95 CI) 119875 value





1400(120ndash16337) 0035lowast



806(441ndash1473) 0000lowast




Alcoholconsumption


1200(120ndash14373) 0035lowast


156(091ndash265) 0105













(027ndash1966) 0451

Alcoholconsumption






109(013ndash912) 0936












Alcoholconsumption


207(047ndash306) 0547


genotype

175(018ndash286) 0084


genotype

292(041ndash2090) 0287










5 Conclusion




Acknowledgments


References























































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology









5 Conclusion




Acknowledgments


References























































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology




















































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

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