An association study of IL-1 beta -511C/T and IL-1 RN 86 bp VNTR polymorphisms with essential hypertension was carried out in a sample population of 500 Pakistani Pathan subjects selected randomly, comprising groups of 235 subjects with hypertension and 265 controls. The distribution of both genotypes and alleles was not statistically different in cases and controls. In conclusion, IL-1 beta -511C/T and IL-1 RN 86 bp VNTR do not contribute to the aetiology of essential hypertension in the Pakistani Pathan population investigated here

Ahmad, U.

Chohan, M. O.

Frossard, P. M.

Jafar, T.

Khawaja, M. R.

Saleheen, D.

Taj, F.

eCommons@AKU

RESEARCH LETTERAssociation study of two interleukin-1gene loci with essential hypertensionin a Pakistani Pathan populationJournal of Human Hypertension (2008) 22, 60–62;doi:10.1038/sj.jhh.1002257; published online 5 July 2007An association study of IL-1b -511C/T and IL-1 RN86 bp VNTR polymorphisms with essential hyper-tension was carried out in a sample population of500 Pakistani Pathan subjects selected randomly,comprising groups of 235 subjects with hyper-tension and 265 controls. The distribution of bothgenotypes and alleles was not statistically differentin cases and controls. In conclusion, IL-1b -511C/Tand IL-1 RN 86 bp VNTR do not contribute to theaetiology of essential hypertension in the PakistaniPathan population investigated here.Essential hypertension (EHT) is a complex dis-order resulting from interactions between environ-mental and genetic factors. Family and twin studiessuggest that approximately one-third of the variancein blood pressure is attributable to genetic determi-nants.1 Despite extensive efforts and the wide rangeof strategies employed, genetic variants of thiscomplex phenotype still remain elusive. The use ofcandidate genes in case–control study designs toinvestigate complex disorders has become an in-creasingly preferred method owing to its cost-effectiveness and reasonably high success rates.The role of immunological mediators is gainingincreasing interest in the aetiology of cardiovascularphenotypes such as EHT. C-reactive protein (CRP)has been shown to be a significant predictor of thedevelopment of EHT.2,3 Allele -511T in the promoterregion of the human interleukin-1 b (IL-1b) gene wasassociated with higher blood pressure among Chi-nese hypertensive individuals.4 Allele 2 of a VNTR(variable number of tandem repeats) in intron 2 ofthe interleukin-1 receptor antagonist (IL-1 RN) genehas been associated with single-vessel coronaryartery disease in an English population5 and withEHT in an Australian population.6We investigated one single-nucleotide poly-morphism (SNP) in IL-1b (-511C/T) and one VNTRin IL-1RN (86 bp VNTR) in a case–control studydesign to assess their putative associations withEHT in a Pakistani Pathan population. Pathans are aunique ethnicity in terms of their diet, language andsociocultural habits. This ethnicity is also character-ized by high consanguinity rates as a result of itsclosed cultural and social systems. Genetic studiesin such populations are thus less susceptible to thebias of ‘population stratification’. The high degree ofconsanguinity observed in Pathans is hypothesizedto be responsible for the pooling of recessive allelesresulting in the overexpression of certain diseases.These include recessive monogenic diseases as wellas complex diseases, for which increasing evidencesuggests a mode of recessive inheritance of under-lying major gene effects. This may explain the 56%prevalence rate of EHT in the Pathan ethnicity,which is twofold higher than in other Pakistanipopulations.7The study was approved by the Ethical ReviewCommittee of Aga Khan University. The sample sizewas calculated to detect an association at 80%power with an a of 5%. Subjects were recruited fromthe Pathan Colony, Karachi (an area of the cityinhabited almost exclusively by Pathans who havemigrated from the North-Western Frontier Provinceof Pakistan), through a two-stage selection process.The population census was used to randomly selecthouseholds. This was followed by another census ofthese selected households. A total of 500 indivi-duals over 40 years of age with almost equal genderdistribution were randomly selected in the secondstage. The two-stage random selection aimed atselecting equal proportions of normotensive (NT)and EHT Pathans.A trained team, including a doctor and commu-nity health workers, carried out the recruitment ofselected subjects. EHT was defined as systolic bloodpressure (SBP) X140 mm Hg or diastolic bloodpressure (DBP) X90 mm Hg, in supine position,after 20 min of rest on 2 separate days, or if theperson was taking antihypertensive treatment. Themean of three readings was used in the analyses. Torule out any secondary cause of hypertension,complete medical history was obtained and physicalexamination was conducted on all subjects afterthey gave their consent. A pre-tested and codedquestionnaire was used to record life style and riskfactor data. NT subjects had no personal history ofEHT (documented as resting SBPso140 mm Hg andDBPs o90 mm Hg on at least three separate occa-sions) and no family history of EHT in directrelatives (parents and siblings). We remain awareof the fact that NTs in such studies may not beproper ‘controls’, as they are indeed simply free ofdisease at the time of study, but may developJournal of Human Hypertension (2008) 22, 60–62& 2008 Nature Publishing Group All rights reserved 0950-9240/08 $30.00www.nature.com/jhhhypertension at a later stage. This is why a betterterm to characterize disease-free subjects would be a‘comparison’ (rather than ‘control’) group.A total of 10 ml of venous blood was taken in twodifferent tubes for DNA extraction and serumanalyses of creatinine, glucose and lipid profile.DNA was extracted from 5 ml venous blood using astandard phenol–chloroform protocol. Genotypingwas carried out by PCR and restriction fragmentlength polymorphism (RFLP) techniques. The IL-1b-511C/T genotype was identified by PCR followedby RFLP using the restriction enzyme AvaI to digestthe mutant allele as described previously.5 IL-1 RNVNTR genotypes were identified by PCR as reportedpreviously.8Data were coded and entered in SPSS Windows(13.0). Mean values7standard deviation (SD) werecalculated for all variables. Alleles and genotypefrequencies were determined by gene counting. Inunivariate analysis, categorical variables were com-pared using w2 tests. A value of Po0.05 wasconsidered statistically significant.This study included 500 Pathan subjects (265 NTsand 235 EHTs) with a mean age of 51.5710.1 years.Genotypes of both cases and controls were inHardy–Weinberg equilibrium for IL-1b -511C/Tpolymorphism. For IL-1 RN 86 bp VNTR polymorph-ism, the genotypes were in Hardy–Weinberg equili-brium after converting it into a bi-allelic marker bymerging all alleles other than A2 into a single allele(Ax). Allele frequencies for IL-1b -511C/T poly-morphism in the overall population were 63.0% for -511C and 37.0% for -511T. For IL-1 RN VNTRpolymorphism, allelic frequencies were 64.6% forA1, 32.5% for A2, 2.1% for A3 and 0.8% for A4. NoA5 allele was found in this population. The allelicfrequencies on both polymorphisms in our popula-tion were similar to those reported previously insome Caucasian populations.6–8 Table 1 shows thedistribution of genotype and allele frequencies forIL-1b -511C/T and IL-1 RN VNTR polymorphisms inNT and EHT groups. No significant association wasobserved upon comparison of either genotype dis-tribution or allele frequencies between EHTs andNTs.Our aim was to make use of the relative genetichomogeneity of our population to explore putativeassociations of two cytokine gene polymorphismswith EHT. Investigating the Pathan consanguineousethnic group minimizes the influence of bothselection bias and population stratification.The role of immunological reactivity in theaetiology of cardiovascular pathology is gainingincreasing interest. With the recognition that athero-sclerosis is an inflammatory process,9,10 the atten-tion is now focused on the role of inflammatorypathways in the aetiology of cardiovascular dis-eases, including EHT. In a cohort of 20 525 subjectswith no history of cardiovascular events or malig-nancies, CRP was found to be a significant predictorof developing hypertension independently of othercoronary risk factors.2,3 These results stronglyimplicate that the aetiology of hypertension has asignificant inflammatory basis. Some investigatorsbelieve that inflammation within the arterial treeowing to the effects of atherogenic lipoproteins, lowhigh-density lipoprotein, high plasminogen activa-tor inhibitor-1, insulin resistance and smoking,releases cytokines that increase CRP levels.3,11Another explanation for this phenomenon is thatCRP may play a direct role in the causation ofhypertension through its proinflammatory influ-ence.2,3 In either case, cytokines seem to play anessential role in this pathway.In this study, we investigated the association bet-ween two cytokine gene polymorphisms (IL-1b -511C/TTable 1 Distribution of genotypes and allele frequencies among HT and NT Pathan subjectsTotal (500) N (rel. freq) NT (265) N (rel. freq) HT (235) N (rel. freq) Univariate OR (95% CI) P-valueIL-1b -511 C/TGenotypesCC 193 (0.39) 103 (0.39) 90 (0.38) 1.00CT 243 (0.49) 130 (0.49) 113 (0.48) 0.90 (0.52-1.56) 0.722TT 63 (0.12) 32 (0.12) 31 (0.14) 0.90 (0.51-1.59) 0.702AllelesC 629 (0.63) 336 (0.63) 293 (0.62) 1.00T 369 (0.37) 194 (0.37) 175 (0.38) 0.97 (0.75-1.25) 0.797IL-1 RN 86bp VNTRGenotypesAxAx 234 (0.47) 115 (0.44) 119 (0.51) 1.00A2Ax 203 (0.41) 112 (0.43) 91 (0.39) 0.79 (0.54-1.15) 0.209A2A2 60 (0.12) 32 (0.13) 24 (0.10) 0.64 (0.36-1.15) 0.135AllelesAx 671 (0.67) 342 (0.65) 329 (0.70) 1.00A2 323 (0.33) 184 (0.35) 139 (0.30) 0.79 (0.60-1.03) 0.076Abbreviations: CI, confidence interval; HT, hypertensive; IL, interleukin; NT, normotensive; OR, odds ratio; rel. freq, relative frequency; VNTR,variable number of tandem repeats.Research Letter61Journal of Human Hypertensionand IL-1 RN VNTR) and EHT. None of the studiedgenetic markers showed statistically significantdifferences in the distribution of genotypes or allelesamong NT and EHT groups. Moreover, none of thepolymorphisms was associated with diabetes, smok-ing status or body mass index. As a number of EHTsubjects were on blood pressure lowering medica-tions, association analyses of genetic markers withblood pressure levels could not be performed.The results of this study are different from thosereported in some other populations.4,6 A possiblereason could be that these genes may have a minorrole in the causation of EHT; to evidence sucheffects, larger sample sizes would be needed.Alternatively, this genetic effect could be popula-tion-specific only. Moreover, the SNPs investigatedhere may not sufficiently cover the functionalhaplotype in all the ethnicities, and thus thoroughinvestigations on linkage disequilibria among dif-ferent variants within these genes should be per-formed in further association studies. To gain moreinsight into the disease pathogenesis, circulatinglevels of inflammatory markers will also need to beassessed in study participants.In conclusion, our study does not support therelevance of the IL-1b -511C/T and IL-1 RN VNTRpolymorphisms in the aetiology of EHT amongPakistani Pathans. More studies, however, areneeded to study the role of other variants in genesof these markers and other cytokines to decipher thecomplex immunologic pathways contributing to theaetiology of EHT.MR Khawaja1, F Taj1, D Saleheen2, U Ahmad2, MOChohan2, T Jafar3 and PM Frossard21Medical College, Faculty of Health Sciences, AgaKhan University Medical College, Karachi, Pakistan;2Department of Biological and Biomedical Sciences,Faculty of Health Sciences, Aga Khan UniversityMedical College, Karachi, Pakistan and3Departments of Medicine and Community HealthSciences, Faculty of Health Sciences, Aga KhanUniversity Medical College, Karachi, PakistanE-mail: philippe.frossard@aku.eduPublished online 5 July 2007References1 Agarwal A, Williams GH, Fisher ND. Genetics ofhuman hypertension. Trends Endocrinol Metab 2005;16: 127–133.2 Sesso DH, Buring JE, Nader R, Blake GJ, Gaziano JM,Ridker PM. C reactive protein and risk of developinghypertension. JAMA 2003; 290: 2945–2951.3 Grundy SM. Inflammation, hypertension and meta-bolic syndrome. JAMA 2003; 290: 3000–3002.4 Huang G, Niu T, Peng S, Ling D, Liu J, Zhang X et al.Association between the interleukin-1beta C(-511)Tpolymorphism and blood pressure in a Chinesehypertensive population. Immunol Lett 2004; 91:159–162.5 Francis SE, Camp NJ, Dewberry RM, Gunn J, Syrris P,Carter ND et al. Interleukin-1 receptor antagonist genepolymorphism and coronary artery disease. Circula-tion 1999; 99: 861–866.6 Lin RC, Morris BJ. Association analysis of polymorph-isms at the interleukin-1 locus in essential hyperten-sion. Am J Med Genet 2002; 107: 311–316.7 Pakistan Medical Research Council. National HealthSurvey of Pakistan 1990–94. Pakistan Medical Re-search Council, Network Publication Service: Islama-bad, Pakistan, 1998.8 Tarlow JK, Blakemore AI, Lennard A, Solari R, HughesHN, Steinkasserer A et al. Polymorphism in humanIL-1 receptor antagonist gene intron 2 is caused byvariable numbers of an 86-bp tandem repeat. HumGenet 1993; 91: 403–404.9 Ross R. Atherosclerosis – an inflammatory disease. NEngl J Med 1999; 340: 115–126.10 Ridker PM, Hennekens CH, Buring JE, Rifai N.C-reactive protein and other markers of inflammationin the prediction of cardiovascular disease. N Engl JMed 2000; 342: 836–843.11 Schwartz RS, Bayes-Genis A, Lesser JR, Sangiorgi M,Henry TD, Conover CA. Detecting vulnerable plaqueusing peripheral blood: inflammatory and cellularmarkers. J Interv Cardiol 2003; 16: 231–242.What is known about the topicK After the recognition of atherosclerosis as an inflammatoryprocess, role of inflammatory pathways in the aetiology ofhypertension is increasingly being decipheredK IL-1b and IL-1 RN polymorphisms have been reported to beassociated with variation in blood pressureWhat this study addsK IL-1b -511C/T and IL-1 RN variable number of tandemrepeats polymorphisms are not associated with essentialhypertension in a Pakistani Pathan populationResearch Letter62Journal of Human Hypertension

Association study of two interleukin-1 gene loci with essential hypertension in a Pakistani Pathan population

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Association study of two interleukin-1 gene loci with essential hypertension in a Pakistani Pathan population

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