Footprints of genetic susceptibility to pulmonary tuberculosis: Cytokine gene variants in north Indians pdf

Footprintsofgeneticsusceptibilitytopulmonarytuberculosis: CytokinegenevariantsinnorthIndians Abhimanyu,MridulaBose,PankajJha * &IndianGenomeVariationConsortium Department of Microbiology, Vallabhbhai Patel Chest Institute, University of Delhi & * Genomics & Molecular Medicine, CSIR-Institute of Genomics & Integrative Biology, Delhi, India ReceivedMarch1,2011 Background & objectives: Tuberculosis is (TB) responsible for high morbidity and mortality worldwide. Cytokines play a major role in defense against Mycobacterium tuberculosis infection. Polymorphisms in the genes encoding the various pro- and anti-inammatory cytokines have been associated with tuberculosis susceptibility. In this study we examined association of 25 sequence polymorphisms in six candidate cytokine genes namely IFNG, TNFB, IL4, IL1RA, IL1B and IL12 and their related haplotypes with risk of developing pulmonary tuberculosis (PTB) among north Indians. Methods: Pulmonary TB (n=110) patients and 215 healthy controls (HC) from north India were genotyped. Puried multiplex PCR products were subjected to mass spectrometry using Sequenom MassARRAY platform to generate the genotypes in a population-based case-control study. Results: Using multiple corrections, signicant overall risk against PTB was observed at seven loci which included variants in IFNG at rs1861493 and rs1861494; IL1RA at rs4252019, IL4 variant rs2070874, IL12 variants rs3212220, rs2853694 and TNFB variant rs1041981. Analysis of gene structure revealed two haplotype blocks formed by IFNG variants rs1861493 and rs1861494. The TA haplotype was signicantly over-represented (P=0.011) in the cases showing a two-fold risk in the current population (Odds ratio=1.59 CI=1.101 to 2.297) and TNFB variants at rs2229094 and rs1041981 contributed to two haplotypes which were in strong linkage disequilibrium (LD) with AT haplotype showing a three-fold risk (P=0.0011, Odds ratio=3, CI=0.1939 to 0.7445) of developing PTB in north Indians. Interpretation & conclusions: Our study showed six novel associations of cytokine gene variants with susceptibility to PTB in north Indians. Variants of IFNG and TNFB emerged as factors imposing a signicant risk of developing PTB in north Indians apart from risk indicated by IL1RA, IL4 and IL12. Key wordsCytokinegenevariant-haplotype-Mycobacterium tuberculosis-pulmonarytuberculosis-singlenucleotidepolymorphisms 763 IndianJMedRes135,May2012,pp763-770  Tuberculosis (TB) causes signicant morbidity and mortality throughout the world 1 . The vast majorityof individualsinfected withMycobacterium tuberculosis (up to 95%) remain healthy, probably because of mounting an effective immune response against M. tuberculosis. In 1949, Haldane proposed that the maintenance of multiple genes that confer relative susceptibilities on the host to infectious diseases wouldbe favoured byevolution. Insupport of this hypothesis, certain populations appear to be 764 INDIANJMEDRES,MAY 2012 at risk for both increased susceptibility to infection 2 andprogressiveclinicaldiseaseduetomycobacteria 3 . Severalcase-controlstudieshaveidentiedassociation betweenTBandcandidategenespotentiallyinvolved in immune response to TB 4,5 . A growing body of evidencesupports arole ofhost geneticcomponents inthedevelopmentoftuberculosis.Theobservationof familialclusteringofdiseasewithhigherconcordance oftuberculosisdiseaseinmonozygoticversusdizygotic twins 6 , the ethnic clustering of tuberculosis disease withahigherprevalenceoftuberculosisinindividuals ofrecentAfricandescent 2 ,aswellasthedemonstration of both common polymorphisms and rare mutations which confer susceptibility to mycobacterial species inhumans 7 pointsignicantlyinthisdirection.These studies suggest that unique environment and natural selectivefactorsmayberesponsibleforthedevelopment ofethnic-specichostgeneticfactorsassociatedwith TB.  The rst step in innate host defense is cellular uptake of M. tuberculosis, which involves different cellularreceptorsandhumoralfactors.Thesubsequent inammatoryresponseisregulatedbytheproductionof pro-andanti-inammatorycytokinesandchemokines. Interferon-gamma (IFN-γ one of the most important cytokines involved in macrophage activation, stimulatinganti-tumourandanti-microbicidalactivities as well as expression of MHC-II 8,9 . Interleukin-4 (IL-4), an anti-inammatory cytokine has been implicated to downregulate IFN-γ, and thus has a deleterious effect on TB patients 10 . It also promotes the induction of Th2 cells 11 . IL-12, a heterodimeric pro-inammatory cytokine produced by activated macrophages,monocytes,β-lymphocytesanddendritic cellsistheprincipalTh1responseinducingcytokine 11 . Thiscytokine isimportant forsustaining asufcient number of memory/effector Th1 cells to mediate long-term protection to intracellular pathogen. Like tumournecrosisfactor-alpha(TNF-a),IL-1bismainly produced by monocytes, macrophages, anddendritic cells 12 . Intuberculosispatients,IL-1bisexpressedin excess 13 andatthesiteofdisease 14 .Implicatedmainly intuberculosis pleurisy, ausually self-resolvingtype ofprimarytuberculosis,onemayhypothesizethatan increasedIL-1b/IL-1Raratioprotectsagainstamore severeformoftuberculosis.  TNF-b or lymhotoxin-alpha (LTa) is considered to be a proinammatory cytokine and it is shown that secreted LTa is essential for the control of an intracellularbacterialinfection 15 .RecentlyAllieet al 16 suggestedthatLTαmightnothaveacriticalroleinhost defensetoacutemycobacterialinfection,independent ofTNF,butcertainlyacontributionofLTαinthecontrol ofchronic M. tuberculosis infectionisobserved 17 .  Association studies from north India probing multiplelociacrossthespectrumofcandidatecytokine genesarescanty.Thepresentstudy,therefore,wasaimed to bring in focus certain unexplored polymorphisms in the context of tuberculosis susceptibility in north Indianpopulation.Theroleandimportanceofgenetic backgroundintuberculosishasnowbecomeunivocal withethnicityplayingacrucialrole.Probingnewloci relating to tuberculosis susceptibility could suggest novel approach in pharmacogenomics and therapy to combat this pathogen. Also it could provide an insightinto predictingindividual’sgeneticproneness totuberculosisandofbeingfuturediagnostictoolfor preventivetherapyagainsttuberculosis. Material & Methods Study population: PTB patients above 18 yr of age (n=110)wereenrolledrandomlyinthestudybetween 2010-11 from Rajan Babu Institute of Pulmonary Medicine and Tuberculosis (RBIPMT), Kingsway Camp,NewDelhi(India).Thestudywascarriedout in Department of Microbiology, V.P. Chest Institute, University of Delhi, Delhi. Enrolled patients were category I cases, clinically and radiologically (chest X-ray) diagnosed for pulmonary tuberculosis and conrmed by sputum microscopy and culture for Mycobacterium following the guidelines of Revised NationalTBControlProgramme(RNCTP),Ministry of Health and FamilyWelfare, Government of India (http://www.tbcindia.nic.in). All patients were given free anti-tuberculosis drugs under DOTS (Directly Observed Treatment, short course) regimen of the GovernmentofIndia.ThemeanageofPTBcaseswas 31.89 ± 2.6yr while the ratioof male : femalewas 47:53.  Patients having any immunosuppressive presentation such as diabetes mellitus or HIV co- infection which areconsidered to be risk factorsfor tuberculosis development, and patients suspected to have extra-pulmonary tuberculosis along with pulmonarytuberculosiswereexcludedfromthestudy. Structured questionnaires were usedto document all otherrelevantinformationsuchasage,sex,ethnicity, socio-economic status, BCG vaccinations, and previous family history of tuberculosis. The healthy control(HC)groupconsistedof215randomlychosen nonconsanguineous BCG vaccinated students and laboratorypersonnelfromthevariousdepartmentsof theUniversityofDelhiwhowerewillingtoparticipate in the study with no signs, symptoms or history of previous mycobacterial infection. For HC mean age was29.31±.82yrandtheratioofmale:femalewas 43:57. Analysis of population stratication: Serious effort was made to avoid any false-positives arising as a result of population stratication. The self reported ethnicity of each subject and his/her parents was carefully considered. In addition, the genotype data weresubjectedtoEIGENSTRATprincipalcomponent analysis for population stratication correction as illustratedbyPriceet al 18 .  Allindividualswerebriefedaboutthestudyanda signedinformedconsentwasobtainedfromthepatient or his or her guardians before sample collection. The study was approved by the ethics committee of VallabhbhaiPatelChestInstitute,UniversityofDelhi, India. DNA extraction: Three ml of venous blood was collected in BD vacutainers containing ethylene diaminetetraaceticacid(EDTA)asanticoagulantand kept frozen until use. Genomic DNA was extracted from frozen whole blood using QiaAMP DNA kit (Qiagen,Germany).ExtractedDNAwasquantiedby spectrophotometery, checkedfor purityand storedat -20 o Cuntilfurtheranalyses. SNP selection and genotyping:Sixcandidatecytokine genes namely IFNG, TNFB, IL4, IL1RA, IL1B and IL12B, were selected owing to their suggested role in tuberculosis pathogenesis. All single nucleotide polymorphisms (SNPs) selected for genotyping were accessed from the public dbSNP (http://www. ncbi.nih.gov) and the HapMap (http://www.hapmap. org/).MostoftheselectedSNPsarefromtheintronic regionsofthecorrespondinggenes.Wereasonedthat notonlythechangesinthepromoterbutalsoofother unexploredregionsofthegenemayhamperitsnormal functioningleading todisease.The parameterstaken intoaccountwhileSNPselectionwerethefrequency of<0.01indbSNP,reportedallelefrequencyofatleast 20percentintwoworldpopulations(fromHapmap), average spacing 1 kb but in closely spaced minor allelefrequencywascarefullyconsidered.Inaddition, reportedheterozygositywasconsideredinaneffortto minimizeselectionofhomozygousloci.  All SNPs were genotyped using the matrix- assisted laser desorption/ionization time-of-ight (MALDI-TOF) mass spectrometry (Sequenom Inc., USA). Assays for all SNPs were designed using SpectroDESIGNER software (Sequenom Inc., USA) All SNPs were genotyped using the iPLEX assays (www.sequenom.com/iplex). Briey, as template, 5 ng of genomic DNA was used in a multiplex PCR reaction.ThePCRproductwasfurtherpuriedbefore theprimerextensionreactiontogenerateallele-specic baseextensionproducts.Thebase-extensionproducts weredetectedintheMALDI-TOFmassspectrometer to determinegenotypes. Genetic and statistical analyses: Hardy-Weinberg equilibriumwascalculatedinbothPTBcasesandHC separatelytoensurethatthesampleswerewithinallelic populationequilibriumbyusingHaploviewv4.2(http:// www.broad.mit.edu/mpg/haploview/).A stringent cut- offofferedbytheHaploviewv4.2wasusedforfurther analysis (minimum genotype =75% and minimum minor allele frequency 0.0010). The samples and variationsfailingthistestwerenotselectedforfurther analysis.PLINKv1.07(http://pngu.mgh.harvard.edu/ purcell/plink/)wasusedtotestformultiplecomparison andPvalueafterBonferronicorrectionswasconsidered signicant.Haplotypeblockgenerationwasperformed using the algorithm by Gabriel et al 19  implemented in the Haploview software which was also used for initial association testing. The statistical signicance ofPvalueofhaplotypeswasassessedbypermutation analysis(N=10,000)withHaploviewv4.2.  Geneticassociationtestingwasdoneusinga2x2 contingencytable.Oddsratio,twotailedPvaluewas calculatedforalleles.2x2Computationsweredone using GraphPad Prism (version 5.00 for Windows, Graph Pad Software, San Diego California, USA; www.graphpad.com). Two-tailed P<0.05 was consideredstatisticallysignicant. Results  Table Ishows thelocation and characteristicsof theSNPsincludedinthestudyandTableIIshowsthe associationsaftermultiplecorrectionscarriedoutusing PLINK (http://pngu.mgh.harvard.edu/purcell/plink/) whichwerefoundtobeassociatedwithsusceptibility toPTBinnorthIndiansinthisstudy. Population stratication correction: To access any underlyingstructureinthestudypopulationthatcould ABHIMANYUet al:CYTOKINEGENEVARIANTSINPTB 765 Table II.Allelicassociationsinafteradjustmentformultipletesting Gene dbSNP a rsID Case (n=110), control (n=215)frequencies Oddsratio (95%CI) Chisquare Pvalue * P bonferroni # IFNG rs1861493 0.962,0.869 3.8(1.7-8.6) 12.089 5.00E-04 0.00659 rs1861494 0.946,0.859 3.0(1.5-5.6) 10.466 0.0012 0.01581 IL4 rs2070874 0.387,0.255 1.8(1.3-2.6) 10.708 0.0011 0.01387 TNFB rs1041981 0.356,0.238 1.7(1.2-2.6) 8.649 0.0033 0.03618 IL12 rs2853694 0.607,0.478 1.6(1.2-2.4) 8.854 0.0029 0.0399 rs3212220 0.419,0.263 2.0(1.4-2.9) 14.572 1.00E-04 0.00175 IL1RA rs4252019 1.000,0.935 14.0(1.8-103.5) 13.643 2.00E-04 0.00287 SNP,singlenucleotidepolymorphism; * unadjustedP-value; # Pvalueafterbonferronimultipletestingcorrection; a dbSNP,theSNP database(http://www.ncbi.nlm.nih.gov/projects/SNP);P<0.05wasconsideredsignicant Table I. Locationandbase-pairpositionsofsingleneucleotidepolymorphisms(SNPs)ofvariouscytokinegenespassingtheexclusion criteriaandminorallelefrequency(MAF)incontrols Gene name dbSNP a rsID Basechange Chromosome position Location MAF controls References IFNG rs1861493 A/G 68551196 Intron4 0.13 New;thisstudy rs1861494 C/T 68551409 Intron4 0.14 New;thisstudy IL4 rs2070874 C/T 132009710 5’-UTR 0.25 Molleret al,2010 20 TNFB rs1041981 A/C 31540784 Exon4 0.26 New;thisstudy IL12 rs2853694 A/C 158749088 Intron4 0.5 New;thisstudy rs3212220 G/T 158754195 Intron4 0.28 Molleret al,2010 20 IL1RA rs4252019 C/T 113889119 Intron5 0.05 New;thisstudy a dbSNP,theSNPdatabase(http://www.ncbi.nlm.nih.gov/projects/SNP) confoundtheapparentgeneticassociationpopulation straticationcorrectionwascarriedoutusingEigenstrat PrincipalComponentanalysismethodasillustratedby Priceet al 18 .The methodmodels ancestrydifference between cases and controls and any other compared groupbasedonthesuppliedgenotypedata.Ourcases andcontrolsformedahomogenousgroupdevoidofany stratication.According to Indian Genome Variation Consortium (IGVC) 20  north Indians fall into Indo- European lineage. Our cases and controls matched withsuppliedmarkerdataofIndo-Europeanancestry therebyrulingoutcompletelyanyunderlyingstructure inthepopulation. Allelic association of cytokine SNPs and the risk of pulmonary tuberculosis:Amongthe25studiedSNPs, from six candidate cytokine genes the variants of IFNG, IL1RA, IL4, IL12 andTNFBwerefoundtobe associatedwithsusceptibilitytoPTBinnorthIndians. Allstudiedvariantspassingtheexclusioncriteriawere in Hardy-Weinberg equilibrium in both cases and controls.Allelic associationwhen probed invariants passingtheexclusioncriteriayieldedsixlocishowing highriskforPTBsusceptibility. IFNG polymorphism and PTB susceptibility: After adjusting for multiple testing corrections the IFNG intronic variants at rs1861493 [χ 2 =12.089, P bonferroni =0.006593,oddsratio(95%CI)=3.8(1.7-8.6)] andrs1861494(χ 2 =10.466,P bonferroni =0.01581,odds ratio(95%CI)=3.0(1.5-5.6)]showedasignicantrisk ofdevelopingpulmonarytuberculosisinnorthIndians with over-representation of the associated A and T allelesamongPTBpatients,respectively.Investigation of the gene structure and linkage disequilibrium patternshowed haplotypesformed byIFNG variants rs1861493andrs1861494whichwereinhighlinkage disequilibrium (LD) (Fig.). Three combinations of haplotypewereseennamelyTC,CCandTA,ofwhich TA haplotype was over-represented in the cases and imposed a two-fold risk of developing pulmonary tuberculosisinnorthIndians(TableIII). 766 INDIANJMEDRES,MAY 2012 Table III.Heplotypeblocksandfrequencies Blocks Haplotype frequency Case(n=110), control(n=215) frequencies Chisquare Permutations Pvalue # Oddsratio (95%CI) Block1 TC 0.41 0.36,0.43 2.82 0.093 0.75 (0.53-1.1) CC 0.32 0.29,0.33 0.86 0.353 0.85 (0.59-1.2) TA 0.27 0.34,0.24 6.46 0.04 1.59 (1.1-2.3) Block2 AT 0.88 0.95,0.86 10.66 0.005 2.9 (1.5-5.6) GC 0.10 0.05,0.13 8.85 0.017 0.38 (0.2-0.7) # Pvalueafterperformingpermutation(n=10,000);P<0.05wasconsideredsignicant Fig. Linkagedisequilibrium(LD)plotandhaplotypestructureof cytokinegenevariantsinPTBcases.D’valuesaredisplayedwithin eachdiamond,missingvaluesindicateD’=100%.Colourscheme gradientindicatesr 2 values.Lengthofeachblock,inkilobases(kb), isshowninbrackets. IL4 polymorphism and PTB susceptibility:IL4variant rs2070874[x 2 =10.708,P bonferroni =0.01387,oddsratio (95%CI)=1.8(1.3-2.6)]showedatwo-foldriskbyT alleleinnorthIndians.TheotherstudiedIL-4variant rs2243270passingtheexclusioncriteriadidnotshow any association towards susceptibility to pulmonary tuberculosisinthispopulation. IL1RA polymorphism and PTB susceptibility: The signicantlyassociatedlocusofIL1RAincludedintronic variantatrs4252019[χ 2 =13.643,P bonferroni =0.00287, Oddsratio(95%CI)=14.0(1.8-103.5)]showinga14- foldrisk.Othervariantsuchasrs315919andrs380092 didnotshowanyassociationtowardssusceptibilityto pulmonarytuberculosisinthispopulation. IL12 polymorphism and PTB susceptibility: IL12 variants rs3212220 [χ 2 =14.572, P bonferroni  = 0.00175, Oddsratio(95%CI)=2.0(1.4-2.9)]andrs2853694 [χ 2 =8.854,P bonferroni = 0.0399,odds ratio(95%CI) = 1.6(1.2-2.4)]showedatwo-foldriskassociatedwith TandAalleles,respectively. IL1B polymorphism and PTB susceptibility: The selectedIL1Bvariantsdidnotshowanydirectinuence onPTBsusceptibilityinnorthIndians. TNFB polymorphism and PTB susceptibility: TNFB variantsatrs1041981[χ 2 =8.649,P bonferroni =0.03618, Oddsratio(95%CI)=1.7(1.2-2.6)]asynonymous changeshowedatwo-foldriskofassociationforPTB innorthIndians. Interestinglyrs1041981 contributed to a haplotype block with rs2229094 conrming the importanceofthislocusinriskofdevelopingPTBin northIndians.ThetwohaplotypesobservedwereAT and GC of which AT was over-represented in PTB casesandimposedathree-foldriskofdevelopingPTB innorthIndians. Discussion  Thehostgeneticbiascontributingtosusceptibility and progression of pulmonary tuberculosis might ABHIMANYUet al:CYTOKINEGENEVARIANTSINPTB 767 involve interactions between multiple alleles located on different genes and chromosomes 21 . In order to overcome this drawback we planned selection of differentcytokinegeneandmultiplelocitocoverawide spectrumofimmuneresponseassociatedcytokines.  Case-control studies involving carefully chosen locus across ethnicities are valiant means of identifying novel associations pertaining to disease susceptibility.Associationthatarisesmaybe aresult ofthepolymorphisminquestionbeingfunctionalorit beinginlinkagedisequilibriumwithanotherfunctional allele or a result of confounding association due to population stratication. To overcome such false positives, we carefully considered the self reported ethnicityofthestudygroupsandfurthercheckedfor any genetic heterogeneity in our data by Eigenstrat principal component analysis illustrated by Price et al 18 andfoundthatthepresentdatawerefreefromany underlying population structure. Thus, this uniform datarepresentnorthIndianpopulationforassociation analysis.  The IFN-γ being a crucial cytokine in immunopathogenesisofTBhasbeensubjecttoseveral polymorphisms studies for pulmonary tuberculosis susceptibility. The locus probed here namely rs1861494 has not been studied in susceptibility to PTB but extensively studied in many other diseases suchasleprosy 22 andasthma 23 .Kumaret al 24 foundan associationofthislocuswithsusceptibilitytoasthma inIndians andcould identifya haplotype.They also showed that alleles of rs1861494 A/G have differential afnitytobindtoputativenuclearfactor.Inthepresentstudy, wefoundsignicantriskforthelocusinsusceptibility toPTB.The otherprobed locusrs1861493 hasbeen studied in idiopathic inammatory myopathy 24  and asthma 23  but notin pulmonary tuberculosis.We also identied a risk haplotypecontributed by rs1861493 and rs1861494 emphasizing the importance of the above mentioned loci as risk factors for developing pulmonarytuberculosisinnorthIndians. IL4locusrs2070874hasbeenanimportantlocus ofinvestigationinvariousdiseasesincluding asthma andrheumatoidarthritis 25 .ItsroleinTBwasreported nottobesignicantinIranianpulmonaryTBpatients 26 and recently in South Africans TB patients also the locus did not show any association 27 . In the present study this locusshowed a two-fold riskin the north Indianpopulation. IL1RAlocusrs4252019hasshownsignicantrisk ofdevelopmentofpulmonaryTBinnorthIndians.The variantrs4252019hasbeenshowntobeassociatedwith prostatecancerrisk 28 butnotpulmonarytuberculosis. Interestingly, the variant showed a 14-fold risk of developing PTB in the population studied here and emerged as a major locus to look out for in further studies. IL12 variantsrs3212220andrs2853694showeda signicantriskassociatedwithdevelopmentofPTBin northIndians.Thevariantrs321220hasbeenshownto contributetoahaplotypebyMolleret al 20 .Wehavealso predicteditsimportanceinourpreviousstudy 29 .Based ontheanalysisofserumIL-12level,wedemonstrated that forIL12 variant rs3212220TTgenotype among active PTB cases showed signicantly higher serum IL-12levelwhencomparedtoeitherGTorGG.The present study revealed T allele to be a risk allele in the present population. Similarly, rs2853694 a novel variantinthecontextofdevelopingtuberculosis 29 was predictedtobeofimportanceandwasvalidatedinthe presentstudy.Forrs2853694amongactivePTBcases AA genotype showed a trend towards higher serum IL-12levelincontrasttoareversetrendobservedin HCwhereAAaccountedforlowserumIL-12 29 .The presentstudyshowedAalleleatrs2853694tobearisk allele for the north Indian population in the context of PTB susceptibility. An interesting observation was that both the higher serum cytokine producers i.e.TTgenotypeforrs3212220andAAgenotypefor rs2853694 emerged as respective risk alleles T and A for thispopulation, indicating that overproduction of IL-12 by these individuals might be interfering withthe cytokinehomeostasis andthus affectingthe immunefunctionofthecytokineintheseindividuals makingthempronetoinfection.Ourobservationwas furthersupportedbytheworkofLeandroet al 30 ,who indicatedthatroleofIL-12aspotentinducerofIFN-γ liedinitsefcacyatlowconcentrations.Inthepresent studyitis observedthat thePTB patientswith IL12 riskallelegenotypesarenotefcientinducersofIFN-γ whichinturninterfereswiththeprotectiveimmunity intheseindividuals,whereasalowproleofIL-12in HCelicitsaneffectiveandoptimalimmuneresponse renderingtheseindividualshealthy. TNFB though not usually considered for PTB associationstudies,wastakenupinthecurrentstudy becauseofitsroleincontrolofintracellularbacterial infection 15 . The variant rs1041981 emerged as a 768 INDIANJMEDRES,MAY 2012 signicantrisk locusfor PTBsusceptibility innorth Indians. The variant also contributed to a haplotype with rs2229094 and reinstated the role of TNFB polymorphismsinPTB. Overall, ve of the loci namely rs1861493 and rs1861494 (IFNG), rs4252019 (IL1RA) rs1041981 (TNFB) and rs2853694 (IL12) studied in patients of pulmonary tuberculosis showed a signicant risk towards susceptibility to pulmonary tuberculosis in northIndians.Wealsoreportherethesignicantrisk imposedbyIL4 variantrs2070874 intheactivePTB patients.Sixnewassociationsandthreenewassociated haplotypescontributingtothespectrumofcytokinegene polymorphismsandriskofdevelopingtuberculosisin generalandnorthIndiansinparticular,weredetected. Acknowledgment  Theauthorsthankallpatientsandvolunteersforparticipating inthisstudy.ThesupportoftheMedicalSuperintendentandstaff atRajanBabuInstituteofPulmonaryMedicineandTuberculosis (RBIPMT), Kingsway Camp,New Delhi(India) forthe helpin sample collection is acknowledged. Authors acknowledge the CouncilofScienticandIndustrialResearch(CSIR),NewDelhi, for nancial support.The rst author was the Junior Research Fellow(JRF)intheCSIRproject. 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GeneticpolymorphismsinvitaminDreceptor,vitaminD-binding protein, Toll-like receptor 2, nitric oxide synthase 2, and interferon-gammagenesanditsassociationwithsusceptibility totuberculosis.Braz J Med Biol Res 2009;42 :312-22. 770 INDIANJMEDRES,MAY 2012 . CI=0.1939 to 0.7445) of developing PTB in north Indians. Interpretation & conclusions: Our study showed six novel associations of cytokine gene variants with susceptibility to PTB in north Indians. . Footprints of genetic susceptibility to pulmonary tuberculosis: Cytokine gene variants in north Indians Abhimanyu,MridulaBose,PankajJha * &IndianGenomeVariationConsortium Department. different cellularreceptorsandhumoralfactors.Thesubsequent in ammatoryresponseisregulatedbytheproduction of pro-andanti -in ammatorycytokinesandchemokines. Interferon-gamma (IFN-γ one of the most important cytokines involved in