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
(). 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 (map.
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( />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 ( />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( />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( />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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 UniversityofDelhi,Delhi110007,India

 e-mail:
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