NRAMP1 and VDR Gene Polymorphisms in Susceptibility to Tuberculosis in Venezuelan Population

Research ArticleNRAMP1 and VDR Gene Polymorphisms in Susceptibility toTuberculosis in Venezuelan Population

Mercedes Fernaacutendez-Mestre1 Aacutengel Villasmil1

Howard Takiff2 and Zhenia Fuentes Alcalaacute3

1Laboratorio de Fisiopatologıa Centro de Medicina Experimental ldquoMiguel Layrisserdquo Instituto Venezolano de InvestigacionesCientıficas Kilometro 11 Carretera Panamericana Apartado 21827 Caracas 1020A Distrito Capital Venezuela2Laboratorio de Genetica Molecular Centro de Microbiologıa y Biologıa Celular Instituto Venezolano de Investigaciones CientıficasKilometro 11 Carretera Panamericana Apartado 21827 Caracas 1020A Distrito Capital Venezuela3Unidad de Torax Hospital Dr Jose Ignacio Baldo ldquoEl Algodonalrdquo Avenida Intercomunal de AntımanoLa Yaguara Apartado 1000 Caracas 1020A Distrito Capital Venezuela

Correspondence should be addressed to Mercedes Fernandez-Mestre mfernandezmestregmailcom

Received 5 March 2015 Accepted 17 September 2015

Academic Editor Irene Rebelo

Copyright copy 2015 Mercedes Fernandez-Mestre et al This is an open access article distributed under the Creative CommonsAttribution License which permits unrestricted use distribution and reproduction in any medium provided the original work isproperly cited

Natural resistance-associated macrophage protein (Nramp1) and the vitamin D receptor (VDR) are central components ofthe innate and adaptive immunity against Mycobacterium tuberculosis and associations between susceptibility to tuberculosisand polymorphisms in the genes NRAMP and VDR have been sought in geographically diverse populations We investigatedassociations ofNRAMP1 andVDR gene polymorphisms with susceptibility to TB in the Venezuelan populationThe results suggestthe absence of any association between VDR variants FokI ApaI and TaqI and susceptibility to tuberculosis In contrast theNRAMP1 31015840UTR variants were associated with susceptibility toM tuberculosis infection as seen in the comparisons between TST+and TSTminus controls and also with progression to TB disease as shown in the comparisons between TB patients and TST+ controlsThis study confirms the previously described association of the NRAMP1 31015840UTR polymorphism withM tuberculosis infection anddisease progression

1 Introduction

Tuberculosis (TB) is the second cause of death worldwideamong infectious diseases In theWorld Health Organization(WHO) Global Tuberculosis Report for 2014 there were 90million new TB cases in 2013 and 15 million TB deaths (11million among HIV negative and 04 million among HIVpositive) of which 218875 new cases and relapses occurredin the Americas In Venezuela according to the WHOthe incidence of new and relapse TB cases is between 20and 49 per 100000 inhabitants [1] The number of subjectsinfected with Mycobacterium tuberculosis (Mtb) is muchhigher but the greatmajority of those infected are able to keepthe pathogen under control and never develop the diseaseMultiple evidence suggests that there is a genetic componentinvolved in determining resistance or susceptibility to TB

patients who are infected but do not develop the diseaseand who developed the disease but it is difficult to conductgenetic studies on susceptibility to infectious diseases becauseof the multifactorial influences of the host the pathogenand environmental variables that differ for each disease andeven each individual studied The association of host geneticfactorswith susceptibility or resistance toTBhas been studiedextensively using various methods including case-controlstudies candidate gene approaches and family-based andgenome-wide linkage analyses that have revealed severalcandidate genes involved in susceptibility (reviewed in [2])These studies have been performed in different ethnic groupswith large discrepancies between groups regarding the effectof the different candidate genes Two of the genes that haveshown the most robust associations are NRAMP1 and VDR

Hindawi Publishing CorporationDisease MarkersVolume 2015 Article ID 860628 7 pageshttpdxdoiorg1011552015860628

2 Disease Markers

In this study we investigated associations ofNRAMP1 andVDR gene polymorphisms with susceptibility to TB in theVenezuelan population

2 Material and Methods

21 Subjects Thestudy included one hundred andninety-five(195) unrelated and ethnically mixed Venezuelan individualsfrom the northern region of country divided into twocohorts patients and controls

Patients The ninety-three individuals all had a clinicaldiagnosis of pulmonary tuberculosis and were seen by thePneumology Service of the Hospital Jose Ignacio BaldoAlgodonal Caracas There were 52 men (56) and 41 women(44) with an age range of 17ndash70 years Patients were selectedbased on the diagnostic criteria of the National StandardIntegrated Venezuela Tuberculosis Control Program twosputum smears positive for acid fast bacilli by microscopyclinical symptoms characteristic of TB and a chest X rayconsistent with active TB disease

ControlsThere were one hundred and two apparently healthyindividuals who were known to be exposed to patients withactive TB This group was composed of staff members whohad worked at Hospital Dr Jose Ignacio Baldo AlgodonalCaracas for more than 3 years and included 25 men (245)and 77 women (755) whose age range was 20ndash67 yearsThecontrols were classified according to the tuberculin skin testldquoTSTrdquo as follows positiveTST (51102) negativeTST (19102)or without information (32102) All controls were withoutclinical manifestations of TB at the time of blood sampling

Individuals who were HIV positive or known to have anyautoimmune chronic inflammatory or other disease wereexcluded from the study Participants in the study signedan informed consent form previously approved by the IVICBioethics Committee

22 NRAMP1 and VDR Genotype Analysis Genomic DNAwas extracted fromblood samples according to the proceduredescribed by Bunce [3] The polymorphic variants of theNRAMP1 gene were studied by PCR-RFLP technique usingprimers and restriction enzymes reported by Taype et al2006 [4] INT4 (469 + 14GC) D543 (codon 543 Arg rarrAsp) and 31015840UTR (deletion of TGTG in the 31015840UTR 55 nt 31015840to the last codon in exon 15) The polymorphic variants ofthe VDR gene were studied by PCR-RFLP using primers andrestriction enzymes reported by Curran et al 1999 [5] FokIApaI and TaqI

23 Statistical Analysis Allele and genotype frequencieswere determined by direct counting The Hardy-Weinbergequilibrium was calculated with the exact test The statisticalsignificance of allele frequency differences between patientsand controls was estimated by Fisherrsquos exact test using 2 times2 contingency tables The Bonferroni corrected 119901 values(119901c) were obtained by multiplying the 119901 values by the totalnumber of variables analyzed andwere considered significant

when 119901 lt 005 [6] Relative risk with corresponding 95confidence intervals (95 CI) was calculated as odds ratios(OR) according to Woolf rsquos formula [7]

3 Results

31 Frequency of NRAMP1 Polymorphisms in Patients withTuberculosis and Controls Table 1 shows the frequencies ofNRAMP genotypes and alleles in controls and TB patientsThe allelic and genotypic frequencies of the NRAMP1 poly-morphisms showed Hardy-Weinberg equilibrium for INT4(119901 = 0832) D543 (119901 = 0296) and 31015840UTR (119901 =0594) polymorphisms in the control cohort There wasa significantly increased frequency of homozygous 31015840UTRTGTG++ genotype in the healthy controls compared tothe TB patients (79 versus 64 resp OR = 04 95 CI02478ndash09045 119901 = 001 119901c = 003) However the frequencyof the heterozygous 31015840UTR SNP TGTG+del (326 versus21 resp OR = 18 95 CI 09452ndash34976 119901 = 003)and homozygous 31015840UTR SNP deldel genotypes (34 versus0 resp OR = 813 95 CI 04143ndash1596275 119901 = 002)was higher in the patient group than in the control groupalthough the corrected 119901 values (119901c) were not significant Inaddition there was a significant difference in the distributionof the allele frequencies (TGTG+ and TGTG del) betweenthe controls and the TB patients (119901c = 0018) There was nosignificant association between INT4 and D543 genotypeswith tuberculosis

32 Frequency of the VDR Polymorphism in Patients withTuberculosis and Controls Table 2 shows the frequenciesof VDR genotypes and alleles in controls and TB patientsThere was Hardy-Weinberg equilibrium for the genotypedistributions of FokI (119901 = 0074) TaqI (119901 = 0066) andApaI (119901 = 0545) polymorphisms in apparently healthyindividuals The data showed that the frequency of the FFSNP genotype of FokI was higher in the patients than inthe control group (366 versus 255 resp OR = 17 95CI 09120ndash31109 119901 = 004) although the corrected 119901 valuewas not significant No significant difference in the allelefrequencies was observed between the TB patients and thecontrols

33 Genotype and Allele Distribution of NRAMP-31015840UTRVariants in Patients with Tuberculosis and Healthy ControlsClassified by the Tuberculin Skin Test (TST) In order toinvestigate the possible influence of NRAMP1-31015840UTR vari-ants in the development of tuberculosis we compared thegenotype and allele frequencies between the different groupsTB patients versus TST positive controls TB patients versusTST negative controls and TST positive controls versus TSTnegative controls (Table 3)There were statistically significantdifferences between TB patients and TST negative controlsfor TGTG++ (64 versus 947 OR 01 95 CI 00126ndash07760 119901 = 0004 119901c = 0012) and TGTG+del genotypes(326 versus 53 OR 87 95CI 11069ndash683801 119901 = 0008119901c = 0016) These same differences were conserved whenthe comparison was made between TST positive controls

Disease Markers 3

Table 1 Genotype and allele frequency distribution of NRAMP1 gene in the controls and TB patients

INT4 CC GC GG TotalGenotypesControls (number of individuals) 14 45 39 98 of total 749 2406 2086 5241 within condition 1429 4592 3980 within INT4 6087 5625 4643TB patients (number of individuals) 9 35 45 89 of total 481 1872 2406 4759 within condition 1011 3933 5056 within INT4 3913 4375 5375Total 23 80 84 187 of total 1230 4278 4492 100

120594

2 (df 2) = 234 119901 = 03107 Cramerrsquos 119881 = 01118D543 AA AG GG TotalGenotypesControls (number of individuals) 1 10 89 100 of total 052 521 4635 5208 within condition 1 10 89 within D543 25 5556 5235TB patients (number of individuals) 3 8 81 92 of total 156 417 4219 4792 within condition 33 87 88 within D543 75 4444 4765Total 4 18 170 192 of total 208 938 8854 100

120594

2 (df 2) = 127 119901 = 0529 Cramerrsquos 119881 = 0081331015840UTR TGTGdeldel TGTG+del TGTG++ TotalGenotypesControls (number of individuals) 0 21 79 100 of total 000 1111 4180 5291 within condition 000 2100 7900 within 31015840UTR 000 4200 5809TB patients (number of individuals) 3 29 57 89 of total 159 1534 3016 4709 within condition 337 3258 6404 within 31015840UTR 10000 5800 4191Total 3 50 136 189 of total 7196 2646 159 100

120594

2 (df 2) = 722 119901 = 00270 Cramerrsquos 119881 = 01955Note 119901 probability values + = presence of TGTG del = absence of these four bases df degree freedom Cramerrsquos 119881 measure of association between twovariables

versus TST negative controls although the significance waslost with the correction of 119901 values Additionally there was asignificant increase in the frequency of the TGTGdel alleleamong TB patients (OR 90 95 CI 12010ndash682837 119901 =0005 119901c = 0010) and TST positive controls (OR 50 95CI 06330ndash402130119901 = 0046119901c =not significant) comparedto TST negative controls

4 Discussion

The aim of the present study was to look for associationsbetween polymorphisms in VDR and NRAMP1 genes andsusceptibility to infection and disease with Mycobacteriumtuberculosis as indicated by a positive TST and the develop-ment of TB in the Venezuelan population Vitamin D is an

4 Disease Markers

Table 2 Genotype and allele frequency distribution ofVDR gene inthe controls and TB patients

Fok1 ff Ff FF TotalGenotypesControls (number of individuals) 16 60 26 102 of total 82 308 133 523 within condition 157 588 255 within Fok1 571 561 433TB patients (number of individuals) 12 47 34 93 of total 62 241 174 477 within condition 129 505 366 within Fok1 429 439 567Total 28 107 60 195 of total 144 549 307 100120594

2 (df 2) = 281 119901 = 02454 Cramerrsquos 119881 = 012Taq1 tt Tt TT TotalGenotypesControls (number of individuals) 1 38 58 97 of total 05 208 317 53 within condition 1 392 598 within Taq1 333 535 532TB patients (number of individuals) 2 33 51 86 of total 11 18 279 47 within condition 23 384 593 within Taq1 667 465 468Total 3 71 109 183 of total 16 388 596 100120594

2 (df 2) = 048 119901 = 07866 Cramerrsquos 119881 = 00512Apa1 aa Aa AA TotalGenotypesControls (number of individuals) 18 54 29 101 of total 95 284 153 532 within condition 178 535 287 within Apa1 474 563 518TB patients (number of individuals) 20 42 27 89 of total 105 221 142 468 within condition 225 472 303 within Apa1 667 465 468Total 38 96 56 190 of total 20 505 295 100120594

2 (df 2) = 092 119901 = 06313 Cramerrsquos 119881 = 00696Note 119901 probability values df degree freedom Cramerrsquos 119881 measure ofassociation between two variables

immune-modulator molecule that via its receptor VDR canmodulate cytokine responses by T cells [8] Although severalpublications have reported an association between the VDRpolymorphisms and tuberculosis in different populations [8ndash26] our study similar to others [27ndash31] did not observea significant association between the ApaI TaqI or FokIvariants and susceptibility to either infection or developmentof tuberculosis

Nramp1 (natural resistance-associated macrophage pro-tein) is an integralmembrane protein expressed exclusively inthe lysosomal compartment of monocytes and macrophagesAfter phagocytosis Nramp1 is targeted to the membrane ofthe microbe-containing phagosome where it may modifythe intraphagosomal milieu to affect microbial replication[32] Some polymorphisms in the NRAMP1 gene appear tofavor bacterial replicationwithinmacrophages and have beenassociated not only with increased susceptibility to infectionby Mycobacterium tuberculosis but also with an increasedtendency to develop severe disease [33ndash35]

However different studies have found conflicting resultsThere was a positive association between the NRAMP1 gene31015840UTR polymorphism and susceptibility to tuberculosis inWest Africans [36] Koreans [37] Chinese Han [23] andChinese Kazak populations [38] but there was no associationfound in Taiwanese [39] Thai [40] Moroccan [41] Danish[42] Brazilian [43] and Indonesian [44] populations and itwas associated with resistance to TB in Cambodians [31]Theresults presented here found that in the Venezuelan popula-tion the 31015840UTRvariants were associatedwith susceptibility toM tuberculosis infection as seen in the comparisons betweenTST+ and TSTminus controls and also with progression to TBdisease as shown in the comparisons between TB patientsand TST+ controls

The 31015840UTR polymorphism consists of a 4 bp TGTGdeletion located 55 nucleotides downstream the last codon inexon 15 of theNRAMP1 gene in a regionwhere sequence vari-ation can affect mRNA stability andor efficiency of proteintranslation Therefore to explain the increased susceptibilitypromoted by the TGTG+del genotype and TGTGdel alleletwo essential aspects should be considered (1) the proteinencoded by the NRAMP1 gene plays an important role inthe phagolysosomal function of pulmonary macrophagesand in antigen presentation The Nramp1 protein becomesactivated and fused with lysosomes to digest the engulfedmycobacteria (reviewed in [45]) (2) Nramp1 pumps ironout of macrophages thereby reducing iron levels withinboth the cytoplasm and the phagolysosome rendering themetal less available for the iron-requiring intracellular bacilli[46] As a consequence a mutation or polymorphism inthe NRAMP1 gene that results in a nonfunctional Nramp1protein or decreases production of the protein could causea reduction in Nramp1 function or even a complete absenceof the protein Decreased Nramp1 action could lead toincreased bacterial availability of iron thereby promotingmycobacterial replication within macrophages Because theiron is also required by the cell to generate reactive oxygenand nitrogen intermediates the loss of Fe2+ ion transporterfunction of Nramp1 protein could increase availability of ironfor intramacrophage bacteria and simultaneously weakenantimicrobial activity thus favoring infection withM tuber-culosis and progression to tuberculosis disease Furthermorealthough elevated iron may increase susceptibility to TB itmay also predispose an individual to greater morbidity afterTB has developed due to its role in generating ROS causedoxidative stress which is greater in active TB comparedwith historical TB or healthy controls (reviewed in [46])

Disease Markers 5

Table 3 Genotype and allele frequency distribution of NRAMP-31015840UTR in TB patients and healthy controls grouped according to the TST

31015840UTR TGTGdeldel TGTG+del TGTG++ TotalGenotypesControls TST+ (number of individuals) 0 12 38 50 of total 000 86 273 36 within condition 000 2400 7600 within 31015840UTR 000 293 40TB patients (number of individuals) 3 29 57 89 of total 22 209 41 64 within condition 34 326 64 within 31015840UTR 10000 707 60Total 3 41 95 139 of total 22 295 683 100

120594

2 (df 2) = 315 119901 = 0207 Cramerrsquos 119881 = 0150531015840UTR TGTGdeldel TGTG+del TGTG++ TotalGenotypesControls TSTminus (number of individuals) 0 1 18 19 of total 000 09 167 176 within condition 000 53 947 within 31015840UTR 000 33 24TB patients (number of individuals) 3 29 57 89 of total 28 269 528 824 within condition 34 326 64 within 31015840UTR 10000 967 76Total 3 30 75 108 of total 28 278 694 100

120594

2 (df 2) = 697 119901 = 00307 Cramerrsquos 119881 = 025431015840UTR TGTGdeldel TGTG+del TGTG++ TotalGenotypesControls TSTminus (number of individuals) 0 1 18 19 of total 000 14 261 275 within condition 000 53 947 within 31015840UTR 000 77 321Controls TST+ (number of individuals) 0 12 38 50 of total 000 174 551 725 within condition 000 2400 7600 within 31015840UTR 000 923 679Total 0 13 56 69 of total 0 188 812 100

120594

2 (df 2) = 316 119901 = 0206 Cramerrsquos 119881 = 0214Note 119901 probability values df degree freedom Cramerrsquos 119881 measure of association between two variables

In conclusion the NRAMP1 gene 31015840UTR polymorphismmight play an important role in the host defense to the devel-opment of tuberculosis

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

The authors are grateful to the persons that participatedin this study This study was supported by CRP ICGEB

Research Grant CRPVEN11-01 and by FONACIT Project G-2005000393

References

[1] World Health Organization Global Tuberculosis Report 2014World Health Organization Geneva Switzerland 2014

[2] F Wu W Zhang L Zhang et al ldquoNRAMP1 VDR HLA-DRB1 and HLA-DQB1 gene polymorphisms in susceptibilityto tuberculosis among the Chinese kazakh population a case-control studyrdquo BioMed Research International vol 2013 ArticleID 484535 8 pages 2013

[3] M Bunce ldquoPCR-SSP typing in histocompatibility testingrdquo inBidwell and Navarrette C pp 149ndash186 Imperial Collage PressLondon UK 2000

6 Disease Markers

[4] C A Taype J C Castro R A Accinelli P Herrera-Velit MA Shaw and J R Espinoza ldquoAssociation between SLC11A1polymorphisms and susceptibility to different clinical forms oftuberculosis in the Peruvian populationrdquo Infection Genetics andEvolution vol 6 no 5 pp 361ndash367 2006

[5] J E Curran T Vaughan R A Lea S R Weinstein N AMorrison and L R Griffiths ldquoAssociation of a vitaminD recep-tor polymorphism with sporadic breast cancer developmentrdquoInternational Journal of Cancer vol 83 no 6 pp 723ndash726 1999

[6] A Svejgaard and L P Ryder ldquoHLA and disease associationsdetecting the strongest associationrdquo Tissue Antigens vol 43 no1 pp 18ndash27 1994

[7] B Woolf ldquoOn estimating the relation between blood group anddiseaserdquo Annals of Human Genetics vol 19 no 4 pp 251ndash2531955

[8] N Arji M Busson G Iraqi et al ldquoGenetic diversity of TLR2TLR4 and VDR loci and pulmonary tuberculosis in moroccanpatientsrdquo Journal of Infection in Developing Countries vol 8 no4 pp 430ndash440 2014

[9] M Y Areeshi R K Mandal A K Panda and S HaqueldquoVitamin D receptor apai gene polymorphism and tuberculosissusceptibility a meta-analysisrdquo Genetic Testing and MolecularBiomarkers vol 18 no 5 pp 323ndash329 2014

[10] L Joshi M Ponnana S R Penmetsa P Nallari V Valluri andS Gaddam ldquoSerum vitamin D levels and VDR polymorphisms(BsmI and FokI) in patients and their household contacts sus-ceptible to tuberculosisrdquo Scandinavian journal of immunologyvol 79 no 2 pp 113ndash119 2014

[11] C Chen Q Liu L Zhu H Yang and W Lu ldquoVitamin Dreceptor gene polymorphisms on the risk of tuberculosis ameta-analysis of 29 case-control studiesrdquo PLoS ONE vol 8 no12 Article ID e83843 2013

[12] Y-J Wu X Yang X-X Wang et al ldquoAssociation of vitamin Dreceptor BsmI gene polymorphism with risk of tuberculosis ameta-analysis of 15 studiesrdquo PLoS ONE vol 8 no 6 Article IDe66944 2013

[13] A Simon-Gruita G Duta Cornescu N Constantin M DPojoga T Saitan andV Stoian ldquoApa I andTaq I polymorphismsof VDR (vitamin D receptor) gene in association with suscep-tibility to tuberculosis in the Romanian populationrdquo RomanianBiotechnological Letters vol 8 no 1 pp 7956ndash7962 2013

[14] O Ates B Dolek L Dalyan B Musellim G Ongen andA Topal-Sarikaya ldquoThe association between BsmI variant ofvitamin D receptor gene and susceptibility to tuberculosisrdquoMolecular Biology Reports vol 38 no 4 pp 2633ndash2636 2011

[15] S M Marashian P Farnia S Seyf S Anoosheh and A AVelayati ldquoEvaluating the role of vitamin D receptor polymor-phisms on susceptibility to tuberculosis among Iranian patientsa case-control studyrdquo Tuberkuloz ve Toraks vol 58 no 2 pp147ndash153 2010

[16] L Gao Y Tao L Zhang andQ Jin ldquoVitaminD receptor geneticpolymorphisms and tuberculosis updated systematic reviewand meta-analysisrdquo The International Journal of Tuberculosisand Lung Disease vol 14 no 1 pp 15ndash23 2010

[17] Z-Z Zhao T-Z Zhang Y-M Gao and F-M Feng ldquoMeta-analysis of relationship of vitamin D receptor gene polymor-phism and tuberculosis susceptibilityrdquo Zhonghua Jie He He HuXi Za Zhi vol 32 no 10 pp 748ndash751 2009

[18] M Merza P Farnia S Anoosheh et al ldquoThe NRAMPIVDR and TNF-120572 gene polymorphisms in Iranian tuberculosispatients the study on host susceptibilityrdquo Brazilian Journal ofInfectious Diseases vol 13 no 4 pp 252ndash256 2009

[19] R Olesen C Wejse D R Velez et al ldquoDC-SIGN (CD209)pentraxin 3 and vitamin D receptor gene variants associatewith pulmonary tuberculosis risk in West Africansrdquo Genes andImmunity vol 8 no 6 pp 456ndash467 2007

[20] A KWilbur L S Kubatko AM Hurtado K R Hill and A CStone ldquoVitamin D receptor gene polymorphisms and suscepti-bility M tuberculosis in Native Paraguayansrdquo Tuberculosis vol87 no 4 pp 329ndash337 2007

[21] Z Lombard D-L Dalton P A Venter R C Williams andL Bornman ldquoAssociation of HLA-DR -DQ and vitamin Dreceptor alleles and haplotypes with tuberculosis in the Vendaof South Africardquo Human Immunology vol 67 no 8 pp 643ndash654 2006

[22] L Bornman S J Campbell K Fielding et al ldquoVitamin Dreceptor polymorphisms and susceptibility to tuberculosis inWest Africa a case-control and family studyrdquo The Journal ofInfectious Diseases vol 190 no 9 pp 1631ndash1641 2004

[23] W Liu W-C Cao C-Y Zhang et al ldquoVDR and NRAMP1 genepolymorphisms in susceptibility to pulmonary tuberculosisamong the Chinese Han population a case-control studyrdquo TheInternational Journal of Tuberculosis and Lung Disease vol 8no 4 pp 428ndash434 2004

[24] P Selvaraj G Chandra S M Kurian A M Reetha and P RNarayanan ldquoAssociation of vitamin D receptor gene variantsof BsmI ApaI and FokI polymorphisms with susceptibility orresistance to pulmonary tuberculosisrdquo Current Science vol 84no 12 pp 1564ndash1568 2003

[25] R J Wilkinson M Llewelyn Z Toossi et al ldquoInfluence ofvitamin D deficiency and vitamin D receptor polymorphismson tuberculosis among Gujarati Asians in west London a case-control studyrdquoThe Lancet vol 355 no 9204 pp 618ndash621 2000

[26] R Bellamy C Ruwende T Corrah et al ldquoTuberculosis andchronic hepatitis B virus infection in Africans and variation inthe vitamin D receptor generdquoThe Journal of Infectious Diseasesvol 179 no 3 pp 721ndash724 1999

[27] B Y Sinaga M Amin Y Siregar and S M SarumpaetldquoCorrelation between vitamin D receptor gene FOKI and BSMIpolymorphisms and the susceptibility to pulmonary tubercu-losis in an indonesian batak-ethnic populationrdquo Acta MedicaIndonesiana vol 46 no 4 pp 275ndash282 2014

[28] M Y Areeshi R K Mandal N Akhter A K Panda andS Haque ldquoEvaluating the association between taqi variant ofvitamin D receptor gene and susceptibility to tuberculosis ameta-analysisrdquo Toxicology International vol 21 no 2 pp 140ndash147 2014

[29] T J Kang S H Jin C E Yeum et al ldquoVitamin D receptor geneTaqI BsmI and FokI polymorphisms in Korean patients withtuberculosisrdquo Immune Network vol 11 no 5 pp 253ndash257 2011

[30] S J Lewis I Baker and G D Smith ldquoMeta-analysis of vitaminD receptor polymorphisms and pulmonary tuberculosis riskrdquoInternational Journal of Tuberculosis and Lung Disease vol 9no 10 pp 1174ndash1177 2005

[31] J C Delgado A Baena S Thim and A E Goldfeld ldquoEthnic-specific genetic associations with pulmonary tuberculosisrdquoTheJournal of Infectious Diseases vol 186 no 10 pp 1463ndash14682002

[32] F Canonne-Hergaux S Gruenheid G Govoni and P GrosldquoThe Nramp1 protein and its role in resistance to infectionand macrophage functionrdquo Proceedings of the Association ofAmerican Physicians vol 111 no 4 pp 283ndash289 1999

[33] S Searle and J M Blackwell ldquoEvidence for a functionalrepeat polymorphism in the promoter of the human NRAMP1

Disease Markers 7

gene that correlates with autoimmune versus infectious diseasesusceptibilityrdquo Journal of Medical Genetics vol 36 no 4 pp295ndash299 1999

[34] R Bellamy ldquoNRAMP and susceptibility to tuberculosisrdquo TheInternational Journal of Tuberculosis and Lung Disease vol 6no 9 p 747 2002

[35] R Bellamy C Ruwende T Corrah K P W J McAdam HC Whittle and A V S Hill ldquoVariations in the NRAMP1 geneand susceptibility to tuberculosis in West Africansrdquo The NewEngland Journal of Medicine vol 338 no 10 pp 640ndash644 1998

[36] S Ryu Y-K Park G-H Bai S-J Kim S-N Park and S Kangldquo31015840UTR polymorphisms in the NRAMP1 gene are associatedwith susceptibility to tuberculosis in Koreansrdquo InternationalJournal of Tuberculosis and Lung Disease vol 4 no 6 pp 577ndash580 2000

[37] F Wu W Zhang L Zhang et al ldquoNRAMP1 VDR HLA-DRB1 and HLA-DQB1 gene polymorphisms in susceptibilityto tuberculosis among the Chinese Kazakh population a case-control studyrdquo BioMed Research International vol 2013 ArticleID 484535 8 pages 2013

[38] Y-S Liaw J-J Tsai-Wu C-H Wu et al ldquoVariations in theNRAMP1 gene and susceptibility of tuberculosis in TaiwaneserdquoInternational Journal of Tuberculosis and Lung Disease vol 6no 5 pp 454ndash460 2002

[39] S Vejbaesya N Chierakul P Luangtrakool andC Sermduang-prateep ldquoNRAMP1 andTNF-120572polymorphisms and susceptibil-ity to tuberculosis in Thaisrdquo Respirology vol 12 no 2 pp 202ndash206 2007

[40] J El Baghdadi N Remus A Benslimane et al ldquoVariants ofthe human NRAMP1 gene and susceptibility to tuberculosisin Moroccordquo International Journal of Tuberculosis and LungDisease vol 7 no 6 pp 599ndash602 2003

[41] C Soslashborg A B Andersen H O Madsen A Kok-Jensen PSkinhoslashj and P Garred ldquoNatural resistance-associated macro-phage protein 1 polymorphisms are associated with micros-copy-positive tuberculosisrdquo The Journal of Infectious Diseasesvol 186 no 4 pp 517ndash521 2002

[42] M A Shaw A Collins C S Peacock et al ldquoEvidencethat genetic susceptibility to Mycobacterium tuberculosis in aBrazilian population is under oligogenic control linkage studyof the candidate genes NRAMP1 and TNFArdquo Tubercle and LungDisease vol 78 no 1 pp 35ndash45 1997

[43] J Nugraha and R Anggraini ldquoNRAMP1 polymorphism andsusceptibility to lung tuberculosis in Surabaya IndonesiardquoSoutheast Asian Journal of Tropical Medicine and Public Healthvol 42 no 2 pp 338ndash341 2011

[44] Y-H Hsu C-W Chen H S Sun R Jou J-J Lee and I-J Su ldquoAssociation of NRAMP 1 gene polymorphism withsusceptibility to tuberculosis in Taiwanese aboriginalsrdquo Journalof the FormosanMedical Association vol 105 no 5 pp 363ndash3692006

[45] M Nairz D Haschka E Demetz and G Weiss ldquoIron at theinterface of immunity and infectionrdquo Frontiers in Pharmacol-ogy vol 5 article 152 10 pages 2014

[46] J MMcDermid and AM Prentice ldquoIron and infection effectsof host iron status and the iron-regulatory genes haptoglobinand NRAMP1 (SLC11A1) on host-pathogen interactions intuberculosis and HIVrdquo Clinical Science vol 110 no 5 pp 503ndash524 2006

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014


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EndocrinologyInternational Journal of



Disease Markers


BioMed Research International

OncologyJournal of



Oxidative Medicine and Cellular Longevity


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The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

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Computational and Mathematical Methods in Medicine

OphthalmologyJournal of


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Research and TreatmentAIDS


Gastroenterology Research and Practice


Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

2 Disease Markers

In this study we investigated associations ofNRAMP1 andVDR gene polymorphisms with susceptibility to TB in theVenezuelan population

2 Material and Methods

21 Subjects Thestudy included one hundred andninety-five(195) unrelated and ethnically mixed Venezuelan individualsfrom the northern region of country divided into twocohorts patients and controls

Patients The ninety-three individuals all had a clinicaldiagnosis of pulmonary tuberculosis and were seen by thePneumology Service of the Hospital Jose Ignacio BaldoAlgodonal Caracas There were 52 men (56) and 41 women(44) with an age range of 17ndash70 years Patients were selectedbased on the diagnostic criteria of the National StandardIntegrated Venezuela Tuberculosis Control Program twosputum smears positive for acid fast bacilli by microscopyclinical symptoms characteristic of TB and a chest X rayconsistent with active TB disease

ControlsThere were one hundred and two apparently healthyindividuals who were known to be exposed to patients withactive TB This group was composed of staff members whohad worked at Hospital Dr Jose Ignacio Baldo AlgodonalCaracas for more than 3 years and included 25 men (245)and 77 women (755) whose age range was 20ndash67 yearsThecontrols were classified according to the tuberculin skin testldquoTSTrdquo as follows positiveTST (51102) negativeTST (19102)or without information (32102) All controls were withoutclinical manifestations of TB at the time of blood sampling

Individuals who were HIV positive or known to have anyautoimmune chronic inflammatory or other disease wereexcluded from the study Participants in the study signedan informed consent form previously approved by the IVICBioethics Committee

22 NRAMP1 and VDR Genotype Analysis Genomic DNAwas extracted fromblood samples according to the proceduredescribed by Bunce [3] The polymorphic variants of theNRAMP1 gene were studied by PCR-RFLP technique usingprimers and restriction enzymes reported by Taype et al2006 [4] INT4 (469 + 14GC) D543 (codon 543 Arg rarrAsp) and 31015840UTR (deletion of TGTG in the 31015840UTR 55 nt 31015840to the last codon in exon 15) The polymorphic variants ofthe VDR gene were studied by PCR-RFLP using primers andrestriction enzymes reported by Curran et al 1999 [5] FokIApaI and TaqI

23 Statistical Analysis Allele and genotype frequencieswere determined by direct counting The Hardy-Weinbergequilibrium was calculated with the exact test The statisticalsignificance of allele frequency differences between patientsand controls was estimated by Fisherrsquos exact test using 2 times2 contingency tables The Bonferroni corrected 119901 values(119901c) were obtained by multiplying the 119901 values by the totalnumber of variables analyzed andwere considered significant

when 119901 lt 005 [6] Relative risk with corresponding 95confidence intervals (95 CI) was calculated as odds ratios(OR) according to Woolf rsquos formula [7]

3 Results

31 Frequency of NRAMP1 Polymorphisms in Patients withTuberculosis and Controls Table 1 shows the frequencies ofNRAMP genotypes and alleles in controls and TB patientsThe allelic and genotypic frequencies of the NRAMP1 poly-morphisms showed Hardy-Weinberg equilibrium for INT4(119901 = 0832) D543 (119901 = 0296) and 31015840UTR (119901 =0594) polymorphisms in the control cohort There wasa significantly increased frequency of homozygous 31015840UTRTGTG++ genotype in the healthy controls compared tothe TB patients (79 versus 64 resp OR = 04 95 CI02478ndash09045 119901 = 001 119901c = 003) However the frequencyof the heterozygous 31015840UTR SNP TGTG+del (326 versus21 resp OR = 18 95 CI 09452ndash34976 119901 = 003)and homozygous 31015840UTR SNP deldel genotypes (34 versus0 resp OR = 813 95 CI 04143ndash1596275 119901 = 002)was higher in the patient group than in the control groupalthough the corrected 119901 values (119901c) were not significant Inaddition there was a significant difference in the distributionof the allele frequencies (TGTG+ and TGTG del) betweenthe controls and the TB patients (119901c = 0018) There was nosignificant association between INT4 and D543 genotypeswith tuberculosis

32 Frequency of the VDR Polymorphism in Patients withTuberculosis and Controls Table 2 shows the frequenciesof VDR genotypes and alleles in controls and TB patientsThere was Hardy-Weinberg equilibrium for the genotypedistributions of FokI (119901 = 0074) TaqI (119901 = 0066) andApaI (119901 = 0545) polymorphisms in apparently healthyindividuals The data showed that the frequency of the FFSNP genotype of FokI was higher in the patients than inthe control group (366 versus 255 resp OR = 17 95CI 09120ndash31109 119901 = 004) although the corrected 119901 valuewas not significant No significant difference in the allelefrequencies was observed between the TB patients and thecontrols

33 Genotype and Allele Distribution of NRAMP-31015840UTRVariants in Patients with Tuberculosis and Healthy ControlsClassified by the Tuberculin Skin Test (TST) In order toinvestigate the possible influence of NRAMP1-31015840UTR vari-ants in the development of tuberculosis we compared thegenotype and allele frequencies between the different groupsTB patients versus TST positive controls TB patients versusTST negative controls and TST positive controls versus TSTnegative controls (Table 3)There were statistically significantdifferences between TB patients and TST negative controlsfor TGTG++ (64 versus 947 OR 01 95 CI 00126ndash07760 119901 = 0004 119901c = 0012) and TGTG+del genotypes(326 versus 53 OR 87 95CI 11069ndash683801 119901 = 0008119901c = 0016) These same differences were conserved whenthe comparison was made between TST positive controls

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NRAMP1 and VDR Gene Polymorphisms in Susceptibility to Tuberculosis in Venezuelan Population

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Transcript of NRAMP1 and VDR Gene Polymorphisms in Susceptibility to Tuberculosis in Venezuelan Population