Clinical Study Role of Toll-Interacting Protein Gene ...Genetic Division Department, Biomedical Research Center West, Instituto Mexicano del Seguro Social, Sierra Mojada , Colonia

Hindawi Publishing CorporationBioMed Research InternationalVolume 2013 Article ID 459169 7 pageshttpdxdoiorg1011552013459169

Clinical StudyRole of Toll-Interacting Protein Gene Polymorphisms inLeprosy Mexican Patients

Margarita Montoya-Buelna12 Mary Fafutis-Morris23 Alvaro J Tovar-Cuevas2

Anabell Alvarado-Navarro23 Yeminia Valle2 Jorge R Padilla-Gutierrez2

Jose F Muntildeoz-Valle2 and Luis E Figuera-Villanueva4

1 Centro Universitario de Tonala Universidad de Guadalajara Tonala Jal Mexico2 Centro Universitario de Ciencias de la Salud Universidad de Guadalajara Guadalajara Jal Mexico3 CIINDE Universidad de GuadalajaraInstituto Dermatologico de Jalisco ldquoDr Jose Barba Rubiordquo Sierra Mojada 950Edificio P Primer Nivel Colonia Independencia 44340 Guadalajara Jal Mexico

4Genetic Division Department Biomedical Research Center West Instituto Mexicano del Seguro Social Sierra Mojada 800Colonia Independencia 44340 Guadalajara Jal Mexico

Correspondence should be addressed to Mary Fafutis-Morris mfafutisgmailcomand Luis E Figuera-Villanueva luisfiguerayahoocom

Received 30 April 2013 Revised 9 September 2013 Accepted 14 September 2013

Academic Editor Yoshio Ishibashi

Copyright copy 2013 Margarita Montoya-Buelna 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

Background Leprosy is a debilitating infectious disease of human skin and nerves Genetics factors of the host play an importantrole in the disease susceptibility Toll-interacting protein (TOLLIP) is an inhibitory adaptor protein within the toll-like receptor(TLR) pathway which recognizes structurally conserved molecular patterns of microbial pathogens initiating immune responsesThe objective of this study was to investigate the association of variants in the TOLLIP gene with susceptibility to leprosy inMexican patientsMethodsTOLLIP polymorphismswere studied using a case-control design ofMexican patients with lepromatousleprosy (LL) The polymorphisms of TOLLIP at loci minus526 CgtG (rs5743854) 1309956CgtT (rs3750920) 1298430CgtA (rs5744015)and 1292831 GgtA (rs3750919) were analyzed by PCR with sequence-specific primers in LL patients and healthy subjects (HS) ascontrols Results Genotype distributions were in Hardy Weinberg equilibrium for all sites except for rs3750920 Neither genotypenor allele frequencies were statistically different between LL patients and controls (119875 gt 005)Themaximum pairwise Drsquo coefficientreached was 044 of linkage (119875 = 001) for all the polymorphisms except for rs5743854 The three loci haplotype comparisonyielded no significant differences between groups Conclusions Just the individuals with genotype CC of rs3750920 have a trendof protective effect to developing LL

1 Background

Leprosy (L) or Hansenrsquos disease is a chronic infection causedby the intracellular pathogen Mycobacterium leprae [1] Theregistered prevalence globally at the beginning of 2012 was181941 leprosy patients [2] Leprosy is a disease that presentsas a spectrum of clinical manifestations that depend on thehost cell-mediated immune response against the M leprae[3] One end of the disease spectrum patients with tubercu-loid leprosy (TT) shows a strong cellular immune responseresulting in resistance responses that restrict the growth of

the pathogen The number of lesions is few and bacilli rarealthough tissue and nerve damage are frequent At the oppo-site end of this spectrum patients with lepromatous leprosy(LL) have a limited cellular immune response making themsusceptible to disseminated infection Skin lesions are numer-ous and growth of the pathogen is unabated The factors thatinfluence which type of leprosy develops are not well under-stoodMany epidemiological studies which had the objectiveof identifying susceptibility genes [4] have shown how hostgenetic factors play a role in the variability of clinical responseto infection caused byM leprae

2 BioMed Research International

Human toll-like receptors (TLRs) are a family of patternrecognition receptors capable of initiating innate immuneresponses and influencing subsequent adaptive immuneresponses [5] Mycobacterial TLR ligands include moleculesof bacterial cell wall components like that found inM lepraebacilli (lipomannan lipoarabinomannan phosphatidylinos-itol-dimannoside and a 19-kDa lipoprotein) [6ndash11] Theiractivation in response to microbial infection and inflamma-tion triggers NF-120581B and mitogen-activated proteins kinase(MAPK) signaling and culminates in the induction of hostdefense genes including the production of numerous cyto-kines chemokines adhesion molecules and enzymes How-ever excessive and prolonged activation of innate immunityis harmful to the host and may even be fatal [12] Signalingmust therefore be tightly controlled to ensure a beneficialresponse with the appropriate magnitude and duration Mul-tiple mechanisms exist to halt IL1RI and TLR signaling path-ways including the activity of several inducible signalingsuppressors (ie MyD88 and IRAK-M) Toll-interactingprotein (TOLLIP) is an adaptor protein and acts as an inhib-itory factor in the TLR-signaling cascade overexpression ofTOLLIP impairs IL1RI- TLR2- and TLR4-triggered NF-120581Band JNK signaling pathways [12ndash15] Following the stimula-tion of these receptors TOLLIP associates directly with thecytoplasmic TIR domain [12 16] suppressing the phosphory-lation and kinase activity of interleukin-1 receptor associatedkinase 1 (IRAK-1) [16] and in this way controls themagnitudeof inflammatory response to endotoxin [17]

TOLLIP is therefore thought to function mainly to main-tain immune cells in a quiescent state and to facilitate the ter-mination of IL-1RTLR-induced cell signaling during inflam-mation via suppression of IRAK-1rsquos activity [13 16] TOLLIPmoreover deregulated inhibition of this pathwaymight resultin attenuated responses to the proinflammatory cytokines(IL-6 and TNF-120572) suppression of TLR-mediated cellularresponses and a role in the leprosy

Several single-nucleotide polymorphisms in the TLR sig-naling pathways and their negative regulators genes havebeen reported to influence the production of inflammatorycytokines and implicated in the pathogenesis of allergicautoimmune as well as to infectious diseases [18ndash26] How-ever effects of genetic variation in TLRs downstream signal-ing pathways have not been amply studied yet

The TOLLIP gene is located on chromosome 11p15 (MIM606 277) and comprises 6 exon encoding 274 amino acidtranscripts It is a negative regulator of TLR signaling cas-cade and was implicated in suppression of the TLR2 [12]and TLR4 pathways [14] We compared the distribution offour SNPs at TOLLIP gene minus526 CG (rs5743854) exon 4Pro139Pro (rs3750920) exon 6 Ala222Ser (rs5744015) and31015840UTR (rs3750919) in a population-based design study of LLMexican patients

2 Methods

21 Patients and Controls Leprosy patients (119899 = 93) attendedin the Instituto Dermatologico de Jalisco (SSA) and tookpart in this study All patients were residents in the westernof Mexico They were classified according to clinical and

laboratorial (Mitsudarsquos test and histology exams) observa-tionsmade by dermatologist responsible for leprosy diagnosisas LL patients summarized in Table 1

The control group was composed of 152 healthy indi-viduals of Mexican mestizo ethnicity selected according todemographic parameters The mestizo ethnicity is the resultof genetic crossing between Spanish and Amerindian people[27] and represents 80 of the overall population of Mexico[28] Matching patients minimized the risk of populationstratification bias due to differences in ethnic backgroundbetween patients and controls and variations of allele fre-quencies according to ethnic background with control indi-viduals of the same ethnic backgrounds and residence inthe same geographical areas of leprosy prevalence Ethicscommittee of the Instituto Dermatologico de Jalisco (SSA)approved the study and Declarations of Helsinki protocolswere followed Samples were taken only after participants vol-untarily signed an informed consent form Blood sampleswere collected from patients and healthy subjects (HS) inethylenediamine tetra-acetic acid anticoagulant DNA wasextracted using a standard salting-out method [29]

22 Genotyping All four polymorphisms in the TOLLIPgene were genotyped by restriction enzyme digestion (seeTable 1) Patient and control samples were amplified usingthe same primers and conditions as previously described bySchimming et al [30] After digestion with the respectiveenzymes for at least three hours the fragmentswere separatedon polyacrylamide gel electrophoresed in 1X TBE buffer(Native PAGE) and viewed by staining with 02 silvernitrate according to Sanguinetti et al [31]

23 Statistical Analysis Allele frequency was obtained bydirect counting The agreement of Hardy-Weinberg equilib-rium was evaluated using the SAS software v8 (SAS InstituteInc Cary NC USA) Statistical significance was indicated by119875 lt 005 The odds ratios (OR) with 95 confidence inter-vals (95 CI) were calculated using SISA statistics online(httphomeclaranetsisa) to evaluate the risk of the indi-vidual developing the disease while having a particularTOLLIP type Haplotypes frequencies were inferred usingEMHAPFREQ software [32] Pairwise linkage disequilibrium(LD) was expressed as Lewontinrsquos D1015840 corrected coefficient[33] Comparison data was evaluated by 1205942 test or the Fisherexact-test when applicable

3 Results

A total of 98 LL patients were recruited and 152 unrelated oth-erwise healthy individual as a control Analysis of genotypeand allele frequencies of TOLLIP was done in both groupssummarized in Table 2 In controls genotype distributionswere in Hardy Weinberg equilibrium for all sites except forrs3750920 (exon 4)

31 Leprosy Susceptibility To identify a genetic relation bet-ween TOLLIP SNPs and lepromatous pole of clinical spec-trum of leprosy four previously published TOLLIP poly-morphisms at loci minus526 CgtG (rs5743854) 1309956CgtT

BioMed Research International 3

Table 1 Clinical and demographic characteristics of patients with LL

Characteristic LL 119899 = 98Disease duration (years) mean plusmn SD 10 plusmn 79Age mean plusmn SD 5463 plusmn 1592Gender

Female () 36Male () 62

Family history () 38Bacilloscopy ( +++++) 100Mitsuda test ( negatives) 100Treatment PCT () 56New cases (non-Tx ) 44Quantitative variables are expressed asmeansplusmn standard deviations (SD) and qualitative variables as frequencies and percentages () as noted LL lepromatousleprosy PCT polychemotherapy (Rifampin Clofazimine Dapsone) Tx treatment Family history refers to at least one first grade family who has been infectedwith leprosy bacillus Bacilloscopy samples were taken from ear smear

(rs3750920) 1298430CgtA (rs5744015) and 1292831 GgtA(rs3750919) were screened [30] Our results showed that thegenotypes frequencies of tagging SNPs rs5743854 (119875 = 076)rs3750920 (119875 = 005) rs5744015 (119875 = 076) and rs3750919(119875 = 076) were nonsignificant differences of LL patientsversus HS However in LL patients a decreased frequency inCC genotype of rs3750920 (25) in comparison with HS(37) was observed (119875 = 005) These trends could suggesta protective effect in western population from Mexico Alsowhen we analyzed the allele frequencies no significant dif-ferences were found (rs5743854 119875 = 015 rs3750920 119875 =009 rs5744015 119875 = 100 rs3750919 119875 = 029) The alleleand genotype frequencies of the four tagging SNPs in the LLpatients and control subjects and the statistical analysisresults are listed in Table 2

32 Haplotype and Linkage Disequilibrium Estimation Hap-lotypes were estimated based on the genotype frequencies ofthe LL patients and control group (Table 3) Regarding tolinkage disequilibrium analysis (Table 3) only the polymor-phisms located at exon 4 exon 6 and 31015840UTR showed nonran-dom segregation where the maximumD1015840 coefficient reacheda moderate value of LD (LD = 044 Bonferroni correction 119875value = 001) [34]The haplotype comparison for these linkedsites (rs3750920 rs5744015 and rs3750919) showed similardistributions between LL patients and the control groupConversely Schimming et al [30] found linkage evidencebetween the promoter (rs5743854) and 31015840UTR (rs3750919)and other intermediate polymorphisms Since the chromoso-mal position of the polymorphisms was tested in this studywe expected similar results however as it has been describedLD is influenced by population type [35] among other factors[34]

4 Discussion

In leprosy a variety of host immune-genetic factors seemto influence subjectsrsquo susceptibility to M leprae and diseasecourse Human regulation and nature of an optimal inflam-matory response to M leprae remain poorly understood

Mycobacterial cell wall contains a number of toll-like recep-tors (TLRs) ligands including lipoproteins mycolylarabino-galactan-peptidoglycan complexes lipids and lipoarabino-mannan (LAM) Although TLRs are crucial for host defenseit has become apparent that loss of negative regulation ofTLR signaling is strongly linked to acute and chronic inflam-mation as well as systemic autoimmune diseases [18ndash25]In leprosy patients TLR activation may promote excessiveinflammation and apoptosis contributing to pathologicalsigns such as nerve damage [36] A balance of TLR expressionand activation could be crucial to avoid an excessive inflam-matory response and lessen disease severity Negative regula-tion of TLR-induced response is important for suppressinginflammation and deleterious immune responses TOLLIPhas been identified as a negative regulator that suppressesTLR signaling at multiple levels and thus high levels coulddiminish TLR signaling this phenomenon has been observedin response to pathogen challenge [13 37 38] Some bacte-rial components stimulate TLR signaling activating NF120581BJNK and p38 signaling cascades leading to transcriptionand translation of IL-6 and TNF120572 [39] Thus beneficial ordeleterious effects of TOLLIP in leprosy patients depend onexpression level and time of exposure to proinflammatorycytokine It is hypothesized that in leprosy patients highTOLLIP levels could generate a low inflammatory responseagainst mycobacteria however low TOLLIP levels could pro-duce a sustained inflammatory response which is associatedwith tissue damage Screening ofTOLLIP gene by sequencingand SSCP analysis [26 30 40] has shown several muta-tions which affect gene expression and biological functionmodifying inflammatory-cytokine productionTherefore weevaluated whether some of these mutations could influencethe susceptibility to LL We analyze four TOLLIP SNPsrs5743854 at promoter region rs3750920 and rs5744015 in thecoding region and rs3750919 at 31015840UTR

Our study shows that there is no significant associationbetween genotype and allele frequencies of TOLLIP variationminus526GgtC (rs5743854) with LL (Table 2) However it has beenobserved that the same variation of TOLLIP gene showedborderline associationwith atopic dermatitis which is a com-mon chronic inflammatory skin disorder We observed in


Table 2 Genotype and allele distributions of minus526 exon 4 exon 6 and 31015840UTR TOLLIP polymorphisms in LL and HS

Polymorphism LL (119899 = 98)n ()

HS (119899 = 152)n () OR CI (95) 119875 value

rs5743854(minus526 CgtG)Genotype

CC 84 (86) 127 (84)CG 13 (13) 22 (14) 089 (043ndash187) 119875 = 076

GG 1 (1) 3 (2) 050 (005ndash493) 119875 = 055

AlleleC 181 (92) 276 (91)G 15 (8) 28 (9) 082 (042ndash016) 119875 = 054

HWE=043rs3750920(1309956 CgtT)Genotype

CC 24 (25) 56 (37)CT 63 (64) 83 (54) 177 (099ndash316) 119875 = 005

TT 11 (11) 13 (9) 197 (0772ndash502) 119875 = 015

AlleleC 111 (57) 195 (64)T 85 (43) 109 (36) 137 (095ndash198) 119875 = 009

HWE=004rs5744015(1298430 CgtA)Genotype

CC 96 (98) 148 (97)CA 2 (2) 4 (3) 080 (014ndash429) 119875 = 076

AA 0 (0) 0 (0) 154 (003ndash782) 119875 = 100

AlleleC 194 (99) 300 (99)A 2 (1) 4 (1) 077 (014ndash426) 119875 = 100

HWE= 100rs3750919(1292831 GgtA)Genotype

GG 48 (49) 81 (53)GA 40 (41) 62 (41) 109 (064ndash186) 119875 = 076

AA 10 (10) 9 (6) 187 (071ndash494) 119875 = 019

AlleleG 136 (69) 224 (74)A 60 (31) 80 (26) 124 (083ndash184) 119875 = 029

HWE=081LL lepromatous leprosy HS healthy subjects OR odds ratio C confidence interval HWE Hardy-Weinberg equilibrium 119875 value codominant model ispresented

healthy heterozygous subjects that genotype rs5743854 GCshowed a similar frequency (14) to that reported in Cau-casian German control subjects (154) [30] Furthermoreallele frequency in our control group (C allele 90 A allele10) showed a similar allele frequency compared to Germancontrols (C allele 923 A allele 77)

We observed that rs3750920 CT genotype was predom-inant in LL patients (64) and controls (54) comparedto CC (LL patients (25) and controls (37)) and TT(LL patients (11) and controls (9)) genotypes Allele Cfrequency was also more common in LL (57) and controls(63) groups Moreover LL patients showed a decreased


Table 3 Haplotype frequencies of the rs3750920 rs5744015 and rs3750919 TOLLIP polymorphisms in LL and HS

Haplotype LL (2119899 = 196)n ()

HS (2119899 = 304)n ()

ORCI (95) 119875 value

CCG 96 (489) 160 (527) lowast

CCA 15 (768) 32 (105) 078 (040ndash152) 119875 = 047

CAA 0 (0) 1 (0002) 055 (002ndash137) 119875 = 072

CAG 0 (0) 2 (0008) 033 (002ndash700) 119875 = 048

TCG 38 (194) 61 (202) 103 (064ndash167) 119875 = 088

TCA 45 (229) 47 (154) 159 (099ndash258) 119875 = 006

TAA 0 (0) 1 (0002) 166 (003ndash845) 119875 = 080

TAG 2 (102) 0 (0) 83 (040ndash175) 119875 = 017

LL lepromatous leprosy HS healthy subjects OR odds ratio CI confidence interval lowastReference haplotype

frequency in rs3750920 CC genotype (25) in comparisonto HS (37) (119875 = 005)These tendencies suggest a protectiveeffect to eventually development LL disease (OR 177) inMex-ican western population However Shah et al [26] observedthat rs3750920 SNPs were associated with protection fromtuberculosis and increased levels of TOLLIPmRNA and thiswas the first association of TOLLIP polymorphisms with anyinfectious disease These differences could be due to differ-ent genetic backgrounds (Amerindians Spanish and WestAfrican predominantly) from analyzed subjects which havedifferent admixture grades depending on Mexico regionTherefore this genetic heterogeneity (also described as asym-metric admixture) could explain intra- and interpopula-tion differences in significant gene-diseases associations andprevalence for some diseases Although the best solution todescribe these associations would be the analysis of admix-ture informative markers (AIMs) to probe the ancestralhomogeneity between case and control population samplesa practical solution could be done to demonstrate that thelocus of interest has similar allele frequency in parental pop-ulations avoiding population structures problems to achieveconvincing inferences

We also evaluated rs5744015 and rs3750919 polymor-phisms variants and none of them showed significant differ-ences in allelic and genotypic frequencies from patients ver-sus control group We observed that rs5744015 CC genotypewas predominant in LL patients (98) and controls (97)compared to CA (LL patients (2) and controls (3)) andAA (LL patients (0) and controls (0)) genotypes AlleleC frequency was also more common in LL (99) and control(99) groups The AA genotype was not present in patientsand controls which correlates with results found in studies[30] where AA genotype was also absent in patients andpresent in 05 in subjects from the control group Finallyfor rs3750919 region GG genotype was predominant in LLpatients (49) and controls (53) compared to GA (LLpatients (41) and controls (41)) and AA (LL patients(10) and controls (6)) genotypes Allele G frequency wasalso more common in LL (69) and control (74) groupsTherefore GG genotype frequencies as G allele were themost common in our patients and controls and these obser-vations were similar to those observed in previous reports onpatients with atopic dermatitis [30] Exhaustive sequencing

is required to find or rule out the possibility of an as-yet-unknown causal SNP in LL with rs5744015 and rs3750919Further functional evaluation of novel or associated SNPs isalso needed

Concerning to linkage disequilibrium analysis (Table 3)only the polymorphisms located at exon 4 exon 6 and 31015840UTRshowed nonrandom segregation [34] The haplotype com-parison for these linked sites (rs3750920 rs5744015 andrs3750919) showed similar distributions between LL patientsand control group Therefore even when alleles show linkagedisequilibrium this circumstance has no association with riskto develop leprosy in Mexican patients

The reasons for nonreplication of association studiesare numerous and largely reflect inadequate sample sizesalthough differences may also be attributed to populationstratification variation in study design confounding sam-pling bias and missclassification of phenotypes We cannotexclude the possibility that the association result founded forTOLLIP genotype frequencies with LL could be related toother TOLLIP SNPs or neighboring genes not evaluated inthis study which presents LD with this variation As addi-tional studies we contemplate including more patients eval-uating cytokine levels (IL-10 TNF-120572 and IL-6) and mRNAexpression of TOLLIP

Taken together these findings represent new insightsfor a better comprehension of negative regulation of TLR4and TLR 2 signaling pathway and effective mechanisms fortherapeutic intervention and treatment of inflammatory dis-eases We need to consider the recent and impacting findingsreported by Masaki et al in their in vitro studies where theydemonstrate that M leprae is able to induce cellular repro-gramming though epigenetics changes which take part inthe migration plasticity and immunomodulatory functionswhich are finally employed byM leprae to avoid the immuneresponse [41]

5 Conclusion

Individuals with genotype CC of rs3750920 have a trend ofprotective effect to developing LL and rs5743854 rs5744015and rs3750919 SNPs are not associated with LL In the globaltest haplotypes analyses were not associated with LL risk inMexican patients Genetic analysis of genes involved in


susceptibility to leprosy phenotypes will give us the clues ofgeneticmechanisms fromMexican leprosy patients and otherethnic or racial backgrounds

Abbreviations

CI Confidence intervalHS Healthy subjectsIRAK-1 Interleukin-1 receptor associated kinase 1L LeprosyLD Linkage disequilibriumLL Lepromatous leprosyOR Odds ratioPAMP Pathogen-associated molecular patternPCR Polymerase chain reactionRFLP Restriction fragment length polymorphismsSNP Single nucleotide polymorphismsTLR Toll-like receptorTOLLIP Toll-interacting proteinTT Tuberculoid leprosyM leprae Mycobacterium leprae

Conflict of Interests

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

Acknowledgments

The authors thank all patients involved in this research andstaff of the Instituto Dermatologico de Jalisco Dr Jose BarbaRubio SSA for their participation

References

[1] I M B Goulart and L R Goulart ldquoLeprosy diagnostic andcontrol challenges for a worldwide diseaserdquo Archives of Derma-tological Research vol 300 no 6 pp 269ndash290 2008

[2] ldquoGlobal leprosy situationrdquo The Weekly Epidemiological Recordvol 87 no 34 pp 317ndash328 2012

[3] D S Ridley andWH Jopling ldquoClassification of leprosy accord-ing to immunity A five-group systemrdquo International Journal ofLeprosy and Other Mycobacterial Diseases vol 34 no 3 pp255ndash273 1966

[4] E A Misch W R Berrington J C Vary Jr and T R HawnldquoLeprosy and the human genomerdquoMicrobiology and MolecularBiology Reviews vol 74 no 4 pp 589ndash620 2010

[5] R Medzhitov ldquoRecognition of microorganisms and activationof the immune responserdquo Nature vol 449 no 7164 pp 819ndash826 2007

[6] T K Means E Lien A Yoshimura S Wang D T Golenbockand M J Fenton ldquoThe CD14 ligands lipoarabinomannanand lipopolysaccharide differ in their requirement for toll-likereceptorsrdquo Journal of Immunology vol 163 no 12 pp 6748ndash6755 1999

[7] H D Brightbill D H Libraty S R Krutzik et al ldquoHost defensemechanisms triggered by microbial lipoproteins through toll-like receptorsrdquo Science vol 285 no 5428 pp 732ndash736 1999

[8] DMUnderhill A Ozinsky K D Smith andA Aderem ldquoToll-like receptor-2 mediates mycobacteria-induced proinflamma-tory signaling in macrophagesrdquo Proceedings of the NationalAcademy of Sciences of the United States of America vol 96 no25 pp 14459ndash14463 1999

[9] S R Krutzik M T Ochoa P A Sieling et al ldquoActivation andregulation of Toll-like receptors 2 and 1 in human leprosyrdquoNature Medicine vol 9 no 5 pp 525ndash532 2003

[10] N Court S Rose M-L Bourigault et al ldquoMycobacterial PIMsinhibit host inflammatory responses through CD14-dependentand CD14-independent mechanismsrdquo PLoS ONE vol 6 no 9Article ID e24631 2011

[11] P K Dagur B Sharma R Upadhyay et al ldquoPhenolic-glycolipid-1 and lipoarabinomannan preferentially modulateTCR- and CD28-triggered proximal biochemical events lead-ing to T-cell unresponsiveness inmycobacterial diseasesrdquo Lipidsin Health and Disease vol 11 p 119 2012

[12] Y Bulut E Faure L Thomas O Equils and M Arditi ldquoCoop-eration of Toll-like receptor 2 and 6 for cellular activation by sol-uble tuberculosis factor and Borrelia burgdorferi outer surfaceprotein a lipoprotein role of Toll-interacting protein and IL-1receptor signaling molecules in Toll-like receptor 2 signalingrdquoJournal of Immunology vol 167 no 2 pp 987ndash994 2001

[13] K Burns J Clatworthy L Martin et al ldquoTollip a new com-ponent of the IL-1RI pathway links IRAK to the IL-1 receptorrdquoNature Cell Biology vol 2 no 6 pp 346ndash351 2000

[14] T Li J Hu and L Li ldquoCharacterization of Tollip protein uponLipopolysaccharide challengerdquo Molecular Immunology vol 41no 1 pp 85ndash92 2004

[15] W Piao C Song H Chen et al ldquoEndotoxin tolerance dysregu-latesMyD88- andTollIL-1R domain-containing adapter induc-ing IFN-120573-dependent pathways and increases expression ofnegative regulators of TLR signalingrdquo Journal of LeukocyteBiology vol 86 no 4 pp 863ndash875 2009

[16] G Zhang and S Ghosh ldquoNegative regulation of toll-likereceptor-mediated signaling by TolliprdquoThe Journal of BiologicalChemistry vol 277 no 9 pp 7059ndash7065 2002

[17] A Didierlaurent B Brissoni D Velin et al ldquoTollip regulatesproinflammatory responses to interleukin-1 and lipopolysac-chariderdquoMolecular and Cellular Biology vol 26 no 3 pp 735ndash742 2006

[18] W-Q Song H-H Li H-B Chen J-S Yuan and X-J YinldquoRelationship between polymorphism sites of IRF5 TLR-9 andSLE patients in Shandong Han populationrdquo Zhonghua Yi XueZa Zhi vol 89 no 43 pp 3038ndash3042 2009

[19] D N Cook J W Hollingsworth II and D A Schwartz ldquoToll-like receptors and the genetics of innate immunityrdquo CurrentOpinion in Allergy and Clinical Immunology vol 3 no 6 pp523ndash529 2003

[20] A Soylu S Kizildag S Kavukcu et al ldquoTLR-2 Arg753GlnTLR-4 Asp299Gly and TLR-4 Thr399Ile polymorphisms inHenoch Schonlein purpura with and without renal involve-mentrdquo Rheumatology International vol 30 no 5 pp 667ndash6702010

[21] S Pandey R D Mittal M Srivastava et al ldquoImpact of Toll-like receptors [TLR] 2 (-196 to -174 del) and TLR 4 (Asp299GlyThr399Ile) in cervical cancer susceptibility in North IndianwomenrdquoGynecologic Oncology vol 114 no 3 pp 501ndash505 2009

[22] E G Pamer ldquoTLR polymorphisms and the risk of invasivefungal infectionsrdquo The New England Journal of Medicine vol359 no 17 pp 1836ndash1838 2008


[23] P-Y Bochud T R Hawn M R Siddiqui et al ldquoToll-like recep-tor 2 (TLR2) polymorphisms are associated with reversal reac-tion in leprosyrdquo Journal of Infectious Diseases vol 197 no 2 pp253ndash261 2008

[24] P-Y Bochud J W Chien K A Marr et al ldquoToll-like receptor 4polymorphisms and aspergillosis in stem-cell transplantationrdquoThe New England Journal of Medicine vol 359 no 17 pp 1766ndash1777 2008

[25] NW J Schroder C Hermann L Hamann U B Gobel T Har-tung and R R Schumann ldquoHigh frequency of polymorphismArg753Gln of the Toll-like receptor-2 gene detected by a novelallele-specific PCRrdquo Journal of Molecular Medicine vol 81 no6 pp 368ndash372 2003

[26] J A Shah C Vary T H Chau et al ldquoHuman TOLLIP regulatesTLR2 andTLR4 signaling and its polymorphisms are associatedwith susceptibility to tuberculosisrdquoThe Journal of Immunologyvol 189 no 4 pp 1737ndash1746 2012

[27] C Gorodezky C Alaez M N Vazquez-Garcıa et al ldquoThegenetic structure of Mexican Mestizos of different locationstracking back their origins through MHC genes blood groupsystems and microsatellitesrdquo Human Immunology vol 62 no9 pp 979ndash991 2001

[28] I Silva-Zolezzi AHidalgo-Miranda J Estrada-Gil et al ldquoAnal-ysis of genomic diversity in Mexican Mestizo populations todevelop genomic medicine in Mexicordquo Proceedings of theNational Academy of Sciences of the United States of Americavol 106 no 21 pp 8611ndash8616 2009

[29] S A Miller D D Dykes and H F Polesky ldquoA simple saltingout procedure for extracting DNA from human nucleated cellsrdquoNucleic Acids Research vol 16 no 3 p 1215 1988

[30] T T Schimming Q Parwez E Petrasch-ParwezMNothnagelJ T Epplen and S Hoffjan ldquoAssociation of toll-interactingprotein gene polymorphisms with atopic dermatitisrdquo BMCDermatology vol 7 article 3 2007

[31] C J Sanguinetti E D Neto and A J G Simpson ldquoRapid silverstaining and recovery of PCR products separated on polyacry-lamide gelsrdquo BioTechniques vol 17 no 5 pp 914ndash921 1994

[32] L Excoffier and M Slatkin ldquoMaximum-likelihood estimationof molecular haplotype frequencies in a diploid populationrdquoMolecular Biology and Evolution vol 12 no 5 pp 921ndash927 1995

[33] R C Lewontin ldquoThe Interaction of Selection and Linkage IiOptimumModelsrdquo Genetics vol 50 pp 757ndash782 1964

[34] E Lander and L Kruglyak ldquoGenetic dissection of complextraits guidelines for interpreting and reporting linkage resultsrdquoNature Genetics vol 11 no 3 pp 241ndash247 1995

[35] K L OrsquoConnor C R Hill P M Vallone and J M ButlerldquoLinkage disequilibrium analysis of D12S391 and vWA in USpopulation and paternity samplesrdquo Forensic Science Interna-tional vol 5 no 5 pp 538ndash540 2011

[36] R B Oliveira M T Ochoa P A Sieling et al ldquoExpression oftoll-like receptor 2 on human schwann cells a mechanism ofnerve damage in leprosyrdquo Infection and Immunity vol 71 no 3pp 1427ndash1433 2003

[37] G Melmed L S Thomas N Lee et al ldquoHuman intestinalepithelial cells are broadly unresponsive to toll-like receptor2-dependent bacterial ligands implications for host-microbialinteractions in the gutrdquo Journal of Immunology vol 170 no 3pp 1406ndash1415 2003

[38] J-M Otte E Cario and D K Podolsky ldquoMechanisms of crosshyporesponsiveness to toll-like receptor bacterial ligands inintestinal epithelial cellsrdquo Gastroenterology vol 126 no 4 pp1054ndash1070 2004

[39] S Akira and K Takeda ldquoToll-like receptor signallingrdquo NatureReviews Immunology vol 4 no 7 pp 499ndash511 2004

[40] Z Song J Yin C Yao et al ldquoVariants in the Toll-interactingprotein gene are associated with susceptibility to sepsis in theChineseHan populationrdquoCritical Care vol 15 no 1 article R122011

[41] T Masaki J Qu J Cholewa-Waclaw K Burr R Raaum andA Rambukkana ldquoReprogramming adult Schwann cells to stemcell-like cells by leprosy bacilli promotes dissemination of infec-tionrdquo Cell vol 152 no 1 pp 51ndash67 2013

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014


MEDIATORSINFLAMMATION

of


Behavioural Neurology

EndocrinologyInternational Journal of



Disease Markers


BioMed Research International

OncologyJournal of



Oxidative Medicine and Cellular Longevity


PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of



Computational and Mathematical Methods in Medicine

OphthalmologyJournal of


Diabetes ResearchJournal of



Research and TreatmentAIDS


Gastroenterology Research and Practice


Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom


Human toll-like receptors (TLRs) are a family of patternrecognition receptors capable of initiating innate immuneresponses and influencing subsequent adaptive immuneresponses [5] Mycobacterial TLR ligands include moleculesof bacterial cell wall components like that found inM lepraebacilli (lipomannan lipoarabinomannan phosphatidylinos-itol-dimannoside and a 19-kDa lipoprotein) [6ndash11] Theiractivation in response to microbial infection and inflamma-tion triggers NF-120581B and mitogen-activated proteins kinase(MAPK) signaling and culminates in the induction of hostdefense genes including the production of numerous cyto-kines chemokines adhesion molecules and enzymes How-ever excessive and prolonged activation of innate immunityis harmful to the host and may even be fatal [12] Signalingmust therefore be tightly controlled to ensure a beneficialresponse with the appropriate magnitude and duration Mul-tiple mechanisms exist to halt IL1RI and TLR signaling path-ways including the activity of several inducible signalingsuppressors (ie MyD88 and IRAK-M) Toll-interactingprotein (TOLLIP) is an adaptor protein and acts as an inhib-itory factor in the TLR-signaling cascade overexpression ofTOLLIP impairs IL1RI- TLR2- and TLR4-triggered NF-120581Band JNK signaling pathways [12ndash15] Following the stimula-tion of these receptors TOLLIP associates directly with thecytoplasmic TIR domain [12 16] suppressing the phosphory-lation and kinase activity of interleukin-1 receptor associatedkinase 1 (IRAK-1) [16] and in this way controls themagnitudeof inflammatory response to endotoxin [17]

TOLLIP is therefore thought to function mainly to main-tain immune cells in a quiescent state and to facilitate the ter-mination of IL-1RTLR-induced cell signaling during inflam-mation via suppression of IRAK-1rsquos activity [13 16] TOLLIPmoreover deregulated inhibition of this pathwaymight resultin attenuated responses to the proinflammatory cytokines(IL-6 and TNF-120572) suppression of TLR-mediated cellularresponses and a role in the leprosy

Several single-nucleotide polymorphisms in the TLR sig-naling pathways and their negative regulators genes havebeen reported to influence the production of inflammatorycytokines and implicated in the pathogenesis of allergicautoimmune as well as to infectious diseases [18ndash26] How-ever effects of genetic variation in TLRs downstream signal-ing pathways have not been amply studied yet

The TOLLIP gene is located on chromosome 11p15 (MIM606 277) and comprises 6 exon encoding 274 amino acidtranscripts It is a negative regulator of TLR signaling cas-cade and was implicated in suppression of the TLR2 [12]and TLR4 pathways [14] We compared the distribution offour SNPs at TOLLIP gene minus526 CG (rs5743854) exon 4Pro139Pro (rs3750920) exon 6 Ala222Ser (rs5744015) and31015840UTR (rs3750919) in a population-based design study of LLMexican patients

2 Methods

21 Patients and Controls Leprosy patients (119899 = 93) attendedin the Instituto Dermatologico de Jalisco (SSA) and tookpart in this study All patients were residents in the westernof Mexico They were classified according to clinical and

laboratorial (Mitsudarsquos test and histology exams) observa-tionsmade by dermatologist responsible for leprosy diagnosisas LL patients summarized in Table 1

The control group was composed of 152 healthy indi-viduals of Mexican mestizo ethnicity selected according todemographic parameters The mestizo ethnicity is the resultof genetic crossing between Spanish and Amerindian people[27] and represents 80 of the overall population of Mexico[28] Matching patients minimized the risk of populationstratification bias due to differences in ethnic backgroundbetween patients and controls and variations of allele fre-quencies according to ethnic background with control indi-viduals of the same ethnic backgrounds and residence inthe same geographical areas of leprosy prevalence Ethicscommittee of the Instituto Dermatologico de Jalisco (SSA)approved the study and Declarations of Helsinki protocolswere followed Samples were taken only after participants vol-untarily signed an informed consent form Blood sampleswere collected from patients and healthy subjects (HS) inethylenediamine tetra-acetic acid anticoagulant DNA wasextracted using a standard salting-out method [29]

22 Genotyping All four polymorphisms in the TOLLIPgene were genotyped by restriction enzyme digestion (seeTable 1) Patient and control samples were amplified usingthe same primers and conditions as previously described bySchimming et al [30] After digestion with the respectiveenzymes for at least three hours the fragmentswere separatedon polyacrylamide gel electrophoresed in 1X TBE buffer(Native PAGE) and viewed by staining with 02 silvernitrate according to Sanguinetti et al [31]

23 Statistical Analysis Allele frequency was obtained bydirect counting The agreement of Hardy-Weinberg equilib-rium was evaluated using the SAS software v8 (SAS InstituteInc Cary NC USA) Statistical significance was indicated by119875 lt 005 The odds ratios (OR) with 95 confidence inter-vals (95 CI) were calculated using SISA statistics online(httphomeclaranetsisa) to evaluate the risk of the indi-vidual developing the disease while having a particularTOLLIP type Haplotypes frequencies were inferred usingEMHAPFREQ software [32] Pairwise linkage disequilibrium(LD) was expressed as Lewontinrsquos D1015840 corrected coefficient[33] Comparison data was evaluated by 1205942 test or the Fisherexact-test when applicable

3 Results

A total of 98 LL patients were recruited and 152 unrelated oth-erwise healthy individual as a control Analysis of genotypeand allele frequencies of TOLLIP was done in both groupssummarized in Table 2 In controls genotype distributionswere in Hardy Weinberg equilibrium for all sites except forrs3750920 (exon 4)

31 Leprosy Susceptibility To identify a genetic relation bet-ween TOLLIP SNPs and lepromatous pole of clinical spec-trum of leprosy four previously published TOLLIP poly-morphisms at loci minus526 CgtG (rs5743854) 1309956CgtT








4 Discussion







HS (119899 = 152)n () OR CI (95) 119875 value


CC 84 (86) 127 (84)CG 13 (13) 22 (14) 089 (043ndash187) 119875 = 076

GG 1 (1) 3 (2) 050 (005ndash493) 119875 = 055

AlleleC 181 (92) 276 (91)G 15 (8) 28 (9) 082 (042ndash016) 119875 = 054


CC 24 (25) 56 (37)CT 63 (64) 83 (54) 177 (099ndash316) 119875 = 005

TT 11 (11) 13 (9) 197 (0772ndash502) 119875 = 015

AlleleC 111 (57) 195 (64)T 85 (43) 109 (36) 137 (095ndash198) 119875 = 009


CC 96 (98) 148 (97)CA 2 (2) 4 (3) 080 (014ndash429) 119875 = 076

AA 0 (0) 0 (0) 154 (003ndash782) 119875 = 100

AlleleC 194 (99) 300 (99)A 2 (1) 4 (1) 077 (014ndash426) 119875 = 100


GG 48 (49) 81 (53)GA 40 (41) 62 (41) 109 (064ndash186) 119875 = 076

AA 10 (10) 9 (6) 187 (071ndash494) 119875 = 019

AlleleG 136 (69) 224 (74)A 60 (31) 80 (26) 124 (083ndash184) 119875 = 029






Haplotype LL (2119899 = 196)n ()

HS (2119899 = 304)n ()


CCG 96 (489) 160 (527) lowast

CCA 15 (768) 32 (105) 078 (040ndash152) 119875 = 047

CAA 0 (0) 1 (0002) 055 (002ndash137) 119875 = 072

CAG 0 (0) 2 (0008) 033 (002ndash700) 119875 = 048

TCG 38 (194) 61 (202) 103 (064ndash167) 119875 = 088

TCA 45 (229) 47 (154) 159 (099ndash258) 119875 = 006

TAA 0 (0) 1 (0002) 166 (003ndash845) 119875 = 080

TAG 2 (102) 0 (0) 83 (040ndash175) 119875 = 017








5 Conclusion




Abbreviations




Acknowledgments


References
















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of























4 Discussion







HS (119899 = 152)n () OR CI (95) 119875 value


CC 84 (86) 127 (84)CG 13 (13) 22 (14) 089 (043ndash187) 119875 = 076

GG 1 (1) 3 (2) 050 (005ndash493) 119875 = 055

AlleleC 181 (92) 276 (91)G 15 (8) 28 (9) 082 (042ndash016) 119875 = 054


CC 24 (25) 56 (37)CT 63 (64) 83 (54) 177 (099ndash316) 119875 = 005

TT 11 (11) 13 (9) 197 (0772ndash502) 119875 = 015

AlleleC 111 (57) 195 (64)T 85 (43) 109 (36) 137 (095ndash198) 119875 = 009


CC 96 (98) 148 (97)CA 2 (2) 4 (3) 080 (014ndash429) 119875 = 076

AA 0 (0) 0 (0) 154 (003ndash782) 119875 = 100

AlleleC 194 (99) 300 (99)A 2 (1) 4 (1) 077 (014ndash426) 119875 = 100


GG 48 (49) 81 (53)GA 40 (41) 62 (41) 109 (064ndash186) 119875 = 076

AA 10 (10) 9 (6) 187 (071ndash494) 119875 = 019

AlleleG 136 (69) 224 (74)A 60 (31) 80 (26) 124 (083ndash184) 119875 = 029






Haplotype LL (2119899 = 196)n ()

HS (2119899 = 304)n ()


CCG 96 (489) 160 (527) lowast

CCA 15 (768) 32 (105) 078 (040ndash152) 119875 = 047

CAA 0 (0) 1 (0002) 055 (002ndash137) 119875 = 072

CAG 0 (0) 2 (0008) 033 (002ndash700) 119875 = 048

TCG 38 (194) 61 (202) 103 (064ndash167) 119875 = 088

TCA 45 (229) 47 (154) 159 (099ndash258) 119875 = 006

TAA 0 (0) 1 (0002) 166 (003ndash845) 119875 = 080

TAG 2 (102) 0 (0) 83 (040ndash175) 119875 = 017








5 Conclusion




Abbreviations




Acknowledgments


References
















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of



















HS (119899 = 152)n () OR CI (95) 119875 value


CC 84 (86) 127 (84)CG 13 (13) 22 (14) 089 (043ndash187) 119875 = 076

GG 1 (1) 3 (2) 050 (005ndash493) 119875 = 055

AlleleC 181 (92) 276 (91)G 15 (8) 28 (9) 082 (042ndash016) 119875 = 054


CC 24 (25) 56 (37)CT 63 (64) 83 (54) 177 (099ndash316) 119875 = 005

TT 11 (11) 13 (9) 197 (0772ndash502) 119875 = 015

AlleleC 111 (57) 195 (64)T 85 (43) 109 (36) 137 (095ndash198) 119875 = 009


CC 96 (98) 148 (97)CA 2 (2) 4 (3) 080 (014ndash429) 119875 = 076

AA 0 (0) 0 (0) 154 (003ndash782) 119875 = 100

AlleleC 194 (99) 300 (99)A 2 (1) 4 (1) 077 (014ndash426) 119875 = 100


GG 48 (49) 81 (53)GA 40 (41) 62 (41) 109 (064ndash186) 119875 = 076

AA 10 (10) 9 (6) 187 (071ndash494) 119875 = 019

AlleleG 136 (69) 224 (74)A 60 (31) 80 (26) 124 (083ndash184) 119875 = 029






Haplotype LL (2119899 = 196)n ()

HS (2119899 = 304)n ()


CCG 96 (489) 160 (527) lowast

CCA 15 (768) 32 (105) 078 (040ndash152) 119875 = 047

CAA 0 (0) 1 (0002) 055 (002ndash137) 119875 = 072

CAG 0 (0) 2 (0008) 033 (002ndash700) 119875 = 048

TCG 38 (194) 61 (202) 103 (064ndash167) 119875 = 088

TCA 45 (229) 47 (154) 159 (099ndash258) 119875 = 006

TAA 0 (0) 1 (0002) 166 (003ndash845) 119875 = 080

TAG 2 (102) 0 (0) 83 (040ndash175) 119875 = 017








5 Conclusion




Abbreviations




Acknowledgments


References
















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of


















Haplotype LL (2119899 = 196)n ()

HS (2119899 = 304)n ()


CCG 96 (489) 160 (527) lowast

CCA 15 (768) 32 (105) 078 (040ndash152) 119875 = 047

CAA 0 (0) 1 (0002) 055 (002ndash137) 119875 = 072

CAG 0 (0) 2 (0008) 033 (002ndash700) 119875 = 048

TCG 38 (194) 61 (202) 103 (064ndash167) 119875 = 088

TCA 45 (229) 47 (154) 159 (099ndash258) 119875 = 006

TAA 0 (0) 1 (0002) 166 (003ndash845) 119875 = 080

TAG 2 (102) 0 (0) 83 (040ndash175) 119875 = 017








5 Conclusion




Abbreviations




Acknowledgments


References
















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of


















Abbreviations




Acknowledgments


References
















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of









































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of





















of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















Documents

Clinical Study Role of Toll-Interacting Protein Gene ...Genetic Division Department, Biomedical Research Center West, Instituto Mexicano del Seguro Social, Sierra Mojada , Colonia