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Lack of association of TIM3 polymorphisms and allergic phenotypes Zhang, Jian; Daley, Denise; Akhabir, Loubna; Stefanowicz, Dorota; Chan-Yeung, Moira; Becker, Allan B; Laprise, Catherine; Paré, Peter D; Sandford, Andrew J Jun 30, 2009

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ralssBioMed CentBMC Medical GeneticsOpen AcceResearch articleLack of association of TIM3 polymorphisms and allergic phenotypesJian Zhang1, Denise Daley1, Loubna Akhabir1, Dorota Stefanowicz1, Moira Chan-Yeung2, Allan B Becker3, Catherine Laprise4, Peter D Paré1 and Andrew J Sandford*1Address: 1James Hogg iCAPTURE Center for Cardiovascular and Pulmonary Research, St. Paul's Hospital, Vancouver, BC, Canada, 2Respiratory Division, Department of Medicine, the University of Hong Kong, Hong Kong SAR, 3The Section of Allergy and Clinical Immunology, Department of Pediatrics, University of Manitoba, Winnipeg, Canada and 4Université du Québec à Chicoutimi, Saguenay, QC, CanadaEmail: Jian Zhang - zjs015195@yahoo.com; Denise Daley - ddaley@mrl.ubc.ca; Loubna Akhabir - lakhabir@mrl.ubc.ca; Dorota Stefanowicz - dstefanowicz@mrl.ubc.ca; Moira Chan-Yeung - mmwchan@hku.hk; Allan B Becker - becker@cc.umanitoba.ca; Catherine Laprise - Catherine_Laprise@uqac.ca; Peter D Paré - ppare@mrl.ubc.ca; Andrew J Sandford* - asandford@mrl.ubc.ca* Corresponding author    AbstractBackground: T-cell immunoglobulin mucin-3 (TIM3) is a TH1-specific type 1 membrane proteinthat regulates TH1 proliferation and the development of immunological tolerance. TIM3 and itsgenetic variants have been suggested to play a role in regulating allergic diseases. Polymorphisms inthe TIM3 promoter region have been reported to be associated with allergic phenotypes in severalpopulations. The aims of this study were to examine whether genetic variation in the promoterregion of TIM3 influenced transcription of the gene and risk for allergic phenotypes.Methods: We performed 5' rapid amplification of cDNA ends and reverse transcription-polymerase chain reaction. We screened for polymorphisms in the promoter region. Deletionanalysis was used to localize the promoter region of TIM3. Genotyping was performed by TaqManassays in three asthma/allergy population samples.Results: We found two regions with promoter activity in TIM3. One region was from -214 bp to+58 bp and the other from -1.6 kb to -914 bp relative to the transcription start site. None of thesingle nucleotide polymorphisms (SNPs) or haplotypes affected the transcriptional activity inreporter gene assays. No association between the SNPs and any phenotype was observed in thestudy cohorts.Conclusion: Our findings indicate that SNPs and haplotypes in the TIM3 promoter region do nothave a functional effect in vitro and are not associated with allergic diseases. These data suggest thatpolymorphisms in the TIM3 promoter region are unlikely to play an important role in susceptibilityto allergic diseases.Background common disease caused by interactions between multiplePublished: 30 June 2009BMC Medical Genetics 2009, 10:62 doi:10.1186/1471-2350-10-62Received: 27 February 2009Accepted: 30 June 2009This article is available from: http://www.biomedcentral.com/1471-2350/10/62© 2009 Zhang et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.Page 1 of 10(page number not for citation purposes)Asthma is a chronic inflammatory disease of the airwaysthat is a major cause of morbidity in developed countriesand has been increasing in prevalence [1,2]. Asthma is agenes of small to modest effect and equally importantenvironmental factors. Asthma susceptibility has beenlinked to several loci e.g. chromosomes 5, 6, 11, 12 and 14BMC Medical Genetics 2009, 10:62 http://www.biomedcentral.com/1471-2350/10/62[3]. Among these linkages, chromosome 5q23-35 hasbeen replicated in several genome-wide studies in differ-ent populations [3].McIntire et al. identified a chromosomal region that regu-lated TH2 cytokine production as well as airway hyperre-sponsiveness (AHR) using a congenic mouse model ofasthma [4]. This region was distinct from the IL4 cytokinegene cluster and other nearby cytokine genes [4]. Theregion is homologous to human chromosome 5q33 andcontains the TIM (T cell immunoglobulin domain andmucin domain) gene family [4]. There are two genes inthis family (TIM1 and TIM3) that are biologically plausi-ble atopy susceptibility genes. TIM1 (also known as thehepatitis A virus cellular receptor, HAVCR1) is expressedpreferentially on TH2 cells and TIM3 (HAVCR2) isexpressed preferentially on TH1 cells after activation ofnaive CD4+ T-helper cells. TH1 cells mediate immuneresponses to intracellular pathogens, delayed-type hyper-sensitivity reactions, and produce cytokines such as inter-feron-γ, IL2, tumour-necrosis factor-α and lymphotoxin.TH2 cells mediate immune responses to extracellular path-ogens and produce cytokines such as IL4, IL10 and IL13which promote atopic and allergic diseases [5]. TIM1 pro-motes TH2 cytokine production and proliferation. In amurine model of asthma, stimulation of TIM1 in the pres-ence of antigen prevented the development of respiratorytolerance and increased pulmonary inflammation [6].TIM3 inhibits TH1-mediated auto- and alloimmuneresponses and acts via its ligand, galectin-9, to induce celldeath in TH1 but not TH2 cells [7-9]. Considering theirimmunological function and chromosomal location bothTIM1 and TIM3 are good candidate genes for asthma.Recent association studies suggested that polymorphismsin the TIM3 promoter region may be associated withasthma-related phenotypes in both Caucasian and Asianpopulation samples [10-12]. Other studies have demon-strated associations of TIM1 polymorphisms with asthmaand related traits [11,13,14]. In the present study, we per-formed an association study in three asthma/allergy pop-ulation samples to investigate the role of polymorphismsin the TIM3 promoter region and determined whetherthese polymorphisms affected TIM3 transcriptional activ-ity.MethodsStudy populationsWe used three independent asthma/allergy populationsamples: the Canadian Asthma Primary Prevention Study(CAPPS) cohort, the Study of Asthma Genes and the Envi-ronment (SAGE) birth cohort and the Saguenay-Lac-St-Jean (SLSJ)/Québec City (QC) Familial Collection (TableThe CAPPS cohort was initiated in 1995 and recruitedfrom two Canadian cites, Vancouver and Winnipeg[15,16]. Infants were recruited who were at high risk forthe development of asthma, defined as those who had atleast one first-degree relative with asthma or two first-degree relatives with other allergic diseases. In total, therewere 545 families recruited into this study (549 infants, 4sets of twins). At the 7-year time point loss to follow-upwas minimal, with 86% of the families completing a ques-tionnaire. Spirometry and methacholine challenge testingwere performed at the 7-year time point. The diagnoses ofasthma and other atopic disorders were made by a pediat-ric allergist based on a detailed history and physical exam-ination. Atopy was defined as at least one positive skinprick test. Methacholine challenge testing was carried outaccording to Cockcroft et al. [17]. The provocative concen-tration of methacholine that induced a 20% decrease inFEV1 from post-saline value (PC20) was determined. AHRTable 1: Sample sizes by study, phenotype and ethnic backgroundCohortsCAPPS SAGE SLSJ/QC CombinedFamilies 545 723 306 1573Genotyped 1316 1466 1234 4016Caucasian Samples (complete trios)PhenotypeAsthma 51 109 379 539Atopy 105 145 362 612AHR 142 96 278 516Atopic Asthma 37 71 305 413Non Caucasian Samplesa(complete trios)Asthma 3 28 na 31Atopy 18 44 na 62AHR 14 22 na 36Atopic Asthma 3 20 na 23Combined Analysisb (complete trios)Asthma 57 139 379 575Atopy 135 190 362 687AHR 170 120 278 568Atopic Asthma 43 92 305 440aNon Caucasian samples included individuals of Asian (Chinese, Korean and Japanese) and Canadian First Nations descent.bCombined Analysis also contains trios with mixed and unknown ethnicityCAPPS – Canadian Asthma Primary Prevention Study Cohort; SAGE Page 2 of 10(page number not for citation purposes)1). The study protocols were approved by ethical reviewboards at all participating institutions. Informed consentwas obtained from each individual or his/her guardian.– Study of Asthma Genes and the Environment birth cohort; SLSJ/QC – Saguenay – Lac-St-Jean/Québec City Familial Collection; AHR – airway hyperresponsivenessBMC Medical Genetics 2009, 10:62 http://www.biomedcentral.com/1471-2350/10/62for this cohort and the SAGE cohort was defined as a PC20of less than 3.2 mg/ml methacholine [18,19].SAGE is a population-based sample of 16,320 children,born in the province of Manitoba, Canada in 1995 [20].In 2002, the families were sent a questionnaire to deter-mine their health and home environment exposure. Chil-dren were classified according to the presence of asthma(n = 392), hay fever/food allergy (n = 192) or neither (n =3002). All the children in the asthma and allergy groupswere invited to participate in the study, together with arandom sample (n = 200) of children with neither condi-tion. A pediatric allergist assessed the presence of asthmabased on a detailed history and physical examination, amethacholine challenge test was administered and skinprick tests for common allergens were performed. In total,725 families were recruited into the study, including 247with an asthmatic child and 328 with an atopic child.The SLSJ/QC Familial Collection is comprised of 306 fam-ilies from the Saguenay-Lac-Saint-Jean (n = 227) andQuébec City (n = 79) regions of Québec, Canada [21,22].There is at least one adult asthmatic proband in each fam-ily. Asthma was assessed using a respiratory health ques-tionnaire and pulmonary function tests. AHR was definedas a PC20 < 8 mg/ml at the time of recruitment. If PC20 wasnot measurable, a 15% increase in FEV1 after inhalation ofa bronchodilator or a variation in PEF of at least 12%within a 2-week period was also considered diagnostic ofAHR. Participants were defined as having asthma if theyhad a reported history of asthma that was validated by aphysician, or they showed asthma-related symptoms anda positive PC20 at the time of recruitment. Subjects weredefined as atopic if they had at least one positive responseto a skin prick test. Subjects with a PC20 > 8 mg/ml wereconsidered not to have AHR; non-asthmatics were thosewho had no history of physician-diagnosed asthma, nosymptoms of asthma and a PC20 greater than 8 mg/ml;non-atopics were those who had no positive response onskin prick test.Expression of TIM3 in tissuesThe Human Multiple Tissue, Human Immune SystemcDNA Panels and Human Blood Fraction Panel (BD Bio-sciences/Clontech, Palo Alto, CA, USA) were used to ana-lyze expression of TIM3 in various tissues. The PCRprimers for the gene expression study are listed in Table 2.Resting CD14+ (monocytes), CD4+ (T helper/inducercells), CD8+ (T suppressor/cytotoxic cells) and CD19+ (Blymphocytes) cells were positively selected from mononu-clear cells from healthy donors by immunomagnetic sep-aration with Dynabeads M-450 (Dynal, Oslo, Norway).Cells were activated with pokeweed mitogen (Invitrogen,San Diego, CA, USA) and concanavalin A (ICN, CostaTable 2: Sequence of primers used in reverse transcriptase-polymerase chain reaction, 5' Rapid Amplification of cDNA ends (5'RACE) and (RT-PCR) and plasmid constructsTIM3 RT-PCR primers Forward 5'-tgctgctgctgctactacttaca-3'Reverse 5'-aggttggccaaagagatgag-3'5'RACE First round forward 5'-gctggggtgtagaagcagggcagat-3'First round reverse 5'-ccatcctaatacgactcactatagggc-3'Nested PCR forward 5'-tgtctgtgtctctgctgggccatgt-3'Nested PCR reverse 5'-actcactatagggctcgagcggc-3'Plasmid constructs primers Common reverse primer 5'-attatctcgagtggactgggtacttcttccaaForward primer +63 bp 5'-attatggtacctgactgtagacctggcagtgtt-3'Forward primer -241 bp 5'-attatggtaccggacatgctccatttcaggt-3'Forward primer -452 bp 5'-attatggtacctgaggcttatgctgggagtt-3'Forward primer -914 bp 5'-attatggtaccaaaccactcagcctgtgagc-3'Forward primer -1702 bp 5'-attatggtaccgccttgaccaagttcatgct-3'Page 3 of 10(page number not for citation purposes)Forward primer -2220 bp 5'-attatggtaccccagctccctacacacacaa-3'BMC Medical Genetics 2009, 10:62 http://www.biomedcentral.com/1471-2350/10/62Mesa, CA, USA) by standard methods, and the degree ofactivation of lymphocytes was estimated on the basis ofmorphological criteria (blast morphology and mitoses)and expression of two activation markers, CD25 (inter-leukin-2 receptor) and CD71 (transferrin receptor). Weused glycerol-3-phosphate dehydrogenase (G3PDH) as aninternal control for PCR. Amplification conditions werean initial denaturation step at 94°C for 10 min followedby 34, and 30 cycles of denaturation at 94°C for 30 s,annealing at 60°C for 30 s and extension at 72°C for 30 sfor primer pairs amplifying TIM3 and G3PDH, respec-tively.5' Rapid Amplification of cDNA ends (5'RACE)We performed 5' RACE experiments using commerciallyavailable RACE-ready human leukocyte and spleencDNAs (Marathon Ready cDNA, BD Biosciences/Clon-tech) according to the manufacturer's instructions. Prim-ers used for amplification for the first round PCR and forthe nested PCR are shown in Table 2. The amplified RACEproduct was cloned into pCR2.1 TOPO-TA cloning vector(Invitrogen). Plasmids were purified by column chroma-tography (Invisorb Spin Plasmid Mini Kit, Invitek GmbH,Berlin) and subjected to direct sequencing with M13primers.Single nucleotide polymorphism (SNP) screening and genotypingApproximately 2500 bp of the 5' flanking region upstreamof the transcription initiation site of TIM3 was amplifiedby PCR from genomic DNA of 19 unrelated healthy Cau-casians. Subsequently, the products were subjected todirect sequencing with a Big-Dye Terminator Kit (AppliedBiosystems, Foster City, CA, USA). Genotyping of the twotag SNPs was done by TaqMan Assay-on-Demand™ SNPtyping (Applied Biosystems).Plasmid construction, transfection and luciferase assayGenomic fragments of the 5' flanking region of exon 1 ofTIM3 were amplified. PCR products were digested withXhoI and KpnI overnight at 37°C and then subcloned intothe pGL3-Basic vector (Promega, Madison, WI, USA)digested with XhoI and KpnI. The clones were sequencedto confirm that the inserts were correct. The YT human T/NK cell line provided by Dr. Zacharie Brahmi as a gift wasresuspended in RPMI 1640 (Sigma-Aldrich Co, St. Louis,MO, USA) with 20% FBS. Approximately 1 × 107 YT cellswere cotransfected with 30 μg of test construct and 150 ngof pPL-TK (Promega) by electroporation with a Gene Pul-sar II (Bio-Rad, Hercules, CA) set at 300 V and 975 μF.Transfected cells were harvested 24 h after transfection.Cells were lysed by the addition of 200 μl of lysis buffer(Promega). Twenty μl of each lysate was used for luci-which were determined at the same time. The signal wasread using a POLARstar OPTIMA (BMG, Alexandria, VA,USA) fluorimeter. Reporter activity is presented as themean of at least five independent measurements.Statistical analysisDifferences in transcriptional activity in the reporter geneassays were analyzed by ANOVA and unpaired t-tests. Wetested for association with asthma, atopy, atopic asthmaand airway hyper- responsiveness phenotypes using theFamily based Association Test (FBAT) software [23].ResultsTissue expression of TIM3Expression of TIM3 was analyzed by PCR-based methods(Figure 1). TIM3 was strongly expressed in placenta, lung,kidney, spleen, and leukocytes. In the Human Blood Frac-tion Panel TIM3 was more highly expressed in activeCD4+ cells than resting CD4+ cells. However it was morehighly expressed in resting CD8+ cells than in active CD8+cells. TIM3 was also strongly expressed in resting CD14+cells. No splicing variants were found.Isolation of 5' full-length TIM3 transcripts and structure of the human TIM3 geneCurrent information at the time of the experiment (Janu-ary 2007) in the NCBI database http://www.ncbi.nlm.nih.gov indicated that TIM3 was com-posed of seven exons and the translational start site wascontained within exon 1. TIM3 was highly expressed inleukocytes and spleen and therefore 5' RACE experimentswere performed with cDNAs derived from these cell types.We were able to identify an additional 25 bp of sequenceon the 5' side of the known cDNA sequence (Figure 2). Noadditional novel exons were detected.Polymorphism screenWe screened for polymorphisms in the TIM3 promoterregion using DNA from 19 unrelated normal subjects andfound six polymorphisms -574 G/T (rs10515746), -882C/T (rs4704853), -1516 G/T (rs10053538), -1571delC, -1766G/T (rs10061463) and -1922 G/A (rs12186731) inTIM3 (Figure 2). Among the six polymorphisms four (-574 G/T, -882 C/T, -1571delC and -1766G/T) were in per-fect linkage disequilibrium (r2 = 1). There were only threehaplotypes formed by the six polymorphisms.Transcriptional activity of the 5' flanking region of TIM3To examine the transcriptional activity in the 5' flankingregion of TIM3, we constructed plasmids that containedsequences from -2220, -1702, -914, -452, -241 and +63 rel-ative to the transcription initiation site. The primers usedfor plasmid construction are listed in Table 2. The expres-Page 4 of 10(page number not for citation purposes)ferase assay with the Dual-Luciferase Reporter Assay Sys-tem (Promega). The firefly luciferase values werenormalized to the Renilla luciferase values of pRL-TK,sion of TIM3 in YT cells was confirmed by RT-PCR (data notshown). The constructs were then transiently transfectedinto YT cells. Deletion analysis revealed that promoterBMC Medical Genetics 2009, 10:62 http://www.biomedcentral.com/1471-2350/10/62Page 5 of 10(page number not for citation purposes)Expression of TIM3 in multiple human tissuesFigure 1Expression of TIM3 in multiple human tissues. Results of PCR amplification of cDNA from different organs (A), the immune system (B), and blood fractions (C) are shown. G3PDH was included as an internal control. MC, mononuclear cells; R, resting; A, activated; NC, non-template control.Genomic structure of the human TIM3 geneFigure 2Genomic structure of the human TIM3 gene. The open boxes represent the positions of exons 1–7. The shaded box is the region we extended in our 5'RACE experiment. TIM3 contains seven exons and the coding sequence (CDS) starts in exon 1. The downward arrows indicate the SNPs in the promoter region. The four red arrows indicate SNPs that are in perfect LD.BMC Medical Genetics 2009, 10:62 http://www.biomedcentral.com/1471-2350/10/62activity of TIM3 in YT cells required at least 241 bp ofupstream sequence and the maximal reporter gene expres-sion was observed with the -1702 bp construct (Figure 3).There were five polymorphisms, -574 G/T, -882 C/T, -1516G/T, -1571delC and -1766G/T in this region. To determinewhether the five polymorphisms and their haplotypes werefunctional, we generated luciferase reporter gene constructsthat contained the 5' flanking region of TIM3 from exon 1to -1702 bp with three different haplotypes, i.e., haplotypeGCGCG, haplotype GCTCG and haplotype TTG-T. Theresults showed there was no difference in expression levelbetween each haplotype (Figure 3).FBAT analysisTo determine whether the previously reported TIM3 associ-ations [10-12] were present in the CAPPS, SAGE and SLSJ/QC populations, we chose -882 C/T and -1922 G/A as tagSNPs. However -1922 G/A was in a region of repetitivesequence. Therefore, rs13170556, which was in perfectlinkage disequilibrium (LD) with -1922 G/A, was geno-typed in our samples. Both polymorphisms (rs13170556and rs10061463) were in Hardy-Weinberg equilibrium (p> 0.1) in all cohorts. We performed FBAT analysis to test forassociation with asthma, atopy, atopic asthma and AHR.The results were corrected by the number of SNPs testedwithin TIM3 (n = 2) and the effective number of independ-ent phenotypes (n = 3). We found that rs13170556 wasassociated with asthma in the CAPPS cohort in both theCaucasians only analysis and in the combined analysis ofthe Caucasian families with the non Caucasian families (p= 0.0138 and 0.0085, respectively) (Table 3). However,after correction for multiple testing we found no evidencefor association in any of the three cohorts individually or injoint analysis of all the cohorts (Table 3). Similarly, therewas no association found for -882C/T with any phenotypein any of the analyses (Table 4).DiscussionIn the present study, we determined the expression pat-tern of TIM3 in human cells. We investigated the genomicstructure and transcriptional activity of TIM3 and investi-gated polymorphisms in the promoter region of TIM3 inmultiple cohorts. We isolated the full-length genomicregion of TIM3 and characterized its promoter region. Wefound six polymorphisms in TIM3, but none was associ-ated with asthma or the transcriptional activity of the genein vitro.TIM3 was initially cloned as a TH1-specific cell-surfacemarker. In our results, TIM3 was expressed on activatedCD4+ cells as well as resting CD8+ cells and CD14+ cells,consistent with previous reports. In the mouse, TIM3 wasexpressed in both CD4+ and CD8+ cells [24,25] and inexpressed in NK and NTK (NK-like T) cells [26,27]. In ourresults, TIM3 was expressed at a higher level in activatedCD4+ cells than in resting CD4+ cells but converselyexpression was higher in resting CD8+ than in activatedCD8+ cells. Our results demonstrate that the expressionlevel of TIM3 is not only differentially regulated in subsetsof T cells but is also determined by the activation state ofthe cell.TIM3 is expressed in human NK cells both at the mRNAand protein levels [26,27]. We found that TIM3 was alsoexpressed in one type of NK cell line, the YT cell line,which was used in the reporter gene assays. We identifiedTIM3 promoter activity in the -241 bp and -1702 kbregions relative to the transcription initiation site. Con-served non-coding sequences may contain transcriptionalregulatory elements participating in the temporal and tis-sue-specific expression patterns of genes [28,29]. In theUCSC website http://genome.ucsc.edu/ there are threeconserved regions in the TIM3 promoter (Figure 3B) andthe first conserved region contributes to the -241 bp pro-moter region and the last two regions contribute to the -1702 bp promoter region. There are five SNPs in the -1.7kb region and the -1516 G/T, -1571delC and -1766G/TSNPs flank the conserved sequence. However, the haplo-type formed by these SNPs did not affect the promoteractivity (Figure 3C). We also stimulated the YT cell linewith IL-2 at different concentrations but we found no dif-ference in promoter activity among the different haplo-types after the stimulation (data not shown).There are discrepant reports concerning the associationbetween TIM3 polymorphisms and allergic phenotypes[10-14]. Graves et al. [11] studied a mixed Caucasian/His-panic population. The two TIM3 SNPs that showed asso-ciation with eczema and atopy were rs1036199 andrs4704853. However, in our sample these two SNPs werein perfect LD and rs4704853 was not associated with anyphenotype. This discrepancy may be due to the differentethnic group studied in the previous report [11]. Twoother studies reported associations in Asian samples[10,12]. The -574G > T (rs10515746) polymorphism wasassociated with asthma and rhinitis in a Korean popula-tion although the -574T allele was found in less than 2%of the patients [10]. Therefore, our study may not havebeen adequately powered if this association is limited tothe Asian population.In Caucasian and African-American populations no asso-ciation of TIM3 polymorphisms was seen with asthma orrelated phenotypes [13,14]. The three cohorts used in thisstudy were family-based and there were more than 1000individuals in each cohort. There were non-CaucasianPage 6 of 10(page number not for citation purposes)human peripheral blood mononuclear cells TIM3 wasexpressed at a higher level on CD14+ cells and CD8+ cellsthan on CD4+ cells [26]. TIM3 was also reported to besamples in both SAGE and CAPPS but we analyzed thedata separately to avoid possible loss of power due togenetic heterogeneity. Correction for multiple compari-BMC Medical Genetics 2009, 10:62 http://www.biomedcentral.com/1471-2350/10/62Page 7 of 10(page number not for citation purposes)Promoter activity assay of the human TIM3 gene promoter constructsFigure 3Promoter activity assay of the human TIM3 gene promoter constructs. (A) Luciferase activity is presented relative to the PGL3 basic vector after each construct was transfected into YT cells. All values are the mean ± SD of at least five inde-pendent experiments. (B) Comparison of the promoter activity and the conserved regions in the human genome. (C) Compar-ison of the promoter activity between the haplotypes.BMC Medical Genetics 2009, 10:62 http://www.biomedcentral.com/1471-2350/10/62sons was performed to avoid false positive results.Although a nominal association of rs13170556 wasfound in the CAPPS cohort it was not significant after cor-rection for multiple comparisons. Moreover, the associa-tion was not replicated in the other two cohorts and in thecombined analysis of all three cohorts. Therefore, theassociation was likely a statistical artifact rather than a truepositive result.We did not analyze other phenotypes such as total or spe-cific serum IgE in this study. We did not analyze haplo-types in the patient cohorts as we believe that this wouldthere is little benefit of exhaustive haplotype testing [30].In addition, we used the most powerful approach givenour study design, there is high LD in the region, themarker coverage was not dense and our single SNP maineffects were negative. All these factors made it unlikelythat we would have benefited from haplotype tests.The power to detect an association in this study variedwith the phenotype, allele frequency and cohort consid-ered. Power was calculated using the TDT Power Calcula-tor [31]. For a major allele 'A' and minor allele 'a', weassumed the penetrance of the three genotypes was AA =Table 3: Allele frequencies of the rs13170556 polymorphism in the three study cohorts for each phenotypeCohort Phenotype Caucasian only Combined analysisaFrequency OR p cpc Frequency OR p cpcCAPPS Asthma 0.13 0.31 0.0138 0.0822 0.13 0.29 0.0085 0.0508Atopy 0.73 0.3294 1.0000 0.62 0.1070 0.6359AHR 0.90 0.7455 1.0000 0.86 0.6393 1.0000Atopic Asthma 0.40 0.1031 0.6127 0.36 0.0653 0.3882SAGE Asthma 0.18 1.60 0.1477 0.8707 0.17 1.30 0.3757 1.0000Atopy 1.11 0.6961 1.0000 1.06 0.8084 1.0000AHR 1.33 0.3972 1.0000 1.15 0.6472 1.0000Atopic Asthma 1.80 0.1279 0.7540 1.43 0.3022 1.0000SLSJ/QC Asthma 0.15 0.95 0.7456 1.0000 0.15 0.95 0.7456 1.0000Atopy 0.86 0.3669 1.0000 0.86 0.3669 1.0000AHR 0.71 0.0749 0.4350 0.71 0.0749 0.4350Atopic Asthma 0.88 0.4758 1.0000 0.88 0.4758 1.0000Combined analysisb Asthma 0.15 0.94 0.6802 1.0000 0.15 0.91 0.5001 1.0000Atopy 0.89 0.3718 1.0000 0.86 0.2179 1.0000AHR 0.84 0.2402 1.0000 0.83 0.1775 1.0000Atopic Asthma 0.93 0.6434 1.0000 0.90 0.4961 1.0000aCombined analysis of the Caucasian families with the non Caucasian familiesbCombined analysis of the three cohorts (CAPPS, SAGE and SLSJ/QC)ccorrected p valueCAPPS – Canadian Asthma Primary Prevention Study Cohort; SAGE – Study of Asthma Genes and the Environment birth cohort; SLSJ/QC – Saguenay – Lac-St-Jean (SLSJ)/Québec City (QC) Familial Collection; AHR – airway hyperresponsivenessPage 8 of 10(page number not for citation purposes)have been inappropriate since we used tag SNPs fromHapMap and it has been suggested that in this scenario0.1, Aa = 0.2 and aa = 0.5. For an allele frequency of 0.13and the phenotype of allergic asthma in the CAPPS cohortBMC Medical Genetics 2009, 10:62 http://www.biomedcentral.com/1471-2350/10/62(i.e. 37 trios) the power to detect an association was only0.41. However, for a sample size of 96 trios (e.g. AHR inthe SAGE cohort) the power was 0.80 and was >0.80 forall other phenotypes in all cohorts in the Caucasians.ConclusionOur findings indicate that SNPs and haplotypes in theTIM3 promoter region do not have a functional effect invitro and are not associated with allergic diseases. Thesedata suggest that polymorphisms in the TIM3 promoterregion are unlikely to play an important role in suscepti-bility to allergic diseases.Authors' contributionsJZ participated in the genotyping, performed the remain-der of the molecular analysis and produced the first draftof the manuscript, DD performed the analysis of thegenetic epidemiological data and helped to draft the man-uscript, LA participated in the genotyping, DS participatedin the genotyping, MC-Y participated in the recruitment ofthe patient cohorts and helped to draft the manuscript, ABparticipated in the recruitment of the patient cohorts andhelped to draft the manuscript, CL participated in therecruitment of the patient cohorts and helped to draft themanuscript, PDP participated in the recruitment of theTable 4: Allele frequencies of the rs10061463 polymorphism in the three study cohorts for each phenotypeCohort Phenotype Caucasian only Combined analysisaFrequency OR p cpc Frequency OR p cpcCAPPS Asthma 0.18 0.85 0.6829 1.0000 0.16 1.00 1.0000 1.0000Atopy 0.78 0.3859 1.0000 0.80 0.4137 1.0000AHR 0.87 0.5528 1.0000 0.86 0.4967 1.0000Atopic Asthma 0.55 0.2218 1.0000 0.73 0.4904 1.0000SAGE Asthma 0.17 1.10 0.7576 1.0000 0.15 1.13 0.6743 1.0000Atopy 1.18 0.5635 1.0000 1.14 0.6113 1.0000AHR 1.25 0.5045 1.0000 1.10 0.7576 1.0000Atopic Asthma 1.36 0.4319 1.0000 1.33 0.3972 1.0000SLSJ/QC Asthma 0.25 0.97 0.7902 1.0000 0.25 0.97 0.7902 1.0000Atopy 1.03 0.8263 1.0000 1.03 0.8263 1.0000AHR 1.12 0.4232 1.0000 1.12 0.4232 1.0000Atopic Asthma 1.00 1.0000 1.0000 1.00 1.0000 1.0000Combined analysisb Asthma 0.20 0.97 0.8149 1.0000 0.18 0.99 0.9542 1.0000Atopy 1.01 0.9526 1.0000 1.01 0.9542 1.0000AHR 1.07 0.5610 1.0000 1.05 0.6904 1.0000Atopic Asthma 0.99 0.9454 1.0000 1.02 0.8937 1.0000aCombined analysis of the Caucasian families with the non Caucasian familiesbCombined analysis of the three cohorts (CAPPS, SAGE and SLSJ/QC)ccorrected p valueCAPPS – Canadian Asthma Primary Prevention Study Cohort; SAGE – Study of Asthma Genes and the Environment birth cohort; SLSJ/QC – Saguenay – Lac-St-Jean (SLSJ)/Québec City (QC) Familial Collection; AHR – airway hyperresponsivenessPage 9 of 10(page number not for citation purposes)Competing interestsThe authors declare that they have no competing interests.patient cohorts, the design of the study and helped todraft the manuscript, AJS participated in the design of theBMC Medical Genetics 2009, 10:62 http://www.biomedcentral.com/1471-2350/10/62study and helped to draft the manuscript. All authors readand approved the final manuscript.AcknowledgementsThis study was supported by grants from the Canadian Institutes of Health Research and the AllerGen NCE. JZ was supported by a Canadian Lung Association Fellowship Award. AS was supported by a Tier 2 Canada Research Chair and a Michael Smith Foundation for Health Research Senior Scholar Award.References1. Hopes E, McDougall C, Christie G, Dewar J, Wheatley A, Hall IP,Helms PJ: Association of glutamine 27 polymorphism of β2adrenoceptor with reported childhood asthma: populationbased study.  Bmj 1998, 316:664.2. Gergen PJ, Weiss KB: The increasing problem of asthma in theUnited States.  Am Rev Respir Dis 1992, 146:823-824.3. Hoffjan S, Ober C: Present status on the genetic studies ofasthma.  Curr Opin Immunol 2002, 14:709-717.4. 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