Tissue Antigens ISSN 0001-2815

L E T T E R T O T H E E D I T O R

G2848A and T-1237C polymorphisms of the TLR9 geneand susceptibility to inflammatory bowel disease in patientsfrom southern BrazilJ. M. Valverde-Villegas1, B. P. dos Santos1, M. B. Machado2, M. Jobim3, L. F. Jobim3, C. Flores4,D. C. Damin5, P. Portela3 & J. A. B. Chies1

1 Laboratorio de Imunogenetica, PPGBM – Programa de Pos Graduacao em Genetica e Biologia Molecular, Universidade Federal do Rio Grande doSul, Porto Alegre, Brazil2 Hospital Sao Lucas, Pontifıcia Universidade Catolica do Rio Grande do Sul, Porto Alegre, Brazil3 Department of Immunology, Hospital de Clınicas de Porto Alegre, Porto Alegre, Brazil4 Department of Gastroenterology, Hospital de Clınicas de Porto Alegre, Porto Alegre, Brazil5 Department of Coloproctology, Hospital de Clınicas de Porto Alegre, Porto Alegre, Brazil

Key words: inflammatory bowel disease; polymorphisms; susceptibility; toll-like receptor 9

Inflammatory bowel disease (IBD) can be classified in Crohn’sdisease (CD) and ulcerative colitis (UC) depending on clin-ical parameters, including the affected intestinal area (1). Itis widely accepted that a critical determinant for IBD devel-opment in genetically susceptible individuals is the disrup-tion of the homeostasis between the commensal bacteria andthe intestinal mucosal immunity (2). This homeostasis is theresult of a rigorous balance that leads to equilibrium betweenimmune response and tolerance.

Toll-like receptor 9 (TLR9) is a receptor that recognizesconserved molecular patterns such as DNA molecules fromboth normal flora and pathogenic intestinal organisms (3). Astudy suggested that gut flora DNA acts as a natural adjuvantand that TLR9 critically controls the balance between thefrequency and the function of T-regulatory and T-effectorcells on such environment (3). The TLR9 gene is located onthe chromosome 3p21.3, and a recently performed screeningof single nucleotide polymorphisms (SNPs) associated withdifferent diseases, in three different ethnic groups, lead to theconclusion that two TLR9 SNPs, rs5743836 (T-1237C) andrs352140 (G2848A or G1635A), are enough to distinguishthe four commonest TLR9 haplotypes (4). The rs5743836polymorphism is located in the promoter region at position−1237 and corresponds to a T to C exchange, creating apotential NF-κB-binding site (5). This polymorphism has beenimplicated in chronic inflammatory diseases, including asthma(4), systemic lupus erythematosus (6) and CD (7, 8). Thers352140 polymorphism is located at position +2848, in exon2, and corresponds to a G to A exchange, although it does notalters the amino acid sequence (5). Nevertheless, in a recentmeta-analysis, both the A allele from this polymorphism aswell as the AA genotype were associated with an increasedrisk of cancer in Caucasians (9).

Considering the potential impact of TLR9 polymorphismson diseases, this study analyzed the allelic and genotypicfrequencies of the rs352140 and rs5743836 polymorphismsin IBD patients from southern Brazil.

We analyzed a total of 253 IBD patients of which 105 werefrom the Hospital Sao Lucas at the Pontifıcia UniversidadeCatolica do Rio Grande do Sul (PUCRS) and 148 fromHospital de Clınicas de Porto Alegre (HCPA), both centerswere from the city of Porto Alegre, the capital of Rio Grandedo Sul, the southernmost state of Brazil. The control groupwas composed of 239 healthy individuals, all donors at theBlood donor center of Porto Alegre. All patients and controlswere similar according to gender and age. Both groups wereethnically matched and identified as European-derived. Issuesregarding the skin color-based classification criteria usedin Brazil are well documented (10). The diagnosis of IBDwas based on standard clinical, radiological and histologicalcriteria, and individuals were clinically classified as CD orUC patients depending on clinical parameters. One hundredand thirty-four patients were diagnosed as CD and 110 asUC, although nine IBD patients could not be assigned to anyof these two groups. The protocol of this study was approvedby the Sao Lucas Hospital and by the HCPA Ethics Com-mittee, and written informed consent was obtained from allindividuals studied according to the Declaration of Helsinki.

The DNA was isolated using a salting out method and wasstored at −20◦C. The rs352140 polymorphism was detectedby PCR-restriction fragment length polymorphism (PCR-RFLP), and the rs5743836 polymorphism was genotyped bybi-directional PCR allele-specific amplification (BI-PASA),both protocols were previously adapted by dos Santos et al.(6). We used the chi-squared test or Fisher exact test in thecomparison between the presence and absence of polymorphicvariants. A test of accordance to Hardy–Weinberg equilibrium(HWE) was held in cases and controls using the chi-squaredtest. Data were analyzed with spss 15.0 and Winpepi version11.1. Two-tailed value of P < 0.05 was taken to indicatestatistical significance. A formal Bonferroni correction for thenumber of the tests performed (considering the comparison ofalleles and genotypes) would require a significance thresholdof P = 0.008 (P0/N, P0=0.05, N = 6 tests).

190 © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons LtdTissue Antigens, 2014, 83, 190–192

J. M. Valverde-Villegas et al. Letter to the Editor

Considering that both polymorphic variants in this studypresented quite similar allelic frequencies in both cohorts(PUCRS and HCPA groups) and considering that both carecenters are located at the same city, all data were evaluated asa single sample. These same patients were already evaluatedby our group with respect to other polymorphic variants (11).Allelic and genotypic frequencies were in Hardy–Weinbergequilibrium except concerning the rs352140 polymorphism,among patients. Table 1 shows the allelic and genotypic fre-quencies of the two polymorphisms. We observed that thers352140 genotypic frequencies were different between IBDpatients and controls (P = 0.0037). Also, an increased fre-quency of heterozygous was observed when IBD patientswere compared with controls (P = 0.0013). When homozy-gous individuals were grouped in one category and com-pared against the GA genotype (GA vs GG + AA), theresults suggested heterozygosity as an IBD susceptibility fac-tor (P = 0.0013, OR: 1.79, 95% CI: 1.25–2.57). When strat-ified in UC and CD, an increased frequency of heterozygouswas observed among the CD patients (P = 0.00057). Whenhomozygous genotypes were joined (GG + AA vs GA), het-erozygosity appears as a risk factor (P = 0.00067, OR: 2.14,95% CI: 1.38–3.31). These P values were statistically sig-nificant after Bonferroni correction (P < 0.008). No differ-ences between the IBD, CD or UC patients and controls wereobserved concerning the rs5743836 polymorphism.

In regard to the rs352140 polymorphism, recently, a studyby Kim et al. (12) associated the GA genotype with lowerestimated glomerular filtration rate (eGFR) in kidney graft

recipients after a year of renal transplantation. These authorssuggested that this genotype was associated with acute rejec-tion through the interference with signaling cascades or byreducing the effects of glucocorticoids (12). Although to date,there are no functional assays regarding this polymorphism,the work from both Kim et al. and our results associates het-erozygosis of the TLR9 rs352140 with a negative outcome.This is the first time that this polymorphism is associated withIBD or CD, and these results can support the hypothesis thatalleles on the same locus can act synergistically to confersusceptibility to a disease.

Concerning the rs5743836 polymorphism, no associationswere observed in this study. Nevertheless, as TLR9 playsimportant roles in the balance of T regulatory and T effectorcells (2, 3), these results do not rule out this polymorphismas a candidate to IBD susceptibility. Sample size is a limita-tion of this study. It is necessary to replicate this evaluationusing other cohorts with larger samples. Of note, the ethnicbackground – meaning the extent of European, African andAmerindian admixture – varies in different Brazilian regions,and therefore this should be taken into consideration in sub-sequent studies. Also, phenotype analyses would be importantfor determining the real involvement of both TLR9 gene andmolecule in IBD development.

In conclusion, in this study, significantly different rs352140genotypic frequencies, with an increased frequency of het-erozygous, were observed comparing IBD patients and con-trols, suggesting that IBD susceptibility is associated withTLR9 gene variants in Caucasians individuals.

Table 1 Genotypic and allelic frequencies of TLR9 polymorphisms in IBD patients and controlsa

Genotype/allelePolymorphisms

TLR9IBDb

N (frequency)CD

N (frequency)UC

N (frequency)Controls

N (frequency)

T-1237C 248 132 107 239

GenotypesTT 166 (0.67) 86 (0.65) 73 (0.68) 171 (0.72)TC 70 (0.28) 39 (0.30) 30 (0.28) 62 (0.26)CC 12 (0.05) 7 (0.05) 4 (0.04) 6 (0.02)

AllelesT 402 (0.81) 211 (0.80) 176 (0.82) 404 (0.85)C 94 (0.19) 53 (0.20) 38 (0.18) 74 (0.15)

G2848A 253 134 110 239

GenotypesGG 39 (0.15)a 18 (0.13)b 20 (0.18)c 58 (0.24)d

GA 152 (0.60)a 86 (0.64)b 59 (0.54)c 109 (0.46)d

AA 62 (0.25)a 30 (0.23)b 31 (0.28)c 72 (0.30)d

AllelesG 230 (0.45) 122 (0.46) 99 (0.45) 225 (0.47)A 276 (0.55) 146 (0.54) 121 (0.55) 253 (0.53)

a × d (χ2): P = 0.0037

Residual: GA: P = 0.0013 OR: 1.79, CI 95%: 1.25–2.57, P = 0.0013 (GA vs GG + AA)

b × d (χ2): P = 0.0019

Residual: GA: P = 0.00057 OR: 2.14, CI 95%: 1.38–3.31, P = 0.00067 (GA vs GG + AA)

c × d (χ2): P = 0.308

CD, Crohn’s disease; IBD, inflammatory bowel disease; UC, ulcerative colitis; CI, confidence interval and OR, odds ratio are shown.aThe chi-squared test was used for genotypic frequencies and Fisher exact test for allelic frequencies. In bold are indicated P values <0.008(significant threshold after Bonferroni correction).bNine patients could not be assigned to CD or UC.

© 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd 191Tissue Antigens, 2014, 83, 190–192

Letter to the Editor J. M. Valverde-Villegas et al.

Acknowledgments

This work was supported by the Conselho Nacional de Desen-volvimento Cientifico e Tecnologico (CNPQ) and Fundacaode Amparo a Pesquisa do Estado do Rio Grande do Sul(FAPERGS). We appreciated and thank the collaboration ofLenin Maturrano H. from Universidad Nacional Mayor deSan Marcos in Lima, Peru, for the critical review of themanuscript.

Conflict of interest

The authors have declared no conflicting interests.

Correspondence

Jose Artur Bogo ChiesDepartamento de GeneticaInstituto de BiocienciasUniversidade Federal do Rio Grande do SulAv. Bento Goncalves 9500Predio 43323Lab. 212 AgronomiaCEP 91501-970 Porto Alegre, RSBrazilTel: +51 3308 6740Fax: +51 3308 7311e-mail: jabchies@terra.com.br

doi: 10.1111/tan.12302

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192 © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons LtdTissue Antigens, 2014, 83, 190–192