Interleukin-10 gene promoter polymorphisms in celiac patients from north-eastern Italy

Human Immunology xxx (2014) xxx–xxx

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Interleukin-10 gene promoter polymorphisms in celiac patientsfrom north-eastern Italy

http://dx.doi.org/10.1016/j.humimm.2014.04.0110198-8859/� 2014 American Society for Histocompatibility and Immunogenetics. Published by Elsevier Inc. All rights reserved.

⇑ Corresponding author. Fax: +39 040 3785540.E-mail address: [email protected] (L. Segat).

Please cite this article in press as: Zupin L et al. Interleukin-10 gene promoter polymorphisms in celiac patients from north-eastern Italy. Hum Im(2014), http://dx.doi.org/10.1016/j.humimm.2014.04.011

Luisa Zupin a, Vania Polesello a, Eulalia Catamo b, Sergio Crovella a,b, Ludovica Segat a,⇑a Institute for Maternal and Child Health, IRCCS ‘‘Burlo Garofolo’’, Via dell’Istria 65/1, 34137 Trieste, Italyb University of Trieste, Piazzale Europa 1, 34127 Trieste, Italy

a r t i c l e i n f o

Article history:Received 7 October 2013Accepted 6 April 2014Available online xxxx

Keywords:Interleukin-10Celiac diseasePolymorphismGenetic susceptibility

a b s t r a c t

Celiac disease is a complex chronic intestinal disorder driven by an immune response against thegliadin fraction of gluten: many factors are involved in the pathogenesis of the disease, andamong these Interleukin-10 could play an important role. In the present study, the �1082A>G,�819T>C and �592A>C IL10 functional polymorphisms were analyzed in 565 celiac patients and576 healthy controls from north-eastern Italy, stratified for HLA class II celiac disease risk haplo-types. No significant differences were observed for the three IL10 polymorphisms distributionbetween celiac patients and controls with the exception of a slightly increased risk for the�1082A allele in HLA-DQ8 male individuals. Although our findings suggest that the IL10 geneticvariants analyzed do not have a major role in the susceptibility to the development of celiacdisease in north-eastern Italian patients, we think that the possible involvement of IL10 genein CD should deserve further investigation and that large-scale studies are recommended toconfirm our findings.� 2014 American Society for Histocompatibility and Immunogenetics. Published by Elsevier Inc. All rights

reserved.

1. Introduction

Celiac disease (CD) is the most common enteropathy induced bygluten intake in humans. The disease is influenced by the interplaybetween immune, genetic and environmental factors [1]. SpecificMHC II alleles that map to the HLA-DQ locus are known to be themajor genetic factors involved in CD [2].

Most of CD patients (near 90%) carry the HLA-DQ2.5 haplotype(encoded by DQA1⁄05, DQB1⁄02 and DRB1⁄03 alleles), whereas asmaller percent of subjects carry the HLA-DQ8 haplotype (encodedby DQA1⁄03, DQB1⁄0302 and DRB1⁄04 alleles). A smaller percent ofCD patients may present the HLA-DQ2.2 haplotype (HLADQA1⁄0201, DQB1⁄02, DRB1⁄07) known to confer only a minor riskfor CD development with respect to HLA-DQ2.5. Indeed, this haplo-type is able to present only few different gluten peptides, while themore predisposing haplotype (HLA-DQ2.5) can expose a greatervariety of them [3].

In CD patients the ingestion of gluten promotes immuneresponses with inflammatory reaction in the upper small intes-

tine, characterized by infiltration of inflammatory cells withinthe lamina propria and the epithelium with villous atrophy [4].An important regulatory cytokine in the intestinal mucosa isInterleukin 10 (IL-10): it is an inhibitor of Th1 cell developmentand so underproduction of IL-10 may contribute to an increasedTh1-driven inflammation, responsible for the intestinal lesionstypical of CD [5]. Low levels of IL-10 have been associated withanti-tissue transglutaminase (anti tTG) antibodies in CD patients[6] and Salvati et al. [7] have also observed a suppression of gli-adin-specific T cell activation using recombinant human IL-10(rhIL-10).

The IL10 gene maps on the long arm of chromosome 1(1q31–q32) a locus previously identified by different genome wideassociation studies as possibly involved in CD [8–10].

Three single nucleotide polymorphisms (SNPs) at positions�1082 (A>G), �819 (T>C) and �592 (A>C) in the promoter regionof the gene may affect the IL10 gene expression [11–14]; previousstudies have analyzed the influence of the three IL10 SNPs on CDsusceptibility with discordant results [15–17].

The aim of this study is to evaluate the possible associationbetween IL10 and CD, by analyzing the �1082A>G, �819T>C and�592A>C promoter polymorphisms in CD patients and healthycontrols from north-eastern Italy.

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2 L. Zupin et al. / Human Immunology xxx (2014) xxx–xxx

2. Methods

2.1. Patients and controls

The 565 CD Italian patients (European–Caucasian; meanage = 21.75, standard deviation = 15.70, range = 4–85; sex, 338females and 227 males) analyzed in this study, represented aselected subset of a bigger historical cohort (around 2500 samples)of celiac individuals that were recruited at the GastroenterologyService of IRCCS Burlo Garofolo (Trieste, Italy) from July 2000 toDecember 2009.

The whole cohort was initially genotyped for HLA class II (DQ)haplotypes and patients were classified as presenting, in homozy-gosis or heterozygosis, HLA CD high risk haplotypes (HLA-DQ2.5and /or HLA-DQ8) (HR HLA) or the low risk HLA-DQ2.2 haplotype;then we have randomly selected from the these groups 304 HLA-DQ2.5, 93 HLA-DQ8 and 168 HLA-DQ2.2 patients to be further ana-lyzed for IL10 polymorphisms.

CD diagnosis was made according to the European Society forPediatric Gastroenterology, Hepatology and Nutrition guidelines[18].

As healthy controls, we enrolled a total of 576 European–Cauca-sian individuals (European–Caucasian, mean age = 40.56, standarddeviation = 10.30, range = 18–78, sex, 319 females and 257 males),213 carrying in homozygosis or heterozygosis high risk haplotypes(148 HLA-DQ2.5 and 66 HLA-DQ8), 74 HLA-DQ2.2, 292 not carryingany of the HLA risk haplotype (nor, HLA-DQ2.5, nor HLA-DQ2.2, norHLA-DQ8) (NR HLA). All had no clinical signs related to the diseaseand no familiar history of CD. CD was excluded by testing the sub-jects for the presence of anti-tTG antibodies.

Table 1IL10–1082A>G, �819T>C and �592A>C polymorphisms allele, genotype and haplotype freq(HC). Hardy–Weinberg v2 and p values are also reported.

IL10 SNPs Celiac patients

Total Females Malesn = 565 n = 338 n = 227

�1082A>GA 0.60 (682) 0.59 (402) 0.62 (280)G 0.40 (448) 0.41 (274) 0.38 (174)A/A 0.36 (202) 0.35 (118) 0.37 (84)G/A 0.49 (278) 0.49 (166) 0.49 (112)G/G 0.15 (85) 0.16 (54) 0.14 (31)

v2 = 0.45 v2 = 0.12 v2 = 0.43p = 0.50 p = 0.73 p = 0.51

�819C>TC 0.73 (823) 0.73 (493) 0.73 (330)T 0.27 (307) 0.27 (183) 0.27 (124)C/C 0.53 (299) 0.54 (183) 0.51 (116)T/C 0.40 (225) 0.38 (127) 0.43 (98)T/T 0.07 (41) 0.08 (28) 0.06 (13)

v2 = 0.02 v2 = 0.79 v2 = 1.73p = 0.88 p = 0.37 p = 0.19

�592C>AC 0.73 (821) 0.72 (487) 0.74 (334)A 0.27 (309) 0.28 (189) 0.26 (120)C/C 0.53 (300) 0.53 (180) 0.53 (120)C/A 0.39 (221) 0.38 (127) 0.41 (94)A/A 0.08 (44) 0.09 (31) 0.06 (13)

v2 = 0.14 v2 = 1.53 v2 = 0.95p = 0.71 p = 0.22 p = 0.33

HaplotypesGCC 0.39 (443) 0.40 (270) 0.38 (173)ACC 0.51 (370) 0.32 (216) 0.34 (154)ATA 0.26 (296) 0.26 (179) 0.26 (117)Others 0.02 (21) 0.02 (11) 0.02 (10)

Please cite this article in press as: Zupin L et al. Interleukin-10 gene promoter(2014), http://dx.doi.org/10.1016/j.humimm.2014.04.011

A written free and informed consent was obtained from all sub-jects (or their parents in case of minor age).

The study was approved by the local ethical committee (BurloGarofolo protocol no. CE/V-71).

2.2. HLA and IL10 genotyping

Genomic DNAs were extracted using the Wizard Genomic Puri-fication kit (Promega, Madison, WI, USA). Patients and controlswere screened for the presence of HLA class II risk haplotypes byusing the Eu-Gen Risk kit (Eurospital).

IL10–1082A>G (rs1800896), �819C>T (rs1800871) and�592C>A (rs1800872) polymorphisms were detected using,respectively, C_1747360_10, C_1747362_10 and C_1747363_10fluorogenic TaqMan SNP Genotyping Assay (Applied Biosystem �Life Technologies, Carlsbad, California, USA) on the 7500 Fast DXReal Time PCR Instrument (Applied Biosystem).

2.3. Statistical analysis

IL10 gene polymorphisms, allele and genotype frequencies werecalculated by direct counting, while haplotype frequencies andlinkage disequilibrium were computed using the Arlequin software(version 3.1) (http://cmpg.unibe.ch/software/arlequin3/). The Fish-er’s exact test was used for pair wise comparison of allele, geno-type and haplotype frequencies using contingency tables asappropriate and only p-values < 0.05 were considered to be signif-icant. All the statistical analyses were carried out using the open-source R package, available at the http://www.r-project.org site.

uencies (and counts) in all celiac disease (CD) patients and totality of healthy controls

Healthy controls CD vs. HC

Total Females Malesn = 576 n = 319 n = 257

0.61 (699) 0.63 (400) 0.58 (299) p-Value = 0.900.39 (453) 0.37 (238) 0.42 (215)0.36 (205) 0.37 (119) 0.33 (86) p-Value = 0.920.50 (289) 0.51 (162) 0.49 (127)0.14 (82) 0.12 (38) 0.17 (44)v2 = 1.52 v2 = 2.34 v2 = 0.06p = 0.22 p = 0.13 p = 0.80



0.39 (451) 0.37 (238) 0.41 (213) p-Value = 0.420.33 (376) 0.35 (223) 0.30 (153)0.27 (313) 0.27 (171) 0.28 (142)0.01 (12) 0.01 (6) 0.01 (6)

polymorphisms in celiac patients from north-eastern Italy. Hum Immunol

http://cmpg.unibe.ch/software/arlequin3/

http://www.r-project.org


L. Zupin et al. / Human Immunology xxx (2014) xxx–xxx 3

3. Results

The three polymorphisms in the promoter region of the IL10gene at position �1082 (A>G), �819 (T>C) and �592 (A>C) wereanalyzed. All SNPs frequencies were in Hardy–Weinberg equilib-rium in both CD patients and controls with the exception of the�1082A>G polymorphism in the group of HLA-DQ8 healthy con-trols. The �1082A>G, �819T>C and �592A>C polymorphismsresulted to be in linkage disequilibrium (p-value < 0.001,D0 P 0.97, r2 P 0.23) and combined to form three major haplo-types (Tables 1 and 2).

No statistically significant differences were evidenced whencomparing the IL10 allele, genotype and haplotype frequenciesbetween the totality of CD patients and healthy controls (Table 1).

The samples were then stratified according to their HLA class IICD risk haplotypes. For any SNP studied, no statistically significantdifferences between patients and matching HLA controls werefound.

An association was found when the samples were stratifiedaccording to gender: the �1082A allele and A/A genotype were sig-nificantly more frequent in male CD subjects with HLA-DQ8 haplo-type when compared to HLA-DQ8 male controls and associatedwith an increased risk of developing CD (p-value = 0.03,OR = 2.24, CI = 1.05–4.870.20–0.95 and p-value = 0.01, OR = 3.74,CI = 1.27–11.80, respectively). These results were compatible witha recessive model for the A allele (A/A vs. A/G plus G/G, p-value = 0.009, OR = 3.81, CI = 1.31–11.83). Similarly the haplotypefrequencies showed a different distribution: the �1082A allele car-rying haplotypes (ACC plus ATA plus ACA plus ATC) were signifi-cantly more frequent among CD male patients compared tohealthy male controls, where the GCC haplotype was more repre-sented (p-value = 0.03, OR = 0.45, CI = 0.21–0.95) (Table 3). Theseassociations were not evident in female subjects.

4. Discussion

CD is a pathology where gluten proteins intake triggers an auto-immune response and levels of cytokines are altered. The IL-10 isan anti-inflammatory cytokine produced by T-helper 2 cells, ableto downregulate the production of IFN, IL2 and TNF by Th1 cells,causing the characteristic intestinal lesions in CD patients [19].

In the current study no statistically significant differences werefound in the distribution of IL10–1082A>G, �819T>C and �592A>Cpolymorphisms when comparing alleles, genotypes and haplo-types frequencies either between the totality of CD patients andhealthy individuals, or between patients and controls stratifiedaccordingly to their HLA-DQ haplotypes. The stratification for HLAwas performed since HLA-DQ2.5 or HLA-DQ8 are the main knowngenetic risk factors for CD development and could mask the effectsof other genes possibly involved in the pathology. For example,Boniotto et al. [20] have previously reported an associationbetween the MBL2 (Mannose Binding Lecitin 2) gene and risk ofCD in the patients lacking one of the main risk factors such asHLA-DQ8 or HLA-DQ2.5.

However, in our study, also when comparing the IL10 frequen-cies of HLA-DQ2.2 patients, an HLA haplotype associated with onlya slightly increased risk of CD almost comparable to that of otherNR haplotypes, with HLA-DQ2.2 controls and/or controls character-ized by no-CD risk haplotypes, significant differences were notobserved. In our study, none of the CD patient was characterizedby NR HLA haplotype, known to be relatively rare in CD patientsas reported by Karell et al. [21] so we could not compare NR HLAcontrols with NR HLA patients.

Our results are not in agreement with the study by Garrote et al.[15] that found an increased frequency of the GCC haplotype in


Spanish HLA-DQ2 positive patients compared to controls. However,the study of Garrote et al. [15] included a reduced number of sam-ples, 51 CD individuals and 99 healthy controls, while in our studya total of 565 CD patients and 576 healthy controls were tested.

Our data are in accordance with other studies that lacked to findany association between IL10 polymorphisms and CD in two differ-ent Italian groups [16,17].

Contradictory results have been reported concerning the IL-10production levels in CD. [22–25]. Although Forsberg et al. [23] haveobserved an increase of IL-10 mRNA levels in the intestine of CDpatients, nonetheless this could be a consequence of the disease,and not a cause of the pathology; moreover, Cataldo et al. [22] haveshown that, after gluten removal from the diet, the IL-10 levels inCD patients returns to normal levels. The only biological samplesavailable to us for this study were the patients’ DNAs, so it wasnot possible to measure the IL10 levels in patients, although itwould have been of extreme interest.

The three IL10 promoter SNPs analyzed can hypotheticallyaffect the IL10 gene expression [11–14]: the �1082A>G occurwithin a putative Ets transcription factor-binding site, the�819T>C within a putative positive regulatory region, and the�592A>C within a putative STAT 3 binding site and a negative reg-ulatory region [26]. Nonetheless, IL-10 levels are determined bycomplex interactions between cytokines and regulatory elementsin the IL10 gene [14,27,28] and the expression can vary in differentcell types [29]. The lack of association between IL10 promoter poly-morphism and CD, may suggests that they do not have an impor-tant role in the IL-10 production in the intestinal mucosa, or thatthe polymorphisms, in spite of being functional, are not relatedwith the risk of CD development.

However, an association was found when CD samples andhealthy controls were stratified by sex. In male subjects carryingHLA-DQ8 haplotype, the G allele and A/G genotype at position –1082 were more represented among healthy controls than CD sub-jects where the A allele and A/A genotype were more frequent: Gallele and A/G genotype were associated with a protective effectwhereas A allele and A/A genotype at position �1082 were relatedwith an increased predisposition to CD development in the malegroup, but not in females. A similar association between�1082A>G SNP and CD was found by Hahn-Zoric et al. [6]:although not exclusively in males, the authors showed a higher fre-quency of the �1082A>G A/A genotype in Swedish CD patientsthan healthy controls. The association between �1082A>G andincreased CD risk only in the male group could be due to the factthat in women the HLA haplotype influences the development ofCD more than other factors (for example IL-10). Females in factcarry the HLA-DQ2/DQ8 more frequently than males, and maleCD patients develop more often CD in absence of HLA predisposinghaplotype; the presence of HLA-DQ2 and/or HLA-DQ8 molecules isa risk factor stronger in female than in male [30]. This hypothesishowever would not explain why this association is present onlyin males carrying HLA-DQ8 and not in male with HLA-DQ2.2. It isnot excluded that the association we evidenced in HLA-DQ8 malecould be due to a type I error caused by the reduced number ofsamples characterized by this HLA-DQ haplotype. On the otherhand, it has also to be considered that the reduced number of sam-ples could have negatively influenced the power of the study todetect some association, so although our findings suggest thatthe IL10 genetic variants analyzed do not have a major role inthe susceptibility to the development of CD in north eastern Italy,(with maybe the exception of a slightly increased risk for the�1082A allele in HLA-DQ8 male individuals) we cannot completelyexclude that IL-10 cytokine can influence the pathophysiology ofthe disease. For this reason we think that the possible involvementof IL10 gene in CD should deserve further investigation and thatlarge-scale studies are recommended to confirm our findings.



Table 2IL10–1082A>G, �819T>C and �592A>C allele, genotype and haplotype frequencies (and counts) in HLA-DQ2.5, HLA-DQ2.2 and HLA-DQ8 CD patients and HC; HLA-DQ2.5 and HLA-DQ8 individuals were also grouped together as high risk-HLA (HLA HR); healthy individuals with HLA no-risk haplotype were designed as NR HLA; HLA-DQ2.2 and NR HLA controls were also grouped together as HLA-DQ2.2 + NR HLA controls. Hardy–Weinberg v2 and p values are also reported.

IL10 Celiac patients Healthy controls

HLA-DQ2.5 HLA-DQ2.2 HLA-DQ8 HR HLA HLA-DQ2.5 HLA-DQ2.2 HLA-DQ8 HR HLA NR HLA HLA-DQ2.2 + NR HLAn = 304 n = 168 n = 93 n = 397 n = 148 n = 71 n = 66 n = 214 n = 291 n = 362

�1082A>GA 0.60 (363) 0.57 (193) 0.68 (126) 0.62 (489) 0.59 (174) 0.58 (82) 0.63 (83) 0.59 (253) 0.63 (364) 0.62 (446)G 0.40 (245) 0.43 (143) 0.32 (60) 0.38 (305) 0.41 (122) 0.42 (60) 0.37 (49) 0.41 (175) 0.37 (218) 0.38 (278)A/A 0.35 (106) 0.33 (55) 0.44 (41) 0.37 (147) 0.34 (50) 0.37 (26) 0.33 (22) 0.33 (71) 0.37 (108) 0.37 (134)G/A 0.50 (151) 0.49 (83) 0.47 (44) 0.49 (195) 0.50 (74) 0.42 (30) 0.59 (39) 0.52 (111) 0.51 (148) 0.49 (178)G/G 0.15 (47) 0.18 (30) 0.09 (8) 0.14 (55) 0.16 (24) 0.21 (15) 0.08 (5) 0.15 (32) 0.12 (35) 0.14 (50)

v2 = 0.32 p = 0.57 v2 = 0.02 p = 0.89 v2 = 0.63 p = 0.43 v2 = 0.58 p = 0.45 v2 = 0.15 p = 0.70 v2 = 1.28 p = 0.26 v2 = 4.66 p = 0.03 v2 = 1.14 p = 0.28 v2 = 2.13 p = 0.14 v2 = 0.56 p = 0.45

�819C>TC 0.74 (448) 0.73 (246) 0.69 (128) 0.73 (577) 0.73 (215) 0.70 (100) 0.73 (97) 0.73 (313) 0.72 (421) 0.72 (521)T 0.26 (160) 0.27 (90) 0.31 (58) 0.27 (217) 0.27 (81) 0.30 (42) 0.27 (35) 0.27 (115) 0.28 (161) 0.28 (203)C/C 0.54 (163) 0.54 (91) 0.48 (45) 0.52 (208) 0.51 (76) 0.51 (36) 0.53 (35) 0.52 (112) 0.53 (154) 0.52 (190)T/C 0.40 (123) 0.38 (64) 0.41 (38) 0.41 (161) 0.43 (63) 0.39 (28) 0.41 (27) 0.42 (89) 0.39 (113) 0.39 (141)T/T 0.06 (18) 0.08 (13) 0.11 (10) 0.07 (28) 0.06 (9) 0.10 (7) 0.06 (4) 0.06 (13) 0.08 (24) 0.09 (31)


�592C>AC 0.74 (448) 0.74 (247) 0.68 (127) 0.72 (574) 0.73 (217) 0.71 (101) 0.74 (98) 0.74 (316) 0.71 (415) 0.71 (516)A 0.26 (160) 0.26 (89) 0.32 (59) 0.28 (220) 0.27 (79) 0.29 (41) 0.26 (34) 0.26 (112) 0.29 (167) 0.29 (208)C/C 0.53 (163) 0.55 (92) 0.49 (46) 0.52 (208) 0.52 (77) 0.52 (37) 0.54 (36) 0.53 (114) 0.52 (151) 0.52 (188)C/A 0.40 (123) 0.38 (63) 0.38 (35) 0.40 (158) 0.43 (63) 0.38 (27) 0.40 (26) 0.41 (89) 0.39 (113) 0.39 (140)A/A 0.06 (19) 0.08 (13) 0.13 (12) 0.08 (31) 0.05 (8) 0.10 (7) 0.06 (4) 0.06 (12) 0.09 (27) 0.09 (34)


HaplotypeGCC 0.40 (242) 0.42 (141) 0.32 (60) 0.38 (302) 0.40 (120) 0.42 (60) 0.37 (49) 0.40 (173) 0.37 (218) 0.38 (278)ACC 0.33 (201) 0.31 (104) 0.35 (65) 0.33 (266) 0.32 (95) 0.28 (40) 0.36 (48) 0.33 (140) 0.34 (196) 0.33 (236)ATA 0.25 (153) 0.26 (87) 0.30 (56) 0.26 (209) 0.27 (79) 0.29 (41) 0.26 (34) 0.26 (112) 0.27 (160) 0.28 (201)Others 0.02 (12) 0.01 (4) 0.03 (5) 0.02 (17) 0.01 (2) 0.01 (1) 0.01 (1) 0.01 (3) 0.01 (8) 0.01 (9)

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Table 3IL10–1082A>G, �819T>C and �592A>C allele, genotype and haplotype frequencies (and counts) in HLA-DQ8-CD patients and HLA-DQ8-HC stratified by sex. Hardy–Weinberg v2

and p values are also reported.

IL10 Celiac patients HLA-DQ8 Healthy controls HLA-DQ8 CD-DQ8 vs. HC-DQ8 CD-DQ8 vs. HC-DQ8

Females Males Females Males Females Malesn = 54 n = 39 n = 30 n = 36

�1082A>GA 0.61 (66) 0.77 (60) 0.67 (40) 0.60 (43) p-Value = 0.51 p-Value = 0.03*

G 0.39 (42) 0.23 (18) 0.33 (20) 0.40 (29)A/A 0.35 (19) 0.56 (22) 0.43 (13) 0.25 (9) p-Value = 0.77 p-Value = 0.01*

G/A 0.52 (28) 0.41 (16) 0.47 (14) 0.69 (25)G/G 0.13 (7) 0.03 (1) 0.10 (3) 0.06 (2)HW v2 = 0.45 p = 0.50 v2 = 0.94 p = 0.33 v2 = 0.07 p = 0.78 v2 = 7.08 p = 0.01

�819C>TC 0.72 (78) 0.64 (50) 0.73 (44) 0.74 (53) p-Value = 1.00 p-Value = 0.22T 0.28 (30) 0.36 (28) 0.27 (16) 0.26 (19)C/C 0.56 (30) 0.38 (15) 0.53 (16) 0.53 (19) p-Value = 0.79 p-Value = 0.44T/C 0.33 (18) 0.51 (20) 0.40 (12) 0.42 (15)T/T 0.11 (6) 0.10 (4) 0.07 (2) 0.06 (2)HW v2 = 1.55 p = 0.21 v2 = 0.51 p = 0.48 v2 = 0.02 p = 0.90 v2 = 0.19 p = 0.66

�592C>AC 0.71 (77) 0.64 (50) 0.75 (45) 0.74 (53) p-Value = 0.72 p-Value = 0.22A 0.29 (31) 0.36 (28) 0.25 (15) 0.26 (19)C/C 0.56 (30) 0.41 (16) 0.57 (17) 0.53 (19) p-Value = 0.72 p-Value = 0.52C/A 0.31 (17) 0.46 (18) 0.37 (11) 0.42 (15)A/A 0.13 (7) 0.13 (5) 0.07 (2) 0.06 (2)HW v2 = 2.88 p = 0.09 v2 = 0.0003 p = 0.99 v2 = 0.01 p = 0.90 v2 = 0.19 p = 0.66

HaplotypeGCC 0.39 (42) 0.23 (18) 0.33 (20) 0.40 (29) p-Value = 0.69 p-Value = 0.04*

ACC 0.32 (35) 0.38 (30) 0.40 (24) 0.33 (24)ATA 0.28 (30) 0.33 (26) 0.25 (15) 0.26 (19)Others 0.01 (1) 0.05 (4) 0.02 (1) 0.00 (0)

* Statistically significant p-values.

L. Zupin et al. / Human Immunology xxx (2014) xxx–xxx 5

Disclosure statement

The authors declare that they have no conflict of interest.

Authors contribution

L.Z. performed IL10 genotyping, conducted the statistical analy-ses and drafted the manuscript; V.P. participated in statistical anal-yses and writing the manuscript; E.C. performed DNA extraction;S.C. assessed the HLA genotyping and critically revised the manu-script; L.S. conceived the study and revised the manuscript.

Acknowledgment

This work has been supported by RC13/12 grants from IRCCSBurlo Garofolo Trieste (Italy).

References

[1] Green PHR, Cellier C. Celiac disease. N Engl J Med 2007;357(17):1731–43.[2] Kagnoff MF. Celiac disease: pathogenesis of a model immunogenetic disease. J

Clin Invest 2007;117(1):41–9.[3] Vader W, Stepniak D, Kooy Y, Mearin L, Thompson A, van Rood JJ, et al. The

HLA-DQ2 gene dose effect in celiac disease is directly related to the magnitudeand breadth of gluten-specific T cell response. Proc Natl Acad Sci USA2003;100(21):12390–5.

[4] Sollid LM, Markussen G, Ek J, Gjerde H, Vartdal F, Thorsby E. Evidence for aprimary association of celiac disease to a particular HLA-DQ alpha/betaheterodimer. J Exp Med 1989;169(1):345–50.

[5] Núñez C, Alecsandru D, Varadé J, Polanco I, Maluenda C, Fernández-Arquero M,et al. Interleukin-10 haplotypes in celiac disease in the Spanish population.BMC Med Genet 2006;7:32.

[6] Hahn-Zoric M, Hytonen AM, Hanson LA, Nilsson LA, Padyukov L. Association of–1087 IL10 and –308 TNFA gene polymorphisms with serological markers ofcoeliac disease. J Clin Immunol 2003;23(4):291–6.

[7] Salvati VM, Mazzarella G, Gianfrani C, Levings MK, Stefanile R, De Giulio B,et al. Recombinant human interleukin 10 suppresses gliadin dependent T cell


activation in ex vivo cultured coeliac intestinal mucosa. Gut2005;54(1):46–53.

[8] Plaza-Izurieta L, Castellanos-Rubio A, Irastorza I, Fernandez-Jimenez N,Gutierrez G, CEGEC. Revisiting genome wide association studies (GWAS) incoeliac disease: replication study in Spanish population and expressionanalysis of candidate genes. J Med Genet 2011;48(7):493–6.

[9] Romanos J, Barisani D, Trynka G, Zhernakova A, Bardella MT, Wijmenga C. Sixnew coeliac disease loci replicated in an Italian population confirm associationwith coeliac disease. J Med Genet 2009;46(1):60–3.

[10] Hunt KA, Zhernakova A, Turner G, Heap GAR, Franke L, Bruinenberg M, et al.Newly identified genetic risk variants for celiac disease related to the immuneresponse. Nat Genet 2008;40(4):395–402.

[11] Gallager PM, Lowe G, Fitzgerald T, Bella A, Greene CM, McElvaney NG, et al.Association of IL-10 polymorphism with severity of illness in communityacquired pneumonia. Thorax 2003;58(2):154–6.

[12] Rad R, Dossumbekova A, Neu B, Lang R, Bauer S, Saur D, et al. Cytokine genepolymorphisms influence mucosal cytokine expression, gastric inflammation,and host specific colonisation during Helicobacter pylori infection. Gut2004;53(8):1082–9.

[13] Ramkumar HL, Shen de F, Tuo J, Braziel RM, Coupland SE, Smith JR, et al. IL-10 -1082 SNP and IL-10 in primary CNS and vitreoretinal lymphomas. Graefes ArchClin Exp Ophthalmol 2012;250(10):1541–8.

[14] Turner DM, Williams DM, Sankaran D, Lazarus M, Sinnot PJ, Hutchinson IV. Aninvestigation of polymorphism in the IL-10 gene promoter. Eur J Immunogen1997;24(1):1–8.

[15] Garrote J, Arranz E, Gomez-Gonzalez E, Leon A, Farre C, Calvo C, et al. IL6, IL10and TGFB1 gene polymorphisms in coeliac disease: differences between DQ2positive and negative patients. Allergol Immunopathol 2005;33(5):245–9.

[16] Lio D, Scola L, Forte GI, Accomando S, Giacalone A, Crivello A, et al. AlimentaryTract TNF, IFN and IL-10 gene polymorphisms in a sample of Sicilian patientswith coeliac disease. Dig Liver Dis 2005;37(10):756–60.

[17] Cataldo F, Lio D, Marino V, Scola L, Crivello A, Corazza GR, et al. Cytokinegenotyping (TNF and IL-10) in patients with celiac disease and selective IgAdeficiency. Am J Gastroenterol 2003;98(4):850–6.

[18] Walker-Smith JA, Guandalini S, Schmitz J, Shmerling DH, Visakorpi JK. Revisedcriteria for diagnosis of celiac disease. Report of working group of EuropeanSociety of paediatric gastroenterology and nutrition. Arch Dis Child1990;65(8):909–11.

[19] Fiorentino DF, Bond MW, Mosmann TR. Two types of mouse T helper cell: IV.Th2 clones secrete a factor that inhibits cytokine production by Th1 clones. JExp Med 1989;170(6):2081–95.

[20] Boniotto M, Braida L, Baldas V, Not T, Ventura A, Vatta S, et al. Evidence of acorrelation between mannose binding lectin and celiac disease: a model forother autoimmune diseases. J Mol Med 2005;83(4):308–15.


http://refhub.elsevier.com/S0198-8859(14)00122-0/h0005































































6 L. Zupin et al. / Human Immunology xxx (2014) xxx–xxx

[21] Karell K, Louka AS, Moodie SJ, Ascher H, Clot F, Greco L, et al. HLA types inceliac disease patients not carrying the DQA1*05-DQB1*02 (DQ2)heterodimer: results from the European genetics cluster on celiac disease.Hum Immunol 2003;64(4):469–77.

[22] Cataldo F, Lio D, Marino V, Scola L, Crivello A, Corazza GR. Plasma cytokineprofiles in patients with celiac disease and selective IgA deficiency. PediatrAllergy Immunol 2003;14(4):320–4.

[23] Forsberg G, Hernell O, Melgar S, Israelsson A, Hammarstrom S, HammarstromML. Paradoxical coexpression of proinflammatory and down-regulatorycytokines in intestinal T cells in childhood celiac disease. Gastroenterology2002;123(3):667–78.

[24] Mizrachi A, Broide E, Buchs A, Kornberg A, Aharoni D, Bistritzer T, et al. Lack ofcorrelation between disease activity and decreased stimulated secretion of IL-10 in lymphocytes from patients with celiac disease. Scand J Gastroenterol2002;37(8):924–30.

[25] Koss K, Satsangi J, Fanning GC, Welshe KI, Jewell DP. Cytokine (TNF alpha, LTalpha and IL-10) polymorphisms in inflammatory bowel diseases and normalcontrols: differential effects on production and allele frequencies. GenesImmun 2000;1(3):185–90.


[26] Kube D, Platzer C, von Knethen A, Straub H, Bohlen H, Hafner M, et al. Isolationof the human interleukin 10 promoter: characterization of the promoteractivity in Burkitts lymphoma cell lines. Cytokine 1995;7(1):1–7.

[27] Sato K, Nagayama H, Tadokoro K, Juji T, Takahashi TA. Extracellularsignal regulated kinase, stress-activated protein kinase/c-Jun N-terminalkinase, and p38mapk are involved in IL-10-mediated selectiverepression of TNF-alpha-induced activation and maturation of humanperipheral blood monocyte-derived dendritic cells. J Immunol 1999;162(7):3865–72.

[28] Eskdale J, Keijsers K, Huizinga T, Gallagher G. Microsatellite alleles and singlenucleotide polymorphisms (SNP) combine to form four major haplotypefamilies at the human interleukin10 (IL-10) locus. Genes Immun1999;1(2):151–5.

[29] Hedrich CM, Bream JH. Cell type-specific regulation of IL-10 expression ininflammation and disease. Immunol Res 2010;47(1–3):185–206.

[30] Megiorni F, Mora B, Bonamico M, Barbato M, Montuori M, Viola F, et al.HLA-DQ and susceptibility to celiac disease: evidence for genderdifferences and parent-of-origin effects. Am J Gastroenterol 2008;103(4):997–1003.









































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Interleukin-10 gene promoter polymorphisms in celiac patients from north-eastern Italy