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wWFÈk Faculdade de Ciências da Nutrição e Alimentação UNIVERSIDADE DO PORTO Pro-inflammatory genetic polymorphisms and risk of developing Celiac Disease Fábio Pires Pereira Julho 2004 - Porto

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wWFÈk Faculdade de Ciências da Nutrição e Alimentação

UNIVERSIDADE DO PORTO

Pro-inflammatory genetic polymorphisms and risk of developing

Celiac Disease

Fábio Pires Pereira

Julho 2004 - Porto

DM - °! 1

2

CONTENTS

1. ABSTRACT 04

2. INTRODUCTION 05

3. AIM 11

4. MATERIALS AND METHODS 11

5. RESULTS 15

6. DISCUSSION 20

7. CONCLUDING REMARKS 23

8. REFERENCES 24

J

ABBREVIATIONS

CD - Celiac Disease

CI - Confidence Interval

DNA - Deoxyribonucleic Acid

HLA - Human Leukocyte Antigen

IFN-y - Interferon gamma

IFNGR1 - Interferon Gamma Receptor 1 gene

IL-1 - lnterleukin-1

IL-8 - lnterleukin-8

IL-1ra - lnterleukin-1 Receptor Antagonist

IL-1B — lnterlukin-1 beta

IL1B - lnterleukin-1 beta gene

IL1RN - lnterleukin-1 Receptor Antagonist gene

MDE - Mutation Detection Enhancement

MHC - Major Histocompatibility Complex

MIF - Macrophage Migration Inhibitory Factor

OR - Odds Ratio

PCR - Polymerase Chain Reaction

RFLP - Restriction Fragment Length Polymorphism

SSCP - Single Strand Conformation Polymorphism

TNF- - Tumor Necrosis Factor alpha

TNFA - Tumor Necrosis Factor Alpha gene

VNTR - Variable Number of Tandem Repeats

4

1. ABSTRACT

Introduction

Environmental and genetic factors play an important role in Celiac disease (CD). The

relationship between HLA-genes and this disease is now well established, but it is

also clear that other factors have a role in susceptibility. The aim of this study was to

determine the association between polymorphisms in the TNFA, IFNGR1, IL8, IL1B,

MIF and IL1RN genes and risk of development of CD in a Portuguese population.

Materials and Methods:

In a case-control study including 60 CD patients and 930 controls (313 adults and

617 children), the TNFA (-308G/A and -857CAT), IFNGR1 (-56C/T), IL1B (-511 CAT),

IL8 (-251 C/T), IL1RN (intron 2 VNTR), and MIF (-797 VNTR) gene polymorphisms

were genotyped.

Results

A significant association between CD and both the heterozygous GA genotype and

the homozygous AA genotype of the TNFA-308 polymorphism was observed, with an

odds-ratio (OR) of 3.1 (95% confidence interval [CI] = 1.79-5.37) and 10.6 (95% CI =

3.47-32.1), respectively. No relevant associations were found with the TNFA-857,

IFNGR1-56, IL8-251, IL1B-511, IL1RN VNTR and the MIF-797 VNTR

polymorphisms.

Conclusions

These findings suggest that TNFA-308 polymorphism may be associated and

contribute to the risk of developing CD.

5

2. INTRODUCTION

Celiac disease (CD) is a complex and chronic inflammatory intestinal disorder with a

multifactorial etiology, in which genetic and environmental factors play a major role

(1, 2). In susceptible persons, ingestion of one of several proteins found in wheat

(gliadins), barley (hordeins) and rye (secalins), triggers an auto-immune condition,

resulting in infiltration of the intestinal mucosa by both intraepithelial CD8+

lymphocytes and CD4+ lamina propria lymphocytes (3), and, in due course, to crypt

hyperplasia, villous atrophy and flattening of the mucosa (2, 3, 4).

CD was thought to be rare and to occur only in childhood. However, the disease is

now recognized as a common condition in western societies, with a high prevalence

in Caucasians (1 in 200 individuals) (1, 2, 5), but only 20-50% of those affected

individuals present gastrointestinal symptoms (2). This symptomatic presentation

(classical form) is commonly diagnosed in early childhood and is typically

characterized by chronic diarrhea, anorexia, abdominal extension and failure to

thrive. Atypical forms do not present with gastrointestinal symptoms and silent celiac

disease patients are thought to be at risk of developing the same long-term

complications experienced by individuals with typical forms. These include metabolic

bone disease, anemia, chronic hepatitis, other auto-immune diseases and lymphoma

(6).

To date, the cornerstone of treatment is a total lifelong adherence to a gluten

exclusion-diet, and poor diet compliance is associated with increased morbidity and

mortality (2, 6). Wheat, rye and barley should be avoided, but it should not be

forgotten that many oat products are not free of contamination by wheat gluten or

other grains (7). Some patients have a poor clinical response to treatment with a

6

gluten-free diet (6), and steroidal and immunosuppressant treatment therapies may

be necessary (6, 8).

CD is a strongly heritable disease that clusters in families. The disease risk for a

sibling of an affected individual is 20 to 60 times higher than a member of the general

population (2). A high concordance between monozygotic twins was found (75%)

compared to dyzigotic twins (11%) (9). Studies have implicated human leukocyte

antigen (HLA) in disease propensity, and thus, a relationship between CD and major

histocompatibility complex (MHC) molecules is now well established (10, 11, 12). The

HLA-DQ2 heterodimer (DQA1*0501/DQB1*0201) is encoded by more than 95% of

celiac patients, either in cis - DR3-DQ2 haplotype - or in trans - DR5-DQ7 and DR7-

DQ2 hétérozygotes - form, and the remaining sharing DQ8 protein

(DQA1*0301/DQB1*0302) (10, 11,13, 14). Patients who lack HLA-DQ2 or HLA-DQ8

are remarkably rare (11) and unlikely to have celiac disease (1), since these

molecules appear to be necessary, although not sufficient, for the development of the

disease (11). The calcium-dependent enzyme tissue transglutaminase expressed on

the subepithelial layer of intestinal epithelium, deaminates the positively charged

glutamine residues present in gliadin to negatively charged glutamic acids. The

deaminated peptides adhere strongly to the DQ2 or DQ8 positively charged binding

groves, eliciting a strong CD4+T cell response and inflammatory cascade initiation (1,

3).

Little is known about non-HLA linked genes and their associations with the disease

development, but since the DQ markers are also present in 20-30% of normal

individuals (5, 15), it is likely that other genetic factors also play an important role in

the etiopathogenesis of this disease. It has been demonstrated that human genetic

polymorphisms within some inflammation cytokine genes are associated with risk of

7

several diseases. Examples of such cases include polymorphisms in the tumor

necrosis factor alpha gene (TNFA-308A allele), interleukin-1 beta gene (IL1B-511T

allele) and lnterleukin-1 receptor antagonist gene (IL1RN*2 allele) (11, 16, 17, 18, 19,

20, 21, 22, 24, 25). The putative explanation for such associations is that these

polymorphisms could influence the expression of the gene since most of them are

located in the gene's promoter region (26). Thus, polymorphisms in genes that are

associated with an enhanced chronic inflammatory condition may play an important

role in the susceptibility to the development of CD.

Tumor Necrosis Factor-alpha (TNF- ) is a member of a large family of proteins and

receptors that are involved in immune regulation (24). TNF- is a potent pro­

inflammatory cytokine produced mainly by monocyte/macrophage lineage but also T-

cells, neutrophils and mast cells, and has been implicated in the pathogenesis of

several conditions, including malaria, rheumatoid arthritis, infection, systemic lupus

erythematosus and insulin dependent diabetes (16, 24, 26, 27). Many of the

biological properties of TNF- , like fever and insulin resistance (8), are mediated in

synergy with other cytokines such as IL-1 and IFN-y, and it has been suggested that

TNF- in vivo coordinates the cytokine response (24). The TNFA gene is located in

chromosome 6 within the class III region of the highly polymorphic major

histocompatibility complex (MHC), where TNF production is regulated at

transcriptional and posttranscriptional level (26, 27). It has been shown that TNF-

expression is up-regulated in epithelial cells and intraepithelial lymphocytes in the

mucosa of patients with CD (29). Several studies have suggested TNFA promoter

polymorphisms as possible candidates involved in determining CD pathogenesis and

susceptibility (17, 18, 19, 27, 28, 30). Such polymorphisms include the -308A allele,

which may have a direct increasing effect on transcriptional activity leading to a

8

higher production of TNF- , as reported in in vitro experiments (26, 31), as well as in

serum levels measurement experiments (18). This pro-inflammatory phenotype could

predispose to a persistent and/or a more severe form of inflammation, thus

influencing the initiation and/or progress of CD.

Interferon-y (IFN-y) is a pleiotropic cytokine with a major role in host defenses against

infectious agents and in overall immunomodulation (32). IFN-y regulates the action of

mononuclear phagocytes and the production of the pro-inflammatory cytokines IL-12

and TNF- (32). Several findings support a major role of IFN-y in CD pathogenesis:

(a) expression of IFN-y is remarkably expressed in the duodenal mucosa of patients

after gluten exposure in vitro and in vivo (33); (b) patients with untreated disease

contain increased number of IFN-y-positive lamina propria cells (33, 34); and (c)

secreting IFN-y isolated T cells are located in the duodenal epithelium of patients with

classical and refractory disease (35). Thus, if gluten induces an intestinal cytokine

response dominated by IFN-y in CD, and considering that IFN-y is also an important

macrophage activator, it is likely that these activated macrophages might produce

TNF- and probably other factors. Additionally, it was hypothesized that

macrophages secrete metalloproteinases that could disintegrate the mucosal matrix,

thereby causing the typical crypt hyperplasia observed in CD (33). The polymorphism

-56 C/T in the gene encoding IFN-y receptor I (IFNGR1) was reported to influence the

level of gene expression and overexpression of this receptor might contribute to the

exacerbation of an inflammatory response. Thus, a lower level of expression of

IFNGR1, that would lead to a reduced IFN-y-mediated response, could translate into

a protective effect (36).

lnterleukin-8 (IL-8) is known to be a chemotatic cytokine, capable of activating

neutrophils and T-lymphocytes, and has been implicated in a variety of inflammatory

9

diseases (37, 38). IL-8 is produced in high amounts by granulocytes, but also by

epithelial and endothelial cells, macrophages and fibroblasts (37). Considered as a

potent mitogen to human intestinal cells, the mitogenic action of IL-8 is apparent

even at low concentration, and although limited information is available, the overall

findings suggest that this cytokine may be involved in the regulation of cell

proliferation and normal mucosal homeostasis (37, 38). A relationship between TNF-

and IL-8 is proposed by a report showing IL-8 production after TNF- stimulation in

colonic epithelial cells (39). Furthermore, the IL8-251 A/T polymorphism has been

found to have an effect in changing the in vitro levels of IL-8, and a decreased risk of

colorectal cancer was found for the allele A in this promoter position (40). Thus,

considering that CD condition is characterized by an enhanced cell proliferation, it

would be acceptable that IL-8 could contribute, at least to some extent, to cell

kinetics in human small intestinal mucosa.

Interleukin-1G (encoded by IL1B), a strong inducer of inflammation, plays an

important role in initiating and amplifying the inflammatory response (41), with

enhanced levels of this cytokine being reported in the mucosa of patients with active

inflammatory bowel disease (21). lnterleukin-1 receptor antagonist (encoded by

IL1RN) is an endogenous antagonist of IL-1B that competitively binds to IL-1R

receptors without inducing any cellular response, and thus modulating the potentially

injurious effects of IL-1B (22). A penta-allelic 86 base pair tandem repeat

polymorphism is present in intron 2 of the IL1RN gene, of which the 2 repeat allele is

associated with a wide range of inflammatory conditions (22, 42, 43), as does IL1B-

511 C/T promoter polymorphism (42, 21). IL1B and the IL1RN gene polymorphisms,

which are putatively associated with increased levels of IL-1B production (44, 45, 46)

were also associated with inflammatory bowel disease (21, 46).

10

The macrophage migration inhibitory factor (MIF) is a potent pro-inflammatory

mediator, but is now emerging as an immuno-neuroendocrine modulator (20). MIF is

released by cells of the anterior pituitary gland, but macrophages are significant

sources as well (20, 47). Once released, MIF is directly pro-inflammatory through

activating or promoting cytokine expression: TNF- , IL1-R, IL-8, IL-6 and IFN-y (20,

47). In the case of the macrophage, MIF promotes nitric oxide release by interacting

with IFN-y (29) and TNF- enhanced production leads to further MIF release (48).

Activated T cells also secrete MIF, which, in an autocrine way, enhances IL-2 and

IFN-y production (49). Within the inflammatory setting, MIF can also override or

counteract the anti-inflammatory and immunosupressive action of endogenous and

exogenous glucocorticoids on downstream the pro-inflammatory cytokine cascade

(20, 49). MIF gene-promoter polymorphism consists of 5 to 8 tetranucleotide CATT

repeats located at position -797 (20, 47). This promoter polymorphism has been

shown to be functionally active in in vitro assays, as the 5-CATT allele showed lower

transcriptional activity (47). MIF has been associated with several inflammatory

clinical conditions including rheumatoid arthritis (47), multiple sclerosis (48), lung

disease and inflammatory bowel disease (25). However, data regarding the

inflammatory role of MIF in gastrointestinal disease is scarce.

The molecular basis of CD is of enormous complexity, involving genetic and

environmental factors. It is widely accepted that the number of genes that play a role

in the development and the pathogenesis of the disease, may be large. Promoter's

genes are involved in initiating transcription and might harbor functionally relevant

polymorphisms that alter cytokine production and expression. However, the

combined importance of several pro-inflammatory cytokines may play a role not yet

understood in the context of gluten intolerance.

11

3. AIM

The aim of this study was to determine the association between polymorphisms in

the TNFA, IFNGR1, IL8, IL1B, MIF and IL1RN genes and risk of development of CD

in a Portuguese population.

4. MATERIALS AND METHODS

Study population

This case control study was performed in a series of patients with CD (n=60) and in a

control group (n=312). All cases and controls were collected in northern Portugal.

The control group consists of healthy blood-donors (mean age 37 years; median age

35 years; range 18-64 years; male.female ratio 1.7:1). Individuals with CD (mean age

2.7 years; median age 2 years; range 1-14 years; male:female ratio 0.7:1) were

recruited at the Inflammatory Bowel Disease outpatient clinic of the Pediatric

Department of the Hospital São João, Porto, Portugal. For the analysis of the TNFA-

308, TNFA-857, IFNGR1, IL8 and IL1RN polymorphisms an additional group of

samples was available as control (n=617). This group consists of healthy children

and young adults recruited from schools and available from the Pediatric Department

of the Hospital São João, Porto, Portugal (mean age 14 years; median age 14 years;

range 6-21 years; male:female ratio 0.7:1). Genomic DNA from all the individuals

was isolated from blood samples.

The procedures followed in the present study were in accordance with the

institutional ethical standards. All the samples included in the present study were

delinked and unidentified from their donors. Written informed consent was obtained

from all subjects.

12

Genotyping of the TNFA, IFNGR1, IL8, IL1B, MIF and IL1RN polymorphisms.

Genomic DNA was retrieved from blood samples using standard proteinase k

digestion and phenol/chloroform extraction. For controls, the IL1B-511 polymorphism

was genotyped by cold polymerase chain reaction-single-strand conformation

polymorphism (PCR-SSCP) analysis. PCR amplifications were performed in a 25 ^L

volume containing 200 p.mol/L each of deoxynucleoside triphosphate, 20 pmol each

of the forward and reverse primers, 50 mmol/L KCI, 10 mmol/L Tris-HCI (pH 9.0), 1.5

mmol/L MgCb, and 1 U of Taq DNA polymerase (Amersham Biosciences). The

oligonucleotides 5'-GCCTGAACCCTGCATACCGT-3' and 5'-GCCAATAGCCCTCC-

CTGTCT-3' were used as primers in the PCR. Cycling conditions were as follows: 30

seconds at 94°C, 30 seconds at 58°C and 30 seconds at 72°C, for 35 cycles. For

SSCP analysis, PCR reaction products were diluted 1:1 with loading buffer (95%

formamide, 0.05% xylene cyanol and 0.05% bromophenol blue), denatured at 99°C

for 2 minutes, and cooled on ice for 5 minutes. Electrophoresis of the denatured PCR

products was performed in non-denaturing 0.8X MDE gels (BMA, Rockland, ME) and

run at 160 volts, 20°C for 15 hours. PCR-SSCP products were visualized by standard

DNA silver staining. For cases, a PCR-restriction fragment length polymorphism

(RFLP) approach was used. A fragment of 155 bp containing the Aval polymorphic

site at position -511 was amplified by PCR. The oligonucleotides 5'-

GCCTGAACCCTGCATACCGT-3' and 5'-GCCAATAGCCCTCCCTGTCT-3', flanking

this region were used as primers. PCR amplifications were performed in a 25 |al_

volume containing 200 |j.mol/L each of deoxynucleoside triphosphate, 20 pmol each

of the forward and reverse primers, 50 mmol/L KCI, 10 mmol/L Tris-HCI (pH 9.0), 1.5

mmol/L MgCI2, and 1 U of Taq DNA polymerase (Amersham Biosciences). PCR

conditions were as follows: 30 seconds at 94°C, 30 seconds at 58°C and 30 seconds

13

at 72°C, for 35 cycles. The PCR products were digested with 5U of Aval at 37° for 12

hours. Fragments were separated by electrophoresis on 1,5% agarose gels and

stained with ethidium bromide. The alleles were designated as follows: C allele with 2

bands of 90 and 65 bp, T allele with a single band of 155 bp, and the C/T allele with 3

bands of 155, 90 and 65 bp.

The TNFA-308, TNFA-857, and IFNGR1-56 polymorphisms were genotyped by the

Taqman system (Applied Biosystems) using assays-on-demand provided by Applied

Biosystems (C_7514879_10, C_11918223_10 and C_11693991_10, respectively).

The IL8-251 polymorphism was also genotyped by TaqMan system but the

oligonucleotides 5'-TAAAATACTGAAGCTCCACAATTTGG-3' and 5'-ATCTTGTTCT-

AACACCTGCCACTCT-3' were used as primers, and 5'-CATACAATTGATAATTCA-

MGB-3' and 5'-CATACATTTGATAATTCA-MGB-3', as probes. PCR amplifications

were performed in a 25 ^L volume containing TaqMan Universal Master Mix 1x,

900nM of the forward and reverse primer, 200nM of the VIC and FAM probes.

MIF-797 VNTR was genotyped by PCR-GeneScan analysis (Abi Prism 310 Genetic

Analyser). PCR amplifications were performed in a 25 (iL volume containing 200

[4.mol/L each of deoxynucleoside triphosphate, 20 pmol each of the forward and

reverse primers, 50 mmol/L KCI, 10 mmol/L Tris-HCI (pH 9.0), 1.5 mmol/L MgCI2,

and 1,5 U of Taq DNA polymerase (Amersham Biosciences). The oligonucleotides

5'-Tet-GTTGCTGCCTTGTCCTCTTC-3' and 5'-CAGGCATATCAAGAGACATTGA-3'

were used as primers in the PCR. Cycling conditions were as follows: 45 seconds at

94°C, 45 seconds at 58°C and 45 seconds at 72°C, for 35 cycles. PCR products were

prepared to GeneScan analysis with deionized formamide (Amresco) and TAMRA

(Applied Biosystems).

14

The IL1RN penta-allelic intron 2 VNTR was genotyped by PCR-standard agarose gel

electrophoresis. PCR amplifications were performed in a 25 |uL volume containing

200 nmol/L each of deoxynucleoside triphosphate, 20 pmol each of the forward and

reverse primers, 50 mmol/L KCI, 10 mmol/L Tris-HCI (pH 9.0), 1.5 mmol/L MgCI2,

and 1 U of Taq DNA polymerase (Amersham Biosciences). The oligonucleotides 5'-

CCCCTCAGCAACACTCC-3' and 5'-GGTCAGAAGGGCAGAGA-3' were used as

primers in the PCR. Cycling conditions were as follows: 30 seconds at 94°C, 30

seconds at 57°C and 1 minute at 72°C, for 35 cycles. PCR products were separated

by electrophoresis on 2% agarose gels and stained with ethidium bromide. PCR

products were sized relative to a 1-kilobase ladder. The IL1RN alleles were coded as

follows: allele 1=4 repeats, allele 2=2 repeats, allele 3=5 repeats, allele 4=3 repeats,

and allele 5=6 repeats. For the purpose of statistical analysis and due to the rarity of

alleles 3, 4 and 5, this polymorphism was treated as bi-allelic by dividing alleles into

short and long categories, the short allele being those with 2 repeats (allele 2) and

the long allele being those with 3 repeats or more (alleles 1, 3, 4, and 5).

Statistical analysis

Evidence for deviation from Hardy-Weinberg equilibrium of alleles at individual loci

was assessed by exact tests using the program GENEPOP (available from

ftp://ftp.cefe.cnrs-mop.fr/pub/pc/msdos/genepop/). Comparison of genotype

frequencies between the different groups of samples was also assessed by exact

tests using the program GENEPOP. Odds ratios (OR) with 95% confidence intervals

(CI) were estimated by logistic regression analysis. ORs and unconditional logistic

regression models were computed using the SPSS software program (SPSS

Science). Differences were considered to be significant at P<0.05. All statistical tests

were two-sided.

15

5. RESULTS

For the TNFA-308, TNFA-857, IFNGR1 and MIF-797 VNTR polymorphisms two

control groups were available for genotyping. In the group of healthy blood donors

the genotypic distribution was the following: TNFA-308G/G, 235 (76.0%); TNFA-

308G/A, 70 (22.7%); TNFA-308A/A, 4 (1.3%); TNFA-857C/C, 252 (86.0%); TNFA-

857C/T, 39 (13.3%); TNFA-857T7T, 2 (0.7%); IFNGR1-56 C/C, 61 (24.3%); IFNGR1-

56 CAT, 111 (44.2%); IFNGR1-56 T7T, 79 (31.5%); MIF-797 VNTR allele 3 non-

carrier, 197 (83.1%); MIF-797 VNTR allele 3 carrier, 40 (16,9.%). In the group of

healthy children and young adults recruited from schools the genotypic distribution

was the following: TNFA-308G/G, 449 (72.9%); TNFA-308G/A, 159 (25.8%); TNFA-

308A/A, 8 (1.3%); TNFA-857C/C, 497 (82.3%); TNFA-857C/T, 101 (16.7%); TNFA-

857T/T, 6 (1.0%); IFNGR1-56 C/C, 116 (19.0%); IFNGR1-56 C/T, 294 (48.2%);

IFNGR1-56 T/T, 200 (32.8%); MIF-797 VNTR allele 3 non-carrier, 62 (74.7%); MIF-

797 VNTR allele 3 carrier, 21 (25.3%). No significant differences in genotypic

distribution were observed among these two groups (P=0.3 for TNFA-308, P=0.2 for

TNFA-857, P=0.2 for IFNGR1-56 and P=0.06 for MIF-797 VNTR). These two groups

were therefore pooled together in all subsequent analysis. Genotype frequencies of

the TNFA-308, TNFA-857, IFNGR1-56, IL8-251, IL1B-511, MIF-797 VNTR and

IL1RN VNTR polymorphisms in the control group did not deviate significantly from

those expected under Hardy-Weinberg equilibrium (P=0.2, P=0.5, P=0.2, P=0.3,

P=0.2, P=0.7 and P=0.4, respectively; Table 1). The genotype frequencies among

control subjects and CD patients of all the individual loci studied are summarized in

Table 1.

16

Table 1. TNFA (-308 and -857), IFNGR1-56, IL8-251, IL1B-511, MIF-797 VNTR and

IL1RN VNTR genotype frequencies in controls and CD cases.

Controls (%) CD (%)

TNFA-308 G/G

G/A

A/A

Total

TNFA-857

C/C

C/T

T/T

Total

IFNGR1-56

C/C

C/T

TAT

Total

IL8-251

A/A

T/A

T/T

Total

IL1B-511

C/C

C/T

T/T

Total

MIF-797 VNTR allele 3 non-carrier

allele 3 carrier

Total

IL1RNVNTR

L/L

L/2

2/2

Total

684 (73.9%)

229 (24.8%)

12(1.3%)

925*

749 (83.5%)

140(15.6%)

8 (0.9%)

897*

279 (32.4%)

405 (47%)

177(20.6%)

861*

85(18.1%)

218(46.4%)

167(35.5%)

470

140 (45.3%)

128(41.4%)

41 (13.3%)

309

259 (80,9%)

61 (19,1%)

320*

160(51.6%)

121 (39.0%)

29 (9.4%)

310

27 (45.0%)

28 (46.7%)

5 (8.3%)

60

49 (90.7%)

5 (9.3%)

0 (0.0%)

54

19(31.7%)

26 (43.3%)

15(25%)

60

9(15.8%)

21 (36.8%)

27 (47.4%)

57

14 (35.0%)

20 (50.0%)

6(15.0%)

40

35(76,1%)

11 (23,9%)

46

27 (54.0%)

17 (34.0%)

6(12.0%)

50

*The two control cohorts were pooled together for the TNFA-308, TNFA-857, IFNGR1-56 and MIF-797 polymorphisms.

17

In the TNFA gene two distinct promoter polymorphisms (-308 and -857) were

analyzed. The genotypic frequencies distribution of these two polymorphisms among

CD patients and controls is summarized in table 2. No significant differences were

observed for the TNFA-857 polymorphism (P=1.0). For the TNFA-308 polymorphism

the frequency of G/A hétérozygotes and A/A homozygotes were significantly higher

in the group of CD patients (46.7% and 8.3%, respectively) in comparison with the

control group (24.8% and 1.3%, respectively). The risk of developing CD was

therefore significantly increased for both the heterozygous GA genotype, with an OR

of 3.1 (95% confidence interval [CI] = 1.79-5.37), and the homozygous AA genotype

with an OR of 10.6 (95% CI = 3.47-32.1) (Table 2).

For IFNGR1-56, the heterozygous C/T genotype was present in 47% of controls and

43.3% of patients with an OR of 0.9 (95% CI = 0.51 - 1.74). The homozygous T/T

allele was found to be more frequent in patients (25%) than controls (20.6%), though

this difference did not attain the threshold of statistical significance (OR = 1.2; 95% CI

= 0.62-2.51) (Table 2).

IL8-251 A/T allele was present in 46.4% of controls versus 36.8% of patients (OR =

0.9; 95% CI = 0.40 - 2.07). The homozygous allele T/T was found in 13.3% of

controls and 15% of patients (OR = 1.5; 95% CI = 0.53-4.05) (Table 2).

IL1B-511 hétérozygotes and IL1B-511 T homozygotes showed no significant

increase in CD risk (OR of 1.6 (95% CI = 0.76 - 3.22) and 1.5 (95% CI = 0.53 -

4.05), respectively) (Table 2).

MIF-797 VNTR allele 3 non-carriers included 80.9% and 76.1% of controls and

patients, respectively. This allele was more frequent in patients (23.9%) when

compared to controls (19.1%), but with no statistical significance (OR = 1.3; 95% CI =

0.64 - 2.77) (Table 2).

18

After reclassifying the IL1RN VNTR alleles into long and short, no significant

associations were observed between IL1RN genotype and risk of developing CD

(Table 2). The OR for 2/L hétérozygotes was 0.8 (95% CI = 0.43 - 1.60) and for 2/2

homozygotes 1.2 (95% CI = 0.47 - 3.23).

Thus, regarding the IFNGR1-56, IL8-251, IL1B-511, MIF-797 VNTR and the IL1RN

VNTR polymorphisms no significant differences in genotypic distribution could be

observed between CD patients and control individuals (Table 2).

19

Table 2: ORs according to TNFA (-308 and -857), IFNGR1-56, IL8-251, IL1B-511,

MIF-797 VNTR and IL1RN VNTR genotypes.

Controls (%) CD (%) OR (95%CI)

TNFA-308

G/G 684 (73.9%) 27 (45.0%) 1 (Referent)

G/A 229 (24.8%) 28 (46.7%) 3.1 (1.79-5.37)*

A/A 12(1.3%) 5 (8.3%) 10.6(3.47-32.1)*

Total 925* 60

TNFA-857

C/C 749 (83.5%) 49 (90.7%) 1 (Referent)

C/T 140(15.6%) 5 (9.3%) 0.6(0.21-1.39)

T/T 8 (0.9%) 0 (0.0%) ns

Total 897* 54

IFNGR1-56

C/C 279 (32.4%) 19(31.7%) 1 (Referent)

C/T 405 (47%) 26 (43.3%) 0.9(0.51 -1.74)

T/T 177(20.6%) 15(25%) 1.2(0.62-2.51)

Total 861* 60

IL8-251 A/A 85(18.1%) 9(15.8%) 1 (Referent)

T/A 218(46.4%) 21 (36.8%) 0.9(0.40-2.07)

T/T 167(35.5%) 27 (47.4%) 1.5(0.69-3.39)

Total 470 57

IL1B-511 C/C 140 (45.3%) 14 (35.0%) 1 (Referent) C/T 128(41.4%) 20 (50.0%) 1.6(0.76-3.22)

T/T 41 (13.3%) 6(15.0%) 1.5(0.53-4.05)

Total 309 40

MIF-797 VNTR allele 3 non-carrier 259 (80,9%) 35(76,1%) 1 (Referent)

allele 3 carrier 61 (19,1%) 11 (23,9%) 1.3(0.64-2.77)

Total 320* 46

IL1RNVNTR

L/L 160(51.6%) 27 (54.0%) 1 (Referent)

L/2 121 (39.0%) 17(34.0%) 10.8(0.43-1.60)

2/2 29 (9.4%) 6 (12.0%) 1.2(0.47-3.23)

Total 310 50

*The two control cohorts were pooled together for the TNFA-308, TNFA-857, IFNGR1-56 and MIF-797 polymorphisms; T P<

0.0001; ns, not significant; L, long allele (alleles 1, 3, 4, and 5); OR, Odds ratio; CI, Confidence interval.

20

6. DISCUSSION

Celiac disease, or gluten sensitive enteropathy, is an inflammatory intestinal disorder

triggered by an environmental agent, gluten, in genetically susceptible individuals.

The genetics of CD is complex, with evidence for the involvement of multiples genes.

The extensive interplay between intrinsic (genetic) and (extrinsic) environmental

factors makes it difficult to identify core pathogenic mechanisms.

TNF- is a product of activated monocytes/macrophages and other cells and may

have a pivotal role in the progression of inflammatory diseases (16). The results

obtained in this study suggest that the TNFA-308 polymorphism is associated with

risk of CD development. According to these findings, individuals carrying the TNFA-

308G/A genotype showed an increased risk of CD with an OR of 3.1. The

homozygous AA genotype of the TNFA-308 polymorphism also showed an increased

risk of CD with an OR of 10.6 (95% CI = 3.47 - 32.1). For the TNFA-857

polymorphism no significant association was observed. The association between the

TNFA gene and CD is not new and it has been reported in other studies (17, 18, 19,

30, 50). The TNFA gene is located in chromosome 6, in a region compassing the

HLA complex and implicated in elevate disease propensity (50). Although the

majority of the studies demonstrate an association between increased risk of CD and

TNFA promoter polymorphisms, the implicated region may contain multiple

susceptibility loci. In fact, this is the most polymorphic region of the genome and

contains hundreds of genes that encode proteins potentially involved in the regulation

of inflammatory and immune responses (26, 51). Linkage desiquilibrium is strong in

this area and it may be difficult to study the role of a SNP in isolation (27).

Consequently, it is not easy to make general statements about the association of the

TNF polymorphisms and diseases ethiopathogenesis.

21

De la Concha et al. found TNF-308A to be independent of its linkage disequilibrium

with the DQA1*0501/DQB1*0201 genes, concluding that in individuals carrying the

DQA1*0501/DQB1*0201 alleles, the risk of developing CD is further increased by the

presence of adenine at the position -308 of the promoter region of TNFA gene, or of

another allele in very high linkage disequilibrium with it. They suggest that, at least,

another gene, in addition to the known HI_A-DQ, is associated with susceptibility to

CD, which should be either the TNF- itself or a gene in strong linkage disequilibrium

with TNF- . Moreover, they favor the TNFA gene responsibility, regarding the

association with the TNF-308A allele and the increased production of TNF-

previously described and the role played by this cytokine in immune mediated

diseases and particularly in CD (19).

Nevertheless, despite the strong association between HLA and CD, no HLA-

associated gene has been identified as the true origin for the intrinsic genetic nature

of CD, and the precise role of TNFA-308A allele for this susceptibility has not been

well established (17, 18, 27, 30).

In the current study no significant relationships were obtained between the IFNGR1-

56, IL8-251, IL1B-511, MIF-797 and IL1RN VNTR polymorphisms and CD risk.

Although information is scarce on this topic, one report by Nemetz et al. (21) showed

a relationship with increased risk of inflammatory bowel disease and IL1B gene

polymorphisms. Further studies in larger series are probably necessary to make clear

these issues. However, the absence of a significant relationship between these

polymorphisms and CD does not diminish the role of these cytokines in the

pathophisiology of inflammatory diseases, in particular, CD. An activated cell

produces a large spectrum of cytokines and this is not only under the control of the

promoter region of each single cytokine gene. Tough, it is very likely that the

22

dysregulation of the cytokine network, ensuing from the interaction between pro- and

anti-inflammatory stimuli, is central and contributes in a major way to the

pathomechanisms of inflammatory diseases.

Further studies aiming at the clarification of the association between TNFA-308

polymorphisms and risk of CD are needed in order to elucidate the etiopathogenic

mechanisms present in CD. Understanding to which extent this and other factors

affect the risk of developing CD may provide new insights into the pathophysiology

and therapeutic intervention alternatives for this disease.

23

7. CONCLUDING REMARKS

The genome outside HLA class II region seems to carry a considerable amount of

risk, and yet no such genes have been identified. The results obtained in this study

are in agreement with other reports (17, 18, 19, 30), confirming that TNFA-308A is a

strong candidate risk factor for CD development, or another allele in very high linkage

disequilibrium with it. Thus, it would be interesting search for causative genes in the

HLA class III region that encodes the TNF family. Linkage disequilibrium mapping

with SNP using a relatively small set of common sequence variants in the genome

would allow the detection of association between a particular genomic region and this

disease.

Another alternative is to study families, where haplotypes can be more easily

interpreted and methods such as the transmission disequilibrium test can be applied

to check which polymorphism/haplotype is being inherited more frequently. This

would also help explain the association between TNFA polymorphisms and CD in

different populations, since haplotypes are frequently influenced by population

history.

Functional demonstration of the relationship between the candidate gene and

etiopathogenesis of CD will be required and would lead, ultimately, to the clarification

of this issue. The prognostic value of these and other similar genetic markers may be

of utter importance for translating this type of study into clinically relevant

interventions.

24

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