Celiac Disease

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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

J

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

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 DR7DQ2

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 Tcells, 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 TNFexpression 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 TNFand 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 IL1B511 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 TNFA308, 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'-GCCAATAGCCCTCCCTGTCT-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'-ATCTTGTTCTAACACCTGCCACTCT-3' were used as primers, and 5'-CATACAATTGATAATTCAMGB-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