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
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
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
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.
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
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
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 T-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
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
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).
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.
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.
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
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-5'-CATACAATTGATAATTCA-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).
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.
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: 308G/G, 235 (76.0%); 308G/A, 70 (22.7%); 308A/A, 4 (1.3%); 857C/C, 252 (86.0%); TNFA-857C/T, 39 (13.3%); TNFA-857T7T, 2 (0.7%); 56 C/C, 61 (24.3%); IFNGR1-56 CAT, 111 (44.2%); IFNGR1-IFNGR1-56 T7T, 79 (31.5%); MIF-797 VNTR allele 3 non-carrier, 197 (83.1%); MIF-797 VNTR allele 3 non-carrier, 40 (16,9.%). In the group of healthy children and young adults recruited from schools the genotypic distribution was the following: 308G/G, 449 (72.9%); 308G/A, 159 (25.8%); 308A/A, 8 (1.3%); 857C/C, 497 (82.3%); 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%); 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.
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
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).
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).
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<
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.
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 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
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.
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.
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