faculdade de ciências da nutrição e alimentação ... · wwfÈk faculdade de ciências da...
TRANSCRIPT
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
8. REFERENCES
1. Green PHR, Jabri B. Coeliac disease. Lancet 2003;362:383-391.
2. Sollid LM. Coeliac disease: dissecting a complex inflammatory disorder.
Nature Reviwes Immunology 2002;2:647-655.
3. McManus R, Kelleher D. Celiac disease - the villain unmasked? N Eng J Med
2003; 348:2573-2574.
4. Schuppan D. Current concepts of celiac disease pathogenesis.
Gastroenterology 2000; 119:234-242.
5. Mowat AM. Coeliac disease - a meeting point for genetics, immunology and
protein chemistry. Lancet 2003; 361:1290-1292.
6. Fasano A, Catassi C. Current approaches to diagnosis and treatment of celiac
disese: an evolving spectrum. Gastroenterology 2001;120:636-651.
7. American Gastroenterological Association. American Gastroenterological
Association medical position statement: celiac sprue. Gastroenterology
2001;120:1522-1525.
8. Gillet HR, Arnott ID, Mclntyre M, Campbell S, Dahele A, Priest M, Jackson R,
Ghosh S. Successful infliximat treatment for steroid-refractory celiac disase: a
case report. Gastroenterology 2002;122:800-805.
9. Greco L, Romino R, Coto I, Di Cosmo N, Percopo S, MAglio M, Paparo F,
Gasperi V, Limongelli MG, Cotichini R, D'Agate C, Tinto N, Sachetti L, Tosi R,
Stazi MA. The first large population based twin study of celiac disease. Gut
2002;50:624-628.
10.Fernandez-Arquero M, Caldés T, Casado E, Maluenda C, Figueredo MA, de
la Concha EG. Polymorphism within the HLA-DQB1*02 promoter associated
with susceptibility to coeliac disease. Eur J Immunogenetics 1998;25:1-3.
H.Kagnoff MF. Celiac disease pathogenesis: the plot thickens. Gastroenterology
2002;123:939-941.
12. Sollid LM. Molecular basis of celiac disease. Annu Rev Immunol 2000;18:53-
81.L. Lopez-Vasquez A, Rodrigo L, Fuentes D, Riestra S, Bousono C, Garcia-
Fernandez S, Martinez-Borra J, Gonzalez S, Lopez-Larrea C. MHC class I
chain related gene A (MICA) modulates the development of celiac disease in
patients with the high risk heterodimer DQA1*0501/DQB1*0201. Gut
2002;50:336-340.
25
13. Polvi A, Arranz E, Fernandez-Arquero M, Collin P, Maki M, Sanz A, Calvo C,
Maluenda C, Westman P, de la Concha EG, Partanen J. HLA-DQ2-negative
celiac disease in Finland and Spain. Human Immunology 1998, 59:169-175.
14.Londei M, Quarantino S, Maiuri L. Celiac disease: a model autoimmune
disease with gene therapy applications. Gene Therapy 2003;10:835-843.
15.Dieterich W, Esslinger B, Schuppan D. Pathomechanisms in celiac disease.
Int Arch Allergy Immunol 2003;132:98-108.
16.Strieter RM, Kunkel SL, Bone RC. Role of tumor necrosis factor-alpha in
diseases states and inflammation. Crit Care Med 1993;21:S447-S463.
17. Garrote JA, Arranz E, Telleria JJ, Castro J, Calvo C, Blanco-Quirós J. TNF
and LT gene polymorphisms as additional markers of celiac disease
susceptibility in a DQ2-positive population. Immunogenetics 2002;54:551-555.
18.Cataldo F, Lio D, Marino V, Scola L, Crivello A, Mulè M, Corazza GR.
Cytokine genotyping (TNF and IL-10) in patients with celiac disease and
selective IgA deficiency. Am J Gastroenterol 2003;98:850-856.
19.de la Concha EG, Fernandez-Arquero M, Virgil P, Rubio A, Maluenda C,
Polanco I. Fernandez C, Figueredo MA. Celiac disease and TNF promoter
polymorphisms. Human Immunology 2000;61:513-517.
20.Baugh JA, Donnelly SC. Macrophage migration inhibitory factor; a
neuroendocrine modulator of chronic inflammation. J of Endocrinology
2003:179:15-23.
21.Nemetz A, Nosti-Escanilla MP, Moinar T, Kope A, Kovacs A, Feher J,
Tulassay Z, Nagy F, Garcia-Gonzalez MA, Pena AS. IL1B gene
polymorphisms influence the course and severity of inflammatory bowel
disease. Immunogenetics 1999;49:527-531.
22.Arend WP, Malyak M, Guthridge CJ, Gabay C. lnterleukin-1 receptor
antagonist: role in biology. Annu Rev Immunol 1998;16:27-55.
23.Kagnoff MF. Celiac disease pathogenesis: the plot thickens. Gastroenterology
2002;123:939-941.
24.Feldmann M, Maini RN. Anti-TNF theapy of rheumatoid arthritis: ehat have
we learned? Annu Rev Immunol 2001 ; 19:163-196.
25. De Jong YP, Abadia-Molina AC, Satoskar AR, Clarke K, Rietdijk ST, Faubion
WA. Development of chronic colitis is depent on the cytokine MIF. Nature
Immunol 2001;2:1061-1066.
26
26. Wilson AG, Symons JA, McDowell TL, McDevitt HO, Duff GW. Effects of a
polymorphism in the human tumor necrosis factor alpha promoter on
transcriptional activation. Proc Natl Acad Sci U S A 1997;94:3195-3199.
27.0. Hajeer AH, Hutchinson IV. Influence of TNF gene polymorphism on TNF
production and disease. Human Immunology 2001;62:1191-1199.
28. Fernandez L, Femandez-Arquero M, Gual L, Lazaro F, Maluenda C, Polanco
I, Figueredo MA, de la Concha EG. Triplet repeat polymorphism in the
transmembrane region of the mica gene in celiac disease. Tissue Antigens
2002;59:219-222.
29.0'Keeffe J, Lynch S, Whelan A, Jackson J, Kennedy NP, Weir DG, Feighery
C. Flow cytometric measurement of intracellular migration inhibition factor and
tumour necrosis factor alpha in the mucosa of patients with coeliac disease.
Clin Exp Immunol 2001;125:376-382.
30. Q. Louka AS, Lie BA, Talseth B, Ascher H, Ek J, Gudjónsdóttir AH, Sollid LM.
Coeliac disease patients carry conserved HLA-DR3-DQ2 haplotypes revealed
by association of TNF alleles. Immunogentics 2003;55:339-343.
31.Strieter RM, Kunkel SL, Bone RC. Role of tumor necrosis factor-alpha in
diseases states and inflammation. Crit Care Med 1993;21:S447-S463.
32. Bach EA, Aguet M, Schreiber RD. The IFNy receptor: a paradigm for cytokine
receptor signalling. Annu Rev Immunol 1997;15:563-591.
33.Nilsen EM, Jahnsen FL, Lundin KE, Johansen FE, Fausa O, Sollid LM,
Jahnsen J, Scott H, Brandtzaeg P. Gluten induces an intestinal cytokine
response strongly dominated by interferon gamma in patients with celiac
disease. Gastroenterology 1998; 115:551-563.
34.Westerholm-Ormio M, Garioch J, Ketola I, Savilahti E. Inflammatory cytokines
in small intestinal mucosa of patients with potential celiac disease. Clin Exp
Immunol 2002;128:94-101.
35.0laussen RW, Johansen Fe, Lundin KE, Jahnsen J, Brandtzaeg P, Farstad
IN. Interferon-gamma-secreting T cells localize to the epithelium in coeliac
disease. Scand J Immunol 2002;56:652-664.
36.Juliger S, Bongartz M, Luty AJF, Kremsner, PG, Kun JFJ. Functional analysis
of a promoter variant of the gene encoding the interferon-gamma receptor
chain I. Immunogenetics 2003;54:675-680.
27
37.Zachrisson K, Neopikhanov V, Wretlind B, Uribe A. Mitogenic action of tumour
necrosis factor-alpha and interleukin-8 on expiants of human duodenal
mucosa. Cytokine 2001;15:148-155.
38. Weber M, Sydlik C, Quirling M, Nothdurfter C, Zwergal A, Heiss P, Bell S,
Neumeier D, Loms Ziegler-Heitbrock HW, Brand K. Transcriptional inhibition of
interleukin-8 expression in tumor necrosis factor-tolerant cells. The J Biol
Chemistry 2003;278:23586-23593.
39.Eckman L, Jung CH, Schurer-Maly C, Panja A, Morzycka-Wrobleska E,
Kagnoff FM. Differential cytokine expression by human intestinal epithelial cell
lines: regulated expression of interleukin-8. Gastroenterology, 1993; 105:1689-
1697.
40. Landi S, Moreno V, Gioia-Patricola L, Guino E, Navarro M, de Oca J, Capella
G, Canzian F. Association of common polymoprhisms in inflammatory genes
IL6, IL8, tumor necrosis factor alpha, NFKB1, and peroximssome proliferator-
activated receptor y with colorectal cancer. Cancer Res 2003;63:3560-3566.
41.Dinarello CA. Biologic basis for interleukin-1 in disease. Blood 1996;87:2095-
2147.
42.Furuta T, El-Omar EM, Xiao F, Shirai N, Takashima M, Sugimurra H.
Interleukin 113. polymorphisms increase the risk of hypochlorhydria and
atrophic gastritis and reduce the risk of duodenal ulcer recurrence in Japan.
Gastroenterology 2002; 123:92-105.
43. Machado JC, Pharoah P, Sousa S, Carvalho R, Oliveira C, Figueiredo C,
Amorim A, Seruca R, Caldas C, Carneiro F, Sobrinho-Simoes M. Interleukin
1B and interleukin 1RN polymorphisms are associated with increased risk of
gastric carcinoma. Gastroenterology 2001;121:823-829.
44.Pociot F, Molvig J, Wogensen L, Worsaae H, Nerup J. A Taql polymorphism in
the human interleukin-1 beta (IL-1 beta) gene correlates with IL-1 beta
secretion in vitro. Eur J Clin Invest 1992;22:396-402.
45.Danis VA, Millington M, Hyland VJ, Grennan D. Cytokine production by normal
human monocytes: inter-subject variation and relationship to an IL-1 receptor
antagonist (IL-1 Ra) gene polymorphism. Clin Exp Immunol 1995;99:303-310.
46.Santtila S, Savinainen K, Hurme M. Presence of the IL-1RA allele 2 (IL1RN*2)
is associated with enhanced IL-1 B production in vitro. Scand J Immunol
1998;47:195-198.
28
47.Baugh JA, Chitnis S, Donnelly SC, Monteiro J, Lin X, Plant BJ, Wolfe F,
Gregersen PK, Bucala R. Genes and Immunity 2002;3:170-176.
48.Gregersen PK, Bucala R. Macrophage migration inhibitory factor, MIF alleles,
and the genetics of inflammatory disorders: incorporating disease outcome
into the definiton of phenotype. Arthritis and Rheumatism 2003;48:1171-1176.
49. Bâcher M, Metz CN, Calandra T, Mayer K, Chesney J, Lohoff M. An essential
regulatory role for macrophage migration inhibitory factor in T-cell activation.
Proc Natl Acad Sci USA 1996;93:7849-7854.
50. Louka AS, Sollid LM. HLA in coeliac disease: unravelling the complex genetics
of a complex disorder. Tissue Antigens 2003;61:105-117.
51. Cooke GS, Hill AV. Genetics of susceptibility to human infectious disease. Nat
Rev Genet 2001 ;2:967-977.