ARTICLE
Auteur(s) : Maha Kammoun-Krichen1, Noura
Bougacha-Elleuch1, Kaouthar Makni1, Maha
Rebai1, Ahmed Rebai2, Mouna Mnif3,
Mohamed Abid3, Joumaa Jouida4, Hammadi
Ayadi1
1Unité cibles diagnostiques et thérapeutiques en
pathologies humaines, Centre de biotechnologie de Sfax, Tunisie
2Unité de bioinformatique, Centre de biotechnologie de
Sfax, Tunisie
3Service d’endocrinologie, CHU Hedi Chaker, Sfax,
Tunisie
4Dispensaire Bir El-Hfai-Sidi Bouzid, Tunisie
accepté le 1 Janvier 2007
Cytokines, a large group of non-enzymatic protein hormones, are
involved in the induction and effector phases of all inflammatory
and immune responses, and are therefore likely to play a critical
role in the development of autoimmune diseases (AIDs) [1]. The
proinflammatory cytokines interleukin (IL)-1α and β (OMIM 147760
and OMIM 147720), and their receptor antagonist (IL-1Ra) (OMIM
147679) play major roles in initiating and modulating immune
responses [2]. IL-1 is a family of three proteins IL-1α, IL-1β and
IL-1Ra, which are encoded by different genes (IL-1A for IL-1α,
IL-1B for IL-1β and IL-1RN for IL-1Ra), spanning a 430 kb region on
chromosome 2q13-21 [3]. These genes are highly polymorphic,
encompassing both single nucleotide polymorphisms “SNPs”, and
length variants (microsatellites and variable number tandem repeats
“VNTR”). IL-1 genes have been reported to be involved in the
development of various inflammatory and immune diseases, including
Graves’ disease (GD), a major, autoimmune thyroid disease (AITD)
[4, 5]. AITDs also include autoimmune hypothyroidism (AH): primary
idiopathic myxoedema (PIM) and Hashimoto’s thyroiditis (HT). It is
known that IL-1 influences the function of the thyroid cells [6].
In fact, IL-1 downregulates the expression of thyroid-specific
proteins such as thyroglobulin [7] and thyroperoxidase [8],
inhibits iodide organification [9] and the Na+/I- symporter NIS
[10], and reduces the delivery of thyroid hormone to the
circulation [11]. These inhibitory effects are demonstrated both in
apparently normal human thyrocytes adjacent to adenomas or cancers,
in thyrotoxic cells, and, to some extent, in FRTL5 cells.In the
present study, we examined the genetic association of four
polymorphic loci in the IL-1 gene cluster with AITDs in a large
Tunisian family (Akr family), and with GD in unrelated Tunisian
patients. Two of these polymorphisms were found to be associated
with AITDs in family and case-control studie.
Methods and patients
Patients and controls
Patients were recruited from a large family in South Tunisia which
had a high prevalence of AITDs (Akr family) [12]. This family
consists of more than 400 members spanning 10 generations and
including 176 controls and 65 patients. The latter are subdivided
into 34 patients affected with GD and 31 patients with AH (9
HT + 21 Mxp [[Please, could you explane this
abbreviation?]]). The case-control study included 131 GD
patients and 225 controls (healthy subjects with no history of
AITDs). The diagnosis of GD and AH was performed as previously
described [13].
Genotype analysis
DNA was extracted from peripheral blood as previously described
[14]. PCR was used to identify the genotypic pattern of the
different IL-1 genes (table 1). The four
polymorphisms studied are located on chromosome 2q14. The IL-1B and
IL-1A gene polymorphisms : IL1B-511 C/T, IL1B+3954 C/T and IL1A-889
C/T were analysed by PCR followed by restriction fragment length
polymorphism (PCR-RFLP) as previously described [15-17]. The VNTR
located in intron 2 of IL1RN was amplified as previously described
[18]. Alleles are conventionally defined as follows: allele 1 (412
bp, representing 4 repeats); allele 2 (240 bp, 2 repeats); allele 3
(498 bp, 5 repeats); allele 4 (326 bp, 3 repeats) [19].
Table 1 Characteristics of the four polymorphisms
investigated
|
Set
|
Position
|
|
PCR product (bp)
|
Restriction site
|
Reference
|
|
IL1A promoter
|
-889 C/T
|
rs1800587
|
116
|
NcoI
|
[17]
|
|
IL1B promoter
|
-511 C/T
|
rs16944
|
304
|
AvaI
|
[15]
|
|
+3954 C/T
|
rs1143634
|
249
|
TaqI
|
[16]
|
|
IL1RN
|
Intron 2
|
-
|
240, 325, 410 and 500
|
-
|
[18]
|
Statistical analysis
In the familial study, we used the FBAT program (Family-Based
Association Test) [20] to test for association in the presence of
linkage [21]. We used three diagnostic models; (i) the AITD model:
all AITDs patients were considered as affected, (ii) the GD model:
only GD patients were considered to be affected and AH patients
were considered as unaffected, and (iii) the AH model: only AH
patients were considered to be affected. We used Version 1.5 of
FBAT, which provides a haplotypic test of association [22].
The distribution of alleles in unrelated patients affected with
GD versus controls was compared by a standard, chi-square in a 2x2
contingency table. A corrected p-value (pc) < 0.05
was considered significant, where corrected p-values were
calculated according to the Bonferroni correction. Odds ratios (OR)
were calculated according to Woolf’s formula, with 95% confidence
intervals (95% CI). IL-1 haplotypes were estimated from population
genotype data by PHASE version 2.02 software [23, 24]. The power of
the association study was evaluated using functions from the
Genetics R package (available on the URL http://cran.r-project.org)
based on the method of Long and Langley [25].
Results
Family study
The investigation of the four polymorphisms in the Akr family
showed only an association of the IL-1B+3954 polymorphism with
AITDs model (multi-allelic mode, recessive model; p = 0.02). No
association was found with IL-1RN VNTR, IL-1B-511 C/T SNP or
IL-1A-889 C/T SNP with the three models of the FBAT package
(p > 0.05). In order to search for a haplotype associated with
AITDs, the haplotype-based association test (hbat) was used. The
haplotypes were given as follows:
IL-1RN/IL1B-511/IL-1B+3954/IL-1A-889. This analysis showed that the
IL-1RN*3/C/T/T and IL-1RN*1/C/T/T haplotypes were found to be
associated with AITDs (χ2 = 3.95, p = 0.047;
χ2 = 6.8, p = 0.009) respectively, but associated with
neither GD nor AH models.
Case-control study
The genotype and allele frequencies of the IL-1RN VNTR, IL-1B-511
C/T, IL-1B+3954 C/T and IL-1A-889 C/T polymorphisms in unrelated GD
patients and healthy controls are shown in table
2. Departure from Hardy-Weinberg equilibrium (HWE) was
tested for each polymorphism investigated using a chi-square test.
Only IL-1A-889 C/T SNP was found not to be in HWE in the control
population (table 2). The two SNPs in HWE show very high
heterozygosity as compared to expected value.
Four alleles were observed for the IL-1RN VNTR, both in
case-control samples and the Akr family. IL-1RN*1 and IL-1RN*2
alleles were the most frequent (table 2). There was no significant
difference in IL-1RN allele frequencies between GD and the control
group (χ2 = 3.16; df = 3; p = 0.367). Also, allele
frequencies of the IL-1B-511 and IL-1B+3954 showed no significant
difference between GD and the control group (χ2 = 2.18,
p = 0.14; χ2 = 0.01; p = 0.98 respectively). However, we
found a significant increase in the allele and genotype frequencies
of the IL-1A-889 C/T polymorphism, in GD patients as compared to
controls (χ2=9.46; p = 0.0021). As this SNP was not in
HWE in controls, we calculated a chi-square test of association
that corrected for deviation from HWE [26]. We found that the
association of IL-1A-889 C/T is highly significant after correction
(χ2 = 20.63; p = 0.000005). Haplotype analysis of the
four IL-1 polymorphisms showed no haplotype associated with GD
(p > 0.05) in the case-control study.
Linkage disequilibrium analysis between the four markers showed
a linkage only between two SNPs: IL-1B-511 and IL-1B+3954
(χ2 = 8.9; p = 0.0028).
Following the recommendations of Kharrat et al. [27], we
calculated the power of our study at a significance level of 5
10-5 for values of genetic risk ranging from 1.5 to 3,
and a risk-allele frequency ranging from 0.1 to 0.5 under the
multiplicative model. The power of our sample for a gene with a 1.5
risk ranged from 83.1% to 98.7% depending on allele frequency. For
a gene having a relative risk of 2 or more, power is close to 1 for
all allele frequencies.
Table 2 Allele and genotype frequencies of the
polymorphisms investigated in both controls and patients, and the
corresponding Hardy-Weinberg equilibrium test
|
Polymorphism
|
|
|
Hobsb
|
Hthc
|
HWEd
|
|
Allele frequencies
|
|
|
|
|
|
|
IL1RN*1
|
356 (79.1)
|
200 (76.3)
|
|
|
|
|
IL1RN*2
|
72 (16)
|
43 (16.4)
|
|
|
|
|
IL1RN*3
|
10 (2.2)
|
12 (4.6)
|
|
|
|
|
IL1RN*4
|
12 (2.7)
|
7 (2.7)
|
|
|
|
|
IL1B-511 C
|
216 (48)
|
110 (42)
|
|
|
|
|
IL1B-511 T
|
234 (52)
|
152 (58)
|
|
|
|
|
IL1B+3954 C
|
249 (55.3)
|
144 (55)
|
|
|
|
|
IL1B+3954 T
|
201 (44.7)
|
118 (45)
|
|
|
|
|
IL1A-889 T
|
413 (91.8)
|
220 (84)
|
|
|
|
|
IL1A-889 C
|
37 (8.2)
|
42 (16)
|
|
|
|
|
<vsp sp=’0.5’>
|
|
Genotype frequencies
|
|
|
|
|
|
|
(IL1RN*1/ IL1RN*1)
|
140 (62.2)
|
79 (60.3)
|
|
|
|
|
(IL1RN*1/ IL1RN*2)
|
62 (27.5)
|
33 (25.2)
|
|
|
|
|
(IL1RN*1/ IL1RN*3)
|
8 (3.5)
|
4 (3.0)
|
|
|
|
|
(IL1RN*1/ IL1RN*4)
|
7 (3.1)
|
5 (3.8)
|
|
|
|
|
(IL1RN*2/ IL1RN*2)
|
4 (1.8)
|
4 (3.1)
|
|
|
|
|
(IL1RN*2/ IL1RN*3)
|
0
|
2 (1.5)
|
0.346
|
0.348
|
25.06 (1.5 10-5)
|
|
(IL1RN*2/ IL1RN*4)
|
1 (0.5)
|
0
|
|
|
|
|
(IL1RN*3/ IL1RN*3)
|
1 (0.5)
|
3 (2.3)
|
|
|
|
|
(IL1RN*3/ IL1RN*4)
|
0
|
0
|
|
|
|
|
(IL1RN*4/ IL1RN*4)
|
2 (0.9)
|
1 (0.8)
|
|
|
|
|
ARRAY(0x2800e8)
|
|
IL1B-511C/C
|
26 (11.6)
|
17 (13)
|
|
|
|
|
IL1B-511T/C
|
165 (73.3)
|
86 (65.7)
|
0.733
|
0.500
|
49.39 (< 10-6)
|
|
IL1B-511T/T
|
34 (15.1)
|
28 (21.3)
|
|
|
|
|
ARRAY(0x282b1c)
|
|
IL1B+3954 T/T
|
23 (10.2)
|
13 (9.9)
|
|
|
|
|
IL1B+3954 T/C
|
155 (68.9)
|
92 (70.2)
|
0.689
|
0.495
|
34.86 (< 10-6)
|
|
IL1B+3954 C/C
|
47 (20.9)
|
26 (19.9)
|
|
|
|
|
ARRAY(0x284564)
|
|
IL1A-889 T/T
|
188 (83.6)
|
89 (67.9)
|
|
|
|
|
IL1A-889 T/C
|
37 (16.4)
|
42 (32.1)
|
0.164
|
0.151
|
1.80 (0.18)
|
|
IL1A -889 C/C
|
0
|
0
|
|
|
|
aNumber (frequency in %) of the alleles or genotypes.
bHobs: observed heterozygosity.
cHth: expected heterozygosity under HWE
(1-Σpi2 where pi are allele
frequencies).
dChi-square (p-value) for the Hardy-Weinberg
equilibrium test.
Discussion
The etiology of AITDs is complex and involves multiple genetic and
environmental influences. Genetic susceptibility to AITDs is
controlled both by genes implicated in thyroid physiology and in
the immune reaction [28]. Of the interleukins, the TNF-α gene was
found to be implicated in AITD pathogenesis in the Akr family [29].
The IL-1RN gene has been reported to be associated with GD [30].
However, to our knowledge, to date only few studies have evaluated
the relationship between the IL-1B or IL-1A gene polymorphisms and
AITDs and they yielded contradictory results [30-32]. We have
reported a novel finding that demonstrates a significant
association between the IL-1A-889 polymorphism and susceptibility
to GD (p = 0.0021) in a case-control study. The C/C genotype was
absent in both GD patients and controls (table 2) even though the
effector allele was C. The IL-1A-889 C/C genotype has been
associated with significantly lower transcriptional activity of the
IL-1A gene and lower levels of IL-1A in plasma compared with the
T/T genotype [33]. The mechanism of interaction between IL-1A-889
polymorphism and the level of protein synthesis has not yet been
elucidated. It is still unclear whether the IL-1A-889 polymorphism
has a direct influence on protein expression or is in linkage
disequilibrium with the effector gene in the IL1 cluster. As
regards the IL-1B gene, we focused on two C/T SNPs: the first
located at position -511 and the second at position +3954.
Investigation of these polymorphisms revealed an association of the
IL-1B+3954 C/T with AITDs (multi-allelic mode, recessive model:
p = 0.02) in the Akr family. No IL-1B polymorphisms was found to be
associated with GD in the case-control study (p > 0.05).
However, Chen et al. [34] showed that the IL-1B-511 C/T
polymorphism was associated with susceptibility to GD (p=0.038)
rather than the IL-1B+3954 C/T polymorphism. The discrepancy
between our results and those of others may reflect the different
genetic pools represented in the different ethnicities. On the
other hand, we were also interested in a VNTR in intron 2 of the
IL-1RN gene. Our study showed no association between the IL-1RN
polymorphism and AITDs in either familial or the case-control
study. Studies that examined the association of this gene
polymorphism with susceptibility to GD are limited and
contradictory [30-32] (table 3)
[[please check]].
In conclusion, in the Tunisian population, IL-1 gene
polymorphisms seem to be associated with AITD pathogenesis in both
familial and case-control cohorts, with different polymorphisms
involved (IL-1B+3954 and IL-1A-889 SNPs respectively). In the
family studied, two major haplotypes seem to predispose to AITDs.
The discrepancy between results in the case-control and familial
studies may be explained by the genetic heterogeneity of AITDs and
by the fact that the Akr family is an isolated and consanguine
population. Whether these polymorphisms have a direct, functional
effect on gene expression or this association was due to linkage
disequilibrium with another disease-causing polymorphism within or
close to the IL-1 gene cluster, remains to be investigated.
Table 3 Statistical results for the case-control
association study
|
Polymorphism
|
χ2
|
|
|
|
Allelic
|
Genotypic
|
Allelic
|
Genotypic
|
|
|
IL1RN
|
3.16
|
7.55
|
0.367
|
0.48
|
---
|
|
IL1B-511 C/T
|
2.18
|
2.70
|
0.14
|
0.26
|
---
|
|
IL1B+3954 C/T
|
0.01
|
0.07
|
0.985
|
0.964
|
---
|
|
IL1A-889 C/T
|
9.46
|
10.81
|
0.0021
|
0.0014
|
|
Acknowledgements
This work was supported by the Tunisian Ministry of Higher
Education, the Scientific Research and Technology, and the
International Centre for Genetic Engineering and Biotechnology
ICGEB (Italy). We thank Mrs Zineb Hachicha-Elloumi for the critical
reading of the manuscript.
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