Clinical Immunology (2008) 127, 298–302
a v a i l a b l e a t w w w. s c i e n c e d i r e c t . c o m
w w w. e l s e v i e r. c o m / l o c a t e / y c l i m
RAPID COMMUNICATION
Association of the interleukin (IL)-16 gene
polymorphisms with Graves' disease
Xue-Jiang Gu a,b,c,1 , Bin Cui a,c,1 , Ze-Fei Zhao a , Hao-Yan Chen a,c ,
Xiao-Ying Li a,c,d , Shu Wang a,c , Guang Ning a,c,d,⁎, Yong-Ju Zhao a,⁎
a
Shanghai Clinical Center for Endocrine and Metabolic Diseases, Shanghai Institute of Endocrinology and Metabolism,
Shanghai Key Laboratory for Endocrine Tumors, Ruijin Hospital, Shanghai JiaoTong University School of Medicine,
197 RuiJin Er Lu, Shanghai 200025, P.R. China
b
Department of Endocrine and Metabolic Diseases, The First Affiliated Hospital of Wenzhou Medical college, 2 Fuxue Lane,
Zhejiang 325000, P.R. China
c
Laboratory Endocrine and Metabolic Diseases of Institute of Health Science,
Shanghai JiaoTong University School of Medicine and Shanghai Institutes for Biological Sciences,
Chinese Academy of Sciences, 225 South ChongQing Road, Shanghai 200025, P.R. China
d
Division of Endocrine and Metabolic Diseases, E-Institute of Shanghai Universities, Ruijin Hospital,
Shanghai JiaoTong University School of Medicine, Shanghai, P.R. China
Received 25 August 2007; accepted with revision 24 January 2008
Available online 3 April 2008
KEYWORDS
IL-16;
Graves'disease;
Polymorphism
Abstract Interleukin (IL)-16 was one of the cytokines with the function of T helper cell
recruitment, whose expression in the thyrocyte and orbital fibroblast of Graves' disease (GD)
patients was increased. Recently association of IL-16 gene polymorphisms with autoimmune
diseases had been reported. However, there was little known about the impact of IL-16 gene
polymorphisms on GD. In this study, we performed a case-control association study of three tagSNPs
(rs4778889–rs1131445–rs4778641) within the IL-16 gene on 258 patients with GD and 208 healthy
subjects in the Chinese population. Our data showed that common IL-16 variants were associated
with GD (P = 0.013–0.0186) and Graves' disease associated ophthalmopathy (GO) (P = 0.0033–0.041).
A novel protective haplotype containing the three tagSNPs (C–T–C) was observed in association with
GO (P = 0.013). In conclusion, IL-16 gene was significantly associated with susceptibility to Graves'
disease and Graves' disease associated ophthalmopathy in the Chinese population.
© 2008 Elsevier Inc. All rights reserved.
⁎ Corresponding authors. Y.‐J. Zhao is to be contacted at Shanghai Clinical Center for Endocrine and Metabolic Diseases, RuiJin Hospital,
Shanghai JiaoTong University School of Medicine, 197 Ruijin Er Lu, Shanghai 200025, P.R.China. Fax: +86 21 64373514. G. Ning, Laboratory
Endocrine and Metabolic Diseases of Institute of Health Science, Shanghai JiaoTong University School of Medicine and Shanghai Institutes for
Biological Sciences, Chinese Academy of Sciences, 225 South ChongQing Road, Shanghai 200025, P.R.China.
E-mail addresses: [email protected] (G. Ning), [email protected] (Y.-J. Zhao).
1
Xue-Jiang Gu and Bin Cui contributed equally to this work.
1521-6616/$ – see front matter © 2008 Elsevier Inc. All rights reserved.
doi:10.1016/j.clim.2008.01.017
Rapid Communication
Introduction
Graves' disease (GD), in common with most other autoimmune
disorders, had a substantial genetic component to their pathogenesis [1]. Data from twin studies suggested that about 80%
of the susceptibility to GD was determined by genetic factors,
with the remainder influenced by environmental or other
factors [2]. GD was characterized by lymphocytic infiltration
into the thyroid gland, comprising CD4+ T helper cells [3,4].
T cells trafficked to the gland are thought to orchestrate
inflammation and tissue remodeling through their expressions
and release of cytokines. Important to the process of
lymphocyte recruitment was the chemoattractants that
activated target cells and enhanced their migration toward
sites of inflammation [5].
As a cytokine with chemotactic properties, IL-16 is believed
to be a T helper cell chemoattractant factor. IL-16 gene,
located in chromosome 15q26.35, is initially translated into a
631-amino acid precursor protein and subsequently cleaved by
caspase-3 to form the active C-terminal domain, which is comprised of 121 amino acids [6–10]. By binding to the CD4 molecule, IL-16 can activate T-cells, monocytes, macrophages, and
dendritic cells [11], and stimulate the production of proinflammatory cytokines such as tumor necrosis factor (TNF)-α, IL1β, IL-6, and IL-15 by monocytes [12]. The serum levels of IL-16
had been reported to associate with chronic inflammatory
diseases such as allergen-induced bronchial asthma, rheumatoid arthritis, and inflammatory bowel disease [13–15]. Jane
Pritchard et al. had also observed that IL-16 protein level was
increased by GD-IgG in fibroblasts of GD patients [16,17].
Recently, a new functional polymorphism in the promoter
region at position −295 of the human IL-16 gene was found
[18], which had been reported to be associated with other
autoimmune diseases [19–21]. However, it remains to be determined whether the polymorphism in IL-16 gene is associated with GD in Chinese populations. In our study, we not only
performed the population-based case-control association
study analysis on the promoter single nucleotide polymorphism
(SNP) of IL-16 gene, but also assessed the association between
IL-16 haplotypes and GD by genotyping tagging SNPs (tagSNPs),
which covered at least 95% of the entire common variations in
this gene. Our data revealed SNPs and haplotypes of the IL-16
gene were significantly associated with susceptibilities to GD
and GO (Graves' disease associated ophthalmopathy) in the
Chinese population.
Subjects and methods
Subjects
Totally 258 patients with GD (67 males and 191 females; aged
8–73 years; mean age, 36.4 ± 12.8 years) being treated at
299
Ruijin Hospital were enrolled in this study. GD was diagnosed
on the basis of clinical manifestations, biochemical criteria of
thyrotoxicosis [TSH b 0.05 mIU/L; free T3 N 6.49 pmol/L (normal range: 2.62–6.49 pmol/L) and/or free T4 N 19.04 pmol/L
(normal range: 9.01–19.04 pmol/L), Abbott Laboratories,
USA], and the presence of TSH receptor antibodies [22].One
hundred and thirty six GO were diagnosed by experienced
ophthalmologists and were classified using the NOSPECS classification [23]. For the statistical analysis, patients with
classes 2–6 were considered as having GO. Two hundred and
eight healthy Chinese volunteers (45 males and 163 females;
aged 20–71 years; mean age 37.5 ± 8.9 years), from the same
region of China without family history of GD or other
autoimmune diseases, served as the control group. Informed
consent was obtained from all participants, and the study was
approved by the Institutional Ethics Board of the RuiJin
Hospital affiliated to Shanghai JiaoTong University School of
Medicine.
SNP selection and genotype
For the selection of SNPs, Haploview software (http://www.
broad.mit.edu/mpg/haploview) was applied to conduct
linkage disequilibrium and haplotype block analyses by
using Hapmap phase genotype data for the chromosomal
region 15:79,371,522–79,395,156 (CHB database, Hapmap
release 22a [Apr.2007]). The amplicon of interest was a 23 kb
region, with IL-16 gene and approximately 3 kb upstream and
3 kb downstream of IL-16gene. The selection of tagSNPs was
performed by running the tagger program implemented
in Haploview [24]. The criteria for r2 was set at N 0.8. Any
marker that was not eventually chosen as a tagging marker
was considered strongly correlated with at least one of the
tagging markers with r2 N 0.8. One SNP (rs4778889) located at
position − 295 in the promoter region was forced as tagSNP
because it had been reported previously. Another two
tagSNPs were chosen from HapMap SNPs with minor allele
frequencies (MAF) N 10% (rs1131445 and rs4778641).Using
the three SNPs (r2 N 0.8), we were able to capture ∼ 95%
(19/20SNPs) of the Hapmap phase CHB common variation in
IL-16.
Genomic DNA from peripheral blood was extracted using a
commercially available kit (Qiagen, Hilden, Germany) according to the manufacturer's instruction and all three selected
tagSNPs were genotyped in the 466 participants by PCR-RFLP.
Supplementary Material Table 1 showed the primers and conditions. PCR products were digested and analyzed by agarose
gel electrophoresis. To ensure the accuracy of the method
employed, a homozygous wild-type sample and a homozygous
mutant one were used as controls when the PCR-RFLP was
performed. The two samples had been genotyped by direct
sequencing. To confirm the PCR-RFLP results, 30% of the
samples were sequenced using the CEQ 8800 Genetic Analysis
Figure 1 Structures of IL-16 and location of SNPs analyzed in this study. Coding and untranslated regions of IL-16 are represented by
and respectively.
300
Table 1A
SNP
rs4778889
rs1131445
rs4778641
Rapid Communication
IL-16 Gene Variations and Associations with GD
Major/minor allele
T/C
T/C
C/T
MAF
Genotype a (Control/case)
Control/case
11
12
22
0.26/0.19
0.34/0.29
0.43/0.49
113/170
85/132
67/75
80/76
104/102
104/118
14/12
19/23
37/63
Pallele
Padditive
Pdominant
Precessive
0.0186
0.080
0.139
0.016
0.076
0.15
0.013
0.024
0.5
0.33
0.94
0.074
Values indicate number of subjects except for MAF.
Pallele refers to allelic association of IL-16 variations with GD. Padditive, Pdominant, and Precessive show genotypic association for IL-16
variations under additive, dominant, and recessive mode of inheritance. Significant P values b 0.05 are marked in bold.
SNP are listed in the order of the position of the gene.
GD: Graves' disease; MAF: Minor allele frequency.
a
The major allele was always referred to as allele 1and the minor allele as allele 2.
System (Beckman Coulter, Fullerton, CA, USA). The results of
both methods were identical.
Results
Single variant association
Statistics
The Hardy–Weinberg Equilibrium was performed using Excel
(Microsoft Office Excel; Microsoft Corp., Redmond, WA, USA).
Allelic association for individual IL-16 SNPs with GD was
performed by χ2 or Fisher exact test within the SPSS for
Windows version 11.0 statistical package (SPSS, Chicago, IL,
USA). The strength of the association between the presence of
the disease and the constitutionally determined alleles was
estimated by the odds ratio (OR). The OR and 95% confidence
intervals (CIs) were calculated using the Mantel–Haenszel
method. The associations of genotype and frequencies with GD
were estimated using the software program SNPStats [25]
separately under additive, dominant, and recessive model.
The software program SNPStats was also adopted for haplotype
analysis. In a first approximation, all haplotypes identified
were computed but, in order to minimize loss of power, haplotypes with a frequency lower than 5% were excluded from
further analysis.
We used QUANTO software (http://hydra.usc.edu/gxe) to
calculate the power of association studies. At the 0.05 level
of significance with the two-sided test for the rs4778889
(rs1131445; rs4778641) polymorphism, our study had 93%
(95%; 91%) power to detect an effect with a relative risk of
2.0 in the group of GD patients and healthy controls, and 84%
(87%; 88%) power in the group of GO patients and healthy
controls, under a recessive genetic model.
Table 1B
SNP
rs4778889
rs1131445
rs4778641
The IL-16 gene consists of 8 exons spanning approximately
17 kb of genomic DNA. The organization of the IL-16 gene is
shown in Figure. 1. A total of 3 SNPs were identified in this study
and all SNPs were in Hardy–Weinberg equilibrium. Table 1A
showed the full genotype and allele frequency data. There was
evidence that the rs4778889 allele resulted in increased
susceptibility to GD [OR = 1.446 (95%CI, 1.063–1.968)].GD
patients presented with significantly higher portions of Tallele
and TT genotypes compared with the control subjects [Pallele =
0.0186, Pdominant = 0.013 respectively]. The SNP (rs4778889)
was also significantly associated with GO at even lower level of
P-value [Pallele = 0.0033, Pdominant = 0.0043] (Table 1B). Interestingly, we observed that another variant (rs4778641) was significantly associated with GO [Pallele = 0.041, Precessive = 0.012], but
not associated with GD.
Haplotype analysis
Haplotypes analyses were estimated using the SNPStats
software program. Rare haplotypes (frequencies b0.05) were
combined. The most common haplotype (T–T–T) was served as
reference haplotype in our analysis (Tables 2A and 2B). There
was a significant difference of the global haplotype profile
between GD and control group (P = 0.0093). None of the
individual haplotype effects reached statistical significance
except one (C–T–C), which showed close to significance
IL-16 Gene Variations and Associations with GO
Major/minor allele
T/C
T/C
C/T
MAF
Genotype a (Control/case)
Control/case
11
12
22
0.26/0.17
0.34/0.30
0.43/0.51
113/95
85/67
67/38
80/37
104/56
104/58
14/4
19/13
37/40
Pallele
Padditive
Pdominant
Precessive
0.0033
0.275
0.041
0.003
0.26
0.046
0.0043
0.13
0.4
0.11
0.89
0.012
Values indicate number of subjects except for MAF.
Pallele refers to allelic association of IL-16 variations with GD. Padditive, Pdominant, and Precessive show genotypic association for IL-16 variations
under additive, dominant, and recessive mode of inheritance. Significant P values b 0.05 are marked in bold.
SNP are listed in the order of the position of the gene.
GO: Graves' disease associated ophthalmopathy; MAF: Minor allele frequency.
a
The major allele was always referred to as allele 1and the minor allele as allele 2.
Rapid Communication
Table 2A
301
Three marker haplotype frequencies in a case control cohort of patients with GD
No
rs4778889
rs1131445
rs4778641
Controls/cases
P-value
OR
95% CI
1
2
3
4
Rarea
T
T
C
T
T
C
T
T
T
C
C
C
0.39/0.46
0.28/0.26
0.20/0.16
0.05/0.08
0.08/0.04
0.15
0.058
0.54
0.0038
1
0.79
0.71
1.19
0.39
(0.57–1.09)
(0.49–1.01)
(0.68–2.08)
(0.21–0.74)
Global haplotype association P-value: 0.0093.
Abbreviations: GD: Graves' disease; OR: odd ratio; CI: confidence interval.
The most common haplotype is reference.
Data are presented as frequency. P values b 0.05 are considered significant.
a
Rare haplotype combined. Each haplotype has frequency below 5% among the controls.
(P=0.058). With regard to GO, the frequency of haplotype (C–
T–C) was significantly lower in GO individuals compared to those
in control [0.14 vs 0.20; P=0.013; OR=0.57, 95%CI (0.36–0.89)],
suggesting that this haplotype might play a protective role in
GO. However, the global association of haplotype was also found
to be significant (P=0.012) between GO and control individuals.
Discussion
In the present study, we investigated the role of three
tagSNPs of IL-16 gene in the risk of GD and GO susceptibility in
Chinese GD patients. Our results revealed the variations of IL16 gene and the global haplotype association with GD and GO.
A haplotype C–T–C (rs4778889–rs1131445–rs4778641) was
negatively correlated with GO and might played a protective
role in GO.
The rs4778889, located at the position − 295 of the
promoter region, might be a functional polymorphism that
had been reported by Kristin M et al. In in vitro experiments,
Kristin M et al found that the location of the −295 SNP could be
at the GATA-3 binding site, and the −295T resulted in reduced
promoter activity compared with the C allele. They also found
that the elevated level of T allele at the −295 position was
significantly associated with all asthma phenotypes (21).
Similar association-study results had also been found in other
autoimmune diseases, such as Crohn's disease (19). In our
study, we also discovered a significant association of the
rs4778889 SNP with GD. The T allele and TT genotype were
significantly higher in GD individuals than those in control
(Pallele = 0.0186; OR = 1.446; Pdominant = 0.013; OR = 1.61). Association of the rs4778889 polymorphism with GO appeared as a
stronger statistical association between IL-16 gene and GO
Table 2B
with a lower P-value (Pallele = 0.0033, OR = 1.78; Pdominant =
0.0043, OR = 1.92).
Additionally, another variant (rs4778641) presented a
significant different distribution between GO and control
individuals (Pallele = 0.041, Precessive = 0.012). The rs4778641
polymorphism was located in 3′untranslated region (3′-UTR),
a region that played a pivotal role in the control of gene
expression by binding proteins that regulated mRNA processing, translation, or degradation [26]. To identify the function
of the rs4778641 polymorphism, we performed a functional
prediction of 3′-UTR of the IL-16 gene by using the online
software targetscan [27].We found only a single target for
microRNA (miR-125/351), however there was no polymorphism found in this region, suggesting that this SNP could not
directly influence IL-16 gene transcription and function, and
maybe other factors had played a role in the longer conserved
3′-UTR regions of the IL-16 gene.
Haplotype analysis of the rs4778889–rs1131445–rs4778641
combination revealed four main and rare haplotypes. The
common haplotype T–T–T, which shared the same three
nucleotides, displayed a different frequency between GD and
control individuals (0.46 vs. 0.39), as well between GO and
control individuals (0.49 vs. 0.39). Compared to the common
haplotype, the haplotype C–T–C showed a highly significant
negative correlation between GO and control individuals (0.14
vs. 0.20, P = 0.013, OR = 0.57), suggesting that it might be a
protective haplotype. Herein, haplotype analysis reconfirmed
the results of rs4778889T and rs4778641T being the major risk
variants for GO in the Chinese population.
However, there are some inadequacies in this study. First,
the sample size in our study is relatively small, which may not
have the efficient statistical power to detect a weak genetic
effect, resulting in a fluctuated estimation. Second, we could
Three marker haplotype frequencies in a case control cohort of patients with GO
No
rs4778889
rs1131445
rs4778641
Control/case
P-value
OR
95% CI
1
2
3
4
Rarea
T
T
C
T
T
C
T
T
T
C
C
C
0.39/0.49
0.28/0.28
0.20/0.14
0.05/0.06
0.08/0.03
0.26
0.013
0.78
0.0073
1
0.80
0.57
0.91
0.34
(0.54–1.18)
(0.36–0.89)
(0.47–1.77)
(0.15–0.74)
Global haplotype association P-value: 0.012.
Abbreviations: GO: Graves' disease associated ophthalmopathy; OR: odd ratio; CI: confidence interval.
The most common haplotype is reference.
Data are presented as frequency. P values b 0.05 are considered significant.
a
Rare haplotype combined. Each haplotype has frequency below 5% among the controls.
302
only reveal limited polymorphisms of IL-16 gene associated
with susceptibility to GD and GO, there might be still other
unidentified SNPs which influenced the development of GD
and GO. Therefore, further association studies utilizing a large
sample size from different ethnic origins and biological
research should be carried out to verify this association.
In conclusion, our study revealed that SNPs and haplotypes of the IL-16 gene were significantly associated with
susceptibility to GD and GO in the Chinese population.
Acknowledgments
The present study would not have been possible without the
participation of the patients and healthy volunteers. The
study is supported by the grants from the National Nature
Science Foundation of China (No. 30370666) and Shanghai
Leading Academic Discipline Project (Project Number:
Y0204).
Appendix A. Supplementary data
Supplementary data associated with this article can be found,
in the online version, at doi:10.1016/j.clim.2008.01.017.
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