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Toll-Like Receptor Gene Polymorphisms are Associated with Susceptibility to Graves' Ophthalmopathy in Taiwan Males
BMC Medical Genetics 2010, 11:154 doi:10.1186/1471-2350-11-154 Wen-Ling Liao ([email protected])
Rong-Hsing Chen ([email protected]) Hui-Ju Lin ([email protected]) Yu-Huei Liu ([email protected]) Wen-Chi Chen ([email protected])
Yuhsin Tsai ([email protected]) Lei Wan ([email protected]) Fuu-Jen Tsai ([email protected])
ISSN 1471-2350 Article type Research article Submission date 23 March 2010 Acceptance date 5 November 2010
Publication date 5 November 2010
Article URL http://www.biomedcentral.com/1471-2350/11/154
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Toll-Like Receptor Gene Polymorphisms are Associated with Susceptibility to Graves’ Ophthalmopathy in Taiwan Males
Wen-Ling Liao
1,2, Rong-Hsing Chen
3, Hui-Ju Lin
4, Yu-Huei Liu
1,2, Wen-Chi Chen
5,
Yuhsin Tsai
2, Lei Wan
1,2,6§, Fuu-Jen Tsai
1,2,6§1
Genetic Center, China Medical University Hospital, Taichung, Taiwan;
2
School of Chinese Medicine, China Medical University, Taichung, Taiwan;
3
School of Post Baccalaureate Chinese Medicine; China Medical University,
Taichung, Taiwan;
4
Department of ophthalmology, China Medical University Hospital, Taichung,
Taiwan;
5Graduate Institute of Integrated Medicine; China Medical University,
Taichung, Taiwan;
6
Department of Biotechnology, Asia University, Taichung, Taiwan.
§
Corresponding author
Email addresses:
WLL: [email protected]
RHC: [email protected]
HJL: [email protected]
YHL: [email protected]
WCC: [email protected]
YHT: [email protected]
LW: [email protected]
FJT: [email protected]
Abstract
Background: Toll-like receptors (TLRs) are a family of pattern-recognition receptors,
which plays a role in eliciting innate/adaptive immune responses and developing
chronic inflammation. The polymorphisms of TLRs have been associated with the
risk of various autoimmune diseases, including systemic lupus erythematosus (SLE),
multiple sclerosis and rheumatorid arthritis. The aim of this study was to evaluate
whether TLR genes could be used as genetic markers for the development of Graves’
ophthalmopathy (GO).
Methods: 6 TLR-4 and 2 TLR-9 gene polymorphisms in 471 GD patients (200
patients with GO and 271 patients without GO) from a Taiwan Chinese population
were evaluated.
Results: No statistically significant difference was observed in the genotypic and
allelic frequencies of TLR-4 and TLR-9 gene polymorphisms between the GD
patients with and without GO. However, sex-stratified analyses showed that the
association between TLR-9 gene polymorphism and GO phenotype was more
pronounced in the male patients. The odds ratios (ORs) was 2.11 (95% confidence
interval [CI] = 1.14–3.91) for rs187084 A G polymorphism and 1.97 (95% CI =
1.07–3.62) for rs352140 A G polymorphism among the male patients. Increasing
one G allele of rs287084 and one A allele of rs352140 increased the risk of GO (p
values for trend tests were 0.0195 and 0.0345, respectively). Further, in haplotype
analyses, the male patients carrying the GA haplotype had a higher risk of GO (odds
ratio [OR] = 2.02, 95% confidence interval [CI] = 1.09–3.73) than those not carrying
the GA haplotype.
Conclusion: The present data suggest that TLR-9 gene polymorphisms were
significantly associated with increased susceptibility of ophthalmopathy in male GD
patients.
Background
Graves’ disease (GD) is an organ-specific autoimmune thyroid disease, one of the
manifestations of which is ophthalmopathy [1]. Graves’ ophthalmopathy (GO) is
characterized by inflammation and fat deposition in the eye muscles and the
connective tissue surrounding the eye. It is known that multiple factors contribute to
the etiology and severity of GD, including the host’s genetic factors as well as
environmental factors [2-3]. Female sex, old age, and smoking history are common
risk factors for GD [4-8]. With regard to genetic factors, the classical major
histocompatibility complex class II genes and cytotoxic T cell antigen-4 genes
(CTLA-4) [9-11] have been consistently reported to be associated with GD. Also,
there were published studies on association of GO and genes such as CD103 [12],
CTLA-4 and IL-13 [13]. However, the findings of most genes effect in GD or GO
were inconsistent. Recently, toll-like receptors (TLRs) which play important roles in
eliciting human innate/adaptive immune responses and developing chronic
inflammation [14] are a new area of basic immunological investigation and could be
associated with autoimmune thyroiditis [15].
TLRs are a family of pattern-recognition receptors and can be expressed in
several types of cells and tissues such as macrophages, dendritic cells (DCs), B cells,
T cells and monocytes. A total of 10 human TLRs have been identified and the
functions of human TLR-1 to TLR-9 have been characterized [16-17][14-15]. TLRs
are well known to recognize a variety of microbial molecules such as TLR-4 can
recognize lipopolysaccharides (LPSs) in gram-negative bacteria and TLR-9 activation
can be triggered by unmethylated CpG DNA of bacteria. Once TLRs are activated by
microbial molecules, downstream signalling pathway via myeloid differentiation
factor 88 (MyD88) and interleukin-1R (IL-1R)-associated kinase (IRAK) activates
nuclear factor κB (NF-κB), leading to cytokine production, and even to apoptosis.
TLRs could response to not only exogenous, microbe derived pathogen associated
molecular patterns (PAMPs) but also endogenous or self ligands. Recently, the ability
of TLRs to recognize host-derived danger signals, which are produced on cell damage
and necrosis, was also recognized [18-19]. Moreover, studies have found that the
immune complexes containing self-RNA and/or self-DNA can act as endogenous
triggers for the activation of TLRs [20-22].
Recently, release of endogenous TLR ligands during inflammation and the
consequent activation of TLR signaling pathways have been indicated and
polymorphism of the TLR4 and TLR9 gene have been reported to be associated with
many autoimmune diseases, such as systemic lupus erythematosus (SLE),
atherosclerosis, asthma, type 1 diabetes, multiple sclerosis, and rheumatoid arthritis
(RA) [14, 23-31]. However, to the best of our knowledge, there were no reports about
TLR4 and TLR9 in GO. Therefore, our aim in the present study was to investigate the
potential association between GO and single-nucleotide polymorphisms (SNPs) of
TLR genes—TLR-4 and TLR-9 genes—in a Chinese population in Taiwan.
Methods
Patients and data collection
Four hundred and seventy one GD patients at China Medical University Hospital
in Taiwan were enrolled in present study. All GD patients were examined by
experienced endocrinologist and identified using following criteria: having
hyperthyroidism, having diffused goiter and presence at least one of
thyroid-stimulating hormone (TSH) receptor antibody, diffusely increased
131I
(iodine-131) uptake in the thyroid gland or presence of exophthalmos. The proptosis
was quantified with an exophthalmometer. The data regarding age, sex, history of
tobacco use, thyroid gland pathology and affected anatomic sites were extracted from
the questionnaire and blood samples were collected by venipuncture for genomic
DNA isolation and serological test at the enrollment in study. Informed consent was
obtained from each participant before his/her inclusion in this study and the ethics
committee of China Medical University Hospital gave its approval for the study.
Genomic DNA extraction and genotyping
The genomic DNA was extracted from peripheral blood leukocytes of GD patients
using the Genomic DNA kit (Qiagen) according to the manufacturer’s instructions. To
select the most representative single nucleotide polymorphisms (SNPs) by capturing
the majority genetic variation, SNP genotype information was downloaded in
December 2008 from the HapMap Han Chinese in Beijing (HCB) and Japanese in
Tokyo (JPT) population. HapMap genotypes were analyzed within Haploview and
Tag SNPs were selected using the Tagger function. The following criteria were used
to select Tag SNPs: (1) a threshold minor allele frequency (MAF) of 0.10 (2)
multimarker method with r
2threshold > 0.8 and logarithm of odds threshold 3.0 (3)
probe or primers that pass the qualification as recommended by the manufacturer
(Applied Biosystems Inc., Foster City, CA) to ensure a high genotyping success rate.
The mean max r
2were 0.949 and 1 for TLR4 and TLR9 SNPs, respectively. A total of six SNPs for TLR-4 and 2 SNPs for TLR-9
,were selected in present study.
Genotyping was achieved using an assay on-demand allelic discrimination assay and
detection system according to the manufacturer’s instructions (Applied Biosys tems).
Briefly, polymerase chain reaction (PCR) was performed in the presence of 10 ng
genomic DNA, 10µl TaqMan master mix, and 0.125µl of 40x assay mix. Polymerase
chain reaction analysis was performed in 96-well plates on a thermal cycler (ABI
9700; Applied Biosystems). Reaction conditions were 50° C for 2 minutes and 95° C
for 10 minutes, followed by 40 cycles at 95° C for 15 seconds and 60° C for 1 minute.
Real-time detection of fluorescence signals was performed using the ABI Prism 7900
Real-Time PCR System. The frequency of SNPs’ genotype for HCB + JPT population
was extracted from PubMed [32-36].
Statistical analysis
The difference of genotypic and allelic frequencies distributions between GD
patients with and without ophthalmopathy and between GD patients and individuals
information from HCB-HapMap were analyzed by the χ
2test or Fisher exact test. The
odds ratio (OR) was calculated from genotypic and allelic frequencies with 95%
confidence interval (CI) by using unconditional logistical regression adjusting for age
of diagnosis, gender and smoking history. We further stratified data into subgroups by
sex and homogeneity test were performed to determine whether the association
between genotype/haplotype frequencies and GO phenotype is similar in males and
females two groups. If the homogeneity test was significant, the different association
in males and female was indicated. Then males and females two groups should be
analyzed separately. All statistical analyses were conducted using SAS statistical
software, version 9.1 (SAS Institute Inc., Cary, NC). All tests were two-sided, and a p
value less than 0.05 was used as the level of significance.
Power calculation
The power calculation was based on the following assumptions: the allele
frequencies of the investigated SNPs within the population were based on the
information from HapMap-CHB (0.422 to 0.675 for TLR9 SNPs and 0.067 to
0.633for TLR4 SNPs) and the size of present study were 271 for control group and
200 for case group. Therefore, the estimated effect size of OR, 0.578 to 0.592 for
TLR9 and 1.63 to 2.25 for TLR4 of the investigated SNPs in case group can be
observed using a two independent sample test with a power of 80% at two-sided type
I error rate 0.05 (Additional file 1: table S1).
For the effect size of TLR on GO, no formal data are available. Therefore, we
compare the observable effect sizes from our design power calculation with the effect
sizes (OR=0.55 to 0.56 for TLR9 and 1.78 to 2.85 for TLR4) from previous study in
RA [37]. The magnitude of effect size in previous study in RA is bigger than our
estimated effect size. Therefore, the power will be bigger than 80% if our effect size
could be the same with the effect size from previous study in RA. Based upon above
information, our study design could be justified.
Results
A total of 471 GD patients comprising 200 patients with GO and 271 patients
without GO were enrolled in this study. The female to male ratio was 3.75 and the
mean age was 39.41 ± 12.42 years. There were statistically significant differences in
the GO phenotype and smoking history between the male and female patients. The
percentage of male and female GD patients with GO was 52% and 40%, respectively
(p = 0.04). None of the frequency distribution for other anatomic sites (goiter, nodular
hyperplasia, myxedema and vitiligo) was significant different between two genders. A
higher percentage of male patients had smoking history than female patients (66.67%
vs. 12.10%, p < 0.0001) (Table 1).
Association between TLR gene polymorphism and disease severity
We compared the allelic and genotypic frequencies of TLR-4 and TLR-9 gene
polymorphisms in the Taiwanese GD patients, with the information regarding the
populations of Han Chinese in Beijing (HCB) and Japanese in Tokyo (JPT) extracted
from the PubMed SNP database. The TLR-4 and TLR-9 gene polymorphisms in the
Taiwanese GD patients were not statistically different from those in normal HCB and
JPT. Further, we classified the GD patients on the basis of the presence of GO
(Yes/No) in order to determine whether TLR-4 and TLR-9 genes were associated with
the GO phenotype. The results did not indicate any significant differences in the
TLR-4 and TLR-9 gene polymorphisms between the GD patients with and without
GO (Additional file 2: table S2 and additional file 3: table S3). All tested SNPs were
in Hardy-Weinberg equilibrium (p > 0.05).
Sex-specific effects of TLR gene polymorphisms on susceptibility to GO
In the present study, 52% male GD patients had GO as compared to 40% female
GD patients (p = 0.04). However, there was no sex-based difference in the genotypic
distribution of TLR-4 and TLR-9 gene polymorphisms (data not shown). Therefore,
we performed sex-stratified analyses to investigate the association between TLR gene
polymorphisms and GO phenotype in the 2 sex-based subgroups. Statistically
significant differences were observed in the allelic frequencies of 2 SNPs of the
TLR-9 gene between the male patients with and without GO but not in the female
patients (Table 2). The odds ratios (ORs) for rs187084 A G polymorphism were 2.11 (95% confidence interval [CI] = 1.14–3.91) and 0.79 (95% CI = 0.57–1.08) for
the male and female patients, respectively. The p value for homogeneity test was 0.01.
For rs352140 A G polymorphism, the ORs were 1.97 (95% CI = 1.07–3.62) and 0.76 (95% CI = 0.55–1.05) for the male and female patients, respectively. The p value
for homogeneity test was 0.01. Among the male patients, increasing one G allele of
rs287084 and one A allele of rs352140 increased the risk of GO (p values for trend
tests were 0.0195 and 0.0345, respectively). Furthermore, we investigated the
association between the TLR-9 haplotype and susceptibility to GO in the male and
female patients. AG haplotype was significantly inversely associated with
susceptibility to GO (OR = 0.49, 95% CI = 0.26–0.89), while GA haplotype was
significantly associated with a high risk of GO (OR = 2.02, 95% CI = 1.09–3.73) in
the male patients (Table 3). However, this sex-specific association was not found in
the case of even one of the SNPs of the TLR-4 gene. Furthermore, we investigated the
association between various phenotype parameter and GO among male but no
significant association was found (Additional file 4: table S4).
Discussion
In this study, we investigated the effect of TLR-4 and TLR-9 gene
polymorphisms on the organ-specific autoimmune disease GD in a Taiwanese
population. TLR-9 polymorphisms and haplotype were significant associated with
susceptibility to GO in males. However, none of the TLR-4 and TLR-9 polymorphism
or haplotypes was associated with overall GO risk. The TLR4 is known to activate the
NF-κB and subsequent gene expression such as cytokines and adhesion molecules. A
TLR-4 polymorphism that prevents ligands binding and subsequent cellular signaling
would result in lower NF-κB activation and subsequent NF-κB-dependent
proinflammatory gene expression. However, we did not find any association between
TLR4 polymorphism and GO in present study. Low statistical power may be the
reason for this non significant results in TLR4 association in GO. For TLR9, our
results were consistent with those of the previous studies with regard to the
association between TLR9 gene polymorphisms and systemic autoimmune diseases,
such as SLE in Japan [30][28] and China [38][36]. From SLE model, Tao et al study
[30][28] found diminished expression of TLR9 could increase anti-dsDNA antibody
in SLE mice model which may increase the risk of developing autoimmune disease.
Also, it has been hypothesized that nucleic acid from dying cells may act as ligands
for TLR9 to trigger IFN-1 production in SLE [39][37]. In this study, 2 TLR-9 SNPs,
one (–1486T C, rs187084) is in the promoter region and the other (1635AG, rs352140) is in exon 2. The variation of TLR-9 gene in those region may down
regulate TLR9 expression and involved in production of autoantibodies or IFN-1
which may increase the risk of GO. Therefore, future studies are required to
investigate if variation of TLR9 gene, especially in exon region may affect TLR9
protein expression or function which increase the risk of GO.
TLR-9 polymorphisms were associated with increase the risk of GO in males in
present study. In general, GD is more common among women; however, male sex
also faces the risk of developing progressive and severe thyroid-associated
orbitopathy [4, 7][4, 7] or responds poorly to treatment [40][38]. A previous study has
reported a female to male ratio of 9.3 for patients with mild GO and of 3.2 for those
with moderate GO, with the ratio being 1.4 for patients with severe GO [4][4]. Our
data suggest that the role of TLR-9, which plays an important role in conferring innate
immunity, would be more critical in men with GO. The susceptibility factor seems to
play a different role depending on sex, or the effect could be more apparent in men
and not in women because of the difference in the basic immune responses between
the 2 sexes [41][39]. Further studies are required to investigate that if the TLR-9
activity and its potential relevance cytokines being more prone to male with GO.
As the smoking is potential confounding factor for susceptibility of GO, we
adjusted the variable of smoking history in the multivariate model to eliminate the
smoking effect on susceptibility of GO. However, the significant association between
TLR-9 gene polymorphism and susceptibility to GO in the male patients was no
changed. Furthermore, the strength association between TLR-9 polymorphism and
GO in male was modest in present study. Therefore, a larger sample sized, especially
in male population will be needed in the future study to confirm the importance of
these polymorphisms as genetic markers of GD in Taiwanese population. Furthermore,
the interpretation of our study results is limited because the patients were recruited
from Taiwan only. Studies in ethnically disparate populations are needed before firm
conclusions can be drawn.
Conclusion
In conclusion, our findings suggested that TLR-9 gene polymorphisms were
significantly associated with susceptibility to GO in the male GD patients in Taiwan.
Competing interests
The authors declare that they have no competing interests.
Authors’ contributions
WLL carried out statistical analyses, results interpretation and drafted the manuscript.
RHC, HJL and WCC recruited and maintained the clinical information of participants.
YHL and YHT supervised genotyping experiments. LW and FJT carried out study
design and coordination and have given final approval of the version to be published.
All authors read and approved the final manuscript.
Acknowledgments
This study was supported by a grant from the National Science Council
(98-2320-B-039-008-MY3), Taipei, Taiwan, and a grant from the China Medical
University Hospital (DMR-93-45), Taichung, Taiwan. The author also thank Dr. Li
Tsai-Chung for her advice in the statistical analysis.
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Table 1. Characters of Graves’ Disease Patients between Male and Female.
Male (n = 99)
Female
(n = 372) P-value
*Ophthamology
Yes 51 (51.52) 149 (40.05)
No 48 (48.48) 223 (59.95) 0.04
Goiter
Yes 95 (95.96) 345(92.74)
No 4 (4.04) 27 (7.26) 0.25
Nodular hyperplasia
Yes 8 (8.08) 39 (10.48)
No 91 (91.92) 333 (89.52) 0.48
Myxedema
Yes 2 (2.02) 4 (1.08)
No 97 (97.98) 368 (98.92) 0.46
Vitiligo
Yes 1 (1.01) 3 (0.81)
No 98 (98.99) 369 (99.19) 0.84
Age at enrollment
mean ± SD† 40.25 (10.62) 39.81 (12.57) 0.55†
Age at diagnosis
mean ± SD† 35.69 (10.34) 34.57 (12.25) 0.16†
Smoking history
Never 33 (33.33) 327 (87.90)
Ever 66 (66.67) 45 (12.10) <0.001
Data are no. (%)
*
