Association of Interleukin 1 beta (IL1B) polymorphisms with Grave’s Ophthalmopathy in Taiwan Chinese patients

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09-4965 3626932

Association of Interleukin-1|gb (IL1B) Polymorphisms with Graves\' Ophthalmopathy in Taiwan Chinese Patients Wan Lei

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Yu-Huei Liu, Rong-Hsing Chen, Hsin-Hung Wu, Wen-Ling Liao, Wen-Chi Chen, Yuhsin Tsai, Chang-Hai Tsai, Lei Wan, and Fuu-Jen Tsai

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Association of Interleukin-1

␤ (IL1B) Polymorphisms

with Graves’ Ophthalmopathy in Taiwan

Chinese Patients

Yu-Huei Liu,

1,2

Rong-Hsing Chen,

3

Hsin-Hung Wu,

4

Wen-Ling Liao,

1,2

Wen-Chi Chen,

5

Yuhsin Tsai,

2

Chang-Hai Tsai,

6,7

Lei Wan,

*

,1,2,8

and Fuu-Jen Tsai

*

,1,2,3,6,9,10

PURPOSE. To evaluate whether variations in the IL1B gene could

be associated with Graves’ ophthalmopathy (GO) in patients with Graves’ disease (GD).

METHOD. This case– control study included 471 Taiwan Chinese patients with GD (200 with GO and 271 without GO) and 160 healthy volunteers. Eight single-nucleotide polymorphisms (SNPs) in IL1B were genotyped with an allele-specific exten-sion and ligation assay.

RESULTS. In the IL1B SNPs examined, the C allele of rs1143634

was associated with GD, whereas the T/T genotype of the SNPs rs1143634 and rs16944 were less associated with the disease. The A/A genotype of the SNPs rs3917368 and rs1143643, which had the strongest interaction, may increase the risk of GO (P ⫽ 0.024 and P ⫽ 0.017, respectively). Several GD susceptibility and insusceptibility IL1B haplotypes have been identified, and the Ht4-GCGCCTCC haplotype, composed of eight SNPs and associated with low circulating IL1␤ levels, may be protective against the development of GO (P ⫽ 0.025). Moreover, that the GO-susceptible genotype was associated with lower plasma IL1␤ concentrations implies that the origin of GO may go beyond the IL1B polymorphism-associated ele-vation of circulating IL1␤.

CONCLUSIONS. The data for IL1B polymorphisms and the

asso-ciation of GD and GO with plasma IL1␤ levels show that IL1B polymorphisms may be associated with the development of GD and GO. (Invest Ophthalmol Vis Sci. 2010;51:000 – 000) DOI:10.1167/iovs.09-4965

G

raves’ disease (GD), with or without Graves’ ophthalmop-athy (GO), is an autoimmune disease characterized by hyperthyroidism, diffuse goiter, thyroid-specific autoantibod-ies, and dermopathy due to circulating autoantibodies.1

GO is the most common extrathyroid manifestation of GD and affects 25% to 50% of GD patients.2–5Approximately 28% of patients with GO present as severe cases, with restricted mobility, diplopia, keratopathy, and optic neuropathy.6,7

Several genes have been reported that promote the development of GO, including human leukocyte antigen (HLA) class I and class II molecules.8

For example, the ⫹49G allele of cytotoxic T-lymphocyte-associated antigen-4 (CTLA4) confers genetic sus-ceptibility to GO, although meta-analyses did not support this finding. CT60A/G of CTLA4 is one of the GD-associated poly-morphisms that await further studies that examine their asso-ciation with GO. In addition, polymorphisms in several immu-nomodulatory genes, such as the intron 1 (CA) repeat in interferon-␥ (IFNG); G238A, C863A, and T1031C in tumor necrosis factor-␣ (TNFA); and A1405G in intracellular adhesion molecule-1 (ICAM1), have been reported to increase suscepti-bility to GO.8

The combination of specific alleles among these genes would make the patient susceptible to GO.

Interleukin-1 beta (IL1B), a proinflammatory cytokine ex-pressed by activated macrophages and several other types of cells, is thought to play a crucial role in the pathogenesis of autoim-mune diseases.9,10

IL1␤ was initially known as one of the lym-phocyte activating factors (LAFs), owing to its role in the induc-tion of T-cell proliferainduc-tion and maturainduc-tion.9,10

Recent studies have demonstrated that IL1␤ released by macrophages and fibroblasts can induce adipogenesis and accumulation of glycosaminoglycans (GAGs) and prostaglandin E2 (PGE2), which may result in the development of GO.8,11,12

One study revealed that the single-nucleotide polymorphism (SNP) ⫺511C of IL1B is associated with GO, whereas others did not.13–15

Although the connection between IL1B polymorphism and GO remains controversial, sev-eral studies have demonstrated that polymorphisms of IL1B may correlate with IL1␤ expression in other diseases.16 –18

In addition, IL1␤ promotes the accumulation of GAGs through the upregula-tion of hyaluronan synthesis and accumulaupregula-tion of PGE2, stimu-lates adipogenesis, and hyperinduces the expression of interleu-kin (IL)-6 and -8 and macrophage chemoattractant protein (MCP)-1 in orbital fibroblasts derived from healthy individuals and patients with GO.8,19 –23

Moreover, the polymorphisms of the IL1 family are involved in several autoimmune diseases such as sys-temic lupus erythematosus (SLE),24 –26

rheumatoid arthritis,27,28

autoimmune hemolytic anemia,29

and GD.30

These reports support that IL1B is a potential candidate gene in the development of GO.

Although previous reports have suggested that IL1B polymor-phisms and expression lead to autoimmune diseases,24 –30

the genetic role of IL1B in GO remains to be elucidated. In the present study, we investigated SNPs in IL1B that may be protective against or causative of GO in Taiwan Chinese patients with GD.

From the Departments of1_{Medical Genetics and Medical Research}

and6_{Pediatrics, China Medical University Hospital, Taichung, Taiwan;}

the2_{School of Chinese Medicine, the}3_{School of Post-Baccalaureate}

Chinese Medicine, and the5_{Graduate Institute of Integrated Medicine,}

China Medical University, Taichung, Taiwan; the4_{Department of}

Busi-ness Administration, National Changhua University of Education, Chan-ghua, Taiwan; 7_{Asia University, Taichung, Taiwan; and the}

Depart-ments of8_{Health and Nutrition Biotechnology,} 9_{Biotechnology, and} 10_{Biotechnology and Bioinformatics, Asia University, Taichung,}

Tai-wan.

Supported by Grant CMU96-081 from China Medical University and Grant DMR-95-007 from China Medical University Hospital, Taichung, Taiwan.

Submitted for publication November 23, 2009; revised May 3, 2010; accepted July 8, 2010.

Disclosure:Y.-H. Liu, None; R.-H. Chen, None; H.-H. Wu, None; W.-L. Liao, None; W.-C. Chen, None; Y. Tsai, None; C.-H. Tsai,

None;L. Wan, None; F.-J. Tsai, None

*Each of the following is a corresponding author: Lei Wan, Genetic Center, China Medical University Hospital, No.2 Yuh-Der Road, 404 Taichung, Taiwan; [email protected].

Fuu-Jen Tsai, Genetic Center, China Medical University Hospital, No.2 Yuh-Der Road, 404 Taichung, Taiwan; [email protected].

Clinical and Epidemiologic Research

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ARTICLE TYPE

: CLEP

Investigative Ophthalmology & Visual Science, Month 2010, Vol. 51, No. 0

Copyright © Association for Research in Vision and Ophthalmology 1

OK

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[email protected] [email protected] Table 5 1. Add asterisk* TO READ:Diplotypes* . 2. Please change the "100/100" in the same line of "0.008†" and "1.650 (1.141–2.384)‡". Table 6 Delete dagger TO READ: P = 0.377‡ 7,8 9 7,8

Delete and Insert

Please reivse the e-mail addresses for the corresponding authors (Lei Wan, [email protected] for Lei Wan and [email protected] for Fuu-Jen Tsai)

TO READ: [email protected]; [email protected] Delete and Insert

TO READ: and the Departments of 7 Biotechnology, 8 Biotechnology and

Bioinformatics, and 9 Health and Nutrition Biotechnology, Asia University, Taichung, Taiwan.

M

ETHODS

Patients and Healthy Individuals

A group of 484 patients with a confirmed diagnosis of GD and a control group of 160 healthy volunteers at China Medical University Hospital in Taiwan were enrolled and actively observed. All individuals in this study provided informed consent, as approved by the ethics commit-tee of China Medical University Hospital and in accordance with the guidelines in the Declaration of Helsinki.

Patients.Diagnosis of GD was based on the typical clinical features of hyperthyroidism: diffuse enlargement of the thyroid gland, increased free thyroxine or triiodothyronine levels, suppressed thyroid-stimulating hormone levels, positive thyrotrophin-receptor autoantibodies, and the presence (or absence) of anti-thyroid peroxidase (anti-TPO) antibodies or antithyroglobulin antibodies. Information regarding sex, age at onset of GD, treatment of hyperthyroidism, personal history of cigarette smoking, history of systemic diseases, and family history of autoimmune thyroid disease was obtained. The inclusion criteria were (1) meeting the diag-nostic criteria of GD at the time of examination; (2) being willing to participate and capable of giving informed consent; and (3) being a self-reported nonaboriginal Taiwanese with no parent or grandparent having an aboriginal background. The exclusion criteria were (1) being unable to understand or give informed consent or (2) being pregnant or having given birth within 1 year, to exclude the possibility of including subjects with postpartum thyroiditis. Patients with GO (GD/GO) were

identified according to the following criteria: Normal upper eyelid posi-tion was 1.5 mm below the superior limbus, and normal lower eyelid position was at the level of the inferior limbus in primary gaze. Proptosis was measured by a Hertel exophthalmometer and was defined as the anteroposterior protrusion of the globe⬎19 mm from the lateral orbital rim in either eye or any discrepancy in the degree of protrusion of the two eyes by⬎1 mm. All individuals classified as affected were interviewed and examined by experienced clinicians. A full medical record review was conducted to obtain demographics (age and sex); history of tobacco use; recurrence of GD (patients with GD who have accepted medical treat-ment); and progression (patients with ongoing GD), treatment, and clin-ical features of the condition.

Healthy Individuals.The healthy group was matched for sex according to the female predominance of GD, including 32 men (20.0%) and 128 women (80.0%). Age was significantly different be-tween the groups of healthy volunteers (27.4⫾ 6.4 years) and patients with GD (39.9⫾ 12.2 years; P ⫽ 1.028 ⫻ 10⫺34).

All blood samples were collected from consenting individuals by venipuncture for subsequent genomic DNA isolation.

SNP Selection

IL1BSNP genotype information was downloaded in December 2008 from the HapMap CHB⫹JPT population. HapMap genotypes were analyzed in Haploview (Haploview software (http://www.broad.mit. edu/mpg/haploview/ provided in the public domain by The Broad

TABLE1. Background and Demographic Characteristics of Healthy Individuals and Graves’ Patients with or without GO

Patients’ Characteristics Healthy (nⴝ 160) GD/GO (nⴝ 200) GD/non-GO (nⴝ 271) P GD vs. Healthy P GD/GO vs. GD/nonGO

Age at diagnosis (mean⫾ SD) 27.4⫾ 6.4 37.5⫾ 10.8 41.7⫾ 12.8 1.028⫻ 10⫺34* 2.978⫻ 10⫺4* Sex Male 32 (20.0) 51 (25.5) 48 (17.7) 0.784 0.040† Female 128 (80.0) 149 (74.5) 223 (82.3) Smoking status‡ Smoking 57 (28.5) 54 (19.9) 0.030† Nonsmoking 146 (71.5) 217 (80.1) Recurrence Yes 99 (49.5) 128 (47.2) NS† No 101 (50.5) 143 (52.8) Treatment Radioiodine Yes 15 (7.5) 6 (2.2) 0.006† No 185 (92.5) 265 (97.8)

Thyroid gland surgery

Yes 24 (12.0) 23 (8.5) NS† No 176 (88.0) 248 (91.5) Clinical features Goiter Grade 1 14 (7.0) 17 (6.3) NS§ Grade 2 5 (2.5) 21 (7.7) Grade 3 22 (11.0) 32 (11.8) Grade 4 129 (64.5) 169 (62.4) Grade 5 30 (15.0) 32 (11.8) Nodular hyperplasia Yes 22 (11.0) 25 (9.2) NS† No 178 (89.0) 246 (90.8) Myxedema Yes 5 (2.5) 1 (0.4) 0.042* No 195 (97.5) 270 (99.6) Vitiligo Yes 2 (1.0) 2 (0.7) NS† No 198 (99.0) 269 (99.3)

Data are n (%), unless otherwise indicated. Bold probabilities are statistically significant.

* Age and myxedema were determined by Mann-Whitney U test. †All characteristics except age, goiter, and myxedema were determined by ␹2_{test using 2⫻ 2 contingency tables.}

‡ Smoking category includes former, current, and ever smoker categories.

§ Goiter was determined by chi-square test using 2⫻ 5 contingency tables. P ⬍ 0.05 was statistically significant.

2 Liu et al. IOVS,Month 2010, Vol. 51, No. 0

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: CLEP

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Insert dagger TO READ: 0.784†

Institute, Massachusetts Institute of Technology, Cambridge, MA), and Tag SNPs were selected by using the Tagger function and applying the following additional criteria: (1) a threshold minor allele frequency (MAF) in the HapMap CHB⫹JPT population of 0.10 for tag SNPs and (2) a genotyping score (Illumina, Inc., San Diego, CA) more than or equal to 0.6, as recommended by the manufacturer, to ensure a high genotyping success rate. Eight polymorphisms in the IL1B gene met the criteria and were selected, including the SNPs rs3917368 (A/G at 3⬘ UTR), rs2853550 (C/T at 3⬘ UTR), rs1143643 (A/G at intron 6), rs1143634 (C/T at exon 5, known as ⫹3954A/G and ⫹3962A/G), rs1143630 (A/C at intron 3), rs1143627 (C/T at 5⬘-UTR, known as ⫺31C/T), rs16944 (C/T at 5⬘-UTR, known as ⫺511C/T), and rs12621220 (C/T at 5⬘-UTR).

Genomic DNA Extraction and Genotyping

All blood samples from individuals were collected by venipuncture for genomic DNA isolation. The genomic DNA was extracted from periph-eral blood leukocytes (Genomic DNA kit; Qiagen, Valencia, CA) in accordance with the manufacturer’s instructions. DNA concentration was quantified in all samples before genotyping. Thirteen of the 484 samples were excluded in this study because the amount of DNA was not enough to perform the assay. All eight single-nucleotide polymor-phisms (SNPs) in IL1B were genotyped with an allele-specific exten-sion and ligation assay according to the manufacturer’s instructions (Illumina).

IL1

␤ Quantitative Measurement

The plasma IL1␤ level was measured by using a quantitative enzyme-linked immunosorbent assay according to the manufacturer’s instruc-tions (eBioscience, San Diego, CA).

Statistical Analysis

Associations between each SNP and disease were assessed by␹2_test.

Allele and genotype frequencies in cases and controls were compared

and odds rations (ORs) per SNP were estimated by applying uncondi-tional logistic regression. Alleles and genotype frequencies of alleles, genotypes, haplotypes, and diplotypes were expressed as a percentage of the total number of alleles, genotypes, haplotypes, and diplotypes. Results reaching P⬍ 0.05 were statistically significant. The OR, with the 95% confidence interval (CI), was calculated from the genotype and allelic frequencies. Associations between each SNP and IL1␤ plasma levels were assessed by Student’s t-test (for two-category vari-able) or ANOVA (for a three or more category variable; SPSS for Windows, ver., 14.0; Chicago, IL). Haplotypes were inferred by using Phase 2.1, a computational tool based on Bayesian methods.31_Linkage

disequilibrium (LD) was performed with Haploview 4.1.31_The

multi-factor dimensionality reduction (MDR) 1.1.0 of the open-source MDR software package (Dartmouth Medical School, Hanover, NH) was used to detect the best locus–locus interaction models with an estimated testing accuracy of⬎50% consistency. The interaction dendrogram was established according to a hierarchical clustering algorithm.32–35

R

ESULTS

Basic Characteristics of Patients with GD

and Correlation between the Factors

The demographics and clinical information of the participants are summarized in Table 1. We also examined the association of GO with age, sex, smoking status, recurrence, treatment, and clinical features. The ␹2

test and Mann-Whitney U test revealed that sex, smoking status, and radioiodine therapy were significantly associated with GO among patients with GD.

Allele and Genotype Frequencies

of the IL1B Polymorphisms

To identify the SNPs associated with GO, we genotyped eight SNPs in IL1B. As comparing with healthy individuals, the

TABLE2. Allele Frequencies of IL1B Single-Nucleotide Polymorphism in Healthy Individuals and Patients with or without GO

Alleles Healthy (nⴝ 320) n(%) GD/non-GO (nⴝ 542) n(%) GD/GO (nⴝ 400) n(%) P* (OR, 95% CI)†

GD vs. Healthy GD/GO vs. Healthy

GD/GO vs. GD/non-GO rs3917368 A allele 171 (53.4) 297 (54.8) 234 (58.5) 0.362 0.174 0.257 G allele 149 (46.6) 245 (45.2) 166 (41.5) rs2853550 C allele 300 (93.8) 505 (93.2) 367 (91.8) 0.478 0.307 0.410 T allele 20 (6.3) 37 (6.8) 33 (8.3) rs1143643 A allele 170 (53.1) 297 (54.8) 235 (58.8) 0.297 0.131 0.226 G allele 150 (46.9) 245 (45.2) 165 (41.3) rs1143634 T allele 168 (52.5) 8 (1.5) 5 (1.3) 1.655⫻ 10⫺112 1.414⫻ 10⫺57 0.769 C allele 152 (47.5) 534 (98.5) 395 (98.8) (78.984, 43.795–142.446) (87.316, 35.184–216.689) rs1143630 C allele 266 (83.1) 453 (83.6) 332 (83.0) 0.931 0.965 0.814 A allele 54 (16.9) 89 (16.4) 68 (17.0) rs1143627 T allele 182 (56.9) 309 (57.0) 228 (57.0) 0.967 0.973 0.997 C allele 138 (43.1) 233 (43.0) 172 (43.0) Rs16944 C allele 166 (51.9) 309 (57.0) 229 (57.3) 0.090 0.150 0.968 T allele 154 (48.1) 233 (43.0) 171 (42.8) rs12621220 C allele 201 (62.8) 334 (61.6) 256 (64.0) 0.954 0.742 0.456 T allele 119 (37.2) 208 (38.4) 144 (36.0)

* Allele frequencies were determined by␹2_{test using 2⫻ 2 contingency tables.}

† OR and 95% CI per allele were estimated by applying unconditional logistic regression. The major allele was used as the reference. P⬍ 0.05 is statistically significant.

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T ABLE 3. Genotype Frequencies of IL1B SNPs in Healthy Individuals and Patients with or without GO Genotypes Healthy _(nⴝ 160) n (%) GD/non-GO (n ⴝ 271) n (%) GD/GO (n ⴝ 200) n (%) P * OR (95% CI)† GD vs. Healthy GO vs. Healthy GD/GO vs. GD/non-GO GD vs. Healthy GO vs. Healthy GD/GO vs. GD/non-GO rs3917368 A/A 44 (27.5) 70 (25.8) 71 (35.5) 0.594 0.27 0.030 1 A/G 83 (51.9) 157 (57.9) 92 (46.0) 0.578 (0.380–0.878) G/G 33 (20.6) 44 (16.2) 37 (18.5) 0.829 (0.479–1.434) A/G ⫹ G/G 0.559 0.106 0.024 0.633 (0.425–0.941) rs2853550 C/C 141 (88.1) 234 (86.3) 167 (83.5) 0.123 0.202 0.391 C/T 18 (11.3) 37 (13.7) 33 (16.5) T/T 1 (0.6) 0 (0) 0 (0) C/T ⫹ T/T 0.086 0.263 — rs1143643 A/A 44 (27.5) 70 (25.8) 72 (36.0) 0.495 0.229 0.022 1 A/G 82 (51.3) 157 (57.9) 91 (45.5) 0.564 (0.371–0.856) G/G 34 (21.3) 44 (16.2) 37 (18.5) 0.818 (0.473–1.413) A/G ⫹ G/G 0.526 0.086 0.017 0.619 (0.416–0.921) rs1143634 T/T 53 (33.1) 0 (0) 0 (0) 11 C/T 31 (19.4) 263 (97.0) 195 (97.5) 1.065 ⴛ 10 ⴚ 91 2.993 ⴛ 10 ⴚ 51 0.767 2.387 ⫻ 10 10 (0.0–0.0) 10.162 ⫻ 10 9(0.0–0.0) C/C 76 (47.5) 8 (3.0) 5 (2.5) 2.763 ⫻ 10 8(0.0–0.0) 1.063 ⫻ 10 8(0.0–0.0) C/T ⫹ C/C 6.283 ⴛ 10 ⴚ 39 1.207 ⴛ 10 ⴚ 18 0.767 7.111 ⫻ 10 9(0.0–0.0) 3.020 ⫻ 10 9(0.0–0.0) rs1143630 C/C 112 (70.0) 186 (68.6) 138 (69.0) 0.447 0.877 0.496 A/C 42 (26.3) 81 (29.9) 56 (28.0) A/A 6 (3.8) 4 (1.5) 6 (3.0) A/C ⫹ A/A 0.775 0.838 0.933 rs1143627 T/T 46 (28.8) 83 (30.6) 65 (32.5) 0.528 0.379 0.710 C/T 90 (56.3) 143 (52.8) 98 (49.0) C/C 24 (15.0) 45 (16.6) 37 (18.5) C/T ⫹ C/C 0.481 0.379 0.592 rs16944 C/C 48 (29.9) 84 (31.0) 66 (33.0) 0.041 0.21 0.638 1 C/T 70 (43.8) 143 (52.8) 97 (48.5) 0.911 (0.599–1.387) T/T 42 (26.3) 44 (16.2) 37 (18.5) 0.563 (0.356–0.889) C/T ⫹ T/T 0.664 0.543 0.645 rs12621220 C/C 58 (36.3) 101 (37.3) 83 (41.5) 0.358 0.296 0.642 C/T 85 (53.1) 132 (48.7) 90 (45.0) T/T 17 (10.6) 38 (14.0) 27 (13.5) C/T ⫹ T/T 0.527 0.311 0.352 Bold data indicate statistical significance. * Genotype frequencies were determined by ␹ 2 test using 2 ⫻ 3o r2 ⫻ 2 contingency tables. † O R and 95% CI per genotype were estimated by applying unconditional logistic regression. P ⬍ 0.05 was statistically significant.

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presence of the C allele of SNP rs1143643 may increase the risk of GD (P⫽ 1.655 ⫻ 10⫺112) and GO (P⫽ 1.414 ⫻ 10⫺57), although all eight allele distributions of the IL1B polymor-phisms did not differ significantly between GD patients with or without GO (Table 2). Table 3 summarizes the genotype dis-tributions of the IL1B polymorphisms in all individuals. The T/T genotype of SNP rs1143634 was present only in healthy individuals, and the T/T genotype of SNP rs16944 was less frequent in patients with GD (P⫽ 0.041). In addition, the A/A genotype of SNPs rs3917368 and rs1143643 may increase the risk of GO among patients with GD (P⫽ 0.024 and P ⫽ 0.017, respectively). The interaction dendrogram of the eight SNPs in the IL1B gene were constructed with the MDR software, and the results revealed a strong interaction of the rs3917368 and rs1143643 loci in the IL1B gene in modulating the risk of GO (Fig. 1). These results suggest that patients with the T/T geno-type at SNPs rs1143634 and rs16944 have a lower risk of developing GD. In addition, patients with the A/A genotype of SNPs rs3917368 and rs1143643 may have a higher risk of developing GO.

Haplotype Frequencies

of the IL1B Polymorphisms

Combination of the eight selected SNPs in IL1B by tagging SNPs in the HapMap CHB⫹JPT population may represent different IL1B haplotypes. In addition, MDR analysis indi-cated that the best interaction model for predicting the development of GO is the eight-locus model composed by all eight SNPs analyzed in the present study (testing accu-racy, 63.9%; cross-validation consistency, 100/100; P ⬍ 0.0001). Therefore, we determined the haplotypes in the eight SNPs that had frequencies of⬎5% and identified the 13 haplotypes shown in Table 4). Ht3-GCGCACTT, Ht5-ACACACTT, and Ht6-GTGCCCTC were found only in pa-tients with GD, whereas Ht9-ACATCTTC, Ht10-ACATCTCC, Ht11-GCGCCCCT, Ht12-ACACCTTC, and HT13-GCGTCCCT were found only in the healthy individuals. Haplotype-specific analysis showed that the Ht1-ACACCTCC and Ht2-GCGCCCTT haplotypes may increase the risk of GD (P ⫽ 1.241 ⫻ 10⫺44 and 7.388⫻ 10⫺8; OR, 21.599 and 3.917; 95% CI, 12.221–38.175 and 2.304 – 6.658, respectively) com-pared with the risk in healthy individuals. Ht4-GCGCCTCC may reduce the risk of GO among the patients with GD (P⫽ 0.025; OR, 0.502; 95% CI, 0.273– 0.925). Table 5 showed that 141 patients with GD bore the diplotype A-A/A-A, and it appeared more frequently in patients with GO than did A-A/G-G or G-G/G-G (P⫽ 0.008; OR, 1.650; 95% CI, 1.141–

2.384). In 419 patients with GD, the non-Ht4/non-Ht4 dip-lotype was less frequently found in the patients who also had GO compared with at least one Ht4 haplotype (diplo-types Ht4/Ht4 and Ht4/non-Ht4, P⫽ 0.007; OR, 0.414; 95% CI, 0.214 – 0.797). These findings confirm that the results from genotype and haplotype analysis. In addition, the LD plots of IL1B in the healthy individuals and GD patients with or without GO showed an apparent variation in these poly-morphisms (Fig. 2). These observations suggest that Ht1-ACACCTCC and Ht2-GCGCCCTT haplotypes put the in-dividual at risk for the development of GD, whereas Ht4-GCGCCTCC may play a protective role against the develop-ment of GO.

GO-Associated IL1B Polymorphisms Related to

Circulating IL1

␤ Levels among Patients with GD

We performed enzyme-linked immunosorbent assays on plasma proteins from 432 of 471 GD patients and examined their association with IL1B genotypes and diplotypes (Table 6). The mean plasma IL1␤ concentration in patients with GO was 181.5⫾ 580.1 pg/mL, which was significantly higher than in those without GO (88.8⫾ 190.9 pg/mL, P ⫽ 0.038). Stratified analysis showed that patients with Ht4-GCGCCTCC had much lower concentrations of IL1␤ (P ⫽ 0.042). Although patients with the A-A/G-G and G-G/G-G diplotypes of the SNPs rs3917368 and rs1143643 are less susceptible to GO (GO versus nonGO, 45.9% vs. 57.8%, respectively), they had unex-pectedly higher IL1␤ concentrations than did those with the A-A/A-A diplotype (P⫽ 0.029). Genotype analysis is consistent with the observation. Our results demonstrated that higher circulating IL1␤ concentrations may correlate with GO devel-opment. The GO-protective haplotype Ht4-GCGCCTCC may be used to predict IL1␤-induced GO in patients with GD. How-ever, patients with A-A/G-G and G-G/G-G, although less suscep-tible to GO, may have higher plasma IL1␤ concentrations than do those with A-A/A-A. These findings imply that the involve-ment of IL1␤ in GO development may be more than the IL1B polymorphism-associated circulating IL1␤ elevation in patients with GD.

D

ISCUSSION

We found that the SNPs rs3917368 and rs1143643 in the 3⬘ UTR and intron regions of IL1B, respectively, may be risk genotypes for development of GO. Persons with the genotypes containing both rs3917368 A/A and rs1143643 A/A may bear a

FIGURE 1. Interaction dendrogram. The location of the longitudinal con-necting bars indicates the strength of the dependence: left is weaker and

rightis stronger. The hierarchical

clus-ter analysis placed IL1B rs3917368 and rs1143643 on the same branch, demonstrating the strong interaction between these two SNPs. There were interactions between IL1B rs3917368-rs1143643 and other SNPs as shown in the dendrogram. IL1B, interleukin-1␤ gene; SNP, single nucleotide polymor-phism.

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T ABLE 4. Haplotypes from SNPs of IL1B in Healthy Individuals and Patients with or without GO GD P, Global† P, Individual‡ OR (95% CI)§ Haplotypes* Healthy n (%) Non-GO n (%) GO n (%) GD vs. Healthy GD/GO vs. Healthy GD/GO vs. GD/nonGO GD vs. Healthy GD/GO vs. Healthy GD/GO vs. GD/nonGO Ht1 ACACCTCC 13 (4.1) 251 (46.3) 199 (49.8) 1.241 ⴛ 10 ⫺ 44 9.684 ⴛ 10 ⫺ 41 0.296 21.599 (12.221–38.175) 23.380 (12.979–42.118) Ht2 GCGCCCTT 16 (5.0) 97 (17.9) 64 (16.0) 7.388 ⴛ 10 ⫺ 8 3.058 ⴛ 10 ⫺ 6 0.445 3.917 (2.304–6.658) 3.619 (2.048–6.395) Ht3 GCGCACTT 0 (0.0) 53 (9.8) 37 (9.3) 9.598 ⫻ 10 ⫺ 9 2.232 ⫻ 10 ⫺ 8 0.785 6.068 ⫻ 10 8(0.0–0.0) 1.424 ⫻ 10 9(0.0–0.0) Ht4 GCGCCTCC 13 (4.1) 39 (7.2) 15 (3.8) 0.250 0.829 0.025 0.502 (0.273–0.925) Ht5 ACACACTT 0 (0.0) 36 (6.6) 31 (7.8) 9.454 ⫻ 10 ⫺ 7 3.566 ⫻ 10 ⫺ 7 0.513 5.908 ⫻ 10 8(0.0–0.0) 1.401 ⫻ 10 9(0.0–0.0) Ht6 GTGCCCTC 0 (0.0) 14 (2.6) 15 (3.8) 1.496 ⫻ 10 ⫺ 3 4.639 ⫻ 10 ⫺ 4 0.305 5.662 ⫻ 10 8(0.0–0.0) 1.343 ⫻ 10 9(0.0–0.0) Ht7 GTGCCCTT 1 (0.3) 9 (1.7) 9 (2.3) 1.643 ⫻ 10 ⫺ 173 1.793 ⫻ 10 ⫺ 100 0.348 0.043 0.273 0.514 6.214 (0.826–46.737) Ht8 GCGCCCTC 5 (1.6) 12 (2.2) 12 (3.0) 0.310 0.207 0.449 Ht9 ACATCTTC 39 (12.2) 0 (0.0) 0 (0.0) 1.370 ⫻ 10 ⫺ 27 7.009 ⫻ 10 ⫺ 13 — 0.000 (0.0–0.0) 0.000 (0.0–0.0) Ht10 ACATCTCC 35 (10.9) 0 (0.0) 0 (0.0) 7.485 ⫻ 10 ⫺ 25 1.191 ⫻ 10 ⫺ 11 — 0.000 (0.0–0.0) 0.000 (0.0–0.0) Ht11 GCGCCCCT 29 (9.1) 0 (0.0) 0 (0.0) 8.971 ⫻ 10 ⫺ 21 7.954 ⫻ 10 ⫺ 10 — 0.000 (0.0–0.0) 0.000 (0.0–0.0) Ht12 ACACCTTC 23 (7.2) 0 (0.0) 0 (0.0) 1.003 ⫻ 10 ⫺ 16 5.047 ⫻ 10 ⫺ 8 — 0.000 (0.0–0.0) 0.000 (0.0–0.0) Ht13 GCGTCCCT 23 (7.2) 0 (0.0) 0 (0.0) 1.003 ⫻ 10 ⫺ 16 5.047 ⫻ 10 ⫺ 8 — 0.000 (0.0–0.0) 0.000 (0.0–0.0) Remaining 123 (38.4) 31 (5.7) 18 (4.5) —— — Total 320 542 400 * Order of SNPs comprising the IL1B haplotypes: rs3917368, rs2853550, rs1143643, rs1143634, rs1143630, rs1143627, rs16944, and rs12621220. The haplotypes were identified by the Bay esian statistical method available in the program Phase 2.1. † Global test for haplotype frequency in relation to GD or GO development was determined by ␹ 2 test using 2 ⫻ n contingency tables (where n is 13 ⫹ 1 (haplotype ⬎ 5% plus remaining haplotypes). P ⬍ 0.05 is statistically significant. ‡ Individual haplotype frequency in relation to GD or GO development was determined by ␹ 2test using 2 ⫻ 2 contingency tables. P ⬍ 0.05 is statistically significant. § O R and 95% CI for genotypes were estimated by applying unconditional logistic regression.

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higher risk of developing GO. Clinical association studies showed that the presence of the A-A/A-A diplotype was signif-icantly associated with a higher risk of GO, but the presence of the diplotype along with the Ht4-GCGCCTCC haplotype may be protective against GO. In addition, the circulating IL1␤ concentrations have been analyzed in GD patients and the association with GO as well as genotypes have been made. Our results showed that IL1B polymorphisms may be associated with the level of circulating IL1␤ as well as GO development in patients with GD.

Several reports have predicted the genetic association of

IL1B with the development of GD and GO. Hayashi et al.36

found a significant association between GD and the ⫺31T (rs1143627) allele in the promoter region in their study of the Japanese population. Liu et al.13

found an association between ⫺511C/T (rs16944) and GD with GO in China, whereas Lacka et al.14

and Khalilzadeh et al.15

did not find an association

between the IL1B polymorphisms –511C/T (rs16944) and ⫹3954 C/T (rs1143634) and GO in their studies in Iran and Poland, respectively. We found that the C allele of ⫹3954 (rs1143634) is related to the development of GD and GO. In addition, the T/T genotype of⫺511 (rs16944) is related to less susceptibility to GD. However, in the present study, the geno-types⫺31C/T (rs1143627) and –511C/T (rs16944) were not associated with the development of GO. The effect of popula-tion differences in the determinapopula-tion of such associapopula-tions should not be underestimated. In the present study, for the first time, we found the association between A/A genotypes of rs3917368 and rs1143643 and the risk for GO, although the significance was weak. Although we identified haplotypes with the Phase program, we found much more diverse haplotype distribution in healthy individuals than in the patients with GD. In addition, patients with GO had more similar haplotype distribution than did those without GO. This observation is TABLE5. Distribution of IL1B Diplotypes and Their Associations with GO

Diplotypes Without GO (nⴝ 271) n(%) With GO (nⴝ 200) n(%) Cross Validation Consistency P OR (95% CI) A™A/A™A 70 (25.8) 71 (35.5) 1 A™A/G™G 157 (57.9) 92 (44.0) 0.030§ 0.578 (0.380–0.878)㛳 G™G/G™G 44 (14.2) 37 (18.5) 0.830 (0.480–1.434)㛳 A™A/G™G⫹ G™G/G™G 201 (74.2) 129 (64.5) 100/100 0.024§ 0.633 (0.425–0.941)㛳 0.008† 1.650 (1.141–2.384)‡ Non-Ht4/non-Ht4 232 (85.6) 187 (93.5) 1 Ht4/non-Ht4 39 (14.4) 11 (5.5) 0.002§ 0.350 (0.174–0.702)㛳 Ht4/Ht4 0 (0.0) 2 (1.0) 2.004⫻ 109_{(0.0–0.0)㛳} Ht4/Ht4⫹ Ht4/non-Ht4 39 (75.0) 13 (25.0) 0.007§ 0.414 (0.214–0.797)㛳

* The order of the SNPs comprising the IL1B haplotype was rs3917368 and rs2853550. The haplotypes were identified by the Bayesian statistical method available in the program Phase 2.1.

† Significance of diplotype in relation to GO development was determined by applying multifactor dimensionality reduction (MDR) models.

P⬍ 0.05 was statistically significant.

‡ ORs and 95% CIs for A™A/A™A and non A™A/A™A was estimated by applying multifactor dimensionality reduction (MDR) models. § Genotype frequencies were determined by␹2_{test using 2⫻ 2 or 2 ⫻ 3 contingency tables. P ⬍ 0.05 was statistically significant.} 㛳_{ORs and 95% CIs for non-Ht4/non-Ht4 (reference) and Ht4/Ht4 and Ht4/non-Ht4 was estimated by applying unconditional logistic regression.}

FIGURE2. Pairwise LD measures of D⬘ (top) and r2_{(bottom) for the SNPs of the IL1B locus. The scale above the figures indicates the site of each}

SNP around the IL1B gene region in healthy individuals (A), patients with Graves’ disease without ophthalmopathy (B), and patients with Graves’

disease with ophthalmopathy (C).

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*

consistent with our results from LD analysis. These results may provide new information for prediction of development of GD and GO by different IL1B haplotypes.

Recent studies have shown that a positive feedback cycle composed of mechanical, immunologic, and cellular processes is involved in the development of GO.8,21,22Interstitial accu-mulation of GAGs, PGE2, and adipogenesis in the orbital tissue contributes to GO in GD patients and these observations and a series of thyroid-related factors, such as the thyrotropin recep-tor antigen, are thought to be a consequence of the release of certain cytokines, including IL1␤, from T cells, monocytes, and activated fibroblasts.8,21–23,37Polymorphisms may cause alter-nations in the expression and function of IL1B,16 –18 which may affect the downregulation of T-cell activation and the subsequent inflammatory diseases, autoimmune diseases, can-cer, and GAG accumulation. Given the important role of T cells in the pathogenesis of GO, IL1B may be a candidate gene for the induction of these autoimmune reactions. Our results dem-onstrated that GO patients have higher plasma IL1␤ levels than those without GO, which supports this hypothesis.

Although our hypothesis was that polymorphisms of IL1B relate to the development of GO in patients with GD, the ⫺31C and ⫺511T of IL1B, the polymorphism associated with decreased and increased transcriptional activity,38,39were as-sociated with neither IL1␤ levels nor GO development in our study. We found that the Ht4-GCGCCTCC is related to lower circulating IL1␤ concentration and less susceptibility toward GO, indicating that Ht4-GCGCCTCC may be used to predict the IL1␤-induced GO in patients with GD. However, the A-A/G-G and G-G/G-G diplotypes (less susceptible for GO were related to high circulating levels of IL1␤. It is notable that the IL1␤ concentration examined in our patients (0 –3.843 ng/mL) was lower than the IL1␤ dosage most used in the in vitro experi-ments (10 ng/mL),19,20,40,41and the outliers (IL1␤ ⬎1 ng/mL)

were only the patients with the A-A/G-G genotype. Although plasma IL1␤ in healthy individuals is yet to be determined, it would be interesting to know whether there are critical dose-dependent levels of IL1␤ in protection against or promotion of the development of GO. The linkage among the IL1B polymor-phisms, IL1␤ level, and GO development should be further confirmed in studies with larger samples.

In the present study, our results suggest that IL1B geno-types are associated with the development of GO, the most common orbital disease in GD. This report provides evidence from examination of patient outcomes that polymorphisms of the IL1B gene may predict the development of GO.

Acknowledgments

The authors thank Yu-Huei Liang and Su-Ching Liu for technical assis-tance in analyzing the polymorphisms.

References

1. Mishra A, Mishra SK. Multicentre study of thyroid nodules in patients with Graves’ disease (comment on Br J Surg 2000;87: 1111–1113). Br J Surg. 2001;88:313.

2. Gianoukakis AG, Khadavi N, Smith TJ. Cytokines, Graves’ disease, and thyroid-associated ophthalmopathy. Thyroid. 2008;18:953– 958.

3. Perros P, Neoh C, Dickinson J. Thyroid eye disease. BMJ. 2009; 338:560.

4. Kuriyan AE, Phipps RP, Feldon SE. The eye and thyroid disease.

Curr Opin Ophthalmol.2008;19:499 –506.

5. Khoo TK, Bahn RS. Pathogenesis of Graves’ ophthalmopathy: the role of autoantibodies. Thyroid. 2007;17:1013–1018.

6. Kloprogge S, Kowal L, Wall J, Frauman AG. The clinicopathologic basis of Graves’ ophthalmopathy: a review. Eur J Ophthalmol. 2005;15:315–323.

TABLE6. Stratified Analysis of Plasma IL1␤ Levels on GO Risk by IL1B Genotypes

Variables GD/non-GO GD/GO P* P†

— 88.8⫾ 190.9 (249) 181.5⫾ 580.1 (183) 0.038 rs3917368 A/A 62.5⫾ 80.9 (63) 65.7⫾ 124.8 (65) 0.868 A/G 102.1⫾ 224.8 (144) 302.8⫾ 825.9 (84) 0.030 0.032 G/G 81.0⫾ 180.1 (42) 103.7⫾ 200.3 (34) 0.606 P⫽ 0.377†‡ Pⴝ 0.031‡ A/G⫹G/G 97.3⫾ 215.2 (186) 245.4⫾ 709.5 (118) 0.029 rs1143643 A/A 62.5⫾ 80.9 (63) 65.8⫾ 123.9 (66) 0.859 A/G 102.1⫾ 224.8 (144) 305.5⫾ 830.6 (83) 0.029 0.031 G/G 81.0⫾ 180.1 (42) 103.7⫾ 200.3 (34) 0.606 P⫽ 0.377‡ Pⴝ 0.029‡ A/G⫹G/G 97.3⫾ 215.2 (186) 246.8⫾ 712.4 (117) 0.029 A™A/A™A 62.5⫾ 80.9 (63) 65.6⫾ 124.8 (65) 0.868 A™A/G™G 102.1⫾ 224.8 (144) 302.8⫾ 825.9 (84) 0.030 0.032 G™G/G™G 81.0⫾ 180.1 (42) 103.7⫾ 200.3 (34) 0.606 P⫽ 0.377‡ Pⴝ 0.031‡ A™A/G™G⫹ G™G/G™G 97.3⫾ 215.2 (186) 245.4⫾ 709.5 (118) 0.029 Non-Ht4/non-Ht4 91.1⫾ 202.0 (213) 189.0⫾ 598.1 (171) 0.042 Ht4/non-Ht4 73.1⫾ 103.7 (36) 86.6⫾ 178.5 (10) 0.604 0.042 Ht4/Ht4 — 14.2⫾ 10.9 (2) 0.758 P⫽ 0.402‡ P⫽ 0.547‡ Ht4/non-Ht4⫹ Ht4/Ht4 73.1⫾ 103.7 (36) 74.6⫾ 163.9 (12) 0.042

Data are expressed as mean⫾ SD plasma IL1␤ levels (in picograms/milliliter), with the number of subjects in parentheses. P⬍ 0.05 is statistically significant (data in bold).

* Significance of genotypes and haplotypes toward plasma IL1␤ levels or plasma IL1␤ levels in relation to GO development in all patients with GD were determined by Student’s t-test or ANOVA, as suitable.

† Significance of each genotype or haplotype in relation to plasma IL1␤ levels in all patients with GD were determined by Student’s t-test.

‡ Significance of genotypes and haplotypes in relation to plasma IL1␤ levels in patients with or without GO were determined by ANOVA.

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