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G single nucleotide polymorphism in Chinese Han patients with ocular Behçet's disease
Human Immunology 71 (2010) 79 – 82
Contents lists available at ScienceDirect
Research article
Monocyte chemoattractant protein–1 ⫺2518 A/G single nucleotide polymorphism in Chinese Han patients with ocular BehÈet’s disease Shengping Hou a, Peizeng Yang a,*, Liping Du a, Zhengxuan Jiang a, Liming Mao a, Qinmeng Shu a, Hongyan Zhou b, Aize Kijlstra c a
First Affiliated Hospital, Chongqing Medical University, Chongqing Key Laboratory of Ophthalmology, Chongqing Eye Institute, Chongqing, China Uveitis Study Center, Sun Yat-sen University, Guangzhou, China c Eye Research Institute Maastricht, Department of Ophthalmology, University Hospital, Maastricht, Maastricht, The Netherlands b
A R T I C L E
I N F O
Article history: Received 25 June 2009 Received in revised form 30 August 2009 Accepted 17 September 2009 Available online 25 September 2009
Keywords: BehÈet’s disease MCP-1 Gene polymorphism Association
A B S T R A C T
Recent studies in Caucasian uveitis patients have shown an association with the ⫺2518 A/G polymorphism of the monocyte chemoattractant protein (MCP)–1 gene. It is unknown whether this polymorphism is also associated with ocular BehÈet’s disease (BD) in Chinese populations. The aim of the current study was therefore to investigate the possible involvement of MCP-1 in the susceptibility to ocular BD in Chinese Han individuals. A case control association study was performed in 296 ocular BD patients and 319 geographically and age-matched healthy controls using polymerase chain reaction–restriction fragment length polymorphism (PCR-RFLP). Binary logistic regression analysis revealed a decreased frequency of the homozygous AA genotype and an increased frequency of AG genotype of the MCP-1 ⫺2518 polymorphism in ocular BD patients compared with healthy controls, when adjusted for gender (p ⫽ 0.048, p ⫽ 0.028, respectively). However, when segregated on the basis of several clinical findings, no any association was found between this polymorphism and ocular BD. In conclusion, the present study suggests that the MCP-1 ⫺2518 AA genotype seems to display a protective association with ocular BD, whereas the ⫺2518 AG genotype might be a susceptible factor for ocular BD in the Chinese Han population. Crown copyright 䉷 2010 Published by Elsevier Inc. on behalf of American Society for Histocompatibility and Immunogenetics. All rights reserved.
1. Introduction BehÈet’s disease (BD) is well known as a refractory multisystem disorder characterized by recurrent oral ulceration, genital ulceration, recurrent uveitis, and skin lesions [1–3]. It is also now recognized as a systemic vasculitis that can affect joints, blood vessels, the central nervous system, and the gastrointestinal tract [4 –7]. Although BD is thought to be mediated by complex mechanisms, including genetic, environmental and immunologic factors, its precise pathogenesis is not yet clear. An imbalance of immune responses, including the recruitment of leukocytes to the site of inflammation [8,9], increased interleukin (IL)– 8 level [10], and increased proinflammatory cytokines, such as IL-1, IL-6, tumor necrosis factor (TNF)–␣, interferon (IFN)–␥ and IL-17 in BD patients [11,12], all might play critical roles in the development of BD. The recruitment of macrophages and monocytes to the sites of inflammation suggests that chemokines might be involved in the inflammatory process of BD. Chemokines are a group of secreted proteins that contain 70 –90 amino acids and have a molecular weight of approximately 8 –10
* Corresponding author. E-mail address: [email protected] (P. Yang).
kDa. They are classically divided into four subfamilies according to the presence of four cysteine residues in conserved locations of the primary structure and whereby two amino terminal cysteine residues are immediately adjacent or separated by one amino acid. A typical example of a so-called CC type chemokine is monocyte chemotactic protein (MCP)–1 [13]. MCP-1 is a potent chemokine released by lymphocyte, monocytes, mast cells, and eosinophils during inflammation [14,15]. Ocular cells, such as the retinal pigment epithelial cell cultured in vitro, have also been shown to produce MCP-1 [16]. The elevated MCP-1 concentration in aqueous humor of active anterior uveitis patients [17] and ocular fluids and tissues during ocular inflammation [17–21], combined with recent reports showing an association of MCP-1 polymorphism with uveitis [22–24] and other autoimmune diseases [25–27], suggest that MCP-1 may be involved in the development of BD, a common seen uveitis in Chinese population [5]. In view of the crucial role of MCP-1 in autoimmune disease, the present study was therefore designed to examine the association of MCP-1 polymorphism with ocular BD using a case-control association study. Our results showed that a genetic susceptibility to ocular BD was conferred by the AG genotype of MCP-1 ⫺2518 A/G polymorphism, whereby the AA genotype shows a protective effect with regard to ocular BD development.
0198-8859/10/$32.00 - see front matter Crown copyright 䉷 2010 Published by Elsevier Inc. on behalf of American Society for Histocompatibility and Immunogenetics. All rights reserved. doi:10.1016/j.humimm.2009.09.354
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S. Hou et al. / Human Immunology 71 (2010) 79 – 82
2. Subjects and methods 2.1. Subjects The present study was approved by the Ethics Committee of our hospitals and adhered to the tenets of the Declaration of Helsinki. In addition, informed consent was obtained from each participant or each participant’s guardian. The diagnosis of ocular BD was based on the criteria of the International Study Group [28]. After giving informed consent, blood samples were obtained from 296 ocular BD patients and 319 healthy controls, who were recruited from Zhongshan Ophthalmic Center, Sun Yat-sen University, Guangzhou, China, and the First Affiliated Hospital, Chongqing Medical University, Chongqing, People’s Republic of China. 2.2. DNA extraction Genomic DNA was extracted from peripheral blood mononuclear cells (PBMCs) using the QIAamp DNA Mini Blood Kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. DNA samples were collected in 1.5-ml Eppendorf tubes and stored at ⫺20⬚C until use. 2.3. Genotyping Genotyping for the MCP-1 polymorphism (⫺2518 A/G, rs1024611 in dbSNP database) was performed using polymerase chain reaction–restriction fragment length polymorphism (PCRRFLP). The forward primer 5=-CCGCATTCAATTTCCCTTTAT-3= and reverse primer 5=-TTCCAAAGCTGCCTCCTCA-3= were designed using primer Premier 5.0 software (Premier Biosoft International, Palo Alto, CA). The amplification was performed using initial denaturation at 94⬚C for 10 minute followed by 35 cycles at 94⬚C for 45 seconds, 55⬚C for 45 seconds, 72⬚C for 45 seconds, and 72⬚C for 5 minutes on a GeneAmp 9700 thermal cycler (PerkinElmer Applied Biosystems, Foster City, CA). The PCR products were incubated with PvuII at 37⬚C (New England Biolabs, Burlington, Ontario, Canada) for at least 3 hours and separated on 2% agarose gels (Fig. 1). A 10% quantity of the PCR samples was directly sequenced to confirm the PCR-RFLP results (Invitrogen Biotechnology Co., Shanghai, China) (Fig. 1). 2.4. Statistical analysis Hardy-Weinberg equilibrium (HWE) was tested using the test. A binary logistic regression analysis was performed to assess the influence of the MCP-1 polymorphism and patient gender on the susceptibility to ocular BD, using SPSS version 17.0 for Windows (SPSS Inc., Chicago, IL). All statistical tests were two-sided, and statistical significance was set at p ⬍ 0.05. 2
3. Results Clinical findings of ocular BD patients and baseline characteristics of controls are presented in Table 1. The average age of the ocular BD patients was 31.3 ⫾ 7.2 and that of healthy controls was 34.7 ⫾ 11.6 (Table 1). No statistically significant difference in the distribution of age was observed between ocular BD patients and controls (p ⬎ 0.05). A total of 296 ocular BD patients and 319 healthy controls were genotyped for the MCP-1 ⫺2518 A/G polymorphism, and the result of genotyping was matched with that of direct sequencing for 10% samples. The distribution of the genotypic frequency of this SNP in all subjects did not show a significant deviation from the HWE (p ⬎ 0.05). The distribution of the genotypic and allelic frequency of the MCP-1 ⫺2518 A/G polymorphism is shown in Table 2. A decreased frequency of the homozygous AA genotype of the MCP-1 ⫺2518 A/G polymorphism was observed in ocular BD patients compared with healthy controls (18.9% vs. 27.9%, p ⫽ 0.048, odds ratio 0.64,
Fig. 1. Identification of the MCP-1 ⫺2518 A/G polymorphism in the Chinese Han population. (A) Sequencing results of the AA genotype. (B) Sequencing result of AG genotype. (C) Sequencing result of GG genotype. (D) Results of PCR-RFLP of the MCP-1 ⫺2518 A/G polymorphism. PvuII digestion of PCR products of the AA genotype gives one fragment, the AG genotype gives three fragments, and the AG genotype gives two fragments.
95% confidence interval 0.42– 0.99) (Table 2). The frequency of the AG genotype of the MCP-1 ⫺2518 A/G polymorphism was significantly increased in ocular BD patients compared with healthy controls (48.7% vs. 42.3%, p ⫽ 0.028, odds ratio 1.51, 95% confidence interval 1.05–2.17) (Table 2). In addition, our results did not indicate a significant difference when allelic and genotypic frequencies were analyzed according to clinical findings, including oral ulcer, genital ulcer, hypopyon, skin lesions, positive pathergy test results, and arthritis (p ⬎ 0.05). 4. Discussion In this study we focused on the ⫺2518 A/G polymorphism of MCP-1, the polymorphism of which has been shown to affect its expression level and the clinical presentation of various immunemediated diseases [27], and we performed a case-control association study for this polymorphism with ocular BD. A decreased MCP-1 ⫺2518 AA genotypic frequency and an increased AG genotypic frequency was found in ocular BD patients compared with healthy controls, suggesting that this gene may be involved in the pathogenesis of ocular BD. BehÈet’s disease is an endemic multifactorial genetic disease [1]. Whereas the disease is rare in the Western countries [3,29], it occurs with high prevalence in the countries of the ancient Silk Road [30,31], which extends from eastern Asia to the Mediterranean basin [32–34]. Epidemiologic investigations have shown that BD has a striking geographic and ethnic distribution, with a particularly high prevalence observed in individuals from Turkish, Japa-
S. Hou et al. / Human Immunology 71 (2010) 79 – 82
Table 1 Clinical characteristic of patients with ocular BehÈet’s disease (BD) Phenotype
Ocular BD patients
Age (y) at onset (mean ⫾ SD) Male gender Female gender Uveitis Oral ulcer Genital ulcer Hypopyon Skin lesions Positive pathergy test results Arthritis
Total (n ⫽ 296)
%
31.3 ⫾ 7.2 247 49 296 286 124 68 148 102 78
83.4 16.6 100 96.6 41.9 23.0 50.0 34.5 26.4
nese, or Chinese descent [3,4] and mounting evidence suggests that genetic predisposition might play a crucial role in the development of BD. At present, the contribution of genes encoding for human leucocyte antigens (HLA) to the genetic risk of BD is best characterized [35–39]. The association with HLA-B51 [39,40] is also well known. However, these identified genes only account for a small portion of the overall estimated genetic risk for BD, and further studies on other susceptible genes have therefore been initiated by various groups. Karasneh et al. identified 16 non-HLA susceptible loci in Turkish patients using a whole-genome screening approach, and our recent studies revealed several susceptible genes for BD in the Chinese Han population, including SUMO4 andFCRL3 [41,42]. These studies suggest that multiple immune-associated factors might be involved in the pathogenesis of this disease, and that the currently observed genetic risk factors are only a small part of the possible susceptibility genes associated with this disease. Largescale association studies are needed to address the full range of susceptible genes for BD. MCP-1 is a potent chemokine that plays an important role in the recruitment of leukocytes to the site of inflammation. Recently a ⫺2518 A/G polymorphism in the promoter region of MCP-1 has been described that was shown to affect the transcriptional activation of this gene [27] and that has therefore been considered as a good candidate in the genetic predisposition to autoimmune disease [23,43,44]. Furthermore, MCP-1 concentrations have been found to be increased in serum of BD patients [45]. Based on these earlier results, we specifically tested whether the MCP-1 ⫺2518 A/G polymorphism was associated with ocular BD in Chinese Han population. Our results showed that the AG genotype of MCP-1 ⫺2518 A/G polymorphism was positively associated with the susceptibility to ocular BD. In addition, the AA genotype of MCP-1 ⫺2518 A/G polymorphism contributes to a protective effect against ocular BD development. In accordance with our results, the MCP-1 ⫺2518 A/G polymorphism has been shown to have a significant association with uveitis and various autoimmune diseases [22–24]. As BD is a general autoimmune disorder characterized by ocular and multiple extraocular findings, we therefore performed an association study of MCP-1 polymorphism with these extraocular disease findings. However, we were not able to show an association
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of the tested polymorphism with the clinical presentation of BD. A recent study has indicated that monocytes from AA homozygotic individuals released lesser MCP-1 protein compared with individuals carrying the G allele [27]. Consequently, it would be reasonable to speculate that, under the same stimulatory conditions, AA homozygotic individuals, compared with individuals carrying the G allele, may display a lesser inflammatory response by recruitment of few monocytes, T lymphocytes, and eosinophils to the inflammation sites [46,47]. Taken together, these data suggest a possible implication of MCP-1 polymorphism in the pathogenesis of various autoimmune diseases, including BD. It is worth pointing out that our results are discordant with those reported by Korean investigators. Cho et al. [45] were not able to find an association between the MCP-1 polymorphism and the occurrence of BD in Korean patients. The high dominance of the G allele in Korea may explain the observed difference with our study. The frequency of the G allele in our study was 50.9%, whereas the G allelic frequency reported by Cho et al. was 65% [45]. Genetic heterogeneity between two different ethnic populations may partially explain the discrepant findings. The sample size used in studies may also have influenced the results. In the Cho et al. study, only 104 patients were tested, whereas in our study 296 patients were enrolled. The third possibility could be the origin of the patients. In the Cho et al. study, BD patients were recruited from a rheumatology research center, whereas in our study the tested patients came from an ophthalmology department. It is noteworthy that a few factors, such as population composition and stratification, might influence the result of an association study. In the present study, the following attempts were made to ensure that the analysis results were valid. First, genotype distribution of MCP-1 polymorphism in healthy controls matches the Hardy-Weinberg equilibrium. Second, the controls and patients were strictly matched according to the places where these individuals were born to exclude the possible influence of stratification of the population. Third, the number of donors enrolled in this study (296 ocular BD patients and 319 age-, gender-, and ethnicity-matched healthy controls) was large enough to avoid a bias of the results. Finally, direct sequencing for 10% samples was performed to validate the result of genotyping. Several possible limitations of the present study merit particular consideration. The patients enrolled in our study were recruited from an ophthalmic center. Because BD is an autoimmune disease that affects multiple systems, the patients enrolled in this study might therefore represent a separate disease population. Gender might also influence the results in a case-control association study. In our study a much higher ratio of male to female patients might have resulted in bias, although we did a binary logistic regression analysis. A study using a well-matched gender ratio in case and control populations is needed to clarify our results. Furthermore, the patients enrolled in this study were recruited only from among Chinese Han individuals and excluded other Chinese ethnic subpopulations. Therefore, the results presented here need to be confirmed using different ethnic populations. Finally, the functional
Table 2 Allelic and genotypic frequencies of the MCP-1 polymorphism in ocular BehÈet disease (BD) patients and controls SNP
Genotype allele
Ocular BD (n ⫽ 296)
Controls (n ⫽ 319)
p Value
Odds ratio (95% CI)
p Valuea
Odds ratio (95% CI)b
MCP-1 ⫺2518 A/G
AA AG GG A G
56 (18.9%) 144 (48.7%) 96 (32.4%) 256 (43.2%) 336 (56.8%)
89 (27.9%) 135 (42.3%) 95 (29.8%) 313 (49.1%) 325 (50.9%)
0.009 0.115 0.478 0.041 0.041
0.60 (0.41–0.88) 1.29 (0.94–1.78) 1.13 (0.80–1.59) 0.79 (0.63–0.99) 1.26 (1.01–1.58)
0.048 0.028 0.566 0.404 0.404
0.64 (0.42–0.99) 1.51 (1.05–2.17) 0.89 (0.59–1.33) 0.90 (0.69–1.16) 1.26 (0.86–1.45)
CI, confidence interval; SNP, single nucleotide polymorphism. a
Adjusted gender p value. Adjusted gender OR (95% CI).
b
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studies were not performed in this study. Since a functional study might reveal how a genetic variant translates into physiologic processes that affect disease susceptibility, the association results presented should be further investigated using functional experiments to understand the mechanisms by which the susceptible genes interact with certain environmental triggers and initiate the development of BD. In summary, the present study suggests that the MCP-1 gene seems to be one of the genetic markers involved in the genetic susceptibility of ocular BD. A large number of well-matched samples, multiple ethnic groups, and further functional studies will contribute to the understanding of genetic risks predisposing to BD, identification of disease-causing variants, and knowledge of the underlying mechanisms by which disease-causing variants affect disease susceptibility.
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Acknowledgments [27]
This work was supported by the following: Project of International Cooperation in Science and Technology, Guangdong Province (2006A50107001), Key Project of Natural Science Foundation (30630064), National Supporting Project of P. R. China, Key Project of Health Bureau of Chongqing (2008/1/15), Key Project of Natural Science Foundation of Chongqing (CSTC, 2009BA5037) and Project of Chongqing Key Laboratory of Ophthalmology (CSTC, 2008CA5003). The authors express thanks to all individuals enrolled in the present study.
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Contents lists available at ScienceDirect
Research article
Monocyte chemoattractant protein–1 ⫺2518 A/G single nucleotide polymorphism in Chinese Han patients with ocular BehÈet’s disease Shengping Hou a, Peizeng Yang a,*, Liping Du a, Zhengxuan Jiang a, Liming Mao a, Qinmeng Shu a, Hongyan Zhou b, Aize Kijlstra c a
First Affiliated Hospital, Chongqing Medical University, Chongqing Key Laboratory of Ophthalmology, Chongqing Eye Institute, Chongqing, China Uveitis Study Center, Sun Yat-sen University, Guangzhou, China c Eye Research Institute Maastricht, Department of Ophthalmology, University Hospital, Maastricht, Maastricht, The Netherlands b
A R T I C L E
I N F O
Article history: Received 25 June 2009 Received in revised form 30 August 2009 Accepted 17 September 2009 Available online 25 September 2009
Keywords: BehÈet’s disease MCP-1 Gene polymorphism Association
A B S T R A C T
Recent studies in Caucasian uveitis patients have shown an association with the ⫺2518 A/G polymorphism of the monocyte chemoattractant protein (MCP)–1 gene. It is unknown whether this polymorphism is also associated with ocular BehÈet’s disease (BD) in Chinese populations. The aim of the current study was therefore to investigate the possible involvement of MCP-1 in the susceptibility to ocular BD in Chinese Han individuals. A case control association study was performed in 296 ocular BD patients and 319 geographically and age-matched healthy controls using polymerase chain reaction–restriction fragment length polymorphism (PCR-RFLP). Binary logistic regression analysis revealed a decreased frequency of the homozygous AA genotype and an increased frequency of AG genotype of the MCP-1 ⫺2518 polymorphism in ocular BD patients compared with healthy controls, when adjusted for gender (p ⫽ 0.048, p ⫽ 0.028, respectively). However, when segregated on the basis of several clinical findings, no any association was found between this polymorphism and ocular BD. In conclusion, the present study suggests that the MCP-1 ⫺2518 AA genotype seems to display a protective association with ocular BD, whereas the ⫺2518 AG genotype might be a susceptible factor for ocular BD in the Chinese Han population. Crown copyright 䉷 2010 Published by Elsevier Inc. on behalf of American Society for Histocompatibility and Immunogenetics. All rights reserved.
1. Introduction BehÈet’s disease (BD) is well known as a refractory multisystem disorder characterized by recurrent oral ulceration, genital ulceration, recurrent uveitis, and skin lesions [1–3]. It is also now recognized as a systemic vasculitis that can affect joints, blood vessels, the central nervous system, and the gastrointestinal tract [4 –7]. Although BD is thought to be mediated by complex mechanisms, including genetic, environmental and immunologic factors, its precise pathogenesis is not yet clear. An imbalance of immune responses, including the recruitment of leukocytes to the site of inflammation [8,9], increased interleukin (IL)– 8 level [10], and increased proinflammatory cytokines, such as IL-1, IL-6, tumor necrosis factor (TNF)–␣, interferon (IFN)–␥ and IL-17 in BD patients [11,12], all might play critical roles in the development of BD. The recruitment of macrophages and monocytes to the sites of inflammation suggests that chemokines might be involved in the inflammatory process of BD. Chemokines are a group of secreted proteins that contain 70 –90 amino acids and have a molecular weight of approximately 8 –10
* Corresponding author. E-mail address: [email protected] (P. Yang).
kDa. They are classically divided into four subfamilies according to the presence of four cysteine residues in conserved locations of the primary structure and whereby two amino terminal cysteine residues are immediately adjacent or separated by one amino acid. A typical example of a so-called CC type chemokine is monocyte chemotactic protein (MCP)–1 [13]. MCP-1 is a potent chemokine released by lymphocyte, monocytes, mast cells, and eosinophils during inflammation [14,15]. Ocular cells, such as the retinal pigment epithelial cell cultured in vitro, have also been shown to produce MCP-1 [16]. The elevated MCP-1 concentration in aqueous humor of active anterior uveitis patients [17] and ocular fluids and tissues during ocular inflammation [17–21], combined with recent reports showing an association of MCP-1 polymorphism with uveitis [22–24] and other autoimmune diseases [25–27], suggest that MCP-1 may be involved in the development of BD, a common seen uveitis in Chinese population [5]. In view of the crucial role of MCP-1 in autoimmune disease, the present study was therefore designed to examine the association of MCP-1 polymorphism with ocular BD using a case-control association study. Our results showed that a genetic susceptibility to ocular BD was conferred by the AG genotype of MCP-1 ⫺2518 A/G polymorphism, whereby the AA genotype shows a protective effect with regard to ocular BD development.
0198-8859/10/$32.00 - see front matter Crown copyright 䉷 2010 Published by Elsevier Inc. on behalf of American Society for Histocompatibility and Immunogenetics. All rights reserved. doi:10.1016/j.humimm.2009.09.354
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2. Subjects and methods 2.1. Subjects The present study was approved by the Ethics Committee of our hospitals and adhered to the tenets of the Declaration of Helsinki. In addition, informed consent was obtained from each participant or each participant’s guardian. The diagnosis of ocular BD was based on the criteria of the International Study Group [28]. After giving informed consent, blood samples were obtained from 296 ocular BD patients and 319 healthy controls, who were recruited from Zhongshan Ophthalmic Center, Sun Yat-sen University, Guangzhou, China, and the First Affiliated Hospital, Chongqing Medical University, Chongqing, People’s Republic of China. 2.2. DNA extraction Genomic DNA was extracted from peripheral blood mononuclear cells (PBMCs) using the QIAamp DNA Mini Blood Kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. DNA samples were collected in 1.5-ml Eppendorf tubes and stored at ⫺20⬚C until use. 2.3. Genotyping Genotyping for the MCP-1 polymorphism (⫺2518 A/G, rs1024611 in dbSNP database) was performed using polymerase chain reaction–restriction fragment length polymorphism (PCRRFLP). The forward primer 5=-CCGCATTCAATTTCCCTTTAT-3= and reverse primer 5=-TTCCAAAGCTGCCTCCTCA-3= were designed using primer Premier 5.0 software (Premier Biosoft International, Palo Alto, CA). The amplification was performed using initial denaturation at 94⬚C for 10 minute followed by 35 cycles at 94⬚C for 45 seconds, 55⬚C for 45 seconds, 72⬚C for 45 seconds, and 72⬚C for 5 minutes on a GeneAmp 9700 thermal cycler (PerkinElmer Applied Biosystems, Foster City, CA). The PCR products were incubated with PvuII at 37⬚C (New England Biolabs, Burlington, Ontario, Canada) for at least 3 hours and separated on 2% agarose gels (Fig. 1). A 10% quantity of the PCR samples was directly sequenced to confirm the PCR-RFLP results (Invitrogen Biotechnology Co., Shanghai, China) (Fig. 1). 2.4. Statistical analysis Hardy-Weinberg equilibrium (HWE) was tested using the test. A binary logistic regression analysis was performed to assess the influence of the MCP-1 polymorphism and patient gender on the susceptibility to ocular BD, using SPSS version 17.0 for Windows (SPSS Inc., Chicago, IL). All statistical tests were two-sided, and statistical significance was set at p ⬍ 0.05. 2
3. Results Clinical findings of ocular BD patients and baseline characteristics of controls are presented in Table 1. The average age of the ocular BD patients was 31.3 ⫾ 7.2 and that of healthy controls was 34.7 ⫾ 11.6 (Table 1). No statistically significant difference in the distribution of age was observed between ocular BD patients and controls (p ⬎ 0.05). A total of 296 ocular BD patients and 319 healthy controls were genotyped for the MCP-1 ⫺2518 A/G polymorphism, and the result of genotyping was matched with that of direct sequencing for 10% samples. The distribution of the genotypic frequency of this SNP in all subjects did not show a significant deviation from the HWE (p ⬎ 0.05). The distribution of the genotypic and allelic frequency of the MCP-1 ⫺2518 A/G polymorphism is shown in Table 2. A decreased frequency of the homozygous AA genotype of the MCP-1 ⫺2518 A/G polymorphism was observed in ocular BD patients compared with healthy controls (18.9% vs. 27.9%, p ⫽ 0.048, odds ratio 0.64,
Fig. 1. Identification of the MCP-1 ⫺2518 A/G polymorphism in the Chinese Han population. (A) Sequencing results of the AA genotype. (B) Sequencing result of AG genotype. (C) Sequencing result of GG genotype. (D) Results of PCR-RFLP of the MCP-1 ⫺2518 A/G polymorphism. PvuII digestion of PCR products of the AA genotype gives one fragment, the AG genotype gives three fragments, and the AG genotype gives two fragments.
95% confidence interval 0.42– 0.99) (Table 2). The frequency of the AG genotype of the MCP-1 ⫺2518 A/G polymorphism was significantly increased in ocular BD patients compared with healthy controls (48.7% vs. 42.3%, p ⫽ 0.028, odds ratio 1.51, 95% confidence interval 1.05–2.17) (Table 2). In addition, our results did not indicate a significant difference when allelic and genotypic frequencies were analyzed according to clinical findings, including oral ulcer, genital ulcer, hypopyon, skin lesions, positive pathergy test results, and arthritis (p ⬎ 0.05). 4. Discussion In this study we focused on the ⫺2518 A/G polymorphism of MCP-1, the polymorphism of which has been shown to affect its expression level and the clinical presentation of various immunemediated diseases [27], and we performed a case-control association study for this polymorphism with ocular BD. A decreased MCP-1 ⫺2518 AA genotypic frequency and an increased AG genotypic frequency was found in ocular BD patients compared with healthy controls, suggesting that this gene may be involved in the pathogenesis of ocular BD. BehÈet’s disease is an endemic multifactorial genetic disease [1]. Whereas the disease is rare in the Western countries [3,29], it occurs with high prevalence in the countries of the ancient Silk Road [30,31], which extends from eastern Asia to the Mediterranean basin [32–34]. Epidemiologic investigations have shown that BD has a striking geographic and ethnic distribution, with a particularly high prevalence observed in individuals from Turkish, Japa-
S. Hou et al. / Human Immunology 71 (2010) 79 – 82
Table 1 Clinical characteristic of patients with ocular BehÈet’s disease (BD) Phenotype
Ocular BD patients
Age (y) at onset (mean ⫾ SD) Male gender Female gender Uveitis Oral ulcer Genital ulcer Hypopyon Skin lesions Positive pathergy test results Arthritis
Total (n ⫽ 296)
%
31.3 ⫾ 7.2 247 49 296 286 124 68 148 102 78
83.4 16.6 100 96.6 41.9 23.0 50.0 34.5 26.4
nese, or Chinese descent [3,4] and mounting evidence suggests that genetic predisposition might play a crucial role in the development of BD. At present, the contribution of genes encoding for human leucocyte antigens (HLA) to the genetic risk of BD is best characterized [35–39]. The association with HLA-B51 [39,40] is also well known. However, these identified genes only account for a small portion of the overall estimated genetic risk for BD, and further studies on other susceptible genes have therefore been initiated by various groups. Karasneh et al. identified 16 non-HLA susceptible loci in Turkish patients using a whole-genome screening approach, and our recent studies revealed several susceptible genes for BD in the Chinese Han population, including SUMO4 andFCRL3 [41,42]. These studies suggest that multiple immune-associated factors might be involved in the pathogenesis of this disease, and that the currently observed genetic risk factors are only a small part of the possible susceptibility genes associated with this disease. Largescale association studies are needed to address the full range of susceptible genes for BD. MCP-1 is a potent chemokine that plays an important role in the recruitment of leukocytes to the site of inflammation. Recently a ⫺2518 A/G polymorphism in the promoter region of MCP-1 has been described that was shown to affect the transcriptional activation of this gene [27] and that has therefore been considered as a good candidate in the genetic predisposition to autoimmune disease [23,43,44]. Furthermore, MCP-1 concentrations have been found to be increased in serum of BD patients [45]. Based on these earlier results, we specifically tested whether the MCP-1 ⫺2518 A/G polymorphism was associated with ocular BD in Chinese Han population. Our results showed that the AG genotype of MCP-1 ⫺2518 A/G polymorphism was positively associated with the susceptibility to ocular BD. In addition, the AA genotype of MCP-1 ⫺2518 A/G polymorphism contributes to a protective effect against ocular BD development. In accordance with our results, the MCP-1 ⫺2518 A/G polymorphism has been shown to have a significant association with uveitis and various autoimmune diseases [22–24]. As BD is a general autoimmune disorder characterized by ocular and multiple extraocular findings, we therefore performed an association study of MCP-1 polymorphism with these extraocular disease findings. However, we were not able to show an association
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of the tested polymorphism with the clinical presentation of BD. A recent study has indicated that monocytes from AA homozygotic individuals released lesser MCP-1 protein compared with individuals carrying the G allele [27]. Consequently, it would be reasonable to speculate that, under the same stimulatory conditions, AA homozygotic individuals, compared with individuals carrying the G allele, may display a lesser inflammatory response by recruitment of few monocytes, T lymphocytes, and eosinophils to the inflammation sites [46,47]. Taken together, these data suggest a possible implication of MCP-1 polymorphism in the pathogenesis of various autoimmune diseases, including BD. It is worth pointing out that our results are discordant with those reported by Korean investigators. Cho et al. [45] were not able to find an association between the MCP-1 polymorphism and the occurrence of BD in Korean patients. The high dominance of the G allele in Korea may explain the observed difference with our study. The frequency of the G allele in our study was 50.9%, whereas the G allelic frequency reported by Cho et al. was 65% [45]. Genetic heterogeneity between two different ethnic populations may partially explain the discrepant findings. The sample size used in studies may also have influenced the results. In the Cho et al. study, only 104 patients were tested, whereas in our study 296 patients were enrolled. The third possibility could be the origin of the patients. In the Cho et al. study, BD patients were recruited from a rheumatology research center, whereas in our study the tested patients came from an ophthalmology department. It is noteworthy that a few factors, such as population composition and stratification, might influence the result of an association study. In the present study, the following attempts were made to ensure that the analysis results were valid. First, genotype distribution of MCP-1 polymorphism in healthy controls matches the Hardy-Weinberg equilibrium. Second, the controls and patients were strictly matched according to the places where these individuals were born to exclude the possible influence of stratification of the population. Third, the number of donors enrolled in this study (296 ocular BD patients and 319 age-, gender-, and ethnicity-matched healthy controls) was large enough to avoid a bias of the results. Finally, direct sequencing for 10% samples was performed to validate the result of genotyping. Several possible limitations of the present study merit particular consideration. The patients enrolled in our study were recruited from an ophthalmic center. Because BD is an autoimmune disease that affects multiple systems, the patients enrolled in this study might therefore represent a separate disease population. Gender might also influence the results in a case-control association study. In our study a much higher ratio of male to female patients might have resulted in bias, although we did a binary logistic regression analysis. A study using a well-matched gender ratio in case and control populations is needed to clarify our results. Furthermore, the patients enrolled in this study were recruited only from among Chinese Han individuals and excluded other Chinese ethnic subpopulations. Therefore, the results presented here need to be confirmed using different ethnic populations. Finally, the functional
Table 2 Allelic and genotypic frequencies of the MCP-1 polymorphism in ocular BehÈet disease (BD) patients and controls SNP
Genotype allele
Ocular BD (n ⫽ 296)
Controls (n ⫽ 319)
p Value
Odds ratio (95% CI)
p Valuea
Odds ratio (95% CI)b
MCP-1 ⫺2518 A/G
AA AG GG A G
56 (18.9%) 144 (48.7%) 96 (32.4%) 256 (43.2%) 336 (56.8%)
89 (27.9%) 135 (42.3%) 95 (29.8%) 313 (49.1%) 325 (50.9%)
0.009 0.115 0.478 0.041 0.041
0.60 (0.41–0.88) 1.29 (0.94–1.78) 1.13 (0.80–1.59) 0.79 (0.63–0.99) 1.26 (1.01–1.58)
0.048 0.028 0.566 0.404 0.404
0.64 (0.42–0.99) 1.51 (1.05–2.17) 0.89 (0.59–1.33) 0.90 (0.69–1.16) 1.26 (0.86–1.45)
CI, confidence interval; SNP, single nucleotide polymorphism. a
Adjusted gender p value. Adjusted gender OR (95% CI).
b
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studies were not performed in this study. Since a functional study might reveal how a genetic variant translates into physiologic processes that affect disease susceptibility, the association results presented should be further investigated using functional experiments to understand the mechanisms by which the susceptible genes interact with certain environmental triggers and initiate the development of BD. In summary, the present study suggests that the MCP-1 gene seems to be one of the genetic markers involved in the genetic susceptibility of ocular BD. A large number of well-matched samples, multiple ethnic groups, and further functional studies will contribute to the understanding of genetic risks predisposing to BD, identification of disease-causing variants, and knowledge of the underlying mechanisms by which disease-causing variants affect disease susceptibility.
[21]
[22]
[23]
[24]
[25]
[26]
Acknowledgments [27]
This work was supported by the following: Project of International Cooperation in Science and Technology, Guangdong Province (2006A50107001), Key Project of Natural Science Foundation (30630064), National Supporting Project of P. R. China, Key Project of Health Bureau of Chongqing (2008/1/15), Key Project of Natural Science Foundation of Chongqing (CSTC, 2009BA5037) and Project of Chongqing Key Laboratory of Ophthalmology (CSTC, 2008CA5003). The authors express thanks to all individuals enrolled in the present study.
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