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BJO Online First, published on May 6, 2015 as 10.1136/bjophthalmol-2015-306677 Laboratory science
The association between toll-like receptor 4 polymorphisms and diabetic retinopathy in Chinese patients with type 2 diabetes Yuxin Xu,1 Zhengxuan Jiang,1 Jinhai Huang,2 Qianli Meng,3 Paul Coh,4 Liming Tao1 1
Department of Ophthalmology, The Second Affiliated Hospital of Anhui Medical University, Hefei, Anhui, China 2 School of Optometry and Ophthalmology and Eye Hospital, Wenzhou Medical University, Wenzhou, Zhejiang, China 3 Department of Ophthalmology, Guangdong Eye Institute, Guangdong General Hospital, Guangdong Academy of Medical Sciences, Guangzhou, Guangdong, China 4 Department of Ophthalmology, University of California San Francisco, San Francisco, California, USA Correspondence to Dr Zhengxuan Jiang, Department of Ophthalmology, The Second Affiliated Hospital of Anhui Medical University, 678 Furong Road, Hefei, Anhui 230601, China; [email protected] and Pro Liming Tao, Department of Ophthalmology, The Second Affiliated Hospital of Anhui Medical University, 678 Furong Road, Hefei, Anhui 230601, China; [email protected] Received 23 January 2015 Revised 30 March 2015 Accepted 20 April 2015
ABSTRACT Purpose Diabetic retinopathy (DR) is one of the secondary microvascular complications of type 2 diabetes mellitus. Persistent inflammation and impaired neovascularisation may be important contributors to the development of DR. A recent study showed that toll-like receptor 4 (TLR4) polymorphisms were associated with DR. The present study was designed to determine whether single-nucleotide polymorphisms (SNPs) in the TLR4 gene were associated with DR in a Chinese Han population. Materials and methods Three SNPs (rs10759931, rs1927911 and rs1927914) in the TLR4 gene were chosen as candidate SNPs. Genomic DNA from type 2 diabetes patients and healthy controls were genotyped for the above-mentioned genetic variations through the use of PCR restriction fragment length polymorphism assay. Data were analysed by χ2 analysis. Results The results showed that the three analysed polymorphisms in the TLR4 gene were in Hardy– Weinberg equilibrium, both in the patients and in the controls. In the type 2 diabetes group, a significantly higher frequency of the C allele of rs1927914 was observed in patients with type 2 diabetes than that in controls. The result showed that the frequencies of the TT genotype and the T allele of rs1927914 were significantly decreased in patients with type 2 diabetes. Significantly increased frequencies of the CC genotype and the C allele of rs1927911 were observed in patients with type 2 diabetes. In the DR group, the C allele of rs1927914 was significantly increased in the DR group compared with that of the control. The frequencies of the CC genotype and the C allele of rs1927911 tended to be higher in patients with DR than in the healthy controls. However, no difference was found when the Bonferroni correction was applied. No difference was detected between patients and controls with regard to all haplotypes. Conclusions This study suggested that rs1927914 and rs1927911 were associated with type 2 diabetes mellitus and that rs1927914 was associated with susceptibility to DR in a Han Chinese population.
INTRODUCTION
To cite: Xu Y, Jiang Z, Huang J, et al. Br J Ophthalmol Published Online First: [ please include Day Month Year] doi:10.1136/bjophthalmol2015-306677
Diabetic retinopathy (DR) is one of the most common microvascular complications of diabetic mellitus.1 Estimated to be the main cause of new blindness in working-age adults residing in developed countries,2 DR is the most severe of the several ocular complications of diabetes.1 3 Epidemiology studies have revealed that there are 93 million people with DR worldwide, about 17 million of whom have proliferative DR and 28
million of whom have vision-threatening DR.4 With an ageing population, these numbers will continue to increase. Considering the projected increase in the worldwide prevalence of diabetes, 380 million people will be diagnosed with DR by 2025.5 While the aetiology of DR continues to be largely unknown, it is thought to be a multifactorial disease. It has been estimated that genetic predisposition and environmental elements are involved in the development of the disease.6 On one hand, some evidence supports the principle of inherited genetic susceptibility as an important risk factor for DR. Previous familial aggregation studies have shown that siblings and relatives of diabetics with DR have as high as a threefold increased risk for DR compared with siblings and relatives of diabetics without DR in either type 1 or type 2 diabetes and across different ethnicities.7–10 On the other hand, a chronic low-grade subclinical inflammation is an important contributor to the development of DR.11 Inflammation in the diabetic retina is mediated by leucocyte adhesion to the retinal vasculature and alteration of the blood–retinal barrier.12 Increased levels of inflammatory mediators may lead to a persistent chronic inflammatory condition in the diabetic retina, resulting in leucocyte activation, adhesion to the vascular endothelium and extravasations into the retinal tissues.13 14 A toll-like receptor (TLR) is an important pathogen recognition receptor involved in an immune response to exogenous or endogenous ligands.15 TLR4 initiates the production of proinflammatory cytokines, including interleukin-1 and tumour necrosis factor-α, through interaction with its ligand.16 The activity and function of TLR4 may be modulated by genetic variations and single-nucleotide polymorphisms (SNPs). Therefore, polymorphisms in the TLR4 gene seem to modify the signalling of the immune response in the pathogenesis of DR.17 Recent studies have shown that the TLR4 gene is associated with several diseases, such as chronic obstructive pulmonary disease, gastric cancer and DR.18–20 Much evidence has shown that genetic heterogeneity exists in different ethnic cohorts, even within the same population that comes from a different region. Therefore, we designed this case–control study to determine whether TLR4 gene polymorphisms are associated with susceptibility to DR in a Han Chinese population.
METHODS Subjects A total of 236 patients with type 2 diabetes and 274 age-matched, sex-matched and ethnicity-
Xu Y, et al. Br J Ophthalmol 2015;0:1–5. doi:10.1136/bjophthalmol-2015-306677
Copyright Article author (or their employer) 2015. Produced by BMJ Publishing Group Ltd under licence.
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Laboratory science matched healthy controls were recruited from the second affiliated hospital of Anhui Medical University (Anhui, P.R. China). The distribution of age and gender is shown in table 1. The patients with type 2 diabetes were classified as patients with DR (139 patients) and patients with type 2 diabetes without retinopathy (97 patients). All the patients and healthy controls underwent a full ophthalmic examination. Diabetes was diagnosed according to WHO criteria. Retinopathy was diagnosed according to the Early Treatment Diabetic Retinopathy study criteria.21
in the allele and genotype distribution were evaluated with the use of the χ2 test, which was performed using SPSS (V.11.0, SPSS, Chicago, Illinois, USA). Fisher’s exact test was used in the condition with the number of genotypes or alleles fewer than five. The p values were corrected with the Bonferroni correction by multiple comparison with the numbers of analyses performed. A p value of 0.05). In the type 2 diabetes group, 139 patients were diagnosed with retinopathy. The distribution of genotypes and alleles of TLR4 SNPs in the patients with DR and the controls is shown in table 4. The frequencies of the rs1927914 CC genotype and C allele were significantly increased in the DR group compared with those of the control group (χ2=5.01, p=0.03, OR 2.13, 95% CI 1.09 to 4.16 and χ2=7.54, p=0.006, OR 1.51, 95% CI 1.13 to 2.03, respectively). After Bonferroni correction, no difference in the rs1927914 CC genotype was found between two groups. The frequency of the CC genotype of rs1927911 tended to be higher in the patients with DR than in the healthy controls (χ2=4.75, p=0.03, OR 1.59, 95% CI 1.05 to 2.41). However, no difference was found when the Bonferroni correction was applied. Haplotype analysis was performed using the SHEsis software platform.23 No differences were detected between patients and controls with regard to all haplotypes.
DISCUSSION In this study, we conducted an investigation to determine whether polymorphisms of the TLR4 gene are associated with
DR in a Chinese Han population. The results showed that two SNPs of the TLR4 gene, rs1927914 and rs1927911, were associated with susceptibility to type 2 diabetes. There was no association between rs10759931 and type 2 diabetes. Since many patients with type 2 diabetes have retinopathy, we further analysed the association between the tested SNPs of the TLR4 gene and DR. rs1927911 was associated with susceptibility to DR in Chinese Han population. Haplotype analysis revealed that no significant difference was found between type 2 diabetes, DR and controls with regard to these haplotypes in this study. DR is a significant cause of blindness, and treatment options have limitations. Although the aetiology of DR remains unknown, several hypotheses have been proposed, including genetic factors and inflammation.11 24 25 On the one hand, the major role genetic factors play in DR has been supported by ethnic differences in frequency, increased familial aggregation and a higher concordance in monozygotic versus dizygotic twins.7–9 To date, the candidate gene approach has been used in the genetic research of DR.26 A number of hypothesised candidate genes in several pathways, which have been proposed to involve the pathogenesis of DR, have been identified to be associated with DR. Many genes have been reported to have associations with DR, including angiotensin I converting enzyme,
Table 4 Frequencies of alleles and genotypes of toll-like receptor 4 polymorphisms in patients with diabetic retinopathy (DR) and controls SNP
Genotype allele
DR (N=139)
Controls (N=274)
χ2
p Value
Pc Value
OR (95% CI)
rs10759931
AA AG GG A G TT TC CC T C TT TC CC T C
55 (39.5%) 81 (58.3%) 3 (2.2%) 191 (68.7%) 87 (31.3%) 37 (26.6%) 83 (59.7%) 19 (13.7%) 157 (56.4%) 121 (43.6%) 9 (6.5%) 67 (48.2%) 63 (45.3%) 85 (30.6%) 193 (69.4%)
115 (42.0%) 151 (55.1%) 8 (2.9%) 381 (69.5%) 167 (30.5%) 108 (39.4%) 147 (53.6%) 19 (7.0%) 363 (66.2%) 185 (33.8%) 22 (8.1%) 158 (57.7%) 94 (34.2%) 202 (36.9%) 346 (63.1%)
0.22 0.38 0.21 0.06 0.06 6.63 1.37 5.01 7.54 7.54 0.32 2.96 4.75 3.21 3.21
0.64 0.54 0.65 0.81 0.81 0.01 0.24 0.03 6.00×10−3 6.00×10−3 0.57 0.09 0.03 0.07 0.07
NS NS NS NS NS NS NS NS 0.018 0.018 NS NS NS NS NS
0.91 1.14 0.73 0.96 1.04 0.56 1.28 2.13 0.66 1.51 0.79 0.68 1.59 0.75 1.33
rs1927914
rs1927911
(0.60 (0.75 (0.19 (0.70 (0.76 (0.36 (0.85 (1.09 (0.49 (1.13 (0.36 (0.45 (1.05 (0.55 (0.97
to 1.37) to 1.72) to 2.81) to 1.32) to 1.42) to 0.87) to 1.94) to 4.16) to 0.89) to 2.03) to 1.77) to 1.03) to 2.41) to 1.03) to 1.81)
NS, not significant; SNPs, single-nucleotide polymorphisms.
Xu Y, et al. Br J Ophthalmol 2015;0:1–5. doi:10.1136/bjophthalmol-2015-306677
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Laboratory science angiotensin II type 1 receptor, angiotensinogen, vascular endothelial growth factor, aldose reductase, receptor for advanced glycation end products and apolipoprotein E.26–29 However, the results were not consistent among different populations; therefore, these genes need to be examined in other populations. On the other hand, inflammation and angiogenesis are involved in the pathogenesis of DR. The causal relationship between inflammation and angiogenesis in DR is widely accepted.11 TLR signal transduction is a central component of primary innate immune response to pathogens. As an important member of the TLR family, TLR4 is highly expressed in many cell types, including macrophages, endothelial and smooth muscle cells. It is a transmembrane receptor and involved in regulating innate immunity.16 After banding with a ligand, the activation of TLR4 initiates the production of proinflammatory cytokines and chemokines through the mitogen-activated protein kinase and nuclear factor-κB pathways. The neutrophils and monocytes are then recruited to the site of infection.16 20 As persistent inflammation occurs during the early stages of DR, TLR4 may have an important role in this disease. Genetic variations in TLR4 have been reported to be associated with several inflammatory diseases, including DR.18 19 24 25 It has been suggested that any deregulation of the TLR4 signal, which is associated with certain SNP alleles in this gene, could influence the development of the disease. Based on the genetic factor and inflammation in the development of DR, as well as the association of TLR4 polymorphisms with inflammatory diseases, we selected TLR4 as a candidate gene in the present study. Since there are many SNPs in one candidate gene, and some of them may be associated with disease, it is extremely important to choose the relevant SNPs. In the present study, SNP candidates were selected based on previous studies. Five SNPs in the TLR4 (rs4986790, rs4986791, rs10759931, rs1927914 and rs1927911) were found to be associated with diseases in different populations.20 24 25 The rs4986790 and rs4986791 have been widely studied in inflammatory diseases, such as Crohn’s disease and gastric cancer.30 31 Since two of them, rs4986790 and rs4986791, are not polymorphic in the Chinese population according to International HapMap data (http://www.hapmap. org), they were not included in this study. In order to ensure the results, the following steps were implemented: first, the patients were diagnosed using Early Treatment Diabetic Retinopathy study criteria, and the controls were from the same geographical regions as the patients with type 2 diabetes (age matched and gender matched); second, to validate the results of the PCR-RFLP method, 15% of the samples were randomly chosen and tested by direct sequencing. The results were consistent with that of the PCR-RFLP method. In the present study, we investigated the association of DR with rs10759931, rs1927914 and rs1927911 of the TLR4 gene. These three SNPs have been identified as having an association with diabetic foot ulcers and DR in the Indian population.20 24 The results of this study revealed that the rs1927914 C allele was associated with susceptibility to DR and type 2 diabetes. This result is consistent with the study that reported that the rs1927914 variant was associated with DR in an Indian population.20 This SNP was also found to be associated with diabetic foot ulcers in an Indian population.20 24 These results revealed that rs1927914 may be a common predisposed SNP of DR in both Chinese Han and Indian populations. However, the previous study found no significant association between rs1927914 and normal tension glaucoma in a South Korean population.32 The disease heterogeneity may explain these differences. The C allele and CC genotype of rs1927911 were 4
found to be associated with type 2 diabetes. We further analysed whether there was an association between the DR group and the controls. However, no significant difference was observed between the two groups after Bonferroni correction. A similar result was found in the previous study, which indicated that rs1927911 was not associated with DR in an Indian and a South Korean population.20 32 Contrary to our observation, rs1927911 was found to be associated with a lower risk of myocardial infarction in an American population, and the SNP was reported to have an increased risk of developing chemotherapy-induced neutropenia in a Caucasus population.33 34 With respect to the TLR4 variant rs10759931, no significant difference between the two groups was revealed concerning the allele and genotype of this SNP. A similar result was reported in Gram-negative bacterial infections in Han Chinese neonates.35 A conflicting study result showed that this SNP was associated with DR in an Indian population.20 Taken together, these contradictory results may be explained by different ethnicities and different sample sizes. Like other studies on the associations of candidate genes, there are some limitations in our study.36 37 First, as a lot of putative genes and genetic variants have been found in some studies but few of these have been consistently replicated; the results of the present study need to be confirmed in other ethnic populations, and with larger sample sizes. Second, the present study tested only four SNPs in the TLR4 gene. It is necessary to investigate some other SNPs of the TLR4 to understand its precise association with DR. In addition, the functional role of rs1927914 polymorphism is unknown. As it may influence the expression of TLR4 and be linked to disequilibrium with the causative variants in this gene, further studies will resolve these issues. In conclusion, the study showed that rs1927914 was associated with susceptibility to DR in a Han Chinese population. The other two tested SNPs were not found to be associated with the risk of DR. Hence, the study is a step towards improving the understanding of the possible relationship between innate immunity and the complications of type 2 diabetes, including DR. Contributors Study design: YX, ZJ and LT; data collection and management: PC and JH; interpreting the data: ZJ and QM; preparation of the initial manuscript: ZJ and LT; critical revisions to the initial manuscript: PC; YX assumed full responsibility for the integrity of the data and the accuracy of the data analysis. The authors are very grateful to all members for their participation in this study. Funding This work was supported by the National Natural Science Foundation Project of China (grant number 81300755 and 81371031) and the key project of Natural Science Foundation of Higher Educational Bureau of Anhui Province (grant number KJ2013A147). Competing interests None declared. Patient consent Obtained. Ethics approval The study was approved by the local institutional ethnics committee of the second affiliated hospital of Anhui Medical University. All procedures were conducted in accordance with the Declaration of Helsinki. Provenance and peer review Not commissioned; externally peer reviewed.
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Shi YY, He L. SHEsis, a powerful software platform for analyses of linkage disequilibrium, haplotype construction, and genetic association at polymorphism loci. Cell Res 2005;15:97–8. Singh K, Singh VK, Agrawal NK, et al. Association of Toll-like receptor 4 polymorphisms with diabetic foot ulcers and application of artificial neural network in DFU risk assessment in type 2 diabetes patients. Biomed Res Int 2013;2013:318686. Song J, Kim DY, Kim CS, et al. The association between Toll-like receptor 4 (TLR4) polymorphisms and the risk of prostate cancer in Korean men. Cancer Genet Cytogenet 2009;190:88–92. Liew G, Klein R, Wong TY. The role of genetics in susceptibility to diabetic retinopathy. Int Ophthalmol Clin 2009;49:35–52. Ng DP. Human genetics of diabetic retinopathy: current perspectives. J Ophthalmol 2010;2010:pii: 172593. Abhary S, Hewitt AW, Burdon KP, et al. A systematic meta-analysis of genetic association studies for diabetic retinopathy. Diabetes 2009;58:2137–47. Zhao T, Zhao J. Association between the -634C/G polymorphisms of the vascular endothelial growth factor and retinopathy in type 2 diabetes: a meta-analysis. Diabetes Res Clin Pract 2010;90:45–53. Hold GL, Rabkin CS, Chow WH, et al. A functional polymorphism of toll-like receptor 4 gene increases risk of gastric carcinoma and its precursors. Gastroenterology 2007;132:905–12. Achyut BR, Ghoshal UC, Moorchung N, et al. Association of Toll-like receptor-4 (Asp299Gly and Thr399Ileu) gene polymorphisms with gastritis and precancerous lesions. Hum Immunol 2007;68:901–7. Suh W, Kim S, Ki CS, et al. Toll-like receptor 4 gene polymorphisms do not associate with normal tension glaucoma in a Korean population. Mol Vis 2011;17:2343–8. Enquobahrie DA, Smith NL, Bis JC, et al. Cholesterol ester transfer protein, interleukin-8, peroxisome proliferator activator receptor alpha, and Toll-like receptor 4 genetic variations and risk of incident nonfatal myocardial infarction and ischemic stroke. Am J Cardiol 2008;101:1683–8. Miedema KG, te Poele EM, Tissing WJ, et al. Association of polymorphisms in the TLR4 gene with the risk of developing neutropenia in children with leukemia. Leukemia 2011;25:995–1000. Zhou JG, Zhang JP, Lee BH, et al. Toll-like receptor 4 polymorphisms in gram-negative bacterial infections of Han Chinese neonates. Am J Perinatol 2015;32:363–70. Jiang Z, Yang P, Hou S, et al. IL-23R gene confers susceptibility to Behcet’s disease in a Chinese Han population. Ann Rheum Dis 2010;69:1325–8. Jiang Z, Yang P, Hou S, et al. Polymorphisms of IL23R and Vogt-Koyanagi-Harada syndrome in a Chinese Han population. Hum Immunol 2010;71:414–17.
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The association between toll-like receptor 4 polymorphisms and diabetic retinopathy in Chinese patients with type 2 diabetes Yuxin Xu, Zhengxuan Jiang, Jinhai Huang, Qianli Meng, Paul Coh and Liming Tao Br J Ophthalmol published online May 6, 2015
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BJO Online First, published on May 6, 2015 as 10.1136/bjophthalmol-2015-306677 Laboratory science
The association between toll-like receptor 4 polymorphisms and diabetic retinopathy in Chinese patients with type 2 diabetes Yuxin Xu,1 Zhengxuan Jiang,1 Jinhai Huang,2 Qianli Meng,3 Paul Coh,4 Liming Tao1 1
Department of Ophthalmology, The Second Affiliated Hospital of Anhui Medical University, Hefei, Anhui, China 2 School of Optometry and Ophthalmology and Eye Hospital, Wenzhou Medical University, Wenzhou, Zhejiang, China 3 Department of Ophthalmology, Guangdong Eye Institute, Guangdong General Hospital, Guangdong Academy of Medical Sciences, Guangzhou, Guangdong, China 4 Department of Ophthalmology, University of California San Francisco, San Francisco, California, USA Correspondence to Dr Zhengxuan Jiang, Department of Ophthalmology, The Second Affiliated Hospital of Anhui Medical University, 678 Furong Road, Hefei, Anhui 230601, China; [email protected] and Pro Liming Tao, Department of Ophthalmology, The Second Affiliated Hospital of Anhui Medical University, 678 Furong Road, Hefei, Anhui 230601, China; [email protected] Received 23 January 2015 Revised 30 March 2015 Accepted 20 April 2015
ABSTRACT Purpose Diabetic retinopathy (DR) is one of the secondary microvascular complications of type 2 diabetes mellitus. Persistent inflammation and impaired neovascularisation may be important contributors to the development of DR. A recent study showed that toll-like receptor 4 (TLR4) polymorphisms were associated with DR. The present study was designed to determine whether single-nucleotide polymorphisms (SNPs) in the TLR4 gene were associated with DR in a Chinese Han population. Materials and methods Three SNPs (rs10759931, rs1927911 and rs1927914) in the TLR4 gene were chosen as candidate SNPs. Genomic DNA from type 2 diabetes patients and healthy controls were genotyped for the above-mentioned genetic variations through the use of PCR restriction fragment length polymorphism assay. Data were analysed by χ2 analysis. Results The results showed that the three analysed polymorphisms in the TLR4 gene were in Hardy– Weinberg equilibrium, both in the patients and in the controls. In the type 2 diabetes group, a significantly higher frequency of the C allele of rs1927914 was observed in patients with type 2 diabetes than that in controls. The result showed that the frequencies of the TT genotype and the T allele of rs1927914 were significantly decreased in patients with type 2 diabetes. Significantly increased frequencies of the CC genotype and the C allele of rs1927911 were observed in patients with type 2 diabetes. In the DR group, the C allele of rs1927914 was significantly increased in the DR group compared with that of the control. The frequencies of the CC genotype and the C allele of rs1927911 tended to be higher in patients with DR than in the healthy controls. However, no difference was found when the Bonferroni correction was applied. No difference was detected between patients and controls with regard to all haplotypes. Conclusions This study suggested that rs1927914 and rs1927911 were associated with type 2 diabetes mellitus and that rs1927914 was associated with susceptibility to DR in a Han Chinese population.
INTRODUCTION
To cite: Xu Y, Jiang Z, Huang J, et al. Br J Ophthalmol Published Online First: [ please include Day Month Year] doi:10.1136/bjophthalmol2015-306677
Diabetic retinopathy (DR) is one of the most common microvascular complications of diabetic mellitus.1 Estimated to be the main cause of new blindness in working-age adults residing in developed countries,2 DR is the most severe of the several ocular complications of diabetes.1 3 Epidemiology studies have revealed that there are 93 million people with DR worldwide, about 17 million of whom have proliferative DR and 28
million of whom have vision-threatening DR.4 With an ageing population, these numbers will continue to increase. Considering the projected increase in the worldwide prevalence of diabetes, 380 million people will be diagnosed with DR by 2025.5 While the aetiology of DR continues to be largely unknown, it is thought to be a multifactorial disease. It has been estimated that genetic predisposition and environmental elements are involved in the development of the disease.6 On one hand, some evidence supports the principle of inherited genetic susceptibility as an important risk factor for DR. Previous familial aggregation studies have shown that siblings and relatives of diabetics with DR have as high as a threefold increased risk for DR compared with siblings and relatives of diabetics without DR in either type 1 or type 2 diabetes and across different ethnicities.7–10 On the other hand, a chronic low-grade subclinical inflammation is an important contributor to the development of DR.11 Inflammation in the diabetic retina is mediated by leucocyte adhesion to the retinal vasculature and alteration of the blood–retinal barrier.12 Increased levels of inflammatory mediators may lead to a persistent chronic inflammatory condition in the diabetic retina, resulting in leucocyte activation, adhesion to the vascular endothelium and extravasations into the retinal tissues.13 14 A toll-like receptor (TLR) is an important pathogen recognition receptor involved in an immune response to exogenous or endogenous ligands.15 TLR4 initiates the production of proinflammatory cytokines, including interleukin-1 and tumour necrosis factor-α, through interaction with its ligand.16 The activity and function of TLR4 may be modulated by genetic variations and single-nucleotide polymorphisms (SNPs). Therefore, polymorphisms in the TLR4 gene seem to modify the signalling of the immune response in the pathogenesis of DR.17 Recent studies have shown that the TLR4 gene is associated with several diseases, such as chronic obstructive pulmonary disease, gastric cancer and DR.18–20 Much evidence has shown that genetic heterogeneity exists in different ethnic cohorts, even within the same population that comes from a different region. Therefore, we designed this case–control study to determine whether TLR4 gene polymorphisms are associated with susceptibility to DR in a Han Chinese population.
METHODS Subjects A total of 236 patients with type 2 diabetes and 274 age-matched, sex-matched and ethnicity-
Xu Y, et al. Br J Ophthalmol 2015;0:1–5. doi:10.1136/bjophthalmol-2015-306677
Copyright Article author (or their employer) 2015. Produced by BMJ Publishing Group Ltd under licence.
1
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Laboratory science matched healthy controls were recruited from the second affiliated hospital of Anhui Medical University (Anhui, P.R. China). The distribution of age and gender is shown in table 1. The patients with type 2 diabetes were classified as patients with DR (139 patients) and patients with type 2 diabetes without retinopathy (97 patients). All the patients and healthy controls underwent a full ophthalmic examination. Diabetes was diagnosed according to WHO criteria. Retinopathy was diagnosed according to the Early Treatment Diabetic Retinopathy study criteria.21
in the allele and genotype distribution were evaluated with the use of the χ2 test, which was performed using SPSS (V.11.0, SPSS, Chicago, Illinois, USA). Fisher’s exact test was used in the condition with the number of genotypes or alleles fewer than five. The p values were corrected with the Bonferroni correction by multiple comparison with the numbers of analyses performed. A p value of 0.05). In the type 2 diabetes group, 139 patients were diagnosed with retinopathy. The distribution of genotypes and alleles of TLR4 SNPs in the patients with DR and the controls is shown in table 4. The frequencies of the rs1927914 CC genotype and C allele were significantly increased in the DR group compared with those of the control group (χ2=5.01, p=0.03, OR 2.13, 95% CI 1.09 to 4.16 and χ2=7.54, p=0.006, OR 1.51, 95% CI 1.13 to 2.03, respectively). After Bonferroni correction, no difference in the rs1927914 CC genotype was found between two groups. The frequency of the CC genotype of rs1927911 tended to be higher in the patients with DR than in the healthy controls (χ2=4.75, p=0.03, OR 1.59, 95% CI 1.05 to 2.41). However, no difference was found when the Bonferroni correction was applied. Haplotype analysis was performed using the SHEsis software platform.23 No differences were detected between patients and controls with regard to all haplotypes.
DISCUSSION In this study, we conducted an investigation to determine whether polymorphisms of the TLR4 gene are associated with
DR in a Chinese Han population. The results showed that two SNPs of the TLR4 gene, rs1927914 and rs1927911, were associated with susceptibility to type 2 diabetes. There was no association between rs10759931 and type 2 diabetes. Since many patients with type 2 diabetes have retinopathy, we further analysed the association between the tested SNPs of the TLR4 gene and DR. rs1927911 was associated with susceptibility to DR in Chinese Han population. Haplotype analysis revealed that no significant difference was found between type 2 diabetes, DR and controls with regard to these haplotypes in this study. DR is a significant cause of blindness, and treatment options have limitations. Although the aetiology of DR remains unknown, several hypotheses have been proposed, including genetic factors and inflammation.11 24 25 On the one hand, the major role genetic factors play in DR has been supported by ethnic differences in frequency, increased familial aggregation and a higher concordance in monozygotic versus dizygotic twins.7–9 To date, the candidate gene approach has been used in the genetic research of DR.26 A number of hypothesised candidate genes in several pathways, which have been proposed to involve the pathogenesis of DR, have been identified to be associated with DR. Many genes have been reported to have associations with DR, including angiotensin I converting enzyme,
Table 4 Frequencies of alleles and genotypes of toll-like receptor 4 polymorphisms in patients with diabetic retinopathy (DR) and controls SNP
Genotype allele
DR (N=139)
Controls (N=274)
χ2
p Value
Pc Value
OR (95% CI)
rs10759931
AA AG GG A G TT TC CC T C TT TC CC T C
55 (39.5%) 81 (58.3%) 3 (2.2%) 191 (68.7%) 87 (31.3%) 37 (26.6%) 83 (59.7%) 19 (13.7%) 157 (56.4%) 121 (43.6%) 9 (6.5%) 67 (48.2%) 63 (45.3%) 85 (30.6%) 193 (69.4%)
115 (42.0%) 151 (55.1%) 8 (2.9%) 381 (69.5%) 167 (30.5%) 108 (39.4%) 147 (53.6%) 19 (7.0%) 363 (66.2%) 185 (33.8%) 22 (8.1%) 158 (57.7%) 94 (34.2%) 202 (36.9%) 346 (63.1%)
0.22 0.38 0.21 0.06 0.06 6.63 1.37 5.01 7.54 7.54 0.32 2.96 4.75 3.21 3.21
0.64 0.54 0.65 0.81 0.81 0.01 0.24 0.03 6.00×10−3 6.00×10−3 0.57 0.09 0.03 0.07 0.07
NS NS NS NS NS NS NS NS 0.018 0.018 NS NS NS NS NS
0.91 1.14 0.73 0.96 1.04 0.56 1.28 2.13 0.66 1.51 0.79 0.68 1.59 0.75 1.33
rs1927914
rs1927911
(0.60 (0.75 (0.19 (0.70 (0.76 (0.36 (0.85 (1.09 (0.49 (1.13 (0.36 (0.45 (1.05 (0.55 (0.97
to 1.37) to 1.72) to 2.81) to 1.32) to 1.42) to 0.87) to 1.94) to 4.16) to 0.89) to 2.03) to 1.77) to 1.03) to 2.41) to 1.03) to 1.81)
NS, not significant; SNPs, single-nucleotide polymorphisms.
Xu Y, et al. Br J Ophthalmol 2015;0:1–5. doi:10.1136/bjophthalmol-2015-306677
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Laboratory science angiotensin II type 1 receptor, angiotensinogen, vascular endothelial growth factor, aldose reductase, receptor for advanced glycation end products and apolipoprotein E.26–29 However, the results were not consistent among different populations; therefore, these genes need to be examined in other populations. On the other hand, inflammation and angiogenesis are involved in the pathogenesis of DR. The causal relationship between inflammation and angiogenesis in DR is widely accepted.11 TLR signal transduction is a central component of primary innate immune response to pathogens. As an important member of the TLR family, TLR4 is highly expressed in many cell types, including macrophages, endothelial and smooth muscle cells. It is a transmembrane receptor and involved in regulating innate immunity.16 After banding with a ligand, the activation of TLR4 initiates the production of proinflammatory cytokines and chemokines through the mitogen-activated protein kinase and nuclear factor-κB pathways. The neutrophils and monocytes are then recruited to the site of infection.16 20 As persistent inflammation occurs during the early stages of DR, TLR4 may have an important role in this disease. Genetic variations in TLR4 have been reported to be associated with several inflammatory diseases, including DR.18 19 24 25 It has been suggested that any deregulation of the TLR4 signal, which is associated with certain SNP alleles in this gene, could influence the development of the disease. Based on the genetic factor and inflammation in the development of DR, as well as the association of TLR4 polymorphisms with inflammatory diseases, we selected TLR4 as a candidate gene in the present study. Since there are many SNPs in one candidate gene, and some of them may be associated with disease, it is extremely important to choose the relevant SNPs. In the present study, SNP candidates were selected based on previous studies. Five SNPs in the TLR4 (rs4986790, rs4986791, rs10759931, rs1927914 and rs1927911) were found to be associated with diseases in different populations.20 24 25 The rs4986790 and rs4986791 have been widely studied in inflammatory diseases, such as Crohn’s disease and gastric cancer.30 31 Since two of them, rs4986790 and rs4986791, are not polymorphic in the Chinese population according to International HapMap data (http://www.hapmap. org), they were not included in this study. In order to ensure the results, the following steps were implemented: first, the patients were diagnosed using Early Treatment Diabetic Retinopathy study criteria, and the controls were from the same geographical regions as the patients with type 2 diabetes (age matched and gender matched); second, to validate the results of the PCR-RFLP method, 15% of the samples were randomly chosen and tested by direct sequencing. The results were consistent with that of the PCR-RFLP method. In the present study, we investigated the association of DR with rs10759931, rs1927914 and rs1927911 of the TLR4 gene. These three SNPs have been identified as having an association with diabetic foot ulcers and DR in the Indian population.20 24 The results of this study revealed that the rs1927914 C allele was associated with susceptibility to DR and type 2 diabetes. This result is consistent with the study that reported that the rs1927914 variant was associated with DR in an Indian population.20 This SNP was also found to be associated with diabetic foot ulcers in an Indian population.20 24 These results revealed that rs1927914 may be a common predisposed SNP of DR in both Chinese Han and Indian populations. However, the previous study found no significant association between rs1927914 and normal tension glaucoma in a South Korean population.32 The disease heterogeneity may explain these differences. The C allele and CC genotype of rs1927911 were 4
found to be associated with type 2 diabetes. We further analysed whether there was an association between the DR group and the controls. However, no significant difference was observed between the two groups after Bonferroni correction. A similar result was found in the previous study, which indicated that rs1927911 was not associated with DR in an Indian and a South Korean population.20 32 Contrary to our observation, rs1927911 was found to be associated with a lower risk of myocardial infarction in an American population, and the SNP was reported to have an increased risk of developing chemotherapy-induced neutropenia in a Caucasus population.33 34 With respect to the TLR4 variant rs10759931, no significant difference between the two groups was revealed concerning the allele and genotype of this SNP. A similar result was reported in Gram-negative bacterial infections in Han Chinese neonates.35 A conflicting study result showed that this SNP was associated with DR in an Indian population.20 Taken together, these contradictory results may be explained by different ethnicities and different sample sizes. Like other studies on the associations of candidate genes, there are some limitations in our study.36 37 First, as a lot of putative genes and genetic variants have been found in some studies but few of these have been consistently replicated; the results of the present study need to be confirmed in other ethnic populations, and with larger sample sizes. Second, the present study tested only four SNPs in the TLR4 gene. It is necessary to investigate some other SNPs of the TLR4 to understand its precise association with DR. In addition, the functional role of rs1927914 polymorphism is unknown. As it may influence the expression of TLR4 and be linked to disequilibrium with the causative variants in this gene, further studies will resolve these issues. In conclusion, the study showed that rs1927914 was associated with susceptibility to DR in a Han Chinese population. The other two tested SNPs were not found to be associated with the risk of DR. Hence, the study is a step towards improving the understanding of the possible relationship between innate immunity and the complications of type 2 diabetes, including DR. Contributors Study design: YX, ZJ and LT; data collection and management: PC and JH; interpreting the data: ZJ and QM; preparation of the initial manuscript: ZJ and LT; critical revisions to the initial manuscript: PC; YX assumed full responsibility for the integrity of the data and the accuracy of the data analysis. The authors are very grateful to all members for their participation in this study. Funding This work was supported by the National Natural Science Foundation Project of China (grant number 81300755 and 81371031) and the key project of Natural Science Foundation of Higher Educational Bureau of Anhui Province (grant number KJ2013A147). Competing interests None declared. Patient consent Obtained. Ethics approval The study was approved by the local institutional ethnics committee of the second affiliated hospital of Anhui Medical University. All procedures were conducted in accordance with the Declaration of Helsinki. Provenance and peer review Not commissioned; externally peer reviewed.
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The association between toll-like receptor 4 polymorphisms and diabetic retinopathy in Chinese patients with type 2 diabetes Yuxin Xu, Zhengxuan Jiang, Jinhai Huang, Qianli Meng, Paul Coh and Liming Tao Br J Ophthalmol published online May 6, 2015
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