Journal of Diabetes and Its Complications xxx (2014) xxx–xxx
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Original Article
Chimerin 2 genetic polymorphisms are associated with non-proliferative diabetic retinopathy in Taiwanese type 2 diabetic patients Marcelo Chen a, b, c, Wun-Rong Lin a, Chieh-Hsiang Lu d, Ching-Chu Chen e, f, Yu-Chuen Huang f, g, Wen-Ling Liao h, i, 1,⁎, Fuu-Jen Tsai f, g, 1,⁎ a
Department of Urology, Mackay Memorial Hospital, Taipei, Taiwan Mackay Medicine, Nursing and Management College, Taipei, Taiwan c School of Medicine, Mackay Medical College, New Taipei City, Taiwan d Division of Endocrinology and Metabolism of Internal Medicine, Ditmanson Medical Foundation Chia-Yi Christian Hospital, Chia-Yi, Taiwan e Division of Endocrinology and Metabolism, Department of Medicine, China Medical University Hospital, Taichung, Taiwan f School of Chinese Medicine, China Medical University, Taichung, Taiwan g Genetics Center, Department of Medical Research, China Medical University Hospital, Taichung, Taiwan h Center for Personalized Medicine, China Medical University Hospital, Taichung, Taiwan i Graduate Institute of Integrate Medicine, School of Chinese Medicine, China Medical University, Taichung, Taiwan b
a r t i c l e
i n f o
Article history: Received 20 December 2013 Received in revised form 1 April 2014 Accepted 11 April 2014 Available online xxxx Keywords: Single nucleotide polymorphism CHN2 Diabetic retinopathy Type 2 diabetes Taiwanese
a b s t r a c t Aim: To investigate whether chimerin 2 (CHN2) genetic polymorphisms were associated with the susceptibility to diabetic retinopathy (DR) in Taiwanese individuals with type 2 diabetes. Methods: This case-control study comprised of 171 individuals with DR and 548 without DR. Four rs39059, rs2023908, rs1002630 and rs1362363 polymorphism of CHN2 were genotyped for each subjects. All subjects underwent a complete ophthalmologic examination, and basic information (age, gender, age at diagnosis of diabetes, and ocular history of the patient) was record. Several clinical parameters (systolic and diastolic blood pressure, waist and hip circumferences, body mass index levels, fasting glucose and HbA1c) were measured. Results: Logistic regressions were used to analyze odds ratios between SNPs and DR after controlling for gender, systolic blood pressure, waist and hip ratio, duration of diabetes, serum HbA1c levels and nephropathy classification. A protective effect of rs1002630 (GA + AA) and rs1362363 (AG + GG) [odds ratio (OR) (95% confidence interval) = 0.45 (0.22–0.88), 0.66 (0.44–0.99), respectively) was observed. Furthermore, the protective effect of rs1002630 was observed when compared subjects with non-proliferative DR with subjects without DR [OR = 0.25 (95%C.I. = 0.09–0.73)]. Conclusions: This study showed that the rs1002630 of CHN2 were associated with DR risk and non-proliferative DR risk in Taiwanese individuals with type 2 diabetes. Variations at this locus may contribute to the pathogenesis of DR. © 2014 Elsevier Inc. All rights reserved.
1. Introduction Diabetic retinopathy (DR) is one of the common microvascular complications in diabetic patients (Sheetz & King, 2002), and it is the
Conflict of Interest: There is no conflict of interest. Grant support: This study was supported by research grants from China Medical University Hospital (DMR-102-045) and China Medical University (CMU102-N-01), Taichung City, Taiwan and from Ditmanson Medical Foundation Chia-Yi Christian Hospital Research Program (R101-14), Chia-Yi City, Taiwan. ⁎ Corresponding authors: Genetic Center, China Medical University Hospital, No. 2, YuhDer Road, Taichung 404, Taiwan. Tel.: +886 4 22052121x2041; fax: +886 4 22053425. E-mail addresses: [email protected] (W-L. Liao), [email protected] (F-J. Tsai). 1 Wen-Ling Liao and Fuu-Jen Tsai contributed equally to this work.
leading cause of blindness in adults (Thylefors et al., 1995). The development of DR is associated with long duration of diabetes, elevated glycosylated hemoglobin levels, high blood pressure, and chronic hyperglycemia (Stratton et al., 2000). The pathogenesis of DR is complex, having a multifactorial biochemical pathogenesis, primarily due to altered glucose metabolism (Brownlee, 2001; Jerneld & Algvere, 1986). In diabetic patients, high glucose concentration can directly impair retinal endothelial cell barrier function, resulting in an increase in permeability, edema, and loss of blood retinal barrier integrity. Sequential events in this damage induce leukocyte infiltration, and retinal proteins become exposed to the immune system (Cai & Boulton, 2002). Several interconnecting biochemical pathways that are implicated in the progression of the DR have been raised, such as increased expression of growth factor-1 (IGF-1) and vascular
http://dx.doi.org/10.1016/j.jdiacomp.2014.04.009 1056-8727/© 2014 Elsevier Inc. All rights reserved.
Please cite this article as: Chen, M., et al., Chimerin 2 genetic polymorphisms are associated with non-proliferative diabetic retinopathy in Taiwanese type 2 diabetic patients, Journal of Diabetes and Its Complications (2014), http://dx.doi.org/10.1016/j.jdiacomp.2014.04.009
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M. Chen et al. / Journal of Diabetes and Its Complications xxx (2014) xxx–xxx
permeability factor vascular endothelial growth factor (VEGF), accelerated formation of advanced glycation endproducts (AGEs) and subclinical inflammation and capillary occlusion (Ellis et al., 2013). However, the mechanism of how DR develops remains unclear. Moreover, it has become evident through familial aggregation studies that susceptibility to complications of diabetes such as DR also has a heritable component independent of glycemic control and duration of diabetes (Hallman et al., 2005; Rema et al., 2002). Since 1997, the Diabetes Control and Complications Trial Research Group first reported that the severity of DR may be influenced by familial (Anonymous, 1997); the possibly genetic effect on DR has been raised. To date, candidate gene association studies or genome wide association studies have reported that many genes are associated with the development of DR. VEFG is potent angiogenic, and vascular permeability factor and the polymorphism of the promoter and 5’untranslated region of VEGF gene are reported to be associated with DR (Awata et al., 2002; Petrovic, 2013). Chimerin 2 (CHN2) gene which plays a role in the proliferation and migration of smooth muscle cells (Maeda et al., 2006) has been reported to be associated with diabetic nephropathy (DN) in type 1 diabetes in European descent (Pezzolesi et al., 2009) and DN in T2D patients in Japanese (Maeda et al., 2010) and DR in T2D Chinese patient (Hu et al., 2011). In the present study, we investigated whether the polymorphism of CHN2 (Maeda et al., 2006) was associated with the susceptibility to DR in Taiwanese type 2 diabetic (T2D) patients. 2. Materials and methods 2.1. Subjects and data collection This is a cross-sectional study that involved 719 T2D patients aged over 20 years who were recruited from the China Medical University Hospital (CMUH), Taichung, Taiwan. Diabetes was diagnosed according to the medical records and fasting plasma glucose levels, by using the American Diabetes Association Criteria (Anonymous, 2013). Subjects with type 1 diabetes, gestational diabetes, and maturity onset diabetes of the young (MODY) were excluded from this study. All the participants were of Han Chinese origin, as the Han Chinese account for 98% of the population of Taiwan. All T2D subjects underwent a complete ophthalmologic examination, including corrected visual acuity, fundoscopic examination, and fundus photography. An expert ophthalmologist graded DR according to the American Academy of Ophthalmology proposed international scales for severity of clinical DR. Among 171 DR subjects, 70 subjects with proliferative DR (PDR) and 101 subjects with non-proliferative DR (NPDR) were identified. Data regarding age, gender, age at diagnosis of diabetes, and ocular history of the patient were collected from questionnaires. For each patient, systolic and diastolic blood pressure, waist and hip circumferences, and body mass index levels were determined, and blood samples were collected by venipuncture for genomic DNA isolation and serological tests, including fasting glucose, HbA1c at the time of enrollment in the study. Subjects with estimated glomerular filtration rate (eGFR) less than 60 were considered with diabetic nephropathy. The study was approved by the CMUH Institutional Review Board, and informed consent was obtained from all the study participants. 2.2. SNP selection and genotyping SNPs rs39059 which has been reported to be associated with diabetic complication from previous studies (Hu et al., 2011; Maeda et al., 2010) and three other SNPs rs2023908, rs1002630, rs1362363 of CHN2 were selected for genotyping. The minor allele frequency for these SNPs is more than 5% in our population, and the deviation from Hardy–Weinberg equilibrium was not observed for any SNP. For genotyping, the genomic DNA was extracted from peripheral blood leukocytes using the Genomic DNA kit (Qiagen), in accordance with the manufacturer's instructions. Genotypes of the patients were
determined using the ABI TaqMan® SNP genotyping assays (Applied Biosystems, Foster City, CA, USA) by using the commercial genotyping assays (ABI Assay ID: C_2568295_10 for rs2023908; ABI assay ID: C_11805351_10 for rs1002630; ABI assay ID: C_7601381_20 for rs1362363 and ABI assay ID: C_525465_10 for rs39059; Applied Biosystems). The reaction mixture for the real-time polymerase chain reaction (PCR) contained 10 ng of genomic DNA, 10 μ L TaqMan master mix, and 0.125 μ L of 40 × assay mix. PCR was performed in 96well plates on a thermal cycler (ABI 7700; Applied Biosystems). Reaction conditions were 60 °C for 30 s and 95 °C for 10 min, followed by 40 cycles at 95 °C for 15 s and 60 °C for 1 min. 2.3. Statistical analysis Characteristics and clinical data of subjects with DR and without DR were compared using the Student t-test for continuous variables and chi-square test for categorical variables. The genotype and allele frequency distributions in the polymorphisms between T2D subjects with and without were analyzed by the χ 2 test or Fisher exact test for differences in proportions. The odds ratio (OR) was calculated from genotype frequencies and allelic frequencies with 95% confidence interval (95% CI) by using logistical regression. The potential confounding factors, including the common risk factors, diabetes duration and serum HbA1c level and the one which was significant associated with outcome in univaraite models (gender, systolic blood pressure, waist–hip ratio and DN) were added in multivariate analysis. All statistical analyses were conducted using SPSS statistical software, version 17.0 (SPSS Inc., Chicago, IL, USA), and p value less than 0.05 (two-sided) was used as the level of significance. 3. Results Of the 719 T2D subjects, 171 (23.8%) had DR. Comparison of subjects with and without DR showed that the male to female ratios were similar (p = 0.418). Males with DR were diagnosed with T2D at a younger age than those without DR (p = 0.009). Subjects with DR were diabetic for a longer period of time than those without DR (p b 0.001). The serum HbA1c level and systolic blood pressure of subjects with DR were higher than those of subjects without DR (p b 0.001). Foveal thickness was higher in subjects with DR for both eyes (p b 0.001). The waist and hip ratio was significant higher in subjects with DR than those without DR (p = 0.013), but body mass index was not different between the two groups. The higher percentage of DR subjects with eGFR less than 60 compared to the subjects without DR (18.1% vs. 8.4%, p = 0.001) (Table 1). Table 2 shows the genotypic distribution of the SNPs in subject with and without DR. A higher frequencies of GG homozygotes at the rs1002630 variant was observed in the subjects with DR compared with the subjects without DR (91.8% vs. 85.9%, p value for chi square test was 0.04). After controlling for gender, duration of diabetes, serum HbA1c levels, SBP, WHR and DN in multivariate analysis, a protective effect of homozygotes and heterozygotes containing the A allele of the rs1002630 variants (adjusted OR = 0.45, 95% CI = 0.22–0.88) and the homozygotes and heterozygotes containing the G allele of rs362363 variant (adjusted OR = 0.66, 95% CI = 0.44–0.99) were observed when compared the subjects without DR with the subject with DR. Furthermore, the DR subjects were grouped into the subjects with NPDR and the subjects with PDR according to the scales for severity of clinical DR. And the genotypic distribution of the susceptibility SNPs was compared in the subjects with NPDR and PDR with the subjects without DR (Table 3). When compared with subjects without DR, subjects with NPDR had statistically significant lower frequencies of variant homozygotes and heterozygotes for A allele of rs1002630 (5.0% vs. 14.1%, p values for chi square test was 0.01). In multivariate analysis, the protective effect of the risk allele for rs1002630 were observed when compared the subjects with NPDR with the subjects
Please cite this article as: Chen, M., et al., Chimerin 2 genetic polymorphisms are associated with non-proliferative diabetic retinopathy in Taiwanese type 2 diabetic patients, Journal of Diabetes and Its Complications (2014), http://dx.doi.org/10.1016/j.jdiacomp.2014.04.009
M. Chen et al. / Journal of Diabetes and Its Complications xxx (2014) xxx–xxx Table 1 Characteristics and the clinical profile of type 2 diabetic patients with and without retinopathy.
Gender Male Female Age at diagnosis Male Female Diabetes duration (year) HbA1c (%) BMI (kg/m2) Systolic BP (mmHg) Diastolic BP (mmHg) W/R ratio Waist (cm) Hip (cm) Foveal thickness (μm) Oculus dexter Oculus sinister Retinopathy classification NPDR PDR DN classification eGFR≧60 eGFR b 60
T2D with retinopathy (n = 171)
T2D without retinopathy (n = 548)
86 (50.3%) 85 (49.7%)
295 (53.8%) 253 (46.2%)
46.7 ± 9.33 48.74 ± 9.28 14.83 ± 8.40 8.27 ± 1.42 25.27 ± 4.02 135.01 ± 18.50 74.61 ± 11.40 0.93 ± 0.07 89.89 ± 10.37 97.01 ± 8.13
49.8 ± 9.45 50.45 ± 8.52 8.34 ± 6.49 7.74 ± 1.40 25.10 ± 3.80 127.40 ± 16.05 76.37 ± 10.88 0.91 ± 0.06 88.64 ± 10.03 97.09 ± 7.59
0.009a 0.119a b0.001a b0.001a 0.617a b0.001a 0.075 a 0.013 a 0.159 a 0.908 a
207.37 ± 85.14 209.30 ± 81.72
182.38 ± 44.34 182.78 ± 42.15
b0.001a b0.001a
502 (91.6) 46(8.4)
b0.001
p-Value 0.418b
101 (59.1%) 70 (40.9%) 140(81.9) 31(18.1)
b
Abbreviation:T2D, type 2 diabetic; SD, standard deviation; BMI, body mass index; BP, blood pressure; W/R, waist over hip; DR, diabetes retinopathy; NPDR, non-proliferative DR; PDR, proliferative DR; DN, diabetes nephropathy; eGFR, estimated glomerular filtration rate a student's t-test. b chi-square test.
without DR in dominant model OR = 0.25, 95% CI = 0.09–0.73. However, the frequency of the risk allele (in recessive model) for these four markers was not statistically and significantly different when we compared the subjects with PDR with the subjects without DR.
4. Discussion DR is the common micro-vascular complication of T2D and the duration of T2D and serum HbA1c levels are known to be two major risk factors for DR. In our study, DR subjects were with a longer duration of T2D, higher serum HbA1c levels, higher systolic blood pressure, higher W/H ratio and higher percentage with DN; therefore, the variables were adjusted during statistical analysis. CHN2 gene, located on chromosome 7p15.3 is a member of the chimerin family which is ligand-activated Rac-specific GTPase-
3
activating proteins (Caloca et al., 2008). Chimerins are expressed in many human tissues, especially brain, pancreas, and insulin-sensitive tissues (Suliman et al., 2009) and could regulate the proliferation and migration of vascular smooth muscle cells which are important aspects of atherogenesis (Maeda et al., 2006). Its expression has been associated with high-grade gliomas (Yuan et al., 1995), breast cancer (Yang et al., 2005), and lymphoma (Nishiu et al., 2002). Furthermore, the CHN2 gene was identified to be a key element of proximal insulin signaling in vivo study (Suliman et al., 2009). Polymorphisms of the CHN2 gene have been reported to be associated with schizophrenia in men (Hashimoto et al., 2005), smoking (Barrio-Real et al., 2013) and diabetic complications (Hu et al., 2011; Maeda et al., 2010; Pezzolesi et al., 2009). In the present study, we reported that the CHN2 rs1002630 was significantly associated with DR and NPDR after adjusting for confounding factors. Although we failed to find the associations between these four SNPs with severity of DR in our population, it is possible due to small sample size in PDR group. Furthermore, one loci (rs39059) at CHN2 was reported to be associated with nephropathy in type I diabetic patients from previous genome-wide association study in European descent (Pezzolesi et al., 2009) and be replicated in DN in Japanese T2D subjects (Maeda et al., 2010). Hu et al. (Hu et al., 2011) showed that the rs39059 was associated with DR in Chinese T2D subjects in China (Hu et al., 2011). In the present study, we analyzed CHN2 rs39059 and rs2023908 which are highly linkage in our population. There is no significant association between these two SNPs with DR in our Taiwanese population. Also, no significant associations between these two SNPs with DN were observed in our limited diabetic nephropathy (DN) samples (no. of DN = 77) (results not shown). Further studies with larger sample size are necessary to elucidate the role of CHN2 in DR. Recent genetic epidemiologic studies have shown associations with DR risk in different ethnic groups. UTS2 (Okumus et al., 2012), CEP135 and NPY2R (Grassi et al., 2012), UCP1 (Brondani et al., 2012) and PPARgamma2 (Ma et al., 2012) have been reported to be associated with DR risk in Caucasians, while ACE (Lu et al., 2012), RXR-gamma (Hsieh et al., 2011) and the genes TMEM217, MRPL14 and GRIK2 on chromosome 6 (Lin et al., 2013) have been reported to be associated with DR in Asians. For MTHFR (Niu & Qi, 2012) and eNOS (Cilensek et al., 2012; Ezzidi et al., 2008) and VEGF gene (Awata et al., 2002; Bonnefond et al., 2013; Suganthalakshmi et al., 2006), the gene effect in DR risk were observed in both Caucasians and Asians. Our study investigated 4 SNPs of CHN2, and we found that rs1002630, but not rs39059 which has been reported to be associated with DR in Chinese T2D subjects in China (Hu et al., 2011) was associated with DR risk in Chinese individuals in Taiwan. Because the subjects in these two studies were Han Chinese, the association of these SNPs with DR in other ethnicities is not known. Therefore, these study results need to be validated in other ethnic groups.
Table 2 Genotypic distribution and adjusted OR of the DR susceptibility SNPs in T2D subjects with and without retinopathy. Database SNP ID
Risk allele
Genotype (dominate model)
Subject with DR n (%)
Subject without DR n (%)
rs2023908
C
rs1002630
A
rs1362363
G
rs39059
G
AA AC + CC GG GA + AA AA AG + GG AA AG + GG
116 (67.5) 55 (32.2) 157 (91.8) 14 (8.2) 67 (39.2) 104 (60.8) 89 (52.0) 82 (48.0)
337 (61.5) 211 (38.5) 471 (85.9) 77 (14.1) 175 (31.9) 373 (68.1) 283 (51.6) 265 (48.4)
OR (95% CI) 1.00 (ref.) 0.78 (0.53–1.09) 1.00 (ref.) 0.545 (0.30–0.99)⁎ 1.00 (ref.) 0.73 (0.51–1.04) 1.00 (ref.) 0.98 (0.70–1.39)
aOR (95% CI) 1.00 (ref.) 0.66 (0.43–1.00) 1.00 (ref.) 0.45 (0.22–0.88)⁎ 1.00 (ref.) 0.66 (0.44–0.99)⁎ 1.00 (ref.) 1.07 (0.72–1.59)
aOR = adjusted OR after controlling for gender, SBP, WHR, duration of diabetes, HbA1C and DN. rs = nomenclature of single nucleotide polymorphisms. Abbreviation: T2D, type 2 diabetic; DR, diabetic retinopathy; SNP, single-nucleotide polymorphism; OR, odds ratio; CI, confidence interval; aOR, adjusted OR; SBP, systolic blood pressure; WHR, waist/hip ratio; DN, diabetic nephropathy. ⁎ p value b 0.05.
Please cite this article as: Chen, M., et al., Chimerin 2 genetic polymorphisms are associated with non-proliferative diabetic retinopathy in Taiwanese type 2 diabetic patients, Journal of Diabetes and Its Complications (2014), http://dx.doi.org/10.1016/j.jdiacomp.2014.04.009
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M. Chen et al. / Journal of Diabetes and Its Complications xxx (2014) xxx–xxx
Table 3 Genotype distribution and adjusted odds ratios of CHN2 gene in type 2 diabetic subjects with and without retinopathy. Risk allele
Genotype (dominate model)
T2D with DR NPDR n = 101 (%)
PDR n = 70 (%)
rs2023908
C
rs1002630
A
rs1362363
G
rs39059
G
AA AC + CC GG GA + AA AA AG + GG AA AG + GG
72 (71.3) 29 (28.7) 96 (95.0) 5 (5.0) 41 (40.6) 60 (59.4) 58 (57.4) 43 (42.6)
44 (62.9) 26 (37.1) 61 (87.1) 9 (12.9) 26 (37.1) 44 (62.9) 31 (44.3) 39 (55.7)
db SNP ID
T2D without DR n = 548(%)
aOR (95% CI) T2D with NPDR vs. without DR
T2D with PDR vs. without DR
337 (61.5) 211 (38.5) 471 (85.9) 77 (14.1) 175 (31.9) 373 (68.1) 283 (51.6) 265 (48.4)
1.00 (ref.) 0.60 (0.36–1.00) 1.00 (ref.) 0.25 (0.09–0.73)⁎ 1.00 (ref.) 0.63 (0.39–1.01) 1.00 (ref.) 0.87 (0.55–1.39)
1.00 0.77 1.00 0.87 1.00 0.68 1.00 1.44
(ref.) (0.42–1.41) (ref.) (0.38–2.03) (ref.) (0.37–1.25) (ref.) (0.80–2.60)
aOR = adjusted OR after controlling for gender, SBP, WHR, duration of diabetes, HbA1C and DN. rs = nomenclature of single nucleotide polymorphisms. Abbreviation: T2D, type 2 diabetic; DR, diabetic retinopathy; SNP, single-nucleotide polymorphism; OR, odds ratio; CI, confidence interval. aOR, adjusted OR; SBP, systolic blood pressure; WHR, waist/hip ratio; DN, diabetic nephropathy; NPDR, nonproliferative DR. PDR, proliferative DR. ⁎ p value b 0.05.
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Please cite this article as: Chen, M., et al., Chimerin 2 genetic polymorphisms are associated with non-proliferative diabetic retinopathy in Taiwanese type 2 diabetic patients, Journal of Diabetes and Its Complications (2014), http://dx.doi.org/10.1016/j.jdiacomp.2014.04.009
Contents lists available at ScienceDirect
Journal of Diabetes and Its Complications journal homepage: WWW.JDCJOURNAL.COM
Original Article
Chimerin 2 genetic polymorphisms are associated with non-proliferative diabetic retinopathy in Taiwanese type 2 diabetic patients Marcelo Chen a, b, c, Wun-Rong Lin a, Chieh-Hsiang Lu d, Ching-Chu Chen e, f, Yu-Chuen Huang f, g, Wen-Ling Liao h, i, 1,⁎, Fuu-Jen Tsai f, g, 1,⁎ a
Department of Urology, Mackay Memorial Hospital, Taipei, Taiwan Mackay Medicine, Nursing and Management College, Taipei, Taiwan c School of Medicine, Mackay Medical College, New Taipei City, Taiwan d Division of Endocrinology and Metabolism of Internal Medicine, Ditmanson Medical Foundation Chia-Yi Christian Hospital, Chia-Yi, Taiwan e Division of Endocrinology and Metabolism, Department of Medicine, China Medical University Hospital, Taichung, Taiwan f School of Chinese Medicine, China Medical University, Taichung, Taiwan g Genetics Center, Department of Medical Research, China Medical University Hospital, Taichung, Taiwan h Center for Personalized Medicine, China Medical University Hospital, Taichung, Taiwan i Graduate Institute of Integrate Medicine, School of Chinese Medicine, China Medical University, Taichung, Taiwan b
a r t i c l e
i n f o
Article history: Received 20 December 2013 Received in revised form 1 April 2014 Accepted 11 April 2014 Available online xxxx Keywords: Single nucleotide polymorphism CHN2 Diabetic retinopathy Type 2 diabetes Taiwanese
a b s t r a c t Aim: To investigate whether chimerin 2 (CHN2) genetic polymorphisms were associated with the susceptibility to diabetic retinopathy (DR) in Taiwanese individuals with type 2 diabetes. Methods: This case-control study comprised of 171 individuals with DR and 548 without DR. Four rs39059, rs2023908, rs1002630 and rs1362363 polymorphism of CHN2 were genotyped for each subjects. All subjects underwent a complete ophthalmologic examination, and basic information (age, gender, age at diagnosis of diabetes, and ocular history of the patient) was record. Several clinical parameters (systolic and diastolic blood pressure, waist and hip circumferences, body mass index levels, fasting glucose and HbA1c) were measured. Results: Logistic regressions were used to analyze odds ratios between SNPs and DR after controlling for gender, systolic blood pressure, waist and hip ratio, duration of diabetes, serum HbA1c levels and nephropathy classification. A protective effect of rs1002630 (GA + AA) and rs1362363 (AG + GG) [odds ratio (OR) (95% confidence interval) = 0.45 (0.22–0.88), 0.66 (0.44–0.99), respectively) was observed. Furthermore, the protective effect of rs1002630 was observed when compared subjects with non-proliferative DR with subjects without DR [OR = 0.25 (95%C.I. = 0.09–0.73)]. Conclusions: This study showed that the rs1002630 of CHN2 were associated with DR risk and non-proliferative DR risk in Taiwanese individuals with type 2 diabetes. Variations at this locus may contribute to the pathogenesis of DR. © 2014 Elsevier Inc. All rights reserved.
1. Introduction Diabetic retinopathy (DR) is one of the common microvascular complications in diabetic patients (Sheetz & King, 2002), and it is the
Conflict of Interest: There is no conflict of interest. Grant support: This study was supported by research grants from China Medical University Hospital (DMR-102-045) and China Medical University (CMU102-N-01), Taichung City, Taiwan and from Ditmanson Medical Foundation Chia-Yi Christian Hospital Research Program (R101-14), Chia-Yi City, Taiwan. ⁎ Corresponding authors: Genetic Center, China Medical University Hospital, No. 2, YuhDer Road, Taichung 404, Taiwan. Tel.: +886 4 22052121x2041; fax: +886 4 22053425. E-mail addresses: [email protected] (W-L. Liao), [email protected] (F-J. Tsai). 1 Wen-Ling Liao and Fuu-Jen Tsai contributed equally to this work.
leading cause of blindness in adults (Thylefors et al., 1995). The development of DR is associated with long duration of diabetes, elevated glycosylated hemoglobin levels, high blood pressure, and chronic hyperglycemia (Stratton et al., 2000). The pathogenesis of DR is complex, having a multifactorial biochemical pathogenesis, primarily due to altered glucose metabolism (Brownlee, 2001; Jerneld & Algvere, 1986). In diabetic patients, high glucose concentration can directly impair retinal endothelial cell barrier function, resulting in an increase in permeability, edema, and loss of blood retinal barrier integrity. Sequential events in this damage induce leukocyte infiltration, and retinal proteins become exposed to the immune system (Cai & Boulton, 2002). Several interconnecting biochemical pathways that are implicated in the progression of the DR have been raised, such as increased expression of growth factor-1 (IGF-1) and vascular
http://dx.doi.org/10.1016/j.jdiacomp.2014.04.009 1056-8727/© 2014 Elsevier Inc. All rights reserved.
Please cite this article as: Chen, M., et al., Chimerin 2 genetic polymorphisms are associated with non-proliferative diabetic retinopathy in Taiwanese type 2 diabetic patients, Journal of Diabetes and Its Complications (2014), http://dx.doi.org/10.1016/j.jdiacomp.2014.04.009
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permeability factor vascular endothelial growth factor (VEGF), accelerated formation of advanced glycation endproducts (AGEs) and subclinical inflammation and capillary occlusion (Ellis et al., 2013). However, the mechanism of how DR develops remains unclear. Moreover, it has become evident through familial aggregation studies that susceptibility to complications of diabetes such as DR also has a heritable component independent of glycemic control and duration of diabetes (Hallman et al., 2005; Rema et al., 2002). Since 1997, the Diabetes Control and Complications Trial Research Group first reported that the severity of DR may be influenced by familial (Anonymous, 1997); the possibly genetic effect on DR has been raised. To date, candidate gene association studies or genome wide association studies have reported that many genes are associated with the development of DR. VEFG is potent angiogenic, and vascular permeability factor and the polymorphism of the promoter and 5’untranslated region of VEGF gene are reported to be associated with DR (Awata et al., 2002; Petrovic, 2013). Chimerin 2 (CHN2) gene which plays a role in the proliferation and migration of smooth muscle cells (Maeda et al., 2006) has been reported to be associated with diabetic nephropathy (DN) in type 1 diabetes in European descent (Pezzolesi et al., 2009) and DN in T2D patients in Japanese (Maeda et al., 2010) and DR in T2D Chinese patient (Hu et al., 2011). In the present study, we investigated whether the polymorphism of CHN2 (Maeda et al., 2006) was associated with the susceptibility to DR in Taiwanese type 2 diabetic (T2D) patients. 2. Materials and methods 2.1. Subjects and data collection This is a cross-sectional study that involved 719 T2D patients aged over 20 years who were recruited from the China Medical University Hospital (CMUH), Taichung, Taiwan. Diabetes was diagnosed according to the medical records and fasting plasma glucose levels, by using the American Diabetes Association Criteria (Anonymous, 2013). Subjects with type 1 diabetes, gestational diabetes, and maturity onset diabetes of the young (MODY) were excluded from this study. All the participants were of Han Chinese origin, as the Han Chinese account for 98% of the population of Taiwan. All T2D subjects underwent a complete ophthalmologic examination, including corrected visual acuity, fundoscopic examination, and fundus photography. An expert ophthalmologist graded DR according to the American Academy of Ophthalmology proposed international scales for severity of clinical DR. Among 171 DR subjects, 70 subjects with proliferative DR (PDR) and 101 subjects with non-proliferative DR (NPDR) were identified. Data regarding age, gender, age at diagnosis of diabetes, and ocular history of the patient were collected from questionnaires. For each patient, systolic and diastolic blood pressure, waist and hip circumferences, and body mass index levels were determined, and blood samples were collected by venipuncture for genomic DNA isolation and serological tests, including fasting glucose, HbA1c at the time of enrollment in the study. Subjects with estimated glomerular filtration rate (eGFR) less than 60 were considered with diabetic nephropathy. The study was approved by the CMUH Institutional Review Board, and informed consent was obtained from all the study participants. 2.2. SNP selection and genotyping SNPs rs39059 which has been reported to be associated with diabetic complication from previous studies (Hu et al., 2011; Maeda et al., 2010) and three other SNPs rs2023908, rs1002630, rs1362363 of CHN2 were selected for genotyping. The minor allele frequency for these SNPs is more than 5% in our population, and the deviation from Hardy–Weinberg equilibrium was not observed for any SNP. For genotyping, the genomic DNA was extracted from peripheral blood leukocytes using the Genomic DNA kit (Qiagen), in accordance with the manufacturer's instructions. Genotypes of the patients were
determined using the ABI TaqMan® SNP genotyping assays (Applied Biosystems, Foster City, CA, USA) by using the commercial genotyping assays (ABI Assay ID: C_2568295_10 for rs2023908; ABI assay ID: C_11805351_10 for rs1002630; ABI assay ID: C_7601381_20 for rs1362363 and ABI assay ID: C_525465_10 for rs39059; Applied Biosystems). The reaction mixture for the real-time polymerase chain reaction (PCR) contained 10 ng of genomic DNA, 10 μ L TaqMan master mix, and 0.125 μ L of 40 × assay mix. PCR was performed in 96well plates on a thermal cycler (ABI 7700; Applied Biosystems). Reaction conditions were 60 °C for 30 s and 95 °C for 10 min, followed by 40 cycles at 95 °C for 15 s and 60 °C for 1 min. 2.3. Statistical analysis Characteristics and clinical data of subjects with DR and without DR were compared using the Student t-test for continuous variables and chi-square test for categorical variables. The genotype and allele frequency distributions in the polymorphisms between T2D subjects with and without were analyzed by the χ 2 test or Fisher exact test for differences in proportions. The odds ratio (OR) was calculated from genotype frequencies and allelic frequencies with 95% confidence interval (95% CI) by using logistical regression. The potential confounding factors, including the common risk factors, diabetes duration and serum HbA1c level and the one which was significant associated with outcome in univaraite models (gender, systolic blood pressure, waist–hip ratio and DN) were added in multivariate analysis. All statistical analyses were conducted using SPSS statistical software, version 17.0 (SPSS Inc., Chicago, IL, USA), and p value less than 0.05 (two-sided) was used as the level of significance. 3. Results Of the 719 T2D subjects, 171 (23.8%) had DR. Comparison of subjects with and without DR showed that the male to female ratios were similar (p = 0.418). Males with DR were diagnosed with T2D at a younger age than those without DR (p = 0.009). Subjects with DR were diabetic for a longer period of time than those without DR (p b 0.001). The serum HbA1c level and systolic blood pressure of subjects with DR were higher than those of subjects without DR (p b 0.001). Foveal thickness was higher in subjects with DR for both eyes (p b 0.001). The waist and hip ratio was significant higher in subjects with DR than those without DR (p = 0.013), but body mass index was not different between the two groups. The higher percentage of DR subjects with eGFR less than 60 compared to the subjects without DR (18.1% vs. 8.4%, p = 0.001) (Table 1). Table 2 shows the genotypic distribution of the SNPs in subject with and without DR. A higher frequencies of GG homozygotes at the rs1002630 variant was observed in the subjects with DR compared with the subjects without DR (91.8% vs. 85.9%, p value for chi square test was 0.04). After controlling for gender, duration of diabetes, serum HbA1c levels, SBP, WHR and DN in multivariate analysis, a protective effect of homozygotes and heterozygotes containing the A allele of the rs1002630 variants (adjusted OR = 0.45, 95% CI = 0.22–0.88) and the homozygotes and heterozygotes containing the G allele of rs362363 variant (adjusted OR = 0.66, 95% CI = 0.44–0.99) were observed when compared the subjects without DR with the subject with DR. Furthermore, the DR subjects were grouped into the subjects with NPDR and the subjects with PDR according to the scales for severity of clinical DR. And the genotypic distribution of the susceptibility SNPs was compared in the subjects with NPDR and PDR with the subjects without DR (Table 3). When compared with subjects without DR, subjects with NPDR had statistically significant lower frequencies of variant homozygotes and heterozygotes for A allele of rs1002630 (5.0% vs. 14.1%, p values for chi square test was 0.01). In multivariate analysis, the protective effect of the risk allele for rs1002630 were observed when compared the subjects with NPDR with the subjects
Please cite this article as: Chen, M., et al., Chimerin 2 genetic polymorphisms are associated with non-proliferative diabetic retinopathy in Taiwanese type 2 diabetic patients, Journal of Diabetes and Its Complications (2014), http://dx.doi.org/10.1016/j.jdiacomp.2014.04.009
M. Chen et al. / Journal of Diabetes and Its Complications xxx (2014) xxx–xxx Table 1 Characteristics and the clinical profile of type 2 diabetic patients with and without retinopathy.
Gender Male Female Age at diagnosis Male Female Diabetes duration (year) HbA1c (%) BMI (kg/m2) Systolic BP (mmHg) Diastolic BP (mmHg) W/R ratio Waist (cm) Hip (cm) Foveal thickness (μm) Oculus dexter Oculus sinister Retinopathy classification NPDR PDR DN classification eGFR≧60 eGFR b 60
T2D with retinopathy (n = 171)
T2D without retinopathy (n = 548)
86 (50.3%) 85 (49.7%)
295 (53.8%) 253 (46.2%)
46.7 ± 9.33 48.74 ± 9.28 14.83 ± 8.40 8.27 ± 1.42 25.27 ± 4.02 135.01 ± 18.50 74.61 ± 11.40 0.93 ± 0.07 89.89 ± 10.37 97.01 ± 8.13
49.8 ± 9.45 50.45 ± 8.52 8.34 ± 6.49 7.74 ± 1.40 25.10 ± 3.80 127.40 ± 16.05 76.37 ± 10.88 0.91 ± 0.06 88.64 ± 10.03 97.09 ± 7.59
0.009a 0.119a b0.001a b0.001a 0.617a b0.001a 0.075 a 0.013 a 0.159 a 0.908 a
207.37 ± 85.14 209.30 ± 81.72
182.38 ± 44.34 182.78 ± 42.15
b0.001a b0.001a
502 (91.6) 46(8.4)
b0.001
p-Value 0.418b
101 (59.1%) 70 (40.9%) 140(81.9) 31(18.1)
b
Abbreviation:T2D, type 2 diabetic; SD, standard deviation; BMI, body mass index; BP, blood pressure; W/R, waist over hip; DR, diabetes retinopathy; NPDR, non-proliferative DR; PDR, proliferative DR; DN, diabetes nephropathy; eGFR, estimated glomerular filtration rate a student's t-test. b chi-square test.
without DR in dominant model OR = 0.25, 95% CI = 0.09–0.73. However, the frequency of the risk allele (in recessive model) for these four markers was not statistically and significantly different when we compared the subjects with PDR with the subjects without DR.
4. Discussion DR is the common micro-vascular complication of T2D and the duration of T2D and serum HbA1c levels are known to be two major risk factors for DR. In our study, DR subjects were with a longer duration of T2D, higher serum HbA1c levels, higher systolic blood pressure, higher W/H ratio and higher percentage with DN; therefore, the variables were adjusted during statistical analysis. CHN2 gene, located on chromosome 7p15.3 is a member of the chimerin family which is ligand-activated Rac-specific GTPase-
3
activating proteins (Caloca et al., 2008). Chimerins are expressed in many human tissues, especially brain, pancreas, and insulin-sensitive tissues (Suliman et al., 2009) and could regulate the proliferation and migration of vascular smooth muscle cells which are important aspects of atherogenesis (Maeda et al., 2006). Its expression has been associated with high-grade gliomas (Yuan et al., 1995), breast cancer (Yang et al., 2005), and lymphoma (Nishiu et al., 2002). Furthermore, the CHN2 gene was identified to be a key element of proximal insulin signaling in vivo study (Suliman et al., 2009). Polymorphisms of the CHN2 gene have been reported to be associated with schizophrenia in men (Hashimoto et al., 2005), smoking (Barrio-Real et al., 2013) and diabetic complications (Hu et al., 2011; Maeda et al., 2010; Pezzolesi et al., 2009). In the present study, we reported that the CHN2 rs1002630 was significantly associated with DR and NPDR after adjusting for confounding factors. Although we failed to find the associations between these four SNPs with severity of DR in our population, it is possible due to small sample size in PDR group. Furthermore, one loci (rs39059) at CHN2 was reported to be associated with nephropathy in type I diabetic patients from previous genome-wide association study in European descent (Pezzolesi et al., 2009) and be replicated in DN in Japanese T2D subjects (Maeda et al., 2010). Hu et al. (Hu et al., 2011) showed that the rs39059 was associated with DR in Chinese T2D subjects in China (Hu et al., 2011). In the present study, we analyzed CHN2 rs39059 and rs2023908 which are highly linkage in our population. There is no significant association between these two SNPs with DR in our Taiwanese population. Also, no significant associations between these two SNPs with DN were observed in our limited diabetic nephropathy (DN) samples (no. of DN = 77) (results not shown). Further studies with larger sample size are necessary to elucidate the role of CHN2 in DR. Recent genetic epidemiologic studies have shown associations with DR risk in different ethnic groups. UTS2 (Okumus et al., 2012), CEP135 and NPY2R (Grassi et al., 2012), UCP1 (Brondani et al., 2012) and PPARgamma2 (Ma et al., 2012) have been reported to be associated with DR risk in Caucasians, while ACE (Lu et al., 2012), RXR-gamma (Hsieh et al., 2011) and the genes TMEM217, MRPL14 and GRIK2 on chromosome 6 (Lin et al., 2013) have been reported to be associated with DR in Asians. For MTHFR (Niu & Qi, 2012) and eNOS (Cilensek et al., 2012; Ezzidi et al., 2008) and VEGF gene (Awata et al., 2002; Bonnefond et al., 2013; Suganthalakshmi et al., 2006), the gene effect in DR risk were observed in both Caucasians and Asians. Our study investigated 4 SNPs of CHN2, and we found that rs1002630, but not rs39059 which has been reported to be associated with DR in Chinese T2D subjects in China (Hu et al., 2011) was associated with DR risk in Chinese individuals in Taiwan. Because the subjects in these two studies were Han Chinese, the association of these SNPs with DR in other ethnicities is not known. Therefore, these study results need to be validated in other ethnic groups.
Table 2 Genotypic distribution and adjusted OR of the DR susceptibility SNPs in T2D subjects with and without retinopathy. Database SNP ID
Risk allele
Genotype (dominate model)
Subject with DR n (%)
Subject without DR n (%)
rs2023908
C
rs1002630
A
rs1362363
G
rs39059
G
AA AC + CC GG GA + AA AA AG + GG AA AG + GG
116 (67.5) 55 (32.2) 157 (91.8) 14 (8.2) 67 (39.2) 104 (60.8) 89 (52.0) 82 (48.0)
337 (61.5) 211 (38.5) 471 (85.9) 77 (14.1) 175 (31.9) 373 (68.1) 283 (51.6) 265 (48.4)
OR (95% CI) 1.00 (ref.) 0.78 (0.53–1.09) 1.00 (ref.) 0.545 (0.30–0.99)⁎ 1.00 (ref.) 0.73 (0.51–1.04) 1.00 (ref.) 0.98 (0.70–1.39)
aOR (95% CI) 1.00 (ref.) 0.66 (0.43–1.00) 1.00 (ref.) 0.45 (0.22–0.88)⁎ 1.00 (ref.) 0.66 (0.44–0.99)⁎ 1.00 (ref.) 1.07 (0.72–1.59)
aOR = adjusted OR after controlling for gender, SBP, WHR, duration of diabetes, HbA1C and DN. rs = nomenclature of single nucleotide polymorphisms. Abbreviation: T2D, type 2 diabetic; DR, diabetic retinopathy; SNP, single-nucleotide polymorphism; OR, odds ratio; CI, confidence interval; aOR, adjusted OR; SBP, systolic blood pressure; WHR, waist/hip ratio; DN, diabetic nephropathy. ⁎ p value b 0.05.
Please cite this article as: Chen, M., et al., Chimerin 2 genetic polymorphisms are associated with non-proliferative diabetic retinopathy in Taiwanese type 2 diabetic patients, Journal of Diabetes and Its Complications (2014), http://dx.doi.org/10.1016/j.jdiacomp.2014.04.009
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M. Chen et al. / Journal of Diabetes and Its Complications xxx (2014) xxx–xxx
Table 3 Genotype distribution and adjusted odds ratios of CHN2 gene in type 2 diabetic subjects with and without retinopathy. Risk allele
Genotype (dominate model)
T2D with DR NPDR n = 101 (%)
PDR n = 70 (%)
rs2023908
C
rs1002630
A
rs1362363
G
rs39059
G
AA AC + CC GG GA + AA AA AG + GG AA AG + GG
72 (71.3) 29 (28.7) 96 (95.0) 5 (5.0) 41 (40.6) 60 (59.4) 58 (57.4) 43 (42.6)
44 (62.9) 26 (37.1) 61 (87.1) 9 (12.9) 26 (37.1) 44 (62.9) 31 (44.3) 39 (55.7)
db SNP ID
T2D without DR n = 548(%)
aOR (95% CI) T2D with NPDR vs. without DR
T2D with PDR vs. without DR
337 (61.5) 211 (38.5) 471 (85.9) 77 (14.1) 175 (31.9) 373 (68.1) 283 (51.6) 265 (48.4)
1.00 (ref.) 0.60 (0.36–1.00) 1.00 (ref.) 0.25 (0.09–0.73)⁎ 1.00 (ref.) 0.63 (0.39–1.01) 1.00 (ref.) 0.87 (0.55–1.39)
1.00 0.77 1.00 0.87 1.00 0.68 1.00 1.44
(ref.) (0.42–1.41) (ref.) (0.38–2.03) (ref.) (0.37–1.25) (ref.) (0.80–2.60)
aOR = adjusted OR after controlling for gender, SBP, WHR, duration of diabetes, HbA1C and DN. rs = nomenclature of single nucleotide polymorphisms. Abbreviation: T2D, type 2 diabetic; DR, diabetic retinopathy; SNP, single-nucleotide polymorphism; OR, odds ratio; CI, confidence interval. aOR, adjusted OR; SBP, systolic blood pressure; WHR, waist/hip ratio; DN, diabetic nephropathy; NPDR, nonproliferative DR. PDR, proliferative DR. ⁎ p value b 0.05.
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Please cite this article as: Chen, M., et al., Chimerin 2 genetic polymorphisms are associated with non-proliferative diabetic retinopathy in Taiwanese type 2 diabetic patients, Journal of Diabetes and Its Complications (2014), http://dx.doi.org/10.1016/j.jdiacomp.2014.04.009
