CPVL/CHN2 Genetic Variant Is Associated With Diabetic Retinopathy in Chinese Type 2 Diabetic Patients

We used a two-stage approach for this study. In stage 1, we recruited 1,276 type 2 diabetic patients from the Shanghai Diabetes Institute Inpatient Database of Shanghai Jiao Tong University Affiliated Sixth People’s Hospital (10,11) and examined them for diabetic retinopathy and nephropathy. These patients included 378 individuals with diabetic nephropathy but no retinopathy, 374 with diabetic retinopathy but no nephropathy, 244 with both diabetic retinopathy and nephropathy, and 280 control subjects. The control subjects were defined as having normoalbuminuria, no retinopathy, and having diabetes for >10 years. In stage 2, we recruited 590 type 2 diabetic patients from the Shanghai Diabetic Complications Study (12) and Shanghai Diabetes Institute Inpatient Database, including 209 patients with diabetic retinopathy and 381 patients with diabetes for >5 years and without retinopathy. The basic characteristics of the study population were shown in Table 1.

This study was approved by the institutional review board of Shanghai Jiao Tong University Affiliated Sixth People’s Hospital and in accordance with the principle of the Helsinki Declaration II. Written informed consent was obtained from each participant.

The 24-h albumin excretion rates (AERs) were measured in 3 consecutive days, and the mean value was recorded for each patient. Patients with AER <30 mg/24 h, 30 mg/24 h ≤ AER <300 mg/24 h, or AER ≥300 mg/24 h were classified as having normoalbuminuria, microalbuminuria, or proteinuria, respectively. Estimated glomerular filtration rate was calculated by using a formula developed by the Modification of Diet in Renal Disease study group with adjustment for Chinese ethnicity: 186 × (serum creatinine in μmol/L × 0.011)^–1.154 × (age in years)^–0.203 × (0.742 if female) × (1.233 if Chinese) (13).

Fundus photography was performed following a standardized protocol at the Department of Ophthalmology, Shanghai Jiao Tong University Affiliated Sixth People’s Hospital. Both eyes of each participant were photographed with a 45-degree 6.3-megapixel digital nonmydriatic camera (Canon CR6-45NM, Lake Success, NY). Level of retinopathy was defined according to the International Classification of Diabetic Retinopathy (14): mild nonproliferative diabetic retinopathy, moderate nonproliferative diabetic retinopathy, severe nonproliferative diabetic retinopathy, or proliferative diabetic retinopathy. The worse eye was recorded for each patient.

In stage 1, we genotyped 55 tagging SNPs that capture 95% of the common variants, including one reported SNP from each locus (CPVL/CHN2 rs39059, FRMD3 rs10868025, CARS rs451041, and IRS2 rs1411766). The SNPs showed associations to diabetic nephropathy or retinopathy and were genotyped in stage 2 samples. The genotyping was performed by using primer extension of multiplex products with detection by matrix-assisted laser desorption ionization – time of flight mass spectroscopy using a MassARRAY Compact Analyzer (Sequenom, San Diego, CA).

The Hardy-Weinberg equilibrium test was performed before the association analysis. The allelic frequencies between the diabetic patients with or without complications were compared by χ² tests using PLINK (v1.07) (15), and odd ratios (ORs) with 95% CIs were presented. Combined ORs from different studies were calculated by using a Comprehensive Meta Analysis (v2.2.057) with a fixed- or random-effect model after testing for heterogeneity. The test for homogeneity was assessed by the Cochran Q test. The genotype-disease association analyses were performed under the additive model by logistic regression with adjustment of confounding factors. The effects of SNPs on the levels of retinopathy severity were analyzed by trend analysis. Skewly distributed quantitative traits (estimated glomerular filtration rate and AER) were logarithmically transformed to approximate univariate normality before analysis. Quantitative traits were analyzed by linear regression with adjustment of confounding factors under an additive genetic model. The statistical analyses were performed using SAS for Windows (version 8.0; SAS Institute, Cary, NC) unless specified otherwise. A two-tailed P value of <0.05 was considered statistically significant.

On the basis of the previously reported effect size of these loci (∼1.40) (8), our stage 1 samples (∼600 case subjects vs. 600 control subjects) had >90% power to replicate the reported effects of SNPs with minor allele frequencies >0.2 and 75% power to replicate the effect SNP with minor allele frequency of 0.1 at a level of significance of 0.05.

We firstly analyzed the effects of these SNPs on diabetic retinopathy and nephropathy in stage 1 samples (Supplementary Tables 1 and 2). As shown in Table 2, four CPVL/CHN2 SNPs (rs39059, rs17756941, rs245955, and rs245962) and FRDM3 rs10868025 were nominally associated with diabetic retinopathy (P < 0.05), with rs39059 and rs10868025 showing the strongest association within each locus (OR 0.774, 95% CI 0.657–0.912, P = 0.002, for rs39059 G allele; 0.833, 0.703–0.987, P = 0.034, for rs10868025 G allele). However, none of the genotyped SNPs showed a significant association to diabetic nephropathy.

To further validate the effects of rs39059 and rs10868025 on diabetic retinopathy, we genotyped both SNPs in stage 2 samples. We found only CPVL/CHN2 rs39059 showed similar effects to diabetic retinopathy, as identified in the first stage (OR 1.269, 95% CI 0.989–1.628, P = 0.061, for rs39059; 1.014, 0.789–1.302, P = 0.9147, for rs10868025). We then performed a meta-analysis with the fixed-effect model and found rs39059 was associated with diabetic retinopathy, with an OR of 1.285 (1.120–1.474, P = 0.0003) (Table 3). This association remained significant after adjusting confounding factors, including hemoglobin A_1c levels, duration of diabetes, systolic and diastolic blood pressure, and BMI (OR 1.242, 1.074–1.437, P = 0.0034).

We then analyzed the effects of CPVL/CHN2 rs39059 on the disease severity of diabetic retinopathy in all the samples. As shown in Fig. 1, CPVL/CHN2 rs39059 showed an association to the levels of diabetic retinopathy, with the risk allele more frequent in the more severe retinopathy patients (P = 0.0007 for trend analysis).

In the current study, we analyzed the effects of SNPs from four loci on diabetic retinopathy and nephropathy in Chinese type 2 diabetic patients. We first reported that CPVL/CHN2 rs39059 was associated with diabetic retinopathy. Although the association we observed was solid by replication in independent samples and the P value remained significant after Bonferroni correction of analysis on multiple SNPs (corrected P = 0.033, on the basis of the association analysis between 55 SNPs and two traits), we still cannot fully exclude the possibility that the association detected was false positive because of the relatively small sample size of this study. But considering we also found an association between rs39059 and disease severity in our samples, which supported the role of this locus in diabetic retinopathy from another aspect, the chance of a false-positive finding was limited. Although this locus was originally identified to be associated with diabetic nephropathy in a previous genome-wide association study (since the concordance rate of retinopathy and nephropathy was high in diabetic patients [16] and the genome-wide association study in the GoKinD samples (8) did not exclude the patients with retinopathy), CPVL/CHN2 may be a susceptible locus of diabetic retinopathy other than nephropathy. However, whether this effect is restricted to type 2 diabetes is still unknown and needs to be investigated in studies with type 1 diabetic patients.

The SNP rs39059 locates in the intron of CHN2, which encodes β-2 chimerin that have been shown to regulate cell growth, proliferation, and migration (17). Previous studies showed decreased expression of CHN2 is associated with high-grade malignant gliomas, breast cancer, and duodenal adenocarcinoma, whereas increased expression of CHN2 is reported to be associated with lymphomas (17,18). One other gene within the same haplotype block of rs39059 is CPVL. It encodes a carboxypeptidase that cleaves a single amino acid from the COOH termini of peptides (19). However, the function of this gene is still largely unknown. Although hemoglobin A_1c levels and systolic blood pressure were also associated with diabetic retinopathy in our samples (P = 0.0065 for systolic blood pressure and P = 7.0 × 10⁻⁹ for hemoglobin A_1c), since rs39059 showed association to none of these traits (P = 0.5031 for systolic blood pressure and P = 0.1561 for hemoglobin A_1c), our data suggest this locus did not participate in the pathogenesis of diabetic retinopathy through the effects on blood pressure and glucose levels. Thus, the mechanism how this locus affected diabetic retinopathy susceptibility remains to be investigated. And the causal variant also remains to be identified by fine mapping studies.

In this study, we failed to replicate the associations between these four loci and diabetic nephropathy in Chinese type 2 diabetic patients, although we had enough statistical power. Because we used a tagging SNP approach and captured most of the common variants within these loci, it was unlikely that causal variants were captured by different SNPs in different populations. One possible explanation may be the complicated phenotypes in type 2 diabetic patients. In our study, >60% of the diabetic nephropathy patients also suffered from hypertension; although we statistically adjusted blood pressure as a confounding factor, the impact of hypertension cannot be ignored. In a previous replication study in Japanese type 2 diabetic patients, Maeda et al. (9) also failed to replicate the effects of most of these loci in four independent samples; thus, it is highly possible that the effects of these loci on diabetic nephropathy are restricted in the European descent population or type 1 diabetic patients. Further studies with type 1 diabetic patients are needed to confirm the effects of these loci.

In summary, we found CPVL/CHN2 rs39059 was associated with diabetic retinopathy in Chinese type 2 diabetic patients. Further studies are needed to replicate this finding.

This work was supported by grants from the National 973 Program (2011CB504001), the Project of National Natural Science Foundation of China (30800617), the major program of Shanghai Municipality for Basic Research (08dj1400601), the National 863 Program (2006AA02A409), the Shanghai Rising-Star Program (09QA1404400), “Chen Guang” Project (09CG07), and the Young Talent Program of Shanghai Municipal Health Bureau.

No potential conflicts of interest relevant to this article were reported.

C.H. researched data, contributed to discussion, and wrote the manuscript. R.Z. and W.Y. researched data. J.W. contributed to discussion and wrote the manuscript. C.W. researched data. C.P. contributed to discussion. X.M. researched data. Y.B. and K.X. contributed to discussion. W.J. contributed to discussion and reviewed and edited the manuscript.

The authors thank all the research participants and the nursing and medical staff at Shanghai Clinical Center for Diabetes for their dedication and professionalism in conducting this study.