Associations between erythropoietin polymorphisms and risk of diabetic microvascular complications

We conducted a meta-analysis to evaluate the relationship between erythropoietin (EPO) polymorphisms and diabetic microvascular complications. We searched the PubMed, Embase, Cochrane library, Web of Science, Wanfang, and Chinese National Knowledge Infrastructure databases for appropriate studies. Odds ratios (ORs) with 95% confidence intervals (CIs) were calculated to evaluate the associations. Ultimately, eight studies consisting of 2,861 cases and 2,136 controls were identified and included in our meta-analysis. Results with our genotype model indicated an association between rs1617640 polymorphisms and diabetic microvascular complications (TT vs. GG: OR = 1.544, 95% CI = 1.089–2.189, P = 0.015). No clear associations between the rs1617640 and rs507392 polymorphisms and diabetic retinopathy were observed. By contrast, rs551238 polymorphisms were associated with increased diabetic retinopathy risk (allele model: OR = 0.774, 95% CI = 0.658–0.911, P = 0.002; genotype model: AC vs. CC: OR = 0.598, 95% CI = 0.402–0.890, P = 0.011; dominant model: OR = 0.561, 95% CI = 0.385–0.817, P = 0.003; recessive model: OR = 0.791, 95% CI = 0.643–0.973, P = 0.026). These results indicate that EPO polymorphisms are a risk factor for diabetic microvascular complications.

Erythropoietin (EPO) is a kidney-derived peptide hormone that plays a major role in the stimulation of bone marrow stem cells and erythropoiesis [5][6][7]. EPO can promote retinal angiogenesis independently of VEGF in proliferative diabetic retinopathy [8,9]. In diabetic retinas, EPO protects retinal cells by upregulating ZnT8 via ERK pathway activation and HIF-1α expression inhibition [10], and EPO may slow progression of diabetic nephropathy (DN) [11,12]. EPO can also improve cardiac function by suppressing endoplasmic reticulum stress and inducing SERCA2a expression [13].
Several studies have examined associations between EPO polymorphisms and diabetic complications; however, the results have been inconsistent. Fan, et al. suggested that erythropoietin polymorphisms increased diabetic retinopathy risk [14]. Conversely, Balasubbu, et al. found no significant association [15]. Thus, our study investigated relationships between single-nucleotide polymorphisms (SNPs) in the EPO gene and diabetic complications. Figure 1 shows our study selection process. In total, 688 studies were retrieved from the PubMed, Embase, Cochrane library, Web of Science, Wanfang, and Chinese National Knowledge Infrastructure databases. Of these, 151 duplicates were excluded from this study. Another
Hardy-Weinberg equilibrium (HWE) was assessed via Chi-square test in the control subjects of each study. Genotype distributions among controls were consistent with HWE in only eight studies [15][16][17][18][19][20]. These eight studies, which investigated three SNPs (rs1617640, rs507392 and rs551238), were included in the final metaanalysis. The eight studies included 2,861 cases and 2,136 controls. Five studies focused on diabetic retinopathy, and three focused on diabetic retinopathy and end-stage renal disease. Four examined patients with type 2 diabetes mellitus (T2DM), and one examined patients with type 1 diabetes mellitus (T1DM). Four included patients of Asian descent, three included patients of American descent, and one included patients of Australian descent. According to the Newcastle-Ottawa quality assessment scale (NOS), quality scores ranged from 6-7, and the median score of the case-control studies was 6.5.

EPO rs551238 quantitative analyses
Three studies were eligible for the EPO rs551238 meta-analysis [14,16,20]. An association was found between EPO rs551238 polymorphisms and diabetic retinopathy (allele model:

Publication bias and sensitivity analysis
Begg's funnel plot and Egger's test were employed to detect publication bias in this meta-analysis. We found no evidence of publication bias, with the exception of rs1617640 (TT vs. GG, TT vs. TG+GG), which exhibited a slight publication bias ( Figure 5, Table 3). A sensitivity analysis was performed to examine the influence of excluding each study on the pooled ORs. The overall effects were not altered when each study was omitted, suggesting that our meta-analysis results were reliable ( Figure 6).

DISCUSSION
EPO is located on chromosome 7q21 and encodes a potent angiogenic factor expressed in the retina and kidney [23][24][25]. Several studies have assessed the impacts of EPO single-nucleotide polymorphisms (SNPs) on diabetic microvascular complications; however, the results have been inconsistent. We performed this meta-analysis to obtain conclusive results regarding the relationship between EPO polymorphisms and diabetic microvascular complications. We evaluated the most commonly investigated EPO polymorphisms: rs1617640, rs507392, and rs551238. Our results indicate that EPO polymorphisms are a risk factor for diabetic microvascular complications.
We searched several databases for relevant studies assessing the association between the EPO polymorphisms and diabetic complications. Our analysis included eight studies with a combined total of 2,861 cases and 2,136 controls. These studies were conducted between 2008 and 2015 to investigate the association between EPO polymorphisms and diabetic retinopathy and nephropathy. Tong, et al. suggested an association between the T allele of SNP rs1617640 and proliferative diabetic retinopathy as well as end-stage renal disease [19]. Gong associated higher diabetic retinopathy risk with carrying the TT/GT genotype and T allele at the EPO gene site rs1617640 [20]. Abhary, et al. concluded that all three EPO SNPs (rs507392, rs1617640, and rs551238) were associated with diabetic retinopathy risk independent of duration of DM, degree of glycemic control, and nephropathy [16]. However, some studies found no associations [15]. In our study, the pooled results indicated a relationship between rs1617640 polymorphisms and diabetic complications (genotype model: TT vs. GG: OR = 1.544, 95% CI = 1.089-2.189, P = 0.015).
Our subgroup study stratified by complication type showed that rs1617640 polymorphisms were associated with diabetic retinopathy and end-stage renal disease. However, the association with diabetic retinopathy was not significant in any model. Only one study evaluated T1DM in Australian populations, so we were not able to evaluate statistical significance in this case.
Only one study investigated rs507392 polymorphisms, and found no association between these polymorphisms and diabetic retinopathy. Three studies investigated the association between EPO rs551238 polymorphisms and diabetic retinopathy risk, with inconsistent results. We pooled the results and found a relationship between rs551238 polymorphisms and diabetic retinopathy.
Our meta-analysis had some limitations. First, the number of included studies was relatively small. rs1617640 polymorphisms were examined in eight studies, and rs507392 and rs551238 polymorphisms were investigated in one and three studies, respectively. Previous studies demonstrated that EPO variations played roles in DR and DN development [19,26], and different results were found for T2DM and T1DM. Thus, we performed a subgroup study stratified by complication type, DM type, and ethnicity. However, some of our stratified analyses were limited by small sample sizes. Three studies focused on DR and DN. Only one study assessed T1DM in an Australian population. There were some differences between cases and controls, including patient age, gender, duration of DM, HbA1c level and BMI (Table 2). Stratification analyses based on differences in DM duration, gender, or other factors could not be performed because of limited sample sizes. Data from large, multi-center studies, including DM severity, ethnicity, and other complications and factors, are needed to confirm these relationships. Second, due to the limited number of studies, we did not investigate publication bias for the rs551238 polymorphism. A small publication bias was detected for the rs1617640 polymorphism in some models (TT vs. GG, TT vs. TG + GG), indicating that some unpublished studies might have been overlooked. Third, heterogeneity was found between the rs1617640 polymorphism studies; however, we could not conduct subgroup analyses to investigate the heterogeneity source due to the limited number of studies. Finally, we did not examine other risk factors, such as environmental effects and genetic factors.
In summary, we found that EPO polymorphisms are a risk factor for diabetic microvascular complications. rs1617640 polymorphisms are associated with increased risk of diabetic retinopathy and nephropathy, and rs551238 polymorphisms are associated with increased risk of diabetic retinopathy.

Inclusion and exclusion criteria
All selected studies in this analysis met the following criteria: (1) case-control studies; (2) explored the correlation between EPO polymorphisms and diabetic microvascular complications; and (3) contained sufficient data for calculating odds ratios (ORs) and 95% confidence intervals (95% CIs). Exclusion criteria were as follows: (1) studies without Hardy-Weinberg equilibrium (HWE) in the control groups; (2) studies not relevant to diabetic complications or lacking a control population; and (3) studies lacking sufficient data for quantitative analyses.

Data extraction and quality assessment
Two investigators (Li H and Xu HP) independently reviewed all articles and extracted available data from individual studies. Any discrepancies were resolved by discussion between these two investigators. The following information was extracted from each eligible study: (1) first author, (2) year of publication, (3) country of origin and ethnicity of study participants, (5) type of diabetes mellitus and complications, (6) genotyping method, (7) number of cases and controls, (8) polymorphism and genotype distribution, and HWE for controls. We assessed the quality of each eligible study according to the Newcastle-Ottawa quality assessment scale (NOS) [27]. Quality score ranged from 0-9.

Statistical analysis
HWE was assessed via Chi-square test in the control populations of each study. Pooled ORs and corresponding 95% CIs were calculated to estimate associations between EPO polymorphisms and diabetic microvascular complications. Z test was used to assess the overall effect and P < 0.05 was considered statistically significant. The strength of the association was determined using the following models: allele model (T vs. G, T vs. C, A vs. C), genotype model (TG vs. GG, TT vs. GG, TC vs. CC, TT vs. CC, AC vs. CC, AA vs. CC), dominant model (TT/TG vs. GG, TT/ TC vs. CC, AA/AC vs. CC), and recessive model (TT vs. TG/GG, TT vs. TC/CC, AA vs. AC/CC). Subgroup analyses were conducted according to ethnicity, type of diabetes mellitus, and complication. We used the Q-statistic and I 2 statistic to evaluate statistical heterogeneity among studies [27]. P ≥ 0.1 and I 2 < 50% suggested a lack of heterogeneity among studies. A random-effect model was used through the DerSimonian and Laird method in the presence of heterogeneity (P < 0.1 or I 2 > 50%); otherwise, a fixed-effect model based on the Mantel-Haenszel method was employed (P ≥ 0.1 or I 2 < 50%). We conducted a sensitivity analysis to assess the stability of the results. Egger's test and Begg's funnel plot were used to evaluate publication bias. Analyses were performed using STATA version 11.0 software (Stata Corporation, College Station, TX, USA).