In our case-control study, we did not find evidence of an association between EPO gene polymorphisms and DR in patients with T2DM from Southern Brazil. However, the meta-analysis showed that the G allele of the rs1617640 was associated with protection for both PDR and NPDR. In the subgroup analyses by type of diabetes and ancestry, the G allele was also associated with a decreased risk of DR (PDR + NPDR) among patients with T1DM and among those of non-Asian ancestry. No other associations were detected.
Regarding the rs1617640 polymorphism, the findings of our case-control study are in accordance with most of the previous individual studies, which reported no association between this genetic variant and DR in Indian , Chinese [14, 16], and Italian T2DM patients , as well as in five different cohorts of subjects of white European ancestry with T1DM [11, 13, 15]. The rs1617640 polymorphism was also not associated with time to development of severe DR in a large cohort of T1DM patients followed for over 15 years (from the WESDR + DCCT/EDIC studies) . However, other studies have found opposing results [10, 11, 17, 19, 20]. The T allele was associated with an increased risk of PDR in three European-American cohorts of T2DM and T1DM patients from different geographic areas in the United States  and with DR in North Indians with T2DM . On the other hand, the G allele was associated with the increased risk of DR in T2DM patients from Australia , China , and Slovenia . Thus, differences in ethnicity and type of diabetes do not seem to explain the discrepancies between these studies.
When all the available genotype data were pooled in the meta-analysis, the between-heterogeneity was substantial, and the random-effects model revealed no association between the rs1617640 polymorphism and DR. The asymmetry seen in the funnel plots, and not confirmed by statistical test, can be attributed to neither publication bias nor small study effects [34−37] because most of the studies did not find an association between the rs1617640 variant and DR, and two of the subject sets that lied outside the majority of funnel plots and had shown the strongest association had sample sizes of more than 1100 individuals [set #2 of 10, 17]. Apart from this, genotype frequencies were not in HWE among controls in three studies [set #1 of 11, 17, 19]. Departures from HWE may occur due to several reasons other than genotyping errors, such as population stratification and selection bias in the enrollment of controls [34, 35]. Despite these considerations, HWE has been used as the main parameter of post-genotyping quality control in association studies.
Therefore, following the standard recommendations for meta-analyses of gene-disease associations [34−36], we removed the sets in which genotype frequencies were in Hardy–Weinberg disequilibrium to repeat the overall analysis and perform the subgroup analyses for the rs1617640 polymorphism to identify the possible causes of between-study heterogeneity. Heterogeneity was still moderate to high for most genetic models in the PDR, NPDR, and non-Asian subgroup analyses, while it was low or null among T2DM, T1DM, and Asian patients. The subject set #2 of Tong et al. , which contributed 20.6% of the total sample size in the overall meta-analysis, seemed to be the main factor contributing to the heterogeneity across the studies and also contributed to a statistically significant association between the rs1617640 polymorphism and DR in PDR, NPDR, T1DM, and non-Asian subgroups.
An issue that may raise some criticism regarding the results is the fact that our case-control study did not detect an association between the rs1617640 polymorphism and DR, whereas our meta-analysis revealed an association of the G allele with a reduced risk of DR. This is not unexpected. First, the meta-analysis involves a larger number of subjects, therefore it is more powerful than a single study to detect an association of low magnitude. Second, the association with either one of the two alleles, or the lack of it, may be population-specific. Third, although the type of diabetes did not seem to explain the discrepant results across the individual studies, the meta-analysis showed that the G allele of the rs1617640 polymorphism was associated with a decreased risk of DR under all the genetic models in T1DM patients, while no association was observed in T2DM patients. Thus, the results of our case-control study are actually in agreement with those obtained in the meta-analysis.
Moreover, the findings of our meta-analysis regarding the rs1617640 polymorphism are in line with those reported in a previous study, in which the TT genotype was associated with an increased risk of DR, as compared to the GG genotype, with a similar magnitude of association . In their analyses, without the cohorts of Tong et al. , no association between the rs1617640 polymorphism and DR was observed in any genetic model, as well as in T2DM and Asian populations . In addition to four peer-reviewed studies published between 2008 and 2014, also included in our meta-analysis [10−12, 14], the overall analysis performed by Li et al.  considered the data from two theses for master’s degrees but did not include studies published from 2015 onwards.
Although both meta-analyses indicate the existence of an association between the rs1617640 polymorphism and DR, the actual biological model that describes such relationship is yet to be defined. The G allele was associated with DR under a recessive model in four studies [11, 17, 19, 20]. However, in our overall meta-analysis, the G allele was associated with a decreased risk of DR under the dominant, heterozygous additive, and overdominant genetic models. This is in line with the original report by Tong et al. , which suggested an additive allele–dosage effect for the rs1617640 polymorphism. To the best of our knowledge, those authors were the only ones who performed functional assays and prediction analysis to evaluate the effect of the rs1617640 variant on gene expression. The T allele markedly increased the EPO expression in cultured human embryonic kidney (HEK) 293 cells, and the computational analysis suggested that the T allele creates a transcriptional binding site, which likely accounted for the enhanced expression as compared with the G allele. Moreover, vitreous levels of EPO were much higher in non-diabetic subjects with the TT genotype than in those with the GG genotype . Taken together, these findings suggest that high levels of EPO are associated with DR, especially PDR, and the T allele of the rs1617640 is likely a risk factor for DR as it increases the gene expression. This is consistent with experimental evidence showing that exogenous EPO protects against early DR, but it is detrimental in PDR [4−6].
In relation to the rs507392 and rs551238 polymorphisms, available data are scarcer [10, 11, 14, 16−19]. Not all the studies discussed so far have investigated the association of these two genetic variants with DR and, among those that examined such an association, not all reported the genotype data. The findings of the previous studies are varied, even in populations with the same ethnicity. Under the recessive and homozygous additive models, the C allele of both polymorphisms was strongly associated with an increased risk of DR in Australian  and Chinese  T2DM patients, whereas it was strongly associated with a decreased risk of DR in another population of Chinese T2DM patients . In addition, the C allele of the rs507392 polymorphism was associated with a decreased risk of DR in North Indian T2DM patients, whereas the C allele of the rs551238 was not associated with this complication . In contrast, the C allele of the rs551238 polymorphism was less frequent in patients with DR than in those without this complication, while the rs507392 polymorphism was not associated with DR in the cohort of Utahns (USA) of European ancestry with T2DM . Similar to our case-control study, the rs507392 and rs551238 polymorphisms were not associated with DR in T1DM patients from Australia  and in T2DM patients from China  and Italy . Our meta-analysis, including either all the studies or only the subject sets in which the control groups met the HWE, revealed no association of the rs507392 and rs551238 variants with DR. It is worth mentioning that Li et al.  did not perform the meta-analysis for the rs507392 polymorphism because they considered only the study by Abhary et al.  as having DR as an outcome. As for the rs551238 polymorphism, the pooled analysis of three studies (only one common to ours) showed that the A allele was associated with a reduced risk of DR .
Although the rs1617640, rs507392, and rs551238 polymorphisms were in high LD with each other in T1DM and T2DM patients [10, 11, 14, 16, 19, present study], findings regarding an additional combined effect of the three EPO polymorphisms in the susceptibility of DR are also inconclusive. No evidence of an association between EPO haplotypes and DR was found in our case-control study as well as in two Chinese T2DM populations [14, 16]. However, the GCC haplotype was reported to be independently associated with an increased risk of DR under a recessive model in T2DM, but not in T1DM, in a white Australian population . In another study of Chinese T2DM patients, the strongest relationship was observed for the carriership of at least one copy of the minor allele of each polymorphism (GCC) in comparison to the homozygosity for the three major alleles . It is worth noting that risk and protective haplotypes were identified in the cohorts studied by Tong et al. , and the main difference between them was the rs1617640 polymorphism. Risk haplotypes carried the T allele, while the G allele was present in the protective haplotypes, irrespective of the alleles at the other two polymorphisms . On the other hand, a recent study on North Indians with T2DM reported that the main source of the association between the TTA haplotype and DR was the T allele of the rs507392 polymorphism . However, our meta-analysis detected no association between the GCC haplotype and DR.
In general, the studies included in our meta-analysis can be considered of good quality as suggested by the scoring scale used for this purpose (NOS). However, specific guidelines for genetic association studies have focused on the HWE test in controls as a means of assessing study quality [34−36] and on the phenotyping, blinding, validity of genotyping method, and population stratification [21, 34]. A critical aspect related to the methodological quality of the previous studies is the lack of blinding and re-genotyping as quality control procedures in half of them [16, 17, 19, 20]. In the other studies, at least one procedure to improve the genotyping accuracy was reported, such as the re-genotyping of part of the samples by sequencing , sequencing of some samples for each genotype at each polymorphism , and genotype reading by two investigators blinded to the sample phenotypes . Other authors, who genotyped the samples for EPO polymorphisms using the Sequenom technology, described a battery of quality control tests [14, 18]. In addition, retinopathy grading was reported to have been performed without prior knowledge of genotypes in one study .
Population stratification is unlikely to have been a confounding factor [21, 42] in the previous studies since the authors enrolled subjects from populations with a majority ethnic group (> 90%) [10, 11, 14, 16, 17, 19, 20], used the ancestry from a given region as one of the inclusion criteria [10, 18] or matched the cases and controls by ethnicity . In the study by Abhary et al. , there was no difference in the allele frequencies of EPO polymorphisms among white subjects of European ancestry and non-white subjects of Asian and Middle Eastern ancestry. In our case-control study, the genotype and allele frequencies were virtually identical in white and non-white subjects.
On the other hand, the unavailability of all genotype data that could be incorporated in the quantitative synthesis is the main limitation of our meta-analysis. Some of the previous original studies were published without reporting the genotype frequencies of the polymorphisms under investigation. In addition, data reported in some papers were unclear, inaccurate, or did not match the sample size described in the text or in the tables. Despite our attempt to obtain all the missing genotype data by e-mail, they were still missing for the rs1617640 [13, 15], rs507392, and rs551238  polymorphisms. Hence, we included most, but not all the previous studies that examined the association of EPO polymorphisms with DR and met the screening criteria. Although we could perform subgroup analyses by the severity of DR, considering NPDR, and PDR as separate outcomes, it was also limited by the lack of this information in most of the eligible studies. In future studies, authors, as well as the reviewers and editors, should be aware that the complete description of genotype frequencies and outcomes is essential to allow for comparisons across the studies and perform pooled association analyses.