https://orcid.org/0000-0002-6977-2928
ABSTRACT
Diabetes-related retinopathy (DR) is one of the sight-threatening diseases. The present case–control study screened 134 patients with type 2 diabetes and 36 healthy ones without diabetes. The genotyping of SNPs rs2010963 and rs699947 of the VEGF gene were done by tetra primers ARMS-PCR. The mutant GG genotype of rs2010963 was significantly associated with DR (OR = 10.29; 95% CI = 2.20–59.06; p = 0.004). No significant associations existed between patients with DR and those with NDR or controls in the genotype or allele frequencies of rs699947 polymorphism. The haplotype analysis for rs2010963 and rs699947 shows the CG haplotype was significantly different between DR and NDR OR 2.0 (95% CI:1.02–3.93), p = 0.043. and between DR and control OR 2.42 (95% CI: 1.2–4.65), p = 0.0108. The two SNPs showed moderate Linkage disequilibrium (LD) between DR and control, D’ = 0.64 also, moderate LD between DR cases and NDR, D’ = 0.75. No significant importance for rs699947 SNP with diabetic retinopathy. The 2010963 SNP in the VEGF gene is associated with the risk of NPDR and PDR. The findings also suggest a moderate LD in the two SNPs and their CG haplotype associated with DR progression.
Introduction
Diabetes mellitus is a widespread disease associated with high morbidity rates [Citation1,Citation2]. Diabetes results in various consequences, classified as macrovascular (in a large blood vessel) problems, such as heart disease and stroke, and microvascular (in a small blood vessel) diseases, such as kidney disease. Diabetic retinopathy (DR), a microvascular complication of diabetes, is the leading cause of vision loss among the general population in different countries, notably among the working-age population and the elderly [Citation3,Citation4]. The disease of DR occurs due to damage to the retina as a consequence of diabetic mellitus complications that lead to permanent damage to the eyes and sometimes even vision loss [Citation5]. About 415 million individuals across the globe were diagnosed with diabetes in 2015, with estimates indicating that the number would increase to 642 million by the year 2040 [Citation6]. Due to the rise in the number of individuals with diabetes and the increased duration of the disease, diabetic retinopathy, and eye disease are growing globally [Citation7,Citation8].
Diabetic Retinopathy (DR) is a leading cause of blindness. DR mutilates a diabetic patient’s retinal blood vessels. The DR is classified into two main subtypes: non-proliferative diabetic retinopathy disease (NPDR) and proliferative diabetic retinopathy disease (PDR) [Citation9]. During the early stages, the disease is known as NPDR, which is classified into three subcategories: mild, moderate, and severe. One microaneurysm (MA), a tiny red dot on the terminal of blood vessels, is seen in the mild stage. The MAs ascend to a deeper layer of the retina in the Moderate stage and create a flame-shaped hemorrhage. The severe stage is characterized by more than 20 intraretinal hemorrhages in each of the four quadrants, with obvious venous bleeding and significant intraretinal microvascular changes in the affected retina. When DR progresses to the advanced stage of the disease, it results in neovascularization, the natural development of new blood vessels in the form of functioning microvascular networks that grow on the surface of the retina, and the disease known as PDR [Citation10].
The VEGF (VEGF) gene is located on chromosome 6 p 21.1. About 4 kb, the size of its coding region. The VEGF gene has eight exons that may be combined in different ways to produce a wide variety of mRNAs via alternative splicing. This gene’s polymorphism exceeds the 150 SNPs in exons or promoters [Citation11]. These SNPs are primarily clustered in the VEGF gene’s promoter and 5’-UTR [Citation12]. The 5’-UTR has many different binding sites for transcription factors, and the polymorphisms in this region cause gene expression changes [Citation13]. This study aimed to investigate whether DR patients had an association with genotypes of two SNPs in the VEGF gene and whether a specific set of variants of these SNPs inherited together.
Methods
Subject. Recruit 134 patients with diabetes and 36 healthy controls in a case–control study. The ophthalmologist evaluated the patient’s eyes, sought the presence of any signs of DR, and grouped them into 64 subjects with NPDR, 39 subjects with PDR, and 31 diabetic patients without retinopathy (NDR). The sociodemographic characteristics of patients were presented in our previous study [Citation14].
Selection criteria. Participants included those 18 years or older with a medical history consistent with type 2 diabetes under the criteria of the World Health Organization in its 2019 categorization standards [Citation15]. One or two eyes with any signs of the DR were included. Individuals affected by inflammatory retinal disorders (uveitis, retinal vasculitis) and all type 1 diabetes were excluded. Eight samples were excluded from statistical analysis because they did not give any variation; it is suspected they showed genotypes differently for rs2010963 (C > T instead of C > G).
Genotyping. Genomic DNA was extracted from whole blood in EDTA tubes using G-spin™ Total DNA Extraction Mini Kit (iNtRON Biotechnology, Korea) and stored at −20°C in the biotechnology laboratory of Babylon University until the genotyping. The genotyping of the SNPs in the study were tetra primers ARMS-PCR technique. In brief, the outer primers of rs2010963 and rs699947 SNPs generate the confirmative amplicon for the 5’ UTR and promoter region where the SNPs resides. The inner reverse primers were designed for the mutant allele by mismatching the 3’ end of the primer. Also, the third nucleotide of the 3’ end was mismatched to increase the specificity. The PCR steps were as follows: 40 cycles of denaturation at 95°C for 35 s and annealing at 65°C decreased gradually every cycle to reach the lowest annealing at 58°C for 40 s and 72°C for extension and final extension for 40 s and 7 min respectively. The PCR cycles are preceded by initial denaturation at 95°C for 2 min for activation. Followed by agarose gel electrophoresis (2% and 75 V) for 1 hour. The SNPgen® tool was used to design rs2010963 primers, whereas rs699947 primers were obtained from an Elfaki et al. study [Citation16], as shown in .
Table 1. Primers utilized in the detection of SNPs by the tetra primers ARMS–PCR.
Statistical analysis. The SPSS version 26.0 for Windows was used (Chicago, IL, USA). Genotype and allele frequency of VEGF polymorphisms were compared between the control group and the case groups using the Chi-square test [Citation17]. The binary and multinominal logistics were used to estimate the risk allele and genotype associated with DR, NPDR, and PDR. The haploview 4.2 (Broad Institute) program was used to calculate the haplotype frequencies of the VEGF rs2010963 and rs699947 SNPs and to build linkage disequilibrium (LD).
Results
The genotyping of rs2010963 and rs699947 using the tetra primer-ARMS PCR. The bands were presented as follows the band of the C allele is 140 bp., and the G allele band is 177 bp. The heterozygous (CG) is shown in the presence of the two bands (140 bp. and 177 bp.) for rs2010963. The bands of rs699947 were presented as follows the band of the A allele is 243 bp. The C allele band is 149 bp. The heterozygous (AC) is shown in the presence of the two bands (149 bp. and 243 bp.) .
Figure 1. Agarose gel electrophoresis for the tetra primer-ARMS PCR product for genotyping rs2010963 SNP. (75 V for 60 min, M: DNA marker 50–500 bp, 2% Agarose).
Figure 2. Agarose gel electrophoresis of the tetra primer-ARMS PCR for genotyping rs699947 SNP (75 V for 60 min, M: DNA marker 50–500 bp, 2% Agarose).
The genotypic and allelic frequencies of rs2010963 in the healthy control (HC), NDR, and DR groups are detailed in . The genotype distribution of rs2010963 was in Hardy–Weinberg equilibrium (HWE) in the control group and cases groups using Yate’s correction (p-value ≥ 0.054). The Chi-square test results demonstrated that the frequencies of genotypes and alleles among study groups were statistically significant, with p-value = 0.027 and 0.024, respectively. The results revealed that the heterozygote genotype (CG) has a higher frequency among all of the controls, NDR, NPDR, and PDR; the lowest frequency is for the CC genotype. The G Allele was a higher frequency in case groups; in contrast, the C allele was a higher frequency than the G allele in the control group (P = 0.024). The genotypic and allelic frequencies of rs699947 in the HC, NDR, and DR groups are detailed in . Neither the NPDR nor the PDR groups showed any significant differences in genotype or allele frequency for the rs699947 polymorphism compared to the control or NDR groups. The C allele frequency among case groups was 64.8% and 70.5%, whereas in the controls and NDR groups was 63.6% and 56.9%, respectively; thus, no significant differences were found between controls and cases regarding the C allele.
Table 2. Distribution of Alleles and genotypes of rs2010963 and rs699947 between Controls, NDR, NPDR and PDR.
The VEGF polymorphisms were selected for logistic regression analysis for the genetic association study. First; we made a binary logistic regression analysis of the comparison between control and DR subjects and NDR and DR subjects; for rs2010963, the results revealed that the homozygous GG genotype of minor alleles was significantly associated with DR (OR = 10.29; 95% CI = 2.20–59.06; p = 0.004). No statistically significant association with DR for the heterozygous CG genotype (). The second is a multinomial logistic regression analysis between groups of DR (NPDR and PDR), which once compared with the controls and then for the NDR group. The analysis showed that minor alleles (GG) were also strongly associated with NPDR and PDR (OR = 6.33; 95% CI = 1.20–33.39; p = 0.030) and (OR = 34; 95% CI = 2.9–392.94; p = 0.005) respectively (). However, the heterozygous CG genotype was unrelated to NPDR or PDR state (). In contrast, for rs699947, the heterozygous AC and homozygous (AA & CC) genotypes in binary logistic regression and multinomial logistic regression were statistically insignificant in DR status, and subtypes of DR (NPDR & PDR) , respectively.
Table 3. Binary Logistic Regression for rs2010963 & rs699947 SNP between Controls, NDR and DR.
Table 4. Multinomial Logistic Regression for rs2010963 & rs699947 SNP between Controls, NDR, NPDR and PDR.
Moreover, in the case of rs2010963, the GG genotype and recessive model (CC+CG vs. GG) frequencies were significantly higher in the DR compared with controls (OR = 0.10; 95% CI = 0.02–0.46; p = 0.002) and (OR = 5.63; 95% CI = 1.75–18.38; p = 0.003), respectively. Interestingly, when analyzing the association between groups of DR (NPDR and PDR) with controls and NDR, the dominant model (CC vs. CG+GG) was statistically significant only with PDR (OR = 8.44; 95% CI = 1.20–99.07; p = 0.026). In contrast, the recessive model (CC+CG vs. GG) was associated with NPDR and PDR subjects (p = 0.016 and 0.001, respectively) (). In contrast, for rs699947, the comparisons between DR status and subtypes of DR (NPDR & PDR) with controls regarding dominant, recessive, codominant, and heterozygous models revealed similar results (p-value > 0.05) for all models . Thus, our samples have no significant importance for this SNP with diabetic retinopathy.
Table 5. Association of Different Genetic Models for rs2010963 & rs699947 SNP between Controls, NDR, and DR.
Table 6. Association of Different Genetic Models for rs2010963 & rs699947 SNP between Controls, NDR, NPDR, and PDR.
Haplotype analysis and linkage disequilibrium. The haplotype analysis results and the Linkage disequilibrium pattern (LD) between VEGF polymorphisms (rs2010963 and rs699947) are shown in . and , respectively. The predicted haplotypes with frequencies ≥0.03 were compared between the DR, NDR, and control groups. The frequency of CG haplotype was statistically significant between DR and NDR (OR = 2.0; 95% CI = 1.02–3.93; p = 0.043), and also there were significant differences between DR and controls (OR = 2.42; 95% CI = 1.2–4.65; p = 0.0108). The CC haplotype appears to have significant differences between DR and control groups only (OR = 0.56; 95% CI = 0.33–0.99; p = 0.0479) (). Pairwise LD parameter D’ was 0.75 with r2 = 0.17 for DR and NDR groups. In contrast, D’ was 0.64 with r2 = 0.14 for DR and control groups. The findings suggest a moderate LD in two SNPs (rs2010963 and rs699947).
Table 7. Haplotyping of rs2010963 and rs699947 of VEGF among DR and NDR.
Table 8. Haplotyping of rs2010963 and rs699947 of VEGF among DR and control.
Figure 3. Linkage disequilibrium (LD) of rs2010963 and rs699947 SNPs in DR and NDR.(a)The pairwise LD between SNPs, using D’ (red color) value, (b)The pairwise LD between SNPs, using r2 (gray color) value, LD is represented by D’ or r2 multiplied by 100.
Figure 4. Linkage disequilibrium (LD) of rs2010963 and rs699947 SNPs in DR and control. (a)The pairwise LD between SNPs, using D’ (red color) value, (b)The pairwise LD between SNPs, using r2 (gray color) value, LD is represented by D’ or r2 multiplied by 100.
Discussion
In this study, we looked at the possibility that VEGF polymorphisms are linked to the existence of DR in a well-defined group of Iraqi patients with type 2 diabetes. VEGF serves as the primary regulator in both normal and abnormal vascular development. It has the potential to enhance retinal vascular permeability, destroy the blood-retinal barrier, and generate new blood vessels in DR, all of which are directly linked to the emergence and progression of the disease [Citation18]. The VEGF polymorphism (rs2010963) was the one that received the most attention from researchers. Previous studies have demonstrated that rs2010963 is one of many SNPs associated with increased or decreased VEGF protein synthesis and may alter protein conformation [Citation19]. We’ve already shown solid evidence linking VEGF SNP rs2010963 to DR severity or existence. The rs2010963 locate on 5’ UTR of the VEGF gene; in the 5ʹUTR of the VEGF gene, the G + C content (up to 83%) upstream of the transcription start point is relatively high. The 5’-untranslated region of the VEGF gene (5ʹUTR) plays a key part in VEGF production. Change in the sequence of this region is thought to be linked to alterations in protein expression of the VEGF gene, which in turn may increase the risk of developing retinopathy [Citation20,Citation21] as well as NPDR in the Asian population [Citation22]. Yang et al. concluded that rs2010963 was a risk factor for PDR in most groups but not Caucasians, where no link was found between rs2010963 and PDR development [Citation23]. According to our findings, those with the GG genotype had a higher probability of developing PDR than those with the CC genotype. Compared with other studies, CG lowered the risk of PDR [Citation24]. On the other hand, a study by Fan showed that gene polymorphisms such
as rs2010963 have been strongly linked with Recent meta-analysis findings indicated that rs2010963 was associated with PDR in the overall population VEGF protein in the blood [Citation25]. The VEGF protein is a potent angiogenic agent in various diseases. It has been established that elevated serum and vitreous levels of VEGF in the presence of retinopathy are closely associated with proliferative diabetic retinopathy [Citation26]. A study from Egypt explored the association between rs699947 SNP and the susceptibility of DR, and it found no significant association between them. This result is consistent with our findings [Citation27]. Different studies also revealed no association between the presence of rs699947 SNP and the risk of DR [Citation28]. In a previous meta-analysis, the rs699947 in Asian individuals with type 2 diabetes are more likely to be associated with DR but not in white people [Citation29]. While a meta-analysis from China recognizes the reverse association that rs699947 polymorphism is strongly connected with DR after adjusting for outliers, rs2010963 polymorphism may not be linked to DR [Citation30]. Different meta-analyses on rs2010963; one revealed an association while the other did not [Citation31,Citation32].
In contrast, many others showed a positive association of rs699947 SNP with the susceptibility of DR [Citation12,Citation13,Citation33]. A recent meta-analysis demonstrated that rs699947 might be implicated in the emergence and progression of DR [Citation22]. Numerous studies have discovered an association between various genetic polymorphisms and DR, whereas others have failed to do so [Citation34]. This outcome discrepancy might be due to a sample size disparity and clinical or methodological heterogeneity, and sampling of allele inheritance but not genotype. The degree of heterogeneity is likely influenced by several factors, including ethnicity, environmental factors, and social relationships that govern the cross-mating between families. Also, it might be that these polymorphisms convey sex depending on where there are different proportions of males/females in various studies; the discrepancy occurs. Howover the two SNPs (rs699947 & rs2010963) among DR patinets revealed no association with gender, and since majority of included studies did not report the association of the genotypes of the rs699947 & rs2010963 SNPs with gender, we are unable to perform the further analysis. Only few studies found that sex may a risk factor for DR, but not polymorphisms [Citation35,Citation36]. Yet we drew in our account a conception; the two SNPs (rs699947 & rs2010963) among NPDR and PDR need to be studied further to identify the link between the two.
The findings suggest a moderate LD in two SNPs (rs2010963 and rs699947). The rs699947, as mentioned early in the study, it was observed to have no significant association with DR; however, the C allele shared with the G allele of rs2010963; both alleles are minor alleles (mutant) and contributed significantly >2 folds to DR progression. Thus, it is valuable to measure the LD, which confers a piece of more information if statistical genetics fails to find an association. LD occur when nonrandom gametic alleles are associated at various loci in a population [Citation37]. The haplotype has an effect that differs from genotypes on disease, and the locus is still of clinical importance even though there is no link between genotype and clinical condition [Citation38]. The VEGF gene SNPs located at the promoter region and 5’ UTR and
the presence of the CG haplotype association confer upregulation or downregulation of VEGF expression, eventually leads worsen normal vision. A recent study shows different haplotypes (CD) from rs699947 and rs35569394 polymorphisms have a non-significant association with DR. Simultaneously, their alleles C and D have strong LD [Citation33]. One explanation is differences in allele and genotype frequencies of SNPs on one side and, on the other, the differences between different studies. Also, the patterns of LD
are from each other have strong LD. The haplotype and LD are important for large-scale association-mapping studies [Citation39]. unpredictable; two loci near each other might have weak LD, whereas loci of different SNPs that are so far from each other have strong LD. The haplotype and LD are important for large-scale association mapping studies [Citation40].
Conclusion
The GG genotype of rs2010963 in the VEGF gene had a higher probability of developing either NPDR or PDR. No significant importance for rs699947 SNP with diabetic retinopathy. The findings suggest a moderate LD in two SNPs (rs2010963 and rs699947) and their CG haplotype associated with DR progression by 2 folds.
Abbreviations
CI: Confidence interval; DM: Diabetes mellitus DR: Diabetic retinopathy; HC: Healthy controls; HWE: Hardy–Weinberg equilibrium; MA: Microaneurysm; LD: Linkage disequilibrium; NDR: Non diabetic retinopathy;NPDR: Non-proliferative diabetic retinopathy; OR: Odds ratio; PDR: Proliferative diabetic retinopathy; SNPs: Single nucleotide polymorphisms; T-ARMS PCR: Tetra primer amplication refractory mutation system polymerase chain reaction; T1DM: Type 1 diabetes mellitus; T2DM: Type 2 diabetes mellitus; VEGF: Vascular endothelial growth factor; UTR: untranslated region.
Ethical approval
Ethical approval for this study was granted by the human ethics committee (IRB) at the University of Kufa ((347/2021). we are following the ethical standards of the Helsinki Declaration.
Acknowledgments
We would like to thank Dr. Giyath Al-Deen Thegil Neamah for his kind words and moral support.
Disclosure statement
No potential conflict of interest was reported by the author(s).
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