|Year : 2016 | Volume
| Issue : 1 | Page : 75-78
Vascular endothelial growth factor gene polymorphism is not associated with diabetic retinopathy in Egyptian Patients
Rana Ahmed Abdel Fattah1, Rasha Mounir Eltanamly1, Mostafa Hamed Nabih1, Manal Mohammed Kamal2
1 Department of Ophthalmology, Cairo University, Cairo, Egypt
2 Department of Clinical Pathology, Cairo University, Cairo, Egypt
|Date of Web Publication||4-Jan-2016|
Rasha Mounir Eltanamly
34, Elnadi Street, Maadi, 11431, Cairo
Source of Support: None, Conflict of Interest: None
| Abstract|| |
Purpose: Vascular endothelial growth factor (VEGF) was implicated as a major contributor to the development of diabetic retinopathy (DR). This study investigated whether single nucleotide polymorphisms of A allele of rs699947 or G allele of rs10434 in the VEGF gene were associated with DR in Egyptian patients with type 2 diabetes mellitus (DM).
Methods: This is a case–controlled study which was performed at Cairo University Hospital in 2012 on Egyptian patients with type 2 DM with and without DR. Healthy adults without diabetes comprised the comparison group. Patients underwent an ophthalmological examination and fundus photography. Genotyping was performed for the A allele of rs699947 and the G allele of rs10434 polymorphisms using real-time polymerase chain reaction.
Results: A total of 128 patients were enrolled in this study and divided into three groups: Group A included 46 patients with type 2 DM and DR; Group B included 41 patients with type 2 DM without DR; and Group C included 41 healthy controls. There was no significant association between rs699947 or rs10434 and any of the three groups (P = 0.5, P = 0.7, respectively). Allelic frequency in the three groups was not statistically significant for rs699947 or rs10434 (P = 0.6, P = 0.6, respectively).
Conclusion: Rs699947 or rs10434 polymorphism was not associated with the presence of DR in Egyptian patients. Further studies are required before genetic testing for polymorphism can be used clinically to correlate with DR.
Keywords: Diabetic Retinopathy, Gene, Polymorphism
|How to cite this article:|
Abdel Fattah RA, Eltanamly RM, Nabih MH, Kamal MM. Vascular endothelial growth factor gene polymorphism is not associated with diabetic retinopathy in Egyptian Patients. Middle East Afr J Ophthalmol 2016;23:75-8
|How to cite this URL:|
Abdel Fattah RA, Eltanamly RM, Nabih MH, Kamal MM. Vascular endothelial growth factor gene polymorphism is not associated with diabetic retinopathy in Egyptian Patients. Middle East Afr J Ophthalmol [serial online] 2016 [cited 2022 Aug 19];23:75-8. Available from: http://www.meajo.org/text.asp?2016/23/1/75/171760
| Introduction|| |
The severity and progression of diabetic retinopathy (DR) are influenced by genetic and environmental factors. Vascular endothelial growth factor (VEGF) is implicated as a major contributor to the development of DR., Both hypoxia and hyperglycemia are potent stimuli for VEGF protein expression. Several single nucleotide polymorphisms (SNPs) could be associated with differential expression of VEGF., Among them, the A allele of rs699947 and G allele of rs10434 are the main loci. The mechanism through which SNPs affect susceptibility to DR remains unknown. However, promoter SNPs rs699947 have been reported to alter the VEGF promoter activity or affect the levels of mRNA expression.,
This study investigates whether SNPs of A allele of rs699947 or G allele of rs10434 in the VEGF gene are associated with DR in a cohort of Egyptian patients with type 2 diabetes mellitus (DM).
| Methods|| |
This case–control type of study was performed from March 2012 to March 2013 at Kasr Alainy Cairo University Hospitals. The study was approved by the institutional review board, and informed consent was obtained from all patients. Real-time polymerase chain reaction (RT-PCR) for rs699947 and rs10434 SNPs was performed. Participants were assigned to 1 of 3 groups: Group A included 46 patients with type 2 DM of seven or more years duration and DR; Group B included 41 patients with type 2 DM and no DR; and Group C included 41 healthy adults without diabetes.
All patients underwent a complete ophthalmic examination including the measurement of best-corrected visual acuity, slit lamp bio-microscopy (Topcon Corp., Tokyo, Japan) and fundus examination with + 90 D or + 78 D lens, and indirect ophthalmoscopy (Keeler all pupil, UK). Fundus fluorescein angiography was performed with the TRC50DX retinal camera (Topcon Corp, Tokyo, Japan), after dilation of the pupils with tropicamide (0.5%). Presence and stage of DR was based on indirect ophthalmoscopy and angiography.
DR was classified according to the early treatment diabetic retinopathy study as proliferative DR (PDR) and nonproliferative DR (NPDR). Signs consistent with NPDR included microaneurysms, hemorrhages, hard exudates, cotton wool spots, venous abnormality, and peripheral ischemia on fluorescein angiography. PDR was identified by the presence of neovascularization, vitreous hemorrhage, and fibrovascular proliferation.
Phlebotomy was performed to collect 5 ml of venous blood from each participant and stored at −80°C prior to DNA extraction. Genomic DNA was isolated from venous blood leukocytes using a genomic DNA extraction and purification kit (Roche Molecular Systems, Inc., CA, USA). Amplification and detection of the polymorphisms were performed with RT-PCR. The LightCycler ® 2.0 (Roche, CA, USA) was programmed with the following parameters: 48 cycles, denaturation at 95°C for 10 min, followed by 45 cycles of 95°C for 10 s, followed by 60°C with a holding time of 10 s, followed by 72°C with a holding time of 15 s, and followed by melting and cooling. A Allele of rs699947 was detected at a melting point of 62.61°C ± 2.5°C, while C allele was detected at a melting point of 55.78°C ± 2.5°C. In a heterozygous sequence, when both alleles were present, two peaks were detected.
For rs10434, G allele was detected at a melting point of 62.79°C ± 2.5°C, and A allele was detected at a melting point of 68.19°C ± 2.5°C. For a heterozygous sequence, two peaks were detected.
Data are presented as mean ± standard deviation, median and range, or frequencies (number of cases) and percentages when appropriate. Comparison of numerical variables between the groups was performed with one-way analysis of variance with post-hoc multiple three group comparisons. For comparing categorical data, the Chi-square test was performed. Fisher's exact test was performed when the expected frequency was <5. Odds ratio (OR) was calculated for each genotype between each two groups. A two-sided P < 0.05 was considered statistically significant. All statistical calculations were performed with SPSS (Statistical Package for the Social Science; IBM Corp., Armonk, New York, USA) version 15 for Microsoft Windows.
| Results|| |
A total of 128 patients were enrolled in this study (50% were males). However, two-third of the diabetic patients was females. The mean age of Groups A-C was 58.9 ± 6.0 years, 58 ± 7.2 years, and 55 ± 5.3 years, respectively. Mean duration of DM was 15 ± 4.4 years in Group A and 10.7 ± 5.1 years in Group B. Hypertension (HTN) was the comorbidity in 46% of Group A participants and 54.3% of Group B participants, Group C participants were all healthy with no comorbidities.
The distribution of rs699947 genotypes in the three groups is presented in [Table 1]. The association of rs699947 to any of the three groups was not statistically significant (P = 0.5, all comparisons).
OR of AA/CC alleles of rs699947 in each group was compared against the two other groups. Furthermore, OR of AA/AC and AC/CC in each group was compared against the two other groups [Table 2]. All genotypes of rs699947 SNP were not a significant risk factor for DR.
Distribution of rs10434 genotypes in the three groups is presented in [Table 3]. The association of rs10434 to any of the three groups was statistically not significant (P = 0.7).
Allele frequency of both rs699947 (P = 0.6) and rs10343 (P = 0.6) in the three groups was not significantly different [Table 4].
OR of GG/AA alleles of rs10434 in each group was compared against the two other groups. The OR of GG/AG and AG/AA in each group was compared against the two other groups [Table 5]. The genotypes of rs10434 SNP are not risk factors for DR.
| Discussion|| |
The relationship between VEGF gene polymorphism and DR has been the subject of recent studies. These studies have reported a possible association between VEGF gene polymorphisms and various diseases, in which angiogenesis might be critical in disease development. The association between VEGF polymorphisms and the presence or severity of DR is inconclusive, and results are the same or conflicting either in the same or different populations. Due to the disparity of results among different populations, we evaluated this association in an Egyptian cohort of type 2 DM patients. We selected rs699947 SNP at the promoter region because the conflicting results from these studies, and rs10434 has not been thoroughly evaluated.
This study involved 128 participants divided into three groups: 46 in the DR group, 41 in the no DR group, and 41 in controls. All candidates were genotyped for rs699947 and res10434 SNPs using RT-PCR. There was no statistically significant association between rs699947 or rs10434 and any of the three groups in our sample (P = 0.462) (P = 0.686), respectively. Allele frequency in the three groups was not statistically significant (P = 0.616) (P = 0.572), respectively.
We found that the comparison of the genotype ratio in each group with the other two groups indicated that no genotype is a risk factor for DR. In addition, there were no statistically significant correlations of any genotype to sex, age, or HTN in the three groups.
The promoter region of rs699947 is strongly associated with DR in Korean and Chinese populations., However, it was marginally associated with DR in the Japanese , and Indian populations. The promoter region of rs699947 was not associated with DR in Caucasians., Thus, there seems to be a strong racial influence on the study outcomes.
A study by Abhary et al. reported that in type 2 DM patients, rs3025021 and rs10434 were both significantly associated with DR after controlling for gender, HbA1c, and duration of disease (P = 0.002, both associations). The genotypes GG, GA for rs10434 appear to confer the greatest risk for DR (OR = 2.6; 95% CI, 1.3–5.3). They showed that certain haplotype contains the risk allele of the two individually associated SNPs (C for rs3025021 and G for rs10434), but several other haplotypes also contain these alleles with no significant risk of DR, indicating that the true risk allele that is tagged by these SNPs is likely in the background of that particular haplotype, rather than being these specific SNPs., Homozygosity of the VEGF GG genotype of rs10434 SNP was associated with PDR, and heterozygosity for the rs10434 SNP is associated with HTN.
The results of this current study contradict other studies. The differences between this study and previous studies could be related to the different ethnicity of the study cohorts. In general, the frequency of the susceptibility allele differs among populations. Thus, the contradictory reports may be due to the difference in allele frequency among populations.
A limitation of this study is small sample size. Perhaps, statistically significant differences could be found in a larger sample. In addition, identification of haplotypes would have given different results. These results can be explained by the fact that a combined haplotype play a greater role than an individual SNP, in influencing gene function and DR susceptibility. Haplotypes might be more predictive and informative than individual SNPs.
More studies with larger number of patients evaluating different polymorphisms are needed before genetic testing can be clinically used and correlated with DR.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
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[Table 1], [Table 2], [Table 3], [Table 4], [Table 5]
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