Lack of association of VEGF gene 3’- UTR polymorphisms (C702T, C936T and G1612A) and the risk of developing advanced retinopathy of prematurity (ROP)
- First Online:
- Cite this article as:
- Gismondi, D., Ndoja, L., Qu, X. et al. Graefes Arch Clin Exp Ophthalmol (2013) 251: 413. doi:10.1007/s00417-011-1913-4
- 177 Views
Retinopathy of prematurity (ROP) is a well-known visual impairment in premature children. It is a disease of developing retinal blood vessels. The condition is a multifactorial disease, and is clinically similar to familial exudative vitreoretinopathy (FEVR) which occurs in full-term infants. Both of these disorders can cause blindness in young children. While the pathogenesis and etiology of ROP are currently unknown, candidate gene and twin studies suggest that this unpredictability of ROP could be due to genetic factors in addition to environmental factors [1, 2]. However, at present there is no reliable biomarker to predict which premature infant will develop retinal detachment and blindness. The ability to identify a biomarker might allow fewer at-risk babies to be screened at the early stages of the disease.
The blinding complication of ROP is strongly associated with the development of retinal neovascularization. In this process, vascular endothelial growth factor plays a major role [3, 4]. Additionally it has been reported that VEGF haplotypes are associated with proliferative ROP . Because of these, and to continue our efforts to identify a potential biomarker that can predict the high-risk infants, in this study we have analyzed genetic polymorphisms (C702T, C936T and G1612A) in the 3’-UTR of VEGF gene.
Distribution of genotypic and allelic frequencies of VEGF 3’-UTR polymorphisms (C702T, C936T and G1612A) between the control, ROP and FEVR patients
n = 49
n = 33
n = 10
n = 53
n = 43
n = 12
n = 48
n = 34
n = 10
For genetic analysis, blood samples were collected from each patient, and leukocyte DNA was isolated. Genomic DNA was amplified by the polymerase chain reaction (PCR) method with three commercially synthesized pairs of primers designed to amplify the 3’-untranslated region comprising the polymorphism (C702T, C936T and G1612A) as described previously . Briefly, 40 ng of genomic DNA was annealed with respective primers at 60°C for 1 min and extended at 72°C for 2 min with 32 cycles, using the FailSafe PCR buffer G system supplied by Epicenter (Madison, WI,USA). The amplified product was subjected to restriction enzyme digestion with 10 units of Msp I (C702T), Nla III (C936T) and Mnl I (G1612A) at 37°C for overnight. The digested products were electrophoresed on a 9% polyacrylamide gel (PAG), and the banding pattern was visualized by the ethidium bromide staining. The significance of allele frequency differences was tested by using a chi-square test (X2) with Yates correction and confirmed by Fisher's exact test. Because there is only a limited amount of information on the frequency in the pre-term population, study size estimations are difficult to make. However, the power of Fisher's exact test must show that if the frequency of the allele in the ROP group is increased by 10%, we would have a 94% chance of detecting a statistically significant difference with 43 infants. A result is considered significant when a p-value is less than 0.05.
The genotypic and allelic frequencies were presented in Table 1. Our analyses suggest that the C702T polymorphism is very rare in this cohort, and there is no significant difference between allelic frequency distribution among three groups of patients with a C allelic frequency is almost 1.0. This result is consistent with other reports [7, 8], and therefore it is not analyzed further. In the case of C936T polymorphism, allele and genotype frequency distribution are very similar to those reported by others [7, 8]. In the case of ROP, both the X2 test with Yates correction and Fisher's exact test have given a p-value of 0.11. The 95% confidence interval for the odds ratio is 0.88 and 4.36. Similarly in the case of FEVR, the X2 test with Yates correction has given a p-value of 0.12, and the Fisher exact test has a p-value of 0.10. The 95% confidence interval for the odds ratio is 0.83 and 34.83. Similar results were obtained in the case of G1612A polymorphism. Because these p-values are large, we conclude that there is no association between the progression of ROP, FEVR and the 3’ – UTR polymorphism in the VEGF gene. The 95% confidence interval for the odds ratio supports this conclusion. These results are consistent with another study using a different cohort of patients with respect to C936T polymorphism [9, 10]. Although we cannot draw a strong conclusion from this small-scale study, the odds ratio in both cases supports our conclusion. Further studies are needed to uncover the role of increased expression of VEGF gene in both avascular retina and intravitreous neovascularization.
We would like to thank Dr. Michael T. Trese of William Beaumont Hospital, Royal Oak, MI, USA for providing blood samples from patients and normal individuals. This work was supported by Oakland University Research Grant GRC # 23580.