Association Between The Gene Polymorphisms Of Retinal Neovascularization Factors And Retinopathy Of Premature

BackgroundRetinopathy of prematurity (ROP) is a vasoproliferative disorder affecting preterm infants with low gestational age and birth weight. Retinopathy of prematurity (ROP) is a leading cause of impaired vision and blindness in children throughout the world. It is a retinal vascular disease that occurs in infants with low gestational age and birth weight. ROP is becoming more frequent due to the improved survival of extremely premature infants.ROP is characterized by abnormal retinal neovascularisation, which possibly leads to retinal detachment, macular folds, myopia, refractive amblyopia, or strabismic amblyopia and may result in visual loss or blindness. However, retinal detachment occurs and leads to visual loss in only a few percent of infants with stage3or more severe ROP, and in most cases, spontaneously regresses. In general more than50%of preterm infants weighing less than1250g at birth show evidence of ROP and about10%of the infants develop stage3ROP. Many causative factors, like low birth weight, low gestational age and supplemental oxygen therapy are associated with ROP. The incidence of ROP is more frequent in white than in black infants and in males than in females. And the most conspicuous question is why ROP in some premature infants progress despite rigorous and timely intervention while in other cases with similar clinical characteristics it regresses. Genetic differences between the infants could be an explanation. As is well known, genetic factors such as polymorphisms of different genes may alter not only the risk but also the progression of ROP Genetic polymorphism may alter the function of the genes which normally control retinal vascularization, such as vascular endothelial growth factor (VEGF), which may also be involved in pathogenesis of ROP. Evaluation of candidate genetic polymorphism influencing the outcome of ROP may provide new information about the pathogenesis of the disease. Screening of genetic polymorphisms may also help to identify and treat the high risk infants in time. Although a significant amount of information regarding these genetic polymorphisms has been published, mostly in developed countries in recent years. These studies should be interpreted with some degree of caution. However, variations of candidate genes were present in small subsets of subjects and/or were limited geographically to specific ethnic populations, making the generalizability of the results uncertain. No previous definitive study has confirmed that a genetic susceptibility to ROP exists. Also, information regarding the extent of that genetic contribution has not been identified previously.Objective:Retinopathy of prematurity (ROP) is a most common disease result in children’s blindness which is characterized by abnormal retinal neovascularisation. This study was designed to investigate the association between the mutiple gene polymorphismof several retinal neovascularization factors and the development of ROP in Chinese Han population. Candidate gene strategy, case-control study and mass spectrum genotyping technique were adopted in the study.The results will be significantly important to guide the prevention and management of ROP and will establish the theoretical and experimental foundation of exploring the pathogenesis of ROP, screening for susceptible genotye and gene therapy in future.Methods:1. A case-control study was performed from January,2011to December,2012. Cases in this study were all from the department of neonatology, Shenzhen maternal and Child Health Hospital. ROP screening was carried out by the same Ophthalmology physician with binocular indirect ophthalmoscope or Retcam. Diagnosis and staging of ROP were made according to the guideline of Chinese Medical Association in 2004:oxygen usage, treatment and prevention of ROP in preterm infants. After that, infants with a birth weight less than1500g or gestation age of less than32weeks were choosed.90infants without ROP were randomly recruited in the control group, while infants with ROP were randomly recruited in severe group and mild group according to the degree of ROP. Severe group included73infants with pre-threshold type1, threshold, aggressive posterior ROP and stage4/5ROP who need laser photocoagulation or vitrectomy. Mild group included78infants with stage1, stage2and non-threshold stage3ROP, scar ROP who needn’t surgery. Infants were excluded with family history such as Exudative Retinopathy fundus lesions, Norrie’s disease, diabetes, heart disease, respiratory disease, liver and kidney dysfunction and other systemic vascular abnormalities. All of the patients have no blood relationships from Han Chinese population. All patients (parents) were informed of the purpose of the study and it was approved by the ethics committee of our hospital.2. Blood samples (0.5ml) were collected from the peripheral vein of each patient, them they were placed in EDTA anticoagulant tubes before being stored in the-80℃refrigerator. DNA was extracted with DNA Blood Mini Kit. Subsequently, genomic DNA was amplified by the PCR method with primers designed by Primer Premier5.0software.3. SNPs were genotyped by the MassArray time of flight mass spectrometry system, PCR gel electrophoresis or direct sequencing respectively.4. In this study, Independent samples T test was used to compare the difference of gestational age, birth weight between groups; Chi-square test was used to compare the difference of gender between groups; Hardy-Weinberg equilibrium of the SNPs were calculated using the software online (http://www.oege.org/software/hwe-mr-calc.shtml).Chi-square test was used to compare allelic and genotype frequencies among the groups. For the adjustment for known risk factors of ROP (gestational age, birth weight), logistic regression analysis was used to compare genotype frequencies between groups. Odds ratios (Odds, ratio, OR) and95%confidence intervals (confidence, interval, CI) were calculated. The level of significance was set at p<0.05.The statistical power was calculated for all comparisons (alpha=0.05). All the data was recorded by Microsoft Excel software and all calculations were performed with the statistical software package SPSS13.0. Two-sided probability test was used for all statistical tests.Results:1. Comparisons of general data:SNP typing was successfully performed in230cases. The success rate is95.4%. Among them,85cases were in control group,71cases in severe group and74cases in mild group.11cases were excluded from the study. Chi-square test was used to compare the difference of gender. There was no significant difference in gender distribution among the three groups (χ2=0.7478, P=0.787,>0.05); Independent samples T test was used to compare the difference of gestational age and birth weight respectively among the three groups. Comparisions of gestational age and birth weight were significantly different between control group and mild group, control group and sever group, mild group and severe group (P<0.05).2. Hardy-Weinberg equilibrium test:No significant deviations from the Hardy-Weinberg equilibrium were observed for polymorphisms among three groups. All genotypes were in Hardy-Weinberg equilibrium (P>0.05). Proving that the datas were from the same Mendel group and the results of sampling and genotyping were reliable. The allele frequencies in our population represented the allele frequencies in the general population3. Comparisons of the genotypes and allele distributions for the polymorphisms among three groups.3.1Comparisons of the genotypes and allele distributions of VEGF polymorphisms among groupsGenotype distributions of VEGF+405and+936polymorphisms were significantly different among three groups (P=0.000, P=0.010,<0.05) while those of VEGF-460and-2578polymorphisms showed no significant difference between the three groups (P=0.87, P=0.98,<0.05). Among them, VEGF+405genotype distributions between severe group and control group, severe group and mild group had significant difference (P=0.000, P=0.000,<0.017), while genotype distributions between mild group and control group showed no significant difference (P=0.873,>0.017). Genotype distributions of VEGF+936polymorphism showed remarkable difference between severe group and control group (P=0.010,<0.017), while no significant difference between mild group and control group, severe group and mild group(P=0.552, P=0.055,>0.017).The allele distributions of VEGF+405,+936polymorphisms had significant difference between the three groups (P=0.000, P=0.002,<0.05), while those of-460,-2578locus showed no significant difference between three groups (P=0.990, P=0.429,<0.05). Among them, VEGF+405allele distributions between severe group and control group, severe group and mild group had significant difference (P=0.000, P=0.015, P<0.017). Between severe group and control group, C allele was significantly high in severe group (OR:2.711,95%CI:1.709-4.302); Between severe group and mild group, C allele was significantly high in severe group(OR:1.798,95%CI:1.121-2.885). The allele distributions of VEGF+936polymorphism had significant difference between severe group and control group (P=0.001,<0.017), T allele carrier was significantly high in severe group (OR:2.684,95%CI:1.521-4.739).3.2Comparisons of the genotypes and allele distribution of PEDF polymorphisms among groupsPEDF-5376genotype distribution were significantly different among three groups (P=0.003,<0.05). Among them, there were significant differences between severe group and the control group (P=0.000,<0.017); and there was no significant difference between mild group and control group (P=0.472,<0.017), severe group and mild group (P=0.24,<0.017). Genotype distributions of+311polymorphism among groups had no significant difference (P=0.182,0.05).The PEDF-5376allele distributions was significantly different among three groups (P=0.003, P<0.05), while the+311allele distributions showed no significant difference among groups (P=0.383,>0.05). Among them, the PEDF-5376allele distributions showed significant difference between severe group and control group, severe group and mild group (P=0.001, P=0.009, P<0.017), and no significant difference was found between mild group and control group(P=0.531,0.017). Between severe group and control group, T allele carrier was significantly high in severe group (OR:2.213,95%CI:1.371-3.572); Between severe group and mild group, T allele was significantly high in severe group (OR:1.919,95%CI:1.170-2.883.1485).3.3Comparisons of the genotypes and allele frequencies of eNOS polymorphisms among groupsThe genotype distribution of eNOS-786and+894polymorphisms showed no significant difference among groups (P=0.590, P=0.834,>0.05). There were also no significantly difference with the allele distribution of the polymorphisms among three groups (P=0.231, P=0.410,>0.05).3.4Comparisons of the genotypes and allele frequencies of IGF-1R polymorphism among groupsThe genotype and allele distribution of IGF-1R+3174polymorphism showed no significant difference among groups (P=0.25, P=0.661,>0.05).3.5Comparisons of the genotypes and allele frequencies of Ang-2polymorphism among groupsThe genotype and allele distribution of Ang-2+35polymorphism showed no significant difference among groups (P=0.951, P=0.892,>0.05).3.6Comparisons of the genotypes and allele frequencies of ACE polymorphism among groupsThe genotype and allele distribution of ACE I/D polymorphism showed no significant difference among groups (P=0.777, P=0.773,>0.05).3.7Comparisons of the genotypes and allele frequencies of ND polymorphisms among groupsAll the allele of ND Val60Glu was T, Ala105Thr was G and+597C/A C. No polymorphisms were found among groups.3.8Asociation between the SNPs and ROP by logistic regression analysisBetween control group and severe group, binary logistic regression analysis was performed to evaluate the association between the genotypes of VEGF+405,+936, PEDF-5376polymorphisms and ROP. After the adjustment of the association on gestational age and birth weight, significant difference was found for each polymorphism (P=0.010; P=0.023; P=0.045, P<0.05), Among them, compared with CC genotype, the VEGF+405GG genotype carrier had significantly decreased risk to develop severe ROP (OR:0.140,95%CI:0.039-0.505, P=0.003); Compared with CC genotype,the VEGF+936CT genotype carrier had increased risk to develop severe ROP (OR:3.281,95%CI:1.272-8.462, P=0.014); Compared with TT genotype, the PEDF-5376CT carrier had decreased risk to develop severe ROP (OR:0.315,95%CI:0.121-0.817,P=0.018)Between mid group and severe group, binary logistic regression analysis was performed to evaluate the association between the genotypes of VEGF+405polymorphism and sever ROP. After the adjustment of the association on gestational age and birth weight, significant association of the VEGF+405polymorphism and severe ROP was (P=0.000,<0.05). Among them, compared to GG genotype, both GC and CC genotype carrier had decreased risk to develop severe ROP (OR:0.088,95%CI:0.028-0.279; OR:0.276,95%CI:0.110-0.693). Both were with statistical significance (P=0.000; P=0.006; P<0.05).Binary logistic regression analysis was performed to evaluate the association between VEGF460/+405haplotypes and ROP. After the adjustment of the association on gestational age and birth weight,-460T/+405C homozygote was significantly associated with increased likelihood of suffering severe ROP not only between severe group and control group (OR:5.319,95%CI:2.034-13.904, P=0.001) but also between severe group and mild group (OR:3.878,95%CI:1.693-8.885, P=0.001). To the contrary, VEGF-460T/+405G homozygote significantly was significantly associated with decreased likelihood of suffering severe ROP not only between severe group and control group (OR:0.121,95%CI:0.022-0.660, P=0.015) but also between severe group and mild group (OR:0.289,95%CI:0.123-0.680, P=0.019).Conclusions: Our study found that VEGF, PEDF SNPs were significantly associated with severe ROP but not mild ROP. VEGF+405CC, VEGF+936CT, PEDF-5376TT may be susceptible genotypes;The VEGF+405C allele, VEGF+936T allele, PEDF-5376T allele may be risk alleles; The VEGF-460T/+405C homozygote may be risk haplotype combination. It is hypothesized that the normal expression of vascular factors were influenced by related gene polymorphisms at the level of gene transcription. And it is also found that the VEGF+405genotype and allele distribution, PEDF-5376allele distribution showed significant differences between severe group and mild group, proving that genetic variance exists between mild and severe ROP, suggesting that genetic variance may play an important role in the pathogenesis of severe ROP while mild ROP may be mainly effected by the environmental factors.The results of our study contribute to the knowledge on ROP susceptibility molecular mechanism. The identified risk genotypes or haplotypes in our study could be used, if validated, as molecular markers to improve our ability to define high-risk populations who may be susceptible to severe ROP in Chinese Han population.