VEGF-A 基因变异对新生血管性年龄相关性黄斑变性及雷珠单抗疗效的影响

Effect of VEGF-A gene mutation on neovascular age-related macular degeneration and the response to ranibizumab

目的探讨中国人群血管内皮生长因子A(VEGF-A)基因变异对新生血管性年龄相关性黄斑变性(nAMD)及雷珠单抗治疗疗效的影响。方法采用病例对照研究方法,纳入2017年2月至2018年1月在北京医院确诊的nAMD患者127例和健康对照组101人。nAMD患者雷珠单抗治疗后3个月,视力提高5个ETDRS字母数及以上者为视力改善组,视力改善低于5个ETDRS字母数者为视力欠佳组。每人取外周血5 ml,采用酚氯仿法提取DNA,采用Sanger法对VEGF-A外显子及上下游各2 kb片段进行测序并筛选变异位点。使用限制性内切酶片段长度多态性对测序发现的变异进行基因分型。采用Hardy-Weinberg平衡(HWE)检验研究样本群体代表性。分析并比较病例组与对照组、视力改善组与视力欠佳组在等位基因分布频率及基因型分布频率的差异。结果VEGF-A基因变异位点rs3025018位于第7内含子,该位点VEGF-A的等位基因包括C、G和T,基因型为CC、CT、CG、TT和TG。病例组与对照组不同等位基因分布比较,差异有统计学意义(χ2=7.492,P=0.024);其中2个组间G与C+T分布频率比较,差异有统计学意义(χ2=7.490,P=0.006,OR=0.407,95%CI=0.210~0.695)。病例组与对照组各基因型分布频率比较,差异有统计学意义(χ2=13.376;P=0.010);其中基因型CG+GT与CC+CT+TT分布频率比较,差异有统计学意义(χ2=8.335,P=0.004,OR=0.367,95%CI=0.183~0.802)。视力改善组与视力欠佳组在等位基因及基因型频率上差异均无统计学意义(均P>0.05)。结论携带VEGF-A基因的rs3025018位点G等位基因人群患nAMD的可能性较携带C或T等位基因的小。VEGF-A基因位点rs3025018变异对雷珠单抗治疗黄斑变性的疗效无明显影响。

Objective To investigate the effect of vascular endothelial growth factor-A(VEGF-A)gene mutation to neovascular age-related macular degeneration(nAMD)and the response to ranibizumab with this mutation in Chinese.Methods This was a case-control study.We recruited 127 cases(diagnosed as nAMD)and 101 ethical,age and geographical area matched unrelated healthy controls in Beijing Hospital from February 2017 to January 2018.The patients with nAMD were divided into two subgroups:good response to intravitreal ranibizumab(IVR)and poor response to IVR based on whether gain 5 letters 3 months after therapy.Phenol chloroform method was used in purification of genomic DNA in the peripheral venous blood of each individual.All exons and 2 kb upstream and downstream sequence of VEGF-A was sequenced by using Sanger Sequenced method,and candidate variations were screened out.Restriction fragment length polymorphism(RFLP)method was used in genotyping of the case-control study.Hardy-Weinberg equilibrium was used to test the representativeness of the sample group.The differences of allele distribution frequency and genotype distribution frequency between the case group and control group,good response group and poor response group were compared.Written informed consent was obtained from each subject prior to entering the study cohort.The study protocol was approved by the Ethics Committee of Beijing Hospital(No.2017S-012).Results The mutation(rs3025018)was located in 7th intron of VEGF-A.The allele were C,G,T and the genotype were CC,CT,CG,TT and TG.The allele distribution frequency between the case and control group were significantly different(χ2=7.492,P=0.024).The allele G vs.C+T distribution frequency between the case and control group were significantly different(χ2=7.490,P=0.006).The genotype distribution frequency between the case group and control group were significantly different(χ2=13.376,P=0.010).The genotype(CG+GT vs.CC+CT+TT)distribution frequency between the case group and control group were significantly different(χ2=8.335,P=0.004).The allele frequencies or genotype frequency were not significantly different between the good response group and poor response group(all at P>0.05).Conclusions G allele of VEGF-A(rs3025018)carriers were less possible to occur nAMD compared with C and T allele.However,there is no effect of VEGF-A gene mutations(rs3025018)on response to ranibizumab for nAMD.