Assessing Susceptibility To Age-Related Macular Degeneration With Genetic Makers And Environmental Factors

Chapter1Association analysis of LOC387715/HTRA1region with Age-related Macular DegenerationObjective:To investigate the association of2single nucleotide polymorphisms (SNPs) in the LOC387715/HTRA1region at Chromosome10q26with different phenotypes of Advanced AMD and lay the foundation for the prediction model for prevalence and incidence of advanced AMD.Methods:1. All1844participants were recruited from Caucasian cohort, including1335advanced AMD patients and509age and ethnicity matched normal controls. DNA was extracted from their blood.2. Two SNPs rs10490924and rs11200638, in the LOC387715/HTRA1region at Chromosome10q26, were genotyped in all participants.3. The chi-squared test and Fisher exact tests over alleles were performed to assess evidence for association.Results:1. SNP rs10490924and rs11200638were found significant associations with both forms of advanced AMD (GA and CNV) with the p<1.0E-17.2. The minor alleles of SNP rs10490924and rs11200638were T and A respectively. The Odd Ratios and the minimum of95%CI of these2minor alleles in each case group were greater than1.3. Neither rs10490924nor rs11200638was found significant difference between GA and CNV.4. SNP rs10490924and rs11200638in the LOC387715/HTRA1region show almost complete linkage disequilibrium.Conclusions:1. Two SNPs rs10490924and rs11200638, in the LOC387715/HTRA1region at Chromosome10q26, show significant association with both forms of advanced AMD.2. The minor alleles, also the risk alleles in the pathogenesis of AMD, of SNP rs10490924and rs11200638are T and A respectively.3. The LOC387715/HTRA1region is the major genetic marker of advanced AMD. Chapter2Association analysis of CFH, C3, CFB and C2gene with Age-related Macular DegenerationObjective:To investigate the association of6SNPs in4genes of CFH, C3, CFB and C2with different phenotypes of Advanced AMD and lay the foundation for the prediction model for prevalence and incidence of advanced AMD.Methods:1. All1844participants were recruited from Caucasian cohort, including1335advanced AMD patients and509age and ethnicity matched normal controls. DNA was extracted from their blood.2. SNP rs1061170, rs2274700and rs1410996from CFH, rs2230199from C3, rs641153from CFB, and rs9332739from C2were chosen and genotyped in all participants.3. The chi-squared test and Fisher exact tests over alleles were performed to assess evidence for association.Results:1. SNP rs1061170, rs2274700and rs1410996from CFH, rs2230199from C3, rs641153from CFB, and rs9332739from C2were found significant associations with both forms of advanced AMD with the p<0.05.2. The risk alleles of SNP rs1061170, rs2274700, rs1410996and rs2230199were C, C, C and G respectively. The Odd Ratios and the minimum of95%CI of these4risk alleles in each case group were greater than1. The protective alleles of SNP rs641153and rs9332739were T and C respectively. The Odd Ratios and the maximum of95%CI of these2protective alleles in each case group were less than1.3. Except SNP rs2230199, SNP rs1061170, rs2274700, rs1410996, rs641153and rs9332739was found no significant difference between GA and CNV.4. Three SNPs in CFH gene show almost complete linkage disequilibrium.Conclusions:1. Six SNP from4genes, CFH rs1061170, CFH rs2274700, CFH rs1410996, C3rs2230199, CFB rs641153and C2rs9332739, show significant association with both forms of advanced AMD.2. The risk alleles of SNP rs1061170, rs2274700, rs1410996and rs2230199are C, C, C and G respectively. The protective alleles of SNP rs641153and rs9332739are T and C respectively.3. CFH, C3, CFB and C2genes are the major genetic markers of advance AMD. The alternative complement pathway and the classical complement pathway of the complement system may play the important roles in the pathogenesis of AMD.Chapter3Assessing susceptibility to Age-related Macular Degeneration with multiple genetic markers and environmental factorsObjective:To evaluate the independent and joint effects of multiple genetic factors and environmental variables on AMD and to develop a predictive model with both genetic and environmental factors included.Methods:1. All1844participants were recruited from Caucasian cohort. Demographic information, including enroll age, smoking status and BMI, was collected.2. DNA was evaluated for eight variants in five genes related to AMD, including SNP rs10490924from LOC387715, rs11200638from HTRA1, rs1061170, rs2274700and rs1410996from CFH, rs2230199from C3, rs641153from CFB, and rs9332739from C2.3. Unconditional logistic regression analyses were performed to evaluate the independent and joint effects of multiple genetic factors and environmental variables related to AMD and generate the risk predictive model.Results:1. SNP rs10490924and rs11200638in LOC387715/HTRA1region show significant association only between bilateral CNV and unilateral CNV, while SNP rs2274700and rs1410996on CFH show significant association only between bilateral GA and unilateral GA.2. Smoking status was independently associated with advanced AMD (for former smokers OR=1.80,95%CI=1.32-2.45; for current smokers OR=3.71,95%CI=2.02-6.80). BMI was shown boundary association (P=0.093and0.081) with combined AMD, although it was barely significant (P=0.038and0.03) in CNV subgroup.3. No significant interaction was found among any of the genotypes, smoking or BMI. In terms of interaction between genotypes, only rs1061170-CT× rs10490924-TT and rs2274700-CT×rs10490924-TG were found weak effect of interaction, with the P=0.029and0.027respectively.4. The risk model of AMD showed that the odds ratio of a current smoker with the genotype of rs2274700-CC, rs2230199-GG, rs10490924-TT was as about450times as the odds ratio of a non-smoker with the genotype of rs2274700-TT, rs2230199-CC, rs10490924-GG.5. The ROC curve showed that the cutoff of0.73yielded approximately75%of sensitivity and75%of specificity as well. The model has78%discrimination accuracy.Conclusions:1. CFH may play a more important role in the pathogenesis of GA than CNV and so does LOC387715/HTRA1in CNV than GA.2. Smoking is as an independently risk factor in the pathogenesis of AMD. BMI, as another risk factor, shows much weaker contribution than smoking to the occurrence of AMD.3. There is no significant interaction among smoking, BMI and any of the genotypes.4. The best fit model is achieved with age, smoking and six genetic markers (CFH rs1061170, CFH rs2274700, C2rs9332739, CFB rs641153, LOC387715/HTRA1rs10490924, and C3rs2230199) with multiplicative model. It indicates that the potential for individual prediction of risk for AMD may become reality.Chapter4Retinal structural changes of the HTRA1Knockout MiceObjective:To investigate the function of HTRA1gene in the formation of mice retina and analysis the influence of gene deletion to the structure of mice retina.Methods:1. HTRA1knockout mice and C57BL/6wild type mice were collocted. DNA was extracted from their tails. PCR was used to genotype the mice.2. The eyeballs of HTRA1knockout mice and C57BL/6wild type mice in different age groups were enucleated. Retinal flat-mount immunofluorescence was performed to compare the changes of the retinal structure in each group.Results:The communicating arteries in the retina of the HTRA1 knockout mice were found obviously decreased by comparing with the wild type mice. It was observed in3and6-month old mice, but not in1-month old mice.Conclusions:1. HTRA1knockout mice are important animal models for investigating the role of HTRA1gene and lay the foundation of further study of HTRA1gene.2. HTRA1gene may have the function of maintaining the normal appearance of the retinal vessels and playing a key role in their pathological changes.