Whole-Exome Sequencing in Age-Related Macular Degeneration Identifies Rare Variants in COL8A1, a Component of Bruch's Membrane

Purpose: Genome-wide association studies and targeted sequencing studies of candidate genes have identified common and rare variants that are associated with age-related macular degeneration (AMD). Whole-exome sequencing (WES) studies allow a more comprehensive analysis of rare coding variants across all genes of the genome and will contribute to a better understanding of the underlying disease mechanisms. To date, the number of WES studies in AMD case-control cohorts remains scarce and sample sizes are limited. To scrutinize the role of rare protein-altering variants in AMD cause, we performed the largest WES study in AMD to date in a large European cohort consisting of 1125 AMD patients and 1361 control participants. Design: Genome-wide case-control association study of WES data. Participants: One thousand one hundred twenty-five AMD patients and 1361 control participants. Methods: A single variant association test of WES data was performed to detect variants that are associated individually with AMD. The cumulative effect of multiple rare variants with 1 gene was analyzed using a gene-based CMC burden test. Immunohistochemistry was performed to determine the localization of the Col8a1 protein in mouse eyes. Main Outcome Measures: Genetic variants associated with AMD. Results: We detected significantly more rare protein-altering variants in the COL8A1 gene in patients (22/2250 alleles [

A large genome-wide association study of age-related macular degeneration highlights contributions of rare and common variants.

This is the author accepted manuscript. The final version is available from Nature Publishing Group via http://dx.doi.org/10.1038/ng.3448Advanced age-related macular degeneration (AMD) is the leading cause of blindness in the elderly, with limited therapeutic options. Here we report on a study of >12 million variants, including 163,714 directly genotyped, mostly rare, protein-altering variants. Analyzing 16,144 patients and 17,832 controls, we identify 52 independently associated common and rare variants (P < 5 × 10(-8)) distributed across 34 loci. Although wet and dry AMD subtypes exhibit predominantly shared genetics, we identify the first genetic association signal specific to wet AMD, near MMP9 (difference P value = 4.1 × 10(-10)). Very rare coding variants (frequency <0.1%) in CFH, CFI and TIMP3 suggest causal roles for these genes, as does a splice variant in SLC16A8. Our results support the hypothesis that rare coding variants can pinpoint causal genes within known genetic loci and illustrate that applying the approach systematically to detect new loci requires extremely large sample sizes.We thank all participants of all the studies included for enabling this research by their participation in these studies. Computer resources for this project have been provided by the high-performance computing centers of the University of Michigan and the University of Regensburg. Group-specific acknowledgments can be found in the Supplementary Note. The Center for Inherited Diseases Research (CIDR) Program contract number is HHSN268201200008I. This and the main consortium work were predominantly funded by 1X01HG006934-01 to G.R.A. and R01 EY022310 to J.L.H

Analysis of Risk Alleles and Complement Activation Levels in Familial and Non-Familial Age-Related Macular Degeneration.

AIMS:Age-related macular degeneration (AMD) is a multifactorial disease, in which complement-mediated inflammation plays a pivotal role. A positive family history is an important risk factor for developing AMD. Certain lifestyle factors are shown to be significantly associated with AMD in non-familial cases, but not in familial cases. This study aimed to investigate whether the contribution of common genetic variants and complement activation levels differs between familial and sporadic cases with AMD. METHODS AND RESULTS:1216 AMD patients (281 familial and 935 sporadic) and 1043 controls (143 unaffected members with a family history of AMD and 900 unrelated controls without a family history of AMD) were included in this study. Ophthalmic examinations were performed, and lifestyle and family history were documented with a questionnaire. Nine single nucleotide polymorphisms (SNPs) known to be associated with AMD were genotyped, and serum concentrations of complement components C3 and C3d were measured. Associations were assessed in familial and sporadic individuals. The association with risk alleles of the age-related maculopathy susceptibility 2 (ARMS2) gene was significantly stronger in sporadic AMD patients compared to familial cases (p = 0.017 for all AMD stages and p = 0.003 for advanced AMD, respectively). ARMS2 risk alleles had the largest effect in sporadic cases but were not significantly associated with AMD in densely affected families. The C3d/C3 ratio was a significant risk factor for AMD in sporadic cases and may also be associated with familial cases. In patients with a densely affected family this effect was particularly strong with ORs of 5.37 and 4.99 for all AMD and advanced AMD respectively. CONCLUSION:This study suggests that in familial AMD patients, the common genetic risk variant in ARMS2 is less important compared to sporadic AMD. In contrast, factors leading to increased complement activation appear to play a larger role in patients with a positive family history compared to sporadic patients. A better understanding of the different contributions of risk factors in familial compared to non-familial AMD will aid the development of reliable prediction models for AMD, and may provide individuals with more accurate information regarding their individual risk for AMD. This information is especially important for individuals who have a positive family history for AMD

Complement Activation Levels Are Related to Disease Stage in AMD

PURPOSE. To study the levels of complement activation in different disease stages of AMD and the influence of genetic polymorphisms in complement genes. METHODS. We included 797 patients with AMD and 945 controls from the European Genetic Database. Patients were grouped into five AMD stages: early AMD, intermediate AMD, central geographic atrophy, active choroidal neovascularization or inactive choroidal neovascularization. Differences in complement activation, as defined by the systemic C3d/C3 ratio, between AMD stages were evaluated using general linear modeling. In addition, we evaluated the influence of 18 genetic AMD polymorphisms in complement genes and their effect on complement activation. Differences in complement activation between stages were evaluated stratifying by complement associated haplotypes. RESULTS. Complement activation levels differed significantly between AMD disease stages. As compared with controls, the C3d/C3 ratio was higher in patients with intermediate AMD (P < 0.001) and central geographic atrophy (P = 0.001). Two polymorphisms in CFH (rs10922109 and rs570618) and one in CFB (rs116503776) were significantly associated with complement activation. The association between AMD disease stage and complement activation was more pronounced in patients with haplotypes associated with the highest complement activation. CONCLUSIONS. In general, consecutive AMD disease stages showed increasing levels of complement activation, especially in individuals with a genetic burden in complement genes. These findings contribute to the discussion on the pathogenesis of AMD in relation to complement activation and might suggest refinement in patient selection and the optimum window of treatment with complement inhibitors. Prospective studies are needed to confirm these results

Analysis of rare variants in the CFH gene in patients with the cuticular drusen subtype of age-related macular degeneration

Purpose: Age-related macular degeneration (AMD) and cuticular drusen (CD), a clinical subtype of AMD, have been linked to genetic variants in the complement factor H (CFH) gene. In this study, we aimed to investigate the frequency of rare variants in the CFH gene in 180 cases with CD. In addition, we aimed to determine the frequency of a previously reported rare, highly penetrant CFH variant (p. Arg1210Cys) in a Dutch-German non-CD-type AMD case-control cohort, and to describe the phenotype of patients carrying the p. Arg1210Cys variant. Methods: Study subjects were selected from the European Genetic Database (EUGENDA), a joint AMD database of the Radboud University Medical Centre and the University Hospital of Cologne, and graded at the Cologne Image Reading Centre and Laboratory (CIRCL). Additionally, two CD cases were recruited from the VU Medical Centre in Amsterdam. The CFH gene was analyzed in 180 CD cases with Sanger sequencing. All identified variants were analyzed for potential damaging effects with prediction software tools Sorting Intolerant from Tolerant (SIFT) and Polymorphism Phenotyping (PolyPhen). In addition, we genotyped the p. Arg1210Cys variant in 813 non-CD type AMD cases and 1175 controls. Results: Sequencing identified 11 rare, heterozygous missense variants, one frameshift variant, and one splice acceptor site variant in 16 CD cases. The p. Arg1210Cys variant was identified in two CD cases but was not identified in our Dutch-German non-CD-type AMD case-control cohort. Conclusions: The present study identified the presence of rare variants in the CFH gene in 16 (8.8%) of 180 patients with the CD subtype of AMD. The carriers of rare CFH variants displayed a significantly earlier age at onset than non-carriers (p=0.016). The rare missense variant p. Arg1210Cys was identified in two CD cases, but was not detected in 813 non-CD type AMD cases or in the 1,175 controls of our Dutch-German cohort. The current study suggests that the p. Arg1210Cys variant may be restricted to a subset of patients with the CD subtype of AMD. Detailed clinical pheno-typing, including fluorescein angiography, of patients with AMD carrying the p. Arg1210Cys variant in other cohorts is required to confirm this finding

Clinical Characteristics of Familial and Sporadic Age-Related Macular Degeneration: Differences and Similarities

PURPOSE. We describe the differences and similarities in clinical characteristics and phenotype of familial and sporadic patients with age-related macular degeneration (AMD). METHODS. We evaluated data of 1828 AMD patients and 1715 controls enrolled in the European Genetic Database. All subjects underwent ophthalmologic examination, including visual acuity testing and fundus photography. Images were graded and fundus photographs were used for automatic drusen quantification by a machine learning algorithm. Data on disease characteristics, family history, medical history, and lifestyle habits were obtained by a questionnaire. RESULTS. The age at first symptoms was significantly lower in AMD patients with a positive family history (68.5 years) than in those with no family history (71.6 years, P = 1.9 x 10(-5)). Risk factors identified in sporadic and familial subjects were increasing age (odds ratio [OR], 1.08 per year; P = 3.0 x 10(-51), and OR, 1.15; P = 5.3 x 10(-36), respectively) and smoking (OR, 1.01 per pack year; P = 1.1 x 10(-6) and OR, 1.02; P = 0.005). Physical activity and daily red meat consumption were significantly associated with AMD in sporadic subjects only (OR, 0.49; P = 3.7 x 10(-10) and OR, 1.81; P = 0.001). With regard to the phenotype, geographic atrophy and cuticular drusen were significantly more prevalent in familial AMD (17.5% and 21.7%, respectively) compared to sporadic AMD (9.8% and 12.1%). CONCLUSIONS. Familial AMD patients become symptomatic at a younger age. The higher prevalence of geographic atrophy and cuticular drusen in the familial AMD cases may be explained by the contribution of additional genetic factors segregating within families

Clinical Characteristics of Familial and Sporadic Age-Related Macular Degeneration: Differences and Similarities

PURPOSE. We describe the differences and similarities in clinical characteristics and phenotype of familial and sporadic patients with age-related macular degeneration (AMD). METHODS. We evaluated data of 1828 AMD patients and 1715 controls enrolled in the European Genetic Database. All subjects underwent ophthalmologic examination, including visual acuity testing and fundus photography. Images were graded and fundus photographs were used for automatic drusen quantification by a machine learning algorithm. Data on disease characteristics, family history, medical history, and lifestyle habits were obtained by a questionnaire. RESULTS. The age at first symptoms was significantly lower in AMD patients with a positive family history (68.5 years) than in those with no family history (71.6 years, P = 1.9 x 10(-5)). Risk factors identified in sporadic and familial subjects were increasing age (odds ratio [OR], 1.08 per year; P = 3.0 x 10(-51), and OR, 1.15; P = 5.3 x 10(-36), respectively) and smoking (OR, 1.01 per pack year; P = 1.1 x 10(-6) and OR, 1.02; P = 0.005). Physical activity and daily red meat consumption were significantly associated with AMD in sporadic subjects only (OR, 0.49; P = 3.7 x 10(-10) and OR, 1.81; P = 0.001). With regard to the phenotype, geographic atrophy and cuticular drusen were significantly more prevalent in familial AMD (17.5% and 21.7%, respectively) compared to sporadic AMD (9.8% and 12.1%). CONCLUSIONS. Familial AMD patients become symptomatic at a younger age. The higher prevalence of geographic atrophy and cuticular drusen in the familial AMD cases may be explained by the contribution of additional genetic factors segregating within families

Risk estimates and risk differences of allele frequencies of AMD-associated SNPs and serum complement activation levels for advanced AMD based on family history.

<p>Risk estimates and risk differences of allele frequencies of AMD-associated SNPs and serum complement activation levels for advanced AMD based on family history.</p

Demographics in familial and sporadic individuals.

<p>Demographics in familial and sporadic individuals.</p