HighlightsThe complement system plays a central role in age-related macular degeneration (AMD).Common and rare genetic variants in complement genes have been identified in AMD.Several of the rare variants affect the functioning of the complement system.However, a genetic association with AMD cannot always be proven.Functional assays can help identify patients for complement inhibiting therapies.

In age-related macular degeneration (AMD), rare variants in the complement system have been described, but their functional consequences remain largely unexplored. OBJECTIVES To identify new rare variants in complement genes and determine the functional effect of identified variants on complement levels and complement regulation in serum samples from carriers and noncarriers. DESIGN, SETTING, AND PARTICIPANTS This study evaluated affected (n = 114) and unaffected (n = 60) members of 22 families with AMD and a case-control cohort consisting of 1831 unrelated patients with AMD and 1367 control individuals from the European Genetic Database from March 29, 2006, to April 26, 2013, in Nijmegen, the Netherlands, and Cologne, Germany. Exome sequencing data of families were filtered for rare variants in the complement factor H (CFH), complement factor I (CFI), complement C9 (C9), and complement C3 (C3) genes. The case-control cohort was genotyped with allele-specific assays. Serum samples were obtained from carriers of identified variants (n = 177) and age-matched noncarriers (n = 157). Serum concentrations of factor H (FH), factor I (FI), C9, and C3 were measured, and C3b degradation ability was determined. MAIN OUTCOMES AND MEASURES Association of rare variants in the CFH, CFI, C9, and C3 genes with AMD, serum levels of corresponding proteins, and C3b degradation ability of CFH and CFI variant carriers. RESULTS The 1831 unrelated patients with AMD had a mean (SD) age of 75.0 (9.4) years, and 60.5% were female. The 1367 unrelated control participants had a mean (SD) age of 70.4 (7.0), and 58.7% were female. All individuals were of European descent. Rare variants in CFH, CFI, C9, and C3 contributed to an increased risk of developing AMD (odds ratio, 2.04; 95% CI, 1.47-2.82; P < .001). CFI carriers had decreased median FI serum levels (18.2 μg/mL in Gly119Arg carriers and 16.2 μg/mL in Leu131Arg carriers vs 27.2 and 30.4 μg/mL in noncarrier cases and controls, respectively; both P < .001). Elevated C9 levels were observed in Pro167Ser carriers (10.7 μg/mL vs 6.6 and 6.1 μg/mL in noncarrier cases and controls, respectively; P < .001). The median FH serum levels were 299.4 μg/mL for CFH Arg175Gln and 266.3 μg/mL for CFH Ser193Leu carriers vs 302.4 and 283.0 μg/mL for noncarrier cases and controls, respectively. The median C3 serum levels were 943.2 μg/mL for C3 Arg161Trp and 946.7 μg/mL for C3 Lys155Gln carriers vs 874.0 and 946.7 μg/mL for noncarrier cases and controls, respectively. The FH and FI levels correlated with C3b degradation in noncarriers (R 2 = 0.35 and R 2 = 0.31, respectively; both P < .001). CONCLUSIONS AND RELEVANCE Reduced serum levels were associated with C3b degradation in carriers of CFI but not CFH variants, suggesting that CFH variants affect functional activity of FH rather than serum levels. Carriers of CFH (Arg175Gln and Ser193Leu) and CFI (Gly119Arg and Leu131Arg) variants have an impaired ability to regulate complement activation and may benefit more from complement-inhibiting therapy than patie...

T h e ne w e ngl a nd jou r na l o f m e dicine n engl j med 372;6 nejm.org february 5, 2015

IMPORTANCE Rare variants in the complement genes CFH, CFI, C9, and C3 have been found to be highly associated with age-related macular degeneration (AMD); however, the effect on clinical characteristics and familial segregation by these variants is lacking. OBJECTIVES To determine the contribution of rare CFH Arg1210Cys, CFI Gly119Arg, C9 Pro167Ser, and C3 Lys155Gln variants in the development of AMD in 22 multiplex families and to describe clinical differences in carriers vs noncarriers in these families and a large case-control cohort. DESIGN, SETTING, AND PARTICIPANTS This retrospective case-control study included 114 affected and 60 unaffected members of 22 multiplex families with AMD as well as 1589 unrelated patients with AMD and 1386 unrelated control individuals enrolled in the European Genetic Database (EUGENDA). Patients were recruited from March 29, 2006, to April 26, 2013, and data were collected from April 20, 2012, to May 7, 2014. All participants underwent an extensive ophthalmic examination and completed a questionnaire. Venous blood samples were obtained from all participants for genetic analysis, including whole-exome sequencing and measurements of complement activation. Data were analyzed from September 23, 2014, to November 4, 2015. MAIN OUTCOMES AND MEASURES Differences between carriers and noncarriers of rare variants in age at onset of symptoms, the family history of AMD, complement activation levels (C3d:C3 ratio), the presence of reticular pseudodrusen, and AMD phenotype. RESULTS Among the 114 affected and 60 unaffected members of 22 multiplex families with AMD and the 1598 unrelated patients with AMD and 1386 controls in the EUGENDA cohort who underwent analysis, the presence of the CFI Gly119Arg, C9 Pro167Ser, or C3 Lys155Gln variant was confirmed in 18 individuals in 5 families but did not completely segregate with the disease. In the case-control cohort, the 91 affected carriers of these variants were younger at symptom onset (mean [SD] age, 67.4 [8.5] vs 71.3 [8.9] years; P = .01) and more often reported a positive family history (35 of 79 [44.3%] vs 367 of 1201 [30.6%]; P = .008) compared with the 1498 noncarriers. Patients with advanced atrophic AMD carried these rare variants more frequently than patients with neovascular AMD (11 of 93 [11.8%] vs 40 of 835 [4.8%]; P = .04). CONCLUSIONS AND RELEVANCE Previously reported rare variants do not completely segregate within families with AMD. However, patients carrying these rare variants differ clinically from noncarriers by an earlier age at symptom onset, higher prevalence of a positive family history, and AMD phenotype. These results suggest that genetic tests for AMD might be designed to detect common and rare genetic variants, especially in families, because rare variants contribute to the age at onset and progression of the disease.

The SNP rs3753396 in CFH and SNP rs6685931 in CFHR4 are associated with systemic complement activation levels. The SNP rs6685931 in CFHR4 and its linked haplotype H1-2 also conferred a risk for AMD development, and therefore could be used to identify AMD patients who would benefit most from complement-inhibiting therapies.