Serum levels of FH (A), FI (B), C9 (C), and C3 (D) measured in carriers and noncarriers in corresponding genes. Significance values are on the left (vs noncarrier cases) and right (vs noncarrier controls). Lines indicate median; error bars, interquartile range. C3 indicates complement 3 gene; C9, complement 9 gene; CFH, complement factor H gene; and CFI, complement factor I gene.
aP < .001.
C3b degradation depicted as ratio of 43-kDa degradation product over the α′ chain. Significance values are on the left (vs noncarrier cases) and right (vs noncarrier controls). Lines indicate median; error bars, interquartile range.
bP < .01.
cP = .39.
Correlation between the FH (A) and FI (B) serum levels and the ability to degrade C3b. Complement factor H (CFH) gene carriers had normal serum levels but functional defect. Complement factor I (CFI) gene variants had reduced serum levels.
eMethods. Additional Analyses
eFigure. Pedigrees of 7 AMD Families in Which Rare Variants Were Identified
eTable 1. Number and Percentage of Carriers of Rare Variants in the Case-Control Cohort
eTable 2. Median Serum Levels of FH, FI, C9, and C3 in Carriers and Noncarriers of Rare Variants
eTable 3. Median C3b Degradation (43-kDA Product Over the α-Chain) and Quartiles
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Geerlings MJ, Kremlitzka M, Bakker B, et al. The Functional Effect of Rare Variants in Complement Genes on C3b Degradation in Patients With Age-Related Macular Degeneration. JAMA Ophthalmol. 2017;135(1):39–46. doi:10.1001/jamaophthalmol.2016.4604
Is there a functional effect of rare genetic variants in the complement system on complement levels and activity in serum?
In this study, carriers of complement factor I gene variants had decreased factor I levels; carriers of the complement 9 Pro167Ser gene had increased C9 levels, whereas C3 and factor H levels were not altered. Carriers of complement factor H and complement factor I gene variants had a reduced ability to degrade C3b, which for the complement factor I gene was associated with reduced serum factor I levels.
This study suggests that carriers of rare variants in the complement factor I and complement factor H genes are less able to inhibit complement activation and may benefit more from complement-inhibiting therapy than patients with age-related macular degeneration in general.
In age-related macular degeneration (AMD), rare variants in the complement system have been described, but their functional consequences remain largely unexplored.
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.
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 (R2 = 0.35 and R2 = 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 patients with AMD in general.
Age-related macular degeneration (AMD) is caused by a combination of environmental and genetic factors. Although aging and smoking confer the strongest nongenetic risk, genetic alterations account for 45% to 70% of the variability in disease risk.1 Genetically, AMD is heterogeneous, with 34 genomic loci implicated in disease pathogenesis. Susceptibility genes that reside in these loci are grouped into 4 main pathways: (1) complement system, (2) high-density lipoprotein metabolism, (3) angiogenesis, and (4) extracellular matrix remodeling.2,3
The complement system is part of the innate immune system, and tight regulation of this system is needed to protect the body’s own cells from tissue damage. The central component of the system is C3, which is cleaved into C3b and C3a. C3b is a crucial component of C3 and C5 convertases that catalyze further steps in the cascade. The final step is the formation of the membrane attack complex, which includes several copies of C9. Factor H (FH) is one of the main inhibitors of complement through binding of C3b and aiding its degradation by serine protease factor I (FI).4,5
In AMD, the complement system is highly burdened by genetic variations.6 Most of these genetic variants are relatively common in the population and have a modest to low effect on AMD development.3 Recently, rare genetic variants (defined by a minor allele frequency <1%) in the complement system were also described to play an important role in AMD. Such rare variants were described in the complement factor H (CFH) (NM_000186),7-10 complement factor I (CFI) (NM_000204),11,12 complement factor 9 (C9) (NM_001737),12 and complement factor 3 (C3) (NM_000064) genes.12-15 Carriers of these rare genetic variants presented with a younger age at disease onset and more often progressed to end-stage AMD compared with noncarriers.10,16-19
Only a limited number of studies10-12,18-21 have investigated functional effects of rare variants on activity of the affected protein and the complement system overall. The reported effects of rare CFH variants on FH levels are inconsistent. Although one study19 reported reduced serum FH levels in rare variants carriers, others10,20 did not observe this effect. Lower FI serum and plasma levels were found in carriers of CFI variants compared with controls.11,18 In addition, the CFI variant Gly119Arg resulted in a lower ability to degrade C3b.11 Similarly, carriers of the rare variant Lys155Gln in C3 had reduced C3b cleavage.12 The C3 variant Arg161Trp was reported to affect the ability of FH to inhibit C3 convertase.21C9 variants were previously associated with AMD12,22; however, the functional effect of these variants has not been studied.
In this study, we aimed to identify novel rare genetic variants in complement genes previously associated with AMD. We intended to determine the effect of rare genetic variants on levels of complement components in serum and analyze the ability to degrade C3b in serum samples from rare variant carriers compared with noncarriers.
We evaluated 22 severely affected AMD families with at least 4 affected siblings, resulting in 114 affected and 60 unaffected family members, from March 29, 2006, to April 26, 2013, in Nijmegen, the Netherlands, and Cologne, Germany. In addition, 1831 unrelated patients with AMD and 1367 unrelated control individuals from the European Genetic Database (EUGENDA) were studied. Control individuals were 60 years or older. All patients underwent clinical evaluation and were graded for AMD according to the Cologne Image Reading Center protocol.17,23 Serum samples were obtained by a standard coagulation and centrifugation protocol, after which they were stored at −80°C within 1 hour after collection. Genomic DNA was isolated from peripheral blood samples according to standard procedures. This study was approved by local ethics committees on research involving human subjects, namely, the Commissie Mensgebonden Onderzoek Regio Arnhem-Nijmegen and the local committee of University Hospital Cologne, and met the criteria of the Declaration of Helsinki.24 Before enrollment in EUGENDA, all participants provided written informed consent and were assigned a database identifier code for anonymization.
Whole exome sequencing analysis was implemented to uncover the coding regions (eMethods in the Supplement) of selected complement genes previously reported to harbor rare variants associated with AMD, namely, CFH, CFI, C9, and C3.7-15 From the candidate genes, we selected variants that would induce an amino acid change. Frequency filters from the public databases 1000 Genomes Project and Exome Variant Server database ensured selection of rare variants only. Variants with a minor allele frequency less than 1% were considered rare. Variants found in multiple individuals were selected for Sanger sequencing as confirmation and segregation. Primer sets used for Sanger sequencing were designed manually using Primer3Plus.25 Predicted effect of each variation was examined using PolyPhen2 and SIFT (Sorting Intolerant From Tolerant).26,27
Genotyping of rare genetic variants CFH Ser193Leu, CFH Arg175Gln, CFI Pro553Ser, CFI Leu131Arg, C9 Arg118Trp, and C3 Arg161Trp was performed for participants of the EUGENDA case-control cohort by custom-made competitive allele-specific polymerase chain reaction assays (Kompetitive Allele Specific Single-Nucleotide Polymorphism Genotyping System; LGC Ltd) according to the manufacturer’s recommendations.
We collected serum samples of rare variant carriers (n = 157) and available family members (n = 93). Two comparison groups with similar mean age had available serum samples: (1) 77 patients with AMD who did not carry any of the selected variants and (2) 80 control individuals who did not have any of the selected variants. The total number of serum samples encompassed 407, of whom 201 individuals carry a rare variant.
The concentrations of FH, FI, C9, and C3 in serum samples were measured by enzyme-linked immunosorbent assay in triplicate.28 In addition, the degradation of C3b in fluid phase was analyzed to assess how CFH and CFI variants affect the proteins' ability to degrade C3b in the fluid phase. Details are in the eMethods in the Supplement.
Because of the low frequency of rare variants, asymptotic statistics can be inaccurate. Therefore, we used exact statistics to test association for individual variants. This analysis included an aggregate meta-analysis, which included close family members. To include the first-degree siblings (second-degree siblings for families A and E), we calculated statistical significance using a binominal distribution. When family members were included, the chance for siblings to inherit similar rare genetic variation was 50%. The calculation of this statistical aggregation score has been replicated as described in detail by Raychaudhuri et al7 and a previously rare variant analysis.12
Serum levels and carrier status were analyzed using Kruskal-Wallis with the Dunn post hoc comparison adjustment. Within figures, median values with interquartile ranges are depicted, and differences at P < .05 were considered statistically significant. For correlations, the covariates AMD status and rare variant status were included. Spearman ρ correlation coefficient was used for nonparametric correlations, and P values were evaluated using Bonferroni correction (P < .007 was considered significant).
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 controls had a mean (SD) age of 70.4 (7.0), and 58.7% were female. All individuals were from European descent. We found CFI Gly119Arg, C9 Pro167Ser, and C3 Lys155Gln, which have been previously associated with AMD,7,11-14 and we described their familial segregation in detail previously.17 We identified 6 additional rare genetic variants, namely, CFH Ser193Leu, CFH Arg175Gln, CFI Pro553Ser, CFI Leu131Arg, C9 Arg118Trp, and C3 Arg161Trp (Table 1). Although the identified variants were highly prevalent within these families, a perfect segregation with disease phenotype was not observed (eFigure in the Supplement).
The 6 new variants clustered in 5 AMD families (families A, B, D, E, and F). Furthermore, we identified 2 smaller families, both consisting of 3 siblings, carrying the same variants (families C [CFH Ser193Leu] and G [C3 Arg161Trp]). Newly identified rare variants were present more frequently in affected members compared with unaffected individuals (28 of 37 [75.7%] and 4 of 10 [40.0%], respectively; P = .07).
Next, we investigated whether the rare variants identified in our families were associated with AMD in a case-control cohort of 1831 patients with AMD and 1367 control individuals. We identified 194 carriers of novel and previously identified variants in complement genes CFH, CFI, C9, or C3. Carrying one of these rare variants was significantly associated with AMD status because 139 carriers (72.4%) were AMD case patients and 53 (27.6%) were control individuals (odds ratio, 2.04; 95% CI, 1.47-2.82; P < .001). In the meta-analysis, which combined results of the family and case-control cohorts, all variants had a nominal association with AMD (Table 2 and eTable 1 in the Supplement).
To determine the effect of newly identified rare variants (CFH Ser193Leu, CFH Arg175Gln, CFI Pro553Ser, CFI Leu131Arg, C9 Arg118Trp, and C3 Arg161Trp) and of previously identified variants (CFI Gly119Arg, C9 Pro167Ser, and C3 Lys155Gln)17 on protein expression, we performed serum measurements. Levels of FH, FI, C3, and C9 were determined by enzyme-linked immunosorbent assay in serum samples of 314 individuals, of which 157 carried a rare variant. Carriers of different variants were grouped per gene and included both patients with AMD and controls. To assess differences between cases and controls, noncarriers were split based on AMD status (Figure 1). Significant differences in serum levels of FI and C9 were observed (eTable 2 in the Supplement). Carriers of CFI Gly119Arg had a significantly decreased median serum FI level (18.2 µg/mL) compared with noncarrier cases and controls (27.2 and 30.4 µg/mL, both P < .001), as did carriers of CFI Leu131Arg (16.2 µg/mL) vs noncarrier cases (27.2 µg/mL, P = .005) and controls (30.4 µg/mL, P = .001) (Figure 1B). Carriers of C9 Pro167Ser had an elevated C9 serum level (10.7 µg/mL) compared with noncarriers (6.6 µg/mL in cases and 6.1 µg/mL in controls, P < .001) (Figure 1C). Median FI serum levels for noncarriers were 27.2 and 30.4 µg/mL (cases and controls, respectively). Median C9 serum levels for noncarriers were 6.6 and 6.1 µg/mL (cases and controls, respectively). 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 (Figure 1A and D and eTable 2 in the Supplement).
Next, we assessed C3b degradation in serum samples from individuals carrying CFH or CFI mutations and compared them with noncarriers. As illustrated in Figure 2, carriers of rare variants in CFH and CFI had a lower capacity to degrade C3b when compared with noncarriers, except for CFI Pro553Ser carriers (eTable 3 in the Supplement).
Finally, we determined whether FH and FI levels affect an individual’s ability to degrade C3b irrespective of rare variant status (Figure 3). Linear regression revealed a positive correlation between both FH and FI serum levels with C3b degradation ratios in noncarrier cases and controls (r2 = 0.34 for FH serum levels and r2 = 0.31 for F1 serum levels; P < .001). Carriers of CFH and CFI variants were plotted in these graphs, revealing lower serum concentrations and/or a reduced ability to degrade C3b.
We identified 6 new rare variants in complement genes in 5 of 22 densely affected families with AMD. Serum measurements revealed altered serum levels for individuals carrying some of these variants compared with controls. In addition, serum samples from carriers of rare variants in CFH and CFI revealed a diminished ability to degrade C3b, suggesting that the variants result in impaired complement regulation.
To our knowledge, this is the first description of variants CFI Leu131Arg and C9 Arg118Trp in the literature. Variants Ser193Leu and Arg175Gln in CFH were both previously identified by our group.8CFI Pro553Ser has been described earlier in atypical hemolytic uremic syndrome (aHUS), a severe rare renal disease, but was also noted as a possible risk variant for AMD.12,18,29,30 The Arg161Trp variant in C3, another aHUS variant,31 was previously reported.15
In our case-control cohort, the newly and previously described rare variants were predominantly found in cases and contributed to an increased risk of developing AMD. Similarly, the carrier status within the families was higher in cases compared with unaffected family members. Even though the variants did not perfectly segregate with the AMD phenotype, the combined P value of the case-control and family cohorts supports an association of these rare variants with AMD. Our findings further strengthen the notion that rare variants in complement genes play an important role in AMD development and that family studies are a useful approach to identify rare variants.3,10,32
By analyzing the effect of rare variants on protein expression, we found that carriers of CFH mutations have normal serum FH concentration. Although no functional analysis has been described for either of these variants, a previous study19 measured FH levels in 5 patients with AMD carrying other rare variants in CFH (Cys192Phe, Tyr277*, Cys431Ser, 2 splice site variants). Lower median FH concentrations were observed in carriers compared with noncarriers. In another study,10 carriers of CFH variants Arg53Cys and Asp90Gly were found to have normal FH concentrations. Similarly, measurement of serum levels of CFH Gln950His carriers revealed FH levels within the reference range.20 These results indicate that not all rare variants in CFH lead to lower FH levels. Variants group into 2 major mutation types. Type 1 mutations cause low protein levels as a result of misfolding or degradation, whereas type 2 mutations result in reduced functionality with normal protein levels. The CFH variants identified in our study (CFH Ser193Leu and Arg175Gln) are most likely type 2.
The FI levels were measured in the serum of carriers of CFI Gly119Arg, Leu131Arg, and Pro553Ser. It was previously reported that individuals with advanced AMD carrying CFI variants have reduced FI concentration.18 In particular, Gly119Arg had high odds ratios for AMD and significantly reduced FI levels in plasma.11 We confirm this finding and report that novel variant Leu131Arg in CFI also leads to a strong reduction of FI serum levels. The third variant, CFI Pro553Ser (10 cases and 2 controls), did not alter FI levels compared with noncarriers, which is consistent with previous studies.12,18
C3 levels of carriers of C3 Lys155Gln or Arg161Trp variants were normal compared with noncarriers, in line with a previous report.33 Another study12 found that carriers of rare variant Lys155Gln failed to degrade C3b properly and hypothesized that this was caused by reduced binding to FH. Although C3 Lys155Glnis was highly associated with AMD, median serum level of C3 Lys155Gln did not differ from that of noncarriers, supporting that the variant influences protein functionality rather than level. In aHUS, Arg161Trp has been described to be pathogenic because of a hyperactive C3 convertase formation attributable to increased binding to factor B, accompanied by increased C3a, C5a, and membrane attack complex. The Arg161Trp variant leads to reduced binding to FI cofactors, such as FH.21,31,34,35 Although C3 variants result in a lower C3 level in most aHUS and AMD patients (70%-80%), very low C3 levels are observed only in patients with homozygous CFH or gain-of-function mutations in CFB or C3.21,33,36 One individual in family E carrying the Arg161Trp (indicated by an asterisk) reported reduced renal function attributable to hypertension and urolithiasis.
To our knowledge, we are the first to report significantly elevated C9 serum levels in carriers of C9 Pro167Ser in AMD. We hypothesize this increased C9 level results from elevated complement activation in patients with AMD, which, through lysis of the target cells, may directly contribute to retinal destruction observed in the disease pathogenesis. For C9 only 2 variants have been described in AMD: C9 Pro167Ser12 and Arg95*,22 the latter being inherent to Asian populations. Our study indicates that other rare variants in the C9 gene, such as Arg118Trp, are also associated with AMD, although this variant did not affect C9 serum concentration.
In this study, all carriers of rare variants in CFH and CFI had reduced ability to degrade C3b compared with noncarriers. Furthermore, carriers of CFI variants (Gly119Arg and Leu131Arg) had decreased FI serum level. A previous study,11 using recombinant FI (Gly119Arg), reported that both expression and secretion of mutant protein were reduced compared with wild-type protein. Consequently, impaired levels led to reduced C3b degradation. The FH levels remained stable, suggesting that CFH variants affect complement activation independent of FH serum levels by its inability to properly serve as a cofactor in the cleavage of C3b to inactive C3b. This finding might be explained by the variants’ location at the N-terminus, where a C3b-binding site is located.37
We detected a natural correlation of FH and FI levels with the ability to degrade C3b in noncarrier individuals. Carriers of rare variants in CFH and CFI group outside the linear curve of noncarriers. This finding suggests that carriers of rare variants in the CFH and CFI genes have a decreased ability to degrade C3b and thus higher levels of complement activation compared with noncarriers and may benefit more from complement-inhibiting therapy than patients with AMD in general.
Several clinical trials are currently evaluating complement-inhibiting treatments in AMD,38 and 2 clinical trials have been completed. The Complement Inhibition with Eculizumab for the Treatment of Nonexudative Age-Related Macular Degeneration (COMPLETE) study involved eculizumab, an antibody that binds to C5 and inhibits cleavage of C5 to C5a.39 The trial results suggested eculizumab was not effective in treating AMD because the growth of geographic atrophy (advanced AMD) did not decrease after 6 months of treatment. The Safety, Tolerability, and Evidence of Activity of FCFD4514S Administered Monthly or Every Other Month to Patients With Geographic Atrophy (MAHALO) study with lampalizumab, an antibody directed against complement factor D, which is a rate-limiting enzyme involved in the activation of the alternative pathway,40 had promising results because progression of the geographic atrophy lesion had a 20% reduction after 18 months of treatment. Lampalizumab has been suggested to be most effective in a subpopulation of patients because an even higher reduction rate was seen in patients with a specific CFI genotype.40 Selection of such patients would lead to more effective clinical trials, requiring smaller patient groups to reveal an effect of the drug being tested. Screening of individuals for genetic (eg, rare variants in CFH and CFI) or serum (eg, reduced FI levels) biomarkers will enable treatment in an early phase of the disease, before substantial tissue damage has occurred. Personalized treatment could be provided for patients with rare genetic variants in the CFH and CFI genes or reduced FI levels, linked to the functional inability to degrade C3b efficiently, and act on this effect by using complement inhibitors.
A limitation of the study is that functional effects of variants were only assessed for those variants that were significantly associated with AMD. Private variants were not included in this study. Enlarging the study cohort would improve the power to detect rare variants.
We identified multiple rare variants in complement genes encoding FH, FI, C9, or C3. Carriers of CFI (Gly119Arg; Leu131Arg) had decreased FI levels, whereas individuals with the C9 Pro167Ser variant had elevated serum concentrations compared with noncarriers. Carrying a CFH or C3 variant did not change FH or C3 levels. Carriers of rare variants in CFH and CFI had a reduced ability to degrade C3b compared with noncarriers. For CFI, this effect was linked to reduced serum FI levels, but CFH affects C3b degradation independent of FH serum levels. Carriers of rare variants in CFI and CFH are less able to inhibit complement activation and may benefit more from complement-inhibiting therapy than patients with AMD in general. Our results suggest that patients with AMD should be screened using a functional complement assay and should be tested for rare genetic variants and corresponding serum levels to apply the most proper therapeutic regimen for disease treatment.
Corresponding Author: Anneke I. den Hollander, PhD, Department of Ophthalmology, Donders Institute for Brain, Cognition, and Behaviour, Radboud University Medical Center, PO Box 9101, 6500 HB Nijmegen, the Netherlands (email@example.com).
Accepted for Publication: September 19, 2016.
Published Online: December 1, 2016. doi:10.1001/jamaophthalmol.2016.4604
Author Contributions: Dr den Hollander had full access to all the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis.
Study concept and design: Geerlings, Kremlitzka, Saksens, Blom, de Jong, den Hollander.
Acquisition, analysis, or interpretation of data: Geerlings, Kremlitzka, Bakker, Nilsson, Saksens, Lechanteur, Pauper, Corominas, Fauser, Hoyng, de Jong, den Hollander.
Drafting of the manuscript: Geerlings, Kremlitzka.
Critical revision of the manuscript for important intellectual content: All authors.
Statistical analysis: Geerlings, Kremlitzka, Saksens.
Obtained funding: Blom, den Hollander.
Administrative, technical, or material support: Geerlings, Kremlitzka, Bakker, Nilsson, Saksens, Pauper, Corominas, Fauser.
Conflict of Interest Disclosures: All authors have completed and submitted the ICMJE Form for Disclosure of Potential Conflicts of Interest and none were reported.
Funding/Support: The research leading to these results has received funding from the European Research Council under the European Union’s Seventh Framework Programme (FP/2007-2013)/ERC Grant Agreement n. 310644 (MACULA) (Dr den Hollander), grant C-GE-0811-0548-RAD04 from the Foundation Fighting Blindness USA (Dr den Hollander), grant K2012-66S-14928-09-5 from the Swedish Research Council (Dr Blom), and a grant for clinical research (Avtal om Läkarutbildning och Forskning [ALF]) (Dr Blom).
Role of the Funder/Sponsor: The funding sources had no role in the design and conduct of the study; collection, management, analysis, and interpretation of the data; preparation, review, or approval of the manuscript; and decision to submit the manuscript for publication.
Additional Contributions: Joannes M. M. Groenewoud, MSc, Department for Health Evidence, Radboud University Medical Center, Nijmegen, the Netherlands, provided support in the statistical analyses. He received no additional compensation for this role.
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