Functional analysis of rare genetic variants in complement factor I in advanced age-related macular degeneration.

Factor I (FI) is a serine protease inhibitor of the complement system. Heterozygous rare genetic variants in complement factor I (CFI) are associated with advanced age-related macular degeneration (AMD). The clinical impact of these variants is unknown since a majority have not been functionally characterized and are classified as 'variants of uncertain significance' (VUS). This study assessed the functional significance of VUS in CFI. Our previous cross-sectional study using a serum-based assay demonstrated that CFI variants in advanced AMD can be categorized into three types. Type 1 variants cause a quantitative deficiency of FI. Type 2 variants demonstrate a qualitative deficiency. However, Type 3 variants consist of VUS that are less dysfunctional than Types 1 and 2 but are not as biologically active as wild type (WT). In this study, we employed site-directed mutagenesis followed by expression of the recombinant variant and a comprehensive set of functional assays to characterize nine Type 3 variants that were identified in 37 individuals. Our studies establish that the expression of the recombinant protein compared with WT is reduced for R202I, Q217H, S221Y and G263V. Further, G362A and N536K, albeit expressed normally, have significantly less cofactor activity. These results led to re-categorization of CFI variants R202I, Q217H, S221Y and G263V as Type 1 variants and to reclassification of N536K and G362A as Type 2. The variants K441R, Q462H and I492L showed no functional defect and remained as Type 3. This study highlights the utility of an in-depth biochemical analysis in defining the pathologic and clinical implications of complement variants underlying AMD.

Long-term risk of subsequent cancer incidence among hereditary and nonhereditary retinoblastoma survivors.

BACKGROUND: Increased sarcoma and melanoma risks after hereditary retinoblastoma are well established, whereas less is known about epithelial subsequent malignant neoplasms (SMNs) and risks for multiple (≥2) SMNs. METHODS: Leveraging long-term follow-up and detailed histologic information, we quantified incident SMN risk among 1128 hereditary and 924 nonhereditary retinoblastoma survivors (diagnosed 1914-2006; follow-up through 2016). Standardised incidence ratios (SIRs) compared cancer risk after retinoblastoma relative to the general population. We estimated cumulative incidence accounting for competing risk of death. RESULTS: Hereditary survivors had statistically significantly increased SMN risk (N = 239; SIR = 11.9; 95% confidence interval [CI] 10.4-13.5), with SIRs >80-fold for sarcomas, nasal cavity tumours and pineoblastoma. Significantly increased risks were also observed for melanoma and central nervous system, oral cavity and breast SMNs (SIRs = 3.1-17), but not the uterus, kidney, lung, bladder, pancreas or other types. Cumulative incidence 50 years following hereditary retinoblastoma was 33.1% (95% CI 29.0-37.2) for a first SMN and 6.0% (95% CI 3.8-8.2) for a second SMN. SMN risk was not increased after nonhereditary retinoblastoma (N = 25; SIR = 0.8; 95% CI 0.5-1.2). CONCLUSION: Beyond the established sarcoma and melanoma risks after hereditary retinoblastoma, we demonstrate increased risk for a more limited number of epithelial malignancies than previously suggested. Cumulative incidence estimates emphasise long-term SMN burden after hereditary retinoblastoma.

Choriocapillaris dropout in early age-related macular degeneration.

Loss of choriocapillaris (CC) in advanced age-related macular degeneration (AMD) is well documented but changes in early AMD have not been quantified. Postmortem eyes from donors with clinically documented early AMD were examined in choroidal whole mounts to determine the area, pattern, and severity of CC loss. Choroids from postmortem human eyes without AMD (n = 7; mean age = 86.1) and from eyes with a Grade 2 clinical classification of early AMD (n = 7; mean age = 87) were immunolabeled with Ulex europaeus agglutinin (UEA) lectin-FITC to stain blood vessels. Whole mounts were imaged using confocal microscopy and image analysis was performed to determine the area of vascular changes and density of vasculature (percent vascular area, %VA). All areas evaluated had a complete RPE monolayer upon gross examination. In age-matched control eyes, the CC had broad lumens and a homogenous pattern of freely interconnecting capillaries. The mean %VA ± standard deviation in submacula of control subjects was 78.1 ± 3.25%. In eyes with early AMD, there was a significant decrease in mean %VA to 60.1 ± 10.4% (p < 0.0001). The paramacular %VA was not significantly different in eyes with or without AMD. The area of submacular choroid affected by CC dropout was 0.04 ± 0.09 mm2 in control eyes. In eyes with early AMD, the mean area affected by CC dropout was significantly increased (10.4 ± 6.1 mm2; p < 0.001). In some cases, incipient neovascular buds were observed at the border of regions with CC dropout in early AMD choroids. In conclusion, UEA lectin-labeled choroidal whole mounts from donors with clinically documented early AMD has provided a unique opportunity to examine regional changes in vascular pathology associated with choriocapillaris. The study demonstrated attenuation of submacular CC in early AMD subjects but no vascular pathology was observed outside the submacular region. While the affected area in some eyes was quite extensive histologically, these changes may not be detectable clinically using standard in vivo imaging.

Protective coding variants in CFH and PELI3 and a variant near CTRB1 are associated with age-related macular degeneration†.

Although numerous common age-related macular degeneration (AMD) alleles have been discovered using genome-wide association studies, substantial disease heritability remains unexplained. We sought to identify additional common and rare variants associated with advanced AMD. A total of 4,332 cases and 25,268 controls of European ancestry from three different populations were genotyped using the Illumina Infinium HumanExome BeadChip. We performed meta-analyses to identify associations with common variants, and single variant and gene-based burden tests to identify rare variants. Two protective, low-frequency, non-synonymous variants were significantly associated with a decrease in AMD risk: A307V in PELI3 (odds ratio [OR] = 0.14, P = 4.3 × 10-10) and N1050Y in CFH (OR = 0.76, P = 6.2 × 10-12). The new variants have a large effect size, similar to some rare mutations we reported previously in a targeted sequencing study, which remain significant in this analysis: CFH R1210C (OR = 18.82, P = 3.5 × 10-07), C3 K155Q (OR = 3.27, P = 1.5 × 10-10) and C9 P167S (OR = 2.04, P = 2.8 × 10-07). We also identified a strong protective signal for a common variant (rs8056814) near CTRB1 associated with a decrease in AMD risk (logistic regression: OR = 0.71, P = 1.8 × 10-07). Suggestive protective loci were identified in the COL4A3 and APOH genes. Our results support the involvement of common and low-frequency protective variants in this vision-threatening condition. This study expands the roles of the innate immune pathway as well as the extracellular matrix and high-density lipoprotein pathways in the aetiology of AMD.

Rare genetic variants in the CFI gene are associated with advanced age-related macular degeneration and commonly result in reduced serum factor I levels.

To assess a potential diagnostic and therapeutic biomarker for age-related macular degeneration (AMD), we sequenced the complement factor I gene (CFI) in 2266 individuals with AMD and 1400 without, identifying 231 individuals with rare genetic variants. We evaluated the functional impact by measuring circulating serum factor I (FI) protein levels in individuals with and without rare CFI variants. The burden of very rare (frequency <1/1000) variants in CFI was strongly associated with disease (P = 1.1 × 10(-8)). In addition, we examined eight coding variants with counts ≥5 and saw evidence for association with AMD in three variants. Individuals with advanced AMD carrying a rare CFI variant had lower mean FI compared with non-AMD subjects carrying a variant (P < 0.001). Further new evidence that FI levels drive AMD risk comes from analyses showing individuals with a CFI rare variant and low FI were more likely to have advanced AMD (P = 5.6 × 10(-5)). Controlling for covariates, low FI increased the risk of advanced AMD among those with a variant compared with individuals without advanced AMD with a rare CFI variant (OR 13.6, P = 1.6 × 10(-4)), and also compared with control individuals without a rare CFI variant (OR 19.0, P = 1.1 × 10(-5)). Thus, low FI levels are strongly associated with rare CFI variants and AMD. Enhancing FI activity may be therapeutic and measuring FI provides a screening tool for identifying patients who are most likely to benefit from complement inhibitory therapy.

Whole-exome sequencing identifies rare, functional CFH variants in families with macular degeneration.

We sequenced the whole exome of 35 cases and 7 controls from 9 age-related macular degeneration (AMD) families in whom known common genetic risk alleles could not explain their high disease burden and/or their early-onset advanced disease. Two families harbored novel rare mutations in CFH (R53C and D90G). R53C segregates perfectly with AMD in 11 cases (heterozygous) and 1 elderly control (reference allele) (LOD = 5.07, P = 6.7 × 10(-7)). In an independent cohort, 4 out of 1676 cases but none of the 745 examined controls or 4300 NHBLI Exome Sequencing Project (ESP) samples carried the R53C mutation (P = 0.0039). In another family of six siblings, D90G similarly segregated with AMD in five cases and one control (LOD = 1.22, P = 0.009). No other sample in our large cohort or the ESP had this mutation. Functional studies demonstrated that R53C decreased the ability of FH to perform decay accelerating activity. D90G exhibited a decrease in cofactor-mediated inactivation. Both of these changes would lead to a loss of regulatory activity, resulting in excessive alternative pathway activation. This study represents an initial application of the whole-exome strategy to families with early-onset AMD. It successfully identified high impact alleles leading to clearer functional insight into AMD etiopathogenesis.

Idiopathic preretinal glia in aging and age-related macular degeneration.

During analysis of glia in wholemount aged human retinas, frequent projections onto the vitreal surface of the inner limiting membrane (ILM) were noted. The present study characterized these preretinal glial structures. The amount of glial cells on the vitreal side of the ILM was compared between eyes with age-related macular degeneration (AMD) and age-matched control eyes. Retinal wholemounts were stained for markers of retinal astrocytes and activated Müller cells (glial fibrillary acidic protein, GFAP), Müller cells (vimentin, glutamine synthetase) and microglia/hyalocytes (IBA-1). Retinal vessels were labeled with UEA lectin. Images were collected using a Zeiss LSM 710 confocal microscope. Retinas were then cryopreserved. Laminin labeling of cryosections determined the location of glial structures in relation to the ILM. All retinas investigated herein had varied amounts of preretinal glia. These glial structures were classified into three groups based on size: sprouts, blooms, and membranes. The simplest of the glial structures observed were focal sprouts of singular GFAP-positive cells or processes on the vitreal surface of the ILM. The intermediate structures observed, glial blooms, were created by multiple cells/processes exiting from a single point and extending along the vitreoretinal surface. The most extensive structures, glial membranes, consisted of compact networks of cells and processes. Preretinal glia were observed in all areas of the retina but they were most prominent over large vessels. While all glial blooms and membranes contained vimentin and GFAP-positive cells, these proteins did not always co-localize. Many areas had no preretinal GFAP but had numerous vimentin only glial sprouts. In double labeled glial sprouts, vimentin staining extended beyond that of GFAP. Hyalocytes and microglia were detected along with glial sprouts, blooms, and membranes. They did not, however, concentrate in the retina below these structures. Cross sectional analysis identified small breaks in the ILM above large retinal vessels through which glial cells exited the retina. Preretinal glial structures of varied sizes are a common occurrence in aged retinas and, in most cases, are subclinical. While all retinal glia are found in blooms, vimentin labeling suggests that Müller cells form the leading edge. All retinas investigated from eyes with active choroidal neovascularization (CNV) had extensive glial membranes on the vitreal surface of the ILM. Although these structures may be benign, they may exert traction on the retina as they spread along the vitreoretinal interface. In cases with CNV, glial cells in the vitreous could bind intravitreally injected anti-vascular endothelial growth factor. These preretinal glial structures indicate the remodeling of both astrocytes and Müller cells in aged retinas, in particular those with advanced AMD.

Contribution of the Nurses' Health Study to the Epidemiology of Cataract, Age-Related Macular Degeneration, and Glaucoma.

OBJECTIVES: To review the contribution of the Nurses' Health Study (NHS) to understanding the genetic and lifestyle factors that influence the risk of cataract, age-related macular degeneration, and glaucoma. METHODS: We performed a narrative review of the publications of the NHS between 1976 and 2016. RESULTS: The NHS has helped to elucidate the roles of genetics, lifestyle factors (e.g., cigarette smoking associated with cataract extraction and age-related macular degeneration), medical conditions (e.g., diabetes associated with cataract extraction and glaucoma), and dietary factors (e.g., greater carotenoid intake and lower glycemic diet associated with lower risk of age-related macular degeneration) in the etiology of degree and progression of lens opacities, cataract extraction, age-related macular degeneration, primary open-angle glaucoma, and exfoliation glaucoma. CONCLUSIONS: The findings from the NHS, combined with those of other studies, have provided compelling evidence to support public health recommendations for helping to prevent age-related eye diseases: abstinence from cigarette smoking, maintenance of healthy weight and diabetes prevention, and a healthy diet rich in fruits and vegetables.

(Epi)Genetic analyses of age-related macular degeneration: case-control and discordant twin studies.

BACKGROUND/AIMS: Phenotypic discordance in monozygotic (MZ) twin pairs can have an epigenetic or genetic basis. Although age-related macular degeneration (AMD) has a strong genetic component, few studies have addressed its epigenetic basis. METHODS: Using SNP arrays, we evaluated differences in copy number variation (CNV) and allele-specific methylation (ASM) patterns (via methyl-sensitive restriction enzyme digestion of DNA) in MZ twin pairs from the US Twin Study of AMD. Further analyses examined the relationship between ASM and CNVs with AMD by both case/control analysis of ASM at candidate regions and by analysis of ASM and CNVs in twins discordant for AMD. RESULTS: The frequency of ASM sites differs between cases and controls in regions surrounding the AMD candidate genes CFH, C2 and CFB. While ASM patterns show a substantial dependence on local sequence polymorphisms, we observed dissimilar patterns of ASM between MZ twins. The genes closest to the sites where discordant MZ twins have dissimilar patterns of ASM are enriched for genes implicated in gliosis, a process associated with neovascular AMD. Similar twin-based analyses revealed no AMD-associated CNVs. CONCLUSIONS: Our results provide evidence of epigenetic influences beyond the known genetic susceptibility and implicate inflammatory responses and gliosis in the etiology of AMD.

Common variation in three genes, including a noncoding variant in CFH, strongly influences risk of age-related macular degeneration.

Age-related macular degeneration (AMD) is a common, late-onset disease with seemingly typical complexity: recurrence ratios for siblings of an affected individual are three- to sixfold higher than in the general population, and family-based analysis has resulted in only modestly significant evidence for linkage. In a case-control study drawn from a US-based population of European descent, we have identified a previously unrecognized common, noncoding variant in CFH, the gene encoding complement factor H, that substantially increases the influence of this locus on AMD, and we have strongly replicated the associations of four other previously reported common alleles in three genes (P values ranging from 10(-6) to 10(-70)). Despite excellent power to detect epistasis, we observed purely additive accumulation of risk from alleles at these genes. We found no differences in association of these loci with major phenotypic categories of advanced AMD. Genotypes at these five common SNPs define a broad spectrum of interindividual disease risk and explain about half of the classical sibling risk of AMD in our study population.

The Role of Nutritional Factors in Transitioning between Early, Mid and Late Stages of Age-Related Macular Degeneration: prospective longitudinal analysis.

BACKGROUND: Transitions between different stages of age-related macular degeneration (AMD) are not completely captured by traditional survival models with an endpoint of advanced AMD. OBJECTIVE: This study aimed to explore the transitions from early and intermediate AMD to higher non-advanced and advanced stages and determine the contributions of nutritional factors to these outcomes. METHODS: Eyes with early or intermediate AMD at baseline, classified according to the Age-Related Eye Disease Study severity score, were included in this prospective longitudinal analysis. Foods and the biologically active nutrients associated with AMD [green leafy vegetables, fish, lutein/zeaxanthin (LZ), and ω-3 fatty acids] were determined by a baseline food frequency questionnaire. Progression was defined as eyes transitioning to higher severity groups including non-advanced and advanced stages over 5 years, confirmed at 2 consecutive visits. Cox proportional hazards models for foods and nutrients were analyzed adjusting for demographics, lifestyle, baseline macular status, family history of AMD, caloric intake and genetic risk. RESULTS: Among 2697 eyes, 616 (23%) progressed to higher severity groups. In the food group model, higher intake of green leafy vegetables reduced incidence of transitions [hazard ratio (HR) (≥ 2.7 servings/week vs. none) = 0.75 (95% confidence interval 0.59 - 0.96), P = 0.02]. Higher fish intake was also protective [HR (≥ two 4-ounce servings/week vs. < 2) = 0.79 (0.65 - 0.95), P = 0.01]. In the nutrient model, LZ intake was protective [HR (≥ 2 mg/day vs. < 2) = 0.76 (0.60 - 0.96), P = 0.02]. Higher intake of ω-3 fatty acids also tended to be beneficial [HR (≥ 0.7 g/week vs. < 0.7) = 0.85 (0.71 - 1.01), P = 0.06]. CONCLUSIONS: Increased consumption of green leafy vegetables, LZ, and fish nutritionally rich in ω-3 fatty acids during the initial stages of AMD may reduce rates of progression to higher severity of this debilitating disease. CLINICAL TRIAL: NCT00594672.

Common variants near FRK/COL10A1 and VEGFA are associated with advanced age-related macular degeneration.

Despite significant progress in the identification of genetic loci for age-related macular degeneration (AMD), not all of the heritability has been explained. To identify variants which contribute to the remaining genetic susceptibility, we performed the largest meta-analysis of genome-wide association studies to date for advanced AMD. We imputed 6 036 699 single-nucleotide polymorphisms with the 1000 Genomes Project reference genotypes on 2594 cases and 4134 controls with follow-up replication of top signals in 5640 cases and 52 174 controls. We identified two new common susceptibility alleles, rs1999930 on 6q21-q22.3 near FRK/COL10A1 [odds ratio (OR) 0.87; P = 1.1 × 10(-8)] and rs4711751 on 6p12 near VEGFA (OR 1.15; P = 8.7 × 10(-9)). In addition to the two novel loci, 10 previously reported loci in ARMS2/HTRA1 (rs10490924), CFH (rs1061170, and rs1410996), CFB (rs641153), C3 (rs2230199), C2 (rs9332739), CFI (rs10033900), LIPC (rs10468017), TIMP3 (rs9621532) and CETP (rs3764261) were confirmed with genome-wide significant signals in this large study. Loci in the recently reported genes ABCA1 and COL8A1 were also detected with suggestive evidence of association with advanced AMD. The novel variants identified in this study suggest that angiogenesis (VEGFA) and extracellular collagen matrix (FRK/COL10A1) pathways contribute to the development of advanced AMD.

Age-related macular degeneration.

Age-related macular degeneration is a major cause of blindness worldwide. With ageing populations in many countries, more than 20% might have the disorder. Advanced age-related macular degeneration, including neovascular age-related macular degeneration (wet) and geographic atrophy (late dry), is associated with substantial, progressive visual impairment. Major risk factors include cigarette smoking, nutritional factors, cardiovascular diseases, and genetic markers, including genes regulating complement, lipid, angiogenic, and extracellular matrix pathways. Some studies have suggested a declining prevalence of age-related macular degeneration, perhaps due to reduced exposure to modifiable risk factors. Accurate diagnosis combines clinical examination and investigations, including retinal photography, angiography, and optical coherence tomography. Dietary anti-oxidant supplementation slows progression of the disease. Treatment for neovascular age-related macular degeneration incorporates intraocular injections of anti-VEGF agents, occasionally combined with other modalities. Evidence suggests that two commonly used anti-VEGF therapies, ranibizumab and bevacizumab, have similar efficacy, but possible differences in systemic safety are difficult to assess. Future treatments include inhibition of other angiogenic factors, and regenerative and topical therapies.

Dietary omega-3 fatty acids, other fat intake, genetic susceptibility, and progression to incident geographic atrophy.

OBJECTIVE: To investigate associations between dietary omega-3 fatty acids and other fat intake, genes related to age-related macular degeneration (AMD), and progression to geographic atrophy (GA). DESIGN: Observational analysis of a prospective cohort. PARTICIPANTS: A total of 2531 individuals from the Age-Related Eye Disease Study, among which 525 eyes progressed to GA and 4165 eyes did not. METHODS: Eyes without advanced AMD at baseline were evaluated for progression to GA. Behavioral data, including smoking and body mass index measurements, were collected at baseline using questionnaires. Dietary data were collected from food frequency questionnaires (FFQs) at baseline. Omega-3 fatty acids (docosahexaenoic acid [DHA] and eicosapentaenoic acid [EPA]), omega-6 fatty acids, monounsaturated, saturated, polyunsaturated, and total fat were adjusted for sex and calories and divided into quintiles (Q). Eight single nucleotide polymorphisms in 7 genes (CFH, ARMS2/HTRA1, CFB, C2, C3, CFI, and LIPC) were genotyped. Cox proportional hazards models were used to test for associations between incident GA and intake of dietary lipids and interaction effects between dietary fat intake and genetic variation on risk of GA. MAIN OUTCOME MEASURES: Associations between dietary fat intake reported from FFQs, genetic variants, and incident GA. RESULTS: Increased intake of DHA was significantly associated with reduced risk of progression to GA in models with behavioral factors (model A) plus genetic variants (model B) (P trend = 0.01 and 0.03, respectively). Total omega-3 long chain polyunsaturated (DHA + EPA) fatty acid intake was significantly associated with reduced risk of progression in model B (P trend = 0.02). Monounsaturated fat was associated with increased risk in model A (P trend = 0.05). DHA intake was significantly associated with reduced risk of incident GA among those with the ARMS2/HTRA1 homozygous risk genotype (hazard ratio [HR] Q5 vs Q1, 0.4; P = 0.002; P for interaction between gene and fat intake = 0.05). DHA was not associated with reduced risk of GA among those with the homozygous ARMS2/HTRA1 nonrisk genotype (HR, 1.0; P = 0.90). CONCLUSIONS: Increased self-reported dietary intake of omega-3 fatty acids is associated with reduced risk of GA and may modify genetic susceptibility for progression to GA. FINANCIAL DISCLOSURE(S): The author(s) have no proprietary or commercial interest in any materials discussed in this article.

Genome-wide association study of advanced age-related macular degeneration identifies a role of the hepatic lipase gene (LIPC).

Advanced age-related macular degeneration (AMD) is the leading cause of late onset blindness. We present results of a genome-wide association study of 979 advanced AMD cases and 1,709 controls using the Affymetrix 6.0 platform with replication in seven additional cohorts (totaling 5,789 unrelated cases and 4,234 unrelated controls). We also present a comprehensive analysis of copy-number variations and polymorphisms for AMD. Our discovery data implicated the association between AMD and a variant in the hepatic lipase gene (LIPC) in the high-density lipoprotein cholesterol (HDL) pathway (discovery P = 4.53e-05 for rs493258). Our LIPC association was strongest for a functional promoter variant, rs10468017, (P = 1.34e-08), that influences LIPC expression and serum HDL levels with a protective effect of the minor T allele (HDL increasing) for advanced wet and dry AMD. The association we found with LIPC was corroborated by the Michigan/Penn/Mayo genome-wide association study; the locus near the tissue inhibitor of metalloproteinase 3 was corroborated by our replication cohort for rs9621532 with P = 3.71e-09. We observed weaker associations with other HDL loci (ABCA1, P = 9.73e-04; cholesterylester transfer protein, P = 1.41e-03; FADS1-3, P = 2.69e-02). Based on a lack of consistent association between HDL increasing alleles and AMD risk, the LIPC association may not be the result of an effect on HDL levels, but it could represent a pleiotropic effect of the same functional component. Results implicate different biologic pathways than previously reported and provide new avenues for prevention and treatment of AMD.

Deep ensemble learning for automated non-advanced AMD classification using optimized retinal layer segmentation and SD-OCT scans.

BACKGROUND: Accurate retinal layer segmentation in optical coherence tomography (OCT) images is crucial for quantitatively analyzing age-related macular degeneration (AMD) and monitoring its progression. However, previous retinal segmentation models depend on experienced experts and manually annotating retinal layers is time-consuming. On the other hand, accuracy of AMD diagnosis is directly related to the segmentation model's performance. To address these issues, we aimed to improve AMD detection using optimized retinal layer segmentation and deep ensemble learning. METHOD: We integrated a graph-cut algorithm with a cubic spline to automatically annotate 11 retinal boundaries. The refined images were fed into a deep ensemble mechanism that combined a Bagged Tree and end-to-end deep learning classifiers. We tested the developed deep ensemble model on internal and external datasets. RESULTS: The total error rates for our segmentation model using the boundary refinement approach was significantly lower than OCT Explorer segmentations (1.7% vs. 7.8%, p-value = 0.03). We utilized the refinement approach to quantify 169 imaging features using Zeiss SD-OCT volume scans. The presence of drusen and thickness of total retina, neurosensory retina, and ellipsoid zone to inner-outer segment (EZ-ISOS) thickness had higher contributions to AMD classification compared to other features. The developed ensemble learning model obtained a higher diagnostic accuracy in a shorter time compared with two human graders. The area under the curve (AUC) for normal vs. early AMD was 99.4%. CONCLUSION: Testing results showed that the developed framework is repeatable and effective as a potentially valuable tool in retinal imaging research.

Family History of Age-Related Macular Degeneration and Genetics Predict Progression to Advanced Age-Related Macular Degeneration Adjusting for Macular Status, Demographic, and Lifestyle Factors.

PURPOSE: To determine if a family history of age-related macular degeneration (AMD) and genetic variants identify eyes at higher risk for progression to advanced AMD (AAMD), after controlling for baseline demographics, behavioral factors, and macular status. DESIGN: Prospective, longitudinal cohort study. METHODS: Eyes were classified using the Age-Related Eye Disease Study severity scale. Non-genetic and genetic predictors for progression to AAMD, geographic atrophy, and neovascular disease were evaluated. Cox proportional hazards models using the eye as the unit of analysis were used to calculate hazard ratios (HRs), accounting for correlated data. Discrimination between progressing and non-progressing eyes was assessed using C-statistics and net reclassification improvement (NRI). RESULTS: Among 4910 eyes, 863 progressed to AAMD over 12 years. Baseline AMD severity scale and status of the fellow eye were important predictors; genes provided additional discrimination. A family history of AMD also independently predicted progression after accounting for genetic and other covariates: 1 family member versus none (HR 1.21 [95% confidence interval {CI} 1.02-1.43]; P = 0.03); ≥2 family members versus none (HR 1.55 [95% CI 1.26-1.90]; P < 0.001). A composite risk score calculated using ß estimates of both non-genetic and significant genetic factors predicted progression to AAMD (HR 5.57; 90th vs 10th percentile; area under the receiver operating characteristic curve [AUC] = 0.92), providing superior fit compared with other models, including one with only ocular variables (NRI = 0.34; P < 0.001; AUC = 0.87, ΔAUC 0.05 ± 0.005; P < 0.001). CONCLUSION: Genetic variants and family history provided additional discrimination for predicting progression to AAMD, after accounting for baseline macular status and other covariates.

Identifying Novel Genes and Variants in Immune and Coagulation Pathways Associated with Macular Degeneration.

Purpose: To select individuals and families with a low genetic burden for age-related macular degeneration (AMD), to inform the clinical diagnosis of macular disorders, and to find novel genetic variants associated with maculopathies. Design: Genetic association study based on targeted and whole-exome sequencing. Participants: A total of 758 subjects (481 individuals with maculopathy and 277 controls), including 316 individuals in 72 families. Methods: We focused on 150 genes involved in the complement, coagulation, and inflammatory pathways. Single-variant tests were performed on 7755 variants shared among ≥ 5 subjects using logistic regression. Gene-based tests were used to evaluate aggregate effects from rare and low-frequency variants (at minor allele frequency [MAF] ≤ 5% or ≤ 1%) in a gene using burden tests. For families whose affected members had a low burden of genetic risk based on known common and rare variants related to AMD, we searched for rare variants (MAF < 0.001) whose risk alleles occurred in ≥ 80% of affected individuals but not in controls. Immunohistochemistry was performed to determine the protein expression of a novel gene (coagulation factor II thrombin receptor-like 2 [F2RL2]) in retinal tissues. Main Outcome Measures: Genotypes and phenotypes of macular degeneration. Results: We confirmed the association of a synonymous variant in complement factor H (Ala473, rs2274700, proxy to intronic rs1410996, r 2  = 1) with maculopathy (odds ratio, 0.64; P = 4.5 × 10-4). Higher AMD polygenic risk scores (PRSs) were associated with intermediate and advanced AMD. Among families with low PRSs and no known rare variants for maculopathy, we identified 2 novel, highly penetrant missense rare variants in ADAM15, A disintegrin and metalloprotease, metallopeptidase domain 15 (p.Arg288Cys) and F2RL2 (p.Leu289Arg). Immunohistochemistry analyses revealed F2RL2 protein expression in cone photoreceptor outer segments and Müller glia cells of human and pig retinas. Coagulation factor II thrombin receptor-like 2 expression appeared increased in fibrotic areas in advanced AMD samples with neovascularization, suggesting that F2RL2 may play a role in the progression to advanced macular disease. Conclusions: New missense rare variants in the genes ADAM15 and F2RL2 were associated with maculopathies. Results suggest that novel genes related to the coagulation and immune pathways may be involved in the pathogenesis of macular diseases.

Rare Dysfunctional Complement Factor I Genetic Variants and Progression to Advanced Age-Related Macular Degeneration.

Purpose: To evaluate associations between rare dysfunctional complement factor I (CFI) genetic variant status and progression to advanced age-related macular degeneration (AAMD), geographic atrophy (GA), and neovascular disease (NV). Design: Prospective, longitudinal study. Participants: Patients aged 55 to 80 years at baseline identifying as White with non-AAMD in 1 or both eyes at baseline were included. Follow-up grades were assigned as early, intermediate, or AAMD (GA or NV). CFI variants were categorized using genotyping and sequencing platforms. Methods: Analyses were performed using the Seddon Longitudinal Cohort Study (N = 2116 subjects, 3901 eyes, and mean follow-up of 8.3 years) and the Age-Related Eye Disease Study (N = 2837 subjects, 5200 eyes, and mean follow-up of 9.2 years). CFI rare variants associated with low serum factor I (FI) protein levels and decreased FI function (type 1), other AMD genetic variants, and demographic, behavioral, and ocular factors were evaluated. Generalized estimating equations methods were used to assess the association between CFI rare variants and progression, independent of other genetic variants and covariates. Main Outcome Measures: Progression to AAMD, GA, or NV. Results: In the prospective cohort of 4953 subjects (9101 eyes with non-AAMD at baseline), 1% were type 1 rare CFI carriers. Over 12 years, progression to AAMD was 44% for carriers and 20% for noncarriers (P < 0.001), 30% of carriers versus 10% of noncarriers progressed to GA (P < 0.001), and 18% of carriers compared with 11% of noncarriers progressed to NV (P = 0.049). CFI carriers were more likely to have a family history of AMD (P for trend = 0.035) and a higher baseline AMD grade (P < 0.001). After adjusting for all covariates, CFI carrier status was associated with progression to GA (odds ratio [OR] = 1.91; 95% confidence interval [CI] = 1.03, 3.52) but not NV (OR = 0.96). Higher body mass index was associated with progression among CFI carriers (body mass index ≥ 25 vs. < 25; OR = 5.8; 95% CI 1.5, 22.3) but not for noncarriers (OR = 1.1; 95% CI = 0.9, 1.3), with P_interaction = 0.011. Conclusions: Results suggest that carriers of rare dysfunctional type 1 CFI variants are at higher risk for progression to AAMD with GA. Financial Disclosures: Proprietary or commercial disclosure may be found after the references.

Evaluation of Risk Prediction with Hierarchical Data: Dependency Adjusted Confidence Intervals for the AUC.

The area under the true ROC curve (AUC) is routinely used to determine how strongly a given model discriminates between the levels of a binary outcome. Standard inference with the AUC requires that outcomes be independent of each other. To overcome this limitation, a method was developed for the estimation of the variance of the AUC in the setting of two-level hierarchical data using probit-transformed prediction scores generated from generalized estimating equation models, thereby allowing for the application of inferential methods. This manuscript presents an extension of this approach so that inference for the AUC may be performed in a three-level hierarchical data setting (e.g., eyes nested within persons and persons nested within families). A method that accounts for the effect of tied prediction scores on inference is also described. The performance of 95% confidence intervals around the AUC was assessed through the simulation of three-level clustered data in multiple settings, including ones with tied data and variable cluster sizes. Across all settings, the actual 95% confidence interval coverage varied from 0.943 to 0.958, and the ratio of the theoretical variance to the empirical variance of the AUC varied from 0.920 to 1.013. The results are better than those from existing methods. Two examples of applying the proposed methodology are presented.