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

Abstract

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.

Keywords: AMD, age-related macular degeneration; ATP, adenosine triphosphate; C3, complement component 3; C9, complement component 9; CADD, Combined Annotation Dependent Depletion; CFH, complement factor H; CFI, complement factor I; Coagulation pathway, Immune pathways; ENG, endoglin; F2RL2, coagulation factor II thrombin receptor-like 2; FANTOM5, functional annotation of the mammalian genome; GS, glutamine synthetase; GWAS, genome-wide association studies; MAF, minor allele frequency; Macular degeneration; Maculopathy; PECAM1, Platelet Endothelial Cell Adhesion Molecule 1; PRS, polygenic risk score; SKAT, sequence kernel association testing; SNP, single nucleotide polymorphism; TPM, tags per million; Targeted sequencing; WES, whole-exome sequencing; Whole-exome sequencing.

Figure 1

Overall study design. In the discovery phase, single-variant and gene-based association analyses were performed for 758 samples. We also searched for highly penetrant variants in families with ≥ 2 affected members, low PRS among affected family members, and no known complement rare variants (26 families). Two rare variants displayed high penetrance in >80% of affected members in 2 families. Immunochemistry was performed to determine the expression of a new gene F2RL2 in pig and human retinas. AMD = age-related macular degeneration; CFH = complement factor H; F2RL2 = coagulation factor II thrombin receptor-like 2; MAF = minor allele frequency; PRS = polygenic risk score; SKAT = sequence kernel association testing.

Figure 2

Polygenic risk score (PRS) distribution in 5581 samples. A, Polygenic risk score distribution in advanced AMD (n = 2485), early and intermediate AMD (n = 1208), and controls (n = 1888). B, Summary of PRS values in advanced AMD (median PRS = 1.4), early and intermediate disease (median PRS = 0.61), and controls (median PRS = 0.06). Genotypes previously measured by HumanCoreExome arrays were used to calculate the PRS, using 26 genotyped variants known to be associated with AMD.^,, , , , AMD = age-related macular degeneration; PRS = polygenic risk score based on known variants associated with age-related macular degeneration.

Figure 3

Pedigree A, carrying the ADAM15 rare variant rs757672473 (p.Arg288Cys). A, The pedigree diagram. B, Multimodal retinal imaging of subjects in pedigree A. II:1 had macular atrophy with temporal and peripheral drusen in both eyes (OU) and fibrosis consistent with neovascular age-related macular degeneration (AMD) in the left eye (OS). II:2, brother of II:1, had atrophy OU seen on infrared imaging OU (II:2-a) with progression to neovascular disease (NV) (II:2-b). III:1, son of II:2, had few drusen OU (III:1-a), which progressed to geographic atrophy (GA) OD and NV OS (III:1-b-d). The son, III:2, a noncarrier, had large drusen that progressed to GA in the right eye (OD) and NV OS (III:2-a-d). The son III:3 had large drusen OU with progression to drusenoid retinal pigment epithelial detachment OD. III:4, daughter of II:2, had drusenoid retinal pigment epithelial detachment OU that progressed to GA OU (III:4-a-d). Individual III:6, also son of II:2, progressed from early AMD to large drusen OU and GA OD.

Figure 4

Pedigree B, carrying the F2RL2 rare variant rs147969213 (p.Leu289Arg). A, The pedigree diagram. B, Multimodal retinal imaging of subjects in pedigree B. Individual II:1, a female aged 77 years (II:1-a), with progression to a circular pattern of atrophy shown in fundus color both eyes (OU) 16 years later (II:1-b) and atrophy seen on OCT at the same time (II:1-c). The sister, II:2, had atrophy right eye (OD) and atrophy left eye (OS) with fibrosis and pigment mottling with a history of laser treatment for neovascular disease (NV), seen on fundus color photograph OU at age 82. III:1, the daughter of II:1, had large drusen with drusenoid retinal pigment epithelial detachment seen on fundus color OU at age 65 years (III;1a), which progressed to larger retinal pigment epithelial detachment seen on infrafred imaging OU (III:1-b) and OCT OU, and 3 years later had neovascular disease (NV) OD and received anti-VEGF injections (III:1-c). III-4, the son of individual II:2, had drusen and central geographic atrophy (GA) with foveal sparing OU beginning at age 58 years, seen on fundus photo, OCT, and fluorescein angiography OU at age 82 (III:4-a-c). F2RL2 = coagulation factor II thrombin receptor-like 2.

Figure 5

F2RL2 expression patterns in human retinas. A, Superior-oriented peripheral retinas of 3 nondiseased (top row; left to right: 70-year-old woman, 97-year-old woman, and 78-year-old man) and 3 neovascular patients with AMD (bottom row; left to right: 92-year-old women, 94-year-old women, and 94-year-old man) stained for expression of F2RL2 by immunohistochemistry. Expression seems high in a subset of photoreceptor outer segments and intermediate where the first retinal vascular plexus and the ganglion cells are located. A diffuse pattern across the retina resembling Müller glia staining is seen as well. B, Immunofluorescence staining for F2RL2 expression (red signal) in retina of an eye with neovascular AMD (94-year-old woman as shown in panel A). Retina was also stained with an antibody against glutamine synthetase (green signal) to visualize Müller glia cells, peanut agglutinin lectin (white signal) to detect cones, and nuclear 4’,6-diamidino-2-phenylindole (blue signal) to visualize nuclei. The cone outer segment signal of F2RL2 (arrows) can be seen on top of cone inner segments (white arrowheads; top right panel). Yellow signal throughout the retina confirms the expression of F2RL2 in Müller glia cells. Higher magnification of region where Müller glia cell bodies reside is shown in lower right panel. Scale bars: 25 mm. AMD = age-related macular degeneration; F2RL2 = coagulation factor II thrombin receptor-like 2; GCL = ganglion cell layer; INL = inner nuclear layer; IS = inner segments; ONL = outer nuclear layer; OS = outer segments.

Figure 6

F2RL2 expression in pig retinas and regions of vascular pathology. A-C, Immunofluorescence for F2RL2 expression (red signal) in pig retinas. Like in humans, F2RL2 expression is primarily seen in cone outer segments (OS, arrow) and Müller glia cells (green signal). Müller glia cell bodies (arrowheads) show clear expression of F2RL2 (magenta: peanut agglutinin lectin marking cone segments; red: F2RL2; green: glutamine synthetase [GS] marking Müller glia cells; blue: nuclear 4’,6-diamidino-2-phenylindole [DAPI] marking nuclei). A, Shows all 4 colors. B, Shows F2RL2, GS, and DAPI. C, Shows F2RL2 and GS. D-K, Images of immunohistochemistry (D-G) and immunofluorescence (H-K) staining from regions of human retinas with neovascular pathology and fibrotic scars. D, Regions of fibrotic scar in the outer nuclear layer (ONL), where photoreceptors used to reside, show the strongest expression of F2RL2 protein. Expression in Müller glia cell bodies is seen as a band of cells (arrowheads) across the INL. Expression of F2RL2 is also seen around the choroidal and retinal vasculature (arrows). Boundaries of different retinal layers are demarcated by dotted lines or vertical lines (for choroid). E, F, F2RL2 expression outside region of vascular pathology on same retina as shown in (D). Müller glia (arrowheads) cell morphology is discernable by the strong expression of F2RL2. F, Shows higher magnification of a region shown in (E) with dotted line tracing the outlines of a Müller glia cell. Weak expression is also seen in photoreceptor cells (asterisks). Outer segment expression in cones is indicated by arrow. G, Absence of primary antibody control staining shows no signal. Incubation was performed in parallel, omitting the primary antibody. H, Immunofluorescence for F2RL2 (red signal) and PECAM1 (green signal) to determine if F2RL2 is also expressed in retinal (arrowhead) and/or choroidal (arrows) endothelial cells. No apparent overlap is seen between the red and green signal. Fibrotic scar tissue in ONL shows few photoreceptor nuclei (blue: DAPI) left. I, Omission of primary antibody as a control for the secondary antibodies used in (H). J, K, Higher magnification of same staining shown in (H), showing choroidal (J, arrow) and retinal (K, arrowhead) vasculature (green signal) surrounded by F2RL2 but not overlapping with F2RL2 (blue: nuclear DAPI). Human retina in (D-K) is from a 94-year-old woman (same as shown in Fig 5) with neovascular AMD pathology. AMD = age-related macular degeneration; F2RL2 = coagulation factor II thrombin receptor-like 2; GCL = ganglion cell layer; INL = inner nuclear layer; IPL = inner plexiform layer; IS = inner segments; ONL = outer nuclear layer; OS = outer segments; PECAM1 = Platelet Endothelial Cell Adhesion Molecule 1.