Levels of the HtrA1 Protein in Serum and Vitreous Humor Are Independent of Genetic Risk for Age-Related Macular Degeneration at the 10q26 Locus.

Purpose: The purpose of this study was to determine if levels of the HtrA1 protein in serum or vitreous humor are influenced by genetic risk for age-related macular degeneration (AMD) at the 10q26 locus, age, sex, AMD status, and/or AMD disease severity, and, therefore, to determine the contribution of systemic and ocular HtrA1 to the AMD disease process. Methods: A custom-made sandwich ELISA assay (SCTM ELISA) for detection of the HtrA1 protein was designed and compared with three commercial assays (R&D Systems, MyBiosource 1 and MyBiosource 2) using 65 serum samples. Concentrations of HtrA1 were thereafter determined in serum and vitreous samples collected from 248 individuals and 145 human donor eyes, respectively. Results: The SCTM ELISA demonstrated high specificity, good recovery, and parallelism within its linear detection range and performed comparably to the R&D Systems assay. In contrast, we were unable to demonstrate the specificity of the two assays from MyBioSource using either recombinant or native HtrA1. Analyses of concentrations obtained using the validated SCTM assay revealed that genetic risk at the 10q26 locus, age, sex, or AMD status are not significantly associated with altered levels of the HtrA1 protein in serum or in vitreous humor (P > 0.05). Conclusions: HtrA1 levels in serum and vitreous do not reflect the risk for AMD associated with the 10q26 locus or disease status. Localized alteration in HTRA1 expression in the retinal pigment epithelium, rather than systemic changes in HtrA1, is the most likely driver of elevated risk for developing AMD among individuals with risk variants at the 10q26 locus.

Progression of Age-Related Macular Degeneration Among Individuals Homozygous for Risk Alleles on Chromosome 1 (CFH-CFHR5) or Chromosome 10 (ARMS2/HTRA1) or Both.

IMPORTANCE: Age-related macular degeneration (AMD) is a common cause of irreversible vision loss among individuals older than 50 years. Although considerable advances have been made in our understanding of AMD genetics, the differential effects of major associated loci on disease manifestation and progression may not be well characterized. OBJECTIVE: To elucidate the specific associations of the 2 most common genetic risk loci for AMD, the CFH-CFHR5 locus on chromosome 1q32 (Chr1) and the ARMS2/HTRA1 locus on chromosome 10q26 (Chr10)-independent of one another and in combination-with time to conversion to late-stage disease and to visual acuity loss. DESIGN, SETTING, AND PARTICIPANTS: This case series study included 502 individuals who were homozygous for risk variants at both Chr1 and Chr10 (termed Chr1&10-risk) or at either Chr1 (Chr1-risk) or Chr10 (Chr10-risk) and who had enrolled in Genetic and Molecular Studies of Eye Diseases at the Sharon Eccles Steele Center for Translational Medicine between September 2009 and March 2020. Multimodal imaging data were reviewed for AMD staging, including grading of incomplete and complete retinal pigment epithelium and outer retinal atrophy. MAIN OUTCOMES AND MEASURES: Hazard ratios and survival times for conversion to any late-stage AMD, atrophic or neovascular, and associated vision loss of 2 or more lines. RESULTS: In total, 317 participants in the Chr1-risk group (median [IQR] age at first visit, 75.6 [69.5-81.7] years; 193 women [60.9%]), 93 participants in the Chr10-risk group (median [IQR] age at first visit, 77.5 [72.2-84.2] years; 62 women [66.7%]), and 92 participants in the Chr1&10-risk group (median [IQR] age at first visit, 71.7 [68.0-76.3] years; 62 women [67.4%]) were included in the analyses. After adjusting for age and AMD grade at first visit, compared with 257 participants in the Chr1-risk group, 56 participants in the Chr1&10-risk group (factor of 3.3 [95% CI, 1.6-6.8]; P < .001) and 58 participants in the Chr10-risk group (factor of 2.6 [95% CI, 1.3-5.2]; P = .007) were more likely to convert to a late-stage phenotype during follow-up. This difference was mostly associated with conversion to macular neovascularization, which occurred earlier in participants with Chr1&10-risk and Chr10-risk. Eyes in the Chr1&10-risk group (median [IQR] survival, 5.7 [2.1-11.1] years) were 2.1 (95% CI, 1.1-3.9; P = .03) times as likely and eyes in the Chr10-risk group (median [IQR] survival, 6.3 [2.7-11.3] years) were 1.8 (95% CI, 1.0-3.1; P = .05) times as likely to experience a visual acuity loss of 2 or more lines compared with eyes of the Chr1-risk group (median [IQR] survival, 9.4 [4.1-* (asterisk indicates event rate did not reach 75%)] years). CONCLUSIONS AND RELEVANCE: These findings suggest differential associations of the 2 major AMD-related risk loci with structural and functional disease progression and suggest distinct underlying biological mechanisms associated with these 2 loci. These genotype-phenotype associations may warrant consideration when designing and interpreting AMD research studies and clinical trials.

Protective chromosome 1q32 haplotypes mitigate risk for age-related macular degeneration associated with the CFH-CFHR5 and ARMS2/HTRA1 loci.

BACKGROUND: Single-variant associations with age-related macular degeneration (AMD), one of the most prevalent causes of irreversible vision loss worldwide, have been studied extensively. However, because of a lack of refinement of these associations, there remains considerable ambiguity regarding what constitutes genetic risk and/or protection for this disease, and how genetic combinations affect this risk. In this study, we consider the two most common and strongly AMD-associated loci, the CFH-CFHR5 region on chromosome 1q32 (Chr1 locus) and ARMS2/HTRA1 gene on chromosome 10q26  (Chr10 locus). RESULTS: By refining associations within the CFH-CFHR5 locus, we show that all genetic protection against the development of AMD in this region is described by the combination of the amino acid-altering variant CFH I62V (rs800292) and genetic deletion of CFHR3/1. Haplotypes based on CFH I62V, a CFHR3/1 deletion tagging SNP and the risk variant CFH Y402H are associated with either risk, protection or neutrality for AMD and capture more than 99% of control- and case-associated chromosomes. We find that genetic combinations of CFH-CFHR5 haplotypes (diplotypes) strongly influence AMD susceptibility and that individuals with risk/protective diplotypes are substantially protected against the development of disease. Finally, we demonstrate that AMD risk in the ARMS2/HTRA1 locus is also mitigated by combinations of CFH-CFHR5 haplotypes, with Chr10 risk variants essentially neutralized by protective CFH-CFHR5 haplotypes. CONCLUSIONS: Our study highlights the importance of considering protective CFH-CFHR5 haplotypes when assessing genetic susceptibility for AMD. It establishes a framework that describes the full spectrum of AMD susceptibility using an optimal set of single-nucleotide polymorphisms with known functional consequences. It also indicates that protective or preventive complement-directed therapies targeting AMD driven by CFH-CFHR5 risk haplotypes may also be effective when AMD is driven by ARMS2/HTRA1 risk variants.

Macular retinal thickness differs markedly in age-related macular degeneration driven by risk polymorphisms on chromosomes 1 and 10.

The two most common genetic contributors to age-related macular degeneration (AMD), a leading cause of irreversible vision loss worldwide, are variants associated with CFH-CFHR5 on chromosome 1 (Chr1) and ARMS2/HTRA1 on chromosome 10 (Chr10). We sought to determine if risk and protective variants associated with these two loci drive differences in macular retinal thickness prior and subsequent to the onset of clinically observable signs of AMD. We considered 299 individuals (547 eyes) homozygous for risk variants or haplotypes on Chr1 or Chr10 exclusively (Chr1-risk and Chr10-risk, respectively) or homozygous for a neutral haplotype (Chr1-neu), for the protective I62 tagged haplotype (Chr1-prot-I62) or for the protection conferring CFHR1/3 deletion haplotype (Chr1-prot-del) on Chr1 without any risk alleles on Chr10. Among eyes with no clinically observable signs of AMD, the deletion of CFHR1/3, which is strongly protective against this disease, is associated with significantly thicker retinas in the perifovea. When controlling for age, Chr10-risk eyes with early or intermediate AMD have thinner retinas as compared to eyes from the Chr1-risk group with similar disease severity. Our analysis indicates that this difference likely results from distinct biological and disease initiation and progression events associated with Chr1- and Chr10-directed AMD.

Fundus autofluorescence characteristics of nascent geographic atrophy in age-related macular degeneration.

PURPOSE: We examined the fundus autofluorescence (FAF) characteristics of nascent geographic atrophy (nGA), pathological features preceding the development of drusen-associated atrophy in eyes with age-related macular degeneration (AMD) that can be visualized using high-resolution optical coherence tomography (OCT). METHODS: Spectral-domain OCT (SD-OCT) and FAF imaging were performed longitudinally in 221 eyes with intermediate AMD (having at least drusen >125 µm), and seven areas that developed drusen-associated atrophy in five eyes were examined and categorized with respect to FAF characteristics. These categories then were used to characterize 49 areas of nGA or drusen-associated atrophy on SD-OCT identified in a cross-sectional study with 230 participants with bilateral intermediate AMD. RESULTS: Sequential imaging revealed that FAF characteristics in the atrophic areas could be grouped into three categories: predominantly hyperautofluorescent (hyperAF), presence of both hyper- and hypoautofluorescence (mixed AF), or predominantly hypoautofluorescent (hypoAF). In the cross-sectional study, the FAF characteristics were significantly dependent on the type of atrophic area (P = 0.002), where areas of nGA appeared most commonly as being mixed AF (63%), while areas of drusen-associated atrophy most commonly as hypoAF (86%). CONCLUSIONS: Fundus autofluorescence imaging revealed that areas of nGA were most commonly characterized by both hyper- and hypoautofluorescent changes, which differs from areas of drusen-associated atrophy that most often appeared hypoautofluorescent. These findings provide important insights into the FAF characteristics of areas undergoing atrophic changes in eyes still considered to be in the early stages of AMD by current methods, and thus assist in the characterization of disease severity in these early stages.

Optical coherence tomography-defined changes preceding the development of drusen-associated atrophy in age-related macular degeneration.

PURPOSE: To characterize the pathological changes preceding the development of drusen-associated atrophy in eyes with age-related macular degeneration (AMD) using spectral-domain optical coherence tomography (SD-OCT). DESIGN: Longitudinal and cross-sectional retrospective observational study. PARTICIPANTS: A total of 181 participants with intermediate AMD in at least 1 eye (141 unilateral, 40 bilateral) were assessed longitudinally. A total of 230 participants with bilateral intermediate AMD (40 longitudinal participants with an additional 190 participants) were analyzed cross-sectionally. METHODS: Spectral-domain OCT, color fundus photography (CFP), near-infrared reflectance, and fundus autofluorescence imaging were performed in all participants at cross-section and every 3 months for up to 30 months in the longitudinal study. Spectral-domain OCT volume scans were examined for features that portend the development of drusen-associated atrophy, and the topography, prevalence, and risk factors of these features were determined through cross-sectional analysis. MAIN OUTCOME MEASURES: The pathological features on SD-OCT preceding the development of drusen-associated atrophy and the characteristics of these features. RESULTS: Twenty areas from 16 eyes of 16 participants developed drusen-associated atrophy after an average of 20 months (range, 8-30 months). Spectral-domain OCT features unique in these areas included: subsidence of the outer plexiform layer (OPL) and inner nuclear layer (INL), and development of a hyporeflective wedge-shaped band within the limits of the OPL. These characteristics were termed "nascent geographic atrophy" (nGA), describing features that portend the development of drusen-associated atrophy. Cross-sectional examination of participants with bilateral intermediate AMD revealed that independent risk factors for the presence of nGA included the presence of pigmentary changes (odds ratio [OR], 16.84; 95% confidence interval [CI], 2.42-117.24) and nGA in the fellow eye (OR, 4.15; 95% CI, 1.12-15.34); nGA was present in 21.9% of participants with drusen >125 µm and pigmentary changes in both eyes. CONCLUSIONS: This study identified pathological changes occurring before the development of drusen-associated atrophy using SD-OCT, which we defined as nGA. Although nGA is undetectable on CFP, it is important for determining the risk of future vision loss in AMD and could be used as an earlier surrogate end point in interventional trials targeting the early stages of AMD.

Reticular pseudodrusen: a risk factor for geographic atrophy in fellow eyes of individuals with unilateral choroidal neovascularization.

PURPOSE: To determine whether reticular pseudodrusen (RPD) confer an increased risk of progression to late-stage age-related macular degeneration (AMD) in fellow eyes of those recently diagnosed with unilateral choroidal neovascularization (CNV). DESIGN: Retrospective study. PARTICIPANTS: Two hundred consecutive participants with CNV secondary to AMD in 1 eye and no signs of late-stage AMD in the fellow eye. METHODS: Clinical examination and comprehensive retinal imaging, including spectral-domain optical coherence tomography, near-infrared reflectance (NIR), and color fundus photography, at baseline and every follow-up visit. MAIN OUTCOME MEASURES: Incidence of geographic atrophy (GA) and CNV in the fellow eye. RESULTS: Mean age ± standard deviation was 77±7 years, and 61% of the cohort were female. Fifty-eight percent (n = 116) had RPD, 68% had drusen of 125 µm or more, 36% had pigmentary changes, 10% had both drusen of 125 µm or more and pigmentary changes, and 17% had only RPD in their fellow eyes. After a mean follow-up of 2.3 years, CNV developed in 36% of patients and GA developed in 14% of patients. Those with RPD demonstrated late-stage AMD (61% vs. 33.4%; P <0.001) and GA (22.4% with RPD vs. 2.4% without RPD; P <0.001) more often. The presence of reticular pseudodrusen was an independent risk factor for the development of GA (hazard ratio [HR], 4.93; P = 0.042), but not for CNV (HR, 1.19; P = 0.500), at least within the follow-up of this study. Both drusen of 125 µm or more and pigmentary changes at baseline were significant risk factors for the development of CNV and GA (HR, 1.96-11.73; P ≤0.020). CONCLUSIONS: Reticular pseudodrusen seem to confer an increased risk of progression to GA, in addition to drusen and pigmentary changes. The presence of RPD needs to be taken into account when discussing a patient's prognosis and planning management.

A common haplotype in the complement regulatory gene factor H (HF1/CFH) predisposes individuals to age-related macular degeneration.

Age-related macular degeneration (AMD) is the most frequent cause of irreversible blindness in the elderly in developed countries. Our previous studies implicated activation of complement in the formation of drusen, the hallmark lesion of AMD. Here, we show that factor H (HF1), the major inhibitor of the alternative complement pathway, accumulates within drusen and is synthesized by the retinal pigmented epithelium. Because previous linkage analyses identified chromosome 1q25-32, which harbors the factor H gene (HF1/CFH), as an AMD susceptibility locus, we analyzed HF1 for genetic variation in two independent cohorts comprised of approximately 900 AMD cases and 400 matched controls. We found association of eight common HF1 SNPs with AMD; two common missense variants exhibit highly significant associations (I62V, chi2 = 26.1 and P = 3.2 x 10(-7) and Y402H, chi2 = 54.4 and P = 1.6 x 10(-13)). Haplotype analysis reveals that multiple HF1 variants confer elevated or reduced risk of AMD. One common at-risk haplotype is present at a frequency of 50% in AMD cases and 29% in controls [odds ratio (OR) = 2.46, 95% confidence interval (1.95-3.11)]. Homozygotes for this haplotype account for 24% of cases and 8% of controls [OR = 3.51, 95% confidence interval (2.13-5.78)]. Several protective haplotypes are also identified (OR = 0.44-0.55), further implicating HF1 function in the pathogenetic mechanisms underlying AMD. We propose that genetic variation in a regulator of the alternative complement pathway, when combined with a triggering event, such as infection, underlie a major proportion of AMD in the human population.