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.

The vascular geometry of the choriocapillaris is associated with spatially heterogeneous molecular exchange with the outer retina.

Vision relies on the continuous exchange of material between the photoreceptors, retinal pigment epithelium and choriocapillaris, a dense microvascular bed located underneath the outer retina. The anatomy and physiology of the choriocapillaris and their association with retinal homeostasis have proven difficult to characterize, mainly because of the unusual geometry of this vascular bed. By analysing tissue dissected from 81 human eyes, we show that the thickness of the choriocapillaris does not vary significantly over large portions of the macula or with age. Assessments of spatial variations in the anatomy of the choriocapillaris in three additional human eyes indicate that the location of arteriolar and venular vessels connected to the plane of the choriocapillaris is non-random, and that venular insertions cluster around arteriolar ones. Mathematical models built upon these anatomical analyses reveal that the choriocapillaris contains regions where the transport of passive elements is dominated by diffusion, and that these diffusion-limited regions represent areas of reduced exchange with the outer retina. The width of diffusion-limited regions is determined by arterial flow rate and the relative arrangement of arteriolar and venular insertions. These analyses demonstrate that the apparent complexity of the choriocapillaris conceals a fine balance between several anatomical and functional parameters to effectively support homeostasis of the outer retina. KEY POINTS: The choriocapillaris is the capillary bed supporting the metabolism of photoreceptors and retinal pigment epithelium, two critical components of the visual system located in the outer part of the retina. The choriocapillaris has evolved a planar multipolar vascular geometry that differs markedly from the branched topology of most vasculatures in the human body. Here, we report that this planar multipolar vascular geometry is associated with spatially heterogenous molecular exchange between choriocapillaris and outer retina. Our data and analyses highlight a necessary balance between choriocapillaris anatomical and functional parameters to effectively support homeostasis of the outer retina.

Modeling complex age-related eye disease.

Modeling complex eye diseases like age-related macular degeneration (AMD) and glaucoma poses significant challenges, since these conditions depend highly on age-related changes that occur over several decades, with many contributing factors remaining unknown. Although both diseases exhibit a relatively high heritability of >50%, a large proportion of individuals carrying AMD- or glaucoma-associated genetic risk variants will never develop these diseases. Furthermore, several environmental and lifestyle factors contribute to and modulate the pathogenesis and progression of AMD and glaucoma. Several strategies replicate the impact of genetic risk variants, pathobiological pathways and environmental and lifestyle factors in AMD and glaucoma in mice and other species. In this review we will primarily discuss the most commonly available mouse models, which have and will likely continue to improve our understanding of the pathobiology of age-related eye diseases. Uncertainties persist whether small animal models can truly recapitulate disease progression and vision loss in patients, raising doubts regarding their usefulness when testing novel gene or drug therapies. We will elaborate on concerns that relate to shorter lifespan, body size and allometries, lack of macula and a true lamina cribrosa, as well as absence and sequence disparities of certain genes and differences in their chromosomal location in mice. Since biological, rather than chronological, age likely predisposes an organism for both glaucoma and AMD, more rapidly aging organisms like small rodents may open up possibilities that will make research of these diseases more timely and financially feasible. On the other hand, due to the above-mentioned anatomical and physiological features, as well as pharmacokinetic and -dynamic differences small animal models are not ideal to study the natural progression of vision loss or the efficacy and safety of novel therapies. In this context, we will also discuss the advantages and pitfalls of alternative models that include larger species, such as non-human primates and rabbits, patient-derived retinal organoids, and human organ donor eyes.

Retinal and Choroidal Thickness in an Indigenous Population from Ghana: Comparison with Individuals with European or African Ancestry.

Purpose: To evaluate the thickness of the macular retina and central choroid in an indigenous population from Ghana, Africa and to compare them with those measured among individuals with European or African ancestry. Design: Cross-sectional study, systematic review, and meta-analyses. Participants: Forty-two healthy Ghanaians, 37 healthy individuals with European ancestry, and an additional 1427 healthy subjects with African ancestry from previously published studies. Methods: Macular retinal thickness in the fovea, parafovea, and perifovea and central choroidal thickness were extracted from OCT volume scans. Associations with ethnicity, age, and sex were assessed using mixed-effect regression models. Monte Carlo simulations were performed to determine the sensitivity of significant associations to additional potential confounders. Pooled estimates of retinal thickness among other groups with African ancestry were generated through systematic review and meta-analyses. Main Outcome Measures: Macular retinal thickness and central choroidal thickness and their association with ethnicity, age, and sex. Results: When adjusted for age and sex, the macular retina and central choroid of Ghanaians are significantly thinner as compared with subjects with European ancestry (P < 0.001). A reduction in retinal and choroidal thickness is observed with age, although this effect is independent of ethnicity. Meta-analyses indicate that retinal thickness among Ghanaians differs markedly from that of African Americans and other previously reported indigenous African populations. Conclusions: The thickness of the retina among Ghanaians differs not only from those measured among individuals with European ancestry, but also from those obtained from African Americans. Normative retinal and choroidal parameters determined among individuals with African or European ancestry may not be sufficient to describe indigenous African populations. Financial Disclosures: Proprietary or commercial disclosure may be found in the Footnotes and Disclosures at the end of this article.

Variability in Retinal Neuron Populations and Associated Variations in Mass Transport Systems of the Retina in Health and Aging.

Aging is associated with a broad range of visual impairments that can have dramatic consequences on the quality of life of those impacted. These changes are driven by a complex series of alterations affecting interactions between multiple cellular and extracellular elements. The resilience of many of these interactions may be key to minimal loss of visual function in aging; yet many of them remain poorly understood. In this review, we focus on the relation between retinal neurons and their respective mass transport systems. These metabolite delivery systems include the retinal vasculature, which lies within the inner portion of the retina, and the choroidal vasculature located externally to the retinal tissue. A framework for investigation is proposed and applied to identify the structures and processes determining retinal mass transport at the cellular and tissue levels. Spatial variability in the structure of the retina and changes observed in aging are then harnessed to explore the relation between variations in neuron populations and those seen among retinal metabolite delivery systems. Existing data demonstrate that the relation between inner retinal neurons and their mass transport systems is different in nature from that observed between the outer retina and choroid. The most prominent structural changes observed across the eye and in aging are seen in Bruch's membrane, which forms a selective barrier to mass transfers at the interface between the choroidal vasculature and the outer retina.

Characterization of West African Crystalline Macular Dystrophy in the Ghanaian Population.

OBJECTIVE: West African crystalline maculopathy (WACM) is characterized by the presence of macular hyperrefractile crystal-like deposits. Although the underlying pathophysiology has not been elucidated, a few biologic drivers have been proposed. We analyzed a large WACM case series to gain a more robust understanding of its features and etiology. DESIGN: Prospective, cross-sectional cohort study. SUBJECTS: Participants with WACM were selected from the large cohort recruited in the Ghana Age-Related Macular Degeneration Study. METHODS: Demographic and detailed medical histories, full ophthalmic examinations, digital color fundus photographs, and OCT images were obtained. All cases with WACM were evaluated by 3 retina experts. Crystal numbers, location, and distribution were determined. Associations between WACM and White age-related macular degeneration (AMD) risk variants were assessed using Firth's bias-reduced logistic regression, including age and sex as covariates. MAIN OUTCOME MEASURES: Phenotypic features of, and genetic associations with, WACM. RESULTS: West African crystalline maculopathy was identified in 106 eyes of 53 participants: 22 were bilateral and 24 were unilateral. Grading for AMD was not possible in 1 eye in 7 participants with WACM; therefore, laterality was not assessed in these subjects. Thirty-eight participants were women and were 14 men; sex was unrecorded for 1 participant. The mean age was 68.4 years (range, 45-101 years). Typical WACM crystals were demonstrated on OCT, which were more easily identified at high contrast and predominantly located at the inner limiting membrane. In eyes with copathology, crystals localized deeper in the inner retina, with wider retinal distribution over copathology lesions. There was no association with age or sex. A significant association was observed between the complement factor H (CFH) 402H risk variant and WACM. CONCLUSIONS: This study confirms the localization of crystals adjacent to the inner limiting membrane and distribution over lesions in eyes with copathology. The evaluation of OCT images under high contrast allows improved identification. West African crystalline maculopathy may be associated with the CFH-CFHR5 AMD risk locus identified among Whites; however, it is also possible that the combination of crystals and the CFH 402H allele increases the risk for developing late AMD. Further analyses using larger sample sizes are warranted to identify causalities between genotype and WACM phenotype.

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.

Chromosome 10q26-driven age-related macular degeneration is associated with reduced levels of HTRA1 in human retinal pigment epithelium.

Genome-wide association studies have identified the chromosome 10q26 (Chr10) locus, which contains the age-related maculopathy susceptibility 2 (ARMS2) and high temperature requirement A serine peptidase 1 (HTRA1) genes, as the strongest genetic risk factor for age-related macular degeneration (AMD) [L.G. Fritsche et al., Annu. Rev. Genomics Hum. Genet. 15, 151-171, (2014)]. To date, it has been difficult to assign causality to any specific single nucleotide polymorphism (SNP), haplotype, or gene within this region because of high linkage disequilibrium among the disease-associated variants [J. Jakobsdottir et al. Am. J. Hum. Genet. 77, 389-407 (2005); A. Rivera et al. Hum. Mol. Genet. 14, 3227-3236 (2005)]. Here, we show that HTRA1 messenger RNA (mRNA) is reduced in retinal pigment epithelium (RPE) but not in neural retina or choroid tissues derived from human donors with homozygous risk at the 10q26 locus. This tissue-specific decrease is mediated by the presence of a noncoding, cis-regulatory element overlapping the ARMS2 intron, which contains a potential Lhx2 transcription factor binding site that is disrupted by risk variant rs36212733. HtrA1 protein increases with age in the RPE-Bruch's membrane (BM) interface in Chr10 nonrisk donors but fails to increase in donors with homozygous risk at the 10q26 locus. We propose that HtrA1, an extracellular chaperone and serine protease, functions to maintain the optimal integrity of the RPE-BM interface during the aging process and that reduced expression of HTRA1 mRNA and protein in Chr10 risk donors impairs this protective function, leading to increased risk of AMD pathogenesis. HtrA1 augmentation, not inhibition, in high-risk patients should be considered as a potential therapy for AMD.

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.

Comparison of the Morphology of the Foveal Pit Between African and Caucasian Populations.

Purpose: The purpose of this study was to characterize foveal pit morphology in an African (Ghanaian) population, to compare it to that of a Caucasian group and to determine if it varied with age in the two populations. Methods: The depth, diameter, slope, and volume of the foveal pit were interpolated from optical coherence tomography volume scans recorded in 84 Ghanaian and 37 Caucasian individuals. Their association with age, sex, and ethnicity was investigated using multilevel regression models. Results: The foveal pit differed significantly in width, slope, and volume between Ghanaian men and women (P < 0.001), but only in width and volume between Caucasian men and women (P < 0.01). In Ghanaians, age was associated with a narrowing of the foveal depression and a reduction of its volume. Overall, these changes were more pronounced in women as compared to men and were largely absent from the Caucasian group. When controlled for age, the foveal pit of Ghanaians was significantly wider and larger in volume as compared to the Caucasian group (P < 0.001). Conclusions: The morphology of the foveal pit differs between African and Caucasian individuals. These anatomic differences should be considered when examining differences in prevalence and clinical features of vitreoretinal disorders involving the fovea between the two populations. Translational Relevance: Differences in retinal anatomy may partly explain variations in the prevalence and clinical features of retinal diseases between Africans and Caucasians. Such differences should be adequately considered in diagnoses and monitoring of ocular diseases in patients with African ancestry.

Prevalence of Retinal Diseases and Associated Risk Factors in an African Population From Mwanza, Tanzania.

BACKGROUND AND OBJECTIVE: To determine the prevalence of retinal disease among a population in Mwanza, Tanzania, and to identify relevant risk factors for retinal disorders in this cohort. PATIENTS AND METHODS: A cross-sectional population-based study was conducted in Mwanza, Tanzania, among patients older than 18 years. Participants completed a demographics survey and underwent an ophthalmic examination that included fundus photography. RESULTS: Complete data were available for 1,007 (93.8%) of the 1,073 persons examined. The prevalence of vitreoretinal disorders was 22.8% (230/1,007). The leading retinal diseases were age-related macular degeneration (7.0%), hypertensive retinopathy (4.5%), and macular scars (2.7%). CONCLUSION: This study is the first population-based study of retinal disease in Mwanza. The findings reveal a considerable burden of retinal disease in this region, suggesting a need for trained local ophthalmic personnel and resources. [Ophthalmic Surg Lasers Imaging Retina. 2020;51:S17-S25.].

Allometry and Scaling of the Intraocular Pressure and Aqueous Humour Flow Rate in Vertebrate Eyes.

In vertebrates, intraocular pressure (IOP) is required to maintain the eye into a shape allowing it to function as an optical instrument. It is sustained by the balance between the production of aqueous humour by the ciliary body and the resistance to its outflow from the eye. Dysregulation of the IOP is often pathological to vision. High IOP may lead to glaucoma, which is in man the second most prevalent cause of blindness. Here, we examine the importance of the IOP and rate of formation of aqueous humour in the development of vertebrate eyes by performing allometric and scaling analyses of the forces acting on the eye during head movement and the energy demands of the cornea, and testing the predictions of the models against a list of measurements in vertebrates collated through a systematic review. We show that the IOP has a weak dependence on body mass, and that in order to maintain the focal length of the eye, it needs to be an order of magnitude greater than the pressure drop across the eye resulting from gravity or head movement. This constitutes an evolutionary constraint that is common to all vertebrates. In animals with cornea-based optics, this constraint also represents a condition to maintain visual acuity. Estimated IOPs were found to increase with the evolution of terrestrial animals. The rate of formation of aqueous humour was found to be adjusted to the metabolic requirements of the cornea, scaling as Vac(0.67), where Vac is the volume of the anterior chamber. The present work highlights an interdependence between IOP and aqueous flow rate crucial to ocular function that must be considered to understand the evolution of the dioptric apparatus. It should also be taken into consideration in the prevention and treatment of glaucoma.