Demonstration of the pathogenicity of a common non-exomic mutation in ABCA4 using iPSC-derived retinal organoids and retrospective clinical data.

Mutations in ABCA4 are the most common cause of Mendelian retinal disease. Clinical evaluation of this gene is challenging because of its extreme allelic diversity, the large fraction of non-exomic mutations, and the wide range of associated disease. We used patient-derived retinal organoids as well as DNA samples and clinical data from a large cohort of patients with ABCA4-associated retinal disease to investigate the pathogenicity of a variant in ABCA4 (IVS30 + 1321 A > G) that occurs heterozygously in 2% of Europeans. We found that this variant causes mis-splicing of the gene in photoreceptor cells such that the resulting protein contains 36 incorrect amino acids followed by a premature stop. We also investigated the phenotype of 10 patients with compound genotypes that included this mutation. Their median age of first vision loss was 39 years, which is in the mildest quintile of a large cohort of patients with ABCA4 disease. We conclude that the IVS30 + 1321 A > G variant can cause disease when paired with a sufficiently deleterious opposing allele in a sufficiently permissive genetic background.

Choroidal endothelial and macrophage gene expression in atrophic and neovascular macular degeneration.

The human choroid is a heterogeneous, highly vascular connective tissue that dysfunctions in age-related macular degeneration (AMD). In this study, we performed single-cell RNA sequencing on 21 human choroids, 11 of which were derived from donors with early atrophic or neovascular AMD. Using this large donor cohort, we identified new gene expression signatures and immunohistochemically characterized discrete populations of resident macrophages, monocytes/inflammatory macrophages and dendritic cells. These three immune populations demonstrated unique expression patterns for AMD genetic risk factors, with dendritic cells possessing the highest expression of the neovascular AMD-associated MMP9 gene. Additionally, we performed trajectory analysis to model transcriptomic changes across the choroidal vasculature, and we identified expression signatures for endothelial cells from choroidal arterioles and venules. Finally, we performed differential expression analysis between control, early atrophic AMD, and neovascular AMD samples, and we observed that early atrophic AMD samples had high expression of SPARCL1, a gene that has been shown to increase in response to endothelial damage. Likewise, neovascular endothelial cells harbored gene expression changes consistent with endothelial cell damage and demonstrated increased expression of the sialomucins CD34 and ENCM, which were also observed at the protein level within neovascular membranes. Overall, this study characterizes the molecular features of new populations of choroidal endothelial cells and mononuclear phagocytes in a large cohort of AMD and control human donors.

Sensitive quantification of m.3243A>G mutational proportion in non-retinal tissues and its relationship with visual symptoms.

The m.3243A>G mutation in the mitochondrial genome commonly causes retinal degeneration in patients with maternally inherited diabetes and deafness and mitochondrial encephalopathy, lactic acidosis and stroke-like episodes. Like other mitochondrial mutations, m.3243A>G is inherited from the mother with a variable proportion of wild type and mutant mitochondrial genomes in different cells. The mechanism by which the m.3243A>G variant in each tissue relates to the manifestation of disease phenotype is not fully understood. Using a digital PCR assay, we found that the % m.3243G in skin derived dermal fibroblasts was positively correlated with that of blood from the same individual. The % m.3243G detected in fibroblast cultures remained constant over multiple passages and was negatively correlated with mtDNA copy number. Although the % m.3243G present in blood was not correlated with severity of vision loss, as quantified by Goldmann visual field, a significant negative correlation between % m.3243G and the age of onset of visual symptoms was detected. Altogether, these results indicate that precise measurement of % m.3243G in clinically accessible tissues such as skin and blood may yield information relevant to the management of retinal m.3243A>G-associated disease.

Transcriptomic and Chromatin Accessibility Analysis of the Human Macular and Peripheral Retinal Pigment Epithelium at the Single-Cell Level.

Some human retinal diseases are characterized by pathology that is restricted to specific cell types and to specific regions of the eye. Several disease entities either selectively affect or spare the macula, the retina region at the center of the posterior pole. Photoreceptor cells in the macula are involved in high-acuity vision and require metabolic support from non-neuronal cell types. Some macular diseases involve the retinal pigment epithelium (RPE), an epithelial cell layer with several metabolic-support functions essential for the overlying photoreceptors. In the current study, the ways in which RPE confers region-specific disease susceptibility were determined by examining heterogeneity within RPE tissue from human donors. RPE nuclei from the macular and peripheral retina were profiled using joint single-nucleus RNA and ATAC sequencing. The expression of several genes differed between macular and peripheral RPE. Region-specific ATAC peaks were found, suggesting regulatory elements used exclusively by macular or peripheral RPE. Across anatomic regions, subpopulations of RPE were identified that appeared to have differential levels of expression of visual cycle genes. Finally, loci associated with age-related macular degeneration were examined for a better understanding of RPE-specific disease phenotypes. These findings showed variations in the regulation of gene expression in the human RPE by region and subpopulation, and provide a source for a better understanding of the molecular basis of macular disease.

A Retrospective Longitudinal Study of 460 Patients with ABCA4-Associated Retinal Disease.

PURPOSE: To investigate the distribution of genotypes and natural history of ABCA4-associated retinal disease in a large cohort of patients seen at a single institution. DESIGN: Retrospective, single-institution cohort review. PARTICIPANTS: Patients seen at the University of Iowa between November 1986 and August 2022 clinically suspected to have disease caused by sequence variations in ABCA4. METHODS: DNA samples from participants were subjected to a tiered testing strategy progressing from allele-specific screening to whole genome sequencing. Charts were reviewed, and clinical data were tabulated. The pathogenic severity of the most common alleles was estimated by studying groups of patients who shared 1 allele. Groups of patients with shared genotypes were reviewed for evidence of modifying factor effects. MAIN OUTCOME MEASURES: Age at first uncorrectable vision loss, best-corrected visual acuity, and the area of the I2e isopter of the Goldmann visual field. RESULTS: A total of 460 patients from 390 families demonstrated convincing clinical features of ABCA4-associated retinal disease. Complete genotypes were identified in 399 patients, and partial genotypes were identified in 61. The median age at first vision loss was 16 years (range, 4-76 years). Two hundred sixty-five families (68%) harbored a unique genotype, and no more than 10 patients shared any single genotype. Review of the patients with shared genotypes revealed evidence of modifying factors that in several cases resulted in a > 15-year difference in age at first vision loss. Two hundred forty-one different alleles were identified among the members of this cohort, and 161 of these (67%) were found in only a single individual. CONCLUSIONS: ABCA4-associated retinal disease ranges from a very severe photoreceptor disease with an onset before 5 years of age to a late-onset retinal pigment epithelium-based condition resembling pattern dystrophy. Modifying factors frequently impact the ABCA4 disease phenotype to a degree that is similar in magnitude to the detectable ABCA4 alleles themselves. It is likely that most patients in any cohort will harbor a unique genotype. The latter observations taken together suggest that patients' clinical findings in most cases will be more useful for predicting their clinical course than their genotype. FINANCIAL DISCLOSURE(S): Proprietary or commercial disclosure may be found in the Footnotes and Disclosures at the end of this article.

Characterization of a novel Pde6b-deficient rat model of retinal degeneration and treatment with adeno-associated virus (AAV) gene therapy.

In humans, mutations in the beta subunit of cGMP-phosphodiesterase type 6 (PDE6B) cause autosomal recessive retinitis pigmentosa (RP), which typically has an aggressive clinical course of early-onset severe vision loss due to rapid photoreceptor degeneration. In this study, we describe the generation of a novel Pde6b-deficient rat model using CRISPR-Cas9 genome editing. We characterize the model at multiple time points using clinical imaging modalities as well as histology with immunohistochemistry to show rapid photoreceptor degeneration compared to wild-type and heterozygous animals. We describe the manufacture of two different adeno-associated viral (AAV) vectors (AAV2/1, AAV2/5) under current Good Manufacturing Practices (cGMP) and demonstrate their ability to drive human PDE6B expression in vivo. We further demonstrate the ability of AAV-mediated subretinal gene therapy to delay photoreceptor loss in Pde6b-deficient rats compared to untreated controls. However, severe progressive photoreceptor loss was noted even in treated eyes, likely due to the aggressive nature of the disease. These data provide useful preclinical data to guide the development of potential human gene therapy for PDE6B-associated RP. In addition, the rapid photoreceptor degeneration of the Pde6b-deficient rat with intact inner retina may provide a useful model for the study of cell replacement strategies.

Human photoreceptor cells from different macular subregions have distinct transcriptional profiles.

The human neural retina is a light sensitive tissue with remarkable spatial and cellular organization. Compared with the periphery, the central retina contains more densely packed cone photoreceptor cells with unique morphologies and synaptic wiring. Some regions of the central retina exhibit selective degeneration or preservation in response to retinal disease and the basis for this variation is unknown. In this study, we used both bulk and single-cell RNA sequencing to compare gene expression within concentric regions of the central retina. We identified unique gene expression patterns of foveal cone photoreceptor cells, including many foveal-enriched transcription factors. In addition, we found that the genes RORB1, PPFIA1 and KCNAB2 are differentially spliced in the foveal, parafoveal and macular regions. These results provide a highly detailed spatial characterization of the retinal transcriptome and highlight unique molecular features of different retinal regions.

Human iPSC Modeling Reveals Mutation-Specific Responses to Gene Therapy in a Genotypically Diverse Dominant Maculopathy.

Dominantly inherited disorders are not typically considered to be therapeutic candidates for gene augmentation. Here, we utilized induced pluripotent stem cell-derived retinal pigment epithelium (iPSC-RPE) to test the potential of gene augmentation to treat Best disease, a dominant macular dystrophy caused by over 200 missense mutations in BEST1. Gene augmentation in iPSC-RPE fully restored BEST1 calcium-activated chloride channel activity and improved rhodopsin degradation in an iPSC-RPE model of recessive bestrophinopathy as well as in two models of dominant Best disease caused by different mutations in regions encoding ion-binding domains. A third dominant Best disease iPSC-RPE model did not respond to gene augmentation, but showed normalization of BEST1 channel activity following CRISPR-Cas9 editing of the mutant allele. We then subjected all three dominant Best disease iPSC-RPE models to gene editing, which produced premature stop codons specifically within the mutant BEST1 alleles. Single-cell profiling demonstrated no adverse perturbation of retinal pigment epithelium (RPE) transcriptional programs in any model, although off-target analysis detected a silent genomic alteration in one model. These results suggest that gene augmentation is a viable first-line approach for some individuals with dominant Best disease and that non-responders are candidates for alternate approaches such as gene editing. However, testing gene editing strategies for on-target efficiency and off-target events using personalized iPSC-RPE model systems is warranted. In summary, personalized iPSC-RPE models can be used to select among a growing list of gene therapy options to maximize safety and efficacy while minimizing time and cost. Similar scenarios likely exist for other genotypically diverse channelopathies, expanding the therapeutic landscape for affected individuals.

Automating iPSC generation to enable autologous photoreceptor cell replacement therapy.

BACKGROUND: Inherited retinal degeneration is a leading cause of incurable vision loss in the developed world. While autologous iPSC mediated photoreceptor cell replacement is theoretically possible, the lack of commercially available technologies designed to enable high throughput parallel production of patient specific therapeutics has hindered clinical translation. METHODS: In this study, we describe the use of the Cell X precision robotic cell culture platform to enable parallel production of clinical grade patient specific iPSCs. The Cell X is housed within an ISO Class 5 cGMP compliant closed aseptic isolator (Biospherix XVivo X2), where all procedures from fibroblast culture to iPSC generation, clonal expansion and retinal differentiation were performed. RESULTS: Patient iPSCs generated using the Cell X platform were determined to be pluripotent via score card analysis and genetically stable via karyotyping. As determined via immunostaining and confocal microscopy, iPSCs generated using the Cell X platform gave rise to retinal organoids that were indistinguishable from organoids derived from manually generated iPSCs. In addition, at 120 days post-differentiation, single-cell RNA sequencing analysis revealed that cells generated using the Cell X platform were comparable to those generated under manual conditions in a separate laboratory. CONCLUSION: We have successfully developed a robotic iPSC generation platform and standard operating procedures for production of high-quality photoreceptor precursor cells that are compatible with current good manufacturing practices. This system will enable clinical grade production of iPSCs for autologous retinal cell replacement.

Local factor H production by human choroidal endothelial cells mitigates complement deposition: implications for macular degeneration.

Activation of the alternative complement pathway is an initiating event in the pathology of age-related macular degeneration (AMD). Unchecked complement activation leads to the formation of a pro-lytic pore, the membrane attack complex (MAC). MAC deposition is observed on the choriocapillaris of AMD patients and likely causes lysis of choroidal endothelial cells (CECs). Complement factor H (FH, encoded by the gene CFH) is an inhibitor of complement. Both loss of function of FH and reduced choroidal levels of FH have been reported in AMD. It is plausible that reduced local FH availability promotes MAC deposition on CECs. FH is produced primarily in the liver; however, cells including the retinal pigment epithelium can produce FH locally. We hypothesized that CECs produce FH locally to protect against MAC deposition. We aimed to investigate the effect of reduced FH levels in the choroid to determine whether increasing local FH could protect CECs from MAC deposition. We demonstrated that siRNA knockdown of FH (CFH) in human immortalized CECs results in increased MAC deposition. We generated AMD iPSC-derived CECs and found that overexpression of FH protects against MAC deposition. These results suggest that local CEC-produced FH protects against MAC deposition, and that increasing local FH protein may be beneficial in limiting MAC deposition in AMD. © 2022 The Pathological Society of Great Britain and Ireland.

MACULAR ATROPHY AND PHENOTYPIC VARIABILITY IN AUTOSOMAL DOMINANT STARGARDT-LIKE MACULAR DYSTROPHY DUE TO PROM1 MUTATION.

PURPOSE: To describe the phenotypic variability and rates of progression of atrophy in patients with PROM1 -associated macular dystrophy. METHODS: Patients in this retrospective, longitudinal case series from a tertiary center had clinical examination and multimodal imaging performed. Areas of retinal pigment epithelium and ellipsoid zone loss over time by optical coherence tomography were calculated by two independent graders. RESULTS: Fifteen patients from five kindreds with an Arg373Cys mutation in PROM1 were studied. The average age was 39 years, and 80% were women. The visual acuity was 20/40 at presentation and 20/57 at last follow-up (average 4.8 years). Three distinct macular phenotypes were observed: 1) central geographic atrophy (13%), 2) multifocal geographic atrophy (20%), and 3) bull's eye maculopathy (67%). The overall rate of atrophy progression was 0.36 mm 2 /year, but the average rate of atrophy progression varied by macular phenotype: 1.08 mm 2 /year for central geographic atrophy, 0.53 mm 2 /year for multifocal geographic atrophy, and 0.23 mm 2 /year for bull's eye maculopathy. CONCLUSION: Patients with PROM1 -associated macular dystrophy demonstrate distinct phenotypes, with bull's eye maculopathy being the most common. The average rate of atrophy progression may be similar to reported rates for ABCA4 -related Stargardt disease and less than age-related macular degeneration. These results provide important measures for following treatment response in future gene and stem cell-based therapies.

Development and biological characterization of a clinical gene transfer vector for the treatment of MAK-associated retinitis pigmentosa.

By combining next generation whole exome sequencing and induced pluripotent stem cell (iPSC) technology we found that an Alu repeat inserted in exon 9 of the MAK gene results in a loss of normal MAK transcript and development of human autosomal recessive retinitis pigmentosa (RP). Although a relatively rare cause of disease in the general population, the MAK variant is enriched in individuals of Jewish ancestry. In this population, 1 in 55 individuals are carriers and one third of all cases of recessive RP is caused by this gene. The purpose of this study was to determine if a viral gene augmentation strategy could be used to safely restore functional MAK protein as a step toward a treatment for early stage MAK-associated RP. Patient iPSC-derived photoreceptor precursor cells were generated and transduced with viral vectors containing the MAK transcript. One week after transduction, transcript and protein could be detected via rt-PCR and western blotting respectively. Using patient-derived fibroblast cells and mak knockdown zebra fish we demonstrate that over-expression of the retinal MAK transgene restored the cells ability to regulate primary cilia length. In addition, the visual defect in mak knockdown zebrafish was mitigated via treatment with the retinal MAK transgene. There was no evidence of local or systemic toxicity at 1-month or 3-months following subretinal delivery of clinical grade vector into wild type rats. The findings reported here will help pave the way for initiation of a phase 1 clinical trial for the treatment of patients with MAK-associated RP.

Disruption of RPGR protein interaction network is the common feature of RPGR missense variations that cause XLRP.

Retinitis pigmentosa (RP) is an inherited retinal degenerative disease with severe vision impairment leading to blindness. About 10-15% of RP cases are caused by mutations in the RPGR gene, with RPGR mutations accounting for 70% of X-linked RP cases. The mechanism by which RPGR mutations cause photoreceptor cell dysfunction is not well understood. In this study, we show that the two isoforms of RPGR (RPGR1-19 and RPGRORF15) interact with endogenous PDE6D, INPP5E, and RPGRIP1L. The RPGR1-19 isoform contains two PDE6D binding sites with the C-terminal prenylation site being the predominant PDE6D binding site. The C terminus of RPGR1-19 that contains the prenylation site regulates its interaction with PDE6D, INPP5E, and RPGRIP1L. Only the RPGR1-19 isoform localizes to cilia in cultured RPE1 cells. Missense variations found in RPGR patients disrupt the interaction between RPGR isoforms and their endogenous interactors INPP5E, PDE6D, and RPGRIP1L. We evaluated a RPGR missense variation (M58K) found in a family with X-linked retinitis pigmentosa (XLRP) and show that this missense variation disrupts the interaction of RPGR isoforms with their endogenous interactors. The M58K variation also disrupts the ciliary localization of the RPGR1-19 isoform. Using this assay, we also show that some of the RPGR missense variants reported in the literature might not actually be disease causing. Our data establishes an in vitro assay that can be used to validate the potential pathogenicity of RPGR missense variants.

Single-cell transcriptomics of the human retinal pigment epithelium and choroid in health and macular degeneration.

The human retinal pigment epithelium (RPE) and choroid are complex tissues that provide crucial support to the retina. Disease affecting either of these supportive tissues can lead to irreversible blindness in the setting of age-related macular degeneration. In this study, single-cell RNA sequencing was performed on macular and peripheral regions of RPE-choroid from 7 human donor eyes in 2 independent experiments. In the first experiment, total RPE/choroid preparations were evaluated and expression profiles specific to RPE and major choroidal cell populations were identified. As choroidal endothelial cells represent a minority of the total RPE/choroidal cell population but are strongly implicated in age-related macular degeneration (AMD) pathogenesis, a second single-cell RNA-sequencing experiment was performed using endothelial cells enriched by magnetic separation. In this second study, we identified gene expression signatures along the choroidal vascular tree, classifying the transcriptome of human choriocapillaris, arterial, and venous endothelial cells. We found that the choriocapillaris highly and specifically expresses the regulator of cell cycle gene (RGCC), a gene that responds to complement activation and induces apoptosis in endothelial cells. In addition, RGCC was the most up-regulated choriocapillaris gene in a donor diagnosed with AMD. These results provide a characterization of the human RPE and choriocapillaris transcriptome, offering potential insight into the mechanisms of choriocapillaris response to complement injury and choroidal vascular disease in age-related macular degeneration.

Exome-based investigation of the genetic basis of human pigmentary glaucoma.

BACKGROUND: Glaucoma is a leading cause of visual disability and blindness. Release of iris pigment within the eye, pigment dispersion syndrome (PDS), can lead to one type of glaucoma known as pigmentary glaucoma. PDS has a genetic component, however, the genes involved with this condition are largely unknown. We sought to discover genes that cause PDS by testing cohorts of patients and controls for mutations using a tiered analysis of exome data. RESULTS: Our primary analysis evaluated melanosome-related genes that cause dispersion of iris pigment in mice (TYRP1, GPNMB, LYST, DCT, and MITF). We identified rare mutations, but they were not statistically enriched in PDS patients. Our secondary analyses examined PMEL (previously linked with PDS), MRAP, and 19 other genes. Four MRAP mutations were identified in PDS cases but not in controls (p = 0.016). Immunohistochemical analysis of human donor eyes revealed abundant MRAP protein in the iris, the source of pigment in PDS. However, analysis of MRAP in additional cohorts (415 cases and 1645 controls) did not support an association with PDS. We also did not confirm a link between PMEL and PDS in our cohorts due to lack of reported mutations and similar frequency of the variants in PDS patients as in control subjects. CONCLUSIONS: We did not detect a statistical enrichment of mutations in melanosome-related genes in human PDS patients and we found conflicting data about the likely pathogenicity of MRAP mutations. PDS may have a complex genetic basis that is not easily unraveled with exome analyses.

Choriocapillaris Degeneration in Geographic Atrophy.

Early age-related macular degeneration (AMD) is characterized by degeneration of the choriocapillaris, the vascular supply of retinal photoreceptor cells. We assessed vascular loss during disease progression in the choriocapillaris and larger vessels in the deeper choroid. Human donor maculae from controls (n = 99), early AMD (n = 35), or clinically diagnosed with geographic atrophy (GA; n = 9, collected from outside the zone of retinal pigment epithelium degeneration) were evaluated using Ulex europaeus agglutinin-I labeling to discriminate between vessels with intact endothelial cells and ghost vessels. Morphometric analyses of choriocapillaris density (cross-sectional area of capillary lumens divided by length) and of vascular lumen/stroma ratio in the outer choroid were performed. Choriocapillaris loss was observed in early AMD (Bonferroni-corrected P = 0.024) with greater loss in GA (Bonferroni-corrected P < 10-9), even in areas of intact retinal pigment epithelium. In contrast, changes in lumen/stroma ratio in the outer choroid were not found to differ between controls and AMD or GA eyes (P > 0.05), suggesting choriocapillaris changes are more prevalent in AMD than those in the outer choroid. In addition, vascular endothelial growth factor-A levels were negatively correlated with choriocapillaris vascular density. These findings support the concept that choroidal vascular degeneration, predominantly in the microvasculature, contributes to dry AMD progression. Addressing capillary loss in AMD remains an important translational target.

Bulk and single-cell gene expression analyses reveal aging human choriocapillaris has pro-inflammatory phenotype.

The human choroidal vasculature is subject to age-related structural and gene expression changes implicated in age-related macular degeneration (AMD). In this study, we performed both bulk and single-cell RNA sequencing on infant (n = 4 for bulk experiments, n = 2 for single-cell experiments) and adult (n = 13 for bulk experiments, n = 6 for single-cell experiments) human donors to characterize how choroidal gene expression changes with age. Differential expression analysis revealed that aged choroidal samples were enriched in genes encoding pro-inflammatory transcription factors and leukocyte transendothelial cell migration adhesion proteins. Such genes were observed to be differentially expressed specifically within choroidal endothelial cells at the single-cell level. Immunohistochemistry experiments support transcriptional findings that CD34 is elevated in infant choriocapillaris endothelial cells while ICAM-1 is enriched in adults. These results suggest several potential drivers of the pro-inflammatory vascular phenotype observed with advancing age.

Spectacle: An interactive resource for ocular single-cell RNA sequencing data analysis.

Single-cell RNA sequencing has revolutionized ocular gene expression studies. This technology has enabled researchers to identify expression signatures for rare cell types and characterize how gene expression changes across biological conditions, such as topographic region or disease status. However, sharing single-cell RNA sequencing results remains a major obstacle, particular for individuals without a computational background. To address these limitations, we developed Spectacle, an interactive web-based resource for exploring previously published single-cell RNA sequencing data from ocular studies. Spectacle is powered by a locally developed R package, cellcuratoR, which utilizes the Shiny framework in R to generate interactive visualizations for single-cell expression data. Spectacle contains five pre-processed ocular single-cell RNA sequencing data sets and is accessible via the web at OcularGeneExpression.org/singlecell. With Spectacle, users can interactively identify which cell types express a gene of interest, detect transcriptomic subpopulations within a cell type, and perform highly flexible differential expression analyses. The freely-available Spectacle system reduces the bioinformatic barrier for interacting with rich single-cell RNA sequencing studies from ocular tissues, making it easy to quickly identify cell types that express a gene of interest.

Primary congenital and developmental glaucomas.

Glaucoma is the leading cause of irreversible blindness worldwide. Although most glaucoma patients are elderly, congenital glaucoma and glaucomas of childhood are also important causes of visual disability. Primary congenital glaucoma (PCG) is isolated, non-syndromic glaucoma that occurs in the first three years of life and is a major cause of childhood blindness. Other early-onset glaucomas may arise secondary to developmental abnormalities, such as glaucomas that occur with aniridia or as part of Axenfeld-Rieger syndrome. Congenital and childhood glaucomas have strong genetic bases and disease-causing mutations have been discovered in several genes. Mutations in three genes (CYP1B1, LTBP2, TEK) have been reported in PCG patients. Axenfeld-Rieger syndrome is caused by mutations in PITX2 or FOXC1 and aniridia is caused by PAX6 mutations. This review discusses the roles of these genes in primary congenital glaucoma and glaucomas of childhood.

The ARMS2 A69S Polymorphism Is Associated with Delayed Rod-Mediated Dark Adaptation in Eyes at Risk for Incident Age-Related Macular Degeneration.

PURPOSE: To examine the association between sequence variants in genetic risk factors for age-related macular degeneration (AMD) and delayed rod-mediated dark adaptation (RMDA), the first functional biomarker for incident AMD, in older adults with normal macular health and early AMD. DESIGN: Cross-sectional. PARTICIPANTS: Adults 60 years of age or older showing normal macular health (defined as both eyes at step 1 on the Age-Related Eye Disease Study 9-step AMD classification system) and those with AMD in one or both eyes (defined as steps 2-9). METHODS: Single nucleotide polymorphisms were genotyped in the complement factor H (CFH) and ARMS2 genes using a Taqman assay. Rod-mediated dark adaptation was assessed in 1 eye after photobleach with targets centered at 5° on the inferior vertical meridian. Rate of dark adaptation was defined by rod intercept time (RIT), duration (in minutes) required for sensitivity to reach a criterion sensitivity level in the latter half of the second component of rod recovery. Associations between CFH and ARMS2 polymorphisms and RMDA were adjusted for age and smoking. MAIN OUTCOME MEASURE: Rod intercept time. RESULTS: The sample consisted of 543 participants having both genotype and RIT determination; 408 showed normal macular health and 135 demonstrated AMD, most having early AMD (124 of 135). For the combined sample, higher RIT (slower RMDA) was observed for both the A69S variant in ARMS2 and the Y402H variant in CFH (adjusted P = 0.0001 and P = 0.0023, respectively). For healthy participants, the A69S variant in ARMS2 was associated with higher RIT (adjusted P = 0.0011), whereas the Y402H variant in CFH was not (adjusted P = 0.2175). For AMD patients, the A69S variant of ARMS2 and the Y402H variant of CFH were associated with higher RIT (adjusted P = 0.0182 and P = 0.0222, respectively). Those with a larger number of high-risk ARMS2 and CFH alleles showed higher RIT, in both healthy and AMD groups (adjusted P = 0.0002 and P < 0.0001, respectively). CONCLUSIONS: We report a novel association wherein older adults with high-risk ARMS2 and CFH genotypes are more likely to demonstrate delayed RMDA, the first functional biomarker for incident early AMD. Before the AMD clinical phenotype is present, those showing normal macular health with the ARMS2 A69S allele demonstrate delayed RMDA. Understanding ARMS2 function is a research priority.