A multi-cohort genome-wide association study in African ancestry individuals reveals risk loci for primary open-angle glaucoma.

Primary open-angle glaucoma (POAG), the leading cause of irreversible blindness worldwide, disproportionately affects individuals of African ancestry. We conducted a genome-wide association study (GWAS) for POAG in 11,275 individuals of African ancestry (6,003 cases; 5,272 controls). We detected 46 risk loci associated with POAG at genome-wide significance. Replication and post-GWAS analyses, including functionally informed fine-mapping, multiple trait co-localization, and in silico validation, implicated two previously undescribed variants (rs1666698 mapping to DBF4P2; rs34957764 mapping to ROCK1P1) and one previously associated variant (rs11824032 mapping to ARHGEF12) as likely causal. For individuals of African ancestry, a polygenic risk score (PRS) for POAG from our mega-analysis (African ancestry individuals) outperformed a PRS from summary statistics of a much larger GWAS derived from European ancestry individuals. This study quantifies the genetic architecture similarities and differences between African and non-African ancestry populations for this blinding disease.

A Novel Mouse Model for Late-Onset Retinal Degeneration (L-ORD) Develops RPE Abnormalities Due to the Loss of C1qtnf5/Ctrp5.

Late-onset retinal degeneration (L-ORD) is an autosomal dominant macular dystrophy resulting from mutations in the gene CTRP5/C1QTNF5. A mouse model (Ctrp5+/-) for the most common S163R developed many features of human clinical disease. We generated a novel homozygous Ctrp5 gene knock-out (Ctrp5-/-) mouse model to further study the mechanism of L-ORD. The retinal morphology of these mice was evaluated by retinal imaging, light microscopy, and transmission electron microscopy (TEM) at 6, 11, and 18.5 mo. Expression of Ctrp5 was analyzed using immunostaining and qRT-PCR. The Ctrp5-/- mice showed lack of both Ctrp5 transcript and protein. Presence of a significantly larger number of autofluorescent spots was observed in Ctrp5-/- mice compared to the WT (P < 0.0001) at 19 mo. Increased RPE stress with vacuolization and thinning was observed as early as 6 mo in Ctrp5-/- mice. Further, ultrastructural analyses revealed a progressive accumulation of basal laminar sub-RPE deposits in Ctrp5-/- mice from 11 mo. The Ctrp5-/- mice shared retinal and RPE pathology that matches with that previously described for Ctrp5+/- mice suggesting that pathology in these mice results from the loss of functional CTRP5 and that the presence of CTRP5 is critical for normal RPE and retinal function.

Mouse Choroid Proteome Revisited: Focus on Aging.

Age is a major risk factor for age-related macular degeneration (AMD), and age has a role in the disease phenotypes of heritable macular dystrophies. The proteomes of C57Bl6/J mouse choroids at 2 ages were analyzed to identify biochemical processes affected by aging. Proteins of interest were identified as those contributing most to the variance in principal component analysis and those showing the largest significant differences between ages. These proteins implicated altered ECM composition, immune system function, and lipid metabolism.

Single cell RNA sequencing confirms retinal microglia activation associated with early onset retinal degeneration.

Mutations in the Membrane-type frizzled related protein (Mfrp) gene results in an early-onset retinal degeneration associated with retinitis pigmentosa, microphthalmia, optic disc drusen and foveal schisis. In the current study, a previously characterized mouse model of human retinal degeneration carrying homozygous c.498_499insC mutations in Mfrp (MfrpKI/KI) was used. Patients carrying this mutation have retinal degeneration at an early age. The model demonstrates subretinal deposits and develops early-onset photoreceptor degeneration. We observed large subretinal deposits in MfrpKI/KI mice which were strongly CD68 positive and co-localized with autofluorescent spots. Single cell RNA sequencing of MfrpKI/KI mice retinal microglia showed a significantly higher number of pan-macrophage marker Iba-1 and F4/80 positive cells with increased expression of activation marker (CD68) and lowered microglial homeostatic markers (TMEM119, P2ry13, P2ry13, Siglech) compared with wild type mice confirming microglial activation as observed in retinal immunostaining showing microglia activation in subretinal region. Trajectory analysis identified a small cluster of microglial cells with activation transcriptomic signatures that could represent a subretinal microglia population in MfrpKI/KI mice expressing higher levels of APOE. We validated these findings using immunofluorescence staining of retinal cryosections and found a significantly higher number of subretinal Iba-1/ApoE positive microglia in MfrpKI/KI mice with some subretinal microglia also expressing lowered levels of microglial homeostatic marker TMEM119, confirming microglial origin. In summary, we confirm that MfrpKI/KI mice carrying the c.498_499insC mutation had a significantly higher population of activated microglia in their retina with distinct subsets of subretinal microglia. Further, studies are required to confirm whether the association of increased subretinal microglia in MfrpKI/KI mice are causal in degeneration.

A Mouse Model with Ablated Asparaginase and Isoaspartyl Peptidase 1 (Asrgl1) Develops Early Onset Retinal Degeneration (RD) Recapitulating the Human Phenotype.

We previously identified a homozygous G178R mutation in human ASRGL1 (hASRGL1) through whole-exome analysis responsible for early onset retinal degeneration (RD) in patients with cone-rod dystrophy. The mutant G178R ASRGL1 expressed in Cos-7 cells showed altered localization, while the mutant ASRGL1 in E. coli lacked the autocatalytic activity needed to generate the active protein. To evaluate the effect of impaired ASRGL1 function on the retina in vivo, we generated a mouse model with c.578_579insAGAAA (NM_001083926.2) mutation (Asrgl1mut/mut) through the CRISPR/Cas9 methodology. The expression of ASGRL1 and its asparaginase activity were undetectable in the retina of Asrgl1mut/mut mice. The ophthalmic evaluation of Asrgl1mut/mut mice showed a significant and progressive decrease in scotopic electroretinographic (ERG) response observed at an early age of 3 months followed by a decrease in photopic response around 5 months compared with age-matched wildtype mice. Immunostaining and RT-PCR analyses with rod and cone cell markers revealed a loss of cone outer segments and a significant decrease in the expression of Rhodopsin, Opn1sw, and Opn1mw at 3 months in Asrgl1mut/mut mice compared with age-matched wildtype mice. Importantly, the retinal phenotype of Asrgl1mut/mut mice is consistent with the phenotype observed in patients harboring the G178R mutation in ASRGL1 confirming a critical role of ASRGL1 in the retina and the contribution of ASRGL1 mutations in retinal degeneration.

Deciphering the genetic architecture and ethnographic distribution of IRD in three ethnic populations by whole genome sequence analysis.

Patients with inherited retinal dystrophies (IRDs) were recruited from two understudied populations: Mexico and Pakistan as well as a third well-studied population of European Americans to define the genetic architecture of IRD by performing whole-genome sequencing (WGS). Whole-genome analysis was performed on 409 individuals from 108 unrelated pedigrees with IRDs. All patients underwent an ophthalmic evaluation to establish the retinal phenotype. Although the 108 pedigrees in this study had previously been examined for mutations in known IRD genes using a wide range of methodologies including targeted gene(s) or mutation(s) screening, linkage analysis and exome sequencing, the gene mutations responsible for IRD in these 108 pedigrees were not determined. WGS was performed on these pedigrees using Illumina X10 at a minimum of 30X depth. The sequence reads were mapped against hg19 followed by variant calling using GATK. The genome variants were annotated using SnpEff, PolyPhen2, and CADD score; the structural variants (SVs) were called using GenomeSTRiP and LUMPY. We identified potential causative sequence alterations in 61 pedigrees (57%), including 39 novel and 54 reported variants in IRD genes. For 57 of these pedigrees the observed genotype was consistent with the initial clinical diagnosis, the remaining 4 had the clinical diagnosis reclassified based on our findings. In seven pedigrees (12%) we observed atypical causal variants, i.e. unexpected genotype(s), including 4 pedigrees with causal variants in more than one IRD gene within all affected family members, one pedigree with intrafamilial genetic heterogeneity (different affected family members carrying causal variants in different IRD genes), one pedigree carrying a dominant causative variant present in pseudo-recessive form due to consanguinity and one pedigree with a de-novo variant in the affected family member. Combined atypical and large structural variants contributed to about 20% of cases. Among the novel mutations, 75% were detected in Mexican and 50% found in European American pedigrees and have not been reported in any other population while only 20% were detected in Pakistani pedigrees and were not previously reported. The remaining novel IRD causative variants were listed in gnomAD but were found to be very rare and population specific. Mutations in known IRD associated genes contributed to pathology in 63% Mexican, 60% Pakistani and 45% European American pedigrees analyzed. Overall, contribution of known IRD gene variants to disease pathology in these three populations was similar to that observed in other populations worldwide. This study revealed a spectrum of mutations contributing to IRD in three populations, identified a large proportion of novel potentially causative variants that are specific to the corresponding population or not reported in gnomAD and shed light on the genetic architecture of IRD in these diverse global populations.

Detection and validation of novel mutations in MERTK in a simplex case of retinal degeneration using WGS and hiPSC-RPEs model.

Inherited retinal degenerations (IRDs) are a group of genetically heterogeneous conditions with a broad phenotypic heterogeneity. Here, we report detection and validation of the underlying cause of progressive retinal degeneration in a nuclear family of European descent with a single affected individual. Whole genome sequencing of the proband and her unaffected sibling identified a novel intron 8 donor splice site variant (c.1296 + 1G>A) and a novel 731 base pair deletion encompassing exon 9 (Chr2:g.112751488_112752218 del) resulting in c.1297_1451del; p.K433_G484fsTer3 in the Mer tyrosine kinase protooncogene (MERTK), which is highly expressed in the retinal pigment epithelium (RPE). The proband carried both variants in the heterozygous state, which segregated with disease in the pedigree. These MERTK variants are predicted to result in the defective splicing of exon 8 and loss of exon 9 respectively. To evaluate the impact of these novel variants, peripheral blood mononuclear cells of the proband and her parents were reprogrammed to humaninduced pluripotent stem cell (hiPSC) lines, which were subsequently differentiated to hiPSC-RPE. Analysis of the proband's hiPSC-RPE revealed the absence of both MERTK transcript and its respective protein as well as abnormal phagocytosis when compared with the parental hiPSC-RPE. In summary, whole genome sequencing identified novel compound heterozygous variants in MERTK as the underlying cause of progressive retinal degeneration in a simplex case. Further, analysis using an hiPSC-RPE model established the functional impact of novel MERTK mutations and revealed the potential mechanism underlying pathology in the proband.

Reticular Pseudodrusen in Late-Onset Retinal Degeneration.

PURPOSE: To characterize the association of reticular pseudodrusen (RPD) with late-onset retinal degeneration (L-ORD) using multimodal imaging. DESIGN: Prospective, 2-center, longitudinal case series. PARTICIPANTS: Twenty-nine patients with L-ORD. METHODS: All patients were evaluated within a 3-year interval with near-infrared reflectance, fundus autofluorescence, and spectral-domain OCT. In addition, a subset of patients also underwent indocyanine green angiography, fundus fluorescein angiography, mesopic microperimetry, and multifocal electroretinography. MAIN OUTCOME MEASURES: Prevalence, topographic distribution, and temporal phenotypic changes of RPD in L-ORD. RESULTS: A total of 29 patients with molecularly confirmed L-ORD were included in this prospective study. Reticular pseudodrusen was detected in 18 patients (62%) at baseline, 10 of whom were men. The prevalence of RPD varied with age. The mean age of RPD patients was 57.3 ± 7.2 years. Reticular pseudodrusen was not seen in patients younger than the fifth decade of life (n = 3 patients) or in the eighth decade of life (n = 5 patients). Reticular pseudodrusen were found commonly in the macula with relative sparing of the fovea and also were identified in the peripheral retina. The morphologic features of RPD changed with follow-up. Two patients (3 eyes) demonstrated RPD regression. CONCLUSIONS: Reticular pseudodrusen is found frequently in patients with L-ORD and at a younger age than in individuals with age-related macular degeneration (AMD). Reticular pseudodrusen exhibits quick formation and collapse, change in type and morphologic features with time, and relative foveal sparing and also has a peripheral retinal location in L-ORD.

Investigation of associations between Piezo1 mechanoreceptor gain-of-function variants and glaucoma-related phenotypes in humans and mice.

Glaucoma disproportionately affects individuals of African descent. Prior studies of the PIEZO1 mechanoreceptor have suggested a possible role in glaucoma pathophysiology. Here, we investigated associations between a Piezo1 gain-of-function variant common in individuals of African descent with glaucoma-related phenotypes. We analyzed whole genome sequences to identify Piezo1 variants and their frequencies among 1565 human participants. For the most common variant (e756del), we compared phenotypes between heterozygotes, homozygotes, and wildtypes. Longitudinal mixed effects models of visual field mean deviation (MD) and retinal nerve fiber layer (RNFL) thickness were used to evaluate progression. Based on trends in the models, further investigation was conducted using Piezo1 gain-of-function mice. About 30% of African descent individuals had at least one e756del allele. There were trends suggesting e756del was associated with higher IOPs, thinner RNFLs, lower optic nerve head capillary densities, and greater decreases in MD and RNFL thickness over time, but these did not reach statistical significance. Among mice, increased Piezo1 activity was not significantly associated with IOP or retinal ganglion cell density. Our study confirms that the Piezo1 e756del gain-of-function variant is a frequent polymorphism present in African descent individuals but is unrelated to examined differences in glaucoma phenotypes. Ongoing work is needed to elucidate the role of Piezo1-mediated mechanotransduction in glaucoma.

Whole genome sequencing data of multiple individuals of Pakistani descent.

Here we report whole genome sequencing of four individuals (H3, H4, H5, and H6) from a family of Pakistani descent. Whole genome sequencing yielded 1084.92, 894.73, 1068.62, and 1005.77 million mapped reads corresponding to 162.73, 134.21, 160.29, and 150.86 Gb sequence data and 52.49x, 43.29x, 51.70x, and 48.66x average coverage for H3, H4, H5, and H6, respectively. We identified 3,529,659, 3,478,495, 3,407,895, and 3,426,862 variants in the genomes of H3, H4, H5, and H6, respectively, including 1,668,024 variants common in the four genomes. Further, we identified 42,422, 39,824, 28,599, and 35,206 novel variants in the genomes of H3, H4, H5, and H6, respectively. A major fraction of the variants identified in the four genomes reside within the intergenic regions of the genome. Single nucleotide polymorphism (SNP) genotype based comparative analysis with ethnic populations of 1000 Genomes database linked the ancestry of all four genomes with the South Asian populations, which was further supported by mitochondria based haplogroup analysis. In conclusion, we report whole genome sequencing of four individuals of Pakistani descent.

Mutations in CERKL and RP1 cause retinitis pigmentosa in Pakistani families.

This study was conducted to identify the genetic basis of retinal dystrophies in consanguineous Pakistani families. We recruited two families with retinitis pigmentosa (RP) displaying visual difficulties, including nyctalopia and constricted visual fields. Linkage analysis and Sanger sequencing resulted in the identification of a previously reported nonsense mutation, c.847C > T, in exon 5 of CERKL in one family and a novel four-base pair deletion in exon 4 of RP1, c.delAGAA4218_4221, leading to premature protein termination in the second family. Here, we report two RP-causing mutations extending the genetic heterogeneity of the disease.

Novel mutations in LTBP2 identified in familial cases of primary congenital glaucoma.

Purpose: Primary congenital glaucoma (PCG) is a genetically heterogeneous disorder caused by developmental defects in the anterior chamber and trabecular meshwork. This disease is an important cause of childhood blindness. In this study, we aim to identify the genetic determinants of PCG in three consanguineous families of Pakistani descent. Methods: Affected members of all three families underwent detailed ophthalmological examination including slit-lamp biomicroscopy. Blood samples were collected from affected and healthy members of all three families, and genomic DNA was extracted. Linkage analysis was performed for the known or reported loci of PCG to localize the disease interval, and logarithm of odds (LOD) scores were calculated. All protein-coding exons of the candidate gene, latent transforming growth factor-beta binding protein 2 (LTBP2), were bidirectionally sequenced to identify the disease-causing mutation. Results: Short tandem repeat (STR) marker-based linkage analysis localized the critical interval to chromosome 14q with a maximum two-point LOD score of 2.86 (PKGL076), 2.8 (PKGL015), and 2.92 (PKGL042). Bidirectional Sanger sequencing of LTBP2 revealed three novel pathogenic variants, i.e., c.3028G>A (p.Asp1010Asn), c.3427delC (p.Gln1143Argfs*35), and c.5270G>A (p.Cys1757Tyr) in PKGL076, PKGL015, and PKGL042, respectively. All three mutations segregated with the disease phenotype in their respective families and were absent in 200 ethnically matched normal chromosomes. Conclusions: We identified three novel mutations, p.D1010N, p.Q1143Rfs*35, and p.C1757Y, in LTBP2 responsible for PCG.

Role of sorbitol-mediated cellular stress response in obesity-associated retinal degeneration.

PURPOSE: Obesity is a global health problem associated with several diseases including ocular complications. Earlier we reported progressive retinal degeneration because of obesity in a spontaneous obese rat (WNIN/Ob) model. In the current study, we examined the molecular mechanisms leading to retinal degeneration in WNIN/Ob rat. METHODS: Sorbitol was estimated by the fluorometric method in the retina of WNIN/Ob rats at different age (3-, 6- and 12- months), along with their respective lean rats. Immunoblotting was performed in the retina to assess the status of the insulin signaling pathway, ER stress and cellular stress (p38MAPK and ERK1/2). Human SK-N-SH cells were treated with 0.5 and 1.0 M sorbitol for 30 min to study insulin signaling, ER stress, and cellular stress. TUNEL assay was done to measure apoptosis. The retinal function in the rats was determined by electroretinogram. RESULTS: A gradual but significantly higher intracellular sorbitol accumulation was observed in the retina of obese rats from 3- to 12-months. The cellular osmotic stress has activated the insulin signaling mechanism without activating AKT and also triggered ER stress. Both the stresses activated the ERK and p38MAPK signaling causing apoptosis in the retina leading to retinal degeneration. Retinal dysfunction was confirmed by altered scotopic and photopic electroretinogram responses. These in vivo results were mimicked in SK-N-SH cells when exposed to sorbitol in vitro. CONCLUSIONS: These results suggest cellular stress due to sorbitol accumulation impairing the ER function, thereby leading to progressive retinal degeneration under obese conditions.

Association of Genetic Variants With Primary Open-Angle Glaucoma Among Individuals With African Ancestry.

Importance: Primary open-angle glaucoma presents with increased prevalence and a higher degree of clinical severity in populations of African ancestry compared with European or Asian ancestry. Despite this, individuals of African ancestry remain understudied in genomic research for blinding disorders. Objectives: To perform a genome-wide association study (GWAS) of African ancestry populations and evaluate potential mechanisms of pathogenesis for loci associated with primary open-angle glaucoma. Design, Settings, and Participants: A 2-stage GWAS with a discovery data set of 2320 individuals with primary open-angle glaucoma and 2121 control individuals without primary open-angle glaucoma. The validation stage included an additional 6937 affected individuals and 14 917 unaffected individuals using multicenter clinic- and population-based participant recruitment approaches. Study participants were recruited from Ghana, Nigeria, South Africa, the United States, Tanzania, Britain, Cameroon, Saudi Arabia, Brazil, the Democratic Republic of the Congo, Morocco, Peru, and Mali from 2003 to 2018. Individuals with primary open-angle glaucoma had open iridocorneal angles and displayed glaucomatous optic neuropathy with visual field defects. Elevated intraocular pressure was not included in the case definition. Control individuals had no elevated intraocular pressure and no signs of glaucoma. Exposures: Genetic variants associated with primary open-angle glaucoma. Main Outcomes and Measures: Presence of primary open-angle glaucoma. Genome-wide significance was defined as P < 5 × 10-8 in the discovery stage and in the meta-analysis of combined discovery and validation data. Results: A total of 2320 individuals with primary open-angle glaucoma (mean [interquartile range] age, 64.6 [56-74] years; 1055 [45.5%] women) and 2121 individuals without primary open-angle glaucoma (mean [interquartile range] age, 63.4 [55-71] years; 1025 [48.3%] women) were included in the discovery GWAS. The GWAS discovery meta-analysis demonstrated association of variants at amyloid-ß A4 precursor protein-binding family B member 2 (APBB2; chromosome 4, rs59892895T>C) with primary open-angle glaucoma (odds ratio [OR], 1.32 [95% CI, 1.20-1.46]; P = 2 × 10-8). The association was validated in an analysis of an additional 6937 affected individuals and 14 917 unaffected individuals (OR, 1.15 [95% CI, 1.09-1.21]; P < .001). Each copy of the rs59892895*C risk allele was associated with increased risk of primary open-angle glaucoma when all data were included in a meta-analysis (OR, 1.19 [95% CI, 1.14-1.25]; P = 4 × 10-13). The rs59892895*C risk allele was present at appreciable frequency only in African ancestry populations. In contrast, the rs59892895*C risk allele had a frequency of less than 0.1% in individuals of European or Asian ancestry. Conclusions and Relevance: In this genome-wide association study, variants at the APBB2 locus demonstrated differential association with primary open-angle glaucoma by ancestry. If validated in additional populations this finding may have implications for risk assessment and therapeutic strategies.

Association of severity of primary open-angle glaucoma with serum vitamin D levels in patients of African descent.

Purpose: To study the relationship between primary open-angle glaucoma (POAG) in a cohort of patients of African descent (AD) and serum vitamin D levels. Methods: A subset of the AD and glaucoma evaluation study III (ADAGES III) cohort, consisting of 357 patients with a diagnosis of POAG and 178 normal controls of self-reported AD, were included in this analysis. Demographic information, family history, and blood samples were collected from all the participants. All the subjects underwent clinical evaluation, including visual field (VF) mean deviation (MD), central cornea thickness (CCT), intraocular pressure (IOP), and height and weight measurements. POAG patients were classified into early and advanced phenotypes based on the severity of their visual field damage, and they were matched for age, gender, and history of hypertension and diabetes. Serum 25-Hydroxy (25-OH) vitamin D levels were measured by enzyme-linked immunosorbent assay (ELISA). The association of serum vitamin D levels with the development and severity of POAG was tested by analysis of variance (ANOVA) and the paired t-test. Results: The 178 early POAG subjects had a visual field MD of better than -4.0 dB, and the 179 advanced glaucoma subjects had a visual field MD of worse than -10 dB. The mean (95% confidence interval [CI]) levels of vitamin D of the subjects in the control (8.02 ± 6.19 pg/ml) and early phenotype (7.56 ± 5.74 pg/ml) groups were significantly or marginally significantly different from the levels observed in subjects with the advanced phenotype (6.35 ± 4.76 pg/ml; p = 0.0117 and 0.0543, respectively). In contrast, the mean serum vitamin D level in controls was not significantly different from that of the subjects with the early glaucoma phenotype (p = 0.8508). Conclusions: In this AD cohort, patients with advanced glaucoma had lower serum levels of vitamin D compared with early glaucoma and normal subjects.

Late-onset retinal degeneration pathology due to mutations in CTRP5 is mediated through HTRA1.

Late-onset retinal degeneration (L-ORD) is an autosomal dominant macular degeneration characterized by the formation of sub-retinal pigment epithelium (RPE) deposits and neuroretinal atrophy. L-ORD results from mutations in the C1q-tumor necrosis factor-5 protein (CTRP5), encoded by the CTRP5/C1QTNF5 gene. To understand the mechanism underlying L-ORD pathology, we used a human cDNA library yeast two-hybrid screen to identify interacting partners of CTRP5. Additionally, we analyzed the Bruch's membrane/choroid (BM-Ch) from wild-type (Wt), heterozygous S163R Ctrp5 mutation knock-in (Ctrp5S163R/wt ), and homozygous knock-in (Ctrp5S163R/S163R ) mice using mass spectrometry. Both approaches showed an association between CTRP5 and HTRA1 via its C-terminal PDZ-binding motif, stimulation of the HTRA1 protease activity by CTRP5, and CTRP5 serving as an HTRA1 substrate. The S163R-CTRP5 protein also binds to HTRA1 but is resistant to HTRA1-mediated cleavage. Immunohistochemistry and proteomic analysis showed significant accumulation of CTRP5 and HTRA1 in BM-Ch of Ctrp5S163R/S163R and Ctrp5S163R/wt mice compared with Wt. Additional extracellular matrix (ECM) components that are HTRA1 substrates also accumulated in these mice. These results implicate HTRA1 and its interaction with CTRP5 in L-ORD pathology.

Human iPSC-Derived Retinal Pigment Epithelium: A Model System for Prioritizing and Functionally Characterizing Causal Variants at AMD Risk Loci.

We evaluate whether human induced pluripotent stem cell-derived retinal pigment epithelium (iPSC-RPE) cells can be used to prioritize and functionally characterize causal variants at age-related macular degeneration (AMD) risk loci. We generated iPSC-RPE from six subjects and show that they have morphological and molecular characteristics similar to those of native RPE. We generated RNA-seq, ATAC-seq, and H3K27ac ChIP-seq data and observed high similarity in gene expression and enriched transcription factor motif profiles between iPSC-RPE and human fetal RPE. We performed fine mapping of AMD risk loci by integrating molecular data from the iPSC-RPE, adult retina, and adult RPE, which identified rs943080 as the probable causal variant at VEGFA. We show that rs943080 is associated with altered chromatin accessibility of a distal ATAC-seq peak, decreased overall gene expression of VEGFA, and allele-specific expression of a non-coding transcript. Our study thus provides a potential mechanism underlying the association of the VEGFA locus with AMD.

The African Descent and Glaucoma Evaluation Study (ADAGES) III: Contribution of Genotype to Glaucoma Phenotype in African Americans: Study Design and Baseline Data.

PURPOSE: To describe the study protocol and baseline characteristics of the African Descent and Glaucoma Evaluation Study (ADAGES) III. DESIGN: Cross-sectional, case-control study. PARTICIPANTS: Three thousand two hundred sixty-six glaucoma patients and control participants without glaucoma of African or European descent were recruited from 5 study centers in different regions of the United States. METHODS: Individuals of African descent (AD) and European descent (ED) with primary open-angle glaucoma (POAG) and control participants completed a detailed demographic and medical history interview. Standardized height, weight, and blood pressure measurements were obtained. Saliva and blood samples to provide serum, plasma, DNA, and RNA were collected for standardized processing. Visual fields, stereoscopic disc photographs, and details of the ophthalmic examination were obtained and transferred to the University of California, San Diego, Data Coordinating Center for standardized processing and quality review. MAIN OUTCOME MEASURES: Participant gender, age, race, body mass index, blood pressure, history of smoking and alcohol use in POAG patients and control participants were described. Ophthalmic measures included intraocular pressure, visual field mean deviation, central corneal thickness, glaucoma medication use, or past glaucoma surgery. Ocular conditions, including diabetic retinopathy, age-related macular degeneration, and past cataract surgery, were recorded. RESULTS: The 3266 ADAGES III study participants in this report include 2146 AD POAG patients, 695 ED POAG patients, 198 AD control participants, and 227 ED control participants. The AD POAG patients and control participants were significantly younger (both, 67.4 years) than ED POAG patients and control participants (73.4 and 70.2 years, respectively). After adjusting for age, AD POAG patients had different phenotypic characteristics compared with ED POAG patients, including higher intraocular pressure, worse visual acuity and visual field mean deviation, and thinner corneas (all P < 0.001). Family history of glaucoma did not differ between AD and ED POAG patients. CONCLUSIONS: With its large sample size, extensive specimen collection, and deep phenotyping of AD and ED glaucoma patients and control participants from different regions in the United States, the ADAGES III genomics study will address gaps in our knowledge of the genetics of POAG in this high-risk population.

Long-Term Effects of Gene Therapy in a Novel Mouse Model of Human MFRP-Associated Retinopathy.

Patients harboring homozygous c.498_499insC mutations in MFRP demonstrate hyperopia, microphthalmia, retinitis pigmentosa, retinal pigment epithelial atrophy, variable degrees of foveal edema, and optic disc drusen. The disease phenotype is variable, however, with some patients maintaining good central vision and cone function till late in the disease. A knock-in mouse model with the c.498_499insC mutation in Mfrp (Mfrp KI/KI) was developed to understand the effects of these mutations in the retina. The model shares many of the features of human clinical disease, including reduced axial length, hyperopia, retinal degeneration, retinal pigment epithelial atrophy, and decreased electrophysiological responses. In addition, the eyes of these mice had a significantly greater refractive error (p < 0.01) when compared to age-matched wild-type control animals. Administration of recombinant adeno-associated virus-mediated Mfrp gene therapy significantly prevented thinning from retinal neurodegeneration (p < 0.005) and preserved retinal electrophysiology (p < 0.001) when treated eyes were compared to contralateral sham-treated control eyes. The Mfrp KI/KI mice will serve as a useful tool to model human disease and point to a potential gene therapeutic approach for patients with preserved vision and electrophysiological responses in MFRP-related retinopathy.

Genetic Architecture of Primary Open-Angle Glaucoma in Individuals of African Descent: The African Descent and Glaucoma Evaluation Study III.

PURPOSE: To find genetic contributions to glaucoma in African Americans. DESIGN: Cross-sectional, case-control study. PARTICIPANTS: One thousand eight hundred seventy-five primary open-angle glaucoma (POAG) patients and 1709 controls, self-identified as being of African descent (AD), from the African Descent and Glaucoma Evaluation Study (ADAGES) III and Wake Forest School of Medicine. METHODS: MegaChip genotypes were imputed to Thousand Genomes data. Association of single nucleotide polymorphisms (SNPs) with POAG and advanced POAG was tested by linear mixed model correcting for relatedness and population stratification. Genetic risk scores were tested by receiver operator characteristic curves (ROC-AUCs). MAIN OUTCOME MEASURES: Primary open-angle glaucoma defined by visual field loss without other nonocular conditions (n = 1875). Advanced POAG was defined by age-based mean deviation of visual field (n = 946). RESULTS: Eighteen million two hundred eighty-one thousand nine hundred twenty SNPs met imputation quality of r2 > 0.7 and minor allele frequency > 0.005. Association of a novel locus, EN04, was observed for advanced POAG (rs185815146 ß, 0.36; standard error, 0.065; P < 3×10-8). For POAG, an AD signal was observed at the 9p21 European descent (ED) POAG signal (rs79721419; P < 6.5×10-5) independent of the previously observed 9p21 ED signal (rs2383204; P < 2.3×10-5) by conditional analyses. An association with POAG in FNDC3B (rs111698934; P < 3.9×10-5) was observed, not in linkage disequilibrium (LD) with the previously reported ED SNP. Additional previously identified loci associated with POAG in persons of AD were: 8q22, AFAP1, and TMC01. An AUC of 0.62 was observed with an unweighted genetic risk score comprising 11 SNPs in candidate genes. Two additional risk scores were studied by using a penalized matrix decomposition with cross-validation; risk scores of 50 and 400 SNPs were identified with ROC of AUC = 0.74 and AUC = 0.94, respectively. CONCLUSIONS: A novel association with advanced POAG in the EN04 locus was identified putatively in persons of AD. In addition to this finding, this genome-wide association study in POAG patients of AD contributes to POAG genetics by identification of novel signals in prior loci (9p21), as well as advancing the fine mapping of regions because of shorter average LD (FNDC3B). Although not useful without confirmation and clinical trials, the use of genetic risk scores demonstrated that considerable AD-specific genetic information remains in these data.