Alterations of the Ganglion Cell Complex in Age-Related Macular Degeneration: an AMISH Eye Study Analysis.

PURPOSE: To compare the Ganglion Cell Complex (GCC) thickness in eyes with age-related macular degeneration (AMD) versus healthy controls in an elderly Amish population DESIGN: Cross-sectional study METHODS: This is a post hoc analysis of the family-based prospective study of Amish subjects. Study subjects were imaged by the Cirrus HD-OCT (Carl Zeiss Meditec Inc, Dublin, CA) using a macular cube protocol of 512 × 128 scans (128 horizontal B-scans, each compromising 512 A-scans) over a 6 mm x 6 mm region centered on the fovea. The ganglion cell analysis algorithm calculated the GCC thickness by segmenting the outer boundaries of the retinal nerve fiber layer (RNFL) and inner plexiform layer (IPL) in all B-scans of the volume, with the region between these boundaries representing the combined thickness of the GCL and the IPL layer. A number of parameters were used to evaluate the GCC thickness: the average GCC thickness, minimum (lowest GCC thickness at a single meridian crossing the elliptical annulus), and sectoral (within each of six sectoral areas: superior, superotemporal, superonasal, inferior, inferonasal, and inferotemporal). The stage of AMD was graded on color fundus photographs in accordance with the Beckman Initiative for Macular Research classification system. RESULTS: Of 1339 subjects enrolled in the Amish eye study, a total of 1294 eyes of twelve hundred and ninety-four subjects had all required imaging studies of sufficient quality and were included in the final analysis, and of these 798 (62%) were female. Following age adjustment, the average GCC thickness was significantly (p<0.001) thinner in AMD subjects (73.71 ± SD; 13.77 µm) compared to normals (77.97 ± 10.42 µm). Independent t test showed that, early (75.03 ± 12.45 µm), and late AMD (61.64 ± 21.18 µm) groups (among which GA eyes had the lowest thickness of 58.10 ± 20.27 microns) had a statistically significant lower GCC thickness compared to eyes without AMD. There was no significant difference in average GCC thickness between early AMD and intermediate AMD (76.36 ± 9.25 µm) eyes. CONCLUSIONS: The GCC thickness in AMD eyes is reduced compared to normal eyes, but the relationship is complex with the greatest reduction in late AMD eyes (particularly GA eyes) but no difference between early and intermediate AMD eyes.

Longitudinal Evaluation of the Distribution of Intraretinal Hyper-Reflective Foci in Eyes with Intermediate Age-Related Macular Degeneration.

PURPOSE: Intraretinal hyper-reflective foci (IHRF) are optical coherence tomography (OCT) risk factors for progression of age-related macular degeneration (AMD). In this study we assess the change in the number and distribution of IHRF over two years. METHODS: The axial distribution of IHRF were quantified in eyes with intermediate AMD (iAMD) at baseline and 24 months, using a series of 5 sequential equidistant en face OCT retinal slabs generated between the outer border of the internal limiting membrane (ILM) and the inner border of the retinal pigment epithelium (RPE). Following thresholding and binarization, IHRF were quantified in each retinal slab using ImageJ. The change in IHRF number in each slab between baseline and month 24 was calculated. RESULTS: Fifty-two eyes showed evidence of IHRF at baseline, and all continued to show evidence of IHRF at 24 months (M24). The total average IHRF count/eye increased significantly from 4.67 ± 0.63 at baseline to 11.62 ± 13.86 at M24 (p < 0.001) with a mean increase of 6.94 ± 11.12 (range: - 9 to + 60). Overall, at M24, 76.9% eyes showed an increase in IHRF whereas 15.4% of eyes showed a decrease (3 eyes [5.7%] showed no change). There was a greater number of IHRF and a greater increase in IHRF over M24 in the outer slabs. CONCLUSIONS: IHRF are most common in the outer retinal layers and tend to increase in number over time. The impact of the distribution and frequency of these IHRF on the overall progression of AMD requires further study.

Assessment of intraretinal hyperreflective foci using multimodal imaging in eyes with age-related macular degeneration.

PURPOSE: This study aimed to investigate the correspondence between intraretinal hyperreflective foci (IHRF) identified on optical coherence tomography (OCT) B-scans with hyperpigmentation on colour fundus photography (CFP) or hyperreflectivity on infrared reflectance (IR) images in eyes with age-related macular degeneration (AMD). METHODS: Flash CFP, IR images and OCT B-scans obtained at the same visit were evaluated. Individual IHRF identified on OCT B-scans were assessed for the qualitative presence or absence of a hypotransmission tail into the choroid. The corresponding IR image obtained at the time of OCT acquisition was analysed for the presence or absence of hyperreflectivity in this region. The IR images were manually registered to the CFP image, and CFP images were inspected for the presence or absence of hyperpigmentation at the location of IHRF. RESULTS: From 122 eyes, a total of 494 IHRF were evaluated. For the primary analysis of qualitative presence or absence of hyperpigmentation on CFP and hyperreflectivity on IR at the locations corresponding to IHRF on OCT, 301 (61.0%) of the IHRFs demonstrated evidence of hyperpigmentation on CFP, while only 115 (23.3%) showed evidence of hyperreflectivity on IR. The qualitative determination of the presence or absence of an abnormality on CFP or IR were significantly different (p < 0.0001). 327 (66.2%) of the IHRF showed hypotransmission, and 80.4% of these IHRF showed hyperpigmentation on CFP, though only 23.9% (p < 0.0001) demonstrated hyperreflectivity on IR. CONCLUSIONS: Less than two-thirds of IHRF evident on OCT manifest as hyperpigmentation on colour photos, though IHRF with posterior shadowing are more likely to be evident as pigment. IR imaging appears to be even more poorly sensitive for visualizing IHRF.

Whole genome sequencing of 4,787 individuals identifies gene-based rare variants in age-related macular degeneration.

Genome-wide association studies have contributed extensively to the discovery of disease-associated common variants. However, the genetic contribution to complex traits is still largely difficult to interpret. We report a genome-wide association study of 2394 cases and 2393 controls for age-related macular degeneration (AMD) via whole-genome sequencing, with 46.9 million genetic variants. Our study reveals significant single-variant association signals at four loci and independent gene-based signals in CFH, C2, C3, and NRTN. Using data from the Exome Aggregation Consortium (ExAC) for a gene-based test, we demonstrate an enrichment of predicted rare loss-of-function variants in CFH, CFI, and an as-yet unreported gene in AMD, ORMDL2. Our method of using a large variant list without individual-level genotypes as an external reference provides a flexible and convenient approach to leverage the publicly available variant datasets to augment the search for rare variant associations, which can explain additional disease risk in AMD.

Patterns of Gene Expression, Splicing, and Allele-Specific Expression Vary among Macular Tissues and Clinical Stages of Age-Related Macular Degeneration.

Age-related macular degeneration (AMD) is a leading cause of blindness, and elucidating its underlying disease mechanisms is vital to the development of appropriate therapeutics. We identified differentially expressed genes (DEGs) and differentially spliced genes (DSGs) across the clinical stages of AMD in disease-affected tissue, the macular retina pigment epithelium (RPE)/choroid and the macular neural retina within the same eye. We utilized 27 deeply phenotyped donor eyes (recovered within a 6 h postmortem interval time) from Caucasian donors (60-94 years) using a standardized published protocol. Significant findings were then validated in an independent set of well-characterized donor eyes (n = 85). There was limited overlap between DEGs and DSGs, suggesting distinct mechanisms at play in AMD pathophysiology. A greater number of previously reported AMD loci overlapped with DSGs compared to DEGs between disease states, and no DEG overlap with previously reported loci was found in the macular retina between disease states. Additionally, we explored allele-specific expression (ASE) in coding regions of previously reported AMD risk loci, uncovering a significant imbalance in C3 rs2230199 and CFH rs1061170 in the macular RPE/choroid for normal eyes and intermediate AMD (iAMD), and for CFH rs1061147 in the macular RPE/choroid for normal eyes and iAMD, and separately neovascular AMD (NEO). Only significant DEGs/DSGs from the macular RPE/choroid were found to overlap between disease states. STAT1, validated between the iAMD vs. normal comparison, and AGTPBP1, BBS5, CERKL, FGFBP2, KIFC3, RORα, and ZNF292, validated between the NEO vs. normal comparison, revealed an intricate regulatory network with transcription factors and miRNAs identifying potential upstream and downstream regulators. Findings regarding the complement genes C3 and CFH suggest that coding variants at these loci may influence AMD development via an imbalance of gene expression in a tissue-specific manner. Our study provides crucial insights into the multifaceted genomic underpinnings of AMD (i.e., tissue-specific gene expression changes, potential splice variation, and allelic imbalance), which may open new avenues for AMD diagnostics and therapies specific to iAMD and NEO.

Longitudinal evaluation of the distribution of intraretinal hyper-reflective foci in eyes with intermediate age-related macular degeneration.

Purpose: Intraretinal hyper-reflective foci (IHRF) are optical coherence tomography (OCT) risk factors for progression of age-related macular degeneration (AMD). In this study we assess the change in the number and distribution of IHRF over two years. Methods: The axial distribution of IHRF were quantified in eyes with intermediate AMD (iAMD) at baseline and 24 months, using a series of 5 sequential equidistant en face OCT retinal slabs generated between the outer border of the internal limiting membrane (ILM) and the inner border of the retinal pigment epithelium (RPE). Following thresholding and binarization, IHRF were quantified in each retinal slab using ImageJ. The change in IHRF number in each slab between baseline and month 24 was calculated. Results: Fifty-two eyes showed evidence of IHRF at baseline, and all continued to show evidence of IHRF at 24 months (M24). The total average IHRF count/eye increased significantly from 4.67 ± 0.63 at baseline to 11.62 ± 13.86 at M24 (p<0.001) with a mean increase of 6.94 ± 11.12 (range: - 9 to + 60). Overall, at M24, 76.9% eyes showed an increase in IHRF whereas 15.4% of eyes showed a decrease (4 eyes [7.6%] showed no change). There was a greater number of IHRF and a greater increase in IHRF over M24 in the outer slabs. Conclusions: IHRF are most common in the outer retinal layers and tend to increase in number over time. The impact of the distribution and frequency of these IHRF on the overall progression of AMD requires further study.

Relationship between the distribution of intra-retinal hyper-reflective foci and the progression of intermediate age-related macular degeneration.

PURPOSE: To assess the relationship between the distribution of intra-retinal hyper-reflective foci (IHRF) on optical coherence tomography (OCT) and progression of intermediate age-related macular degeneration (iAMD) over 2 years. METHODS: Cirrus OCT volumes of the macula of subjects enrolled in the Amish Eye Study with 2 years of follow-up were evaluated for the presence of iAMD and IHRF at baseline. The IHRF were counted in a series of 5 sequential en face slabs from outer to inner retina. The number of IHRF in each slab at baseline and the change in IHRF from baseline to year 2 were correlated with progression to late AMD at 2 years. RESULTS: Among 120 eyes from 71 patients with iAMD, 52 eyes (43.3%) of 42 patients had evidence of both iAMD and IHRF at baseline. Twenty-three eyes (19.0%) showed progression to late AMD after 2 years. The total IHRF count increased from 243 at baseline to 604 at 2 years, with a significant increase in the IHRF number in each slab, except for the innermost slab 5 which had no IHRF at baseline or follow-up. The IHRF count increased from 121 to 340 in eyes that showed progression to late AMD. The presence of IHRF in the outermost retinal slabs 1 and 2 was independently associated with a significant risk of progression to late AMD. A greater increase in IHRF count over 2 years in these same slabs 1 and 2 was also associated with a higher risk of conversion to late AMD. CONCLUSIONS: The risk of progression to late AMD appears to be significantly associated with the distribution and extent of IHRF in the outermost retinal layers. This observation may point to significant pathophysiologic differences of IHRF in inner versus outer layers of the retina.

Drusen morphometrics on optical coherence tomography in eyes with age-related macular degeneration and normal aging.

PURPOSE: To compare drusen size metrics (apical height and basal width) on optical coherence tomography (OCT) B-scans with their size assessed on color photos in eyes with age-related macular degeneration (AMD) and normal aging. METHODS: A total of 508 drusen were evaluated in this analysis. Flash color fundus photos (CFP), infrared reflectance (IR) images, and OCT B-scans obtained at the same visit were evaluated. Individual drusen were identified on CFPs and the diameters of the drusen were measured in planimetric grading software. CFPs were manually registered to the IR image with their corresponding OCT volume. After confirming correspondence between the CFP and OCT, the apical height and basal width of the same drusen were measured on OCT B-scans. RESULTS: Drusen were divided into small, medium, large, and very large categories based on their diameter on the CFP images (< 63, 63 to 124, 125 to 249, and [Formula: see text] 250 µm, respectively). The OCT apical height of small drusen on CFP ranged from 20 to 31 µm, while medium drusen ranged from 31 to 46 µm, large drusen ranged from 45 µm to 111 µm, and very large drusen ranged from 55 µm to 208 µm. The OCT basal width measured < 99 µm in small drusen, from 99 to 143 µm in medium drusen, from 141 to 407 µm in large drusen, and > 209 µm in very large drusen. CONCLUSION: Drusen of different size categories on color photographs may also be separated according to their apical height and basal width on OCT. The apical height and basal width ranges defined in this analysis may be of value in the design of an OCT-based grading scale for AMD.

Rare variant analyses across multiethnic cohorts identify novel genes for refractive error.

Refractive error, measured here as mean spherical equivalent (SER), is a complex eye condition caused by both genetic and environmental factors. Individuals with strong positive or negative values of SER require spectacles or other approaches for vision correction. Common genetic risk factors have been identified by genome-wide association studies (GWAS), but a great part of the refractive error heritability is still missing. Some of this heritability may be explained by rare variants (minor allele frequency [MAF] ≤ 0.01.). We performed multiple gene-based association tests of mean Spherical Equivalent with rare variants in exome array data from the Consortium for Refractive Error and Myopia (CREAM). The dataset consisted of over 27,000 total subjects from five cohorts of Indo-European and Eastern Asian ethnicity. We identified 129 unique genes associated with refractive error, many of which were replicated in multiple cohorts. Our best novel candidates included the retina expressed PDCD6IP, the circadian rhythm gene PER3, and P4HTM, which affects eye morphology. Future work will include functional studies and validation. Identification of genes contributing to refractive error and future understanding of their function may lead to better treatment and prevention of refractive errors, which themselves are important risk factors for various blinding conditions.

Risk Factors for Progression of Age-Related Macular Degeneration: Population-Based Amish Eye Study.

Objective: To evaluate the optical coherence tomography (OCT)-based risk factors for progression to late age-related macular degeneration (AMD) in a population-based study of elderly Amish. Methods: A total of 1332 eyes of 666 consecutive subjects who completed a 2-year follow-up visit were included in this multicenter, prospective, longitudinal, observational study. Imaging features were correlated with 2-year incidence of late AMD development. Odds ratios for imaging features were estimated from logistic regression. Baseline OCT images were reviewed for the presence of drusen volume ≥0.03 mm3 in the central 3 mm ring, intraretinal hyperreflective foci (IHRF), hyporeflective drusen cores (hDC), subretinal drusenoid deposits (SDD), and drusenoid pigment epithelium detachment (PED). Subfoveal choroidal thickness, drusen area, and drusen volume within 3 and 5 mm circles centered on the fovea were also assessed. Results: Twenty-one (1.5%) of 1332 eyes progressed to late AMD by 2 years. The mean age of the study subjects was 65 ± 10.17 (±SD) years and 410 subjects were female. Univariate logistic regression showed that drusen area and volume in both 3 mm and 5 mm circles, subfoveal choroidal thickness, drusen volume ≥ 0.03 mm3 in the 3 mm ring, SDD, IHRF, and hDC were all associated with an increased risk for development of late AMD. The multivariate regression model identified that drusen volume in the 3 mm ring (OR: 2.59, p = 0.049) and presence of IHRF (OR: 57.06, p < 0.001) remained as independent and significant risk factors for progression to late AMD. Conclusions: This population-based study confirms previous findings from clinic-based studies that high central drusen volume and IHRF are associated with an increased risk of progression to late AMD. These findings may be of value in risk-stratifying patients in clinical practice or identifying subjects for early intervention clinical trials.

Systems genomics in age-related macular degeneration.

Genomic studies in age-related macular degeneration (AMD) have identified genetic variants that account for the majority of AMD risk. An important next step is to understand the functional consequences and downstream effects of the identified AMD-associated genetic variants. Instrumental for this next step are 'omics' technologies, which enable high-throughput characterization and quantification of biological molecules, and subsequent integration of genomics with these omics datasets, a field referred to as systems genomics. Single cell sequencing studies of the retina and choroid demonstrated that the majority of candidate AMD genes identified through genomic studies are expressed in non-neuronal cells, such as the retinal pigment epithelium (RPE), glia, myeloid and choroidal cells, highlighting that many different retinal and choroidal cell types contribute to the pathogenesis of AMD. Expression quantitative trait locus (eQTL) studies in retinal tissue have identified putative causal genes by demonstrating a genetic overlap between gene regulation and AMD risk. Linking genetic data to complement measurements in the systemic circulation has aided in understanding the effect of AMD-associated genetic variants in the complement system, and supports that protein QTL (pQTL) studies in plasma or serum samples may aid in understanding the effect of genetic variants and pinpointing causal genes in AMD. A recent epigenomic study fine-mapped AMD causal variants by determing regulatory regions in RPE cells differentiated from induced pluripotent stem cells (iPSC-RPE). Another approach that is being employed to pinpoint causal AMD genes is to produce synthetic DNA assemblons representing risk and protective haplotypes, which are then delivered to cellular or animal model systems. Pinpointing causal genes and understanding disease mechanisms is crucial for the next step towards clinical translation. Clinical trials targeting proteins encoded by the AMD-associated genomic loci C3, CFB, CFI, CFH, and ARMS2/HTRA1 are currently ongoing, and a phase III clinical trial for C3 inhibition recently showed a modest reduction of lesion growth in geographic atrophy. The EYERISK consortium recently developed a genetic test for AMD that allows genotyping of common and rare variants in AMD-associated genes. Polygenic risk scores (PRS) were applied to quantify AMD genetic risk, and may aid in predicting AMD progression. In conclusion, genomic studies represent a turning point in our exploration of AMD. The results of those studies now serve as a driving force for several clinical trials. Expanding to omics and systems genomics will further decipher function and causality from the associations that have been reported, and will enable the development of therapies that will lessen the burden of AMD.

Consequences of a Rare Complement Factor H Variant for Age-Related Macular Degeneration in the Amish.

Purpose: Genetic variants in the complement factor H gene (CFH) have been consistently implicated in age-related macular degeneration (AMD) risk. However, their functional effects are not fully characterized. We previously identified a rare, AMD-associated variant in CFH (P503A, rs570523689) in 19 Amish individuals, but its functional consequences were not investigated. Methods: We performed genotyping for CFH P503A in 1326 Amish individuals to identify additional risk allele carriers. We examined differences for age at AMD diagnosis between carriers and noncarriers. In blood samples from risk allele carriers and noncarriers, we quantified (i) CFH RNA expression, (ii) CFH protein expression, and (iii) C-reactive protein (CRP) expression. Potential changes to the CFH protein structure were interrogated computationally with Phyre2 and Chimera software programs. Results: We identified 39 additional carriers from Amish communities in Ohio and Indiana. On average, carriers were younger than noncarriers at AMD diagnosis, but this difference was not significant. CFH transcript and protein levels in blood samples from Amish carriers and noncarriers were also not significantly different. CRP levels were also comparable in plasma samples from carriers and noncarriers. Computational protein modeling showed slight changes in the CFH protein conformation that were predicted to alter interactions between the CFH 503 residue and other neighboring residues. Conclusions: In total, we have identified 58 risk allele carriers for CFH P503A in the Ohio and Indiana Amish. Although we did not detect significant differences in age at AMD diagnosis or expression levels of CFH in blood samples from carriers and noncarriers, we observed modest structural changes to the CFH protein through in silico modeling. Based on our functional and computational observations, we hypothesize that CFH P503A may affect CFH binding or function rather than expression, which would require additional research to confirm.

As in Real Estate, Location Matters: Cellular Expression of Complement Varies Between Macular and Peripheral Regions of the Retina and Supporting Tissues.

The cellular events that dictate the initiation of the complement pathway in ocular degeneration, such as age-related macular degeneration (AMD), is poorly understood. Using gene expression analysis (single cell and bulk), mass spectrometry, and immunohistochemistry, we dissected the role of multiple retinal and choroidal cell types in determining the complement homeostasis. Our scRNA-seq data show that the cellular response to early AMD is more robust in the choroid, particularly in fibroblasts, pericytes and endothelial cells. In late AMD, complement changes were more prominent in the retina especially with the expression of the classical pathway initiators. Notably, we found a spatial preference for these differences. Overall, this study provides insights into the heterogeneity of cellular responses for complement expression and the cooperation of neighboring cells to complete the pathway in healthy and AMD eyes. Further, our findings provide new cellular targets for therapies directed at complement.

Genomewide Association Study of Retinal Traits in the Amish Reveals Loci Influencing Drusen Development and Link to Age-Related Macular Degeneration.

Purpose: The purpose of this study was to identify genetic risk loci for retinal traits, including drusen, in an Amish study population and compare these risk loci to known risk loci of age-related macular degeneration (AMD). Methods: Participants were recruited from Amish communities in Ohio, Indiana, and Pennsylvania. Each participant underwent a basic health history, ophthalmologic examination, and genotyping. A genomewide association analysis (GWAS) was conducted for the presence and quantity of each of three retinal traits: geographic atrophy, drusen area, and drusen volume. The findings were compared to results from a prior large GWAS of predominantly European-ancestry individuals. Further, a genetic risk score for AMD was used to predict the presence and quantity of the retinal traits. Results: After quality control, 1074 participants were included in analyses. Six single nucleotide polymorphisms (SNPs) met criteria for genomewide significance and 48 were suggestively associated across three retinal traits. The significant SNPs were not highly correlated with known risk SNPs for AMD. A genetic risk score for AMD provided significant predictive value of the retinal traits. Conclusions: We identified potential novel genetic risk loci for AMD in a midwestern Amish study population. Additionally, we determined that there is a clear link between the genetic risk of AMD and drusen. Further study, including longitudinal data collection, may improve our ability to define this connection and improve understanding of the biological risk factors underlying drusen development.

Inflammatory adipose activates a nutritional immunity pathway leading to retinal dysfunction.

Age-related macular degeneration (AMD), the leading cause of irreversible blindness among Americans over 50, is characterized by dysfunction and death of retinal pigment epithelial (RPE) cells. The RPE accumulates iron in AMD, and iron overload triggers RPE cell death in vitro and in vivo. However, the mechanism of RPE iron accumulation in AMD is unknown. We show that high-fat-diet-induced obesity, a risk factor for AMD, drives systemic and local inflammatory circuits upregulating interleukin-1ß (IL-1ß). IL-1ß upregulates RPE iron importers and downregulates iron exporters, causing iron accumulation, oxidative stress, and dysfunction. We term this maladaptive, chronic activation of a nutritional immunity pathway the cellular iron sequestration response (CISR). RNA sequencing (RNA-seq) analysis of choroid and retina from human donors revealed that hallmarks of this pathway are present in AMD microglia and macrophages. Together, these data suggest that inflamed adipose tissue, through the CISR, can lead to RPE pathology in AMD.

Effect of OCT B-Scan Density on Sensitivity for Detection of Intraretinal Hyperreflective Foci in Eyes with Age-Related Macular Degeneration.

PURPOSE: To evaluate the impact of reducing the density of B-scans in an optical coherence tomography (OCT) volume on the sensitivity for detecting intraretinal hyperreflective foci (IHRF) in eyes with intermediate age-related macular degeneration (AMD). METHODS: A total of 165 eyes with intermediate AMD and IHRF were evaluated in this retrospective analysis. For each case, Cirrus HD-OCT volumes were imported into the reading center 3 D-OCTOR software. The number of IHRF cases was assessed based on all 128 B-scans (spaced 47 µm apart), using a categorical scale (graded as 1-4, 5-9, 10-14, 15-19, and >20). Additionally, the B-scan densities in the volume were lowered to 64 B-scans (spaced 94 µm apart), 43 B-scans (spaced 140 µm apart), and 32 B-scans (spaced 188 µm apart). The number of eyes with any IHRF and the numerical category of IHRF in the eye were used to compare the sensitivity at each reduced B-scan density against the reference 128 B-scan volume. RESULTS: In the primary analysis for the qualitative presence or absence of any IHRF, the sensitivity decreased to 98.2% (p = .32) with 64 B-scans, 92.7% (p = .001) with 43 B-scans, and 75.2% (p = .001) with 32 B-scans, compared with the 128 B-scan reference. With regard to the number of IHRF per eye, there was a significant difference (with a lower level chosen on the scale) when the B-scan density was reduced to 43 or 32 B-scans (p = .002 and p < .001, respectively). CONCLUSION: Increasing the inter-B-scan spacing from 47 to 188 microns significantly reduced the ability to accurately determine whether IHRF were present in an eye. An increase in inter-B-scan spacing to 140 microns was associated with a significant misclassification of the IHRF quantity. These findings may be relevant in the design of OCT scanning protocols for studies utilizing these biomarkers for AMD progression.

Implication of specific retinal cell-type involvement and gene expression changes in AMD progression using integrative analysis of single-cell and bulk RNA-seq profiling.

Age-related macular degeneration (AMD) is a blinding eye disease with no unifying theme for its etiology. We used single-cell RNA sequencing to analyze the transcriptomes of ~ 93,000 cells from the macula and peripheral retina from two adult human donors and bulk RNA sequencing from fifteen adult human donors with and without AMD. Analysis of our single-cell data identified 267 cell-type-specific genes. Comparison of macula and peripheral retinal regions found no cell-type differences but did identify 50 differentially expressed genes (DEGs) with about 1/3 expressed in cones. Integration of our single-cell data with bulk RNA sequencing data from normal and AMD donors showed compositional changes more pronounced in macula in rods, microglia, endothelium, Müller glia, and astrocytes in the transition from normal to advanced AMD. KEGG pathway analysis of our normal vs. advanced AMD eyes identified enrichment in complement and coagulation pathways, antigen presentation, tissue remodeling, and signaling pathways including PI3K-Akt, NOD-like, Toll-like, and Rap1. These results showcase the use of single-cell RNA sequencing to infer cell-type compositional and cell-type-specific gene expression changes in intact bulk tissue and provide a foundation for investigating molecular mechanisms of retinal disease that lead to new therapeutic targets.

Myopia in African Americans Is Significantly Linked to Chromosome 7p15.2-14.2.

Purpose: The purpose of this study was to perform genetic linkage analysis and association analysis on exome genotyping from highly aggregated African American families with nonpathogenic myopia. African Americans are a particularly understudied population with respect to myopia. Methods: One hundred six African American families from the Philadelphia area with a family history of myopia were genotyped using an Illumina ExomePlus array and merged with previous microsatellite data. Myopia was initially measured in mean spherical equivalent (MSE) and converted to a binary phenotype where individuals were identified as affected, unaffected, or unknown. Parametric linkage analysis was performed on both individual variants (single-nucleotide polymorphisms [SNPs] and microsatellites) as well as gene-based markers. Family-based association analysis and transmission disequilibrium test (TDT) analysis modified for rare variants was also performed. Results: Genetic linkage analysis identified 2 genomewide significant variants at 7p15.2 and 7p14.2 (in the intergenic region between MIR148A and NFE2L3 and in the noncoding RNA LOC401324) and 2 genomewide significant genes (CRHR2 and AVL9) both at 7p14.3. No genomewide results were found in the association analyses. Conclusions: This study identified a significant linkage peak in African American families for myopia at 7p15.2 to 7p14.2, the first potential risk locus for myopia in African Americans. Interesting candidate genes are located in the region, including PDE1C, which is highly expressed in the eyes, and known to be involved in retinal development. Further identification of the causal variants at this linkage peak will help elucidate the genetics of myopia in this understudied population.

Automated identification of clinical features from sparsely annotated 3-dimensional medical imaging.

One of the core challenges in applying machine learning and artificial intelligence to medicine is the limited availability of annotated medical data. Unlike in other applications of machine learning, where an abundance of labeled data is available, the labeling and annotation of medical data and images require a major effort of manual work by expert clinicians who do not have the time to annotate manually. In this work, we propose a new deep learning technique (SLIVER-net), to predict clinical features from 3-dimensional volumes using a limited number of manually annotated examples. SLIVER-net is based on transfer learning, where we borrow information about the structure and parameters of the network from publicly available large datasets. Since public volume data are scarce, we use 2D images and account for the 3-dimensional structure using a novel deep learning method which tiles the volume scans, and then adds layers that leverage the 3D structure. In order to illustrate its utility, we apply SLIVER-net to predict risk factors for progression of age-related macular degeneration (AMD), a leading cause of blindness, from optical coherence tomography (OCT) volumes acquired from multiple sites. SLIVER-net successfully predicts these factors despite being trained with a relatively small number of annotated volumes (hundreds) and only dozens of positive training examples. Our empirical evaluation demonstrates that SLIVER-net significantly outperforms standard state-of-the-art deep learning techniques used for medical volumes, and its performance is generalizable as it was validated on an external testing set. In a direct comparison with a clinician panel, we find that SLIVER-net also outperforms junior specialists, and identifies AMD progression risk factors similarly to expert retina specialists.

COMPARISON OF SPECTRALIS AND CIRRUS OPTICAL COHERENCE TOMOGRAPHY FOR THE DETECTION OF INCOMPLETE AND COMPLETE RETINAL PIGMENT EPITHELIUM AND OUTER RETINAL ATROPHY.

PURPOSE: To evaluate and compare the detection of incomplete and complete retinal pigment epithelial and outer retinal atrophy (iRORA and cRORA) using Spectralis and Cirrus optical coherence tomography (OCT) devices. METHODS: Subjects with late age-related macular degeneration were imaged on the same day with Spectralis and Cirrus OCT. Two, masked, independent, and experienced retina specialist graders evaluated each case for the presence of cRORA and iRORA lesions. RESULTS: A significantly higher number of lesions were observed using Spectralis compared with Cirrus (239 vs. 226 and 223 vs. 209). Higher number of iRORA lesions were identified with Spectralis (105 vs. 90 and 96 vs. 82), and no significant difference was observed between devices for cRORA lesions (134 vs. 136 and 128 vs. 126). When considering the presence or absence of iRORA or cRORA, the agreement between devices for both graders was excellent for cRORA and good for iRORA. CONCLUSION: Spectralis and Cirrus OCT identified a similar number of cRORA lesions, although more iRORA lesions could be detected with Spectralis OCT. These findings may have implications for developing acquisition protocols for trials based on the intended atrophy targets and highlight the importance of using a consistent OCT instrument across a study.