High throughput functional profiling of genes at intraocular pressure loci reveals distinct networks for glaucoma.

INTRODUCTION: Primary open angle glaucoma (POAG) is a leading cause of blindness globally. Characterized by progressive retinal ganglion cell degeneration, the precise pathogenesis remains unknown. Genome-wide association studies (GWAS) have uncovered many genetic variants associated with elevated intraocular pressure (IOP), one of the key risk factors for POAG. We aimed to identify genetic and morphological variation that can be attributed to trabecular meshwork cell (TMC) dysfunction and raised IOP in POAG. METHODS: 62 genes across 55 loci were knocked-out in a primary human TMC line. Each knockout group, including five non-targeting control groups, underwent single-cell RNA-sequencing (scRNA-seq) for differentially-expressed gene (DEG) analysis. Multiplexed fluorescence coupled with CellProfiler image analysis allowed for single-cell morphological profiling. RESULTS: Many gene knockouts invoked DEGs relating to matrix metalloproteinases and interferon-induced proteins. We have prioritized genes at four loci of interest to identify gene knockouts that may contribute to the pathogenesis of POAG, including ANGPTL2, LMX1B, CAV1, and KREMEN1. Three genetic networks of gene knockouts with similar transcriptomic profiles were identified, suggesting a synergistic function in trabecular meshwork cell physiology. TEK knockout caused significant upregulation of nuclear granularity on morphological analysis, while knockout of TRIOBP, TMCO1 and PLEKHA7 increased granularity and intensity of actin and the cell-membrane. CONCLUSION: High-throughput analysis of cellular structure and function through multiplex fluorescent single-cell analysis and scRNA-seq assays enabled the direct study of genetic perturbations at the single-cell resolution. This work provides a framework for investigating the role of genes in the pathogenesis of glaucoma and heterogenous diseases with a strong genetic basis.

Deep Learning-Based Identification of Intraocular Pressure-Associated Genes Influencing Trabecular Meshwork Cell Morphology.

Purpose: Genome-wide association studies have recently uncovered many loci associated with variation in intraocular pressure (IOP). Artificial intelligence (AI) can be used to interrogate the effect of specific genetic knockouts on the morphology of trabecular meshwork cells (TMCs) and thus, IOP regulation. Design: Experimental study. Subjects: Primary TMCs collected from human donors. Methods: Sixty-two genes at 55 loci associated with IOP variation were knocked out in primary TMC lines. All cells underwent high-throughput microscopy imaging after being stained with a 5-channel fluorescent cell staining protocol. A convolutional neural network was trained to distinguish between gene knockout and normal control cell images. The area under the receiver operator curve (AUC) metric was used to quantify morphological variation in gene knockouts to identify potential pathological perturbations. Main Outcome Measures: Degree of morphological variation as measured by deep learning algorithm accuracy of differentiation from normal controls. Results: Cells where LTBP2 or BCAS3 had been perturbed demonstrated the greatest morphological variation from normal TMCs (AUC 0.851, standard deviation [SD] 0.030; and AUC 0.845, SD 0.020, respectively). Of 7 multigene loci, 5 had statistically significant differences in AUC (P < 0.05) between genes, allowing for pathological gene prioritization. The mitochondrial channel most frequently showed the greatest degree of morphological variation (33.9% of cell lines). Conclusions: We demonstrate a robust method for functionally interrogating genome-wide association signals using high-throughput microscopy and AI. Genetic variations inducing marked morphological variation can be readily identified, allowing for the gene-based dissection of loci associated with complex traits. Financial Disclosures: Proprietary or commercial disclosure may be found in the Footnotes and Disclosures at the end of this article.

Diagnostic Accuracy of Artificial Intelligence in Glaucoma Screening and Clinical Practice.

PURPOSE: Artificial intelligence (AI) has been shown as a diagnostic tool for glaucoma detection through imaging modalities. However, these tools are yet to be deployed into clinical practice. This meta-analysis determined overall AI performance for glaucoma diagnosis and identified potential factors affecting their implementation. METHODS: We searched databases (Embase, Medline, Web of Science, and Scopus) for studies that developed or investigated the use of AI for glaucoma detection using fundus and optical coherence tomography (OCT) images. A bivariate random-effects model was used to determine the summary estimates for diagnostic outcomes. The Preferred Reporting Items for Systematic Reviews and Meta-Analysis of Diagnostic Test Accuracy (PRISMA-DTA) extension was followed, and the Quality Assessment of Diagnostic Accuracy Studies-2 (QUADAS-2) tool was used for bias and applicability assessment. RESULTS: Seventy-nine articles met inclusion criteria, with a subset of 66 containing adequate data for quantitative analysis. The pooled area under receiver operating characteristic curve across all studies for glaucoma detection was 96.3%, with a sensitivity of 92.0% (95% confidence interval: 89.0-94.0) and specificity of 94.0% (95% confidence interval: 92.0-95.0). The pooled area under receiver operating characteristic curve on fundus and OCT images was 96.2% and 96.0%, respectively. Mixed data set and external data validation had unsatisfactory diagnostic outcomes. CONCLUSION: Although AI has the potential to revolutionize glaucoma care, this meta-analysis highlights that before such algorithms can be implemented into clinical care, a number of issues need to be addressed. With substantial heterogeneity across studies, many factors were found to affect the diagnostic performance. We recommend implementing a standard diagnostic protocol for grading, implementing external data validation, and analysis across different ethnicity groups.