Optometry Commons^™

Exaggerated False Positives By Popular Differential Expression Methods When Analyzing Human Population Samples, Yumei Li, Xinzhou Ge, Fanglue Peng, Wei Li, Jingyi Jessica Li

Faculty, Staff and Students Publications

When identifying differentially expressed genes between two conditions using human population RNA-seq samples, we found a phenomenon by permutation analysis: two popular bioinformatics methods, DESeq2 and edgeR, have unexpectedly high false discovery rates. Expanding the analysis to limma-voom, NOISeq, dearseq, and Wilcoxon rank-sum test, we found that FDR control is often failed except for the Wilcoxon rank-sum test. Particularly, the actual FDRs of DESeq2 and edgeR sometimes exceed 20% when the target FDR is 5%. Based on these results, for population-level RNA-seq studies with large sample sizes, we recommend the Wilcoxon rank-sum test.

Genetic Subtyping And Phenotypic Characterization Of The Immune Microenvironment And Myc/Bcl2 Double Expression Reveal Heterogeneity In Diffuse Large B-Cell Lymphoma, Zijun Y Xu-Monette, Li Wei, Xiaosheng Fang, Qingyan Au, Harry Nunns, Máté Nagy, Alexandar Tzankov, Feng Zhu, Carlo Visco, Govind Bhagat, Karen Dybkaer, April Chiu, Wayne Tam, Youli Zu, Eric D Hsi, Fredrick B Hagemeister, Xiaoping Sun, Xin Han, Heounjeong Go, Maurilio Ponzoni, Andrés J M Ferreri, Michael B Møller, Benjamin M Parsons, J Han Van Krieken, Miguel A Piris, Jane N Winter, Yong Li, Bing Xu, Maher Albitar, Hua You, Ken H Young

Faculty, Staff and Students Publications

PURPOSE: Diffuse large B-cell lymphoma (DLBCL) is molecularly and clinically heterogeneous, and can be subtyped according to genetic alterations, cell-of-origin, or microenvironmental signatures using high-throughput genomic data at the DNA or RNA level. Although high-throughput proteomic profiling has not been available for DLBCL subtyping, MYC/BCL2 protein double expression (DE) is an established prognostic biomarker in DLBCL. The purpose of this study is to reveal the relative prognostic roles of DLBCL genetic, phenotypic, and microenvironmental biomarkers.

EXPERIMENTAL DESIGN: We performed targeted next-generation sequencing; IHC for MYC, BCL2, and FN1; and fluorescent multiplex IHC for microenvironmental markers in a large cohort of …

Visual Pigment-Deficient Cones Survive And Mediate Visual Signaling Despite The Lack Of Outer Segments, Hui Xu, Nange Jin, Jen-Zen Chuang, Zhao Zhang, Xiaoyue Zhong, Zhijing Zhang, Ching-Hwa Sung, Christophe P Ribelayga, Yingbin Fu

Faculty, Staff and Students Publications

Rhodopsin and cone opsins are essential for light detection in vertebrate rods and cones, respectively. It is well established that rhodopsin is required for rod phototransduction, outer segment disk morphogenesis, and rod viability. However, the roles of cone opsins are less well understood. In this study, we adopted a loss-of-function approach to investigate the physiological roles of cone opsins in mice. We showed that cones lacking cone opsins do not form normal outer segments due to the lack of disk morphogenesis. Surprisingly, cone opsin-deficient cones survive for at least 12 mo, which is in stark contrast to the rapid rod …

Abnormal Perception Of Pattern-Induced Flicker Colors In Subjects With Glaucoma, Padmapriya Ramamoorthy, Nicole L Alexander, Benjamin J Frankfort

Faculty, Staff and Students Publications

Pattern-induced flicker colors (PIFCs) are subjective colors that can be elicited with rotation of an achromatic stimulus such as the Benham disk. The perceptive mechanisms underlying PIFCs are not well-understood, but are thought to be generated primarily by retinal cell types which may be dysfunctional in glaucoma. Using a custom computer-based system, we tested PIFC perception across several Benham disk parameters, including the rates of acceleration and deceleration, rotational direction, and image contrast in both control and glaucoma subjects. We defined the Benham perception limit (BPL) during acceleration as the rotational speed at which PIFCs were first detected (Benham perception …

Review Of Gene Therapies For Age-Related Macular Degeneration, Arshad M Khanani, Mathew J Thomas, Aamir A Aziz, Christina Y Weng, Carl J Danzig, Glenn Yiu, Szilárd Kiss, Nadia K Waheed, Peter K Kaiser

Faculty, Staff and Students Publications

Gene therapies aim to deliver a therapeutic payload to specified tissues with underlying protein deficiency. Since the 1990s, gene therapies have been explored as potential treatments for chronic conditions requiring lifetime care and medical management. Ocular gene therapies target a range of ocular disorders, but retinal diseases are of particular importance due to the prevalence of retinal disease and the current treatment burden of such diseases on affected patients, as well as the challenge of properly delivering these therapies to the target tissue. The purpose of this review is to provide an update on the most current data available for …

Progressive, Long-Term Hearing Loss In Congenital Cmv Disease After Ganciclovir Therapy, Tatiana M Lanzieri, Alison Chantal Caviness, Peggy Blum, Gail Demmler-Harrison

Faculty, Staff and Students Publications

BACKGROUND: Long-term hearing outcomes among children with symptomatic congenital cytomegalovirus (CMV) disease who received 6-week ganciclovir therapy early in life are unknown.

METHODS: Longitudinal study of 76 children with symptomatic congenital CMV disease, born 1983-2005, who were categorized into three groups: group A treated with ganciclovir; group B untreated who had microcephaly, chorioretinitis, or sensorineural hearing loss (SNHL; ≥25 dB) diagnosed in the first month of life (congenital); and group C untreated who did not meet criteria for group B.

RESULTS: Patients in groups A (n = 17), B (n = 27), and C (n = 32) were followed to …

Secretogranin Iii Stringently Regulates Pathological But Not Physiological Angiogenesis In Oxygen-Induced Retinopathy, Chang Dai, Prabuddha Waduge, Liyang Ji, Chengchi Huang, Ye He, Hong Tian, Elizabeth Zuniga-Sanchez, Amit Bhatt, Iok-Hou Pang, Guanfang Su, Keith A Webster, Wei Li

Faculty, Staff and Students Publications

Conventional angiogenic factors, such as vascular endothelial growth factor (VEGF), regulate both pathological and physiological angiogenesis indiscriminately, and their inhibitors may elicit adverse side effects. Secretogranin III (Scg3) was recently reported to be a diabetes-restricted VEGF-independent angiogenic factor, but the disease selectivity of Scg3 in retinopathy of prematurity (ROP), a retinal disease in preterm infants with concurrent pathological and physiological angiogenesis, was not defined. Here, using oxygen-induced retinopathy (OIR) mice, a surrogate model of ROP, we quantified an exclusive binding of Scg3 to diseased versus healthy developing neovessels that contrasted sharply with the ubiquitous binding of VEGF. Functional immunohistochemistry visualized …

3’Aqtl-Atlas: An Atlas Of 3’Utr Alternative Polyadenylation Quantitative Trait Loci Across Human Normal Tissues, Ya Cui, Fanglue Peng, Dan Wang, Yumei Li, Jason Sheng Li, Lei Li, Wei Li

Faculty, Staff and Students Publications

Genome-wide association studies (GWAS) have identified thousands of non-coding single-nucleotide polymorphisms (SNPs) associated with human traits and diseases. However, functional interpretation of these SNPs remains a significant challenge. Our recent study established the concept of 3' untranslated region (3'UTR) alternative polyadenylation (APA) quantitative trait loci (3'aQTLs), which can be used to interpret ∼16.1% of GWAS SNPs and are distinct from gene expression QTLs and splicing QTLs. Despite the growing interest in 3'aQTLs, there is no comprehensive database for users to search and visualize them across human normal tissues. In the 3'aQTL-atlas (https://wlcb.oit.uci.edu/3aQTLatlas), we provide a comprehensive list of 3'aQTLs containing …

Retrobulbar Hematoma In A Patient With Thyroid Eye Disease, Christina Y Weng

Faculty, Staff and Students Publications

A 37-year-old female with a history of thyroid eye disease presented after trauma to the right side of her face. Visual acuity was light perception and a relative afferent pupillary defect was detected. There was gross proptosis of the unruptured right globe with diffuse conjunctival hemorrhage. Computed tomography revealed an intraconal retrobulbar hematoma (RBH) with anterior herniation of the globe. The extraocular muscles were thickened, consistent with her thyroid eye disease, and her superior rectus was avulsed. The optic nerve was on stretch, but there was no evidence of transection. The patient ultimately underwent enucleation with the plan for an …

Initial Experiences Using Plasma Rich In Growth Factors To Treat Keratoneuralgia, Margaret Wang, Sowmya Yennam, Stephen Pflugfelder

Faculty, Staff and Students Publications

Keratoneuralgia, a clinical diagnosis of sensitized corneal pain without visible ocular surface damage, generally has minimal response to conventional therapies. Causes include refractive surgery and chronic dry eye. We evaluated the efficacy of Plasma Rich in Growth Factors (PRGF), a novel treatment prepared using a commercially available kit, in patients with keratoneuralgia. A retrospective chart review identified patients who had the clinical diagnosis of keratoneuralgia and were treated with PRGF for at least 3 months from October 2015 to April 2020 at a single academic institution. Both objective eye exam findings and concurrent treatments were obtained at baseline, 3 months, …

Il-17 Producing Lymphocytes Cause Dry Eye And Corneal Disease With Aging In Rxrα Mutant Mouse, Jehan Alam, Ghasem Yazdanpanah, Rinki Ratnapriya, Nicholas Borcherding, Cintia S De Paiva, Dequan Li, Rodrigo Guimaraes De Souza, Zhiyuan Yu, Stephen C Pflugfelder

Faculty, Staff and Students Publications

PURPOSE: To investigate IL-17 related mechanisms for developing dry eye disease in the Pinkie mouse strain with a loss of function RXRα mutation.

METHODS: Measures of dry eye disease were assessed in the cornea and conjunctiva. Expression profiling was performed by single-cell RNA sequencing (scRNA-seq) to compare gene expression in conjunctival immune cells. Conjunctival immune cells were immunophenotyped by flow cytometry and confocal microscopy. The activity of RXRα ligand 9-cis retinoic acid (RA) was evaluated in cultured monocytes and γδ T cells.

RESULTS: Compared to wild type (WT) C57BL/6, Pinkie has increased signs of dry eye disease, including decreased tear …

Problems In Csf And Ophthalmic Disease Research, Ryan Machiele, Benjamin Jay Frankfort, Hanspeter Esriel Killer, David Fleischman

Faculty, Staff and Students Publications

There has been significant interest and progress in the understanding of cerebrospinal fluid pressure and its relationship to glaucoma and other ophthalmic diseases. However, just as every physiologic fluid pressure fluctuates, cerebrospinal fluid pressure (CSFP) is similarly dynamic. Coupling this with the difficulty in measuring the pressure, there are many obstacles in furthering this field of study. This review highlights some of the difficulties in CSFP research, including fluid compartmentalization, estimation equations, and pressure fluctuation. Keeping these limitations in mind will hopefully improve the quality and context of this burgeoning field.

Systematic Profiling Of Dnmt3a Variants Reveals Protein Instability Mediated By The Dcaf8 E3 Ubiquitin Ligase Adaptor, Yung-Hsin Huang, Chun-Wei Chen, Venkatasubramaniam Sundaramurthy, Mikołaj Słabicki, Dapeng Hao, Caroline J Watson, Ayala Tovy, Jaime M Reyes, Olga Dakhova, Brielle R Crovetti, Christina Galonska, Minjung Lee, Lorenzo Brunetti, Yubin Zhou, Katrina Tatton-Brown, Yun Huang, Xiaodong Cheng, Alexander Meissner, Peter J M Valk, Lionel Van Maldergem, Mathijs A Sanders, Jamie R Blundell, Wei Li, Benjamin L Ebert, Margaret A Goodell

Faculty, Staff and Students Publications

Clonal hematopoiesis is a prevalent age-related condition associated with a greatly increased risk of hematologic disease; mutations in DNA methyltransferase 3A (DNMT3A) are the most common driver of this state. DNMT3A variants occur across the gene with some particularly associated with malignancy, but the functional relevance and mechanisms of pathogenesis of the majority of mutations are unknown. Here, we systematically investigated the methyltransferase activity and protein stability of 253 disease-associated DNMT3A mutations, and found that 74% were loss-of-function mutations. Half of these variants exhibited reduced protein stability and, as a class, correlated with greater clonal expansion and acute …

Gut Microbiota From Sjögren Syndrome Patients Causes Decreased T Regulatory Cells In The Lymphoid Organs And Desiccation-Induced Corneal Barrier Disruption In Mice, Laura Schaefer, Claudia M Trujillo-Vargas, Firas S Midani, Stephen C Pflugfelder, Robert A Britton, Cintia S De Paiva

Faculty, Staff and Students Publications

Sjögren syndrome (SS) is an autoimmune inflammatory disorder characterized by secretory dysfunction in the eye and mouth; in the eye, this results in tear film instability, reduced tear production, and corneal barrier disruption. A growing number of studies show that homeostasis of the ocular surface is impacted by the intestinal microbiome, and several 16S sequencing studies have demonstrated dysbiosis of the intestinal microbiota in SS patients. In this study, we utilized metagenomic sequencing to perform a deeper analysis of the intestinal microbiome using stools collected from sex- and age-matched healthy (n = 20), dry eye (n = 4) …

Selective Vulnerability Of The Intermediate Retinal Capillary Plexus Precedes Retinal Ganglion Cell Loss In Ocular Hypertension, Priyamvada M Pitale, Guofu Shen, Rohini R Sigireddi, Maria Polo-Prieto, Yong H Park, Solomon E Gibson, Peter D Westenskow, Roomasa Channa, Benjamin J Frankfort

Faculty, Staff and Students Publications

Introduction: Glaucoma, a disease of retinal ganglion cell (RGC) injury and potentially devastating vision loss, is associated with both ocular hypertension (OHT) and reduced ocular blood flow. However, the relationship between OHT and retinal capillary architecture is not well understood. In this project, we studied microvasculature damage in mice exposed to mild levels of induced OHT.

Methods: Mild OHT was induced with the microbead model for 2 weeks. At this time point, some retinas were immunostained with CD31 (endothelium), Collagen IV (basement membrane), and RBPMS (RGCs) for z-stack confocal microscopy. We processed these confocal images to distinguish the three retinal …

Selectively Targeting Disease-Restricted Secretogranin Iii To Alleviate Choroidal Neovascularization, Liyang Ji, Prabuddha Waduge, Lili Hao, Avinash Kaur, Wencui Wan, Yan Wu, Hong Tian, Jinsong Zhang, Keith A Webster, Wei Li

Faculty, Staff and Students Publications

Choroidal neovascularization (CNV), a leading cause of blindness in the elderly, is routinely treated with vascular endothelial growth factor (VEGF) inhibitors that have limited efficacy and potentially adverse side effects. An unmet clinical need is to develop novel therapies against other angiogenic factors for alternative or combination treatment to improve efficacy and safety. We recently described secretogranin III (Scg3) as a disease-selective angiogenic factor, causally linked to diabetic retinopathy and acting independently of the VEGF pathway. An important question is whether such a disease-selective Scg3 pathway contributes to other states of pathological angiogenesis beyond diabetic retinopathy. By applying a novel …

New, Potent, Small Molecule Agonists Of Tyrosine Kinase Receptors Attenuate Dry Eye Disease, Zhiyuan Yu, Shaon Joy, Tianxiong Mi, Ghasem Yazdanpanah, Kevin Burgess, Cintia S De Paiva

Faculty, Staff and Students Publications

Nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), and neurotrophin3 (NT-3) bind to tyrosine kinase (Trk) receptors, TrkA, TrkB, and TrkC, respectively. This study investigated the efficacy of novel molecule agonists of Trk receptors in an in vivo model of dry eye disease (DED). Small molecule TrkC agonist (C1) and a pan-Trk agonist (pan) were synthesized for this. C57BL/6J mice were subjected to desiccating stress (DS) and received bilateral eye drops of C1, pan, or vehicle (2x/day). Dry eye signs, inflammation and expression of corneal barrier function, and conjunctival goblet cell (GC) densities were measured as part of the DED …

Cathepsin S Is A Novel Target For Age-Related Dry Eye, Zhiyuan Yu, Jinmiao Li, Gowthaman Govindarajan, Sarah F Hamm-Alvarez, Jehan Alam, De-Quan Li, Cintia S De Paiva

Faculty, Staff and Students Publications

Cathepsin S (CTSS) is a protease that is proinflammatory on epithelial cells. The purpose of this study was to investigate the role of CTSS in age-related dry eye disease. Ctss−/− mice [in a C57BL/6 (B6) background] of different ages were compared to B6 mice. CTSS activity in tears and lacrimal gland (LG) lysates was measured. The corneal barrier function was investigated in naïve mice or after topical administration of CTSS eye drops 5X/day for two days. Eyes were collected, and conjunctival goblet cell density was measured in PAS-stained sections. Immunoreactivity of the tight junction proteins, ZO-1 and occludin, was …

Spatial Transcriptomics Of The Lacrimal Gland Features Macrophage Activity And Epithelium Metabolism As Key Alterations During Chronic Inflammation, Olivier Mauduit, Vanessa Delcroix, Takeshi Umazume, Cintia S De Paiva, Darlene A Dartt, Helen P Makarenkova

Faculty, Staff and Students Publications

The lacrimal gland (LG) is an exocrine gland that produces the watery part of the tear film that lubricates the ocular surface. Chronic inflammation, such as Sjögren’s syndrome (SS), is one of the leading causes of aqueous-deficiency dry eye (ADDE) disease worldwide. In this study we analyzed the chronic inflammation in the LGs of the NOD.B10Sn-H2b/J (NOD.H-2b) mice, a mouse model of SS, utilizing bulk RNAseq and Visium spatial gene expression. With Seurat we performed unsupervised clustering and analyzed the spatial cell distribution and gene expression changes in all cell clusters within the LG sections. Moreover, for the …

Super-Resolution Microscopy Reveals Photoreceptor-Specific Subciliary Location And Function Of Ciliopathy-Associated Protein Cep290, Valencia L Potter, Abigail R Moye, Michael A Robichaux, Theodore G Wensel

Faculty, Staff and Students Publications

Mutations in the cilium-associated protein CEP290 cause retinal degeneration as part of multiorgan ciliopathies or as retina-specific diseases. The precise location and the functional roles of CEP290 within cilia and, specifically, the connecting cilia (CC) of photoreceptors, remain unclear. We used super-resolution fluorescence microscopy and electron microscopy to localize CEP290 in the CC and in the primary cilia of cultured cells with subdiffraction resolution and to determine effects of CEP290 deficiency in 3 mutant models. Radially, CEP290 localizes in close proximity to the microtubule doublets in the region between the doublets and the ciliary membrane. Longitudinally, it is distributed throughout …

Structure And Dynamics Of Photoreceptor Sensory Cilia, Theodore G Wensel, Valencia L Potter, Abigail Moye, Zhixian Zhang, Michael A Robichaux

Faculty, Staff and Students Publications

The rod and cone photoreceptor cells of the vertebrate retina have highly specialized structures that enable them to carry out their function of light detection over a broad range of illumination intensities with optimized spatial and temporal resolution. Most prominent are their unusually large sensory cilia, consisting of outer segments packed with photosensitive disc membranes, a connecting cilium with many features reminiscent of the primary cilium transition zone, and a pair of centrioles forming a basal body which serves as the platform upon which the ciliary axoneme is assembled. These structures form a highway through which an enormous flux of …

Single-Cell Transcriptomics Identifies Limbal Stem Cell Population And Cell Types Mapping Its Differentiation Trajectory In Limbal Basal Epithelium Of Human Cornea, De-Quan Li, Sangbae Kim, Jin-Miao Li, Qianmiao Gao, Jongsu Choi, Fang Bian, Jiaoyue Hu, Yun Zhang, Jin Li, Rong Lu, Yumei Li, Stephen C Pflugfelder, Hongyu Miao, Rui Chen

Faculty, Staff and Students Publications

PURPOSE: This study aimed to uncover novel cell types in heterogenous basal limbus of human cornea for identifying LSC at single cell resolution.

METHODS: Single cells of human limbal basal epithelium were isolated from young donor corneas. Single-cell RNA-Sequencing was performed using 10x Genomics platform, followed by clustering cell types through the graph-based visualization method UMAP and unbiased computational informatic analysis. Tissue RNA in situ hybridization with RNAscope, immunofluorescent staining and multiple functional assays were performed using human corneas and limbal epithelial culture models.

RESULTS: Single-cell transcriptomics of 16,360 limbal basal cells revealed 12 cell clusters belonging to three lineages. …

Addressing Artificial Intelligence Bias In Retinal Diagnostics, Philippe Burlina, Neil Joshi, William Paul, Katia D Pacheco, Neil M Bressler

Faculty, Staff and Students Publications

PURPOSE: This study evaluated generative methods to potentially mitigate artificial intelligence (AI) bias when diagnosing diabetic retinopathy (DR) resulting from training data imbalance or domain generalization, which occurs when deep learning systems (DLSs) face concepts at test/inference time they were not initially trained on.

METHODS: The public domain Kaggle EyePACS dataset (88,692 fundi and 44,346 individuals, originally diverse for ethnicity) was modified by adding clinician-annotated labels and constructing an artificial scenario of data imbalance and domain generalization by disallowing training (but not testing) exemplars for images of retinas with DR warranting referral (DR-referable) from darker-skin individuals, who presumably have greater …

Identification Of Deep-Intronic Splice Mutations In A Large Cohort Of Patients With Inherited Retinal Diseases, Xinye Qian, Jun Wang, Meng Wang, Austin D Igelman, Kaylie D Jones, Yumei Li, Keqing Wang, Kerry E Goetz, David G Birch, Paul Yang, Mark E Pennesi, Rui Chen

Faculty, Staff and Students Publications

High throughput sequencing technologies have revolutionized the identification of mutations responsible for a diverse set of Mendelian disorders, including inherited retinal disorders (IRDs). However, the causal mutations remain elusive for a significant proportion of patients. This may be partially due to pathogenic mutations located in non-coding regions, which are largely missed by capture sequencing targeting the coding regions. The advent of whole-genome sequencing (WGS) allows us to systematically detect non-coding variations. However, the interpretation of these variations remains a significant bottleneck. In this study, we investigated the contribution of deep-intronic splice variants to IRDs. WGS was performed for a cohort …

Loss Of Class Iii Phosphoinositide 3-Kinase Vps34 Results In Cone Degeneration, Ammaji Rajala, Feng He, Robert E Anderson, Theodore G Wensel, Raju V S Rajala

Faculty, Staff and Students Publications

The major pathway for the production of the low-abundance membrane lipid phosphatidylinositol 3-phosphate (PI(3)P) synthesis is catalyzed by class III phosphoinositide 3-kinase (PI3K) Vps34. The absence of Vps34 was previously found to disrupt autophagy and other membrane-trafficking pathways in some sensory neurons, but the roles of phosphatidylinositol 3-phosphate and Vps34 in cone photoreceptor cells have not previously been explored. We found that the deletion of Vps34 in neighboring rods in mouse retina did not disrupt cone function up to 8 weeks after birth, despite diminished rod function. Immunoblotting and lipid analysis of cones isolated from the cone-dominant retinas of the …

Mtor-Initiated Metabolic Switch And Degeneration In The Retinal Pigment Epithelium, Young-Mi Go, Jing Zhang, Jolyn Fernandes, Christopher Litwin, Rui Chen, Theodore G Wensel, Dean P Jones, Jiyang Cai, Yan Chen

Faculty, Staff and Students Publications

The retinal pigment epithelium (RPE) is a particularly vulnerable tissue to age-dependent degeneration. Over the life span, the RPE develops an expanded endo-lysosomal compartment to maintain the high efficiency of phagocytosis and degradation of photoreceptor outer segments (POS) necessary for photoreceptor survival. As the assembly and activation of the mechanistic target of rapamycin complex 1 (mTORC1) occur on the lysosome surface, increased lysosome mass with aging leads to higher mTORC1 activity. The functional consequences of hyperactive mTORC1 in the RPE are unclear. In the current study, we used integrated high-resolution metabolomic and genomic approaches to examine mice with RPE-specific deletion …