John H. Fingert, Ph.D. - US grants

[unreadable] DESCRIPTION (provided by applicant): The goal of this application is to develop Dr. John Fingert into an independent clinician-scientist. Drs. Edwin Stone and Wallace Alward will assume responsibility as mentors to ensure success in his development in the fields of genetic research and ophthalmology. The core of this proposal is intensive training in genetic approaches to studying inherited forms of glaucoma. Glaucoma associated with pigment dispersion syndrome (PDS) is of particular interest for research due to its unique clinical features. PDS is common (affecting up to 2.5% of Americans) and due to the early age of onset, its effects on individuals and society may be felt for decades more than many other eye diseases. The defining characteristic of PDS is the release of pigment from the iris that causes many patients to develop pigmentary glaucoma and vision loss. Although structural and anatomical factors clearly contribute to the development of PDS, epidemiological and animal studies have provided strong evidence for a significant genetic component to the pathophysiology of this condition. The genetic basis of PDS is unknown, however, the University of Iowa has unique resources to facilitate the discovery of PDS-causing genes including a world class ophthalmic genetics research laboratory and a large collection of clinically-characterized PDS patients and pedigrees. The principle hypothesis of this proposal is that PDS may be caused by defects in genes involved in iris pigment production. To test this hypothesis we will search for PDS disease genes with three approaches: First, we will use positional cloning to identify the disease-causing gene in a large PDS pedigree. Second, we will genotype large cohorts of PDS patients and controls with genetic markers in search of an association between alleles of these markers and PDS. Third, we will screen our cohorts of PDS patients and controls for disease-causing mutations in genes associated with pigment production. These diverse approaches will facilitate the discovery of PDS-causing genes and provide insight into the pathogenesis of this disease, with the ultimate goal of facilitating improvements in the diagnosis and treatment of this condition. "Glaucoma is a common, blinding condition with genetic risk factors. One form of disease (pigmentary glaucoma) is caused by release of iris pigment within the eye. We are studying genes involved in pigment production as a cause of this form of glaucoma." [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): Glaucoma is a leading cause of blindness and visual disability that has a major impact on the quality of life and productivity of millions of Americans. Glaucoma is defined by quantitative traits including, optic nerve cupping, visual field loss, and central corneal thickness. Intraocular pressure is another crucial quantitative trait that is monitored in glaucoma patients. The cascade of events that lead to glaucoma is not well known, which has hindered efforts for early detection and treatment of this condition. One way to investigate the pathogenesis of glaucoma is to identify disease-causing genes. Glaucoma is a heterogeneous disease and is likely caused by the interactions of many genes. This complexity has challenged efforts to identify genes that cause glaucoma. As a result, there is a critical need for research that focuses on glaucoma phenotypes with reduced complexity to facilitate the discovery of disease-causing genes. Our long-term goal is to identify and define pathways that lead from DNA variations in the genome to the vision loss of glaucoma. As stages of glaucoma pathogenesis are discovered, new interventions to prevent vision loss will become possible. Several quantitative features of the glaucoma phenotype (cup-to-disc ratio, corneal thickness, and intraocular pressure) are each independently associated with glaucoma and highly heritable. We hypothesize that the same genes that determine the magnitudes of these features of glaucoma will also be important in the pathogenesis of glaucoma overall. Furthermore, a single feature of glaucoma has less complexity than the whole disease. This reduced complexity will facilitate discovery of genetic factors for components of the glaucoma phenotype. Genes that control quantitative traits of glaucoma will be identified with studies of human cohorts and inbred mice. The Ocular Hypertension Treatment Study (OHTS) is a large treatment trial designed to show the efficacy of treating patients with high intraocular pressure. We will identify genetic factors that control the magnitude of cup-to-disc ratio, intraocular pressure, and corneal thickness by conducting a genome-wide association study of the patients in this trial (Specific Aim 1). Quantitative traits are often determined by the actions of many genetic factors. Studies of inbred mice with fixed genetic backgrounds have the advantage of reducing the complexity of these traits. By crossing inbred strains, specific risk alleles that determine a quantitative trait may be more easily recognized against the uniform genetic background. Therefore, we will study the genetic basis of an important quantitative feature of glaucoma (central corneal thickness) with crosses of inbred mice (Specific Aim 2). We expect that our proposed studies will identify new risk alleles for quantitative features of glaucoma. The discovery of such risk alleles will have an important impact on the future clinical management of glaucoma by facilitating early diagnosis and enabling the development of new sight- saving treatments. PUBLIC HEALTH RELEVANCE: Although glaucoma is a heritable cause of blindness that affects millions of Americans, little is known about the genetic risk factors that contribute to this condition. We propose experiments that will identify genes that control component features of the overall glaucoma phenotype. These discoveries will provide insight into the biological pathways that lead to disease and will also facilitate improved diagnosis, prognosis, and treatments for those with this debilitating condition.

DESCRIPTION (provided by applicant): Optic nerve diseases are a major public health problem. The defining feature of several optic nerve diseases, including glaucoma and cavitary optic disc anomaly (CODA), is excavation or cupping of the head of the optic nerve. The biological pathways that lead to excavation of the optic disc in these diseases, however, are incompletely understood. Consequently, there is a critical need to clarify the biological mechanisms that lead to loss of tissue (excavation) of the optic nerve to improve our understanding of disease processes and to develop new treatments for optic nerve diseases including CODA and glaucoma. We recently discovered that mutation of the matrix metallopeptidase 19 (MMP19) gene is associated with CODA. Specifically, we discovered a 6 kb DNA sequence upstream of the MMP19 gene that is triplicated in patients with CODA. Moreover, we have proven that this sequence is an active enhancer and upregulates expression of MMP19. We have also shown that MMP19 is specifically expressed in the optic nerve head, where the abnormalities of CODA occur. Moreover, gene expression data shows that MMP19 expression is greatly increased in the optic nerves of both human patients and animals with glaucoma. Together these findings support our hypothesis that abnormal regulation of MMP19 promotes congenital excavation of the optic disc in CODA. We will test this hypothesis with the following research aims: Aim 1: Test glaucoma patients for MMP19 mutations. We will test a cohort of glaucoma patients for copy number variations, regulatory sequence mutations, and coding sequence mutations in MMP19 with next generation sequencing. Aim 2: Identify the specific enhancer element and transcription factors that regulate MMP19 expression. We will 1) identify the specific DNA sequence of the MMP19 enhancer element via mutagenesis studies and 2) isolate transcription factor(s) that bind to the MMP19 enhancer using a DNA pull down assay and mass spectrometry. This proposal will clarify mechanisms of disease in CODA and provide insights in the process of optic nerve cupping in glaucoma. These investigations may provide the foundation for future studies to develop new sight-saving therapies.

DESCRIPTION (provided by applicant): Glaucoma is a common disease of the optic nerve that affects over 60 million people worldwide and is a leading cause of blindness and visual disability in the United States. However, the biological pathways that lead to glaucoma are not well understood, and this has hindered efforts for early detection and treatment of this condition. Consequently, there is great need to clarify the causes of glaucoma at the molecular level. We identified a new glaucoma gene, TANK binding kinase 1 (TBK1) and discovered that duplication of the TBK1 gene is associated glaucoma. TBK1 has been studied extensively in non-ocular tissues and has well- defined roles in the innate immune system. Activated TBK1 stimulates assembly of a phagosome and engulfment / elimination of bacteria, proteins, and organelles (a process known as autophagy). Three autophagy genes (TBK1, OPTN, and TLR4) encode interacting proteins and have also been described as NTG genes. Moreover, autophagy has been implicated in the retinal ganglion cell death in experimental animal models of glaucoma. Our discovery that TBK1 is an NTG gene provides additional evidence that autophagy has an important role in the pathogenesis of NTG. The convergence of data from human genetic studies of NTG and from experimental glaucoma model systems provides strong evidence that autophagy may be a central process in the pathogenesis of retinal ganglion cell death in glaucoma. Our central hypothesis is that TBK1 influences autophagy at the key site of pathology in glaucoma, the retinal ganglion cells that form the optic nerve. Dysregulation of this pathway (e.g. by duplication of TBK1) may start a cascade of events that leads to apoptosis of the retinal ganglion cells, vision loss, and glaucoma. We propose to test our hypothesis with three specific aims that use our unique and powerful collection of glaucoma patient cohorts, human donor eyes, and transgenic TBK1 mice that have the same genetic defect as human patients. We will identify new genes that cause glaucoma by testing large cohorts of glaucoma patients for mutations in autophagy genes using next generation DNA sequencing strategies. We will determine the effect of TBK1 mutation (gene duplication) on the development of glaucoma and activation of autophagy in the retina of transgenic TBK1 mice (which we have made and are ready to study). We will also test drugs that stimulate or block autophagy for their ability to prevent glaucoma in these mice. We will investigate the pathway(s) by which TBK1 defects lead to glaucoma by identifying interacting proteins in the retina. With these experiments, we will begin to characterize the biological pathway by which defects in the TBK1 gene lead to glaucoma, validate an animal model of glaucoma, and begin to translate our discoveries into new approaches to diagnosis and treatment of disease.

? DESCRIPTION (provided by applicant): Glaucoma is a common disease of the optic nerve that affects over 60 million people worldwide and is a leading cause of blindness and visual disability in the United States. However, the biological pathways that lead to glaucoma are not well understood, and this has hindered efforts for early detection and treatment of this condition. Consequently, there is great need to clarify the causes of glaucoma at the molecular level. We discovered that duplication of the TANK binding kinase 1 (TBK1) gene is associated with glaucoma that occurs without elevation of eye pressure - termed normal tension glaucoma (NTG). TBK1 function hasn't been studied in the eye although TBK1 has been shown to have a role in autophagy in non-ocular tissue. Autophagy is a cellular process that delivers cytosolic proteins, organelles, and even intracellular pathogens to lysosomes for clearance. Remarkably, three known NTG genes (TBK1, OPTN, TLR4) all interact with each other and have important roles in activating autophagy, which suggests that autophagy may be an important common pathway in NTG pathogenesis. Moreover, autophagy has also been implicated in the retinal ganglion cell death and optic nerve damage in animal models of glaucoma. Based on these data, we hypothesize that TBK1 gene duplication alters autophagy and leads to the retinal ganglion cell death and vision loss of NTG. We have created a unique set of resources to study TBK1, autophagy, and glaucoma: cultured retinal ganglion cells (using induced stem cell technology) that have an extra dose of the TBK1 gene - the same defect as our patients with TBK1-associated glaucoma. Preliminary studies of these cells have provided more evidence that duplication of TBK1 gene activates autophagy and causes glaucoma. To test our hypothesis and investigate the role of autophagy in glaucoma we propose this two-part aim: DETERMINE THE ROLE OF TBK1 GENE DUPLICATION AND AUTOPHAGY IN THE PATHOGENESIS OF NTG USING IPSC-DERIVED RGC-LIKE NEURONS ? Aim 1A: Test cells cultured from patients with NTG due to a TBK1 gene duplication (skin fibroblasts, iPSCs and iPSC-derived RGC-like neurons) for altered autophagy molecules with microscopic, biochemical, and autophagic flux assays. ? Aim 1B: Determine the effect of TBK1 gene duplication on the phosphorylation of its substrate OPTN at key residues necessary for its function in autophagy with immunoprecipitation and western blot analysis with anti-phospho-OPTN antibodies. With these experiments, we will begin to characterize the biological pathway by which defects in the TBK1 gene alter autophagy and lead to glaucoma. These studies will also provide the basis for development of novel glaucoma therapies that are targeted to autophagy and glaucoma that occurs without elevation of eye pressure.

ABSTRACT Glaucoma is the leading cause of irreversible blindness worldwide. Pigmentary glaucoma is caused by a release of iris pigment known as pigment dispersion syndrome. Pigment dispersion syndrome is a common disorder that affects about 1 in 40 (2.5%) Americans. As many as 10 to 20% of those with pigment dispersion syndrome develop high intraocular pressure (ocular hypertension) and a secondary glaucoma - pigmentary glaucoma. Young to middle-aged people are affected by pigmentary glaucoma and consequently the visual disability it causes may lead to decades more loss of function and productivity than late-onset forms of glaucoma. Thus, research to determine the cause of pigmentary glaucoma is an important public concern. Strong evidence indicates that pigmentary glaucoma has a genetic basis. DBA/2J mice are an inbred strain of mice that develop pigmentary glaucoma that is a clearly inherited trait. Large human pedigrees have also been reported, in which pigmentary glaucoma is dominantly inherited. Cases of pigmentary glaucoma are also frequently clustered within families and ethnic groups. These data provide strong evidence for the existence of genes that cause or predispose pigmentary glaucoma. However, no genes that cause pigmentary glaucoma have been identified to date. The largely unknown genetic basis of pigmentary glaucoma has been an obstacle to discovery of the mechanisms that cause this disease and has also hindered development of new more effective therapies for this potentially blinding disease. As a result, there is critical need to identify and study the genes that cause pigmentary glaucoma. Aberrant release of iris pigment is a defining feature of pigmentary glaucoma. Moreover, mutations in genes involved in melanin synthesis and melanosomes (Gpnmb and Typr1) are known to cause pigmentary glaucoma in DBA/2J mice. Based on these observations, we hypothesize that some cases of pigmentary glaucoma are caused by mutations in genes involved in melanin synthesis. We will evaluate our hypothesis by studying whole exome data collected from 5 pigmentary glaucoma pedigrees and a cohort of pigmentary glaucoma patients (n=214) and matched controls (n=354) with the aims described below: AIM 1 PEDIGREE-BASED exome analysis of several large pigmentary glaucoma pedigrees. AIM 2 POPULATION-BASED exome analysis of a cohort of pigmentary glaucoma patients (n=214) and matched controls (n=254) We will validate our human studies with analyses of pigmentary glaucoma in mice using 1) publicly available strains of mice from The Jackson Laboratory and 2) our inducible pigmentary glaucoma mouse model. Identifying the first human pigmentary glaucoma genes will provide new insights into the pathogenesis of this important disease and may advance development of sight-saving therapies.

Exfoliation syndrome is common and may affect 5-10% of Americans and up to 50% in some populations (i.e. Icelanders). As many as 10 to 50% of those with exfoliation syndrome develop high intraocular pressure and a secondary glaucoma - exfoliative glaucoma, which causes blindness and visual disability in millions worldwide. Exfoliative syndrome has a strong genetic basis, but complex inheritance. Genome-wide association studies (GWAS) of exfoliation syndrome have successfully identified 7 genetic factors that increase risk for disease: LOXL1, CACNA1A, POMP, TMEM136, AGPAT1, RBMS3, and SEMA6A. However, an animal model has not yet been built based upon these gene discoveries. There is a critical need for these human GWAS discoveries to be translated into animal models. We previously reported mice, B6-Lystbg-j, with a mutation in the Lyst gene, that have some features of exfoliation syndrome, but produce only tiny amounts of exfoliation material and do not develop glaucoma. We hypothesize that inactivating the 7 exfoliation syndrome risk factor genes using CRISPR/Cas9 methods will generate animal models of exfoliation syndrome and glaucoma. We further hypothesize that introducing these mutations on the B6-Lystbg-j background strain will take advantage of its subtle exfoliation syndrome features and facilitate developing a new line with pronounced features, high pressure and glaucoma. We have created CRISPR reagents to inactivate the seven exfoliation syndrome risk factors and we have begun injecting mouse embryos. We are injecting pools of CRISPR reagents for our target genes in order to get founder mice with one or more inactivated risk factor genes. All mutations are made in B6-Lystbg-j mice. AIM 1A: ASSESS FOUNDER MICE. We will assess the eyes of founder mice (in vivo) that have one or more inactivated exfoliation syndrome risk factor genes for exfoliation material. Those with exfoliative material in their eyes will be aged to 1 year, assessed for high intraocular pressure, and glaucomatous optic neuropathy. AIM 1B: GENERATE MICE WITH MULTIPLE INACTIVATED RISK FACTORS. We will conduct crosses to produce mice with multiple inactivated exfoliation syndrome risk factor genes on a B6-Lystbg-j background: 1) To produce mice with 2 heterozygously inactivated risk factor genes (with extreme individual phenotypes). 2) To produce mice with 2 risk factors (Loxl1, Cacn1a1) heterozygously inactivated (top GWAS hits). AIM 2A DETERMINE EXPRESSION PATTERNS OF RISK FACTOR GENES IN B6-Lystbg-j MICE. We will assess expression of the 7 risk factor genes at the mRNA and protein level in ocular tissues of B6-Lystbg-j mice relevant to exfoliation syndrome pathophysiology (lens, iris, and iridocorneal angle). AIM 2B DETERMINE EXPRESSION PATTERNS OF RISK FACTOR GENES IN OUR CRISPR/Cas9 MICE. INNOVATION. Pooling CRISPR guide RNAs to make mice with inactivated risk factor genes (alone and in combination). Creating the 1st animal model of exfoliation syndrome from GWAS hits as a step towards a cure.

Abstract Glaucoma is a group of age-related neurodegenerative diseases characterized by loss of retinal ganglion cells and their axons. Because RGCs are post-mitotic neurons, their loss is permanent and causes a gradual decline leading toward irreversible blindness. Unfortunately, glaucoma manifests in a way that damages US society more broadly?it disproportionately affects certain racial groups. Glaucoma is a worse problem amongst African Americans than Caucasians. Though socioeconomic factors often contribute to this disparity, a large part is biological and driven by genomic differences between racial groups. A recent breakthrough points to APBB2 as central to glaucoma in African Americans. A large genome-wide association study (GWAS) has found a single nucleotide polymorphism in APBB2 (rs59892895) with genome-wide significance (P=2x10-8; Odds ratio=1.33), and successfully replicated the finding in additional cohorts. identified a variant in the APBB2 gene as an important cause of glaucoma in people of African descent. ABPP2 encodes a cytoplasmic adaptor protein with multiple protein-protein interaction domains. Remarkably, the high-risk allele is present in approximately 21% of African Americans and apparently absent from Caucasians. Our team has contributed to this discovery of APBB2 as a gene of importance to glaucoma in African Americans, and as additionally shown in our Preliminary Data, have developed a hypothesis that overexpression of APBB2 is the disease-causing mechanism. To test this new hypothesis, we recently generated and now have in hand, a mouse model on a pure C57BL/6J genetic background that is overexpressing Apbb2. The strain was created by the University of Iowa Genome Editing Facility and features a full-length mouse Apbb2 (transcript variant 1) under control of a ubiquitous promotor (CAG) and includes a 6His/3XFlag attached to the N-terminus (abbreviated as B6- Tg(Apbb2). The experiments of this proposal utilize this new strain as a central resource to complete the important, but somewhat high risk, test via manipulation that over-expression of Apbb2 promotes glaucoma. In Specific Aim 1, we propose to test the anatomical and physiological consequences over time of Apbb2 overexpression in mice. This Aim will also complete a thorough characterization of the B6-Tg(Apbb2) strain. Specific Aim 2 will study APBB2 from a molecular perspective, identifying retinal APBB2 binding partners that will guide future mechanistic and candidate-driven genetic experiments.