University of Iowa

Iowa Research Online Theses and Dissertations

Spring 2015

Molecular genetics of optic nerve disease using patients with cavitary optic disc anomaly Ralph Jeremiah Hazlewood II University of Iowa

Copyright 2015 Ralph Jeremiah Hazlewood II This dissertation is available at Iowa Research Online: http://ir.uiowa.edu/etd/1622 Recommended Citation Hazlewood II, Ralph Jeremiah. "Molecular genetics of optic nerve disease using patients with cavitary optic disc anomaly." PhD (Doctor of Philosophy) thesis, University of Iowa, 2015. http://ir.uiowa.edu/etd/1622.

Follow this and additional works at: http://ir.uiowa.edu/etd Part of the Genetics Commons

MOLECULAR GENETICS OF OPTIC NERVE DISEASE USING PATIENTS WITH CAVITARY OPTIC DISC ANOMALY

by Ralph Jeremiah Hazlewood II

A thesis submitted in partial fulfillment of the requirements for the Doctor of Philosophy degree in Genetics in the Graduate College of The University of Iowa May 2015 Thesis Supervisor: Associate Professor: John H. Fingert

Copyright by RALPH JEREMIAH HAZLEWOOD II 2015 All Rights Reserved

Graduate College The University of Iowa Iowa City, Iowa

CERTIFICATE OF APPROVAL ____________________________ PH.D. THESIS _________________ This is to certify that the Ph.D. thesis of Ralph Jeremiah Hazlewood II has been approved by the Examining Committee for the thesis requirement for the Doctor of Philosophy degree in Genetics at the May 2015 graduation. Thesis Committee:

____________________________________________ John H. Fingert, Thesis Supervisor ____________________________________________ Michael G. Anderson ____________________________________________ Robert F. Mullins ____________________________________________ Maurine Neiman ____________________________________________ Andrew F. Russo

To my mother Queen Marion, thank you for always being there; may you continue to rest in peace and to my son Ralph J. Hazlewood III

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Without struggle, there is no progress. Frederick Douglas “West India Emancipation”

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ACKNOWLEDGEMENTS The journey to this point has been an exceptional one that would not have been possible without the guidance and support of family, friends, and colleagues whom in one way or another have contributed to the completion of my thesis. First and foremost, I would like to thank my parents Ralph Hazlewood I and Marion Hazlewood. Mom, your sacrifices and unwavering perseverance has motivated and allowed me to pursue my dreams. Although your time on this Earth was cut short before you could see me achieve my goals, I know you are there in spirit and I am eternally grateful for you. I love you! I would like to thank my mentor and advisor Dr. John Fingert. You have been the best mentor during my tenure at Iowa and have been extremely supportive of my development as a graduate student and independent scientist and have been one of my biggest allies. I admire your work ethic and enthusiasm with which you approach your research work and deeply appreciate you for being approachable and inspiring creativity. I am extremely thankful for your guidance, great words of encouragement, and warmth particularly when I experienced a loss. For that, I will forever be grateful. I also want to thank Benjamin Roos, Frances Solivan-Timpe, Kathy Miller and current and previous research staff in the Glaucoma Genetics Lab, who have contributed in various ways toward my thesis. Many thanks to Benjamin for being there to listen whenever I needed to bounce ideas off of someone, helping with troubleshooting experiments, providing technical insight, and teaching me the

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techniques used in my thesis research. You have all welcomed me in the lab making it a very supportive environment and it was a pleasure working with you. Thank you to my thesis dissertation committee Dr. Robert Mullins, Dr. Michael Anderson, Dr. Maurine Neiman, Dr. Andrew Russo, and ad hoc committee member Dr. Markus Kuehn for your time, valuable insights, and vested involvement in my research projects as well as your helpful discussions on research directions. I would also like to thank research collaborators intramural and extramural who have provided research support/reagents, examined patients, and other resources for my thesis research. Finally, to my friends and family, I would like to thank you for supporting me throughout graduate school. I want to also recognize my fiancée Tiffany Fagan for supporting me throughout the process, being there to listen while I practice presentations and giving me encouragement when times were rough as well as helping with microscopy. Many thanks to all.

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ABSTRACT Glaucoma is the second leading cause of irreversible blindness in the United States and is the leading cause of blindness in African Americans. Cupping or excavation of the optic nerve, which sends the visual signal from the photoreceptors in the eye to the brain, is a chief feature of glaucoma. A similar excavated appearance of the optic nerve is also the primary clinical sign of other congenital malformations of the eye including optic nerve head coloboma, optic pit, and morning glory disc anomaly collectively termed cavitary optic disc anomaly (CODA). Clinical similarities between CODA and glaucoma have suggested that these conditions may have overlapping pathophysiology. Although risk factors are known, such as the elevated intraocular pressure (IOP) observed in some glaucoma subjects, the biological pathways and molecular events that lead to excavation of the optic disc in glaucoma and in CODA are incompletely understood, which has hindered efforts to improve diagnosis and treatment of these diseases. Consequently, there is a critical need to clarify the biological mechanisms that lead to excavation of the optic nerve, which will lead to improvements in our understanding of these important disease processes. Because of their similar clinical phenotypes and the limited therapy geared at lowering IOP in glaucoma patients, our central hypothesis is that genes involved in Mendelian forms of CODA would also be involved in a subset of glaucoma cases and may provide insight into glaucomatous optic neuropathy. The purpose of my research project has been to identify and functionally characterize the gene that causes congenital autosomal dominant CODA in a multiplex family with 17 affected members. The gene that causes CODA was previously mapped to chromosome 12q14 and following screening of candidate genes within the region that did not yield any plausible coding sequence mutations, a triplication of a 6 Kbp segment of DNA upstream of the matrix

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metalloproteinase 19 (MMP19) gene was subsequently identified using comparative genomic hybridization arrays and qPCR. This copy number variation (CNV) was present in all affected family members but absent in unaffected family members, a panel of 78 normal control subjects, and the Database of Genomic Variants. In a case-control study of singleton CODA subjects, CNVs were also detected; we detected the same 6 Kbp triplication in 1 of 24 subjects screened. This subject was part of another 3-generation autosomal dominant CODA pedigree where affected members each have the same CNV identified in the larger CODA pedigree. A separate case-control study with 172 glaucoma cases (primary open angle glaucoma = 84, normal tension glaucoma = 88) was evaluated for MMP19 CNVs, however none were detected. Although our cohort of CODA patients is small limiting our ability to accurately determine the proportion of CODA caused by MMP19 mutations, our data indicates that the MMP19 CNV is not an isolated case and additional CODA subjects may have MMP19 defects. Because of the location of the CNV, we evaluated its effect on downstream gene expression with luciferase reporter gene assays. These assays revealed that the 6 Kbp sequence spanned by the CNV in CODA subjects functioned as a transcriptional enhancer; in particular, a 773bp segment had a strong positive influence (8-fold higher) on downstream gene expression. MMP19, a largely understudied gene, was further characterized by expression studies in the optic nerve and retina. Using frozen sections from normal donor eyes, we demonstrated that MMP19 is predominantly localized to the optic nerve head in the lamina cribrosa region with moderate labeling in the postlaminar region, and weak labeling in the prelaminar region and retina. We also evaluated MMP19 expression in relation to the cell types that populate the optic nerve such as astrocytes and retinal ganglion cells. The pattern of expression is consistent with MMP19 being a secreted protein accumulating in the extracellular spaces and basement membranes of the optic nerve. Our studies

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have identified the first gene associated with CODA and future research is focused on recapitulating CODA phenotypes in animal models and assessing the mechanism of MMP19 involvement during development.

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PUBLIC ABSTRACT Diseases of the optic nerve, which conveys the visual signal from the retina to the brain, are recognized by degeneration (loss of tissue) of the nerves. Glaucoma, the most common form of optic nerve disease, is a leading cause of blindness affecting up to 80 million people worldwide. Another optic nerve disease called cavitary optic disc anomaly (CODA) is very similar to glaucoma in clinical appearance of optic nerves upon eye examination. Although risk factors such as pressure buildup in the eye are known, the events that lead to degeneration are poorly understood, hindering development of treatments. For this reason, we studied the genes of CODA patients in hopes of gaining insight into more common blinding disorders such as glaucoma. Prior search for changes in DNA sequences inherited with CODA disease revealed a micro-repeated region called a copy number variation (CNV) on chromosome 12 near the matrix metalloproteinase 19 (MMP19) gene. My thesis work shows that this CNV regulates gene expression. I show that it functions as a strong enhancer of gene expression and that MMP19 is located in human optic nerves where the abnormalities of CODA and glaucoma occur. I further identified similar CNVs near MMP19 in other unrelated CODA patients. Overall, this research shows that CNVs near MMP19 found in CODA subjects regulate gene expression e.g. MMP19, may cause a significant fraction of CODA cases, and provides a new target for future therapies. Further studies are needed to understand the impact of MMP19 activity in the optic nerve.

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TABLE OF CONTENTS LIST OF TABLES ............................................................................................................ xii LIST OF FIGURES ......................................................................................................... xiii LIST OF ABBREVIATIONS .......................................................................................... xiv PREFACE ...................................................................................................................... xvi CHAPTER I INTRODUCTION ..........................................................................................1 Optic nerve and the visual system ....................................................................1 Etiology and epidemiology of Glaucoma .........................................................1 Glaucoma classifications ..................................................................................3 Causes and risk factors of glaucoma ................................................................3 Rare optic nerve diseases, congenital optic disc anomaly ................................5 Molecular genetic approaches to optic nerve diseases .....................................8 Genetic investigations using linkage analysis ...........................................8 Genome wide association studies ............................................................13 Genetic evidence of CODA ............................................................................14 Significance and Focus of Dissertation Studies..............................................15 CHAPTER II HETEROZYGOUS TRIPLICATION OF UPSTREAM REGULATORY SEQUENCES LEADS TO DYSREGULATION OF MATRIX METALLOPROTEINASE 19 (MMP19) IN PATIENTS WITH CAVITARY OPTIC DISC ANOMALY (CODA) ..............................................................20 Introduction.....................................................................................................20 Materials and methods ....................................................................................22 Study sample subjects and controls .........................................................22 Candidate gene screening and exome sequencing...................................22 CNV analysis, array CGH, and quantitative PCR ...................................23 Cloning and Promoter constructs ............................................................24 Cell culture and transient transfection with Luciferase assays ................27 Immunohistochemistry ............................................................................27 Results.............................................................................................................28 Candidate gene screening ........................................................................28 Copy number variation (CNV) analysis ..................................................30 Testing glaucoma patients for the chromosome 12q CNV .....................33 Transcription activity of the chromosome 12q14 CNV ..........................34 Immunohistochemistry ............................................................................37 Discussion .......................................................................................................38 Model of MMP19 and CODA .................................................................40 MMP19 and retinal disease of CODA.....................................................43 CHAPTER III EVALUATION OF A NEW CODA PEDIGREE ....................................52 Introduction.....................................................................................................52 Methods ..........................................................................................................54 Human subjects and Clinical studies .......................................................54 Copy Number Analysis ...........................................................................55 Results.............................................................................................................56 x

Discussion .......................................................................................................59 CHAPTER IV PATTERN OF MMP19 EXPRESSION IN THE OPTIC NERVE...........62 Introduction.....................................................................................................62 Extracellular Matrix.................................................................................63 Matrix metalloproteinases .......................................................................64 MMP19 expression in other tissues .........................................................65 MMP19 expression in the optic nerve .....................................................66 Methods ..........................................................................................................67 Ocular tissue preparations .......................................................................67 Immunolabeling and immunohistochemistry ..........................................67 Results.............................................................................................................68 Astrocytes ................................................................................................68 Retinal ganglion cell axons .....................................................................72 Microglia .................................................................................................74 Extracellular matrix components .............................................................76 Discussion .......................................................................................................80 CHAPTER V CONCLUSIONS AND FUTURE DIRECTIONS .....................................82 Conclusions.....................................................................................................82 CODA mutation identified ......................................................................82 Reporter gene assays indicate MMP19 CNVs alter gene expression ......84 MMP19 is the CODA gene .....................................................................87 Future Studies .................................................................................................89 REFERENCES ..................................................................................................................93

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LIST OF TABLES Table 1. Loci associated with glaucoma ............................................................................12 Table 2. TaqMan Copy Number assays and sequencing primers ......................................45 Table 3. List of genes within the chromosome 12q locus evaluated for mutations in CODA subjects......................................................................................................................48 Table 4. Genotype Analysis ...............................................................................................49 Table 5. Variations detected in unrelated CODA subjects within the 6Kbp region upstream of MMP19 .................................................................................................................50 Table 6. Haplotyping SNP data .........................................................................................58

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LIST OF FIGURES Figure 1. Schematic diagram of glaucoma. .........................................................................3 Figure 2. Fundus images of patients with cavitary optic disc anomaly (CODA). ...............6 Figure 3. Similarities between the optic nerves of patients with cavitary optic disc anomaly (CODA) and glaucoma ................................................................................8 Figure 4. CODA pedigrees. ...............................................................................................18 Figure 5. Chromosome 12 triplication upstream of MMP19 gene ....................................25 Figure 6. Quantitative PCR assay for chromosome 12q14 CNV. .....................................31 Figure 7. Triplicated MMP19 promoter sequence has strong enhancer activity. ..............36 Figure 8. MMP19 is localized in the human optic nerve head ..........................................37 Figure 9. Model of MMP19 expression and optic disc excavation ..................................42 Figure 10. Plasmid vector used for Luciferase reporter assays .........................................51 Figure 11. Pedigree 081-E .................................................................................................55 Figure 12. TaqMan Copy number qPCR plot ....................................................................57 Figure 13. MMP19 in cross sections of the optic nerve ....................................................69 Figure 14. GFAP and MMP19 ...........................................................................................70 Figure 15. Astrocyte morphology in the optic nerve. ........................................................71 Figure 16. βIII Tubulin and MMP19 .................................................................................73 Figure 17. CD45 and MMP19 ...........................................................................................75 Figure 18. COL IV and MMP19 ........................................................................................77 Figure 19. Elastin and MMP19 ..........................................................................................79 Figure 20. Fibrillin-1 and MMP19.....................................................................................80

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LIST OF ABBREVIATIONS CODA

Cavitary optic disc anomaly

RGC

Retinal ganglion cells

CNS

Central nervous system

POAG

Primary open angle glaucoma

IOP

Intraocular pressure

NTG

Normal tension glaucoma

JOAG

Juvenile open angle glaucoma

RPE

Retinal pigment epithelium

SNP

Single nucleotide polymorphism

GWAS

Genome-wide association studies

iPSC

Induced pluripotent stem cells

MMP19

Matrix metalloproteinase 19

CNV

Copy number variation

qPCR

Quantitative polymerase chain reaction

SSCP

Single stranded conformation polymorphism

HRM

High resolution melt

CGH

Comparative genomic hybridization

GFAP

Glial fibrillary acidic protein

COL IV

Collagen type IV

FBN1

Fibrillin-1

TIMP

Tissue inhibitors of matrix metalloproteinase

ONH

Optic nerve head xiv

ECM

Extracellular matrix

NFH

Neurofilament heavy chain

CRA

Central retinal artery

OCT

Optical coherence tomography

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PREFACE This dissertation consists of both published and unpublished data describing the identification and characterization of genetic factors leading to cavitary optic disc anomaly. A version of Chapter 2 material has been published as Hazlewood, R.J., Roos, B.R., SolivanTimpe, F., Honkanen, R.A., Jampol, L.M., Gieser, S.C., Meyer, K.J., Mullins, R.F., Kuehn, M.H., Scheetz, T.E., Kwon, Y.H., Alward, W.L., Stone, E.M. and Fingert, J.H. (2015) Heterozygous Triplication of Upstream Regulatory Sequences Leads to Dysregulation of Matrix Metalloproteinase 19 (MMP19) in Patients with Cavitary Optic Disc Anomaly (CODA). Human mutation, 36:369-378. Human donor eyes used in immunohistochemical experiments in this thesis were obtained by generous gifts by Dr. Markus Kuehn and Dr. Robert Mullins of the Wynn Institute for Vision Research (WIVR) at the University of Iowa. CONTRIBUTIONS CHAPTER 1: Ralph Hazlewood wrote all content in chapter 1, except for illustrations taken from publically available content on the internet and were referenced as such. CHAPTER 2: Ralph performed the cell culture, functional Luciferase assays, histology and immunohistochemistry of human optic nerves. Ralph and Benjamin Roos performed cloning, TaqMan copy number assays, and DNA sequencing. Kaci Meyer performed initial CGH array and Todd Scheetz performed the exome analysis. Ralph Hazlewood and John Fingert wrote the paper with co-authors Lee Jampol, Wallace Alward, and Robert Honkanen contributing to editing. CHAPTER 3: Ralph Hazlewood performed all experiments and wrote all content in chapter 3. CHAPTER 4: Ralph Hazlewood performed all experiments and wrote chapter 4. Ralph Hazlewood, Rob Mullins, and John Fingert analyzed the data. CHAPTER 5: Ralph Hazlewood wrote chapter 5.

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CHAPTER I INTRODUCTION Optic nerve and the visual system The optic nerve relays the visual signals received from the photoreceptors in the retina to the brain via the retinal ganglion cell (RGC) axons. These nerve fibers coming from the retina turn at the front of the optic nerve and continue posteriorly through the back of the nerve. Made up of about 1 million nerve fibers, the optic nerve leaves the globe at the back of the eye and travels to the lateral geniculate nucleus of the thalamus where information is relayed to the visual cortex. The RGC axons coming from the retina converge at the posterior pole of the eye at the optic nerve head (ONH) or optic disc. The ONH serves as the beginning of the optic nerve and has a small depression at its center called the optic cup. In addition to retinal ganglion cells axons, the primary cell types that populate the optic nerve are astrocytes, which are neuroepithelial derived from the optic stalk; microglia which are hemapoetic derived; and oligodendrocytes, which are precursor cells migrating into the nerve(Burne, Staple et al. 1996). Astrocytes are the most abundant cell type in the ONH and have been extensively studied having roles in regulating blood flow, maintaining extracellular homeostasis, controlling synaptic plasticity, and providing structural support (Ullian, Sapperstein et al. 2001, Nedergaard, Ransom et al. 2003, Takano, Tian et al. 2006, Sun, Lye-Barthel et al. 2009). Because of its developmental origin, the eye is considered to be part of the central nervous system (CNS) and is a major substrate for scientific discovery.

Etiology and epidemiology of Glaucoma Optic nerve atrophy (damage to the optic nerve) may result from a wide range of pathologies, which can include trauma, age-related effects, inherited optic neuropathy, or 1

congenital disease (Riordan-Eva 2004). Cupping or excavation of the optic nerve results in the loss of RGC axons and neural tissue and is the chief feature of optic neuropathies, most commonly glaucoma. Glaucoma is a complex neurodegenerative disorder and is one of the leading causes of irreversible blindness worldwide. Recent estimates suggest that glaucoma may affect as little as 5.2 million(Roodhooft 2002) to as much as 80 million people worldwide revealing the enormous impact of glaucoma on human health(Quigley and Broman 2006). Glaucoma more commonly affects people of African or Asian descent and is the leading cause of blindness in these populations in the United States. The glaucomas are an acquired optic neuropathy that along with progressive optic nerve damage and deterioration of the neuroretinal rim of the ONH, leads to RGC axon death resulting in an insidious loss of vision. Colloquially called the “silent thief of sight”, glaucoma’s effect on the optic nerve typically leads to loss of peripheral vision that may also progress to involve central vision (Figure 1).

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Figure 1. Schematic diagram of glaucoma. (A) Damage to the optic nerve is a chief feature of glaucoma leading to gradual loss of peripheral vision and subsequent reduction of central field. (B) Optic nerve damage is frequently associated with increases in intraocular pressure (IOP). (A.D.A.M. Medical images online and National Eye Institute)

Glaucoma classifications The glaucomas have been grouped using several strategies: primary vs. secondary and open-angle vs. closed-angle. Secondary glaucoma is disease that is associated with other ocular disorders or known events such as trauma or developmental abnormalities and systemic diseases. Primary glaucoma, on the other hand, occurs in their absence and is an isolated disease of the optic nerve. Changes to the junction between the iris and the cornea (iridiocorneal angle) dictate open-angle versus closed-angle; closed angle glaucoma occurs when there is a blockage of this angle. The most common form of glaucoma is primary open-angle glaucoma (POAG), which presents in approximately 70% of all glaucoma cases in the United States(Quigley and Broman 2006).

Causes and risk factors of glaucoma Elevated intraocular pressure (IOP) in the eye is an important risk factor for development of primary open-angle glaucoma (Figure 1). The ciliary body in the eye produces aqueous humor fluid. This fluid flows from the posterior chamber to the anterior chamber emptying out of the 3

eye through the trabecular meshwork. Anything that obstructs this drainage or outflow of aqueous humor leads to an increase in IOP and thus can lead to glaucoma(Schwartz 1978). Alternatively, glaucoma can occur at any IOP. Normal tension glaucoma (NTG) presents with progressive optic nerve degeneration leading to retinal ganglion cell death, but this degeneration occurs at IOPs at or below 21mmHg. This illustrates that elevated IOP increases risk for glaucoma but is typically not sufficient to cause disease on its own. Other major risk factors for glaucoma include age, race, family history and quantitative traits such as central corneal thickness (Armaly 1965, Armaly 1966, Sheffield, Stone et al. 1993, Chang, Congdon et al. 2005, van Koolwijk, Despriet et al. 2007). As stated before, POAG is more prevalent in African Americans and prevalence can be as high as four to five times higher in this population than that of European Americans(Tielsch, Sommer et al. 1991). Additionally, the prevalence of glaucoma is higher in people over age 40; presentation at younger than age 40 is characterized by definition as juvenile open-angle glaucoma (JOAG)(Leske 1983). One study also indicated that glaucoma is 7-10 times more prevalent in first degree relatives than in the general population (Wolfs, Klaver et al. 1998) demonstrating a significant role family history plays in glaucoma pathogenesis. There has been strong evidence of glaucoma’s heritability. Evidence of the familial nature of glaucoma stems from twin studies(Merin and Morin 1972, Teikari 1987, Teikari, Airaksinen et al. 1987, Gray 1992), familial pedigrees of families showing Mendelian inheritance(Beiguelman and Prado 1963, Crombie and Cullen 1964, Harris 1965), and epidemiological studies(Tielsch 1996). Familial cases have shown higher prevalence in first degree cases than in the general population indicating that there is a strong genetic component to glaucoma (Armaly 1965, Paterson 1970, Tielsch, Katz et al. 1994, Leske, Nemesure et al. 2001,

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Weih, Nanjan et al. 2001, Gordon, Beiser et al. 2002). Moreover, twin studies have shown concordance(Gray 1992) and discordance (Teikari, Airaksinen et al. 1987), which indicate that non-genetic components may play a role in glaucoma and in fact may be a complex or multifactorial disease. There is also evidence of quantitative traits associated with glaucoma such as intraocular pressure, cup-to-disc ratio, and outflow facility segregating in families (Armaly 1966, Klein, Klein et al. 2004, Chang, Congdon et al. 2005, van Koolwijk, Despriet et al. 2007). Complex (genetic and environmental) risk factors of optic nerve disease have led to an incomplete understanding of the biological pathways that lead to the excavation of the optic disc in glaucoma and optic nerve diseases.

Rare optic nerve diseases, congenital optic disc anomaly A similar excavated appearance of the optic disc, observed in glaucoma subjects, is also the primary clinical sign of congenital malformations of the eye including optic nerve head coloboma, optic pit, and morning glory disc anomaly(Fingert, Honkanen et al. 2007, Honkanen, Jampol et al. 2007). These congenital conditions have overlapping clinical features and many have suggested that these optic nerve diseases represent a single spectrum of abnormalities. This range of optic nerve abnormalities has been termed cavitary optic disc anomaly (CODA) (Figure 2).

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Figure 2. Fundus images of patients with cavitary optic disc anomaly (CODA). CODA presents with a spectrum of congenital optic nerve malformations including optic nerve coloboma (left), morning glory disc anomaly (center), and optic pits (right). Photos courtesy of John Fingert The component features of CODA are described below: • Optic pits are focal deep excavations or regional depressions of the ONH where the normal optic tissue is absent; these pits are usually located in the temporal optic disc though some are centrally located. They are associated with serous retinal detachments or posterior vitreous detachments where the retina detaches from the choroid layer causing patients with excellent vision to have severe vision loss. Most optic pits are unilateral and sporadic, but some cases have autosomal dominant inheritance. • Morning glory disc anomaly is a malformation of the optic nerve in which the optic disc has an elongated funnel shape surrounded by elevated chorioretinal pigmentation (Fingert, Honkanen et al. 2007) that resembles a morning glory flower (Fingert, Honkanen et al. 2007). Other features of morning glory disc anomaly are anomalous retinal vessels that are arranged in a radial distribution originating from ciliary circulation and an abnormal tuft of glial tissue anterior to the optic disc (Lee and Traboulsi 2008). Most patients with morning glory disc anomaly have

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poor visual acuity that may be associated with retinal and macula detachments. Morning glory disc anomalies are typically sporadic and unilateral. • Optic nerve coloboma are malformations in which there is missing tissue caused by incomplete closure of the embryonic fissure during development(Georgalas, Ladas et al. 2011) and may occur in isolation or as part of syndromes in which abnormalities of other ocular structures are also present including the choroid, iris, retina, cornea, and eyelid. Colobomas occur in 2.6 per 10,000 live births in the US and affect 3.2-11% of blind children worldwide (Hornby, Gilbert et al. 2000, Lee, Lee et al. 2013). Colobomas typically occur as part of larger systemic syndromes such as CHARGE syndrome, which presents with heart defects, growth retardation and ear abnormalities. Some of the syndromic forms of coloboma have known genetic causes, however, genes for isolated optic nerve coloboma have not yet been identified. As with optic pits and morning glory disc anomaly, optic nerve coloboma is associated with serous retinal and macula detachments. Peripapillary pigmentary changes and retinal schisis (retinal splitting) are also associated with CODA (Brodsky 1994, Honkanen, Jampol et al. 2007). Also, there has been evidence of retinal pigment epithelium (RPE) changes within CODA subjects (Munk, Simjanoski et al. 2014). Examination of CODA eyes with OCT identified RPE extensions far beyond the termination of the choroid and into the optic cup. The impact of this observation is yet to be determined. Large pedigrees have been reported by us (Honkanen, Jampol et al. 2007) and others (Savell and Cook 1976, Slusher, Weaver et al. 1989) that exhibit autosomal dominant inheritance of CODA. Both CODA and glaucoma have similar appearing excavated optic nerve heads (Figure 3).

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Figure 3. Similarities between the optic nerves of patients with cavitary optic disc anomaly (CODA) and glaucoma. Both CODA patients (A) and glaucoma patients (B) have excavated optic discs compared with a healthy individual (C) and loss of the normal rim of neural tissue (arrows). (Figure courtesy of John Fingert) Interestingly, some patients with CODA have progressive worsening of optic nerve excavation, which is a hallmark of glaucoma (Moore, Salles et al. 2000, Honkanen, Jampol et al. 2007). In fact this progression occurs in the absence of elevated IOP, a feature that resembles NTG. The similarities between the optic nerve appearance of those with CODA and glaucoma have suggested that these conditions may have overlapping pathophysiology (Savell and Cook 1976, Slusher, Weaver et al. 1989, Honkanen, Jampol et al. 2007).

Molecular genetic approaches to optic nerve diseases Genetic investigations using linkage analysis It has long been recognized that glaucoma pathogenesis has a genetic basis as several studies have shown genetic defects confer increased risk to glaucoma (Merin and Morin 1972, Teikari 1987, Tielsch 1996, Wolfs, Klaver et al. 1998). Positional cloning or linkage analysis is used to identify the chromosome location of potential disease genes using single nucleotide polymorphism (SNP) markers. Disease-causing genes are identified using large families transmitting disease in a Mendelian fashion. With linkage analysis, we compare the inheritance of thousands of SNPs to the disease in a large family to identify those that are co-inherited with 8

disease in the family. The idea is that SNPs that are in close proximity and flank the diseasecausing gene would be co-inherited with disease since their close proximity hinders the chance of a meiotic crossover event. Linkage analysis also does not rely on any a prioi hypotheses or information about the biological mechanism of pathogenesis so it has been used as a great tool to study diseases that are poorly understood like optic nerve disease and glaucoma. Following evidence of genetic components involved in the pathogenesis of glaucoma, the first genetic linkage of primary glaucoma was identified on chromosome 1q in a large autosomal dominant pedigree (Sheffield, Stone et al. 1993). This novel locus, designated GLC1A, was found to be coinherited with juvenile open-angle glaucoma and found to account for other primary glaucomas such as POAG (Morissette, Cote et al. 1995). Subsequently, further examination identified myocilin (MYOC, OMIM 601652) as having a variety of mutations responsible for a range of glaucoma disease from JOAG to adult onset POAG, as well as accounting for approximately 34% of the POAG cases worldwide (Stone, Fingert et al. 1997, Alward, Fingert et al. 1998). Since then, genetic studies of glaucoma pedigrees mapped several disease-causing genes to other chromosomal loci. GLC1B was mapped to 2cen-q13 for adult-onset POAG in an autosomal dominant family(Stoilova, Child et al. 1996); GLC1C was mapped to 3q21-q24 in a third, large autosomal dominant pedigree for POAG(Wirtz, Samples et al. 1997). GLC1D and GLC1E was mapped to 8q23 (Trifan, Traboulsi et al. 1998) and 10p15-p14 (Sarfarazi, Child et al. 1998), respectively. Subsequently, optineurin (OPTN, OMIM 602432) was shown to cause 1-2% of NTG cases (Rezaie, Child et al. 2002, Alward, Kwon et al. 2003) a sixth locus, GLC1F, was mapped to 7q35-q36(Wirtz, Samples et al. 1999); GLC1G, after some controversy, was mapped to 5q22.1(Monemi, Spaeth et al. 2005, Fan, Wang et al. 2006); and GLC1J and GLC1K were mapped by Wiggs and colleagues by a genome-wide scan of 25 JOAG families(Wiggs, Lynch et

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al. 2004). A new glaucoma locus, GLC1P was mapped to 12q14 where tank-binding kinase 1 (TBK1, OMIM 604834) has been associated with 1% of NTG cases (Fingert, Robin et al. 2011). To date, at least 39 genetic loci have been associated with varying types of glaucoma demonstrating the genetic heterogeneity and complex nature of glaucoma (Fan, Wang et al. 2006, Challa 2011, van Koolwijk, Bunce et al. 2013) (Table 1).

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Locus Name

Chromosome Location 1q21-q31

Gene

Condition

MYOC

1-4% POAG & JOAG

TMCO1

GLC1B

1q24 2cen-q13

OAG POAG

GLC1C(Wirtz, Samples et al. 1997) GLC1D(Trifan, Traboulsi et al. 1998) GLC1E(Sarfarazi, Child et al. 1998) GLC1F(Wirtz, Samples et al. 1999) GLC1G(Samples, Kitsos et al. 2004, Monemi, Spaeth et al.

3q21-q24 8q23 10p15-p14 7q35-q36 5q22.1

GLC1A(Sheffield, Stone et al. 1993, Stone, Fingert et al. 1997)

(Stoilova, Child et al. 1996)

OPTN WDR36

POAG POAG POAG & NTG POAG POAG (controversial)

2005)

GLC1H(Lin, Liu et al. 2008)

2p16.3-p15

POAG

GLC1I(Allingham, Wiggs et al. 2005, Woodroffe, Krafchak 15q11-q13

POAG

et al. 2006)

GLC1J(Wiggs, Lynch et al. 2004) GLC1K(Wiggs, Lynch et al. 2004) GLC1L(Baird, Foote et al. 2005) GLC1M(Pang, Fan et al. 2006) GLC1N(Wiggs, Allingham et al. 2000, Wang, Fan et al.

9q22 20p12 3p21-p22 5q221-q32 15q22-q24

JOAG JOAG POAG & NTG JOAG JOAG

2006)

GLC1O(Pasutto, Matsumoto et al. 2009)

19q12-q14

NTF4

GLC1P(Fingert, Robin et al. 2011) GLC3A(Sarfarazi, Akarsu et al. 1995) GLC3B(Akarsu, Turacli et al. 1996)

12q14 2p21 1p36

TBK1 CYP1B1

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POAG & NTG (controversial) 1.3% NTG Congenital glaucoma Congenital glaucoma

Additional loci 15q22 7q31 2p21 6p21.1-p12.1 9p21

LOXL1(Thorleifsson, Magnusson et al. 2007) CAV1, CAV2(Thorleifsson, Walters et al. 2010) SRBD1(Meguro, Inoko et al. 2010) ELOVL5(Meguro, Inoko et al. 2010) CDKN2B-AS1(Ramdas, van Koolwijk et al. 2010, Burdon,

Exfoliation glaucoma POAG NTG NTG OAG

Macgregor et al. 2011)

19p13 1q24 14q22.3-q23 17p13.1 10q21.3-q22.1 14q31 3q25.31 9q31.1 14q24.3

ADAMTS10(Kuchtey, Olson et al. 2011) TMCO1(Burdon, Macgregor et al. 2011) SIX1/SIX6(Ramdas, van Koolwijk et al. 2010) GAS7(van Koolwijk, Ramdas et al. 2012) ATOH7(Ramdas, van Koolwijk et al. 2010) GALC(Liu, Gibson et al. 2011) FNDC3B(Hysi, Cheng et al. 2014)

Glaucoma OAG Optic disc parameters POAG Optic disc parameters POAG IOP

ABCA1(Chen, Lin et al. 2014, Gharahkhani, Burdon et al. 2014) LTBP2(Ali, McKibbin et al. 2009)

POAG Congenital glaucoma

Table 1. Loci associated with glaucoma. Genetics studies that have identified glaucoma loci show that several genes have been implicated but only a few have been replicated or shown as causative. Not all loci are listed. (Table generated by Ralph Hazlewood)

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Although at least 39 additional loci have been linked to glaucoma, only a handful have been validated, most notably MYOC and OPTN. Monemi and colleagues found variants in WDR36 within the GLC1G locus segregated in all affected members of 130 families sampled with POAG and these variants were absent in normal controls(Monemi, Spaeth et al. 2005). However, our laboratory and others did not confirm WDR36’s association with glaucoma as mutations in WDR36 were not detected in subsequent studies (Jia, Tam et al. 2009, van Koolwijk, Bunce et al. 2013). Additionally, putative POAG-causing gene NTF4 is controversial as very rare mutations found conferring risk to POAG in certain populations could not be replicated broadly (Pasutto, Matsumoto et al. 2009, Liu, Liu et al. 2010, Rao, Kaur et al. 2010, Vithana, Nongpiur et al. 2010) and (unpublished data). While several genes have been implicated in glaucoma, only MYOC, OPTN, and TBK1 have been directly ascertained as causative while the role of others like NTF4, ASB10, and WDR36 remain controversial (Hauser, Allingham et al. 2006, Hewitt, Dimasi et al. 2006, Fingert, Alward et al. 2007, Liu, Liu et al. 2010, Rao, Kaur et al. 2010, Fingert, Roos et al. 2012).

Genome wide association studies Like linkage analysis, genome wide association studies (GWAS) try to identify the chromosomal location of disease-causing genes, but instead assess genetic risk at a population level. Similarly, GWAS have the power to identify common ancestral variants, look for specific genetic variants that are associated with disease in the population, and/or identify those markers that are in linkage disequilibrium with the causal variant. The association may or may not point to the disease-causing gene and as a result, it is referred to an association rather than causative. Several GWAS have been performed to identify optic nerve disease and glaucoma genes (Table 1). Utilizing a genome-wide association scan, common genetic variants were mapped close to 13

CAV1 and CAV2 in POAG patients on 7q31(Thorleifsson, Walters et al. 2010, Wiggs, Kang et al. 2011) though members of our lab and collaborators did not replicate the findings in our cohort of POAG patients(Kuehn, Wang et al. 2011). Several other association studies have mapped susceptibility loci for disease or quantitative traits of glaucoma (Nakano, Ikeda et al. 2009, Meguro, Inoko et al. 2010, Burdon, Macgregor et al. 2011, van Koolwijk, Bunce et al. 2013) but replication in other populations and functional studies are needed to confirm these findings. Several glaucoma loci have been found, however, only few genes have been identified and replicated in independent studies and populations as causative. Moreover, Mendelian genetic loci account for approximately 5% of the genetic variation that predispose glaucoma worldwide; more work is needed to elucidate the cause of the remaining 95%.

Genetic evidence of CODA Although most cases of optic pits, optic nerve coloboma, and morning glory disc anomaly are sporadic, there are some rare pedigrees that show these collective conditions segregating as a Mendelian trait (Savell and Cook 1976, Corbett, Savino et al. 1980, Slusher, Weaver et al. 1989). Stefko and colleagues reported autosomal dominant inheritance of optic pits (Stefko, Campochiaro et al. 1997). Also many cases of coloboma show evidence of heritability with the larger syndromes and several genes have been identified for a subset of cases (Ferda Percin, Ploder et al. 2000, Jamieson, Perveen et al. 2002, Azuma, Yamaguchi et al. 2003, Schimmenti, de la Cruz et al. 2003, Gregory-Evans, Williams et al. 2004, Fingert, Honkanen et al. 2007). However there is no known gene for isolated coloboma. Prior molecular and genetic studies have shown mutations in PAX2 and PAX6 genes in patients with optic nerve head abnormalities (Sanyanusin, Schimmenti et al. 1995); PAX6 was implicated for a single bilateral case of morning glory disc anomaly (Azuma, Yamaguchi et al. 2003). However, it is unknown if 14

these genes are risk alleles for the collective spectrum of congenital malformations associated with the heritable form of CODA.

Significance and Focus of Dissertation Studies Optic nerve diseases are a major public health problem affecting over 60 million people worldwide. These diseases have a complex genetic basis including strong evidence for genetic heterogeneity. Because the eye is considered part of the CNS, many congenital anomalies of the optic disc involve neurodevelopmental problems that can be life-threatening. The biological pathways that lead to excavation of the optic disc in glaucoma and in CODA, however, are incompletely understood. Consequently, novel investigations of the biological mechanisms that lead to loss of tissue (excavation) of the optic nerve will ultimately lead to improvements in our understanding of disease processes and to developments of new treatments for optic nerve diseases including CODA and glaucoma. Studies designed to clarify the genetic basis of optic nerve disease and to identify disease-causing genes may provide clinicians with powerful tools to improve diagnostic precision via genetic testing. Further, genetics studies may also lead to more accurate prognoses that are based on genotype-phenotype correlations in which genetic tests predict the likely course of a disease. Finally, knowing the disease gene may also lead to better treatments and improved or novel therapeutics. Once the molecular and genetic basis of disease is discovered, custom treatments may be developed based on an individual’s genome. Such therapies may target the genetic defects at the molecular level and directly correct or compensate for the cause of a disease. The Glaucoma Genetics Laboratory has studied the genetic basis of several congenital and adolescent optic nerve diseases such as CODA and JOAG(Savell and Cook 1976, Slusher, Weaver et al. 1989, Stone, Fingert et al. 1997, Alward, Fingert et al. 1998, Fingert, Heon et al. 1999, Fingert, Honkanen et al. 2007, Honkanen, Jampol et al. 2007, Fingert, 15

Robin et al. 2011, Kawase, Allingham et al. 2012, Seo, Solivan-Timpe et al. 2013, Fingert, Darbro et al. 2014) as well as in age-related or adult-onset conditions such as ischemic optic neuropathy and POAG(Fingert, Grassi et al. 2007, Hayreh, Fingert et al. 2008) (Stone, Fingert et al. 1997, Alward, Fingert et al. 1998, Fingert, Heon et al. 1999, Fingert, Robin et al. 2011, Kawase, Allingham et al. 2012, Seo, Solivan-Timpe et al. 2013, Fingert, Darbro et al. 2014), which along with investigations by other groups, have provided insights into the pathophysiology of optic nerve disease (Kwon, Fingert et al. 2009, Fingert 2011, Zode, Kuehn et al. 2011, Zode, Bugge et al. 2012). In a previous experiment, a new glaucoma locus, GLC1P on 12q14, was identified for low pressure glaucoma from studies of a large African America pedigree (Fingert, Robin et al. 2011). This novel locus revealed a 300 kbp duplication that was inherited with all affected family members and glaucoma patients but absent in controls. This region spans several genes, notably TBK1, which is expressed in the retina and retinal ganglion cells. The extra copy of TBK1 may be responsible for glaucoma. Subsequent analysis confirmed the TBK1 duplication and revealed that TBK1 duplication leads to increased autophagy in induced pluripotent stem cell-derived retinal cells from patients with normal tension glaucoma(Tucker, Solivan-Timpe et al. 2014). Furthermore, the role of copy number variations and chromosomal rearrangements in optic nerve disease such as those recently identified in glaucoma is largely unknown. The focus of this dissertation is to elucidate the underlying genetic factors responsible for optic nerve diseases such as glaucoma and CODA. Patients with CODA are of genetic importance for several reasons. First, in addition to optic pits, coloboma, and morning glory disc anomaly, CODA patients have congenital excavations and degeneration of the optic nerve. Though there is congenital optic nerve degeneration, in some cases these abnormal optic nerves

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have been shown to progressively deteriorate with time as is seen in glaucoma. Prominent and progressive ONH cupping is observed with normal IOP (Honkanen, Jampol et al. 2007) which resembles NTG. Second, there is evidence that CODA is a heritable trait as there is evidence of optic nerve disease segregating in families in a Mendelian fashion (Fingert, Honkanen et al. 2007). Third, the heritability of CODA in rare families is significant as it allows for genetic studies aimed at following familial mutations throughout generations. The distinct clinical appearance of CODA facilitates the study of the genetic factors predictive of CODA and allows for the study of CODA as a model for the optic nerve damage seen in more common blinding conditions such as glaucoma. Finally, because some CODA patients have progressive optic nerve damage similar to NTG subjects, the principle hypothesis of this thesis project is that identification of the CODA gene will provide insight into more common glaucomatous optic neuropathy. Prior experiments mapped the gene that causes autosomal dominant CODA to chromosome 12q14 using positional cloning and linkage analysis of a large CODA pedigree with 17 affected family members (Fingert, Honkanen et al. 2007) (Figure 4A).

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Figure 4. CODA pedigrees. Those family members that have been clinically diagnosed with CODA (i.e. exhibiting features of optic pits, optic nerve coloboma, and/or morning glory disk anomaly) are indicated with black symbols, while clinically unaffected family members are indicated with white symbols. Grey symbols represent family members who are obligate carriers. A. Pedigree 981-R which was described in previous reports (Fingert et al. 2007; Honkanen et al. 2007) B. Pedigree IP-09-47.(Figure adapted from Hazlewood et al 2015).

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This locus contained ~245 known genes. Candidate gene screening of ~80 genes did not yield any plausible coding-sequence mutations more prevalent in affected members than in normal subjects. Subsequent investigation of this novel locus through array comparative genomic hybridization revealed a copy number variation (CNV), a triplication of a 6 Kbp DNA segment upstream of the matrix metalloproteinase 19 (MMP19) gene. This finding represents the discovery of new disease-causing gene for CODA. Based on these data, the focus of this thesis project is to study the molecular genetics of CODA and functionally determine the mechanism of MMP19 activity in optic nerve disease.

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CHAPTER II HETEROZYGOUS TRIPLICATION OF UPSTREAM REGULATORY SEQUENCES LEADS TO DYSREGULATION OF MATRIX METALLOPROTEINASE 19 (MMP19) IN PATIENTS WITH CAVITARY OPTIC DISC ANOMALY (CODA) Introduction The optic nerve (cranial nerve II) conveys visual signals from the retina to the brain. The inner most cells of the retina, the retinal ganglion cells, project axons that converge to form the optic nerve as they exit the eye en route to the brain. Diseases of the optic nerve may be recognized and categorized in part by characteristic changes to the appearance of the optic nerve head that are visible during an eye examination. One group of optic nerve diseases (the glaucomas) is defined largely by progressive loss of optic nerve fibers and the resulting excavated or cupped contour of the optic nerve head. Glaucoma typically has an onset later in life and affects up to 60 million adults worldwide (Roodhooft 2002, Quigley and Broman 2006). A spectrum of congenital malformations of the eye (optic nerve coloboma, megalopapilla, optic pit, and morning glory disc anomaly) also have an excavated optic nerve head (Brodsky 1994) that resembles the nerve appearance in glaucoma. Some syndromic cases of optic nerve head malformations have been associated with mutations in PAX2 (OMIM 167409) (Dureau, AttieBitach et al. 2001) or PAX6 (OMIM 607108) (Azuma, Yamaguchi et al. 2003). Optic nerve colobomas and renal disease may be caused by mutations in the PAX2 gene (Dureau, AttieBitach et al. 2001), while other sporadic cases of optic nerve disease have been attributed to PAX6 mutations (Azuma, Yamaguchi et al. 2003). Finally, rare families have been reported with autosomal dominant inheritance of congenitally excavated optic nerves as well as other features of optic pit, optic nerve coloboma, and morning glory disc anomaly, which has been termed 20

cavitary optic disc anomaly (CODA)(Savell and Cook 1976, Slusher, Weaver et al. 1989, Honkanen, Jampol et al. 2007). Clinical similarities between CODA and normal tension glaucoma, glaucoma that occurs without elevated intraocular pressure, have suggested that these conditions may have overlapping pathophysiology. Both conditions have similar appearing optic nerves that have an excavated topography and both conditions develop without elevated intraocular pressure. In some cases, patients with CODA have had progressive worsening of optic nerve excavation, which is a hallmark of glaucoma (Moore, Salles et al. 2000, Honkanen, Jampol et al. 2007). However, there are some clear differences between CODA and glaucoma. Patients with CODA have a strong predilection for retinal detachments and/or separation of the retinal layers (retinoschisis) that lead to profound central vision loss, while such retinal abnormalities are less commonly seen in glaucoma patients with large optic cups and no signs of congenital optic pits (Spaide, Costa et al. 2003, Hollander, Barricks et al. 2005, Kahook, Noecker et al. 2007, Zumbro, Jampol et al. 2007). More than half of patients with CODA develop retinal detachments and/or retinoschisis in one or both eyes (Honkanen, Jampol et al. 2007). The same genetic defect that causes CODA also greatly increases risk for retinal disease. We previously mapped the disease-causing gene for familial autosomal dominant CODA to a novel genetic locus on chromosome 12q with linkage analysis of a large autosomal dominant pedigree (Fingert, Honkanen et al. 2007). This CODA locus spans 13.5 Mbp and more than 200 genes. Members of the pedigree were initially sequenced for disease-causing mutations in three candidate genes (GDF-11, WIF1, and NEUROD4) within the locus, but no variations were detected (Fingert, Honkanen et al. 2007). Here we report additional studies to identify and characterize the mutation in the chromosome 12q locus that causes CODA in our large pedigree

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using DNA sequencing, copy number variation (CNV) analysis, immunohistochemistry, and a reporter gene assay to assess mutation effects on transcription.

Materials and methods Study sample subjects and controls Seventeen clinically affected family members and one obligate carrier for CODA in a large pedigree (Figure 4A) were previously described (Fingert, Honkanen et al. 2007, Honkanen, Jampol et al. 2007). Patients received complete ophthalmic examinations by one of the authors (L.M.J., W.L.M.A., or R.A.H.) or fellowship trained glaucoma-specialists. Affected family members have cavitary optic disc anomalies with clinical criteria previously described (Fingert, Honkanen et al. 2007, Honkanen, Jampol et al. 2007). Additional CODA patients (Figure 4B), patients with glaucoma, and matched control subjects were recruited from the clinics at the University of Iowa using previously described criteria (Fingert, Robin et al. 2011). Informed consent was obtained for study participants and was conducted with the approval of the University of Iowa’s Institutional Review Board. DNA was prepared from blood samples from each study participant as previously described (Buffone and Darlington 1985).

Candidate gene screening and exome sequencing Testing candidate genes for disease-causing mutations was facilitated with the TrAPSS software program, which prioritized gene segments for sequencing based on annotation and homology (O'Leary, Davis et al. 2007). Candidate genes in the CODA locus were PCR amplified and analyzed for mutations by single stranded conformation polymorphism (SSCP), high resolution melt (HRM) analysis and/or bi-directional sequencing as previously described (Fingert, Heon et al. 1999, Fingert, Robin et al. 2011).

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One family member with CODA (Patient IV-8, Figure 4A) was additionally tested for disease-causing mutations by conducting whole exome sequencing and restricting our analysis to the variants lying within the linked chromosome 12q locus. Exome capture was conducted using SureSelect Human All Exon v2 (Agilent, Santa Clara, CA) using the manufacturer’s protocol and paired-end, 50 nucleotide reads were obtained using a HiSeq2000 (Illumina, San Diego, CA) which achieved an average exon coverage of 65X. Identified sequence variants were filtered to include only those variants located within the chromosome 12 CODA locus (defined by genetic markers D12S1618 and D12S1702), non-synonymous coding sequence variants, and variants that occurred at a frequency of less than 1% in 1000 genomes and less than 0.6% in the Exome Sequence Project public databases (Genomes Project, Abecasis et al. 2012)(EVS).

CNV analysis, array CGH, and quantitative PCR Family members of Pedigree IP-09-47 and 981-R were genotyped with a custom Sureprint G3 8x60K Human CGH microarray (Sureselect, Agilent, CA USA). The genomic interval (NCBI37/hg19) chr12:53,586,000-58,865,000 was assayed with approximately 100bp probe spacing and processed according to the manufacturer’s protocol. Array CGH was performed using 0.5µg of patient DNA and 0.5µg of human reference DNA (Promega, WI). Patient genomic DNA and reference DNA from normal control were labeled in parallel, each with a distinct fluorescently labeled probe and co-hybridized to the array. Data was analyzed using Agilent CytoGenomics v2.7 software. TaqMan Copy Number Assays were used in real time quantitative PCR (qPCR) done in triplicate and analyzed with Copy Caller Software v1.0 (Applied Biosystems, CA) in accordance to manufacturer’s conditions and protocols. The coordinates for the TaqMan probes used are

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shown in Table 2. Detected and confirmed variants were submitted to ClinVar (http://www.ncbi.nlm.nih.gov/clinvar/).

Cloning and Promoter constructs The triplicated 6 Kbp upstream sequence of MMP19 (RefSeq NM_002429.4) was PCR amplified from RPCI11-559I11 BAC clone (Empire Genomics, NY) and cloned into TOPO-XL shuttle vector (Invitrogen, CA). The CNV DNA sequence in TOPO-XL vector was then digested with KpnI and XhoI and cloned upstream of the Luciferase gene into the pGL3-Promoter vector (Promega, WI, USA, Figure 10) to generate the vector now called “pGL3 (Full Length 6Kb Fragment)”. The pGL3-Promoter vector contains the SV40 promoter but no enhancer element. Clones were bi-directionally sequenced to confirm orientation, size and validate sequence. Seven subclones of the 6 Kbp fragment, each less than 1 Kbp in size were generated from the Full Length vector by PCR with overlapping primer pairs using the same restriction digestions enzymes KpnI and XhoI. These smaller fragments were then subcloned into pGL3-Promoter vectors. These constructs were listed as pGL3-ProA, pGL3-ProB, pGL3-ProC, pGL3-ProD, pGL3-ProE, pGL3-ProF, and pGL3-ProG (Figure 5A).

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Figure 5. Chromosome 12 triplication upstream of MMP19 gene. A. This schematic shows the results of the CNV analysis of the 981-R and IP-09-47 CODA pedigrees. A heterozygous triplication (2 extra copies) of a 6 Kbp DNA segment of chromosome 12q14 was detected upstream of MMP19 in members of pedigrees 981-R and IP-09-47 with CODA. The extent of the triplication in members of pedigrees IP-09-47 and 981-R is represented with a grey box labeled “981-R & IP-09-47 CNV Analysis” and the location of the transcription start site of MMP19 is indicated with a bent arrow. The portion of the 6 Kbp DNA segment that was used in the Luciferase reporter assay is indicated with the grey box labeled “Full Length Fragment pGL3 reporter clone” The segments of the CNV that were subcloned to assess for transcription activity are indicated by boxes labeled ProA-ProG. The location of the TaqMan qPCR probes used to asses copy number is shown below promoter fragments (black bars). B. The locations of Alurepeat domains flanking the triplicated sequence are indicated with black or grey boxes representing AluS and AluJb subtypes respectively. C. The triplicated sequence coincides with a region of H3K27 acetylation sites (indicated by the peaks above the horizontal line) and DNaseI hypersensitivity sites indicated by boxes below the horizontal line. Potential transcription factor binding sites identified by UCSC browser are shown below the horizontal line. (From Hazlewood et al 2015)

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Cell culture and transient transfection with Luciferase assays Luciferase transfection assays were performed in HEK293T cells. Cells were maintained in Dulbecco’s Modified Eagle’s medium supplemented with 10% fetal bovine serum. Low passage cells were plated onto 6-well tissue culture plates (Corning, NY, USA) at a density of 500,000 cells/well until 70-90% confluent. Cells were transfected in triplicate with 2.5µg of pGL3 (Full Length 6Kb Fragment) plasmid and 200ng of CMV-Renilla plasmid and permeated with 8µl Lipofectamine 2000 reagent (Invitrogen, CA, USA). Co-transfection with Renilla served as an internal control to assess transfection efficiencies. DNA and Lipofectamine complexes were added to each well and incubated for 24 hours at 37°C in a CO2 incubator. Following incubation, cells were lysed and Luciferase activity measured using Dual-Glo Luciferase assay system (Promega, WI, USA). Luminescence was recorded using NOVOstar microplate reader (BMG LABTECH, Germany). Firefly Luciferase activity for each DNA fragment was normalized to empty vector and relative Luciferase activity was calculated. Data is shown as fold change compared to empty vector control, pGL3 (Control Promoter). All experiments were repeated at least three times and statistical significance calculated with oneway ANOVA and two-tailed Student’s t test.

Immunohistochemistry Human donor eyes were obtained from the Iowa Lions Eye Bank following informed consent from the donor’s families. Human donor eyes without eye disease were fixed with 4% paraformaldehyde in phosphate buffered solution (PBS) within 8 hours of death. Wedges of retina and optic nerve were cryopreserved in sucrose solution and embedded in optimal cutting temperature solution (Barthel and Raymond 1990). Polyclonal antibodies directed against MMP19 (1:100, ab2 AV32315; Sigma Aldrich, St. Louis, MO, USA) were used as previously 27

described (Fingert, Robin et al. 2011). Co-labeling experiments were performed using antiMMP19 and a polyclonal antibody against glial fibrillary acidic protein (GFAP 1:100, SAB2500462; Sigma Aldrich, St. Louis, MO USA). Briefly, cryostat sections were collected, prepared, and blocked in 1% BSA/0.1% Triton X-100 for 15 minutes followed by incubation with anti-MMP19 overnight at 4°C. For co-labeling experiments, sections were incubated with anti-MMP19 and anti-GFAP overnight at 4°C. Following primary incubation with anti-MMP19 and anti-GFAP, sections were washed in 3x 5 minute washes in PBS, and then incubated with Alexa-488 conjugated donkey anti-rabbit and Alexa-546 conjugated donkey anti-goat secondary antibodies (1:200, Invitrogen, CA, USA) and 4’6-diamidino-2-phenylindole (DAPI) for 30 minutes. Sections were washed again 3 times for 15 minutes, mounted in Aqua-Mount (Thermo Scientific MI, USA), and observed under fluorescence microscopy.

Results In our previous reports we described the clinical features of CODA in 17 members of a five-generation pedigree with autosomal dominant inheritance of disease (Pedigree 981-R, Figure 4A) (Moore, Salles et al. 2000, Fingert, Honkanen et al. 2007, Honkanen, Jampol et al. 2007). Linkage analysis of this family mapped the mutation that causes CODA to a 13.47 Mbp segment of chromosome 12q between genetic markers D12S1618 (53,892,649) bp and D12S1702 (67,359,260 bp) (Fingert, Honkanen et al. 2007).

Candidate gene screening We previously tested members of pedigree 981-R for CODA-causing mutations in the entire coding sequence of three genes in the linked chromosome 12q14 locus (GDF-11, WIF1, and NEUROD4), however, none were detected (Fingert, Honkanen et al. 2007). We selected additional candidate genes in the linked region with known gene function and/or ocular 28

expression that are consistent with a role in CODA pathogenesis (i.e. function in neurogenesis and expression in the optic nerve or retina). DNA from two members of pedigree 981-R (Figure 4A, III-10 and IV-8) was tested for CODA-causing mutations by sequencing the entire coding sequence of seven additional candidate genes (TBK1, SILV, ZNF1A4, ADMR, STAC3, BLOC1S1, and SLC6A7). Functional domains and conserved sequences were identified in an additional 47 candidate genes (Table (S2)3) using the TrAPSS software package (O'Leary, Davis et al. 2007). Using a combination of single strand conformation polymorphism (SSCP), high-resolution melt (HRM) analysis, and bi-directional Sanger sequencing, these high interest gene segments were also tested for CODA-causing mutations. Several non-synonymous sequence variations were detected in these candidate genes, but no plausible coding sequence mutations were found that are both co-inherited with disease in pedigree 981-R and absent from control subjects (data not shown). We subsequently used whole exome sequence analysis of one CODA patient in pedigree 981-R (Patient IV-8, Figure 4A) as a more comprehensive search for disease-causing mutations within the chromosome 12q locus. Two rare heterozygous, non-synonymous coding sequence variants were detected, missense mutation R464Q in ITGA5 and frame-shift mutation c110111insA in CD63. However, when the entire 981-R pedigree was tested with Sanger sequencing, the R464Q variant in ITGA5 was not co-inherited with disease, ruling it out as a potential cause of CODA. The presence of the other variant detected by exome sequencing, the CD63 frameshift mutation, could not be confirmed with Sanger sequencing, suggesting that its initial discovery was an exome sequencing error. Overall, the analysis of the exome data did not identify any plausible disease-causing variants in genes within the linked chromosome 12q locus.

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Copy number variation (CNV) analysis We also tested CODA pedigree 981-R (Figure 4A) for the presence of CNVs within the linked chromosome 12q locus. DNA from 2 affected members of CODA pedigree 981-R (Figure 4A, III-10 and IV-8) was genotyped for CNVs in a comparative genomic hybridization (CGH) experiment using a custom chromosome 12 Agilent CGH array with 60,000 probes distributed across the chromosome 12q locus according to the manufacturer’s protocol. CNVs in this region of chromosome 12q have been previously detected spanning the normal tension glaucoma gene, TANK binding kinase 1 (TBK1)(Fingert, Robin et al. 2011, Kawase, Allingham et al. 2012, Ritch, Darbro et al. 2014, Awadalla, Fingert et al. 2015), however no TBK1 duplications or deletions were identified in DNA samples from our CODA patients. Both CODA patients were found to carry 2 extra copies (4X) of a 6 Kbp DNA segment, chr12:g.56238827_56244961 (3), within the CODA locus and 2.1 Kbp upstream of the matrix metalloproteinase 19 (MMP19 OMIM 601807) gene (Figure 5A). The presence of the CNV was further confirmed with a TaqMan quantitative PCR (qPCR) assay. When the entire pedigree was tested with the qPCR assay, we discovered that this CNV is co-inherited with CODA in all 17 affected family members and absent from unaffected members. The autosomal dominant inheritance pattern of the 4X CNV indicates that it is a heterozygous 6 Kbp triplication. Also, this triplication was not detected in 78 normal control subjects (Figure 6), nor was it previously reported in the online Database of Genomic Variants (http://dgv.tcag.ca/dgv/app/home).

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Figure 6. Quantitative PCR assay for chromosome 12q14 CNV. A TaqMan qPCR assay was used to test 24 unrelated CODA patients for the chromosome 12q14 triplication upstream of MMP19. A triplication was detected in one of these subjects, a member of pedigree IP-09-47. The results of testing members of CODA pedigrees 981-R and IP-09-47 and 78 control subjects with the qPCR assay are shown above. (From Hazlewood et al 2015) An additional 24 unrelated patients with a clinical diagnosis of CODA or its component features (optic pit, optic nerve coloboma, or morning glory disc anomaly) were tested for evidence of a CNV upstream of MMP19 using our qPCR assay. One of these 24 patients was found to have a DNA triplication upstream of MMP19, which suggests that MMP19 CNVs may contribute some fraction of additional cases of CODA. This patient (Figure 4B, Pedigree IP-09-47, patient III-1) is a member of a three-generation pedigree with two other family members affected with CODA including his mother and maternal grandmother. Testing DNA from his affected mother (Figure 4B, Pedigree IP-09-47, II-1) with our qPCR assay showed that she also carries a triplicated DNA sequence upstream of MMP19. DNA was not available from the patient’s maternal grandmother

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(Figure 4B, Pedigree IP-09-47, I-2). Analysis of IP-09-47 family members with a custom Agilent CNV microarray identified a 6 Kbp triplication with the same borders as the CNV detected in pedigree 981-R (Figure 5A). CNVs with the same borders were detected in two CODA pedigrees (981R and IP-09-47) that are not known to be genealogically related. Consequently, we investigated the possibility that the CNVs in each family arose independently on different chromosome 12q haplotypes by genotyping both families at genetic markers flanking the CNVs. Single nucleotide polymorphism (SNP) markers and short tandem repeat polymorphism (STRP) markers closely flanking the CNV were typed in both families (Table (S3)4). Allele sharing was observed between the two pedigrees at markers most closely flanking the CNV. These data do not provide evidence to suggest the CNVs in pedigree 981-R and IP-09-47 arose independently. However, the power of this analysis was limited by the informativity of the flanking markers and by the small size of pedigree IP-09-47. Finally, the entire coding sequence of 13 of our cohort of 24 unrelated CODA patients was evaluated for mutations. A synonymous Val176Val change was detected in one individual with CODA and no non-synonymous changes were detected. Overall, these data show that a CNV upstream of MMP19 is associated with CODA. We further explored the DNA sequence spanned by this CNV for a role in CODA pathogenesis by testing a cohort of patients for mutations. Our cohort of 24 unrelated CODA patients was tested for mutations in the 6 Kbp region upstream of MMP19 that was encompassed by the CNV. Approximately 71% of this region was tested for mutations using unidirectional Sanger sequencing, while 29% of the region was composed of highly repetitive sequences and could not

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be assessed reliably with this method. Five variations were detected (Table 5), however, four of these variants were judged to be implausible causes of CODA due to their relatively high frequency in 1000 Genomes Project (Genomes Project, Abecasis et al. 2012) and control subjects. The fifth variant, which was detected in a single CODA patient and was absent from control populations, has unknown significance. These data suggest that currently detectable DNA sequence variations within the 6 Kbp region upstream of MMP19 are unlikely to be common causes of CODA. Additionally, no obvious difference in optic nerve appearance was noted between those CODA subjects found to carry the MMP19 CNV and those without the CNV. One mechanism for the generation of CNVs is non-allelic homologous recombination due to the presence of repetitive DNA sequences. Consequently, we analyzed DNA sequence upstream of MMP19 in search of repetitive sequences that might have been involved with the development of the CNV in our CODA patients. We identified numerous Alu elements that are members of the short interspersed elements (SINE) family of repetitive sequences upstream of the MMP19 (Figure 5B). A high density of Alu elements are located on either side of the 6 Kbp DNA sequence that is triplicated in patients with CODA in pedigrees 981-R and IP-09-47. Further sequence analysis showed that this 6 Kbp sequence spans a region of DNaseI hypersensitivity and H3K27 acetylation sites that are often found near regulatory sequences (Figure 5C). The positions of several potential binding sites for transcription factors are also clustered on this region upstream of MMP19 (Figure 5C).

Testing glaucoma patients for the chromosome 12q CNV The chief feature of CODA is an excavated optic nerve head that resembles damage caused by glaucoma. Consequently, we tested cohorts of patients with primary open angle glaucoma that 33

occurs at normal intraocular pressure (n = 84) or at elevated intraocular pressure (n = 88) to see if a similar genetic defect might be involved in the pathogenesis of glaucoma. These 172 glaucoma patients were tested for CNVs using our qPCR assay, however, none were detected.

Transcription activity of the chromosome 12q14 CNV The location of the 6 Kbp triplication, which is 2.1 Kbp upstream of MMP19, suggests that it might contain transcription regulatory elements that alter gene expression. Consequently, this DNA sequence was investigated for transcriptional activity using a luciferase reporter gene assay. A single copy of the 6 Kbp DNA sequence spanned by the CNV was transfected upstream of the Luciferase gene in a vector with a control SV40 promoter (pGL3, Promega) (Figure 10). The transcriptional activity of this DNA segment was measured in HEK293T cells 24 hours after transfection. Cells transfected with this DNA segment produced a 1.3-fold increase (not significant) in Luciferase activity compared with cells transfected with pGL3 vector containing no enhancer sequences (Figure 7A). Large DNA sequence such as the 6 Kbp CNV identified in this study may harbor multiple enhancer and/or silencer elements and motifs (Kleinjan, Bancewicz et al. 2008, Ochi, Tamai et al. 2012, Sanyal, Lajoie et al. 2012). In order to fine map the location of individual enhancer elements, the DNA segment spanned by the CNV was subcloned into 7 smaller overlapping fragments pGL3-ProA, pGL3-ProB, pGL3-ProC, pGL3ProD, pGL3-ProE, pGL3-ProF, and pGL3-ProG (Figure 5A) and transfected into HEK293T cells. Cells transfected with the vector containing DNA fragment pGL3-ProF produced the greatest Luciferase activity. These cells generated 8-fold more Luciferase activity than cells transfected with the vector containing no enhancer sequences, p G

T

4 (16.7%)

2 (10%)

6.70%

5%

p>0.99

rs7296597

NC_000012.12:g.56241955A>G

A

4 (16.7%)

2 (10%)

10%

5%

p>0.99

rs7296830

NC_000012.12:g.56242017C>T

T

4 (16.7%)

2 (10%)

8%

5%

p>0.99

rs56180965

NC_000012.12:g.56241588 T>C het

C

2 (8.33%)

2 (20%)

10%

5%

p>0.99

g.56241951 T>C het

NC_000012.12:g.56241951 T>C het

C

1 (4.17%)

0 (0%)

N/A

N/A

p>0.99

Table 5. Variations detected in unrelated CODA subjects within the 6Kbp region upstream of MMP19. Four of the five detected variants were judged as implausible causes of CODA because of their high frequency in the 1000 Genomes Project and/or normal control cohort. The fifth variant was seen in 1 of 24 CODA subjects and was absent from controls and from the 1000 Genomes Project. The significance of this variant is currently unknown. Coordinates are taken from GRCh37/hg19. P-values were calculated using Fisher’s exact test. MAF = minor allele frequency. N/A = not available (From Hazlewood et al 2015)

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Figure 10. Plasmid vector used for Luciferase reporter assays. DNA sequences were cloned into the multiple cloning site (shown in gray) using restriction enzymes KpnI and XhoI upstream of the SV40 Promoter. (From Hazlewood et al 2015) "Copyright (2015) Wiley. Used with permission from (Human Mutation, John Wiley and Son Inc)."

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CHAPTER III EVALUATION OF A NEW CODA PEDIGREE Introduction Optic nerve head excavation (loss of tissue) is the defining feature of common and rare optic nerve diseases such as glaucoma and cavitary optic disc anomaly (CODA), respectively. CODA is a rare eye disease associated a spectrum of congenital malformations of the optic nerve including optic pit, optic nerve coloboma, and morning glory disc anomaly. CODA patients also present with optic nerve defects in the absence of elevated intraocular pressure. There are striking similarities in the appearance of the optic nerves of patients with CODA and patients with glaucoma (Figure 3). Two hallmarks of glaucoma (excavated optic discs and progressive worsening of optic nerve damage) have also been documented in CODA. Some patients have had progressive worsening of optic nerve degeneration, a hallmark of normal tension glaucoma (Moore, Salles et al. 2000, Honkanen, Jampol et al. 2007). These similarities suggest that the same genetic defect that causes CODA may also be involved in the pathophysiology of optic nerve cupping in glaucoma. CODA subjects also have a predilection to retinal detachments and retinoschisis (splitting of the retinal layers), though these retinal features are not frequently observed in glaucoma subjects (Spaide, Costa et al. 2003, Zumbro, Jampol et al. 2007). Over the past 15 years, family-based linkage and genetic association studies have identified several genes associated with glaucoma. These include MYOC, TBK1, OPTN, WDR36, CDKN2B-AS1, TMCO1, and CAV1/CAV2 and are reviewed elsewhere (Fingert 2011, Mackey and Hewitt 2014). Similarly genes such as PAX2 and PAX6 have been implicated in ONH abnormalities (Amiel, Audollent et al. 2000, Azuma, Yamaguchi et al. 2003). Though several genes have been identified, mutations in these genes account for less than 10% of cases. This

52

points to the notion that the missing heritability may lie in areas where traditional screening techniques fall short. Copy number variations (CNV) were first recently identified in glaucoma subjects. These subjects had a duplication that spanned the TBK1 gene on chromosome 12q14 (Fingert, Robin et al. 2011). Subsequent studies have since confirmed this duplication in several populations indicating that copy number variations may play an important role in glaucoma pathogenesis (Kawase, Allingham et al. 2012, Liu, Garrett et al. 2014, Ritch, Darbro et al. 2014, Awadalla, Fingert et al. 2015). Recently, the first copy number variations (triplication) associated with CODA was reported. A copy number variation (triplication of a 6 Kbp DNA segment) was discovered upstream of the matrix metalloproteinase 19 (MMP19) gene in a rare, large pedigree with CODA (Hazlewood, Roos et al. 2015). In addition, the DNA sequences spanned by the triplication exhibit transcriptional activity and functions as a strong enhancer. MMP19 protein is also strongly expressed in the ONH where abnormalities of CODA occur (Hazlewood, Roos et al. 2015). The association of MMP19 gene and CODA is supported by several lines of evidence. First, CNVs have been identified and implicated in influencing gene expression in several disease processes including glaucoma(Liu, Gibson et al. 2011), triphalangeal thumbpolysyndactylly syndrome(Klopocki, Ott et al. 2008), Liebenberg syndrome(Spielmann, Brancati et al. 2012), cancer(Jaeger, Leedham et al. 2012, Sun, Shi et al. 2014), and others(Verdin, D'Haene et al. 2013, Chen, Alvarez et al. 2014, Ibn-Salem, Kohler et al. 2014). Second, along with MMP19 protein expression in the optic nerve from our recent report, MMP19 mRNA expression is increased in glaucoma mouse models (Howell, Walton et al. 2011). Finally, the population based-segment of our recent report identified triplications upstream of MMP19 in 1 of

53

24 (4.2%) sporadic CODA subjects suggesting that chromosome 12q14 CNVs may be responsible for a fraction of all CODA cases. This subject was part of another small autosomal dominant CODA pedigree in which all affected members assayed were found to have triplications upstream of MMP19 (Hazlewood, Roos et al. 2015). Together, these data strongly indicate an important role for MMP19 in the pathogenesis of CODA. We subsequently identified another small CODA pedigree and aimed to investigate the presence of any MMP19 upstream CNVs in this affected members of this pedigree.

Methods Human subjects and Clinical studies Patients and families with CODA have been actively recruited and ascertained at the University of Iowa Hospitals and Clinics as well as through collaborations with clinicians at other institutions via referrals from neighboring states. The CODA family in this study (081-E) was recruited by Dr. Robert Lesser at Lakeshore Eye Surgery Center (Figure 11). The Institutional Review Board (IRB) of the University of Iowa approved this study. Informed consent was obtained from participants before enrolling in the study. All subjects received complete ophthalmoscopic examinations including optic nerve exams, visual acuity tests, measurements of intraocular pressure (tonometry), slit-lamp biomicroscopy, and dilated fundus exams. These exams were performed by board-certified or fellowship-trained clinician and glaucoma specialists. Clinical criteria for subjects affected with CODA have been previously described (Honkanen, Jampol et al. 2007). Briefly, patients were diagnosed with CODA if they had abnormal optic nerve heads with atypical central retinal arteries, atypical optic nerve coloboma, optic pits, and/or morning glory disc anomaly. Complete ophthalmic examinations could not be obtained from other members of 081-E. 54

Figure 11. Pedigree 081-E. The proband (081-E-1) was tested for copy number variations in the chromosome 12q14 region with TaqMan qPCR assays. Black bars represent affected status and white bars represent unaffected/unknown. DNAwas not available for affected member 081-E-2. The affection statuses of other family members are unknown. (Pedigree generated by Ralph Hazlewood)

Copy Number Analysis Whole blood was collected from both affected subjects of pedigree 081-E using nonorganic procedures (Buffone and Darlington 1985). In short genomic DNA was extracted from leukocytes in the samples in a vacutainer containing EDTA-stabilized blood using Qiagen DNeasy blood and tissue kit (Qiagen, Hilden, Germany) according to manufacturer’s protocol. Isolated DNA was re-suspended in elution buffer/TE buffer or de-ionized water and concentrations determined using the Nanodrop spectrophotometer (Thermo Scientific, Wilmington, DE). TaqMan Copy Number Assays were used in real time quantitative PCR experiments. DNA from CODA patients and normal control subjects were PCR amplified in 55

triplicate using our validated probes designed to hybridize within the MMP19 upstream sequence to assess copy number (Hazlewood, Roos et al. 2015). An RnaseP control reference amplicon was used for normalization. CNVs were detected using CopyCaller software (Applied BioSystems, Carlsbad, CA) according to manufacturer’s protocol. DNA from CODA subjects was also genotyped at genetic single nucleotide polymorphism (SNP) and short tandem repeat polymorphic (STRP) markers closely flanking the CNV to detect any areas of allele sharing or a shared haplotype that may indicate relatedness between different CODA families.

Results Using quantitative PCR, the proband of pedigree 081-E (081-E-1) was found to carry chromosome 12q14 CNVs upstream of MMP19. In fact, this subject had 2 extra copies (triplication) of DNA sequences upstream of MMP19 (Figure 12).

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Copy Number

MMP19 Copy Number Assay 5 4 3 2 1 0 081-E-1

081-E-2

NL-661

NL-716-1

NL-808

NL-825-1

NL-843

NL-867

Figure 12. TaqMan Copy number qPCR plot. Members of CODA pedigree 081-E and several normal control subjects were assayed for chromosome 12q14 copy number variations in triplicate experiments. The graph show that affected members 081-E-1 and 081-E-2 both have 2 extra copies (triplication) of DNA sequences upstream of MMP19. (Data generated by Ralph Hazlewood) This triplication overlaps the CNV identified in our recent report of a large autosomal dominant CODA pedigree (981-R) (Hazlewood, Roos et al. 2015) and may be the same in its extent. No triplications were detected in normal control subjects. We further detected CNVs upstream of MMP19 in her affected cousin (081-E-2). These data provide additional independent evidence that CNVs upstream of MMP19 are involved in the pathogenesis of CODA. Similar MMP19 CNVs were detected in affected members of this pedigree although it is not known if they are genealogically related to pedigree 981-R from our recent report. We consequently investigated this occurrence by genotyping pedigree 081-E and 981-R at several unique SNP and STRP genetic markers closely flanking the CNV. The haplotype analysis revealed that both affected members of pedigree 081-E share alleles closely flanking the CNV with affected members of 981-R (Table 6). In fact members of pedigree 081-E share alleles with pedigree 981-R at all markers tested which is consistent with a founder effect and suggests that both pedigrees may have a common ancestor.

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Pedigree 981-R Marker D12S1586 rs998401 rs950166 rs3847674 rs3782240 rs2293413 rs2293414 rs3759097 rs1137900 MMP19 rs1052206 rs11171710 rs705698 rs705699

Position (hg19) 54,146,806 55,355,233 56,056,312 56,076,266 56,082,785 56,096,816 56,097,025 56,103,803 56,182,393 56,229,21456,236,735 56,348,028 56,368,078 56,384,687 56,384,804

Pedigree 081-E

981-R IV-8

981-R II-3

981-R III-7

981-R IV-2

Shared Allele

1,208,427 701,079 19,954 6,519 14,031 209 6,778 25,285

2,3 CC AG TT CG CA CC AG TT

3,3 CC AA TT GG CC CC AA TT

3,3 CC AA CT CG CA CC AG TT

3,4 CC AA CT CG CA CC AG TT

3 C A T G C C A T

CC AA CT GG CC CC AA TT

CC AA CT CG CT CA AG TT

C A C or T G C C A T

Allele sharing between pedigrees C A UNKNOWN G C C A T

46,821

CNV

CNV

CNV

CNV

CNV

CNV

CNV

CNV

CNV

111,293 20,050 16,609 117

TT GG CT TT

TC GG CC TT

TC AG CT CT

TT AG CT CT

T G C T

TT GG CC TT

TC GG CC TT

T G C T

T G C T

Separation Distance (bp)

081-E1

081-E2

Shared allele

Variation (CA)n C/T A/G C/T C/G C/T A/C A/G C/T

MAF NA 8% 38% 27% 44% 47% 46% 45% 39%

C/T A/G C/T C/T

24% 46% 31% 40%

Table 6. Haplotyping SNP data. Members of Pedigree 981-R and pedigree 081-E who all carried MMP19 CNVs were genotyped at single nucleotide polymorphisms (SNPs) surrounding MMP19 gene. Areas of allele sharing in which tested members of both pedigrees with CODA carry the same allele (consistent with an ancestral genotype) are indicated. MAF = minor allele frequency. (Table generated by Ralph Hazlewood)

58

Discussion Identification of gene(s) responsible for a spectrum of cavernous defects of the optic nerve such as glaucoma and the nerve findings part of CODA (optic pits, optic nerve coloboma, and morning glory disc anomaly) have largely been elucidated by linkage-based studies. These known genes have commonly been associated with other systemic diseases such as CHARGE syndrome in which subjects can present with growth defects and heart abnormalities. The gene that causes congenital autosomal dominant CODA was previously mapped to chromosome 12q14 and we recently reported that a 6 Kbp triplication of DNA sequences upstream of MMP19 was associated with CODA. In that report, we discovered a heterozygous triplication approximately 2.1 Kbp upstream of MMP19 in Pedigree 981-R. Subsequent analysis of sporadic CODA patients identified an additional subject with chromosome 12q triplications with the same borders as pedigree 981-R(Hazlewood, Roos et al. 2015). Our studies of an additional CODA pedigree have identified a chromosome 12q triplication upstream of MMP19. Quantitative PCR data show that the affected members of pedigree 081-E have 2 extra copies (triplication) of DNA sequences upstream of MMP19. Two TaqMan copy number experiments with unique probes both identified this triplication in affected members. The finding of the triplication in this CODA family and absent in normal control subjects does lends greater support for the importance of MMP19 in the pathogenesis of CODA. Similarly, haplotyping analysis revealed that all genetic markers tested showed areas of allele sharing between 081-E-1 and pedigree 981-R. Interestingly, the patients in both pedigrees described in this report have independently settled in the Midwestern United States and are descendants of Russian immigrants. In addition, the surnames of both pedigrees are strikingly similar, differing in only 3 letters. These data are consistent with a founder effect and that these 59

two families may be distantly related by a common ancestor. Future studies aimed determining the borders of the CNV in pedigree 081-E with comparative genomic hybridization arrays would add further support for this founding effect. Since no mutations have been detected in unaffected CODA subjects’ family members or normal control subjects to date, this TaqMan qPCR method may be a useful screening tool of CODA disease in a subset of the patient population. Familial CODA subjects or relatives of affected CODA subjects with known MMP19 CNVs and/or suspected carriers of disease will be candidate populations of subjects that may benefit from screening, which may ultimately lead to improved diagnosis and treatment options for CODA. Because CODA and glaucoma share important clinical features, additional screening in glaucoma subjects would be beneficial to ascertain the role of MMP19 in glaucomatous optic nerve damage. Defects in other MMPs have been correlated with open-angle glaucoma in beagles (Kuchtey, Olson et al. 2011) and primary angle-closure glaucoma in humans (Wang, Chiang et al. 2006, Cong, Guo et al. 2009). Moreover, matrix metalloproteinases were elevated in the lamina cribrosa of monkeys with experimental glaucoma (Agapova, Kaufman et al. 2003) and MMP19 expression in the optic nerve is significantly correlated with glaucoma in both DBA/2J mice and in humans (Howell, Walton et al. 2011). Optic nerve gene expression signatures between DBA/2J mice with glaucoma and matched control mice were compared using an online resource from Jackson Labs (Howell, Macalinao et al. 2011, Howell, Walton et al. 2011). Not only does this resource show that expression of Mmp19 is significantly increased in mice with glaucoma (3.2-fold increase, p < 0.0001), but Mmp19 has the third most significant change in expression associated with glaucoma in the entire genome.

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In summary with our previous study that shows that the 6 Kbp triplication has transcriptional activity and that MMP19 is expressed in the ONH, the identification of possibly another Mendelian CODA family with CNVs upstream of MMP19 in this study provides greater evidence that MMP19 has a role in the pathogenesis of CODA, at least in a subset of patients. Moreover, the similar optic nerve degeneration in CODA and glaucoma subjects along with MMP19 expression in glaucoma models suggests that this defect may have a role in a glaucoma pathogenesis as well. Consequently, our investigation of the molecular cause of CODA may also provide key insights into the causes of glaucoma as well. These studies may also provide new insights into the basic biology of optic nerve cupping in glaucoma that occurs at low intraocular pressure (IOP). In particular, MMP19 provides new mechanistic insights and a new of direction for optic nerve research and studies of the molecular biology of optic nerve cupping, which may eventually lead to desperately needed therapies for CODA and glaucoma patients that frequently continue to lose vision despite achieving low IOP. More work is needed with in vivo animal models to confirm and recapitulate the optic nerve defects observed in CODA subjects.

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CHAPTER IV PATTERN OF MMP19 EXPRESSION IN THE OPTIC NERVE Introduction The optic nerve or cranial nerve II, which transmits the visual signal from the photoreceptors in the retina to the brain, is considered to be part of the central nervous system. It is primarily made up of glial cells (astrocytes and microglia), axons projecting from the retinal ganglion cell (RGCs), oligodrendrocytes, connective tissue, macrophages, and extracellular matrix and can be divided in four parts: 1) the optic nerve head (ONH), which houses the nerve fiber layer, the prelaminar region, lamina cribrosa/laminar region, and postlaminar/retrolaminar region, 2) intraorbital part, 3) intracanalicular part and 4) intracranial part. The length of the optic nerve varies from person to person and even between eye to eye of the same person ranging between 35-55mm from eyeball to chiasma and with the ONH averaging about 1 mm long and 1.5 mm in diameter (Hayreh S. 2011). RGC axons project from the retina, turn and pass through the scleral canal en route to the vision center of the cortex. The lamina cribrosa is an important collagenous structure as it supports the RGC axons as they pass through the posterior of the eye. It is important to note that there is no clear demarcation between the relative regions of the ONH. Since the main defects of ocular optic nerve diseases such as CODA and glaucoma occur in anterior optic nerve, my thesis has focused on this region encompassing the ONH also called optic disc. The identification of CNVs upstream of MMP19 in several CODA family pedigrees points to an important role of MMP19 in the pathogenesis of CODA. Luciferase assays to tease out the functional importance of the CNV indicated that DNA sequences spanned by the CNV caused a robust increase in downstream gene expression (i.e. MMP19) and functioned as a strong

62

transcriptional enhancer (Figure 7) (Hazlewood, Roos et al. 2015). Moreover, I investigated MMP19 protein localization and found that MMP19 was expressed in the ONH in human donor eyes. Since this finding represents a new mechanistic discovery that may yield important insights into novel therapeutics, I sought to understand and further describe MMP19 expression in the optic nerve.

Extracellular Matrix The extracellular matrix (ECM) has a very important function in the optic nerve. It has a structural role and is responsible for mechanical stability as well as serves to mediate interactions with cells and nerve fiber bundles(Anderson 1969) and influence functions such as tissuespecific gene expression(Fukuchi, Sawaguchi et al. 1992). Disruptions to this ECM connection may affect the strength of laminar beams within the lamina cribrosa (Quigley, Addicks et al. 1981, Quigley, Hohman et al. 1983). Astrocytes make up the majority of the glial cell population accounting for more than half of its volume and have a role in maintaining the extracellular matrix environment in the optic nerve (Hernandez 2000, Morgan 2000). They become reactive in response to injury in the central nervous system such as glaucomatous damage in which an increased expression of glial fibrillary protein (GFAP) is observed(Hernandez 2000). Studies have shown that reactive astrocytes lead to the production of ECM macromolecules reviewed by Hernandez in ref (Hernandez 2000). The ECM of the human optic nerve is composed of collagen type I (COL I), COL III, COL IV, COL V, laminin, elastin, tenascin, fibrillin, glycosaminoglycans, fibronectin and basement membranes (Goldbaum, Jeng et al. 1989, Morrison, L'Hernault et al. 1989, Fukuchi, Sawaguchi et al. 1992, Fukuchi, Sawaguchi et al. 1994, Pena, Varela et al. 1999, Stracke, Fosang et al. 2000). The degree to which these ECM components are expressed during optic atrophy associated with glaucomatous damage or optic 63

nerve degeneration varies, but reports have shown that most have enhanced expression during injury(Hernandez, Andrzejewska et al. 1990, Morrison, Dorman-Pease et al. 1990, Fukuchi, Sawaguchi et al. 1992, Pena, Mello et al. 2000).

Matrix metalloproteinases MMPs are zinc-dependent extracellular proteases that are required for the breakdown of the ECM in many normal processes such as embryonic development, cell surface receptor cleavage, apoptosis, and tissue remodeling as well as having a role in disease states such as arthritis, cardiovascular disease, and angiogenesis (Sedlacek, Mauch et al. 1998, Liu, Sun et al. 2006, Page-McCaw, Ewald et al. 2007, Van Lint and Libert 2007). Moreover, MMPs degrade many kinds of ECM proteins, many of which are found within the optic nerve. MMPs are also expressed in a wide variety of tissues and cell types as secreted proteases such as MMP2 and MMP9 that are activated when cleaved by extracellular proteinases, or membrane-associated such as MMP14 and MMP17. Because of their important activity in normal and disease states, MMPs are highly regulated enzymes. Tissue inhibitors of matrix metalloproteinases (TIMPs) are specialized proteins that inhibit the activity of MMPs but also have roles in cell differentiation, cell migration and synaptic plasticity(Brew and Nagase 2010). There are several TIMPs: TIMP1, TIMP2, TIMP3, and TIMP4, in addition to membrane-bound TIMPs such as MT-TIMP1 and MT-TIMP2. MMP19 has only been recently cloned within the last decade. MMP19 is different than the other MMPs not only in its location on chromosome 12q14 but it lacks several structural domains and subgroups found in other MMPs (Pendas, Knauper et al. 1997). MMP19 has an insertion of a cysteine in the catalytic domain, a C-terminal threonine-rich tail, and a different latency motif (Cossins, Dudgeon et al. 1996, Pendas, Knauper et al. 1997, Sedlacek, Mauch et al. 1998, Mueller, Mauch et al. 2000, Beck, Ruckert et al. 2008). Additionally, there is evidence of 64

broad MMP19 expression in human tissues although most MMPs have minimal expression in adult cells (Pendas, Folgueras et al. 2004). This makes MMP19 in a separate class from other MMPs and MMP19 regulation and activity in normal and disease processes are incompletely understood.

MMP19 expression in other tissues Data on the expression of MMP19 in the eye is currently very limited as studies have focused on its role in cancer and more recently arthritis. However, studies have shown that MMP19 is expressed in human epidermis, heart and blood vessels walls, macrophages, spleen, thymus, prostate, pancreas, placenta, ovary, small intestine, and lung but absent from brain and peripheral blood leukocytes in some datasets. The expression discrepancy could be explained by the differences at the mRNA and protein level. MMP19 mRNA is expressed in numerous tissues (Cossins, Dudgeon et al. 1996, Pendas, Knauper et al. 1997) but MMP19 protein is more restricted highlighting greater regulation of this important enzyme (Beck, Ruckert et al. 2008). MMP19 is expressed in astroglial tumors but the importance of this expression is shown it its regulation. Lampert et al. described low expression of MMP19 in tumors using qPCR (Lampert, Machein et al. 1998) but others have shown increased expression in glioma tumors at the protein level (Stojic, Hagemann et al. 2008, Lettau, Hattermann et al. 2010). This phenomenon underscores the importance of regulating MMP19 post-transcriptionally and post-translationally. Its activity in normal tissues underscores its importance in normal tissue remodeling (Stracke, Hutton et al. 2000). Nuclear MMP19 labeling has also been identified in dorsal root ganglion (DRG) neurons cultured on laminin substrates and cryosections of DRG neurons (Fudge and Mearow 2013). MMP19 can also hydrolyze laminin-5(van Horssen, Vos et al. 2006), a vascular basement membrane protein (van Horssen, Bo et al. 2005). Similarly, MMP19 has been shown to 65

digest members of the ECM including collagen type I (COL I), COL IV, fibronectin, casein, gelatin I, tenascin, aggrecan, nidogen, and cartilage oligomeric protein (COMP), laminin 5γ2, and insulin-like growth factor binding protein 3 (Stracke, Fosang et al. 2000, Stracke, Hutton et al. 2000, Sternlicht and Werb 2001, Sadowski, Dietrich et al. 2003, Titz, Dietrich et al. 2004, Sadowski, Dietrich et al. 2005). Most, if not all of these ECM components, have been found in the optic nerve.

MMP19 expression in the optic nerve Because of MMP19’s role in the pathogenesis of CODA, I investigated MMP19 localization in the optic nerve. From our recent report, in longitudinal or sagittal cryosections we observed MMP19 localization throughout the ONH. Some weak labeling was observed in the retina, possibly within the plexiform layers but this observation was not consistently observed in several eyes (data not shown). However, MMP19 localized consistently throughout the glial lamina and postlaminar regions of the ONH in 5 eyes (Figure 8). Weaker labeling was observed in the anterior prelaminar region. MMP19 had an irregular and interrupted expression pattern with bright punctate-like spots as well as linear or fibrous-like immunoreactivity in the ONH. Specifically, labeling was observed, toward the edges of the nerve where the sclera canal and nerve connect, then tapered off toward the center of the nerve near the central retinal artery (CRA). MMP19 immunoreactivity was also observed along the basement membranes of the CRA. Also, this same localization pattern was concentrated along the edges of the nerve where the pia mater attaches to it. The localization of MMP19 in this region is consistent with our model for CODA and optic nerve degeneration (Figure 9) (Hazlewood, Roos et al. 2015). Over activity of MMP19 caused by the triplication of enhancer sequences in CODA subjects may lead to increased tissue remodeling of the support structures in the nerve. An increase in protease 66

activity and degradation of extracellular matrix in the lamina cribrosa leads to a disinsertion and collapse of the lamina cribrosa leading to the ONH appearance and malformations characteristic of CODA. To get a better understanding of MMP19 localization and expression in the optic nerve, I investigated MMP19 expression in the relation to the cells that populate the nerve.

Methods Ocular tissue preparations Human donor eyes were obtained from Dr. Markus Kuehn and Dr. Robert Mullins of the Wynn Institute for Vision Research (WIVR) and the Iowa Lions Bank following informed consent from the donor’s families. Within 8 hours of death, human donor eyes without eye disease were fixed with 4% paraformaldehyde (PFA) in PBS. Globes were then grossed and wedges of the retina and optic nerve were cryopreserved through graded sucrose solutions (5% and 20%), embedded in 20% sucrose solution in optimal cutting temperature solution (TissueTek OCT compound; Sakura, Torrence CA, USA) and frozen at -80°C (Barthel and Raymond 1990). For immunohistochemistry, 12-10 micron sections were cut on a cryostat and mounted on Superfrost Plus microscope slides and stored at 4°C. Only sections prepared within 1 month were used for experiments.

Immunolabeling and immunohistochemistry Polyclonal antibodies directed against MMP19 (1:100, ab2 AV32315; Sigma Aldrich, St. Louis, MO, USA) were used as previously described (Fingert et al. 2011)(Hazlewood, Roos et al. 2015). Anti-MMP19 co-labeling experiments were performed using the following antibodies: polyclonal antibody against glial fibrillary acidic protein (GFAP 1:100, SAB2500462; Sigma Aldrich, St. Louis, MO USA); monoclonal anti-neuron specific beta III Tubulin (TU-20 1:200, ab7751; Abcam, Cambridge, MA USA); monoclonal anti-neurofilament (SMI-312 1:1200, 67

ab24574; Abcam, Cambridge, MA USA); polyclonal anti-collagen type IV (1:200, AB769; Millipore EMD, Temecula, CA USA), monoclonal anti-fibrillin-1 (1:200, MAB2502; Millipore EMD, Temecula, CA USA), monoclonal anti-CD45 (1:200, 555480; BD Biosciences, San Jose CA USA), and monoclonal anti-elastin (1:100, E4013; Sigma Aldrich, St. Louis, MO USA). Briefly, cryostat sections were collected, prepared, and blocked in 1% BSA/1% Triton X100/0.05% Tween-20 for 1 hour followed by incubation with anti-MMP19 overnight at 4°C. All steps were performed in a humidified chamber. For co-labeling experiments, sections were coincubated with anti-MMP19 overnight at 4°C. Following primary incubations, sections were washed in 3x 5 minute washes in 1X PBS, and then incubated with the appropriate Alexa fluor secondary antibodies (1:200, Invitrogen, CA, USA) and 4’6-diamidino-2-phenylindole (DAPI) for 30 minutes in the dark protected from light. Secondary antibodies used were Alexa-546 conjugated donkey anti-rabbit (for MMP19) and Alexa-488 conjugated donkey anti-goat (GFAP & COL IV) or Alexa-488 goat anti-mouse highly cross absorbed (all other antibodies). Sections were washed again 3 times for 15 minutes, mounted in Aqua-Mount (Thermo Scientific MI, USA), and observed under fluorescence microscopy. Results Astrocytes In cross or en face sections of the optic nerve and ONH, localization was similar to the longitudinal sections in that MMP19 labeling had a fibrous-like pattern concentrated toward the edges of the nerve. These linear-appearing processes resembled the processes that astrocytes project (Figure 13).

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Figure 13. MMP19 in cross sections of the optic nerve. MMP19 labeling is observed concentrated around the edges of the ONH with linear-like projections going into the nerve. CRA = central retinal artery (Micrographs generated by Ralph Hazlewood)

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This prompted an examination of MMP19’s expression with astrocyte marker GFAP. GFAP reliably labels both type 1A and 1B astrocytes within the retina and optic nerve. Following co-incubation with anti-MMP19 and anti-GFAP, we did not observe significant colocalization. GFAP labeled astrocytes within the neural retina and ONH but MMP19 was localized only within the optic nerve. Under examination of confocal microscopy, there was no significant overlap with between the process of the astrocytes and the linear-like localization of MMP19 (Figure 14).

Figure 14. GFAP and MMP19. GFAP (green) was incubated with Anti-MMP19 (red) at 4°C overnight then visualized by confocal microscopy. DAPI (blue) stains nuclei. GFAP labels astrocytes within the ONH while MMP19 displays an irregular labeling pattern. There are some areas have co-labeling between GFAP and MMP19 (arrows). Bottom panel shows no primary control sections. (Micrographs generated by Ralph Hazlewood) The robust expression of GFAP in these transverse sections indicates that there is an abundance of astrocytes within the glial lamina.

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However, there appeared to be some overlap or coexpression between MMP19 and GFAP in some areas of the nerve, especially in the regions where MMP19 labeling was most pronounced. In fact, much of MMP19 immunolabeling mirrored the GFAP labeling of the astrocyte projections following side-by-side, which may indicate secretion of MMP19 in the extracellular spaces of nerve. Astrocytes within the optic nerve, especially within the prelaminar and laminar regions have a very distinct pattern. Jakobs and colleagues vividly demonstrate that astrocytes in the glial lamina have long processes that extend the width of the optic nerve (Sun, Lye-Barthel et al. 2009). These processes also contact the pia and blood vessels while crossing over axon bundles and overlay the collagenous plates in the lamina cribrosa. Similarly, astrocytes vary morphologically in the extent and amount of processes that cell bodies project between the different regions of the nerve (Figure 15).

Figure 15. Astrocyte morphology in the optic nerve. Astrocytes have long processes that traverse the width of the optic nerve. (A) Schematic drawing of astrocyte projections in the optic nerve. (B) IHC image of transgenic mice that express eGFP in the astrocytes and colabeled with axon marker SMI-32. Copyright (2009) Wiley. Used with permission from (Sun et al, The morphology and spatial arrangement of astrocytes in the optic nerve head of the mouse, J Comp Neurol, John Wiley and Son Inc).

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The localization of MMP19 in the glial lamina appearing disorganized and interrupted in transverse sections is consistent with a distribution of MMP19 protein in the extracellular spaces between the morphological varieties of astrocytes in this area (Figure 14). The spatial organization of the astrocytes allows retinal ganglion cells axons to pass through- although the shear density of astrocytes in the glial lamina causes considerable overlap between astrocytes. Also, there is no line of demarcation or sharp transition zone between anterior laminar glia and posterior glia so glial cells in this region can vary widely in size and shape. This makes the unorganized and irregular labeling of MMP19 appear in the ECM in the ONH.

Retinal ganglion cell axons As stated previously, RGC axons from the retina exit the posterior of eye through the lamina cribrosa. The axons then become myelinated as they pass through the retrolaminar region forming axon bundles. To examine MMP19 expression in relation to the axons and neurons in the optic nerve, MMP19 was co-incubated with several markers for RGC axons. βIII Tubulin and neurofilament heavy chain (NFH) have been established as markers for RGC axons and will label the axon bundles within the optic nerve. In combination with a neuron-specific βIII Tubulin antibody on transverse sections, MMP19 continued to have fibrous like expression pattern which did not have an appreciable colocalization with βIII Tubulin (Figure 16). βIII Tubulin could also distinguish and label axons within the different regions of the ONH where the nerve fibers are becoming grouped/bundled together en route to myelination and the optic nerve major. Pia septa divide the nerve fibers as it runs to the sclera allowing them to form axon bundles. Interestingly, MMP19 immunolabeling did have irregular fibrous-like projections that traverse the axons bundles. Using an NFH antibody, similar immunolabeling of axons and axon bundles was

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observed as the βIII Tubulin antibody (data not shown). Together these data show that MMP19 does not have significant overlap or colocalization with RGC axons.

Figure 16. βIII Tubulin and MMP19. Anti-βIII Tubulin (green) was incubated with AntiMMP19(red) at 4°C overnight then visualized by confocal microscopy. DAPI (blue) stains nuclei. βIII Tubulin labels the axons and axon bundles within the ONH while MMP19 displays an irregular labeling pattern, sometimes across the axon bundles. (Micrographs generated by Ralph Hazlewood)

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Microglia Following an absence of significant colocalization in RGC axons and astrocytes, microglial expression was next investigated. MMP19 was co-incubated with Iba1 (data not shown) and CD45, markers for microglial cells in the optic nerve. Microglia was observed in the lamina cribrosa region and even within the nerve bundles in agreement with previous reports (Neufeld 1999, Yuan and Neufeld 2001). However, there was no colocalization with MMP19 in either region of the ONH (Figure 17).

Figure 17. CD45 and MMP19. Anti-CD45 (green) was incubated with Anti-MMP19 (red) at 4°C overnight then visualized by confocal microscopy. DAPI (blue) stains nuclei. CD45 labels macrophages and microglia within the ONH while MMP19 displays an irregular labeling pattern, sometimes across the axon bundles. Scale bar = 50um (Micrographs generated by Ralph Hazlewood)

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Extracellular matrix components The irregular pattern of MMP19 labeling in the ONH and its lack of significant coexpression with astrocytes and RGC axons suggest that it is a secreted protein expressed in ECM of the nerve. Several ECM components were investigated; these include COLIV, Elastin, and Fibrillin-1. COLIV was observed within the extracellular spaces between the axon bundles and basement membranes. Expression was also observed around fascicles and following the connective tissue that forms the bridge between the nerve and sclera passing through the lamina cribrosa (Figure 18). MMP19 displayed its irregular labeling pattern but did not have significant colocalization with COLIV. In some areas of the nerve, MMP19 labeling did appear to originate at the basement membranes and extracellular spaces and is located around COLIV expression. This irregular expression is similar to that observed by others. In examination of sulfated proteoglycan in the lamina cribrosa in monkey eyes, Fukuchi and colleagues have found fine fibrillar material located around collagen bundles and/or filling the spaces between collagen fibrils within the basal lamina (Fukuchi, Sawaguchi et al. 1994). This linear fibril-like material is strikingly similar to the localization pattern of MMP19, particularly large, punctate and linearlike structures within the laminar regions.

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Figure 18. COL IV and MMP19. Col IV (green) was incubated with Anti-MMP19 (red) at 4°C overnight then visualized by confocal microscopy. DAPI (blue) stains nuclei. COL IV labels collagen in the extracellular matrix within the ONH while MMP19 displays an irregular labeling pattern. There are limited areas of co-labeling between COL IV and MMP19 (arrows) and others where MMP19 appears to originate (arrowheads). (Micrographs generated by Ralph Hazlewood)

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We next looked at the coexpression MMP19 with elastin and elastic fibers. Cryosections were first interrogated for elastic fibers with a Verhoeff-van Gieson (VVG) stain (Hann and Fautsch 2011, Kazlouskaya, Malhotra et al. 2013).VVG stain is a modified van Gieson stain which instead of identifying connective tissue fibers such as collagen only, it can distinguish elastic fibers as well. With this stain, elastic fibers are stained blue to black, connective tissues such as collagen are stained red, nuclei are stained black, and other tissue elements such as cytoplasm and muscle are stained yellow. Following VVG staining, elastin was observed throughout the optic nerve as small, thin elastic fibers. The appearance of the fibers did not resemble separate MMP19 labeling using immunohistochemistry. Therefore we labeled cryosections with an antibody to Elastin with co-incubation with MMP19. Upon close examination, MMP19 appeared to surround elastic fibers similar to what was observed in colabeling experiments with COLIV (Figure 19).

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Figure 19. Elastin and MMP19. Elastin (green) was incubated with Anti-MMP19 (red) at 4°C overnight then visualized by confocal microscopy. DAPI (blue) stains nuclei. Elastin labels elastic fibers in the extracellular matrix within the ONH while MMP19 displays an irregular labeling pattern. Some areas have co-labeling between Elastin and MMP19 where it appears that MMP19 originates at the region of highest Elastin labeling (arrows). (Micrographs generated by Ralph Hazlewood) Recent reports have suggested a link between microfibrils and glaucoma (Kuchtey and Kuchtey 2014). Microfibrils are fine extracellular filaments that form sheaths of fibrillar material in both elastic and non elastic tissue (Kuchtey and Kuchtey 2014). Sequence variants in microfibril-associated genes ADAMTS10 (Kuchtey, Olson et al. 2011), LOXL1 (Thorleifsson, Magnusson et al. 2007), and LTBP2 (Ali, McKibbin et al. 2009, Narooie-Nejad, Paylakhi et al. 2009) are correlated with glaucoma. In addition, patients with Marfan syndrome have increased incidence of glaucoma because of mutations in FBN1, the main component of microfibrils. FBN1 has also been shown to be expressed in the eye (Kuchtey, Chang et al. 2013). The similarities between FBN1 localization in the aqueous humor outflow facility structures and MMP19 motivated an examination of MMP19 and FBN1 in the optic nerve. Following co-

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incubation, FBN1 was localized to the ECM of the optic nerve with labeling of fine extracellular material in the prelaminar region. However, there was no significant colocalization between FBN1 and MMP19 in this region with MMP19 concentrated on the periphery of the nerve (Figure 20).

Figure 20. Fibrillin-1 and MMP19. FBN1 (green) was incubated with Anti-MMP19 (red) at 4°C overnight then visualized by fluorescence microscopy. DAPI (blue) stains nuclei. FBN1 labels microfibrils in the extracellular matrix within the ONH while MMP19 displays an irregular labeling pattern. (Micrographs generated by Ralph Hazlewood)

Discussion Immunofluorescence microscopy studies in cryosections of normal human donor eyes indicate that MMP19 is expressed in an irregular and interrupted pattern in the optic nerve. This expression was more pronounced around the edges of the nerve at the site of the sclera canal and pia mater. MMP19 stained linear-like projections concentrated at the edges of the nerve that appear to resemble the processes that astrocytes project. In colocalization experiments, localization was observed within the basement membranes and ECM spaces between connective tissue fibers. This was especially observed with examinations with GFAP as MMP19 labeled the

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extracellular spaces between astrocytes. Further, MMP19 did not significantly colocalize with markers for astrocytes, RGC axons, microglia, and connective tissue components of the ECM. However, there were small areas of costaining corresponding to intracellular and extracellular expression of MMP19. The observed localization is consistent with other previous reports which show a varied expression of MMP19. For example, Fudge and colleagues have shown extracellular MMP19 expression in sensory neurons (Fudge and Mearow 2013), while others have observed intracellular expression of MMP19 (Kolb, Mauch et al. 1999, Djonov, Hogger et al. 2001). For this reason care must be taken when correlating immunohistochemical expression and enzymatic activity. For example, there was minimal, if any, overlap between microglial marker CD45 and Iba1 (data not shown) with MMP19, however other reports have shown that MMP19 is expressed by microglia of multiple sclerosis lesions (van Horssen, Vos et al. 2006). Since the distribution of MMP19 closely corresponded to localization of extracellular molecules such as COLIV as well as astrocytes and to some extent RGC neurons, MMP19 may have a stronger and complex role in regulation of ECM turnover within the optic nerve. Additional studies with an exhaustive panel of markers or electron microscopy to ascertain MMP19 positive cellular or extracellular structures might provide evidence of MMP19 enzymatic activity in the nerve. Alternately, localization experiments with primary cell cultures of astrocytes, RGCs, macrophages and/or microglia may clarify the relationship between MMP19 varied intracellular and secreted expressions in the optic nerve.

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CHAPTER V CONCLUSIONS AND FUTURE DIRECTIONS Conclusions Excavation (cupping) of the optic nerve is a chief feature of optic neuropathies. It results in the loss of RGC axons and neural rim tissue. Glaucoma, the most common optic neuropathy, is considered to be a complex heterogeneous group of neurodegenerative disorders (Allingham, Liu et al. 2009, Liu and Allingham 2011, Tham, Li et al. 2014) primarily characterized by progressive optic nerve degeneration, which leads to an insidious loss of vision and is one of the leading causes of blindness worldwide. Rare diseases of the optic nerve have been identified in which patients present with congenital malformations of the optic nerve including morning glory disc anomaly, optic pits, and optic nerve colobomas. These conditions have been observed segregating in a Mendelian fashion and are collectively called cavitary optic disc anomaly (CODA). CODA subjects have a similar ONH appearance as glaucoma subjects and in fact, some have progressive optic nerve degeneration, a hallmark of glaucoma. Previous work in a large autosomal dominant CODA pedigree has resulted in the identification of a novel CODA locus on chromosome 12q14 (Fingert, Honkanen et al. 2007). In this thesis, I sought to identify and functionally characterize the gene that causes CODA.

CODA mutation identified In investigations of the previously identified CODA locus, a 6 Kbp repeated segment of DNA upstream of matrix metalloproteinase 19 (MMP19) was recently identified. The copy number variation (CNV) was identified in all 17 affected members of our large autosomal dominant CODA pedigree (981-R), absent in unaffected members and not found in the Database of Genomic Variants (DGV). Confirmation by qPCR revealed that the 6 Kbp CNV is a 82

heterozygous triplication segregating in an autosomal dominant manner. This data represents the first identification of the CODA gene(Hazlewood, Roos et al. 2015). Many diseases are not explained by traditional studies of genetic variation such as direct sequencing and genome-wide SNP association studies, which often exclude CNVs from detection. Other options are needed to detect structural changes as CNVs may be critical contributors to human disorders. From the advent and development of methods to detect them, CNVs have been recognized as a major component of global human genetic variation. Between any two individuals, there may be up to 1000 different CNVs that could be as large as several megabases of DNA sequence accounting for phenotypic variability (Hurles, Dermitzakis et al. 2008, Zhang, Gu et al. 2009). We are now beginning to unravel the phenotypic variability of CNVs as part of natural human variation and those associated with Mendelian or complex genetic disorders. A literature search for CNVs having a role in disease yields 2,326 PubMed entries as of February 2015. These studies include a range of disorders such as schizophrenia and autism to cancer and HIV/AIDS susceptibility (Xu, Roos et al. 2008, Glessner, Wang et al. 2009, Ni, Zhuo et al. 2013). Copy number changes however have not been extensively studied in glaucoma and optic nerve disease etiology. Few copy number mutations have been shown to play a role in vision disorders. Nishimura et al. identified chromosomal abnormalities in two patients with Axenfeld-Rieger syndrome on chromosome 6p25 upstream of FOXC1 (Nishimura, Swiderski et al. 1998). Deletions of genes such as LMXB1 and 4q34, and duplications such as TBK1 and CNTN4 have been implicated in glaucoma and related disorders (Cohn, Kearns et al. 2005, Connell, Brosnahan et al. 2007, Bongers, de Wijs et al. 2008, Chanda, Asai-Coakwell et al. 2008, Sakata, Usui et al. 2008, Fingert, Robin et al. 2011). In particular, there is substantial and reproducible evidence that TBK1 duplications cause glaucoma and is the subject of further

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investigation with animal models examining the impact of gene copy number changes on glaucoma etiology.

Reporter gene assays indicate MMP19 CNVs alter gene expression The genetic mutation in CODA subjects was identified as a 6 Kbp heterozygous triplication within the regulatory region of the MMP19 gene. Discussed in the review by Zhang et al., CNVs can be located outside of genes in intergenic regions disrupting regulatory elements that can affect the expression of neighboring genes(Klopocki, Ott et al. 2008, Zhang, Gu et al. 2009, Ott, Hein et al. 2012, Verdin, D'Haene et al. 2013, Knight 2014, Kim, Sock et al. 2015). Since the 6 Kbp triplication is located upstream of MMP19, we hypothesized that it may alter MMP19 expression. This led to experiments designed to investigate if the 6 Kbp sequence spanned by the CNV had any functional activity. Using Luciferase reporter gene assays of transcriptional activity, we looked for any changes in Luciferase transcription in vectors transiently transfected with different fragments of the 6 Kbp repeated sequence in HEK293T cells. One segment, approximately 773bp within the 6 Kbp triplication, caused an 8-fold increase in downstream gene expression compared to empty vector controls. These data indicate that the 6 Kbp sequence spanned by the CNV causes dysregulation of downstream gene expression, i.e. MMP19. Though an artificial system, HEK293T cells have been extensively used to study transcription as they represent all cell types. There are several interpretations of these Luciferase experiments that future studies can seek to clarify. 1) Based on preliminary qPCR data, MMP19 appears to have a relatively lower expression in fibroblasts of CODA subjects than normal controls. However, western blot experiments to assess MMP19 protein in fibroblasts appear to show similar levels of protein expression between CODA subjects and normal controls. This difference in RNA and protein 84

expression could be a result of a feedback loop mechanism via accumulation of MMP19 protein leading to regulation of transcription and low abundant RNA. This phenomenon has been demonstrated quite extensively in particular the bistability of the Sonic Hedgehog (Shh) signaling pathway. Cells regulate their state based on Shh concentration in which Shh activate Gli transcription factors to activate downstream gene targets and alternately lead to activation of transcription repressors by the same Gli factors(Graham, Tabei et al. 2010). MMP19 protein in conjunction with its substrate may act to form a negative feedback loop to decrease RNA transcripts in fibroblasts allowing a return to homeostasis throughout the body since CODA subjects do not have systemic diseases and the phenotype is restricted to the eye. Similarly, an autoregulatory feedback mechanism in aniridia subjects as described by Bhatia et al may be intact in fibroblasts but disrupted only in ocular tissues leading to CODA phenotypes(Bhatia, Bengani et al. 2013). Since bioinformatic tests show an enrichment of eye-specific POU domain transcription factors within the CNV, it would be interesting to investigate the degradation rates of protein and RNA by the use inhibitors or transgenic animal models to ablate and introduce the MMP19 variations identified in humans. Similarly, it would be beneficial to re-examine the mRNA and protein expression of MMP19 in CODA fibroblasts to learn more about its systemic regulation. 2) In conjunction with the possibility of a feedback loop and coordinated transcription factor binding leading to ocular phenotypes, the MMP19 mutations identified in CODA patients may have introduced or disrupted transcription factor binding sites within the promoter or enhancer regions of MMP19. Distant enhancers are known to dictate temporal, spatial, and dosage-specific gene expression (Nord, Blow et al. 2013). The Luciferase expression data may indicate the enhancer sequence located within the 773bp ProF Sequence is mediated by

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transcription factors leading to chromosome looping and subsequent binding to the transcription start site of MMP19 leading to expression. It is plausible that heterozygous triplication of these sequences introduces more transcription factor binding sites and leads to overexpression of MMP19. Because the CNV is surrounded by highly repetitive elements, we could not determine its breakpoint and it is unknown if MMP19 overexpression is due to introduction of binding sites or disruption of repressor elements. More work with animal models is needed to confirm and understand the impact of the CNV in vivo, although studies of evolutionary conserved enhancers may produce contradictory findings as some are more conserved than others in different tissues(Nord, Blow et al. 2013). 3) One important finding is that the CNV in CODA subjects is surrounded and flanked by numerous highly repetitive Alu elements. This leads to the conclusion that the CNV is Alumediated and arose by replication or movement of these elements in the genome. Reports have shown that mobile elements can regulate gene expression and lead to disease progression (Schiaffino, Bassi et al. 1995, Szmulewicz, Novick et al. 1998, Huopaniemi, Tyynismaa et al. 2000, Hasler and Strub 2006, Gallus, Cardaioli et al. 2010, Tucker, Scheetz et al. 2011, Holdt, Hoffmann et al. 2013, Quinn and Bubb 2014). Because of the high degree of repetitive Alu elements surrounding the CNV hindering cloning and amplification of the CNV, the entire 6 Kbp sequence could not be captured for the Luciferase transcription assays. For this same reason, the Alu elements flanking the CNV were not included in the Luciferase assays for their impact on downstream gene expression. Although these elements were not included in the Luciferase analyses, the DNA sequences spanned by the CNV does regulate downstream gene expression and function as a transcriptional enhancer, which is supported by H3K27 acetylation marks and DNaseI hypersensitivity sites within the region. Additionally it is known that methylation

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suppresses proviral DNA and intragenomic parasites such Alu elements(Yoder, Walsh et al. 1997). That leaves the possibility of potential therapeutic intervention aimed at suppressing the activity of the Alu elements in transmitting the 6 Kbp sequence by increasing methylation at the locus. It would be interesting to investigate this new area of epigenetic regulation; the area of H3K27 acetylation within the CNV would be a good target to manipulate the methylation status in CODA subjects. Similarly, H3K27 acetylation marks identified by the ENCODE consortium were found in endothethial, embryonic, mesodermal, ectodermal, and endodermal cell types (Rosenbloom, Dreszer et al. 2012) (illustrated in Figure 5). These sites are found in within the DNA sequences spanned by the triplication upstream on MMP19. The presence of these marks of active regulatory regions within the triplication lends support for its role in regulating downstream, gene expression. It is also possible that the CNV identified in CODA patients may alter other nearby genes because of the high degree of Alu repetitive elements. An examination of co-expressed genes in CODA subjects with an RNA Seq experiment may provide information on the importance of other genes. 4) Another important interpretation for MMP19 as the plausible cause of CODA is its protein structure. MMP19 is very structurally different that other MMPs. MMP19 is located on a different chromosome than the other MMPs, and lacks several structural domains identified in other MMPs (Cossins, Dudgeon et al. 1996, Pendas, Knauper et al. 1997, Sedlacek, Mauch et al. 1998, Mueller, Mauch et al. 2000, Beck, Ruckert et al. 2008). Its unique structure may hint at a reason why it affects CODA subjects causing optic nerve disease and no other systemic ailments.

MMP19 is the CODA gene The discovery of copy number variations upstream of MMP19 represents the first gene identified for Mendelian cavitary optic disc anomaly and its associated spectrum of nerve 87

findings. Immunohistochemical studies show that MMP19 has a very distinct localization pattern within the optic nerve. In longitudinal or sagittal sections, we observed that MMP19 is expressed as bright punctate-like spots in the ONH. This labeling was localized and concentrated toward the edges of the nerve where the sclera canal and pia mater connects to the optic nerve. In cross or en face transverse sections, MMP19 had a fibrous-like expression pattern with similar localization around the edges of the nerve at the site of the pia mater and sclera canal. This expression pattern is striking for several reasons. The pia mater is a meningeal envelope of thin fibrous connective tissue that ensheathes the brain and optic nerve. It is anchored to the brain and optic nerve by astrocyte processes, which again are responsible for maintaining the extracellular space of the nerve. Additionally, basement membranes derived from endothelial cells of the vasculature and glia line the pia and optic nerve septa as well as the astrocytes within the laminar beams in the laminar cribrosa (Morrison, L'Hernault et al. 1989). Localization of MMP19 resulted in a labeling pattern consistent with expression in the basement membranes of the pia (Figures 14, 18 and (Anderson 1969)). Similarly, extracellular molecules are abundant in the laminar region as evidenced by co-labeling IHC experiments with MMP19 and consistent with previous reports (Morrison, L'Hernault et al. 1989, Fukuchi, Sawaguchi et al. 1992, Fukuchi, Sawaguchi et al. 1994) Upon close inspection, MMP19 was distributed evenly and localized on the basement membranes of the central retinal artery. Moreover, labeling was observed along basements membranes and extracellular spaces between the astrocytes (Figure 14). Since astrocytes can have very different morphology in the extent and amount of projections between the different regions of the nerve and the shear density of astrocytes in the glial lamina causes considerable overlap between them, the irregular distribution of MMP19 is consistent with expression in the extracellular spaces and basements membranes between these cells as shown in

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Figures 13, 14, and 19. The density of nuclei and astrocyte processes in the different regions of the nerve is also consistent with previous reports of the human optic nerve (Balaratnasingam, Kang et al. 2014). Together, these data indicate that MMP19 is expressed in the relevant ocular tissues to cause CODA. Immunofluorescence microscopy demonstrated that MMP19 is a secreted protein in the extracellular spaces in the nerve. Longitudinal and transverse sections support our model showing that copy number variations upstream of MMP19 leads to MMP19’s dysregulation in the optic nerve head. This dysregulation leads to a mechanism of increased enzymatic activity in the basement membrane and ECM leading to remodeling of the support structures in the nerve, i.e. glial lamina and cells within this region. Since astrocytes have a role in maintaining neuronal health and the extracellular environment possibly through gap junctions(Morgan 2000), expression of MMP19 in the basement membranes and ECM of these cells provide evidence of a mechanism of action for optic nerve degeneration. Also, RGC axons may be particularly sensitive to this laminar remodeling as studies have shown RGCs deviate from their original pathway when going between the plates of the lamina cribrosa(Morgan, Jeffery et al. 1998). Thus, the remodeling by MMP19 is a plausible cause for the optic nerve head degeneration observed in CODA patients.

Future Studies CODA is a congenital disease of the optic nerve in which it is believed that the initial insult leading to optic nerve degeneration is occurs during development. We have identified 3 autosomal dominant CODA pedigrees with chromosome 12q14 triplications. All affected members had 2 extra copies (triplication) of a 6 Kbp segment of DNA approximately 2.1 Kbp upstream of MMP19 gene (Hazlewood, Roos et al. 2015). Luciferase promoter bashing assays in HEK293T revealed that one copy of the 6 Kbp CNV functioned as a transcriptional enhancer of 89

downstream gene expression. These data support the role of MMP19 in the pathogenesis of CODA. Since the entire 6 Kbp region and the surrounding Alu motifs couldn’t be interrogated with the Luciferase assays, future corroborating studies with animal models that contain a triplicated 6 Kbp sequence or triplication of the ProF region identified as having the strongest positive influence on downstream gene expression (Figure 7) is needed to show definitive causation. Efforts are underway to generate transgenic zebrafish animals overexpressing human MMP19 in the developing ocular tissues. Examination of optic nerve phenotypes during development in these animals will provide an opportunity to recapitulate the phenotypes observed in CODA subjects. In addition, studies to examine the importance of MMP19 dysregulation in transgenic rodent models as well as rodent models with local MMP19 overespression by subretinal or intravitreal injection are planned and would allow confirmation of CODA pathogenesis. Collection of skin biopsies from CODA subjects would allow reprogramming of keratinocytes to induced pluripotent stem (iPS) cells. These cells would allow for an investigation of MMP19 activity in the relevant ocular tissues not currently available in living CODA subjects. Currently, we with support from department collaborations are generating iPS cells and differentiating them to retinal progenitors, retinal ganglion cells, and/or astrocytes which would provide suitable cell types to study the activity of MMP19 during development in CODA patient ocular cells. Additional experiments aimed at understanding the mechanism of MMP19 dysregulation in CODA patients are warranted. Since MMP19 has not been extensively studied, there are many areas for future studies to tease apart the importance of MMP19 in CODA pathogenesis; studies that may allow for the interrogation of novel therapeutics based on a clearer understanding of

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MMP19. Some additional areas of interest include the discovery of MMP19’s substrate in the optic nerve. Another important interpretation of MMP19’s mechanism is action is through its substrate specificity. As stated earlier, MMP19 has been shown to digest members of the extracellular matrix including COL I, COL IV, fibronectin, casein, gelatin I, tenascin, aggrecan, nidogen, and cartilage oligomeric protein (COMP), laminin 5γ2, and insulin-like growth factor binding protein 3 (Stracke, Fosang et al. 2000, Stracke, Hutton et al. 2000, Sternlicht and Werb 2001, Sadowski, Dietrich et al. 2003, Titz, Dietrich et al. 2004, Sadowski, Dietrich et al. 2005). ECM components COL I, COL III, COL IV, COL V, laminin, elastin, and fibronectin have been observed in the optic nerve (Goldbaum, Jeng et al. 1989, Morrison, L'Hernault et al. 1989). Although it has been shown that MMP19 is capable of degrading several ECM components, it is unknown if this holds true in the optic nerve. One-dimensional and two-dimensional mass spectrometry experiments are planned to examine MMP19 substrates in retina and optic nerve lysates. As such, broad-spectrum inhibitors that are capable of inhibiting matrix metalloproteinases may be a novel treatment option for CODA subjects. Additionally, since MMPs are tightly regulated proteases, future studies aimed at examining the impact of MMP regulators such as TIMPs will provide information about MMP19 dysregulation. Finally, because MMP19 is expressed at the site of injury in CODA subjects and the optic nerve head appearance in CODA subjects is strikingly similar to glaucoma patients, testing glaucoma patients for MMP19 mutations may help solve some of the missing genetic influences of glaucoma. Recent studies by our laboratory identified duplications of TBK1 in chromosome 12q14 in glaucoma subjects. The fact that CODA and glaucoma subjects have CNVs on the same chromosome 12q14 arm (Fingert 2011, Fingert, Robin et al. 2011) indicates that this region may be a hotspot for mutations leading to optic nerve disease. Therefore, future and on-going studies

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continue to test additional CODA and glaucoma subjects for copy number variations and coding sequence variations in MMP19.

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