Dissertation
Investigations of human genetics and mouse models in the study of glaucoma
University of Iowa
Doctor of Philosophy (PhD), University of Iowa
Spring 2021
DOI: 10.17077/etd.006227
Abstract
Glaucoma is the leading cause of irreversible vision loss worldwide. The primary feature of glaucoma is the progressive degeneration of retinal ganglion cell (RGC) axons of the optic nerve, accompanied by the death of RGCs in the retina. Elevated intraocular pressure (IOP) is a major risk factor for glaucoma. Cases with elevated IOP and no identifiable etiology are termed primary open angle glaucoma (POAG). Alternatively, secondary forms of glaucoma are caused by readily observable anatomical abnormalities that reduce fluid outflow and increase IOP. The etiology of glaucoma is complex, influenced by the combined actions of many genetic and environmental risk factors including elevated IOP, African ethnicity, advanced age and family history. Genetic studies have identified three genes, myocilin, optineurin, and TBK1, that cause rare, heritable forms of glaucoma with little influence from other genes or the environment. Moreover, there are at least 14 genes that have been associated with an increased risk of POAG. Genetic discoveries provide insight into the molecular pathways affected by glaucoma and can guide the approach to developing innovative therapy to prevent vision loss. My investigations aim to: 1. Identify novel glaucoma-causing gene candidates; 2. Test hypotheses for pathogenic mechanisms of glaucoma-associated genes; and 3. Study RGC degeneration using genetic mouse models of glaucoma.
I identified a novel gene candidate for pigmentary glaucoma, a secondary form of glaucoma caused by pigment dispersion syndrome (PDS). PDS is caused by the abnormal release of pigment from the iris. Subsequently, pigment accumulates in the anterior segment, including the trabecular meshwork (TM), the primary aqueous humor outflow pathway. Pigment deposition is hypothesized to increase IOP by damaging the TM. In mice, five genes are known to cause PDS (Tyrp1, Gpnmb, Tyrp2, Lyst, and Mitf). I examined these five genes in a cohort of patients with PDS and found that loss of function mutations within these genes are not a common cause of PDS in humans. In secondary analyses, I identified a new gene candidate for pigmentary glaucoma, MRAP, which exhibited a higher mutation burden in a PDS patient cohort compared to normal control subjects. MRAP is similar to the five genes that cause PDS in mice in that it encodes a protein involved in melanogenesis.
In addition to identifying new gene candidates, I set out to investigate the pathogenic mechanisms of previously identified glaucoma-associated genes. Myocilin (MYOC) mutations often cause elevated IOP and are the most common genetic cause of glaucoma. The MYOC Tyr437His mutation causes juvenile open angle glaucoma (JOAG), diagnosed between age 3-40 and presenting with markedly high IOP, autosomal dominant inheritance, and strong family history. I performed immunohistochemical analyses of a human eye with a MYOC Tyr437His mutation and observed myocilin protein abnormally accumulated within cells of the TM. These results support the currently proposed hypothesis that the MYOC Tyr437His mutation causes decreased secretion of myocilin and leads to TM cell damage and subsequent IOP elevation.
I investigated another MYOC mutation, Gln368Stop, which is the most common genetic cause of POAG (diagnosed after age 40 and IOP > 21mm Hg). Although MYOC mutations are typically associated with elevated IOP, I found that a significant proportion of patients having glaucoma with normal IOP, termed normal tension glaucoma (NTG), harbor the MYOC Gln368Stop mutation. While the MYOC Gln368Stop mutations is more prevalent in POAG, I showed that this mutation is also associated with NTG. These findings show that MYOC mutations are not exclusive to glaucoma cases with maximum IOP > 21 mm Hg.
In contrast to MYOC, there are several other genes that have been identified which do not cause glaucoma on their own but are associated with increased POAG risk. APBB2 was recently identified to increase POAG risk exclusively in people of African ancestry. APBB2 promotes the processing of amyloid precursor protein (APP) into neurotoxic β-amyloid and is hypothesized to act in RGCs to increase vulnerability to glaucoma. I studied retinal tissue sections from human donor eyes with and without the APBB2 risk allele and found greater immunoreactivity of APBB2 and β-amyloid in RGCs of African individuals with the risk allele compared to those without. These findings are limited to a small sample size but are supportive of increased production of APBB2 and subsequent increase of neurotoxic β-amyloid formation within RGCs as a possible mechanism of increased POAG risk conferred by APBB2.
Finally, to facilitate future studies of RGC degeneration and pave the way for testing of neuroprotective therapy, I characterized a genetic mouse model of congenital glaucoma. Unlike other mouse models of inherited glaucoma which require extensive aging for disease to manifest, nee mice exhibit elevated IOP and features of glaucoma within the first three months of life. I defined the specific time course of RGC death by quantifying RGCs in cohorts of young nee mice from 4-15 weeks of age. Nee mice showed a 49% reduction in the number of RGCs expressing the RGC-specific transcription factor BRN3A by 8 weeks of age. I also used morphometric data quantified from BRN3A+ nuclei to test whether RGCs of a certain size are more vulnerable to glaucoma. I found that RGC nucleus size changes dynamically with disease stage, whereby all RGCs increase in size in the early stages of disease, while small RGCs compose a larger proportion of the total RGC population in later stages of disease. These studies show that RGC size is dynamic and dependent on disease stage, presenting a challenge in addressing the longstanding question of whether there is a relationship between cell size and vulnerability to death.
Details
- Title: Subtitle
- Investigations of human genetics and mouse models in the study of glaucoma
- Creators
- Carly Jane van der Heide
- Contributors
- Michael G Anderson (Advisor)John H Fingert (Advisor)Wallace L Alward (Committee Member)Robert C Piper (Committee Member)Mark Stamnes (Committee Member)
- Resource Type
- Dissertation
- Degree Awarded
- Doctor of Philosophy (PhD), University of Iowa
- Degree in
- Molecular Physiology and Biophysics
- Date degree season
- Spring 2021
- Publisher
- University of Iowa
- DOI
- 10.17077/etd.006227
- Number of pages
- xvii, 177 pages
- Copyright
- Copyright 2019 Carly Jane van der Heide
- Language
- English
- Description illustrations
- color illustrations
- Description bibliographic
- Includes bibliographical references
- Public Abstract (ETD)
Glaucoma is the leading cause of irreversible vision loss worldwide. Glaucoma risk is influenced by a combination of environmental factors and genetics. Some genes have strong effects, where a single mutation can cause disease on its own with little influence from other genes or the environment. Conversely, other genes have small effects on glaucoma risk, not causing disease by themselves but acting together in combination to increase risk. By studying the function of these genes, we can identify which biological processes are disrupted in glaucoma and target them with treatment.
In this thesis, I performed several studies using genetics to understand glaucoma. First, I analyzed DNA from a large group of patients with a subtype of glaucoma to identify new gene candidates for causing disease. I also studied two mutations in a previously identified gene, myocilin, which often causes glaucoma with high intraocular pressure, a major risk factor. In subsequent studies, I investigated the biology of another gene, APBB2, which was recently identified to be a risk factor in individuals with African ancestry and is hypothesized to promote the death of neurons in a manner like that seen in Alzheimer’s disease. Finally, I studied the features of glaucoma in a mouse strain with a genetic mutation that causes disease in the first few months of life. These mice with inherited glaucoma are a valuable tool for research because they are severely affected when they are young and can be used to test therapy to protect against neurodegeneration caused by glaucoma.
- Academic Unit
- Molecular Physiology and Biophysics
- Record Identifier
- 9984188579502771
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