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Patient-based research powers the revealing of disease mechanisms

Digital EditionOphthalmology Times: March 15, 2021
Volume 46
Issue 5

Approach can unravel causes in MYOC and TBK1 glaucoma.

This article was reviewed by John H. Fingert, MD, PhD

Patient-based research is proving to be a powerful approach to unraveling the causes of diseases in affected patients.

John H. Fingert, MD, PhD, a professor of ophthalmology and visual sciences at Carver College of Medicine, University of Iowa, in Iowa City, described how patient-based research led to identification of a mutation that caused juvenile open-angle glaucoma (JOAG) resulting from abnormal accumulation of mutant myocilin (MYOC) protein in the trabecular meshwork (TM) cells and to identification of how the TANK-binding kinase 1 (TBK1) gene causes normal tension glaucoma (NTG).

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The research in both cases led to an understanding of the mechanism of the mutations and potential treatments.

The vast majority of glaucoma cases have complex genetics and result from the combined action of many genes and environmental factors; no one gene is responsible for causing this type of glaucoma.

More than 47 risk factors genes for glaucoma have been identified, Fingert pointed out.

In contrast are the cases with simple genetics, which represent about 5% of all glaucoma cases. Glaucoma cases with simple genetics are caused primarily by mutations in single genes.

The 3 genes identified as causes of these cases are MYOC, OPTN (the optineurin gene), and TBK1.

“Mutations in MYOC glaucoma are more common in patients with higher intraocular pressure [IOP], that is, over 21 mm Hg. Mutations in OPTN and TBK1 glaucomas are more common in patients with primary open-angle glaucoma [POAG] with IOPs of 21 mm Hg or lower,” Fingert explained.

Related: Exploring the evolution of interventional glaucoma

He and his colleagues set out to investigate the cause of glaucoma in 2 large families that have disease caused by MYOC and TBK1 mutations by creating animal models—animals engineered to carry each family’s glaucoma-causing mutation.

MYOC mutation
Case 1 was a boy who had been diagnosed with JOAG when he was aged 16 years, when his IOP exceeded 50 mm Hg.

He had a strong family history of glaucoma. Genetic testing detected a Tyr437His mutation in the MYOC gene. Studies of this family were used to show that MYOC mutations cause glaucoma.

The prevalence of MYOC glaucoma has been reported in many case-control studies ranging from 8% to 63% of JOAG cases, 3% to 4% of POAG, and 1% of NTG.

“These data showed that the MYOC mutation is the most common molecular-defined glaucoma,” Fingert said.

Glaucoma seems to develop as the result of abnormal intracellular accumulation of mutant MYOC protein in the TM cells, as seen in an autopsy eye of a patient with MYOC glaucoma.

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When cells in the TM were compared between healthy eyes and eyes with POAG, the activity in the cells differed markedly.

In healthy eyes, the normal MYOC gene with a normal DNA sequence directs production of MYOC protein, which leaves the cell via the secretory pathway.

In an eye with POAG, a mutant MYOC gene produces mutant protein that folds improperly and accumulates in the cell. This protein may be toxic to TM cells, causing them to become dysfunctional with elevated IOP and glaucoma, Fingert explained.

Investigators at the University of Iowa developed a transgenic murine model with the same mutation as the patient in case 1.

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The mice developed high IOP and optic nerve damage.1 Using the murine model of MYOC glaucoma, a possible therapy was identified.

4-phenylbutyrate (PBA) was theorized to help MYOC protein fold properly, thus preventing accumulation in the TM cells.

When the mice were treated with PBA, the IOP remained normal despite the MYOC mutation and no retinal ganglion cell (RGC) loss or optic nerve damage occurred. The outcome was that PBA cured MYOC glaucoma in mice caused by the Tyr437His mutation in MYOC glaucoma.

Gene editing using CRISPR/Cas9 technology also has been tested as a potential glaucoma therapy in the transgenic MYOC mice, with the goal of silencing the mutant MYOC gene.2

“The gene editing was successful in lowering IOP in young MYOC mice and reduced high IOPs in older mice. Gene editing was able to cure mice with a Tyr437His mutation,” he reported.

Related: Multi-pressure dial: A new approach to lowering IOP and treating glaucoma

TBK1 mutation
Case 2 was a 30-year-old patient diagnosed with NTG with IOPs of about 15 mm Hg, severe visual loss bilaterally, and a strong family history of glaucoma.

Genetic testing identified duplication of the TBK1 gene. Studies of this family showed that gene duplication causes glaucoma.

Fingert and colleagues conducted case-control studies that showed that about 1% of 5 patient cohorts had duplicate or triplicate TBK1 genes that caused NTG. A close look at the generations of family members showed that in some patients IOPs below 10 mm Hg were necessary to slow or prevent disease progression.

Fingert and colleagues created a transgenic murine model of TBK1 glaucoma with an extra TBK1 gene and found that more TBK1 was produced in the RGCs.3

The mice developed the same features of human TBK1 glaucoma, that is, normal IOP and progressive RGC death with aging as in humans.

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The investigators hypothesized the following mechanism of TBK1 glaucoma. TBK1 stimulates autophagy, a process that breaks down cellular structures.

In the murine model, TBK1 gene duplication causes RGC death by promoting excess autophagy.

“The ultimate loss of too many vital cell structures favors RGC death in NTG in this model,” Fingert said.

To test this, he and his colleagues used pluripotent stem cells induced from a skin biopsy from the patient in case 2.

The stem cells then were differentiated into RGCs that expressed key markers of the neurons. They found a large increase in LC3-II, a key marker for activation of autophagy in cells from patients with TBK1 gene duplication, suggesting that this duplication may stimulate autophagy in RGCs.4

Therapies to inhibit TBK1 have not been tested in a TBK1 murine mode.

Related: Exploring the evolution of interventional glaucoma

However, amlexanox (Selleck USA) is a drug that blocks TBK1 activity and may also block stimulation of autophagy.

Amlexanox has been investigated as a potential therapy for glaucoma using a different animal model (OPTN transgenic mice) and has shown some promise.

“Amlexanox has great potential to treat glaucoma in TBK1 transgenic mice and possibly humans with TBK1 glaucoma,” he said. “These 2 cases of MYOC and TBK1 glaucoma show the great power of patient-based research,” Fingert concluded.


John H. Fingert, MD, PhD
e: john-fingert@uiowa.edu
This article was adapted from Fingert’s presentation at the 2020 virtual meeting of the American Academy of Ophthalmology. He is a consultant for Perfuse Therapeutics.


1. Zode GS, Kuehn MH, Nishimura DY, et al. Reduction of ER stress via a chemical chaperone prevents disease phenotypes in a mouse model of primary open angle glaucoma. J Clin Invest. 2011;121(9):3542-3553. doi:10.1172/JCI58183

2. Jain A, Zode G, Kasetti RB, et al. CRISPR-Cas9-based treatment of myocilin-associated glaucoma. Proc Natl Acad Sci U S A. 2017;114(42):11199-11204. doi:10.1073/pnas.1706193114

3. Fingert JH, Miller K, Hedberg-Buenz A, et al. Transgenic TBK1 mice have features of normal tension glaucoma. Hum Mol Genet. 2017;26(1):124-132. doi:10.1093/hmg/ddw372.

4. Tucker BA, Solivan-Timpe F, Roos BR, et al. Duplication of TBK1 stimulates autophagy in iPSC-derived retinal cells from a patient with normal tension glaucoma. J Stem Cell Res Ther. 2014;3(5):161. doi:10.4172/2157-7633.1000161

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