1 An RNA Sequencing Analysis of Glaucoma Genesis in Mice

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An RNA Sequencing Analysis of Glaucoma

Genesis in Mice

Jai Sharma1and Vidhyacharan Bhaskar2,*

1Student Intern Researcher, Robinson Lab, Stanford University;

269 Campus Drive, Palo Alto, CA 94305; Email: jaisharmaus@gmail.com

2Professor, School of Engineering, San Francisco State University;

1600 Holloway Avenue, San Francisco, CA 94132

*Corresponding Author’s Email: vcharan@gmail.com

Abstract—Glaucoma is the leading cause of irreversible blind-

ness in people over the age of 60, accounting for 6.6 to 8%

of all blindness in 2010, but there is still much to be learned

about the genetic origins of the eye disease. With the modern

development of Next-Generation Sequencing (NGS) technologies,

scientists are starting to learn more about the genetic origins

of Glaucoma. This research uses differential expression (DE)

and gene ontology (GO) analyses to study the genetic differ-

ences between mice with severe Glaucoma and multiple control

groups. Optical nerve head (ONH) and retina data samples

of genome-wide RNA expression from NCBI (NIH) are used

for pairwise comparison experimentation. In addition, principal

component analysis (PCA) and dispersion visualization methods

are employed to perform quality control tests of the sequenced

data. Genes with skewed gene counts are also identiﬁed, as they

may be marker genes for a particular severity of Glaucoma.

The gene ontologies found in this experiment support existing

knowledge of Glaucoma genesis, providing conﬁdence that the

results were valid. Future researchers can thoroughly study the

gene lists generated by the DE and GO analyses to ﬁnd potential

activator or protector genes for Glaucoma in mice to develop drug

treatments or gene therapies to slow or stop the progression of

the disease. The overall goal is that in the future, such treatments

can be made for humans as well to improve the quality of life for

human patients with Glaucoma and reduce Glaucoma blindness

rates.

Index Terms—mRNA Sequence Data; Statistical Analysis;

Differential Expression Analysis; Gene Ontology Analysis; Glau-

coma; Ophthalmology

I. INTRODUCTION

A. Background

The neurodegenerative eye disease Glaucoma is a part of a

group of eye diseases which can cause blindness. Glaucoma

in particlar is one of the leading causes of blindness in people

over the age of 60 [1]. The most common type of Glaucoma,

Primary Open Angle Glaucoma (POAG)1, is caused when

resistance in the Trabecular Meshwork leads to increased

Intraocular Pressure (IOP) in the eye. As a result, aqueous

humor, which supplies nutrients to the cornea, is unable to

ﬂow from the Posterior Chamber to the Anterior Chamber,

through the Trabecular Meshwork, and back into the body.

1This paper will be discussing only Primary Open Angle Glaucoma, so

the terms “Glaucoma” and “Primary Open Angle Glaucoma” will be used

interchangeably unless otherwise speciﬁed.

In POAG, a gradual increase in IOP leads to damage to

the optic nerve and the chronic and irreversible onset of

Glaucoma. As POAG progresses, the IOP increase leads

to cupping of the optic disc; while the ratio between the

diameter of the optic cup to the diameter of the optic disc

is 0.5 in a normal eye, this ratio increases during cupping

in POAG [1]. Left untreated, Glaucoma takes on average 25

years to progress from normal vision to complete blindness.

Even with treatment, 9% to 19% of patients reach complete

blindness, implying a need for treatment improvements [2].

Glaucoma in general is characterized by the cupping

and atrophy of the optic nerve head, visual ﬁeld loss of the

patient, and often the increased IOP [3]. Thapa, et al. studied

a subset of the Nepalese population and found that the

occurrence rate of Glaucoma was 1.9%. Approximately 68%

of these cases were Primary Open Angle Glaucoma, 22.67%

of these cases were Primary Angle Closure Glaucoma,

and 9.33% of these cases were secondary Glaucoma [4].

Casson, et al. highlighted that while Glaucoma used to be

characterized by high IOP, many cases of Glaucoma without

high IOP and high IOP without Glaucoma disputed this

assumption. Now, high IOP is simply considered a risk factor

rather than a deﬁning feature, and reducing IOP is currently

the only treatment strategy for Glaucoma [5].

B. Risk Factors and Symptoms

It is known that old age, family history, Black ethnic

origin, and Myopia (nearsightedness) are all risk factors for

Glaucoma. Once a patient is diagnosed with Glaucoma, routine

screening by an optometrist is used to ensure the disease’s slow

progression. As POAG progresses, the patient experiences

a loss of peripheral vision (tunnel vision), ﬂuctuating pain,

headaches, and blurred vision. Symptoms are usually worse at

nighttime or in dark spaces [1].

C. Current Diagnosis and Treatment Methods

While 111.8 million people are expected to have Glaucoma

by 2040, there is much to be learned about the genetic

origins and development of this disease. Glaucoma (all types

collectively) have a 10% diagnosis rate worldwide and less

arXiv:2210.01546v1 [q-bio.GN] 2 Oct 2022

than 50% diagnosis rate in developed nations [2].

Currently, there are several ways to diagnose Glaucoma.

First, Non-Contact Tonometry can be used to shoot puffs

of air and measure puff response from the cornea. A small

reaction to the puff of air would be associated with high

IOP, and an according Glaucoma diagnosis can be made.

This method is relatively less accurate, but it is a good

estimation for eye pressure. The industrial standard for

Glaucoma diagnosis is the Goldmann Applanation Tonometry

method, which utilizes advanced equipment to make contact

with the cornea and apply different amounts of pressure

to ﬁnd the IOP, from which a Glaucoma diagnosis can be

made. Other forms of diagnosis include Fundoscopy, which

can help examine the eye for cupping, and the Visual Field

Assessment Test, which tests for peripheral vision loss [1].

Glaucoma is asymptomatic until it reaches an advanced stage,

so it is recommended that patients undergo regular screening

examinations from age 40 onward. The rate of false positives

for Glaucoma diagnosis is high, so it is also recommended

that any positive ﬁnding is followed up with further testing [6].

The IOP for a normal eye ranges from 10 mmHg

to 21 mmHg, and treatment is often started when IOP

reaches 24 mmHg. Current treatments include Prostaglandin

Analogues (e.g. Latanoprost) which increase uveoscleral

outﬂow, Topical Beta-Blockers (e.g. Timolol) and Carbonic

Anhydrase Inhibitors (e.g. Dorzolamide) which reduce

the production of aqueous humor, and Sympathomimetics

(e.g. Brimonidine) which increase uveoscleral outﬂow and

reduce the production of aqueous humor. Surgical methods

of treatment include Trabeculectomy which creates a new

channel for aqueous humor to drain from the eye; however,

this procedure is risky and its effects are only temporary

[1]. Conlon, et al. analyzed trends in Glaucoma treatments,

concluding that there has been a recent increase in Glaucoma

medications and the use of laser trabeculoplasty over the past

decade. Additionally, there has been a noticable decrease in

the frequency of invasive incisional surgery. New methods

such as microinvasive Glaucoma surgery have also been

introduced [7]. Despite advances in laser and incisional

surgery, Schwartz, et al. claim that medical therapy is still the

primary method of treatment and that there is good evidence

that laser trabeculoplasty is just as good as initial medical

therapy [8].

D. Research Goal

Crucial to the development of new cures for POAG is

the more sophisticated understanding of the disease at the

genetic level. The goal of this research is to ﬁnd the genes

responsible for the genesis of Glaucoma in mice so that

this information may be used to develop new treatments in

the future which target these genes or corresponding gene

pathways in mice. The overall goal is that future research can

develop new and more effective POAG treatments for humans.

This research is focused on generating candidate gene lists

which may contain potential activator or protector genes for

Glaucoma in mice. However, it should be noted that doing

intensive biological studies to study these gene lists manually

is outside the scope of this research.

Note that mice were used here because mice have

eyes which are similar to human eyes in that they develop

hereditary Glaucoma and high IOP, as will be explained in the

next section. This research and similar research on Glaucoma

have taken advantage of this unique property of mice.

II. LITERATURE REVIEW

Research has been conducted to explore the function

of ncRNA, transcriptional mRNA, and similar biological

molecules in Glaucoma, Cataracts, and different types of

cancers. This research has shown to successfully identify

targets for further therapeutic research to develop treatments

for the corresponding disease.

Chen, et al. explored time-series circRNA, lncRNA,

miRNA, and mRNA expression proﬁles of developing mice

retina samples. The aim of this research was to ﬁnd the

key functional ncRNA and ceRNA which regulated retinal

neurogenesis. The research found that several ncRNAs in

the circRNA/lncRNA-miRNA-mRNA network, including

circCDYL, circATXN1, circDYM, circPRGRIP, lncRNA

Meg3, and lncRNA Vax2os, involved neurotransmitter

transport and multicellular organism growth during retinal

development [13]. Zhou, et al. aimed to identify differing

biological mechanisms involved in each Glaucoma subtype.

Results from the research indicated that human participants

with high tension Glaucoma had an enrichment of genes

associated with unfolded protein response. Further, the

research identiﬁed the differential expression of genes in

certain IOP regulating tissues [2]. Howell, et al. used house

mice mRNA data set to investigate the genetic origins of

Glaucoma using temporally ordered sequences of Glaucoma

states. The overall goal was to identify molecular events

which can be targeted to provide effective new treatments

for human glaucoma. A major ﬁnding from this study was

that mice with a mutation in the C1QA gene were protected

from Glaucoma. Additionally, it was found that inhibition

of endothelin system with bosentan, an endothelin receptor

antagonist, was strongly protective against damage from

Glaucoma [14].

Current research methods perform genetic comparisons

between mice with Glaucoma and mice without Glaucoma,

but one of the drawbacks of this research is that it fails to

stratify these comparison based on speciﬁc features such

as age or strain. Thus, we lack insight into how these

features speciﬁcally contribute to or protect from Glaucoma.

The research presented in this paper intends to solve this issue.

This research focuses on ﬁnding potential activator

or protector genes for Primary Open Angle Glaucoma in

house mice. This research is signiﬁcant because one control

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1AnRNASequencingAnalysisofGlaucomaGenesisinMiceJaiSharma1andVidhyacharanBhaskar2,*1StudentInternResearcher,RobinsonLab,StanfordUniversity;269CampusDrive,PaloAlto,CA94305;Email:jaisharmaus@gmail.com2Professor,SchoolofEngineering,SanFranciscoStateUniversity;1600HollowayAvenue,SanFrancisco,CA94132*Corr...

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