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© 2016. Published by The Company of Biologists Ltd.

This is an Open Access article distributed under the terms of the Creative Commons Attribution License

(http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution and reproduction

in any medium provided that the original work is properly attributed.

YBR/EiJ mice: a new model of glaucoma caused by genes on chromosomes 4 and

17

K Saidas Nair2, Mihai Cosma3, Narayanan Raghupathy3, Michael A Sellarole2, Nicholas G Tolman3, Wilhelmine de Vries3, Richard S Smith3, and Simon WM John1,3,4*

1 Howard Hughes Medical Institute, The Jackson Laboratory, Bar Harbor, ME, USA. 2 Department of Ophthalmology, University of California, San Francisco, USA. 3 The Jackson Laboratory, Bar Harbor, ME, USA. 4 Department of Ophthalmology, Tufts University School of Medicine Boston, MA, USA

*Author for Correspondence:

Simon W.M. John

The Howard Hughes Medical Institute

The Jackson Laboratory

600 Main St.

Bar Harbor, ME 04609

simon.john@jax.org

voice: 207-288-6496 fax: 207-288-6671

Key Words: Mouse model, Glaucoma, Genetics, Ocular Diseases

http://dmm.biologists.org/lookup/doi/10.1242/dmm.024307Access the most recent version at DMM Advance Online Articles. Posted 9 June 2016 as doi: 10.1242/dmm.024307http://dmm.biologists.org/lookup/doi/10.1242/dmm.024307Access the most recent version at

First posted online on 9 June 2016 as 10.1242/dmm.024307

http://dmm.biologists.org/lookup/doi/10.1242/dmm.024307http://dmm.biologists.org/lookup/doi/10.1242/dmm.024307

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Summary Statement: We introduce and characterize the YBR/EiJ mouse strain as a

model of glaucoma, exhibiting high intraocular pressure and glaucomatous

neurodegeneration. Utilizing this model, we have identified genetic loci contributing to

glaucoma phenotypes.

Abstract

A variety of inherited animal models with different genetic causes and distinct genetic

backgrounds are needed to help dissect the complex genetic etiology of glaucoma. The

scarcity of such animal models has hampered progress in glaucoma research. Here, we

introduce a new inherited glaucoma model: the inbred mouse strain YBR/EiJ (YBR). YBR

mice develop a form of pigmentary glaucoma. They exhibit a progressive age-related

pigment dispersing iris disease characterized by iris stromal atrophy. Subsequently, these

mice develop elevated intraocular pressure (IOP) and glaucoma. Genetic mapping studies

utilizing YBR as a glaucoma susceptible and C57BL/6J as a glaucoma resistant strain

was performed to identify genetic loci responsible for the iris disease and high IOP. A

recessive locus linked to Tyrp1b on Chr4 contributes to iris stromal atrophy and high IOP.

However, this is not the only important locus. A recessive locus on YBR Chr17 causes

high IOP independent of the iris stromal atrophy, and in eyes with angles (location of the

ocular drainage tissue) that are largely open. The YBR alleles of genes on Chromosomes

4 and 17 underlie the development of high IOP and glaucoma but do so by independent

mechanisms. Together, these two loci act in an additive manner to increase the

susceptibility of YBR mice to developing high IOP. The Chromosome 17 locus is

important not only as it causes IOP elevation in mice with largely open-angles but also

because it exacerbates IOP elevation and glaucoma induced by pigment dispersion.

Therefore, YBR mice are a valuable resource for studying the genetic etiology of IOP

elevation and glaucoma, as well as for testing new treatments.

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Introduction

Glaucoma is a heterogeneous group of diseases characterized by death of

retinal ganglion cells, specific visual field deficits, and optic nerve degeneration. It is a

leading cause of blindness worldwide and affects over 70 million people (Quigley, 1996).

Elevated intraocular pressure (IOP) is often associated with glaucoma and is one of the

strongest known risk factors(Gordon et al., 2002). Normal IOP is controlled by a balance

between aqueous humor (AqH) production by the ciliary body and its drainage through

the trabecular and uveoscleral drainage pathways(Fautsch and Johnson, 2006; Shields,

1992). Decreased AqH drainage is believed to be responsible for elevation of IOP in most

cases. Experimentally elevating IOP can cause glaucoma in animals, supporting a

harmful role for elevated IOP in human glaucoma (Morrison et al., 1990; Quigley et al.,

1980).

Some autosomal dominant and recessive mutations causing glaucoma have

been uncovered (Liu and Allingham, 2011). However, the mutations important for IOP

elevation and susceptibility to neurodegeneration in most individuals with glaucoma are

largely unknown. Glaucoma, in general is thought to be a complex disease. The disease

outcome in most glaucoma cases does not depend on a single genetic mutation but is

instead controlled by mutations in multiple genes. The underlying complexity makes it

difficult to identify glaucoma genes. Recent advances in genome-wide studies have led to

the identification of multiple genes associated with glaucoma in humans (Bailey et al.,

2016; Burdon et al., 2011; Ramdas et al., 2011; Wiggs et al., 2012). A remaining

challenge is to prove the importance of the glaucoma-associated genes in disease

pathogenesis.

Use of mouse models is a viable approach for dissecting the complex genetics

of IOP elevation and glaucoma for several reasons. First, there are striking similarities

between the human and mouse ocular drainage structures. The development and

organization of these structures, including the trabecular meshwork, closely resemble

each other in mice and humans (Civan and Macknight, 2004; Gould et al., 2004; Smith et

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al., 2001; Tamm, 2013). Second, the dynamics of aqueous humor production and outflow

are similar between mice and humans (Aihara et al., 2003; Crowston et al., 2004). Thus,

mice provide a valuable model for validating genes associated with risk of high IOP and

glaucoma in human studies. Furthermore, mice provide an important experimental

platform for elucidating molecular mechanisms and discovering new glaucoma genes.

Glaucoma gene discovery using mice requires phenotypically affected mouse

strains that are often inbred (Anderson et al., 2002; Chang et al., 1999; John et al., 1998),

chemically mutagenized (Cross et al., 2014; Gould et al., 2007; Lachke SA, 2011; Nair

KS, 2011) or mice with other induced or spontaneous mutations (Buys et al., 2014; Chang

et al., 2001; Mao et al., 2011). This strategy of using mutant mice with glaucoma-relevant

phenotypes has been effective for identifying causative genes and understanding the

underlying disease mechanism. For example, the inbred mouse strain DBA/2J (D2) is a

widely used glaucoma model. These mice spontaneously develop a chronic, age-related

glaucoma. Mutations in two D2 genes [tyrosinase-related protein 1, Tyrp1, and

glycoprotein (transmembrane) nmb, Gpnmb] were shown to induce a depigmenting iris

disease that disperses pigment into the anterior chamber (Anderson et al., 2002; Chang

et al., 1999). After pigment dispersion, these mice develop high IOP, and pressure-

induced glaucomatous neurodegeneration (Libby et al., 2005a). The D2 model has

contributed significantly to our understanding of mechanisms that underlie pigment

dispersion, IOP elevation and optic nerve degeneration (Howell GR et al., 2011; Howell et

al., 2007a; Howell et al., 2007b; Howell et al., 2012; Libby et al., 2005b; Nickells et al.,

2012).

A variety of animal models with different genetic causes and genetic

backgrounds are needed to fully understand the complex etiology of glaucoma. Given

their relative affordability and experimental power, a variety of new mouse models are

needed for both gene identification/validation and mechanistic studies. A first step to

gene discovery using mice is to characterize disease phenotypes. Here we characterize

an inbred mouse strain, YBR/EiJ (YBR), as a new model of glaucoma. Identifying genetic

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factors contributing to glaucoma in YBR mice has potential to further our understanding of

mechanisms that underlie high IOP and glaucoma.

Results

YBR mice exhibit iris stromal atrophy and pigment dispersion

YBR mice were aged and, using a slit lamp, their eyes were assessed for iris

phenotypes and pupillary abnormalities every 2 to 3 months between 3 to 16 months of

age. YBR mice develop an age-related iris disease that primarily affects the iris stroma

and resembles the iris stromal atrophy phenotype of DBA/2J mice (Chang et al., 1999)

(Fig. 1 A-J). By 6 months of age, a mild iris stromal atrophy is evident (Fig. 1 B). With

increasing age, the iris depigmentation becomes more prominent and transillumination

defects are evident (areas of depigmented iris that allow light to pass through, Fig. 1). Iris

depigmentation is further corroborated by a histological examination of the iris (Fig. 1 K-

M). The iris depigmentation and cell loss is restricted to the iris stroma with no obvious

loss of iris pigment epithelium cells (Fig. 1 L and M).

YBR mice develop elevated IOP

We next determined the IOPs of YBR mice between 3 and 15 months of age.

Since 95% of values fall within 2 standard deviations (SD) of the mean and 18 mmHg is 2

(SD) from the mean of young YBR mice, we regard an IOP value >18 mmHg as high IOP.

As is common, we regard IOP values greater than 21 mmHg as glaucoma relevant, due

to the further increased risk for developing glaucoma at these values. Starting at 6-7

months, the number of YBR mice developing high IOP increases. With advancing age

there is a further increase in the proportion of mice with high IOP (Fig. 2 A, B and C). The

iris atrophy and pigment dispersion precede the IOP elevation and are likely to contribute

to inefficient AqH drainage. Histologic analyses show that following deterioration of the

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YBR iris stroma, the released pigment, and cellular debris initially accumulate at the

iridocorneal angle. The angle contains the ocular drainage structures (trabecular

meshwork and Schlemm’s canal) and runs for 360 degrees around the limbus of the eye.

With age, the pigment accumulation induces damage to the angle, which progresses to

the formation of focal tissue adhesions (anterior synechiae and angle closure) that block

AqH drainage (Fig. 2 D, and E).

Glaucomatous neurodegeneration in YBR eyes

To determine if high IOP leads to optic neuropathy, we performed a histological

assessment of the retinas and optic nerves of YBR eyes. Characteristic of glaucoma, the

retinal layers of the older YBR eyes were indistinguishable from those of the young YBR

eyes, except for the loss of retinal ganglion cells (RGCs) and thinning of the nerve fiber

layer, which contains RGC axons (Fig. 3 A). Aged YBR eyes also displayed optic nerve

excavation/remodeling a classic hallmark of glaucomatous neurodegeneration (Fig. 3 B).

Assessment of RGC axons in optic nerve cross sections at 8 months (an age before

significant elevation in IOP) demonstrated that essentially all axons had a healthy

morphology (Fig. 3 D). By 14 months, about 60% of YBR eyes had severe axon loss with

extensive gliosis and optic nerve damage (Fig. 3 C and D). Together, our data suggest

that YBR mice develop a pressure-induced optic nerve degeneration that is characteristic

of glaucoma.

Identification of genetic loci contributing to pigment dispersion and IOP elevation.

To identify genetic factors that contribute to the pigment-dispersing iris disease and

high IOP, we performed gene-mapping experiments. For these experiments, the

glaucoma susceptible YBR strain of mice was crossed to C57BL/6J (B6), a control strain

that does not develop glaucoma. The F1 progeny were backcrossed to YBR to allow

random segregation of chromosomal regions in the resulting N2 progeny. To determine if

iris disease was present and correlated with elevated IOP, N2 progeny underwent slit

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lamp examination and IOP measurements between the ages of 8 and 17 months. N2

progeny were genotyped using single nucleotide polymorphic (SNP) marker analysis.

Slit lamp examination of the N2 progeny revealed a spectrum of severity of iris phenotypes

that broadly formed 2 groups: mice with normal irides and mice with prominent iris disease

characterized by stromal atrophy and abnormal pigment dispersion (Fig. 4 A-D). Only mice with a

brown coat color exhibited the iris disease phenotype, but only 86% of mice with a brown coat

color had the iris disease. A recessive Tyrp1b allele on Chr4 is responsible for the brown coat

color present in various inbred mouse strains (Bennett et al., 1990), and we previously

demonstrated this Tyrp1b allele as responsible for the iris stromal atrophy phenotype in D2 mice (

Anderson et al., 2002; Chang et al., 1999). Our allele-specific and sequence analyses confirmed

that the YBR strain had the Tyrp1b allele. Tyrp1b allele encodes a mutant protein containing two

missense mutations resulting in the Cys to Tyr substitution at position 86 and the Arg to His

substitution at position 302 compared with the Tyrp1 allele of C57BL/6J mice. Tyrp1b results in a

brown coat color. It has been hypothesized that Tyrp1b alters the melanosomal membrane

allowing cytotoxic intermediates of pigment production to induce the iris stromal atrophy disease

(Anderson et al., 2002). These findings suggest that Tyrp1b underlies the iris disease in YBR

eyes.

Consistent with IOP elevation caused by the depigmenting iris disease and angle

closure, high IOP was common in eyes with iris disease. Interestingly, however, some

mice without any sign of iris disease developed high IOP (Fig. 4 E-F). It is well established

that within an eye the angle can have local regions that are either normal or abnormal

(Chang et al., 2001; Libby et al., 2003; Smith et al., 2000). Unlike the mice with extensive

iris disease and secondary angle closure, a subset of progeny develop high IOP

independent of the iris disease (normal iris, Fig. 4 H) and with largely open angles as

assessed by histology (>60% open or normal, Fig. 4 I-J). Thus, iris disease and high IOP

are sometimes genetically separable, indicating that factor(s) other than iris disease may

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contribute to high IOP. We, therefore, performed QTL analysis to identify genetic loci

contributing to high IOP. Our QTL analysis using IOP values as a continuous trait

detected two significant chromosomal regions: one on Chr4 (likely Tyrp1b) and one on

Chr17 (Fig. 5 A and Table 1). Next and to delineate the effect of iris disease from other

factors contributing to high IOP, we performed a QTL analysis on a cohort of mice with a

black coat color, hence only heterozygous for the Tyrp1b region and not exhibiting iris

disease. This strategy increased the likelihood of detecting additional genes acting

independently of the iris disease that could have been missed in our earlier analysis. This

QTL analysis detected only the YBR Chr17 locus as significantly contributing to high IOP

(Fig. 5 B). This statistically demonstrates that a gene within the Chr17 locus contributes to

high IOP in the absence of the iris disease.

Iris disease and the YBR Chr17 locus induce high IOP

Further analysis of the implicated chromosomal regions (YBR Chr4 and Chr17)

using correlation analysis (linear regression) demonstrates that the mice homozygous for

recessive YBR loci (Y/Y) on either Chr4 or Chr17 develop significantly higher IOP

compared to mice heterozygous for each of the YBR loci (Y/B) (Fig. 6 A and B). To further

understand the role of the YBR Chr4 and Chr17 loci, we tested if these two loci interact to

induce high IOP using an ANOVA full model. Results of our analysis demonstrated that

the peak markers on Chr4 and Chr17 do not interact (p = 0.285). Next, we employed an

additive model to test the combined effect of markers on Chr4 and Chr17 on IOP

outcome. Exclusion of either the Chr4 or Chr17 region from the additive model caused a

significant decrease in the magnitude of IOP and frequency of mice developing high IOP

(p=0.0042 for effect of Chr4 exclusion and p= 2.2125 x 10-5 for the effect of Chr17

exclusion). In addition, a correlation analysis demonstrates that mice carrying both the

recessive YBR Chr4 and Chr17 loci develop significantly higher IOP compared to mice

heterozygous for a recessive allele in any one of the loci (Fig. 6 C), supporting recessive

effects for each gene.

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Discussion

Glaucoma is a complex and heterogeneous disease affected by many genetic

factors. Thus, the study of a variety of genetically diverse glaucoma models is warranted.

Here, we introduce and characterize the YBR/EiJ mouse strain as a new model of

glaucoma. YBR mice develop an age-related pigment dispersing iris disease. They

subsequently develop high IOP and hallmarks of glaucomatous neurodegeneration.

Additionally, we identify genetic loci that contribute to iris disease and high IOP in the

YBR strain. These mice will be important for identifying new genes and mechanisms that

cause high IOP and glaucoma.

Iris stromal atrophy in YBR mice has similarities to Essential Iris Stromal Atrophy.

The iris disease in YBR mice is characterized by iris stromal atrophy (Fig. 1).

Some aspects of the YBR phenotype have strong similarities to human essential iris

atrophy. In essential iris atrophy (EIA), iris stromal atrophy is often accompanied by

formation of abnormal adherence of the iris to cornea and trabecular meshwork

(peripheral anterior synechiae, PAS) and abnormal proliferation of corneal endothelium

over the trabecular meshwork and iris (iridodocorneal endothelial syndrome, ICE)

(Shields, 1979; Shields, 1997; Shields and Bourgeois, 1996). This leads to blockage of

aqueous humor access to the drainage pathway (secondary angle closure), and elevation

of IOP. In YBR eyes, the released iris stromal pigment and cellular debris accumulate

within the ocular drainage structures, subsequently leading to angle closure, IOP

elevation, and glaucoma. The etiology of EIA is not well understood. It is suggested to be

primarily an acquired disease, mediated by viral infection causing subclinical inflammation

around the corneal endothelium. However, some familial cases of EIA have been reported

and an interplay of genetic and environmental factors likely participate in disease

pathogenesis (Shields, 1979).

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Genetics of iris stromal atrophy

Phenotypic analyses of the segregating progeny from our mapping cross

demonstrate that the iris disease in these mice is linked to a gene responsible for brown

coat color. The Tyrp1b allele underlies a brown coat in various inbred mouse strains

(Bennett et al., 1990). Both YBR and D2 mice share the same allele of Tyrp1 (Tyrp1b).

Therefore, as is well established for glaucomatous D2 mice (Anderson et al., 2002; Chang

et al., 1999), the Tyrp1b allele is likely responsible for iris stromal atrophy and IOP

elevation in YBR mice. However, compared to the D2 mice, the degree of iris

depigmentation is less severe in age-matched YBR mice. This is likely because in D2

mice a digenic combination of mutant Tyrp1 and mutant Gpnmb is responsible for an

earlier onset and more severe iris disease, which impacts both the iris pigment epithelium

and the iris stroma (Chang et al., 1999). However, in YBR mice the iris pigment epithelium

remains largely intact and only the iris stroma undergoes the depigmenting disease (Fig.

1 K-M). Our genetic analysis failed to detect any additional factor(s) that contribute to iris

disease in the YBR mice. Moreover, YBR mice have a wild-type form of Gpnmb, ruling it

out as a factor contributing to their iris disease (unpublished data). It remains possible that

in YBR mice Tyrp1b interacts with multiple small effect genes that were undetected in our

analysis but may contribute to the iris disease phenotype. Interestingly, only 86% of

homozygous Tyrp1b N2 mice in the current study developed iris disease, compared to a

fully penetrant effect of Tyrp1b on iris disease in all of our previous studies on different

genetic backgrounds (Anderson et al., 2001; Chang et al., 1999; John et al., 1998),

suggesting a possible influence of other modifier gene(s).

Genetics of High IOP and glaucoma

Our results indicate that iris disease plays an integral role in the pathogenesis of

IOP elevation in YBR mice (Fig. 6 A). However, a distinct YBR locus on Chr17 induces

high IOP independent of the iris disease phenotype (Fig. 6 B). Based on statistical

modeling, we interpret our results as follows: the YBR Chr4 locus primarily dictates the iris

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disease phenotype and the deposited pigment and debris impact the drainage tissue,

contributing to angle closure and high IOP. The YBR Chr17 locus acts through a distinct

mechanism (not involving iris disease) to induce high IOP. Although these loci underlie

distinct pathogenic processes, they act in an additive fashion to promote an elevation of

IOP.

Interestingly, some of the mapping progeny with high IOP but no iris disease

(homozygous YBR Chr17 but not for YBR Chr4 locus) have largely open angles (>60% of

analyzed angle open and

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Materials and methods Animal husbandry

Mice were obtained from The Jackson Laboratory (Bar Harbor, ME, USA). All

experiments were conducted in accordance with the Association for Research in Vision

and Ophthalmology’s statement on the use of animals in ophthalmic research. The

Jackson Laboratory’s Institutional Animal Care and Use Committee approved all

procedures described here. The YBR/EiJ mice were maintained on a NIH31 diet (6% fat)

and HCl acidified water (pH 2.8-3.2). To avoid obesity, C57BL/6J (B6) mice and mapping

progeny were fed with the same NIH 31 diet but with a 4% fat content. Diets were

provided ad libitum. Mice were housed in cages with pine wood shavings as bedding and

covered with polyester filters. The cages were maintained in an environment kept at 21°C

with a 14-hour light: 10-hour dark cycle.

Clinical examination

Eyes were examined with a slit-lamp biomicroscope and photographed with a 40×

objective lens. All photographs were taken using identical camera settings. Phenotypic

evaluation included iris structure, pupillary abnormalities, cataracts, and the overall

dimensions of the anterior chamber. Iris disease was evaluated by indices of iris atrophy

and dispersed pigment following previously described criteria (John et al., 1998).

Transillumination assays were performed to assess the extent of iris depigmentation.

Briefly, a narrow beam of light passed through the pupil is reflected back through

depigmented areas of iris tissue, and are visualized as reddish light. Examination of at

least 30 mice of each genotype at 3, 6, 9, 12, and 14 months of age was performed. All

cohorts included male and female mice. Additionally, smaller groups of mice (10–15) were

analyzed every month between 3 and 16 months of age.

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IOP Measurement IOP was measured using the microneedle method as previously described in

detail (John et al., 1998). Briefly, mice were anesthetized by intraperitoneal injection of a

mixture of ketamine (99 mg/kg; Ketalar, Parke-Davis, Paramus, NJ) and xylazine (9

mg/kg; Rompun, Phoenix Pharmaceutical, St. Joseph, MO) prior to IOP assessment - a

procedure that does not alter IOP in the experimental window (Savinova et al., 2001). All

cohorts included male and female mice. In all our experiments, B6 mice were used along

with experimental mice as a methodological control to ensure proper equipment

calibration and function.

Optic Nerve Assessment

The optic nerves were collected from mice within 48 hours of IOP measurement. Each

age group contained samples from males and females, as well as left and right nerves.

Intracranial portions of the optic nerves were dissected and assessed for glaucomatous

damage as previously described (Howell et al., 2007a). Briefly, the optic nerve cross-

sections were stained with para-phenylenediamine (PPD) and examined for

glaucomatous damage. PPD stains the myelin sheaths of a healthy axon, but stains both

the myelin sheaths and the axoplasm of sick or dying axons, thus allowing very sensitive

detection and quantification of axon damage and loss according to previously validated

and published criteria [29].

Assessment of the drainage tissue

Hematoxylin and eosin stained ocular sections from 3 mapping progeny (6 eyes)

with black coat color (heterozygous for the Tyrp1b) and exhibiting high IOP were

assessed to determine if the angle were open or closed as a result of synechiae. We

evaluated 10 similarly spaced (15 to 20 microns) sections for 3 ocular regions (peripheral,

mid- peripheral and central). Both angles of a section were considered for evaluation.

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Thus, a total 60 angle locations were analyzed in each eye (10 sections X 2 angles X 3

regions). Thereafter, we calculated the percentage of angles that were open.

Gene Mapping and QTL studies

To map genes controlling iris disease and high IOP, glaucoma susceptible YBR

mice were crossed to glaucoma resistant B6 mice. All the F1 progeny exhibited no signs

of iris disease or high IOP. We utilized a backcross strategy by crossing F1 generation

progeny to the parent YBR strain. A total of more than 90 N2 progeny were aged up to 17

months and clinical examination of their eyes performed every 2 months between 3 and

17 months. IOP was measured at multiple time points between the ages of 8 and 17

months. For each test animal, the highest IOP value detected at any of the ages was

considered for subsequent QTL analysis. Initial genotyping was performed using over 116

genome-wide single nucleotide polymorphic markers that differentiate the YBR from the

B6 genome and spaced approximately at about 20 MB interval (SNPs, KBioscience, UK).

Y and B denote YBR and B6 alleles throughout the manuscript. We performed a genome-

wide one-dimensional QTL scan to identify the chromosomal loci regulating IOP. R/QTL

version 1.14-2 was used for QTL analysis (Broman et al., 2003). Inverse transformed

IOP values were used in the analysis since they showed a normal distribution. Pseudo-

markers were generated at 2 cM spacing for each chromosome, and a whole genome

scan was performed using 256 imputations. One thousand permutations were performed

to determine the thresholds for QTL detection. Three thresholds at 1%, 5%, and 10%

were calculated from the permutation results. A QTL with LOD score above the 1%

threshold was classified as a strong QTL, while those at or above 10% were classified as

a suggestive QTL. Genotyping quality assessment was done by evaluating the

recombination fraction plot and map plot Fine mapping of a critical YBR Chr 17 region (69

MB to 86 MB) was performed using MIT markers distinguishing YBR from B6 genome

(NCBI Build 37).

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Correlation analysis

We used rank-Z transformed IOP for the following statistical analysis. We tested

for the interaction effect between the markers on Chr 4 and Chr 17 on IOP. For this

purpose, we used a full model (IOP~Chr4+Chr17+Chr4:Chr17) including additive

main effects and their interactions and a reduced model (IOP~Chr4+Chr17) with only

additive main effects. We compared these two models using ANOVA and evaluated the

significance of interaction with F-statistic using R. After finding no interaction effect, we

used the additive model (IOP~Chr4+Chr17) and tested for each main effect by dropping

the corresponding term and computed the F-statistic. In addition, we performed t-tests

(two tail) to compare mice with various genotypic combinations of the implicated YBR

Chr4 and Chr17 loci. For data in Fig 6C, a significant p-value threshold 0.05 is equivalent

to 0.05/6=0.0083 by using Bonferroni correction for multiple comparisons (we

performed six pairwise comparisons).

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Acknowledgements

Weidong Zhang for computational components of QTL analysis. Jesse Hammer and

Suling Wang for preparing the figures. Amy Bell for IOP measurements. Histology core of

The Jackson Laboratory prepared the ocular histological sections.

Competing interests

No competing interests declared.

Authors' contributions

KSN, SWMJ conceived experiments, analyzed the data and prepared the manuscript.

SWMJ oversaw all aspects of the study. MC bred the mice and performed clinical analysis

and IOP measurement of the mice. MS, NT contributed towards colony management,

genotyping and histological analysis. WNdV contributed to colony management and

manuscript preparation. NR performed the QTL and statistical analysis, and contributed to

manuscript preparation. RS performed angle and optic nerve assessment.

Funding

This work was supported in part NIH P30 core grant for vision research (UCSF,

Ophthalmology), Research to Prevent Blindness unrestricted grant (UCSF

Ophthalmology) and NEI grants, EY011721 (SWMJ), EY022891 (KSN), National

Glaucoma Research Program of the Bright Focus Foundation (KSN), the Partridge

Foundation (SWMJ) and the Barbara and Joseph Cohen Foundation. SWMJ is an

Investigator of The Howard Hughes Medical Institute.

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Figures

Figure 1. YBR mice exhibit a form of pigment dispersing iris disease: YBR mice exhibit a

progressive iris disease with age, characterized by iris stromal atrophy and pigment dispersion.

(A-E) The top row of panels shows broad beam illumination of YBR eyes at each indicated age to

assess the presence of dispersed pigment within the anterior chamber and iris stromal

morphology. (F-J) The bottom row of panels shows transillumination to assess the degree of iris

depigmentation as observed by areas where reflected light passes through the iris (white

arrows). Pupils are outlined by red dotted lines. In transillumination, reflected light passes

through the iris, after reflecting off the posterior pole of the eye. Normally, this reflected

light would be blocked by the iris.

(A, F) Three-month old YBR eyes have normal iris morphology with clearly evident iris

details. (B, G) By 6 months, mutant mice exhibit mild iris disease characterized by the

mild accumulation of iris pigment in the peripupillary region. (C, H) By 9 months, YBR

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eyes exhibit iris stromal atrophy characterized by thinning of the iris stroma (black

arrowhead) and mild transillumination defects (white arrow, bottom panel). The degree of

iris atrophy progressively worsens with age. Accumulation of dispersed pigment is

greatest in the inferior angle (black arrow, top panel) due to gravity. (D, I) At 12 mo, some

of the YBR eyes exhibited vascularization of the cornea (black asterisk), and most eyes

show worsening transillumination defects (white arrow). (K) In a young YBR mouse, the

iris is well developed and has an intact stroma (S) and pigment epithelium (P). (L) The iris

of an old YBR mouse exhibiting moderately atrophied stroma (black arrow) and intact

pigment epithelium (open arrow). (M) In severe cases, the stroma is largely atrophied with

areas where it is essentially non-existent (black arrow). The iris pigment epithelium of

these mice remains remarkably intact considering the overall condition of the iris (open

arrows). Scale=50m

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Figure 2. YBR mice exhibit elevated IOP subsequent to iris disease

(A) The IOP values at each of the indicated ages are represented as a scatter plot.

Dashed line indicates IOP at 21 mm of Hg (regarded as glaucoma relevant). (B). The

same IOP values are also represented as a bar graph of the mean IOP ± SEM. (C)

Percent of eyes with IOP at each binned level at the indicated ages. All groups contained

at least 30 mice and included both males and females in similar proportions. With age,

YBR eyes develop high IOP. By 12-13 months, the IOP values level out, as there is no

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further increase in mean IOP with advancing age. (D) Young YBR mice have normal,

open iridocorneal angles. The angle has a well-formed trabecular meshwork (TM,

asterisk) and an open Schlemm's Canal (SC). (E) In an aged 14 mo old YBR mice, there

is variability in the angle morphology. Some angle regions exhibit anterior synechia

(arrow) occluding the drainage tissue. Schlemm’s canal and the trabecular meshwork are

occluded and not readily identifiable. Scale=100m.

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Figure 3. YBR mice develop glaucomatous neuropathy: (A) Retinas of representative

YBR mice. Eight month old (without any sign of glaucoma; left panel) and 14 month old

(exhibiting glaucoma; right panel). Both retinas have a normal outer segment (OS), outer

nuclear layer (ONL) and inner nuclear layer (INL). However, the retina from the14 month

old YBR mouse has fewer retinal ganglion cells in the ganglion cell layer (GCL). (B) YBR

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eyes at 8 months have a normal optic nerve head characterized by a thick nerve fiber

layer entering the optic nerve (arrows facing each other) and a central vessel (V). By 14

months, YBR eyes develop advanced optic nerve excavation and extreme thinning of the

nerve fiber layer (arrows facing each other). Scale=50 m (C) Optic nerve degeneration

was analyzed using PPD-stained optic nerve cross sections. Nerves from 8 months old

mice (representative image - left panel) had no detectable axonal damage and axons

have a clear axoplasm and darkly stained myelin sheaths. By 14 months, however,

nerves had severe damage and showed extensive axon loss and glial scarring

(representative image – right panel). (D) A bar graph representing the degree of optic

nerve damage at indicated ages. There were at least 30 eyes in each group with both

sexes in similar proportion. At 8 months, none of the YBR eyes exhibits glaucomatous

neurodegeneration. By 14 months, about 60% of YBR eyes exhibited severe

glaucomatous nerve damage and axonal loss. Scale=50m.

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Figure 4. Iris disease and high IOP are genetically separable: (A-F) Representative

images (broad beam illumination) of young control YBR mice (4 mo) and old affected N2

mice (14 mo) that are heterozygous or homozygous for the YBR derived Tyrp1b allele. Y

and B denote YBR and B6 alleles respectively. (A-D) Mice that are homozygous for

Tyrp1b (Tyrp1Y/Y) develop age-related iris stromal atrophy and their anterior chambers

become deepened due to high IOP. (E,F) Some heterozygous mice (Tyrp1Y/B) despite

having an intact iris develop high IOP (range 21.1 – 32.1 mmHg) and exhibit deep anterior

chambers. This suggests that iris disease and high IOP are genetically separable. (G-H)

Representative image of the iris from N2 mice (14 mo) that are homozygous for the Chr17

(Y/Y) locus but are either homozygous (Tyrp1Y/Y) or heterozygous (Tyrp1Y/B) for Tyrp1. ( I)

Representative images from a histological evaluation of angles spanning much of

thickness of the eyes of Tyrp1Y/B mice (Methods). Despite high IOP, a normal iris and

open angle is observed in most regions of this eye. Open arrows indicate an iris process,

structures known to be in close proximity to the drainage tissue even in normal mouse

eyes. Despite the sectioning-based distortion that displaced the iris processes towards

the drainage structures, this section clearly shows a normal, open mouse angle. (J)

Although >60% of the angle was open in each of these eyes, there were local regions

with angle closure due to anterior synechia (arrow). Local angle closure is common in

non-glaucomatous mouse strains with normal IOP at this age, likely due to their naturally

very narrow angles. Scale=100m.

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Figure 5. QTL analyses identify genetic regions contributing to high IOP in YBR

mice: (A) Genome-wide scan using IOP values as a quantitative trait in N2 mapping

progeny identify two significant QTLs: on Chr4 and Chr17. The blue, green and red lines

indicate the significance thresholds at 1%, 5%, and 10% respectively. Both the Chr4 and

Chr17 regions show LOD scores above the 1% threshold. (B) A genome-wide scan of

only black mice (Tyrp1Y/B, with no iris disease) identify a significant QTL on Chr17.

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Figure 6. Factors independent of the iris disease contribute to high IOP: Graphs

showing the relationship between IOP (transformed values, see methods) and the

genotypes at the two significant QTL markers at Chr 4 and Chr17. (A, B) Mice

homozygous for either a recessive YBR Chr4 or YBR Chr17 loci have significantly higher

IOP than mice carrying their respective heterozygous alleles (p-value = 4.239 X 10-3 for

YBR Chr4 comparison and p-value = 2.125 X 10-6 for YBR Chr17 comparison). (C) Mice

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homozygous for both the recessive YBR Chr4 and YBR Chr17 loci respectively (Y/Y,

Y/Y), have significantly higher IOP compared to mice carrying heterozygous alleles on

both the loci (Chr4 Y/B, Chr 17 Y/B) or a recessive allele on any one of the loci (Chr4 Y/Y,

Chr17 Y/B or Chr4 Y/B, Chr17 Y/Y). p=1.05 X 10-7 for “Chr4 Y/Y, Chr17 Y/Y” vs “Chr4

Y/B, Chr17 Y/B” comparison. p=5.75 X 10-4 for “Chr4 Y/Y, Chr17 Y/Y” vs “Chr4 Y/Y,

Chr17 Y/B” comparison and p=1.07 X 10-3 for “Chr4 Y/Y, Chr17 Y/Y” vs “Chr4 Y/B, Chr17

Y/Y” comparison.

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Table 1

Chromosome Critical Interval (bp) LOD Risk Allele Inheritance

4 80,834123 - 93,224754 3.64 YBR Recessive

17 73,461956 - 86,112456 5.14 YBR Recessive