Glaucoma Microarray Analysis of Iris Gene Expression in Mice with Mutations Influencing Pigmentation Colleen M. Trantow,1 Tryphena L. Cuffy,2 John H. Fingert,2,3 Markus H. Kuehn,2,3 and Michael G. Anderson1,2,3 PURPOSE. Several ocular diseases involve the iris, notably includ- lak syndrome, Chediak-Higashi syndrome, Horner’s syndrome, ing oculocutaneous albinism, pigment dispersion syndrome, Waardenburg syndrome, and Fuchs’ heterochromic iridocycli- and exfoliation syndrome. To screen for candidate genes that tis. In addition, other ocular diseases, such as pigment disper- may contribute to the pathogenesis of these diseases, genome- sion syndrome and exfoliation syndrome, involve disease-re- wide iris gene expression patterns were comparatively ana- lated morphologic changes to the pigmented tissues of the iris. lyzed from mouse models of these conditions. Each of these diseases involves strong hereditary links, but METHODS. Iris samples from albino mice with a Tyr mutation, much remains unknown concerning the underlying genetic pigment dispersion–prone mice with Tyrp1 and Gpnmb mu- pathways. In this study, we focused on three of these condi- tations, and mice resembling exfoliation syndrome with a Lyst tions: albinism, pigment dispersion syndrome, and exfoliation mutation were compared with samples from wild-type mice. syndrome. All mice were strain (C57BL/6J), age (60 days old), and sex In oculocutaneous albinism (OCA), there is reduced or (female) matched. Microarrays were used to compare tran- absent pigmentation of the skin, hair, and eyes. Decreased scriptional profiles, and differentially expressed transcripts melanin in the eyes can give rise to several ocular abnormali- were described by functional annotation clustering using ties, including foveal hypoplasia and decreased visual acuity; DAVID Bioinformatics Resources. Quantitative real-time PCR retinal ganglion cell axon misrouting; and strabismus, nystag- was performed to validate a subset of identified changes. mus, iris translucency, color vision impairment, and photopho- bia.1 The hereditary basis of OCA is complex. There are at least RESULTS. Compared with wild-type C57BL/6J mice, each dis- 4 genes that contribute to classic forms of OCA and at least ease context exhibited a large number of statistically significant another 12 associated with syndromic forms. The best under- changes in gene expression, including 685 transcripts differ- stood form of OCA, and the most common in many popula- entially expressed in albino irides, 403 in pigment dispersion– tions, is OCA1.2,3 OCA1 is caused by mutations in the tyrosi- prone irides, and 460 in exfoliative-like irides. nase (TYR) gene, which encodes the rate-limiting enzyme CONCLUSIONS. Functional annotation clusterings were particu- necessary for melanin synthesis. Most people with OCA1 are larly striking among the overrepresented genes, with albino believed to be compound heterozygotes, although in 15% of and pigment dispersion–prone irides both exhibiting overall OCA1 cases, the second mutation remains unidentified.4 Inter- evidence of crystallin-mediated stress responses. Exfoliative- estingly, TYR appears to also influence many traits beyond like irides from mice with a Lyst mutation showed overall pigmentation. For example, tyrosinase mutation is capable of evidence of involvement of genes that influence immune sys- rescuing a mouse model of pigment dispersion,5 but acts to tem processes, lytic vacuoles, and lysosomes. These findings worsen disease in mouse models of developmental glaucoma.6 have several biologically relevant implications, particularly Clearly, much remains unknown concerning TYR and its influ- with respect to secondary forms of glaucoma, and represent a ences on ocular disease. useful resource as a hypothesis-generating dataset. (Invest Oph- In pigment dispersion syndrome, liberated pigment from thalmol Vis Sci. 2011;52:237–248) DOI:10.1167/iovs.10-5479 the iris pigment epithelium becomes aberrantly deposited throughout the anterior chamber. As pigment accumulates in he iris plays an essential role in regulating the amount of the iridocorneal angle, aqueous humor outflow resistance and Tlight passing to the retina and is also important in many intraocular pressure can become elevated.7,8 Although pig- human diseases. Several diseases change iris pigmentation, ment dispersion syndrome has strong hereditary links,9,10 the including forms of oculocutaneous albinism, Hermansky-Pud- genetic risk factors remain to be identified. DBA/2J mice de- velop a form of pigmentary glaucoma involving a pigment- dispersing iris disease, elevated intraocular pressure, and optic nerve damage.11,12 Mutations in two genes encoding melano- From the 1Department of Molecular Physiology and Biophysics, 2 3 somal proteins, Tyrp1 and Gpnmb, are responsible for initia- the Interdisciplinary Graduate Program in Genetics, and the Depart- 13 ment of Ophthalmology and Visual Sciences, University of Iowa, Iowa tion of the DBA/2J disease process. To date, genetic studies City, Iowa. of TYRP1 and GPNMB in human pigment dispersion patients Supported by National Eye Institute Grant EY017673 and Supple- have not detected mutations,13,14 suggesting that other genes mentary Grant EY017673-02S2 (MGA) and a Grant from The Glaucoma in a pathway linked to TYRP1 and GPNMB may be the next Foundation (MGA, JHF). MHK is supported by National Eye Institute most logical candidates worthy of consideration. Grant EY019485. In exfoliation syndrome, a primary diagnostic feature is the Submitted for publication March 5, 2010; revised July 27, 2010; presence of fibrillar exfoliative material throughout the ante- accepted August 8, 2010. rior chamber of the eye.15 The disease often also involves the Disclosure: C.M. Trantow, None; T.L. Cuffy, None; J.H. Fingert, dispersion of iris pigment and morphologic changes to the None; M.H. Kuehn, None; M.G. Anderson, None 16,17 Corresponding author: Michael G. Anderson, Department of Mo- structure of the iris pigment epithelium. As with pigment lecular Physiology and Biophysics, 6-430 Bowen Science Building, 51 dispersion syndrome, accumulations of material within the Newton Road, Iowa City, IA 52242; [email protected]. iridocorneal angle can obstruct aqueous humor outflow, result- Investigative Ophthalmology & Visual Science, January 2011, Vol. 52, No. 1 Copyright 2011 The Association for Research in Vision and Ophthalmology, Inc. 237 Downloaded from iovs.arvojournals.org on 09/30/2021 238 Trantow et al. IOVS, January 2011, Vol. 52, No. 1 ing in elevated intraocular pressure and glaucoma. Recently, mice. Enucleated eyes were dissected in phosphate-buffered saline genetic variations in the LOXL1 gene have been linked with with both irides from each mouse pooled to form one sample; three exfoliation syndrome.18 Because the same LOXL1 alleles asso- samples (mice) were analyzed per strain. Iris samples were homoge- ciated with exfoliation syndrome also occur in the general nized, and RNA was extracted, treated with DNase I, and purified population at a very high frequency, additional risk factors are (Aurum Total RNA Mini Kit; Bio-Rad Laboratories; Hercules, CA). RNA presumed to exist. B6-Lystbg-J mice exhibit multiple ocular was subsequently purified by EtOH precipitation and quantified features resembling exfoliation syndrome, including the pres- (Quant-iT RiboGreen RNA Assay Kit; Molecular Probes, Eugene, OR) ence of an exfoliative-like material, pigment dispersion, and iris and the integrity confirmed on a bioanalyzer (model 2100; Agilent transillumination defects caused by an apparent loss of cell– Technologies, Inc., Palo Alto, CA). RNA samples were converted to cRNA cell adhesions within the iris pigment epithelium.17 Accord- compatible with gene microarrays according to the manufacturer’s standard ingly, LYST and other genes within the LYST genetic pathway protocols and hybridized (Mouse Genome 2.0 arrays; Affymetrix, Santa are candidates that are likely to contribute to exfoliation syn- Clara, CA). Raw data were normalized by using the RMA (robust drome in humans. multichip average) algorithm, and quality was assessed with PLM 21 We report global gene expression patterns of the iris in four (probe level model) methodology. Normalized data were log2 trans- strains of mice with identical genetic backgrounds: wild-type formed and filtered to remove probesets that did not display expres- C57BL/6J mice with normal irides, albino mice with Tyr mu- sion levels above 5.0 in at least two samples and those that did not tation, pigment dispersion–prone mice with Tyrp1 and display at least a 1.8-fold difference between the highest and lowest Gpnmb mutations, and exfoliative-like mice with Lyst muta- expression values. The remaining probesets were then evaluated using tion. In each comparison between these strains, transcriptional the significance analysis for microarray algorithm to identify significant changes are presented for select genes of functionally anno- expression changes (SAM; ver. 3.05; Excel Add-In; Microsoft, Red- tated clusters and are also presented according to the magni- mond, WA).22 Significance was determined by using a two-class, un- tude of the ratio of change. paired Wilcoxon rank sum test with 100 permutations. In the compar- ison of C57BL/6J versus B6.Tyrc-2J, the delta value was 0.373, resulting in the identification of 4304 probesets with an estimated false discov- METHODS ery rate (FDR) of 3.7%. In the comparison of C57BL/6J versus B6.Tyrp1b GpnmbR150X, the delta value was 0.329, resulting