Pathogenesis and treatments of TGFBI corneal dystrophies.

Progress in Retinal and Eye Research xxx (2015) 1e22

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Progress in Retinal and Eye Research journal homepage: www.elsevier.com/locate/prer

Pathogenesis and treatments of TGFBI corneal dystrophies Kyung Eun Han a, Seung-il Choi b, Tae-im Kim b, c, Yong-Sun Maeng b, R. Doyle Stulting d, Yong Woo Ji b, c, Eung Kweon Kim b, c, e, * a

Department of Ophthalmology, Ewha Womans University, School of Medicine, Seoul, South Korea Corneal Dystrophy Research Institute, Department of Ophthalmology, Yonsei University College of Medicine, Seoul, South Korea c Institute of Vision Research, Department of Ophthalmology, Yonsei University College of Medicine, Seoul, South Korea d Stulting Research Center, Woolfson Eye Institute, Atlanta, GA, USA e BK21 Plus Project for Medical Science and Severance Biomedical Science Institute, Yonsei University College of Medicine, Seoul, South Korea b

a r t i c l e i n f o

a b s t r a c t

Article history: Received 5 August 2015 Received in revised form 12 November 2015 Accepted 16 November 2015 Available online xxx

Transforming growth factor beta-induced (TGFBI) corneal dystrophies are a group of inherited progressive corneal diseases. Accumulation of transforming growth factor beta-induced protein (TGFBIp) is involved in the pathogenesis of TGFBI corneal dystrophies; however, the exact molecular mechanisms are not fully elucidated. In this review article, we summarize the current knowledge of TGFBI corneal dystrophies including clinical manifestations, epidemiology, most common and recently reported associated mutations for each disease, and treatment modalities. We review our current understanding of the molecular mechanisms of granular corneal dystrophy type 2 (GCD2) and studies of other TGFBI corneal dystrophies. In GCD2 corneal fibroblasts, alterations of morphological characteristics of corneal fibroblasts, increased susceptibility to intracellular oxidative stress, dysfunctional and fragmented mitochondria, defective autophagy, and alterations of cell cycle were observed. Other studies of mutated TGFBIp show changes in conformational structure, stability and proteolytic properties in lattice and granular corneal dystrophies. Future research should be directed toward elucidation of the biochemical mechanism of deposit formation, the relationship between the mutated TGFBIp and the other materials in the extracellular matrix, and the development of gene therapy and pharmaceutical agents. © 2015 Elsevier Ltd. All rights reserved.

Keywords: Transforming growth factor beta-induced corneal dystrophies Transforming growth factor beta-induced protein Granular corneal dystrophy type 2 Molecular mechanism Genetics

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Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.1. What is TGFBIp and what is its function? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.2. What are TGFBI corneal dystrophies? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Epidemiology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Classification and mutations of TGFBI corneal dystrophies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.1. Dystrophies with deposits primarily in Bowman layer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.1.1. ReiseBücklers corneal dystrophy (RBCD) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.1.2. ThieleBehnke corneal dystrophy (TBCD) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2. Dystrophies with deposits primarily in the stroma . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2.1. Lattice corneal dystrophy with TGFBI mutation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2.2. Granular corneal dystrophy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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Abbreviations: ALKP, anterior lamellar keratoplasty; BAC, benzalkonium chloride; DALK, deep anterior lamellar keratoplasty; ECM, extracellular matrix; FAS1, fasciclin 1; FD-OCT, Fourier-domain optical coherence tomography; GCD1, granular corneal dystrophy type 1; GCD2, granular corneal dystrophy type 2; KE, keratoepithelin; LASEK, laser epithelial keratomileusis; LASIK, laser in situ keratomileusis; LCD, lattice corneal dystrophy; MMC, mitomycin C; mTOR, mammalian target of rapamycin; PKP, penetrating keratoplasty; PRK, photorefractive keratectomy; PTK, phototherapeutic keratectomy; RBCD, Reis-Bücklers corneal dystrophy; RGD, arginineeglycineeaspartate; RK, radial keratotomy; ROS, reactive oxygen species; TBCD, Thiel-Behnke corneal dystrophy; TGFBI, transforming growth factor beta-induced; TGFBIp, transforming growth factor betainduced protein; UPS, ubiquitin/proteasome system; WT, wild-type. * Corresponding author. Department of Ophthalmology, Yonsei University College of Medicine, 50 Yonsei-ro, Seodaemun-gu, 120-752 Seoul, South Korea. E-mail address: [email protected] (E.K. Kim). http://dx.doi.org/10.1016/j.preteyeres.2015.11.002 1350-9462/© 2015 Elsevier Ltd. All rights reserved.

Please cite this article in press as: Han, K.E., et al., Pathogenesis and treatments of TGFBI corneal dystrophies, Progress in Retinal and Eye Research (2015), http://dx.doi.org/10.1016/j.preteyeres.2015.11.002

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3.3. Variations of phenotypes in atypical TGFBI corneal dystrophies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Corneal deposits of TGFBIp in TGFBI corneal dystrophies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.1. The origin of TGFBIp and location of deposits . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.2. Metabolism of wild and mutated TGFBIps . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.2.1. Degradation of wild and each mutated TGFBIp with amyloidogenicity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.2.2. Difference of protein profiles of deposits and adjacent tissues in different mutations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.2.3. Alteration of conformational structure and stability of TGFBIp . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.3. Mutated TGFBIp accumulates in the cornea specifically . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.3.1. Localization of TGFBI deposits within the cornea . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.3.2. Formation of granular deposits . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.3.3. Sudden morphologic changes of the corneal deposits . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.4. Phenotypic differences from the same mutation in TGFBI corneal dystrophies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.4.1. The production of TGFBIp differs among individuals and family members . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.4.2. Two mutations of TGFBI in single allele or in both alleles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.4.3. Possible effects of other genomic factors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Molecular pathogenesis of GCD2 and TGFBI corneal dystrophies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.1. Characteristics of corneal fibroblasts of GCD2 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.1.1. Morphological properties of GCD2 corneal fibroblasts . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.1.2. Oxidative stress in GCD2 corneal fibroblasts . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.1.3. Mitochondrial dysfunction in GCD2 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.1.4. Mutant-TGFBIp accumulates in the cell because of defective autophagy in GCD2 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.1.5. Altered cell cycle arrest and reduced proliferation of GCD2 corneal fibroblasts . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.2. Regulation of TGFBIp expression in corneal fibroblasts . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.2.1. Role of TGF-b in TGFBIp expression . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.2.2. TGFBI gene regulation: epigenetics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.2.3. Factors affecting production of TGFBIp in corneal fibroblasts; LPA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . Treatment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.1. Surgical approaches . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.1.1. Laser ablation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.1.2. Keratoplasty . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.1.3. Corneal electrolysis and corneal epithelial debridement . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.1.4. Stage-related therapy of corneal dystrophies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Perspectives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7.1. Animal models . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7.2. Potential therapeutic approaches . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7.2.1. Agents that might be used to reduce production or enhance clearance of TGFBI expression . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7.2.2. Avoid agents that deteriorate disease . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7.2.3. Possible roles of blood vessels . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7.2.4. Gene therapy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conclusions and future directions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Disclosure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

1. Introduction 1.1. What is TGFBIp and what is its function? Transforming growth factor beta-induced gene (TGFBI; BIGH3;

bigh3) encodes transforming growth factor beta-induced protein (TGFBIp), a 68-kDa extracellular matrix (ECM) protein composed of 683 amino acid residues. TGFBIp is also described as keratoepithelin (KE), BIGH3, big-h3, or bigh3 depending on the major production site or the time of description. TGFBIp contains a secretory signal peptide sequence, a cysteine-rich EMI domain, four homologous fasciclin 1 (FAS1) domains which each contain 140 amino acid residues at the N-terminus, and an arginine-glycine-aspartate (RGD) motif which binds to integrin at the C-terminus. TGFBIp was first isolated from an adenocarcinoma cell line, where it was upregulated after TGF-b treatment (Skonier et al., 1992). TGFBIp is known to exist ubiquitously in various organs including heart, liver, pancreas (Skonier et al., 1992), skin (Skonier et al., 1992), bone (Kitahama et al., 2000), tendon (Ferguson et al., 2003a, 2003b), endometrium (Carson et al., 2002), kidney (Lee et al., 2003) and

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blood plasma (Klintworth et al., 1998). The role of TGFBIp is not understood completely. TGFBIp is thought to play pivotal roles in physiologic and pathologic responses by mediating cell adhesion (Nam et al., 2005; Park et al., 2004, 2009; Skonier et al., 1992), migration (Nam et al., 2005; Park et al., 2004), proliferation and differentiation (Park et al., 2009). In vitro, TGFBIp is reported to mediate cell adhesion and/or spreading through integrins a1b1, a3b1, anb3, anb5, a6b4, and amb2 (Kim and Kim, 2008; Kim et al., 2002, 2003; Nam et al., 2006; Ohno et al., 1999; Park et al., 2004). TGFBIp is also associated with progression, dissemination, metastasis and suppression of malignant tumors (Fan et al., 2014; Massague, 2008; Nam et al., 2005). Recently, TGFBIp was reported to increase adhesion, migration and morphologic differentiation of human lymphatic endothelial cells so that inhibition of TGFBIp expression resulted in reduction of tumor lymphangiogenesis followed by reduction of metastasis of TGFBIp-producing tumors (Maeng et al., 2015a). TGFBIp is also known to increase the adhesion and migration of endothelial progenitor cells through integrins a4 and a5 (Maeng et al., 2015b). During development, TGFBIp has been detected in mouse corneal epithelium and stroma



from 15.5 to 18.5 days after conception (Schorderet et al., 2000; Ferguson et al., 2003a, 2003b). In the cornea, TGFBIp is expressed mainly in the epithelium (Escribano et al., 1994), in association with collagen type VI in the stroma, at the stromal/Descemet membrane interface in Fuchs dystrophy (Hirano et al., 1996), and in retrocorneal fibrous membranes (El-Shabrawi et al., 1998; Leung et al., 2000). About 60% of corneal TGFBIp is found covalently linked to collagen type XII (Runager et al., 2013). During wound healing of the rabbit cornea, the expression of TGFBIp is up-regulated significantly (Rawe et al., 1997). It appears that TGFBIp plays a role both in corneal wound healing and maintenance of the ECM. The precise role, however, is still largely unknown. 1.2. What are TGFBI corneal dystrophies? Corneal dystrophies are inherited corneal diseases that are typically slowly progressive, symmetric, and without relationship to systemic or environmental factors (American Academy of Ophthalmology (2007)). Bilateral corneal deposits can cause tearing, photophobia, pain, and eventually reduce visual acuity. In 1890, Groenouw (1890) first described two patients with ‘noduli corneae.’ In 1938, Bücklers (1938) first classified corneal dystrophies as granular, macular and lattice dystrophy on the basis of their appearance. Once genetic analysis became available, specific genetic defects began to be associated with corneal dystrophies. Munier et al. (1997) recognized the following relationships between TGFBI mutations and specific corneal dystrophies: p.Arg124Leu for ReisBücklers corneal dystrophy (RBCD), p.Arg555Gln for Thiel-Behnke corneal dystrophy (TBCD), p.Arg124Cys for lattice corneal dystrophy type 1 (LCD1), p.Arg555Trp for granular corneal dystrophy type 1 (GCD1), and p.Arg124His for granular corneal dystrophy type 2 (GCD2). As of the date of this review, at least 30 mutations in the TGFBI gene have been associated with corneal dystrophies, and most cases demonstrate a conserved mutation in one of two hot spots at codon Arg124 in first FAS1 domain or Arg555 in fourth FAS1 domain. Information of all mutations and variants in the TGFBI corneal dystrophies are available at the Leiden Open Variation Database (LOVD) locus specific database (http://databases.love.nl/ shared/genes/TGFBI). In this review, clinical manifestations, epidemiology, inheritance patterns, pathogenesis, and therapeutic strategies for TGFBI corneal dystrophies will be discussed. 2. Epidemiology Most estimates of the prevalence of TGFBI corneal dystrophies have been derived from clinical observations, and there are only a few epidemiologic studies. According to published studies, p.Arg124His is the most frequently observed mutation in the Asian population. Previous Japanese studies reported that the p.Arg124His mutation was the most common, accounting for up to 72% of patients with corneal dystrophies (Fujiki et al., 2001; Mashima et al., 2000). The second most frequently observed mutation is probably LCD1 (Fujiki et al., 2001) or GCD1 (Mashima et al., 2000). Our group found 21 homozygotes in Korea and reported that the prevalence of GCD2 heterozygote is 1 out of every 870 Korean people (Lee et al., 2010). In another study, among 268 Korean patients with TGFBI corneal dystrophy, GCD2 was the most frequent mutation followed by LCD1 and variant LCD (p.Leu527Arg and p.Pro542Arg) (Cho et al., 2012). In China, GCD1 was the most frequent mutation followed by LCD1 and GCD2 (Yang et al., 2010). Unlike the finding in Asian populations, LCD1 was more common in Western countries. Among the types of TGFBI corneal dystrophies, novel mutations which are responsible for atypical LCD variants are frequently reported. Since these data did not include unpublished

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cases, the prevalence of TGFBI corneal dystrophies might be underestimated. Reported mutations and their phenotypes are listed in Table 1. 3. Classification and mutations of TGFBI corneal dystrophies Even after the publication of Munier et al.'s milestone paper (1997) associating genotypes and phenotypes for corneal dystrophies, classification of TGFBI corneal dystrophies by clinical appearance remains difficult because there is not a one-to-one correspondence between genetic mutations and clinical findings. The International Committee for Classification of Corneal Dystrophies (IC3D) anatomically organized corneal dystrophies by the corneal layer primarily affected and defined a new classification for individual corneal dystrophies (Weiss et al., 2015), based on genetics. In this paper, every variant of each mutation was categorized following the new IC3D classification (Weiss et al., 2015), abnormal genetic locus. Additional information about variants of each mutation are provided below under the heading “Notes.” 3.1. Dystrophies with deposits primarily in Bowman layer 3.1.1. ReiseBücklers corneal dystrophy (RBCD) RBCD is an autosomal dominant corneal dystrophy that affects primarily Bowman layer. It is also known as corneal dystrophy of Bowman layer type I (CDB1). Reis (1917) first described this dystrophy in 1917, and Bücklers (1949) reported similar familial findings of subepithelial opacities and recurrent corneal erosions in 1949. Irregular geographic subepithelial grayewhite deposits begin to appear predominantly at Bowman layer and extend to the anterior stroma in the central and mid-peripheral cornea in the first and second decades (Fig. 1). Vision can be impaired in early childhood. RBCD may not be distinguishable from TBCD by slit lamp examination (Weiss et al., 2008; Werner et al., 1999). Light microscopy reveals an irregular, thinned epithelium and absence of Bowman layer, which is mostly replaced by subepithelial deposits. These deposits are eosinophilic on hematoxylin and eosin (H&E) staining and red with Masson's trichrome. Transmission electron microscopy reveals electron-dense rod-shaped and trapezoidal deposits surrounded by microfibrillar material and large proteoglycan filaments. The major associated gene of RBCD is p.Arg124Leu in TGFBI. Notes for RBCD p.Arg124Leu was reported to be associated with a superficial variant of granular corneal dystrophy in a Japanese family (Mashima et al., 1999). p.[(Arg124Leu; Thr125_Glu126del)] were associated with a superficial variant of GCD1 (Dighiero et al., 2000a). Afshari et al. (2001) reported a case of subepithelial corneal opacities in a geographic pattern which did not stain with periodic acid-Schiff, Masson's trichrome or Congo red stains with p.Gly623Asp mutation. Cases with p.Pro540del (Rozzo et al., 1998) and p.His626Pro (Wheeldon et al., 2008) have also been reported. 3.1.2. ThieleBehnke corneal dystrophy (TBCD) TBCD is an autosomal dominant corneal dystrophy of Bowman layer. TBCD shows honeycomb-shaped subepithelial reticular opacities (Fig. 2). The opacification appears during the first or second decade of life. Initially, visual impairment may be mild, but with age, as the deposits coalesce, vision gradually decreases (Thiel and Behnke, 1967). Because of the similarity of location and depth of deposits, TBCD was considered to be an atypical type of RBCD in the 1980s. Clinically, TBCD is distinguished from RBCD by the older age of disease onset, less severity of the corneal opacity, and gradual deterioration of visual acuity compared to RBCD (Kuchle et al., 1995; Weiss et al., 2008). In many cases, however, these clinical differences were not distinct. Transmission electron


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Table 1 Reported mutations in TGFBI corneal dystrophies. Phenotype

Mutations

Countries (references)

RBCD

p.Arg124Leu p.Phe540del p.Gly623Asp p.His626Pro p.[(Arg124Leu; Thr125_Glu126del)] p.Arg555Gln p.Arg124Cys p.Val505Asp p.Leu509Arg p.Leu509Pro p.Leu518Pro p.Ile522Asn p.Thr538Pro p.Val539Asp p.Leu569Arg p.His572Arg

Worldwide Italy (Rozzo et al., 1998) USA (Afshari et al., 2001) New Zealand (Wheeldon et al., 2008) France (Dighiero et al., 2000b; Dighiero et al., 2001)

TBCD LCD1

LCD I/IIIA

LCD III LCD IIIA

LCD IV Atypical or undefined

GCD1

GCD2

Worldwide Worldwide China (Tian et al., 2005) France (Niel-Butschi et al., 2011) France (Niel-Butschi et al., 2011) Japan (Endo et al., 1999) China (Zhang et al., 2009) China (Yu et al., 2006; Zhu et al., 2012) India (Chakravarthi et al., 2005) USA (Warren et al., 2003) Thai (Atchaneeyasakul et al., 2006; Lakshminarayanan et al., 2011); Chile (Romero et al., 2010); China (Zhong et al., 2010) p.Val625Asp China (Tian et al., 2007) p.[Arg124Cys]; [Gly470Ter] Japan (Sakimoto et al., 2003) p.[Arg514Pro(;)Phe515Leu] China (Zhong et al., 2010) p.[(Ala546Asp; Pro551Gln)] USA (Klintworth et al., 2004; Poulsen et al., 2014) p.Leu518Arg Italy (Munier et al., 2002) p.Thr538Arg USA (Munier et al., 2002); Ukraine (Pampukha et al., 2008) p.Phe540del Italy (Munier et al., 2002) p.Ala620Asp Singapore (Lakshminarayanan et al., 2011) p.Asn622His UK (Stewart et al., 1999a) p.Gly623Asp Not mentioned (Munier et al., 2002) p.His626Arg Not mentioned (Munier et al., 2002); France (Dighiero et al., 2001; Schmitt-Bernard et al., 2000b); UK (Stewart et al., 1999a) p.His626Pro Not mentioned (Munier et al., 2002) p.Asn629_Pro630ins3 France (Schmitt-Bernard et al., 2000b) p.[(Ala546Asp; Pro551Gln)] USA (Aldave et al., 2004) Not determined Japan (Hida et al., 1987a; Hida et al., 1987b) p.Pro501Thr China (Yu et al., 2006); Japan (Mashima et al., 2000; Yamamoto et al., 1998) p.Phe540Ser Germany (Stix et al., 2005) p.Ala546Thr France (Dighiero et al., 2000b; Dighiero et al., 2001) p.Ala620Pro Korean (Jung et al., 2014) p.Asn622Lys USA (Munier et al., 2002) p.Val627Ter USA (Munier et al., 2002)p.Leu527Arg Japan (Fujiki et al., 1998; Hirano et al., 2001); Korea (Kim et al., 2014) p.Arg496Trp Japan (Kawasaki et al., 2011) p.Pro542Arg Korea (Cho et al., 2012) p.Asn544Ser Japan (Mashima et al., 2000; Yamamoto et al., 1998) p.Ala546Asp USA (Eifrig et al., 2004); Mexico (Correa-Gomez et al., 2007) p.Phe547Ser Hungary (Takacs et al., 2007) p.Leu565Pro Poland (Oldak et al., 2014) p.His572del USA (Aldave et al., 2006) p.Gly594Val India (Chakravarthi et al., 2005) p.Val613Gly Algery (Niel-Butschi et al., 2011) p.Val613_Pro616del China (Yang et al., 2010) P.Val624_625del India (Chakravarthi et al., 2005) P.His626Pro Czech (Liskova et al., 2008) p.Val631Asp Italy (Munier et al., 2002) p.Arg555Trp Worldwide p.Val113Ile Mexico (Zenteno et al., 2009) p.Asp123His Vietnam (Ha et al., 2003) p.Arg124Ser UK (Stewart et al., 1999b); Not mentioned (Munier et al., 2002) p.Ser516Arg India (Paliwal et al., 2010) p.[Arg549Thr]; [Arg555Trp] Germany (Frising et al., 2006) p.Leu559Val India (Paliwal et al., 2010) p.Arg124His Worldwide p.Leu509Pro Germany (Gruenauer-Kloevekorn et al., 2009) p.Leu550Pro Mexico (Zenteno et al., 2009); Indonesia (Lakshminarayanan et al., 2011) p.Met619Lys USA (Aldave et al., 2008) p.[Arg124His(;)c.307_308delCT] China (Pang and Lam, 2002; Yam et al., 2012) p.[Arg124His]; [Asn544Ser] Japan (Yamada et al., 2009)

RBCD, Reis-Bücklers corneal dystrophy; TBCD, Thiel-Behnke corneal dystrophy; LCD, lattice corneal dystrophy; GCD1, granular corneal dystrophy type 1; GCD2, granular corneal dystrophy type 2.

microscopy reveals distinct differences between these two corneal dystrophies. The deposits of TBCD consist of irregular aggregates of ‘curly’ fibrils measuring 9e15 nm in diameter. In contrast, electron

microscopy of RBCD shows electron dense rod- or trapezoidalshaped bodies (Kuchle et al., 1995; Ridgway et al., 2000). After genetic analysis of these patients, TBCD was confirmed to be



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associated with a distinct mutation (p.Arg555Gln) of the TGFBI gene. 3.2. Dystrophies with deposits primarily in the stroma 3.2.1. Lattice corneal dystrophy with TGFBI mutation LCD with TGFBI mutation is inherited as an autosomal dominant trait and is characterized by linear and branching refractile lines, and/or ovoid and whitish dots in the stroma. These changes appear bilaterally in most affected individuals, but some patients show an unusual unilateral phenotype (Aldave et al., 2006; Seitz et al., 1993; Sridhar et al., 2001, 2002; Yellore et al., 2008). LCD is classified as classic lattice corneal dystrophy (type 1), and its variants (type IIIA, I/IIIA, IV, and atypical) according to the clinical characteristics. 3.2.1.1. Classic lattice corneal dystrophy (Lattice corneal dystrophy type 1; LCD1). LCD1 is inherited as an autosomal dominant trait and the associated mutation is p.Arg124Cys. Translucent fine lattice lines appear during the first decade of life in the anterior central corneal stroma, then spread deeper into the stroma and to the periphery with time (Fig. 3). The anterior location of corneal deposits leads to recurrent corneal erosions and irregularity of the corneal surface. Initially, the space between the lattice lines and dots is clear. In the adult, a dense diffuse anterior stromal haze may develop, and this usually affects visual acuity. Histologically, corneal deposits stain with orange with Congo red and reveal green birefringence and dichroism under polarized light. The fine lattice lines consist of highly aligned fibrils with a diameter of 8e10 nm (Francois and Feher, 1972). Notes for LCD1 p.Arg124Cys has also been found to in patients with RBCD-like phenotype (Yang et al., 2011) or TBCD-like phenotype (Chang et al., 2009). The heterozygote compound mutation of p.[Arg124Cys]; [Gly470Ter] was reported, and the phenotype was not more severe than that of p.Arg124Cys alone. A relative heterozygous for p.Gly470Ter alone had normal corneas (Sakimoto et al., 2003). The following mutations are variants of LCD even though they were reported as LCD1 or similar to LCD1: p.Val505Asp (Tian et al., 2005), p.Len509Arg (Niel-Butschi et al., 2011), p.Leu509Pro (NielButschi et al., 2011), p.Leu518Pro (Endo et al., 1999), p.Ile522Asn (Zhang et al., 2009), p.Thr538Pro (Yu et al., 2006; Zhu et al., 2012), p.Val539Asp (Chakravarthi et al., 2005), p.Leu569Arg (Warren et al.,

Fig. 1. Slit-lamp photograph of a 24-year-old female RBCD patient 10 years after phototherapeutic keratectomy (PTK). Irregular geographic subepithelial grayewhite deposits (black arrow) appear predominantly at the Bowman layer outside of the PTK area. A lucid interval (white arrow) is noted between the typical geographic deposits and the limbus. The PTK area shows small deposits due to the recurrence (black arrowheads).

Fig. 2. Slit-lamp photograph of a TBCD patient. Honeycomb-shaped subepithelial reticular opacities are observed.

2003), p.His572Arg (Atchaneeyasakul et al., 2006; Lakshminarayanan et al., 2011; Romero et al., 2010; Zhong et al., 2010), p.Val625Asp (Tian et al., 2007), p.[Arg514Pro(;) Phe515Leu] (Zhong et al., 2010), and p.[(Ala546Asp; Pro551Gln)] (Klintworth et al., 2004; Poulsen et al., 2014). 3.2.1.2. Variants of lattice corneal dystrophy (III, IIIA, I/IIIA, and IV). Nearly all types of lattice dystrophy except classic LCD1 arise from mutations of domain 4 of TGFBI. Variants of LCD1 have been classified as LCD III, IIIA, I/IIIA, and IV based on the location of deposits, age of the onset, and characteristics of lattice lines (Klintworth, 1999). LCD III and IIIA are characterized by thick lattice lines in the mid stroma which appear late in life. Recurrent erosions seldom occur, and visual acuity does not decrease until later. LCD IIIA has autosomal dominant inheritance and the associated mutation is p.Pro501Thr (Mashima et al., 2000; Yamamoto et al., 1998). LCD III has an autosomal recessive inheritance and causes thick lattice lines that extend from limbus to limbus. The responsible gene has yet to be determined (Hida et al., 1987a, 1987b). LCD IV is characterized by a late-onset atypical form with deep stromal amyloid deposits. The associated mutation is p.Leu527Arg (Fujiki et al., 1998). LCD I/IIIA is classified as an intermediate form of LCD I and IIIA, and the associated mutation is p.Leu518Arg (Munier et al., 2002). The p.His626Arg mutation is associated with asymmetric thick lattice lines in the anterior and mid-stroma, with a relatively late onset during the 3rd to 4th decades of life (Dighiero et al., 2001; Munier et al., 2002; Schmitt-Bernard et al., 2000b; Stewart et al., 1999a).

Fig. 3. Slit-lamp photograph of a LCD1 patient. Central branching linear corneal opacities are observed mainly in the anterior stroma.


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Notes for variant LCD Numerous types of variant LCD have been reported to be associated with p.Arg496Trp (Kawasaki et al., 2011), p.Pro501Thr (Mashima et al., 2000; Yamamoto et al., 1998; Yu et al., 2006), p.Thr538Arg (Munier et al., 2002; Pampukha et al., 2008), p.Phe540del (Munier et al., 2002), p.Phe540Ser (Stix et al., 2005), p.Pro542Arg (Cho et al., 2012), p.Asn544Ser (Mashima et al., 2000; Yamada et al., 2009), p.Ala546Asp (Correa-Gomez et al., 2007; Eifrig et al., 2004), p.Ala546Thr (Dighiero et al., 2000b, 2001), p.Phe547Ser (Takacs et al., 2007), p.Leu565Pro (Oldak et al., 2014), p.His572del (Aldave et al., 2006), p.Gly594Val (Chakravarthi et al., 2005), p.Val613Gly (Niel-Butschi et al., 2011), p.Val613_Pro616del (Yang et al., 2010), p.Ala620Asp (Lakshminarayanan et al., 2011), p.Ala620Asp (Lakshminarayanan et al., 2011), p.Ala620Pro (Jung et al., 2014), p.Asn622His (Stewart et al., 1999a), p.Asn622Lys (Munier et al., 2002), p.Gly623Asp (Munier et al., 2002), p.His626Arg (Dighiero et al., 2001; Munier et al., 2002; SchmittBernard et al., 2000b; Stewart et al., 1999a), p.His626Pro (Munier et al., 2002), p.Val624_Val625del (Chakravarthi et al., 2005), p.Val627Ter (Munier et al., 2002), p.Asn629_Pro630ins3 (SchmittBernard et al., 2000b), and p.Val631Asp (Munier et al., 2002). 3.2.2. Granular corneal dystrophy 3.2.2.1. Granular corneal dystrophy type 1 (GCD1). GCD1 is an autosomal dominant disease, which was first described by Groenouw (Groenouw, 1890). The p.Arg555Trp mutation is associated with this phenotype. Multiple, small, well-defined, grayish-white granules appear in the anterior stroma, sparing the limbus, during the first or second decade of life (Fig. 4). The granules vary in shape and are classified as drop-shaped, crumb-shaped, and ring-shaped (Weidle and Lisch, 1984). In the early stage, vision is usually not affected because the stroma between the opacities remains clear. Glare, photophobia, and painful recurrent erosions often occur. With advancing age, the opacities become confluent in the anterior corneal stroma, and the earlier ones become larger and denser. Finally, visual acuity decreases significantly. Histopathologically, GCD1 is characterized by eosinophilic, rod-shaped hyaline deposits which stain bright red with Masson's trichrome. Electron microscopy shows stromal deposits are made up of rod-shaped or trapezoidal bodies 100e500 nm in diameter, with a filamentous, ‘motheaten,’ homogeneous morphology in their inner structure (Akiya and Brown, 1970; Sornson, 1965). Three patients with a severe phenotype, proven to be homozygotes, were reported in a family in Japan. Clinical manifestations appeared at the early age of 6 years, and severe placoid corneal deposits and early recurrence after surgery were observed (Okada et al., 1998b). Notes for GCD1 p.Val113Ile (Zenteno et al., 2006), p.Asp123His (Ha et al., 2003), p.Arg124Ser (Munier et al., 2002; Stewart et al., 1999b), p.Ser516Arg (Paliwal et al., 2010), p.[Arg549Thr]; [Arg555Trp] (Frising et al., 2006), p.Leu559Val (Chakravarthi et al., 2005) have been reported to be associated with the GCD1 phenotype. 3.2.2.2. Granular corneal dystrophy type 2 (GCD2, Avellino CD, Combined granular-lattice CD). Granular corneal dystrophy type 2 (GCD2) is an autosomal dominant disease that shares clinical and histologic characteristics of both granular and lattice dystrophy (Fig. 5). Substitution of arginine for histidine at codon 124 (p.Arg124His) is associated with this disease. In 1988, Folberg et al. (1988) reported the histopathological features of hyaline and amyloid deposits in these patients. In 1992, Holland et al. (1992) reported two related American families whose origin was Avellino, Italy, and since then, GCD2 has also been called “Avellino corneal dystrophy.” Initially, slit lamp examination shows small, faint superficial stromal opacities in the first or second decade of life. These

granular deposits are the earliest manifestations (Holland et al., 1992; Munier et al., 1997). More deposits accumulate with age. In some patients, lattice-like amyloid lesions appear in the deeper stroma with superficial granular lesions. In some older patients, superficial, translucent diffuse anterior stromal haze appears, and coalescence of the opacities may impair visual acuity. When the superficial granular deposits involve the Bowman layer, painful corneal erosions develop and after the erosive attack, some corneal deposits disappear partially or totally. The disappearance of the center of drop- or crumb-shaped granular deposits leads to the formation of ring-shaped opacities (Han et al., 2010). There are typically fewer granular deposits in patients with GCD2 than in patients those with GCD1. Histologically, granular deposits in the anterior stroma stain with Masson's trichrome, and the lattice-like lesions in the deep stroma stain with Congo red. Electron microscopy reveals rod-shaped or trapezoidal central electron-dense areas surrounded by 9e10 nm tubular microfibrils (Rodrigues et al., 1983). Homozygous patients have earlier onset and faster progression with grayewhite, discrete opacities in the superficial cornea, compared with heterozygote patients (Mashima et al., 1998; Moon et al., 2007; Okada et al., 1998a). The opacities appear as early as 3 years of age, and these opacities increase in size and shape with age and tend to coalescence earlier. Vision is frequently impaired at an early age, requiring surgery. A distinct form of the homozygous mutation of p.Arg124His showing a reticular pattern with grayish, confluent opacities and translucent intervening spaces in the anterior stroma (type II opacity) has also been reported in Japan (Tsujikawa et al., 2007; Watanabe et al., 2001). Because of these translucent spaces, best-corrected visual acuities of patients with type II opacity were better than those of conventional, or “type I opacity' homozygous patients. Interestingly, the lattice-like deposits do not appear in the corneas of homozygous patients. Notes for GCD2 p.[Arg124His(;)c.307_308delCT] (Yam et al., 2012), p.[Arg124His]; [Asn544Ser] (Yamada et al., 2009), p.Leu509Pro (Gruenauer-Kloevekorn et al., 2009), p.Leu550Pro (Lakshminarayanan et al., 2011; Zenteno et al., 2009), and p.Met619Lys (Aldave et al., 2008) also show the phenotype of GCD2. 3.3. Variations of phenotypes in atypical TGFBI corneal dystrophies Numerous variations of mutations of TGFBI corneal dystrophies with atypical phenotypes have been reported. In the p.Asp123His mutation of a Vietnamese family, smaller and deeper deposits appeared late in life with less severity and penetrance than GCD1 (Ha et al., 2003). A p.Met619Lys mutation has been associated with an atypical and variable phenotype of granular-lattice corneal dystrophy. These deposits were shown to be hyaline and amyloid. Central subepithelial needle-like deposits were observed in younger patients, and polymorphous anterior stromal opacities were observed in older individuals (Aldave et al., 2008). We recently observed a novel p.Ala620Pro mutation, which produced a late-onset, asymmetric progressive LCD IIIA phenotype (Jung et al., 2014). Interestingly, the same stromal deposits stained positively with Masson's trichrome and showed birefringence with Congo red as the deposits of p.Met619Lys did. This suggests that the deposit contained both amyloid and hyaline. A missense mutation p.Leu565Pro in the TGFBI gene has been associated with atypical late-onset LCD in a Polish family (Oldak et al., 2014). In a Czech family, a p.His626Pro mutation in TGFBI showed subepithelial geographical opacities mainly in Bowman layer, which was clinically similar to RBCD; however, the deposits were identified histologically as amyloid but no fuchsinophilic deposits were seen (Liskova et al., 2008). The same mutation, p.His626Pro,



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Fig. 4. Slit-lamp photograph and histologic examination of a 57-year-old GCD1 patient. A, Numerous superficial discoid and annular deposits are observed in the subepithelial and anterior stromal layer. B, Histologic findings after toluidine blue staining of the same patient. The opacities between the basal epithelial layer and Bowman layer (white arrows) may explain the frequent erosions seen in patients with GCD1 (original magnification 200).

Fig. 5. Slit-lamp photograph of a GCD2 patient. Multiple drop-and crumb-shaped granular deposits are seen in the anterior stroma and deep lattice-like deposits are present in deep stroma.

showed a significantly different phenotype that overlaps both RBCD and TBCD. Visual acuity was severely affected, with clinical manifestations beginning at a young age (10.3 ± 1.5 years) and substantial recurrence following penetrating keratoplasty (PKP) (Wheeldon et al., 2008). The p.His626Pro mutation has been reported also as an associated mutation of variant LCD I/IIIA, which shows a late onset, asymmetric, dense haze associated with lattice lines (Munier et al., 2002). The various phenotypes resulting from the same mutation and similar phenotypes arising from different mutations made us realize the complexities of the nomenclature of the TGFBI corneal dystrophies. 4. Corneal deposits of TGFBIp in TGFBI corneal dystrophies 4.1. The origin of TGFBIp and location of deposits Normal TGFBIp is found during corneal embryogenesis (Saika et al., 2001). After birth, TGFBIp can be produced by epithelial cells (Akiya et al., 1999; Escribano et al., 1994; Korvatska et al., 1999), keratocytes (Menasche et al., 1992; Wittebol-Post et al., 1987), or both (Lisch and Seitz, 2014). Under normal circumstances, most of the TGFBIp is produced by the corneal epithelium, as keratoepithelin which is the alternative name for TGFBIp, suggests. During wound healing of the normal human cornea, TGFBIp is

found both in the epithelium and the keratocytes near the wound, suggesting that keratocytes can also produce TGFBIp (Takacs et al., 1999). In RBCD and TBCD, the mutated TGFBIp preferentially aggregates in the Bowman layer. In GCD2, most of the deposits are in the superficial stroma (hyaline) and deep stroma (amyloid). In LCD1 and other LCD variants, most of the deposits are located in the superficial and deep stroma. In GCD1, many granular deposits are found not only in the superficial stroma but also between the basal cell layer of epithelium and Bowman layer, explaining the frequent epithelial erosions of GCD1 (Akhtar et al., 1999). In contrast, diffuse superficial opacities in the epithelium and subepithelium were observed in three LCD1 patients after PKP (Lisch and Seitz, 2014). Since none of the patients showed lattice lines in the stroma, the authors hypothesized that the origin of superficial diffuse opacities in the graft was the epithelial cells, while the absence of lattice lines was an indirect indication that stromal lattice lines originate from keratocytes. Some authors suggest that mutated protein may have an epithelial origin because reappearance of corneal deposits near the epithelial layer is more prominent in recurrent GCD after PKP (Frising et al., 2006; Johnson et al., 1981; Lyons et al., 1994). 4.2. Metabolism of wild and mutated TGFBIps 4.2.1. Degradation of wild and each mutated TGFBIp with amyloidogenicity Several disease-associated proteins are abnormally degraded under physiological conditions (Fandrich et al., 2001; Munishkina et al., 2004). Alterations of the proteolytic process and alterations of protein turnover have been reported to participate in the pathogenesis of TGFBI corneal dystrophies. In 2000, Korvatska et al. (2000) first verified that the typical amyloid deposits of p.Arg124Cys and non-amyloid (granular) deposits of p.Arg124His and p.Arg124Leu were associated with abnormal turnover and degradation of mutant TGFBIp. Examination of normal cornea revealed the major corneal proteins of 68kDa TGFBIp and the minor species of 64-, 57-, 47- and 29-kDa fragments when western blotting was done with two species of antibodies, anti-KE 39-364 (N-terminal) and anti-KE 426-682 (Cterminal). In p.Arg124Cys corneas, the amount of 68-kDa full-size TGFBIp did not significantly differ from the wild type (WT). The N-terminal 44-kDa fragments, however, were the major and


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excessively accumulated species while the sum of TGFBIp and its fragments was 3-fold greater than that of WT. The authors hypothesized that these fragments were amyloidogenic units. In p.Arg124His corneas, the amount of 68-kDa protein was twice than that of WT, and the major species was 66-kDa fragment. In contrast, p.Arg124Leu corneas showed ~2.5-fold excess of 68-kDa TGFBIp compared to WT, whereas the pattern of other fragments did not significantly differ from that of WT. The authors showed that the proteolytic processing of TGFBIp is altered, and the degradation patterns are specific for each mutation. These data are confirmed by Karring et al. (2010) who found that the c-terminal fragments were retained in the cornea after highly orchestrated N-terminal proteolytic processing. Unique fragments were also observed in the cell lysates of recombinant TGFBIp produced in transfected HEK293FT cells (Han et al., 2011, 2012b). By using different antibodies, 47-kDa protein fragments of p.Leu518Pro and 43-kDa fragments of p.Arg124Cys and p.Leu527Arg cell lysates were detected (Han et al., 2011). Because these fragments were not found in the WT, p.Arg124His, the 47- and 43-kDa fragments were considered to be possible candidates for the amyloid aggregates in LCD. In similar experiments by the same investigators, 35-kDa fragments were detected in p.Arg124His and p.Arg555Trp, and the fragments were not present in WT or p.Arg124Cys. These fragments may account for the non-amyloid (granular) deposits in corneas with GCD1 and GCD2 (Han et al., 2012b). These studies also imply that abnormal proteolysis is involved in the pathogenesis of this group of corneal dystrophies. It remains to be determined why the same residues, such as Arg124, form amyloid in certain mutations, but not in others. Using a synthetic peptide and in vitro dialysis approach, a model of amyloidogenicity of synthetic 22-amino acid peptides covering 110e131 of the native TGFBIp sequence and its mutated form Cys124 were evaluated (Schmitt-Bernard et al., 2000a). The Cys124 peptides showed >30-fold Thioflavin T fluorescence than the native form (Arg124), suggesting a higher capacity for amyloidogenesis of the Arg124Cys form. In a subsequent study, Schmitt-Bernard et al. (2002) synthesized 8 different peptides in an attempt to elucidate the mechanisms involved in the amyloidogenesis: four 22 amino acid-peptides (Arg110-131, Cys110-131, His110-131, Ser110-131) including native and mutated-peptides representing the normal condition, LCD1, GCD2, and GCD1; two 18 amino acid-peptides (Arg114-131, Cys114-131) missing 110e113 amino acids (Leu-GlyVal-Val) and N-terminal ends; one 13 amino acid-peptide (110e122) missing 9 (from 123 to 131) amino acids and C-terminal ends; and one 22 amino-acids peptide with a blocked SH-group (Cys110-131Acm) to inhibit the formation of disulfide bonds. Blocking the SH-group by preventing disulfide bonds in Cys124 peptides and deleting the 4 N-terminal residues including codons 112 and 113 (ValeVal) decreased amyloid fibril formation; whereas, deletion of the 9 C-terminal residues increased the production of amyloid fibrils. Amyloid fibril formation has been attributed to the ValeVal hydrophobic interaction, cysteineedisulfide bonds, and hydrogen bonding. Consistent with this hypothesis, a synthetic peptide, BB1, which was designed to counteract the role of Val112eVal113 showed a 35% reduction in the formation of amyloid fibrils. In addition to the Arg124 residue, a putative amyloidogenic motif (515e532) has been suggested. Yuan et al. (2007a) synthesized peptides containing amino acid peptides of 515e532 of normal TGFBIp and discovered that these peptides had a high propensity to form b-sheet amyloid fibrils under physiologic condition in vitro. The authors performed N-methylation by substituting methyl groups for the hydrogen atoms, and this completely abolished formation and extension of amyloid b-sheets.

These studies suggest that inhibiting the accumulation of abnormal deposits is a potential therapeutic modality. 4.2.2. Difference of protein profiles of deposits and adjacent tissues in different mutations Protein profiles of deposits and adjacent tissues are dependent on the types of deposits and associated mutations. Karring et al. (2012) analyzed the protein profiles from homozygous LCD1 (p.Val624Met; LCD variant) and non-amyloid (granular) deposits from heterozygous GCD2 (p.Arg124His) patients. Granular deposits, amyloid deposits, and disease-free tissue from normal corneas were isolated by laser capture microdissection. Results of liquid chromatography-tandem mass spectrometry (LC-MS/MS) revealed that intact full-size TGFBIp, serum amyloid P-component, clusterin, type III collagen, keratin 3, and histone H3-like protein accumulated in granular deposits. The amyloid deposits were composed of serum amyloid P-component, clusterin, and a C-terminal fragment of TGFBIp containing residues Tyr571-Arg588, apolipoprotein E, and apolipoprotein A-IV. Interestingly, the serine protease hightemperature requirement (Htr) A1 was abundant in amyloid deposits. Alignment analysis revealed that numerous proteolytic sites in the fourth FAS1 domain were associated with the activity of HtrA1. The same research group also investigated the proteomic profiles of amyloid deposits associated with the heterozygous LCD1 (p.Ala546Asp positioned before the Tyr571-Arg588 sequence; LCD variant), periamyloid corneal tissue (disease-free corneal stroma of the LCD1 cornea) and healthy stroma from normal patients (Karring et al., 2013). As expected, similar proteomic profiles were observed in amyloid deposits. In contrast, even though the adjacent corneal tissue also had a similar protein profile, C-terminal fragments including Tyr571-Arg588 residues were less abundant than in the amyloid deposits or healthy corneal stroma. The authors suggested that the C-terminal TGFBIp fragment encompassing residues Tyr571-Arg588 from the fourth FAS1 domain, serum amyloid P-component, apolipoprotein A-IV, clusterin, and serine protease HtrA1 accumulate in the corneal amyloid deposits and the residues contains the amyloid core. The Tyr571-Arg588 peptide was also identified as the amyloid core of in vitro fibrillated p.Ala546Thr substitution (Sorensen et al., 2015). When comparing the two phenotypically different lattice corneal dystrophies, a single p.Ala546Asp substitution or a p.[(Ala546Asp; Pro551Gln)] cis compound heterozygous mutation substitution in TGFBIp, LC-MS/MS results revealed that the protein profile of amyloid deposits in these different mutations were similar: TGFBIp, serum amyloid P-component, apolipoprotein A-I, apolipoprotein A-IV, apolipoprotein E, apolipoprotein D, clusterin and HtrA1. These results were similar with the previous two reports of Karring et al. (Poulsen et al., 2014). The single p.Ala546Asp mutation leads to bilateral polymorphic, polygonal, refractile, whitish-gray opacities with a chipped ice appearance (CorreaGomez et al., 2007; Eifrig et al., 2004), while the p.[(Ala546Asp; Pro551Gln)] cis compound heterozygous mutation creates branching and non-branching lattice-like stromal deposits resembling pipe stems (Klintworth et al., 2004). Stability data indicate that the fourth FAS1 domain of p.Pro551Gln resembles that of WT in stability; however, p.[(Ala546Asp; Pro551Gln)] cis compound heterozygous mutation shows structural changes when compared to the p.Ala546Asp single mutation. Therefore, phenotypical differences between the p.Ala546Asp mutation and the p.[(Ala546Asp; Pro551Gln)] cis compound heterozygous mutation may be related to stability differences between TGFBIp's, rather than differences between protein profiles. 4.2.3. Alteration of conformational structure and stability of TGFBIp Protein mis- or unfolding and its structural change has been



proposed as the core mechanism of amyloid-fibril formation in TGFBI corneal dystrophies and many other diseases including Alzheimer's disease, Parkinson's disease, and Huntington's disease (Clout and Hohenester, 2003; Zerovnik, 2002). TGFBIp and Drosophila fasciclin I are members of a protein superfamily containing FAS1 domains that closely resemble each other. The FAS1 domain has a compact globular structure that consists of a-helices and b-sheets. Using the homology model, Clout and Hohenester (2003) suggested that there were differences between common mutations and less common mutations. They hypothesized that the common mutations at codons Arg124 and Arg555 might affect proteineprotein interactions resulting in alteration of stability. They suggested that it is less likely that the mutation of either arginine would lead to protein misfolding. Arg124 is located between the a1 and a2 helices of first FAS1, while Arg555 is located in the linker of a3 and a4 helices of the fourth FAS1 domain and both arginine at codons 124 and 555 are anticipated to be exposed to solvent. Meanwhile, the less common mutations in the fourth FAS1 domain may induce protein misfolding which can interfere with the secretion of protein or cause protein destabilization. Since residues in the fourth FAS1 domain such as Leu518, Leu527, Thr538, Asn544, Ala546, Asn622, Gly623, His626, and Val623 are buried inside and surrounded by hydrophobic residues, their mutations are likely to disrupt the packing and induce protein unfolding (Clout and Hohenester, 2003). The changes in conformational stability also differed according to the location of individual mutations. Transverse urea gradient gel electrophoresis, which measures susceptibility to chemical denaturation, showed that mutations within the first FAS1 domain (p.Arg124His, p.Arg124Leu, and p.Arg124Cys) did not affect stability. In contrast, mutations in the fourth FAS1 domain (p.Ala546Thr, p.Arg555Gln, and p.Arg555Trp) affected stability according to the following order: p.Arg555Trp > WT > p.Arg555Gln > p.Ala546Thr. Both an increase (p.Arg555Trp) and a decrease (p.Arg555Gln and p.Ala546Thr) in stability caused protein aggregation which might be a factor for different phenotypes. Significantly, when the deposits were incubated under physiologic conditions, the mutant p.Ala546Thr displayed a 6-fold higher Thioflavin T signal than the fourth FAS1 domain of WT, while mutant p.Arg555Gln and p.Arg555Trp displayed no significant increase in fluorescence over the time, suggesting no amyloid fibril formation. These findings suggest that different aggregation mechanisms are involved in during protein deposition in the TGFBI corneal dystrophies (Runager et al., 2011). The mutant GCD1 (p.Arg555Trp) increased the propensity to aggregate and showed less susceptibility to proteolysis than WT. The cleavage site most susceptible to thermolysin and trypsin was located between residues Arg557 and Leu558 in the fourth FAS1 domain of WT, while the p.Arg555Trp mutant in the fourth FAS1 domain showed resistance to proteolysis. Liquid-state nuclear magnetic resonance (NMR) spectroscopy revealed that the Arg555 residue was exposed in the WT, while the Trp555 mutation was buried in the core in an empty hydrophobic cavity in the fourth FAS1 domain. Molecular dynamics (MD) simulation showed that the C-terminus of the a3 helix which contained this cleavage site was less flexible in the mutant domain, resulting in proteolytic resistance (Underhaug et al., 2013).

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marrow, urinary bladder, adrenal gland, parathyroid gland, muscle, and prostate (El Kochairi et al., 2006). This may be due to the high concentration of TGFBIp in cornea (Dyrlund et al., 2012). Among 1679 proteins in the corneal stoma, TGFBIp is the second most abundant protein (17.6 ± 3.3%). Other hypotheses have been proposed to explain the origin of the corneal deposits (Korvatska et al., 2000; Schmitt-Bernard et al., 2002). Some cornea-specific factors may be involved in the pathogenesis. This hypothesis is supported by the observation that cultured corneal fibroblasts which bear the p.Arg124Cys mutation do not produce amyloid spontaneously (Korvatska et al., 2000). Alternatively, heparin sulfate which is one of ECM proteoglycan accelerate fibrillation of mutated protein in p.Ala546Thr mutation in vitro (Andreasen et al., 2012). Specific constitutive and/or physiological factors in the cornea may lead spontaneous aggregation of mutant TGFBIp and partially degraded peptides. Usually there is a clear zone (lucid interval) between the limbus and deposits. We, the authors, observed a clear zone near blood vessels in GCD2 corneas. For example, the deposits disappear spontaneously near the advancing edge of pterygia, maintaining the clear zone between the deposit and the tip of pterygia (Lee et al., 2006). No deposits appear at perilimbal cataract incisions, while central corneal wounds usually result in the dense exacerbation of the deposits (Feizi et al., 2007; Jun et al., 2004; Kim et al., 2008a; Lee et al., 2006; Roh et al., 2006a). Deposits are more severe near the central portion of radial keratotomy (RK) incisions in GCD1 corneas, compared to the peripheral portion of incisions (Fig. 6) (Feizi et al., 2007). These findings suggest to us that blood vessels may inhibit the deposition of abnormal TGFBIp. 4.3.1. Localization of TGFBI deposits within the cornea TGFBIp is a 68-kDa secretory protein that is subsequently degraded in tissue. The degraded TGFBIp is different from one to another type of mutation. Localization of deposits within the cornea is related to degradation conditions and products of mutated TGFBIp. In GCD2, two-fold differences were detected in the expression of 555 genes between WT and homozygous GCD2 primary cultured corneal fibroblasts. Of these, 319 genes were upregulated, and 236 genes were down-regulated in the homozygous GCD2 primary cultured corneal fibroblasts (Choi et al., 2010). The different ECM condition of each mutation might influence the localization of TGFBI deposits within the cornea.

4.3. Mutated TGFBIp accumulates in the cornea specifically Interestingly, the cornea is known to be the only affected tissue in patients with TGFBI corneal dystrophy. Autopsy of a 79 year-old patient with LCD1 and Alzheimer's disease revealed pathologic TGFBIp in cornea, but not in the other 17 organs or tissues examined, including brain, heart, lung, kidney, liver, lymph nodes, bone

Fig. 6. Slit-lamp photograph reveals exacerbation of granular deposits in a GCD1 patient who underwent radial keratectomy (RK) for the correction of myopia. Deposits around the central portion of the incisions were thicker and denser than those near the peripheral portion of the incisions (courtesy of Sepehr Feizi, MD, MSc Shaheed Beheshti University, Iran).


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4.3.2. Formation of granular deposits As noted earlier, the main source of the TGFBIp in the granular deposits is epithelial cells and keratocytes. When we observed the very early deposits in the cornea of a GCD2 heterozygote, an isolated single deposit was surrounded by a clear zone while the clear zone was surrounded again by a diffuse, hazy subepithelial deposit. This suggests that the subepithelial tiny, central deposit might be the nidus for accumulation of deposits, similar to the nucleation process in chemical crystallization. An early deposit surrounded by a clear zone has also been observed 4 years after PKP in the donor cornea in a homozygote with GCD2 (Fig. 7) (Choi et al., 2011b). Since transplanted central stromal keratocytes can survive more than 10 years after PKP (Kim et al., 2009b), these deposits may have been produced by recipient epithelium, which covers the donor cornea soon after transplantation. More definitive data, however, are needed to fully explain the origin and deposition of opacities related to TGFBIp. 4.3.3. Sudden morphologic changes of the corneal deposits Most corneal deposits increase in size and number slowly with time. Sudden disappearance of preexisting corneal deposits, however, can occur in some cases. We studied the disappearance of deposits in GCD1 and GCD2 patients by serial examination of the same corneas over time (Han et al., 2013). Only superficial deposits tended to disappear, suggesting that loosely attached superficial deposits might be detached by friction from the lid. Serial photos show that the clear central part of the annular corneal deposit created by drop-out of GCD1 and GCD2 opacities fills up again with aggregated materials over time. We correlated the prevalence of discoid granular deposits, annular granular deposits and lattice-like lesions with age in GCD2 heterozygote patients (Han et al., 2013). Discoid granules were the most frequent type of deposit in all age groups, sharply increasing in number with age. Annular granular deposits, presumed to be the result of spontaneous drop-out of central opacities, increased also. Since the disappearance of preexisting corneal deposits has also been observed in a TBCD patient (Kobayashi et al., 2005), this dropout phenomenon might not be a disease-specific event but a universal characteristic of deposits that accumulate near the surface of the cornea. 4.4. Phenotypic differences from the same mutation in TGFBI corneal dystrophies The phenotypes of patients with TGFBI corneal dystrophies can

differ even among members of the same family, and the explanation for this observation is not clear. Corneal deposits may be composed not only of TGFBIp, but also by other extracellular materials. The genetic environment of the ECM of each patient might be different creating different phenotypes. 4.4.1. The production of TGFBIp differs among individuals and family members The severity of deposits in GCD2 varies among individuals and families. Rosenwasser et al. (1993) reported that variable proportions of granular and lattice deposits, whether granular dominant or lattice dominant, were observed within the same family and among different families. Cao et al. (2009) reported reduced penetrance in seven patients in a family, 4 of whom had no corneal deposits at all. We reported strikingly varied phenotypes between individuals and also showed that the productivity of TGFBIp from cultured corneal fibroblasts obtained from normal corneal donor rim varies significantly (Han et al., 2012a). These data suggest that level of production of TGFBIp might partially explain the phenotype, but this hypothesis needs to be confirmed by additional research. 4.4.2. Two mutations of TGFBI in single allele or in both alleles TGFBI compound mutation e two TGFBI mutations in a single individual e can occur in two different ways: two mutations in the same allele (cis form) and one mutation in one allele and the other mutation in opposite allele (trans form). So far, 7 compound heterozygous TGFBI mutations have been reported. The p.[(Arg124Leu; Thr125_Glu126del)] cis compound heterozygous mutation, induced early onset, rapidly spreading round or snowflakes-like opacities and an early ground-glass haze in the intervening stroma, which suggests a superficial variant GCD (Dighiero et al., 2000a, 2001). The p.[(Ala546Asp; Pro551Gln)] cis compound heterozygous mutation showed central retractile anterior stromal opacification and punctate grayewhite deposits as well as branching lattice lines in the mid and peripheral cornea (Aldave et al., 2004). The p.[Arg549Thr]; [Arg555Trp] trans compound heterozygous mutation for GCD1 (Frising et al., 2006) showed severe recurrence after keratoplasty. The p.[Arg124His]; [Asn544Ser] trans compound heterozygous mutation showed a summation of phenotypes of GCD2 and LCD, while p.Arg124His alone did not (Yamada et al., 2009). The p.[Arg124Cys]; [Gly470Ter] trans compound heterozygous mutation has been identified in one family (Sakimoto et al., 2003). The proband's daughter, who carries the heterozygous mutation of p.Gly470Ter alone, was unaffected. The

Fig. 7. Slit-lamp photographs of a 17-year-old female homozygous patient with GCD2 4 years after penetrating keratoplasty (PKP). A, Extensive fine granular deposits are observed primarily in the center of the graft and there are deposit-free intervals between the central recurrent granules and the deposits around the suture tracts. B, Magnified view of Fig. 7A shows tiny, central deposits (arrows) surrounded by a clear zone.



function of p.Gly470Ter in the trans compound heterozygous mutations needs more research. There are other two reports of compound heterozygote mutations in which the form of mutation was not determined. The p.[Arg124His()c.307_308delCT] mutation was associated with large granular flake-ice like opacities involving two-thirds of the cornea without lattice-like deposits (Yam et al., 2012). The p.[Arg514Pro() Phe515Leu] (Zhong et al., 2010) mutation was associated with LCD variants. 4.4.3. Possible effects of other genomic factors Many reports have shown that there are severe and mild phenotypes of TGFBI corneal dystrophies, even though the associated mutation is the same. To evaluate the possible effects of other genetic factors on the expression of TGFBI mutations, a genome-wide linkage analysis with single nucleotide polymorphism (SNP) of a total of 551 Korean GCD2 patients and their relatives from 59 families was performed (Lee et al., 2011a). The authors selected nine candidate loci for GCD2 after single-point linkage analyses and only the 3q26.3 region that includes the neuroligin 1 gene (NLGN1) was supported by empirical statistical significance in simulation analysis. The authors, however, could not find a significant correlation between the 3q26.3 region and the severity of GCD2 phenotypes. More research is needed in this field. 5. Molecular pathogenesis of GCD2 and TGFBI corneal dystrophies Recently, multiple studies of cellular, biochemical, and molecular mechanisms of TGFBI corneal dystrophies have been conducted. Our study group has focused on the pathogenesis of GCD2. GCD2 corneal fibroblasts show several pathologic differences compared to normal corneal fibroblasts such as vulnerability to oxidative stress, age-dependent alteration of mitochondria, and a defective autophagic proteolytic system. 5.1. Characteristics of corneal fibroblasts of GCD2 5.1.1. Morphological properties of GCD2 corneal fibroblasts Keratocytes are the predominant cellular component of the corneal stroma (Otori, 1967). They are responsible for maintaining corneal transparency and the ECM environment (Jester et al., 1999). Unlike normal cells, corneal fibroblasts from GCD2 patients are larger, have multiple vesicles of various sizes containing amorphous material, and show a senescence-like appearance (Fig. 8). In addition, GCD2 corneal fibroblasts showed greater co-localizations of mutant-TGFBIp and cathepsin D (a lysosomal enzyme) than WT cells (Choi et al., 2012). Transmission electron microscopy revealed more elongated or fragmented swollen mitochondria when compared to small, round mitochondria of normal corneal fibroblasts (Choi et al., 2015a; Kim et al., 2008a). Disorganized and dilated mitochondria of decreased numbers were observed especially in GCD2 homozygous cells in late passage. Corneal fibroblasts from GCD2 homozygotes will not survive more than 8 passages in vitro while those from normal donors can be passed more than 24 passages. There are reports that fourth FAS1 p.Ala546Thr oligomers caused leakage from an artificial membrane system (Andreasen et al., 2015, 2012). Since amyloid oligomers in Parkinson's disease and Alzheimer's disease have been reported to be associated with perturbation and changes in the permeability of membranes (Volles and Lansbury, 2002; Sokolov et al., 2006), the relationship between mutated protein and cellular abnormalities observed through the leakage of the cellular membrane needs further research in TGFBI corneal dystrophies.

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In addition, 555 genes showed more than a twofold difference in expression between WT and homozygous GCD2 cultured corneal fibroblasts (Choi et al., 2010). Of these, 319 genes were up-regulated and 236 were down-regulated in the homozygous GCD2 corneal fibroblasts compared to WT. The complexity of several gene functions might be one of the factors accounting for the cellular changes in GCD2 corneal fibroblasts. 5.1.2. Oxidative stress in GCD2 corneal fibroblasts The cornea is consistently exposed to the atmosphere and absorbs most of the ultraviolet (UV) light that strikes the eye (Wenk et al., 2001). Exposure to UV light generates reactive oxygen species (ROS) which can cause oxidative injury. In mammalian cells, antioxidant enzymes (Cu/Zn-superoxide dismutase [SOD], MnSOD), antioxidant defense enzymes (glutathione peroxidase [GPx] and glutathione reductase [GR]) and non-enzymatic ROS scavenging systems all work to regulate the concentration of ROS (Rao et al., 1987). ROS react with intracellular lipids, proteins, and DNA and release the following by-products: malondialdehyde (MDA) (Esterbauer et al., 1991; Gutteridge and Halliwell, 1990) and 4hydroxy-2-nonenal (HNE) (Esterbauer et al., 1991; Gutteridge and Halliwell, 1990) from lipids, protein carbonyl groups (Berlett and Stadtman, 1997; Dalle-Donne et al., 2003) from proteins, and 8hydroxy-20 -deoxyguanosine (8-OHdG) (Kasai et al., 1984; Kasai and Nishimura, 1984) from DNA. Elevation of these by-products is a sign of oxidative stress. Mitochondria consume a significant amount of oxygen during oxidative phosphorylation reactions for ATP synthesis (Halliwell and Gutteridge, 1989). During the sequential oxygen reduction reactions, oxygen-derived toxic free radicals including superoxide (O 2 ), hydroxyl radicals ( OH), and hydrogen peroxide (H2O2) are produced (Mates et al., 1999). In GCD2 corneal fibroblasts, the levels of the MDA, 4-HNE, and protein carbonyl groups were significantly higher than levels in WT corneal fibroblasts (Choi et al., 2009). MDA immunoreactivity was also significantly higher in the corneal stroma and epithelial layer of GCD2 patients when compared to those of normal corneal tissue (Choi et al., 2009). Intracellular ROS and H2O2 levels were significantly elevated and expression levels of various antioxidant enzymes including Cu/Zn-SOD and Mn-SOD, GPx, and GR were significantly higher in GCD2 corneal fibroblasts than normal ones. In spite of an increased catalase mRNA level, the expression level of catalase protein which is the major enzyme for detoxificating H2O2, was decreased in GCD2 corneal fibroblasts (Choi et al., 2009). Cell viability after H2O2 exposure was significantly lower in GCD2 corneal fibroblasts than normal ones (Choi et al., 2009). These findings suggest that GCD2 corneal fibroblasts not only are in oxidative stress, but also are more susceptible to oxidative damage. 5.1.3. Mitochondrial dysfunction in GCD2 Mitochondria are essential organs for metabolism and cell survival that produce energy via a proton gradient across the membrane. As cells age, deleterious proteins accumulate and mitochondria become dysfunctional. Mitochondrial activity and mitochondrial membrane potential (DJ) fall rapidly during late passages in cultured GCD2 corneal fibroblasts, while levels in WT cells remain constant (Kim et al., 2011). Delayed proliferation, reduced cellular viability, typical apoptotic features, and alteration of the expression of the membrane-bound mitochondrial complex I ~ V were apparent in the late passages of mutant GCD2 cells. The mutation of GCD2 may affect mitochondrial activity via abnormal protein production and increased oxidative stress. Increased intracellular ROS were recovered using application of the antioxidant butylated


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Fig. 8. Morphological characterization of primary cultured GCD2 corneal fibroblasts by phase-contrast microscopy of WT (A and C) and GCD2 homozygote (B and D) corneal fibroblasts (Choi et al., 2012; Choi et al., 2014). The homozygous GCD2 corneal fibroblasts showed intracellular deposits of various sizes.

hydroxyanisole in cultured GCD2 cells. These data suggest that antioxidants may serve to reverse GCD2-induced cellular toxicities. 5.1.4. Mutant-TGFBIp accumulates in the cell because of defective autophagy in GCD2 5.1.4.1. Autophagy and ubiquitin-proteasome in TGFBIp degradation in GCD2. Two proteolytic systems, the ubiquitin-proteasome system (UPS) and the autophagy system, are involved in the process of degrading abnormal proteins and damaged organelles in eukaryoic cells (Ciechanover, 2005). The UPS selectively degrades proteins, while the autophagy system degrades several species of molecules (microautophagy or chaperone-mediated autophagy) and damaged organelles (macroautophagy) such as mitochondria (Cuervo, 2004). Among them, macroautophagy (hereafter referred to as autophagy) has been considered the major system for protein and organelle quality control. In GCD2 corneal fibroblasts, mutant-TGFBIp accumulates in

lysosomes or autophagosomes with LC3-enriched cytosolic vesicles and cathepsin D (Choi et al., 2011b, 2012, 2014; Kim et al., 2009a). The levels of TGFBIp in GCD2 homozygous corneal fibroblasts was not significantly different from that in WT cells, while the level of expression of TGFBI mRNA was lower in GCD2 corneal fibroblasts than it was in WT cells (Choi et al., 2014). When WT and GCD2 homozygous corneal fibroblasts were treated with cyclohexamide, a protein synthesis inhibitor, TGFBIp was nearly absent in WT cells after 40 min, while mutant-TGFBIp remained in GCD2 cells after 60 min. These results suggest that the intracellular accumulation of mutant-TGFBIp is results from either defective degradation or delayed extracellular secretion. This mechanism is further supported by data showing that mutant intracellular TGFBIp accumulated when mutant-TGFBI was transfected to normal corneal fibroblasts (Choi et al., 2014). When the autophagy inhibitor bafilomycin A1 was treated to cultured WT and GCD2 corneal fibroblasts, intracellular TGFBIp



increased significantly while the UPS inhibitor MG132 did not affect the level of TGFBIp in either cell line. This indicates that TGFBIp is degraded by autophagy, rather than UPS. The autophagy markers, LC3-II and SQSTM1 were increased, but BECN1, which is involved in the early stage of autophagosome formation, was unchanged in GCD2 corneal fibroblasts. Bafilomycin A1 increased LC3-II levels similarly in WT and GCD2 corneal fibroblasts. These findings suggest the accumulation of mutant-TGFBIp is a result of delayed fusion between autophagosomes and lysosomes, rather than autophagosome formation at an early stage in the process (Choi et al., 2012). Autophagy is regulated by the mammalian target of rapamycin (mTOR), which is one of the best-characterized downstream pathways. mTORC1, which is sensitive to rapamycin, is composed of mTOR, G-protein beta-subunit like protein (GbL), and regulatoryassociated protein of mTOR (raptor) (Hara et al., 2002). In the unstressed condition, mTORC1 suppresses the downstream kinase complex and blocks autophagy (Fig. 9). As noted above, the autophagy marker LC3-II was significantly elevated and the number of endogenous LC3 puncta, which colocalizes with mutant-TGFBIp, was significantly increased in GCD2 corneal fibroblasts. When the lysosomal protease inhibitor, leupeptin, was added, the LC3-II level increased significantly. In addition, the expression of raptor decreased and autophagy was activated via mTOR pathway in GCD2 cells (Choi et al., 2014). This level of activation, however, was not sufficient to remove mutantTGFBIp from these cells. Several mTORC1 components such as mTOR, GbL, and raptor are known to be degraded via the UPS (Fu et al., 2009), therefore, a reduced raptor level would suggest that the activation of autophagy

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might be related to the UPS. Polyubiquitinated proteins accumulated in GCD2 corneal fibroblasts (Choi et al., 2012). MG132, an UPS inhibitor, significantly increased the levels of ubiquitinated raptor in GCD2 corneal fibroblasts and resulted in increased apoptotic cell death in GCD2 cells (Choi et al., 2014). UPS activation may play a protective role in GCD2 corneal fibroblasts. The crosstalk between the UPS and autophagyelysosome systems may exist in GCD2 and requires further investigation. 5.1.4.2. Endocytosis in TGFBIp degradation. Most secretory proteins are transferred from the nucleus to the extracellular space via the endoplasmic reticulum (ER)-Golgi secretory pathway (Cheng, 2010). To determine whether the ER-Golgi secretory pathway is involved in TGFBIp secretion, corneal fibroblasts were treated with brefeldin-A (which inhibits protein transport from the ER) and monensin (which blocks protein transport to Golgi apparatus). Both brefeldin-A and monensin treatments almost completely blocked TGFBIp secretion in WT and GCD2 corneal fibroblasts. Pulse-chase experiments revealed that newly synthesized WT TGFBIp was almost entirely secreted with only 0.5% remaining in the cells. In contrast, 20% of mutant TGFBIp was retained inside the GCD2 homozygous corneal fibroblasts (Choi et al., 2015b). These findings demonstrate that TGFBIp is secreted via the ER-Golgi secretory pathway, and that this secretion is delayed in GCD2 corneal fibroblasts. Removal of extracellular proteins is essential for cellular processes, including tissue development, remodeling, and repair (Holmbeck et al., 1999; Van Amersfoort and Van Strijp, 1994). Endocytosis is the initiating step of internalization of molecules from ECM. After internalization, molecules are recycled back to the

Fig. 9. The phosphatidylinositol-3-kinase (PI3K)/the serine/threonine kinase AKT signaling pathways block the activation of autophagy via the activation of mTOR. p53 acts as a negative regulator of mTOR. The autophagosome, after formation, fuses with the lysosome to become an autophagolysosome, where lysosomal hydrolases digest the sequestered cytoplasmic derived contents. Autophagy can be blocked with 3-methylamphetamine (3-MA: a class III PI3K inhibitor). Autophagy can be activated with AKT inhibitors and rapamycin, a small molecular inhibitor to mTOR.


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surface or transported to lysosomes for degradation. Genistein (an inhibitor of caveolae-mediated internalization) treatment reduced intracellular TGFBIp. Treatment of inhibitors of clathrin-mediated endocytosis, chlorpromazine, did not reduce the expression level of intracellular TGFBIp (Choi et al., 2015b). Cultured WT corneal fibroblast showed flask-shaped invaginations of the cell membrane and abundant membrane-associated vesicles by electron microscopy. To determine whether internalized TGFBIp is degraded by lysosome, NIH3T3 and ZW13-1 cells, which do not express TGFBIp but internalize TGFBIp, were treated with bafilomycin A1, an inhibitor of lysosomal degradation. Internalized TGFBIp accumulated in lysosomes after this treatment, suggesting that internalized TGFBIp is degraded by lysosome. Co-immunoprecipitation revealed that internalization of TGFBIp is associated with the interaction of RGD-motif and integrin aVb3. The schematic diagram of secretion and internalization of TGFBIp is shown in Fig. 10. 5.1.4.3. ER stress in GCD2. Accumulation of unfolded proteins induces ER stress (Schroder and Kaufman, 2005). Zhu et al. (2012) investigated ER stress in TGFBI mutations using recombinant cDNA of the GCD1 (p.Arg555Trp) and LCD (p.Thr538Pro) in vitro. These studies await confirmation and investigations of the effect of other TGFBI mutations. 5.1.5. Altered cell cycle arrest and reduced proliferation of GCD2 corneal fibroblasts When mammalian cells divide, cells in the quiescent phase (G0) enter the cell division cycle, which consists of the first gap phase (G1), DNA synthesis (S), the second gap phase (G2), and mitosis (M) phases. Cyclin-dependent kinases (CDKs) activate cyclin which has a role in proliferation. Two proteins, the CDK interacting protein/ kinase interacting protein (Cip/Kip) family (p21, p27, and p57) and

inhibitors of CDK family (p15, p16, p18, and p19), regulate cylcin (Sherr and Roberts, 1995). GCD2 homozygote corneal fibroblasts proliferated at lower rate than WT cells at the 7th to 8th passage and morphological changes, such as an increase in size, were observed by phase contrast microscopy (Kim et al., 2011). Fluorescence-activated cell sorting (FACS) analysis revealed that WT cells accumulate in the G1 phase. In contrast, most GCD2 corneal fibroblasts are arrested in the G2 phase. In GCD2 fibroblasts, the expression of several cell cycle activators and inhibitors showed different pattens when compared with normal cells. The expression of Cyclins (A1, B1, D1, and E1), CDK6, p21, and p53 was significantly lower, and the expression of p16 and p27 was higher in GCD2 corneal fibroblasts. Bafilomycin A1, an autophagy inhibitor that interrupts autophagosome-lysosome fusion, reduced Cyclin A1, B1, and D1 and p53 and increased p21 in WT cells as shown in GCD2 cells (Choi et al., 2015a). These findings suggest the insufficient and/or defective autophagy in GCD2 corneal fibroblasts might be an explanation for the reduced rate of proliferation of GCD2 cells. 5.2. Regulation of TGFBIp expression in corneal fibroblasts 5.2.1. Role of TGF-b in TGFBIp expression TGF-b1 induces progressive accumulation of TGFBIp and ECM proteins such as collagen, fibronectin, and keratin in the corneal epithelium and stroma (Jeon et al., 2012; Yellore et al., 2011). TGFb1 can regulate TGFBIp expression through Smad transcription factors and E-box-dependent mechanisms (Kato et al., 2009; Shi and Massague, 2003). The activated TGF-b receptor complex phosphorylates Smad2 and Smad3, and the phospho-Smad2/3 assembles with Smad4. Then, the Smad complex translocates to the nucleus and regulates transcription of target genes (Shi and Massague, 2003).

Fig. 10. Schematic diagram of trafficking and deposition of TGFBIp. TGFBIp is secreted via the ER-Golgi system. Secreted TGFBIp remains in the extracellular matrix (ECM) and would be degraded during ECM remodeling after its life span. In TGFBI corneal dystrophy, secreted mutated TGFBIp will aggregate with other ECM components, such as collagen in the ECM. TGFBIp is secreted by the epithelium and, in some conditions, by keratocytes. At least some of the secreted TGFBIp is constitutively internalized via the caveolae-dependent pathway and is degraded by lysosomes in the cultured corneal fibroblasts (Choi et al., 2015b). It is not known whether TGFBIp internalization occurs in the epithelium.



5.2.2. TGFBI gene regulation: epigenetics The production of TGFBIp from normal human corneal fibroblast has been shown to be variable and dependent on the corneal donor (Han et al., 2012a). Our group searched the promoter of TGFBI gene in white blood cells from severe (n ¼ 3) or mild (n ¼ 3) corneal phenotype GCD2 patients and did not find differences in promoters up to -3K of the DNA sequence between two groups. Lee et al. (2011b) also observed the TGFBI promoter in A549 and MBA-MD-231 cells. They found that TGFBI expression required Sp1 transcription elements that can bind transcription factors Sp1 and Sp3 in vitro. They also observed that occupancy of the TGFBI promoter by Sp1 and Sp3 in vivo was only seen in TGFBI-expressing cells, leading them to conclude that the expression of TGFBI is well correlated with chromatin conformation at the TGFBI promoter, and that factors Sp1 and Sp3 are related for the control of constitutive expression of TGFBI gene. In general, methylation (H3Kme) can be associated with either active or inactive gene promoters depending on the position of modified lysine. Maeng et al. (2015) found that TGF-b1 leads to the enrichment of H3K4me1,3 and depletion of H3K27me3 marks at TGFBIp and ECM-associated gene promoters in corneal fibroblasts. More information is needed to fully characterize novel epigenetic chromatin mechanisms of TGFBIp production in GCD2. 5.2.3. Factors affecting production of TGFBIp in corneal fibroblasts; LPA Normal corneal fibroblasts express two lysophosphatidic acid (LPA) receptors, LPA1 and LPA3 (Wang et al., 2002) and corneal injury increases expression of LPA, as well as that of TGF-b1 (Kaji et al., 2001). Jeon et al. (2012) investigated the effects of LPA on the TGF-b signaling pathway, which may contribute to the increased accumulation of deposits seen after laser ablation. LPA increased TGFBIp expression through JNK-dependent activation of TGF-b signaling pathways in WT and GCD corneal fibroblasts. 6. Treatment 6.1. Surgical approaches 6.1.1. Laser ablation Various therapeutic strategies have been utilized to improve vision by removing corneal deposits and preventing their deposition. Phototherapeutic keratectomy (PTK) is the procedure of choice for the treatment of shallow corneal deposits, improving vision and delaying the need for PKP or deep anterior lamellar keratoplasty (DALK) in the future, when deposition of opacities continues. The procedure can be repeated for patients in whom deposition of opacities continues (Maclean et al., 1996). Unfortunately, in patients with TGFBI corneal dystrophies, the dystrophic deposits typically recur after PTK, DALK, and PKP. Published reports of recurrence rates after PTK and keratoplasty varies according to the many factors, including the definition of recurrence and the length of follow-up. Recurrences after PTK were seen in 0e47% of eyes with RBCD at 12e21.6 months (Dinh et al., 1999; Eggink et al., 2002), 80e100% of eyes with TBCD at 8e108 months (Hieda et al., 2013; Sorour et al., 2005), 14e17% of eyes with an unspecified type of LCD or LCD1 at 6 months to 15 years (Das et al., 2005; Dinh et al., 1999), and 20e100% of eyes with an unspecified type of GCD at 7e40.3 months (Das et al., 2005; Dinh et al., 1999; Dogru et al., 2001). The mean time to recurrence in patients with homozygous GCD2 was 3 months after PTK and it was much shorter and aggressive in eyes of patient with heterozygous GCD2 (Moon et al., 2007). Whether PTK increases the need for PKP is debated. Park et al. (2007) hypothesize that PTK might be a risk factor for the need

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for keratoplasty because surgical trauma and laser ablation can exacerbate the natural course of GCD2. In contrast, others suggest that PTK increases visual acuity without affecting the prognosis of subsequent PKP (Seitz et al., 2004). Refractive corneal surface ablation is one of the most popular procedures for the correction of refractive errors in many countries. However, since the first case report of the exacerbation of GCD2 after laser in situ keratomileusis (LASIK) in Korea in 2002, approximately 100 cases have been reported in South Korea, Japan, the United States, and Iran after photorefractive keratectomy (PRK), RK, LASIK, and laser epithelial keratomileusis (LASEK) (Aldave et al., 2007; Awwad et al., 2008; Banning et al., 2006; Chiu et al., 2007; Feizi et al., 2007; Kim et al., 2008a, 2008b, 2008c; Lee et al., 2007; Roh et al., 2006b; Wan et al., 2002). The exacerbation pattern was different depending on the refractive surgical procedure. After LASIK, multiple small, discrete granular deposits typically appear in the interface of the flap and the stromal bed. These differ from the larger, discrete, granular deposits typically seen in virgin GCD2 corneas. Interface deposits appear 1e5 years after LASIK and are primarily composed of hyaline material (Kim et al., 2008c). Following LASEK and PRK, numerous fine granular deposits are seen in the anterior stroma. Exacerbation of deposits has been reported 1e3 years after LASEK (Kim et al., 2008a), and severe recurrences have been seen 5 and 11 years after PRK (Kim et al., 2008b). More severe recurrences are seen after LASIK than after PRK (Kim et al., 2008b), and scanning electron microscopy shows granular deposits that appear to be adherent to damaged collagen fibrils (Roh et al., 2006b). This observation suggests that more severe recurrences after LASIK than after PRK are explained by the fact that two surfaces with damaged collagen layers are present after LASIK, compared to one after PRK. 6.1.2. Keratoplasty PKP and anterior lamellar keratoplasty (ALKP) have traditionally been used to treat deep deposits from stromal corneal dystrophies, with a more recent trend toward DALK. The recurrence rate after PKP is reported to be 46.4% in GCD1 at 10 years (Ellies et al., 2002), 50e60% in LCD at 48e135 months (Ellies et al., 2002; Marcon et al., 2003), and 88.9% in RBCD at 5.9 years (Ellies et al., 2002), 38.5e100% in TBCD at 3.7e15 years (Ellies et al., 2002; Sorour et al., 2005), and 88% for TBCD or RBCD at 24 months (Marcon et al., 2003). Pandrowala et al. (2004) reported that the recurrence rate of corneal dystrophies was 2.8% in 181 eyes after PKP over a period of 6 years. Because RBCD, TBCD, some cases of GCD1, GCD2, and LCD do not affect the deep corneal stroma, ALKP may be considered for primary cases and cases of recurrence following PTK. ALKP is technically less difficult than DALK; however, visual acuity after ALKP is often less than it would have been after DALK due to irregularity and/or scar formation on donor-to-recipient interface (Lyons et al., 1994; Richard et al., 1978). Ünal et al. (2013) reported that among 17 eyes with LCD and 9 with GCD, the recurrence rate after DALK was 35.3% for LCD and 22% for GCD during the 12e96 months postoperatively. Park et al. (2007) reported that 1 of 4 eyes with GCD showed a recurrence at 13 months after DALK. Salouti et al. (2009) reported the highest recurrence rate: approximately 71% 13e16 months after DALK. This unusually high recurrence rate might be due to a surgical technique which leaves recipient stroma, unlike the conventional DALK procedure. Vajpayee et al. (2007) reported that recurrences were not found up to 11 months in the 2 GCD patients. When comparing ALKP and PKP in GCD patients (the subtype was not reported), Lyons et al. (1994) reported that visual acuities were similar and recurrence was observed after 3 years for both procedures. Kawashima et al. (2006) compared the surgical


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outcomes after DALK and PKP in patients with LCD and macular corneal dystrophy. They concluded that DALK is a safe alternative to PKP for the treatment of LCD and macular corneal dystrophy. Because epithelial cells have been implicated the origin of the deposits, limbal stem cell transplantation has been attempted as a treatment for stromal corneal dystrophies (Dunaief et al., 2001; Reinhard et al., 1999; Spelsberg et al., 2004; Sundmacher et al., 1999). Homologous penetrating central limbokeratoplasty, however, is not effective for long-term treatment because donor epithelial cells are eventually replaced by host epithelial cells (Spelsberg et al., 2004). 6.1.3. Corneal electrolysis and corneal epithelial debridement Removal of corneal opacities by electrolysis reportedly improved the visual acuity of an 11-year old boy with RBCD through 7 years of follow-up (Kamoi et al., 2005). Mashima et al. (2002) applied electrolysis to the surface of corneal grafts in two GCD2 homozygotes and one RBCD patient after PKP and the hostegraft interface in one GCD2 heterozygote after ALKP. It is thought the NaOH which is produced by the electric current might dissolve the corneal deposits. Deposits have been reported to recur 2 or more years after electrolysis (Mashima et al., 2002), and the long-term outcome of electrolysis is yet to be determined. Corneal epithelial debridement, followed by instillation of autologous fibronectin eye drops, has been used to manage the corneal epithelial irregularities that cause visual disturbances in LCD1 patients (Morita et al., 2012). The authors reported that this treatment restored visual acuity in 2e4 months and was likely to be effective for at least 2e3 years. They also pointed out that debridement followed by topical autologous fibronectin eye drops would be more effective in younger patients with more superficial corneal involvement than it would be in older patients with deposits in the deep stroma. 6.1.4. Stage-related therapy of corneal dystrophies Maintaining functional vision for as long as possible and minimizing the extent of surgery is an appropriate strategy for the management of corneal dystrophies. Seitz and Lisch (2011) proposed stage-related therapy of corneal dystrophies based on the depth of opacities: excimer laser PTK for superficial opacities of the epithelium, Bowman layer, and anterior stroma; ALKP or DALK for deeper corneal stromal deposits sparing the corneal endothelium; and PKP for corneal dystrophies involving the entire cornea such as Fuchs endothelial corneal dystrophy. Seitz et al. (2004) estimated the depth and size of corneal deposits in GCD and LCD using light microscopy of corneal buttons obtained from PKP. They reported that hypothetical ablation of 100 mm of anterior stroma would improve visual acuities in most cases. We were able to show that Fourier-domain optical coherence tomography (FD-OCT) provides a much more precise determination of the depth and thickness of corneal deposits in corneas with GCD2 (Kim et al., 2010). We also reported the outcome of PTK for diffuse corneal haze, which appears in older heterozygous GCD2 patients. Using intra-operative slit lamp examination, ablation of about 43.7 ± 6.2 mm of corneal stroma was sufficient to remove diffuse haze in 43 eyes of 30 patients. During a mean follow-up of 21 months, no recurrence was observed. We advocate the use of both preoperative FD-OCT and intraoperative slit lamp examination to maximize the visual outcome while minimizing the depth of ablation (Jung et al., 2013). FD-OCT was also useful for determining whether DALK or PKP would be most appropriate when the lattice-like deposit were located so close to the endothelial layer that slit lamp examination did not allow determination of the depth of the opacity (Kim et al., 2010).

7. Perspectives 7.1. Animal models There have been many attempts to evaluate the physiologic and pathologic roles of the TGFBI gene using transgenic animals with knock-in or knock-out TGFBI genes. Transgenic mice that overexpress hbigh3 under the control of the Albumin enhancer/promoter develop bilateral central corneal opacities (Kim et al., 2007). Changes in all corneal layers including irregular corneal epithelium, disorganization of the stromal layer, and discontinuity of the endothelium, as well as narrow anterior chamber and cataract formation were observed. Another transgenic mouse line which expresses a mutated TGFBI gene (Arg555Trp) revealed an accelerated aging process including lipogenic pigmentation, retinal degeneration, and follicular hyperplasia of the spleen (Bustamante et al., 2008). Recently, Yamazoe et al. (2015) published a genetically established GCD2 mouse model showing granular and lattice deposits in 45.0% of homozygotes and 19.4% of heterozygotes. Establishment of a complete animal model and genome editing using the animal model might provide further insight into the pathogenesis and treatment of TGFBI corneal dystrophies. 7.2. Potential therapeutic approaches 7.2.1. Agents that might be used to reduce production or enhance clearance of TGFBI expression As TGFBI corneal dystrophies progress throughout life and are not completely responsive to surgical intervention, noninvasive pharmacological approaches have been designed to decrease the production of TGFBIp. We reported that lithium chloride (LiCl) inhibited the expression of TGFBIp in normal and GCD2 corneal fibroblasts. Lithium inhibits the phosphorylation of Smad2/3(S423/ 425), which is a downstream regulator of the TGF-b signaling pathway and inhibits the glycogen synthase kinase 3 (GSK-3) (Choi et al., 2011b). Han et al. (2012b) compared the effect of NaCl and LiCl on TGFBIp to evaluate the impact of solute on protein aggregation. Addition of NaCl to the cell lysates increased polymeric TGFBIp and reduced monomeric and dimeric TGFBIp, while LiCl did not increase the TGFBIp polymerization at all tested concentrations (from 0.5 to 2 M). They suggested that LiCl may have a therapeutic effect by inhibiting the aggregation of TGFBIp in vitro. We introduced two molecules, melatonin and rapamycin, as candidate drugs for the treatment of GCD2 (Choi et al., 2011a, 2012, 2013). Both types of melatonin receptors (MT1 and MT2) are upregulated in GCD2 corneal fibroblasts. Melatonin reduced the expression of TGFBIp, which had been elevated by the oxidative stress inducer, paraquat. Melatonin also increased the expression of antioxidants such as Cu/Zn-SOD and GR in WT and GCD2 corneal fibroblasts. In addition, melatonin increased the autophagic degradation of TGFBIp in WT and GCD2 corneal fibroblasts via mTOR-dependent pathway. Another autophagy inducer, rapamycin, also reduced mutant-TGFBIp levels in GCD2 corneal fibroblasts without altering normal TGFBIp levels in WT corneal fibroblasts (Choi et al., 2012). Notably, co-treatment with melatonin and rapamycin reduced mutant-TGFBIp much more than either drug alone (Choi et al., 2013). Direct destruction of amyloid fibrils of TGFBIp by heliumcadmium laser coupled with amyloid-specific thioflavin T has also been suggested. Two synthetic 22-residue peptides C110-131 for p.Arg124Cys and H110-131 for p.Arg124His were successfully degraded by laser-exited thioflavin T in a dose-dependent manner (Ozawa et al., 2011). Chemical chaperoning can stabilize the conformation of proteins by preventing misfolding and promote proper intracellular



trafficking (Kolter and Wendeler, 2003; Ulloa-Aguirre et al., 2004). Trimethylamine N-oxide (TMAO), a chemical osmolyte with chaperoning activity, reduced aggregation of the amyloid-beta (Ab) (1e40) peptides in Arg-124 mutants in a dose-dependent fashion in vitro (Yam et al., 2012). All these data suggest the possibility of medical treatments for TGFBI corneal dystrophies in the future. 7.2.2. Avoid agents that deteriorate disease Mitomycin C (MMC) is an alkylating agent that inhibits DNA synthesis. Topical MMC has been used to reduce corneal scarring after excimer laser PRK because of its anti-proliferative effect (Azar and Jain, 2001; Kim et al., 2004). It has also been used during PTK for the treatment of GCD2 corneal opacities, but recurrences were noted in 2 GCD2 homozygotes during 20 months of follow-up (Kim et al., 2006). In one older RBCD patient, recurrence was not noted up to 12 months after PTK with intraoperative topical MMC (Miller et al., 2004). When corneal fibroblasts from normal, heterozygous and homozygous GCD2 patients were incubated with MMC in vitro, apoptosis was observed in all cell types, with GCD2 homozygous cells being most vulnerable (Kim et al., 2008a). MMC reduced expression of TGFBI mRNA and TGFBIp in the cell and in culture media for all cell types. MMC also decreased Bcl-xL mRNA expression, which inhibits mitochondrial apoptosis and increased Bax mRNA expression, which counteracts to Bcl-xL, in all cell types. Our 3-year follow-up study, however, showed that adjunctive application of MMC during laser ablation did not prevent exacerbation of opacities in heterozygous GCD2 patients (Ha et al., 2010). As the corneal fibroblasts reabsorb secreted TGFBIp and degrade it in lysosomes, apoptotic cell death induced by MMC application during surface ablation would decrease the cleaning action of keratocytes in the dystrophy condition (Choi et al., 2015b). We do not recommend the application of adjuvant MMC treatment during PTK until more careful studies are performed. A deleterious effect of benzalkonium chloride (BAC), which is the most widely used preservative in eye drops, has been suggested (Kato et al., 2013). To compare the effect of BAC on amyloidogenicity and seed-dependent fibril elongation, three synthetic peptides (an R-type peptide associated with wild-type Arg124, a C-type peptide associated with LCD1, and an H-type peptide associated with GCD2) were incubated with various concentration of BAC. They reported that all the 3 synthetic peptides and the WT Arg124 peptide formed amyloid fibrils regardless of the seed. Because the critical micelle concentration of BAC (C14), 0.02 mM, is similar to that in commercially available eye drops (0.001e0.02%; 0.03e0.6 mM), the authors hypothesized that instillation of eye drops what contain BAC might enhance amyloidogenicity and suggested that topical medications containing BAC should be avoided in patients with TGFBI corneal dystrophies (Kato et al., 2013). Further investigation is needed to test the role of BAC and the possible effects of other components in topical medications. 7.2.3. Possible roles of blood vessels Blood vessels are present in the conjunctiva and corneal limbal area but not in the central cornea. The corneal deposits in GCD2 cornea exist at the central cornea but not at the perilimbal area (lucid interval). A deposit-free clear zone was also noted around vascularized pterygia (Lee et al., 2006). As shown in Fig. 6, deposits are more concentrated near the central portion of the incision lines, compared to the peripheral portion of the incisions in eyes that have undergone RK (Feizi et al., 2007). Corneal deposits were not observed around the peripheral corneal incision line made during cataract surgery or phakic intraocular lens insertion, while central corneal deposits were exacerbated by laser ablation of the central cornea (Roh et al., 2006a). Together, these observations suggest that

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deposits of GCD are most dense centrally because blood vessels play a role in preventing their accumulation or absorbing corneal deposits.

7.2.4. Gene therapy Gene therapy, which uses RNA interference (RNAi), has been investigated to treat diseases by silencing the disease-associated mutant allele. In applying RNAi for gene suppression, there are two commonly used methods: small interfering RNAs (siRNAs) consisted of 21e23 nucleotide pairs, and short hairpin siRNAs (shRNAs) generated by RNA polymerase III promoters. Yuan et al. (2007b) generated shRNA which decreases the TGFBIp expression in a transformed HEK 293 cell line transfected with plasmids expressing TGFBIp. Courteny et al. (2014) developed siRNA which is an allele-specific nature of the TGFBI-Arg124Cys LCD1 and this reduced the expression of mutant TGFBI mRNA and TGFBIp and also decreased amyloid aggregate formation in vitro. The non-selective nature of those siRNAs or shRNAs, however, certainly raises concerns regarding the safety of their clinical application, since TGFBIp plays multiple physiological roles. Recently, site-specific genome editing technologies to repair DNA have been introduced. Among the various techniques, clustered regularly interspaced short palindromic repeat/Cas9 (CRISPR/ Cas9) e mediated genome editing is dramatically accelerating progress in the genomic editing area and has already successfully corrected the dystrophic phenotype in a mouse model of Duchenne muscular dystrophy (Long et al., 2014). An animal model of corneal dystrophies would be good candidate for genetic correction through CRISPR/Cas9.

8. Conclusions and future directions Over the years, our understanding of the pathogenesis of TGFBI corneal dystrophies has advanced significantly, but much remains to be learned. TGFBIp is the second most abundant protein in the corneal stroma. The function of TGFBIp and the process of protein metabolism in the normal and mutated cornea have not yet been elucidated. Additional research is also required to explain the biochemical mechanisms that cause mutated proteins to have a greater tendency to deposit. Since the deposits are located in the ECM, the relationship between the mutated proteins and ECM components should also be investigated. More research is needed to identify factors controlling synthesis and removal of abnormal deposits, and the possibility of pharmacologic control of disease. Every typical phenotype has a specific associated TGFBI mutation. However, there are some phenotypic variations for each mutation. The relationship between the mutated TGFBIp and other factors that might explain the phenotypic variation needs to be defined. Differences in the severity of disease among family members suggest the presence of secondary gene(s) in determining the phenotype of TGFBI corneal dystrophies. In fact, a full understanding of the relationship between DNA coding sequences and protein accumulations is yet to be understood. Today, there are several surgical techniques for treating visually significant deposits. With greater experience, the best option for every condition will be determined. Future development of topical medications might prevent the deposition of abnormal TGFBIp and/ or dissolve existing deposits. Eventually, gene therapy such as CRISPR/Cas9emediated genome editing may provide an effective treatment modality to repair the mutated sequence of the gene responsible for TGFBI corneal dystrophies so that treatment to eliminate deposits will no longer be necessary.


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