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Fuchs Corneal Dystrophy
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Fuchs Corneal Dystrophy Allen O. Eghrari, S. Amer Riazuddin, John D. Gottsch1 Wilmer Eye Institute, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA 1 Corresponding author e-mail address: [email protected]
Contents 1. Introduction 2. Structural Changes in the FCD Cornea 2.1 Epithelium 2.2 Bowman's Layer 2.3 Stroma 2.4 Descemet Membrane 2.5 Endothelium 3. Genetic Basis of FCD 3.1 Genetic Linkage Analysis 3.2 Causal Genetic Mutations 3.3 Association Studies 4. Functional Mechanisms in FCD 4.1 Oxidative Damage and Apoptosis 4.2 Mitochondrial Dysregulation 4.3 Epithelial–Mesenchymal Transition 4.4 Unfolded Protein Response 4.5 MicroRNA References
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Abstract Fuchs corneal dystrophy (FCD) is a hereditary, progressive disease of the posterior cornea which results in excrescences of Descemet membrane, endothelial cell loss, corneal edema, and, in late stages, bullous keratopathy. Structural changes are noted principally in Descemet membrane and the endothelium, with thickening of Descemet membrane, loss of barrier function, and increased corneal hydration, although secondary effects occur throughout all layers. Multiple chromosomal loci and, more recently, causal genetic mutations have been identified for this complex disorder, including in TCF8, SLC4A11, LOXHD1, and AGBL1. A trinucleotide repeat in TCF4 correlates strongly with disease status and interacts in common pathways with previously identified genes. Dysregulation of pathways involving oxidative stress and apoptosis, epithelial-tomesenchymal transition, microRNA, mitochondrial genes, and unfolded protein response has been implicated in FCD pathogenesis.
Progress in Molecular Biology and Translational Science ISSN 1877-1173 http://dx.doi.org/10.1016/bs.pmbts.2015.04.005
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2015 Elsevier Inc. All rights reserved.
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1. INTRODUCTION Fuchs corneal dystrophy (FCD) is a progressive, hereditary, bilateral corneal condition first described a century ago by the Austrian ophthalmologist Ernst Fuchs.1 Patients present in the fifth to sixth decade of life with blurry morning vision that increases in duration as the disease progresses. Primarily a condition of the posterior cornea, characteristic features include the formation of focal excrescences of Descemet membrane termed “guttae,” a progressive increase in corneal edema, loss of endothelial cell density over years, and endstage disease manifested by formation of painful epithelial bullae as the cornea decompensates in its ability to maintain stromal dehydration. Recent advances in our understanding of the genetic and pathophysiological mechanisms of the disease as well as the application of new imaging modalities provide unique insight into the development of and potential therapeutic options for FCD.
2. STRUCTURAL CHANGES IN THE FCD CORNEA The characteristic clinical feature of FCD is the presence of guttae, focal nodules in Descemet membrane which are anvil shaped and protrude posteriorly into the endothelial plane, and are several microns in diameter. Guttae are visible clinically with slit-lamp biomicroscopy, and in early stages help to determine the clinical phenotype (Fig. 1). Unlike peripheral, age-associated nodules or pigment deposition on the endothelium from the anterior chamber, these excrescences begin in the center and over the course of decades, begin to coalesce and present peripherally. The grading scale commonly utilized and described by Krachmer 35 years ago reflects this phenomenon; a severity score between one and five is given based on the number and distribution of guttae. A score of one, reflecting minimal and generally asymptomatic disease, is defined as more than 12 central guttae, whereas a cluster of central confluent guttae are graded as two, and higher scores represent increasing diameters of distribution of confluent guttae.2 Although the traditional model of FCD suggested that severe disease is marked by corneal decompensation and diffuse stromal and epithelial edema in the presence of diffuse guttae, assessment of a large number of patients with FCD across various levels of severity suggests that the progressive development of edema is a gradual process that occurs over time,3 with symptoms developing in later stages over the course of decades.
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Figure 1 Direct illumination of guttae with slit-lamp biomicroscopy. In the slit of light seen passing through the cornea from left (anterior surface) to right (posterior surface), the beaten-metal appearance of guttae is appreciated posteriorly in light reflected from Descemet membrane (arrow).
We will subsequently explore pathological structural changes in the FCD-affected cornea.
2.1 Epithelium Prior to the advent of slit-lamp biomicroscopy, FCD was identified in its severe stages, and Fuchs initially described the disease as dystrophia epithelialis corneae.1 The epithelium is generally intact in early stages of classic Fuchs dystrophy, a phenomenon evidenced by a lack of change in anterior keratometry in patients undergoing endothelial keratoplasty, which would be expected if distortion of the epithelium were to occur in the disease process. However, in severe FCD, loss of pump function in the edematous cornea results in anterior migration of fluid through the corneal stroma and the formation of painful epithelial bullae.
2.2 Bowman's Layer In vivo confocal microscopy of patients with FCD reveals an abnormal Bowman’s layer in approximately half of patients with FCD, with diffuse bright reflection and a paucity or absence of nerves.4
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2.3 Stroma Anterior stromal changes occur during periods of corneal edema with formation of stromal haze and visual higher order aberrations, a phenomenon that may persist after endothelial keratoplasty.5 Confocal microscopy suggests that after endothelial transplantation, subepithelial haze rather than interface haze is the predominant source of forward light scatter.6 Collagen fibers in the anterior stroma are tightly packed relative to the posterior stroma, in which fibrils have less connections. As a result, corneal edema results in increased hydration of the posterior cornea and bulging of the posterior stroma into the anterior chamber. This focal thickening in the central corneal relative to the periphery can be appreciated through anterior segment optical coherence tomography or Scheimpflug photography; the latter has been utilized to demonstrate an increased center-to-peripheral thickness ratio in FCD-affected corneas compared to controls.7
2.4 Descemet Membrane Descemet membrane is normally composed of two layers, an anterior banded layer approximately 3 μm in thickness which develops in utero and is relatively constant in thickness over time, and a posterior nonbanded layer which is deposited by the endothelium and which thickens over an individual lifetime to approximately 10 μm in an adult. Descemet membrane in FCD is marked by the presence of a thick posterior banded layer, a posterior-most layer in which collagen fibrils approximately 10–20 nm in diameter and with 110 nm banding are deposited by the endothelium. This posterior banded layer measures approximately 16 μm in thickness and correlates directly with clinical severity of FCD.8 It is in this posterior banded layer that focal excrescences of Descemet membrane produce posteriorly and tent up the endothelium. In some cases, particularly those in which the cornea has suffered severe edema, a fibrillar layer is present, without fibroblasts or cellular elements. This has also been described as a posterior collagenous layer.9,10 Guttae develop in the central cornea in early disease and are formed peripherally in more advanced disease. However, a propensity for the development of guttae in the inferotemporal quadrant has been documented in multiple families using retroillumination photography11,12 and specular microscopy.13 Although the causes for this are unclear, the inferotemporal cornea is associated with a thinner cornea and potentially increased light exposure (Fig. 2).
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Figure 2 Retroillumination photography reveals the distribution of guttae throughout the cornea, a process which is largely central but increases in the periphery over time.
2.5 Endothelium The endothelium in both normal and FCD-affected corneas is a single monolayer of cells which is responsible for maintaining stromal dehydration, and measures approximately 5 μm in thickness. As endothelial cell density decreases, in vivo confocal microscopy reveals increased pleomorphism and polymegethism.14 Polymerase chain reaction array analysis of human Descemet membrane–endothelial complexes reveals upregulation of numerous extracellular matrix-related genes in FCD, including collagen and fibronectin, suggesting significant matrix alterations in FCD pathophysiology.15 Transmission electron microscopy of FCD corneas demonstrates some endothelial cells with cytoplasmic filaments, increased rough endoplasmic reticulum, and cytoplasmic processes, bearing similarity to fibroblasts; intercellular vacuoles remain in areas associated with loss of cells.9 Decreased aquaporin expression has been detected in FCD corneal endothelium and may contribute to corneal edema.16
3. GENETIC BASIS OF FCD Through the passage of a century after Fuchs’ initial description of FCD, an understanding of the genetic basis of disease has largely correlated
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with the knowledge and application of human genetics, with rapid advances made in the twenty-first century. Although the possibility of a hereditary component was proposed within a few years after Fuchs’ publication of his findings, pedigrees presented in the 1970s assisted to establish its autosomal dominant pattern of inheritance.2,17,18
3.1 Genetic Linkage Analysis To date, large family studies of FCD have resulted in the identification of several chromosomal loci associated with FCD. The FCD1 locus, the first to be associated with classic, late-onset FCD, was localized to a 26.4-Mb interval between 13pTel and 13q12.13 through recruitment and analysis of a large Caucasian family.19 A total of 13 affected and 3 unaffected individuals across three generations were recruited for genotyping, and linkage using 399 STR markers from the MD10 genotyping panel revealed significant two-point logarithm of odds scores of 3.91 at D13S1236 and 3.80 at D13S1304, assuming fully penetrant dominant inheritance. Notably, two children in this family whose parents were both affected demonstrated clinical signs of late-onset FCD at a young age. The FCD2 locus was identified in three large families and localized to 18q21.2–18q21.32.20 This was the first locus associated with multiple families, suggesting that this area may represent a common locus. Linkage analyses were performed using 1107 STR markers, and maximum two-point LOD scores among the three families were 3.41, 2.89, and 2.45. Differences in haplotypes among families raised the possibility of heterogeneity of the locus. Using retroillumination photography, progression in FCD2associated families was compared with that in FCD1-related families and found to demonstrate a less marked rate of development of guttae.12 The FCD3 locus was identified through a large family with 17 examined individuals, 10 of which manifested clinical signs of disease. This interval was localized to chromosome 5 using an SNP array, followed by refinement of the region with an STR marker panel to 5q33.1–5q35.2.21 The FCD4 locus was identified in the region 9p22.1–9p24.1 and was the first to demonstrate interaction between two pathologic alleles in this disease. After mutations in TCF8 were identified among some affected members of this family, linkage analyses were performed conditioned to the presence of the TCF8 mutation, and refined using STR markers, with maximum LOD scores of 3.09 at D9S168 and 3.20 at D9S256, and a linkage
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interval spanning 14.3 Mb between recombinations at D91681 and D9S1684.22 While either the pathologic mutation in TCF8 or the causal FCD4 haplotype was associated with clinically significant disease, those with the presence of both genetic inputs demonstrated markedly severe disease, generally progressing to corneal transplantation. A number of additional loci have been identified with large numbers of small pedigrees which, although below the typical LOD threshold of 3.0, may serve as promising targets in the future. Using SNP linkage panels and DNA from 92 individuals from 22 families, four loci were identified on chromosome 1 (rs760594), chromosome 7 (rs257376), chromosome 15 (rs352476), and chromosome 17 (rs938350).23 A follow-up study using 215 individuals from 64 families identified six linkage peaks with maximum HLOD scores over 2, and including two over 3: one in chromosome 10 (rs1889974) with HLOD score of 3.37 and one in chromosome 15 (rs235512) with HLOD score of 3.53.68
3.2 Causal Genetic Mutations Coding mutations in four genes have been identified to be causal for the FCD phenotype: TCF8, SLC4A11, LOXHD1, and AGBL1. Mutations in TCF8 had been previously implicated in posterior polymorphous corneal dystrophy, which similar to FCD primarily affects the posterior cornea. To investigate whether this gene may be associated with the FCD phenotype, an initial screen of 74 Chinese individuals with FCD for TCF8 mutations identified a novel variant, p.N696S, present in 1 sporadic case and absent in 93 controls.24 In a large family also linked to the FCD4 locus, a causal missense mutation in TCF8 was identified, p.Q840P.22 Four additional pathogenic mutations (p.N78T, p.P649A, p.Q810P, and p.A905T) were discovered through sequencing of exonic regions of TCF8 in 384 unrelated FCD-affected individuals. Of these five mutations, three occurred at sites that are highly evolutionarily conserved in vertebrates (p.Q810P, p.Q840P, and p.A905T) and two at moderately conserved sites. In contrast to frameshift, nonsense, or lost-start-codon mutations associated with PPCD, the causal alleles in TCF8 for FCD contain missense mutations. Functional assessment using zebrafish embryos revealed an inability of human mRNA containing each of the FCD-linked mutations to fully rescue developmental abnormalities caused by morpholino oligonucleotide knockdown of endogenous TCF8. While the variants p.N78T and p.Q810P partially rescued the phenotype, the other
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three variants demonstrated phenotypes identical to morpholino injection alone, with successful rescue limited to wild-type mRNA. The second gene for which causal mutations have been associated with FCD, SLC4A11, codes for a sodium-borate cotransporter that can also stimulate cell growth and proliferation through the MAPK pathway.25 In the corneal endothelium, SLC4A11 demonstrates robust Na+-coupled OH transport, but does not transport BðOHÞ4 or HCO3 .26 Expression of this gene was shown to be downregulated in FCD through serial analysis of gene expression.27 Mutations in this gene have been associated with the autosomal recessive form of congenital hereditary endothelial dystrophy28 and systemically in Harboyan syndrome, a condition which includes both corneal dystrophy and deafness. A screen of 89 FCD-affected individuals, 64 of Chinese and 25 of Indian descent, revealed four previously unreported mutations (p.S33SfsX18, p.E399K, p.G709E, and p.T754M) which were not present in 354 ethnically matched controls, and acquired with autosomal dominant inheritance.29 These missense mutations were shown in a biochemical assay of cell surface localization and confocal immunolocalization to demonstrate defective localization to the cell surface. A large study sequencing all coding regions of SLC4A11 in 192 FCD-affected and 192 unaffected individuals resulted in the identification of an additional seven missense mutations (p.E167D, p.R282P, p.Y526C, p.V575M, p.G583D, p.G742R, and p.G834S) among affected individuals, with no such mutations among the control samples.30 Sorting Intolerant from Tolerant (SIFT) and PolyPhen predicted that five of these mutations were deleterious. Loss of SLC4A11 was explored utilizing short hairpin RNA against SLC4A11 in an immortalized human corneal endothelial cell (HCEC) line to downregulate SLC4A11 gene expression; knockdown suppressed growth and reduced viability among HCECs, associated with increased apoptosis in SLC4A11-depleted cells.31 The third gene with FCD-associated causal mutations, LOXHD1, was identified in a family previously linked the FCD2 locus in chromosome 18. LOXHD1 codes for a protein composed of polycystin/lipoxygenase/ alpha-toxin domains, which modulate plasma membrane targeting. Sequencing of 36 Mb on chromosome 18 using a custom exon-capture array in one affected and one unaffected member of three families linked to the FCD2 locus identified a p.R547C mutation in LOXHD1 in one affected individual, a variant not present in either the 1000 Genomes project or 1500 Exomes from the University of Seattle Human Exome Variant database. PolyPhen 2 predicted this variant to be pathogenic and it was absent in
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384 unaffected control individuals. Immunohistochemistry was utilized to identify LOXHD1 in mouse corneas, both in the epithelial and endothelial layers. Immunostaining of the affected proband cornea, one from a FCDaffected individual known not to bear the identified mutation in LOXHD1, and a control with keratoconus revealed increased LOXHD1 staining in Descemet membrane and corneal endothelium in the LOXHD1-associated Fuchs cornea. Three-dimensional reconstruction of the LOXHD1 protein predicted that most mutations resided on the protein surface, likely affecting interaction with other proteins.32 The fourth gene with an identified causal mutation, AGBL1, codes for ATP/GTP-binding protein-like 1. The cytosolic carboxypeptidase family, of which AGBL1 is a member, catalyzes deglutamylation of polyglutamylated proteins. Polyglutamylation is a reversible posttranslational protein modification which generates glutamate side chains on tubulins and other proteins, a process which has been shown to be necessary for neuronal survival.33 In a three-generation family with 12 affected individuals, a genome-wide linkage analysis with a multilocus model was conducted, conditioned on severe disease resulting from two disease alleles, mild disease from a single allele, and offspring of two affected parents receiving the disease allele from the parent otherwise unrelated to the pedigree. Two positive signals were noted on 3p and 15q, which were investigated further with nextgeneration sequencing. A C-to-T transition, c.3082C > T in AGBL1, was identified, which results in a premature stop codon; this nonsense allele segregated with the FCD haplotype under the multilocus model. Sequencing of FCD-affected individuals resulted in the identification of two other individuals with the same nonsense mutation and the identification of a second allele, a heterozygous missense variant, c.2969G > C, neither of which was present among 384 ethnically matched controls. To investigate the role of AGBL1, immunoprecipitated lysates were probed with anti-Myc antibody and variant forms of the protein demonstrated reduced binding affinity with TCF4. The results overall indicated that AGBL1 interacts specifically with TCF4, but not with TCF8.34
3.3 Association Studies A genome-wide association study by Baratz and colleagues among 130 cases and 260 controls matched by age and sex interrogated 338,727 SNPs, of which one reached threshold for genome-wide significance: rs613872, an intronic variant in TCF4. The minor allele (G) frequency was significantly
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enriched in FCD cases (G ¼ 0.37) compared to controls (0.14), a finding replicated in a second group of 150 cases and 150 controls (0.43 among cases and 0.15 among controls). The odds ratio associated with the minor allele was notable in heterozygotes (5.5) and particularly homozygotes (30).35 In the United States, these findings were replicated in a large cohort of 450 cases and 360 controls with odds ratio of 4.5,36 and in a Midwestern population of 82 patients with FCD and 163 control subjects.37 The population of Tangier, an island in the Chesapeake Bay with a high prevalence of FCD, was found to also demonstrate a high minor allele frequency of 0.37, comparable to affected cohorts in the aforementioned studies.38 Globally, findings were replicated in an Australian cohort of 105 cases and 275 controls with odds ratio of 4.05.39 In a Chinese cohort of 57 affected and 121 unaffected individuals, two other SNPs in TCF4 were significantly associated with FCD affectation with an odds ratio over 240; however, the rs613872 minor allele was absent among the affected cohort. Together, these findings indicated strong association but raised the possibility of a yet undetermined causal allele in TCF4. Subsequently, testing of 66 affected and 63 unaffected individuals for an intronic TGC trinucleotide repeat in TCF4 demonstrated, in cases with over 50 TGC repeats, sensitivity of 79% and specificity of 96%, even greater specificity than had been previously determined with the rs613872 single nucleotide polymorphism.41 This association with the CTG 18.1 trinucleotide repeat expansion was replicated in 120 affected Caucasian individuals and 100 controls (p ¼ 6.5 1025) and the two polymorphisms were found to be in linkage disequilibrium. In this cohort, the odds ratio of each copy of the CTG18.1 expanded allele was 32.3, and in 29 families, the expanded allele cosegregated with the trait in 15 families (52%) with complete penetrance.42 Trans-ethnic replication of the CTG18.1 repeat expansion has been described in the Chinese population with 57 affected and 121 control individuals; the expanded CTG18.1 allele was associated (p ¼ 4.7 1014) with FCD with odds ratio of 66.5.43 Replication was also performed in 44 affected and 108 control patients from an Indian population sample, with significance achieved for the CTG18.1 expanded allele (p ¼ 2 104); 34% of affected and 5% of control subjects were found to harbor over 50 CTG repeats.44 A comprehensive assessment of 1866 genetic variants, with sequencing of the TCF4 coding region, introns, and flanking sequence surrounding the two variants, revealed that no single causative allele correlated with all cases of FCD, but the trinucleotide repeat is the most strongly associated of the two TCF4 variants.45
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Clusterin is a molecular chaperone which plays a role in extracellular matrix interaction and is overexpressed in FCD relative to pseudophakic bullous keratopathy and normal control corneas46 and has been shown to colocalize in guttae.47 In the aforementioned Australian cohort exploring association with TCF4, additional SNPs were interrogated and one found to be significant in CLU (rs17466684, p ¼ 0.003) with odds ratio of 1.85.39 Immunohistochemistry revealed anti-CLU labeling in FCD epithelium but absent in control epithelium, while labeling in Descemet membrane was limited to its anterior portion in FCD and more diffusely distributed in controls. Both affected and control endothelium expressed CLU, although endothelial cell density was decreased in affected individuals.39 Polymerase chain reaction array analyses reveal upregulation of clusterin expression in human FCD-affected Descemet membrane– endothelium complexes, which demonstrate increased immunohistochemistry labeling among 40 samples when compared to controls.15 TGF-β1 protein has also been found to be overexpressed in FCD and in addition to clusterin, colocalizes in guttae.47 An association with a single haplotype of TGF-β1 SNPs and FCD affectation reached statistical significance among Caucasian Australians (p ¼ 0.011), with odds ratio of 2.29.39
4. FUNCTIONAL MECHANISMS IN FCD 4.1 Oxidative Damage and Apoptosis Loss of corneal endothelial cell density is a characteristic feature of FCD and is associated with increased apoptotic processes. A study of 47 FCD-affected corneal buttons revealed a significantly higher percentage of apoptotic endothelial cells by nucleus labeling assay relative to controls, a phenomenon also supported by TUNEL assay.48 Subsequent studies with TUNEL assay of entire corneas revealed significantly higher apoptotic cell numbers not only in the corneal endothelium but also in the stroma and epithelium in patients with FCD compared to controls.49 Serial analysis of gene expression reveals decreased transcription levels of antioxidant molecules in FCD-affected corneal endothelium, including glutathione S-transferase-pi.27 A number of apoptosis-related factors are found more frequently in FECD-affected corneas. Immunohistochemical study of FCD corneas reveals a lower number of p27- and survivin-positive epithelial cells, and higher cathepsin-positive epithelial cells in FCD corneas relative to controls.50 Descemet membrane extracted from FCD corneas undergoing
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endothelial keratoplasty reveals abundant advanced glycation end products in Descemet membrane, molecules which are associated with oxidative stress, inflammation, and aging.51 A comparison of immortalized FCD and control corneal endothelial cell lines in which oxidative stress was induced with tert-butyl hydroperoxide (tBHP) reveals significantly increased activation of p53 in FCD cells relative to controls (Fig. 3).52 Peroxiredoxins are expressed in human corneal endothelium and protect against oxidative stress by removing cellular hydrogen peroxide. Proteomic analysis reveals significantly decreased expression of Prx-2, -3, and -5 in FCD cells compared to controls, a finding supported by decreased Prx-2 mRNA detected by real-time PCR analysis.53 Evidence suggests that the oxidant–antioxidant balance in FCD-affected endothelial cells shifts to a pro-oxidant state. In addition to downregulation of peroxiredoxins, PCR array also demonstrates transcriptional downregulation of other antioxidants, including SOD2, MT3, and TXNDR1 in HCECs, without a compensatory increase in other antioxidants such as catalase or glutathione peroxidases and/or transferases.54 These downregulated antioxidants share a common promoter region, the antioxidant response element, to which the transcription factors nuclear factor erythroid 2-related factor-1 (Nrf1) and -2 (Nrf2) bind and induce upregulation of antioxidant enzyme genes in settings of oxidative stress. The identification of a
Figure 3 Descemet membrane removed from a patient at the time of endothelial keratoplasty reveals classic anvil-shaped excrescences which protrude posteriorly, termed guttae.
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significant decrease in Nrf2 protein in FCD endothelial cells compared to controls is further supported by depleted expression of a major Nrf2regulated antioxidant gene, HO-1,54 and significant decrease in the Nrf2 protein stabilizer DJ-1.55 Identification of dysregulated oxidative stress pathways offers potential avenues for future therapeutic targets. Sulforaphane is a naturally occurring glucosinolate found in green leafy vegetables which modifies sulfhydryl residues of Keap1, causing the release and activation of Nrf2. In human unaffected and FCD-affected immortalized corneal endothelial cell lines subjected to oxidative stress with tBHP, pretreatment with sulforaphane decreased intracellular production of reactive oxygen species, enhanced nuclear translocation of Nrf2, and decreased p53 staining.56
4.2 Mitochondrial Dysregulation FCD is principally a disease of the central cornea, and early research suggested regional differences in mitochondrial cytochrome oxidase (CO) activity in FCD-affected corneal endothelium, with decreased CO centrally and increased CO in the periphery.57 Serial analysis of gene expression in FCD and normal corneal endothelium revealed significant underexpression in FCD of multiple mitochondrial genes involved in electron transport and oxidative phosphorylation, including NADH dehydrogenase subunits 1, 2, and 4, cytochrome b, cytochrome c oxidase subunit III, and ATP synthase F0 subunit 6.27 Comparison of oxidative DNA damage in FCD-affected and unaffected corneal endothelium was conducted with high-sensitivity ELISA to detect the concentration of 8-OHdG (an oxidative DNA damage marker). This was significantly higher in FCD-affected corneal endothelium than in controls, and immunolabeling revealed that this marker colocalized to mitochondria.54 To test if FCD was associated with a systemic deficiency in mitochondrial enzymes, a separate study challenged mitochondria in peripheral blood lymphocytes with hydrogen peroxide in 35 FCD patients and 32 controls; increased mitochondrial DNA (mtDNA) damage and a higher ratio of a 4977-bp deletion were found in FCD patients compared to controls.58 In a study of mtDNA variants on FCD susceptibility, a total of 530 cases and 498 controls of European descent were assessed for 10 mtDNA variants defining European haplogroups, and replication analyses conducted in cohorts of 3200 and 3043 individuals. The mtDNA variant A10398G
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was significantly associated with FCD with odds ratio of 0.72, suggesting that this variant confers a protective effect; this persisted even after adjusting for smoking status. Haplogroup I was significantly associated with FCD even after adjusting for smoking or the TCF4 intronic variant rs613872.59
4.3 Epithelial–Mesenchymal Transition Although the corneal endothelium is often considered to be in a state of arrested growth, with endothelial cells migrating to cover defects in the endothelial layer, evidence exists of stem cell markers in the posterior limbus and corneal endothelium, and markers of proliferation are present after wounding of the endothelium.60 Fibroblastic features of some endothelial cells in FCD9 may be suggestive of alterations in the process of maturation and differentiation, such as epithelial–mesenchymal transition (EMT). Several genes associated with FCD such as TCF8 and TCF4 are implicated directly or indirectly in the EMT pathway. In breast cancer cells, TCF8 serves as a transcriptional repressor and downregulates E-cadherin, inducing a transition to a mobile, fibroblastoid appearance. TCF4 modulates multiple pathways, including TGF-β, and upregulates TCF8 expression.61 TGF-βI induces EMT in colon cancer cells62 and silencing of clusterin in lung adenocarcinoma cells appears to induce a mesenchymal-to-epithelial transition through the ERK/Slug pathway.63
4.4 Unfolded Protein Response Several genes implicated in FCD pathogenesis modify protein folding mechanisms, supporting a possible role for the unfolded protein response. CLU is involved with protein folding and colocalizes to guttae, and pathogenic LOXHD1 alleles induce misfolded aggregates in corneal endothelial cells.32 In a comparison of FCD corneas with keratoconus and normal controls, misfolded proteins were found to be increased in FCD.64 Of 10 corneas with FCD and 9 corneas with non-FCD corneal dystrophy, all FCD corneas exhibited prominent rough endoplasmic reticulum, in contrast to only a third of non-FCD corneas. FCD-associated corneas also demonstrated increased immunofluorescent labeling for GRP78, phospho-eIF2a, and CHOP, markers for unfolded protein response relative to both normal and keratoconus corneas. Similarly, the apoptosis markers caspase 3 and caspase 9 were both significantly increased in FCD corneas, suggesting that
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increased misfolded protein in FCD may contribute to an increased unfolded protein response and caspase-dependent apoptosis.64
4.5 MicroRNA MicroRNA (miRNA) dysregulation is implicated in numerous pathophysiological pathways, including those associated with FCD and other corneal dystrophies with similar phenotypes. A mutation in miR-184 has been shown to be causative for EDICT syndrome, which includes a beaten-metal appearance to the corneal endothelium.65 The miR-200 family and miR-205 have been shown to regulate EMT by targeting of TCF8 and SIP1,66 although the nature of miRNA interaction with dysfunctional TCF8 transcripts is unclear at this time. Study of miRNA expression in HCECs reveals significant downregulation of at least 87 miRNAs in FCD compared with normal endothelium, and a significant decrease in DICER1, which encodes an endoribonuclease critical to miRNA biogenesis.67 Evidence of downregulation of three miR-29 family members (miR-29a-3p, miR-29b-2-5p, and miR-29c-5p) is further supported by upregulation of collagen I and collagen IV, mRNA targets which are associated with extracellular matrix production, and increased endothelial laminin protein expression FCD.67
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10. Waring 3rd GO. Posterior collagenous layer of the cornea. Ultrastructural classification of abnormal collagenous tissue posterior to Descemet’s membrane in 30 cases. Arch Ophthalmol. 1982;100(1):122–134. 11. Meadows DN, Eghrari AO, Riazuddin SA, Emmert DG, Katsanis N, Gottsch JD. Progression of Fuchs corneal dystrophy in a family linked to the FCD1 locus. Invest Ophthalmol Vis Sci. 2009;50(12):5662–5666. 12. McGlumphy EJ, Yeo WS, Riazuddin SA, et al. Age-severity relationships in families linked to FCD2 with retroillumination photography. Invest Ophthalmol Vis Sci. 2010;51(12):6298–6302. 13. Fujimoto H, Maeda N, Soma T, et al. Quantitative regional differences in corneal endothelial abnormalities in the central and peripheral zones in Fuchs’ endothelial corneal dystrophy. Invest Ophthalmol Vis Sci. 2014;55(8):5090–5098. 14. Rokita-Wala I, Mrukwa-Kominek E, Gierek-Ciaciura S. Changes in corneal structure observed with confocal microscopy during Fuchs endothelial dystrophy. Klin Oczna. 2000;102(5):339–344. 15. Weller JM, Zenkel M, Schlotzer-Schrehardt U, Bachmann BO, Tourtas T, Kruse FE. Extracellular matrix alterations in late-onset Fuchs’ corneal dystrophy. Invest Ophthalmol Vis Sci. 2014;55(6):3700–3708. 16. Kenney MC, Atilano SR, Zorapapel N, Holguin B, Gaster RN, Ljubimov AV. Altered expression of aquaporins in bullous keratopathy and Fuchs’ dystrophy corneas. J Histochem Cytochem. 2004;52(10):1341–1350. 17. Cross HE, Maumenee AE, Cantolino SJ. Inheritance of Fuchs’ endothelial dystrophy. Arch Ophthalmol. 1971;85(3):268–272. 18. Rosenblum P, Stark WJ, Maumenee IH, Hirst LW, Maumenee AE. Hereditary Fuchs’ dystrophy. Am J Ophthalmol. 1980;90(4):455–462. 19. Sundin OH, Jun AS, Broman KW, et al. Linkage of late-onset Fuchs corneal dystrophy to a novel locus at 13pTel-13q12.13. Invest Ophthalmol Vis Sci. 2006;47(1):140–145. 20. Sundin OH, Broman KW, Chang HH, Vito EC, Stark WJ, Gottsch JD. A common locus for late-onset Fuchs corneal dystrophy maps to 18q21.2-q21.32. Invest Ophthalmol Vis Sci. 2006;47(9):3919–3926. 21. Riazuddin SA, Eghrari AO, Al-Saif A, et al. Linkage of a mild late-onset phenotype of Fuchs corneal dystrophy to a novel locus at 5q33.1-q35.2. Invest Ophthalmol Vis Sci. 2009;50(12):5667–5671. 22. Riazuddin SA, Zaghloul NA, Al-Saif A, et al. Missense mutations in TCF8 cause lateonset Fuchs corneal dystrophy and interact with FCD4 on chromosome 9p. Am J Hum Genet. 2010;86(1):45–53. 23. Afshari NA, Li YJ, Pericak-Vance MA, Gregory S, Klintworth GK. Genome-wide linkage scan in Fuchs endothelial corneal dystrophy. Invest Ophthalmol Vis Sci. 2009;50(3):1093–1097. 24. Mehta JS, Vithana EN, Tan DT, et al. Analysis of the posterior polymorphous corneal dystrophy 3 gene, TCF8, in late-onset Fuchs endothelial corneal dystrophy. Invest Ophthalmol Vis Sci. 2008;49(1):184–188. 25. Park M, Li Q, Shcheynikov N, Zeng W, Muallem S. NaBC1 is a ubiquitous electrogenic Na+-coupled borate transporter essential for cellular boron homeostasis and cell growth and proliferation. Mol Cell. 2004;16(3):331–341. 26. Jalimarada SS, Ogando DG, Vithana EN, Bonanno JA. Ion transport function of SLC4A11 in corneal endothelium. Invest Ophthalmol Vis Sci. 2013;54(6):4330–4340. 27. Gottsch JD, Bowers AL, Margulies EH, et al. Serial analysis of gene expression in the corneal endothelium of Fuchs’ dystrophy. Invest Ophthalmol Vis Sci. 2003;44(2): 594–599.
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28. Vithana EN, Morgan P, Sundaresan P, et al. Mutations in sodium-borate cotransporter SLC4A11 cause recessive congenital hereditary endothelial dystrophy (CHED2). Nat Genet. 2006;38(7):755–757. 29. Vithana EN, Morgan PE, Ramprasad V, et al. SLC4A11 mutations in Fuchs endothelial corneal dystrophy. Hum Mol Genet. 2008;17(5):656–666. 30. Riazuddin SA, Vithana EN, Seet LF, et al. Missense mutations in the sodium borate cotransporter SLC4A11 cause late-onset Fuchs corneal dystrophy. Hum Mutat. 2010;31(11):1261–1268. 31. Liu J, Seet LF, Koh LW, et al. Depletion of SLC4A11 causes cell death by apoptosis in an immortalized human corneal endothelial cell line. Invest Ophthalmol Vis Sci. 2012;53(7):3270–3279. 32. Riazuddin SA, Parker DS, McGlumphy EJ, et al. Mutations in LOXHD1, a recessivedeafness locus, cause dominant late-onset Fuchs corneal dystrophy. Am J Hum Genet. 2012;90(3):533–539. 33. Rogowski K, van Dijk J, Magiera MM, et al. A family of protein-deglutamylating enzymes associated with neurodegeneration. Cell. 2010;143(4):564–578. 34. Riazuddin SA, Vasanth S, Katsanis N, Gottsch JD. Mutations in AGBL1 cause dominant late-onset Fuchs corneal dystrophy and alter protein-protein interaction with TCF4. Am J Hum Genet. 2013;93(4):758–764. 35. Baratz KH, Tosakulwong N, Ryu E, et al. E2-2 protein and Fuchs’s corneal dystrophy. N Engl J Med. 2010;363(11):1016–1024. 36. Li YJ, Minear MA, Rimmler J, et al. Replication of TCF4 through association and linkage studies in late-onset Fuchs endothelial corneal dystrophy. PLoS One. 2011;6(4): e18044. 37. Stamler JF, Roos BR, Wagoner MD, et al. Confirmation of the association between the TCF4 risk allele and Fuchs endothelial corneal dystrophy in patients from the Midwestern United States. Ophthalmic Genet. 2013;34(1–2):32–34. 38. Eghrari AO, McGlumphy EJ, Iliff BW, et al. Prevalence and severity of Fuchs corneal dystrophy in Tangier Island. Am J Ophthalmol. 2012;153(6):1067–1072. 39. Kuot A, Hewitt AW, Griggs K, et al. Association of TCF4 and CLU polymorphisms with Fuchs’ endothelial dystrophy and implication of CLU and TGFBI proteins in the disease process. Eur J Hum Genet. 2012;20(6):632–638. 40. Thalamuthu A, Khor CC, Venkataraman D, et al. Association of TCF4 gene polymorphisms with Fuchs’ corneal dystrophy in the Chinese. Invest Ophthalmol Vis Sci. 2011;52(8):5573–5578. 41. Wieben ED, Aleff RA, Tosakulwong N, et al. A common trinucleotide repeat expansion within the transcription factor 4 (TCF4, E2-2) gene predicts Fuchs corneal dystrophy. PLoS One. 2012;7(11):e49083. 42. Mootha VV, Gong X, Ku HC, Xing C. Association and familial segregation of CTG18.1 trinucleotide repeat expansion of TCF4 gene in Fuchs’ endothelial corneal dystrophy. Invest Ophthalmol Vis Sci. 2014;55(1):33–42. 43. Xing C, Gong X, Hussain I, et al. Transethnic replication of association of CTG18.1 repeat expansion of TCF4 gene with Fuchs’ corneal dystrophy in Chinese implies common causal variant. Invest Ophthalmol Vis Sci. 2014;55(11):7073–7078. 44. Nanda GG, Padhy B, Samal S, Das S, Alone DP. Genetic association of TCF4 intronic polymorphisms, CTG18.1 and rs17089887 with Fuchs’ endothelial corneal dystrophy in Indian population. Invest Ophthalmol Vis Sci. 2014;55(11):7674–7680. 45. Wieben ED, Aleff RA, Eckloff BW, et al. Comprehensive assessment of genetic variants within TCF4 in Fuchs’ endothelial corneal dystrophy. Invest Ophthalmol Vis Sci. 2014;55(9):6101–6107.
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46. Jurkunas UV, Bitar MS, Rawe I, Harris DL, Colby K, Joyce NC. Increased clusterin expression in Fuchs’ endothelial dystrophy. Invest Ophthalmol Vis Sci. 2008;49(7): 2946–2955. 47. Jurkunas UV, Bitar M, Rawe I. Colocalization of increased transforming growth factorbeta-induced protein (TGFBIp) and clusterin in Fuchs endothelial corneal dystrophy. Invest Ophthalmol Vis Sci. 2009;50(3):1129–1136. 48. Borderie VM, Baudrimont M, Vallee A, Ereau TL, Gray F, Laroche L. Corneal endothelial cell apoptosis in patients with Fuchs’ dystrophy. Invest Ophthalmol Vis Sci. 2000;41(9):2501–2505. 49. Szentmary N, Szende B, Suveges I. Epithelial cell, keratocyte, and endothelial cell apoptosis in Fuchs’ dystrophy and in pseudophakic bullous keratopathy. Eur J Ophthalmol. 2005;15(1):17–22. 50. Szentmary N, Szende B, Suveges I. P53, CD95, cathepsin and survivin pathways in Fuchs’ dystrophy and pseudophakic bullous keratopathy corneas. Histol Histopathol. 2008;23(8):911–916. 51. Wang Z, Handa JT, Green WR, Stark WJ, Weinberg RS, Jun AS. Advanced glycation end products and receptors in Fuchs’ dystrophy corneas undergoing Descemet’s stripping with endothelial keratoplasty. Ophthalmology. 2007;114(8):1453–1460. 52. Azizi B, Ziaei A, Fuchsluger T, Schmedt T, Chen Y, Jurkunas UV. p53-regulated increase in oxidative-stress-induced apoptosis in Fuchs endothelial corneal dystrophy: a native tissue model. Invest Ophthalmol Vis Sci. 2011;52(13):9291–9297. 53. Jurkunas UV, Rawe I, Bitar MS, et al. Decreased expression of peroxiredoxins in Fuchs’ endothelial dystrophy. Invest Ophthalmol Vis Sci. 2008;49(7):2956–2963. 54. Jurkunas UV, Bitar MS, Funaki T, Azizi B. Evidence of oxidative stress in the pathogenesis of Fuchs endothelial corneal dystrophy. Am J Pathol. 2010;177(5):2278–2289. 55. Bitar MS, Liu C, Ziaei A, Chen Y, Schmedt T, Jurkunas UV. Decline in DJ-1 and decreased nuclear translocation of Nrf2 in Fuchs endothelial corneal dystrophy. Invest Ophthalmol Vis Sci. 2012;53(9):5806–5813. 56. Ziaei A, Schmedt T, Chen Y, Jurkunas UV. Sulforaphane decreases endothelial cell apoptosis in Fuchs endothelial corneal dystrophy: a novel treatment. Invest Ophthalmol Vis Sci. 2013;54(10):6724–6734. 57. Tuberville AW, Wood TO, McLaughlin BJ. Cytochrome oxidase activity of Fuchs’ endothelial dystrophy. Curr Eye Res. 1986;5(12):939–947. 58. Czarny P, Seda A, Wielgorski M, et al. Mutagenesis of mitochondrial DNA in Fuchs endothelial corneal dystrophy. Mutat Res Fundam Mol Mech Mutagen. 2014;760:42–47. 59. Li YJ, Minear MA, Qin X, et al. Mitochondrial polymorphism A10398G and haplogroup I are associated with Fuchs’ endothelial corneal dystrophy. Invest Ophthalmol Vis Sci. 2014;55(7):4577–4584. 60. McGowan SL, Edelhauser HF, Pfister RR, Whikehart DR. Stem cell markers in the human posterior limbus and corneal endothelium of unwounded and wounded corneas. Mol Vis. 2007;13:1984–2000. 61. Sobrado VR, Moreno-Bueno G, Cubillo E, et al. The class I bHLH factors E2-2A and E2-2B regulate EMT. J Cell Sci. 2009;122(pt 7):1014–1024. 62. Ma C, Rong Y, Radiloff DR, et al. Extracellular matrix protein betaig-h3/TGFBI promotes metastasis of colon cancer by enhancing cell extravasation. Genes Dev. 2008;22(3):308–321. 63. Chou TY, Chen WC, Lee AC, Hung SM, Shih NY, Chen MY. Clusterin silencing in human lung adenocarcinoma cells induces a mesenchymal-to-epithelial transition through modulating the ERK/slug pathway. Cell Signal. 2009;21(5):704–711. 64. Engler C, Kelliher C, Spitze AR, Speck CL, Eberhart CG, Jun AS. Unfolded protein response in Fuchs endothelial corneal dystrophy: a unifying pathogenic pathway? Am J Ophthalmol. 2010;149(2), 194–202.e2.
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65. Iliff BW, Riazuddin SA, Gottsch JD. A single-base substitution in the seed region of miR-184 causes EDICT syndrome. Invest Ophthalmol Vis Sci. 2012;53(1):348–353. 66. Gregory PA, Bert AG, Paterson EL, et al. The miR-200 family and miR-205 regulate epithelial to mesenchymal transition by targeting ZEB1 and SIP1. Nat Cell Biol. 2008;10(5):593–601. 67. Matthaei M, Hu J, Kallay L, et al. Endothelial cell microRNA expression in human lateonset Fuchs’ dystrophy. Invest Ophthalmol Vis Sci. 2014;55(1):216–225. 68. Li YJ, Minear MA, Rimmler J, et al. Replication of TCF4 through association and linkage studies in late-onset Fuchs endothelial corneal dystrophy. PLoS One. 2011;6(4):e18044.
Fuchs Corneal Dystrophy Allen O. Eghrari, S. Amer Riazuddin, John D. Gottsch1 Wilmer Eye Institute, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA 1 Corresponding author e-mail address: [email protected]
Contents 1. Introduction 2. Structural Changes in the FCD Cornea 2.1 Epithelium 2.2 Bowman's Layer 2.3 Stroma 2.4 Descemet Membrane 2.5 Endothelium 3. Genetic Basis of FCD 3.1 Genetic Linkage Analysis 3.2 Causal Genetic Mutations 3.3 Association Studies 4. Functional Mechanisms in FCD 4.1 Oxidative Damage and Apoptosis 4.2 Mitochondrial Dysregulation 4.3 Epithelial–Mesenchymal Transition 4.4 Unfolded Protein Response 4.5 MicroRNA References
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Abstract Fuchs corneal dystrophy (FCD) is a hereditary, progressive disease of the posterior cornea which results in excrescences of Descemet membrane, endothelial cell loss, corneal edema, and, in late stages, bullous keratopathy. Structural changes are noted principally in Descemet membrane and the endothelium, with thickening of Descemet membrane, loss of barrier function, and increased corneal hydration, although secondary effects occur throughout all layers. Multiple chromosomal loci and, more recently, causal genetic mutations have been identified for this complex disorder, including in TCF8, SLC4A11, LOXHD1, and AGBL1. A trinucleotide repeat in TCF4 correlates strongly with disease status and interacts in common pathways with previously identified genes. Dysregulation of pathways involving oxidative stress and apoptosis, epithelial-tomesenchymal transition, microRNA, mitochondrial genes, and unfolded protein response has been implicated in FCD pathogenesis.
Progress in Molecular Biology and Translational Science ISSN 1877-1173 http://dx.doi.org/10.1016/bs.pmbts.2015.04.005
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1. INTRODUCTION Fuchs corneal dystrophy (FCD) is a progressive, hereditary, bilateral corneal condition first described a century ago by the Austrian ophthalmologist Ernst Fuchs.1 Patients present in the fifth to sixth decade of life with blurry morning vision that increases in duration as the disease progresses. Primarily a condition of the posterior cornea, characteristic features include the formation of focal excrescences of Descemet membrane termed “guttae,” a progressive increase in corneal edema, loss of endothelial cell density over years, and endstage disease manifested by formation of painful epithelial bullae as the cornea decompensates in its ability to maintain stromal dehydration. Recent advances in our understanding of the genetic and pathophysiological mechanisms of the disease as well as the application of new imaging modalities provide unique insight into the development of and potential therapeutic options for FCD.
2. STRUCTURAL CHANGES IN THE FCD CORNEA The characteristic clinical feature of FCD is the presence of guttae, focal nodules in Descemet membrane which are anvil shaped and protrude posteriorly into the endothelial plane, and are several microns in diameter. Guttae are visible clinically with slit-lamp biomicroscopy, and in early stages help to determine the clinical phenotype (Fig. 1). Unlike peripheral, age-associated nodules or pigment deposition on the endothelium from the anterior chamber, these excrescences begin in the center and over the course of decades, begin to coalesce and present peripherally. The grading scale commonly utilized and described by Krachmer 35 years ago reflects this phenomenon; a severity score between one and five is given based on the number and distribution of guttae. A score of one, reflecting minimal and generally asymptomatic disease, is defined as more than 12 central guttae, whereas a cluster of central confluent guttae are graded as two, and higher scores represent increasing diameters of distribution of confluent guttae.2 Although the traditional model of FCD suggested that severe disease is marked by corneal decompensation and diffuse stromal and epithelial edema in the presence of diffuse guttae, assessment of a large number of patients with FCD across various levels of severity suggests that the progressive development of edema is a gradual process that occurs over time,3 with symptoms developing in later stages over the course of decades.
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Figure 1 Direct illumination of guttae with slit-lamp biomicroscopy. In the slit of light seen passing through the cornea from left (anterior surface) to right (posterior surface), the beaten-metal appearance of guttae is appreciated posteriorly in light reflected from Descemet membrane (arrow).
We will subsequently explore pathological structural changes in the FCD-affected cornea.
2.1 Epithelium Prior to the advent of slit-lamp biomicroscopy, FCD was identified in its severe stages, and Fuchs initially described the disease as dystrophia epithelialis corneae.1 The epithelium is generally intact in early stages of classic Fuchs dystrophy, a phenomenon evidenced by a lack of change in anterior keratometry in patients undergoing endothelial keratoplasty, which would be expected if distortion of the epithelium were to occur in the disease process. However, in severe FCD, loss of pump function in the edematous cornea results in anterior migration of fluid through the corneal stroma and the formation of painful epithelial bullae.
2.2 Bowman's Layer In vivo confocal microscopy of patients with FCD reveals an abnormal Bowman’s layer in approximately half of patients with FCD, with diffuse bright reflection and a paucity or absence of nerves.4
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2.3 Stroma Anterior stromal changes occur during periods of corneal edema with formation of stromal haze and visual higher order aberrations, a phenomenon that may persist after endothelial keratoplasty.5 Confocal microscopy suggests that after endothelial transplantation, subepithelial haze rather than interface haze is the predominant source of forward light scatter.6 Collagen fibers in the anterior stroma are tightly packed relative to the posterior stroma, in which fibrils have less connections. As a result, corneal edema results in increased hydration of the posterior cornea and bulging of the posterior stroma into the anterior chamber. This focal thickening in the central corneal relative to the periphery can be appreciated through anterior segment optical coherence tomography or Scheimpflug photography; the latter has been utilized to demonstrate an increased center-to-peripheral thickness ratio in FCD-affected corneas compared to controls.7
2.4 Descemet Membrane Descemet membrane is normally composed of two layers, an anterior banded layer approximately 3 μm in thickness which develops in utero and is relatively constant in thickness over time, and a posterior nonbanded layer which is deposited by the endothelium and which thickens over an individual lifetime to approximately 10 μm in an adult. Descemet membrane in FCD is marked by the presence of a thick posterior banded layer, a posterior-most layer in which collagen fibrils approximately 10–20 nm in diameter and with 110 nm banding are deposited by the endothelium. This posterior banded layer measures approximately 16 μm in thickness and correlates directly with clinical severity of FCD.8 It is in this posterior banded layer that focal excrescences of Descemet membrane produce posteriorly and tent up the endothelium. In some cases, particularly those in which the cornea has suffered severe edema, a fibrillar layer is present, without fibroblasts or cellular elements. This has also been described as a posterior collagenous layer.9,10 Guttae develop in the central cornea in early disease and are formed peripherally in more advanced disease. However, a propensity for the development of guttae in the inferotemporal quadrant has been documented in multiple families using retroillumination photography11,12 and specular microscopy.13 Although the causes for this are unclear, the inferotemporal cornea is associated with a thinner cornea and potentially increased light exposure (Fig. 2).
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Figure 2 Retroillumination photography reveals the distribution of guttae throughout the cornea, a process which is largely central but increases in the periphery over time.
2.5 Endothelium The endothelium in both normal and FCD-affected corneas is a single monolayer of cells which is responsible for maintaining stromal dehydration, and measures approximately 5 μm in thickness. As endothelial cell density decreases, in vivo confocal microscopy reveals increased pleomorphism and polymegethism.14 Polymerase chain reaction array analysis of human Descemet membrane–endothelial complexes reveals upregulation of numerous extracellular matrix-related genes in FCD, including collagen and fibronectin, suggesting significant matrix alterations in FCD pathophysiology.15 Transmission electron microscopy of FCD corneas demonstrates some endothelial cells with cytoplasmic filaments, increased rough endoplasmic reticulum, and cytoplasmic processes, bearing similarity to fibroblasts; intercellular vacuoles remain in areas associated with loss of cells.9 Decreased aquaporin expression has been detected in FCD corneal endothelium and may contribute to corneal edema.16
3. GENETIC BASIS OF FCD Through the passage of a century after Fuchs’ initial description of FCD, an understanding of the genetic basis of disease has largely correlated
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with the knowledge and application of human genetics, with rapid advances made in the twenty-first century. Although the possibility of a hereditary component was proposed within a few years after Fuchs’ publication of his findings, pedigrees presented in the 1970s assisted to establish its autosomal dominant pattern of inheritance.2,17,18
3.1 Genetic Linkage Analysis To date, large family studies of FCD have resulted in the identification of several chromosomal loci associated with FCD. The FCD1 locus, the first to be associated with classic, late-onset FCD, was localized to a 26.4-Mb interval between 13pTel and 13q12.13 through recruitment and analysis of a large Caucasian family.19 A total of 13 affected and 3 unaffected individuals across three generations were recruited for genotyping, and linkage using 399 STR markers from the MD10 genotyping panel revealed significant two-point logarithm of odds scores of 3.91 at D13S1236 and 3.80 at D13S1304, assuming fully penetrant dominant inheritance. Notably, two children in this family whose parents were both affected demonstrated clinical signs of late-onset FCD at a young age. The FCD2 locus was identified in three large families and localized to 18q21.2–18q21.32.20 This was the first locus associated with multiple families, suggesting that this area may represent a common locus. Linkage analyses were performed using 1107 STR markers, and maximum two-point LOD scores among the three families were 3.41, 2.89, and 2.45. Differences in haplotypes among families raised the possibility of heterogeneity of the locus. Using retroillumination photography, progression in FCD2associated families was compared with that in FCD1-related families and found to demonstrate a less marked rate of development of guttae.12 The FCD3 locus was identified through a large family with 17 examined individuals, 10 of which manifested clinical signs of disease. This interval was localized to chromosome 5 using an SNP array, followed by refinement of the region with an STR marker panel to 5q33.1–5q35.2.21 The FCD4 locus was identified in the region 9p22.1–9p24.1 and was the first to demonstrate interaction between two pathologic alleles in this disease. After mutations in TCF8 were identified among some affected members of this family, linkage analyses were performed conditioned to the presence of the TCF8 mutation, and refined using STR markers, with maximum LOD scores of 3.09 at D9S168 and 3.20 at D9S256, and a linkage
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interval spanning 14.3 Mb between recombinations at D91681 and D9S1684.22 While either the pathologic mutation in TCF8 or the causal FCD4 haplotype was associated with clinically significant disease, those with the presence of both genetic inputs demonstrated markedly severe disease, generally progressing to corneal transplantation. A number of additional loci have been identified with large numbers of small pedigrees which, although below the typical LOD threshold of 3.0, may serve as promising targets in the future. Using SNP linkage panels and DNA from 92 individuals from 22 families, four loci were identified on chromosome 1 (rs760594), chromosome 7 (rs257376), chromosome 15 (rs352476), and chromosome 17 (rs938350).23 A follow-up study using 215 individuals from 64 families identified six linkage peaks with maximum HLOD scores over 2, and including two over 3: one in chromosome 10 (rs1889974) with HLOD score of 3.37 and one in chromosome 15 (rs235512) with HLOD score of 3.53.68
3.2 Causal Genetic Mutations Coding mutations in four genes have been identified to be causal for the FCD phenotype: TCF8, SLC4A11, LOXHD1, and AGBL1. Mutations in TCF8 had been previously implicated in posterior polymorphous corneal dystrophy, which similar to FCD primarily affects the posterior cornea. To investigate whether this gene may be associated with the FCD phenotype, an initial screen of 74 Chinese individuals with FCD for TCF8 mutations identified a novel variant, p.N696S, present in 1 sporadic case and absent in 93 controls.24 In a large family also linked to the FCD4 locus, a causal missense mutation in TCF8 was identified, p.Q840P.22 Four additional pathogenic mutations (p.N78T, p.P649A, p.Q810P, and p.A905T) were discovered through sequencing of exonic regions of TCF8 in 384 unrelated FCD-affected individuals. Of these five mutations, three occurred at sites that are highly evolutionarily conserved in vertebrates (p.Q810P, p.Q840P, and p.A905T) and two at moderately conserved sites. In contrast to frameshift, nonsense, or lost-start-codon mutations associated with PPCD, the causal alleles in TCF8 for FCD contain missense mutations. Functional assessment using zebrafish embryos revealed an inability of human mRNA containing each of the FCD-linked mutations to fully rescue developmental abnormalities caused by morpholino oligonucleotide knockdown of endogenous TCF8. While the variants p.N78T and p.Q810P partially rescued the phenotype, the other
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three variants demonstrated phenotypes identical to morpholino injection alone, with successful rescue limited to wild-type mRNA. The second gene for which causal mutations have been associated with FCD, SLC4A11, codes for a sodium-borate cotransporter that can also stimulate cell growth and proliferation through the MAPK pathway.25 In the corneal endothelium, SLC4A11 demonstrates robust Na+-coupled OH transport, but does not transport BðOHÞ4 or HCO3 .26 Expression of this gene was shown to be downregulated in FCD through serial analysis of gene expression.27 Mutations in this gene have been associated with the autosomal recessive form of congenital hereditary endothelial dystrophy28 and systemically in Harboyan syndrome, a condition which includes both corneal dystrophy and deafness. A screen of 89 FCD-affected individuals, 64 of Chinese and 25 of Indian descent, revealed four previously unreported mutations (p.S33SfsX18, p.E399K, p.G709E, and p.T754M) which were not present in 354 ethnically matched controls, and acquired with autosomal dominant inheritance.29 These missense mutations were shown in a biochemical assay of cell surface localization and confocal immunolocalization to demonstrate defective localization to the cell surface. A large study sequencing all coding regions of SLC4A11 in 192 FCD-affected and 192 unaffected individuals resulted in the identification of an additional seven missense mutations (p.E167D, p.R282P, p.Y526C, p.V575M, p.G583D, p.G742R, and p.G834S) among affected individuals, with no such mutations among the control samples.30 Sorting Intolerant from Tolerant (SIFT) and PolyPhen predicted that five of these mutations were deleterious. Loss of SLC4A11 was explored utilizing short hairpin RNA against SLC4A11 in an immortalized human corneal endothelial cell (HCEC) line to downregulate SLC4A11 gene expression; knockdown suppressed growth and reduced viability among HCECs, associated with increased apoptosis in SLC4A11-depleted cells.31 The third gene with FCD-associated causal mutations, LOXHD1, was identified in a family previously linked the FCD2 locus in chromosome 18. LOXHD1 codes for a protein composed of polycystin/lipoxygenase/ alpha-toxin domains, which modulate plasma membrane targeting. Sequencing of 36 Mb on chromosome 18 using a custom exon-capture array in one affected and one unaffected member of three families linked to the FCD2 locus identified a p.R547C mutation in LOXHD1 in one affected individual, a variant not present in either the 1000 Genomes project or 1500 Exomes from the University of Seattle Human Exome Variant database. PolyPhen 2 predicted this variant to be pathogenic and it was absent in
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384 unaffected control individuals. Immunohistochemistry was utilized to identify LOXHD1 in mouse corneas, both in the epithelial and endothelial layers. Immunostaining of the affected proband cornea, one from a FCDaffected individual known not to bear the identified mutation in LOXHD1, and a control with keratoconus revealed increased LOXHD1 staining in Descemet membrane and corneal endothelium in the LOXHD1-associated Fuchs cornea. Three-dimensional reconstruction of the LOXHD1 protein predicted that most mutations resided on the protein surface, likely affecting interaction with other proteins.32 The fourth gene with an identified causal mutation, AGBL1, codes for ATP/GTP-binding protein-like 1. The cytosolic carboxypeptidase family, of which AGBL1 is a member, catalyzes deglutamylation of polyglutamylated proteins. Polyglutamylation is a reversible posttranslational protein modification which generates glutamate side chains on tubulins and other proteins, a process which has been shown to be necessary for neuronal survival.33 In a three-generation family with 12 affected individuals, a genome-wide linkage analysis with a multilocus model was conducted, conditioned on severe disease resulting from two disease alleles, mild disease from a single allele, and offspring of two affected parents receiving the disease allele from the parent otherwise unrelated to the pedigree. Two positive signals were noted on 3p and 15q, which were investigated further with nextgeneration sequencing. A C-to-T transition, c.3082C > T in AGBL1, was identified, which results in a premature stop codon; this nonsense allele segregated with the FCD haplotype under the multilocus model. Sequencing of FCD-affected individuals resulted in the identification of two other individuals with the same nonsense mutation and the identification of a second allele, a heterozygous missense variant, c.2969G > C, neither of which was present among 384 ethnically matched controls. To investigate the role of AGBL1, immunoprecipitated lysates were probed with anti-Myc antibody and variant forms of the protein demonstrated reduced binding affinity with TCF4. The results overall indicated that AGBL1 interacts specifically with TCF4, but not with TCF8.34
3.3 Association Studies A genome-wide association study by Baratz and colleagues among 130 cases and 260 controls matched by age and sex interrogated 338,727 SNPs, of which one reached threshold for genome-wide significance: rs613872, an intronic variant in TCF4. The minor allele (G) frequency was significantly
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enriched in FCD cases (G ¼ 0.37) compared to controls (0.14), a finding replicated in a second group of 150 cases and 150 controls (0.43 among cases and 0.15 among controls). The odds ratio associated with the minor allele was notable in heterozygotes (5.5) and particularly homozygotes (30).35 In the United States, these findings were replicated in a large cohort of 450 cases and 360 controls with odds ratio of 4.5,36 and in a Midwestern population of 82 patients with FCD and 163 control subjects.37 The population of Tangier, an island in the Chesapeake Bay with a high prevalence of FCD, was found to also demonstrate a high minor allele frequency of 0.37, comparable to affected cohorts in the aforementioned studies.38 Globally, findings were replicated in an Australian cohort of 105 cases and 275 controls with odds ratio of 4.05.39 In a Chinese cohort of 57 affected and 121 unaffected individuals, two other SNPs in TCF4 were significantly associated with FCD affectation with an odds ratio over 240; however, the rs613872 minor allele was absent among the affected cohort. Together, these findings indicated strong association but raised the possibility of a yet undetermined causal allele in TCF4. Subsequently, testing of 66 affected and 63 unaffected individuals for an intronic TGC trinucleotide repeat in TCF4 demonstrated, in cases with over 50 TGC repeats, sensitivity of 79% and specificity of 96%, even greater specificity than had been previously determined with the rs613872 single nucleotide polymorphism.41 This association with the CTG 18.1 trinucleotide repeat expansion was replicated in 120 affected Caucasian individuals and 100 controls (p ¼ 6.5 1025) and the two polymorphisms were found to be in linkage disequilibrium. In this cohort, the odds ratio of each copy of the CTG18.1 expanded allele was 32.3, and in 29 families, the expanded allele cosegregated with the trait in 15 families (52%) with complete penetrance.42 Trans-ethnic replication of the CTG18.1 repeat expansion has been described in the Chinese population with 57 affected and 121 control individuals; the expanded CTG18.1 allele was associated (p ¼ 4.7 1014) with FCD with odds ratio of 66.5.43 Replication was also performed in 44 affected and 108 control patients from an Indian population sample, with significance achieved for the CTG18.1 expanded allele (p ¼ 2 104); 34% of affected and 5% of control subjects were found to harbor over 50 CTG repeats.44 A comprehensive assessment of 1866 genetic variants, with sequencing of the TCF4 coding region, introns, and flanking sequence surrounding the two variants, revealed that no single causative allele correlated with all cases of FCD, but the trinucleotide repeat is the most strongly associated of the two TCF4 variants.45
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Clusterin is a molecular chaperone which plays a role in extracellular matrix interaction and is overexpressed in FCD relative to pseudophakic bullous keratopathy and normal control corneas46 and has been shown to colocalize in guttae.47 In the aforementioned Australian cohort exploring association with TCF4, additional SNPs were interrogated and one found to be significant in CLU (rs17466684, p ¼ 0.003) with odds ratio of 1.85.39 Immunohistochemistry revealed anti-CLU labeling in FCD epithelium but absent in control epithelium, while labeling in Descemet membrane was limited to its anterior portion in FCD and more diffusely distributed in controls. Both affected and control endothelium expressed CLU, although endothelial cell density was decreased in affected individuals.39 Polymerase chain reaction array analyses reveal upregulation of clusterin expression in human FCD-affected Descemet membrane– endothelium complexes, which demonstrate increased immunohistochemistry labeling among 40 samples when compared to controls.15 TGF-β1 protein has also been found to be overexpressed in FCD and in addition to clusterin, colocalizes in guttae.47 An association with a single haplotype of TGF-β1 SNPs and FCD affectation reached statistical significance among Caucasian Australians (p ¼ 0.011), with odds ratio of 2.29.39
4. FUNCTIONAL MECHANISMS IN FCD 4.1 Oxidative Damage and Apoptosis Loss of corneal endothelial cell density is a characteristic feature of FCD and is associated with increased apoptotic processes. A study of 47 FCD-affected corneal buttons revealed a significantly higher percentage of apoptotic endothelial cells by nucleus labeling assay relative to controls, a phenomenon also supported by TUNEL assay.48 Subsequent studies with TUNEL assay of entire corneas revealed significantly higher apoptotic cell numbers not only in the corneal endothelium but also in the stroma and epithelium in patients with FCD compared to controls.49 Serial analysis of gene expression reveals decreased transcription levels of antioxidant molecules in FCD-affected corneal endothelium, including glutathione S-transferase-pi.27 A number of apoptosis-related factors are found more frequently in FECD-affected corneas. Immunohistochemical study of FCD corneas reveals a lower number of p27- and survivin-positive epithelial cells, and higher cathepsin-positive epithelial cells in FCD corneas relative to controls.50 Descemet membrane extracted from FCD corneas undergoing
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endothelial keratoplasty reveals abundant advanced glycation end products in Descemet membrane, molecules which are associated with oxidative stress, inflammation, and aging.51 A comparison of immortalized FCD and control corneal endothelial cell lines in which oxidative stress was induced with tert-butyl hydroperoxide (tBHP) reveals significantly increased activation of p53 in FCD cells relative to controls (Fig. 3).52 Peroxiredoxins are expressed in human corneal endothelium and protect against oxidative stress by removing cellular hydrogen peroxide. Proteomic analysis reveals significantly decreased expression of Prx-2, -3, and -5 in FCD cells compared to controls, a finding supported by decreased Prx-2 mRNA detected by real-time PCR analysis.53 Evidence suggests that the oxidant–antioxidant balance in FCD-affected endothelial cells shifts to a pro-oxidant state. In addition to downregulation of peroxiredoxins, PCR array also demonstrates transcriptional downregulation of other antioxidants, including SOD2, MT3, and TXNDR1 in HCECs, without a compensatory increase in other antioxidants such as catalase or glutathione peroxidases and/or transferases.54 These downregulated antioxidants share a common promoter region, the antioxidant response element, to which the transcription factors nuclear factor erythroid 2-related factor-1 (Nrf1) and -2 (Nrf2) bind and induce upregulation of antioxidant enzyme genes in settings of oxidative stress. The identification of a
Figure 3 Descemet membrane removed from a patient at the time of endothelial keratoplasty reveals classic anvil-shaped excrescences which protrude posteriorly, termed guttae.
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significant decrease in Nrf2 protein in FCD endothelial cells compared to controls is further supported by depleted expression of a major Nrf2regulated antioxidant gene, HO-1,54 and significant decrease in the Nrf2 protein stabilizer DJ-1.55 Identification of dysregulated oxidative stress pathways offers potential avenues for future therapeutic targets. Sulforaphane is a naturally occurring glucosinolate found in green leafy vegetables which modifies sulfhydryl residues of Keap1, causing the release and activation of Nrf2. In human unaffected and FCD-affected immortalized corneal endothelial cell lines subjected to oxidative stress with tBHP, pretreatment with sulforaphane decreased intracellular production of reactive oxygen species, enhanced nuclear translocation of Nrf2, and decreased p53 staining.56
4.2 Mitochondrial Dysregulation FCD is principally a disease of the central cornea, and early research suggested regional differences in mitochondrial cytochrome oxidase (CO) activity in FCD-affected corneal endothelium, with decreased CO centrally and increased CO in the periphery.57 Serial analysis of gene expression in FCD and normal corneal endothelium revealed significant underexpression in FCD of multiple mitochondrial genes involved in electron transport and oxidative phosphorylation, including NADH dehydrogenase subunits 1, 2, and 4, cytochrome b, cytochrome c oxidase subunit III, and ATP synthase F0 subunit 6.27 Comparison of oxidative DNA damage in FCD-affected and unaffected corneal endothelium was conducted with high-sensitivity ELISA to detect the concentration of 8-OHdG (an oxidative DNA damage marker). This was significantly higher in FCD-affected corneal endothelium than in controls, and immunolabeling revealed that this marker colocalized to mitochondria.54 To test if FCD was associated with a systemic deficiency in mitochondrial enzymes, a separate study challenged mitochondria in peripheral blood lymphocytes with hydrogen peroxide in 35 FCD patients and 32 controls; increased mitochondrial DNA (mtDNA) damage and a higher ratio of a 4977-bp deletion were found in FCD patients compared to controls.58 In a study of mtDNA variants on FCD susceptibility, a total of 530 cases and 498 controls of European descent were assessed for 10 mtDNA variants defining European haplogroups, and replication analyses conducted in cohorts of 3200 and 3043 individuals. The mtDNA variant A10398G
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was significantly associated with FCD with odds ratio of 0.72, suggesting that this variant confers a protective effect; this persisted even after adjusting for smoking status. Haplogroup I was significantly associated with FCD even after adjusting for smoking or the TCF4 intronic variant rs613872.59
4.3 Epithelial–Mesenchymal Transition Although the corneal endothelium is often considered to be in a state of arrested growth, with endothelial cells migrating to cover defects in the endothelial layer, evidence exists of stem cell markers in the posterior limbus and corneal endothelium, and markers of proliferation are present after wounding of the endothelium.60 Fibroblastic features of some endothelial cells in FCD9 may be suggestive of alterations in the process of maturation and differentiation, such as epithelial–mesenchymal transition (EMT). Several genes associated with FCD such as TCF8 and TCF4 are implicated directly or indirectly in the EMT pathway. In breast cancer cells, TCF8 serves as a transcriptional repressor and downregulates E-cadherin, inducing a transition to a mobile, fibroblastoid appearance. TCF4 modulates multiple pathways, including TGF-β, and upregulates TCF8 expression.61 TGF-βI induces EMT in colon cancer cells62 and silencing of clusterin in lung adenocarcinoma cells appears to induce a mesenchymal-to-epithelial transition through the ERK/Slug pathway.63
4.4 Unfolded Protein Response Several genes implicated in FCD pathogenesis modify protein folding mechanisms, supporting a possible role for the unfolded protein response. CLU is involved with protein folding and colocalizes to guttae, and pathogenic LOXHD1 alleles induce misfolded aggregates in corneal endothelial cells.32 In a comparison of FCD corneas with keratoconus and normal controls, misfolded proteins were found to be increased in FCD.64 Of 10 corneas with FCD and 9 corneas with non-FCD corneal dystrophy, all FCD corneas exhibited prominent rough endoplasmic reticulum, in contrast to only a third of non-FCD corneas. FCD-associated corneas also demonstrated increased immunofluorescent labeling for GRP78, phospho-eIF2a, and CHOP, markers for unfolded protein response relative to both normal and keratoconus corneas. Similarly, the apoptosis markers caspase 3 and caspase 9 were both significantly increased in FCD corneas, suggesting that
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increased misfolded protein in FCD may contribute to an increased unfolded protein response and caspase-dependent apoptosis.64
4.5 MicroRNA MicroRNA (miRNA) dysregulation is implicated in numerous pathophysiological pathways, including those associated with FCD and other corneal dystrophies with similar phenotypes. A mutation in miR-184 has been shown to be causative for EDICT syndrome, which includes a beaten-metal appearance to the corneal endothelium.65 The miR-200 family and miR-205 have been shown to regulate EMT by targeting of TCF8 and SIP1,66 although the nature of miRNA interaction with dysfunctional TCF8 transcripts is unclear at this time. Study of miRNA expression in HCECs reveals significant downregulation of at least 87 miRNAs in FCD compared with normal endothelium, and a significant decrease in DICER1, which encodes an endoribonuclease critical to miRNA biogenesis.67 Evidence of downregulation of three miR-29 family members (miR-29a-3p, miR-29b-2-5p, and miR-29c-5p) is further supported by upregulation of collagen I and collagen IV, mRNA targets which are associated with extracellular matrix production, and increased endothelial laminin protein expression FCD.67
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65. Iliff BW, Riazuddin SA, Gottsch JD. A single-base substitution in the seed region of miR-184 causes EDICT syndrome. Invest Ophthalmol Vis Sci. 2012;53(1):348–353. 66. Gregory PA, Bert AG, Paterson EL, et al. The miR-200 family and miR-205 regulate epithelial to mesenchymal transition by targeting ZEB1 and SIP1. Nat Cell Biol. 2008;10(5):593–601. 67. Matthaei M, Hu J, Kallay L, et al. Endothelial cell microRNA expression in human lateonset Fuchs’ dystrophy. Invest Ophthalmol Vis Sci. 2014;55(1):216–225. 68. Li YJ, Minear MA, Rimmler J, et al. Replication of TCF4 through association and linkage studies in late-onset Fuchs endothelial corneal dystrophy. PLoS One. 2011;6(4):e18044.