Comment on "Efficacy of the Rho-Kinase Inhibitor for Corneal Endothelial Protection in Fuchs Endothelial Corneal Dystrophy after Phacoemulsification by Keeratidamkerngsakul et al."

The aim of this review was to provide an evidenced-based review of the genetic basis of the corneal endothelial dystrophies. A review of the English language peer-reviewed literature describing the molecular genetic basis of posterior polymorphous corneal dystrophy (PPCD), congenital hereditary endothelial dystrophy (CHED), Fuchs endothelial corneal dystrophy (FECD) and X-linked endothelial corneal dystrophy (XECD) was performed. Mutations in several genes have been implicated as playing a pathogenic role in the corneal endothelial dystrophies: VSX1 mutations in PPCD1; COL8A2 mutations in PPCD2 and FECD; ZEB1 mutations in PPCD3 and FECD; and SLC4A11 mutations in CHED2 and FECD. However, linkage, association and familial segregation analyses support a role of only one gene in each corneal endothelial dystrophy: ZEB1 in PPCD3, SLC4A11 in CHED2 and COL8A2 in FECD (early onset). In addition, insufficient evidence exists to consider the autosomal dominant form of CHED (CHED1) as distinct from PPCD. An accurate classification of the corneal endothelial dystrophies requires a critical review of the evidence to support the role of each suggested chromosomal locus, gene and genetic mutation associated with a corneal endothelial dystrophy. Only after the separation of evidence from opinion is performed can a critical examination of the molecular pathways that lead to endothelial dysfunction in each of these disorders be accurately performed.

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IntroductionThe pathogenic role of variants in TCF4 and COL8A2 in causing Fuchs’ endothelial corneal dystrophy (FECD) is not controversial and has been confirmed by numerous studies. The causal role of other genes, SLC4A11, ZEB1, LOXHD1, and AGBL1, which have been reported to be associated with FECD, is more complicated and less obvious. We performed a systematic review of the variants in the above-mentioned genes in FECD cases, taking into account the currently available population frequency information, transcriptomic data, and the results of functional studies to assess their pathogenicity.MethodsSearch for articles published in 2005–2022 was performed manually between July 2022 and February 2023. We searched for original research articles in peer-reviewed journals, written in English. Variants in the genes of interest identified in patients with FECD were extracted for the analysis. We classified each presented variant by pathogenicity status according to the ACMG criteria implemented in the Varsome tool. Diagnosis, segregation data, presence of affected relatives, functional analysis results, and gene expression in the corneal endothelium were taken into account. Data on the expression of genes of interest in the corneal endothelium were extracted from articles in which transcriptome analysis was performed. The identification of at least one variant in a gene classified as pathogenic or significantly associated with FECD was required to confirm the causal role of the gene in FECD.ResultsThe analysis included 34 articles with 102 unique ZEB1 variants, 20 articles with 64 SLC4A11 variants, six articles with 26 LOXHD1 variants, and five articles with four AGBL1 variants. Pathogenic status was confirmed for seven SLC4A11 variants found in FECD. No variants in ZEB1, LOXHD1, and AGBL1 genes were classified as pathogenic for FECD. According to the transcriptome data, AGBL1 and LOXHD1 were not expressed in the corneal endothelium. Functional evidence for the association of LOXHD1, and AGBL1 with FECD was conflicting.ConclusionOur analysis confirmed the causal role of SLC4A11 variants in the development of FECD. The causal role of ZEB1, LOXHD1, and AGBL1 variants in FECD has not been confirmed. Further evidence from familial cases and functional analysis is needed to confirm their causal roles in FECD.

The hallmark of Fuchs endothelial corneal dystrophy (FECD) is corneal “guttae”. However, some eyes with cornea guttata will never progress to FECD! Thus, we should talk about FECD not before endothelial decompensation = “swelling” = “increased central corneal thickness” does occur. Early-onset FECD with well-defined genetic cause must be differentiated from late-onset FECD. Differential diagnosis to other endothelial dystrophies is typically an easy task. FECD must be differentiated from pseudoexfoliation (PEX) keratopathy with respect to incidence of secondary glaucoma, therapeutic necessities, and prognosis of corneal transplantation. Concerning microsurgery, FECD is a friend, while PEX keratopathy is a challenge. Thus, differentiation to PEX keratopathy is not just an academic problem! Progression of FECD cannot be defined via endothelial cell counts, because numbers are notoriously measured wrong. Neither manual nor automated endothelial cell count will give valid results in case of unevenly distributed guttae. Progression should be defined via best-corrected visual acuity (worse morning vs afternoon), glare, corneal thickness profile (tomography), and cell morphology. For many decades, symptomatic medical therapy consisted of unpreserved hyperosmolar (5 %) sodium chloride solution. Preclinical causative approaches include lithium, N-acetylcysteine, and the ROCK inhibitor.

PurposeThe most common cause of Fuchs' endothelial corneal dystrophy (FECD) is an intronic CTG repeat expansion in TCF4. Expanded CUG repeat RNA colocalize with splicing factor, muscleblind-like 1 (MBNL1), in nuclear foci in endothelium as a molecular hallmark. Myotonic dystrophy type 1 (DM1) is a neuromuscular disorder caused by a CTG repeat expansion in the 3′-untranslated region (UTR) of DMPK. In this study, we examine for RNA-MBNL1 foci in endothelial cells of FECD subjects with DM1, test the hypothesis that DM1 patients are at risk for FECD, and determine prevalence of TCF4 and DMPK expansions in a FECD cohort.MethodsUsing FISH, we examined for nuclear RNA-MBNL1 foci in endothelial cells from FECD subjects with DM1. We examined 13 consecutive unrelated DM1 patients for FECD using slit-lamp and specular microscopy. We genotyped TCF4 and DMPK repeat polymorphisms in a FECD cohort of 317 probands using short-tandem repeat and triplet repeat-primed PCR assays.ResultsWe detected abundant nuclear RNA foci colocalizing with MBNL1 in endothelial cells of FECD subjects with DM1. Six of thirteen DM1 patients (46%) had slit-lamp and specular microscopic findings of FECD, compared to 4% disease prevalence (P = 5.5 × 10-6 ). As expected, 222 out of 317 (70%) FECD probands harbored TCF4 expansion, while one subject harbored DMPK expansion without prior diagnosis of DM1. ConclusionsOur work suggests that DM1 patients are at risk for FECD. DMPK mutations contribute to the genetic burden of FECD but are uncommon. We establish a connection between two repeat expansion disorders converging upon RNA-MBNL1 foci and FECD.

PurposeCTG trinucleotide repeat (TNR) expansion in an intron of the TCF4 gene is the most common genetic variant associated with Fuchs' endothelial corneal dystrophy (FECD). Although several mechanisms have been implicated in the disease process, their exact pathophysiologic importance is unclear. To understand events leading from TCF4 TNR expansion to disease phenotype, we characterized splicing, gene expression, and exon sequence changes in a rare cohort of patients with TNR expansions but no phenotypic FECD (RE+/FECD−).MethodsCorneal endothelium and blood were collected from patients undergoing endothelial keratoplasty for non-FECD corneal edema. Total RNA was isolated from corneal endothelial tissue (n = 3) and used for RNASeq. Gene splicing and expression was assessed by Mixture of Isoforms (MISO) and MAP-RSeq software. Genomic DNA was isolated from blood mononuclear cells and used for whole genome exome sequencing. Base calling was performed using Illumina's Real-Time Analysis.ResultsThree genes (MBNL1, KIF13A, AKAP13) that were previously identified as misspliced in patients with a CTG TNR expansion and FECD disease (RE+/FECD+) were found normally spliced in RE+/FECD− samples. Gene expression differences in pathways associated with the innate immune response, cell signaling (e.g., TGFβ, WNT), and cell senescence markers were also identified between RE+/FECD− and RE+/FECD+ groups. No consistent genetic variants were identified in RE+/FECD− patient exomes.ConclusionsIdentification of novel splicing patterns and differential gene expression in RE+/FECD− samples provides new insights and more relevant gene targets that may be protective against FECD disease in vulnerable patients with TCF4 CTG TNR expansions.

Aim: a review of miRNA expression connected to epithelial mesenchymal transition studies in Fuchs’ endothelial corneal dystrophy (FECD). Methods: literature search strategy—PubMed central database, using “miRNA” or “microRNA” and “epithelial mesenchymal transition” or “EMT” and “Fuchs’ endothelial corneal dystrophy” or “FECD” as keywords. Experimental or clinical studies on humans published in English regarding miRNA profiles of epithelial mesenchymal transition in Fuchs’ endothelial corneal dystrophy published between 2009 and 2022 were included. Conclusion: The publications regarding the miRNA profiles of epithelial mesenchymal transition in Fuchs’ endothelial corneal dystrophy are scarce but provide some valuable information about the potential biomarkers differentiating aging changes from early disease stages characterized by epithelial mesenchymal transition. In the corneal tissue of FECD patients, miRNA-184 seed-region mutation as well as unidirectional downregulation of total miRNA expression led by the miRNA-29 were demonstrated. For early diagnostics the miRNA of epithelial mesenchymal transition in aqueous humor should be analyzed and used as biomarkers.

Fuchs endothelial corneal dystrophy (FECD) is the most common late-onset, vision-threatening corneal dystrophy in the United States, affecting about 4% of the population. Advanced FECD involves a thickening of the cornea from stromal edema and changes in Descemet membrane. To understand the relationship between FECD and central corneal thickness (CCT), we characterized common genetic variation in COL8A2 and TCF4, genes previously implicated in CCT and/or FECD. Other genes previously associated with FECD (PITX2, ZEB1, SLC4A11), and genes only known to affect CCT (COL5A1, FOXO1, AVGR8, ZNF469) were also interrogated. FECD probands, relatives and controls were recruited from 32 clinical sites; a total of 532 cases and 204 controls were genotyped and tested for association of FECD case/control status, a 7-step FECD severity scale and CCT, adjusting for age and sex. Association of FECD grade with TCF4 was highly significant (OR = 6.01 at rs613872; p = 4.8×10−25), and remained significant when adjusted for changes in CCT (OR = 4.84; p = 2.2×10−16). Association of CCT with TCF4 was also significant (p = 6.1×10−7), but was abolished with adjustment for FECD grade (p = 0.92). After adjusting for FECD grade, markers in other genes examined were modestly associated (p ∼ 0.001) with FECD and/or CCT. Thus, common variants in TCF4 appear to influence FECD directly, and CCT secondarily via FECD. Additionally, changes in corneal thickness due to the effect of other loci may modify disease severity, age-at-onset, or other biomechanical characteristics.

Diagnostic Performance of 3-Dimensional Thickness of the Endothelium–Descemet Complex in Fuchs’ Endothelial Cell Corneal Dystrophy

The pathophysiology of Fuchs' endothelial dystrophy – A review of molecular and cellular insights

Fuchs' endothelial corneal dystrophy (FECD) has been considered a genetically heterogeneous disease but is increasingly associated with the transcription factor 4 (TCF4) gene. This study investigates the prevalence of the cytosine-thymine-guanine (CTG)n repeat expansion in TCF4 among FECD patients in northern Sweden coupled to the phenotype. Blood samples were collected from 85 FECD cases at different stages. Short tandem repeat PCR and triplet repeat-primed PCR were applied in order to determine TCF4 (CTG)n genotype. A (CTG)n repeat expansion (n > 50) in TCF4 was identified in 76 of 85 FECD cases (89.4%) and in four of 102 controls (3.9%). The median (CTG)n repeat length was 81 (IQR 39.3) in mild FECD and 87 (IQR 13.0) in severe FECD (p = 0.01). A higher number of (CTG)n repeats in an expanded TCF4 allele increased the probability of severe FECD. Other ocular surgery was overrepresented in FECD cases without a (CTG)n repeat expansion (44.4%, n = 4) compared with 3.9% (n = 3) in FECD cases with an (CTG)n repeat expansion (p < 0.001). In northern Sweden, the FECD phenotype is associated with (CTG)n expansion in the TCF4 gene, with nearly 90% of patients being hetero- or homozygous for (CTG)n expansion over 50 repeats. Furthermore, the severity of FECD was associated with the repeat length in the TCF4 gene. Ocular surgery might act as an environmental factor explaining the clinical disease in FECD without a repeat expansion in TCF4.

Matrix metalloproteinases and their inhibitors in Fuchs endothelial corneal dystrophy

Mutations in the SLC4A11 gene, which encodes a plasma membrane borate transporter, cause recessive congenital hereditary endothelial corneal dystrophy type 2 (CHED2), corneal dystrophy and perceptive deafness (Harboyan syndrome), and dominant late-onset Fuchs endothelial corneal dystrophy (FECD). We analyzed missense SLC4A11 mutations identified in FECD and CHED2 patients and expressed in transfected HEK 293 cells. Chemical cross-linking and migration in nondenaturing gels showed that SLC4A11 exists as a dimer. Furthermore, co-immunoprecipitation of epitope-tagged proteins revealed heteromeric interactions between wild-type (WT) and mutant SLC4A11 proteins. When expressed alone, FECD- and CHED2-causing mutant SLC4A11 proteins are primarily retained intracellularly. Co-expression with WT SLC4A11 partially rescued the cell surface trafficking of CHED2 mutants, but not FECD mutants. CHED2 alleles of SLC4A11 did not affect cell surface processing of WT SLC4A11. In contrast, FECD mutants reduced WT cell surface processing efficiency, consistent with dominant inheritance of FECD. The reduction in movement of WT protein to the cell surface caused by FECD SLC4A11 helps to explain the dominant inheritance of this disorder. Similarly, the failure of CHED2 mutant SLC4A11 to affect the processing of WT protein, explains the lack of symptoms found in CHED2 carriers and the recessive inheritance of the disorder.

BackgroundThe pathophysiology underlying Fuchs' Endothelial Corneal Dystrophy (FECD), especially in older individuals, remains unclear, with a genetic predisposition being reported as the single best predictor of the disease. Genetic studies have shown that several genes in various loci such as COL8A2, SLC4A11, TCF8/ZEB1 and TCF4 are associated with FECD in different populations and ethnicities. A case–control study was conducted to determine the association between genetic variants and FECD in a tertiary care setting in Malaysia. A total number of 12 patients with clinically diagnosed FECD and 12 age, gender and race matched control subjects were recruited. Extracted genomic DNA were genotyped using Infinium Global Screening Array (GSA)-24 version 1.0 BeadChip with iScan high-throughput system. Illumina GenomeStudio 2.0 Data Analysis and PLINK version 1.9 software were used to perform association tests and determine the distribution of obtained variants among the cases and controls.ResultsA significant novel genetic variant, rs11626651, a variant of the LOC105370676 gene or known as the LINC02320 gene, located at chromosome 14, has been identified as a suggestive association with FECD (p < 5 × 10−6). Further analysis in this study suggested that candidate genes such as COL8A2, ZEB1/TCF8, TCF4 and SLC4A11 had no significant associations with FECD.ConclusionsThe discovery of a novel variant may influence the underlying pathogenic basis of FECD in Malaysia. The current study is the first genetic study on FECD to use Infinium GSA. It is the first comprehensive report in Malaysia to provide genetic information of potential relevance to FECD, which may pave the way for new therapeutic strategies in the future. A detailed analysis with a larger sample size is recommended for further evaluation.

PurposeThis study evaluated the dysregulation of TCF4 isoforms and differential exon usage (DEU) in corneal endothelial cells (CECs) of Fuchs endothelial corneal dystrophy (FECD) with or without trinucleotide repeat (TNR) expansion in the intron region of the TCF4 gene.MethodsThree RNA-Seq datasets of CECs (our own and two other previously published datasets) derived from non-FECD control and FECD subjects were analyzed to identify TCF4 isoforms and DEU events dysregulated in FECD by comparing control subjects to those with FECD with TNR expansion and FECD without TNR expansion.ResultsOur RNA-Seq data demonstrated upregulation of three TCF4 isoforms and downregulation of two isoforms in FECD without TNR expansion compared to the controls. In FECD with TNR expansion, one isoform was upregulated and one isoform was downregulated compared to the control. Additional analysis using two other datasets identified that the TCF4-277 isoform was upregulated in common in all three datasets in FECD with TNR expansion, whereas no isoform was dysregulated in FECD without TNR expansion. DEU analysis showed that one exon (E174) upstream of the TNR, which only encompassed TCF4-277, was upregulated in common in all three datasets, whereas eight exons downstream of the TNR were downregulated in common in all three datasets in FECD with TNR expansion.ConclusionsThis study identified TCF4-277 as a dysregulated isoform in FECD with TNR expansion, suggesting a potential contribution of TCF4-277 to FECD pathophysiology.