Ricardo F. Frausto

Genetics of the Corneal Endothelial Dystrophies: An Evidence-Based Review

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.

Classification of Posterior Polymorphous Corneal Dystrophy as a Corneal Ectatic Disorder Following Confirmation of Associated Significant Corneal Steepening

IMPORTANCE The identification of steep corneal curvatures in a significant percentage of patients with posterior polymorphous corneal dystrophy (PPCD) confirms this previously reported association and suggests a role for the ZEB1 protein in keratocyte function. OBJECTIVE To determine whether PPCD is characterized by significant corneal steepening. DESIGN, SETTING, AND PARTICIPANTS Cross-sectional study at university-based and private ophthalmology practices of 38 individuals (27 affected and 11 unaffected) from 23 families with PPCD. EXPOSURE Slitlamp examination and corneal topographic imaging were performed for individuals with PPCD and unaffected family members. Saliva or blood samples were obtained from each individual for DNA isolation and ZEB1 sequencing. Corneal ZEB1 expression was measured using immunohistochemistry. MAIN OUTCOMES AND MEASURES Percentage of individuals affected with PPCD and controls with an average keratometric value greater than 48.0 diopters (D) in each eye; the mean keratometric value averaged for both eyes of individuals with PPCD and controls; and the correlation of ZEB1 mutation with keratometric value. RESULTS ZEB1 coding region mutations were identified in 7 of the 27 affected individuals. Ten of the 38 individuals (26.3%) had average keratometric values greater than 48.0 D OU: 10 of 27 individuals with PPCD (37.0%; 6 of 7 individuals with ZEB1 mutations [85.7%] and 4 of 20 individuals without ZEB1 mutations [20.0%]) and 0 of 11 unaffected individuals (P = .04 for unaffected vs affected individuals; P = .004 for individuals with PPCD with vs without ZEB1 mutation). The mean keratometric value of each eye of affected individuals (48.2 D) was significantly greater than that of each eye of unaffected family members (44.1 D) (P = .03). Affected individuals with ZEB1 mutations demonstrated a mean keratometric value of 53.3 D, which was significantly greater than that of affected individuals without ZEB1 mutations (46.5 D; P = .004). Fluorescence immunohistochemistry demonstrated ZEB1 expression in keratocyte nuclei. CONCLUSIONS AND RELEVANCE Abnormally steep corneal curvatures are identified in 37% of all individuals with PPCD and 86% of affected individuals with PPCD secondary to ZEB1 mutations. ZEB1 is present in keratocyte nuclei, suggesting a role for ZEB1 in keratocyte function. Therefore, ZEB1 may play a role in both corneal stromal and endothelial development and function, and PPCD should be considered both an endothelial dystrophy and an ectatic disorder.

Posterior Amorphous Corneal Dystrophy Is Associated with a Deletion of Small Leucine-rich Proteoglycans on Chromosome 12

Posterior amorphous corneal dystrophy (PACD) is a rare, autosomal dominant disorder affecting the cornea and iris. Next-generation sequencing of the previously identified PACD linkage interval on chromosome 12q21.33 failed to yield a pathogenic mutation. However, array-based copy number analysis and qPCR were used to detect a hemizygous deletion in the PACD linkage interval containing 4 genes encoding small leucine-rich proteoglycans (SLRPs): KERA, LUM, DCN, and EPYC. Two other unrelated families with PACD also demonstrated deletion of these SLRPs, which play important roles in collagen fibrillogenesis and matrix assembly. Given that these genes are essential to the maintenance of corneal clarity and the observation that knockout murine models display corneal phenotypic similarities to PACD, we provide convincing evidence that PACD is associated with haploinsufficiency of these SLRPs.

Transcriptomic Analysis of Cultured Corneal Endothelial Cells as a Validation for Their Use in Cell Replacement Therapy.

The corneal endothelium plays a primary role in maintaining corneal homeostasis and clarity and must be surgically replaced with allogenic donor corneal endothelium in the event of visually significant dysfunction. However, a worldwide shortage of donor corneal tissue has led to a search for alternative sources of transplantable tissue. Cultured human corneal endothelial cells (HCEnC) have been shown to restore corneal clarity in experimental models of corneal endothelial dysfunction in animal models, but characterization of cultured HCEnC remains incomplete. To this end, we utilized next-generation RNA sequencing technology to compare the transcriptomic profile of ex vivo human corneal endothelial cells (evHCEnC) with that of primary HCEnC (pHCEnC) and HCEnC lines and to determine the utility of cultured and immortalized corneal endothelial cells as models of in vivo corneal endothelium. Multidimensional analyses of the transcriptome data sets demonstrated that primary HCEnC have a closer relationship to evHCEnC than do immortalized HCEnC. Subsequent analyses showed that the majority of the genes specifically expressed in HCEnC (not expressed in ex vivo corneal epithelium or fibroblasts) demonstrated a marked variability of expression in cultured cells compared with evHCEnC. In addition, genes associated with either corneal endothelial cell function or corneal endothelial dystrophies were investigated. Significant differences in gene expression and protein levels were observed in the cultured cells compared with evHCEnC for each of the genes tested except for AGBL1 and LOXHD1, which were not detected by RNA-seq or qPCR. Our transcriptomic analysis suggests that at a molecular level pHCEnC most closely resemble evHCEnC and thus represent the most viable cell culture-based therapeutic option for managing corneal endothelial cell dysfunction. Our findings also suggest that investigators should perform an assessment of the entire transcriptome of cultured HCEnC prior to determination of their potential clinical utility for the management of corneal endothelial cell failure.

Pre-Descemet corneal dystrophy and X-linked ichthyosis associated with deletion of Xp22.31 containing the STS gene.

Purpose: To report the association of X-linked ichthyosis and pre-Descemet corneal dystrophy with a deletion of the steroid sulfatase gene (STS) detected with microarray-based comparative genomic hybridization (aCGH). Methods: A slit-lamp biomicroscopic examination and cutaneous examination were performed, after which a saliva sample was collected as a source of genomic DNA. Polymerase chain reaction amplification of each of the 10 exons of STS was performed, as was aCGH on genomic DNA to detect copy number variation. Results: The slit-lamp examination revealed punctate opacities in the posterior corneal stroma of each eye. The cutaneous examination demonstrated scaling and flaking skin of the arms and legs. Polymerase chain reaction amplification using primers designed to amplify each of the 10 exons of STS failed to produce any amplicons. Subsequently, aCGH performed on genomic DNA revealed a microdeletion in the Xp22.31 cytoband of approximately 1.7 megabases, containing STS. Conclusions: The identification of a microdeletion within Xp22.3 containing STS with aCGH in an individual with suspected pre-Descemet corneal dystrophy and X-linked ichthyosis demonstrates the clinical utility of copy number variation analysis in confirming a presumptive clinical diagnosis.

Posterior polymorphous corneal dystrophy 3 is associated with agenesis and hypoplasia of the corpus callosum

Posterior polymorphous corneal dystrophy (PPCD) is a dominantly inherited disorder of the corneal endothelium that has been associated with mutations in the zinc-finger E-box binding homeobox 1 gene (ZEB1) gene in approximately one-third of affected families. While the corneal dystrophies have traditionally been considered isolated disorders of the corneal endothelium, we have recently identified two cases of maldevelopment of the corpus callosum in unrelated individuals with PPCD. The proband of the first family was diagnosed shortly after birth with agenesis of the corpus callosum and several other developmental abnormalities. Karyotype, FISH and whole genome copy number variant analyses were normal. She was subsequently diagnosed with PPCD, prompting screening of the ZEB1 gene, which identified a novel deletion (c.449delG; p.(Gly150Alafs*36)) present in the heterozygous state that was not identified in either unaffected parent. The proband of the second family was diagnosed several months after birth with thinning of the corpus callosum and PPCD. Whole genome copy number variant analysis revealed a 1.79 Mb duplication of 17q12 in the proband and her father and brother, neither of whom had PPCD. ZEB1 sequencing identified a novel deletion (c.1913-1914delCA; p.(Ser638Cysfs*5)) present in the heterozygous state, which was also identified in the probands affected mother. Thus, we report the first two cases of the association of PPCD with a developmental abnormality of the brain, in this case maldevelopment of the corpus callosum.

Transcriptome analysis of the human corneal endothelium.

PURPOSE To comprehensively characterize human corneal endothelial cell (HCEnC) gene expression and age-dependent differential gene expression and to identify expressed genes mapped to chromosomal loci associated with the corneal endothelial dystrophies posterior polymorphous corneal dystrophy (PPCD)1, Fuchs endothelial corneal dystrophy (FECD)4, and X-linked endothelial dystrophy (XECD). METHODS Total RNA was isolated from ex vivo corneal endothelium obtained from six pediatric and five adult donor corneas. Complementary DNA was hybridized to the Affymetrix GeneChip 1.1ST array. Data analysis was performed using Partek Genomics Suite software, and differentially expressed genes were validated by digital molecular barcoding technology. RESULTS Transcripts corresponding to 12,596 genes were identified in HCEnC. Nine genes displayed the most significant differential expression between pediatric and adult HCEnC: CAPN6, HIST1H3A, HIST1H4E, and HSPA2 were expressed at higher levels in pediatric HCEnC, while ITGBL1, NALCN, PREX2, TAC1, and TMOD1 were expressed at higher levels in adult HCEnC. Analysis of the PPCD1, FECD4 and XECD loci demonstrated transcription of 53/95 protein-coding genes in the PPCD1 locus, 27/40 in the FECD4 locus, and 35/68 in the XECD locus. CONCLUSIONS An analysis of the HCEnC transcriptome reveals the expression of almost 13,000 genes, with less than 1% mapped to chromosomal loci associated with PPCD1, FECD4, and XECD. At least nine genes demonstrated significant differential expression between pediatric and adult HCEnC, defining specific functional properties distinct to each age group. These data will serve as a resource for vision scientists investigating HCEnC gene expression and can be used to focus the search for the genetic basis of the corneal endothelial dystrophies for which the genetic basis remains unknown.

Identification of Potentially Pathogenic Variants in the Posterior Polymorphous Corneal Dystrophy 1 Locus.

Posterior polymorphous corneal dystrophy 1 (PPCD1) is a genetic disorder that affects corneal endothelial cell function and leads to loss of visual acuity. PPCD1 has been linked to a locus on chromosome 20 in multiple families; however, Sanger sequencing of protein-coding genes in the consensus region failed to identify any causative missense mutations. In this study, custom capture probes were utilized for targeted next-generation sequencing of the linked region in a previously reported family with PPCD1. Variants were detected through two bioinformatics pipelines and filtered according to multiple criteria. Additionally, a high-resolution microarray was used to detect copy number variations. No non-synonymous variants in the protein-coding region of annotated genes were identified. However, 12 single nucleotide variants in 10 genes, and 9 indels in 7 genes met the filtering criteria and were considered candidate variants for PPCD1. Eleven single nucleotide variants were confirmed by Sanger sequencing, including 2 synonymous variants and 9 non-coding variants, in 9 genes. One microdeletion was detected in an intron of OVOL2 by microarray but was subsequently not identified by PCR. Using a comprehensive next-generation sequencing approach, a total of 16 genes containing single nucleotide variants or indels that segregated with the affected phenotype in an affected family previously mapped to the PPCD1 locus were identified. Screening of these candidate genes in other families previously mapped to the PPCD1 locus will likely result in the identification of the genetic basis of PPCD1.

Confirmation of the OVOL2 Promoter Mutation c.-307T>C in Posterior Polymorphous Corneal Dystrophy 1

Purpose To identify the genetic basis of posterior polymorphous corneal dystrophy (PPCD) in families mapped to the PPCD1 locus and in affected individuals without ZEB1 coding region mutations. Methods The promoter, 5’ UTR, and coding regions of OVOL2 was screened in the PPCD family in which linkage analysis established the PPCD1 locus and in 26 PPCD probands who did not harbor a ZEB1 mutation. Copy number variation (CNV) analysis in the PPCD1 and PPCD3 intervals was performed on DNA samples from eight probands using aCGH. Luciferase reporter assays were performed in human corneal endothelial cells to determine the impact of the identified potentially pathogenic variants on OVOL2 promoter activity. Results OVOL2 mutation analysis in the first PPCD1-linked family demonstrated segregation of the c.-307T>C variant with the affected phenotype. In the other 26 probands screened, one heterozygous coding region variant and five promoter region heterozygous variants were identified, though none are likely pathogenic based on allele frequency. Array CGH in the PPCD1 and PPCD3 loci excluded the presence of CNV involving either OVOL2 or ZEB1, respectively. The c.-307T>C variant demonstrated increased promoter activity in corneal endothelial cells when compared to the wild-type sequence as has been demonstrated previously in another cell type. Conclusions Previously identified as the cause of PPCD1, the OVOL2 promoter variant c.-307T>C was herein identified in the original family that established the PPCD1 locus. However, the failure to identify presumed pathogenic coding or non-coding OVOL2 or ZEB1 variants, or CNV involving the PPCD1 and PPCD3 loci in 26 other PPCD probands suggests that other genetic loci may be involved in the pathogenesis of PPCD.

Transcriptomic Profiling of Posterior Polymorphous Corneal Dystrophy.

Purpose To investigate the molecular basis of posterior polymorphous corneal dystrophy (PPCD) by examining the PPCD transcriptome and the effect of decreased ZEB1 expression on corneal endothelial cell (CEnC) gene expression. Methods Next-generation RNA sequencing (RNA-seq) analyses of corneal endothelium from two PPCD-affected individuals (one with PPCD3 and one of unknown genetic cause) compared with two age-matched controls, and primary human CEnC (pHCEnC) transfected with siRNA-mediated ZEB1 knockdown. The expression of selected differentially expressed genes was validated by quantitative polymerase chain reaction (qPCR) and/or assessed by in situ hybridization in the corneal endothelium of four independent cases of PPCD (one with PPCD3 and three of unknown genetic cause). Results Expression of 16% and 46% of the 104 protein-coding genes specific to ex vivo corneal endothelium was lost in the endothelium of two individuals with PPCD. Thirty-two genes associated with ZEB1 and 3 genes (BMP4, CCND1, ZEB1) associated with OVOL2 were differentially expressed in the same direction in both individuals with PPCD. Immunohistochemistry staining and RNA-seq analyses demonstrated variable expression of type IV collagens in PPCD corneas. Decreasing ZEB1 expression in pHCEnC altered expression of 711 protein-coding genes, many of which are associated with canonical pathways regulating various cellular processes. Conclusions Identification of the altered transcriptome in PPCD and in a cell-based model of PPCD provided insight into the molecular alterations characterizing PPCD. Further study of the differentially expressed genes associated with ZEB1 and OVOL2 is expected to identify candidate genes for individuals with PPCD and without a ZEB1 or OVOL2 mutation.