Autosomal-Dominant Corneal Endothelial Dystrophies CHED1 and PPCD1 Are Allelic Disorders Caused by Non-coding Mutations in the Promoter of OVOL2.
ABSTRACT: Congenital hereditary endothelial dystrophy 1 (CHED1) and posterior polymorphous corneal dystrophy 1 (PPCD1) are autosomal-dominant corneal endothelial dystrophies that have been genetically mapped to overlapping loci on the short arm of chromosome 20. We combined genetic and genomic approaches to identify the cause of disease in extensive pedigrees comprising over 100 affected individuals. After exclusion of pathogenic coding, splice-site, and copy-number variations, a parallel approach using targeted and whole-genome sequencing facilitated the identification of pathogenic variants in a conserved region of the OVOL2 proximal promoter sequence in the index families (c.-339_361dup for CHED1 and c.-370T>C for PPCD1). Direct sequencing of the OVOL2 promoter in other unrelated affected individuals identified two additional mutations within the conserved proximal promoter sequence (c.-274T>G and c.-307T>C). OVOL2 encodes ovo-like zinc finger 2, a C2H2 zinc-finger transcription factor that regulates mesenchymal-to-epithelial transition and acts as a direct transcriptional repressor of the established PPCD-associated gene ZEB1. Interestingly, we did not detect OVOL2 expression in the normal corneal endothelium. Our in vitro data demonstrate that all four mutated OVOL2 promoters exhibited more transcriptional activity than the corresponding wild-type promoter, and we postulate that the mutations identified create cryptic cis-acting regulatory sequence binding sites that drive aberrant OVOL2 expression during endothelial cell development. Our data establish CHED1 and PPCD1 as allelic conditions and show that CHED1 represents the extreme of what can be considered a disease spectrum. They also implicate transcriptional dysregulation of OVOL2 as a common cause of dominantly inherited corneal endothelial dystrophies.
Project description:<h4>Purpose</h4>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.<h4>Methods</h4>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.<h4>Results</h4>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.<h4>Conclusions</h4>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.
Project description: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 and/or coding regions of OVOL2 were 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 screening 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 when compared to the wild-type sequence. Conclusions: The previously identified and presumed pathogenic OVOL2 promoter variant c.-307T>C was identified in the PPCD 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. Overall design: Eight PPCD probands without a ZEB1 protein-coding region pathogenic variant were analyzed for copy number variation within the PPCD1 (chromosome 20) and the PPCD3 (chromosome 10) loci.
Project description:In a large family of Czech origin, we mapped a locus for an autosomal-dominant corneal endothelial dystrophy, posterior polymorphous corneal dystrophy 4 (PPCD4), to 8q22.3-q24.12. Whole-genome sequencing identified a unique variant (c.20+544G>T) in this locus, within an intronic regulatory region of GRHL2. Targeted sequencing identified the same variant in three additional previously unsolved PPCD-affected families, including a de novo occurrence that suggests this is a recurrent mutation. Two further unique variants were identified in intron 1 of GRHL2 (c.20+257delT and c.20+133delA) in unrelated PPCD-affected families. GRHL2 is a transcription factor that suppresses epithelial-to-mesenchymal transition (EMT) and is a direct transcriptional repressor of ZEB1. ZEB1 mutations leading to haploinsufficiency cause PPCD3. We previously identified promoter mutations in OVOL2, a gene not normally expressed in the corneal endothelium, as the cause of PPCD1. OVOL2 drives mesenchymal-to-epithelial transition (MET) by directly inhibiting EMT-inducing transcription factors, such as ZEB1. Here, we demonstrate that the GRHL2 regulatory variants identified in PPCD4-affected individuals induce increased transcriptional activity in vitro. Furthermore, although GRHL2 is not expressed in corneal endothelial cells in control tissue, we detected GRHL2 in the corneal "endothelium" in PPCD4 tissue. These cells were also positive for epithelial markers E-Cadherin and Cytokeratin 7, indicating they have transitioned to an epithelial-like cell type. We suggest that mutations inducing MET within the corneal endothelium are a convergent pathogenic mechanism leading to dysfunction of the endothelial barrier and disease.
Project description:We have previously described a mouse model of human posterior polymorphous corneal dystrophy (PPCD) and localized the causative mutation to a 6.2 Mbp region of chromosome 2, termed Ppcd1. We now show that the gene rearrangement linked to mouse Ppcd1 is a 3.9 Mbp chromosomal inversion flanked by 81 Kbp and 542 bp deletions. This recombination event leads to deletion of Csrp2bp Exons 8 through 11, Dzank1 Exons 20 and 21, and the pseudogene Znf133. In addition, we identified translocation of novel downstream sequences to positions adjacent to Csrp2bp Exon 7 and Dzank1 Exon 20. Twelve novel fusion transcripts involving Csrp2bp or Dzank1 linked to downstream sequences have been identified. Eight are expressed at detectable levels in PPCD1 but not wildtype eyes. Upregulation of two Csrp2bp fusion transcripts, as well as upregulation of the adjacent gene, Ovol2, was observed. Absence of the PPCD1 phenotype in animals haploinsufficient for Csrp2bp or both Csrp2bp and Dzank1 rules out haploinsufficiency of these genes as a cause of mouse PPCD1. Complementation experiments confirm that PPCD1 embryonic lethality is due to disruption of Csrp2bp expression. The ocular expression pattern of Csrp2bp is consistent with a role for this protein in corneal development and pathogenesis of PPCD1.
Project description: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.
Project description:To investigate the molecular basis of posterior polymorphous corneal dystrophy (PPCD) by examining the transcriptome in an affected individuals with an OVOL2 promoter mutation c.307T>C (PPCD1). Overall design: Cornea endothelial tissue from a patient (P7), age 67, with PPCD were obtained when the patient underwent keratoplasty . The Descemet membrane and endothelium were placed onto a piece of filter paper and submerged in TRI Reagent (Thermo Fisher, Waltham, MA) for RNA isolation. Human cornea tissue from an age-matched donor (C7), age 63, was obtained from an commercial eye bank. Descemet’s membrane and corneal endothelium were stripped from the donor cornea using a published technique and placed into TRI Reagent for RNA isolation.
Project description:Posterior polymorphous corneal dystrophy (PPCD) is an autosomal-dominant disorder of the corneal endothelium associated with visually significant corneal edema and glaucoma. Statistical genetic analysis of 4 families with PPCD has demonstrated linkage to a 2.4 cM common support interval on chromosome 20 bordered by the markers D20S182 and D20S139. We sought to identify the genetic basis of PPCD linked to chromosome 20 (PPCD1) by screening the 26 positional candidate genes between these markers in a family previously mapped to the PPCD1 region.The coding regions of the 26 positional candidate genes mapped to the common PPCD1 support interval were amplified and sequenced in affected and unaffected individuals from a family previously linked to the PPCD1 locus. Nine other genes positioned just outside of the common PPCD1 support interval but within the autosomal-dominant congenital hereditary endothelial dystrophy interval were also screened.Four DNA sequence variants in 3 of the positional candidate genes demonstrated complete segregation with the affected phenotype: p.Thr109Thr (rs6111803) in OVOL2, p.Arg56Gln (novel variant-RPSnovel) in RPS19P1, and p.Thr85Thr (rs1053834) and p.Pro99Ser (rs1053839) in C20orf79. Each of these 4 sequence variants demonstrated significant linkage with the affected phenotype in this family (P = 2.5 x 10 for RPSnovel, rs1053834 and rs1053839; P = 8.6 x 10 for rs6111803). However, we also identified each of these 4 sequence variants in > or = affected control individuals. The haplotype on which the disease-causing mutation is segregating was found to have a population frequency of 4.2% in the CEPH HapMap trios. Although a number of other previously described and novel single nucleotide polymorphisms were identified in the 35 positional candidate genes located within the PPCD1 and congenital hereditary endothelial dystrophy intervals, none segregated with the affected phenotype.We report the absence of a presumed pathogenic coding region mutation in the common PPCD1 support interval. Although minor alleles of 4 single nucleotide polymorphisms were identified that segregated with the affected phenotype, the relatively high frequency of each minor allele in the general population indicates that none is a candidate for the causal variant for PPCD. Instead, the causal variant is most likely a coding region deletion or a variant in a noncoding region of the PPCD1 common support interval.
Project description: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.
Project description:Name of the disease (synonyms) CUGC for posterior polymorphous corneal dystrophy (PPCD).OMIM# of the disease 122000; 609141; 618031.Name of the analysed genes or DNA/chromosome segments OVOL2 (PPCD1); ZEB1 (PPCD3); GRHL2 (PPCD4).OMIM# of the gene(s) 616441; 189909; 608576. Review of the analytical and clinical validity as well as of the clinical utility of DNA-based testing for variants in theOVOL2, ZEB1andGRHL2gene(s) in a diagnostic setting, predictive and parental settings and for risk assesment in relatives.
Project description:The PPCD1 mouse, a spontaneous mutant that arose in our mouse colony, is characterized by an enlarged anterior chamber resulting from metaplasia of the corneal endothelium and blockage of the iridocorneal angle by epithelialized corneal endothelial cells. The presence of stratified multilayered corneal endothelial cells with abnormal patterns of cytokeratin expression are remarkably similar to those observed in human posterior polymorphous corneal dystrophy (PPCD) and the sporadic condition, iridocorneal endothelial syndrome. Affected eyes exhibit epithelialized corneal endothelial cells, with inappropriate cytokeratin expression and proliferation over the iridocorneal angle and posterior cornea. We have termed this the "mouse PPCD1" phenotype and mapped the mouse locus for this phenotype, designated "Ppcd1", to a 6.1 Mbp interval on Chromosome 2, which is syntenic to the human Chromosome 20 PPCD1 interval. Inheritance of the mouse PPCD1 phenotype is autosomal dominant, with complete penetrance on the sensitive DBA/2J background and decreased penetrance on the C57BL/6J background. Comparative genome hybridization has identified a hemizygous 78 Kbp duplication in the mapped interval. The endpoints of the duplication are located in positions that disrupt the genes Csrp2bp and 6330439K17Rik and lead to duplication of the pseudogene LOC100043552. Quantitative reverse transcriptase-PCR indicates that expression levels of Csrp2bp and 6330439K17Rik are decreased in eyes of PPCD1 mice. Based on the observations of decreased gene expression levels, association with ZEB1-related pathways, and the report of corneal opacities in Csrp2bp(tm1a(KOMP)Wtsi) heterozygotes and embryonic lethality in nulls, we postulate that duplication of the 78 Kbp segment leading to haploinsufficiency of Csrp2bp is responsible for the mouse PPCD1 phenotype. Similarly, CSRP2BP haploinsufficiency may lead to human PPCD.