The PPCD1 Mouse: Characterization of a Mouse Model for Posterior Polymorphous Corneal Dystrophy and Identification of a Candidate Gene

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 Csrp2bptm1a(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

Morphological and immunocytochemical characterization of snake-like chromatin cells

Snake-like chromatin (SLC) is a nuclear alteration occurring under various pathological conditions and in different tissues. The aim of this study was the morphological and immunocytochemical characterization of SLC-positive conjunctival epithelial cells from keratoconjunctivitis sicca (KCS) patients. Impression cytology specimens from the upper bulbar conjunctiva of 10 controls and 10 KCS patients with a high incidence of SLC cells were assessed, the morphology of SLC nuclei evaluated by light microscopy, and proliferation markers, nucleolar proteins, lamins and cytokeratin filaments detected immunocytochemically. In KCS patients, SLC cells with a normal nuclear shape, with nuclear membrane notching (2.3% of cells) and with binuclear dumb-bell structures (4.4% of cells) were observed. The most striking features of SLC cells were the absence of an A/C lamin signal, the redistribution of fibrillarin into two spots adjacent to SLC structures and cytokeratin 14 positivity in the strangulation belt of the dumb-bell structures. The deficiency of lamin A/C is the probable reason for the disintegration of chromatin from the nuclear lamina in SLC cells. The occurrence of SLC-positive cells, SLC-positive dumb-bell shaped nuclei and SLCpositive binucleated cells, together with the absence of mitotic markers, leads to the conclusion that the SLC phenomenon might be a form of nuclear segregation

Ex vivo cultivated oral mucosal epithelial cell transplantation for limbal stem cell deficiency: a review

Destruction or dysfunction of limbal epithelial stem cells (LESCs) leads to unilateral or bilateral limbal stem cell deficiency (LSCD). Fifteen years have passed since the first transplantation of ex vivo cultivated oral mucosal epithelial cells (COMET) in humans in 2004, which represents the first use of a cultured non-limbal autologous cell type to treat bilateral LSCD. This review summarizes clinical outcomes from COMET studies published from 2004 to 2019 and reviews results with emphasis on the culture methods by which grafted cell sheets were prepared

Supplementary Material for: Detailed Assessment of Renal Function in a Proband with Harboyan Syndrome Caused by a Novel Homozygous <b><i>SLC4A11 </i></b>Nonsense Mutation

<b><i>Background/Aims:</i></b> To identify the underlying molecular genetic cause of disease in a patient with Harboyan syndrome and to perform a detailed assessment of her renal function. We also assessed the influence of the <i>SLC4A11</i> mutation identified on the corneal endothelium in the heterozygous state. <b><i>Methods:</i></b> A 55-year-old female was examined ophthalmologically, audiologically and nephrologically including 24-hour urine collection. The coding region of <i>SLC4A11</i> was directly sequenced. Specular microscopy was performed in the proband's 21-year-old daughter. <b><i>Results:</i></b> The proband had bilateral iridectomy at the age of 3 months because of an initial diagnosis of congenital glaucoma and since the age of 12 years she underwent several keratoplasties in each eye. Nephrological examination did not reveal any abnormalities. Moderate bilateral sensorineural hearing loss was confirmed by audiometry. A novel homozygous mutation predicted to lead to a premature stop codon at the protein level, c.2188C>T; p.(Arg730*), was identified in <i>SLC4A11</i>. No changes in corneal endothelial cell morphology or density were observed in the heterozygous daughter. <b><i>Conclusion:</i></b> In contrast to the <i>Slc4a11</i>^-/- mouse, no abnormalities in daily renal ion excretion or polyuria were observed in the Harboyan syndrome patient. The mutation identified does not affect corneal endothelial cell morphology or density in the heterozygous state. © 2014 S. Karger AG, Base

Autosomal-dominant corneal endothelial dystrophies CHED1 and PPCD1 are allelic disorders caused by non-coding mutations in the promoter of OVOL2

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&gt;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&gt;G and c.-307T&gt;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