The PPCD1 mouse: characterization of a mouse model for posterior polymorphous corneal dystrophy and identification of a candidate gene

Abstract

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.

Figure 1. Phenotypic appearance of mouse PPCD1.

Enucleated eyes were fixed in 10% formalin in phosphate-buffered saline and photographed. A. Appearance of PPCD1 mouse, showing the enlarged anterior chamber. B. Comparison of normal (left) and affected (right) eyes. Corneal neovascularization can be observed on the surface of the affected cornea. The arrow indicates an anterior synechia. Age, 3 months. C. Affected eye, showing corneal haze and cell growth across the surface of the pupil (arrow). Age, 3 months. D. Affected eye, showing lens subluxation, corneal haze, exudate in the anterior chamber, and anterior synechia. Age, 5 months.

Figure 2. Histology of affected eyes.

Eyes were fixed in 10% formalin in phosphate-buffered saline, processed, and stained with hematoxylin and eosin as described in Materials and Methods. Genotypes are indicated at the upper left. D2, DBA/2J; AC, anterior chamber; c, cornea; ce, corneal epithelium; cn, corneal endothelium; Ir, iris; s, synechia. A. Normal eye, age 2.5 months. B. PPCD1 eye, age 2.5 months. C. Normal iridocorneal angle, age 3 months. D. PPCD1 iridocorneal angle, age 3 months. Arrowheads indicate multilayered, stratified endothelial cells. E. `Normal cornea, age 3 months. F. PPCD1 cornea showing multilayered, stratified endothelial cells on the posterior surface of the central cornea (arrowhead), age 3 months. G. F4/80 immunohistochemistry of normal cornea, age P19. H. F4/80 immunohistochemistry of PPCD1 cornea, age P19. F4/80-positive cells stain reddish-brown. I. Corneal neovascularization (asterisks), age 3 months. J. Anterior synechia, age 5 months.

Figure 3. Cytokeratin AE1/AE3 immunohistochemistry of affected eyes.

Positive cells are reddish-brown. Genotypes are indicated at the upper left. D2, DBA/2J; ce, corneal epithelium; cn, corneal endothelium; AC, anterior chamber; IR, iris. A. Iridocorneal angle of PPCD1 animal, age P19. Arrows indicate epithelialized endothelium. B. Iridocorneal angle of normal littermate, age P19, showing absence of immunoreactivity in the corneal endothelium and iridocorneal angle. C. Central cornea of 3-month old PPCD1 animal showing cytokeratin AE1/AE3 immunoreactivity of both corneal endothelium and epithelium. D. Central cornea of 3-month old normal littermate showing cytokeratin AE1/AE3 immunoreactivity of corneal epithelium only.

Figure 4. Development of the mouse PPCD1 phenotype.

The iridocorneal angle from normal eyes (A, C, and E) is compared with that of PPCD1 littermates (B, D, F, and G). Panel H shows the PPCD1 iridocorneal angle at P5. Genotypes are indicated at the upper left. D2, DBA/2J; AC, anterior chamber; c, cornea. A. Normal, P0. B. PPCD1, P0. C. Normal, P2. D. PPCD1, P2. E. Normal, P3. F. PPCD1, P3. G. PPCD1, P3, enlarged to show layering of corneal endothelial cells (arrow). H. PPCD1, P5.

Figure 5. Chromosomal localization of Ppcd1 locus.

Shaded boxes indicate the129 (PPCD1) allele. Open boxes indicate the DBA/2J (normal eye) allele. SSLP markers analyzed are indicated on the left. Numbers of animals of each haplotype are indicated at the bottom.

Figure 6. CGH Analysis.

Log₂ratio is shown in the lower panel and transcripts (green lines) in the top panel. Coordinates in Mbp are shown at the top. The log₂ratio values are indicated by blue (PPCD1 versus DBA/2J), red (PPCD1 versus R1 ES cells), or green (G1 versus H11) dots. The solid lines indicate segments identified by the Nimblegen CGH-segMNT algorithm. Arrows indicate the direction of transcription. A, Csrp2bp; B, LOC100043552; C, 6330439K17Rik.

Figure 7. qPCR of Csrp2bp and 6330439K17Rik.

Expression levels were determined using IDT Prime Time^TM 5′- nuclease assays. Values are mean +/− SD. Black shows wild-type values and gray, PPCD1 values. P values are 0.01 and 0.02, for Csrp2bp and 6330439K17Rik, respectively.

Figure 8. Comparison of mouse and human PPCD1 loci.

Mouse chromosome 2 is shown on the upper line and human chromosome 20 on the lower line. Chromosomal positions in Mbp are shown at the bottom, with the relative positions on human chromosome 2 shown in parentheses. Microsatellite markers used to define the mouse and human critical intervals are labelled. Bar A indicates the critical interval for mouse Ppcd1 (see below) and bars B and C indicate the PPCD1 critical intervals defined by Gwilliam et al. and Yellore et al., respectively. Mouse genes and markers are indicated by diamonds and human genes and markers are indicated by triangles. Lines connect orthologous genes.