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

Replication of TCF4 through Association and Linkage Studies in Late-Onset Fuchs Endothelial Corneal Dystrophy

Fuchs endothelial corneal dystrophy (FECD) is a common, late-onset disorder of the corneal endothelium. Although progress has been made in understanding the genetic basis of FECD by studying large families in which the phenotype is transmitted in an autosomal dominant fashion, a recently reported genome-wide association study identified common alleles at a locus on chromosome 18 near TCF4 which confer susceptibility to FECD. Here, we report the findings of our independent validation study for TCF4 using the largest FECD dataset to date (450 FECD cases and 340 normal controls). Logistic regression with sex as a covariate was performed for three genetic models: dominant (DOM), additive (ADD), and recessive (REC). We found significant association with rs613872, the target marker reported by Baratz et al.(2010), for all three genetic models (DOM: P = 9.33×10−35; ADD: P = 7.48×10−30; REC: P = 5.27×10−6). To strengthen the association study, we also conducted a genome-wide linkage scan on 64 multiplex families, composed primarily of affected sibling pairs (ASPs), using both parametric and non-parametric two-point and multipoint analyses. The most significant linkage region localizes to chromosome 18 from 69.94cM to 85.29cM, with a peak multipoint HLOD = 2.5 at rs1145315 (75.58cM) under the DOM model, mapping 1.5 Mb proximal to rs613872. In summary, our study presents evidence to support the role of the intronic TCF4 single nucleotide polymorphism rs613872 in late-onset FECD through both association and linkage studies

A Noncoding Point Mutation of Zeb1 Causes Multiple Developmental Malformations and Obesity in Twirler Mice

Heterozygous Twirler (Tw) mice develop obesity and circling behavior associated with malformations of the inner ear, whereas homozygous Tw mice have cleft palate and die shortly after birth. Zeb1 is a zinc finger protein that contributes to mesenchymal cell fate by repression of genes whose expression defines epithelial cell identity. This developmental pathway is disrupted in inner ears of Tw/Tw mice. The purpose of our study was to comprehensively characterize the Twirler phenotype and to identify the causative mutation. The Tw/+ inner ear phenotype includes irregularities of the semicircular canals, abnormal utricular otoconia, a shortened cochlear duct, and hearing loss, whereas Tw/Tw ears are severely malformed with barely recognizable anatomy. Tw/+ mice have obesity associated with insulin-resistance and have lymphoid organ hypoplasia. We identified a noncoding nucleotide substitution, c.58+181G>A, in the first intron of the Tw allele of Zeb1 (Zeb1Tw). A knockin mouse model of c.58+181G>A recapitulated the Tw phenotype, whereas a wild-type knockin control did not, confirming the mutation as pathogenic. c.58+181G>A does not affect splicing but disrupts a predicted site for Myb protein binding, which we confirmed in vitro. In comparison, homozygosity for a targeted deletion of exon 1 of mouse Zeb1, Zeb1ΔEx1, is associated with a subtle abnormality of the lateral semicircular canal that is different than those in Tw mice. Expression analyses of E13.5 Twirler and Zeb1ΔEx1 ears confirm that Zeb1ΔEx1 is a null allele, whereas Zeb1Tw RNA is expressed at increased levels in comparison to wild-type Zeb1. We conclude that a noncoding point mutation of Zeb1 acts via a gain-of-function to disrupt regulation of Zeb1Tw expression, epithelial-mesenchymal cell fate or interactions, and structural development of the inner ear in Twirler mice. This is a novel mechanism underlying disorders of hearing or balance

Genetic characterization of posterior polymorphous corneal dystrophy.

The purpose of this dissertation is to examine the genetic origins of posterior polymorphous corneal dystrophy (PPCD). The proband of a large PPCD family, UM:139, was examined clinically and histopathologically to confirm the diagnosis of PPCD. In addition to the proband, 12 of the 28 individuals sampled in this family were found to have PPCD. We used linkage analysis with an autosomal dominant model to exclude (lod < -2.0) the PPCD1 locus on 20q11, the congenital hereditary endothelial dystrophy (CHED1) locus on 20q11, the loci containing the type IV collagen genes associated with Alport Syndrome (which includes PPCD as a sequela), and the locus containing the collagen type VIII alpha-2 gene. We also used linkage analysis with an autosomal recessive model to exclude the CHED2 locus on 20p13. Linkage analysis of whole-genome scan data identified a previously unknown PPCD locus (PPCD3) on chromosome 10p11 in the 8.55 cM region between D10S213 and D10S578 with a maximum lod score at D10S1780 of 4.35. Informatic examination of the more than 25 genes known to lie within this region revealed a homeodomain transcription factor, TCF8. Sequencing of TCF8 in UM:139 revealed a cosegregating frame-shift mutation. Sequencing of DNA from 10 additional PPCD probands revealed two additional frame-shift and two nonsense mutations. Studies of total RNA isolated from human and mouse tissue confirm the expression of TCF8 in cornea and other bodily tissues. These studies, along with the mutation screening, support the conclusion that variants in TCF8 cause PPCD. We propose two models for the role of TCF8 variants in the PPCD phenotype including ectopic expression of some epithelial characteristics not typical of corneal endothelial cells. The first model supposes a reduced ability to repress the epithelial phenotype and the second model supposes a reduced ability to regulate the expression of corneal collagens. The identification of TCF8 as the PPCD3 gene has implications for the further investigation of PPCD, for the study of similar endothelial cells elsewhere in the body, and for the understanding of the regulation of corneal endothelial cellular morphology and gene expression.Ph.D.Biological SciencesGeneticsHealth and Environmental SciencesPublic healthUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/124850/2/3163853.pd

Ultrasonic Inspection of Thin Walled Composite Tubes

Fiber reinforced composite materials are being used in an increasingly wide range of structural applications. However, many types of composite laminates are extremely susceptible to impact damage [1,2]. Relatively low energy impacts may induce significant internal damage, generally in the form of matrix cracks and delaminations, which are not accompanied by visual evidence [3–5]. This damage has been shown to significantly reduce the strength, stiffness and fatigue life of compression loaded, flat composite laminates [1,2,4–7], as well as the strength of thin walled composite tubes [8]. Residual strength and life are closely related to the nature and extent of damage [6,7]; thus, the capability to perform detailed nondestructive evaluation of damage represents a critical component in the damage tolerant design of composite structures.</p

Posterior polymorphous corneal dystrophy is associated with TCF8 gene mutations and abdominal hernia How to cite this article: Aldave AJ, Yellore VS, Yu F, Bourla N, Sonmez B, Salem AK, Rayner SA, Sampat KM, Krafchak CM, Richards JE. 2007. Posterior polymorphous corneal dystrophy is associated with TCF8 gene mutations and abdominal hernia. Am J Med Genet Part A 143A:2549–2556.

Mutations in the two-handed zinc-finger homeodomain transcription factor gene ( TCF8 ) have been associated with posterior polymorphous corneal dystrophy (PPCD) and extraocular developmental abnormalities. We performed screening of TCF8 in 32 affected, unrelated probands, affected and unaffected family members of probands identified with a TCF8 mutation, and in 100 control individuals. Eight different pathogenic mutations were identified in eight probands: four frameshift (c.953_954insA, c.1506dupA, c.1592delA, and c.3012_3013delAG); three nonsense (Gln12X, Gln214X, Arg325X); and one missense (Met1Arg). Screening of TCF8 in affected and unaffected family members in six families demonstrated that each identified mutation segregated with the disease phenotype in each family; two probands did not have additional family members available for analysis. None of the eight TCF8 mutations was identified in 200 control chromosomes. The prevalence of hernias of the abdominal region in affected individuals with PPCD associated with TCF8 mutations was significantly higher than the prevalence in both individuals with PPCD not associated with a TCF8 mutation and in unaffected individuals. Therefore, PPCD is associated with TCF8 mutations in one quarter of affected families in this study, or about one third of all PPCD families that have been screened thus far. In these families, the presence of apparently causative TCF8 mutations is associated with abdominal and inguinal hernias. © 2007 Wiley-Liss, Inc.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/57405/1/31978_ftp.pd