Date of Degree
PhD (Doctor of Philosophy)
Markus H. Kuehn
Primary angle closure glaucoma (PACG) is a chronic optic neuropathy that results in retinal ganglion cell (RGC) degeneration, cupping of the optic nerve head (ONH) and subsequent loss of vision. In humans, PACG occurs as a result of a plateau iris or more commonly, a pupillary block. Increased Intraocular pressure and reduced axial length are some of the predisposing factors to PACG. The condition occurs in several breeds of dogs and the prognosis for affected animals is typically poor. Unlike PACG in humans, the mechanism of PACG in canines involves the gradual collapse of the ciliary cleft with or without complete collapse of the irido-corneal angle. We have identified and examined several Basset Hound (BH) pedigrees with clinically confirmed PACG that segregates in an autosomal recessive manner. The goal of this proposal is to utilize the Basset Hound PACG model in order to characterize the genetics of PACG. In addition to investigating the underlying genetic mechanisms of PACG, a series of functional studies aimed at improving our understanding of the pathophysiology of PACG, were also performed to investigate the disease.
Extensive clinical phenotyping of all pedigree members was conducted by a veterinary ophthalmologist. We performed a genome-wide logistic regression test for association using 37 PACG cases and 41 unaffected controls. Population stratification and cryptic relatedness were assessed using a multidimensional scaling analysis. The expression of two candidate genes within the target tissues of the Basset Hound eye was assessed by immunohistochemistry. SNP-chip genotyping was additionally conducted in 9 affected and 15 unaffected pedigree members. Two-point and multipoint linkage analyses of genome-wide SNP data were performed using Superlink-Online SNP-1.1. Targeted exome capture was performed using the Agilent SureSelect exon kit.
A primary fibroblast cell culture was established from the sclera of three PACG, two wild type and two obligatory carrier Bassets. Total RNA extracted from fibroblast cells was assayed using the Affymetrix GeneChip Canine Genome 2.0 Array. The Robust Multichip Average expression summary method was used for background adjustment and normalization. A two class, unpaired, Wilcoxon statistical test was conducted to identify differentially expressed genes. qRT-PCR was performed to validate significantly expressed genes. Furthermore, a primary fibroblast cell culture was established from the skin of a PACG and an obligatory carrier BH. Microscope images and cell counts from all cell cultures were established at various time points.
Our findings reveal significant associations at two novel loci: BICF2P31912 in COL1A2 on chromosome 14 with a per-allele odds ratio OR(95% CI) 8.95(1.73-6.51); Pgenome = 3.6 x 10-4 and BICF2P893476 residing in proximity to RAB22A on chromosome 24 with a per-allele odds ratio OR(95% CI) = 12.03(1.78-8.66); Pgenome = 4.9 x 10-4. COL1A2 and RAB22A demonstrated wide-spread localization throughout the eye and were prominently noted in the ciliary body, trabecular meshwork and iris. A 1.82Mbp locus was additionally mapped to chromosome 19 with a maximum LOD score of 3.24. The locus contains 12 Ensemble predicted canine genes and shares synteny to a region on chromosome 2 in the human genome. Using exome-sequencing analysis, a possibly damaging, nonsynonymous variant was found to segregate with PACG in the gene Nebulin (NEB) (g.55885214 A->G, p.2051 K->R), which alters a conserved Lysine. Nebulin, a protein that promotes the contractile function of sarcomeres was found to be prominently expressed in the ciliary muscles of the anterior segment. Primary scleral fibroblast cells derived from PACG animals were found to exhibit severely reduced growth rates when compared to wild type derived cells. Genes with sharply reduced expression levels are of particular interest due to the possible involvement of a loss of- function mutation in PACG. More than 600 genes were found to be significantly under expressed in PACG derived cells. In contrast to unaffected-derived cell, PACG derived cells display significantly altered gene expression patterns for a number of possibly important candidate genes. Furthermore, PACG derived cells display aberrant and reduced motility in PACG versus wild type derived cell cultures.
The identification of two genetic associations supports the potential segregation of PACG risk-conferring variants in the Basset Hound. The genetic associations identified may contribute to mechanisms underlying the pathogenesis of PACG, which remain to be elucidated. Moreover, our studies provide the first evidence of a gene directly linked to PACG in the Basset Hound. Our findings may provide insight into the molecular mechanisms that underlie PACG. The phenotypic similarities of disease presentation in dogs and humans may enable the translation of findings made in this study to patients with PACG. The findings of our functional studies suggest that cellular dysfunction is an important aspect in the pathophysiology of PACG in the dog. The identification of genes with significantly altered expression levels may provide insight into the molecular pathways associated with the development of the disease and aid in the identification of the genetic defect underlying PACG.
Canine, Gene mapping, Genetics, Glaucoma, Human Genetics
xii, 197 pages
Includes bibliographical references (pages 165-197).
Copyright 2014 Dina Ahram