Molecular Biology and Genetics of Fuchs’ Endothelial Corneal Dystrophy

Author: Abraham Kuot

Kuot, Abraham, 2015 Molecular Biology and Genetics of Fuchs’ Endothelial Corneal Dystrophy, Flinders University, School of Medicine

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Fuchs’ endothelial corneal dystrophy (FECD) is a common disease of the corneal endothelium (CE) that is characterised by the formation of guttae, abnormal thickening of the Descemet’s membrane (DM), and gradual loss of corneal endothelial cells (CECs). These features lead to corneal oedema, progressive vision loss, and ultimately blindness, if left untreated. Corneal grafting is the only effective treatment for FECD. In Australia, ~6% of corneal grafts are performed in FECD patients annually.

Currently, the mechanism of FECD is poorly understood. It occurs as a rare familial early-onset or a common late-onset familial or sporadic disease. This project focused on the late-onset disease that shows strong genetic heterogeneity, much of which remains undetermined.

Mutations in the AGBL1, LOXHD1, SLC4A11, and ZEB1 genes contibute to the disease in Caucasian American and Asian populations. Four chromosomal loci are linked to familial disease but the underlying genes remain unknown. Strong genetic association between FECD and nucleotide variants in TCF4 has been identified in Caucasian American, Chinese and Indian cases. Molecular studies suggest involvement of oxidative stress, apoptosis, protein misfolding, corneal endothelial ion transporters loss, and accelerated senescence in the disease pathophysiology.

This project aimed to advance the understanding of the molecular basis of FECD. The specific aims were to identify genetic causes of FECD in Caucasian Australian cases, determine relative abundance of proteins between FECD-affected and unaffected DM, and identify dysregulated genes between FECD-affected and unaffected CE.

Mutation screening of LOXHD1, SLC4A11 and ZEB1 genes in 128 cases by Ion Torrent Next-Generation Sequencing identified novel mutations in LOXHD1 and SLC4A11 as the possible causes of FECD in three cases. Genotyping of 208 cases for TGC repeat expansion in TCF4 by short tandem repeat assay confirmed strong association with FECD.

Genome-wide association study (GWAS) using pooled DNA identified TCF4(transcription factor 4) as a major susceptibility gene for FECD in Australian cases. Genotyping of 190 cases for FECD associated potential novel genes by Sequenom MassArray Spectrometry replicated association of ATP1B1, KANK4 and LAMC1 genes with FECD. These genes were initially identified through GWAS performed in American cases by our collaborators in the USA. Quantitative reverse transcription-polymerase chain reaction (quantitative RT-PCR) indicated down-regulation of ATP1B1 in FECD-affected CE compared to normal CE, and immunohistochemistry showed differential distribution of the encoded protein in diseased cornea compared to normal cornea.

Comparison of relative abundance of proteins between affected and unaffected DM by label-free mass spectrometry revealed reduced abundance of APOE in FECD-affected DM. Immunohistochemistry validated APOE as a component of the DM, with differential distribution between affected and unaffected corneas. Analysis of expression of APOE mRNA levels between FECD-affected and unaffected CE by quantitative RT-PCR revealed down-regulation of the transcript in the disease, suggesting a correlation between reduced abundance of the protein and under-expression of the gene in FECD.

Investigation of dysregulated genes between FECD-affected and unaffected CE by microarray analysis revealed 135 differentially expressed genes. Of these, 123 genes were up-regulated in the disease. Validation of 12 prioritised genes by quantitative RT-PCR confirmed significant up-regulation of ALPK2, BGN, CLIC6, CPAMD8, CST1, CX3CR1, EDN1, HLA-DRA, NOX4 and PPP1R1B genes in FECD.

Ingenuity Pathway Analysis of all genes/proteins identified to be involved in FECD in this project and previous independent studies suggests involvement of activated transcription factor JUN (Jun Proto-Oncogene) and β-catenin/TCF4 complex, tumour suppressor p53, and immune response pathways in the mechanism of FECD.

Overall, this project revealed novel FECD-causing mutations in three cases, confirmed association of TCF4 with FECD in Australian cases, and identified several new genes involved in FECD. Physiologically, the novel genes are involved in protein synthesis (ALPK2 and KANK4) and assembly/structural integrity in the DM (BGN and LAMC1), electrochemical gradient homeostasis (ATP1B1 and CLIC6), premature senescence (CST1), and oxidant-antioxidant imbalance (APOE and NOX4) pathways, known to be involved in FECD. The CX3CR1, HLA-DRA and EDN1 genes are involved in immune response. Involvement of the immune response pathway in FECD is a novel finding.

Keywords: Fuchs' endothelial corneal dystrophy, FECD, Fuchs' dystrophy, Fuchs' endothelial dystrophy, Molecular Biology and Genetics of Fuchs' endothelial corneal dystrophy, Corneal dystrophy, Late-onset Fuchs' endothelial dystrophy

Subject: Medicine thesis

Thesis type: Doctor of Philosophy
Completed: 2015
School: School of Medicine
Supervisor: Dr. Shiwani Sharma