Hafford Tear, Nathaniel; (2022) Unravelling Molecular Mechanisms Underlying Inherited Corneal Endothelial Disease. Doctoral thesis (Ph.D), UCL (University College London).

Preview

Text NHT_Thesis_29.09.22 2.pdf - Other Download (30MB) | Preview

Abstract

Fuchs endothelial corneal dystrophy (FECD) and posterior polymorphous corneal dystrophy (PPCD) are clinically distinct heritable conditions associated with corneal endothelial barrier dysfunction that ultimately result in loss of corneal clarity and subsequent visual impairment. FECD is a common age-related corneal dystrophy that, in up to 80% of patients, is associated with a trinucleotide repeat expansion (termed CTG18.1) within an intronic region of the transcription factor encoding gene TCF4. PPCD is a rare autosomal dominant corneal dystrophy attributed to mutations in three distinct transcription factor encoding genes, (OVOL2 [PPCD1], ZEB1 [PPCD3] and GRHL2 [PPCD4]) that are all established regulators of epithelial-mesenchymal transition (EMT), suggesting a shared mechanisms of dysregulation may underlie distinct genetic subtypes of this disease. In this thesis I present the use of established patient-derived corneal endothelial cell (CEC) culture techniques in combination with next generation sequencing (NGS) based technologies to probe the genetic aetiologies and transcriptomic signatures of dysregulation underlying these diseases. Specifically, a novel amplification-free approach was developed, utilised, and refined to enable the CTG18.1 repeat expansions to be interrogated at the nucleotide level within a FECD patient cohort. This approach revealed striking levels of repeat length instability and mosaicism are associated with CTG18.1 expansion, advancing our understating of FECD pathophysiology in addition to more broadly illustrating the power of this long-read non-amplification dependant sequencing methodology to study repetitive genomic regions. RNA-seq data was generated from PPCD patient- and control-derived CEC cultures to define mechanism of transcriptomic dysregulation underlying disease and advance our understanding of the pathophysiology of this genetically heterogenous disease. Bioinformatic interrogation of these data highlighted dysregulated expression of the PPCD-associated OVOL2/ZEB1/GRHL2 axis and EMT-associated genes, and ectopic expression of corneal progenitor epithelium cell-type markers within the PPCD1 and PPCD3 corneal endothelium. Furthermore, epithelial cell-type- specific gene isoforms were upregulated in PPCD1 and PPCD3 corneal endothelium including targets of the epithelial splicing regulator protein, ESRP1. Over-expression of ESRP1 was subsequently modelled in immortalised endothelial cell line (HCEC12). Consequently, an upregulation of ESRP1 target gene epithelial-cell-type specific isoforms and corneal progenitor epithelium markers was discovered, suggesting a major role of ESRP1 in PPCD pathogenicity. Finally, a refined cohort of genetically unresolved PPCD patients recruited at Moorfields Eye Hospital (MEH) and General University Hospital (GUH), Prague, was established to identify additional genetic causes of PPCD.

Download activity - last month

Export as JSONXMLCSV

Download activity - last 12 months

Export as JSONCSVXML

Downloads by country - last 12 months

Export as JSONCSVXML

Archive Staff Only

View Item