Transforming growth factor βeta induced protein (TGFBIp) is a 68kDa secreted, extracellular matrix (ECM) protein, involved in corneal wound healing (CWH). Mutation of TGFBI is associated with a number of corneal dystrophies. TGFBI-R124H (rs121909211) is a hotspot mutation associated with granular type 2 corneal dystrophy (GCD2) an autosomal dominant-negative, symmetrical, non-inflammatory disease, restricted to the cornea and characterized by snowflake-like amyloid deposits. These appear by the 5th decade of life in humans leading ultimately to corneal blindness. Currently, the rate of corneal dystrophy ranges from high to low prevalence depending on country. As yet, the only effective therapies are surgical, however, the trauma caused leads to recurrent and exacerbated disease symptoms. A transgenic mouse model of GCD2 was generated by inserting a human TGFBI-R124H cDNA sequence into the exon 1 of the Tgfbi genomic sequence. In this investigation, allele-specific gene expression techniques such as RT-qPCR were used to assess the expression dynamics of TGFBI in corneal wound healing following a corneal debridement procedure on transgenic mouse corneas. A histology staining panel was used to characterize amyloid disease. Additional in silico experiments investigated differences within mutant TGFBI-R124H biology in comparison to wild-type. siRNA and CRISPR/Cas9 tools are developed in this work as a way to treat mutant expression induction following injury exacerbations. The injury model recorded a 4x rate of onset increase and a 4x penetrance percentage representation increase in comparison to the non-injury model in heterozygous samples and 2x percentage representation increase in homozygous samples. The corneal wound healing response expression of TGFBI increased 3x baseline (day 0) by 48 hours after injury. Suggesting a role within stromal wound healing repair, other cytokines such as Tgfb1 showed a peak expression by 24 hours post injury suggesting an epithelial wound repair role. Histology confirms the presence of amyloid corneal deposits in the anterior stroma as well as fibrosis. Additionally, results are presented which validate and establish genome altering tool systems such as siRNA and CRISPR/Cas9 for use as a TGFBI-R124H silencing agent.
An investigation into the role of TGFBI in corneal wound healing and homestatis
Donaghy, J. (Author). Nov 2023
Student thesis: Doctoral Thesis