Braddock, Freddie Louis; (2025) Variants and Perturbed Pathways Associated with Keratoconus and a Novel Stromal Corneal Dystrophy. Doctoral thesis (Ph.D), UCL (University College London).

Text Freddie_Braddock_Thesis_Final_Corrected.pdf - Accepted Version Access restricted to UCL open access staff until 1 March 2027. Download (31MB)

Abstract

Keratoconus (KC) is a progressive corneal ectasia typically presenting in early adulthood, with a prevalence of 1.2-3.3%. It is a complex condition and both environmental and genetic factors contribute to KC risk. Family history is a major risk factor for KC, hypothesised to result from rare, highly penetrant variants. To investigate this theory, Whole Genome Sequence (WGS) data was generated for seventeen KC families (2 affected individuals). For each family a filtering strategy was adapted to identify candidate causative variant(s). Rare variants were assessed for potential pathogenicity, and genes were assessed for biological relevance and corneal expression. Candidate gene variants were identified for all 17 KC families, including variants in COL8A2, COL4A4, COL12A1, COL16A1, KRT4 and KRT79. In an analytically powerful family (KC family 62) two partially or fully segregating haplotypes encompassing GWAS-associated loci were identified (chr13q32.1-q34 and chr15q21.3-25). Corneal epithelial-like cells (CEpi) were differentiated from patient-derived induced pluripotent stem cells from one KC family 62 patient and one control line as a pilot functional study. Successful differentiation was demonstrated via characterisation of CEpi markers (K14, K3, P63 and PAX6). Bulk RNA sequencing enabled investigation of the potential effects of variants, highlighting COL4A1 and COL4A2 as candidate genes for this family. Eight affected individuals in three generations of a family were diagnosed with a corneal dystrophy which remained genetically unsolved. Analysis of WGS data excluded established corneal dystrophy genes. A segregating novel heterozygous missense variant was identified in SPARCL1, NM_004684: c.334G > A; p.(Glu112Lys). SPARCL1 regulates decorin, encoded by an established corneal dystrophy gene, DCN. Immunohistochemistry of corneal tissue from an affected individual revealed epithelial retention of SPARCL1 and a reduction in decorin levels compared to control tissue. This study expands our current understanding of the phenotypic and genetic heterogeneity of inherited corneal disease and adds to our knowledge of genomic variants and pathways that may contribute to the development of KC.

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