Inherited Retinal Dystrophies: Genomics and Functional Studies in Novel Variant and Disease Gene Discovery

Abstract

Purpose: Inherited retinal dystrophy (IRD) describes a collection of degenerative retinal disorders, with a prevalence of approximately 1 in 3500. Many of the underlying disease genes and their functions are not known. There is progressive degeneration of the photoreceptors which may result in complete blindness at early ages of life. Although mutations in over 250 genes have been described in IRD, diverse clinical features and the genotypic heterogeneity make the application of conventional sequencing methods very limited. The aims of this study were to: identify the underlying disease-causing gene in a cohort of 12 retinitis pigmentosa (RP) families using genomic approaches and targeted gene analysis; identify the underlying candidate disease gene in a family with a novel syndromic retinal dystrophy and investigate the function of the gene using cell-based assays; and to characterise a mouse model of the novel retinal dystrophy syndrome. Methods: Whole exome sequencing using the TruSeq exome enrichment system (Illumina Inc., San Diego, CA, USA), was applied to 12 probands diagnosed with autosomal dominant retinitis pigmentosa (ADRP, n=6) and autosomal recessive retinitis pigmentosa (ARRP, n=6). Pathogenicity was predicted and detected variants were confirmed and segregation determined using Sanger sequencing. In a family with an inherited novel syndromic retinal dystrophy described in the thesis, genomic, cell-based, and animal model approaches were undertaken to identify and understand the function of the novel disease gene. Functional studies were performed in mutant and control human fibroblasts, and transfected HeLa cells. A CRISPR/Cas9 generated mouse model of the disease was investigated using electroretinography (ERG), histology, and immunohistochemistry studies to understand the impact of the detected variant. Results: Pathogenic variants were identified in 4 families with ADRP families. Clear-cut variants were identified in 2 families with ARRP. A novel candidate retinal disease gene, alpha kinase-1 (ALPK1), has been identified in the newly described ROSAH syndrome which analysis from this thesis implicates in centrosome and cilia biology. Analyses in mouse retina showed expression of Alpk1 in the connecting cilium region of the photoreceptors with a possible role in ciliary trafficking. Cell-based assays revealed ALPK1 localisation in the centrosomes and the basal body of the primary cilium. Immunocytochemistry on skin fibroblasts revealed a higher percentage of cells with abnormal numbers of centrosomes in affected individuals. In HeLa cells transfected with the mutant construct, there were a higher number of multinucleated cells, further suggesting abnormality of centrosome biology. ERG studies revealed significant decrease in scotopic and photopic responses in mice with the orthologous mouse mutation, and histology sections showed thinning of the retinal layers. Further immunohistochemistry studies confirmed significant decrease in Tcp1 (ALPK1 binding partner) expression in the inner segment of photoreceptors and aberration of connecting cilium proteins, Ift88 and centrin in the mouse model. Conclusions: Genomic and functional work in this thesis has led to new knowledge with implications for regulation of centrosome biology and impact on ciliary function in the retina. This study highlights benefits of genomic investigation, phenotype-based bioinformatics, and functional studies to gain insight to the pathophysiology of the disease, paving the way for development of therapeutic strategies in the future.