Molecular and genetic basis of inherited optic neuropathies

Abstract

Inherited optic neuropathies represent an important cause of chronic visual morbidity among children and young adults. This group of disorders is genetically heterogeneous and it can be caused by pathogenic mutations within both the mitochondrial and nuclear genomes. The unifying pathophysiological theme is mitochondrial dysfunction, but the specific disease mechanisms that ultimately precipitate neuronal loss, particularly retinal ganglion cell (RGC) degeneration, still remain unclear. The work presented in this thesis provides further insight into the molecular and genetic basis of two classical forms of inherited optic neuropathy, namely autosomal dominant optic atrophy (DOA) and Wolfram syndrome. Dominant optic atrophy (DOA) secondary to pathogenic OPA1 mutations is the most common inherited optic neuropathy diagnosed in clinical practice. The pathology is characterised by the preferential loss of RGCs within the inner retina and optic nerve degeneration. Although most OPA1 mutation carriers will only develop isolated optic atrophy, a subgroup of patients, referred to as DOA plus (DOA+), will develop more severe neuromuscular complications in addition to visual failure. The complexity of these clinical presentations may be due in part, to the various roles of OPA1 in the mitochondrial compartment such as regulating mitochondrial fusion and cristae structure,sequesterisation of pro-apoptotic molecules, mitochondrial DNA (mtDNA) maintenance,proper functioning of the oxidative phosphorylation system and calcium homoeostasis. To investigate the disease mechanisms that could explain the varying clinical manifestations and severity of OPA1 mutations, I made use of a cohort of eight fibroblast cell lines established from four patients with pure optic atrophy (OA) and four patients with DOA+ phenotypes. OPA1 expression and mitochondrial fragmentation patterns were compared between these two groups. There was no significant disruption in OPA1 transcription, mitochondrial OXPHOS and mtDNA maintenance. DOA primary fibroblasts showed increased fragmentation of the mitochondrial network and cell lines established from patients with DOA+ phenotypes were found to be particularly susceptible to fragmentation under basal conditions, which had not been reported previously. iii To further explore the findings obtained in OPA1 mutant fibroblasts, I made use of a cohort of nine myoblast cell lines that had previously been established from patient muscle biopsies. Interestingly, a similar mitochondrial fragmentation pattern was observed in OPA1 mutant primary myotubes and this was associated with decreased mitochondrial DNA molecule number in DOA+ myotubes. I also investigated two sisters from a consanguineous Arab Muslim family who developed a fatal form of juvenile-encephalopathy complicated by optic atrophy and cardiomyopathy. Exome sequencing identified a putative homozygous OPA1 mutation, which was confirmed by both functional studies and in silico modelling. Whole-exome analysis was carried out on a cohort of fourteen patients with optic atrophy that had previously been found to be OPA1-negative. Pathogenic mutations in the Wolframin (WFS1) gene, which is known to cause Wolfram syndrome, were identified in 3/14 (21%) patients. Based on our results, WFS1 mutations are an important cause of inherited optic atrophy and genetic testing should be considered in OPA1-negative patients. In conclusion, the body of work presented in my PhD thesis has provided further insight into the expanding genotypic and phenotypic spectrum of inherited optic neuropathies, which is highly relevant for clinical diagnosis and patient management.