Investigating amyotrophic lateral sclerosis candidate genes

Empirical thesis.Bibliography: pages 121-134.1. Introduction -- 2. Subjects and materials -- 3. Methods -- 4. Reults -- 5. Discussion -- Appendices -- Abbreviations -- References.Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease of motor neurons.Approximately 10% of cases are classiffed as familial and 90% sporadic, although a proportion of sporadic cases have an unrecognised family history. This thesis aimed o identify novel ALS genes from familial and sporadic cases. Whole-exome and whole genome sequencing analysis identified 22 candidate gene variants in an ALS family,which were investigated using an in silico analysis pipeline including protein predictions,conservation, genic tolerance, tissue expression and gene function. Three top priority variants, in CYB5R3, DCAF7, and SAV1, were assessed using in vitro toxicity and localisation assays, with one candidate, CYB5R3, displaying a differential cytoplasmic protein expression pattern. Whole-genome data from 635 sporadic ALS patients were interrogated for ALS candidate genes (n = 21), and ten novel variants were identified, including a novel CYB5R3 variant, which rated strongly from in silico tools. Lastly, this project sought to develop a CRISPR-Cas9 protocol to generate ALS cell models for future use in the in vitro analysis pipeline. Overall, a combination of genetic, in silico and in vitro pipelines were used to identify candidate genes in an ALS kindred and apparently sporadic patients. Novel ALS genes will further our understanding of disease biology and contribute to development of diagnostics and treatments.Mode of access: World wide web1 online resource (xviii, 134 pages) colour illustration

Genetic and immunopathological analysis of CHCHD10 in Australian amyotrophic lateral sclerosis and frontotemporal dementia and transgenic TDP-43 mice

Objective: Since the first report of CHCHD10 gene mutations in amyotrophiclateral sclerosis (ALS)/frontotemporaldementia (FTD) patients, genetic variation in CHCHD10 has been inconsistently linked to disease. A pathological assessment of the CHCHD10 protein in patient neuronal tissue also remains to be reported. We sought to characterise the genetic and pathological contribution of CHCHD10 to ALS/FTD in Australia. Methods: Whole-exome and whole-genome sequencing data from 81 familial and 635 sporadic ALS, and 108 sporadic FTD cases, were assessed for genetic variation in CHCHD10. CHCHD10 protein expression was characterised by immunohistochemistry, immunofluorescence and western blotting in control, ALS and/or FTD postmortem tissues and further in a transgenic mouse model of TAR DNA-binding protein 43 (TDP-43) pathology. Results: No causal, novel or disease-associated variants in CHCHD10 were identified in Australian ALS and/or FTD patients. In human brain and spinal cord tissues, CHCHD10 was specifically expressed in neurons. A significant decrease in CHCHD10 protein level was observed in ALS patient spinal cord and FTD patient frontal cortex. In a TDP-43 mouse model with a regulatable nuclear localisation signal (rNLS TDP-43 mouse), CHCHD10 protein levels were unaltered at disease onset and early in disease, but were significantly decreased in cortex in mid-stage disease. Conclusions: Genetic variation in CHCHD10 is not a common cause of ALS/FTD in Australia. However, we showed that in humans, CHCHD10 may play a neuron-specific role and a loss of CHCHD10 function may be linked to ALS and/or FTD. Our data from the rNLS TDP-43 transgenic mice suggest that a decrease in CHCHD10 levels is a late event in aberrant TDP-43-induced ALS/FTD pathogenesis