School of Biomedical Sciences - Theses
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ItemA Novel Approach for the Molecular Diagnosis of Mitochondrial DNA-related Diseases Using Massively Parallel Sequencing( 2014)Mitochondrial diseases are a group of clinically and genetically heterogeneous disorders caused when the final stage of mitochondrial energy production, oxidative phosphorylation (OXPHOS), is disrupted. Of all inborn errors of metabolism, inherited OXPHOS disorders are the most common group, with a minimum estimated birth prevalence of ~1 in 5000. The OXPHOS system is encoded by both the mitochondrial and nuclear genomes, and mutations in either can cause disease. In addition to genetic complexity, there is a great deal of observed phenotypic heterogeneity. These disorders can give rise to almost any symptom, in any organ, at any age of onset, with any mode of inheritance. Childhood presentations are typically more severe and progressive, usually resulting in disability and death. Cases of mitochondrial disease due to mutations in mitochondrial DNA (mtDNA) account for up to 30% of childhood cases and 70% of adult cases. The detection and characterization of mtDNA mutations currently relies on a series of time consuming and costly sequential diagnostic tests, as no single test has been able to interrogate every mtDNA nucleotide position, report the proportion of mutant mtDNA in a tissue (degree of heteroplasmy) and detect large deletions. However, a recently proposed novel approach to whole mitochondrial genome analysis using long-range PCR (LR-PCR) enrichment of the whole mtDNA with a single set of back-to-back primers followed by massively parallel sequencing (MPS) has made it possible to achieve molecular diagnosis in a single test. This study aimed to develop a new pipeline based on this approach but using LR-PCR enrichment followed by a NextEra XT (Illumina, USA) library preparation and MPS on the Illumina MiSeq, for routine diagnostic analysis of the mitochondrial genome at the Victorian Clinical Genetics Services (VCGS). The quality of 165 patient genomic DNA (gDNA) samples with independently established mtDNA defects was assessed using the TapeStation gDNA ScreenTape Assay. The final cohort comprised of 118 high quality gDNA samples (from a variety of tissues) representing a range of mtDNA defects and a range of mutant heteroplasmy levels. Two approaches for enriching the whole mitochondrial genome using LR-PCR were developed, a single amplicon approach using one pair of back-to-back primers, and an approach using two sets of primers to create smaller overlapping amplicons. The NextEra XT library preparation was optimized for both LR-PCR approaches and produced good MPS data on the Illumina MiSeq in two sequencing runs. Two commercial software packages were chosen for analysis of the MPS data generated in these runs, the mtDNA Variant Analyser (Illumina, USA) and NextGENe (SoftGenetics, USA), and their performance was evaluated.
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ItemECAP measures predict cochlear implant behavioural thresholds( 2015)Aim: To estimate high rate stimulus behavioural threshold current levels of cochlear implant (CI) users by comparing peak amplitudes and associated latencies of electrically evoked compound action potentials (ECAPs) evoked by different stimulation pulse parameters. Background: CIs create a perception of sound by electrically stimulating the cochlea. The current level for each electrode that corresponds to the minimum amount of stimulation required to create a perception of sound is presently determined through a lengthy and subjective clinical process. This process can be particularly challenging for infants and the long-term deaf. With adequate stimulation, peripheral auditory neurons generate an electrical response called an ECAP. The minimum amount of stimulation required to elicit ECAPs has been assumed to be able to predict behavioural thresholds. This has been found to be untrue, and ECAP-based behavioural thresholds produce sub-optimal speech perception outcomes. There is evidence that differences between ECAP- and behavioural thresholds are caused by differences between individuals’ peripheral auditory neuron survival. Separately, there is evidence that the extent of change in certain ECAP measures with change in stimulation conditions is also affected by peripheral neural survival. We suggest that these ECAP measures may therefore be directly used to estimate differences between ECAP- and behavioural thresholds. Methods: Amplitude growth function series with four different stimulation parameters were created from ECAP thresholds to C-levels for 52 electrodes of 10 adult CI users. ECAPs were recorded intracochlearly through neural response telemetry. The current level difference required to equalise ECAP amplitude, and the change in ECAP peak latency, between stimulation conditions was determined for each electrode. Behavioural thresholds at stimulation rates of 40, 500, 1000 and 2000 pps were determined for each electrode using a 3-interval-forced-choice task. Pearson correlations were performed between equalising CL and low rate changes in behavioural thresholds, both per electrode and per subject mean. Stepwise multiple regression was used to directly estimate higher rate thresholds. Results: ECAP measures significantly improved ECAP threshold based predictions of higher stimulation rate behavioural thresholds (1000 pps: R2 = 0.338, n = 52). Conclusions: This technique is superior to purely ECAP threshold-based fitting, improving predictions of behavioural thresholds. These methods will require validation, but show promise as a new clinical method to create better speech-perception outcomes for certain CI users.