Medicine (RMH) - Theses
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ItemFunctional analysis of sodium channel gene variation in epilepsy( 2013)A genetic etiology of epilepsy is widely accepted in 50-70% of all epilepsy syndromes. With genome sequencing now increasingly efficient and affordable, more and more novel genes and mutations are being discovered that are associated with epilepsy. However, most of the mutations have been discovered in genes that code for ion channels which has led to the theory that the genetic epilepsies are a family of channelopathies. The voltage-gated sodium channel family have been particularly implicated with over 800 variants discovered in this gene family. Given their critical role in regulating neuronal excitability it is not surprising that genetic variations in sodium channels can have functional and potentially devastating consequences. With a focus on the voltage-gated sodium channels, the three chapters in this thesis used high-throughput automated planar patch-clamp technology to try and develop a deeper understanding of genetic risk in epilepsy. Chapter two examines a novel cause in a mouse model of absence epilepsy that harbours a mutation in the Scn8a gene. The phenotype of this mouse is enhanced on the C3H background, as opposed to C57, where the C3H animal also has a mutation in the Scn2a gene. The individual biophysical profiles of these two mutations were examined on the Nanion patchliner, and their potential genetic interaction was investigated in a computer model of a layer 5 pyramidal neuron, to see if this could be explained by a biological interaction at the axon initial segment. The results revealed an overall loss of function of the NaV1.6V752F mutant, and an overall gain of function in the NaV1.2V929F mutant. When these changes were implemented in the computer model, it revealed that the output was dominated by the NaV1.2V929F mutant, which suggests there is not a biological interaction of these two genes at the axon initial segment. Alternative scenarios where there may be an alternative site for biological epistasis will be revealed with future studies using immunohistochemistry and brain slice patch clamp recording in the mice. It may also be the case that the NaV1.2V929F mutant is not a modifier of the NaV1.6V752F mutant, which will be revealed by genetic studies to identify the modifier genes. The third chapter examined the modulation of NaV1.2 and NaV1.1 by the β1 auxiliary subunit. As mutations in the β1-subunit have been detected in patients with epilepsy, understanding their impact on subunits from excitatory and inhibitory neurons is critical for understanding how this variation impacts on risk for epilepsy. There was a differential modulation revealed where β1 had a greater functional effect on the NaV1.2 channel but a greater effect on current density on the NaV1.1 channel. Therefore if a variant in β1 experiences a functional change this suggests differentially altered levels of excitation and inhibition in the brain, which could feasibly result in an epileptic phenotype. The fourth chapter looked at exploiting the high-throughput capabilities of the Nanion patchliner, and examined eight mutations in the β1-subunit co-expressed with NaV1.1 and NaV1.2 that have been associated with epilepsy. With this influx of data we needed to devise a new way to represent this data, and converted all raw measurements to effect size values, and represented them on tornado diagrams. With this measurement we could then more easily directly compare parameters from the individual protocols and calculate averages both across mutations, and across parameters. From this data set it is quite apparent that the β1 mutants modulate the α-subunits quite differently, both comparing α-subunits, and comparing mutations. More importantly however this chapter highlighted a new way of thinking about analysis of high-throughput electrophysiology data. As people continue to look into the genetics of epilepsy and reveal novel genes and novel mutations implicated in the disease, we need to look for new ways to tame the genetic complexity, and look for points of convergence. High-throughput technology allows us to decrease the time lapse between the discovery of the genetic variants and the corresponding functional analysis. And the type of analysis as suggested in chapter four, enables us to start to look for points of convergence in the functional data. This data can then be used to train clustering algorithms to group the variants based on their ‘channelomic’ profile. To do this we need a large volume of functional data obtained from variants that have strong corresponding phenotypic data, and future studies should endeavour to accomplish this.
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ItemIdentification of predictors of epilepsy outcomes( 2013)BACKGROUND: Epilepsy is a group of complex conditions characterized by the occurrence of recurrent spontaneous seizures, associated with high risk of brain abnormalities, co-morbid disorders and premature mortality. These issues in patients with new onset-seizures have not been well characterized. Furthermore, the first anti-epileptic drug (AED) treatment will fail in more than 50% of patients because of inadequate seizure control or intolerable adverse effects. Substitution to a newer AED may result in better outcomes. AIMS: (1) To determine the frequency and type of potentially epileptogenic abnormalities identified on MRI, in patients presenting with a possible new-onset seizure disorder, and their association with abnormal electrical activity on electroencephalogram (EEG), the frequency of co-morbidities and injuries, and the incidence of mortality (2) To examine whether patients who fail their first AED will have better neuropsychiatric, neurocognitive, quality of life (QOL), bone density and content, body composition and metabolism outcomes if substituted to monotherapy treatment with a newer-generation AED, levetiracetam, compared with another older-generation AED, valproate or carbamazepine. METHODS: (1) A consecutive series of 993 patients (597 males [61%], mean (SD) age: 42.2 (18.8) years, range: 14.3-94.3 years) who presented to an adult First Seizure Clinic over a ten year period were studied. The MRI scans were acquired using a dedicated epilepsy protocol or other specific protocols depending on clinical indication on either a 1.5T or 3.0T scanners. The co-morbidities and injuries were obtained from two state-wide administrative datasets, the Victorian Admitted Episodes Dataset (VAED) and the Victorian Emergency Minimum Dataset (VEMD), for the period from June 18, 1999 to June 30, 2010. Mortality data were collated from the National Death Index (NDI) developed by the Australian Institute of Health and Welfare (AIHW). (2) A randomized comparative trial was undertaken. Participants with focal epilepsy who had failed monotherapy with phenytoin sodium, carbamazepine, or valproate sodium were randomized to substitution monotherapy with levetiracetam (n= 51) or a different older AED (n= 48). Assessments were performed at baseline, 3 months, and 12 months using questionnaires measuring neuropsychiatric, QOL, seizure control, AED adverse effects, and neurocognitive outcomes. Assessments of bone density and content, body composition and metabolism were performed at 3- and 15 months after randomization. The assessments included: areal bone mineral density (aBMD) at lumbar spine, total hip, forearm, and femoral neck and total body bone mineral content [dual energy x-ray absorptiometry (DXA); Hologic QDR® 4500A densitometer], body composition [Hologic Software Version 5.73], peripheral quantitative computed tomography at non-dominant radius and tibia [pQCT; Stratec XCT 3000], serum markers of bone turnover, sex and metabolism hormones, questionnaires for bone health and lifestyle, blood pressure and anthropometry. RESULTS: (1) Potentially-epileptogenic lesions were detected in 177 (23%) of the patients in the First Seizure Clinic Cohort. Their frequency was higher in patients who had focal-onset seizures (53%). MRI and EEG were concordant in terms of the presence or absence of epilepsy associated abnormality in 62%, with 18% having an abnormality on both utilities. Almost 65% of patients with epilepsy had, at least, one co-morbid disorder as defined by an epilepsy-specific co-morbidity index, and 32% presented to Emergency Departments after sustaining an injury. Those patients were more likely to be males, have lesional focal epilepsy, frequent seizures and be diagnosed with epilepsy at 65+ years of age. The overall case mortality was 8.3% (82 cases) in the cohort. Standardized mortality ratio (SMR) for patients with new-onset seizures was 2.2 (95% CI 1.73-2.80); p< 0.001. The highest excess mortality was seen in younger patients (< 60 years). Proportionate mortality ratio (PMR) was highest for deaths from cerebrovascular diseases (16%) and malignant neoplasms (16%). Epilepsy was listed as the leading cause of death in 10%. (2) In the RCT, there were no differences in depression scores at 3 months between the treatment groups (improvement in 39.5% of the levetiracetam group vs. 34.1% of the older AED group; p= 0.60), but a greater proportion of the older AED group improved on the 89-item Quality of Life in Epilepsy Inventory (QOLIE-89) compared with the levetiracetam group (71.1% vs. 48.8% respectively; p= 0.04). The QOL, anxiety, and AED adverse effects scores were improved in both groups at 3 and 12 months after randomization. There were significant decreases in both treatment groups in aBMD at the lumbar spine (-9.0%; p< 0.001 in the levetiracetam group and -9.8%; p <0.001 in the older AED group), forearm (-1.46%; p< 0.001 and -0.96%; p< 0.001 respectively) and femoral neck (-0.47%; p= 0.026 and -1.45%; p< 0.001 respectively) on DXA scanning. The total hip aBMD significantly decreased in the older AED group (-0.84%; p< 0.001). The treatment groups did differ only in the change in femoral neck aBMD (p= 0.005). The serum levels of C-terminal telopeptides of type I collagen (βCTX), a marker of bone resorption, decreased in both groups (-16.1%; p= 0.021and -15.2%; p= 0.028 respectively) while procollagen 1 N-terminal peptide (P1NP), a marker of bone formation, significantly decreased in the older AED group (-27.3%; p= 0.008). There was no significant change in percentage abdominal fat in either treatment group. CONCLUSIONS: (1) MRI reveals potentially-epileptogenic lesions in a minority of patients who present following a newly-diagnosed seizure disorder. Lesions are most common in patients who have experienced focal seizures. The presence of a potentially-epileptogenic MRI lesion did not influence the likelihood of having an abnormal EEG. The majority of patients with new-onset seizures who consult a specialized First Seizure Clinic suffer from somatic or psychiatric co-morbid disorders. This finding should provide useful implications in the diagnosis and management of epilepsy and co-existing conditions, as well as in health care provision. Patients with new-onset seizures have a two-fold increased mortality compared with the general population. Cerebrovascular disease, and neoplasms, but not epilepsy, were the leading causes of death in epilepsy. (2) Substitution monotherapy in a patient who is experiencing ongoing seizures or tolerability issues is associated with sustained improvements in measures of QOL, psychiatric and adverse events outcomes. The significant bone loss at the lumbar spine, forearm, and femoral neck seen in both treatment groups and the modest reduction in bone turnover indicates that the adverse effects on bone health of chronic AEDs is likely occur with the newer-generation AEDs as well the older-generation AEDs.
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ItemRadiological and molecular factors associated with seizures in patients with supratentorial gliomas( 2013)Tumour associated epilepsy (TAE) is a common and disabling symptom experienced by patients with supratentorial gliomas. The pathogenesis of TAE is likely to involve a complex interplay between macroscopic anatomical factors, molecular factors and individual patient factors. The overarching hypothesis of this thesis is that patients with TAE have tumours with different radiological, molecular and genetic features compared to those without TAE. Furthermore, identification of these features may allow the identification of patients who are at increased risk of the development of TAE, and provide tailored, individualized treatment of these patients. An improved understanding of the genetic and molecular features associated with TAE may also inform the development of novel therapeutic strategies for these patients. There is an increasing body of evidence implicating glutamate, the most abundant neurotransmitter in the mammalian central nervous system, in the pathogenesis of TAE. Magnetic resonance spectroscopy (MRS) provides a non-invasive method to quantify brain metabolites in-vivo, but has not yet been validated for glutamate. In this thesis we firstly demonstrate that MRS quantification of glutamate has a modest, but statistically significant, correlation with concentrations of glutamate measured from tumour biopsy specimens. We then demonstrate that these MRS measurements of glutamate are useful in predicting not only glioma grade, but also the incidence of TAE. We also demonstrate that patients with low-grade gliomas and TAE are more likely to have larger tumours than patients without TAE. The opposite is found in patients with high-grade gliomas and TAE. There is also evidence that common pathological processes, including glutamate excitotoxicity, may be involved in TAE and neurodegenerative conditions such as Alzheimer’s disease, the sequelae of traumatic head injury and idiopathic epilepsy. A number of molecular factors associated with neurodegeneration and excitotoxicity are therefore investigated for their association with TAE. Phosphorylation of tau was found to be lower in patients with TAE compared to those without TAE. Over-expression of glycogen synthase kinase (GSK3β) was found to correlate with TAE, as was loss of glutamic acid decarboxylase 67 (GAD67), potentially reflecting a selective loss of inhibitory interneurons. Finally, the expression of the IDH1-R132H mutation, the most common mutation in low-grade gliomas, is shown to correlate with TAE. In conclusion, TAE results from a complex interaction of patient, environmental and tumour factors, including glutamate excitotoxicity, selective loss of inhibitory interneurons and IDH1-R132H expression. Not only do the results presented in this thesis suggest that TAE differs from other epileptic syndromes, but also that the mechanisms may differ between low and high-grade gliomas. Collaboration between neurosurgeons, neurologists, radiologists, pathologists and basic scientists will be essential for further investigation of this debilitating disease.
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ItemBioinformatics: the application of multigenic models to predict disease and treatment outcomes( 2011)One of the single most difficult aspects of treating newly diagnosed epilepsy, for patients and clinicians, is managing the uncertainty surrounding whether the anti-epileptic drug (AED) therapy will prevent further seizures. By investigating the pharmacogenomics of a population of Australian newly treated epilepsy patients, this thesis seeks to identify whether it is possible to develop risk prediction models that could improve personalising the treatment care provided to this population of patients by reducing the uncertainty and helping select the “right drug for the right patient”. The thesis explores the premise that developing predictive models for a complex phenotype such as seizure control in newly treated epilepsy, will require the utilization of multiple genomic variants, and also genomic and non-genomic factors need to be integrated. The results of the research demonstrated a proof-of-concept for this premise, showing that using the genetic profiles across five genetic variants improves the ability to significantly predict epilepsy pharmacoresponse in comparison to investigating each of the individual genomic components. This model was developed in an Australian population of newly treated epilepsy patients prospectively followed to determine the outcome of their drug treatment, and then validated in two other Australian cohorts. This model has potentially important clinical implications given that the current standard of care for newly diagnosed epilepsy patients does not provide clinicians with a meaningful tool to determine which of the patients are at a higher risk of not-responding to their initial AED therapy. To understand the broader clinical utility of the model, the Australian derived multigenic model was tested in two newly treated epilepsy cohorts from the United Kingdom. It was found that the Australian population did not significantly predict treatment outcome in the UK newly treated populations overall. However, the combination of five-SNPs identified as being relevant to pharmacoresponse in the Australian population treated with carbamazepine and valproate, were significantly predictive of pharmacoresponse in all the UK patients that were treated with these two drugs. This suggests that these five SNPs have drug specific predictive value. With a focus on developing a more accurate predictive model, non-genomic factors were also investigated and integrated with the genomic predictors into a unified predictive model. This model was found to have high predictive value for seizure control in newly treated epilepsy, and would potentially provide a clinically useful tool to assist in the ability to personalise treatment advice given to newly treated patients. The ability to determine non-responders improved from the currently generic 30% likelihood of not responding to initial carbamazepine or valproate treatment in the Australian newly treated population, to a 75% likelihood of not responding if the integrated, pre-treatment, model predicts the patient to be a “non-responder”, or conversely a 82% likelihood of responding if the model predicts the patient to be a “responder”. As a final task, the model development approach, which resulted in successfully identifying a multigenic predictive model for epilepsy pharmacoresponse based on a limited candidate gene dataset of approximately 4,000 carefully selected genetic markers, was applied to two genome-wide datasets. Based on this attempt to directly upscale the model development from candidate genes to a genome-wide scale, a number of limitations in the marker selection and model development stages of the approach were identified. These limitations emphasize the importance of designing tailored approaches to identifying multigenic models based on the differing contexts of datasets. Here, both in the epilepsy pharmacoresponse and HIV-1 susceptibility genome-wide attempts, the approach did not result in the development of significantly predictive models when applied to independent validation cohorts. However, possible future directions on how to overcome some of these direct upscale limitations are provided. Additionally, potential future directions that could result from this body of work are also explored.
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ItemNeurobiological factors predisposing to tumour associated seizures( 2010)Tumour associated seizures (TAS) are a common, disabling co-morbidity of gliomas. The pathogenesis of TAS and its associated implications for survival remain poorly understood. Elevations in glutamate levels, due to alterations in expression of membrane transporters, have been reported in gliomas in vitro. As glutamate is highly epileptogenic and cytotoxic we examined whether elevations of glutamate levels and alterations in transporters were specifically associated with TAS and survival. Our comprehensive study of two large cohorts of patients (290 in total) is the first human evidence showing associations between preoperative seizure risk and clinicopathological and molecular factors in patients with glioma. We found the clinicopathological factors that were independent predictors for preoperative TAS to be earlier age at diagnosis, temporal lobe location of tumour and tumour types of anaplastic astrocytoma or those with oligodendroglial components. A strong predictor for postoperative TAS was preoperative TAS adding credence to the philosophy “seizures beget seizures”. We found of the molecular factors, raised glutamate levels in tumour and peritumoural tissue as well as altered expression of astrocytic glutamate transporters EAAT2 and xCT all to be significantly associated with preoperative TAS. We also found neuronal HCN channelopathies, particularly downregulation of peritumoural HCN2 expression, to be associated with seizures due to glioma. This is further support for the concept of peritumoural tissue holding the key to TAS pathogenesis and we suggest it should be a subject for further investigation. These findings strongly support the “glutamate hypothesis” which proposes that glioma, due to dysfunction of the glial glutamate transporters among other factors, produces abnormally high levels of glutamate which then leads to excitotoxic changes in surrounding neuronal networks that ultimately manifest as seizures. Looking for further insights into the prognosis of glioma, we have also assessed the effects of preoperative seizures and raised glutamate levels on survival. This has suggested a positive influence. Postoperative seizures on the other hand, were associated with worse survival. Through a pilot study, we also explored the feasibility of using MRS for glutamate as a non invasive clinical diagnostic and monitoring tool in the management of TAS. We did not find any statistical significance of spectroscopically derived glutamate levels with TAS but the study did highlight some improvements in design required for a larger validation study in the future. This is still a promising prospect and could lead the way in the non invasive workup of people with TAS. The importance of the study of epilepsy and tumours hinges on the therapeutic and diagnostic contributions that are possible. Understanding the underlying mechanism of TAS, particularly the significance of the peritumoural tissue in this process, could revolutionize current drug treatments as well as redefine surgical objectives for the patient with TAS.