Medicine (RMH) - Theses
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ItemAntiepileptic drug teratogenicity: a human and laboratory translational study( 2018)Antiepileptic drug (AED) associated teratogenicity has been well documented in the literature. The risk of physical birth defects during the first trimester of pregnancy is increased threefold for most AEDs and over ten-fold for the most teratogenic AED, valproate. Despite this risk, women require long term treatment to stop or reduce the occurrence of seizures and the consequent harm to both mother and foetus, including the possibility of sudden unexpected death in epilepsy. The mechanism resulting in this teratogenicity, and in particular why some women are more susceptible to have children with AED induced birth defects is incompletely elucidated. In recent years there has been emerging evidence that AEDs may be interacting with genomic factors to result in birth defects. These genomic factors may be susceptibility alleles in the mother or father, de novo mutations in the child or epigenetic factors such as alterations in DNA methylation in the mother or child. The studies reported in this thesis aim to a) develop an animal model of valproate induced defects that closely mimics a human clinical setting and can be used to better understand the pathogenesis of AED induced defects, and b) identify genomic markers of AED induced defects using whole genome analysis of human samples and determining if having epilepsy is a contributing factor to the onset of these defects. For aim a) the development of the animal model entailed using an epileptic strain of rats, Genetic Absence Epilepsy Rats from Strasbourg, determining a dose at which dietary valproate is therapeutic, mating the rats and conducting a morphological assessment of both internal and external defects. For aim b) human samples were collected and subjected to whole genome analysis, including whole exome sequencing and DNA methylation scans. Additionally, birth defect rates for non-epileptic women in the Australian Pregnancy Register were also separately quantified. The human samples for investigations were collected from participants and their families enrolled in the Register. Using both human and animal models this study aimed to generate new knowledge, which could ultimately lead to a pharmacogenomic approach to the selection of AEDs for women who wish to become pregnant. This would allow women to make more informed decisions, reduce the risk of having a baby with a birth defect and potentially assist in the formation of new AEDs with lower teratogenic risk.
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ItemGenetic and molecular determinants of acquired and genetic epilepsy( 2016)Introduction: Despite the differences in the pathophysiological mechanisms and clinical features of both genetic and acquired epilepsies, dysfunction of both voltage- and ligand-gated ion channels have been highlighted as major contributors of the epileptic phenotype. In this thesis, the molecular mechanism by which T-type Ca2+ channels, AMPAR and TARPs lead to the development of epilepsy was evaluated. In addition, whole genome sequencing was utilised to identify novel candidate genes that could play a role in the epilepsy phenotype. Results: Anti-epileptogenic effects of selective blocking of T-type calcium channels in models of acquired epilepsy. The anti-epileptogenic effects of Z944, a novel, potent and highly selective T-type Ca2+ was used in the amygdala kindling and post-status epilepticus (SE) model. In the amygdala kindling model, Z944 a novel, potent and highly selective T-type Ca2+, did not suppress seizures in fully kindled rats. However, treatment with Z944 delayed de progression of kindling. In the post-SE model, treatment with Z944 after SE massively reduced the number of spontaneous seizures in comparison to vehicle and levetiracetam treated animals. Moreover, treatment with Z944 showed a strong comorbidity modifier effect in depressive like behaviour and may improve cognition after SE. Effects of the T-type calcium channel CaV3.2 R1584P mutation on seizure susceptibility in congenic rats. The CaV3.2 R1584P mutation was not enough to cause absence seizures in a seizure resistant background in the congenic animals but may be associated with the anxiety phenotype. However, the genetic background of the NEC congenic might contain genes that may suppress the pro-epileptic effect of the R1584P mutation. Role of AMPAR and TARPs in the pathogenesis of genetic generalised epilepsy and acquired epilepsy. The results of this chapter indicate a temporal association between the increased TARPs, Stargazin, γ3, γ4, γ5 and γ8, mRNA expression and the development of absence seizures in GAERS. In the post-SE epilepticus model, there was a significant reduction in mRNA expression in the TARPs γ3 and γ8 in the hippocampus 13 weeks after SE. Whole genome sequencing the GAERS and NEC rat strains. TThrough the proposed screen construct variants were identified in high-seizing F2 rats in the following genes; Cacna1h which codes for the CaV3.2 T-type Ca2+ channel, and F1LVI7_RAT and LOC300024. Similarly, non-seizing F2 rats are heterozygous for the mutations in RGD1308133, D3ZPQ1_RAT, Mkl and BiK. In the NEC and F2 non-seizing variants found in Abat, Cyp11b3 and Cyp11b2. Moreover, the flexibility of this method means that it can be applicable with other models of genetic and acquired epilepsies. Conclusions: Here is shown that blocking T-type Ca2+ channels with Z944 has encouraging positive preclinical evidence for disease-modifying in epileptogenesis and in epilepsy behavioural comorbidities that may be possible to translate to a clinical trial. Moreover, the analysis of T-type Ca2+ channels, AMPAR, TARPs and the discovery of novel potential molecular targets using whole genome sequencing uncovers overlap among genetic and acquired epilepsy. Importantly, the findings in this thesis could lead to discoveries that expand our knowledge of epileptogenesis, opening the door for the development of novel therapies.