Errata pasted onto back end-paper. / Copies of author's previously published articles inserted. / Bibliography : leaves 157-176. / viii, 176, [62] leaves : ill. (col.) ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Thesis (Ph.D.)--University of Adelaide, Dept. of Paediatrics, 1997

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No description available.

 Genome-wide mapping of susceptibility genes was conducted in two complex disorders of hypertension and epilepsy, allowing the dissection of the genetic architecture of these common diseases and related quantitative traits. The study performed comprehensive genetic analyses in a genome-wide scale, using different structure of data – sib-pairs and case-control samples. To identify genes influencing hypertension and blood pressure, a combined linkage and association study was conducted using over half a million SNPs genotyped in 328 siblings. Regions of significant linkage were identified for blood pressure traits on chromosomes 2q22.3 and 5p13.2, respectively. Further family-based association analysis of the linkage peak on chromosome 5 yielded a significant association (rs1605685, P < 7  10-5) for hypertension. One candidate gene, PDC, was replicated in the family-based association tests. A two-stage genome-wide association study (GWAS) was performed in a total of 1,087 cases and 3,444 controls, to identify common susceptibility variants of epilepsy in Chinese. The combined analysis identified two association signals in CAMSAP1L1, rs2292096 [G] (P=1.0×10-8, OR =0.63) and rs6660197 [T] (P=9.9×10-7, OR=0.69), which are highly correlated, achieving genome-wide significance. One SNP (rs9390754, P = 1.7 × 10-5) in GRIK2 was refined as a previously-implicated association. In addition to SNPs, the assessment of CNVs in GWAS was performed, which could provide valuable clues to discover genes contributing to the heritability of epilepsy. A genome-wide scan for epilepsy through the use of DNA pooling also provides an alternative approach to reducing the substantial cost and thus increase efficiency in large-scale genetic association studies. The genome-wide mapping studies in families and unrelated individuals are complementary and together offer a comprehensive catalog of common variations and structural variants implicated for both quantitative and qualitative traits. / published_or_final_version / Psychiatry / Doctoral / Doctor of Philosophy

目的：症狀性癲癇通常是由大腦損傷引起的，但大多數人患有這種腦損傷的人不會進而發展為癲癇，這表明，有些人擁有癲癇的易感基因。全基因組關聯研究（GWAS）是一種找到複雜疾病潛在基因的有效方法。最近關於對中國人口症狀性癲癇的GWAS報導了一個在1q32.1的CAMSAP1L1基因 (也叫做CAMSAP2) 上最顯著的單核苷酸多態性rs2292096 [G] （p=1.0×10⁻⁸，OR=0.63）。除了這個SNP，我們還選擇另一個在ERBB4基因上的潛在SNP rs13021324 [C]（p=5.8×10⁻⁵，OR=0.73）作為我們的研究。 CAMSAP1L1編碼一種細胞骨架蛋白，但是，它是否具有任何神經特定功能尚未知曉。 ERBB4是一個精神分裂症的易感基因，也有文章報導了ERBB4的突變引起了早期肌陣攣性腦病。我們推測，這兩個基因會影響人發展成為症狀性癲癇的易感性，以上的單核苷酸多態性會影響基因的表達或功能。這項研究的目標是，探索CAMSAP1L1的一些功能，和比較基因型之間以及癲癇患者和對照組之間這兩個基因的表達。 / 方法：在癲癇患者樣品中，我們對CAMSAP1L1 的SNP（rs2292096）和ERBB4的SNP（rs13021324）進行基因分型，首先將從癲癇患者的冰凍組織中提取RNA和蛋白質，他們的表達水平將分別通過實時PCR和蛋白印跡來測量，看家基因GAPDH用於測量對照，然後將不同的單核苷酸多態性與相應的表達水平關聯來觀察單核苷酸多態性是否會影響表達水平。同時，我們收集了匹配的癲癇患者和普通人的海馬和顳葉的石蠟包埋樣品，我們將通過免疫組化來測量樣品之間CAMSAP1L1這種蛋白表達水平是否不同。此外，對於基因CAMSAP1L1，我們將使用SH-SY5Y細胞進行雙螢光實驗，嘗試看到它在細胞中的位置，同時通過轉染來研究它對於神經突增長的一些功能。 / 結果：在顳葉有關SNP rs2292096（CAMSAP1L1）的RNA表達相關分析中，GG基因型患者呈現出高表達的趨勢（p=0.024）。而在相同的蛋白關聯分析中，則沒有看到這種趨勢（p=0.568）。在免疫組化試驗中，海馬部位的CASMAP1L1對照組的表達有高於癲癇組的趨勢（p=0.059）。而在顳葉，這兩組中觀察到的差異很小（p=0.12）。 在rs13021324（ERBB4）在RNA關聯表達分析中，攜帶等位基因C的患者的表達有高於T等位基因的一些趨勢, 但是不是很明顯。雙螢光試驗中，我們看到CAMSAP1L1在神經突的表達，它與β-微管蛋白有部分的表達重疊，在CAMSAP1L1轉染實驗中，近60的基因被抑制，在0μM和25μM RA 分化的SH-SY5Y細胞中，CAMSAP1L1的siRNA引起的基因沉默顯著增強了神經突以及分支的生長。 / 結論：在GWAS中發現的CAMSAP1L1基因上SNP的 顯著型p值表明，SNP和癲癇之間的遺傳性關聯可能是由於特定SNP標記的單體型的蛋白質表達水平和功能所決定的。我們的數據表明，在SNP rs2292096中，GG基因型的人倾向于有較高的表達，但需要更多的實驗來證實這一結果。CAMSAP1L1會抑制神經突生長。對於基因ERBB4，在C等位基因型中輕微高ErbB4的表達和我們的GWAS以及和以前的研究相符。 / Purpose: Symptomatic epilepsy is initiated by a brain insult, but most people suffering brain insults do not go on to develop epilepsy, indicating that some people are genetically predisposed to epilepsy after such insults. Genome wide association study (GWAS) is an effective way to find genes that contribute to diseases. The most significant SNP in a recent GWAS of symptomatic epilepsy in the Chinese population was rs2292096 [G] (p=1.0×10⁻⁸, OR=0.63), in the CAMSAP1L1 gene, also known as CAMSAP2. We chose to study this SNP as well as another SNP associated with epilepsy in the same GWAS, rs13021324 [C] (p=5.8×10⁻⁵, OR=0.73), which is in the epilepsy candidate gene ERBB4. CAMSAP1L1 encodes a cytoskeletal protein; however, it is not yet known to have any neurologically-specific function. ERBB4, a schizophrenia-susceptibility gene, has been reported to be mutated in a case of early myoclonic encephalopathy. We hypothesize that these two genes affect the predisposition of people to develop symptomatic epilepsy, and that the above SNPs influence gene expression or function. The objectives of this study are to elucidate some functions of CAMSAP1L1 and to explore whether the expression of the two genes will be affected by different genotypes. / Methods: One CAMSAP1L1 SNP (rs2292096) and one ERBB4 SNP (rs13021324) were genotyped in epilepsy patients. RNA and homogenates were prepared from frozen hippocampus and temporal lobe tissue from epilepsy patients. CAMSAP1L1 and ERBB4 RNA and protein levels were measured by real-time PCR and western blotting with reference to the housekeeping gene GAPDH, and expression levels were compared among genotypes of the above SNPs. We performed immunohistochemistry in paraffin-embedded sections to compare CAMSAP1L1 protein levels between epilepsy and control subjects in hippocampus and temporal lobe. In addition, we used human SH-SY5Y neuroblastoma cells to examine CAMSAP1L1 localization and function, performing double immunofluorescence for CAMSAP1L1 and tubulin and measuring the effect of CAMSAP1L1 knockdown on neurite outgrowth. / Results: In analysis of rs2292096 (CAMSAP1L1) in the temporal lobe, patients with the GG genotype showed higher expression of RNA (p=0.024), but not of protein (p=0.57). Immunohistochemistry showed a tendency toward lower expression of CAMSAP1L1 in epilepsy patients than in control subjects (p=0.059) in hippocampus but not in temporal lobe (p=0.12). Expression analysis of rs13021324 demonstrated non-significant tendencies toward higher expression of ErbB4 with C allele than the T allele for RNA and protein in hippocampus and temporal lobe. Double immunofluorescence showed CAMSAP1L1 expression on neurites, partially overlapping with β-tubulin. CAMSAP1L1 siRNA transfection of SH-SY5Y cells treated with or without retinoic acid reduced the CAMSAP1L1 protein level nearly 60% and stimulated neurite outgrowth, as measured by outgrowths, processes and branches compared with the control siRNA group. / Conclusion: The association of CAMSAP1L1 and ERBB4 SNPs with epilepsy is likely due to linkage disequilibrium with SNPs that affect functions or levels of CAMSAP1L1 or ErbB4. Our data suggest that the rs2292096 GG genotype, which reduces risk of symptomatic epilepsy, tends to increase expression of CAMSAP1L1, but more subjects are needed to confirm this result. CAMSAP1L1 represses neurite outgrowth. A tendency toward higher expression of ErbB4 with the C allele is consistent with the GWAS finding that the C allele decreases risk and with a report that ErbB4 levels are decreased in epilepsy. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Zhang, Shuai. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2012. / Includes bibliographical references (leaves 77-94). / Abstracts also in Chinese. / Abstract --- p.I / 摘要 --- p.III / Acknowledgements --- p.V / List of figures --- p.VI / List of tables --- p.VIII / List of abbreviations --- p.IX / Table of contents --- p.XII / Chapter Chapter One --- Introduction --- p.1 / Chapter 1.1 --- Introduction to epilepsy --- p.1 / Chapter 1.1.1 --- Epilepsy types and epidemiology --- p.1 / Chapter 1.1.2 --- Drug-resistant epilepsy --- p.2 / Chapter 1.1.3 --- Treatment for epilepsy --- p.3 / Chapter 1.2 --- Genetics of epilepsy --- p.5 / Chapter 1.2.1 --- Genetic mechanism underlying epilepsy --- p.5 / Chapter 1.2.2 --- Gene expression in epilepsy --- p.8 / Chapter 1.2.3 --- Genomics in epilepsy --- p.10 / Chapter 1.2.4 --- Animal models --- p.11 / Chapter 1.3 --- Methods to identify variants affecting disease susceptibility --- p.12 / Chapter 1.3.1 --- Traditional methods to identify genetic loci for disease susceptibility --- p.12 / Chapter 1.3.2 --- Genome wide association study (GWAS) --- p.12 / Chapter 1.3.3 --- A GWAS of symptomatic epilepsy in the Chinese population --- p.14 / Chapter 1.4 --- Functions of the CAMSAP family --- p.18 / Chapter 1.5 --- Functions of ErbB4 --- p.21 / Chapter 1.6 --- Hypothesis and proposed plan for expression study of CAMSAP1L1 and ERBB4 --- p.22 / Chapter 1.6.1 --- Proposed hypothesis of CAMSAP1L1 and ERBB4 in symptomatic epilepsy --- p.22 / Chapter 1.6.2 --- Objectives of the study --- p.22 / Chapter 1.6.3 --- Significance of the study --- p.23 / Chapter 1.7 --- Study scheme --- p.23 / Chapter Chapter Two --- RNA expression analysis of SNPs rs2292096 and rs13021324 --- p.24 / Chapter 2.1 --- Introduction --- p.24 / Chapter 2.2 --- Materials and methods --- p.24 / Chapter 2.2.1 --- Materials --- p.24 / Chapter 2.2.2 --- Genotyping --- p.25 / Chapter 2.2.3 --- RNA extraction --- p.26 / Chapter 2.2.4 --- Reverse transcription PCR --- p.27 / Chapter 2.2.5 --- Real-time PCR --- p.27 / Chapter 2.2.6 --- Data analysis --- p.29 / Chapter 2.3 --- Results and discussions --- p.29 / Chapter 2.3.1 --- Genotyping --- p.29 / Chapter 2.3.2 --- Real-time PCR --- p.31 / Chapter 2.4 --- Conclusion --- p.35 / Chapter Chapter Three --- Protein expression analysis of rs2292096 and rs13021324 --- p.37 / Chapter 3.1 --- Introduction --- p.37 / Chapter 3.2 --- Materials and methods --- p.37 / Chapter 3.2.1 --- Materials --- p.37 / Chapter 3.2.1 --- Protein extraction --- p.38 / Chapter 3.2.3 --- Western blot --- p.38 / Chapter 3.2.4 --- Western blot for quantification of protein samples --- p.39 / Chapter 3.3 --- Results and discussion --- p.40 / Chapter 3.3.1 --- CAMSAP1L1 band confirmation --- p.40 / Chapter 3.3.2 --- ErbB4 immunobloting --- p.43 / Chapter 3.4 --- Conclusion --- p.48 / Chapter Chapter Four --- Immunohistochemistry analysis of CAMSAP1L1 --- p.49 / Chapter 4.1 --- Introduction --- p.49 / Chapter 4.2 --- Materials and methods --- p.49 / Chapter 4.2.1 --- Materials --- p.49 / Chapter 4.2.2 --- Immunohistochemistry --- p.50 / Chapter 4.2.3 --- Quantification of FFPE samples by Image Pro-Plus --- p.51 / Chapter 4.3 --- Results and discussion --- p.52 / Chapter 4.3.1 --- CAMSAP1L1 immunohistochemistry --- p.52 / Chapter 4.3.2 --- CAMSAP1L1 protein quantification between epilepsy and control groups --- p.54 / Chapter 4.4 --- Conclusions --- p.57 / Chapter Chapter Five --- Double immunofluorescence and CAMSAP1L1 siRNA transfection in SH-SY5Y cells --- p.58 / Chapter 5.1 --- Introduction --- p.58 / Chapter 5.2 --- Materials and methods --- p.59 / Chapter 5.2.1 --- Materials --- p.59 / Chapter 5.2.2 --- Double immunofluorescence --- p.60 / Chapter 5.2.3 --- CAMSAP1L1 siRNA transfection --- p.60 / Chapter 5.2.4 --- Gene knockdown assay --- p.61 / Chapter 5.2.4 --- MTT cell viability assay --- p.62 / Chapter 5.2.5 --- Neurite outgrowth assay --- p.62 / Chapter 5.3 --- Results and discussion --- p.63 / Chapter 5.3.1 --- Double immunofluorescence --- p.64 / Chapter 5.3.2 --- CAMSAP1L1 knockdown assay by western blot --- p.64 / Chapter 5.3.3 --- MTT cell viability assay --- p.66 / Chapter 5.3.4 --- Neurite outgrowth --- p.67 / Chapter 5.4 --- Conclusion --- p.72 / Chapter Chapter Six --- Overall conclusion and prospects --- p.73 / Chapter 6.1 --- Overall conclusion --- p.73 / Chapter 6.1.1 --- CAMSAP1L1 --- p.73 / Chapter 6.1.2 --- ErbB4 --- p.74 / Chapter 6.2 --- Future work --- p.74 / Chapter 6.3 --- Prospects --- p.75 / References --- p.77

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Developmental and epileptic encephalopathies (DEE) represent a set of rare but devastating and largely intractable childhood epilepsies. While mouse models have made innumerable contributions to understanding the genetic basis of neurological diseases, only a small fraction of missense, gain-of-function DEE variants has been modeled in mice. In this dissertation, we focus on one DEE gene, ARHGEF9. With fewer than fifty ARHGEF9 patients reported to date, ARHGEF9 can be considered one of the rare players in DEE. ARHGEF9 encodes a brain-specific protein also known as collybistin (CB), a guanine nucleotide exchange factor and an essential regulator of inhibitory postsynaptic density. In Chapter 3 and Chapter 4, we present results and ongoing studies from our efforts to unravel the pathological mechanism of ARHGEF9 DEE. We studied the G55A variant on the SH3 domain, which was discovered in one severe case of DEE. Using a novel Arhgef9G55A mouse model, we examined behavioral, cellular, and electrophysiological consequences of Arhgef9G55A. Results demonstrate that the Arhgef9G55A mouse model is an adequate ARHGEF9 DEE model, because it phenocopies key aspects of human ARHGEF9 DEE. We showed the interesting protein aggregation phenotype caused by the G55A variant. Specifically, in Arhgef9G55A/Y neurons, CB forms protein aggregates at the proximal AIS, leading to dramatic disruptions in inhibitory postsynaptic components at the AIS. Furthermore, electrophysiological studies revealed significant changes in intrinsic neuronal excitability and synaptic transmission in Arhgef9G55A/Y brains. The work within this dissertation shows that the G55A variant disrupts axon initial segment structure and functions. In Chapter 2, we review and summarize current understandings on AIS structure and functions. We highlight the central role of the AIS in initiating action potential and integrating synaptic inputs through axo-axonic synapses. Based on our experimental results, we propose that disruptions in AIS function are closely tied to the pathophysiology of ARHGEF9 DEE. Aside from the clinical significance of our study, we demonstrate the important role of CB at the AIS. We propose that CB is a specific stabilizer of axo-axonic synapses. The difference in the requirement of CB in inhibitory synapse formation in different neuronal compartments could be a core molecular machinery underlying the functional diversity of inhibitory inputs.

Thesis (MSc)--Stellenbosch University, 2000. / ENGLISH ABSTRACT: Please see fulltext for abstract / AFRIKAANSE OPSOMMING: Sien asb volteks vir opsomming

The human neocortex is a highly sophisticated and organized brain structure that is thought to mediate some of the most complex cognitive functions in humans including language and abstract thought. As such, environmental and genetic insults to its normal structure or function can result in devastating neurological conditions including severe epilepsy and intellectual disability. Malformations of cortical development are an increasing collection of disorders that cause neocortical abnormalities due to impaired developmental processes. One recently identified disorder in this class is a thin lissencephaly variant (TLIS) associated with several mutations in the C-terminus death domain of the caspase-2 activation adaptor CRADD (also known as RAIDD). Beyond this, little is known about the mechanism underlying TLIS pathophysiology despite an increasing number of identified individuals suffering from it. In order to better understand this disorder, as well as the normal developmental mechanisms that are impaired in its pathogenesis, I have developed and characterized three murine models by introducing one of a number of different genetic perturbations associated with TLIS. These animal models show behavioral and biochemical abnormalities similar to those seen in human TLIS subjects. Focusing future studies on the developmental processes that underlie differences seen in these mouse models could greatly inform understanding of disease mechanism in humans and assist in the development in therapeutic interventions. My work presented in this dissertation thus effectively establishes a translationally relevant animal model of TLIS.

Childhood epileptic encephalopathies (EE) are severe neurodevelopmental diseases that manifest in early development. EE is characterized by abnormal electroencephalographic (EEG) activity, intractable seizures comprising of various seizure types, as well as cognitive, behavioral and neurological defects. Developmental and epileptic encephalopathies (DEEs) are a subclass of EEs where the progressive and permanent cognitive and neurophysiological deterioration is not caused by seizure activity alone, but is caused by the same underlying etiology. Recent advances in whole exome sequencing revealed an important role for synaptic dysregulation in DEE and identified multiple new causative variants in synaptic genes. Indeed, mutations in various genes associated with neuronal functions like synaptic transmission and recycling, including transporters, neurotransmitter receptors, and ion channels, have all been identified as causative of DEE. In total, pathogenic DEE-causing variants in over eighty-five genes have been identified and more are likely to follow as next-generation sequencing becomes widely available. DEEs comprise a large group of genetically and phenotypically heterogenous diseases that have been difficult to treat. While in many cases the etiology is unknown, de novo heterozygous missense mutations have often been identified as the underlying cause of DEE. Existing pharmacological interventions by way of antiepileptic drugs leave approximately seventy-percent of DEE patients with intractable seizures. Moreover, these pharmacological treatments do not address the cognitive impairments and associated comorbidities caused by the underlying pathophysiological mechanism. In fact, treatment with antiepileptic drugs may actually worsen cognitive comorbidities due to side effects. Additionally, there are no pharmacological treatments for these cognitive comorbidities other than mood stabilizers and antipsychotics. Therefore, alternative approaches to treatments that address the underlying genetic etiology are necessary. Indeed, the recent utilization of gene therapeutic approaches in other genetic disease models such as spinal muscular atrophy (SMA) has spurred the investigation of gene therapies to treat DEEs. Here, we executed a molecular, behavioral and functional characterization of three preclinical mouse models of DEE involved in synaptic function (Dnm1) and ion channel function (Kcnq3). The human orthologs of the Dnm1 and Kcnq3 genes cause some of the most severe DEE syndromes. Understanding the pathophysiological mechanisms by which mutations in these genes cause disease, is important in identifying and assessing future gene therapeutic interventions. Patients with heterozygous DNM1 pathogenic mutations present with early onset seizures, severe intellectual disability, developmental delay, lack of speech and ambulation, and hypotonia. For the DNM1 dominant-negative model of DEE, we first characterized the Dnm1Ftfl mouse which phenocopies the key disease-defining phenotypes and comorbidities observed in DNM1 patients. Further, we modelled a gene therapy approach in Dnm1Ftfl mice using an RNA interference-based, virally delivered treatment construct. Dnm1Ftfl homozygous mice showed early onset lethality, seizures, growth deficits, hypotonia, and severe ataxia. Molecular analysis of Dnm1Ftfl homozygous mice showed gliosis, cellular degeneration, increased neuronal activation and aberrant metabolic activity, all indicative of recurrent seizure activity. Importantly, our gene therapy treatment significantly rescued all the severe phenotypes associated with DEE, including seizures, early-onset lethality, growth deficits, and aberrant neuronal phenotypes. Thus, our gene therapy approach provided a proof-of-principle for the efficacy of gene silencing to treat DEEs caused by dominant-negative mutations. Second, a DNM1 human variant modelled in mice was generated and characterized. The Dnm1G359A mutation, unlike the Dnm1Ftfl mouse-specific mutation has been identified in patients suffering from DNM1 DEE. Thus, this model allows for a more clinically relevant assessment of the impact of a human DNM1 mutation in mice. In the long run, this model will help validate gene therapeutic approaches that may be clinically relevant to DNM1 DEE patients. The Dnm1G359A mutation, like the Dnm1Ftfl mutation, led to early onset seizures, growth deficits, and lethality, establishing the Dnm1G359A mouse model as a viable model to study DNM1 DEE. In the gain-of-function KCNQ3 model of DEE, Kcnq3R231H mice were characterized molecularly and behaviorally. Patients with KCNQ3 mutations show electrical status epilepticus during sleep (ESES), as well as cognitive and behavioral impairments. The Kcnq3R231H variant led to severe spike-wave discharge phenotype on EEG, decreased maximal seizure threshold, and anxiety-like behavior. Additionally, Kcnq3R231H led to increased localization of Kcnq3 protein at neuronal membranes, suggesting a role for membrane aggregation on disease phenotypes. Altogether, these findings show the viability of preclinical models of both dominant-negative and gain-of-function mutations in replicating key disease-defining phenotypes associated with severe DEEs. Additionally, the results presented here establish a proof-of-principle demonstration that gene silencing can rescue severe phenotypes caused by dominant-negative mutations in DEE. Future studies on both dominant-negative and gain-of-function models should enable an in-depth understanding of mechanistic implications for each mutation, and lead to gene therapeutic strategies to mitigate the debilitating phenotypes of these DEEs.