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GRIN2A and GRIN2B encode the GluN2A and GluN2B subunits of the NMDA receptor, a subtype of ionotropic glutamate receptor that displays voltage-dependent block by Mg2+ and a high permeability to Ca2+. These receptors play important roles in synaptogenesis, synaptic transmission and synaptic plasticity, as well as contributing to neuronal loss and dysfunction in several neurological disorders. Recently, individuals with a range of childhood onset epilepsies, intellectual disability and other neurodevelopmental abnormalities have been found to carry heterozygous gene-disrupting or protein-altering point mutations in GRIN2A and GRIN2B. This thesis addresses the hypothesis that these point mutations cause key functional disturbances to NMDA receptor properties that contribute to neurodevelopmental disorders. To test this hypothesis, a group of related mutations were selected for functional assessment in heterologous systems: four missense mutations affecting residues in or near the subunit pore regions, all of which are associated with epilepsy and intellectual disability. To model the impact of gene disrupting mutations in GRIN2A, a preliminary analysis of the functional consequences of GluN2A haploinsufficiency in a genetically modified rat was also performed. Three of the four missense mutations were found to be associated with profound alterations in fundamental NMDA receptor properties: compared to wild type, GluN2AN615K was found to reduce Mg2+ block, GluN2BN615I and GluN2BV618G to cause potentiation by Mg2+, and GluN2AN615K and GluN2BN615I showed reduced conductance. GluN2AR586K was not found to influence the parameters assessed. When GluN2AN615K was expressed alongside wild type subunits in the same NMDA receptor, it was found to have a dominant negative effect. Finally, I established successful gene targeting in a new rat Grin2A knock-out model, and observed that heterozygous neurons had lower GluN2A protein expression and current density, making a good model to study human epilepsies associated with loss of a GRIN2A allele. This thesis provides evidence that three missense mutations in GRIN2A and GRIN2B affect physiologically important properties of the NMDA receptor, and that GluN2A haploinsufficiency influences important neural properties in vitro. This is consistent with these mutations causing disease and highlights these and related mutations as potential therapeutic targets in the future.

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

We report on an 8-year- old girl with severe developmental and epileptic encephalopathy due to the compound heterozygous null variants p.(Gln661*) and p.(Leu830Profs*2) in GRIN2A resulting in a knockout of the human GluN2A subunit of the N-methyl- D- aspartate receptor. Both parents had less severe GRIN2A-related phenotypes and were heterozygous carriers of the respective null variant. Functional investigations of both variants suggested a loss-of- function effect. This is the first description of an autosomal recessive, biallelic type of GRIN2A-related disorder. Nonetheless, there are marked parallels to two previously published families with severe epileptic encephalopathy due to homozygous null variants in GRIN1 as well as various knockout animal models. Compared to heterozygous null variants, biallelic knockout of either GluN1 or GluN2A is associated with markedly more severe phenotypes in both humans and mice. Furthermore, recent findings enable a potential precision medicine approach targeting GRIN-related disorders due to null variants.

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