High resolution genotype phenotype correlations for severe paediatric disease caused by mutations in eEF1A2

这个学生项目涉及对单个基因内多个突变的研究，使用单个基因内等位基因异质性的概念作为精准医学方法的范例。以前无法解释的癫痫患儿的外显子组测序已经确定了新的致病突变，导致新的机制见解和病因驱动的治疗策略的前景。其中一个基因编码eEF1A2，这是一种翻译因子，不同寻常的是，它只在神经元、肌肉和心脏中表达。迄今为止，已知约40例重度癫痫和中/重度癫痫患儿存在EEF1A21新发错义杂合突变。许多人还患有自闭症和共济失调；还有一些人只能坐轮椅。此外，最近还描述了一个有三个孩子的家庭患有严重癫痫。这三个人都有一个纯合错义突变，死于儿童早期扩张型心肌病。预计EEF1A2将导致1/500中/重度ID2病例。最近，该基因两侧的EEF1A2和eQTL突变与常见的、通常较轻的癫痫有关。了解EEF1A2的突变如何导致这一系列疾病，将为与其他更常见疾病共享的分子途径提供新的思路，并且可能适用于药物靶向。精准医学，即针对癫痫的潜在遗传原因进行抗癫痫治疗，已经用于治疗其他特定的基因突变，这给这种方法带来了很大的希望。然而，在设计任何治疗策略之前，关键是要通过实验确定错义突变是导致功能丧失还是功能/显性负面效应的增加。目的在本项目中，对患者登记数据的分析将与湿实验室工作相结合。学生将进行基于实验室的突变蛋白分析以及对表达它们的细胞的下游影响。CRISPR/Cas9基因编辑将用于将突变引入神经细胞系，从而实现详细的功能生化分析。细胞系将在IncuCyte活细胞分析系统上进行分析，以检查突变对神经突生长和增殖等定量参数的影响（所有必要的技术都在我们的小组中得到了很好的建立4）。不同突变形式的蛋白质结合伙伴的蛋白质组学分析将用于进一步了解单个突变的病理后果；该项目的这一方面将涉及大型数据集分析方面的培训。我们有初步证据表明，虽然一些错义突变导致失去与蛋白质合成所需的辅助因子的结合，但其他突变不会，这表明该分析可以为单个基因中突变的功能获得或丧失提供证据。这名学生还将获得来自挪威600名癫痫患儿的DNA。通过这种方式，基因型/表型相关性可以为新诊断儿童的家庭提供更好的预后指标。

BackgroundThis studentship project involves the study of multiple mutations within a single gene, using the concept of allelic heterogeneity within a single gene as a paradigm for the precision medicine approach. Exome sequencing in children with previously unexplained epilepsy has identified novel causative mutations, leading to the prospect of new mechanistic insights and aetiologically-driven therapeutic strategies. One such gene encodes eEF1A2, a translation factor that, unusually, is expressed only in neurons, muscle and heart. There are so far ~40 children with severe epilepsy and moderate/severe ID known to have de novo missense heterozygous mutations in EEF1A21. Many also have autism and ataxia; others are wheelchair-bound. Furthermore, a family with three children with severe epilepsy has recently been described. All three had a homozygous missense mutation and died in early childhood from dilated cardiomyopathy. EEF1A2 is predicted to be responsible for 1/500 cases of moderate/severe ID2. More recently, mutations in EEF1A2 and eQTL flanking the egene have been associated with common, often milder, epilepsy3. Understanding how mutations in EEF1A2 can cause this range of disorders will shed new light on molecular pathways that are shared with those affected in other more common disorders, and that might be amenable to drug targeting. Precision medicine, where anti-epileptic treatments are targeted to the underlying genetic cause of epilepsy, is already in use for other specific gene mutations leading to great hope for this approach. However, it is critical to establish experimentally whether the missense mutations result in loss of function or a gain of function/dominant negative effect before any treatment strategy can be designed.AimsIn this project analysis of data from a patient registry will be combined with wet lab work. The student will carry out lab based analysis of the mutant proteins and the downstream effects on cells expressing them. CRISPR/Cas9 gene editing will be used to introduce mutations into neuronal cell lines, enabling detailed functional biochemical analysis. The cell lines will be analysed on the IncuCyte live cell analysis system to examine the effects of the mutations on quantitative parameters such as neurite outgrowth and proliferation (all necessary techniques are well established in our group4). Proteomic analysis of binding partners of the different mutant forms of the protein will be used to understand further the pathological consequences of individual mutations; this aspect of the project will involve training in the analysis of large datasets. We have preliminary evidence that whilst some missense mutations result in loss of binding to co-factors necessary for protein synthesis, other mutations do not, suggesting that this analysis can provide evidence for gain or loss of function of mutations within a single gene. The student will also have access to DNA from a Norwegian cohort of 600 children with well characterised epilepsy. In this way genotype/phenotype correlations could be made with the aim of providing better prognostic indicators for the families of newly diagnosed children.