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

eEF1A2 突变引起的严重儿科疾病的高分辨率基因型表型相关性

• 批准号: 2106381
• 金额: --
• 依托单位: University of Edinburgh
• 依托单位国家: 英国
• 项目类别: Studentship
• 财政年份: 2018
• 资助国家: 英国
• 起止时间: 2018 至 无数据
• 项目状态: 已结题
• 来源: https://gtr.ukri.org/projects?ref=studentship-2106381
• 关键词: resolution; genotype; phenotype; correlations; severe

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.

背景本研究项目涉及单个基因内的多个突变的研究，使用单个基因内等位基因异质性的概念作为精确医学方法的范例。外显子组测序在儿童与以前无法解释的癫痫已经确定了新的致病突变，导致新的机制的见解和病因驱动的治疗策略的前景。一个这样的基因编码eEF 1A 2，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，已知约有40名患有重度癫痫和中度/重度ID的儿童在EEF 1A 21中具有新生错义杂合突变。许多人还患有自闭症和共济失调;其他人则需要坐轮椅。此外，一个有三个孩子患有严重癫痫的家庭最近被描述。这三个人都有一个纯合的错义突变，并在儿童早期死于扩张型心肌病。预计EEF 1A 2与1/500例中度/重度ID 2病例有关。最近，EEF 1A 2和eQTL侧翼的egene突变已与常见的，往往是温和的癫痫3。了解EEF 1A 2中的突变如何导致这一系列疾病，将为与其他更常见疾病中受影响的分子途径提供新的线索，并且可能适合药物靶向。精准医学，其中抗癫痫治疗针对癫痫的潜在遗传原因，已经用于其他特定的基因突变，从而为这种方法带来了巨大的希望。然而，它是至关重要的，以建立实验是否错义突变导致功能丧失或获得功能/显性负效应之前，任何治疗策略可以designed.AimsIn这个项目的数据分析，从患者登记将结合湿实验室工作。学生将进行基于实验室的突变蛋白质和表达它们的细胞的下游效应的分析。CRISPR/Cas9基因编辑将用于将突变引入神经元细胞系，从而实现详细的功能生化分析。将在IncuCyte活细胞分析系统上分析细胞系，以检查突变对定量参数（如神经突生长和增殖）的影响（所有必要的技术均已在我们的组中充分确立4）。蛋白质的不同突变形式的结合伴侣的蛋白质组学分析将用于进一步了解个别突变的病理后果;该项目的这一方面将涉及大型数据集分析的培训。我们有初步证据表明，虽然一些错义突变导致蛋白质合成所需的辅因子结合丧失，但其他突变则不会，这表明这种分析可以为单个基因内突变的功能获得或丧失提供证据。该学生还将获得来自挪威600名癫痫儿童的DNA。通过这种方式，基因型/表型的相关性，可以为新诊断的儿童的家庭提供更好的预后指标。