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

Background

This 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.

Aims

In 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.