Project summary De novo pathogenic variants in CHD2 are one of the most common causes of the neurodevelopmental disorders (NDDs) that include refractory epilepsy, intellectual disability and autism spectrum disorders. In addition, somatic CHD2 variants, acquired later in life, are highly recurrent in certain types of leukemias. For both types of conditions, loss-of-CHD2 function is the likely pathogenic mechanism, and most variants are truncations. However, there is also evidence that missense variants can disrupt or abrogate CHD2 function, leading to loss-of-function, though they are harder to interpret, and many are classified as variants of uncertain significance (VUS). VUS are one of the biggest challenges for human geneticists, and represent the biggest class of variants returned on clinical genetic test reports, in part because we have a poor understanding of how missense variants impact protein function. Our research addresses these challenges by designing a bimodal high-throughput assay to determine the impact of VUS on CHD2 function using downstream protein abundance (Aim 1) and the CHD2 episignature (Aim 2). We will leverage the fact that CHD2 is a chromatin remodeler and thus affects both downstream protein expression and the DNA methylation landscape to develop a high-throughput, multimodal approach to identify variants that alter CHD2 function. We will also implement prime-editing using pools of pegRNAs to create 30 CHD2 variants and multiplex this assays, with a view to scale to hundreds of CHD2 variants in future work. Our work and approach has broad appeal as it can be scaled to include other NDDs and cancers in the future. Finally, the goal of this research is to develop an experimental paradigm that can be scaled to resolve all CHD2 VUS, providing answers for patients and families, and identifying those individuals that will be eligible for precision therapies in the future.