Deep mutational scanning of CHD2 for variant interpretation in neurodevelopmental disorders

Project description 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 developing a multiplex assay of variant effect (MAVE) to discriminate between pathogenic and benign missense variants. We leveraged the fact that CHD2 is a chromatin remodeler and thus affects gene expression, protein abundance and the DNA methylation landscape to attempt to develop high-throughput assays.

We aimed to establish a CHD2 protein signature using proteomics from wildtype and mutant cells in HAP1 cells. While we did identify differentially abundant proteins, these did not validate in additional cell lines using flow cytometry antibody-based assays.

Sample processing protocol: To test for a CHD2 proteomic signature we generated two CHD2 knockout clonal HAP1 cell lines using prime editing. Cells were lysed and run on an SDS PAGE gel, stained with Coomassie blue, purified, digestion with trypsin enzyme and subject to 60-minutes of LC-MS/MS data acquisition for bottom-up proteomics. 

Data processing protocol:  Peptides were identified using the Scaffold 5 software, peptide counts were exported and differentially abundant proteins were defined by a fold change between wildtype (n=2) and mutant (n=2) using a p-value (t-test) cutoff <0.00005.