Kaan Okay, Juan Ouyang, Katrín Möller, Kasper Daniel Hansen and Hans Tómas Björnsson
The most common phenotype in the Mendelian Disorders of the Epigenetic Machinery (EM) is intellectual disability. Previously people have uncovered a DNA methylation (DNAm) signature in the peripheral blood of such patients but it is unclear what happens in a disease relevant cell such as neurons. Here we have analyzed DNAm data from Oxford nanopore technology (ONT) sequencing in neuronal progenitors with EM knockouts (KO). We created 37 EM gene KOs through CRISPR-Cas9 in murine neural progenitor cells (mNPCs). DNA and RNA were isolated in EM-deficient mNPCs to perform ONT sequencing and bulk RNA sequencing, respectively, to identify shared DNAm and shared expression abnormalities among EM. We also characterized these changes during normal in vitro neurodevelopment. We have previously classified EM genes into co-expressed and non-co-expressed groups. We identified five shared differential methylated regions (DMRs) among 37 EM KOs and controls. Surprisingly the non-co-expressed EM KOs have higher number of shared DMRs (41) than co-expressed ones (2). When we compared our results to changes in DNAm during normal differentiation of mNPCs, we identified examples that could be consistent with precocious differentiation (e.g., Tet3 and Emx2). However, the majority of changes (e.g., Fbxl17) do not fit that pattern or occur at a slightly different location. These results suggest that similar to Kabuki syndrome, a known disorder with precocious differentiation, other EM knockouts may also have changes in differentiation rates and thus differentiation rate may be a possible confounder in our analysis.
