Butyrate modifies epigenetic and immune pathways in peripheral mononuclear cells from children with neurodevelopmental disorders associated with chromatin dysregulation

Pathogenic DNA variants in chromatin-related genes cause an important minority of neurodevelopmental disorders (NDDs). Epigenetic mechanisms, including chromatin regulation, are associated with NDD etiopathogenesis. Therapeutic strategies targeting chromatin dysregulation, such as histone deacetylase inhibition with butyrate, show promise; however, its effects remain poorly understood. We performed single-cell RNA sequencing (scRNA-seq) on 101,539 peripheral immune cells from four children with functionally impairing NDDs (median age 11.2 years,; 3 females): three with de novo pathogenic variants in chromatin-related genes (KMT2D, CHD7, and MECP2), and one without a monogenic diagnosis (non-monogenic), compared with two sex-matched healthy controls (median age 12.5 years, 1 female). Patient cells also underwent scRNA-seq after in vitro butyrate treatment. We identified 136 to 6909 differentially expressed genes (DEGs) across patient comparisons. Untreated patient cells showed dysregulation of ribosomal and immune pathways compared to controls. KMT2D, CHD7, and the non-monogenic patients exhibited downregulation of ribosomal pathways, and upregulation of immune pathways, whereas the MECP2 patient displayed upregulation of ribosomal pathways and mixed regulation of immune pathways. Butyrate largely reversed these pathways, normalizing ribosomal and immune pathways in KMT2D, CHD7, and non-monogenic cells, with partial effects in MECP2. Overall, butyrate induced up-regulation of ribosome, GTPase, cytoskeletal, mitochondrial pathways, and down-regulation of epigenetic and immune pathways. This is the first study to demonstrate that butyrate modulates epigenetic and immune gene networks in monogenic and non-monogenic NDDs. The shared ribosomal-immune RNA signature suggests a common downstream mechanism of chromatin dysregulation, positioning butyrate as a promising therapeutic modulator across diverse NDDs.