KCNB1 mutation impairs neuronal differentiation by disrupting gene expression temporal regulation and neuron-specific pathways

Introduction This study aims to rigorously evaluate the consistency and reliability of a pluripotent stem cell (PSC) differentiation system and explore how the KCNB1 mutation disrupts the temporal regulation of gene expression during neuronal differentiation and modulates neuron function-related pathways. Methods Induced pluripotent stem cells (iPSCs) derived from a patient carrying a KCNB1 variant (c.990G > T, p.Glu330Asp) and from a healthy donor were differentiated into neurons. Differentiation and RNA expression were assessed at multiple time points. Immunofluorescence, RNA sequencing, fuzzy c-means clustering, and pathway analyses were performed. Results The differentiation system was successfully established, with cells exhibiting stage-appropriate morphology and maturing into neurons. RNA sequencing revealed consistent gene expression patterns at the neural progenitor cell (NPC) stage but significant differences at the neuron stage between the KCNB1 mutant patient and the healthy donor. Notably, KCNB1 expression was lower in the patient’s neurons. Genes specifically clustered in healthy neurons were enriched in synapse-related pathways, while genes clustered in patient neurons were associated primarily with basic cellular metabolism pathways and abolished neuron-specific pathways. Conclusion Low expression of KCNB1 disrupts the temporal pattern of gene expression and related neuron-specific pathways during neuronal differentiation and impairs neuronal differentiation and maturity.