Global transcriptional changes across multiple isogenic C9orf72 Patient iPSC-derived neurons

Hexanucleotide repeat expansions in C9orf72 are the most common genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal degeneration (FTD), yet the mechanisms underlying selective neuronal vulnerability remain unclear. A major challenge in identifying consistent transcriptomic changes across C9orf72 patient-derived lines has been due to heterogeneous differentiations, lack of isogenic controls and low sequencing depth. Here, we generated homogeneous cortical neurons (iCNs) from multiple isogenic C9orf72 patient iPSCs pairs and performed deep RNA sequencing. We identified robust and reproducible gene expression and splicing alterations in pathways related to cytoskeletal organization, extracellular matrix adhesion and synaptic signaling. Notably, we observed exon 30 skipping in FLNB, leading to the loss of its hinge domain. This was accompanied by altered FLNB localization, disrupted actin crosslinking and impaired mechano-transduction pathway. These findings reveal convergent transcriptomic and functional disruptions across multiple isogenic C9orf72 patient derived iCNs and offer new insights into ALS/FTD pathogenesis.