Decoding the RNA Splicing Network in HNRNPH2-R114W Brain Organoids

ABSTRACT Autism spectrum disorder and related neurodevelopmental diseases are increasingly linked to disrupted RNA processing during corticogenesis, yet resolving variant-specific mechanisms during early human development remains challenging. We generated isogenic cerebral organoids from a patient iPSC line carrying the HNRNPH2-R114W mutation and its CRISPR-corrected counterpart. Developmental validation by time-course imaging, followed by bulk RNA-Seq at day 20, showed comparable early differentiation and corticogenesis. Genotype was the primary determinant of global expression state, with organized remodeling of neuronal and metabolic programs. Splicing analyses demonstrated junction-dominant dysregulation, where 6,310 junctions exhibited significant differential usage. Junction-level resolution revealed a dominant genotype-related signal than exon-centric analysis. A structural docking model shows HNRNPH2 RNA recognition motifs 1-3 within a spliceosomal context and position residue R114 at an RNA-contact cleft, consistent with direct disruption of RNA engagement by R114W. Integrating differential expression with event- and isoform-level evidence prioritized convergent regulatory hubs enriched for axon guidance, synaptic signaling, and extracellular matrix programs. Single-cell RNA-Seq deconvolution revealed modest compositional shifts that do not account for the magnitude of transcriptional and splicing alterations. Collectively, these data support that HNRNPH2-R114W drives pervasive, junction-centered RNA rewiring in human cortical organoids and defines isoform-level endpoints for mechanistic studies and therapeutic testing in an isogenic developmental model.