Heterozygous loss-of-function variants in Neurabin I (PPP1R9A), responsible for encoding a cytoskeletal scaffolding protein essential for synaptic plasticity, are recurrently associated with neurodevelopmental and neuropsychiatric disorders, yet their direct effects on human neuronal maturation remain unclear. Here, we establish the first comprehensive human mechanistic model of PPP1R9A haploinsufficiency using an isogenic CRISPR/Cas9-engineered iPSC system differentiated into cortical neurons to define dosage-dependent functional consequences. PPP1R9A+/- neurons exhibited pronounced hyperspinogenesis and increased neuritic complexity, indicative of aberrant structural maturation; however, whole-cell patch-clamp recordings revealed impaired intrinsic excitability, including reduced action potential firing, altered waveform properties, and defective axo-somatic coupling, uncovering a striking dissociation between neuronal morphology and function. Long-read single-cell transcriptomics and quantitative proteomics identified coordinated downregulation of ion channel and synaptic transmission pathways, including genes essential for sodium channel function and glutamatergic signaling, together with disruption of synaptic vesicle cycling, axon guidance, and neurodevelopmental programs. Pseudotime trajectory analysis further demonstrated delayed neuronal differentiation, with mutant neurons accumulating at intermediate developmental states rather than acquiring mature cortical identities. Importantly, molecular rescue experiments confirmed causality, as restoration of full-length PPP1R9A expression robustly normalized transcriptional and synaptic signaling programs, whereas allele-specific antisense oligonucleotide-mediated suppression of the mutant transcript achieved only partial rescue. Taken together, these findings establish PPP1R9A haploinsufficiency as a driver of impaired molecular, electrophysiological, and developmental maturation in human cortical neurons, providing a human-specific mechanistic framework linking reduced Neurabin I dosage to neurodevelopmental and psychiatric disease risk.
Consanguinity increases the risk of autosomal recessive disorders and may result in the co-segregation of multiple pathogenic variants within the same family. Although most affected families are explained by a single genetic diagnosis, multilocus pathogenic variation can produce complex and overlapping clinical phenotypes. We investigated a consanguineous Pakistani family with three affected siblings, including dizygotic twins presenting with neurodevelopmental disorder and hearing loss, using detailed clinical evaluation, long-read whole-genome sequencing, bulk transcriptomics, protein profiling, and segregation analysis to determine the underlying molecular diagnoses. One sibling presented with isolated non-syndromic hearing loss, whereas the dizygotic twins exhibited severe neurodevelopmental impairment characterized by global developmental delay, spastic quadriplegic cerebral palsy, microcephaly, and white matter abnormalities. Long-read whole-genome sequencing identified a homozygous start-loss variant in HPDL (c.3G>C) in both twins, consistent with HPDL-related neurodevelopmental disorder with progressive spasticity and brain white matter abnormalities (NEDSWMA). In addition, a novel homozygous nonsense variant in EPS8 (c.1294C>T) was identified in one twin and the sibling with isolated hearing loss, explaining the auditory phenotype. Long-read transcriptomic analysis demonstrated absence of detectable EPS8 transcripts in both individuals homozygous for the nonsense variant, providing transcript-level evidence consistent with a loss-of-function mechanism. Genome-wide comprehensive proteomic profiling (SomaScan) identified distinct protein abundance profiles across family members, with the most pronounced alterations observed in the twins affected by HPDL-related neurodevelopmental disease, particularly the individual harboring pathogenic variants in both EPS8 and HPDL. This study expands the mutational spectrum of EPS8 and highlights the independent segregation of two autosomal recessive disorders within the complex consanguineous family, resulting in distinct and blended phenotypes.
Bassam Jamalalail, Ahmed A. Khalifa, Bipin Balan et al.· bioRxiv· 0 citations