ASXL3 truncating patient variants mediate transcriptional gain-of-function and are antisense oligonucleotide-responsive

Truncating variants in the human Additional sex combs (asx) ASXL genes are frequent in clonal hematopoiesis and severe dominant neurodevelopmental syndromes, yet are assumed to represent loss-of-function (LOF) alleles. However, numerous LOF alleles are documented in healthy individuals. Here we show ASXL3 patient truncations in neurodevelopmental condition Bainbridge-Ropers syndrome (BRS), by virtue of their distinct location in the gene body, instead mediates gain-of-function (GOF) by escaping nonsense-mediated decay and Cullin 4-dependent degradation, resulting in aberrant protein accumulation, widespread transcriptional dysregulation, and altered chromatin accessibility. Patient-derived cell lines partnered with CRISPR knock-in of patient versus population truncations excluded simple haploinsufficiency, and instead support this two-hit GOF mechanism. Deletion mapping identified a broad C-terminal destabilizing region, explaining the 3-prime clustering of benign truncations. Haploinsufficiency was further excluded by forced expression of full length ASXL3 in patient lines, which failed to rescue the disease-associated differential gene expression signature. By contrast, antisense oligonucleotides targeting ASXL3 largely normalized this signature, providing a mechanistic rationale for knockdown therapies in ASXL associated disease.