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

Abstract

One Sentence SummaryASXL3 patient truncations cause BRS through GOF protein accumulation, not LOF, and ASO-mediated knockdown reverses disease transcriptional signatures. 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' 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.