Inactivating mutations in polyadenylation factor CSTF2 disrupt intronic polyadenylation patterns and are associated with intellectual disability.

Alternative polyadenylation contributes extensively to transcript diversification and is particularly prominent during neuronal differentiation; however, its contribution to monogenic neurodevelopmental disorders remains incompletely understood. CSTF2 encodes an RNA-binding subunit of the cleavage stimulation factor complex and has previously been associated with X-linked intellectual disability through a single Asp50Ala missense variant with increased RNA-binding activity. Here, we identify seven rare CSTF2 variants in ten hemizygous male probands with intellectual disability, global developmental delay and/or autism spectrum disorder, including missense, nonsense, splice-site, and frameshift variants. Functional analysis of selected variants using RNA sequencing and 3′-end-proximal sequencing in CSTF2–depleted cells identified Phe19Leu, Gln543ter, Leu555Ser as strongly functionally impairing variants. These variants predominantly reduced usage of intronic polyadenylation sites while increasing usage of downstream sites within terminal 3′UTRs. Suppressed intronic sites were enriched for GU-rich sequence elements, were more evolutionarily conserved, and showed higher predicted polyadenylation–site strength, consistent with preferential impairment of CSTF2-dependent polyadenylation. In contrast, the previously reported Asp50Ala variant produced an opposing pattern, with preferential activation of intronic polyadenylation sites, consistent with its increased RNA-binding activity. These findings indicate that both reduced and increased CSTF2 activity disrupt the balance of polyadenylation–site usage and can result in neurodevelopmental disorder. Our data provide genetic and functional support for CSTF2 as an X–linked neurodevelopmental disorder gene, identify inactivating CSTF2 variants as a disease mechanism, and implicate dysregulation of intronic polyadenylation in human intellectual disability.