Pathogenic NLGN4X variants reveal dual roles in synaptic connectivity and cortical development

Abstract Neuroligins are synaptic adhesion molecules important for neurodevelopment, and rare, highly penetrant variants underlie neurodevelopmental disorders (NDDs). Here, we characterize a rare missense variant NLGN4X G243R identified in a pedigree with NDDs and associated with severe protein trafficking deficits. NLGN4X G243R lies within a cluster of rare NLGN4X variants (amino acids 235-245), all of which impair trafficking. Using biochemical, imaging, and electrophysiological approaches, we find that these NLGN4X variants exhibit severe trafficking defects, leading to impaired cell surface localization and synaptogenesis. Structural modeling further suggests that the NLGN4X NDD-variants destabilize protein conformation which is supported by thermal stability assessment. Notably, we uncover a previously unrecognized role for NLGN4X in regulating neuronal migration during early brain development, extending its functional scope beyond synaptogenesis. Collectively, these results show that NDD-associated NLGN4X variants disrupt both synapse formation and neuronal migration, offering mechanistic insight into their contribution to neurodevelopmental pathology and highlighting potential therapeutic avenues.