bioRxiv2026
Neuroligin-3 (NLGN3) was first identified as a risk gene associated with autism spectrum disorder (ASD). The initial variant, p.R451C, associating NLGN3 with ASD has been heavily investigated, yet little is known about the functional consequences of other NLGN3 variants. Furthermore, while most of the identified variants are present in males with maternally inherited variants from unaffected mothers, several de novo variants were observed in females, suggesting a possible functional difference between de novo and maternally inherited variants. To address the functional consequences of NLGN3 variants in vivo, we generated transgenic Drosophila models corresponding to one de novo variant (p.R175W) identified in one female proband, and two maternally inherited variants (p.R451C and p.R597W) identified in male probands. In Drosophila, loss of the fly homolog, Nlg3, altered sleep patterns, synaptic architecture, and vesicle dynamics, which were rescued by the expression of the human NLGN3Ref allele. When comparing the variants, the de novo p.R175W variant and the maternally inherited p.R451C variant altered synapse morphology and sleep patterns, with minimal effects on vesicle dynamics, and the p.R597W variant altered sleep and vesicle dynamics with minimal impact on synapse morphology. Using overexpression models, human NLGN3Ref altered sleep patterns and synaptic morphology. Moreover, the p.R175W variant exacerbated sleep phenotypes, and the p.R175W and p.R451C variants exacerbated synapse morphology phenotypes. Together, our findings suggest that de novo NLGN3 variants identified in females are likely gain-of-function, while maternally inherited variants have mixed loss-and gain-of-function effects. Moreover, the location of the variants may contribute to the distinct functional differences we observed. Some NLGN3 variants disrupt synaptic development, while other variants alter synaptic function, suggesting that NLGN3 variants have differential effects. These functional differences may provide insight into the heterogeneity of individuals with ASD. Author SummaryAutism spectrum disorder (ASD) is a common neurodevelopmental disorder. Mutations in the Neuroligin-3 (NLGN3) gene are associated with ASD but very few of these mutations have been characterized in animal models. Most of these mutations affect male individuals who maternally inherited their genetic mutation; however, more rarely female individuals may present with a genetic mutation that was not identified in either of the parents. Here, we utilized the fruit fly model to investigate how three different mutations, one mutation identified in a female and two mutations identified in males, affect the flys behavior and synapse development. We identified altered sleep patterns in some of our mutants which is consistent with sleep disturbances being highly comorbid with ASD. Additionally, we identified alterations in synapse development and function which is consistent with the role of NLGN3 in synapse formation and maturation. Together, our findings support that NLGN3 is important for regulating the synapse and mutations in this gene can alter its function. However, different mutations can have differential effects. This demonstrates the need to assess multiple variants simultaneously because each variant may have distinct functional significances.