Investigating the role of rare missense variants in RAB11B in Autism Spectrum Disorder

Background Autism spectrum disorder (ASD) is one of the most prevalent developmental disorders worldwide and is characterized by a strong genetic basis. Its clinical and genetic complexity has greatly encouraged the genomic investigation of the disorder, especially in recent years. Our research group recently published a genomic study of a monocentric family-based cohort of 116 ASD families, including 144 autistic children. One of the ASD individuals from this study carried a de novo missense variant in the Rab GTPase RAB11B (p.Arg33His), predicted to be damaging. Given the growing interest around this gene, recently implicated in a rare form of severe intellectual disability (MIM #617807), we decided to functionally characterize the missense variant from our cohort (p.Arg33His), alongside two other missense variants reported in the literature (p.Arg72Cys and p.Asp157Asn), also predicted pathogenic but not functionally tested. Methods First, we performed an in silico analysis of the effect of each variant on active and inactive RAB11B using a known molecular 3D-modeling software. The in-silico assessments were followed by an in-vitro functional study of overexpressed mutant recombinant proteins, which investigated their localization and function during primary cilium outgrowth through a series of immunofluorescence assays. One-way analysis of variance (ANOVA) statistical test, followed by Tukey multiple comparisons, was implemented to evaluate the statistical significance of observations. Results Results from the three-dimensional simulation of the RAB11B missense variant from our cohort (p.Arg33His) further suggested pathogenicity, while the 3D modeling of the other two variants gave inconclusive results. These initial computational data were further validated by immunofluorescence assays, indicating a loss-of-function effect only for the de novo missense variant (p.Arg33His) identified in the autistic individual from our cohort, which resulted in incorrect cellular localization of the Rab protein and interference in primary cilium outgrowth. Conclusions This study highlights the importance of functional characterization of RAB11B missense variants to validate pathogenic computational predictions. Moreover, by highlighting RAB11B as a possible ASD risk gene, it expands the neurodevelopmental spectrum of RAB11B-related disorders.