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

Investigating the role of rare missense variants in RAB11B in Autism Spectrum Disorder | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Investigating the role of rare missense variants in RAB11B in Autism Spectrum Disorder Marta Viggiano, Laura Sandoni, Fabiola Ceroni, Paola Visconti, and 6 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7509053/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 7 You are reading this latest preprint version Abstract 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. RAB11B Rab GTPases Immunofluorescence Autism ASD Neurodevelopment primary cilium Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Background Autism Spectrum Disorder (ASD) is a clinically heterogeneous neurodevelopmental disorder with an early onset, affecting over 1% of worldwide population ( 1 ). It is characterized by a set of core symptoms, including impairments in social communication, as well as repetitive behaviors and restricted interests ( 2 ). Twin and family studies have shown that ASD has a strong genetic basis, with heritability estimates ranging from 40–90% ( 3 ). More recently, large-scale genomic studies have highlighted its complex genetic architecture, resulting from the interaction between common polygenic variation and both inherited and de novo rare variants. Analysis of high-impact de novo mutations, responsible for at least 10–30% of ASD cases, have led to the identification of hundreds of high-confidence ASD risk genes, many of which are also implicated in other neurodevelopmental disorders (NDD) ( 4 , 5 ). This overlap provides evidence of shared genetic risk and molecular mechanisms across disorders and suggests that additional factors, such as polygenic background or stochastic developmental variation, contribute to the phenotypic diversity observed across individuals. RAB11B encodes a small Rab guanosine triphosphatase (GTPase) involved in intracellular transport and endosomal trafficking. It plays essential roles in many neurodevelopmental processes, such as ciliogenesis, neurite outgrowth, and synapse formation ( 6 ). De novo heterozygous missense variants in RAB11B have been recently associated with a neurodevelopmental disorder characterized by severe intellectual disability (ID), ataxic gait, absent speech, and decreased cortical white matter (NDAGSCW, MIM #617807). To date, seven distinct RAB11B disease-associated variants have been identified in 13 NDAGSCW cases ( 7 , 8 ). Among these, three variants (p.Gly21Arg, p.Val22Met, and p.Ala68Thr) are recurrent, reported in 2, 4, and 2 individuals, respectively. Clinical presentation varies among the reported cases, though intellectual disability is the most consistent feature. Molecular modeling has suggested that variants affecting the nucleotide binding pocket result in more severe phenotypes with intellectual disability, while those outside the binding region, are associated with a milder phenotype with epilepsy. ASD is not a commonly reported manifestation in NDAGSCW, and the potential involvement of RAB11B variants in ASD remains unexplored. As part of a previous genomic sequencing study of 435 individuals from 116 ASD families ( 9 ), we identified a de novo missense variant in RAB11B in an individual with ASD and mild ID. Since ASD is not a recognized core feature of NDAGSCW, this finding prompted us to investigate whether rare RAB11B missense variants might contribute to ASD or to a broader spectrum of neurodevelopmental phenotypes beyond severe ID. To explore this hypothesis, we reviewed the literature and public variant databases for additional rare RAB11B missense variants in individuals with ASD and related conditions. We identified two further uncharacterized interesting variants, one in an individual with ASD and another in an individual with mild ID. The aim of this study is to determine whether these three rare RAB11B missense variants may contribute to ASD or related neurodevelopmental phenotypes, affecting RAB11B protein function. Therefore, we combined in silico and in vitro approaches, including protein structure modeling and subcellular localization assays in cells expressing recombinant mutant proteins. Methods Cases Individual 1 is part of an Italian ASD cohort (proband 52.3) ( 9 ). Individual 2 was recruited into the DDD Study (patient 385827) ( 10 ). Individual 3 belongs to the MSSNG ASD dataset (individual REACH000582) ( 5 ). RAB11B variant analysis The three RAB11B missense variants analyzed in this study were previously identified by different whole exome/genome sequencing (WES/WGS) studies ( 5 , 9 , 10 ). Variants were annotated using ANNOVAR ( 11 ) with data from gnomAD v4.1.0 ( 12 ), InterVar ( 13 ) and dbNSFP 4.7 ( 14 ). Their potential pathogenicity was also evaluated using AlphaMissense ( 15 ), considering the mean AM score for protein residue and the variant-specific pathogenicity score. Molecular modeling Structural modeling and simulation of the three RAB11B missense variants were performed using YASARA software ( 16 ), based on the crystallography-resolved 3D structure of active and inactive forms of human RAB11B (PDB entry: 2F9M and 2F9L, respectively). Generation of WT and Mutant Plasmid Constructs The coding sequence of RAB11B (NM_004218.4) was obtained from total cDNA isolated from the SH-SY5Y cell line (ATCC, Rockville, MD) by PCR amplification. This was then cloned into the p3xFLAG-CMV-10 mammalian expression vector, in frame with the 3xFLAG epitope, to generate the recombinant wild-type (WT) RAB11B construct. Mutations [p.(Ser25Asn), p.(Arg33His), p.(Gln70Leu), p.(Arg72Cys), p.(Asp157Asn)] were introduced into the WT RAB11B coding sequence by multi-step site-directed mutagenesis, using the KAPA HiFi HotStart PCR Kit. Primers sequences used for mutagenesis are listed in Supplementary Information (Table S1 ). Both WT and mutant constructs were verified by Sanger Sequencing. Cell culture and transient transfection ARPE-19 cells (ATCC, Rockville, MD) were cultured on glass coverslips in 6-well plate in DMEM/F-12 supplemented with 10% fetal bovine serum and 0.05 mg/ml penicillin-streptomycin, at 37°C in a 5% CO 2 humidified atmosphere. Cells were transiently transfected with 2.5 µg of plasmid constructs using 3.75 µl of Lipofectamine 3000 Transfection Reagent in fresh culture media. Six hours after transfection, media containing lipid nanoparticles was replaced with DMEM/F-12 complete medium and cells were maintained at 37°C in a 5% CO 2 humidified atmosphere. GFP coding plasmid was used as positive transfection control, while a p3xFLAG-CMV-10 empty vector was used as immunofluorescence negative control. Twenty-four hours after transfection, cells were washed three times in DPBS and serum-starved in serum-free DMEM-F12 for forty-eight hours in order to induce cells quiescence and the primary cilium assembly ( 18 ). Immunofluorescence assay Forty-eight hours after serum starvation, cells were fixed for 15 minutes with 4% paraformaldehyde in DPBS, washed twice in DPBS, permeabilized for 15 minutes at room temperature (RT) with 0.1% Triton X-100 in DPBS, and incubated with blocking solution (4% normal donkey serum (Jackson ImmunoResearch) and 0.05% tween in PBS) for 1 hour at RT. For the staining, cells were then incubated over-night at 4°C with specific primary antibody: mouse anti-FLAG M2 antibody (1:1000, Sigma-Aldrich), rabbit anti-GOLGA2 antibody (1:200, Sigma-Aldrich), rabbit anti-PCM1 antibody (1:200, Sigma-Aldrich) or rabbit anti-acetyl-a-tubulin antibody (1:500, Sigma-Aldrich). After three 0.05% tween-DPBS washing step, cells were incubated with secondary antibodies for 1 hour at RT: goat anti-mouse Cy3 antibody and goat anti-rabbit FITC antibody (1:400, Jackson ImmunoResearch). Samples were washed with DPBS and nuclei were stained with 1µg/ml Hoechst. Coverslips were mounted on glass slides using Fluoromount G. Images were taken using a confocal microscope Nikon A1R + and the NIS-elements software (Nikon). Co-localization and statistical analysis Co-localization analysis based on Pearson’s correlation coefficient (r) and the determination of cilium length were performed using the Coloc-2 plugin and the line tool of Fiji’s, respectively. Statistical analysis was performed using the one-way analysis of variance (ANOVA) followed by Tukey’s multiple comparison test. Results Identification of RAB11B missense variants By performing WGS in a cohort of 116 ASD families including 144 affected individuals, we identified a heterozygous de novo missense variant in RAB11B (p.Arg33His) in a male proband of a trio with unaffected parents ( 9 ). In addition to the ASD diagnosis, the proband (Individual 1) had mild ID and speech delay. MRI at 4 years of age did not reveal any cortical anomaly. Detailed phenotypic description of Individual 1 is reported in Supplementary Information. The variant, located in exon 2, affects an arginine residue that is highly conserved in all Rab subfamily members and across species. Multiple in silico tools predict this variant to be highly deleterious. According to InterVar ACMG guidelines, the variant was classified as likely pathogenic (Table 1 ). Table 1 Main clinical features of individuals with RAB11B missense variants. Genomic change a Amino acid change b gnomAD v.4.1.0 AF total (allele count) Variant prediction score Inheritence (Individual ID) ACMG class c Other genetic variants Clinical details d MPC CADD phred AM PhyloP100 vertebrates g.8399920G > A p.Arg33His 6.2x10 − 7 ( 1/1614208) 2.6 34 0.84 10.003 De novo (Individual 1) LP (PS2,PM1,PM2,PP2) Paternal VUS CNV (NC_000024.9: g.(495953_1264792)dup) Autism (ADOS-2 score: 6; CARS2-ST score: 39); Language level: single words; Mild ID (Leiter-R, B-IQ 56, FR 63); MRI: normal; Focal epilepsy g.8400036C > T p.Arg72Cys 6.2x10 − 7 (1/1613548) 2.74 34 0.99 4.892 Paternal (Individual 2) LP (PM1,PM2,PP2,PP3) Maternal VUS CNV (NC_000023.10: g.68348165_68467987dup) Language level: normal speech; Mild ID (QI: 58; QI verbal: 62); MRI: normal; Obesity. g.8402523G > A p.Asp157Asn novel 1.891 26.3 0.82 9.994 De novo (Individual 3) LP (PS2,PM1,PM2,PP2) De novo pathogenic SNV (NM_001308120.2(TOGARAM1): c.5011delT:(p.F1671fs)) ASD; ADHD; Anxiety; Gastrointestinal comorbidities. a Relative to NC_000019.10 (hg38); b (NM_004218.4); c InterVar Classification according to the ACMG guidelines, together with detailed evidence codes, LP likely pathogenic, VUS variant of uncertain significance; d B-IQ Brief IQ; FR Fluid Reasoning. While RAB11B was recently implicated as causative gene in a novel NDD, no direct association with ASD susceptibility has been described so far. To investigate whether other rare RAB11B missense variants have been reported in individuals with ASD, we analyzed the largest WES/WGS studies on ASD cohorts published to date ( 4 , 5 ). We identified an additional de novo missense variant in RAB11B , p.Asp157Asn, novel and predicted to be deleterious (Table 1 ). This variant was reported in one ASD individual from a WGS study of a cohort of 4,258 ASD families ( 5 ). We also explored the public database DECIPHER and identified another RAB11B missense variant, p.Arg72Cys, also predicted to be damaging. This variant was identified in a 12-years old boy with ID and was inherited from his father, who was reported to have mild ID. Notably, the same variant was reported as de novo variant in a 5-years old female with seizures and delayed speech abilities ( 8 ) (Table 1 ). The p.Arg33His and p.Arg72Cys variants are ultra-rare, each present in the gnomAD v.4.1.0 dataset with total Allele Frequency (AF) < 10 − 4 , while the p.Asp157Asn variant is novel. None of the three RAB11B missense variants has been previously functionally characterized. Although the three identified missense variants are located in different regions of the gene, all map within or in close proximity to protein functional domains. This is consistent with previously reported disease-associated missense variants in RAB11B ( 7 , 8 ). Specifically, RAB11B contains five conserved sequence motifs forming the GDP/GTP-binding domain, as well as two switch regions (Switch I and Switch II), which regulate the conformational change between inactive and active forms and mediate interactions with coenzymes and effector proteins ( 17 , 19 , 20 ). The p.Arg33His and p.Arg72Cys variants are both located in the N-terminal half, with p.Arg33His immediately adjacent to the Switch I region and p.Arg72Cys within the Switch II region. Instead, the p.Asp157Asn maps in the C-terminal half, adjacent to the final G box motif involved in the GDP/GTP binding. Moreover, two of the three identified missense variants are located in close proximity to the GDP-blocking p.Ser25Asn (constitutively inactive) and GTP-blocking p.Gln70Leu (constitutively active) variants, which were generated in vitro to replicate known functional mutations of other Rab proteins, and whose disruptive effects have been validated in numerous experimental settings ( 21 , 22 ) (Fig. 1 ). Functional characterization of missense variants in RAB11B In order to identify mutational hot/cold spots within the RAB11B protein, we evaluated the AlphaMissense (AM) predicted average pathogenicity score for each amino acid residue, together with the frequency of missense variants in gnomAD v4.1.0. High AM pathogenicity scores were widely distributed across the N-terminal and the central region of the protein, while C-terminal residues exhibited lower AM scores, suggesting a reduced constraint against missense variation in this region. Consistent with this pattern, only a limited number of RAB11B missense variants reported in the general population involves the N-terminal protein region and its functional domain, while the majority of variants are clustered in the C-terminal region, where the residues tend to have lower mean AM score and high alternate allele counts (Fig. 1 ). Interestingly, the three identified missense variants overlap with regions showing high AM pathogenicity score. However, the specific AM scores for each of these amino acid substitutions are similar, making it difficult to infer relative pathogenicity based on in silico prediction alone. Therefore, we decided to functionally characterize these variants. - Simulation of structural consequences of identified RAB11B missense variants To evaluate the potential effects of the three identified missense variants on RAB11B protein function, we first modeled each amino acid substitution using YASARA software. Specifically, we introduced the three substitutions into the three-dimensional structures of both the active (GTP-bound) and inactive (GDP-bound) forms of human RAB11B protein. The most relevant structural perturbation was caused by the de novo p.Arg33His variant. In the active form, the side chain of arginine 33 forms a salt bridge with glutamate 35, located at the N-terminal end of the Switch I region. This interaction is mediated by the side chain orientation typical of arginine in the active state, which changes upon transition to the inactive form. Replacement of arginine 33 with histidine disrupts this bond, leading to destabilization of the active protein conformation. This likely affects the stabilizing interactions that Switch I domain establishes with GTP and Mg 2+ ( 23 ) (Fig. 2 a). In contrast, the p.Arg72Cys variant primarily affects the inactive, GDP-bound protein form. In this conformation, arginine 72 forms two hydrogen bonds with glycine 69, through the amino group of the backbone and the side chain, respectively. When arginine 72 is replaced by a cysteine, one of this two hydrogen bonds is disrupted, due to the different size and composition of the cysteine side chain. However, the backbone-mediated hydrogen bond is maintained, likely preserving the overall structural conformation of the inactive form (Fig. 2 b). The third missense variant, p.Asp157Asn, does not appear to cause any significant structural perturbations. The hydrogen bond between aspartate 157 and threonine 159 typical of the RAB11B GTP-bound form is maintained when replacing the aspartate with an asparagine, probably due to the chemical and structural similarity between the two residues (Fig. 2 c). To validate our modeling approach, we also simulated the previously characterized dominant negative p.Ser25Asn (GDP-bound) and constitutively active p.Gln70Leu (GTP-bound) RAB11B variants. Substitution of serine 25 with asparagine prevents the interaction with the Mg 2+ ion essential for GTP binding, while replacing glutamine 70 with leucine causes a severe disruption of the inactive-state conformation, consistent with their known functional impact (Supplementary Fig. 2). - Evaluation of functional consequences of identified RAB11B missense variants To assess whether the identified missense variants could affect RAB11B functionality, particularly impairing the protein activation/inactivation cycle, we evaluated variant effect on RAB11B subcellular localization during the primary cilium assembly. Indeed, RAB11B intracellular localization changes according to its active or inactive state, co-localizing with the peri-centrosomal region as GTP-binding RAB11B or with the Golgi apparatus as GDP-binding RAB11B ( 24 , 25 ). We performed immunofluorescence assays by overexpressing 3xFLAG-RAB11B recombinant mutated proteins in ARPE-19 cells and analyzing their co-localization with the Golgi network and the centriole during ciliogenesis. In our analyses we also included the two RAB11B variants of known effect, p.Ser25Asn and p.Gln70Leu, analyzing cellular localization for six different recombinant 3xFLAG-RAB11B proteins and using GOLGA2 and PCM1 as specific marker for the co-localization analysis with the Golgi apparatus and the pericentrosomal region, respectively. Wild-type (WT) recombinant protein showed homogeneous localization throughout the cytoplasm with punctuated distribution, typically due to the association of the active form with endosomes membrane. The same distribution pattern is recognizable for RAB11B p.Gln70Leu and the three identified missense variants, while p.Ser25Asn mutant protein appeared generally diffused in the cytoplasm, as expected for inactive RAB11B (Fig. 3 a- 4 a). Quantitative analysis of co-localization patterns by Pearson’s correlation coefficient, confirmed the altered localization of the constitutively active and inactive variants, as previously reported ( 7 ), and revealed that one of the newly tested variants also leads to altered protein cellular localization. Specifically, both p.Ser25Asn and Gln70Leu displayed a higher co-localization with the Golgi network compared to recombinant WT RAB11B (Fig. 3 b- 4 b), while quantification of the co-localization of FLAG and PCM1 signals highlighted the GDP-blocking p.Ser25Asn variant as the only variant showing reduced centriole co-localization, as previously found ( 7 ) (ANOVA p-value = 1.4x10 − 8 , WT vs p.Ser25Asn Tukey’s multiple comparisons test p-value = 3x10 − 8 ; Fig. 4 b). Looking at the co-localization with GOLGA2, the de novo variant identified in our ASD cohort, p.Arg33His, also caused protein mislocalization with a significant enrichment in the Golgi network (ANOVA p-value = 1.8x10 − 10 , WT vs p.Arg33His Tukey’s multiple comparisons test p-value = 0.004; Fig. 3 b- 4 b). No significant localization difference was identified for the other two tested missense variants found in ASD/NDD patients, in accordance with milder or absent structural perturbations emerged from YASARA prediction (Fig. 3 b- 4 b). Given the central role of RAB11B in the initiation of the primary cilium assembly, we also assessed the effect of the identified missense variants on the primary cilium morphology. Cells transfected with the WT and the other mutated proteins showed a comparable cilium morphology, with no marked alterations, except for cells expressing the constitutively inactive p.Ser25Asn and the de novo p.Arg33His variant, both of which displayed shorter cilia compared to the other tested variants (Fig. 5 a). This observation was confirmed by quantitative analysis of cilium length, which revealed a significantly reduction in the cilium length in cells expressing these two RAB11B mutants (ANOVA p-value = 0.002, WT vs p.Arg33His, Tukey’s multiple comparisons test p-value = 0.03; WT vs p.Ser25Asn, Tukey’s multiple comparisons test p-value = 0.005 Fig. 5 b). Discussion Rab proteins are a family of over 60 different GDP/GTP molecular switches known to be master regulators of membrane protein trafficking in nerve cells ( 26 , 27 ). The RAB11B gene encodes a small Rab protein ubiquitously expressed in human tissues, and particularly enriched in brain, heart and testis ( 28 ). RAB11B is an important modulator of cellular transport, responsible for the delivery of cellular components or signaling molecules to specific locations in the cell ( 29 ). Notably, it acts as positive regulator for relevant aspects of mammalian neurodevelopment, such as neurite outgrowth, migration of cortical neurons, synaptic plasticity, and glial differentiation and myelination ( 6 ). Moreover, RAB11B plays a key role in primary cilium formation, promoting ciliogenesis initiation ( 30 ). The primary cilium is a sensory microtubule-based organelle present on the cell surface of most mammalian cells, including neurons, whose nucleation starts from a modified centrosome surrounded by pericentriolar material and centriolar satellites, and involves Golgi-derived vesicles ( 31 – 33 ). Non-motile primary cilium is essential for intracellular communication, excitability and signaling ( 34 , 35 ). Cilium disfunction due to mutations in primary cilium genes results in a wide range of clinical conditions called ciliopathies. Interestingly, several studies focusing on high-penetrant rare variants have highlighted an overlap between risk genes associated with ciliary disfunction conditions and high-confidence ASD genes, strongly suggesting the relevance of cilium to ASD and other NDD ( 36 ). RAB11B has not yet been directly implicated in ASD, however some of its regulators/interactors were proposed as contributors to the ASD phenotype, further indicating the involvement of the GTPase in ASD pathogenesis ( 37 – 39 ). Due to the identification of two recurrent de novo missense variants (p.Val22Met and p.Ala68Thr) in five unrelated individuals with severe NDD and specific brain abnormalities, RAB11B was linked to NDAGSCW, a new form of neurodevelopmental disorder with intellectual disability, ataxic gait, absent speech and decreased cortical white matter. Both in silico and in vitro studies were performed to fully characterize the two variants, demonstrating their pathogenic effect on the normal protein function by altering the GDP/GTP binding site ( 7 ). Subsequently, Ahmad et al. reported five additional RAB11B missense variants in six NDD individuals and attempted to establish correlations between molecular modeling prediction and phenotype severity. Specifically, variants located in the nucleotide-binding pocket appear to result in a more severe phenotype with ID, whereas those mapping to the Switch II are associated with milder phenotype ( 8 ). However, functional studies are warranted to confirm variants’ effects and the hypothesized correlation between variant location and phenotype severity. We recently identified an ultra-rare de novo RAB11B variant (p.Arg33His) in an individual with ASD, in addition to mild ID and speech delay ( 9 ). A different amino acid change of the same residue (p.Arg33Cys) had been previously reported in a 7-years-old boy with severe ID and significant speech delay with regression, but with no ataxic gait, seizures or brain anomalies ( 8 ). To investigate the contribution of RAB11B variants to ASD, we searched for additional RAB11B variants in ASD/NDD cohorts and identified a de novo missense variant, p.Asp157Asn, in an individual with ASD recruited into the MSSG cohort and an ultra-rare missense variant, p.Arg72Cys, in a family with ID recruited into the DDD study. While p.Asp157Asn is novel, p.Arg72Cys was previously reported as a de novo change in an 5-years-old with seizures and delayed speech abilities, but no formal intellectual disability, microcephaly or brain MRI abnormalities ( 8 ). Combined analysis of the Alpha Missense prediction score and the distribution of RAB11B variants in general population allowed us to identify “missense-intolerant” regions in the protein sequence, containing all three variants. However, structural modeling demonstrated that the p.Arg33His variant, situated in close proximity of the Switch I region, exerts the highest conformational impact on the 3D structure of the active protein, resulting in the loss of a salt bridge with the first residue of the molecular Switch I. This domain is a highly flexible loop that, through the interactions with guanine nucleotide exchange factors (GEFs), changes conformation between the inactive and active structure allowing for the interchange between GDP and GTP ( 19 ). Given the prime importance of this domain for the activation of RAB11B, by altering the switch I region flexibility and consequently reducing the stability of the active form, the de novo variant p.Arg33His is expected to affect nucleotide binding capability, similarly to the effect of a variant impairing the binding pocket. A milder effect was predicted for the other two missense variants. To further clarify their functional consequences, we decide to characterize the three missense variants with additional in vitro studies. Given the known RAB11B involvement in ciliogenesis initiation and its different intracellular localization during this process, we carried out co-localization analysis in ARPE-19 cells transiently expressing mutant Rab GTPase, to assess the impact of the missense variants in the context of primary cilium outgrowth. We detected altered cellular localization for the p.Arg33His variant, leading to a significant protein accumulation at the Golgi network. A similar effect was found in cells overexpressing the total loss-of-function p.Ser25Asn and the constitutively active p.Gln70Leu variant, in line with previous results ( 7 ). During ciliogenesis, RAB11B organizes vesicular trafficking of Rabin8-containing pre-ciliary vesicles from the trans-Golgi to the centrosome ( 40 ), switching from the inactive form bound to Golgi vesicles to the active form bound to pre-ciliary vesicles located at the centriole ( 24 , 25 , 30 , 40 , 41 ). Thus, the accumulation of the recombinant mutated protein at the Golgi apparatus could be interpreted as evidence of a disrupted cellular mechanism, as demonstrated by the dysregulation caused by the two known-effect variants. When examining co-localization with the basal body of the cilium, we observed a significant alteration in localization only in cells overexpressing the dominant negative p.Ser25Asn mutant. This variant, designed in vitro and not previously reported in patients, significantly impacts the nucleotide binding pocket, as the conserved serine in the G1 box is known to coordinate a Mg 2+ interaction essential for GTP binding ( 42 ). To date, a similar effect on centriole colocalization has been reported for only two de novo RAB11B variants (p.Val22Met and p.Ala68Thr) identified in individuals with severe neurodevelopmental symptoms, both directly affecting the conformation of the binding pocket ( 7 ). Finally, we examined primary cilium morphology by measuring cilia length. Interestingly, we found that Arg33His-mutant RAB11B resulted in significantly shorter cilia compared to the WT, similarly to Ser25Asn mutant, though with a weaker effect. The loss of the salt bridge between Arg 33 and Glu 35 caused by p.Arg33His variant may destabilize the otherwise structured GTP-bound Switch I, potentially reducing the stability of GTP binding. This could lead to the accumulation of inactive protein in the trans-Golgi and ultimately slow cilia growth. As further support of this possibility, a recent study demonstrated the critical role of phenylalanine 36 orientation during the transition to the active conformation catalyzed by the RabGEF SH3BP5, where Phe 36, together with Ile 44, forms a hydrophobic cap on the triphosphate nucleotide ( 19 ). Interestingly, a similar variant pathogenicity mechanism involving altered protein localization has recently been proposed for ASD-related proteins localized to neuronal primary cilia, which are implicated in regulating ciliogenesis. Notably, SYNGAP1 is one of these proteins, and it was recently shown that missense variants identified in ASD individuals lead to its mislocalization away from cilia ( 36 , 43 ). These results support the hypothesis that p.Arg33His is likely the causative variant underlying the ASD phenotype observed in individual 1. Moreover, no additional de novo SNV or CNV were identified in this individual. In Table 1 , we reported only a paternally inherited large duplication on the Y chromosome involving SHOX , as a previous study suggested an association between de novo SHOX microduplication and ASD, albeit with low penetrance ( 44 ). While we cannot fully exclude a potential contribution of this duplication to the individual’s overall genetic risk, our data strongly support a primary role for the RAB11B missense variant in the ASD phenotype. For the other two variants (p.Arg72Cys, p.Asp157Asn), we do not observe significant changes in the association of the mutant proteins with vesicular membranes or in cilium morphology, suggesting that they may have a more subtle effect, if any. The likely milder impact of p.Arg72Cys variant is in line with its mode of inheritance and the milder phenotype observed in individual 2 and the previously reported individual ( 8 ). This proband also carries a duplication on the X chromosome involving the first two exons of OPHN1 (Table 1 ). OPHN1 encodes a Rho GTPase-activating protein (RhoGAP) involved in the growth and stabilization of dendritic spines, and its loss of function is associated with Billuart-type X-linked syndromic intellectual developmental disorder (MRXSBL; MIM #300486) ( 45 ). Since OPHN1 is implicated in MRXSBL via a loss of function mechanism, and the Xq12 duplication in p.Arg72Cys proband involves only the first part of the protein without affecting any functional domain, the effect of the CNV remains difficult to interpret, though it may potentially contribute to an increased genetic risk background. Interestingly, by inspecting the genomic background of individual 3 who carries the p.Asp157Asn RAB11B variant, we discovered the presence of de novo frameshift variant in TOGARAM1 (Table 1 ). TOGARAM1 encodes a specialized protein involved in ciliogenesis, essential for the normal structure and function of primary cilia, and has recently been implicated in Joubert syndrome-37 (JBTS37), an autosomal recessive neurodevelopmental ciliopathy characterized by a distinctive hindbrain malformation and additional variable features ( 46 ). Interestingly, individuals with Joubert Syndrome and other genetic ciliopathies frequently show autistic features ( 47 ), suggesting that ciliary disfunction may contribute to a subset of ASD cases. Although the ASD individual from the MSSNG cohort carries a heterozygous TOGARAM1 protein truncating variant, which contrasts with the recessive mechanism underlying Joubert Syndrome and the apparent haplosufficiency of TOGARAM1 (pLI = 0, LOEUF = 0.72), the TOGARAM1 p.Phe1618fs variant is predicted to escape nonsense-mediated decay (NMD) according to the 50bp rule, likely resulting in a truncated protein that may exert a gain of function effect. Moreover, both RAB11B and TOGARAM1 are involved in ciliogenesis, and while no direct interaction emerges from STRING analysis, the two proteins were indirectly linked via RAB11FIP3 when additional nodes were included (Supplementary Fig. 3). Taken together, these findings suggest that neither de novo variant alone may be sufficient to cause the phenotype, but they may act synergically by affecting the same ciliary pathway, thereby contributing to a high-risk genetic background. Conclusion This study presents a combined in silico and in vitro functional characterization of three previously uncharacterized RAB11B missense variants (p.Arg33His, p.Arg72Cys, p.Asp157Asn). Despite similar pathogenicity predictions for all three variants, functional analyses of these changes based on the 3D structure of the protein ( in silico analyses) and its localization within the cell ( in vitro assays) indicated a loss of function effect for one variant (p.Arg33His), while the other two did not display detectable differences compared to the wild type protein. These findings underscore the critical importance of functional characterization for the accurate interpretation of this class of genetic variation. In addition, the identification of a deleterious RAB11B missense variant in a proband with a typical autistic presentation and only mild ID, contrasting with the severe ID and distinctive brain anomalies previously associated with RAB11B missense variants, represents the first association between missense variants in RAB11B and ASD. This expands the phenotypic spectrum and underscores, once again, the shared genetic risk and overlapping etiologies across different neurodevelopmental phenotypes. Moreover, our results bring further support to the involvement of ciliary dysfunction in ASD/NDD pathogenesis, fostering interest in genes involved in cilia biology and potential biomarkers of disrupted ciliary function. Taken together, our data strongly encourage further investigation of the role of RAB11B in neurodevelopment, supporting its broader involvement across the spectrum of NDDs, including autism. Abbreviations ASD: Autism spectrum disorder NDD: Neurodevelopmental disorder ID: Intellectual disability NDAGSCW: Neurodevelopmental disorder characterized by severe intellectual disability (ID), ataxic gait, absent speech, and decreased cortical white matter (MIM #617807) WES: Whole exome sequencing WGS: Whole genome sequencing WT: Wild-type AM: AlphaMissense ANOVA: Analysis of variance GDP: Guanosine diphosphate GTP: Guanosine triphosphate SNV: Single nucleotide variant CNV: Copy number variant Declarations Ethics approval and consent to participate The WGS study on our cohort of ASD families, including Individual 1, was approved by the local Ethical Committee (Comitato Etico di Area Vasta Emilia Centro, CE-AVEC; CE14060) and performed in compliance with all relevant ethical regulations. All participants or substitute decision makers provided a written informed consent to participate to the study. Consent for publication Written consent was obtained for publication of the photographs of individual 1 (Supplementary Fig. 1). Availability of data and materials WGS data generated from our cohort of ASD families, including Individual 1, are included in our previous publication and its supplementary information files (9). The RAB11B variant identified in Individual 2 is publicly available on DECIPHER (https://www.deciphergenomics.org/, ID: 385827). The MSSNG dataset is included in Trost et colleagues’ publication (5). The dataset supporting the conclusions of this article is included within the article and its additional file. Any additional information is available from the corresponding author upon request. Competing interests The authors declare that they have no competing interests. Funding This research was supported by #NEXTGENERATIONEU (NGEU) and funded by the Ministry of University and Research (MUR), National Recovery and Resilience Plan (NRRP), project MNESYS (PE0000006) – A Multiscale integrated approach to the study of the nervous system in health and disease (DN. 1553 11.10.2022) and project National Center for Gene Therapy and Drugs based on RNA Technology (CN00000041) (M4C2 – Action 1.4- Call “Potenziamento strutture di ricerca e di campioni nazionali di R&S” (CUP J33C22001140001)). Authors' contributions Formal analysis: M.V., L.S., E.B.; Funding acquisition: E.M., E.B.; Investigation: M.V., L.S., F.C., P.V., A.P., M.C.S., A.V., J.R.S., E.B., E.M.; Methodology: M.V., L.S., F.C., E.B.; Resources: P.V., A.P., M.C.S., A.V., J.R.S.; Software: M.V., L.S.; Supervision: E.B., E.M.; Visualization: M.V., L.S.; Writing-original draft: M.V., L.S.; Writing-review & editing: M.V., L.S., F.C., P.V., A.P., A.V., E.B., E.M. All authors approved the submission of this manuscript. Acknowledgements We are extremely grateful to all the families who have participated in this study. We gratefully thank Prof. Monica Baiula and Dr. Roberto Bernardoni for advising cell culture and immunofluorescence. This study makes use of data generated by the DECIPHER community. A full list of centres who contributed to the generation of the data is available from https://deciphergenomics.org/about/stats and via email from [email protected] . DECIPHER is hosted by EMBL-EBI and funding for the DECIPHER project was provided by the Wellcome Trust [grant number WT223718/Z/21/Z]. References Shaw KA, Williams S, Patrick ME, Valencia-Prado M, Durkin MS, Howerton EM, et al. Prevalence and Early Identification of Autism Spectrum Disorder Among Children Aged 4 and 8 Years - Autism and Developmental Disabilities Monitoring Network, 16 Sites, United States, 2022. MMWR Surveill Summ. 2025 Apr 17;74(2):1–22. 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01:40:05","extension":"png","order_by":13,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":183341,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-7509053/v1/40aa89159745103bcc283fc5.png"},{"id":94806857,"identity":"debc7e8d-1c3d-491e-baeb-23abba208f10","added_by":"auto","created_at":"2025-10-31 01:40:04","extension":"xml","order_by":14,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":130940,"visible":true,"origin":"","legend":"","description":"","filename":"afcb19cf4e9a4eee80262e12c479e73c1structuring.xml","url":"https://assets-eu.researchsquare.com/files/rs-7509053/v1/269d9b2b3b29a0e64ddd3934.xml"},{"id":94806860,"identity":"9e882c6c-d23c-4aaa-ba5a-b41dcf6ea851","added_by":"auto","created_at":"2025-10-31 01:40:04","extension":"html","order_by":15,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":141555,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-7509053/v1/57337cb9593f5d54879e73ec.html"},{"id":94806845,"identity":"6bdb7f0b-2661-4f13-a0e2-4c63d932d7a0","added_by":"auto","created_at":"2025-10-31 01:40:03","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":286109,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCombined analysis of the AM pathogenicity score and the distribution of RAB11B missense variants in general population. \u003c/strong\u003eThe predicted AM mean pathogenicity score together with the number of missense variants from gnomAD v4.1.0 are reported along the amino acid sequence (Q15907). The schematic representation of the protein structure highlights the overlap between functional domains, high AM scores and low number of reported variants. All tested variants fall within “missense-intolerant” regions. Red and black diamonds indicate variants of known effect and missense variants identified in ASD/NDD individuals, respectively. The AM variant-specific pathogenicity score is reported in bracket.\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-7509053/v1/948c0e755623ae2a13735cbb.png"},{"id":94806862,"identity":"23f2adaf-9e81-445e-a8db-fcc2991c3a48","added_by":"auto","created_at":"2025-10-31 01:40:04","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":796075,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eYasara simulation of missense variants on the tertiary structure of RAB11B.\u003c/strong\u003e The inactive GDP-bound and the active GTP-bound structures are superposed and color-coded in red and green, respectively. a) The substitution Arg33His caused the loss of a salt bridge between Arg 33 and Glu 35 specific of the active conformation. b) The substitution Arg72Cys led to the loss of one of the two hydrogen bonds connecting Arg 72 and Gly 69 in the inactive structure. c) The substitution Asp157Asn did not show to alter interactions with surrounding residues, probably due to the chemical similarity of aspartate and asparagine.\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-7509053/v1/dda21f4ba655ef9810c71c31.png"},{"id":94806855,"identity":"61b661da-c144-4bc6-8f6b-45167dd898f0","added_by":"auto","created_at":"2025-10-31 01:40:04","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":645508,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCo-localization analysis of WT and mutant RAB11B with the Golgi network.\u003c/strong\u003e a) Immunofluorescence assay of 3XFLAG-RAB11B in ARPE-19 cells, showing cellular localization of recombinant protein (marked by mouse\u003c/p\u003e\n\u003cp\u003eanti-FLAG M2 antibody, red) and the Golgi apparatus (marked by rabbit anti-GOLGA2 antibody, green). Nuclei were stained with DAPI (blue). b) Quantitative analysis of co-localization with GOLGA2, reported as Pearson’s Correlation Coefficients (PCC). c) Significance of difference between the constructs. One way ANOVA (p-value =1.8x10\u003csup\u003e-10\u003c/sup\u003e) and Tukey’s multiple comparison test (****: p-value \u0026lt;0.0001; **: p-value \u0026lt;0.01; *: p-value \u0026lt;0.05; N= 23 cells for each construct).\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-7509053/v1/46e937639d5481f633b30706.png"},{"id":94806843,"identity":"015f6a89-c46e-4bfd-bf44-d642eb557f09","added_by":"auto","created_at":"2025-10-31 01:40:03","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":585446,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCo-localization analysis of WT and mutant RAB11B with the centriole.\u003c/strong\u003e a) Immunofluorescence assay of 3XFLAG-RAB11B in ARPE-19 cells, showing cellular localization of recombinant protein (marked by mouse anti-FLAG M2 antibody, red) and the centriole (marked by rabbit anti-PCM1 antibody, green). Nuclei were stained with DAPI (blue). b) Quantitative analysis of co-localization with PCM1, reported as Pearson’s Correlation Coefficients (PCC). c) Significance of difference between the constructs. One way ANOVA (p-value =1.4x10\u003csup\u003e-8\u003c/sup\u003e) and Tukey’s multiple comparison test (****: p-value \u0026lt;0.0001; N= 23 cells for each construct).\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-7509053/v1/09ec632a4c2bdb5f4b893603.png"},{"id":94806846,"identity":"c7f7bbab-b201-4c38-a9ec-2b081290501f","added_by":"auto","created_at":"2025-10-31 01:40:03","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":622264,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCilium morphology of ARPE-19 cells expressing WT and mutant RAB11B.\u003c/strong\u003e a) Immunofluorescence assay of 3XFLAG-RAB11B in ARPE-19 cells, showing cellular localization of recombinant protein (marked by mouse anti-FLAG M2 antibody, red) and the primary cilium (marked by rabbit anti-acetylated tubulin antibody, green). Nuclei were stained with DAPI (blue). b) Quantitative analysis of cilia length, reported in micrometers. c) Significance of difference between the constructs. One way ANOVA (p-value =0.002) and Tukey’s multiple comparison test (**: p-value \u0026lt;0.01; *: p-value \u0026lt;0.05; N= 25 cells for each construct).\u003c/p\u003e","description":"","filename":"floatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-7509053/v1/2361c6b04b6c1111ae13445b.png"},{"id":94827342,"identity":"d68fc2d8-4e60-488c-9c6c-6509e086779d","added_by":"auto","created_at":"2025-10-31 06:57:29","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3991044,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7509053/v1/fff9b677-7e38-4ef4-af3c-4d4e6cb7a8a6.pdf"},{"id":94806844,"identity":"903c5b54-efa0-42b9-aeef-f8d70fd724a4","added_by":"auto","created_at":"2025-10-31 01:40:03","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":3101755,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryInformations.docx","url":"https://assets-eu.researchsquare.com/files/rs-7509053/v1/7bd49a7007564ebb9659bf38.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Investigating the role of rare missense variants in RAB11B in Autism Spectrum Disorder","fulltext":[{"header":"Background","content":"\u003cp\u003eAutism Spectrum Disorder (ASD) is a clinically heterogeneous neurodevelopmental disorder with an early onset, affecting over 1% of worldwide population (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e). It is characterized by a set of core symptoms, including impairments in social communication, as well as repetitive behaviors and restricted interests (\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eTwin and family studies have shown that ASD has a strong genetic basis, with heritability estimates ranging from 40\u0026ndash;90% (\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e). More recently, large-scale genomic studies have highlighted its complex genetic architecture, resulting from the interaction between common polygenic variation and both inherited and \u003cem\u003ede novo\u003c/em\u003e rare variants. Analysis of high-impact \u003cem\u003ede novo\u003c/em\u003e mutations, responsible for at least 10\u0026ndash;30% of ASD cases, have led to the identification of hundreds of high-confidence ASD risk genes, many of which are also implicated in other neurodevelopmental disorders (NDD) (\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e). This overlap provides evidence of shared genetic risk and molecular mechanisms across disorders and suggests that additional factors, such as polygenic background or stochastic developmental variation, contribute to the phenotypic diversity observed across individuals.\u003c/p\u003e\u003cp\u003e\u003cem\u003eRAB11B\u003c/em\u003e encodes a small Rab guanosine triphosphatase (GTPase) involved in intracellular transport and endosomal trafficking. It plays essential roles in many neurodevelopmental processes, such as ciliogenesis, neurite outgrowth, and synapse formation (\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e). \u003cem\u003eDe novo\u003c/em\u003e heterozygous missense variants in \u003cem\u003eRAB11B\u003c/em\u003e have been recently associated with a neurodevelopmental disorder characterized by severe intellectual disability (ID), ataxic gait, absent speech, and decreased cortical white matter (NDAGSCW, MIM #617807). To date, seven distinct \u003cem\u003eRAB11B\u003c/em\u003e disease-associated variants have been identified in 13 NDAGSCW cases (\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e). Among these, three variants (p.Gly21Arg, p.Val22Met, and p.Ala68Thr) are recurrent, reported in 2, 4, and 2 individuals, respectively. Clinical presentation varies among the reported cases, though intellectual disability is the most consistent feature. Molecular modeling has suggested that variants affecting the nucleotide binding pocket result in more severe phenotypes with intellectual disability, while those outside the binding region, are associated with a milder phenotype with epilepsy. ASD is not a commonly reported manifestation in NDAGSCW, and the potential involvement of RAB11B variants in ASD remains unexplored.\u003c/p\u003e\u003cp\u003eAs part of a previous genomic sequencing study of 435 individuals from 116 ASD families (\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e), we identified a \u003cem\u003ede novo\u003c/em\u003e missense variant in \u003cem\u003eRAB11B\u003c/em\u003e in an individual with ASD and mild ID. Since ASD is not a recognized core feature of NDAGSCW, this finding prompted us to investigate whether rare \u003cem\u003eRAB11B\u003c/em\u003e missense variants might contribute to ASD or to a broader spectrum of neurodevelopmental phenotypes beyond severe ID. To explore this hypothesis, we reviewed the literature and public variant databases for additional rare \u003cem\u003eRAB11B\u003c/em\u003e missense variants in individuals with ASD and related conditions. We identified two further uncharacterized interesting variants, one in an individual with ASD and another in an individual with mild ID.\u003c/p\u003e\u003cp\u003eThe aim of this study is to determine whether these three rare \u003cem\u003eRAB11B\u003c/em\u003e missense variants may contribute to ASD or related neurodevelopmental phenotypes, affecting RAB11B protein function. Therefore, we combined \u003cem\u003ein silico\u003c/em\u003e and \u003cem\u003ein vitro\u003c/em\u003e approaches, including protein structure modeling and subcellular localization assays in cells expressing recombinant mutant proteins.\u003c/p\u003e"},{"header":"Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003eCases\u003c/h2\u003e\u003cp\u003eIndividual 1 is part of an Italian ASD cohort (proband 52.3) (\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e). Individual 2 was recruited into the DDD Study (patient 385827) (\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e). Individual 3 belongs to the MSSNG ASD dataset (individual REACH000582) (\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003cb\u003eRAB11B\u003c/b\u003e \u003cb\u003evariant analysis\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe three \u003cem\u003eRAB11B\u003c/em\u003e missense variants analyzed in this study were previously identified by different whole exome/genome sequencing (WES/WGS) studies (\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eVariants were annotated using ANNOVAR (\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e) with data from gnomAD v4.1.0 (\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e), InterVar (\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e) and dbNSFP 4.7 (\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e). Their potential pathogenicity was also evaluated using AlphaMissense (\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e), considering the mean AM score for protein residue and the variant-specific pathogenicity score.\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eMolecular modeling\u003c/h3\u003e\n\u003cp\u003eStructural modeling and simulation of the three \u003cem\u003eRAB11B\u003c/em\u003e missense variants were performed using YASARA software (\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e), based on the crystallography-resolved 3D structure of active and inactive forms of human RAB11B (PDB entry: 2F9M and 2F9L, respectively).\u003c/p\u003e\n\u003ch3\u003eGeneration of WT and Mutant Plasmid Constructs\u003c/h3\u003e\n\u003cp\u003eThe coding sequence of RAB11B (NM_004218.4) was obtained from total cDNA isolated from the SH-SY5Y cell line (ATCC, Rockville, MD) by PCR amplification. This was then cloned into the p3xFLAG-CMV-10 mammalian expression vector, in frame with the 3xFLAG epitope, to generate the recombinant wild-type (WT) RAB11B construct. Mutations [p.(Ser25Asn), p.(Arg33His), p.(Gln70Leu), p.(Arg72Cys), p.(Asp157Asn)] were introduced into the WT RAB11B coding sequence by multi-step site-directed mutagenesis, using the KAPA HiFi HotStart PCR Kit. Primers sequences used for mutagenesis are listed in Supplementary Information (Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). Both WT and mutant constructs were verified by Sanger Sequencing.\u003c/p\u003e\n\u003ch3\u003eCell culture and transient transfection\u003c/h3\u003e\n\u003cp\u003eARPE-19 cells (ATCC, Rockville, MD) were cultured on glass coverslips in 6-well plate in DMEM/F-12 supplemented with 10% fetal bovine serum and 0.05 mg/ml penicillin-streptomycin, at 37\u0026deg;C in a 5% CO\u003csub\u003e2\u003c/sub\u003e humidified atmosphere. Cells were transiently transfected with 2.5 \u0026micro;g of plasmid constructs using 3.75 \u0026micro;l of Lipofectamine 3000 Transfection Reagent in fresh culture media. Six hours after transfection, media containing lipid nanoparticles was replaced with DMEM/F-12 complete medium and cells were maintained at 37\u0026deg;C in a 5% CO\u003csub\u003e2\u003c/sub\u003e humidified atmosphere. GFP coding plasmid was used as positive transfection control, while a p3xFLAG-CMV-10 empty vector was used as immunofluorescence negative control. Twenty-four hours after transfection, cells were washed three times in DPBS and serum-starved in serum-free DMEM-F12 for forty-eight hours in order to induce cells quiescence and the primary cilium assembly (\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e).\u003c/p\u003e\n\u003ch3\u003eImmunofluorescence assay\u003c/h3\u003e\n\u003cp\u003eForty-eight hours after serum starvation, cells were fixed for 15 minutes with 4% paraformaldehyde in DPBS, washed twice in DPBS, permeabilized for 15 minutes at room temperature (RT) with 0.1% Triton X-100 in DPBS, and incubated with blocking solution (4% normal donkey serum (Jackson ImmunoResearch) and 0.05% tween in PBS) for 1 hour at RT. For the staining, cells were then incubated over-night at 4\u0026deg;C with specific primary antibody: mouse anti-FLAG M2 antibody (1:1000, Sigma-Aldrich), rabbit anti-GOLGA2 antibody (1:200, Sigma-Aldrich), rabbit anti-PCM1 antibody (1:200, Sigma-Aldrich) or rabbit anti-acetyl-a-tubulin antibody (1:500, Sigma-Aldrich). After three 0.05% tween-DPBS washing step, cells were incubated with secondary antibodies for 1 hour at RT: goat anti-mouse Cy3 antibody and goat anti-rabbit FITC antibody (1:400, Jackson ImmunoResearch). Samples were washed with DPBS and nuclei were stained with 1\u0026micro;g/ml Hoechst. Coverslips were mounted on glass slides using Fluoromount G. Images were taken using a confocal microscope Nikon A1R\u0026thinsp;+\u0026thinsp;and the NIS-elements software (Nikon).\u003c/p\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003eCo-localization and statistical analysis\u003c/h2\u003e\u003cp\u003eCo-localization analysis based on Pearson\u0026rsquo;s correlation coefficient (r) and the determination of cilium length were performed using the Coloc-2 plugin and the line tool of Fiji\u0026rsquo;s, respectively. Statistical analysis was performed using the one-way analysis of variance (ANOVA) followed by Tukey\u0026rsquo;s multiple comparison test.\u003c/p\u003e\u003c/div\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cb\u003eIdentification of\u003c/b\u003e \u003cb\u003eRAB11B\u003c/b\u003e \u003cb\u003emissense variants\u003c/b\u003e\u003c/p\u003e\u003cp\u003eBy performing WGS in a cohort of 116 ASD families including 144 affected individuals, we identified a heterozygous \u003cem\u003ede novo\u003c/em\u003e missense variant in \u003cem\u003eRAB11B\u003c/em\u003e (p.Arg33His) in a male proband of a trio with unaffected parents (\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e). In addition to the ASD diagnosis, the proband (Individual 1) had mild ID and speech delay. MRI at 4 years of age did not reveal any cortical anomaly. Detailed phenotypic description of Individual 1 is reported in Supplementary Information. The variant, located in exon 2, affects an arginine residue that is highly conserved in all Rab subfamily members and across species. Multiple \u003cem\u003ein silico\u003c/em\u003e tools predict this variant to be highly deleterious. According to InterVar ACMG guidelines, the variant was classified as likely pathogenic (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eMain clinical features of individuals with \u003cem\u003eRAB11B\u003c/em\u003e missense variants.\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"10\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c9\" colnum=\"9\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c10\" colnum=\"10\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003eGenomic change\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003cp\u003eAmino acid change\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003egnomAD v.4.1.0 \u003c/p\u003e\u003cp\u003eAF total (allele count)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colspan=\"4\" nameend=\"c6\" namest=\"c3\"\u003e\u003cp\u003eVariant prediction score\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c7\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003eInheritence\u003c/p\u003e\u003cp\u003e(Individual ID)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c8\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003eACMG class\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c9\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003eOther genetic variants\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c10\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003eClinical details\u003csup\u003ed\u003c/sup\u003e\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eMPC\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eCADD phred\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003eAM\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c6\"\u003e\u003cp\u003ePhyloP100 vertebrates\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eg.8399920G\u0026thinsp;\u0026gt;\u0026thinsp;A\u003c/p\u003e\u003cp\u003ep.Arg33His\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e6.2x10\u003csup\u003e\u0026minus;\u0026thinsp;7\u003c/sup\u003e\u003c/p\u003e\u003cp\u003e\u003csup\u003e(\u003c/sup\u003e1/1614208)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e2.6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e34\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0.84\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e10.003\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e\u003cem\u003eDe novo\u003c/em\u003e\u003c/p\u003e\u003cp\u003e(Individual 1)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003eLP \u003c/p\u003e\u003cp\u003e(PS2,PM1,PM2,PP2)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003ePaternal VUS CNV (NC_000024.9:\u003c/p\u003e\u003cp\u003eg.(495953_1264792)dup)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003eAutism (ADOS-2 score: 6; CARS2-ST score: 39); Language level: single words; Mild ID (Leiter-R, B-IQ 56, FR 63); MRI: normal; Focal epilepsy\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eg.8400036C\u0026thinsp;\u0026gt;\u0026thinsp;T\u003c/p\u003e\u003cp\u003ep.Arg72Cys\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e6.2x10\u003csup\u003e\u0026minus;\u0026thinsp;7\u003c/sup\u003e\u003c/p\u003e\u003cp\u003e(1/1613548)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e2.74\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e34\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0.99\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e4.892\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003ePaternal\u003c/p\u003e\u003cp\u003e(Individual 2)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003eLP\u003c/p\u003e\u003cp\u003e(PM1,PM2,PP2,PP3)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003eMaternal VUS CNV (NC_000023.10:\u003c/p\u003e\u003cp\u003eg.68348165_68467987dup)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003eLanguage level: normal speech; Mild ID (QI: 58; QI verbal: 62); MRI: normal; Obesity.\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eg.8402523G\u0026thinsp;\u0026gt;\u0026thinsp;A\u003c/p\u003e\u003cp\u003ep.Asp157Asn\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003enovel\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e1.891\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e26.3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0.82\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e9.994\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e\u003cem\u003eDe novo\u003c/em\u003e\u003c/p\u003e\u003cp\u003e(Individual 3)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003eLP\u003c/p\u003e\u003cp\u003e(PS2,PM1,PM2,PP2)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e\u003cem\u003eDe novo\u003c/em\u003e pathogenic SNV (NM_001308120.2(TOGARAM1):\u003c/p\u003e\u003cp\u003ec.5011delT:(p.F1671fs))\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003eASD; ADHD; Anxiety; Gastrointestinal comorbidities.\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colspan=\"10\" nameend=\"c10\" namest=\"c1\"\u003e\u003cp\u003e\u003csup\u003ea\u003c/sup\u003e Relative to NC_000019.10 (hg38); \u003csup\u003eb\u003c/sup\u003e (NM_004218.4); \u003csup\u003ec\u003c/sup\u003e InterVar Classification according to the ACMG guidelines, together with detailed evidence codes, LP likely pathogenic, VUS variant of uncertain significance; \u003csup\u003ed\u003c/sup\u003e B-IQ Brief IQ; FR Fluid Reasoning.\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003eWhile \u003cem\u003eRAB11B\u003c/em\u003e was recently implicated as causative gene in a novel NDD, no direct association with ASD susceptibility has been described so far. To investigate whether other rare \u003cem\u003eRAB11B\u003c/em\u003e missense variants have been reported in individuals with ASD, we analyzed the largest WES/WGS studies on ASD cohorts published to date (\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e). We identified an additional \u003cem\u003ede novo\u003c/em\u003e missense variant in \u003cem\u003eRAB11B\u003c/em\u003e, p.Asp157Asn, novel and predicted to be deleterious (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). This variant was reported in one ASD individual from a WGS study of a cohort of 4,258 ASD families (\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e). We also explored the public database DECIPHER and identified another \u003cem\u003eRAB11B\u003c/em\u003e missense variant, p.Arg72Cys, also predicted to be damaging. This variant was identified in a 12-years old boy with ID and was inherited from his father, who was reported to have mild ID. Notably, the same variant was reported as \u003cem\u003ede novo\u003c/em\u003e variant in a 5-years old female with seizures and delayed speech abilities (\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e) (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eThe p.Arg33His and p.Arg72Cys variants are ultra-rare, each present in the gnomAD v.4.1.0 dataset with total Allele Frequency (AF)\u0026thinsp;\u0026lt;\u0026thinsp;10\u003csup\u003e\u0026minus;\u0026thinsp;4\u003c/sup\u003e, while the p.Asp157Asn variant is novel.\u003c/p\u003e\u003cp\u003eNone of the three \u003cem\u003eRAB11B\u003c/em\u003e missense variants has been previously functionally characterized. Although the three identified missense variants are located in different regions of the gene, all map within or in close proximity to protein functional domains. This is consistent with previously reported disease-associated missense variants in \u003cem\u003eRAB11B\u003c/em\u003e (\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e). Specifically, RAB11B contains five conserved sequence motifs forming the GDP/GTP-binding domain, as well as two switch regions (Switch I and Switch II), which regulate the conformational change between inactive and active forms and mediate interactions with coenzymes and effector proteins (\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e). The p.Arg33His and p.Arg72Cys variants are both located in the N-terminal half, with p.Arg33His immediately adjacent to the Switch I region and p.Arg72Cys within the Switch II region. Instead, the p.Asp157Asn maps in the C-terminal half, adjacent to the final G box motif involved in the GDP/GTP binding. Moreover, two of the three identified missense variants are located in close proximity to the GDP-blocking p.Ser25Asn (constitutively inactive) and GTP-blocking p.Gln70Leu (constitutively active) variants, which were generated \u003cem\u003ein vitro\u003c/em\u003e to replicate known functional mutations of other Rab proteins, and whose disruptive effects have been validated in numerous experimental settings (\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\n\u003ch3\u003eFunctional characterization of missense variants in RAB11B\u003c/h3\u003e\n\u003cp\u003eIn order to identify mutational hot/cold spots within the RAB11B protein, we evaluated the AlphaMissense (AM) predicted average pathogenicity score for each amino acid residue, together with the frequency of missense variants in gnomAD v4.1.0. High AM pathogenicity scores were widely distributed across the N-terminal and the central region of the protein, while C-terminal residues exhibited lower AM scores, suggesting a reduced constraint against missense variation in this region. Consistent with this pattern, only a limited number of RAB11B missense variants reported in the general population involves the N-terminal protein region and its functional domain, while the majority of variants are clustered in the C-terminal region, where the residues tend to have lower mean AM score and high alternate allele counts (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Interestingly, the three identified missense variants overlap with regions showing high AM pathogenicity score. However, the specific AM scores for each of these amino acid substitutions are similar, making it difficult to infer relative pathogenicity based on \u003cem\u003ein silico\u003c/em\u003e prediction alone. Therefore, we decided to functionally characterize these variants.\u003c/p\u003e\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003e- Simulation of structural consequences of identified RAB11B missense variants\u003c/h2\u003e\u003cp\u003eTo evaluate the potential effects of the three identified missense variants on RAB11B protein function, we first modeled each amino acid substitution using YASARA software. Specifically, we introduced the three substitutions into the three-dimensional structures of both the active (GTP-bound) and inactive (GDP-bound) forms of human RAB11B protein. The most relevant structural perturbation was caused by the \u003cem\u003ede novo\u003c/em\u003e p.Arg33His variant. In the active form, the side chain of arginine 33 forms a salt bridge with glutamate 35, located at the N-terminal end of the Switch I region. This interaction is mediated by the side chain orientation typical of arginine in the active state, which changes upon transition to the inactive form. Replacement of arginine 33 with histidine disrupts this bond, leading to destabilization of the active protein conformation. This likely affects the stabilizing interactions that Switch I domain establishes with GTP and Mg\u003csup\u003e2+\u003c/sup\u003e (\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eIn contrast, the p.Arg72Cys variant primarily affects the inactive, GDP-bound protein form. In this conformation, arginine 72 forms two hydrogen bonds with glycine 69, through the amino group of the backbone and the side chain, respectively. When arginine 72 is replaced by a cysteine, one of this two hydrogen bonds is disrupted, due to the different size and composition of the cysteine side chain. However, the backbone-mediated hydrogen bond is maintained, likely preserving the overall structural conformation of the inactive form (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb).\u003c/p\u003e\u003cp\u003eThe third missense variant, p.Asp157Asn, does not appear to cause any significant structural perturbations. The hydrogen bond between aspartate 157 and threonine 159 typical of the RAB11B GTP-bound form is maintained when replacing the aspartate with an asparagine, probably due to the chemical and structural similarity between the two residues (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ec). To validate our modeling approach, we also simulated the previously characterized dominant negative p.Ser25Asn (GDP-bound) and constitutively active p.Gln70Leu (GTP-bound) \u003cem\u003eRAB11B\u003c/em\u003e variants. Substitution of serine 25 with asparagine prevents the interaction with the Mg\u003csup\u003e2+\u003c/sup\u003e ion essential for GTP binding, while replacing glutamine 70 with leucine causes a severe disruption of the inactive-state conformation, consistent with their known functional impact (Supplementary Fig.\u0026nbsp;2).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\u003ch2\u003e- Evaluation of functional consequences of identified RAB11B missense variants\u003c/h2\u003e\u003cp\u003eTo assess whether the identified missense variants could affect RAB11B functionality, particularly impairing the protein activation/inactivation cycle, we evaluated variant effect on RAB11B subcellular localization during the primary cilium assembly. Indeed, RAB11B intracellular localization changes according to its active or inactive state, co-localizing with the peri-centrosomal region as GTP-binding RAB11B or with the Golgi apparatus as GDP-binding RAB11B (\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e). We performed immunofluorescence assays by overexpressing 3xFLAG-RAB11B recombinant mutated proteins in ARPE-19 cells and analyzing their co-localization with the Golgi network and the centriole during ciliogenesis. In our analyses we also included the two \u003cem\u003eRAB11B\u003c/em\u003e variants of known effect, p.Ser25Asn and p.Gln70Leu, analyzing cellular localization for six different recombinant 3xFLAG-RAB11B proteins and using GOLGA2 and PCM1 as specific marker for the co-localization analysis with the Golgi apparatus and the pericentrosomal region, respectively. Wild-type (WT) recombinant protein showed homogeneous localization throughout the cytoplasm with punctuated distribution, typically due to the association of the active form with endosomes membrane. The same distribution pattern is recognizable for RAB11B p.Gln70Leu and the three identified missense variants, while p.Ser25Asn mutant protein appeared generally diffused in the cytoplasm, as expected for inactive RAB11B (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea-\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eQuantitative analysis of co-localization patterns by Pearson\u0026rsquo;s correlation coefficient, confirmed the altered localization of the constitutively active and inactive variants, as previously reported (\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e), and revealed that one of the newly tested variants also leads to altered protein cellular localization. Specifically, both p.Ser25Asn and Gln70Leu displayed a higher co-localization with the Golgi network compared to recombinant WT RAB11B (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb-\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eb), while quantification of the co-localization of FLAG and PCM1 signals highlighted the GDP-blocking p.Ser25Asn variant as the only variant showing reduced centriole co-localization, as previously found (\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e) (ANOVA p-value\u0026thinsp;=\u0026thinsp;1.4x10\u003csup\u003e\u0026minus;\u0026thinsp;8\u003c/sup\u003e, WT vs p.Ser25Asn Tukey\u0026rsquo;s multiple comparisons test p-value\u0026thinsp;=\u0026thinsp;3x10\u003csup\u003e\u0026minus;\u0026thinsp;8\u003c/sup\u003e; Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eb). Looking at the co-localization with GOLGA2, the \u003cem\u003ede novo\u003c/em\u003e variant identified in our ASD cohort, p.Arg33His, also caused protein mislocalization with a significant enrichment in the Golgi network (ANOVA p-value\u0026thinsp;=\u0026thinsp;1.8x10\u003csup\u003e\u0026minus;\u0026thinsp;10\u003c/sup\u003e, WT vs p.Arg33His Tukey\u0026rsquo;s multiple comparisons test p-value\u0026thinsp;=\u0026thinsp;0.004; Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb-\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eb). No significant localization difference was identified for the other two tested missense variants found in ASD/NDD patients, in accordance with milder or absent structural perturbations emerged from YASARA prediction (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb-\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eb).\u003c/p\u003e\u003cp\u003eGiven the central role of RAB11B in the initiation of the primary cilium assembly, we also assessed the effect of the identified missense variants on the primary cilium morphology. Cells transfected with the WT and the other mutated proteins showed a comparable cilium morphology, with no marked alterations, except for cells expressing the constitutively inactive p.Ser25Asn and the \u003cem\u003ede novo\u003c/em\u003e p.Arg33His variant, both of which displayed shorter cilia compared to the other tested variants (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea). This observation was confirmed by quantitative analysis of cilium length, which revealed a significantly reduction in the cilium length in cells expressing these two RAB11B mutants (ANOVA p-value\u0026thinsp;=\u0026thinsp;0.002, WT vs p.Arg33His, Tukey\u0026rsquo;s multiple comparisons test p-value\u0026thinsp;=\u0026thinsp;0.03; WT vs p.Ser25Asn, Tukey\u0026rsquo;s multiple comparisons test p-value\u0026thinsp;=\u0026thinsp;0.005 Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eb).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eRab proteins are a family of over 60 different GDP/GTP molecular switches known to be master regulators of membrane protein trafficking in nerve cells (\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eThe \u003cem\u003eRAB11B\u003c/em\u003e gene encodes a small Rab protein ubiquitously expressed in human tissues, and particularly enriched in brain, heart and testis (\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e). RAB11B is an important modulator of cellular transport, responsible for the delivery of cellular components or signaling molecules to specific locations in the cell (\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e). Notably, it acts as positive regulator for relevant aspects of mammalian neurodevelopment, such as neurite outgrowth, migration of cortical neurons, synaptic plasticity, and glial differentiation and myelination (\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e). Moreover, RAB11B plays a key role in primary cilium formation, promoting ciliogenesis initiation (\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e). The primary cilium is a sensory microtubule-based organelle present on the cell surface of most mammalian cells, including neurons, whose nucleation starts from a modified centrosome surrounded by pericentriolar material and centriolar satellites, and involves Golgi-derived vesicles (\u003cspan additionalcitationids=\"CR32\" citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e). Non-motile primary cilium is essential for intracellular communication, excitability and signaling (\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e, \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e). Cilium disfunction due to mutations in primary cilium genes results in a wide range of clinical conditions called ciliopathies. Interestingly, several studies focusing on high-penetrant rare variants have highlighted an overlap between risk genes associated with ciliary disfunction conditions and high-confidence ASD genes, strongly suggesting the relevance of cilium to ASD and other NDD (\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e). \u003cem\u003eRAB11B\u003c/em\u003e has not yet been directly implicated in ASD, however some of its regulators/interactors were proposed as contributors to the ASD phenotype, further indicating the involvement of the GTPase in ASD pathogenesis (\u003cspan additionalcitationids=\"CR38\" citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eDue to the identification of two recurrent \u003cem\u003ede novo\u003c/em\u003e missense variants (p.Val22Met and p.Ala68Thr) in five unrelated individuals with severe NDD and specific brain abnormalities, \u003cem\u003eRAB11B\u003c/em\u003e was linked to NDAGSCW, a new form of neurodevelopmental disorder with intellectual disability, ataxic gait, absent speech and decreased cortical white matter. Both \u003cem\u003ein silico\u003c/em\u003e and \u003cem\u003ein vitro\u003c/em\u003e studies were performed to fully characterize the two variants, demonstrating their pathogenic effect on the normal protein function by altering the GDP/GTP binding site (\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e). Subsequently, Ahmad \u003cem\u003eet al.\u003c/em\u003e reported five additional \u003cem\u003eRAB11B\u003c/em\u003e missense variants in six NDD individuals and attempted to establish correlations between molecular modeling prediction and phenotype severity. Specifically, variants located in the nucleotide-binding pocket appear to result in a more severe phenotype with ID, whereas those mapping to the Switch II are associated with milder phenotype (\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e). However, functional studies are warranted to confirm variants\u0026rsquo; effects and the hypothesized correlation between variant location and phenotype severity.\u003c/p\u003e\u003cp\u003eWe recently identified an ultra-rare \u003cem\u003ede novo RAB11B\u003c/em\u003e variant (p.Arg33His) in an individual with ASD, in addition to mild ID and speech delay (\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e). A different amino acid change of the same residue (p.Arg33Cys) had been previously reported in a 7-years-old boy with severe ID and significant speech delay with regression, but with no ataxic gait, seizures or brain anomalies (\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e). To investigate the contribution of \u003cem\u003eRAB11B\u003c/em\u003e variants to ASD, we searched for additional \u003cem\u003eRAB11B\u003c/em\u003e variants in ASD/NDD cohorts and identified a \u003cem\u003ede novo\u003c/em\u003e missense variant, p.Asp157Asn, in an individual with ASD recruited into the MSSG cohort and an ultra-rare missense variant, p.Arg72Cys, in a family with ID recruited into the DDD study. While p.Asp157Asn is novel, p.Arg72Cys was previously reported as a \u003cem\u003ede novo\u003c/em\u003e change in an 5-years-old with seizures and delayed speech abilities, but no formal intellectual disability, microcephaly or brain MRI abnormalities (\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eCombined analysis of the Alpha Missense prediction score and the distribution of \u003cem\u003eRAB11B\u003c/em\u003e variants in general population allowed us to identify \u0026ldquo;missense-intolerant\u0026rdquo; regions in the protein sequence, containing all three variants. However, structural modeling demonstrated that the p.Arg33His variant, situated in close proximity of the Switch I region, exerts the highest conformational impact on the 3D structure of the active protein, resulting in the loss of a salt bridge with the first residue of the molecular Switch I. This domain is a highly flexible loop that, through the interactions with guanine nucleotide exchange factors (GEFs), changes conformation between the inactive and active structure allowing for the interchange between GDP and GTP (\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e). Given the prime importance of this domain for the activation of RAB11B, by altering the switch I region flexibility and consequently reducing the stability of the active form, the \u003cem\u003ede novo\u003c/em\u003e variant p.Arg33His is expected to affect nucleotide binding capability, similarly to the effect of a variant impairing the binding pocket. A milder effect was predicted for the other two missense variants.\u003c/p\u003e\u003cp\u003eTo further clarify their functional consequences, we decide to characterize the three missense variants with additional \u003cem\u003ein vitro\u003c/em\u003e studies. Given the known RAB11B involvement in ciliogenesis initiation and its different intracellular localization during this process, we carried out co-localization analysis in ARPE-19 cells transiently expressing mutant Rab GTPase, to assess the impact of the missense variants in the context of primary cilium outgrowth. We detected altered cellular localization for the p.Arg33His variant, leading to a significant protein accumulation at the Golgi network. A similar effect was found in cells overexpressing the total loss-of-function p.Ser25Asn and the constitutively active p.Gln70Leu variant, in line with previous results (\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e). During ciliogenesis, RAB11B organizes vesicular trafficking of Rabin8-containing pre-ciliary vesicles from the trans-Golgi to the centrosome (\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e), switching from the inactive form bound to Golgi vesicles to the active form bound to pre-ciliary vesicles located at the centriole (\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e, \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e, \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e). Thus, the accumulation of the recombinant mutated protein at the Golgi apparatus could be interpreted as evidence of a disrupted cellular mechanism, as demonstrated by the dysregulation caused by the two known-effect variants. When examining co-localization with the basal body of the cilium, we observed a significant alteration in localization only in cells overexpressing the dominant negative p.Ser25Asn mutant. This variant, designed \u003cem\u003ein vitro\u003c/em\u003e and not previously reported in patients, significantly impacts the nucleotide binding pocket, as the conserved serine in the G1 box is known to coordinate a Mg\u003csup\u003e2+\u003c/sup\u003e interaction essential for GTP binding (\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e). To date, a similar effect on centriole colocalization has been reported for only two \u003cem\u003ede novo RAB11B\u003c/em\u003e variants (p.Val22Met and p.Ala68Thr) identified in individuals with severe neurodevelopmental symptoms, both directly affecting the conformation of the binding pocket (\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e). Finally, we examined primary cilium morphology by measuring cilia length. Interestingly, we found that Arg33His-mutant RAB11B resulted in significantly shorter cilia compared to the WT, similarly to Ser25Asn mutant, though with a weaker effect.\u003c/p\u003e\u003cp\u003eThe loss of the salt bridge between Arg 33 and Glu 35 caused by p.Arg33His variant may destabilize the otherwise structured GTP-bound Switch I, potentially reducing the stability of GTP binding. This could lead to the accumulation of inactive protein in the trans-Golgi and ultimately slow cilia growth. As further support of this possibility, a recent study demonstrated the critical role of phenylalanine 36 orientation during the transition to the active conformation catalyzed by the RabGEF SH3BP5, where Phe 36, together with Ile 44, forms a hydrophobic cap on the triphosphate nucleotide (\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e). Interestingly, a similar variant pathogenicity mechanism involving altered protein localization has recently been proposed for ASD-related proteins localized to neuronal primary cilia, which are implicated in regulating ciliogenesis. Notably, SYNGAP1 is one of these proteins, and it was recently shown that missense variants identified in ASD individuals lead to its mislocalization away from cilia (\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e, \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eThese results support the hypothesis that p.Arg33His is likely the causative variant underlying the ASD phenotype observed in individual 1. Moreover, no additional \u003cem\u003ede novo\u003c/em\u003e SNV or CNV were identified in this individual. In Table \u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, we reported only a paternally inherited large duplication on the Y chromosome involving \u003cem\u003eSHOX\u003c/em\u003e, as a previous study suggested an association between \u003cem\u003ede novo SHOX\u003c/em\u003e microduplication and ASD, albeit with low penetrance (\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e). While we cannot fully exclude a potential contribution of this duplication to the individual\u0026rsquo;s overall genetic risk, our data strongly support a primary role for the \u003cem\u003eRAB11B\u003c/em\u003e missense variant in the ASD phenotype.\u003c/p\u003e\u003cp\u003eFor the other two variants (p.Arg72Cys, p.Asp157Asn), we do not observe significant changes in the association of the mutant proteins with vesicular membranes or in cilium morphology, suggesting that they may have a more subtle effect, if any. The likely milder impact of p.Arg72Cys variant is in line with its mode of inheritance and the milder phenotype observed in individual 2 and the previously reported individual (\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e). This proband also carries a duplication on the X chromosome involving the first two exons of \u003cem\u003eOPHN1\u003c/em\u003e (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). \u003cem\u003eOPHN1\u003c/em\u003e encodes a Rho GTPase-activating protein (RhoGAP) involved in the growth and stabilization of dendritic spines, and its loss of function is associated with Billuart-type X-linked syndromic intellectual developmental disorder (MRXSBL; MIM #300486) (\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e). Since \u003cem\u003eOPHN1\u003c/em\u003e is implicated in MRXSBL via a loss of function mechanism, and the Xq12 duplication in p.Arg72Cys proband involves only the first part of the protein without affecting any functional domain, the effect of the CNV remains difficult to interpret, though it may potentially contribute to an increased genetic risk background.\u003c/p\u003e\u003cp\u003eInterestingly, by inspecting the genomic background of individual 3 who carries the p.Asp157Asn \u003cem\u003eRAB11B\u003c/em\u003e variant, we discovered the presence of \u003cem\u003ede novo\u003c/em\u003e frameshift variant in \u003cem\u003eTOGARAM1\u003c/em\u003e (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). \u003cem\u003eTOGARAM1\u003c/em\u003e encodes a specialized protein involved in ciliogenesis, essential for the normal structure and function of primary cilia, and has recently been implicated in Joubert syndrome-37 (JBTS37), an autosomal recessive neurodevelopmental ciliopathy characterized by a distinctive hindbrain malformation and additional variable features (\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e). Interestingly, individuals with Joubert Syndrome and other genetic ciliopathies frequently show autistic features (\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e), suggesting that ciliary disfunction may contribute to a subset of ASD cases. Although the ASD individual from the MSSNG cohort carries a heterozygous \u003cem\u003eTOGARAM1\u003c/em\u003e protein truncating variant, which contrasts with the recessive mechanism underlying Joubert Syndrome and the apparent haplosufficiency of \u003cem\u003eTOGARAM1\u003c/em\u003e (pLI\u0026thinsp;=\u0026thinsp;0, LOEUF\u0026thinsp;=\u0026thinsp;0.72), the \u003cem\u003eTOGARAM1\u003c/em\u003e p.Phe1618fs variant is predicted to escape nonsense-mediated decay (NMD) according to the 50bp rule, likely resulting in a truncated protein that may exert a gain of function effect. Moreover, both RAB11B and TOGARAM1 are involved in ciliogenesis, and while no direct interaction emerges from STRING analysis, the two proteins were indirectly linked via RAB11FIP3 when additional nodes were included (Supplementary Fig.\u0026nbsp;3). Taken together, these findings suggest that neither \u003cem\u003ede novo\u003c/em\u003e variant alone may be sufficient to cause the phenotype, but they may act synergically by affecting the same ciliary pathway, thereby contributing to a high-risk genetic background.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eThis study presents a combined \u003cem\u003ein silico\u003c/em\u003e and \u003cem\u003ein vitro\u003c/em\u003e functional characterization of three previously uncharacterized \u003cem\u003eRAB11B\u003c/em\u003e missense variants (p.Arg33His, p.Arg72Cys, p.Asp157Asn). Despite similar pathogenicity predictions for all three variants, functional analyses of these changes based on the 3D structure of the protein (\u003cem\u003ein silico\u003c/em\u003e analyses) and its localization within the cell (\u003cem\u003ein vitro\u003c/em\u003e assays) indicated a loss of function effect for one variant (p.Arg33His), while the other two did not display detectable differences compared to the wild type protein. These findings underscore the critical importance of functional characterization for the accurate interpretation of this class of genetic variation.\u003c/p\u003e\u003cp\u003eIn addition, the identification of a deleterious \u003cem\u003eRAB11B\u003c/em\u003e missense variant in a proband with a typical autistic presentation and only mild ID, contrasting with the severe ID and distinctive brain anomalies previously associated with \u003cem\u003eRAB11B\u003c/em\u003e missense variants, represents the first association between missense variants in \u003cem\u003eRAB11B\u003c/em\u003e and ASD. This expands the phenotypic spectrum and underscores, once again, the shared genetic risk and overlapping etiologies across different neurodevelopmental phenotypes. Moreover, our results bring further support to the involvement of ciliary dysfunction in ASD/NDD pathogenesis, fostering interest in genes involved in cilia biology and potential biomarkers of disrupted ciliary function.\u003c/p\u003e\u003cp\u003eTaken together, our data strongly encourage further investigation of the role of \u003cem\u003eRAB11B\u003c/em\u003e in neurodevelopment, supporting its broader involvement across the spectrum of NDDs, including autism.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eASD: Autism spectrum disorder\u003c/p\u003e\n\u003cp\u003eNDD: Neurodevelopmental disorder\u003c/p\u003e\n\u003cp\u003eID: Intellectual disability\u003c/p\u003e\n\u003cp\u003eNDAGSCW: Neurodevelopmental disorder characterized by severe intellectual disability (ID), ataxic gait, absent speech, and decreased cortical white matter (MIM #617807)\u003c/p\u003e\n\u003cp\u003eWES: Whole exome sequencing\u003c/p\u003e\n\u003cp\u003eWGS: Whole genome sequencing\u003c/p\u003e\n\u003cp\u003eWT: Wild-type\u003c/p\u003e\n\u003cp\u003eAM: AlphaMissense\u003c/p\u003e\n\u003cp\u003eANOVA: Analysis of variance\u003c/p\u003e\n\u003cp\u003eGDP: Guanosine diphosphate\u003c/p\u003e\n\u003cp\u003eGTP: Guanosine triphosphate\u003c/p\u003e\n\u003cp\u003eSNV: Single nucleotide variant\u003c/p\u003e\n\u003cp\u003eCNV: Copy number variant\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe WGS study on our cohort of ASD families, including Individual 1, was approved by the local Ethical Committee (Comitato Etico di Area Vasta Emilia Centro, CE-AVEC; CE14060) and performed in compliance with all relevant ethical regulations. All participants or substitute decision makers provided a written informed consent to participate to the study.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWritten consent was obtained for publication of the photographs of individual 1 (Supplementary Fig. 1).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWGS data generated from our cohort of ASD families, including Individual 1, are included in our previous publication and its supplementary information files (9). The \u003cem\u003eRAB11B\u003c/em\u003e variant identified in Individual 2 is publicly available on DECIPHER (https://www.deciphergenomics.org/, ID: 385827). The MSSNG dataset is included in Trost et colleagues\u0026rsquo; publication (5).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe dataset supporting the conclusions of this article is included within the article and its additional file. Any additional information is available from the corresponding author upon request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThis research was supported by #NEXTGENERATIONEU (NGEU) and funded by the Ministry of University and Research (MUR), National Recovery and Resilience Plan (NRRP), project MNESYS (PE0000006) \u0026ndash; A Multiscale integrated approach to the study of the nervous system in health and disease (DN. 1553 11.10.2022) and project National Center for Gene Therapy and Drugs based on RNA Technology (CN00000041) (M4C2 \u0026ndash; Action 1.4- Call \u0026ldquo;Potenziamento strutture di ricerca e di campioni nazionali di R\u0026amp;S\u0026rdquo; (CUP J33C22001140001)).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026apos; contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFormal analysis: M.V., L.S., E.B.; Funding acquisition: E.M., E.B.; Investigation: M.V., L.S., F.C., P.V., A.P., M.C.S., A.V., J.R.S., E.B., E.M.; Methodology: M.V., L.S., F.C., E.B.; Resources: P.V., A.P., M.C.S., A.V., J.R.S.; Software: M.V., L.S.; Supervision: E.B., E.M.; Visualization: M.V., L.S.; Writing-original draft: M.V., L.S.; Writing-review \u0026amp; editing: M.V., L.S., F.C., P.V., A.P., A.V., E.B., E.M. All authors approved the submission of this manuscript.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe are extremely grateful to all the families who have participated in this study. We gratefully thank Prof. Monica Baiula and Dr. Roberto Bernardoni for advising cell culture and immunofluorescence.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThis study makes use of data generated by the DECIPHER community. A full list of centres who contributed to the generation of the data is available from https://deciphergenomics.org/about/stats and via email from [email protected]. DECIPHER is hosted by EMBL-EBI and funding for the DECIPHER project was provided by the Wellcome Trust [grant number WT223718/Z/21/Z].\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eShaw KA, Williams S, Patrick ME, Valencia-Prado M, Durkin MS, Howerton EM, et al. 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J Comput Chem. 2015 May 15;36(13):996\u0026ndash;1007. \u003c/li\u003e\n\u003cli\u003eScapin SMN, Carneiro FRG, Alves AC, Medrano FJ, Guimar\u0026atilde;es BG, Zanchin NIT. The crystal structure of the small GTPase Rab11b reveals critical differences relative to the Rab11a isoform. Journal of Structural Biology. 2006 June;154(3):260\u0026ndash;8. \u003c/li\u003e\n\u003cli\u003eWang W, Brautigan DL. Phosphatase inhibitor 2 promotes acetylation of tubulin in the primary cilium of human retinal epithelial cells. BMC Cell Biol. 2008 Dec;9(1):62. \u003c/li\u003e\n\u003cli\u003eJenkins ML, Margaria JP, Stariha JTB, Hoffmann RM, McPhail JA, Hamelin DJ, et al. Structural determinants of Rab11 activation by the guanine nucleotide exchange factor SH3BP5. Nat Commun. 2018 Sept 14;9(1):3772. \u003c/li\u003e\n\u003cli\u003eD\u0026rsquo;Adamo P, Masetti M, Bianchi V, Mor\u0026egrave; L, Mignogna ML, Giannandrea M, et al. 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Proc Natl Acad Sci USA. 2010 Apr 6;107(14):6346\u0026ndash;51. \u003c/li\u003e\n\u003cli\u003eChen W, Feng Y, Chen D, Wandinger-Ness A. Rab11 Is Required for Trans-Golgi Network\u0026ndash;to\u0026ndash;Plasma Membrane Transport and a Preferential Target for GDP Dissociation Inhibitor. Pfeffer SR, editor. MBoC. 1998 Nov;9(11):3241\u0026ndash;57. \u003c/li\u003e\n\u003cli\u003eVetter IR, Wittinghofer A. The guanine nucleotide-binding switch in three dimensions. Science. 2001 Nov 9;294(5545):1299\u0026ndash;304. \u003c/li\u003e\n\u003cli\u003eMignogna ML, D\u0026rsquo;Adamo P. Critical importance of RAB proteins for synaptic function. Small GTPases. 2017 Apr 13;9(1\u0026ndash;2):145\u0026ndash;57. \u003c/li\u003e\n\u003cli\u003eBhartur SG, Calhoun BC, Woodrum J, Kurkjian J, Iyer S, Lai F, et al. Genomic structure of murine Rab11 family members. Biochem Biophys Res Commun. 2000 Mar 16;269(2):611\u0026ndash;7. \u003c/li\u003e\n\u003cli\u003eJing J, Prekeris R. 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J Child Neurol. 1999 Oct;14(10):636\u0026ndash;41. \u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"journal-of-neurodevelopmental-disorders","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"jndd","sideBox":"Learn more about [Journal of Neurodevelopmental Disorders](http://jneurodevdisorders.biomedcentral.com/)","snPcode":"11689","submissionUrl":"https://submission.nature.com/new-submission/11689/3","title":"Journal of Neurodevelopmental Disorders","twitterHandle":"@BioMedCentral","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"RAB11B, Rab GTPases, Immunofluorescence, Autism, ASD, Neurodevelopment, primary cilium","lastPublishedDoi":"10.21203/rs.3.rs-7509053/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7509053/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e\u003cp\u003eAutism 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 \u003cem\u003ede novo\u003c/em\u003e missense variant in the Rab GTPase \u003cem\u003eRAB11B\u003c/em\u003e (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.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e\u003cp\u003eFirst, we performed an \u003cem\u003ein silico\u003c/em\u003e analysis of the effect of each variant on active and inactive RAB11B using a known molecular 3D-modeling software. The \u003cem\u003ein-silico\u003c/em\u003e assessments were followed by an \u003cem\u003ein-vitro\u003c/em\u003e 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.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e\u003cp\u003eResults from the three-dimensional simulation of the \u003cem\u003eRAB11B\u003c/em\u003e 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 \u003cem\u003ede novo\u003c/em\u003e 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.\u003c/p\u003e\u003ch2\u003eConclusions\u003c/h2\u003e\u003cp\u003eThis study highlights the importance of functional characterization of \u003cem\u003eRAB11B\u003c/em\u003e missense variants to validate pathogenic computational predictions. Moreover, by highlighting \u003cem\u003eRAB11B\u003c/em\u003e as a possible ASD risk gene, it expands the neurodevelopmental spectrum of \u003cem\u003eRAB11B-related\u003c/em\u003e disorders.\u003c/p\u003e","manuscriptTitle":"Investigating the role of rare missense variants in RAB11B in Autism Spectrum Disorder","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-10-31 01:39:58","doi":"10.21203/rs.3.rs-7509053/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"reviewerAgreed","content":"257867258655895237778356575100561115444","date":"2026-05-15T10:33:03+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"138710537834914968825811088583176721759","date":"2026-05-13T21:24:04+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"321482442534011226877611551655060916836","date":"2026-03-30T14:29:30+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-10-20T16:49:00+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-09-02T07:11:57+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-09-02T07:11:44+00:00","index":"","fulltext":""},{"type":"submitted","content":"Journal of Neurodevelopmental Disorders","date":"2025-09-01T13:35:59+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"journal-of-neurodevelopmental-disorders","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"jndd","sideBox":"Learn more about [Journal of Neurodevelopmental Disorders](http://jneurodevdisorders.biomedcentral.com/)","snPcode":"11689","submissionUrl":"https://submission.nature.com/new-submission/11689/3","title":"Journal of Neurodevelopmental Disorders","twitterHandle":"@BioMedCentral","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"a4ee7ae7-b141-4434-bc9c-a48d48f20a94","owner":[],"postedDate":"October 31st, 2025","published":true,"recentEditorialEvents":[{"type":"reviewerAgreed","content":"257867258655895237778356575100561115444","date":"2026-05-15T10:33:03+00:00","index":114,"fulltext":""},{"type":"reviewerAgreed","content":"138710537834914968825811088583176721759","date":"2026-05-13T21:24:04+00:00","index":111,"fulltext":""}],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2025-10-31T01:39:58+00:00","versionOfRecord":[],"versionCreatedAt":"2025-10-31 01:39:58","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7509053","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7509053","identity":"rs-7509053","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}