A neurodevelopmental disorder associated with a loss-of-function missense mutation in RAB35

Rab35 (Ras-associated binding protein) is a small GTPase that regulates endosomal membrane trafficking and functions in cell polarity, cytokinesis, and growth factor signaling. Altered Rab35 function contributes to progression of glioblastoma, defects in primary cilia formation, and altered cytokinesis. Here, we report a pediatric patient with global developmental delay, hydrocephalus, a Dandy–Walker malformation, axial hypotonia with peripheral hypertonia, visual problems, and conductive hearing impairment. Exome sequencing identified a homozygous missense variant in the GTPase fold of RAB35 (c.80G>A; p.R27H) as the most likely candidate. Functional analysis of the R27H-Rab35 variant protein revealed enhanced interaction with its guanine-nucleotide exchange factor, DENND1A and decreased interaction with a known effector, MICAL1, indicating that the protein is in an inactive conformation. Cellular expression of the variant drives the activation of Arf6, a small GTPase under negative regulatory control of Rab35. Importantly, variant expression leads to delayed cytokinesis and altered length, number, and Arl13b composition of primary cilia, known factors in neurodevelopmental disease. Our findings provide evidence of altered Rab35 function as a causative factor of a neurodevelopmental disorder. Rab35 (Ras-associated binding protein) is a small GTPase that regulates endosomal membrane trafficking and functions in cell polarity, cytokinesis, and growth factor signaling. Altered Rab35 function contributes to progression of glioblastoma, defects in primary cilia formation, and altered cytokinesis. Here, we report a pediatric patient with global developmental delay, hydrocephalus, a Dandy–Walker malformation, axial hypotonia with peripheral hypertonia, visual problems, and conductive hearing impairment. Exome sequencing identified a homozygous missense variant in the GTPase fold of RAB35 (c.80G>A; p.R27H) as the most likely candidate. Functional analysis of the R27H-Rab35 variant protein revealed enhanced interaction with its guanine-nucleotide exchange factor, DENND1A and decreased interaction with a known effector, MICAL1, indicating that the protein is in an inactive conformation. Cellular expression of the variant drives the activation of Arf6, a small GTPase under negative regulatory control of Rab35. Importantly, variant expression leads to delayed cytokinesis and altered length, number, and Arl13b composition of primary cilia, known factors in neurodevelopmental disease. Our findings provide evidence of altered Rab35 function as a causative factor of a neurodevelopmental disorder. Ras-associated binding (Rab) proteins are a family of small GTPases composed of approximately 60 members in humans (1Homma Y. Hiragi S. Fukuda M. Rab family of small GTPases: an updated view on their regulation and functions.FEBS J. 2021; 288: 36-55Crossref PubMed Scopus (190) Google Scholar). Rabs are evolutionarily conserved and expressed in a wide variety of cell types and at multiple cellular locations (2Zerial M. McBride H. Rab proteins as membrane organizers.Nat. Rev. Mol. Cell Biol. 2001; 2: 107-117Crossref PubMed Scopus (2791) Google Scholar). Rabs switch between an active GTP–bound state and an inactive GDP–bound state. While GDP-bound Rabs are primarily located in the cytosol, once GTP bound, Rabs associate with membranes via C-terminal prenyl groups and bind multiple effectors via their switch 1 and 2 regions (3Stenmark H. Rab GTPases as coordinators of vesicle traffic.Nat. Rev. Mol. Cell Biol. 2009; 10: 513-525Crossref PubMed Scopus (2534) Google Scholar). Interaction with effectors allows the Rabs to drive their plethora of membrane trafficking functions. The exchange of GTP for GDP on Rabs is mediated by guanine-nucleotide exchange factors (GEFs). GEFs bind GDP-bound Rabs and catalyze the removal of GDP, remaining bound to the nucleotide-free form of the GTPase as an enzymatic intermediate until GTP binds the Rab, causing the GEF to dissociate. The active state of the Rab ends when the Rab GTPase hydrolyzes GTP to GDP with the catalytic aid of GTPase-activating proteins (GAPs). Rabs are evolutionarily conserved and control multiple fundamental cellular processes, such as vesicle budding and transport, tethering and fusion, organelle structure and function, lipid remodeling, and regulation of cytoskeletal dynamics (4Klinkert K. Echard A. Rab35 GTPase: a central regulator of phosphoinositides and F-actin in endocytic recycling and beyond.Traffic. 2016; 17: 1063-1077Crossref PubMed Scopus (100) Google Scholar). Rabs conduct these functions at basically all compartments in eukaryotic cells. Altered expression levels and mutations of Rab GTPases have been frequently associated with disease, including cancer, neurodegenerative diseases, and various genetic disorders (5Seixas E. Barros M. Seabra M.C. Barral D.C. Rab and Arf proteins in genetic diseases.Traffic. 2013; 14: 871-885Crossref PubMed Scopus (45) Google Scholar, 6Wilson G.R. Sim J.C.H. McLean C. Giannandrea M. Galea C.A. Riseley J.R. et al.Mutations in RAB39B cause X-linked intellectual disability and early-onset Parkinson disease with α-synuclein pathology.Am. J. Hum. Genet. 2014; 95: 729-735Abstract Full Text Full Text PDF PubMed Scopus (189) Google Scholar, 7Kiral F.R. Kohrs F.E. Jin E.J. Hiesinger P.R. Rab GTPases and membrane trafficking in neurodegeneration.Curr. Biol. 2018; 28: R471-R486Abstract Full Text Full Text PDF PubMed Scopus (154) Google Scholar, 8Kulasekaran G. Chaineau M. Piscopo V.E.C. Verginelli F. Fotouhi M. 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Rab35 regulates membrane trafficking in the endosomal system (11Chaineau M. Ioannou M.S. McPherson P.S. Rab35: GEFs, GAPs and effectors.Traffic. 2013; 14: 1109-1117Crossref PubMed Scopus (101) Google Scholar), is broadly expressed in human tissues, and is evolutionarily conserved (12Rozés-Salvador V. González-Billault C. Conde C. The recycling endosome in nerve cell development: one Rab to rule them all?.Front. Cell Dev. Biol. 2020; 8603794Crossref Scopus (12) Google Scholar). This Rab GTPase localizes to the plasma membrane and endosomal vesicles and tubules and is implicated in endocytosis, vesicle recycling, autophagy, exocytosis, and exosome release (13Allaire P.D. Marat A.L. Dall'Armi C. Di Paolo G. McPherson P.S. Ritter B. The connecdenn DENN domain: a GEF for Rab35 mediating cargo-specific exit from early endosomes.Mol. Cell. 2010; 37: 370-382Abstract Full Text Full Text PDF PubMed Scopus (164) Google Scholar, 14Biesemann A. Gorontzi A. Barr F. Gerke V. Rab35 protein regulates evoked exocytosis of endothelial Weibel–Palade bodies.J. Biol. Chem. 2017; 292: 11631-11640Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar, 15Hsu C. Morohashi Y. Yoshimura S. Manrique-Hoyos N. Jung S. Lauterbach M.A. et al.Regulation of exosome secretion by Rab35 and its GTPase-activating proteins TBC1D10A–C.J. Cell Biol. 2010; 189: 223-232Crossref PubMed Scopus (635) Google Scholar, 16Minowa-Nozawa A. Nozawa T. Okamoto-Furuta K. Kohda H. Nakagawa I. Rab35 GTPase recruits NPD52 to autophagy targets.EMBO J. 2017; 36: 2790-2807Crossref PubMed Scopus (59) Google Scholar). Rab35 functions in the successful abscission of the cytokinetic bridge during cell division through its interaction with the effector oculocerebrorenal syndrome of Lowe protein (OCRL) (17Kouranti I. Sachse M. Arouche N. Goud B. Echard A. Rab35 regulates an endocytic recycling pathway essential for the terminal steps of cytokinesis.Curr. Biol. 2006; 16: 1719-1725Abstract Full Text Full Text PDF PubMed Scopus (301) Google Scholar). In addition, it functions in the formation and signaling of primary cilia by regulating ciliary length, function, and membrane composition (18Kuhns S. Seixas C. Pestana S. Tavares B. Nogueira R. Jacinto R. et al.Rab35 controls cilium length, function and membrane composition.EMBO Rep. 2019; 20e47625Crossref PubMed Scopus (27) Google Scholar). Given the wide variety of cellular processes involving Rab35, altered function of the protein through mutations could result in human disease. For example, defects in cytokinesis and cilia formation have been associated with neurodevelopmental disorders (19Seu K.G. Trump L.R. Emberesh S. Lorsbach R.B. Johnson C. Meznarich J. et al.VPS4A mutations in humans cause syndromic congenital dyserythropoietic anemia due to cytokinesis and trafficking defects.Am. J. Hum. Genet. 2020; 107: 1149-1156Abstract Full Text Full Text PDF PubMed Google Scholar, 20Andreu-Cervera A. Catala M. Schneider-Maunoury S. Cilia, ciliopathies and hedgehog-related forebrain developmental disorders.Neurobiol. Dis. 2021; 150105236Crossref PubMed Scopus (52) Google Scholar). Rab35 is required during neuronal development in the hippocampus (21Maejima I. Hara T. Tsukamoto S. Koizumi H. Kawauchi T. Akuzawa T. et al.RAB35 is required for murine hippocampal development and functions by regulating neuronal cell distribution.Commun. Biol. 2023; 6: 1-12Crossref PubMed Scopus (2) Google Scholar), its expression is decreased in glioblastoma (22Allaire P.D. Seyed Sadr M. Chaineau M. Seyed Sadr E. Konefal S. Fotouhi M. et al.Interplay between Rab35 and Arf6 controls cargo recycling to coordinate cell adhesion and migration.J. Cell Sci. 2013; 126: 722-731Crossref PubMed Scopus (95) Google Scholar), and disruption of Rab35 enhances growth and invasiveness of human glioblastoma in mouse xenograft models (8Kulasekaran G. Chaineau M. Piscopo V.E.C. Verginelli F. Fotouhi M. Girard M. et al.An Arf/Rab cascade controls the growth and invasiveness of glioblastoma.J. Cell Biol. 2021; 220e202004229Crossref PubMed Google Scholar). Here, we report a patient with a neurodevelopmental disorder carrying a variant in RAB35 (c.80G>A; p.(Arg27His)), causing the substitution of arginine 27 (R27) for a histidine (H). The Rab35 R27H variant locks the protein in an inactive state. Cells overexpressing this variant have altered Arf6 activation and disruptions in cytokinesis and primary cilia, suggesting that the mutation is a causative factor for the abnormalities observed in the patient. The proband (Fig. 1A, individual III-2) was the first child of consanguineous, first cousin parents of Muslim-Arab origin, who previously had a spontaneous miscarriage. The proband was born at full term after a pregnancy complicated by maternal insulin-dependent diabetes mellitus, which was controlled before and during pregnancy. She had a birthweight of 3090 g (26th %ile) and an Apgar score of 9 at 1 min and 9 at 5 min. After birth, she had cyanosis and required oxygen treatment and had indirect hyperbilirubinemia. She was discharged from the neonatal intensive care unit at 7 days. Developmental milestones were delayed; at 2.5 years of age, she could roll over but could not sit, crawl, or stand. She had speech delay and significant cognitive delay (developmental quotient = 32). Magnetic resonance imaging of the brain at 11 months revealed a large cyst in the posterior fossa, enlarged lateral ventricles, and a severely hypoplastic vermis, consistent with a Dandy–Walker malformation (Fig. 1, B–E). In addition, there was suspected periventricular leukomalacia indicative of white matter disease. At 35 months of age, thinning of the corpus callosum and significant loss of white matter volume were detected. The electroencephalogram was normal. Echocardiogram after birth revealed a cardiac defect that resolved spontaneously. Abdominal and renal ultrasound were normal. Moderate conductive hearing impairment and visual defects including esotropia, amblyopia, hypermetropia, astigmatism, cupping of the optic disc, and limited bilateral eye movements on abduction and upward gaze were observed. At around 2 years of age, she presented with premature thelarche. Hormonal evaluation including luteinizing hormone and follicle-stimulating hormone was normal. Physical examination at 2.5 years of age revealed growth parameters within the normal range for age. Head circumference was 49 cm (73rd %ile), her overall length was 92.5 cm (68th %ile), and her weight was 12.8 kg (45th %ile). She had intermittent eye contact, a widely opened anterior fontanel, deep set eyes, esotropia, arched eyebrows, short philtrum, dental problems with caries, retrognathia, left-sided low set ear, abnormal palmar creases on the left hand, and mild clinodactyly of fifth fingers bilaterally. There was axial hypotonia with peripheral hypertonia. She also had hirsutism and bilateral enlargement of the breasts. Family history was positive for insulin-dependent diabetes in the mother, with onset at approximately 16 years. A paternal aunt had global developmental delay and a seizure disorder, and a maternal uncle died at around age 6 months because of an unknown cause. Genetic evaluation in the proband included a chromosomal microarray followed by trio exome sequencing, revealing a ∼130 kbp duplication (arr[GRCh37] 14q32.31(102941709_103068021)×3) of unknown clinical significance (variant of unknown significance), harboring no known triplosensitive genes. This was subsequently determined to be inherited from her unaffected father and therefore unlikely to be a major contributor to the phenotype. Trio exome sequencing for the proband and her parents demonstrated a homozygous missense variant in RAB35—chr12:120546244[hg19]; NM_006861.7; c.80G>A; p.(Arg27His). Both parents are heterozygous carriers of this variant. The variant is rare (found neither in gnomAD nor in the local database of ∼14,000 exomes), affects a residue conserved in vertebrate Rab35 (GERP 4.83) (Fig. 1F), and is predicted to be deleterious by multiple bioinformatic tools, including MutationTaster, PolyPhen, and SIFT. Combined annotation-dependent depletion score is 24.7, further supporting pathogenicity. The encoded protein, Rab35, functions in abscission of the cytokinetic bridge (17Kouranti I. Sachse M. Arouche N. Goud B. Echard A. Rab35 regulates an endocytic recycling pathway essential for the terminal steps of cytokinesis.Curr. Biol. 2006; 16: 1719-1725Abstract Full Text Full Text PDF PubMed Scopus (301) Google Scholar) and in primary cilia formation and signaling (18Kuhns S. Seixas C. Pestana S. Tavares B. Nogueira R. Jacinto R. et al.Rab35 controls cilium length, function and membrane composition.EMBO Rep. 2019; 20e47625Crossref PubMed Scopus (27) Google Scholar), phenotypes associated with neurodevelopmental disorders. RAB35 was thus considered a candidate gene leading to the disease phenotype. Secondary findings as defined by the American College of Medical Genetics (23Miller D.T. Lee K. Chung W.K. Gordon A.S. Herman G.E. Klein T.E. et al.ACMG Secondary Findings Working GroupACMG SF v3.0 list for reporting of secondary findings in clinical exome and genome sequencing: a policy statement of the American College of Medical Genetics and Genomics (ACMG).Genet. Med. 2021; 23: 1381-1390Abstract Full Text Full Text PDF PubMed Scopus (318) Google Scholar) included a heterozygous, likely pathogenic, variant in RYR2 (NM_001035.3): c.11060dup; p.(Leu3687PhefsTer15) inherited from the proband's mother. Pathogenic variants in RYR2 lead to ventricular arrhythmias (24Li Y. Wei J. Guo W. Sun B. Estillore J.P. Wang R. et al.Human RyR2 (ryanodine receptor 2) loss-of-function mutations: clinical phenotypes and in vitro characterization.Circ. Arrhythm. Electrophysiol. 2021; 14e010013Crossref Scopus (19) Google Scholar), and therefore, the proband and her mother were referred for evaluation and follow-up with cardiology. In addition, the parents were both heterozygous for a pathogenic variant in DARS2 (NM_018122.5): c.788G>A; p.(Arg263Gln) known to cause leukoencephalopathy with brain stem and spinal cord involvement and lactate elevation (Online Mendelian Inheritance in Man: 611105) with autosomal recessive inheritance. While the proband was not homozygous for the variant, this information has implications for prenatal testing or preimplantation genetic diagnosis in future pregnancies. The R27H mutation in Rab35 is located in proximity to the G-domain (Fig. 2A) that is involved in nucleotide binding, but outside switch I and II regions, which are involved in interaction with effectors and GEFs (25Lin L. Shi Y. Wang M. Wang C. Zhu J. Zhang R. Rab35/ACAP2 and Rab35/RUSC2 complex structures reveal molecular basis for effector recognition by Rab35 GTPase.Structure. 2019; 27: 729-740.e3Abstract Full Text Full Text PDF PubMed Scopus (10) Google Scholar). We examined the three-dimensional structure of Rab35 in complex with GTP (Protein Data Bank identifier: 6IF3) and recognized that the R27 is in proximity to the nucleotide-binding site but does not interact directly with the nucleotide itself (Fig. 2B). Interestingly, R27 interacts via hydrogen bonds with alanine (A) 151 of Rab35. In turn, A151 interacts with the guanosine ring of GTP (Fig. 2C). The R27 residue was mutated in silico to H27 using PyMOL and analyzed for the gain or loss of hydrogen bonds. The R27H mutation is predicted to lead to loss of the hydrogen bond with A151 (Fig. 2D). This in turn could destabilize the interaction between A151 and the nucleotide because of conformational changes in the protein. Thus, the R27H mutation may destabilize the nucleotide-binding site. DENN domain proteins function as GEFs for Rabs. The connecdenn (DENND1) family members connecdenn 1–3 (DENND1A-C) exhibit GEF activity toward Rab35 (9Kumar R. Francis V. Kulasekaran G. Khan M. Armstrong G.A.B. McPherson P.S. A cell-based GEF assay reveals new substrates for DENN domains and a role for DENND2B in primary ciliogenesis.Sci. Adv. 2022; 8eabk3088Crossref Scopus (8) Google Scholar, 11Chaineau M. Ioannou M.S. McPherson P.S. Rab35: GEFs, GAPs and effectors.Traffic. 2013; 14: 1109-1117Crossref PubMed Scopus (101) Google Scholar). GEFs bind their substrate GTPases in the GDP-bound inactive form (26Marat A.L. McPherson P.S. The connecdenn family, Rab35 guanine nucleotide exchange factors interfacing with the clathrin machinery.J. Biol. Chem. 2010; 285: 10627-10637Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar). We thus compared Rab35 R27H, Rab35 wt, Rab35 S22N (a constitutively inactive GDP–bound mutant), and Rab35 Q67L (a constitutively active GTP–bound mutant) for binding to DENND1A/B. The DENN domains of DENND1A/B had little interaction with Rab35 wt and Q67L, whereas they interact with Rab35 S22N (Fig. 3A). Interestingly, the Rab35 R27H variant had a similar interaction as the S22N mutant, suggesting that Rab35 R27H is in an inactive conformation (Fig. 3A). Active Rab35 interacts with effectors to mediate numerous cellular functions. We performed an effector-binding assay using a glutathione-S-transferase (GST)-fused coiled coil region of MICAL1 (GST-MICAL1 CC), which binds Rab35 selectively in the GTP-bound form (27Rahajeng J. Panapakkam Giridharan S.S. Cai B. Naslavsky N. Caplan S. MICAL-L1 is a tubular endosomal membrane hub that connects Rab35 and Arf6 with Rab8a.Traffic. 2012; 13: 82-93Crossref PubMed Scopus (85) Google Scholar). GST-MICAL1 CC binds FLAG-Rab35 wt and Q67L but neither FLAG-Rab35 R27H nor S22N, further indicating that the R27H mutation in Rab35 converts the protein to an inactive conformation (Fig. 3B). Rab35 folds into an inactive conformation when GDP bound or nucleotide free. To determine if R27H alters the nucleotide-binding ability, we performed an in vitro nucleotide loading assay using purified Rab35 wt and the R27H variant. Surprisingly, we detected no significant difference in the GTPγS loading efficiency of Rab35 R27H compared with Rab35 wt (Fig. 3C). However, when Rab35 wt and the R27H variant were loaded with [35S]GTPγS in the presence of MgCl2 and the release of nucleotide was assayed by diluting the loaded GTPase, Rab35 wt displayed stable GTP interaction, whereas GTP was rapidly lost from the R27H variant (Fig. 3D). Cytokinesis is the final step of cell division and allows for the physical separation of a cell into 2 daughter cells. Membrane trafficking plays a critical role in cytokinesis (28Frémont S. Echard A. Membrane traffic in the late steps of cytokinesis.Curr. Biol. 2018; 28: R458-R470Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar). Rab35 functions in the stabilization and abscission of the cytokinetic bridge during cell division, in part by driving the depolymerization of F-actin at the cytokinetic bridge through recruitment of the Rab35 effectors OCRL and MICAL1 (17Kouranti I. Sachse M. Arouche N. Goud B. Echard A. Rab35 regulates an endocytic recycling pathway essential for the terminal steps of cytokinesis.Curr. Biol. 2006; 16: 1719-1725Abstract Full Text Full Text PDF PubMed Scopus (301) Google Scholar, 29Dambournet D. Machicoane M. Chesneau L. Sachse M. Rocancourt M. El Marjou A. et al.Rab35 GTPase and OCRL phosphatase remodel lipids and F-actin for successful cytokinesis.Nat. Cell Biol. 2011; 13: 981-988Crossref PubMed Scopus (223) Google Scholar, 30Frémont S. Hammich H. Bai J. Wioland H. Klinkert K. Rocancourt M. et al.Oxidation of F-actin controls the terminal steps of cytokinesis.Nat. Commun. 2017; 814528Crossref PubMed Scopus (103) Google Scholar). We measured the time in which cells transduced with either GFP-Rab35 wt or GFP-R27H divided, from furrow ingression until the abscission of the cytokinetic bridge. Cells overexpressing the Rab35 R27H variant exhibited a delay in cytokinesis as compared with cells overexpressing Rab35 wt, showing an average of 216 ± 13 min to complete cytokinesis as compared with 168 ± 7.2 min, respectively (mean ± SEM) (Fig. 4, A and B). Rab35 controls cytokinesis and the recycling of membrane receptors and adhesion proteins through mutual antagonism with Arf6. Active Rab35 drives the inactivation of Arf6 via ACAP2, a Rab35 effector and an Arf6 GAP (22Allaire P.D. Seyed Sadr M. Chaineau M. Seyed Sadr E. Konefal S. Fotouhi M. et al.Interplay between Rab35 and Arf6 controls cargo recycling to coordinate cell adhesion and migration.J. Cell Sci. 2013; 126: 722-731Crossref PubMed Scopus (95) Google Scholar, 31Chesneau L. Dambournet D. Machicoane M. Kouranti I. Fukuda M. 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Cell Sci. 2013; 126: 722-731Crossref PubMed Scopus (95) Google Scholar). To assess the levels of active Arf6 in cells overexpressing the R27H variant, we performed a pulldown assay with GST-fused GGA3, a known effector of Arf6 (33Takatsu H. Yoshino K. Toda K. Nakayama K. GGA proteins associate with Golgi membranes through interaction between their GGAH domains and ADP-ribosylation factors.Biochem. J. 2002; 365: 369-378Crossref PubMed Scopus (98) Google Scholar). Cells overexpressing the Rab35 R27H variant showed increased levels of active Arf6 as compared with the cells overexpressing Rab35 wt, whereas the total Arf6 levels remained the same, indicating that the R27H variant mimics results seen with Rab35 knockdown (Fig. 4, C and D). Rab35 functions in the formation and membrane composition of primary cilia (18Kuhns S. Seixas C. Pestana S. Tavares B. Nogueira R. 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Using acetylated tubulin (ac-tubulin) as a marker for the cilia body and CEP164 for the centriole, we observed that Rab35 R27H overexpression exerts a dominant-negative effect with reduction in cilia length, which were on average, 3.3 ± 0.1 μm, whereas the cells overexpressing Rab35 wt exhibited longer cilia, with an average length of 5.1 ± 0.13 μm (mean ± SEM) (Fig. 5, A and B). The number of ciliated cells was also significantly reduced in cells overexpressing the Rab35 R27H variant (Fig. 5C). Rab35 is localized to the ciliary membrane when in its GTP-bound active state (18Kuhns S. Seixas C. Pestana S. Tavares B. Nogueira R. Jacinto R. et al.Rab35 controls cilium length, function and membrane composition.EMBO Rep. 2019; 20e47625Crossref PubMed Scopus (27) Google Scholar). We found that the Rab35 R27H variant is poorly localized at the cilia when compared with the wt protein (Fig. 5, D and E). Primary cilia are involved in multiple cellular processes, and their protein composition is tightly controlled. Rab35 interacts with and regulates the levels of Arl13b at the ciliary membrane (18Kuhns S. Seixas C. Pestana S. Tavares B. Nogueira R. Jacinto R. et al.Rab35 controls cilium length, function and membrane composition.EMBO Rep. 2019; 20e47625Crossref PubMed Scopus (27) Google Scholar). Arl13b mediates the release of lipidated protein cargo and facilitates sonic hedgehog signaling (Shh) (36Hor C.H. Goh E.L. Small GTPases in hedgehog signalling: emerging insights into the disease mechanisms of Rab23-mediated and Arl13b-mediated ciliopathies.Curr. Opin. Genet. Dev. 2019; 56: 61-68Crossref PubMed Scopus (9) Google Scholar), a master regulator pathway of neurodevelopment (37Brady M.V. Vaccarino F.M. Role of SHH in patterning human pluripotent cells towards ventral forebrain fates.Cells. 2021; 10: 914Crossref PubMed Scopus (6) Google Scholar). 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