ARL3 Mutations Cause Joubert Syndrome by Disrupting Ciliary Protein Composition
2018; Elsevier BV; Volume: 103; Issue: 4 Linguagem: Inglês
10.1016/j.ajhg.2018.08.015
ISSN1537-6605
AutoresSumaya Alkanderi, Elisa Molinari, Ranad Shaheen, Yasmin ElMaghloob, Louise A. Stephen, Veronica Sammut, Simon A. Ramsbottom, Shalabh Srivastava, George Cairns, Noel Edwards, Sarah J. Rice, Nour Ewida, Amal Alhashem, Kathryn White, Colin Miles, David Steel, Fowzan S. Alkuraya, Shehab Ismail, John A. Sayer,
Tópico(s)Protist diversity and phylogeny
ResumoJoubert syndrome (JBTS) is a genetically heterogeneous autosomal-recessive neurodevelopmental ciliopathy. We investigated further the underlying genetic etiology of Joubert syndrome by studying two unrelated families in whom JBTS was not associated with pathogenic variants in known JBTS-associated genes. Combined autozygosity mapping of both families highlighted a candidate locus on chromosome 10 (chr10: 101569997–109106128, UCSC Genome Browser hg 19), and exome sequencing revealed two missense variants in ARL3 within the candidate locus. The encoded protein, ADP ribosylation factor-like GTPase 3 (ARL3), is a small GTP-binding protein that is involved in directing lipid-modified proteins into the cilium in a GTP-dependent manner. Both missense variants replace the highly conserved Arg149 residue, which we show to be necessary for the interaction with its guanine nucleotide exchange factor ARL13B, such that the mutant protein is associated with reduced INPP5E and NPHP3 localization in cilia. We propose that ARL3 provides a potential hub in the network of proteins implicated in ciliopathies, whereby perturbation of ARL3 leads to the mislocalization of multiple ciliary proteins as a result of abnormal displacement of lipidated protein cargo. Joubert syndrome (JBTS) is a genetically heterogeneous autosomal-recessive neurodevelopmental ciliopathy. We investigated further the underlying genetic etiology of Joubert syndrome by studying two unrelated families in whom JBTS was not associated with pathogenic variants in known JBTS-associated genes. Combined autozygosity mapping of both families highlighted a candidate locus on chromosome 10 (chr10: 101569997–109106128, UCSC Genome Browser hg 19), and exome sequencing revealed two missense variants in ARL3 within the candidate locus. The encoded protein, ADP ribosylation factor-like GTPase 3 (ARL3), is a small GTP-binding protein that is involved in directing lipid-modified proteins into the cilium in a GTP-dependent manner. Both missense variants replace the highly conserved Arg149 residue, which we show to be necessary for the interaction with its guanine nucleotide exchange factor ARL13B, such that the mutant protein is associated with reduced INPP5E and NPHP3 localization in cilia. We propose that ARL3 provides a potential hub in the network of proteins implicated in ciliopathies, whereby perturbation of ARL3 leads to the mislocalization of multiple ciliary proteins as a result of abnormal displacement of lipidated protein cargo. Mutations in genes that are involved in the structure or function of the primary cilium give rise to a range of disorders known as ciliopathies.1Hildebrandt F. Benzing T. Katsanis N. Ciliopathies.N. Engl. J. Med. 2011; 364: 1533-1543Crossref PubMed Scopus (979) Google Scholar These are typically multi-system disorders, as seen in the archetypal ciliopathy Joubert syndrome (JBTS), which is characterized clinically by brain malformations that result in developmental delay, oculomotor apraxia, and hypotonia.2Romani M. Micalizzi A. Valente E.M. Joubert syndrome: congenital cerebellar ataxia with the molar tooth.Lancet Neurol. 2013; 12: 894-905Abstract Full Text Full Text PDF PubMed Scopus (251) Google Scholar In addition to the neurodevelopmental phenotype, retinal and renal diseases are often associated with JBTS.3Srivastava S. Ramsbottom S.A. Molinari E. Alkanderi S. Filby A. White K. Henry C. Saunier S. Miles C.G. Sayer J.A. A human patient-derived cellular model of Joubert syndrome reveals ciliary defects which can be rescued with targeted therapies.Hum. Mol. Genet. 2017; 26: 4657-4667Crossref PubMed Scopus (40) Google Scholar Now more than 35 genes are known to cause JBTS when mutated in an autosomal-recessive or X-linked manner4Akizu N. Silhavy J.L. Rosti R.O. Scott E. Fenstermaker A.G. Schroth J. Zaki M.S. Sanchez H. Gupta N. Kabra M. et al.Mutations in CSPP1 lead to classical Joubert syndrome.Am. J. Hum. Genet. 2014; 94: 80-86Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar, 5Romani M. Micalizzi A. Kraoua I. Dotti M.T. Cavallin M. Sztriha L. Ruta R. Mancini F. Mazza T. Castellana S. et al.Mutations in B9D1 and MKS1 cause mild Joubert syndrome: expanding the genetic overlap with the lethal ciliopathy Meckel syndrome.Orphanet J. Rare Dis. 2014; 9: 72Crossref PubMed Scopus (44) Google Scholar, 6Roosing S. Romani M. Isrie M. Rosti R.O. Micalizzi A. Musaev D. Mazza T. Al-Gazali L. Altunoglu U. Boltshauser E. et al.Mutations in CEP120 cause Joubert syndrome as well as complex ciliopathy phenotypes.J. Med. Genet. 2016; 53: 608-615Crossref PubMed Scopus (39) Google Scholar, 7Van De Weghe J.C. Rusterholz T.D.S. Latour B. Grout M.E. Aldinger K.A. Shaheen R. Dempsey J.C. Maddirevula S. Cheng Y.H. Phelps I.G. et al.University of Washington Center for Mendelian GenomicsMutations in ARMC9, which encodes a basal body protein, cause Joubert syndrome in humans and ciliopathy phenotypes in zebrafish.Am. J. Hum. Genet. 2017; 101: 23-36Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar (also see GeneReviews in Web Resources). Genetic approaches have moved from traditional linkage studies and homozygosity mapping to exome sequencing strategies, protein interaction networks,8Sang L. Miller J.J. Corbit K.C. Giles R.H. Brauer M.J. Otto E.A. Baye L.M. Wen X. Scales S.J. Kwong M. et al.Mapping the NPHP-JBTS-MKS protein network reveals ciliopathy disease genes and pathways.Cell. 2011; 145: 513-528Abstract Full Text Full Text PDF PubMed Scopus (444) Google Scholar and genome-wide small interfering RNA screens,9Wheway G. Schmidts M. Mans D.A. Szymanska K. Nguyen T.T. Racher H. Phelps I.G. Toedt G. Kennedy J. Wunderlich K.A. et al.UK10K ConsortiumUniversity of Washington Center for Mendelian GenomicsAn siRNA-based functional genomics screen for the identification of regulators of ciliogenesis and ciliopathy genes.Nat. Cell Biol. 2015; 17: 1074-1087Crossref PubMed Scopus (157) Google Scholar allowing a rapid rate of gene discovery. Despite these advancements, which made it possible for the majority of JBTS cases to have a genetic diagnosis,10Shaheen R. Szymanska K. Basu B. Patel N. Ewida N. Faqeih E. Al Hashem A. Derar N. Alsharif H. Aldahmesh M.A. et al.Ciliopathy WorkingGroupCharacterizing the morbid genome of ciliopathies.Genome Biol. 2016; 17: 242Crossref PubMed Scopus (92) Google Scholar many cases of JBTS remain genetically unsolved, and critically, the inter-relationships between the proteins encoded by these genes and the underlying disease mechanisms remain poorly understood. Here, we used a combination of autozygosity mapping and whole-exome sequencing (WES)11Otto E.A. Hurd T.W. Airik R. Chaki M. Zhou W. Stoetzel C. Patil S.B. Levy S. Ghosh A.K. Murga-Zamalloa C.A. et al.Candidate exome capture identifies mutation of SDCCAG8 as the cause of a retinal-renal ciliopathy.Nat. Genet. 2010; 42: 840-850Crossref PubMed Scopus (254) Google Scholar, 12Chaki M. Airik R. Ghosh A.K. Giles R.H. Chen R. Slaats G.G. Wang H. Hurd T.W. Zhou W. Cluckey A. et al.Exome capture reveals ZNF423 and CEP164 mutations, linking renal ciliopathies to DNA damage response signaling.Cell. 2012; 150: 533-548Abstract Full Text Full Text PDF PubMed Scopus (260) Google Scholar in two unsolved JBTS-affected families and identified likely deleterious variants in ARL3 (MIM: 60495). We further investigate the mechanistic impact of this mutation and show that the mutant ARL3 is irresponsive to the guanine nucleotide exchange factor (GEF) activity of ARL13B and causes associated defects in ciliary proteins in affected individuals' fibroblasts. Family 1 is a Saudi Arabian family comprising first-cousin healthy parents and six children, including the 5-year-old male index individual (II:5; Figure 1). His clinical features include developmental delay, multicystic dysplastic left kidney, night blindness, and mild dysmorphic features, including ptosis (Figure 1 and Table 1). Magnetic resonance imaging (MRI) of the brain showed severe vermis hypoplasia with abnormal thick cerebellar peduncles configured in the shape of a typical molar tooth sign (Figure 1B), as well as abnormal configuration of the midbrain, thinning of the pontomesencephalic junction and midportion of the midbrain, and mild decreased brain volume with a paucity of white matter in the frontotemporal region and dilated ventricular system. This family is part of a large ciliopathy cohort (enrolled in a research protocol approved by King Faisal Specialist Hospital and Research Center research advisory council 2080006 after providing informed consent). Family 2, originating from Pakistan, is also consanguineous and comprises three affected children with a clinical syndrome in keeping with JBTS (II;1, II:4, and II:5; Table 1 and Figure 1). The eldest sibling (II:1) presented with hypotonia and psychomotor delay. Subsequently, the child developed night blindness and bilateral visual loss by 4 years of age. She also had recurrent urinary-tract infections (Table 1). Clinical investigations revealed the molar tooth sign that is typical of JBTS on brain MRI, as well as retinal dystrophy (Figure 1). The other two affected siblings (II:4 and II:5) had very similar presentations with predominating brain and retinal features (Table 1 and Figure 1). Siblings II:1 and II:4 experienced problems with thermoregulation, which implies brainstem involvement, as well as the known cerebellar defects typical of JBTS. This family was enrolled in a research protocol approved by the National Research Ethics Service (09/H0903/36) after providing informed consent.Table 1Clinical Features of JBTS in Affected Family MembersFamily 1Family 2II:5II:1II:4II:5Age (years)521129Central nervous symptomsdevelopmental delay, ataxiadevelopmental delay, ataxiadevelopmental delay, ataxiadevelopmental delay, ataxiaOcular symptomsptosis, rod-cone dystrophy, night blindness,bilateral visual pathway involvementrod-cone dystrophy, night blindness, progressive visual lossrod-cone dystrophy, night blindness, progressive visual lossrod-cone dystrophy, night blindness, progressive visual loss, oculomotor apraxiaeGFR (mL/min/1.73 m2)NA75>90>90Renal symptomsnonerecurrent UTInonerecurrent UTIUSS renalleft multicystic dysplastic kidney, right grade I hydronephrosisbilateral renal scarringnormal USSunequal kidney sizeOthersingle palmar crease, pectus carinatum, normal ABRthermoregulation problems,episode of transverse myelitisthermoregulation problems,sleep apneanoneAbbreviations are as follows: ABR, auditory brainstem response; eGFR, estimated glomerular filtration rate; NA, not available; USS, ultrasound scan; and UTI, urinary-tract infection. Open table in a new tab Abbreviations are as follows: ABR, auditory brainstem response; eGFR, estimated glomerular filtration rate; NA, not available; USS, ultrasound scan; and UTI, urinary-tract infection. Exome sequencing of the index individual in each family and variant filtering were performed as previously described.7Van De Weghe J.C. Rusterholz T.D.S. Latour B. Grout M.E. Aldinger K.A. Shaheen R. Dempsey J.C. Maddirevula S. Cheng Y.H. Phelps I.G. et al.University of Washington Center for Mendelian GenomicsMutations in ARMC9, which encodes a basal body protein, cause Joubert syndrome in humans and ciliopathy phenotypes in zebrafish.Am. J. Hum. Genet. 2017; 101: 23-36Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar In brief, WES was performed with the TruSeq Exome Enrichment Kit from Illumina. Coding and splicing homozygous variants were considered as candidates only if they were present within the candidate locus, had a frequency < 0.1% in publicly available variant databases (1000 Genomes, NHLBI Exome Sequencing Project Exome Variant Server, and Genome Aggregation Database [gnomAD]) and a database of in-house ethnically matched exomes (Saudi Human Genome Program; totaling 2,379 exomes), and were predicted to be pathogenic in silico. Interestingly, both families were flagged by the corresponding research group because exome sequencing did not reveal a likely deleterious bi-allelic variant in any of the established JBTS-related genes. Through an investigator-initiated collaboration, an attempt was made to exploit the consanguineous nature of both families, which can readily reveal a potentially unifying etiology if they have an overlapping autozygome, as previously described.7Van De Weghe J.C. Rusterholz T.D.S. Latour B. Grout M.E. Aldinger K.A. Shaheen R. Dempsey J.C. Maddirevula S. Cheng Y.H. Phelps I.G. et al.University of Washington Center for Mendelian GenomicsMutations in ARMC9, which encodes a basal body protein, cause Joubert syndrome in humans and ciliopathy phenotypes in zebrafish.Am. J. Hum. Genet. 2017; 101: 23-36Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar In brief, we performed genome-wide genotyping with the Axiom SNP Chip platform from Affymetrix and the Sure Select V4 platform from Agilent Technologies and then determined autozygomes by using HomozygosityMapper on all available family members. This revealed a single critical locus (chr10: 101,569,997–109,106,128, UCSC Genome Browser hg 19) (Figure 2A). This locus spans 57 genes, none of which is known to be linked to a ciliopathy phenotype. After re-analyzing the exome variants by only considering variants within this locus (Tables S1 and S2), we found a single previously unreported variant in ARL3 in each index individual: c.445C>T (p.Arg149Cys) (GenBank: NM_004311.3) in family 1 and c.446G>A (p.Arg149His) (GenBank: NM_004311.3) in family 2 (Figure 2B). Both homozygous variants fully co-segregated with the JBTS phenotype in each family. ARL3 is a highly conserved gene, and its encoded protein, the small G-protein ARL3, localizes to the cilium and is crucial for ciliogenesis and axoneme formation, as well as cargo displacement of lipidated proteins in the cilium.13Hanke-Gogokhia C. Wu Z. Gerstner C.D. Frederick J.M. Zhang H. Baehr W. Arf-like protein 3 (ARL3) regulates protein trafficking and ciliogenesis in mouse photoreceptors.J. Biol. Chem. 2016; 291: 7142-7155Crossref PubMed Scopus (72) Google Scholar ARL3 variants have also been reported in association with retinal dystrophy.14Zhang Q. Hu J. Ling K. Molecular views of Arf-like small GTPases in cilia and ciliopathies.Exp. Cell Res. 2013; 319: 2316-2322Crossref PubMed Scopus (30) Google Scholar Among ARL3 effectors are the GDI-like solubilizing factors (GSFs) PDE6D, UNC119A, and UNC119B, whose interactions are guanosine triphosphate (GTP) dependent. GSFs bind to and solubilize prenylated and myristoylated proteins, which are released by ARL3-GTP acting as an allosteric release factor.15Ismail S.A. Chen Y.X. Miertzschke M. Vetter I.R. Koerner C. Wittinghofer A. Structural basis for Arl3-specific release of myristoylated ciliary cargo from UNC119.EMBO J. 2012; 31: 4085-4094Crossref PubMed Scopus (85) Google Scholar, 16Ismail S.A. Chen Y.X. Rusinova A. Chandra A. Bierbaum M. Gremer L. Triola G. Waldmann H. Bastiaens P.I. Wittinghofer A. Arl2-GTP and Arl3-GTP regulate a GDI-like transport system for farnesylated cargo.Nat. Chem. Biol. 2011; 7: 942-949Crossref PubMed Scopus (201) Google Scholar The cilia-specific protein ARL13B acts as a specific GEF for ARL3, whereas retinitis protein 2 (RP2) functions as an ARL3 GTPase-activating protein (GAP) and is localized in the pre-ciliary compartment.17Veltel S. Gasper R. Eisenacher E. Wittinghofer A. The retinitis pigmentosa 2 gene product is a GTPase-activating protein for Arf-like 3.Nat. Struct. Mol. Biol. 2008; 15: 373-380Crossref PubMed Scopus (137) Google Scholar, 18Grayson C. Bartolini F. Chapple J.P. Willison K.R. Bhamidipati A. Lewis S.A. Luthert P.J. Hardcastle A.J. Cowan N.J. Cheetham M.E. Localization in the human retina of the X-linked retinitis pigmentosa protein RP2, its homologue cofactor C and the RP2 interacting protein Arl3.Hum. Mol. Genet. 2002; 11: 3065-3074Crossref PubMed Scopus (108) Google Scholar This segregation of a GEF and a GAP is proposed to create an ARL3-GTP gradient inside the cilium,19Gotthardt K. Lokaj M. Koerner C. Falk N. Gießl A. Wittinghofer A. A G-protein activation cascade from Arl13B to Arl3 and implications for ciliary targeting of lipidated proteins.eLife. 2015; 4: e11859Crossref PubMed Google Scholar which ensures the destination-specific release of lipid-modified ciliary proteins, solubilized by GSFs.20Fansa E.K. Kösling S.K. Zent E. Wittinghofer A. Ismail S. PDE6δ-mediated sorting of INPP5E into the cilium is determined by cargo-carrier affinity.Nat. Commun. 2016; 7: 11366Crossref PubMed Scopus (60) Google Scholar The ARL3 Arg149 residue is highly conserved throughout evolution (Figure 2C), and in silico prediction tools suggest that either missense change is likely to be pathogenic (Table S3). Homology models of ARL3 reveal that the two variants, which are located in a loop between the α4 and β6 domains (Figure 3A), are predicted to disrupt the interaction of ARL13B with ARL3 because it requires this precise residue (Arg149) for its interaction (Figure 3B). Superimposing all known structures of ARL3 in complex with its effectors, GAP and GEFS, the ARL3 Arg149 residue is exclusively present in the interface between ARL3 and ARL13B and is involved in an ionic interaction with the conserved ARL13B Glu88 residue (Figure 3B). To functionally investigate the effect of the mutation on the interaction with ARL13B, we performed a GEF fluorescence-based polarization experiment.19Gotthardt K. Lokaj M. Koerner C. Falk N. Gießl A. Wittinghofer A. A G-protein activation cascade from Arl13B to Arl3 and implications for ciliary targeting of lipidated proteins.eLife. 2015; 4: e11859Crossref PubMed Google Scholar Wild-type (WT) and mutant p.Arg149His versions of murine ARL3 (98.35% sequence identity to human ARL3) were bound to fluorescently labeled GDP, and an excess of unlabeled GTP was added in the presence or absence of human ARL13B. We then followed the capability for nucleotide exchange of both versions of the protein by recording the fluorescence polarization over time. Upon addition of the ARL13B GEF, WT ARL3 showed a clear acceleration of nucleotide exchange. Under similar conditions, mutant p.Arg149His ARL3 failed to show acceleration of nucleotide exchange in the presence of ARL13B (Figure 3C). The integrity of the mutant protein was confirmed by pull-down, whereby both WT and p.Arg149His ARL3 proteins were pulled down equally by UNC119A (Figure 3F and Figure S1). Furthermore, we confirmed our results by using the highly conserved C. reinhardtii ARL3 (WT and mutant p.Arg148His) and ARL13B (Figure 3D). To further investigate the importance of the ARL3-ARL13B interaction, we carried out the reverse charge variant p.Glu86Arg in ARL13B by using C. reinhardtii proteins. As expected, p.Glu86Arg ARL13B was not able to accelerate the nucleotide exchange of WT ARL3 (Figure 3E). From these experiments, we conclude that p.Arg149His ARL3 disrupts the interaction with ARL13B and is defective in ARL13B-assisted nucleotide exchange. To determine ciliary morphology, we obtained fibroblasts from all three affected individuals in family 2 (II:1, II:4, and II:5) plus control individuals (both parents [I:1 and I:2] and an unaffected sibling [II:3]). Primary cilia identified by ARL13B antibodies were of normal length in affected individuals (mean length = 5.9, 7.8, and 6.8 μm in II:1, II:4, and II:5, respectively) and control individuals (mean length = 5.7 and 6.0 μm in the parents and 6.1 μm in the unaffected sibling), and there were no significant differences between the two groups (Figure S2). There was also no difference in the percentage of ciliation rates between affected and control fibroblasts (Figure S2). Scanning electron microscopy confirmed these findings of no significant changes in cilia length or structural appearance (Figure S3). ARL3 functions as an allosteric release factor of all GSFs members: PDE6D, UNC119A, and UNC119B. Whereas PDE6D is involved in the trafficking of prenylated proteins, UNC119A and UNC119B traffic myristoylated proteins.19Gotthardt K. Lokaj M. Koerner C. Falk N. Gießl A. Wittinghofer A. A G-protein activation cascade from Arl13B to Arl3 and implications for ciliary targeting of lipidated proteins.eLife. 2015; 4: e11859Crossref PubMed Google Scholar Given that ARL3 exerts its releasing function only when bound to GTP, we expected the ciliary localization of the GSF cargo to be impaired. The INPP5E, GRK1, and PDE6 catalytic subunits are among the prenylated GSF ciliary cargo,20Fansa E.K. Kösling S.K. Zent E. Wittinghofer A. Ismail S. PDE6δ-mediated sorting of INPP5E into the cilium is determined by cargo-carrier affinity.Nat. Commun. 2016; 7: 11366Crossref PubMed Scopus (60) Google Scholar whereas the myristoylated ciliary cargo includes NPHP3, GNAT1, and Cystin1.22Jaiswal M. Fansa E.K. Kösling S.K. Mejuch T. Waldmann H. Wittinghofer A. Novel biochemical and structural insights into the interaction of myristoylated cargo with Unc119 protein and their release by Arl2/3.J. Biol. Chem. 2016; 291: 20766-20778Crossref PubMed Scopus (36) Google Scholar To test our hypothesis, we examined cilia for protein content of both the prenylated INPP5E and myristoylated NPHP3. ARL3-mutant cilia demonstrated a significant loss of both INPP5E and NPHP3 content (Figure 4 and Figures S4–S6), indicating that WT ARL3 is required for normal release of these cargos into the ciliary axoneme. To confirm these phenotypes as specific to the loss of ARL3 function, we sought to determine the ciliary content of GLI3 in WT and ARL3-mutant cilia. GLI3 translocation is independent of GSF transport and relies upon intraflagellar transport proteins and Sonic Hedgehog signal transduction.23Haycraft C.J. Banizs B. Aydin-Son Y. Zhang Q. Michaud E.J. Yoder B.K. Gli2 and Gli3 localize to cilia and require the intraflagellar transport protein polaris for processing and function.PLoS Genet. 2005; 1: e53Crossref PubMed Scopus (708) Google Scholar Consistent with morphologically normal cilia in ARL3-mutant fibroblasts, no defect in ciliary GLI3 was observed after stimulation with SAG, a Hedgehog pathway agonist. The amounts of total ciliary GLI3 and ciliary tip GLI3 were unchanged between affected and control individuals (Figure S7), confirming that the ciliary Hedgehog signaling pathway is not disturbed by this particular ARL3 mutation. Together, these data substantiate a role for ARL3 in the release of both prenylated and myristoylated ciliary cargo, which is disrupted by the p.Arg149His ARL3 variant. We present ARL3 as a ciliopathy- and JBTS-associated gene. LdARL-3A, a Leishmania homolog of ARL3, is an essential component of flagellum formation.24Cuvillier A. Redon F. Antoine J.C. Chardin P. DeVos T. Merlin G. LdARL-3A, a Leishmania promastigote-specific ADP-ribosylation factor-like protein, is essential for flagellum integrity.J. Cell Sci. 2000; 113: 2065-2074Crossref PubMed Google Scholar Arl3 knockdown has previously been investigated in a gene-trap murine model, where Arl3 was disrupted after the first exon.25Schrick J.J. Vogel P. Abuin A. Hampton B. Rice D.S. ADP-ribosylation factor-like 3 is involved in kidney and photoreceptor development.Am. J. Pathol. 2006; 168: 1288-1298Abstract Full Text Full Text PDF PubMed Scopus (116) Google Scholar These Arl3−/− mice, which represent a null allele, developed a severe ciliopathy phenotype with pronounced cystic kidney disease, pancreatic hypoplasia, ductal plate malformation within the liver, and retinal dystrophy with impaired photoreceptor development.25Schrick J.J. Vogel P. Abuin A. Hampton B. Rice D.S. ADP-ribosylation factor-like 3 is involved in kidney and photoreceptor development.Am. J. Pathol. 2006; 168: 1288-1298Abstract Full Text Full Text PDF PubMed Scopus (116) Google Scholar The mice died within 3 weeks of age, indicating a severe phenotype, which is much more detrimental than that of our human subjects, who carry a missense mutation. We speculate that nonsense mutations in ARL3 in humans could cause more pronounced ciliopathy phenotypes, such as the perinatally lethal ciliopathy Meckel syndrome,26Hartill V. Szymanska K. Sharif S.M. Wheway G. Johnson C.A. Meckel-Gruber syndrome: An update on diagnosis, clinical management, and research advances.Front Pediatr. 2017; 5: 244Crossref PubMed Scopus (69) Google Scholar and could go some way to explaining why such a fundamental gene has previously not been identified in ciliopathy syndromes. It is noteworthy that the ExAC Browser and gnomAD do not have any homozygous pathogenic variants reported within ARL3 and that the gene is relatively intolerant to variation (positive Z score of 0.44). We did not identify any additional ARL3 pathogenic variants in our WES databases, which are relatively enriched with autozygosity, or in a cohort of 35 unsolved JBTS-affected individuals. In humans, Strom et al. previously reported the heterozygous missense variant c.269A>G (p.Tyr90Cys) in ARL3 in a European-descent pedigree with non-syndromic retinitis pigmentosa.27Strom S.P. Clark M.J. Martinez A. Garcia S. Abelazeem A.A. Matynia A. Parikh S. Sullivan L.S. Bowne S.J. Daiger S.P. Gorin M.B. De novo occurrence of a variant in ARL3 and apparent autosomal dominant transmission of retinitis pigmentosa.PLoS ONE. 2016; 11: e0150944Crossref PubMed Scopus (29) Google Scholar The variant, which was rare, appeared de novo and was predicted to be pathogenic, was confirmed as heterozygous in three affected individuals, and was transmitted in an autosomal-dominant fashion. A second allele was not identified, and mechanistic evaluation was not carried out. On the other hand, here we have identified bi-allelic ARL3 changes that fully segregate with a classical JBTS phenotype, including retinal changes. Thus, although the connection between the de novo ARL3 variant and retinitis pigmentosa remains unexplained, it seems that bi-allelic ARL3 deleterious variants are sufficient to cause JBTS. The involvement of ciliopathy-associated genes in non-syndromic retinitis pigmentosa has been well described, so it would be of interest for the affected individual reported by Strom et al. to be investigated for the possibility of a second deleterious allele in trans in ARL3. It is also possible that, as reported here, bi-allelic mutations in ARL3 give rise to an extended phenotype compared with its reported dominant phenotype. A growing number of genes are known to cause distinct phenotypes according to whether dominant or recessive variants are inherited. For retinitis pigmentosa, mutations (typically nonsense) in RP1 were initially described in an autosomal-dominant pattern,28Pierce E.A. Quinn T. Meehan T. McGee T.L. Berson E.L. Dryja T.P. Mutations in a gene encoding a new oxygen-regulated photoreceptor protein cause dominant retinitis pigmentosa.Nat. Genet. 1999; 22: 248-254Crossref PubMed Scopus (172) Google Scholar followed by autosomal-recessive (homozygous missense) variants.29Khaliq S. Abid A. Ismail M. Hameed A. Mohyuddin A. Lall P. Aziz A. Anwar K. Mehdi S.Q. Novel association of RP1 gene mutations with autosomal recessive retinitis pigmentosa.J. Med. Genet. 2005; 42: 436-438Crossref PubMed Scopus (61) Google Scholar For Gillespie syndrome, a form of non-progressive cerebellar ataxia, both bi-allelic and mono-allelic mutations in ITPR1 (MIM: 147265) have been reported,30Gerber S. Alzayady K.J. Burglen L. Brémond-Gignac D. Marchesin V. Roche O. Rio M. Funalot B. Calmon R. Durr A. et al.Recessive and dominant de novo ITPR1 mutations cause Gillespie syndrome.Am. J. Hum. Genet. 2016; 98: 971-980Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar and the single heterozygous mutations were thought to exert a dominant-negative effect. In addition, variants in genes known only to be related to autosomal-dominant disease have been found in association with recessive mutations, both where the phenotypes are similar but more severe (ACTG2-related visceral myopathy [MIM: 102545]) and where distinctly different phenotypes have been observed (FBN2-related myopathy [MIM: 612570] and CSF1R-related brain malformation [MIM: 164770]).31Monies D. Maddirevula S. Kurdi W. Alanazy M.H. Alkhalidi H. Al-Owain M. Sulaiman R.A. Faqeih E. Goljan E. Ibrahim N. et al.Autozygosity reveals recessive mutations and novel mechanisms in dominant genes: implications in variant interpretation.Genet. Med. 2017; 19: 1144-1150Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar Interestingly, pathogenic variants in the ARL3 interaction partners ARL13B and PDE6D also cause JBTS. ARL13B (MIM: 608922) mutations were reported in individuals with a classical neurodevelopmental JBTS phenotype (JBTS8) without prominent renal phenotypes.32Cantagrel V. Silhavy J.L. Bielas S.L. Swistun D. Marsh S.E. Bertrand J.Y. Audollent S. Attié-Bitach T. Holden K.R. Dobyns W.B. et al.International Joubert Syndrome Related Disorders Study GroupMutations in the cilia gene ARL13B lead to the classical form of Joubert syndrome.Am. J. Hum. Genet. 2008; 83: 170-179Abstract Full Text Full Text PDF PubMed Scopus (273) Google Scholar It is particularly noteworthy that some affected individuals had a small occipital encephalocele, indicating that more severe brain phenotypes could be likely. PDE6D (MIM: 602676) mutations have been reported in three siblings with JBTS (JBTS22) and associated retinal and post-axial polydactyly phenotypes, as well as kidney hypoplasia.33Thomas S. Wright K.J. Le Corre S. Micalizzi A. Romani M. Abhyankar A. Saada J. Perrault I. Amiel J. Litzler J. et al.A homozygous PDE6D mutation in Joubert syndrome impairs targeting of farnesylated INPP5E protein to the primary cilium.Hum. Mutat. 2014; 35: 137-146Crossref PubMed Scopus (93) Google Scholar Furthermore, the disrupted ciliary carg
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