Biochemical characterization of missense mutations in the Arf/Arl-family small GTPase Arl6 causing Bardet–Biedl syndrome

Kobayashi, Tetsuo; Hori, Yuichi; Ueda, Noboru; Kajiho, Hiroaki; Muraoka, Shin; Shima, Fumi; Kataoka, Tohru; Kontani, Kenji; Katada, Toshiaki

doi:10.1016/j.bbrc.2009.02.087

Cited by 25 publications

(24 citation statements)

References 17 publications

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“…We found 48 of 49 mutations predicted to be pathogenic from human genetic analysis to be such, whereas our interpretation of the BBS1 M390R allele as a hypomorph is consistent with the M390R knock-in mouse model (42) and our prediction of BBS11 P130S to be pathogenic is consistent with a previous report of failure of this allele to rescue melanosome transport (15). Further, our in vivo data for BBS3 and BBS6 are in agreement with two recent biochemical studies (29,30).…”

Section: Discussionsupporting

confidence: 91%

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Functional analyses of variants reveal a significant role for dominant negative and common alleles in oligogenic Bardet–Biedl syndrome

Zaghloul

Liu

Gerdes

et al. 2010

Proc. Natl. Acad. Sci. U.S.A.

109

145

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Technological advances hold the promise of rapidly catalyzing the discovery of pathogenic variants for genetic disease. However, this possibility is tempered by limitations in interpreting the functional consequences of genetic variation at candidate loci. Here, we present a systematic approach, grounded on physiologically relevant assays, to evaluate the mutational content (125 alleles) of the 14 genes associated with Bardet-Biedl syndrome (BBS). A combination of in vivo assays with subsequent in vitro validation suggests that a significant fraction of BBS-associated mutations have a dominantnegative mode of action. Moreover, we find that a subset of common alleles, previously considered to be benign, are, in fact, detrimental to protein function and can interact with strong rare alleles to modulate disease presentation. These data represent a comprehensive evaluation of genetic load in a multilocus disease. Importantly, superimposition of these results to human genetics data suggests a previously underappreciated complexity in disease architecture that might be shared among diverse clinical phenotypes.epistasis | ciliopathy | zebrafish | in vivo assays E xome and whole-genome resequencing is likely to catalyze a paradigm shift in the identification of genetic lesions in patients (1). At the same time, even within the confines of the coding genome, such technologies pose an interpretive problem, in that the pathogenic candidacy of mutations can only be derived by narrow genetic models and limited computational predictive tools, both of which are likely to under-and misinterpret the effect of some mutations. Moreover, inter-and intrafamilial variability, a phenomenon prevalent in most genetic traits, remains a major confounding factor because both allelic variation at a single locus and second-site trans modifiers can exert a significant influence on penetrance and expressivity through additive and epistatic effects (2).Bardet-Biedl Syndrome (BBS) is a useful model for dissecting epistasis because most of the 14 BBS genes can also contribute epistatic alleles (3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17). BBS is also a representative of the ciliopathy disease spectrum, a group of disorders characterized by defects in ciliary structure and/or ciliary signal output (18). Hallmarks of BBS include retinal degeneration, obesity, hypogonadism, polydactyly, renal dysfunction, and mental retardation (19).We and others have shown that the zebrafish provides experimentally tractable and physiologically relevant models of important aspects of ciliary dysfunction (2,15,17,(20)(21)(22)(23). Moreover, human mRNA for ciliopathy genes can rescue both morphant and mutant zebrafish phenotypes efficiently, providing a robust platform for interpretation of the pathological relevance of identified missense alleles, whose causal relation to the disorder cannot be proven definitively with genetic arguments alone (15,17,22,23).Here, we have integrated multiple independent in vivo assays, followed by in vitro validations, to int...

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Section: Discussionsupporting

confidence: 91%

“…In vitro assays. Phenotypes related to some of the BBS proteins have been observed in cultured cells (8)(9)(10)(29)(30)(31)(32)(33)(34). Therefore, we asked whether some of the aberrant activity in zebrafish embryos could be explained by defective behavior in mammalian cells.…”

Section: Resultsmentioning

confidence: 99%

Functional analyses of variants reveal a significant role for dominant negative and common alleles in oligogenic Bardet–Biedl syndrome

Zaghloul

Liu

Gerdes

et al. 2010

Proc. Natl. Acad. Sci. U.S.A.

109

145

View full text Add to dashboard Cite

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“…Preparation of cDNA of mouse Rab33a Q95L (a constitutive active mutant) was reported previously (Itoh et al, 2006). cDNA of an inactive rat Rab33a mutant was produced by making a threonine-to-arginine substitution (T50R; Kobayashi et al, 2009) using the QuikChange II Site-Directed Mutagenesis Kit (Stratagene) with the primers 5Ј-TCCAACGTGGGCAAGCGC TGCCTG-3Ј and 5Ј-TCAGGCAGCGCTTGCCCACGTTGG-3Ј. Fulllength cDNA of rat synaptophysin was amplified with the primers 5Ј-TAGG ATCCATGGACGTGGTGAATCAGCTGG-3Ј and 5Ј-GCGGATCCTTAC ATCTGATTGGAGAAGGAG-3Ј.…”

Section: Methodsmentioning

confidence: 99%

Rab33a Mediates Anterograde Vesicular Transport for Membrane Exocytosis and Axon Outgrowth

et al. 2012

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“…Substitution of the first G with V frequently leads to reduced GTP hydrolysis and thus generates a dominant-active protein (Lu et al, 2001). By contrast, mutants with a substitution of N for the T/S site have a higher affinity for GDP and function as a dominant-negative protein (Fan et al, 2004;Kobayashi et al, 2009;Lu et al, 2001). We introduced these two mutations into the corresponding sites of Arl13b and tested their in…”

Section: Multiple Regions Of Arl13b Are Required For Its Ciliary Locamentioning

confidence: 99%

Cilia localization is essential for in vivo functions of the Joubert syndrome protein Arl13b/Scorpion

2009

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arl13b was initially cloned as the novel cystic kidney gene scorpion (sco) in zebrafish and was shown to be required for cilia formation in the kidney duct. In mouse, a null mutant of Arl13b shows abnormal ultrastructure of the cilium and defective sonic hedgehog (Shh) signaling. Importantly, a recent study linked mutations in ARL13B to a classical form of Joubert syndrome (JS), an autosomal recessive disorder characterized by a distinctive cerebellar malformation. In this study, we analyzed the zebrafish arl13b (sco) mutant and gene products in detail. We first demonstrate that Arl13b is a protein that is highly enriched in the cilium and is required for cilia formation in multiple organs in zebrafish, and that knockdown of arl13b leads to multiple cilia-associated phenotypes. We additionally show that multiple regions of Arl13b are required for its localization to the cilium. By means of rescuing experiments with a series of deletion and point mutants, we further demonstrate that the ciliary localization is crucial for the in vivo function of Arl13b. Together, these results strongly support the hypothesis that JS-related disease (JSRD) is a ciliopathy, or a disease caused by ciliary defects, and that Arl13b functions mainly through the cilium.

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Biochemical characterization of missense mutations in the Arf/Arl-family small GTPase Arl6 causing Bardet–Biedl syndrome

Cited by 25 publications

References 17 publications

Functional analyses of variants reveal a significant role for dominant negative and common alleles in oligogenic Bardet–Biedl syndrome

Functional analyses of variants reveal a significant role for dominant negative and common alleles in oligogenic Bardet–Biedl syndrome

Rab33a Mediates Anterograde Vesicular Transport for Membrane Exocytosis and Axon Outgrowth

Cilia localization is essential for in vivo functions of the Joubert syndrome protein Arl13b/Scorpion

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