guanosine-triphosphate and Abnormalities--Multiple

ARL13B encodes for the ADP-ribosylation factor-like 13B GTPase, which is required for normal cilia structure and Sonic hedgehog (Shh) signaling. Disruptions in cilia structure or function lead to a class of human disorders called ciliopathies. Joubert syndrome is characterized by a wide spectrum of symptoms, including a variable degree of intellectual disability, ataxia, and ocular abnormalities. Here we report a novel homozygous missense variant c.[223G>A] (p.(Gly75Arg) in the ARL13B gene, which was identified by whole-exome sequencing of a trio from a consanguineous family with multiple-affected individuals suffering from intellectual disability, ataxia, ocular defects, and epilepsy. The same variant was also identified in a second family. We saw a striking difference in the severity of ataxia between affected male and female individuals in both families. Both ARL13B and ARL13B-c.[223G>A] (p.(Gly75Arg) expression rescued the cilia length and Shh defects displayed by Arl13b

Topics: Abnormalities, Multiple; Adolescent; ADP-Ribosylation Factors; Adult; Animals; Cell Line, Tumor; Cells, Cultured; Cerebellum; Child; Eye Abnormalities; Female; Guanosine Triphosphate; Homozygote; Humans; Kidney Diseases, Cystic; Loss of Function Mutation; Male; Mice; Mutation, Missense; Pedigree; Retina

RAB18, RAB3GAP1, RAB3GAP2 and TBC1D20 are each mutated in Warburg Micro syndrome, a rare autosomal recessive multisystem disorder. RAB3GAP1 and RAB3GAP2 form a binary 'RAB3GAP' complex that functions as a guanine-nucleotide exchange factor (GEF) for RAB18, whereas TBC1D20 shows modest RAB18 GTPase-activating (GAP) activity in vitro. Here, we show that in the absence of functional RAB3GAP or TBC1D20, the level, localization and dynamics of cellular RAB18 is altered. In cell lines where TBC1D20 is absent from the endoplasmic reticulum (ER), RAB18 becomes more stably ER-associated and less cytosolic than in control cells. These data suggest that RAB18 is a physiological substrate of TBC1D20 and contribute to a model in which a Rab-GAP can be essential for the activity of a target Rab. Together with previous reports, this indicates that Warburg Micro syndrome can be caused directly by loss of RAB18, or indirectly through loss of RAB18 regulators RAB3GAP or TBC1D20.

Topics: Abnormalities, Multiple; Animals; Blotting, Western; Case-Control Studies; Cataract; Cells, Cultured; Cornea; Cytosol; Endoplasmic Reticulum; Fibroblasts; Flow Cytometry; Fluorescent Antibody Technique; Gene Expression Regulation; Guanosine Triphosphate; HeLa Cells; Humans; Hydrolysis; Hypogonadism; Intellectual Disability; Mice; Mice, Knockout; Microcephaly; Optic Atrophy; rab GTP-Binding Proteins; rab1 GTP-Binding Proteins; rab3 GTP-Binding Proteins; Real-Time Polymerase Chain Reaction; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger

Costello syndrome (CS) is a rare congenital disorder characterized by failure to thrive, craniofacial dysmorphisms, cardiac and skin abnormalities, mental retardation, and predisposition to malignancies. CS is caused by heterozygous gain-of-function mutations in HRAS that also occur as somatic alterations in human tumors. HRAS is one of the three classical RAS proteins and cycles between an active, GTP- and an inactive, GDP-bound conformation. We used primary human skin fibroblasts from patients with CS as a model system to study the functional consequences of HRAS mutations on endogenous signaling pathways. The GTP-bound form of HRAS was significantly enriched in CS compared with normal fibroblasts. Active HRAS is known to stimulate both the RAF-MEK-ERK and the PI3K-AKT signaling cascade. Phosphorylation of MEK and ERK was normal in CS fibroblasts under basal conditions and slightly prolonged after epidermal growth factor (EGF) stimulation. Interestingly, basal phosphorylation of AKT was increased yet more in CS fibroblasts. Moreover, AKT phosphorylation was diminished in the early and enhanced in the late phase of EGF stimulation. Taken together, these results document that CS-associated HRAS mutations result in prolonged signal flux in a ligand-dependent manner. Our data suggest that altered cellular response to growth factors rather than constitutive activation of HRAS downstream signaling molecules may contribute to some of the clinical features in patients with CS.

Topics: Abnormalities, Multiple; Cells, Cultured; Chromones; Craniofacial Abnormalities; Enzyme Inhibitors; Epidermal Growth Factor; Failure to Thrive; Fibroblasts; Guanosine Triphosphate; Humans; Immunoblotting; Intellectual Disability; MAP Kinase Kinase 1; MAP Kinase Kinase 2; Morpholines; Mutation; Phosphatidylinositol 3-Kinases; Phosphoinositide-3 Kinase Inhibitors; Phosphorylation; Proto-Oncogene Proteins c-akt; Proto-Oncogene Proteins p21(ras); Signal Transduction; Skin; Skin Abnormalities; Syndrome

Kindlin-1 is an epithelial-specific member of the novel kindlin protein family, which are regulators of integrin functions. Mutations in the gene that encodes Kindlin-1, FERMT1 (KIND1), cause the Kindler syndrome (KS), a human disorder characterized by mucocutaneous fragility, progressive skin atrophy, ulcerative colitis, photosensitivity, and propensity to skin cancer. Our previous studies indicated that loss of kindlin-1 resulted in abnormalities associated with integrin functions, such as adhesion, proliferation, polarization, and motility of epidermal cells. Here, we disclosed novel FERMT1 mutations in KS and used them, in combination with small-interfering RNA, protein, and imaging studies, to uncover new functions for kindlin-1 in keratinocytes and to discern the molecular pathology of KS. We show that kindlin-1 forms molecular complexes with beta1 integrin, alpha-actinin, migfilin, and focal adhesion kinase and regulates cell shape and migration by controlling lamellipodia formation. Kindlin-1 governs these processes by signaling via Rho family GTPases, and it is required to maintain the pool of GTP-bound, active Rac1, RhoA and Cdc42, and the phosphorylation of their downstream effectors p21-activated kinase 1, LIM kinase, and cofilin. Loss of these kindlin-1 functions forms the biological basis for the epithelial cell fragility and atrophy in the pathology of KS.

Topics: Abnormalities, Multiple; Adult; Cell Line, Transformed; Cell Movement; Cell Shape; Child; Enzyme Activation; Focal Adhesions; Guanosine Triphosphate; Humans; Keratinocytes; Membrane Proteins; Middle Aged; Models, Biological; Mucous Membrane; Neoplasm Proteins; Phenotype; Phosphorylation; Protein Binding; Pseudopodia; rho GTP-Binding Proteins; RNA, Small Interfering; Skin Abnormalities; Syndrome