RESUMEN
Spastic paraplegia type 10 (SPG10) is an autosomal dominant form of hereditary spastic paraplegia (HSP) due to mutations in KIF5A, a gene encoding the neuronal kinesin heavy chain implicated in anterograde axonal transport. KIF5A mutations were found in both pure and complicated forms of the disease; a single KIF5A mutation was also detected in a CMT2 patient belonging to an SPG10 mutant family. To confirm the involvement of the KIF5A gene in both CMT2 and SPG10 phenotypes and to define the frequency of KIF5A mutations in an Italian HSP patient population, we performed a genetic screening of this gene in a series of 139 HSP and 36 CMT2 affected subjects. We identified five missense changes, four in five HSP patients and one in a CMT2 subject. All mutations, including the one segregating in the CMT2 patient, are localized in the kinesin motor domain except for one, falling within the stalk domain and predicted to generate protein structure destabilization. The results obtained indicate a KIF5A mutation frequency of 8.8% in the Italian HSP population and identify a region of the kinesin protein, the stalk domain, as a novel target for mutation. In addition, the mutation found in the CMT2 patient strengthens the hypothesis that CMT2 and SPG10 are the extreme phenotypes resulting from mutations in the same gene.
Asunto(s)
Enfermedad de Charcot-Marie-Tooth/genética , Cinesinas/genética , Mutación , Dominios y Motivos de Interacción de Proteínas/genética , Paraplejía Espástica Hereditaria/genética , Adolescente , Adulto , Secuencia de Aminoácidos , Secuencia de Bases , Niño , Exones , Humanos , Persona de Mediana Edad , Linaje , Adulto JovenRESUMEN
BACKGROUND: Mutations in GDAP1 associate with demyelinating (CMT4A) and axonal (CMT2K) forms of CMT. While CMT4A shows recessive inheritance, CMT2K can present with either recessive (AR-CMT2K) or dominant segregation pattern (AD-CMT2K), the latter being characterised by milder phenotypes and later onset. The majority of the GDAP1 mutations are associated with CMT4A and AR-CMT2K, with only four heterozygous mutations identified in AD-CMT2K. METHODS: We screened GDAP1 gene in a series of 43 index patients, 39 with CMT2 and 4 with intermediate CMT, with sporadic and familial occurrence of the disease. RESULTS: Three novel mutations were identified in three families with dominant segregation of the disease: two missense changes, p.Arg226Ser and p.Ser34Cys, affecting the GST domain of the GDAP1 protein and a novel deletion (c.23delAG) leading to early truncation of the protein upstream the GST domain. Wide variability in clinical presentation is shared by all three families mostly in terms of age at onset and disease severity. A rare variant p.Gly269Arg, located within the GST domain, apparently acts as phenotype modulator in the family carrying the deletion. CONCLUSION: The results obtained reveal a GDAP1 mutation frequency of 27% in the dominant families analysed, a figure still unreported for this gene, thus suggesting that GDAP1 involvement in dominant CMT2 might be higher than expected.
Asunto(s)
Axones/metabolismo , Enfermedad de Charcot-Marie-Tooth/genética , Glutatión Transferasa/genética , Mutación , Proteínas del Tejido Nervioso/genética , Adolescente , Adulto , Edad de Inicio , Enfermedad de Charcot-Marie-Tooth/fisiopatología , Niño , Preescolar , Análisis Mutacional de ADN , Electrofisiología , Eliminación de Gen , Genes Dominantes , Humanos , Italia , Datos de Secuencia Molecular , Mutación Missense , Proteínas del Tejido Nervioso/química , Linaje , Fenotipo , Análisis de Secuencia de ADN , Adulto JovenAsunto(s)
Hemiplejía/genética , Mutación Missense/genética , ATPasa Intercambiadora de Sodio-Potasio/genética , Adulto , Niño , Análisis Mutacional de ADN/métodos , Femenino , Haplotipos/genética , Hemiplejía/patología , Humanos , Masculino , Linaje , ATPasa Intercambiadora de Sodio-Potasio/fisiologíaRESUMEN
BACKGROUND: Benign familial neonatal convulsion (BFNC) is a rare autosomal dominant disorder caused by mutations in two genes, KCNQ2 and KCNQ3, encoding for potassium channel subunits underlying the M-current. This current limits neuronal hyperexcitability by causing spike-frequency adaptation. METHODS: The authors describe a BFNC family with four affected members: two of them exhibit BFNC only while the other two, in addition to BFNC, present either with a severe epileptic encephalopathy or with focal seizures and mental retardation. RESULTS: All affected members of this family carry a novel missense mutation in the KCNQ2 gene (K526N), disrupting the tri-dimensional conformation of a C-terminal region of the channel subunit involved in accessory protein binding. When heterologously expressed in CHO cells, potassium channels containing mutant subunits in homomeric or heteromeric configuration with wild-type KCNQ2 and KCNQ3 subunits exhibit an altered voltage-dependence of activation, without changes in intracellular trafficking and plasma membrane expression. CONCLUSION: The KCNQ2 K526N mutation may affect M-channel function by disrupting the complex biochemical signaling involving KCNQ2 C-terminus. Genetic rather than acquired factors may be involved in the pathophysiology of the phenotypic variability of the neurologic symptoms associated with BFNC in the described family.
Asunto(s)
Sustitución de Aminoácidos , Epilepsia Benigna Neonatal/genética , Discapacidad Intelectual/genética , Mutación Missense , Mutación Puntual , Canales de Potasio con Entrada de Voltaje/genética , Adulto , Secuencia de Aminoácidos , Animales , Anticonvulsivantes/farmacología , Anticonvulsivantes/uso terapéutico , Células CHO , Cricetinae , Cricetulus , Resistencia a Medicamentos/genética , Epilepsias Parciales/tratamiento farmacológico , Epilepsias Parciales/genética , Epilepsia Benigna Neonatal/tratamiento farmacológico , Femenino , Humanos , Recién Nacido , Activación del Canal Iónico , Transporte Iónico , Canal de Potasio KCNQ2 , Imagen por Resonancia Magnética , Masculino , Datos de Secuencia Molecular , Linaje , Fenotipo , Canales de Potasio con Entrada de Voltaje/química , Canales de Potasio con Entrada de Voltaje/fisiología , Conformación Proteica , Subunidades de Proteína , Cuadriplejía/genética , Relación Estructura-ActividadAsunto(s)
Anomalías Múltiples/genética , Proteínas Portadoras/genética , Proteínas del Citoesqueleto/genética , Atresia Esofágica/patología , Proteínas del Tejido Nervioso/genética , Fístula Traqueoesofágica/patología , Translocación Genética , Anomalías Múltiples/patología , Animales , Secuencia de Bases , Encéfalo/metabolismo , Línea Celular , Preescolar , Bandeo Cromosómico , Cromosomas Humanos Par 15/genética , Cromosomas Humanos Par 6/genética , Trastornos del Conocimiento/patología , Cricetinae , Discapacidades del Desarrollo/patología , Distonina , Femenino , Perfilación de la Expresión Génica , Genotipo , Humanos , Hibridación Fluorescente in Situ , Cariotipificación , Masculino , Músculos/metabolismo , Mutación , Polimorfismo de Nucleótido Simple , Isoformas de Proteínas/genéticaRESUMEN
Dopamine genes are candidate genes for dyslexia in the light of the well-known comorbidity between dyslexia and ADHD. Within-family association and linkage disequilibrium were tested between four genetic markers at DRD4, DRD3, DRD2, and DAT loci, and dyslexia, in a sample of 130 Italian dyslexic children, 16.9% of whom had comorbid ADHD. No evidence of either association or linkage disequilibrium was found, neither in the total sample nor in the comorbid subgroup. Negative results do not support a common genetic basis between these two disorders for these markers.
Asunto(s)
Dislexia/genética , Desequilibrio de Ligamiento , Receptores Dopaminérgicos/genética , Trastorno por Déficit de Atención con Hiperactividad/epidemiología , Trastorno por Déficit de Atención con Hiperactividad/genética , Niño , Comorbilidad , Dislexia/epidemiología , Femenino , Marcadores Genéticos , Humanos , Italia/epidemiología , Masculino , PrevalenciaRESUMEN
Ocular albinism type 1 (OA1) is an X-linked disorder mainly characterized by a severe reduction of visual acuity, hypopigmentation of the retina and the presence of macromelanosomes in the skin and eyes. Various types of mutation have been identified within the OA1 gene in patients with the disorder, including several missense mutations of unknown functional significance. In order to shed light into the molecular pathogenesis of ocular albinism and possibly define critical functional domains within the OA1 protein, we characterized 19 independent missense mutations with respect to processing and subcellular distribution on expression in COS-7 cells. Our analysis indicates the presence of at least two distinct biochemical defects associated with the different missense mutations. Eleven of the nineteen OA1 mutants (approximately 60%) were retained in the endoplasmic reticulum, showing defecNStive intracellular transport and glycosylation, consistent with protein misfolding. The remaining eight of the nineteen OA1 mutants (approximately 40%) displayed sorting and processing behaviours indistinguishable from those of the wild-type protein. Consistent with our recent findings that OA1 represents a novel type of intracellular G protein-coupled receptor (GPCR), we found that most of these latter mutations cluster within the second and third cytosolic loops, two regions that in canonical GPCRs are known to be critical for their downstream signaling, including G protein-coupling and effector activation. The biochemical analysis of OA1 mutations performed in this study provides important insights into the structure-function relationships of the OA1 protein and implies protein misfolding as a major pathogenic mechanism in OA1.
Asunto(s)
Albinismo Ocular/metabolismo , Proteínas del Ojo/metabolismo , Glicoproteínas de Membrana/metabolismo , Albinismo Ocular/genética , Secuencia de Aminoácidos , Animales , Transporte Biológico , Western Blotting , Células COS , Células Cultivadas , Proteínas del Ojo/química , Proteínas del Ojo/genética , Técnica del Anticuerpo Fluorescente , Glicosilación , Aparato de Golgi/metabolismo , Humanos , Melanocitos/fisiología , Glicoproteínas de Membrana/química , Glicoproteínas de Membrana/genética , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Mutación Missense , Conformación ProteicaRESUMEN
G protein-coupled receptors (GPCRs) participate in the most common signal transduction system at the plasma membrane. The wide distribution of heterotrimeric G proteins in the internal membranes suggests that a similar signalling mechanism might also be used at intracellular locations. We provide here structural evidence that the protein product of the ocular albinism type 1 gene (OA1), a pigment cell-specific integral membrane glycoprotein, represents a novel member of the GPCR superfamily and demonstrate that it binds heterotrimeric G proteins. Moreover, we show that OA1 is not found at the plasma membrane, being instead targeted to specialized intracellular organelles, the melanosomes. Our data suggest that OA1 represents the first example of an exclusively intracellular GPCR and support the hypothesis that GPCR-mediated signal transduction systems also operate at the internal membranes in mammalian cells.
Asunto(s)
Albinismo Ocular/genética , Proteínas del Ojo/fisiología , Membranas Intracelulares/fisiología , Glicoproteínas de Membrana/fisiología , Transducción de Señal , Secuencia de Aminoácidos , Animales , Células COS , Proteínas del Ojo/genética , Proteínas de Unión al GTP/fisiología , Humanos , Lisosomas/metabolismo , Melanocitos/citología , Melanocitos/metabolismo , Glicoproteínas de Membrana/genética , Modelos Biológicos , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Mutación , Mutación Missense , Proteínas Recombinantes/metabolismo , Homología de Secuencia de Aminoácido , TransfecciónRESUMEN
Ocular albinism type 1 (OA1) is an inherited disorder characterized by severe reduction of visual acuity, photophobia, and retinal hypopigmentation. Ultrastructural examination of skin melanocytes and of the retinal pigment epithelium reveals the presence of macromelanosomes, suggesting a defect in melanosome biogenesis. The gene responsible for OA1 is exclusively expressed in pigment cells and encodes a predicted protein of 404 aa displaying several putative transmembrane domains and sharing no similarities with previously identified molecules. Using polyclonal antibodies we have identified the endogenous OA1 protein in retinal pigment epithelial cells, in normal human melanocytes and in various melanoma cell lines. Two forms of the OA1 protein were identified by Western analysis, a 60-kDa glycoprotein and a doublet of 48 and 45 kDa probably corresponding to unglycosylated precursor polypeptides. Upon subcellular fractionation and phase separation with the nonionic detergent Triton X-114, the OA1 protein segregated into the melanosome-rich fraction and behaved as an authentic integral membrane protein. Immunofluorescence and immunogold analyses on normal human melanocytes confirmed the melanosomal membrane localization of the endogenous OA1 protein, consistent with its possible involvement in melanosome biogenesis. The identification of a novel melanosomal membrane protein involved in a human disease will provide insights into the mechanisms that control the cell-specific pathways of subcellular morphogenesis.