RESUMEN
The autosomal recessive lysosomal storage disorder, nephropathic cystinosis is characterized by impaired transport of free cystine out of lysosomes. The gene responsible for cystinosis, CTNS, consists of 12 exons and encodes a 55 kDa putative lysosomal membrane protein, called cystinosin. Up to now more than 55 different CTNS mutations have been described in cystinosis. We have analyzed the mutation pattern in a population of 40 cystinosis patients from 35 families of German and Swiss origin. CTNS mutations in 68 out of 70 alleles were identified. The common 57-kb deletion accounted for 65% of the alleles. In five patients we found a known GACT deletion at position 18-21. In two patients we identified a nucleotide substitution at codon 339 and one patient showed a CG insertion at position 697-698. In five patients we observed a G insertion at position 926-927. Moreover, five novel mutations including two deletions involving exon 3 (61-61+2delGGT) and exon 6 (280delG), two insertions in exon 6 (292-293insA) and exon 7 (684insCACTT) and one nucleotide substitution in exon 11 (923G>T) have been identified. These data provide a basis for routine molecular diagnosis of cystinosis in the central European population, especially in cystinosis patients of German and Swiss origin.
Asunto(s)
Cistinosis/genética , Glicoproteínas , Proteínas de la Membrana/genética , Secuencia de Aminoácidos , Sistemas de Transporte de Aminoácidos Neutros , Secuencia de Bases , Estudios de Cohortes , ADN/química , ADN/genética , Análisis Mutacional de ADN , Efecto Fundador , Eliminación de Gen , Alemania , Humanos , Proteínas de Transporte de Membrana , Datos de Secuencia Molecular , Mutagénesis Insercional , Mutación Puntual , Eliminación de Secuencia , SuizaRESUMEN
Mutations in the human spastin gene (SPG4) cause the most prevalent form of autosomal dominant hereditary spastic paraplegia (HSP), a neurodegenerative disorder characterised by progressive weakness and spasticity of the lower limbs. We address the question of intracellular localisation of spastin. Using polyclonal antibodies against N-terminal spastin sequences, we find that the native protein is localised in both the perinuclear cytoplasm and the nucleus. To identify structural motifs within the protein that can explain entry into the nucleus, we developed a reporter system to test nuclear localisation sequence (NLS)-functionality based on four in-frame fused copies of green fluorescent protein. Using this novel tool we demonstrate that spastin carries two NLSs located in exons 1 and 6. Both are independently functional in mediating nuclear entry.
Asunto(s)
Proteínas de Unión al Calcio/química , Proteínas de Unión al Calcio/metabolismo , Núcleo Celular/metabolismo , Proteínas Luminiscentes/genética , Señales de Localización Nuclear/química , Señales de Localización Nuclear/metabolismo , Adenosina Trifosfatasas , Secuencias de Aminoácidos , Anticuerpos/química , Proteínas de Unión al Calcio/genética , Exones/genética , Técnica del Anticuerpo Fluorescente , Vectores Genéticos , Proteínas Fluorescentes Verdes , Células HeLa , Humanos , Espacio Intracelular/metabolismo , Señales de Localización Nuclear/genética , Oligonucleótidos/genética , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Espastina , TransfecciónRESUMEN
Integrin-associated protein (IAP) is a widely expressed membrane protein with multiple functions in immunological and neuronal processes. Having physically mapped the IAP gene into a BAC/PAC contig covering approximately 1 Mb on human chromosome 3ql3.1-q13.2, we determined the genomic organization of the gene, established its expression in skeletal muscle, and identified a novel splice variant. Our expression studies demonstrate expression of integrin-associated protein in the t-tubular system and the euchromatin of skeletal muscle cells where its function thus far is not known.