RESUMEN
The lysosomal storage disorder, mucopolysaccharidosis type I (MPS I), is caused by a deficiency of the enzyme alpha-L-iduronidase, which is involved in the breakdown of dermatan and heparan sulphates. There are three clinical phenotypes, ranging from the Hurler form characterised by skeletal abnormalities, hepatosplenomegaly and severe mental retardation, to the milder Scheie phenotype where there is aortic valve disease, corneal clouding, limited skeletal problems, but no mental retardation. In this study, 85 MPS I families (73 Hurler, 5 Hurler/Scheie, 7 Scheie) were screened for 9 known mutations (Q70X, A75T, 474-2a>g, L218P, A327P, W402X, P533R, R89Q, 678-7g>a). W402X was the most frequent mutation in our population (45.3%) and Q70X was the second most frequent (15.9%). In 30 families, either one or both of the mutations were not identified, which accounted for 25.9% of the total alleles. Therefore, all 14 exons of the alpha-L-iduronidase gene were screened in these patients and 23 different sequence changes were found, 17 of which were previously unknown. The novel sequence changes include 4 deletions (153delC, 628del5, 740delC, 747delG), 5 nonsense mutations (Q60X, Y167X, Q400X, R619X, R628X), 6 missense mutations (C205Y, G208V, H240R, A319V, P496R, S633L), a splice site mutation (IVS12+5g>a), and a rare polymorphism (A591T). The polymorphism and novel missense mutations were transiently expressed in COS-7 cells and all of them except the polymorphism showed complete loss of enzyme activity. In total, 165 of the 170 mutant alleles were identified in this study and despite the high frequency of W402X and Q70X, the identification of many novel mutations unique to individual families further highlights the genetic heterogeneity of MPS I.
Asunto(s)
Frecuencia de los Genes , Mucopolisacaridosis I/genética , Mutación Missense , Animales , Secuencia de Bases , Células COS , Cartilla de ADN , Heterogeneidad Genética , Humanos , Iduronidasa/genética , Mucopolisacaridosis I/fisiopatología , Mutagénesis Sitio-Dirigida , Fenotipo , Reacción en Cadena de la PolimerasaRESUMEN
Herpes simplex virus type 1 represents an ideal candidate for development as a vehicle for gene transfer to postmitotic neurons of the central nervous system. The natural biology of this virus makes it well suited for this purpose as it is capable of infecting a variety of neuronal cell types in the brain where the viral genome can persist indefinitely in a latent state. In latency, the viral lytic genes are transcriptionally silent and a unique set of latency-associated transcripts are expressed. Two impediments to using herpes simplex virus vectors must be overcome: (1) A noncytotoxic mutant virus backbone must be engineered, and (2) a suitable promoter-regulator that stably expresses foreign genes from the vector genome during latency must be constructed. Deletion of specific immediate early genes from the vector can render the virus nontoxic to neurons in culture and in vivo following stereotactic inoculation into specific regions of the brain. Because these viruses cannot replicate, they enter latency on infection of central nervous system neurons. A number of viral and cellular promoters have been tested for their ability to express genes during latency. Strong viral promoters and neurospecific promoters display transient activity. Although the promoter regions for the latency-associated transcripts are highly active in the peripheral nervous system, they show low-level but persistent activity in the brain. Experiments are in progress to exploit RNA polymerase III gene promoters or novel recombinant promoters capable of auto-inducing their own expression in order to increase gene expression during latency in brain neurons.
Asunto(s)
Encéfalo/fisiología , Técnicas de Transferencia de Gen , Vectores Genéticos , Neuronas/fisiología , Simplexvirus/genética , Transfección , Animales , Células Cultivadas , ADN Viral/análisis , Regulación Viral de la Expresión Génica , Regiones Promotoras Genéticas , Proteínas RecombinantesRESUMEN
Deficiency of the lysosomal enzyme iduronate-2-sulfatase (IDS; EC 3.1.6.13) results in the storage of the glycosaminoglycans heparan sulfate and dermatan sulfate, which leads to the lysosomal storage disorder mucopolysaccharidosis type II. Three overlapping genomic clones derived from an X-chromosome-specific library containing the entire IDS gene were isolated and the sequences of the intron boundaries and the 5' promoter region were determined. The IDS gene is split into nine exons spanning approximately 24 kb. The potential promoter for IDS lacks a TATA box but contains GC box consensus sequences, consistent with its role as a housekeeping gene. A polypyrimidine-like repeat is found in intron 1.
Asunto(s)
Iduronato Sulfatasa/genética , Secuencia de Bases , Mapeo Cromosómico , Clonación Molecular , Secuencia de Consenso , ADN Complementario/genética , Exones , Humanos , Intrones , Datos de Secuencia Molecular , Mucopolisacaridosis II/enzimología , Mucopolisacaridosis II/genética , Regiones Promotoras Genéticas , ARN Mensajero/genética , Cromosoma XRESUMEN
A herpes simplex virus (HSV) vector in which the mammalian promoter for neuron-specific enolase (NSE) controls expression of a marker gene was analyzed for its ability to drive expression of this foreign gene in culture and in vivo. In cultured cells, the vector appeared to give neuron-specific expression. Introduction of 10(6) pfu of the virus into the adult rat caudate nucleus by stereotactic injection was not toxic to the animals and yielded beta-galactosidase (beta-gal)-positive neurons for at least 30 days after viral inoculation. This recombinant herpes virus vector is the first described to use a mammalian promoter to yield extended expression of a foreign gene product in the adult mammalian central nervous system (CNS).