Kehitysvammahuollon tietopankki
 | Kirjaesittely |
Isosomppi Juha, Department of Molecular Mrdicine, National Public Heath
Institute, Helsinki, Finland and Department of Medical Genetics, University of
Helsinki Finland, 2003, ISBN 951-740-340-2
Abstract:
Neuronal ceroid lipofuscinoses (NCL) are a group of common progressive
recessively inherited neurodegenerative disorders of childhood. All types of NCL
diseases cause progressive visual and mental decline, motor disturbances,
epilepsy and behavioral changes, and lead to premature death. Prior to this
study the first NCL gene was recently identified using the positional cloning
approach. Mutations in the palmitoyl protein thioesterase (PPT1) were shown to
result in the infantile form of NCL (INCL). At the same time, the positional
cloning of the vLINCL disease gene (CLN5) was in progress. The position of the
CLN5 gene was assigned by linkage analysis to chromosomal region 13q21.1-q32 and
physical mapping of the region was ongoing. Both of these diseases are
especially enriched in the Finnish population.
In this thesis, fluorescent in situ hybridization on DNA fibers (fiber-FISH) was utilized in the physical mapping project of the critical CLN5 region. This visual mapping approach was essential in our efforts to produce a genomic clone contig over the CLN5 region. The fiber-FISH method not only enabled rapid confirmation of the order of genomic clones, but it also allowed the detection of overlaps between various clones. Thus, high-density mapping was possible without the tedious methods traditionally used in physical mapping approaches. In addition, a novel ultra-sensitive tyramide-based amplification system was used successfully to visualize short probes representing transcribed sequences in the critical region.
The physical map of the critical region facilitated the identification of the CLN5 gene, and its expression was analyzed as a part of this thesis. The biosynthesis, post-translational processing and intracellular localization of the CLN5 protein was investigated in transiently transfected BHK-21 cells. Confocal immunofluorescence microscopy and immunoprecipitation analysis showed that wild type CLN5 is a lysosomally targeted 60-kDa glycoprotein, which is partially secreted into the culture medium. Secretion of the polypeptide into the culture medium would imply that CLN5 is a soluble lysosomal glycoprotein, not an integral transmembrane protein as predicted earlier. The most common naturally occurring CLN5 disease mutation represents a premature stop codon that leaves the 16 C-terminal amino acids of the protein untranslated. These polypeptides were not targeted to lysosomes, which would imply that the pathogenesis of vLINCL might be associated with the defective lysosomal trafficking of the corresponding polypeptide.
In order to better understand the destruction of neurons in the central nervous system in the childhood forms of NCL-disorders, we characterized expression of the PPT1 gene in developing mouse brain and embryo. Northern blot analysis, in situ hybridization and immunohistochemistry revealed gradual increase in expression of PPT1 mRNA and protein during mouse development. A notable increase in PPT1 mRNA expression was monitored during a developmental stage of the mouse brain when new synaptic contacts are extensively formed. In addition to that, a relatively high prevalence of PPT protein was observed in the neuronal extensions. Based on these findings, it was suggested that PPT1 might have a role for survival of neural networks, possibly associated with the development and maintenance of the synaptic machinery.
Linkkejä:
Kehitysvamma-alan kirjallisuutta25.12.2005