Gene analysis of the superoxide dismutase gene in amyotrophic lateral screlosis

点击翻译按钮获取中文摘要

We have characterized the Cu-Zn superoxide dismutase gene (SOD1) in six Japanese families with familial amyotrophic lateral screlosis (FALS). Sequence analysis revealed two novel mutations in this gene from two independent families. A T-to-A transversion causes a Val17Glu substitution in one family. A T-to-G mutation leads to a Leu8Val substitutions in another family. Other families had unique clinical symptoms and normal coding sequences of the SOD1 gene, suggesting that FALS is a complex of heterogeneous motor neuron diseases. We studied the gene expression in the PC 12 cell line transfected with the mutant SOD1 cDNA.The mutant cDNA was synthesized by RT-PCR of mRNA from FALS patients. The cDNA clones were ligated into the pRc/RSV expression plasmid vector, transfected into PC12 cells. After selection with neomycin, the established cell line was culture under various condition, ie. low concentration of CSF or various CO2 concentration. No morphological changes indicating the apoptosis occurred. The fibroblasts from the FALS patient were cultured under the various condition, ie. hypoxia, low concentrations of FCS,the presence of oxidant or heat stress. The mRNA was purified from the treated fibroblasts of the patient and controls. The differential display techniques detected several bands that were specifically expressed in the patients. The band was excised out from the gel, PCR amplified, cloned into the vector and sequenced. Thus obtained cDNA contained the previously unreported sequences. The human brain cDNA library was constructed and was screened using the cloned. The identified plaques contained the same sequence as that we detected in the treated fibroblasts of the patients. This results suggests that the fibroblasts from the FALS patients responds to the heat stress by expressing some specific gene.