Molecular, biological analyzes of familial and sporadic amyortrophic lateral sclerosis in San-in -analyzes of DNA,mRNA,content and activity of SOD1 and androgen receptor gene-

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Using single strand conformational polymorphism (SSCP) analysis and activity assay for Cu/Zn superoxide dismutase (SOD1), we performed family analysis in Japanese family with FALS having two basepair deletion in the SOD1 gene. We also analyzed the activity, content and mRNA of SOD1, and copper ion concentration in a patient of this family. Not only two FALS patients but five clinically non-affected members had abnormal SOD1 gene. The reduction of SOD1 activity was about 70% in the pateients and about 30% in the clinically non-affected members. The SOD activity stain and Western blot analysis of red blood cells (RBCs) and brain homogenate from the patient showed the absence of the mutant SOD1 and low level of total SOD1 activity. The SSCP analysis of RT-PCR products from the patient indicated the presence of an additional SOD1 mRNA due to the mutant SOD1 gene. It was found that the SOD1 in the brain but not in RBCs from the patient was resistant toward diethyl dithiocarbamate (DDC) treatment. DDC is a chelator of copper ion and inactivates the activity of SOD1 that contains one copper at active site per subunit. We determined copper ion concentration which can catalyze free radical reaction using copper phenanthroline assay. The copper ion concentration in the brain from the patient was 1.9 fold higher than those from the controls (n=3). Increased free copper ion can induce the production of hydroxyl radical. We propose that the oxidative stress caused by the reduction of SOD1 activity and increased copper ion plays a main role in the pathogenesis in FALS.