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-

山阴市家族性和散发性肌萎缩侧索硬化症的分子生物学分析 -DNA、mRNA、SOD1 和雄激素受体基因的含量和活性分析 -

• 批准号: 06670654
• 负责人: NAKASHIMA Kenji
• 金额: $ 1.09万
• 依托单位: Tottori University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for General Scientific Research (C)
• 财政年份: 1994
• 资助国家: 日本
• 起止时间: 1994 至 1995
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-06670654/
• 关键词: familial amyotrophic lateral sclerosis; SOD1; gene; Cupper; familial amyotrophic lateral sclerosi; superoxide dismutase; multisystem atrophy; amyotrophic lateral sclerosis; genetics; DNA

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.

应用单链构象多态(SSCP)分析和铜/锌超氧化物歧化酶(SOD1)活性测定，对一家系FALS患者进行了SOD1基因两个碱基缺失的家系分析。我们还分析了该家族中的一个患者的SOD1的活性、含量和mRNA，以及铜离子浓度。不仅有两名FALS患者，而且有5名临床未受影响的成员有SOD1基因异常。患者SOD1活性下降约70%，而临床非患者SOD1活性下降约30%。对患者的红细胞和脑组织匀浆进行超氧化物歧化酶活性染色和Western印迹分析，发现无突变的SOD1，总SOD1活性较低。对患者RT-PCR产物的SSCP分析表明，由于突变的SOD1基因，存在额外的SOD1mRNA。研究发现，患者大脑中的SOD1对二乙基二硫代氨基甲酸酯(DDC)治疗有抵抗力，但患者的红细胞中没有。DDC是一种铜离子的螯合剂，它可以钝化SOD1的活性，因为每个亚基的活性位上只有一个铜。用二氮杂菲铜法测定了催化自由基反应的铜离子浓度。患者脑组织中铜离子浓度是对照组的1.9倍。增加游离态铜离子可诱导产生羟基自由基。我们认为SOD1活性降低和铜离子升高引起的氧化应激在FALS的发病机制中起主要作用。

项目成果

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Shimoda M 等人：“肌萎缩侧索硬化症患者的电诱导眨眼反射和临床眨眼能力。”

中島健二: "家族性筋萎縮性側索硬化症-山陰の家系を中心に-" 臨床神経. 35. 1058 (1995)

Kenji Nakajima：“家族性肌萎缩侧索硬化症 - 关注 Sanin 家族 -”《临床神经病学》35. 1058 (1995)。

Nitta T et al.: "DNA deletions in dystrophin gene and clinical phenotype of Duchenne muscular dystrophy in the San-in area,Japan." Yonago Acta Med. 38. 43-48 (1995)

Nitta T 等人：“日本山阴地区肌营养不良蛋白基因 DNA 缺失和杜氏肌营养不良症的临床表型。”

Nakashima K et al.: "Abnormality of Cu/Zn superoxide dismutase(SOD1)activity in Japanese familial amyotrophic lateral sclerosis with two base deletion in SOD1 gene." Neurology. (in press). (1995)

Nakashima K 等人：“日本家族性肌萎缩性脊髓侧索硬化症患者的铜/锌超氧化物歧化酶 (SOD1) 活性异常，SOD1 基因有两个碱基缺失。”

Nakashima K et al.: "Epidemiological and genetic studies of Huntington's disease in the San-in area of Japan." Neuroepidemiology. (in press). (1995)

Nakashima K 等人：“日本山阴地区亨廷顿病的流行病学和遗传学研究。”