Gene analysis of the superoxide dismutase gene in amyotrophic lateral screlosis

我们的特点是铜锌超氧化物歧化酶基因（SOD 1）在6个日本家庭与家族性肌萎缩侧索硬化症（FALS）。序列分析显示，在该基因的两个新的突变，从两个独立的家庭。一个T-to-A颠换在一个家族中引起Val 17 Glu替换。一个T-G突变导致另一个家族中的Leu 8Val取代。其他家族有独特的临床症状和正常的SOD 1基因编码序列，表明FALS是一个复杂的异质性运动神经元疾病。用RT-PCR方法合成突变型SOD 1 cDNA，并将突变型SOD 1 cDNA转染PC 12细胞，观察其基因表达情况。将cDNA克隆连接到pRc/RSV表达质粒载体中，转染PC 12细胞。建立的细胞系经新霉素筛选后，在不同条件下进行培养。低浓度CSF或各种CO2浓度。细胞形态学未见明显凋亡。在各种条件下培养来自FALS患者的成纤维细胞，即。缺氧、低浓度FCS、存在氧化剂或热应激。从患者和对照的经处理的成纤维细胞中纯化mRNA。差异显示技术检测到几条特异性表达于患者的条带。从凝胶上切下条带，PCR扩增，克隆到载体中并测序。由此获得的cDNA含有以前未报道的序列。构建了人脑cDNA文库，并利用克隆的cDNA进行了筛选。所鉴定的斑块含有与我们在患者的经治疗的成纤维细胞中检测到的序列相同的序列。这一结果表明，来自FALS患者的成纤维细胞通过表达某些特定基因来响应热应激。

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.