Molecular analysis of congenital C9 deficiency

先天性 C9 缺乏症的分子分析

• 批准号: 06670770
• 负责人: IGARASHI Takashi
• 金额: $ 1.28万
• 依托单位: The University of Tokyo
• 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-06670770/
• 关键词: Congenital complement component deficiency; Complement 9 (C9); Molecular analysis; C9欠損症; スプライシング異常

1) Northern analysis : Northern analysis of 8 unrelated patients with C9 deficiency was performed resulting with undetected C9 mRNA in two patients.2) Southern analysis : Southern analysis revealed no major gene rearrangement in all patients with the disease.3) Ampulification of C9 exons and SSCP analysis :a) Each exon was amplified by PCR in 2 pateints without C9 mRNA.Each patient revealed to have G to T change in the boundary site of exon 4 suggesting that the splicing of intron 4 was distrubed and synthesis of C9 protein stopped in the intron.b) SSCP analysis showed that each patient with C9 mRNA had abnormal exon 7.4) DNA analysis of abnormal exon 7 : DNA sequence of exon 7 revealed a missense mutaion in codon 360. The base change was GGG to GAG suggesting the amino acid change from glycin to glutamic acid in the cell penetrating portion of C9 protein.5) Oligo-DNA analysis of parents of the 6 patients with G to A change : Oligo-DNAs complementary to normal or wild type were hybidized to DNA of the parents. Both oligo-DNAs were hybridized to the DNA of parents suggesting that the parents were carriers of the same mutation (heterozygote).

1)北方分析：对8例无亲缘关系的C9缺乏症患者进行北方分析，结果在2例患者中未检测到C9 mRNA。2）Southern分析：Southern分析显示所有患者均无主要基因重排。3）C9外显子的截肢和SSCP分析：a）2例C9 mRNA缺失患者的每个外显子均被PCR扩增，每例患者的第4外显子的边界位点均出现G → T的改变，提示内含子4的剪接被破坏，C9蛋白的合成在内含子处停止。B）SSCP分析显示，所有C9 mRNA阳性患者均存在第7外显子异常。4）第7外显子异常DNA分析：第7外显子DNA序列分析显示第360位密码子发生错义突变。碱基变化为GGG → GAG，提示C9蛋白穿细胞部分的氨基酸由甘氨酸变为谷氨酸。5）6例G → A突变患者父母的寡聚DNA分析：与正常或野生型互补的寡聚DNA与父母的DNA杂交。两个寡聚DNA都与父母的DNA杂交，表明父母是同一突变的携带者（杂合子）。