Molecular analysis of congenital C9 deficiency

1)Northern分析：对8例C9缺乏症患者进行Northern分析，其中2例未检测到C9基因。2)Southern分析显示，所有C9缺乏症患者均未检测到主要基因重排。3)扩增C9外显子及SSCP分析：a)2例C9缺乏症患者每个外显子均经聚合酶链式反应扩增，每例患者外显子4边界位置均发生G-T变化，提示内含子4剪接紊乱，C9蛋白合成停止在内含子中。4)异常外显子7的DNA分析：外显子7的DNA序列显示360密码子存在错义突变。碱基改变为GGG到GAG，提示C9蛋白细胞穿透部分的氨基酸由甘氨酸变为谷氨酸。5)6例G to A改变患者父母的寡核苷酸分析：与正常或野生型互补的寡核苷酸与父母的DNA杂交。这两个寡核苷酸都与父母的DNA杂交，表明父母是同一突变(杂合子)的携带者。

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).