Molecular analysis of congenital C9 deficiency

1)北方分析：对8例不相关的C9缺乏症患者进行北方分析，2例患者C9 mRNA未检出。2)南方分析：南方分析显示所有患者无重大基因重排。3) C9外显子的扩增及SSCP分析：a)对2例无C9 mRNA的患者进行PCR扩增各外显子。每例患者在4号外显子边界位点均出现G到T的改变，提示4号内含子剪接受到干扰，C9蛋白的合成在内含子内停止。b) SSCP分析显示每个C9 mRNA患者都有异常外显子7.4)异常外显子7 DNA分析：7外显子DNA序列显示密码子360错义突变。碱基变化为GGG到GAG，表明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转A型患者亲本的Oligo-DNA分析：将正常型或野生型互补的Oligo-DNA与亲本DNA杂交。这两种低聚DNA与亲本DNA杂交，表明亲本是同一突变（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).