73例中国人血友病甲基因突变的分析

Characterization of Genetic Defects in 73 Cases of Haemophilia A of Chinese Origin

我们用Ｓｏｕｔｈｅｒｎｂｌｏｔｔｉｎｇ、ＰＣＲ、变性梯度凝胶电泳（ＤＧＧＥ）和ＤＮＡ测序等方法对７３例血友病甲患者（经上海瑞金医院测定血浆ＦＶⅢ：Ｃ和ｖＷＦ：Ａｇ诊断，其中无亲缘关系患者６５例，按ＦＶⅢ：Ｃ水平分为轻、中、重三型。ＦＶⅢ：Ｃ＜２％为重型，共４７例；ＦＶⅢ：Ｃ２％～５％为中型，共９例；ＦＶⅢ：Ｃ５％～２５％为轻型，共１７例）进行ＦＶⅢ基因突变检测。共检出内含子２２倒位２３例，均为重型，约占重型的４９％，与国外报道相似。余下５０例（其中无亲缘关系者４５）用ＰＣＲ－ＤＧＧＥ分析所有外显子及其侧翼内含子序列，发现异常条带则进行ＤＮＡ测序。在１７例患者中检出突变１３种，其中无义突变５种，均为重型；错义突变６种，除１例外都是轻中型；小缺失２例，都是重型；其中，ＡＡ４６６Ｌｙｓ（ＡＡＧ）－Ｔｈｒ（ＡＣＧ），７１９Ｔｙｒ（ＴＡＣ）－Ｓｔｏｐ（ＴＡＧ），ＡＡ８２６Ａｓｐ（ＧＡＣ）－Ｇｌｕ（ＧＡＡ），３１２Ｉｌｅ（ＡＴＣ）－ｘｘＣ及ＡＡ１５５１－１５５２ｄｅｌ（ＡＧＡＡ）为新发现的突变。有亲缘关系的患者都有相同的基因突变，而在无亲缘关系患者未发现相同突变。基因突变与临床表现基本相符。

We use Southern blotting, PCR, denaturing gradient gel electrophoresis(DGGE) and DNA sequencing to detect gene mutations of haemophilia A in Chinese population. 73 cases(47 severe(FVⅢ: C <2%), 9 moderate(FVⅢ: C 2%～5%), 17 mild(FVⅢ: C 5%～25%)) of haemophilia A were first screened with Southern blotting, 23 were found to be the intron 22 inversion type, all being severe cases. The remaining 50 cases without intron 22 inversion were examined with PCR-DGGE. Genomic DNA were amplified using GC-clamped primers covering all the exons and all flanking intron regions. Abnormal bands were sequenced. 13 different mutations were identified, including 5 nonsense mutations, 6 missense mutations and 2 small deletions. 5 mutations, AA466Lys (AAG)-Thr(ACG), AA719Tyr(TAC)-Stop(TAG), AA826Asp(GAC)-Glu(GAA), AA312Ile(ATC)-xxC and AA1551-1552del(AGAA) have not been reported before. Generally the genetic defects correspond to the clinical conditions.