血友病A患儿62例基因突变研究

Study of gene mutation in 62 hemophilia A children

目的 分析血友病A患儿FⅧ基因突变类型,初步探讨血友病A基因突变谱,以及基因突变与临床表型的关系.方法 收集2015年1月至2017年3月华中科技大学同济医学院附属同济医院儿童血液科就诊的血友病A患儿62例,均为男性,年龄4月龄~7岁,FⅧ活性0.2%~11.0%.重型血友病A50例,中型10例,轻型2例.取患儿外周血进行DNA分离,采用PCR扩增目的基因片段,结合二代测序方法,对患儿进行22和1内含子检测,阴性者进行二代测序,并与国际FⅧ基因突变数据库比对.结果 内含子22倒位20例（32%）,内含子1倒位2例（3%）,错义突变18例（29%）,无义突变5例（8%）,缺失突变7例（11%）,剪切位点突变1例（2%）,大片段缺失2例（3%）,插入突变1例（2%）.未检测到突变2例（3%）,扩增失败4例（7%）.表型与基因型的关系对比显示：重型血友病A中最常见的基因突变为内含子22倒位共20例,占重型患儿的40%,其次为错义突变共11例,占22%;中型血友病A最常见的基因突变为错义突变共6例,占60%.结论 血友病A发生率最高的基因突变类型为内含子22倒位,其次为错义突变,再次为缺失突变.表型与基因型的关系：重型血友病A中最常见的基因突变为内含子22倒位,其次为错义突变;中型血友病A最常见的基因突变为错义突变.

Objective To analyze the mutation type of FⅧ gene in children with hemophilia A and to explore the relationship among hemophilia gene mutation spectrum, gene mutation and clinical phenotype. Method Sixty-two children with hemophilia A from Department of Pediatric Hematology, Tongji Hospital of Tongji Medical College, Huazhong University of Science and Technology between January 2015 and March 2017 were enrolled. All patients were male, aged from 4 months to 7 years and FⅧ activity ranged 0.2%- 11.0%. Fifty cases had severe, 10 cases had moderate and 2 cases had mild hemophilia A. DNA was isolated from peripheral blood in hemophilia A children and the target gene fragment was amplified by PCR, in combination with the second generation sequencing, 22 and 1 introns were detected. Negative cases were detected by the second generation sequencing and results were compared with those of the international FⅧgene mutation database. Result There were 20 cases （32%） of intron 22 inversion, 2 cases （3%） of intron 1 inversion, 18 cases （29%） of missense mutation, 5 cases （8%） of nonsense mutation, 7 cases （11%） of deletion mutation, 1 case（2%）of splice site mutation, 2 cases （3%） of large fragment deletion and 1 case of insertion mutation （2%）. No mutation was detected in 2 cases （3%）, and 4 cases （7%） failed to amplify. The correlation between phenotype and genotype showed that the most common gene mutation in severe hemophilia A was intron 22 inversion （20 cases）, accounting for 40%of severe patients, followed by 11 cases of missense mutation （22%）. The most common mutation in moderate hemophilia A was missense mutation （6 cases）, accounting for 60% of moderate patients. Conclusion The most frequent mutation type in hemophilia A was intron 22 inversion, followed by missense mutation, again for missing mutation. The relationship between phenotype and genotype：the most frequent gene mutation in severe hemophilia A is intron 22 inversion, followed by missense mutation; the most frequent gene mutation in medium hemophilia A is missense mutation.