摘要
目的:检测一例重型血友病A患者(SH9)的基因突变。方法:用PCR、变性梯度凝胶电泳(DGGE)和DNA测序检测因子Ⅷ基因突变。先用Southernbloting排除内含子22倒位,然后应用PCR对凝血因子Ⅷ基因进行扩增。扩增范围包括所有外显子及其侧翼内含子序列。结果:片段142在DGGE中泳动异常。DNA测序证实C2535A导致B区错义突变826Asp(GAC)→Glu(GAA)。结论:该突变可能是导致重型血友病A的原因,但有待进一步研究证实。
Objective:To identify the genetic defect of a patient with severe hemophilia A (SH9). Methods: PCR, denaturing gradient gel electrophoresis (DGGE) and DNA sequencing were used to screen mutations in the factor Ⅷ gene. Intron 22 inversion was excluded previously by Southern blotting with F8A probe. PCR primers were designed to cover all the coding regions and flanking intron sequences. Amplified products were analysed with DGGE, and bands of abnormal mobility were directly sequenced. Results: PCR fragment 14 2 showed slower mobility than normal. A single nucleotide substitution C2535A causing a missense mutation in the B domain, 826Asp(GAC)→Glu(GAA) was identified by DNA sequencing. Conclusion: This nucleotide substitution may be the molecular etiology of SH9. The possible molecular mechanisms underlying this candidate mutation is yet to be clarified.
出处
《中华血液学杂志》
CAS
CSCD
北大核心
1998年第9期458-460,共3页
Chinese Journal of Hematology
基金
国家自然科学基金
卫生部青年基金
上海血液学研究所胡应州基金
关键词
DNA测序
错义突变
凝血因子Ⅷ
血友病A
Denaturing gradient gel electrophoresis DNA sequencing Missense mutation Coagulation factor Ⅷ Hemophilia A