一例凝血因子 ⅧB 区错义突变导致重型血友病 A

A novel missense mutation in the B domain of Factor Ⅷ causes severe hemophilia A

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１４２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.