Department of Medical Genetics, Medical School and Key Laboratory of Tropic Disease Control, Ministry Education, Sun Yat-sen University, 74#, Zhongshan Road 2, Guangzhou, 510080, China.
Cell Biochem Biophys. 2013 Apr;65(3):463-72. doi: 10.1007/s12013-012-9450-2.
Hemophilia A is an x-linked recessive inherited bleeding disorder. So far, more than 1,885 disease-causing mutations of factor VIII gene have been identified. Clinic confers a great challenge for the molecular diagnosis. We aim to make a better strategy for the molecular diagnosis in Hemophilia A. First, factor VIII intron 22 inversion and intron 1 inversion mutations were detected using Inversion-PCR and double-tube multiple PCRs. And then, non-inversion mutations were analyzed by denaturing high performance liquid chromatography and/or direct sequencing. Novel mutations were further analyzed the conservation and 3D structures by a B domain deleted crystallographic model and bioinformatics. Finally, we can indirectly confirm the diagnosis by linkage analysis for the patients with the confusing diagnosis by the techniques mentioned above. Eleven patients with the factor VIII Inv 22 were found, and the remaining 16 patients were found with 11 different mutations, of which 3 was novel mutations affecting A1, B domains and splicing site. Moreover, the prenatal diagnosis was performed on 14 fetuses. Ten fetuses were successfully confirmed to be normal, 1 fetus to be a heterozygote with factor VIII c.3275-3276 ins A and 3 fetuses to be hemizygotes with factor VIII Inv 22 mutation.
血友病 A 是一种 X 连锁隐性遗传性出血性疾病。到目前为止，已经发现了超过 1885 种因子 VIII 基因突变。临床诊断给分子诊断带来了巨大的挑战。我们旨在为血友病 A 的分子诊断制定更好的策略。首先，采用反转录-PCR 和双管多重 PCR 检测因子 VIII 内含子 22 倒位和内含子 1 倒位突变。然后，通过变性高效液相色谱和/或直接测序分析非倒位突变。通过 B 域缺失晶体结构模型和生物信息学分析，对新发现的突变进行保守性和 3D 结构分析。最后，对于通过上述技术进行混淆诊断的患者，通过连锁分析可以间接确认诊断。发现了 11 例因子 VIII Inv 22 患者，其余 16 例患者发现了 11 种不同的突变，其中 3 种是影响 A1、B 结构域和剪接位点的新型突变。此外，对 14 例胎儿进行了产前诊断。10 例胎儿成功证实正常，1 例胎儿为因子 VIII c.3275-3276 ins A 的杂合子，3 例胎儿为因子 VIII Inv 22 突变的半合子。
