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目的通过检测孕妇外周血中的游离胎儿DNA来筛选重型β-地中海贫血胎儿。方法选择行产前基因诊断的夫妇6对,孕妇孕周23-26周。血液学检查:胎儿的父亲均为β-地中海贫血17M/N型,孕妇本人为携带除17M/N型之外的另一β-地中海贫血突变类型。针对CD17(A→T)无义突变,设计β-珠蛋白肽链上该等位基因的一对特异性引物和通过cycling probe法分别设计检测正常基因序列和基因突变位点的两条荧光探针,分别用FAM和HEX荧光标记。结合RT-PCR技术检测孕妇外周血中游离胎儿DNA,诊断胎儿是否遗传了其父亲的β地中海贫血17M/N碱基突变位点。同时与脐血血液学检查所诊断的胎儿地贫基因型对照。结果提取的6例孕妇血浆DNA模板中有3例同时显示FAM和HEX荧光信号值阳性结果,即这3例孕妇的胎儿遗传了父亲β-珠蛋白肽链上CD17位点的突变碱基(A→T)。另外3例孕妇血浆DNA模板的FAM信号值阳性,HEX信号值阴性,即所孕胎儿没有遗传父亲的CD17位点的突变碱基。结论利用RT-PCR和cycling probe技术检测孕妇外周血中的游离胎儿DNA可用来筛选患重型地中海贫血的胎儿。
Objective To screen fetus with severe β-thalassemia by detecting free fetal DNA in peripheral blood of pregnant women. Methods Six pairs of couples diagnosed as prenatal gene were selected, and pregnant women were gestational weeks 23-26 weeks. Hematology: Fetal fathers were both β-thalassemia 17M / N and pregnant women were themselves carrying another β-thalassemic mutation other than type 17M / N. For the nonsense mutation of CD17 (A → T), a pair of specific primers designed for this allele on β-globin peptide chain and two fluorescent probes designed for detecting normal gene sequence and gene mutation by cycling probe were designed Needles, labeled with FAM and HEX fluorescence, respectively. Combined with RT-PCR to detect fetal peripheral blood free fetal DNA to diagnose whether the fetus inherited her father’s beta thalassemia 17M / N base mutation sites. At the same time with the cord blood hematology diagnosis of fetal thalassemia genotype comparison. Results The results of three samples from 6 plasma DNA templates from pregnant women showed positive results of both FAM and HEX fluorescence signals, that is, the fetuses of these 3 pregnant women inherited the mutant base of CD17 on the β-globin peptide of the father (A → T). Another 3 cases of pregnant women plasma DNA template FAM signal value was positive, HEX signal value is negative, that the fetus has no genetic father CD17 mutation base. Conclusion Detection of free fetal DNA in peripheral blood of pregnant women by RT-PCR and cycling probe can be used to screen fetuses with severe thalassemia.