应用表观遗传学方法检测母体循环中游离胎儿DNA

Detection of the fetal DNA in maternal circulation with application of epigenetics

目的:探讨表观遗传学方法检测孕妇血浆中游离胎儿DNA水平的应用价值。方法:随机选择早、中、晚期妊娠孕妇各20例,以非妊娠妇女20例为对照组。提取血浆中的总游离DNA,用甲基化特异性PCR(MSP)方法检测各组血浆标本中目的基因m-maspin(一种肿瘤抑制基因启动子的甲基化序列)、u-maspin(前者的未甲基化序列)的表达水平,再进行相对定量,对妊娠组和对照组样本均数进行统计学分析。结果:(1)m-maspin在妊娠早、中、晚期孕妇血浆中的浓度分别是非妊娠妇女血浆中浓度的1.0、1.1、1.1倍,差异无统计学意义(P>0.05);(2)u-maspin在正常非妊娠妇女血浆中未能检测到,在60例孕妇血浆中57例可检测到,它在孕妇血浆中的浓度随妊娠进展呈升高趋势,中、晚期妊娠妇女血浆中其浓度相对于早期妊娠分别为1.7倍和3.1倍,差异有统计学意义(P<0.01)。结论:m-maspin不是妊娠特异性标志物,u-maspin为妊娠特异性标志物。u-maspin基因可作为孕妇血浆中游离胎儿DNA水平变化的表观遗传学标志,相对于以胎儿性别基因作为遗传学标志的检测方法,它有助于扩大非创伤性产前诊断的临床应用范围。

Objective：To explore the diagnostic value of epigenetic markers to detect fetal DNA in maternal plasma. Methods： Sixty pregnant women including pregnancy of first trimester（ 20 cases）, second trimester（ 20 cases） and third trimester （ 20 cases） were selected as study group,twenty normal nonpregnant women were selected as control group. DNA from plasma samples was extracted. The methylation-specific PCR（MSP） method was used to detect the expression of the target gene m-maspin （ a methylated tumor suppressor gene） , u-maspin （ un- methylated maspin promoter DNA sequence）and the internal standard gene β-actin gene. The relative quantification of target genes were also detected. Results： The M-maspin amount of first,second and third trimester relative to normal women were 1.0,1.1 and 1.1 respectively. There were no statistical significances （ P 〉 0.05 ） among three trimesters. No expression of u- maspin gene was detected in control group. U-maspin was detected in 57 of 60 samples from pregnant women. The u-maspin amount of second and third trimester relative to first trimester was 1.7 and 3.1. There were statistical significances（ P 〈 0.01 ）among early pregnancy, second trimester and later pregnancy. Conclusion： M-maspin is not a marker of fetal DNA. U-maspin in maternal plasma is elevated with the pregnancy. U-maspin can be treated as a marker of fetal DNA. The u-maspin gene may be considered as a epigenetic marker to detect the fetal DNA in maternal plasma, and compared with SRY gene, this technology may help explore the clinic application of noninvasive prenatal diagnosis and monitor.