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目的探讨HLCS、RASSF1A片段在母胎间甲基化状态的差异并评估其在无创性产前诊断中的应用价值。方法收集388例孕妇血浆,其中126例同时收集外周血细胞及胎盘(或绒毛)组织,采用甲基化敏感性限制性酶切联合荧光定量PCR(MSRE+PCR)技术,检测母胎间HLCS基因、RASSF1A基因甲基化状态的差异,分析其影响因素,并根据孕妇血浆中胎源性HLCS/RASSF1A浓度比值判断胎儿21号染色体数目。结果研究证实HLCS及RASSF1A在胎盘或绒毛组织中均呈高甲基化状态,而在母体外周血细胞呈现低甲基化状态,且这种甲基化差异不受孕妇年龄、孕周、胎儿性别的影响。采用MSRE+PCR技术对孕妇血浆中HLCS、RASSF1A片段的检出率分别为97.4%和96.9%。计算274例正常妊娠孕妇血浆中胎源性HLCS/RASSF1A比值,确定其95%的参考值范围为0.34～2.02,以此为标准判断102例胎儿21号染色体数目,其中98例二倍体妊娠的准确率为95.9%(94/98),4例21三体综合征妊娠均获正确诊断。结论高甲基化HLCS、RASSF1A基因可作为孕妇血浆中胎源DNA的标志物,且有望根据孕妇血浆中甲基化HLCS/RASSF1A浓度比值进行21三体综合征的无创性产前诊断。
Objective To investigate the differences of methylation status between HLCS and RASSF1A in maternal fetal status and to evaluate their value in noninvasive prenatal diagnosis. Methods The plasma of 388 pregnant women was collected. Among them, 126 cases of peripheral blood cells and placenta (or villi) were collected at the same time. The methylation-sensitive restriction enzyme cleavage combined with quantitative PCR (MSRE + PCR) was used to detect the HLCS gene, RASSF1A Gene methylation status, analyze its influencing factors, and according to the concentration of fetal HLCS / RASSF1A in pregnant women to determine the ratio of fetal chromosome 21 number. Results The study confirmed that both HLCS and RASSF1A were hypermethylated in placental or chorionic villi while their peripheral blood cells showed hypomethylation, and the difference in methylation was not affected by the age, gestational age and sex of the fetus. The detection rates of HLCS and RASSF1A fragments in pregnant women using MSRE + PCR were 97.4% and 96.9%, respectively. The ratio of fetal HLCS / RASSF1A in 274 normal pregnant women was calculated, and the 95% reference value was 0.34 ~ 2.02. Based on this, the number of fetal chromosome 21 was determined in 102 fetuses, of which 98 diploid pregnancies The accuracy rate was 95.9% (94/98). All 4 cases of trisomy 21 pregnancy were correctly diagnosed. Conclusion The hypermethylated HLCS and RASSF1A genes can be used as biomarkers of fetal DNA in pregnant women. Noninvasive prenatal diagnosis of trisomy 21 is expected based on the ratio of methylated HLCS / RASSF1A in plasma of pregnant women.