C21orf25片段在母胎间的甲基化差异及其临床价值

Differentially methylated regions of C21orf25 between maternal and fetal DNA:implication for noninvasive prenatal diagnosis

目的:阐明C21orf25片段在母胎间甲基化状态的差异,探讨其用于无创性产前诊断的价值。方法:收集388例孕妇血浆,其中126例同时提取外周血细胞及胎盘(或绒毛)组织DNA,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C21orf25基因、RASSF1A基因甲基化状态的差异,分析其影响因素,并分别以胎源性RASSF1A或SRY基因为参照位点,计算孕妇血浆中胎源性C21orf25与参照位点的浓度比,判断胎儿21号染色体数量。结果:C21orf25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。C21orf25在母胎间的甲基化差异程度受孕周的影响,早孕期小于中、晚孕期(P=0.002,P<0.01)。采用MSRE+PCR技术对孕妇血浆中C21orf25、RASSF1A片段的检出率分别为100%和96.9%。计算184例正常妊娠孕妇血浆中胎源性C21orf25/RASSF1A比值,确定其95%的参考值范围为0.20～3.46,以此为标准判断71例胎儿21号染色体数量,其中67例二倍体妊娠的准确率为94.0%(63/67),4例21三体综合征妊娠,2例C21orf25/RASSF1A比值高于参考区间上限而获得诊断。以SRY为参照位点,94例正常二倍体男胎妊娠中甲基化C21orf25/SRY比值为3.16±1.20,95%参考值范围为0.81～5.51,以此为标准判断35例男胎21号染色体数量,准确率为97.1%(34/35),2例21三体妊娠均获准确诊断。结论:高甲基化C21orf25基因可作为孕妇血浆中胎儿DNA的标志物,通过计算该位点与参照基因的浓度比值,有望进行唐氏综合征的无创性产前诊断。

Objective：To study the values of methylated C21orf25 in identification of free fetal DNA in maternal plasma and in noninvasive prenatal diagnosis of Down＇s syndrome.Methods：Maternal plasma samples were collected from 388 singleton pregnancies,placental or chorionic villi tissue was also obtained in 126 cases of them.Methylation sensitive restriction enzyme digestion followed by fluorescence quantitative PCR（MSRE＋PCR）were employed in analyzing the methylation status of C21orf25 and RASSF1A.The possible factors which affect methylation difference were also clarified.Taking fetal-specific RASSF1A or SRY as a reference locus,noninvasive prenatal diagnosis of trisomy 21 was done by calculating the ratio of fetal-specific C21orf25 to a reference sequence in maternal plasma.Results：It was confirmed that C21orf25 and RASSF1A were hypomethylated in maternal blood cells but hypermethylated in placenta or chorionic villi tissues.However,the methylation difference of C21orf25 was affected by pregnancy period,which was less in early trimester than in later trimesters（P=0.002,P0.01）.Using MSRE＋PCR,the positive rates of fetal C21orf25 and RASSF1A in maternal plasma were 100% and 96.9% respectively.Based on the data from 184 euploidy pregnancies,the 95% reference interval of the fetal-derived C21orf25/RASSF1A ratio in maternal plasma was 0.20 to 3.46,which was taken as the reference for determining the numbers of fetal chromosome 21 in 71 pregnancies.The accurate rate in 67 euploidy pregnancies was 94.0%（63/67）.Two of four trisomy 21 pregnancies were confirmed by this method.When SRY performed as the reference gene,the fetal-derived C21orf25/SRY ratio was 3.16±1.20 and the 95% reference interval was 0.81 to 5.51.In the C21orf25/SRY protocol,34 euploidy pregnancies were correctly discerned with an accurate rate of 97.1%（34/35）,two trisomy 21 fetuses were also figured out.Conclusion：Hypermethylated C21orf25 can be used in identification of fetal DNA in maternal plasma and in noninvasive prenatal diagnosis of trisomy 21.