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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 elucidate the difference of methylation status between C21orf25 fragment and maternal fetus, and explore its 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) tissue DNA were extracted at the same time. Methylation - sensitive restriction enzyme cleavage combined with quantitative PCR (MSRE + PCR) was used to detect the C21orf25 gene , RASSF1A gene methylation status, analysis of its influencing factors, respectively, and fetal gene RASSF1A or SRY gene as a reference site, calculate the concentration of fetal fetal C21orf25 pregnant women and reference sites concentration ratio to determine the fetus 21 Chromosome number. Results: Both C21orf25 and RASSF1A were hypermethylated in placental or chorionic tissues and hypomethylated in maternal peripheral blood. The degree of methylation of C21orf25 in maternal fetus was affected by gestational age, which was lower in early pregnancy than in middle and late pregnancy (P = 0.002, P <0.01). The detection rates of C21orf25 and RASSF1A in plasma of pregnant women using MSRE + PCR were 100% and 96.9% respectively. The ratio of fetal C21orf25 / RASSF1A in 184 normal pregnant women was calculated, and 95% of the reference values were determined to be 0.20-3.46. Based on this, the number of 71 fetuses on chromosome 21 was determined, of which 67 were diploid pregnancies The accuracy rate was 94.0% (63/67), 4 cases of trisomy 21 pregnancy, 2 cases of C21orf25 / RASSF1A ratio higher than the reference interval was diagnosed. Using SRY as a reference site, the ratio of methylation C21orf25 / SRY in 94 normal diploid male fetuses was 3.16 ± 1.20,95%, and the reference value ranged from 0.81 to 5.51 The number of chromosomes, the accuracy rate was 97.1% (34/35), 2 cases of trisomy 21 were accurately diagnosed. CONCLUSION: The hypermethylated C21orf25 gene can be used as a marker of fetal DNA in pregnant women. By calculating the concentration ratio of this locus to the reference gene, noninvasive prenatal diagnosis of Down’s syndrome is expected.