正常整倍体及21三体胎盘组织的甲基化标志物研究

Methylation markers in placenta tissues of euploid and trisomy 21

目的:联合应用甲基化特异性多重连接依赖的探针扩增技术(MS-MLPA)及亚硫酸氢盐测序2种方法进行胎盘组织甲基化研究,以寻找可用于无创性产前诊断21三体综合征的甲基化标志物。方法:用MS-MLPA和亚硫酸氢盐测序2种方法对15例正常整倍体孕妇和11例21三体孕妇的胎盘组织,以及13例非孕妇女外周血进行甲基化检测,并计算候选的3个基因(4个位点)CGI149、CGI045、HLCS-1和HLCS-2的甲基化率。结果:(1)MS-MLPA与亚硫酸氢盐测序所测得的甲基化率一致。(2)所选的4个位点,在13例非孕妇女外周血样本中CGI149、CGI045和HLCS-2三个位点均未甲基化;在15例整倍体胎盘中,CGI149有13例(2例未甲基化)、CGI045有11例(4例未甲基化)、HLCS(包括HLCS-1和HLCS-2)有15例甲基化;在11例21三体胎盘中,CGI149有10例(1例未甲基化)、CGI045及HLCS(包括HLCS-1和HLCS-2)均有11例甲基化。只有HLCS-2符合在所有外周血细胞中均未甲基化,在所有胎盘组织中均甲基化。比较4个位点的甲基化率在正常整倍体胎盘与21三体胎盘的不同,只有CGI149有统计学意义,其它3个位点(CGI045、HLCS-1和HLCS-2)均无统计学意义。结论:(1)MS-MLPA可代替亚硫酸氢盐测序用于胎盘的甲基化研究。(2)本研究所选择的CGI045、CGI149、HLCS-1和HLCS-2不适合用作21三体的甲基化标志物,需进一步寻找新的位点。

AIM： To explore the DNA methylation markers for non-invasive prenatal diagnosis of trisomy 21. METHODS： The DNA methylation ratios of 3 genes （4 fragments） on chromosome 21 （CGI149, CGI045, HLCS-1 and HLCS-2） in blood cells from 13 non-pregnant women, and in placental tissues from 15 euploid and 11 trisomy 21 pregnant women were measured using the methods of methylation-specific multiplex ligation-dependent probe amplification （ MS-ML- PA） and bisulfite sequencing in combination. RESULTS： The results obtained from MS-MLPA were consistent with the results from bisulfite sequencing. The fragments of CGI149, CGI045 and HLCS-2 were unmethylated in the non-pregnant woman blood cells. HLCS-1 and HLCS-2 were methylated in all euploid and all trisomy 21 placentae. CGI149 was methylated in 13 （13/15） of the euploid and 10 （10/11 ） of the trisomy 21 placental tissues. CGI045 was methylated in 11 （11/ 15 ） of the euploid and all the Trisomy 21 placental tissues. Only HLCS-2 was found to be methylated in all placental tissues but unmethylated in all non-pregnant woman blood cells. Only the DNA methylation ratio in CGI149 was significantly different between euploid and trisomy 21 placental tissue （P 〈 0.05）. No difference among HLCS-1, HLCS-2 and CGI045 was observed. CONCLUSION： MS-MLPA is an effective alternative to bisulfite sequencing for the assessment of methylation ratios in placental tissue. CGI149, CGI045, HLCS-1 and HLCS-2 are not appropriate DNA methylation markers for non-invasive prenatal diagnosis of trisomy 21.