子痫前期母体外周血中甲基化差异基因检测及其早期预测价值的研究
发布时间：2018-08-22 10:02
【摘要】：子痫前期(Pre-eclampsia, PE)为怀孕前血压正常的孕妇在妊娠20周以后出现高血压、蛋白尿,又称为先兆子痫。PE影响着全世界3%-5%的孕妇,是导致母儿患病率和死亡率的主要原因,分别占产妇及胎儿死因的20%、40%。资料显示我国PE发生率约2-8%,晚近发生率呈逐年增长趋势。PE不仅可影响妊娠结局,也影响产妇及子代的远期预后。PE的新生儿可发生包括早产,胎儿生长受限,缺氧相关的神经损伤,围生儿死亡等并发症,其日后发生心脏病的风险增加；PE孕妇发生心血管和脑血管疾病的机率亦增加等。 目前认为,异常的胎盘形成发生在妊娠早期导致胎盘缺血(Ⅰ期)；缺血的胎盘在妊娠晚期可分泌可溶性因子从而引起系统性内皮功能紊乱和PE综合征(Ⅱ期)。在PE发生之前,孕妇的血浆成分已经出现了诸多变化,因此,有许多针对预测PE的生物标记物如胎盘激素、血管生成因子、脂质进行的研究。但既往这些生物标记物在PE预测方面的研究多为针对可能导致PE发生的信号转导通路分子的研究,且多为单一标记物的预测研究。而且,由于PE是一异质性疾病,临床表现多样化,应用单一标记物预测的可能性不大。故至今为止尚无一个标记物或者几个联合标记物预测能达到临床应用所需要的灵敏度和特异度,因此这些检测均未能在临床常规开展。 DNA的甲基化检测具有稳定性好的优点,因此既往有诸多针对不良妊娠结局DNA甲基化研究,但这些研究多是基于单一位点或胎盘的甲基化改变的检测,尚无对PE母体外周血全基因组所有位点的甲基化检测报道。 在PE的发生过程中存在胎盘DNA甲基化异常,因此通过检测孕妇外周血差异甲基化基因可能成为预测PE发生的新的标记物。本研究采用前瞻性队列研究设计收集孕妇外周血,应用甲基化芯片筛选、焦磷酸测序验证PE差异甲基化DNA；检测母体外周血差异甲基化DNA在正常孕妇与PE孕妇随孕周的甲基化变化情况；研究和探讨外周血差异甲基化基因作为作为预测PE发生的新的标记物的早期预测价值。 第一章PE母体外周血差异甲基化DNA的筛选及检测[目的] 胎盘DNA甲基化异常参与了PE的发生,母体外周血差异甲基化DNA可能做为无创性预测PE发生的新的标记物。应用前瞻性队列研究方法,采用甲基化芯片技术筛选母体外周血差异甲基化DNA,研究其在正常孕妇与PE孕妇随孕周改变的甲基化变化,筛选可预测PE的候选基因。[方法] 研究对象为2012年10月-2013年11月期间在南方医科大学附属深圳妇幼保健院从孕早期建立妊娠期保健手册开始定期产前检查,并最终在本院分娩的孕妇共1752例,分为早发型PE组、晚发型PE组、正常对照孕妇组(对照组)三组。参照入选标准及排除标准,匹配对照组孕妇年龄、孕产次等基本资料,各组3例抽提其孕早期、孕中期及入院分娩时(孕晚期)血标本DNA,应用人全基因组DNA甲基化芯片对其DNA甲基化程度进行测定。对比识别早发型PE组、晚发型PE组和对照组的差异甲基化DNA。以限定的P值(0.05)和M值(1.0或小于-1.0)作为切割值,筛选出各组的差异甲基化DNA。利用DAVID数据库等分析差异基因主要参与影响的信号通路及调控范围；根据聚类分析后各信号通路的相对重要性、基因功能注释及既往文献报道中与PE发病机制相关基因,筛选出外周血中可能参与PE发病的甲基化差异DNA。[结果] 1.在孕早、中、晚三个时间段,正常对照组中的整体甲基化程度略有变化,孕早期血的甲基化程度最低；与对照组比较,早发型PE组、晚发型PE组的孕早期甲基化程度呈增高趋势。 2.孕早期血甲基化结果：(1)与对照组比对,早发型PE组共发现34713个差异甲基化位点,其中矫正后有统计学差异位点1303个；(2)与对照组比对,晚发型PE组共发现24625个差异甲基化位点,其中矫正后有统计学差异的位点648个；(3)早发型PE组、晚发型PE组及对照组同时满足探针位于CpGs岛并分布在5'UTR或TSS区域的共同差异甲基化基因11个(C22orf45、UPB1、MTNR1A、PLEKHG5、BLCAP、HOXA9、TUBGCP5、ZNF492、PSKH2、 MBP及PIWIL1)。 3.孕中期血甲基化结果：(1)与对照组比对,早发型PE组共发现42243个差异甲基化位点,其中矫正后有统计学差异的位点1176个；(2)与对照组比对,晚发型PE组孕中期血共发现24241个差异甲基化位点,其中矫正后有统计学差异的位点共479个；(3)早发型PE组、晚发型PE组及对照组同时满足探针位于CpGs岛并在分布在5'UTR或TSS区域共有17个共同差异甲基化基因(FBXO39、EPHA5、ZNF542、HOXD1、BOLL、HCN1、CHRNB1、GLP1R、GABRG3、 NKAIN3、NTSR2、CCK, C17orf51、UCP1、SNAP25、MTNR1A、LPPR3) 4.孕晚期血甲基化结果：(1)与对照组比对,早发型PE组共发现42847个差异甲基化位点,其中矫正后有统计学差异的位点共1133个；(2)与对照组比对,晚发型PE组共发现25157个差异甲基化位点,其中矫正后有统计学差异的位点共有491个；(3)早发型PE、晚发型PE及对照组同时满足探针位于CpGs岛并在分布在5'UTR或TSS区域的共有19个基因(PAK7、MOSC2、SHE、TDRD10、UNC5D、CHL1、GRM1、PPAP2C、UGT8、CCK、C17orf51、PAX1、 PUS3、DDX25、NPY2R、SLC7A14、HLA-A、DPP6、DSC2)[小结]利用人全基因组DNA甲基化芯片成功检测了孕早、中、晚三期的早发型PE组、晚发型PE及对照组外周血DNA甲基化,结果显示其均存在差异甲基化基因改变。其中从孕早期血中成功筛选了11甲基化差异显著的基因,孕中期血中成功筛选了17甲基化差异显著的基因,孕晚期血中成功筛选了19甲基化差异显著的基因,其可能成为有效的预测指标。根据芯片位点信息及基因功能等,选取孕早期HOXA9、MTNR1A、PIWIL1进行下一步验证。 第二章PE母体外周血中HOXA9、MTNR1A、PIWIL1的验证及其用于PE早期预测的研究 [目的] 从孕早期甲基化芯片筛选的甲基化差异显著的基因中挑选出HOXA9、MTNR1A、PIWIL1共3个候选基因,以焦磷酸测序检验在早发型PE组、晚发型PE组、正常对照孕妇组(对照组)中对应DNA位点的甲基化变化情况,并以RT-qPCR检测各候选基因的mRNA变化情况。探讨这些指标作为PE早期预测的可行性。[方法] 研究对象为2012年10月-2013年11月期间在南方医科大学附属深圳妇幼保健院从孕早期建立妊娠期保健手册开始定期产前检查,并最终在本院分娩的孕妇。分为早发型PE组、晚发型PE组、对照组三组。排除各种合并症和妊娠并发症之后随机抽样每组10例,提取孕早期及分娩期(孕晚期)母体外周血DNA和RNA。焦磷酸测序检测MTNR1A、HOXA9及PIWILl三个筛选的目标基因在早发型PE组、晚发型PE组和对照组不同时期外周血的甲基化程度；RT-qPCR检测目标基因在各组的mRNA的表达情况。[结果] 1.孕早期焦磷酸测序检验结果显示：(1)HOXA9在早发型PE组甲基化率(4.60±0.88%)显著高于晚发型PE组(1.30±0.73%)和对照组(1.44±0.70%)(P0.05),余组间比较无统计差异(P0.05);(2)MINR1A的两个位点在早发型PE组甲基化率显著高于对照组(P0.05),余组间比较无统计差异(P0.05)；(3)PIWILl在各组间的高甲基化无统计学差异(P0.05)。 2.孕晚期焦磷酸测序检验结果显示：(1)HOXA9在各组间未见统计学差异(P0.05);(2)MINR1A两位点在早发型PE组及晚发型PE组均表现出高甲基化；MTNR1A(1)早发型PE组及晚发型PE组甲基化率与对照组差异有统计学意义(P0.05); MTNR1A(2)甲基化率组间无统计学差异(P0.05);(3) PIWIL1在三组间的高甲基化率差异无统计学差异(P0.05)。 3.孕早期HOXA9mRNA的RT-qPCR结果显示其早发型PE组相对量为0.56±0.08,为晚发型PE组的0.70倍及对照组的0.56倍；而MTNRIA mRNA及PIWIL1mRNA在外周血中未检测到。 4.孕晚期HOXA9mRNA的RT-qPCR结果显示其早发型PE组相对表达量为0.77±0.07,为晚发型PE组的0.94倍及对照组的0.77倍；而MTNRIA mRNA及PIWIL1mRNA在外周血中未检测到。 5.外周血中HOXA9mRNA相对表达量在早发型PE组、晚发型PE组、对照组孕晚期的表达量较孕早期均有增加。[小结] 经焦磷酸测序检验和RT-qPCR验证,不同类型PE外周血HOXA9及MTNR1A在不同孕期甲基化程度存在差异,提示其可能与PE的发病及病理过程有关；此种母体外周血中HOXA9及MTNRIA甲基化差异可作为潜在的早发型PE早期预测指标之一。全文小结 1.成功运用人全基因组DNA甲基化芯片(illumina450k)分析了早发型PE、晚发型PE及正常妊娠女性的孕早、中、晚期母体外周血的DNA甲基化差异。 2.在孕早、中、晚期,与对照组比较,早发型PE和晚发型PE母体外周血中均存在甲基化差异表达基因,这些基因涉及细胞发育与增殖、血管生成、胎盘及胚胎的发育等,因此推测这些差异甲基化基因参与了PE的发生、发展。 3.HOXA9在早发型PE孕早期母体外周血中DNA甲基化水平高于晚发型PE组及对照组(P0.05),其mRNA表达亦降低,提示其有望成为早发型PE早期无创性预测指标。 4.MINR1A两位点在PE母体外周血中呈高甲基化；在孕早期母体外周血中两个位点甲基化率早发型PE组与对照组间有统计学差异(P0.05)；在孕晚期母体外周血中MTNR1A(1)早发型PE组、晚发型PE组甲基化率与对照组差异有统计学意义(P0.05),提示其可能作为早发型PE的早期预测指标。 5.孕妇外周血甲基化差异基因检测可作为预测PE发生的方法进行深入研究。
[Abstract]:Pre eclampsia (PE) is a kind of pregnant women with normal blood pressure before pregnancy. After 20 weeks of gestation, preeclampsia (PE) occurs hypertension, proteinuria, also known as preeclampsia. PE affects 3% - 5% of pregnant women worldwide. PE is the main cause of maternal and fetal morbidity and mortality, accounting for 20% and 40% of maternal and fetal mortality respectively. The incidence of late pregnancy is increasing year by year. PE not only affects the outcome of pregnancy, but also affects the long-term prognosis of pregnant women and their offspring. The chance of disease has also increased.
At present, it is believed that abnormal placenta formation occurs in the early pregnancy leading to placental ischemia (stage I); ischemic placentas secrete soluble factors in late pregnancy leading to systemic endothelial dysfunction and PE syndrome (stage II). Before the occurrence of PE, there have been many changes in the plasma composition of pregnant women, therefore, there are many predictions for PE. Biomarkers such as placental hormones, angiogenesis factors, and lipids have been studied. However, previous studies on the prediction of PE by these biomarkers have mainly focused on the signal transduction pathway molecules that may lead to PE, and mostly on the prediction of single biomarkers. So far, there is no single marker or several combined markers to predict the sensitivity and specificity needed for clinical application, so these tests can not be carried out routinely.
DNA methylation detection has the advantages of good stability, so there have been many previous studies on DNA methylation for adverse pregnancy outcomes, but these studies are mostly based on the detection of single site or placental methylation changes.
In this study, a prospective cohort study was designed to collect maternal peripheral blood samples from pregnant women. Methylation chips were used to screen and pyrophosphate sequencing was used to verify the differential methylation DNA of PE. The changes of methylation of differentially methylated DNA in peripheral blood of normal pregnant women and PE pregnant women with gestational weeks were studied and discussed.
Chapter 1 screening and detection of differential methylation of DNA in maternal peripheral blood of PE [purpose]
Placental DNA methylation abnormalities are involved in the occurrence of PE, and maternal peripheral blood differential methylation DNA may be used as a new noninvasive marker for predicting PE. Prospective cohort study was conducted to screen maternal peripheral blood differential methylation DNA by methylation chip technique and to study its methylation changes in normal pregnant women and pregnant women with PE. Screening and predicting candidate genes for PE. [method]
The subjects were 1752 pregnant women who delivered in Shenzhen Maternal and Child Health Hospital affiliated to Southern Medical University from October 2012 to November 2013. They were divided into three groups: early-onset PE group, late-onset PE group and normal control group. Exclusion criteria were matched with the basic data of pregnant women, such as age, pregnant and parturient times. Blood samples of 3 pregnant women, 3 pregnant women, 3 pregnant women, 3 pregnant women, 3 pregnant women, 3 pregnant women, 3 pregnant women, and 3 pregnant women at the time of admission and delivery (late pregnant women) were used to determine the degree of DNA methylation. Differential methylation DNA was screened by using limited P value (0.05) and M value (1.0 or less than - 1.0) as cut values. The signal pathways and regulatory ranges of the main effects of different genes were analyzed by DAVID database. According to the relative importance of signal pathways after clustering analysis, gene function annotations and previous literature reports were issued with PE. Pathogenesis related genes, screening for peripheral blood may be involved in the pathogenesis of PE methylation differences DNA.[results]
1. In the early, middle, and late trimesters of pregnancy, the whole level of methylation in the normal control group changed slightly, and the level of blood methylation in the early pregnancy was the lowest. Compared with the control group, the early-onset PE group and the late-onset PE group showed an increasing trend of methylation in the early pregnancy.
2. Results of blood methylation in early pregnancy: (1) Compared with the control group, 34 713 differentially methylated sites were found in early-onset PE group, of which 1303 were significantly different after correction; (2) Compared with the control group, 24 625 differentially methylated sites were found in late-onset PE group, of which 648 were statistically different after correction; (3) Early-onset PE group. 11 common differential methylation genes (C22orf45, UPB1, MTNR1A, PLEKHG5, BLCAP, HOXA9, TUBGCP5, ZNF492, PSKH2, MBP and PIWIL1) were found in late-onset PE and control groups.
3. Results of blood methylation in the second trimester of pregnancy: (1) Compared with the control group, 42243 differentially methylated sites were found in the early-onset PE group, of which 1176 were statistically different after correction; (2) Compared with the control group, 24241 differentially methylated sites were found in the blood of the late-onset PE group, of which 479 were statistically different after correction. (3) There were 17 common differentially methylated genes (FBXO39, EPHA5, ZNF542, HOXD1, BOLL, HCN1, CHRNB1, GLP1R, GABRG3, NKAIN3, NTSR2, CCK, C17orf51, UCP1, SNAP25, MTNR1A, LPPR3) in the early-onset PE group, late-onset PE group and control group.
4. Blood methylation results in late pregnancy: (1) Compared with the control group, 42 847 differentially methylated sites were found in the early-onset PE group, of which 1133 were statistically different after correction; (2) Compared with the control group, 25 157 differentially methylated sites were found in the late-onset PE group, of which 491 were statistically different after correction; (3) A total of 19 genes (PAK7, MOSC2, SHE, TDRD10, UNC5D, CHL1, GRM1, PPAP2C, UGT8, CCK, C17orf51, PAX1, PUS3, DDX25, NPY2R, SLC7A14, HLA-A, DPP6, DSC2) located on the CpGs island and distributed in the 5'UTR or TSS region were successfully detected by human genome-wide DNA methylation chip. DNA methylation in peripheral blood of early-onset PE group, late-onset PE group and control group at middle and late stages of pregnancy showed that there were differences in DNA methylation gene changes. Genes with significant difference in glycosylation may be an effective predictor. According to the information of microarray sites and gene function, HOXA9, MTNR1A, PIWIL1 were selected for further validation.
The second chapter is the validation of HOXA9, MTNR1A and PIWIL1 in maternal peripheral blood of PE and its application in early prediction of PE.
[Objective]
Three candidate genes, HOXA9, MTNR1A and PIWIL1, were selected from the genes with significant methylation difference screened by methylation chip in early pregnancy. The methylation changes of DNA sites in early-onset PE group, late-onset PE group and normal control pregnant women group (control group) were detected by pyrophosphate sequencing, and the mRNA changes of each candidate gene were detected by RT-qPCR. To explore the feasibility of these indicators as early prediction of PE. [method]
The subjects were divided into three groups: early-onset PE group, late-onset PE group and control group. All complications and pregnancy complications were excluded and randomly selected. DNA and RNA were extracted from maternal peripheral blood in early pregnancy and late pregnancy. MTNR1A, HOXA9 and PIWILl were sequenced to detect the methylation degree of peripheral blood in early-onset PE group, late-onset PE group and control group at different stages.
1. The results of pyrophosphate sequencing test in early pregnancy showed that: (1) the methylation rate of HOXA9 in early PE group (4.60.88%) was significantly higher than that in late PE group (1.30.73%) and control group (1.44.70%) (P 0.05), and there was no statistical difference between the rest groups (P 0.05); (2) the methylation rate of two sites of MINR1A in early PE group was significantly higher than that in control group (P 0.05). There was no statistical difference (P0.05); (3) there was no significant difference in hypermethylation of PIWILl among groups (P0.05).
2. The results of pyrophosphate sequencing test in late pregnancy showed that: (1) HOXA9 had no significant difference among all groups (P 0.05); (2) MINR1A showed hypermethylation in both early-onset PE group and late-onset PE group; MTNR1A (1) the methylation rate of early-onset PE group and late-onset PE group had significant difference compared with the control group (P 0.05); and MTNR1A (2) methylation rate of MTNR1A (2) had significant difference between the two groups. There was no statistical difference (P0.05); (3) there was no significant difference in hypermethylation rate of PIWIL1 between the three groups (P0.05).
3. The results of RT-q PCR of HOXA9 mRNA in early pregnancy showed that the relative amount of HOXA9 mRNA in early-onset PE group was 0.56+0.08, 0.70 times higher than that in late-onset PE group and 0.56 times higher than that in control group, while MTNRIA mRNA and PIWIL1 mRNA were not detected in peripheral blood.
4. The results of RT-q PCR of HOXA9 mRNA in late pregnancy showed that the relative expression of HOXA9 mRNA in early PE group was 0.77 [0.07], 0.94 times as high as that in late PE group and 0.77 times as high as that in control group, while MTNRIA mRNA and PIWIL1 mRNA were not detected in peripheral blood.
5. The relative expression of HOXA9 mRNA in peripheral blood of early-onset PE group, late-onset PE group and control group was higher than that in early pregnancy. [Summary]
The methylation of HOXA9 and MTNR1A in peripheral blood of different types of PE in different gestational stages was confirmed by pyrophosphate sequencing and RT-q PCR, suggesting that the methylation of HOXA9 and MTNRIA in maternal peripheral blood may be related to the pathogenesis and pathological process of PE.
1. Human genome-wide DNA methylation chip (illumina 450k) was successfully used to analyze the differences of DNA methylation in maternal peripheral blood between early-onset PE, late-onset PE and normal pregnant women.
2. In the early, middle and late pregnancy, there were methylation differentially expressed genes in maternal peripheral blood of early-onset PE and late-onset PE, which involved cell development and proliferation, angiogenesis, placenta and embryo development, and so on.
3. The level of DNA methylation in maternal peripheral blood of early-onset PE was higher than that of late-onset PE and control group (P 0.05), and the expression of HOXA9 mRNA was also decreased, suggesting that HOXA9 might be a noninvasive predictor of early-onset PE.
4. MINR1A hypermethylation was found in maternal peripheral blood of PE patients; methylation rate of two sites in maternal peripheral blood of early-onset PE patients was significantly different from that of control group (P 0.05); MTNR1A (1) early-onset PE patients in maternal peripheral blood of late-onset PE patients was significantly different from that of control group (P 0.05), suggesting that methylation rate of late-onset PE patients was significantly different from that of control group (P 0.05). It may be used as an early predictor of early onset PE.
5. the detection of methylation differential gene in maternal blood can be used as a method to predict the occurrence of PE.
【学位授予单位】：南方医科大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R714.244
本文编号：2196767
[Abstract]:Pre eclampsia (PE) is a kind of pregnant women with normal blood pressure before pregnancy. After 20 weeks of gestation, preeclampsia (PE) occurs hypertension, proteinuria, also known as preeclampsia. PE affects 3% - 5% of pregnant women worldwide. PE is the main cause of maternal and fetal morbidity and mortality, accounting for 20% and 40% of maternal and fetal mortality respectively. The incidence of late pregnancy is increasing year by year. PE not only affects the outcome of pregnancy, but also affects the long-term prognosis of pregnant women and their offspring. The chance of disease has also increased.
At present, it is believed that abnormal placenta formation occurs in the early pregnancy leading to placental ischemia (stage I); ischemic placentas secrete soluble factors in late pregnancy leading to systemic endothelial dysfunction and PE syndrome (stage II). Before the occurrence of PE, there have been many changes in the plasma composition of pregnant women, therefore, there are many predictions for PE. Biomarkers such as placental hormones, angiogenesis factors, and lipids have been studied. However, previous studies on the prediction of PE by these biomarkers have mainly focused on the signal transduction pathway molecules that may lead to PE, and mostly on the prediction of single biomarkers. So far, there is no single marker or several combined markers to predict the sensitivity and specificity needed for clinical application, so these tests can not be carried out routinely.
DNA methylation detection has the advantages of good stability, so there have been many previous studies on DNA methylation for adverse pregnancy outcomes, but these studies are mostly based on the detection of single site or placental methylation changes.
In this study, a prospective cohort study was designed to collect maternal peripheral blood samples from pregnant women. Methylation chips were used to screen and pyrophosphate sequencing was used to verify the differential methylation DNA of PE. The changes of methylation of differentially methylated DNA in peripheral blood of normal pregnant women and PE pregnant women with gestational weeks were studied and discussed.
Chapter 1 screening and detection of differential methylation of DNA in maternal peripheral blood of PE [purpose]
Placental DNA methylation abnormalities are involved in the occurrence of PE, and maternal peripheral blood differential methylation DNA may be used as a new noninvasive marker for predicting PE. Prospective cohort study was conducted to screen maternal peripheral blood differential methylation DNA by methylation chip technique and to study its methylation changes in normal pregnant women and pregnant women with PE. Screening and predicting candidate genes for PE. [method]
The subjects were 1752 pregnant women who delivered in Shenzhen Maternal and Child Health Hospital affiliated to Southern Medical University from October 2012 to November 2013. They were divided into three groups: early-onset PE group, late-onset PE group and normal control group. Exclusion criteria were matched with the basic data of pregnant women, such as age, pregnant and parturient times. Blood samples of 3 pregnant women, 3 pregnant women, 3 pregnant women, 3 pregnant women, 3 pregnant women, 3 pregnant women, 3 pregnant women, and 3 pregnant women at the time of admission and delivery (late pregnant women) were used to determine the degree of DNA methylation. Differential methylation DNA was screened by using limited P value (0.05) and M value (1.0 or less than - 1.0) as cut values. The signal pathways and regulatory ranges of the main effects of different genes were analyzed by DAVID database. According to the relative importance of signal pathways after clustering analysis, gene function annotations and previous literature reports were issued with PE. Pathogenesis related genes, screening for peripheral blood may be involved in the pathogenesis of PE methylation differences DNA.[results]
1. In the early, middle, and late trimesters of pregnancy, the whole level of methylation in the normal control group changed slightly, and the level of blood methylation in the early pregnancy was the lowest. Compared with the control group, the early-onset PE group and the late-onset PE group showed an increasing trend of methylation in the early pregnancy.
2. Results of blood methylation in early pregnancy: (1) Compared with the control group, 34 713 differentially methylated sites were found in early-onset PE group, of which 1303 were significantly different after correction; (2) Compared with the control group, 24 625 differentially methylated sites were found in late-onset PE group, of which 648 were statistically different after correction; (3) Early-onset PE group. 11 common differential methylation genes (C22orf45, UPB1, MTNR1A, PLEKHG5, BLCAP, HOXA9, TUBGCP5, ZNF492, PSKH2, MBP and PIWIL1) were found in late-onset PE and control groups.
3. Results of blood methylation in the second trimester of pregnancy: (1) Compared with the control group, 42243 differentially methylated sites were found in the early-onset PE group, of which 1176 were statistically different after correction; (2) Compared with the control group, 24241 differentially methylated sites were found in the blood of the late-onset PE group, of which 479 were statistically different after correction. (3) There were 17 common differentially methylated genes (FBXO39, EPHA5, ZNF542, HOXD1, BOLL, HCN1, CHRNB1, GLP1R, GABRG3, NKAIN3, NTSR2, CCK, C17orf51, UCP1, SNAP25, MTNR1A, LPPR3) in the early-onset PE group, late-onset PE group and control group.
4. Blood methylation results in late pregnancy: (1) Compared with the control group, 42 847 differentially methylated sites were found in the early-onset PE group, of which 1133 were statistically different after correction; (2) Compared with the control group, 25 157 differentially methylated sites were found in the late-onset PE group, of which 491 were statistically different after correction; (3) A total of 19 genes (PAK7, MOSC2, SHE, TDRD10, UNC5D, CHL1, GRM1, PPAP2C, UGT8, CCK, C17orf51, PAX1, PUS3, DDX25, NPY2R, SLC7A14, HLA-A, DPP6, DSC2) located on the CpGs island and distributed in the 5'UTR or TSS region were successfully detected by human genome-wide DNA methylation chip. DNA methylation in peripheral blood of early-onset PE group, late-onset PE group and control group at middle and late stages of pregnancy showed that there were differences in DNA methylation gene changes. Genes with significant difference in glycosylation may be an effective predictor. According to the information of microarray sites and gene function, HOXA9, MTNR1A, PIWIL1 were selected for further validation.
The second chapter is the validation of HOXA9, MTNR1A and PIWIL1 in maternal peripheral blood of PE and its application in early prediction of PE.
[Objective]
Three candidate genes, HOXA9, MTNR1A and PIWIL1, were selected from the genes with significant methylation difference screened by methylation chip in early pregnancy. The methylation changes of DNA sites in early-onset PE group, late-onset PE group and normal control pregnant women group (control group) were detected by pyrophosphate sequencing, and the mRNA changes of each candidate gene were detected by RT-qPCR. To explore the feasibility of these indicators as early prediction of PE. [method]
The subjects were divided into three groups: early-onset PE group, late-onset PE group and control group. All complications and pregnancy complications were excluded and randomly selected. DNA and RNA were extracted from maternal peripheral blood in early pregnancy and late pregnancy. MTNR1A, HOXA9 and PIWILl were sequenced to detect the methylation degree of peripheral blood in early-onset PE group, late-onset PE group and control group at different stages.
1. The results of pyrophosphate sequencing test in early pregnancy showed that: (1) the methylation rate of HOXA9 in early PE group (4.60.88%) was significantly higher than that in late PE group (1.30.73%) and control group (1.44.70%) (P 0.05), and there was no statistical difference between the rest groups (P 0.05); (2) the methylation rate of two sites of MINR1A in early PE group was significantly higher than that in control group (P 0.05). There was no statistical difference (P0.05); (3) there was no significant difference in hypermethylation of PIWILl among groups (P0.05).
2. The results of pyrophosphate sequencing test in late pregnancy showed that: (1) HOXA9 had no significant difference among all groups (P 0.05); (2) MINR1A showed hypermethylation in both early-onset PE group and late-onset PE group; MTNR1A (1) the methylation rate of early-onset PE group and late-onset PE group had significant difference compared with the control group (P 0.05); and MTNR1A (2) methylation rate of MTNR1A (2) had significant difference between the two groups. There was no statistical difference (P0.05); (3) there was no significant difference in hypermethylation rate of PIWIL1 between the three groups (P0.05).
3. The results of RT-q PCR of HOXA9 mRNA in early pregnancy showed that the relative amount of HOXA9 mRNA in early-onset PE group was 0.56+0.08, 0.70 times higher than that in late-onset PE group and 0.56 times higher than that in control group, while MTNRIA mRNA and PIWIL1 mRNA were not detected in peripheral blood.
4. The results of RT-q PCR of HOXA9 mRNA in late pregnancy showed that the relative expression of HOXA9 mRNA in early PE group was 0.77 [0.07], 0.94 times as high as that in late PE group and 0.77 times as high as that in control group, while MTNRIA mRNA and PIWIL1 mRNA were not detected in peripheral blood.
5. The relative expression of HOXA9 mRNA in peripheral blood of early-onset PE group, late-onset PE group and control group was higher than that in early pregnancy. [Summary]
The methylation of HOXA9 and MTNR1A in peripheral blood of different types of PE in different gestational stages was confirmed by pyrophosphate sequencing and RT-q PCR, suggesting that the methylation of HOXA9 and MTNRIA in maternal peripheral blood may be related to the pathogenesis and pathological process of PE.
1. Human genome-wide DNA methylation chip (illumina 450k) was successfully used to analyze the differences of DNA methylation in maternal peripheral blood between early-onset PE, late-onset PE and normal pregnant women.
2. In the early, middle and late pregnancy, there were methylation differentially expressed genes in maternal peripheral blood of early-onset PE and late-onset PE, which involved cell development and proliferation, angiogenesis, placenta and embryo development, and so on.
3. The level of DNA methylation in maternal peripheral blood of early-onset PE was higher than that of late-onset PE and control group (P 0.05), and the expression of HOXA9 mRNA was also decreased, suggesting that HOXA9 might be a noninvasive predictor of early-onset PE.
4. MINR1A hypermethylation was found in maternal peripheral blood of PE patients; methylation rate of two sites in maternal peripheral blood of early-onset PE patients was significantly different from that of control group (P 0.05); MTNR1A (1) early-onset PE patients in maternal peripheral blood of late-onset PE patients was significantly different from that of control group (P 0.05), suggesting that methylation rate of late-onset PE patients was significantly different from that of control group (P 0.05). It may be used as an early predictor of early onset PE.
5. the detection of methylation differential gene in maternal blood can be used as a method to predict the occurrence of PE.
【学位授予单位】：南方医科大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R714.244
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