【摘要】：子癇前期(Pre-eclampsia, PE)為懷孕前血壓正常的孕婦在妊娠20周以后出現(xiàn)高血壓、蛋白尿,又稱為先兆子癇。PE影響著全世界3%-5%的孕婦,是導致母兒患病率和死亡率的主要原因,分別占產(chǎn)婦及胎兒死因的20%、40%。資料顯示我國PE發(fā)生率約2-8%,晚近發(fā)生率呈逐年增長趨勢。PE不僅可影響妊娠結局,也影響產(chǎn)婦及子代的遠期預后。PE的新生兒可發(fā)生包括早產(chǎn),胎兒生長受限,缺氧相關的神經(jīng)損傷,圍生兒死亡等并發(fā)癥,其日后發(fā)生心臟病的風險增加；PE孕婦發(fā)生心血管和腦血管疾病的機率亦增加等。 目前認為,異常的胎盤形成發(fā)生在妊娠早期導致胎盤缺血(Ⅰ期)；缺血的胎盤在妊娠晚期可分泌可溶性因子從而引起系統(tǒng)性內(nèi)皮功能紊亂和PE綜合征(Ⅱ期)。在PE發(fā)生之前,孕婦的血漿成分已經(jīng)出現(xiàn)了諸多變化,因此,有許多針對預測PE的生物標記物如胎盤激素、血管生成因子、脂質進行的研究。但既往這些生物標記物在PE預測方面的研究多為針對可能導致PE發(fā)生的信號轉導通路分子的研究,且多為單一標記物的預測研究。而且,由于PE是一異質性疾病,臨床表現(xiàn)多樣化,應用單一標記物預測的可能性不大。故至今為止尚無一個標記物或者幾個聯(lián)合標記物預測能達到臨床應用所需要的靈敏度和特異度,因此這些檢測均未能在臨床常規(guī)開展。 DNA的甲基化檢測具有穩(wěn)定性好的優(yōu)點,因此既往有諸多針對不良妊娠結局DNA甲基化研究,但這些研究多是基于單一位點或胎盤的甲基化改變的檢測,尚無對PE母體外周血全基因組所有位點的甲基化檢測報道。 在PE的發(fā)生過程中存在胎盤DNA甲基化異常,因此通過檢測孕婦外周血差異甲基化基因可能成為預測PE發(fā)生的新的標記物。本研究采用前瞻性隊列研究設計收集孕婦外周血,應用甲基化芯片篩選、焦磷酸測序驗證PE差異甲基化DNA；檢測母體外周血差異甲基化DNA在正常孕婦與PE孕婦隨孕周的甲基化變化情況；研究和探討外周血差異甲基化基因作為作為預測PE發(fā)生的新的標記物的早期預測價值。 第一章PE母體外周血差異甲基化DNA的篩選及檢測[目的] 胎盤DNA甲基化異常參與了PE的發(fā)生,母體外周血差異甲基化DNA可能做為無創(chuàng)性預測PE發(fā)生的新的標記物。應用前瞻性隊列研究方法,采用甲基化芯片技術篩選母體外周血差異甲基化DNA,研究其在正常孕婦與PE孕婦隨孕周改變的甲基化變化,篩選可預測PE的候選基因。[方法] 研究對象為2012年10月-2013年11月期間在南方醫(yī)科大學附屬深圳婦幼保健院從孕早期建立妊娠期保健手冊開始定期產(chǎn)前檢查,并最終在本院分娩的孕婦共1752例,分為早發(fā)型PE組、晚發(fā)型PE組、正常對照孕婦組(對照組)三組。參照入選標準及排除標準,匹配對照組孕婦年齡、孕產(chǎn)次等基本資料,各組3例抽提其孕早期、孕中期及入院分娩時(孕晚期)血標本DNA,應用人全基因組DNA甲基化芯片對其DNA甲基化程度進行測定。對比識別早發(fā)型PE組、晚發(fā)型PE組和對照組的差異甲基化DNA。以限定的P值(0.05)和M值(1.0或小于-1.0)作為切割值,篩選出各組的差異甲基化DNA。利用DAVID數(shù)據(jù)庫等分析差異基因主要參與影響的信號通路及調控范圍；根據(jù)聚類分析后各信號通路的相對重要性、基因功能注釋及既往文獻報道中與PE發(fā)病機制相關基因,篩選出外周血中可能參與PE發(fā)病的甲基化差異DNA。[結果] 1.在孕早、中、晚三個時間段,正常對照組中的整體甲基化程度略有變化,孕早期血的甲基化程度最低；與對照組比較,早發(fā)型PE組、晚發(fā)型PE組的孕早期甲基化程度呈增高趨勢。 2.孕早期血甲基化結果：(1)與對照組比對,早發(fā)型PE組共發(fā)現(xiàn)34713個差異甲基化位點,其中矯正后有統(tǒng)計學差異位點1303個；(2)與對照組比對,晚發(fā)型PE組共發(fā)現(xiàn)24625個差異甲基化位點,其中矯正后有統(tǒng)計學差異的位點648個；(3)早發(fā)型PE組、晚發(fā)型PE組及對照組同時滿足探針位于CpGs島并分布在5'UTR或TSS區(qū)域的共同差異甲基化基因11個(C22orf45、UPB1、MTNR1A、PLEKHG5、BLCAP、HOXA9、TUBGCP5、ZNF492、PSKH2、 MBP及PIWIL1)。 3.孕中期血甲基化結果：(1)與對照組比對,早發(fā)型PE組共發(fā)現(xiàn)42243個差異甲基化位點,其中矯正后有統(tǒng)計學差異的位點1176個；(2)與對照組比對,晚發(fā)型PE組孕中期血共發(fā)現(xiàn)24241個差異甲基化位點,其中矯正后有統(tǒng)計學差異的位點共479個；(3)早發(fā)型PE組、晚發(fā)型PE組及對照組同時滿足探針位于CpGs島并在分布在5'UTR或TSS區(qū)域共有17個共同差異甲基化基因(FBXO39、EPHA5、ZNF542、HOXD1、BOLL、HCN1、CHRNB1、GLP1R、GABRG3、 NKAIN3、NTSR2、CCK, C17orf51、UCP1、SNAP25、MTNR1A、LPPR3) 4.孕晚期血甲基化結果：(1)與對照組比對,早發(fā)型PE組共發(fā)現(xiàn)42847個差異甲基化位點,其中矯正后有統(tǒng)計學差異的位點共1133個；(2)與對照組比對,晚發(fā)型PE組共發(fā)現(xiàn)25157個差異甲基化位點,其中矯正后有統(tǒng)計學差異的位點共有491個；(3)早發(fā)型PE、晚發(fā)型PE及對照組同時滿足探針位于CpGs島并在分布在5'UTR或TSS區(qū)域的共有19個基因(PAK7、MOSC2、SHE、TDRD10、UNC5D、CHL1、GRM1、PPAP2C、UGT8、CCK、C17orf51、PAX1、 PUS3、DDX25、NPY2R、SLC7A14、HLA-A、DPP6、DSC2)[小結]利用人全基因組DNA甲基化芯片成功檢測了孕早、中、晚三期的早發(fā)型PE組、晚發(fā)型PE及對照組外周血DNA甲基化,結果顯示其均存在差異甲基化基因改變。其中從孕早期血中成功篩選了11甲基化差異顯著的基因,孕中期血中成功篩選了17甲基化差異顯著的基因,孕晚期血中成功篩選了19甲基化差異顯著的基因,其可能成為有效的預測指標。根據(jù)芯片位點信息及基因功能等,選取孕早期HOXA9、MTNR1A、PIWIL1進行下一步驗證。 第二章PE母體外周血中HOXA9、MTNR1A、PIWIL1的驗證及其用于PE早期預測的研究 [目的] 從孕早期甲基化芯片篩選的甲基化差異顯著的基因中挑選出HOXA9、MTNR1A、PIWIL1共3個候選基因,以焦磷酸測序檢驗在早發(fā)型PE組、晚發(fā)型PE組、正常對照孕婦組(對照組)中對應DNA位點的甲基化變化情況,并以RT-qPCR檢測各候選基因的mRNA變化情況。探討這些指標作為PE早期預測的可行性。[方法] 研究對象為2012年10月-2013年11月期間在南方醫(yī)科大學附屬深圳婦幼保健院從孕早期建立妊娠期保健手冊開始定期產(chǎn)前檢查,并最終在本院分娩的孕婦。分為早發(fā)型PE組、晚發(fā)型PE組、對照組三組。排除各種合并癥和妊娠并發(fā)癥之后隨機抽樣每組10例,提取孕早期及分娩期(孕晚期)母體外周血DNA和RNA。焦磷酸測序檢測MTNR1A、HOXA9及PIWILl三個篩選的目標基因在早發(fā)型PE組、晚發(fā)型PE組和對照組不同時期外周血的甲基化程度；RT-qPCR檢測目標基因在各組的mRNA的表達情況。[結果] 1.孕早期焦磷酸測序檢驗結果顯示：(1)HOXA9在早發(fā)型PE組甲基化率(4.60±0.88%)顯著高于晚發(fā)型PE組(1.30±0.73%)和對照組(1.44±0.70%)(P0.05),余組間比較無統(tǒng)計差異(P0.05);(2)MINR1A的兩個位點在早發(fā)型PE組甲基化率顯著高于對照組(P0.05),余組間比較無統(tǒng)計差異(P0.05)；(3)PIWILl在各組間的高甲基化無統(tǒng)計學差異(P0.05)。 2.孕晚期焦磷酸測序檢驗結果顯示：(1)HOXA9在各組間未見統(tǒng)計學差異(P0.05);(2)MINR1A兩位點在早發(fā)型PE組及晚發(fā)型PE組均表現(xiàn)出高甲基化；MTNR1A(1)早發(fā)型PE組及晚發(fā)型PE組甲基化率與對照組差異有統(tǒng)計學意義(P0.05); MTNR1A(2)甲基化率組間無統(tǒng)計學差異(P0.05);(3) PIWIL1在三組間的高甲基化率差異無統(tǒng)計學差異(P0.05)。 3.孕早期HOXA9mRNA的RT-qPCR結果顯示其早發(fā)型PE組相對量為0.56±0.08,為晚發(fā)型PE組的0.70倍及對照組的0.56倍；而MTNRIA mRNA及PIWIL1mRNA在外周血中未檢測到。 4.孕晚期HOXA9mRNA的RT-qPCR結果顯示其早發(fā)型PE組相對表達量為0.77±0.07,為晚發(fā)型PE組的0.94倍及對照組的0.77倍；而MTNRIA mRNA及PIWIL1mRNA在外周血中未檢測到。 5.外周血中HOXA9mRNA相對表達量在早發(fā)型PE組、晚發(fā)型PE組、對照組孕晚期的表達量較孕早期均有增加。[小結] 經(jīng)焦磷酸測序檢驗和RT-qPCR驗證,不同類型PE外周血HOXA9及MTNR1A在不同孕期甲基化程度存在差異,提示其可能與PE的發(fā)病及病理過程有關；此種母體外周血中HOXA9及MTNRIA甲基化差異可作為潛在的早發(fā)型PE早期預測指標之一。全文小結 1.成功運用人全基因組DNA甲基化芯片(illumina450k)分析了早發(fā)型PE、晚發(fā)型PE及正常妊娠女性的孕早、中、晚期母體外周血的DNA甲基化差異。 2.在孕早、中、晚期,與對照組比較,早發(fā)型PE和晚發(fā)型PE母體外周血中均存在甲基化差異表達基因,這些基因涉及細胞發(fā)育與增殖、血管生成、胎盤及胚胎的發(fā)育等,因此推測這些差異甲基化基因參與了PE的發(fā)生、發(fā)展。 3.HOXA9在早發(fā)型PE孕早期母體外周血中DNA甲基化水平高于晚發(fā)型PE組及對照組(P0.05),其mRNA表達亦降低,提示其有望成為早發(fā)型PE早期無創(chuàng)性預測指標。 4.MINR1A兩位點在PE母體外周血中呈高甲基化；在孕早期母體外周血中兩個位點甲基化率早發(fā)型PE組與對照組間有統(tǒng)計學差異(P0.05)；在孕晚期母體外周血中MTNR1A(1)早發(fā)型PE組、晚發(fā)型PE組甲基化率與對照組差異有統(tǒng)計學意義(P0.05),提示其可能作為早發(fā)型PE的早期預測指標。 5.孕婦外周血甲基化差異基因檢測可作為預測PE發(fā)生的方法進行深入研究。
[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.
【學位授予單位】：南方醫(yī)科大學
【學位級別】：博士
【學位授予年份】：2014
【分類號】：R714.244

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