Detection Of Methylation Gene Difference Of Peripheral Blood And Its Value For Predicting Pre-eclampsia In Expectantat Mother

Pre-eclampsia (PE) is disease in expectant mother whom emerging new hypertension (diastolic blood pressure of≥90mm Hg) and substantial proteinuria (≥300mg in24h) at or after20weeks gestation. It is estimated that there are3-5%of pregnancies suffering from PE. PE is a leading cause for maternal mortality all of the world and it accounted for20%of maternal deaths and40%of baby deaths.In China, the incidence of PE was around2-8%and recently this figures have an increased tendency with years. PE can not only exert an influence on pregnancy outcome but also adverse effect on both mother and baby. For baby with PE mother, a lot of complications include premature delievery and intrauterine growth restriction, etc. will occur and also increased risk of suffering heart diseases after birth. For expectant mother with PE, it increased the risk of the occurrence of cardiovascular disease.Recently, it is considered that abnormal placental formation palyed a role in placental ischemia (Ⅰ stage) at first-trimester of pregnancy and resulted in systemic dysfunction of endothelium and PE syndrome (Ⅱ stage) by secreting soluble factors at third-trimester of pregnancy. Furthermore, the components of maternal blood have changed widely before PE. There are many studies focused on the predictive biomarkers including placental hormone, angiogenic factors and lipids for PE. But, most of these previous studies were based on the hypothesis of signaling transduction pathway caused PE and the predictive biomarker was single. Due to the heterogeneity and clinical manifestation diversification of PE, there are some defects of these methods. Up to now, the detection of biomarker for PE prediction was not widely used for the limits of sensitivity and specificity.DNA methylation detection has the advantage of stability and thus there are numerous studies for the outcome of abnormal pregnancy. But, of these studies, the detective site was single site and no genome-wide DNA methylation sites were reported.Due to the DNA methylation and its aberrance in placenta during the PE, it is feasible for detecting differential methylation genes of peripheral blood for predictive PE. Here this study using the design of prospective cohort study to collecting of peripheral blood in expectant mother, and to screen and validate differential methylation genes in PE by using methylation chip and pyrosequencing; to illustrate differences of the DNA differential methylation in maternal blood between normal and PE pregnancy, to investigate the predictive value for predicting preeclampsia of differential methylation genes.Chapter1Screening and Detection of Differential Methylation Genes in Maternal Peripheral Blood of PE Expectant Mother Objective Many recent data suggested that placenta DNA methylation and its aberrance were involved in PE, therefore,the differential methylated DNA in maternal peripheral blood has the potential as a new biomarker for noninvasive prediction of PE. In this prospective cohort study, the methylation chip was used to screen and detect the candidate genes of PE from the peripheral blood at different stage of pregnancy. Methods Total1752study subjects enrolled in this study from Southern Medical University affiliated shenzhen maternity and child care service centre from Oct.2012to Nov.2013, which were divided into three group early-onset PE, late-onset PE and normal control. According to the set standards, three samples of each group were used to screen the differentially genes (P<0.05, m>lor m<-l) between early-onset PE, late-onset PE and normal control by genome-wide DNA methylation chip. DAVID database was used to analyze the candidate genes-related signal pathway. Cluster analysis and previous articles on PE were also used to screen the candidate genes for predicting PE.Results:1. In first-, mid-and last-trimester in normal pregnancy, there were slight fluctuation of the methylation genes in maternal peripheral blood and the lowest degree of methylation was at first-trimester in normal pregnancy. The methylation genes in first-trimester maternal peripheral blood of early-onset PE and late-onset PE pregnancy were increased than that in control pregnancy.2. The results of differentially methylation genes in first-trimester peripheral blood:(1) There were34713differentially methylation genes in early-onset PE compare with control. Of these,1303genes with significant difference;(2) There were24625differentially methylation genes in late-onset PE compare with control. Of these,648genes with significant difference;(3) It was found that differentially methylated sites of11genes (C22orf4, UPB1, MTNR1A, PLEKHG5, BLCAP, HOXA9, TUBGCP5, ZNF492, PSKH2, MBP and PIWIL1) located on5’UTR and TSS regions by analyzing the data of differentially methylation genes in early-onset PE, late-onset PE and normal control.3. The results of differentially methylation genes in mid-trimester peripheral blood:(1) There were42243differentially methylation genes in early-onset PE compare with control. Of these,1176genes with significant difference;(2) There were24241differentially methylation genes in late-onset PE compare with control. Of these,479genes with significant difference;(3) It was found that differentially methylated sites of17genes (FBXO39, EPHA5, ZNF542, HOXD1, BOLL, HCN1, CHRNB1, GLP1R, GABRG3, NKAIN3, NTSR2, CCK, C17orp1, UCP1, SNAP25, MTNR1A, LPPR3) located on5’UTR and TSS regions by analyzing the data of differentially methylation genes in mid-trimester peripheral blood of early-onset PE, late-onset PE and normal control.4. The results of differentially methylation genes in late-trimester peripheral blood:(1) There were42847differentially methylation genes in early-onset PE compare with control. Of these,1133genes with significant difference;(2) There were25157differentially methylation genes in late-onset PE compare with control. Of these,491genes with significant difference;(3) It was found that differentially methylated sites of19genes (PAK7, M0SC2, SHE, TDRD10, UNC5D, CHL1, GRM1, PPAP2C, UGT8, CCK, C17orfS1, TAXI, PUS3, DDX25, NPY2R, SLC7A14, HLA-A, DPP6, DSC2) located on5’UTR and TSS regions by analyzing the data of differentially methylation genes in late-trimester peripheral blood of early-onset PE, late-onset PE and normal control.SummaryUsing the genomic-wide methylation chip, this study has successfully detected DNA methylation in first-, mid-and last-trimester of pregnancy. There were significant differences of DNA methylation between early-onset PE, late-onset PE and control, respectively. Of the differentially candidate genes,11were screened in first-trimester peripheral blood,17were screened in mid-trimester peripheral blood,19were screened in last-trimester peripheral blood, all of these maybe serve as potential biomarker for predicting PE. According the genes sites and functions,3candidate genes (MTNR1A, HOXA9and PIWIL1) were selected for further validation.Chapter2Validation of MTNR1A, HOXA9and PIWIL1and Its Value for Predicting Pre-eclampsia in Expectantat MotherObjectiveThe purposes of this chapter study were to validate MTNR1A, HOXA9and PIWIL1of gene chip’s results by pyrosequencing and RT-qPCR and discuss its value for predicting pre-eclampsia in expectantat mother. MethodsAll the study subjects enrolled in this study from Southern Medical University affiliated shenzhen maternity and child care service centre from Oct.2012to Nov.2013. The subjects were divided into early-onset PE, late-onset PE and normal control. Genomic DNA and RNA of peripheral blood in first trimester pregnancy and at birth were extracted from the10maternal peripheral blood samples in each group using the indicated kit according to the manufacturer’s instructions. Pyrosequencing was used to validate the candidate genes (MTNR1A, HOXA9and PIWIL). After pyrosequencing by BiotagePyromark Q96MD, each CpG island in candidate genes was analyzed by Pyro Q-CpG software. RT-qPCR was used to detect the candidate genes expression of mRNA.Results1. The results of pyrosequencing in first-trimester peripheral blood:(1) The rates of HOXA9methylation were4.60±0.88%in early-onset PE, which was statistical significance than that in late-onset PE (1.30±0.73%) and control(1.44±0.70%), respectively(P<0.05);(2) There were statistical significance of the two sites(1,2) of MTNR1A methylation between early-onset PE and control group (P＜0.05);(3) No significant difference of PIWIL1hypermethylation were observed in early-onset PE, late-onset PE and control group.2. The results of pyrosequencing in last-trimester peripheral blood:(1) There were not statistical significance of HOXA9methylation in early-onset PE, late-onset PE and control group(P>0.05);(2) The two sites(1,2) of MTNR1A hypermethylation were detected, and the difference of MTNR1A(i) hypermethylation between early-onset PE and control group, late-onset PE and control group were significant, respectively(P<0.05), No significant difference of MTNRIA(2) hypermethylation were observed between each group;(3) No significant difference of PIWIL1hypermethylation were observed in early-onset PE, late-onset PE and control group.3. The mRNA expression levels in first-trimester peripheral blood by RT-qPCR: The relative expression of HOXA9mRNA in early-onset PE were0.56±0.08, it was0.70times than that in late-onset PE and0.56times than that in control. No MTNR1A mRNA and PIWIL1mRNA were detectable in early-onset PE, late-onset PE and control group.4. The mRNA expression levels in last-trimester peripheral blood by RT-qPCR: The relative expression of HOXA9mRNA in early-onset PE were0.77±0.07, it was0.94times than that in late-onset PE and0.77times than that in control. No MTNR1A mRNA and PIWIL1mRNA were detectable in early-onset PE, late-onset PE and control group.5. The mRNA expression levels in last-trimester peripheral blood were slightly increased than that in first-trimester peripheral blood either in early-onset PE and late-onset PE or in control group.SummaryAfter validation by pyrosequencing and RT-qPCR, the results showed that there were difference of methylation levels of HoXA9and MTNR1A at first-trimester peripheral blood and last-trimester peripheral blood in early-onset PE and late-onset PE. These finding indicated that the methylation levels of HOXA9and MTNR1A involved in the PE. Using the detection of difference of methylation levels of HoXA9and MTNR1A in maternal peripheral blood, it can serve as potential biomarkers for predictive early-onset PE in expectantat mother.Conclusion1. Using the genomic-wide methylation chip, this study has successfully detected DNA methylation in maternal peripheral blood at first-, mid-and last-trimester of pregnancy inearly-onset PE, late-onset PE and normal control expectantat mother.2. At first-, mid-and last-trimester of pregnancy, there were differential DNA methylation genes in early-onset and late-onset PE when compared with control. These genes involved in cell development and proliferation, angiogenesis, placenta and embryo development, etc. and maybe play the role in PE.3. The methylation levels of HOXA9were dramatically increased and the mRNA of HoXA9was downregulated in first-trimester peripheral blood of early-onset PE than that in late-onset PE and control. These results suggested that HOXA9may be a noninvasive biomarker for predicting early-onset PE.4. The methylation levels of the two sites (1,2) MTNR1A were dramatically increased in PE. There were statistical significance of the two sites (1,2) of MTNR1A methylation in first-trimester peripheral blood between early-onset PE and control group (P<0.05), and the difference of MTNR1A(1) hypermethylation between early-onset PE and control group, late-onset PE and control group were significant, respectively(P<0.05). These findings suggested that it may be a noninvasive biomarker for predicting early-onset PE5. To detect the difference of methylation gene can serve as a potential biomarker for predictive PE and this need to further study.