慢性高血压易感基因（CDH13，ATP2B1，UMOD）与子痫前期发病的关系及作用机制的初步研究

发布时间：2018-05-07 18:33

  本文选题：子痫前期 + 慢性高血压并发子痫前期　； 参考：《山东大学》2014年博士论文

【摘要】：第一部分子痫前期与慢性高血压并发子痫前期妊娠结局的回顾性研究 研究背景：子痫前期是一种妊娠期常见的、严重影响母婴健康的妊娠特异性疾病。子痫前期,特别是重度子痫前期发生胎盘早剥、凝血功能障碍、肝肾功能损害等的风险明显增加。在胎儿生长受限的病因中,子痫前期约占10%；约25%的新生儿死亡与子痫前期相关。慢性高血压患者妊娠后,发生子痫前期的风险增加。在育龄期妇女中,慢性高血压的发病率约为1%-5%。随着妊娠年龄的推迟,以及肥胖患者数目的增加,慢性高血压患者的数目逐年增加。既往的回顾性研究发现,慢性高血压患者妊娠后,发生子痫前期、胎盘早剥、早产、胎儿生长受限等的风险大大增加。但是这些研究,多集中在上个世纪90年代。探讨慢性高血压患者的妊娠结局,特别是对比分析子痫前期与慢性高血压并发子痫前期患者的妊娠结局,可以更好的指导慢性高血压患者的孕期保健工作,预防不良妊娠结局的发生。 研究目的：回顾性分析妊娠期高血压疾病所导致的不良妊娠结局。特别是与子痫前期相比,慢性高血压并发子痫前期,是否增加不良妊娠结局的发生。 研究方法：我们回顾性分析了2011年1月到2012年3月期间,在山东大学附属省立医院住院分娩的单胎妊娠孕妇,统计分析正常孕妇、子痫前期、妊娠合并慢性高血压以及慢性高血压并发子痫前期患者的下列临床资料：孕妇年龄、妊娠分娩史、既往疾病史(包括糖尿病、高血压病等)、本次分娩孕周、分娩方式、胎儿性别,以及胎儿生长受限、死胎、胎盘早剥,胎膜早破等的发生等。 研究结果：共有3691例孕妇纳入到本研究中。其中正常孕妇2993例,子痫前期患者461例,妊娠合并慢性高血压患者91例,慢性高血压并发子痫前期患者146例。妊娠期高血压疾病组患者的年龄较大(P0.01),分娩孕周较小(P0.01),糖尿病患者的比例(P0.01)以及胎膜早破的发生率(P0.01)较高。与正常孕妇相比,子痫前期患者发生胎儿生长受限(OR:4.88;95%CI:2.76-8.63)、死胎(OR;11.84;95%CI:6.75-20.75)、早产(OR;6.69;95%CI:5.30-8.46)、剖宫产(OR;2.32;95%CI:1.89-2.89)、胎盘早剥(OR;21.7;95%CI:7.04-66.79)的风险明显增加。慢性高血压并发子痫前期患者发生死胎(OR;30.71;95%CI:16.59-56.83)、早产(OR;21.28:95%CI:14.79-30.60)、剖宫产(OR;7.33:95%CI:4.55-11.82)、胎盘早剥(OR;10.38:95%CI:1.88-57.13)的风险明显增加。妊娠合并慢性高血压患者发生剖宫产(OR;2.76:95%CI:1.75-4.35)、胎盘早剥(OR;16.79;95%CI:3.04-92.88)的风险明显增加。与子痫前期患者相比,慢性高血压并发子痫前期患者,发生死胎(OR;2.59;95%CI:1.49-4.52)、早产(OR;3.18;95%CI:2.16-4.68)以及剖宫产(OR;3.16;95%CI:1.89-5.26)的风险明显增加。 研究结论：妊娠期高血压疾病,增加不良妊娠结局的发生。与子痫前期相比,慢性高血压并发子痫前期增加死胎、早产以及剖宫产等不了妊娠结局的发生。 第二部分慢性高血压易感基因(CDH13, ATP2B1, UMOD)多态性与子痫前期发病的相关性研究 研究背景：子痫前期,是一种以高血压和蛋白尿为特点的妊娠期特异性疾病,具有明显的家族聚集倾向,遗传因素在其发病过程中发挥了重要作用。慢性高血压患者妊娠后,发生子痫前期的风险明显增加。子痫前期患者今后发生慢性高血压的风险明显高于正常孕妇。全基因组关联分析(Genome wide association study, GWAS)是研究复杂疾病易感基因的一种有效手段。慢性高血压的GWAS研究报道,CDH13基因、ATP2B1基因、UMOD基因为慢性高血压的易感基因,并在随后的许多研究中得到证实。 CDH13基因位于16q24.2,编码钙粘着蛋白-13(也称为T钙粘蛋白或H钙粘蛋白),在心血管系统中广泛表达。钙粘着蛋白-13在血管生成、保护血管内皮细胞免受氧化应激损伤等方面发挥着重要作用。ATP2B1基因位于12q21.3,编码质膜钙ATP酶异构体1(plasma membrane Ca2+ATPase1,PMCA1),属于P-型离子转运ATP酶家族中的一员。PMCA1在维持细胞内钙离子平衡方面发挥着重要作用。UMOD基因位于16p12.3,编码尿调节蛋白,正常人尿液中含有丰富的尿调蛋白,24小时尿排量约为20-70mg。 研究目的：通过病例一对照关联分析的研究方法,探讨慢性高血压易感基因多态性位点(CDH13基因SNP位点rs11646213;ATP2B1基因SNP位点rs2681472；UMOD基因SNP位点rs13333226)与子痫前期发病的关系。 研究方法：(1)选取在山东大学附属省立医院住院分娩的914名孕妇,其中正常孕妇460名,子痫前期患者454名。采用基因测序的方法,对CDH13基因SNP位点rs11646213进行基因分型。 (2)采用taqman-MGB探针的方法对ATP2B1基因SNP位点rs2681472进行基因分型研究。实验分为两个阶段进行：第一阶段为2009年12月至2011年11月,收集在山东大学附属省立医院住院分娩的1218名孕妇的外周血,其中正常孕妇503名,子痫前期患者715名。第二阶段的研究对象为2011年12月至2013年3月,在山东大学附属省立医院住院分娩的344名孕妇,其中早发型子痫前期166例,正常孕妇178例。同时采用随机抽样的方法,抽取5%的标本进行直接测序,以验证taqman-MGB探针的准确性。 (3)选取在山东大学附属省立医院住院分娩的1418名孕妇,其中正常孕妇716例,子痫前期患者702例。采用taqman-MGB探针的方法对UMOD基因SNP位点rs13333226进行基因分型研究。同时采用随机抽样的方法,抽取5%的标本进行直接测序,以验证taqman-MGB探针的准确性。 统计学方法：计量资料采用均数±标准差的方式表示,计数资料以百分率的形式表示。采用Haploview软件检测连锁不平衡(Broad Institute,Cambridge, Massachusetts,USA).等位基因频率的比较采用卡方检验进行计算。非条件logistic回归分析校正孕妇年龄、产次、孕前体重指数等危险因素。数据分析采用SPSS软件进行分析(version17.O；SPSS Inc.,Chicago,IL,USA).检验水平取P0.05。 研究结果：(1)CDH13基因SNP位点rs11646213的最小等位基因频率(minor allele frequency,MAF)在正常孕妇和子痫前期患者之间,存在统计学差异(P=0.017；OR:1.36；95%CI:1.06-1.76).按照疾病的严重程度,将子痫前期患者分为两组：轻度子痫前期、重度子痫前期后发现,重度子痫前期患者与正常孕妇之间的MAF存在统计学差异(P=0.002；OR:1.57；95%CI:1.16-2.13)；轻度子痫前期与正常孕妇之间的MAF不存在统计学差异(P=0.91；OR:1.02,95%CI:0.70-1.50)。按照疾病的发病时间,将子痫前期分为早发型子痫前期、晚发型子痫前期后发现：早发型子痫前期与正常孕妇之间的MAF存在统计学差异(P=0.004； OR:1.54；95%CI:1.17-2.03)；晚发型子痫前期与正常孕妇之间的MAF不存在统计学差异(P=O.27；OR:1.19,95%CI:0.87-1.62). (2)ATP2B1基因SNP位点rs2681472的基因分型在第一阶段的研究结果为：MAF在正常孕妇和子痫前期患者之间不存在统计学差异(P=0.23；OR：1.11,95%CI：0.94-1.31)。按照发病时间将子痫前期患者分为早发型子痫前期、晚发型子痫前期后发现,正常孕妇和早发型子痫前期患者之间的MAF存在统计学差异(P=0.02； OR:1.29,95%CI:1.04-1.59).在第二阶段的验证实验中也发现,ATP2B1基因SNP位点rs2681472的MAF在正常孕妇和早发型子痫前期患者之间存在统计学差异(P=0.011:OR:1.50,95%CI:1.10-2.06). (3)UMOD基因SNP位点rs13333226的MAF在正常孕妇和子痫前期患者之间不存在统计学差异(P=0.45; OR:1.13,95%CI:0.83-1.53),按照发病时间以及疾病的严重程度进行亚组分析,正常孕妇与疾病组之间的MAF不存在统计学差异(P0.05)。 研究结论：在中国汉族妇女中,CDH13基因的SNP位点rs11646213多态性与子痫前期的发病之间存在相关性。ATP2B1基因的SNP位点rs2681472多态性与早发型子痫前期的发病之间存在相关性。 第三部分CDH13基因在胎盘组织中的表达及甲基化水平的初步研究 研究背景：子痫前期是一种严重影响孕产妇健康的疾病,具体发病机制不明,早孕期胎盘绒毛滋养细胞侵袭力下降,子宫螺旋动脉重铸障碍、胎盘灌注不足可能在其发病过程中发挥了关键作用。近年来,表观遗传学在子痫前期发病中的作用受到越来越多的关注,特别是DNA甲基化水平的研究。我们在前期的关联性分析研究中发现,CDH13基因为子痫前期的遗传易感基因。CDH13基因在体内广泛表达,与肿瘤细胞的侵袭、血管生成等密切相关。已有研究报道,在许多肿瘤组织中CDH13基因启动子区域的甲基化水平存在异常改变,并对其表达产生显著影响。但是,CDH13基因是否在胎盘组织中表达,在正常孕妇与子痫前期患者的胎盘组织中CDH13基因的表达水平以及启动子区域的甲基化水平是否存在差异,尚不清楚。 研究目的：为进一步明确CDH13基因在子痫前期发病中的作用,对CDH13基因在胎盘组织中的表达水平、甲基化水平进行研究。比较分析子痫前期与正常孕妇的胎盘组织中,CDH13基因启动子区域的甲基化水平以及mRNA的表达水平是否存在差异。 研究方法：选择留取年龄、孕次、孕前体重指数相匹配的20例正常孕妇,20例子痫前期患者的胎盘组织：(1)将胎盘组织在4%的甲醛中侵泡24小时,石蜡包埋,进行冰冻切片,免疫组化的方法分析CDH13基因在胎盘组织中的表达情况。(2)采用[rizol法,从30g胎盘组织中提取RNA,采用荧光定量PCR的方法分析CDH13基因的mRNA在正常孕妇与子痫前期患者的胎盘组织中的表达水平。(3)提取胎盘组织的DNA,通过重亚硫酸盐测序的方法,分析CDH13基因启动子区域的甲基化水平在正常孕妇与子痫前期患者之间是否存在差异。 研究结果：(1)CDHl3基因在胎盘血管内皮细胞上表达丰富,也在合体滋养细胞上表达。(2)与正常孕妇相比,子痫前期患者组CDH13基因的mRNA表达水平较高,但两组之间的差异无统计学意义(P0.05)。(3)CDH13基因在胎盘组织中呈现明显的去甲基化状态,子痫前期组的甲基化率高于正常孕妇,但差异无统计学意义(P0.05)。 研究结论：CDH13基因在胎盘组织中表达；正常孕妇与子痫前期患者之间的胎盘组织中CDH13基因的表达水平不存在统计学差异；CDH13基因在正常孕妇与子痫前期患者的胎盘中呈现去甲基化状态。
[Abstract]:A retrospective study on the outcome of pre - eclampsia and pregnancy outcome in patients with chronic hypertension

Background : Preeclampsia is a common pregnancy - specific disease which affects mother - to - child health seriously . In the early stage of eclampsia , especially in the early stage of severe pre - eclampsia , there is a significant increase in the risk of placental abruption , coagulation dysfunction , liver and kidney function damage , etc . In the cause of fetal growth restriction , the pre - eclampsia accounts for about 10 % ;
There is a significant increase in the number of patients with chronic hypertension after pregnancy . The incidence of chronic high blood pressure is about 1 % -5 % . In the period of childbearing age , the incidence of chronic hypertension increases . However , the study shows that the pregnancy outcome of patients with chronic hypertension , especially in the early 1990s , can guide the pregnancy outcome of patients with chronic hypertension and to guide the pregnancy health of patients with chronic hypertension , and to prevent the occurrence of adverse pregnancy outcomes .

Objective : To retrospectively analyze the outcome of adverse pregnancy caused by hypertensive disorder complicating pregnancy , especially in the early stage of chronic hypertension complicated with eclampsia , whether or not to increase the outcome of adverse pregnancy .

Methods : We analyzed retrospectively the following clinical data from January 2011 to March 2012 , including the age of pregnant women , the history of pregnancy , the history of previous diseases ( including diabetes , hypertension , etc . ) , the gestational weeks of pregnancy , the mode of delivery , the sex of the fetus , and the limitation of fetal growth , stillbirth , placental abruption , premature rupture of fetal membranes , etc .

Results : A total of 3691 pregnant women were included in the study . There were 2993 normal pregnant women , 461 cases of pre - eclampsia , 91 cases with chronic hypertension complicated with chronic hypertension , 146 cases of chronic hypertension complicated with pre - eclampsia ( OR ; 21.28 : 95 % CI : 1.89 - 2.89 ) , premature delivery ( OR ; 21.28 : 95 % CI : 14.79 - 30.60 ) , cesarean section ( OR ; 7.33 : 95 % CI : 4.55 - 11.82 ) , placental abruption ( OR ; 10.38 : The risk of cesarean section ( OR ; 2.76 : 95 % CI : 1.75 - 4.35 ) , placental abruption ( OR ; 16.79 ; 95 % CI : 3.04 - 92.88 ) increased significantly in pregnant women with chronic hypertension .

Conclusion : Hypertensive disorder complicating pregnancy can increase the outcome of adverse pregnancy . Compared with the pre - eclampsia , there is no pregnancy outcome in patients with chronic hypertension complicated with pre - eclampsia , such as stillbirth , premature delivery and cesarean section .

Study on the relationship between polymorphism of the second part of chronic high blood pressure susceptibility gene ( CD13 , ATP2B1 , UMOD ) and pre - eclampsia

Background : Preeclampsia is a kind of pregnancy - specific disease characterized by high blood pressure and proteinuria . It has obvious family aggregation tendency . The risk of chronic high blood pressure in patients with chronic hypertension is obviously higher than that of normal pregnant women . Genome wide association study ( GWAS ) is an effective way to study the susceptibility genes of complex diseases . The GWAS study of chronic high blood pressure has been reported that the gene of CD13 , ATP2B1 , UMOD is the susceptible gene of chronic high blood pressure , and has been confirmed in many subsequent studies .

Calcium binding protein - 13 plays an important role in maintaining calcium ion balance in cells . The gene of ATP2B1 is located at 12q21.3 and encodes the plasma membrane Ca2 + ATPase1 , PMCA1 , which is a member of P - type ion transport ATP enzyme family .

Objective : To investigate the SNP loci rs11646213 and rs2681472 of the SNP site of chronic high blood pressure susceptible gene ( SNP ) by a case - control correlation analysis .
The relationship between SNP locus rs13333226 of UMOD gene and pre - eclampsia .

Methods : ( 1 ) 914 pregnant women who had been hospitalized in the Affiliated Hospital of Shandong University were selected , including 460 normal pregnant women and 454 patients with pre - eclampsia . Using gene sequencing , rs11646213 was genotyped for SNP locus rs11646213 .

( 2 ) The SNP locus rs2681472 of ATP2B1 gene was genotyped by taqman - MGB probe . The experiment was divided into two stages : Phase I : From December 2009 to November 2011 , we collected the peripheral blood of 1218 pregnant women who had been hospitalized in the affiliated provincial hospital of Shandong University . The second phase consisted of 166 normal pregnant women and 178 normal pregnant women . At the same time , a random sampling method was used to extract 5 % of specimens for direct sequencing to verify the accuracy of taqman - MGB probe .

( 3 ) The gene typing of the SNP locus rs13333226 of UMOD gene was studied by using taqman - MGB probe , and 5 % of specimens were directly sequenced to verify the accuracy of taqman - MGB probe .

Statistical method : The measurement data is expressed by mean 卤 standard deviation , and the count data is expressed as percentage . The chain imbalance is detected using Haploview software ( Broad Institute , Cambridge , Massachusetts , USA ) . The frequency of allele was calculated by means of Chi - square test . Non - conditional logistic regression analysis corrected the risk factors , such as age , birth rate and body mass index of pregnant women . The data analysis was carried out with SPSS software ( version 17.O ;
SPSS Inc.,Chicago,IL,USA). The level of test was P 0 . 05 .

Results : ( 1 ) The minimum allele frequency ( MAF ) of the SNP locus rs11646213 of CD13 gene was significantly different between normal pregnant women and pre - eclampsia ( P = 0 . 017 ) .
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