The Association Study Of Hypertension Susceptibility Gene (CDH13,ATP2B1, UMOD) In The Pathogenesis Of Preeclampsia

PART I Perinatal and maternal outcome in women with preeclampsia, chronic hypertension and superimposed preeclampsiaBackground:Preeclampsia is still a leading cause of maternal and paternal morbidity and mortality. Pregnancies in women with preeclampsia are at an increased risk of adverse maternal and paternal outcomes that included placenta abruption, Disseminated Intravascular Coagulation, live or renal damage. Preeclampsia accounted for about10%of intrauterine fetal growth restrict. And about25%fetal death are associated with preeclampsia. Women with preeclampsia are also at an increased risk to develop chronic hypertension in the late life. The rates of hypertension are expected to rise because of obesity and the age of childbearing continue to increase. Several studies have demonstrated a higher risk of adverse outcome among women with hypertension after pregnancy, which include preeclampsia, placenta abruption, preterm delivery, and intrauterine fetal growth restrict. Interestingly, most of these studies were performed in the1990s. In order to assist physician in the counseling and treatment of women with hypertension disorder in pregnancy, we retrospectively evaluated perinatal and maternal outcomes in women with hypertension, preeclampsia and superimposed preeclampsia.OBJECTIVE:To evaluate pregnancy outcomes in women with hypertension disorders. To determine whether superimposed preeclampsia results in worse perinatal outcomes compares to preeclampsia. METHODS:A retrospective study was performed in Shandong provincial hospital. Maternal and perinatal outcomes among women with preeclampsia (n=461), chronic hypertension (n=91), superimposed preeclampsia (n=146) were compared with outcomes of control subjects (n=2993). Pregnancy and delivery information for all patients were obtained from medical records. Maternal and perinatal outcomes that were examined included delivery week, cesarean section, fetal sex, stillbirth, placenta abruption, intrauterine fetal growth restrict and premature membranes rupture.RESULTS:A total of3691participants were included into this study. Women with hypertension disorder differed from control subjects in age, parity, delivery week, diabetes mellitus and premature membranes rupture. Women with preeclampsia were significantly more likely to have intrauterine fetal growth restrict (OR:4.88;95%CI:2.76-8.63), stillbirth (OR;11.84;95%CI:6.75-20.75), preterm delivery (OR;6.69;95%CI:5.30-8.46), cesarean section (OR;2.32;95%CI:1.89-2.89), and placenta abruption (OR;21.7;95%CI:7.04-66.79). Compared to controls, women with superimposed preeclampsia had a higher risk to have stillbirth (OR;30.71;95%CI:16.59-56.83), preterm delivery (OR;21.28;95%CI:14.79-30.60), cesarean section (OR;7.33;95%CI:4.55-11.82), and placenta abruption (OR;10.38;95%CI:1.88-57.13). The risks for cesarean section (OR;2.76;95%CI:1.75-4.35), and placenta abruption (OR;16.79;95%CI:3.04-92.88) were significantly different between women with chronic hypertension and controls. Compared to women with preeclampsia, superimposed preeclampsia were at significantly higher risk for stillbirth (OR;2.59;95%CI:1.49-4.52), preterm delivery (OR;3.18;95%CI:2.16-4.68), and cesarean section (OR;3.16;95%CI:1.89-5.26).CONCLUSION; Women with hypertension disorders are at a significantly increased risk of several adverse perinatal outcomes. The risk to develop stillbirth, preterm delivery and cesarean section are higher for women with superimposed preeclampsia, compared with women with preeclampsia. PART Ⅱ The association between hypertension susceptibility gene (CDH13, ATP2B1, UMOD) and preeclampsiaBACKGROUNDS:Preeclampsia is a pregnancy induced hypertension, occurring in3-5%of all pregnancies. Data showed that daughters and sisters of preeclampsia women were prone to develop preeclampsia, which indicated that genetic factors play a significant role in its pathogenesis. Genome-wide association study (GWAS) is an effective way to explore the genetic pathogenesis of complex disease. The GWAS of chronic hypertension identified CDH13gene, ATP2B1gene, UMOD gene as hypertension susceptibility genes. CDH13, located on chromosome16q24, encodes cadherin-13and is highly expressed in the cardiovascular system. Cadherin-13plays an important role in many biological processes including vascular wall remodeling, modulating angiogenesis and protecting endothelial cells from oxidative stress-induced apoptosis, most of which are involved in the pathogenesis of preeclampsia. The ATP2B1gene, located at12q21.3, encodes a plasma membrane Ca2+pump (PMCA1) belonging to the family of P-type primary ion transport ATPases. This pump plays a crucial role in the fine regulation of intracellular free calcium concentration. UMOD, locating at16p12.3, encodes uromodulin/Tamm-Horsfall protein, which is the most abundant tubular protein in human urine.OBJECTIVE:To explore the association between hypertension genetic risk factors (the CDH13rs11646213polymorphism; the ATP2B1rs2681472polymorphism; the UMOD rs13333226polymorphism) and preeclampsia.METHODS:(1) A total of454cases and460controls were recruited from Provincial Hospital Affiliated to Shandong University in this study. The SNP rs11646213was genotyped by polymerase chain reaction (PCR) and direct sequencing.(2) We used a TaqMan-MGB probe assay to genotype the ATP2B1rs2681472polymorphism. A two-stage study was conducted to identify the association between rs2681472and preeclampsia in a Northern Han Chinese population. The first stage consisted of1,218unrelated pregnant women (503controls and715cases) who had been recruited from Provincial Hospital Affiliated to Shandong University from December2009to November2011. The second stage of the replication study included166early-onset preeclampsia cases and178controls recruited from January2011to March2013. Randomly selected samples (5%) were further genotyped by direct sequencing to validate the genotyping assays.(3) A total of1418unrelated Chinese Han women were recruited into this study:716controls,702women with preeclampsia. Genotyping for the UMOD rs13333226polymorphism was performed using a TaqMan-MGB probe assay. Randomly selected samples (5%) were further genotyped by direct sequencing to validate the genotyping assays.STATISTIC ANALYSIS:Basic characteristics of cases and controls were expressed as means±SD. Categorical data were expressed as percentage. Linkage disequilibrium (LD) was assessed using the Haploview software (Broad Institute, Cambridge, Massachusetts, USA). Chi-square test was performed to compare allele frequency. Unconditional logistic regression analysis was used for parity, pre-BMI and maternal age adjustment by SPSS statistical software system (version17.0; SPSS Inc., Chicago, IL, USA). P<0.05was regarded as statistically significant.RESULTS:(1) Minor allele frequency (MAF) of rs11646213was significantly different between cases and controls group (P=0.017, OR=1.36,95%CI=1.06-1.76). Comparing separately women with mild and severe preeclampsia to controls, the association with rs11646213was found in severe preeclampsia (P=0.002, OR=1.54,95%CI=1.17-2.03), but not in mild preeclampsia (P=0.91, OR=1.02,95%CI=0.70-1.50). The MAF was also significant different between early onset preeclampsia and controls (P=0.004, OR=1.57,95%CI=1.16-2.13), but no difference was found between late onset preeclampsia and controls (P=0.27, OR=1.19,95%CI=0.87-1.62).(2) In the first stage study, we observed no difference in the allele frequencies between preeclamptic women and controls (P=0.23; OR:1.11;95%CI:0.94-1.31). When early-and late-onset preeclampsia subgroups were considered, the allele frequencies were significantly different between early-onset preeclampsia and controls (P=0.02; OR=1.29;95%CI=1.04-1.59). A significant difference was also detected in the allele frequencies between early-onset preeclampsia and controls in the replication study (P=0.011; OR:1.50;95%CI:1.10-2.06)。(3) The MAF of the UMOD rsl3333226polymorphism were not significantly different between cases and the controls (P=0.45; OR:1.13;95%CI:0.83-1.53) When the patients were divided into subgroups, MAF were also not different between cases and controls.CONCLUSION:Rs11646213upstream of the CDH13is associated with preeclampsia in Han Chinese women. Rs2681472locating in the ATP2B1gene is associated with early-onset preeclampsia in Northern Han Chinese women. PART III Methylation and expression of CDH13in the placentaBACKGROUNDS:Preeclampsia, characterized by hypertension and proteinuia, is still a leading cause of maternal morbidity and mortality. Preeclampsia/eclampsia is also associated with substantial adverse perinatal outcomes, such as preterm delivery, intrauterine growth restriction and stillbirth. The exact pathogenesis of preeclampsia is still unknown. Superficial trophoblast invasion, which leads to inappropriate maternal spiral artery remodeling and then insufficient uteroplacental blood flow, may play a crucial role in the development of preeclampsia. Epigenetics is the study of heritable changes in gene activity that are not caused by changes in the DNA sequence. We identified CDH13as a preeclampsia susceptibility gene in northern Han Chinese women. CDH13gene encodes T-cadherin (also called Cadherin-13or H-cadherin in humans) and is expressed in diverse organs and cell types. The role for T-cadherin in tumor invasion and angiogenesis had been proved in several studies.OBJECTIVE:To further clarify the role of CDH13in the pathogenesis of preeclampsia. The expression and methylation level of CDH13in placenta was compared between controls and preeclampsia.METHODS:Placental tissue samples from twenty women with preeclampsia and twenty controls were collected, matched for maternal age, parity, and prepregnancy-BMI.(1) Placental biopsies (2-3per placenta) were fixed in4%buffered formaldehyde for24h and stored in0.9%saline before embedding in paraffin. Sections were cut at5mm. T-cadherin immunohistochemistry was performed using the GTVision III detection system/Mo&Rb following the manufacturer’s protocol.(2) Total RNA from30mg placental tissue was extracted using Trizol reagent. Real-time PCR analysis was performed to analysis the mRNA level of CDH13in placental tissue.(3) Genomic DNA was obtained from the placental tissue and bisulfite sequencing was performed to examine the methylation levels of CDH13.RESULTS:(1) CDH13was expressed in the vascular endothelia cells and trophoblast cells in the placenta.(2) There was no difference in the mRNA levels between preeclampsia and controls.(3) The target sequences of CDH13has been the demethylation in the placenta. No significant difference was found about the total methylation level between preeclampsia and controls (P<0.05)CONCLUSION:CDH13is expressed in the placenta. CDH13is mostly demethylation in the placenta.