1) ヒト初期絨毛遺伝子の変化に着目した妊娠高血圧症候群発症の予知と予防(シンポジウム2:周産期「妊娠高血圧症候群の基礎と臨床-予防・治療の新戦略に向けて」,第65回日本産科婦人科学会・学術講演会)

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During the pathogenesis of preeclampsia (PE), inadequate trophoblast invasion of the maternal spiral arteries during early gestation due to immunological, genetic and environmental factors causes an insufficiency of maternal blood inflow into the intervillous space, leading to placental ischemia, as well as trophoblast hypoxia. This causes oxidative stress and aberrant quantitative expression of several genes in the trophoblast, then an imbalance in the concentrations of angiogenesis-related factors, such as vascular endothelial growth factor (VEGFA), placental growth factor (PGF), fms-like tyrosine kinase-1 (FLT-1) and endoglin (ENG) occurs in the maternal circulation. Although it is known that PE is associated with maternal endothelial dysfunction due this imbalance in the concentrations of angiogenesis-related factors, the optimal treatment and prevention of PE are still unknown. We thought that knowledge about the genetic alterations of trophoblast in early gestation would be helpful to develop new treatments and preventive measures for PE. Therefore, we investigated the gene expression and DNA methylation patterns of trophoblasts from patients with PE, especially focusing on early gestation. We quantified the mRNA expression of genes encoding angiogenesis-related factors (ENG, FLT-1, VEGFA, PGF and TGF-β1) and oxidative stress-related factors (heme oxygenase-1(HO-1), superoxide dismutase (SOD)) of trophoblasts at 11 weeks' gestation, and compared the findings between pregnant women who had later developed PE (n=5) and those who had normal pregnancies (n=25). In pregnant women who had later developed PE, ENG, FLT-1, VEGF and TGF-β1 were significantly upregulated, and PGF, HO-1 and SOD were down-regulated compared with the control. These results showed that the alterations in the expression of genes encoding angiogenesis-related factors and oxidative stress-related factors are already present at 11 weeks' gestation in pregnant women who later developed PE. Therefore, we decided to determine whether we could predict the later development of PE using these differences in expression. We obtained blood samples from asymptomatic pregnant women (n=660) at 15-20 weeks' gestation. Among them, 62 later developed PE. We quantified the mRNA expression of cellular components in the maternal blood in the samples from the PE subjects and compared them with 310 control subjects. As a result, we found that FLT-1 and ENG were significantly up-regulated and PGF and HO-1 were significantly down-regulated in the women who ultimately experienced PE, compared with the normal pregnant women. The detection rate of the combined marker panel (mRNA of ENG, FLT-1, PGF and parity) with a 10% false positive rate was 66%. We also compared blood collected at 10-14 weeks' gestation from 11 women who ultimately experienced PE with blood from 88 control subjects. The expression levels of FLT-1, ENG, and TGF-β1 were significantly up-regulated in women who ultimately experienced PE, compared with the controls. The detection rate of the combined marker panel (ENG, FLT1 and TGF-β1 mRNA level and parity) with a 5% false positive rate was 72.3%. We therefore demonstrated that the mRNA levels in the cellular components of blood from first trimester pregnant women could be used to predict the future development of PE. Among the genes that were analyzed, FLT-1 and ENG were the best markers to predict the subsequent onset of PE. Next, we analyzed the alterations of mRNA expressions in placental villi associated with the pathogenesis of PE using microarray technology. The gene expression profiles at 11 weeks' gestation were compared between five women who ultimately experienced PE with five control subjects. A total of 118 genes were detected as genes that were significantly differentially expressed. Of them, genes associated with immune tolerance, the invasion of human trophoblasts, inflammatory stress and coagulation factors were included. These findings suggested that these factors were involved in the pathogenesis of PE. Finally, we investigated the villous DNA methylation in PE compared with normal cases in order to understand the epigenetic pathogenesis of PE that might occur before 11 weeks' gestation. Villi were obtained from artificially aborted women after fetal heart movement was detected by ultrasonography and from pregnant women who delivered by cesarean section. Samples of six weeks, 10-11 weeks, normal third trimester and PE villi were examined in this study. We extracted DNA samples from the villi using the QIAamp^[○!R] DNA Mini Kit and generated DNA methylation profiles using the Illumina Human Methylation 450 Bead Chips^[○!R]. The data were compared among the groups. Before comparison, differences among individuals were minimized using the criteria of IQR75. In consequence, 473,864CpG sites were obtained as a dataset. Of these, 15,153CpG sites were differentially methylated between six weeks and 10-11 weeks. A total of 29,422 CpG sites were differentially methylated between 10-11 weeks and normal third trimester villi, and almost all of these methylation sites different from those extracted as different between six weeks and 10-11 weeks. These results showed that normal placentation and placental development require changes of the DNA methylation status in some specific genes, and that the responsible genes differ with the stage of pregnancy. A total of 206CpG sites on CpG islands were detected as the candidate CpG sites that were differentially methylated in PE cases compared with cases in the normal third trimester. The cluster analysis for all samples was done using the results of these 206CpG sites. In consequence, not only normal trimester and PE villi, but also the six weeks and 10-11 weeks samples were accurately classified. These findings suggest that the alterations of villous DNA methylation occurring between six and 11 weeks of gestation are associated with the pathogenesis of PE. Therefore, we focused on the CpG sites that were differentially methylated between both PE and normal third trimester villi, and between the villi at six weeks and 10-11 weeks gestation. Twentynine genes were detected that were associated with the CpG site set. Of these, six genes (AKT3, HOXC4, MAD1L1, NCOR2, NSD1 and ZFP36L2) were related to growth factors and embryonic development. We therefore investigated the expression of these six genes in the villi. For all six of the genes, the expression was the lowest at six weeks, and increased with gestational age, and was lower in PE compared with normal third trimester villi. In particular, HOXC4 and MAD1L1 were downregulated in PE, to a level that was similar to that detected at six weeks. These trends in gene expressions matched the DNA methylation. These findings suggested that the expression of these genes was controlled by the villous DNA methylation. In conclusion, our data indicated that genetic alterations of placental villi in early pregnancy are related to the pathogenesis of PE. Our findings also suggested that the alterations of villous DNA methylation occurring between six and 11 weeks of gestation are associated with the pathogenesis of PE. It was shown that calculation of the mRNA levels of cellular components in the maternal circulation was useful for predicting PE.
2013-11-01

1) ヒト初期絨毛遺伝子の変化に着目した妊娠高血圧症候群発症の予知と予防(シンポジウム2:周産期「妊娠高血圧症候群の基礎と臨床-予防・治療の新戦略に向けて」,第65回日本産科婦人科学会・学術講演会)

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