KR-102963313-B1 - Analytical method for diagnosing preeclampsia using methylation level of the CpG site cg20772657

Abstract

The present invention provides an analysis method comprising the step of measuring the methylation level of a specific CpG site in a sample of a subject in order to provide information necessary for the diagnosis of preeclampsia.

Inventors

• 류현미
• 임지혜

Assignees

• 차의과학대학교 산학협력단
• 의료법인 성광의료재단

Dates

Publication Date

20260511

Application Date

20240429

Claims (1)

1. An analytical method comprising the step of measuring, by methylation-specific quantitative real-time polymerase chain reaction, whether the CpG site of cg20772657 in a maternal placental sample separated in vitro is hypermethylated, in order to provide information necessary for the diagnosis of preeclampsia.

Description

Analytical method for diagnosing preeclampsia using methylation level of the CpG site cg20772657 The present invention relates to an analysis method for providing information necessary for the diagnosis of preeclampsia using the methylation level of a CpG site. Preeclampsia (PE) is a gestational hypertensive disorder affecting 2–8% of all pregnancies worldwide and is one of the major causes of maternal mortality and morbidity. Preeclampsia is a multisystem disorder characterized by newly onset hypertension and proteinuria in the mother after 20 weeks of gestation. In the absence of proteinuria, findings of newly onset hypertension accompanied by maternal organ dysfunction—including thrombocytopenia, renal failure, hepatic impairment, and pulmonary edema—are sufficient for diagnosis. Consequently, clinical phenotypes vary according to the signs of the syndrome, ranging from elevated blood pressure to more severe complications, including renal and hepatic dysfunction and seizures. Therefore, it is now recommended to classify PE based on the presence or absence of severe maternal and fetal characteristics, rather than subdividing it according to symptom severity, which is generally known as mild, moderate, or severe. The etiology and pathophysiology of PE largely remain a mystery. However, genetic, immunological, endocrine, and environmental factors are all involved (Espinoza J. Abnormal fetal-maternal interactions: an evolutionary value, Obstet Gynecol. 2012;120:370-374). Numerous studies have reported changes in gene expression in various mechanisms associated with PE, including trophoblast motility and invasion, angiogenesis, cell adhesion, and immune responses. Epigenetic events play a significant role in these changes in gene expression. This indicates the contribution of epigenetic modifications to the various symptoms and development of PE. Epigenetic modifications regulate gene expression without altering DNA sequences. DNA methylation is the most common epigenetic mechanism, occurring primarily at CpG sites and is important for optimal placental and fetal development. Several studies have investigated changes in overall DNA methylation in the placenta caused by PE, and these studies show that various DNA regions of the epigenome are hypermethylated and/or hypomethylated in PE placentas compared to normal placentas (Blair JD, et al., Mol Hum Reprod. 2013;19:697-708; Ching T, et al., Mol Hum Reprod. 2014;20:885-904; Wang T, et al. Epigenomics. 2019;11:1003-1019; Gao WL, et al. Hypertens Res. 2011;34:655-661; Kulkarni A, et al., DNA Cell Biol. 2011;30:79-84; Leavey K, et al., Clin Epigenetics. 2018;10:28). However, there are few studies on DNA methylation according to severe features of PE. Therefore, comparative DNA methylation profiling analysis in PE placentas according to severe features can improve the understanding of the pathophysiology of this disease. Figure 1 shows the hierarchical clustering of differentially methylated CpG sites (DMCs) in PE. Methylation degree values in the 850K array were evaluated using independent t-tests ( P < 0.05) and the fold-change criterion (|delta mean of methylation degree| ≥ 0.2). To control for false positive results from multiple tests, P values were corrected using the Benjamini and Hochberg false discovery rate method. Methylation degree values for DMCs were subjected to hierarchical clustering. DMCs are plotted on the x-axis and biological samples on the y-axis; strong methylation is indicated in yellow, while weak or absent methylation is indicated in blue. a Control vs. PE. b Control vs. severe PE. Con: Control. PE: Preeclampsia, PES: Preeclampsia with severe features. Figure 2 is a Venn diagram showing the overlap of DMCs with significant changes. NT: Normal pregnant woman, PT: Pregnant woman with preeclampsia, PTS: Pregnant woman with severe preeclampsia PE is an obstetric disease characterized by severe morbidity in both mother and fetus due to placental trophoblast invasion failure. However, its pathophysiology remains unclear. The inventors performed DNA methylation profiling to identify different patterns of DNA methylation in PE placentas regardless of PE severity. DNA was extracted from placental tissues of 13 normal patients, 5 patients with PE, and 8 pregnant women with severe PE. Genome-wide DNA methylation analysis was performed using the Illumina HumanMethylation 850K BeadChip. The β values obtained through the Illumina HumanMethylation 850K BeadChip are acquired through the calibration and quantification of the Illumina array using the intensity ratio between methylated probes and unmethylated probes. That is, the β value is calculated from the ratio of the methylated signal intensity to the sum of the methylated and unmethylated signals after subtracting the background using a negative control on the array, and the β value of 0 to 1 at each CpG site is associated with the methylation percentage from 0 to 100%. Therefore, among placental samp