Ji Hyae Lim

Neuroprotective effect of topiramate on hypoxic ischemic brain injury in neonatal rats.

Perinatal hypoxia-ischemia is one of the most common risk factors for neonatal mortality and permanent neurodevelopmental disability. Topiramate [2,3:4,5-bis-o-(1-methylethylidene) beta-D-fructo-pyranose sulfamate; TPM] is widely used as an antiepileptic agent with multiple targets. In the present study, we found that treatment with TPM reduced the neuronal damage induced by oxygen-glucose deprivation in vitro with strong inhibition of the alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) receptor. Because perinatal hypoxia is mediated, at least in part, by aberrant glutamatergic excitation, we tested whether treatment with TPM was effective against perinatal brain hypoxia-ischemia. Intraperitoneal or oral pretreatment with TPM was found to reduce the brain damage and subsequent cognitive impairments induced by transient hypoxia-ischemia in perinatal rats. A potent neuroprotective effect of TPM was also observed in a post-treatment regime although post-treatment window appears to be relatively narrow (<2 h). These results suggest that TPM treatment may be beneficial for perinatal hypoxia-ischemia and related damage.

Disease specific characteristics of fetal epigenetic markers for non-invasive prenatal testing of trisomy 21.

BackgroundNon-invasive prenatal testing of trisomy 21 (T21) is being actively investigated using fetal-specific epigenetic markers (EPs) that are present in maternal plasma. Recently, 12 EPs on chromosome 21 were identified based on tissue-specific epigenetic characteristics between placenta and blood, and demonstrated excellent clinical performance in the non-invasive detection of fetal T21. However, the disease-specific epigenetic characteristics of the EPs have not been established. Therefore, we validated the disease-specific epigenetic characteristics of these EPs for use in non-invasive detection of fetal T21.MethodsWe performed a high-resolution tiling array analysis of human chromosome 21 using a methyl-CpG binding domain-based protein (MBD) method with whole blood samples from non-pregnant normal women, whole blood samples from pregnant normal women, placenta samples of normal fetuses, and placenta samples of T21 fetuses. Tiling array results were validated by bisulfite direct sequencing and qPCR.ResultsAmong 12 EPs, only four EPs were confirmed to be hypermethylated in normal placenta and hypomethylated in blood. One of these four showed a severe discrepancy in the methylation patterns of T21 placenta samples, and another was located within a region of copy number variations. Thus, two EPs were confirmed to be potential fetal-specific markers based on their disease-specific epigenetic characteristics. The array results of these EPs were consisted with the results obtained by bisulfite direct sequencing and qPCR. Moreover, the two EPs were detected in maternal plasma.ConclusionsWe validated that two EPs have the potential to be fetal-specific EPs which is consistent with their disease-specific epigenetic characteristics. The findings of this study suggest that disease-specific epigenetic characteristics should be considered in the development of fetal-specific EPs for non-invasive prenatal testing of T21.

Genome-wide microRNA expression profiling in placentas of fetuses with Down syndrome

INTRODUCTION Down syndrome (DS) is the most common aneuploidy, caused by an extra copy of all or part of chromosome 21 (chr21). Differential microRNA (miRNA) expression is involved in many human diseases including DS. However, the genome-wide changes in miRNA expression in DS fetal placentas have yet to be determined, and the function of these changes is also unclear. METHODS We profiled genome-wide miRNA expression in placenta samples from euploid or DS fetuses by using microarray technology and predicted the functions of differentially expressed miRNAs using bioinformatics tools. RESULTS Thirty-four miRNAs were significantly differentially expressed in the DS placenta compared with the normal placenta (16 up-regulated and 18 down-regulated). However, expression of chr21-derived miRNAs did not change. Predicted target genes included 7434 genes targeted by up-regulated miRNAs and 6071 genes targeted by down-regulated miRNAs. Seventy-six of these target genes were located on chr21 (10 genes controlled by down-regulated miRNAs and 34 genes by up-regulated miRNAs, and 32 genes by both). Target genes on chr21 were significantly associated with DS and DS-related disorders, such as mental retardation, neurobehavioral manifestations, and congenital abnormalities. DISCUSSION To our knowledge, this is the first genome-wide study to comprehensively survey placental miRNAs in DS fetuses. Our results provide new insight into miRNA expression in placentas of fetuses with DS. Additionally, our findings indicate that the differentially expressed miRNAs in the DS placenta may potentially affect various pathways related to DS pathogenesis.

Non-invasive detection of fetal trisomy 21 using fetal epigenetic biomarkers with a high CpG density.

Abstract Background: Non-invasive prenatal test of trisomy 21 (T21) is being researched using fetal specific epigenetic biomarkers present in maternal plasma. We applied a methyl-CpG binding domain-based protein (MBD) method based on epigenetic characteristics of fetal specific-methylated regions with a high CpG density in HLCS on chromosome 21 and RASSF1A on chromosome 3 for the non-invasive detection of fetal T21 and estimated the diagnostic accuracy of the method. Methods: A nested case-control study was conducted with maternal plasma collected from 50 pregnant women carrying 40 normal and 10 T21 fetuses. A MBD method was used for enrichment of methylated DNA regions in maternal plasma. The levels of methylated HLCS (M-HLCS) and methylated RASSF1A (M-RASSF1A) were simultaneously measured by multiplex qPCR. Results: Levels of M-HLCS and M-RASSF1A were obtained in all cases. Levels were not different according to fetal gender (p>0.05 in both). The level of M-HLCS was significantly increased in women with a T21 fetus compared with controls (p<0.001). The level of M-RASSF1A was not different between two groups (p>0.05). In non-invasive fetal T21 detection, the specificity of M-HLCS level and the epigenetic-epigenetic ratio (EER) using M-HLCS and M-RASSF1A levels were 82.5% and 92.5%, respectively, at 90.0% sensitivity. Conclusions: Our findings suggest that the EER may be useful as a potential biomarker for the non-invasive detection of fetal T21, regardless of fetal gender. The MBD method can be used as an effective tool in the detection of methylated fetal specific markers with a high CpG density in maternal plasma.

Comprehensive investigation of DNA methylation and gene expression in trisomy 21 placenta.

INTRODUCTION Trisomy 21 (T21) is the most common aneuploidy affecting humans and is caused by an extra copy of all or part of chromosome 21 (chr21). DNA methylation is an epigenetic event that plays an important role in human diseases via regulation of gene expression. However, the integrative association between DNA methylation and gene expression in T21 fetal placenta has yet to be determined. METHODS We profiled expression of 207 genes on chr21 and their DNA methylation patterns in placenta samples from normal and DS fetuses using microarray analysis and predicted the functions of differentially expressed genes using bioinformatics tools. RESULTS We found 47 genes with significantly increased expression in the T21 placenta compared to the normal placenta. Hypomethylation of the 47 genes was observed in the T21 placenta. Most of hypomethylated DNA positions were intragenic regions, i.e. regions inside a gene. Moreover, gene expression and hypomethylated DNA position showed significantly positive associations. By analyzing the properties of the gene-disease network, we found that increased genes in the T21 placenta were significantly associated with T21 and T21 complications such as mental retardation, neurobehavioral manifestations, and congenital abnormalities. DISCUSSION To our knowledge, this is the first study to comprehensively survey the association between gene expression and DNA methylation in chr21 of the T21 fetal placenta. Our findings provide a broad overview of the relationships between gene expression and DNA methylation in the placentas of fetuses with T21 and could contribute to future research efforts concerning genes involvement in disease pathogenesis.

MicroRNAs as potential biomarkers for noninvasive detection of fetal trisomy 21

PurposeThe objective of this study was to discover a panel of microRNAs (miRNAs) as potential biomarkers for noninvasive prenatal testing (NIPT) of trisomy 21 (T21) and to predict the biological functions of identified biomarkers using bioinformatics tools.MethodsUsing microarray-based genome-wide expression profiling, we compared the expression levels of miRNAs in whole blood samples from non-pregnant women, whole blood samples from pregnant women with euploid or T21 fetuses, and placenta samples from euploid or T21 fetuses. We analyzed the differentially expressed miRNAs according to disease and tissue type (P value <0.05 and two-fold expression change). To predict functions of target genes of miRNAs, the functional annotation tools were used.ResultsWe identified 299 miRNAs which reasonably separate the whole blood from the placenta. Among the identified miRNAs, 150 miRNAs were up-regulated in the placenta, and 149 miRNAs were down-regulated. Most of the up-regulated miRNAs in the placenta were members of the mir-498, mir-379, and mir-127 clusters. Among the up-regulated miRNAs in the placenta, mir-1973 and mir-3196 were expressed at higher levels in the T21 placenta than in the euploid placenta. The two miRNAs potentially regulate 203 target genes that are involved in development of brain, central nervous system, and nervous system. The genes are significantly associated with T21-related disorder such as congenital abnormalities, mental disorders, and nervous system diseases.ConclusionsOur study indicates placenta-specific miRNAs that may be potential biomarkers for NIPT of fetal T21 and provides new insights into the molecular mechanisms of T21 via regulation of miRNAs.

Quantification and Application of Potential Epigenetic Markers in Maternal Plasma of Pregnancies with Hypertensive Disorders

The aim of this study was to evaluate quantitative aberrations of novel fetal-specific epigenetic markers in maternal plasma of pregnancies with hypertensive disorders. We compared the concentrations of DSCR3, RASSF1A, and SRY as cell-free fetal DNA markers in 188 normal pregnancies, 16 pregnancies with early-onset preeclampsia (EO-PE), 47 pregnancies with late-onset preeclampsia (LO-PE), and 29 pregnancies with gestational hypertension (GH). The concentrations of all markers were significantly correlated with gestational age (p < 0.001 for all). Strong positive correlations were also observed between DSCR3 and SRY (r = 0.471, p < 0.001), as well as between RASSF1A and SRY (r = 0.326, p = 0.015) and between DSCR3 and RASSF1A (r = 0.673, p < 0.001). The concentrations of DSCR3 and RASSF1A in the EO-PE were significantly higher at 24–32 weeks and onwards (p < 0.05 for both). In the LO-PE, DSCR3 and RASSF1A concentrations were significantly higher only at 33–41 weeks compared with the controls. The concentrations of all markers in the GH group were not significantly different from those in the control group. This study is the first demonstration that DSCR3 is a novel epigenetic marker that can be an alternative to the RASSF1A for the prediction of EO-PE.

Novel Epigenetic Markers on Chromosome 21 for Noninvasive Prenatal Testing of Fetal Trisomy 21

Until now, fetal placenta-specific epigenetic markers for noninvasive prenatal testing of fetal trisomy 21 (T21) have been identified based only on differences in tissue-specific epigenetic characteristics between placenta and maternal blood, but these characteristics have not been validated in T21 placenta. We aimed to discover novel epigenetic markers on chromosome 21 that show a hypermethylated pattern in fetal placenta compared with blood, regardless of the presence of T21. We performed a high-resolution tiling array analysis of chromosome 21 using the methylated-CpG binding domain protein-based method. We identified 93 epigenetic regions that showed fetal placenta-specific differential methylation patterns; among these, three regions showed fetal placenta-specific methylation patterns in T21 placenta samples. The methylation patterns of these three regions in the array were confirmed by bisulfite direct sequencing. The three regions were detectable in first-trimester maternal plasma. Moreover, a combination of their methylation ratio achieved high diagnostic accuracy for noninvasive prenatal testing of fetal T21 by further statistical analysis. These three novel regions with fetal placenta-specific differential methylation patterns on chromosome 21 were identified irrespective of the presence of T21. Our findings suggest that epigenetic characteristics of markers according to the presence or absence of T21 should be considered in the development of noninvasive prenatal testing of fetal T21 using fetal placenta-specific epigenetic markers.