Yanyan Qian

LINE-1 methylation status and its association with tetralogy of fallot in infants

BackgroundMethylation levels of long interspersed nucleotide elements (LINE-1) are representative of genome-wide methylation status and play an important role in maintaining genomic stability and gene expression. To derive insight into the association between genome-wide methylation status and tetralogy of fallot (TOF), we compared the methylation status of LINE-1 element between TOF patients and controls. The methylation of the NKX 2–5, HAND 1, and TBX 20 promoter regions was also evaluated.MethodsGenomic DNA from right ventricular tissue samples was obtained from 32 patients with TOF and 15 control subjects. Sequenom MassARRAY platform was performed to examine the methylation levels of LINE-1, NKX2-5, HAND1 and TBX20. Mann–Whitney U test was used to compare differences in methylation levels between two groups.ResultsThe methylation level of LINE-1 was significantly lower in patients with TOF, with a median of 57.95% (interquartile range [IQR]: 56.10%–60.04%), as opposed to 59.70% in controls (IQR: 59.00%–61.30%; P = 0.0021). The highest LINE-1 methylation level was 61.3%. The risk of TOF increased in subjects with the lowest methylation levels (less than or equal to 59.0%; OR = 14.7, 95% CI: 1.8–117.7, P = 0.014) and in those with medium methylation levels (59.0%–61.3%; OR = 2.0, 95% CI: 0.3–14.2, P = 0.65). An ROC curve analysis showed a relatively high accuracy of using the LINE-1 methylation level in predicting the presence of TOF (AUC = 0.78, 95% CI: 0.65–0.91; P = 0.002). The association of the LINE-1 methylation level with TOF was only observed in males (P = 0.006) and not in females (P = 0.25). Neither age nor gender was found to be associated with the LINE-1 methylation level in patients or controls. Higher methylation levels of NKX2-5 and HAND1 and lower methylation levels of TBX20 were also observed in patients with TOF than in controls. No association was found between the methylation levels of NKX2-5, HAND1 and TBX 20 with the LINE-1 methylation level.ConclusionsLower LINE-1 methylation levels are associated with increased risk of TOF and may provide important clues for the development of TOF.

Association of promoter methylation statuses of congenital heart defect candidate genes with Tetralogy of Fallot

BackgroundAlthough a lower methylation level of whole genome has been demonstrated in Tetralogy of Fallot (TOF) patients, little is known regarding changes in specific gene DNA methylation profiles and the possible associations with TOF. In current study, the promoter methylation statuses of congenital heart defect (CHD) candidate genes were measured in order to further understand epigenetic mechanisms that may play a role in the development of TOF.MethodsThe methylation levels of CHD candidate genes were measured using the Sequenom MassARRAY platform. QRT-PCR was used to analyze the mRNA levels of CHD candidate genes in the right ventricular myocardium of TOF cases and normal controls.ResultsMethylation status analysis was performed on the promoter regions of 71 CHD candidate genes (113 amplicons). We found significant differences in methylation status, between TOF cases and controls, in 26 amplicons (26 genes) (p < 0.05). Of the 26 amplicons, 17 were up regulated and 9 were down regulated. Additionally, 14 of them were located in the CpG islands, 7 were located in the CpG island shores, and 5 were covering the regions near the transcription start site (TSS). The methylation status was subsequently confirmed and mRNA levels were measured for 7 represented candidate genes, including EGFR, EVC2, NFATC2, NR2F2, TBX5, CFC1B and GJA5. The methylation values of EGFR, EVC2, TBX5 and CFC1B were significantly correlated with their mRNA levels (p < 0.05).ConclusionsAberrant promoter methylation statuses of CHD candidate genes presented in TOF cases may contribute to the TOF development and have potential prognostic and therapeutic significance for TOF disease.

The Effect of Multiple Single Nucleotide Polymorphisms in the Folic Acid Pathway Genes on Homocysteine Metabolism

Objective. To investigate the joint effects of the single nucleotide polymorphisms (SNPs) of genes in the folic acid pathway on homocysteine (Hcy) metabolism. Methods. Four hundred women with normal pregnancies were enrolled in this study. SNPs were identified by MassARRAY. Serum folic acid and Hcy concentration were measured. Analysis of variance (ANOVA) and support vector machine (SVM) regressions were used to analyze the joint effects of SNPs on the Hcy level. Results. SNPs of MTHFR (rs1801133 and rs3733965) were significantly associated with maternal serum Hcy level. In the different genotypes of MTHFR (rs1801133), SNPs of RFC1 (rs1051266), TCN2 (rs9606756), BHMT (rs3733890), and CBS (rs234713 and rs2851391) were linked with the Hcy level adjusted for folic acid concentration. The integrated SNPs scores were significantly associated with the residual Hcy concentration (RHC) (r = 0.247). The Hcy level was significantly higher in the group with high SNP scores than that in other groups with SNP scores of less than 0.2 (P = 0.000). Moreover, this difference was even more significant in moderate and high levels of folic acid. Conclusion. SNPs of genes in the folic acid pathway possibly affect the Hcy metabolism in the presence of moderate and high levels of folic acid.

microRNA expression profiling of heart tissue during fetal development

microRNAs (miRNAs) are important both in early cardiogenesis and in the process of heart maturation. The aim of this study was to determine the stage-specific expression of miRNAs in human fetal heart in order to identify valuable targets for further study of heart defects. Affymetrix microarrays were used to obtain miRNA expression profiles from human fetal heart tissue at 5, 7, 9 and 23 weeks of gestation. To identify differentially expressed miRNAs at each time-point, linear regression analysis by the R limma algorithm was employed. Hierarchical clustering analysis was conducted with Cluster 3.0 software. Gene Ontology analysis was carried out for miRNAs from different clusters. Commonalities in miRNA families and genomic localization were identified, and the differential expression of selected miRNAs from different clusters was verified by quantitative polymerase chain reaction (qPCR). A total of 703 miRNAs were expressed in human fetal heart. Of these, 288 differentially expressed miRNAs represented 5 clusters with different expression trends. Several clustered miRNAs also shared classification within miRNA families or proximal genomic localization. qPCR confirmed the expression patterns of selected miRNAs. miRNAs within the 5 clusters were predicted to target genes vital for heart development and to be involved in cellular signaling pathways that affect heart structure formation and heart-associated cellular events. In conclusion, to the best of our knowledge, this is the first miRNA expression profiling study of human fetal heart tissue. The stage-specific expression of specific miRNAs suggests potential roles at distinct time-points during fetal heart development.

Association between mRNA levels of DNMT1, DNMT3A, DNMT3B, MBD2 and LINE-1 methylation status in infants with tetralogy of Fallot

DNA methylation is catalyzed and maintained by DNA methyltransferases (DNMTs: DNMT1, DNMT3A and DNMT3B) and methyl-CpG-binding domain protein 2 (MBD2). However, little is known about the biological and clinical significance of the expression changes of DNMTs and MBD2 and their association with the methylation levels of long interspersed nuclear element-1 (LINE-1) in patients with tetralogy of Fallot (TOF). In this study, quantitative RT-PCR (qRT-PCR) was applied to analyze the mRNA levels of DNMTs and MBD2. The methylation status of LINE-1 was measured using the sequenom MassARRAY platform. The mRNA levels of the DNMTs and MBD2 showed a statistically significant decrease in the patients with TOF (P<0.001). The results also showed that patients with TOF had significantly lower global DNA methylation levels with a median of 61.50% [interquartile range (IQR), 59.78-63.77] compared with 63.54% (IQR, 62.49‑64.88) among the controls (P=0.0099). In the controls, only DNMT1 showed a significant positive correlation with the DNMT3A mRNA levels (r=0.718, P=0.002). Of note, the DNMT1, DNMT3A, DNMT3B and MBD2 mRNA levels positively correlated with each other; this was statistically significant (P<0.05). A significant positive correlation with the global DNA methylation status was observed only for MBD2 (r=-0.579, P=0.005) in patients with TOF. In conclusion, lower LINE-1 methylation levels significantly correlate with aberrant MBD2 mRNA levels. The lower expression of DNMT1 and DNMT3B may play an important role in the pathogenesis of TOF.

The roles of SMYD4 in epigenetic regulation of cardiac development in zebrafish

SMYD4 belongs to a family of lysine methyltransferases. We analyzed the role of smyd4 in zebrafish development by generating a smyd4 mutant zebrafish line (smyd4L544Efs*1) using the CRISPR/Cas9 technology. The maternal and zygotic smyd4L544Efs*1 mutants demonstrated severe cardiac malformations, including defects in left-right patterning and looping and hypoplastic ventricles, suggesting that smyd4 was critical for heart development. Importantly, we identified two rare SMYD4 genetic variants in a 208-patient cohort with congenital heart defects. Both biochemical and functional analyses indicated that SMYD4(G345D) was pathogenic. Our data suggested that smyd4 functions as a histone methyltransferase and, by interacting with HDAC1, also serves as a potential modulator for histone acetylation. Transcriptome and bioinformatics analyses of smyd4L544Efs*1 and wild-type developing hearts suggested that smyd4 is a key epigenetic regulator involved in regulating endoplasmic reticulum-mediated protein processing and several important metabolic pathways in developing zebrafish hearts.

Hypomethylation and decreased expression of BRG1 in the myocardium of patients with congenital heart disease.

BACKGROUND BRG1, an ATPase subunit of the SWItch/Sucrose Non-Fermentable complex, is tightly associated with cardiac development. However, little is known about the association between the pathogenesis of CHD and BRG1. METHODS The methylation of a BRG1 promoter and a novel CpG island in the second intron was analyzed in the myocardium of congenital heart disease (CHD) patients (n = 24) and normal controls (n = 11) using pyrosequencing and the MassARRAY platform. BRG1 expression was sketched in the normal fetal and postnatal heart using real-time PCR. BRG1 mRNA and protein expression was detected by means of real-time PCR and immunohistochemistry. The expression of GATA4 was analyzed with real-time PCR. RESULTS The CpG shore in the second intron of BRG1 was hypomethylated in the myocardium of patients (p < 0.05). BRG1 showed a high level of expression in the normal fetal heart in the second trimester (p < 0.01). Compared with that of the normal subjects, BRG1 expression was decreased by 70% in the myocardium of patients (n = 92; p < 0.05). Of note, the expression of GATA4 was significantly correlated with BRG1 expression (r = 0.7475; p = 0.0082) in the myocardium, and it was also decreased by 70% in these patients (n = 92; p < 0.05). CONCLUSION These results suggested that the early high expression of BRG1 in fetal hearts maintained normal cardiac development and that the abnormal hypomethylation and decreased expression of BRG1 in human hearts probably affect the expression of GATA4, which affects the pathogenesis of CHD. Birth Defects Research 109:1183-1195, 2017.