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Wang Y.,Shanghai JiaoTong University | Wang Y.,Fudan University | Wen Z.,Shanghai JiaoTong University | Shen J.,Shanghai JiaoTong University | And 6 more authors.
Journal of Human Genetics | Year: 2014

Semiconductor high-throughput sequencing, represented by Ion Torrent PGM/Proton, proves to be feasible in the noninvasive prenatal diagnosis of fetal aneuploidies. It is commendable that, with less data and relevant cost also, an accurate result can be achieved owing to the high sensitivity and specificity of such kind of technology. We conducted a comparative analysis of the performance of four different Ion chips in detecting fetal chromosomal aneuploidies. Eight maternal plasma DNA samples, including four pregnancies with normal fetuses and four with trisomy 21 fetuses, were sequenced on Ion Torrent 314/316/318/PI chips, respectively. Results such as read mapped ratio, correlation coefficient and phred quality score were calculated and parallelly compared. All samples were correctly classified even with low-throughput chip, and, among the four chips, the 316 chip had the highest read mapped ratio, correlation coefficient, mean read length and phred quality score. All chips were well consistent with each other. Our results showed that all Ion chips are applicable in noninvasive prenatal fetal aneuploidy diagnosis. We recommend researchers or clinicians to use the appropriate chip with barcoding technology on the basis of the sample number. © 2014 The Japan Society of Human Genetics.


Zhang J.,Fudan University | Zhang J.,Shanghai Key Laboratory of Prevention and Intervention of Birth Defects | Ma X.,Fudan University | Ma X.,Shanghai Key Laboratory of Prevention and Intervention of Birth Defects | And 5 more authors.
Pediatric Research | Year: 2014

Background:As an important component of retinoid acid signaling pathway, the retinoid X receptor (RXRA) is considered to play an important role in the pathogenesis of tetralogy of Fallot (TOF).Methods:The expression level of RXRA mRNA and the methylation status of the RXRA promoter region in 26 patients with TOF and 6 controls were detected using real-time PCR and bisulfite-specific PCR and cloning-based sequencing, respectively. Dual-luciferase reporter assays, combined with in vitro methylation assay, were performed to determine the transcriptional regulatory activity of unmethylated and methylated CpG regions in the RXRA promoter.Results:The mRNA expression of RXRA in the right ventricular outflow tract (RVOT) myocardium was significantly decreased in patients with TOF compared with that in the controls. The methylation status of region -1453 to -1000 containing CpG sites 1-23 in the RXRA promoter region was higher in patients with TOF than that in the controls. This region contained several transcription factor sites. In addition, dual-luciferase reporter assays combined with methylation assay in vitro showed that this region had transcriptional regulatory activity, which can be depressed by the methylation of this region.Conclusion:The elevated methylation at RXRA promoter may be responsible for the downregulated mRNA expression in RVOT myocardium of patients with TOF. Copyright © 2014 International Pediatric Research Foundation, Inc.


Sheng W.,Fudan University | Sheng W.,Shanghai Key Laboratory of Prevention and Intervention of Birth Defects | Chen L.,Fudan University | Wang H.,Fudan University | And 7 more authors.
Pediatric Research | Year: 2016

Background:ZFPM2 gene plays an important role in heart morphogenesis and development of coronary vessels from epicardium, however, little is known regarding its epigenetic regulation in the pathogenesis of tetralogy of fallot (TOF).Methods:The methylation levels of ZFPM2 gene were measured by MassArray (Sequenom, San Diego, CA) and bisulfite sequencing polymerase chain reaction (PCR) (BSP). Real-time PCR was performed to analyze the mRNA levels for ZFPM2 gene in the myocardium of TOF.Results:The methylation levels in the CpG island shore of ZFPM2 promoter were significantly higher in patients with TOF, with a median of 80.32% (interquartile range (IQR): 73.54-85.75%, N = 42), as compared to 59.63% in controls (IQR: 44.79-73.83%; P = 0.0186, N = 6). No significant difference was observed in the methylation status at the CpG island of ZFPM2 promoter. The ZFPM2 mRNA levels were significantly lower in patients with TOF compared to that in the controls (P < 0.05). The aberrant methylation values of ZFPM2 were negatively associated with significant changes in its mRNA level (r = -0.40, P = 0.008, N = 42).Conclusion:Aberrant methylation status at the promoter CpG island shore of ZFPM2 gene may be associated with its gene transcription regulation in the TOF patients. © Copyright 2016 International Pediatric Research Foundation, Inc.


Sheng W.,Fudan University | Sheng W.,Shanghai Key Laboratory of Prevention and Intervention of Birth Defects | Qian Y.,Fudan University | Wang H.,Fudan University | And 11 more authors.
BMC Medical Genomics | Year: 2013

Background: NKX2-5, GATA4 and HAND1 are essential for heart development, however, little is known regarding their epigenetic regulation in the pathogenesis of tetralogy of fallot (TOF). Methods. Methylation levels were measured in three regions of NKX2-5 (M1: -1596 bp ∼ -1374 bp, M2: -159 bp ∼ 217 bp and M3: 1058 bp ∼ 1524 bp), one region of GATA4 (M: -392 bp ∼ 107 bp) and three regions of HAND1 (M1: -887 bp ∼ -414 bp, M2: -436 bp ∼ 2 bp and M3: 37 bp ∼ 398 bp) using the Sequenom MassARRAY platform. QRT-PCR was used to analyze NKX2-5 and HAND1 mRNA levels in the right ventricular myocardium of TOF patients. Results: TOF patients had a significantly higher NKX2-5-M3 median methylation level than controls (41.65% vs. 22.18%; p = 0.0074; interquartile range [IQR]: 30.46%-53.35%, N = 30 and 20.07%-24.31%, N = 5; respectively). The HAND1-M1 median methylation level was also significantly higher in TOF patients than controls (30.05% vs. 17.54%; p = 0.0054; IQR: 20.77%-40.89%, N = 30 and IQR: 14.69%-20.64%; N = 6; respectively). The methylation statuses of NKX2-5-M1, NKX2-5-M2, GATA4-M, HAND1-M2 or HAND1-M3 were not significantly different in TOF patients compared to controls. The methylation values for NKX2-5-M3 were negatively correlated with mRNA levels (r = - 0.463, p = 0.010, N = 30) and there was a significant association between HAND1-M1 methylation status and mRNA levels (r = - 0.524, p = 0.003, N = 30) in TOF patients. Conclusions: Aberrant methylation statuses of the NKX2-5 gene body and HAND1 promoter regions are associated with the regulation of gene transcription in TOF patients and may play an important role in the pathogenesis of TOF. © 2013 Sheng et al.; licensee BioMed Central Ltd.

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