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Background: Congenital heart disease (CHD) is a common birth defect, and most cases occur sporadically. Mutations in key genes that are responsible for cardiac development could contribute to CHD. To date, the genetic causes of CHD remain largely unknown.Methods: In this study, twenty-nine candidate genes in CHD were sequenced in 106 patients with Tetralogy of Fallot (TOF) using target exome sequencing (TES). The co-immunoprecipitation (CO-IP) and luciferase reporter gene assays were performed in HEK293T cells, and wild-type and mutant mRNA of ZFPM2 were microinjected into zebrafish embryos.Results: Rare variants in key cardiac transcriptional factors and JAG1 were identified in the patients. Four patients carried multiple gene variants. The novel E1148K variant was located at the eighth Zinc-finger domain of FOG2 protein. The CO-IP assays in the HEK293T cells revealed that the variant significantly damaged the interaction between ZFPM2/FOG2 and GATA4. The luciferase reporter gene assays revealed that the E1148K mutant ZFPM2 protein displayed a significantly greater inhibition of the transcriptional activation of GATA4 than the wild-type protein. The wild-type mRNA and the E1148K mutant mRNA of ZFPM2 were injected into zebrafish embryos. At 48 hpf, in the mutant mRNA injection group, the number of embryos with an abnormal cardiac chamber structure and a loss of left-right asymmetry was increased. By 72 hpf, the defects in the chamber and left-right asymmetry became obvious.Conclusions: We performed TES in sporadic TOF patients and identified rare variants in candidate genes in CHD. We first validated the E1148 K variant in ZFPM2, which is likely involved in the pathogenesis of CHD via GATA4. Moreover, our results suggest that TES could be a useful tool for discovering sequence variants in CHD patients. [ABSTRACT FROM AUTHOR]

Background: Although 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.Methods: The 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.Results: Methylation 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).Conclusions: Aberrant 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. [ABSTRACT FROM AUTHOR]

Background: Congenital heart disease (CHD) is a common birth defect, and most cases occur sporadically. Mutations in key genes that are responsible for cardiac development could contribute to CHD. To date, the genetic causes of CHD remain largely unknown., Methods: In this study, twenty-nine candidate genes in CHD were sequenced in 106 patients with Tetralogy of Fallot (TOF) using target exome sequencing (TES). The co-immunoprecipitation (CO-IP) and luciferase reporter gene assays were performed in HEK293T cells, and wild-type and mutant mRNA of ZFPM2 were microinjected into zebrafish embryos., Results: Rare variants in key cardiac transcriptional factors and JAG1 were identified in the patients. Four patients carried multiple gene variants. The novel E1148K variant was located at the eighth Zinc-finger domain of FOG2 protein. The CO-IP assays in the HEK293T cells revealed that the variant significantly damaged the interaction between ZFPM2/FOG2 and GATA4. The luciferase reporter gene assays revealed that the E1148K mutant ZFPM2 protein displayed a significantly greater inhibition of the transcriptional activation of GATA4 than the wild-type protein. The wild-type mRNA and the E1148K mutant mRNA of ZFPM2 were injected into zebrafish embryos. At 48 hpf, in the mutant mRNA injection group, the number of embryos with an abnormal cardiac chamber structure and a loss of left-right asymmetry was increased. By 72 hpf, the defects in the chamber and left-right asymmetry became obvious., Conclusions: We performed TES in sporadic TOF patients and identified rare variants in candidate genes in CHD. We first validated the E1148 K variant in ZFPM2, which is likely involved in the pathogenesis of CHD via GATA4. Moreover, our results suggest that TES could be a useful tool for discovering sequence variants in CHD patients.

Background: Linear nevus sebaceous syndrome (LNSS) is a multisystem disorder that includes nevus sebaceous and central nervous system, ocular and skeletal anomalies. We report the first case of a KRAS G12D mosaic mutation in a patient diagnosed with LNSS., Case Presentation: A 3-month-old female with a clinical diagnosis of LNSS presented with intermittent epilepsy. Her mother carefully collected a skin lesion sample from scratched-off scurf obtained from the patient's nails. DNA was extracted, and long-range PCR was performed to amplify the KRAS gene, which was then analyzed by next-generation sequencing. The results revealed the presence of a low-level heterozygous mutation in the KRAS gene (c.35C>T; p.G12D, 5 %)., Conclusions: These findings suggest that the KRAS somatic mosaic mutation in this patient may have caused her skin and eye lesions and epilepsy. With this correct diagnosis, the infant can be effectively treated.

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&#95;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&#95;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&#95;M1, NKX2-5&#95;M2, GATA4&#95;M, HAND1&#95;M2 or HAND1&#95;M3 were not significantly different in TOF patients compared to controls. The methylation values for NKX2-5&#95;M3 were negatively correlated with mRNA levels (r = - 0.463, p = 0.010, N = 30) and there was a significant association between HAND1&#95;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.

Background: Methylation 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., Methods: Genomic 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., Results: The 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., Conclusions: Lower LINE-1 methylation levels are associated with increased risk of TOF and may provide important clues for the development of TOF.