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In this study we aimed to establish the genetic cause of a myriad of cardiovascular defects prevalent in individuals from a genetically isolated population, who were found to share a common ancestor in 1728. Trio genome sequencing was carried out in an index patient with critical congenital heart disease (CHD); family members had either exome or Sanger sequencing. To confirm enrichment, we performed a gene-based association test and meta-analysis in two independent validation cohorts: one with 2685 CHD cases versus 4370 . These controls were also ancestry-matched (same as FTAA controls), and the other with 326 cases with familial thoracic aortic aneurysms (FTAA) and dissections versus 570 ancestry-matched controls. Functional consequences of identified variants were evaluated using expression studies. We identified a loss-of-function variant in the Notch target transcription factor-encoding gene HEY2. The homozygous state (n = 3) causes life-threatening congenital heart defects, while 80% of heterozygous carriers (n = 20) had cardiovascular defects, mainly CHD and FTAA of the ascending aorta. We confirm enrichment of rare risk variants in HEY2 functional domains after meta-analysis (MetaSKAT p = 0.018). Furthermore, we show that several identified variants lead to dysregulation of repression by HEY2. A homozygous germline loss-of-function variant in HEY2 leads to critical CHD. The majority of heterozygotes show a myriad of cardiovascular defects.

Congenital heart defects (CHDs) are the leading cause of death in infants under 1 year of age. Aberrations in the expression and function of cardiac transcription factors (TFs) are a major contributor to CHDs. Despite the numerous studies undertaken to functionally characterize these TFs, their exact role in different stages of cardiogenesis is still not fully elucidated. Here we focused on HEY2, a basic helix loop helix transcriptional repressor, and its potential role in human ventricular septal defects. Genetic analysis was performed based on sequencing of DNA and cDNA obtained from post-operational cardiac tissues and blood of 17 Lebanese patients with various CHDs. The screen covered the entire coding regions of the GATA4 , NKX2.5 , TBX5 , TBX20 and HEY2 genes. Our results revealed two novel somatic mutations, namely p.Ala229Thr and p.161_190 del, affecting HEY2 in the diseased cardiac tissues of two patients with VSD. These results suggest a potential role of HEY2 in regulating ventricular septation in humans.

Spatiotemporal gene expression during cardiac development is a highly regulated process. Activation of key signaling pathways involved in electrophysiological programming, such as Notch and Wnt signaling, occurs in early cardiovascular development and these pathways are reactivated during pathologic remodeling. Direct targets of these signaling pathways have also been associated with inherited arrhythmias such as Brugada syndrome and arrhythmogenic cardiomyopathy. In addition, evidence is emerging from animal models that reactivation of Notch and Wnt signaling during cardiac pathology may predispose to acquired arrhythmias, underscoring the importance of elucidating the transcriptional and epigenetic effects on cardiac gene regulation. Here, we highlight specific examples where gene expression dictates electrophysiological properties in both normal and diseased hearts.

Previous works characterized a novel cell population from adult human peripheral blood, designated peripheral blood insulin-producing cells (PB-IPC). PB-IPC displayed the pluripotent potential of differentiations after the treatment with platelet-derived mitochondria and gave rise to three germ layer-derived cells such as the mitochondrion-induced CD34 + hematopoietic stem cells (HSC)-like cells (miCD34 + HSC). To determine the molecular mechanism underlying the differentiation of miCD34 + cells, mechanistic studies established that MitoTracker Deep Red-labeled mitochondria could enter into the PB-IPC in a dose-dependent manner. Blocking Notch signaling pathway with a γ-secretase inhibitor, DAPT, markedly inhibited the proliferation of PB-IPC and improved the differentiation of miCD34 + HSC. Additionally, treatment with platelet-derived mitochondria can reprogram the differentiation of PB-IPC into miCD34 + HSC through inhibition of the Notch/HEY2 signaling pathway, as demonstrated by blocking experiments with HEY2 small interfering RNA (siRNA). The data indicated that Notch signaling pathway contributes to the miCD34 + HSC differentiation, thus advancing our understanding of the mitochondrial reprogramming and the potential treatment of human hematopoietic disease.

Most prostate cancers are treated, although more than 80% remain clinically insignificant and fewer than 3% are fatal. This retrospective study of 240 radical prostatectomy cases with comprehensive follow-up was a search for reliable markers of prostate cancer prognosis evaluable on biopsy specimens to enable minimization of unnecessary treatment, morbidity, and costs. Representative cancer and benign tissue from each prostatectomy specimen was made into tissue microarrays and stained with antibodies targeting 20 gene sequences. Traditional clinical and pathologic prognosticators and the 20 antibody stains were correlated with patient outcomes. By univariable analysis 4 of 20 antibodies (STMN1/stathmin 1, CYP4Z1/cytochrome p450-4z1, CDH1/E-cadherin, and Hey2), Gleason score, perineural invasion, and apical involvement were statistically significant outcome predictors for biopsy tissue. By multivariate analysis, Gleason score, Hey2, and CYP4Z1 were independently predictive. STMN1 and CDH1 were not independent of Gleason score but remain useful because marker interpretation is objective and Gleason scores often differ for biopsy and prostatectomy specimens.

Notch signaling is an important cellular pathway which affects the development and function of many organs. It plays critical roles in maintaining of progenitor stem cell population as well as balancing cell proliferation, survival, differentiation and apoptosis. It has been shown that notch signaling is aberrantly activated during the carcinogenesis of a variety of human cancers. In this study we aimed to explore activation of this signaling pathway in esophageal squamous cell carcinoma (ESCC) through expressional analysis of notch signaling target genes. The mRNA expression of HEY1and HEY2 was comparatively analyzed by real-time PCR in tumor and related margin normal tissues of 50 ESCC patients. Comparative quantitative real-time PCR indicates the overexpression of HEY1 and HEY2 in 54 and 30 % of ESCC samples, respectively. Overexpression of HEY1 was significantly associated with stage of the tumor ( p  = 0.048) and tumor location ( p  = 0.008). HEY2 overexpression was also significantly correlated to node metastasis of tumor cells ( p  = 0.043). Overexpression of HEY1 and HEY2 in ESCC is correlated to different indices of poor prognosis and it is extrapolated that such overexpression is important in progression and development of ESCC tumorigenesis. To the best of our knowledge, this is the first report introducing aberrant activation of notch signaling target genes in ESCC, where it plays roles in development and progression of the malignancy and may be considered in therapeutic modalities to restrict ESCC progression.

Arteries and veins show marked differences in their anatomy, physiology and genetic expression pattern. In this study, we analyzed impact of overexpression or downregulation of arterial marker gene Hey2 and venous marker gene COUP-TFII in human venous and arterial endothelial cells on genes involved in arteriovenous differentiation. Lentiviral overexpression of venous marker gene COUP-TFII in arterial endothelial cells led to downregulation of NICD4, arterial marker gene Hey2 and EphrinB2. Downregulation of Hey2 could be mediated by direct binding of COUP-TFII to Hey2 promoter as shown by ChIP, EMSA and promoter analysis. Downregulation of Hey2 by shRNA causes downregulation of EphrinB2 expression. Overexpression of arterial marker Hey2 in venous endothelial cells did not change expression pattern of COUP-TFII. Downregulation of venous marker gene COUP-TFII in venous endothelial cells resulted in upregulation of VEGF-A, Dll4 and EphrinB2 expression. Our data support an important role of Hey2 and COUP-TFII in arteriovenous differentiation of human endothelial cells.

The basic helix‐loop‐helix (bHLH) transcription factor Hey2 (gridlock) is an important determinant of mammalian heart development, but its role in human ventricular septal defects is unknown. Hey2 functions as a repressor through the bHLH domain. By direct sequencing, we analyzed the sequences encoding the bHLH domain of the human HEY2 in 52 explanted hearts of unrelated patients with complex cardiac malformations, notably ventricular (VSD) and atrioventricular septal defects (AVSD). We found three nonsynonymous mutations, namely, c.286A>G (p.Thr96Ala), c.293A>C (p.Asp98Ala), and c.299T>C (p.Leu100Ser) affecting the second helix of HEY2 in the diseased cardiac tissues of two patients with AVSD. This result suggests a possible role of HEY2 in the regulation of ventricular septation in humans. Since the two AVSD patients carried also binding domain mutations in other cardiac‐specific transcription factors, e.g. NKX2‐5, TBX5, and GATA4, breakdown of combinatorial interactions of transcription factors may have contributed to the complexity of their cardiac malformations. © 2005 Wiley‐Liss, Inc.