The small molecule chemical compound cinobufotalin attenuates resistance to DDP by inducing ENKUR expression to suppress MYH9-mediated c-Myc deubiquitination in lung adenocarcinoma.

The small molecule chemical compound cinobufotalin (CB) is reported to be a potential antitumour drug that increases cisplatin (DDP) sensitivity in nasopharyngeal carcinoma. In this study, we first found that CB decreased DDP resistance, migration and invasion in lung adenocarcinoma (LUAD). Mechanistic studies showed that CB induced ENKUR expression by suppressing PI3K/AKT signalling to downregulate c-Jun, a negative transcription factor of ENKUR. Furthermore, ENKUR was shown to function as a tumour suppressor by binding to ß-catenin to decrease c-Jun expression, thus suppressing MYH9 transcription. Interestingly, MYH9 is a binding protein of ENKUR. The Enkurin domain of ENKUR binds to MYH9, and the Myosin_tail of MYH9 binds to ENKUR. Downregulation of MYH9 reduced the recruitment of the deubiquitinase USP7, leading to increased c-Myc ubiquitination and degradation, decreased c-Myc nuclear translocation, and inactivation of epithelial-mesenchymal transition (EMT) signalling, thus attenuating DDP resistance. Our data demonstrated that CB is a promising antitumour drug and may be a candidate chemotherapeutic drug for LUAD patients.

Silencing MYH9 blocks HBx-induced GSK3ß ubiquitination and degradation to inhibit tumor stemness in hepatocellular carcinoma.

MYH9 has dual functions in tumors. However, its role in inducing tumor stemness in hepatocellular carcinoma (HCC) is not yet determined. Here, we found that MYH9 is an effective promoter of tumor stemness that facilitates hepatocellular carcinoma pathogenesis. Importantly, targeting MYH9 remarkably improved the survival of hepatocellular carcinoma-bearing mice and promoted sorafenib sensitivity of hepatocellular carcinoma cells in vivo. Mechanistic analysis suggested that MYH9 interacted with GSK3ß and reduced its protein expression by ubiquitin-mediated degradation, which therefore dysregulated the ß-catenin destruction complex and induced the downstream tumor stemness phenotype, epithelial-mesenchymal transition, and c-Jun signaling in HCC. C-Jun transcriptionally stimulated MYH9 expression and formed an MYH9/GSK3ß/ß-catenin/c-Jun feedback loop. X protein is a hepatitis B virus (HBV)-encoded key oncogenic protein that promotes HCC pathogenesis. Interestingly, we observed that HBV X protein (HBX) interacted with MYH9 and induced its expression by modulating GSK3ß/ß-catenin/c-Jun signaling. Targeting MYH9 blocked HBX-induced GSK3ß ubiquitination to activate the ß-catenin destruction complex and suppressed cancer stemness and EMT. Based on TCGA database analysis, MYH9 was found to be elevated and conferred poor prognosis for hepatocellular carcinoma patients. In clinical samples, high MYH9 expression levels predicted poor prognosis of hepatocellular carcinoma patients. These findings identify the suppression of MYH9 as an alternative approach for the effective eradication of CSC properties to inhibit cancer migration, invasion, growth, and sorafenib resistance in HCC patients. Our study demonstrated that MYH9 is a crucial therapeutic target in HCC.

ZEB1 promotes invasion and metastasis of endometrial cancer by interacting with HDGF and inducing its transcription.

Zinc finger E-box binding homeobox 1 (ZEB1), as a typical transcription inhibitory factor of E-cadherin, plays a major role in stimulating the invasion and metastasis of tumors via modulating the epithelial-mesenchymal transition (EMT) signal. However, its function and modulatory mechanisms in endometrial carcinoma (EC) remain unclear. In this study, silencing ZEB1 significantly reduced EC cell migration, invasion, and metastasis, as well as enhanced the sensitivity of EC cells to cisplatin (cDDP) in vitro and in vivo. Mechanism analysis indicated that ZEB1 interacts with hepatoma-derived growth factor (HDGF) and co-localizes in the nucleus. In addition, ZEB1 as a transcription factor binds to the promoter of HDGF to stimulate HDGF transcription. Furthermore, suppression of HDGF in ZEB1-overexpressed EC cells not only reduced the expression of ß-catenin, TCF4, and ZEB1, but also repressed ß-catenin translocation from the cytoplasm into the nucleus and further downregulated the combination with TCF4. Interestingly, the ß-catenin/TCF4 signaling feedback stimulates ZEB1 transcription and therefore constitutes a positive feedback loop. In clinical samples, ZEB1 positively correlates with HDGF expression, and co-expression of ZEB1 and HDGF promotes the pathogenesis of EC. In summary, our study demonstrated that the positive feedback loop of ZEB1/HDGF/ß-catenin/TCF4 plays an unfavorable role in the metastasis of endometrial carcinoma.

Chemical renal artery denervation with appropriate phenol in spontaneously hypertensive rats.

OBJECTIVE: To explore the effectiveness of renal denervation (RDN) on blood pressure with the appropriate dosage of phenol/ethanol solution in spontaneously hypertensive rats (SHRs). METHODS: RDN was performed on the bilateral renal artery. Forty SHRs were divided into four groups according on the dosage of phenol (10% phenol in absolute ethanol): sham group, 0.5 mL phenol group, 1 mL phenol group and 1.5 mL phenol group (n = 10 in each group). Blood pressure was measured by tail-cuff plethysmography. Plasma creatinine was determined four weeks after the treatment. The kidneys and renal arteries were collected and processed for histological examination. RESULTS: A sustained decrease in systolic blood pressure (SBP) was only observed after the application of 1 mL phenol for four weeks, while SBP was lowered during the first week after RDN and increased in the following three weeks in the 0.5 mL and 1.5 mL phenol groups compared with the sham group. Renal norepinephrine (NE) was significantly decreased four weeks after RDN in the 1 mL and 1.5 mL phenol group compared with the sham group, but not in the 0.5 ml group. RDN with 1 mL phenol obviously reduced glomerular fibrosis. Histopathological analysis showed that tyrosine hydroxylase immunoreactivity was lower in the 1 mL and 1.5 mL phenol groups compared with the sham group. Moderate renal artery damage occurred in the 1.5 mL phenol group. CONCLUSION: Chemical denervation with 1 ml phenol (10% phenol in absolute ethanol) effectively and safely damaged peripheral renal sympathetic nerves and contributed to the sustained reduction of blood pressure in SHRs.

Nuclear CD133 expression predicts poor prognosis for hepatocellular carcinoma.

Cancer stem cells (CSCs) are responsible for cancer recurrence and metastasis and are related to poor prognosis in patients with hepatocellular carcinoma (HCC). CD133 is one of the most commonly used CSC markers. In this study, expression and the biological significance of CSC marker CD133 was evaluated in HCC, at mRNA and protein levels. We demonstrate that both mRNA and protein levels of CD133 are significantly elevated in HCC relative to that in adjacent non-cancerous tissue based on bioinformatics and immunohistochemical analysis, respectively (P < 0.01). Intriguingly, we detected nuclear distribution of CD133 and found that nuclear CD133 expression was indicative of poor patient prognosis (median survival 12 months versus 34.5 months) (Log-Rank, P = 0.0258). Meanwhile, our findings suggest that nuclear CD133 expression is positively correlated with tumor size and serves as an independent prognostic factor for HCC after surgical resection (HR = 0.564, 95% CI 0.313-1.018, P = 0.057). Nuclear CD133 expression can potentially serve as a biomarker for clinical diagnosis and prognosis of HCC.

Low MYH9 expression predicts a good prognosis for hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is currently one of the most common causes of cancer-related death and one of the most commonly diagnosed cancers. MYH9 is thought to play a critical role in cancer progression. However, the expression and prognostic value of MYH9 in HCC are still unknown. In this study, the expression and biological significance of MYH9 were evaluated in HCC, at mRNA and protein levels. We showed that both mRNA and protein levels of MYH9 were significantly upregulated in HCC relative to the levels in adjacent non-tumor tissues based on the TCGA database and immunohistochemical analysis, respectively (P < 0.001). Additionally, we found that high MYH9 protein levels correlated with poor patient prognosis (median survival 19 months versus 49 months) (Log-Rank, P = 0.0292). Meanwhile, our data suggested that MYH9 expression is an independent prognostic factor for HCC after surgical resection (HR = 0.675, 95% CI 0.452-1.008, P = 0.054) and can potentially serve as a biomarker for the clinical diagnosis and prognosis of HCC.

A coordinated PCP-Cardiologist Telemedicine Model (PCTM) in China's community hypertension care: study protocol for a randomized controlled trial.

BACKGROUND: Hypertension is a major risk factor for cardiovascular disease, and its control rate has remained low worldwide. Studies have found that telemonitoring blood pressure (BP) helped control hypertension in randomized controlled trials. However, little is known about its effect in a structured primary care model in which primary care physicians (PCPs) are partnering with cardiology specialists in electronic healthcare data sharing and medical interventions. This study aims to identify the effects of a coordinated PCP-cardiologist model that applies telemedicine tools to facilitate community hypertension control in China. METHODS/DESIGN: Patients with hypertension receiving care at four community healthcare centers that are academically affiliated to Shanghai Chest Hospital, Shanghai JiaoTong University are eligible if they have had uncontrolled BP in the previous 3 months and access to mobile Internet. Study subjects are randomly assigned to three interventional groups: (1) usual care; (2) home-based BP telemonitor with embedded Global System for Mobile Communications (GSM) module and unlimited data plan, an app to access personal healthcare record and receive personalized lifestyle coaching contents, and proficiency training of their use; or (3) this plus coordinated PCP-cardiologist care in which PCPs and cardiologists share data via a secure CareLinker website to determine interventional approaches. The primary outcome is mean change in systolic blood pressure over a 12-month period. Secondary outcomes are changes of diastolic blood pressure, HbA1C, blood lipids, and medication adherence measured by the eight-item Morisky Medication Adherence Scale. DISCUSSION: This study will determine whether a coordinated PCP-Cardiologist Telemedicine Model that incorporates the latest telemedicine technologies will improve hypertension care. Success of the model would help streamline the present community healthcare processes and impact a greater number of patients with uncontrolled hypertension. TRIAL REGISTRATION: ClinicalTrials.gov, NCT02919033 . Registered on 23 September 2016.

Impact of prior permanent pacemaker on long-term clinical outcomes of patients undergoing percutaneous coronary intervention.

BACKGROUND: The impact of permanent pacemaker (PPM) on long-term clinical outcomes of patients undergoing percutaneous coronary intervention (PCI) has not been studied. HYPOTHESIS: PPM may increase heart failure (HF) burden on patients undergoing PCI. METHODS: We recruited consecutive patients undergoing PCI and carried out a nested case-control study. Patients with confirmed PPM undergoing first PCI were identified and matched by age and sex in 1:1 fashion to patients without PPM undergoing first PCI. Clinical data were collected and analyzed. The primary endpoint outcomes were all-cause mortality and hospitalization for HF. RESULTS: The final analysis included 156 patients. The mean follow-up period was 4.6 ± 2.9 years. The overall all-cause mortality was 21.15%, without significant difference between the 2 groups (21.79% vs 20.51%; P = 0.85). However, the rate of HF-related hospitalization was significantly higher in patients with PPM than in controls (26.92% vs 10.26%; P = 0.008). After adjustment for hypertension, type 2 diabetes mellitus, hyperlipidemia, chronic kidney disease, stroke, left ventricular ejection fraction, brain natriuretic peptide, and acute coronary syndrome (ACS), PCI patients with PPM were still associated with a greater hospitalization rate for HF (odds ratio: 4.31, 95% confidence interval: 0.94-19.80, P = 0.061). Further analysis in the ACS subgroup showed VVI-mode pacing enhanced the risk for HF-associated hospitalization (adjusted odds ratio: 8.27, 95% confidence interval: 1.37-49.75, P = 0.02). CONCLUSIONS: PPM has no effect on all-cause mortality in patients undergoing first PCI but significantly increases the HF-associated hospitalization rate, especially in ACS patients.

The decrease of cyclin B2 expression inhibits invasion and metastasis of bladder cancer.

OBJECTIVE: It has been shown that cyclin B2 is commonly overexpressed in many malignant tumors. This study aimed to investigate the potential role of cyclin B2 in bladder cancer. METHOD AND MATERIAL: Fixed tissues for immunohistochemistry and fresh tissues for western blotting and quantitative real-time polymerase chain reaction assay were randomly selected from Nanfang hospital. Normal bladder urothelial cell and bladder cancer cell lines was stored in our laboratory, the bladder cancer cells were transfected to develop bladder cancer cell clones expressing decreased cyclin B2 levels, the clones were used for cell growth and metastasis experiments in vitro and in vivo. RESULTS: Western blot and immunohistochemical analysis both showed that the cyclin B2 protein expression was higher in bladder urothelial carcinoma than in normal bladder mucosa, especially in invasive cancer. Real-time polymerase chain reaction showed that the cyclin B2 messenger RNA expression exhibited the same trend. Results of cell lines experiments also showed higher cyclin B2 expression in cancer cells. In vitro tests the decrease of cyclin B2 expression that had little effect on cell growth and cell cycling according to the MTT assay and the Edu assay, whereas in the Boyden chamber transwell assay, the cyclin B2 low-expressing clones significantly inhibits the cells׳ invasion and metastatic abilities. This result was consistent with the scratch-wound assay result showing that the target clone needed more time for healing the wound. The in vivo experiment in nude mice produced similar results, the lung and liver target cell metastasis nodules were smaller and less than those of the negative control by the hepatic subcapsular injection assay, and the mice of the target clone group has longer survival time in no intervention observed test. CONCLUSION: These results indicate that the cyclin B2 was overexpressed in bladder cancer, and the down-regulation of cyclin B2 expression in bladder cancer greatly inhibits the cell׳s invasion and metastatic abilities, and it prolonged the survival time of nude mice in vivo.

Comparison of combination therapy of high-dose oral N-acetylcysteine and intravenous sodium bicarbonate hydration with individual therapies in the reduction of Contrast-induced Nephropathy during Cardiac Catheterisation and Percutaneous Coronary Intervention (CONTRAST): A multi-centre, randomised, controlled trial.

INTRODUCTION: N-acetylcysteine (NAC) and sodium bicarbonate (SOB) therapies may prevent contrast-induced nephropathy (CIN). However, the efficacy of using combination over individual therapies was not established, and there was no large randomised study comparing abbreviated SOB therapy with conventional sustained saline pre-hydration with oral NAC. METHODS: In a multi-centre, open-label, randomised, controlled trial (NCT00497328), we prospectively enrolled 548 patients with at least moderate renal impairment undergoing cardiac catheterisation with or without percutaneous coronary intervention. Patients were randomly assigned to 3 groups: 1) NAC: 154 mEq/L sustained sodium chloride regime (1 mL/kg/h 12 h before, during and 6h after the procedure) with oral NAC at 1.2g bid for 3 days (n=185); 2) SOB: 154 mEq/L abbreviated SOB regime at 3 mL/kg/h 1h before the procedure, and 1 mL/kg/h during and 6h after the procedure (n=182); and 3) COM: combination of abbreviated SOB regime and oral NAC (n=181). The primary end point was incidence of CIN. The secondary end points were rise in serum creatinine, hospitalisation duration, haemodialysis, morbidity and mortality within 30 days. RESULTS: The 3 groups had similar baseline characteristics: age 68 ± 10 years, 76% male, 48% diabetic and baseline glomerular filtration rate (GFR) 47.7 ± 13.0 mL/min. There were 41 (8.8%) patients with GFR<30. The CIN incidences were NAC 6.5%, SOB 12.8% and COM 10.6%. The COM regimen was not superior to either the NAC (relative risk (RR)=1.61, 95% confidence interval (CI): 0.76 to 3.45, p=0.225) or SOB (RR=0.83, 95% CI: 0.44 to 1.56, p=0.593) regimens. The CIN incidence was lower in the NAC group than the SOB group (adjusted odds ratio (OR)=0.40, 95% CI: 0.17 to 0.92; p=0.032). Multivariate analysis showed contrast volume (OR=1.99, 95% CI: 1.33 to 2.96, p<0.001 per 100mL), female (OR=2.47, 95% CI: 1.22 to 5.00, p=0.012) and diabetes (OR=2.03, 95% CI: 1.03 to 3.99, p=0.041) were independent risk predictors. There were no differences in the secondary outcomes among the 3 groups. CONCLUSION: The combination regimen was not superior to individual regimens in preventing CIN in patients with baseline renal impairment. There was a trend suggesting that the 12-hour sustained sodium chloride pre-hydration regimen was more protective than the 1-hour abbreviated SOB regimen.

Prevalence and spectrum of LRRC10 mutations associated with idiopathic dilated cardiomyopathy.

Dilated cardiomyopathy (DCM) is the most common form of primary myocardial disease. It is the most common cause of chronic congestive heart failure and the most frequent reason for heart transplantation in young adults. There is increasing evidence demonstrating that genetic defects are involved in the pathogenesis of idiopathic DCM. Recent studies have shown that genetically defective LRRC10 predisposes animals to DCM. However, the association of LRRC10 with DCM in humans has not been reported. In the current study, the whole coding region and flanking splice junction sites of the LRRC10 gene were sequenced in 220 unrelated patients with idiopathic DCM. The available relatives of the index patients harboring identified mutations and 200 unrelated ethnically matched healthy individuals used as controls were also genotyped for LRRC10. The functional effect of the LRRC10 mutations was analyzed in silico. As a result, two novel heterozygous LRRC10 mutations, p.L41V and p.L163I, were identified in two families with DCM, respectively, with a mutational prevalence of ~0.91%. Genetic analyses of the pedigrees showed that in each family, the mutation co-segregated with DCM was transmitted as an autosomal dominant trait with complete penetrance. The missense mutations were absent in 400 control chromosomes and the altered amino acids were completely conserved evolutionarily across various species. Functional analysis in silico indicated that the LRRC10 mutations were causative. This study firstly reports the association of LRRC10 mutations with enhanced susceptibility to DCM in humans, which provides novel insight into the molecular mechanism underpinning DCM, and contributes to the development of novel prophylactic and therapeutic strategies for DCM.

Alpha-enolase promotes cell glycolysis, growth, migration, and invasion in non-small cell lung cancer through FAK-mediated PI3K/AKT pathway.

BACKGROUND: During tumor formation and expansion, increasing glucose metabolism is necessary for unrestricted growth of tumor cells. Expression of key glycolytic enzyme alpha-enolase (ENO1) is controversial and its modulatory mechanisms are still unclear in non-small cell lung cancer (NSCLC). METHODS: The expression of ENO1 was examined in NSCLC and non-cancerous lung tissues, NSCLC cell lines, and immortalized human bronchial epithelial cell (HBE) by quantitative real-time reverse transcription PCR (qRT-PCR), immunohistochemistry, and Western blot, respectively. The effects and modulatory mechanisms of ENO1 on cell glycolysis, growth, migration, invasion, and in vivo tumorigenesis and metastasis in nude mice were also analyzed. RESULTS: ENO1 expression was increased in NSCLC tissues in comparison to non-cancerous lung tissues. Similarly, NSCLC cell lines A549 and SPCA-1 also express higher ENO1 than HBE cell line in both mRNA and protein levels. Overexpressed ENO1 significantly elevated NSCLC cell glycolysis, proliferation, clone formation, migration, and invasion in vitro, as well as tumorigenesis and metastasis in vivo by regulating the expression of glycolysis, cell cycle, and epithelial-mesenchymal transition (EMT)-associated genes. Conversely, ENO1 knockdown reversed these effects. More importantly, our further study revealed that stably upregulated ENO1 activated FAK/PI3K/AKT and its downstream signals to regulate the glycolysis, cell cycle, and EMT-associated genes. CONCLUSION: This study showed that ENO1 is responsible for NSCLC proliferation and metastasis; thus, ENO1 might serve as a potential molecular therapeutic target for NSCLC treatment.

Glucagon-like peptide-1 protects against cardiac microvascular endothelial cells injured by high glucose.

OBJECTIVE: To investigate the protective effect of glucagon-like peptid-1 (GLP-1) against cardiac microvascular endothelial cell (CMECs) injured by high glucose. METHODS: CMECs were isolated and cultured. Superoxide assay kit and dihydroethidine (DHE) staining were used to assess oxidative stress. TUNEL staining and caspase 3 expression were used to assess the apoptosis of CMECs. H89 was used to inhibit cAMP/PKA pathway; fasudil was used to inhibit Rho/ROCK pathway. The protein expressions of Rho, ROCK were examined by Western blot analysis. RESULTS: High glucose increased the production of ROS, the activity of NADPH, the apoptosis rate and the expression level of Rho/ROCK in CMECs, while GLP-1 decreased high glucose-induced ROS production, the NADPH activity and the apoptosis rate and the expression level of Rho/ROCK in CMECs, the difference were statistically significant (P<0.05). CONCLUSIONS: GLP-1 could protect the cardiac microvessels against oxidative stress and apoptosis. The protective effects of GLP-1 are dependent on downstream inhibition of Rho through a cAMP/PKA-dependent manner, resulting in a subsequent decrease in the expression of NADPH oxidase.

Efficacy and safety of a novel multi-electrode radiofrequency ablation catheter for renal sympathetic denervation in pigs.

OBJECTIVE: To investigate the safety and efficacy of a self-developed novel multi-electrode radiofrequency ablation catheter (Spark) for catheter-based renal denervation (RDN). METHODS: A total of 14 experimental miniature pigs were randomly divided into four groups (55°& 5-watt, 55°& 8-watt, 65°& 5-watt, and 65° & 8-watt groups). Spark was used for left and right renal artery radiofrequency ablation. Blood samples collected from renal arteries and veins as well as renal arteriography were performed on all animals before, immediately after, and three months after procedure to evaluate the effects of Spark on the levels of plasma renin, aldosterone, angiotensin I, and angiotensin II as well as the pathological changes of renal arteries. RESULTS: One pig died of an anesthetic accident, 13 pigs successfully underwent the bilateral renal artery ablation. Compared with basic measurements, pigs in all the four groups had significantly decreased mean arterial pressure after procedure. Histopathological analysis showed that this procedure could result in intimal hyperplasia, significant peripheral sympathetic nerve damage in the renal arteries such as inflammatory cell infiltration and fibrosis in perineurium, uneven distribution of nerve fibers, tissue necrosis, severe vacuolization, fragmented and unclear nucleoli myelin degeneration, sparse axons, and interruption of continuity. In addition, the renal artery radiofrequency ablation could significantly reduce the levels of plasma renin, aldosterone, angiotensin I, and angiotensin II in pigs. CONCLUSIONS: The results suggest that this type of multi-electrode catheter-based radiofrequency ablation could effectively remove peripheral renal sympathetic nerves and reduce the activity of systemic renin-angiotensin system in pigs, thus facilitating the control of systemic blood pressure in pigs.

A novel NKX2-5 loss-of-function mutation predisposes to familial dilated cardiomyopathy and arrhythmias.

Dilated cardiomyopathy (DCM) is the most prevalent type of primary myocardial disease, which is the third most common cause of heart failure and the most frequent reason for heart transplantation. Aggregating evidence demonstrates that genetic risk factors are involved in the pathogenesis of idiopathic DCM. Nevertheless, DCM is of remarkable genetic heterogeneity and the genetic defects underpinning DCM in an overwhelming majority of patients remain unknown. In the present study, the whole coding exons and splice junction sites of the NKX2-5 gene, which encodes a homeodomain transcription factor crucial for cardiac development and structural remodeling, were sequenced in 130 unrelated patients with idiopathic DCM. The available relatives of the index patient harboring an identified mutation and 200 unrelated ethnically matched healthy individuals used as controls were genotyped for the NKX2-5 gene. The functional effect of the mutant NKX2-5 was characterized in contrast to its wild-type counterpart using a dual-luciferase reporter assay system. As a result, a novel heterozygous NKX2-5 mutation, p.S146W, was identified in a family with DCM inherited as an autosomal dominant trait, which co-segregated with DCM in the family with complete penetrance. Notably, the mutation carriers also had arrhythmias, such as paroxysmal atrial fibrillation and atrioventricular block. The missense mutation was absent in 400 reference chromosomes and the altered amino acid was completely conserved evolutionarily among species. Functional analysis revealed that the NKX2-5 mutant was associated with a significantly reduced transcriptional activity. The findings expand the mutational spectrum of NKX2-5 linked to DCM and provide novel insight into the molecular mechanisms underlying DCM, contributing to the antenatal prophylaxis and allele-specific management of DCM.

A novel NKX2.5 loss-of-function mutation associated with congenital bicuspid aortic valve.

Bicuspid aortic valve (BAV) is the most common form of congenital cardiovascular defect in humans and is associated with substantial morbidity and mortality. Emerging evidence demonstrates that genetic risk factors play an important role in the pathogenesis of BAV. However, BAV is a genetically heterogenous disorder, and the genetic defects underpinning BAV in most patients remain to be identified. In the present study, the coding exons and flanking introns of the NKX2.5 gene, which encodes a homeodomain-containing transcription factor essential for the normal development of the aortic valve, were sequenced in 142 unrelated patients with BAV. The available relatives of the mutation carrier and 200 unrelated healthy subjects used as controls were also genotyped for NKX2.5. The functional characteristics of the mutation were delineated by using a dual-luciferase reporter assay system. As a result, a novel heterozygous NKX2.5 mutation, p.K192X, was identified in a family with BAV transmitted in an autosomal dominant pattern. The nonsense mutation was absent in 400 control chromosomes. Functional analyses revealed that the mutant NKX2.5 had no transcriptional activity compared with its wild-type counterpart. Furthermore, the mutation abolished the synergistic transcriptional activation between NKX2.5 and GATA5, another transcription factor crucial for the aortic valvular morphogenesis. In conclusion, this study is the first to link an NKX2.5 loss-of-function mutation to enhanced susceptibility to human BAV, providing novel insight into the molecular mechanism of BAV and suggesting potential implications for genetic counseling and clinical care of families presenting with BAV.

NKX2-6 mutation predisposes to familial atrial fibrillation.

Atrial fibrillation (AF) is the most common form of sustained cardiac arrhythmia and is associated with substantially increased morbidity and mortality rates. Aggregating evidence demonstrates that genetic defects are involved in the pathogenesis of AF and a number of AF-associated genes have been identified. Nevertheless, AF is a genetically heterogeneous disorder and the genetic components underpinning AF in an overwhelming majority of patients remain unclear. In this study, the entire coding exons and splice junction sites of the NK2 homeobox 6 (NKX2-6) gene, which encodes a homeodomain transcription factor important for cardiovascular development, were sequenced in 150 unrelated patients with lone AF, and a novel heterozygous NKX2-6 mutation, p.Q175H, was identified in an index patient. Genetic analysis of the available family members of the mutation carrier revealed that the mutation co-segregated with AF transmitted in an autosomal dominant pattern. The missense mutation was absent in the 200 unrelated ethnically matched healthy individuals used as controls and the altered amino acid was completely conserved evolutionarily among species. Due to unknown transcriptional targets of NKX2-6, the functional characteristics of the mutation as regards transcriptional activity were analyzed using NKX2-5 as a surrogate. Alignment between human NKX2-6 and NKX2-5 proteins displayed that the Q175H-mutant NKX2-6 was equivalent to the Q181H-mutant NKX2-5, and the introduction of Q181H into NKX2-5 significantly decreased its transcriptional activity at the atrial natriuretic factor promoter. The present study firstly associates genetically defective NKX2-6 with enhanced susceptibility to AF, providing novel insight into the molecular mechanisms underlying AF and suggesting potential strategies for the antenatal prophylaxis and personalized treatment of AF.

Prevalence and spectrum of Nkx2.6 mutations in patients with congenital heart disease.

Congenital heart disease (CHD) is the most common form of birth defect and is the most prevalent non-infectious cause of infant death. A growing body of evidence documents that genetic defects are involved in the pathogenesis of CHD. However, CHD is a genetically heterogeneous disease and the genetic basis underpinning CHD in an overwhelming majority of patients remain unclear. In this study, the coding exons and flanking introns of the Nkx2.6 gene, which codes for a homeodomain-containing transcription factor important for normal cardiovascular development, were sequenced in 320 unrelated patients with CHD, and two novel heterozygous Nkx2.6 mutations, p.V176M and p.K177X, were identified in two unrelated patients with CHD, respectively, including a patient with tetralogy of Fallot and a patient with double outlet of right ventricle and ventricular septal defect. The mutations were absent in 400 control chromosomes and the altered amino acids were completely conserved evolutionarily across species. Due to unknown transcriptional targets of Nkx2.6, the functional consequences of the identified mutations at transcriptional activity were evaluated by using Nkx2.5 as a surrogate. Alignment between human Nkx2.6 and Nkx2.5 proteins showed that V176M-mutant Nkx2.6 was equivalent to V182M-mutant Nkx2.5 and K177X-mutant Nkx2.6 was equal to K183X-mutant Nkx2.5, and introduction of V182M or K183X into Nkx2.5 significantly diminished its transcriptional activating function when compared with its wild-type counterpart. To our knowledge, this is the first report on the association of Nkx2.6 loss-of-function mutation with increased susceptibility to tetralogy of Fallot or double outlet of right ventricle and ventricular septal defect, providing novel insight into the molecular mechanism of CHD.

GATA6 loss-of-function mutations contribute to familial dilated cardiomyopathy.

Dilated cardiomyopathy (DCM), the most prevalent form of primary heart muscle disease, is the third most common cause of heart failure and the most frequent reason for cardiac transplantation. Mounting evidence has demonstrated that genetic risk factors are crucial in the pathogenesis of DCM. However, DCM is genetically heterogeneous, and the genetic basis of DCM in a large majority of cases remains unclear. In the current study, the coding exons and flanking introns of the GATA6 gene, which encodes a zinc­finger transcription factor essential for cardiogenesis, was sequenced in 140 unrelated patients with DCM, and two novel heterozygous mutations, p.C447Y and p.H475R, were identified in two index patients with DCM, respectively. Analysis of the pedigrees showed that in each family the mutation co-segregated with DCM transmitted in an autosomal-dominant pattern, with complete penetrance. The missense mutations were absent in 400 control chromosomes and predicted to be disease-causing by MutationTaster or probably damaging by PolyPhen-2. The alignment of multiple GATA6 proteins across species revealed that the altered amino acids were completely conserved evolutionarily. The functional assays showed that the mutated GATA6 proteins were associated with significantly reduced transcriptional activation in comparison with their wild-type counterpart. To the best of our knowledge, this is the first study on the association of GATA6 loss-of-function mutations with enhanced susceptibility to familial DCM, which provides novel insight into the molecular mechanism of DCM and suggests potential implications for the antenatal prophylaxis and allele-specific treatment of DCM.