Trafficking-Deficient G572R-hERG and E637K-hERG Activate Stress and Clearance Pathways in Endoplasmic Reticulum

Background: Long QT syndrome type 2 (LQT2) is the second most common type of all long QT syndromes. It is well-known that trafficking deficient mutant human ether-a-go-go-related gene (hERG) proteins are often involved in LQT2. Cells respond to misfolded and trafficking-deficient proteins by eliciting the unfolded protein response (UPR) and Activating Transcription Factor (ATF6) has been identified as a key regulator of the mammalian UPR. In this study, we investigated the role of ER chaperone proteins (Calnexin and Calreticulin) in the processing of G572R-hERG and E637K-hERG mutant proteins. Methods: pcDNA3-WT-hERG, pcDNA3-G572R-hERG and pcDNA3-E637K-hERG plasmids were transfected into U2OS and HEK293 cells. Confocal microscopy and western blotting were used to analyze subcellular localization and protein expression. Interaction between WT or mutant hERGs and Calnexin/Calreticulin was tested by coimmunoprecipitation. To assess the role of the ubiquitin proteasome pathway in the degradation of mutant hERG proteins, transfected HEK293 cells were treated with proteasome inhibitors and their effects on the steady state protein levels of WT and mutant hERGs were examined. Conclusion: Our results showed that levels of core-glycosylated immature forms of G572R-hERG and E637K-hERG in association with Calnexin and Calreticulin were higher than that in WT-hERG. Both mutant hERG proteins could activate the UPR by upregulating levels of active ATF6. Furthermore, proteasome inhibition increased the levels of core-glycosylated immature forms of WT and mutant hERGs. In addition, interaction between mutant hERGs and Calnexin/Calreticulin wa

A preliminary study of the relationship between promoter methylation of the ABCG1, GALNT2 and HMGCR genes and coronary heart disease.

To investigate the association of ABCG1, GALNT2 and HMGCR genes promoter DNA methylation with coronary heart disease (CHD) and explore the interaction between their methylation status and the CHD patients' clinical characteristics in Han Chinese population.Methylation-specific polymerase chain reaction (MSP) technology was used to examine the role of the aberrant gene promoter methylation in CHD in Han Chinese population. A total of 85 CHD patients and 54 participants without CHD confirmed by angiography were recruited. 82.8% of the participants with ABCG1 gene promoter hypermethylation have CHD, while only 17.4% of the participants without hypermethylation have it. The average age of the participants with GALNT2 gene promoter hypermethylation is 62.10 ± 8.21, while that of the participants without hypermethylation is 57.28 ± 9.87; in the former group, 75.4% of the participants have CHD, compared to only 50% in the latter group. As for the HMGCR gene, the average age of the participants with promoter hypermethylation is 63.24 ± 8.10 and that of the participants without hypermethylation is 57.79 ± 9.55; its promoter hypermethylation is likely to be related to smoking. Our results indicated a significant statistical association of promoter methylation of the ABCG1 gene with increased risk of CHD (OR = 19.966; 95% CI, 7.319-54.468; P*<0.001; P*: adjusted for age, gender, smoking, lipid level, hypertension, and diabetes). Similar results were obtained for that of the GALNT2 gene (OR = 2.978; 95% CI, 1.335-6.646; P* = 0.008), but not of HMGCR gene (OR = 1.388; 95% CI, 0.572-3.371; P*  = 0.469).The present work provides evidence to support the association of promoter DNA methylation status with the risk profile of CHD. Our data indicates that promoter DNA hypermethylation of the ABCG1 and GALNT2 genes, but not the HMGCR gene, is associated with an increased risk of CHD. CHD, smoking and aging are likely to be the important factors influencing DNA hypermethylation

Positive Association between GCKR rs780093 Polymorphism and Coronary Heart Disease in the Aged Han Chinese

Objective. Previous studies have confirmed that GCKR rs780093 polymorphism is associated with triglyceride (TG), a known risk factor of coronary heart disease (CHD). The goal of our study is to explore the association of GCKR rs780093 polymorphism with CHD in Han Chinese population. Methods and Results. A total of 568 CHD cases and 494 non-CHD controls were enrolled in the current case-control study. Genotyping was done using melting temperature shift (Tm-shift) approach. Our results also showed that GCKR rs780093 polymorphism was significantly associated with TG level (P=0.0016). Although there was no significant association between cases and controls (P>0.05), a breakdown analysis by age yielded a significant association of GCKR rs780093 polymorphism with CHD in individuals aged 65 and older (genotype: χ2=6.86; df = 2; P=0.03; allele: χ2=4.11; df = 1; P=0.04). Conclusion. Our findings confirmed the contribution of GCKR rs780093 polymorphism to TG metabolism and demonstrated GCKR rs780093 as a risk factor of CHD in individuals aged 65 and older

Meta-Analyses of <i>KIF6</i> Trp719Arg in Coronary Heart Disease and Statin Therapeutic Effect

Aims: The goal of our study is to assess the contribution of KIF6 Trp719Arg to both the risk of CHD and the efficacy of statin therapy in CHD patients.Methods and Results: Meta-analysis of 8 prospective studies among 77,400 Caucasians provides evidence that 719Arg increases the risk of CHD (PP = 0.642, OR = 1.02, 95% CI = 0.95–1.08). This suggests that the contribution of Trp719Arg to CHD varies in different ethnic groups. Additional meta-analysis also shows that statin therapy only benefit the vascular patients carry 719Arg allele (PP = 0.04, χ2 = 4.228, df = 1, odd ratio (OR) = 1.979, 95% confidence interval (CI) = 1.023–3.828). Similar trends are observed for post hoc analysis between female CHD cases and female healthy controls (dominant model: P = 0.04, χ2 = 4.231, df = 1, OR = 2.015, 95% CI = 1.024–3.964). Non-genetic CHD risk factors are not controlled in these analyses.Conclusions: Our meta-analysis demonstrates the role of Trp719Arg of KIF6 gene in the risk of CHD in Caucasians. The meta-analysis also suggests the role of this variant in statin therapeutic response in vascular diseases. Our case-control study suggests that Trp719Arg of KIF6 gene is associated with CHD in female Han Chinese through a post hoc analysis.</p

Detection of Differentially Expressed MicroRNAs in Rheumatic Heart Disease: miR-1183 and miR-1299 as Potential Diagnostic Biomarkers

This study compared microRNA (miRNA) expression profiles between rheumatic heart disease (RHD) patients and healthy controls to investigate their differential expression and help elucidate their mechanisms of action. Microarray analysis was used to measure miRNA expression, and a total of 133 miRNAs were shown to be significantly upregulated in RHD patients compared with controls, including miR-1183 and miR-1299. A total of 137 miRNAs, including miR-4423-3p and miR-218-1-3p, were significantly downregulated in RHD patients. Quantitative real-time-PCR confirmed microarray findings for miR-1183 and miR-1299 in both tissue and plasma. Bioinformatic predictions were also made of differentially expressed miRNAs as biomarkers in RHD by databases and GO/pathway analysis. Furthermore, we investigated miR-1183 and miR-1299 expression in RHD patients with secondary pulmonary hypertension (PAH). Our findings identified an important role for miR-1299 as a direct regulator of RHD, while the observed difference in expression of miR-1183 between RHD-PAH patients with high or low pulmonary artery pressure suggests that miR-1183 overexpression may reflect pulmonary artery remodeling. miR-1183 and miR-1299 appear to play distinct roles in RHD pathogenesis accompanied by secondary PAH and could be used as potential biological markers for disease development

Primers for MSP of the <i>ABCG1</i>, <i>GALNT2</i> and <i>HMGCR</i> genes.

<p>MSP: methylation-specific polymerase chain reaction; M: methylation-specific primers; U: unmethylation-specific primers.</p

Activation of ATF6 by G572R-hERG and E637K-hERG.

<p>HEK293 cells were transfected with WT-hERG, G572R-hERG or E637K-hERG plasmid. Cell lysates were subjected to Western blotting with anti-ATF6 antibody. The cleaved, activated form of ATF6 at 50 kDa is only detected in G572R-hERG and E637K-hERG expressing cells but not in WT-hERG expressing cells.</p

Methylation percent of the <i>ABCG1</i>, <i>GALNT2</i> and <i>HMGCR</i> genes promoter in the CHD cases and Non-CHD controls according to subgroup analysis by total samples and gender.

<p>Methylation percent of the <i>ABCG1</i>, <i>GALNT2</i> and <i>HMGCR</i> genes promoter in the CHD cases and Non-CHD controls according to subgroup analysis by total samples and gender.</p