Yanzong Yang

Functional dominant-negative mutation of sodium channel subunit gene SCN3B associated with atrial fibrillation in a Chinese GeneID population

Atrial fibrillation (AF) is the most common cardiac arrhythmia in the clinic, and accounts for more than 15% of strokes. Mutations in cardiac sodium channel alpha, beta1 and beta2 subunit genes (SCN5A, SCN1B, and SCN2B) have been identified in AF patients. We hypothesize that mutations in the sodium channel beta3 subunit gene SCN3B are also associated with AF. To test this hypothesis, we carried out a large scale sequencing analysis of all coding exons and exon-intron boundaries of SCN3B in 477 AF patients (28.5% lone AF) from the GeneID Chinese Han population. A novel A130V mutation was identified in a 46-year-old patient with lone AF, and the mutation was absent in 500 controls. Mutation A130V dramatically decreased the cardiac sodium current density when expressed in HEK293/Na(v)1.5 stable cell line, but did not have significant effect on kinetics of activation, inactivation, and channel recovery from inactivation. When co-expressed with wild type SCN3B, the A130V mutant SCN3B negated the function of wild type SCN3B, suggesting that A130V acts by a dominant negative mechanism. Western blot analysis with biotinylated plasma membrane protein extracts revealed that A130V did not affect cell surface expression of Na(v)1.5 or SCN3B, suggesting that mutant A130V SCN3B may not inhibit sodium channel trafficking, instead may affect conduction of sodium ions due to its malfunction as an integral component of the channel complex. This study identifies the first AF-associated mutation in SCN3B, and suggests that mutations in SCN3B may be a new pathogenic cause of AF.

Regulation of CARD8 Expression by ANRIL and Association of CARD8 Single Nucleotide Polymorphism rs2043211 (p.C10X) With Ischemic Stroke

Background and Purpose— ANRIL has long been considered as the strongest candidate gene at the 9p21 locus, robustly associated with stroke and coronary artery disease. However, the underlying molecular mechanism remains unknown. The present study works to elucidate such a mechanism. Methods— Using expression quantitative loci analysis, we identified potential genes whose expression may be influenced by genetic variation in ANRIL. To verify the identified gene(s), knockdown and overexpression of ANRIL were evaluated in human umbilical vein endothelial cells and HepG2 cells. Ischemic stroke and coronary artery disease risk were then evaluated in the gene(s) demonstrated to be mediated by ANRIL in 3 populations of Chinese Han ancestry: 2 ischemic stroke populations consisting of the Central China cohort (903 cases and 873 controls) and the Northern China cohort (816 cases and 879 controls) and 1 coronary artery disease cohort consisting of 772 patients and 873 controls. Results— Expression quantitative loci analysis identified CARD8 among others, with knockdown of ANRIL expression decreasing CARD8 expression and overexpression of ANRIL increasing CARD8 expression. The minor T allele of a previously identified CARD8 variant (rs2043211) was found to be significantly associated with a protective effect of ischemic stroke under the recessive model in 2 independent stroke cohorts. No significant association was found between rs2043211 and coronary artery disease. Conclusions— CARD8 is a downstream target gene regulated by ANRIL. Single nucleotide polymorphism rs2043211 in CARD8 is significantly associated with ischemic stroke. ANRIL may increase the risk of ischemic stroke through regulation of the CARD8 pathway.

Molecular Basis of Gene-Gene Interaction: Cyclic Cross-Regulation of Gene Expression and Post-GWAS Gene-Gene Interaction Involved in Atrial Fibrillation

Atrial fibrillation (AF) is the most common cardiac arrhythmia at the clinic. Recent GWAS identified several variants associated with AF, but they account for <10% of heritability. Gene-gene interaction is assumed to account for a significant portion of missing heritability. Among GWAS loci for AF, only three were replicated in the Chinese Han population, including SNP rs2106261 (G/A substitution) in ZFHX3, rs2200733 (C/T substitution) near PITX2c, and rs3807989 (A/G substitution) in CAV1. Thus, we analyzed the interaction among these three AF loci. We demonstrated significant interaction between rs2106261 and rs2200733 in three independent populations and combined population with 2,020 cases/5,315 controls. Compared to non-risk genotype GGCC, two-locus risk genotype AATT showed the highest odds ratio in three independent populations and the combined population (OR=5.36 (95% CI 3.87-7.43), P=8.00×10-24). The OR of 5.36 for AATT was significantly higher than the combined OR of 3.31 for both GGTT and AACC, suggesting a synergistic interaction between rs2106261 and rs2200733. Relative excess risk due to interaction (RERI) analysis also revealed significant interaction between rs2106261 and rs2200733 when exposed two copies of risk alleles (RERI=2.87, P<1.00×10-4) or exposed to one additional copy of risk allele (RERI=1.29, P<1.00×10-4). The INTERSNP program identified significant genotypic interaction between rs2106261 and rs2200733 under an additive by additive model (OR=0.85, 95% CI: 0.74-0.97, P=0.02). Mechanistically, PITX2c negatively regulates expression of miR-1, which negatively regulates expression of ZFHX3, resulting in a positive regulation of ZFHX3 by PITX2c; ZFHX3 positively regulates expression of PITX2C, resulting in a cyclic loop of cross-regulation between ZFHX3 and PITX2c. Both ZFHX3 and PITX2c regulate expression of NPPA, TBX5 and NKX2.5. These results suggest that cyclic cross-regulation of gene expression is a molecular basis for gene-gene interactions involved in genetics of complex disease traits.

Clinical and electrophysiological characterization of a novel mutation R863X in HERG C-terminus associated with long QT syndrome.

We have found a novel nonsense mutation in the C-terminus of HERG in a four-generation Chinese family with long QT syndrome and investigated the molecular mechanism of this mutation in vitro. Six family members, including the proband, were clinically affected. Syncope and ventricular tachycardia of torsades de pointes were triggered by startling or emotional stress, and β-adrenergic blockade treatment was ineffective. Haplotype analysis showed that only LQT2 markers cosegregated with the disease, and sequence analysis revealed a substitution of T with C at nucleotide position 2770 of the HERG gene (U04270), which creates a stop codon at amino acid position 863 (R863X) of the HERG protein, leading to a deletion of 296 amino acids. Whole cell patch clamp studies showed that the R863X HERG could not induce time-dependent current. Coexpression of R863X with wild-type HERG showed reduced current densities and accelerated voltage-dependent inactivation of HERG channels. Subcellular localization of R863X-EGFP revealed that the mutant did not traffic to the cell surface. These data suggest that R863X failed to form functional HERG channels, contributing to a prolongation of the QT interval and long QT syndrome with a dominant phenotype. These findings provide new insights into the structure-function relationships of the HERG C-terminus.

Meta-analysis identifies robust association between SNP rs17465637 in MIA3 on chromosome 1q41 and coronary artery disease

Several large-scale meta-GWAS identified significant association between SNP rs17465637 in the MIA3 gene and coronary artery disease (CAD) in the European ancestry populations. However, three follow-up replication studies in the Chinese populations yielded inconsistent results. In order to unequivocally determine whether SNP rs17465637 is associated with CAD, we performed an independent case control association study in the Chinese Han population and a follow-up large scale meta-analysis for SNP rs17465637. Our study included 2503 CAD patients and 2920 non-CAD controls of the Chinese Han origin. A significant association was found between SNP rs17465637 and CAD (P = 0.01, OR = 1.11). Meta-analysis included 7263 CAD patients and 8347 controls combined from five Asian populations. The association between SNP rs17465637 and CAD became highly significant (P = 4.97 × 10(-5), OR = 1.11). Similar analysis also identified significant association between SNP rs17465637 and MI (2424 cases vs. 6,536controls; P = 5.00 × 10(-3), OR = 1.10). We conclude that SNP rs17465637 in MIA3 is indeed a genetic risk factor for CAD across different ethnic populations.

Candidate Pathway-Based Genome-Wide Association Studies Identify Novel Associations of Genomic Variants in the Complement System Associated With Coronary Artery Disease

Background—Genomic variants identified by genome-wide association studies (GWAS) explain <20% of heritability of coronary artery disease (CAD), thus many risk variants remain missing for CAD. Identification of new variants may unravel new biological pathways and genetic mechanisms for CAD. To identify new variants associated with CAD, we developed a candidate pathway-based GWAS by integrating expression quantitative loci analysis and mining of GWAS data with variants in a candidate pathway. Methods and Results—Mining of GWAS data was performed to analyze variants in 32 complement system genes for positive association with CAD. Functional variants in genes showing positive association were then identified by searching existing expression quantitative loci databases and validated by real-time reverse transcription polymerase chain reaction. A follow-up case–control design was then used to determine whether the functional variants are associated with CAD in 2 independent GeneID Chinese populations. Candidate pathway-based GWAS identified positive association between variants in C3AR1 and C6 and CAD. Two functional variants, rs7842 in C3AR1 and rs4400166 in C6, were found to be associated with expression levels of C3AR1 and C6, respectively. Significant association was identified between rs7842 and CAD (P=3.99×10−6; odds ratio, 1.47) and between rs4400166 and CAD (P=9.30×10−3; odds ratio, 1.24) in the validation cohort. The significant findings were confirmed in the replication cohort (P=1.53×10−5; odds ratio, 1.37 for rs7842; P=8.41×10−3; odds ratio, 1.21 for rs4400166). Conclusions—Integration of GWAS with biological pathways and expression quantitative loci is effective in identifying new risk variants for CAD. Functional variants increasing C3AR1 and C6 expression were shown to confer significant risk of CAD for the first time.

SNP rs3825214 in TBX5 Is Associated with Lone Atrial Fibrillation in Chinese Han Population

Background A prolonged PR interval is a sign of increased risk of cardiac arrhythmia. Recent genome-wide association studies found that the single-nucleotide polymorphism (SNP) rs3825214 in T-box 5 (TBX5) was positively associated with PR interval, QRS duration, QT interval, and common arrhythmia disorders such as atrial fibrillation (AF) and advanced atrioventricular block. However, other independent replication studies are required to validate the result. This study assessed associations between rs3825214 and ECG parameters, AF, and ventricular tachycardia (VT) in a Chinese Han population. Methodology/Principal Findings To assess the association between rs3825214 and AF and VT, we carried out case-control association studies with 692 AF patients (including 275 lone AF patients), 235 VT patients, and 856 controls. Genotyping was performed using a Rotor-Gene TM 6000 High Resolution Melt system. Statistical analyses of associations were adjusted for potential confounding factors. A moderate association was detected between rs3825214 and AF (P adj = 0.036, OR = 0.79) and a highly significant association was detected between the G allele of rs3825214 and lone AF (P adj = 0.001, OR = 0.65; genotypic P = 3.75×10−4 with a dominant model). We also found that rs3825214 showed a significant association with atrial-ventricular block (AVB; P = 0.028; P adj = 0.035, OR = 0.494). Conclusions Our results indicate that rs3825214 conferred a significant risk of lone AF in this Chinese Han population.

Synergistic association of DNA repair relevant gene polymorphisms with the risk of coronary artery disease in northeastern Han Chinese

Evidence is mounting suggesting that DNA damage is implicated in the development and progression of atherosclerosis. To yield more information, we focused on six well-characterized polymorphisms from four DNA repair-relevant candidate genes, viz. XRCC1 (rs1799782 and rs25487), XRCC3 (rs861539), MTHFR (rs1801133 and rs4846049), and NQO1 (rs1800566), to identify and characterize their potential gene-to-gene interactions in susceptibility to coronary artery disease (CAD) in Han Chinese. This was a hospital-based case-control study involving 1142 patients diagnosed with CAD and 1106 age- and gender-matched controls. All participants were angiographically confirmed. Risk estimates were expressed as odds ratio (OR) and 95% confidence interval (95% CI). All six examined polymorphisms met Hardy-Weinberg equilibrium. Overall there were significant differences in the genotype/allele distributions of MTHFR gene rs1801133 and rs4846049 (both P ≤ 0.005), and in the genotype distributions of XRCC1 gene rs1799782 (P = 0.002) between patients and controls. The adjusted risk of having CAD was more evident for rs1799782 (OR = 1.53; 95% CI: 1.16-2.02; P = 0.003), rs1801133 (OR = 1.54; 95% CI: 1.22-1.94; P < 0.001), and rs4846049 (OR = 1.74; 95% CI: 1.13-2.69; P = 0.013) under the recessive model. Interaction analyses indicated that the overall best multifactor dimensionality reduction (MDR) model included rs4846049, rs1801133, and rs1799782, and this model had a maximal testing accuracy of 0.6885 and a cross-validation consistency of 10 out of 10 (P = 0.0030). Further interaction entropy graph bore out the validity of this MDR model. Taken together, our findings demonstrate a contributory role of genetic defects in XRCC1 and MTHFR genes, both individually and interactively, in the development of CAD in Han Chinese.

Genomic variant in CAV1 increases susceptibility to coronary artery disease and myocardial infarction

BACKGROUND The CAV1 gene encodes caveolin-1 expressed in cell types relevant to atherosclerosis. Cav-1-null mice showed a protective effect on atherosclerosis under the ApoE(-/-) background. However, it is unknown whether CAV1 is linked to CAD and MI in humans. In this study we analyzed a tagSNP for CAV1 in intron 2, rs3807989, for potential association with CAD. METHODS AND RESULTS We performed case-control association studies in three independent Chinese Han populations from GeneID, including 1249 CAD cases and 841 controls in Population I, 1260 cases and 833 controls in Population II and 790 cases and 1212 controls in Population III (a total of 3299 cases and 2886 controls). We identified significant association between rs3807989 and CAD in three independent populations and in the combined population (Padj = 2.18 × 10(-5), OR = 1.19 for minor allele A). We also detected significant association between rs3807989 and MI (Padj = 5.43 × 10(-5), OR = 1.23 for allele A). Allele A of SNP rs3807989 was also associated with a decreased level of LDL cholesterol. Although rs3807989 is a tagSNP for both CAV1 and nearby CAV2, allele A of SNP rs3807989 was associated with an increased expression level of CAV1 (both mRNA and protein), but not CAV2. CONCLUSIONS The data in this study demonstrated that rs3807989 at the CAV1/CAV2 locus was associated with significant risk of CAD and MI by increasing expression of CAV1 (but not CAV2). Thus, CAV1 becomes a strong candidate susceptibility gene for CAD/MI in humans.

An Interactive Association of Advanced Glycation End-Product Receptor Gene Four Common Polymorphisms with Coronary Artery Disease in Northeastern Han Chinese

Background Growing evidence indicates that advanced glycation end-product receptor (RAGE) might play a contributory role in the pathogenesis of coronary artery disease (CAD). To shed some light from a genetic perspective, we sought to investigate the interactive association of RAGE gene four common polymorphisms (rs1800625 or T-429C, rs1800624 or T-374A, rs2070600 or Gly82Ser, and rs184003 or G1704A) with the risk of developing CAD in a large northeastern Han Chinese population. Methodology/Principal Findings This was a hospital-based case-control study incorporating 1142 patients diagnosed with CAD and 1106 age- and gender-matched controls. All individuals were angiographically confirmed. Risk estimates were expressed as odds ratio (OR) and 95% confidence interval (CI). Overall there were significant differences in the genotype and allele distributions of rs1800625 and rs184003, even after the Bonferroni correction. Logistic regression analyses indicated that rs1800625 and rs184003 were associated with significant risk of CAD under both additive (OR = 1.20 and 1.23; 95% CI: 1.06–1.37 and 1.06–1.42; P = 0.006 and 0.008) and recessive (OR = 1.75 and 2.39; 95% CI: 1.28–2.40 and 1.47–3.87; P<0.001 and <0.001) models after adjusting for confounders. In haplotype analyses, haplotypes C-T-G-G and T-A-G-T (alleles in order of rs1800625, rs1800624, rs2070600 and rs184003), overrepresented in patients, were associated with 52% (95% CI: 1.19–1.87; P = 0.0052) and 63% (95% CI: 1.14–2.34; P = 0.0075) significant increases in adjusted risk for CAD. Further interactive analyses identified an overall best multifactor dimensionality reduction (MDR) model including rs1800625 and rs184003. This model had a maximal testing accuracy of 0.6856 and a cross-validation consistency of 10 out of 10 (P = 0.0016). The validity of this model was substantiated by classical Logistic regression analysis. Conclusions Our findings provided strong evidence for the potentially contributory roles of RAGE multiple genetic polymorphisms, especially in the context of locus-to-locus interaction, in the pathogenesis of CAD among northeastern Han Chinese.