Rongfeng Zhang

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.

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.

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.

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.

Common Variants in the TBX5 Gene Associated with Atrial Fibrillation in a Chinese Han Population

Background PR interval variations have recently been associated with an increased risk of long-term atrial fibrillation (AF), heart block and all-cause mortality. Genome-wide association studies have linked the PR interval with several common variants in the TBX5 gene. Several variants in the TBX5 gene, including rs7312625 and rs883079, have been associated with AF. The purpose of this study was to determine the association of single-nucleotide polymorphisms (SNPs) in the TBX5 gene, rs7312625 and rs883079, with AF in Chinese Han patients. Methodology/Principal Findings In this case-control association study, large cohorts of AF patients (n = 1132) and controls (n = 1206) were recruited from different hospitals. The genotyping was performed using a Rotor-Gene TM 6000 high-resolution melt system. Rs7312625, rs3825214 and rs883079 were analyzed. We found that SNP 3825214 was significantly associated with AF (P-obs = 0.002, odds ratio [OR] = 0.82), and lone AF (P-obs = 6.77x10-5, odds ratio [OR] = 0.71). SNP rs7312625 was significantly associated with lone AF (P-obs = 0.015, odds ratio [OR] = 1.27), although its association with AF was not significant. No significant association of SNP rs883079 with AF or lone AF was observed. Thus, we analyzed the interaction among these three loci. We demonstrated significant interaction among rs3825214, rs7312625 and rs883079. Four-locus risk alleles showed the highest odds ratio in combined rs3825214 and rs7312625 (P-obs<0.0001, odds ratio [OR] = 2.21). Six-locus risk alleles showed the highest odds ratio in combined rs3825214, rs7312625 and rs 883079(P-obs<0.0001, odds ratio [OR] = 2.35). Significance was established with the trend test (P<0.0001). Conclusions For the first time, we report the strong association of SNP rs3825214 in the TBX5 gene with AF and lone AF in a Chinese Han population. Rs7312625 was significantly associated with lone AF, and snp-snp interaction increased the risk of atrial fibrillation. Our data might provide new insights into understanding AF pathogenesis and designing novel genetic therapies for AF patients.

Candidate Pathway-Based GWAS Identifies 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.

Genomic Variants in NEURL, GJA1 and CUX2 Significantly Increase Genetic Susceptibility to Atrial Fibrillation.

Atrial fibrillation (AF) is the most common arrhythmia. In 2014, two new meta-GWAS identified 5 AF loci, including the NEURL locus, GJA1 locus, CAND2 locus, and TBX5 locus in the European ancestry populations and the NEURL locus and CUX2 locus in a Japanese population. The TBX5 locus for AF was reported by us in 2013 in the Chinese population. Here we assessed the association between AF and SNPs in the NEURL, GJA1, CAND2 and CUX2 loci in the Chinese Han population. We carried out a large case-control association study with 1,164 AF patients and 1,460 controls. Significant allelic and genotypic associations were identified between NEURL variant rs6584555 and GJA1 variant rs13216675 and AF. Significant genotypic association was found between CUX2 SNP rs6490029 and AF. No association was found between CAND2 variant rs4642101 and AF, which may be due to an insufficient power of the sample size for rs4642101. Together with our previous findings, seven of fifteen AF loci (<50%) identified by GWAS in the European ancestry populations conferred susceptibility to AF in the Chinese population, and explained approximately 14.5% of AF heritability. On the other hand, two AF loci identified in the Japanese population were both replicated in the Chinese population.

Atrial fibrillation is related to lower incidence of deep venous thrombosis in patients with pulmonary embolism

Background Atrial fibrillation (AF) is an established risk factor of left atrial thrombosis and systemic embolism. Traditionally pulmonary embolism (PE) is a recognized complication of deep vein thrombosis (DVT). However, whether AF is responsible for right atrial thrombosis and leads to PE has not been examined. Methods We retrospectively analyzed medical records of patients with confirmed diagnosis of PE with AF (study group) from 2002-2015. Patients with PE without AF, matched by age and sex, served as controls (control group). The CHA2DS2-VASc and CHADS2 scores were classified into two categories, low-intermediate (<2 points) and high-risk (≥2 points). Results A total of 330 patients (110 in study group and 220 in control group). The study group had significantly lower incidence of newly diagnosed DVT (21% vs. 44%, P<0.001), previous history of DVT (6% vs. 17%, P=0.006) and recent surgery or trauma (10% vs. 23%, P=0.004) compared to the control group. When stratified by the CHADS2 score, 49 patients (44.5%) were considered low-intermediate risk. This proportion significantly differed when stratified using CHA2DS2-VASc, in which 13 patients (13.6%) were considered low-intermediate risk, P<0.001. Conclusions The incidence of DVT was much lower in the study group, suggesting the possibility of clots originated from the right heart that may increase the risk of PE. The CHA2DS2-VASc scoring system might be more sensitive for prediction and stratification of the PE in AF patients than the CHADS2 score.

Frequency Gradient Within Coronary Sinus Predicts the Long‐Term Outcome of Persistent Atrial Fibrillation Catheter Ablation

Background The coronary sinus (CS), as a junction of the atria, contributes to atrial fibrillation (AF) by developing unstable reentry, and isolating the atria by ablation at the CS could terminate AF. The present study evaluated whether AF activities at the CS in a subset of patients contributed to AF maintenance and predicted clinical outcome of ablation. Methods and Results We studied 122 consecutive patients who had a first‐time radiofrequency ablation for persistent AF. Bipolar electrograms were obtained from multiple regions of the left atrium by a Lasso mapping catheter before ablation. Pulmonary vein isolation terminated AF in 12 patients (9.8%). Sequential stepwise ablation was conducted in pulmonary vein isolation nontermination patients and succeeded in 22 patients (18%). In the stepwise termination group, AF frequency in the proximal CS (CSp) was significantly higher (10.2±2.1 Hz versus 8.3±1.8 Hz, P<0.001), and the ratio of distal CS (CSd) to proximal CS (CSd/CSp ratio, 56.6%±10.11% versus 70.7%±9.8%, P<0.001) was significantly lower than that in the nontermination group. The stepwise logistic regression analysis indicated that the CSd/CSp ratio was an independent predictor with an odds ratio of 1.131 (95%CI 1.053‐1.214; P=0.001). With a cutoff of 67%, the patients with lower CSd/CSp ratios had significantly better index and long‐term outcomes than those with higher ratios during a follow‐up of 46±18 months. Conclusions Rapid repetitive activities in the musculature of the proximal CS may contribute to maintenance of AF after pulmonary vein isolation alone in persistent AF. A cutoff at 67%, of the CSd/CSp frequency ratio might be an indicator to stratify the subset of patients who might benefit from CS ablation.