Chuchu Wang

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.

Significant Association Between CAV1 Variant rs3807989 on 7p31 and Atrial Fibrillation in a Chinese Han Population

Background Recent genome-wide association studies (GWAS) in European ancestry populations revealed several genomic loci for atrial fibrillation (AF). We previously replicated the 4q25 locus (PITX2) and 16q22 locus (ZFHX3) in the Chinese population, but not the KCNN3 locus on 1q21. With single-nucleotide polymorphism rs3807989 in CAV1 encoding caveolin-1, however, controversial results were reported in 2 Chinese replication studies. Methods and Results Six remaining AF genetic loci from GWAS, including rs3807989/CAV1, rs593479/PRRX1, rs6479562/C9orf3, rs10824026/SYNPO2L, rs1152591/SYNE2, and rs7164883/HCN4, were analyzed in a Chinese Han population with 941 cases and 562 controls. Only rs3807989 showed significant association with AF (Padj=4.77×10−5), and the finding was replicated in 2 other independent populations with 709 cases and 2175 controls, 463 cases and 644 controls, and the combined population with a total of 2113 cases and 3381 controls (Padj=2.20×10−9; odds ratio [OR]=1.34 for major allele G). Meta-analysis, together with data from previous reports in Chinese and Japanese populations, also showed a significant association between rs3807989 and AF (P=3.40×10−4; OR=1.24 for allele G). We also found that rs3807989 showed a significant association with lone AF in 3 independent populations and in the combined population (Padj=3.85×10−8; OR=1.43 for major allele G). Conclusions The data in this study revealed a significant association between rs3807989 and AF in the Chinese Han population. Together with the findings that caveolin-1 interacts with potassium channels Kir2.1, KCNH2, and HCN4 and sodium channels Nav1.5 and Nav1.8, CAV1 becomes a strong candidate susceptibility gene for AF across different ethnic populations. This study is the first to show a significant association between rs3807989 and lone AF.

Association of SNP Rs9943582 in APLNR with Left Ventricle Systolic Dysfunction in Patients with Coronary Artery Disease in a Chinese Han GeneID Population.

Heart failure affects 1–2% of the adult population worldwide and coronary artery disease (CAD) is the underlying etiology of heart failure in 70% of the patients. The pathway of apelin and its apelin receptor (APJ) was implicated in the pathogenesis of heart failure in animal models, but a similar role in humans is unknown. We studied a functional variant, rs9943582 (-154G/A), at the 5’-untranslated region, that was associated with decreased expression of the APJ receptor gene (APLNR) in a population consisting of 1,751 CAD cases and 1,022 controls. Variant rs9943582 was not associated with CAD, but among CAD patients, it showed significant association with left ventricular systolic dysfunction (431 CAD patients with left ventricular systolic dysfunction (LV ejection fraction or LVEF< 40%) versus 1,046 CAD patients without LV systolic dysfunction (LVEF>50%) (P-adj = 6.71×10-5, OR = 1.43, 95% CI, 1.20–1.70). Moreover, rs9943582 also showed significant association with quantitative echocardiographic parameters, including left ventricular end-diastolic diameter (effect size: increased 1.67±0.43 mm per risk allele A, P = 1.15×10-4), left atrial size (effect size: increased 2.12±0.61 mm per risk allele A, P = 9.56×10-4) and LVEF (effect size: decreased 2.59±0.32 percent per risk allele A, P = 7.50×10-15). Our findings demonstrate that allele A of rs9943582 was significantly associated with left ventricular systolic dysfunction, left ventricular end-diastolic diameter, the left atrial diameter and LVEF in the CAD population, which suggests an important role of the apelin/APJ system in the pathology of heart failure associated with ischemic heart disease.

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.

Genetic Regulation of the Thymic Stromal Lymphopoietin (TSLP)/TSLP Receptor (TSLPR) Gene Expression and Influence of Epistatic Interactions Between IL-33 and the TSLP/TSLPR Axis on Risk of Coronary Artery Disease

The thymic stromal lymphopoietin (TSLP)/TSLP receptor (TSLPR) axis is involved in multiple inflammatory immune diseases, including coronary artery disease (CAD). To explore the causal relationship between this axis and CAD, we performed a three-stage case-control association analysis with 3,628 CAD cases and 3,776 controls using common variants in the genes TSLP, interleukin 7 receptor (IL7R), and TSLPR. Three common variants in the TSLP/TSLPR axis were significantly associated with CAD in a Chinese Han population [rs3806933T in TSLP, Padj = 4.35 × 10−5, odds ratio (OR) = 1.18; rs6897932T in IL7R, Padj = 1.13 × 10−7, OR = 1.31; g.19646A>GA in TSLPR, Padj = 2.04 × 10−6, OR = 1.20]. Reporter gene analysis demonstrated that rs3806933 and rs6897932 could influence TSLP and IL7R expression, respectively. Furthermore, the “T” allele of rs3806933 might increase plasma TSLP levels (R2 = 0.175, P < 0.01). In a stepwise procedure, the risk for CAD increased by nearly fivefold compared with the maximum effect of any single variant (Padj = 6.99 × 10−4, OR = 4.85). In addition, the epistatic interaction between TSLP and IL33 produced a nearly threefold increase in the risk of CAD in the combined model of rs3806933TT-rs7025417TT (Padj = 3.67 × 10−4, OR = 2.98). Our study illustrates that the TSLP/TSLPR axis might be involved in the pathogenesis of CAD through upregulation of mRNA or protein expression of the referenced genes and might have additive effects on the CAD risk when combined with IL-33 signaling.

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

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.