Yusuke Ebana

Integrating Genetic, Transcriptional, and Functional Analyses to Identify 5 Novel Genes for Atrial Fibrillation

Background— Atrial fibrillation (AF) affects >30 million individuals worldwide and is associated with an increased risk of stroke, heart failure, and death. AF is highly heritable, yet the genetic basis for the arrhythmia remains incompletely understood. Methods and Results— To identify new AF-related genes, we used a multifaceted approach, combining large-scale genotyping in 2 ethnically distinct populations, cis-eQTL (expression quantitative trait loci) mapping, and functional validation. Four novel loci were identified in individuals of European descent near the genes NEURL (rs12415501; relative risk [RR]=1.18; 95% confidence interval [CI], 1.13–1.23; P=6.5×10−16), GJA1 (rs13216675; RR=1.10; 95% CI, 1.06–1.14; P=2.2×10−8), TBX5 (rs10507248; RR=1.12; 95% CI, 1.08–1.16; P=5.7×10−11), and CAND2 (rs4642101; RR=1.10; 95% CI, 1.06–1.14; P=9.8×10−9). In Japanese, novel loci were identified near NEURL (rs6584555; RR=1.32; 95% CI, 1.26–1.39; P=2.0×10−25) and CUX2 (rs6490029; RR=1.12; 95% CI, 1.08–1.16; P=3.9×10−9). The top single-nucleotide polymorphisms or their proxies were identified as cis-eQTLs for the genes CAND2 (P=2.6×10−19), GJA1 (P=2.66×10−6), and TBX5 (P=1.36×10−5). Knockdown of the zebrafish orthologs of NEURL and CAND2 resulted in prolongation of the atrial action potential duration (17% and 45%, respectively). Conclusions— We have identified 5 novel loci for AF. Our results expand the diversity of genetic pathways implicated in AF and provide novel molecular targets for future biological and pharmacological investigation.

Two Susceptibility Loci to Takayasu Arteritis Reveal a Synergistic Role of the IL12B and HLA-B Regions in a Japanese Population

Takayasu arteritis (TAK) is an autoimmune systemic vasculitis of unknown etiology. Although previous studies have revealed that HLA-B*52:01 has an effect on TAK susceptibility, no other genetic determinants have been established so far. Here, we performed genome scanning of 167 TAK cases and 663 healthy controls via Illumina Infinium Human Exome BeadChip arrays, followed by a replication study consisting of 212 TAK cases and 1,322 controls. As a result, we found that the IL12B region on chromosome 5 (rs6871626, overall p = 1.7 × 10(-13), OR = 1.75, 95% CI 1.42-2.16) and the MLX region on chromosome 17 (rs665268, overall p = 5.2 × 10(-7), OR = 1.50, 95% CI 1.28-1.76) as well as the HLA-B region (rs9263739, a proxy of HLA-B*52:01, overall p = 2.8 × 10(-21), OR = 2.44, 95% CI 2.03-2.93) exhibited significant associations. A significant synergistic effect of rs6871626 and rs9263739 was found with a relative excess risk of 3.45, attributable proportion of 0.58, and synergy index of 3.24 (p ≤ 0.00028) in addition to a suggestive synergistic effect between rs665268 and rs926379 (p ≤ 0.027). We also found that rs6871626 showed a significant association with clinical manifestations of TAK, including increased risk and severity of aortic regurgitation, a representative severe complication of TAK. Detection of these susceptibility loci will provide new insights to the basic mechanisms of TAK pathogenesis. Our findings indicate that IL12B plays a fundamental role on the pathophysiology of TAK in combination with HLA-B(∗)52:01 and that common autoimmune mechanisms underlie the pathology of TAK and other autoimmune disorders such as psoriasis and inflammatory bowel diseases in which IL12B is involved as a genetic predisposing factor.

Identification of six new genetic loci associated with atrial fibrillation in the Japanese population

Atrial fibrillation is the most common cardiac arrhythmia and leads to stroke. To investigate genetic loci associated with atrial fibrillation in the Japanese population, we performed a genome-wide association study (GWAS) that included 8,180 atrial fibrillation cases and 28,612 controls with follow-up in an additional 3,120 cases and 125,064 controls. We replicated previously reported loci and identified six new loci, near the KCND3, PPFIA4, SLC1A4–CEP68, HAND2, NEBL and SH3PXD2A genes. Five of the six new loci were specifically associated with atrial fibrillation in the Japanese population after comparing our data to those from individuals of European ancestry, suggesting that there might be different genetic factors affecting susceptibility across ancestry groups. Our study discovered variants in the HAND2, KCND3 and NEBL genes, which are relevant to atrial fibrillation susceptibility. The involvement of PPFIA4 and SH3PXD2A in axon guidance also suggested a role in disease pathogenesis. Our findings may contribute to a better understanding of atrial fibrillation susceptibility and pathogenesis.

A functional SNP in ITIH3 is associated with susceptibility to myocardial infarction

AbstractMyocardial infarction (MI) results from complex interactions of multiple genetic and environmental factors. To disclose genetic backgrounds of MI, we performed a large-scale, case-control association study using 52,608 gene-based single-nucleotide polymorphism (SNP) markers, and identified a candidate SNP located on chromosome 3p21.2-p21.1. Subsequent linkage-disequilibrium mapping indicated very significant association between MI and a SNP in exon 2 of the inter-alpha (globulin) inhibitor 3 gene (ITIH3; χ2= 24.88, P= 6.1 × 10−7, 3,353 affected individuals versus 3,807 controls). In vitro functional analyses showed that this SNP enhanced the transcriptional level of the ITIH3 gene. Furthermore, we found expression of the ITIH3 protein in the vascular smooth muscle cells and macrophages in the human atherosclerotic lesions, suggesting ITIH3 SNP to be a novel genetic risk factor of MI.

Effects of immunosuppressive and biological agents on refractory Takayasu arteritis patients unresponsive to glucocorticoid treatment

BACKGROUND We aimed to investigate the effects of immunosuppressive and biological agents on refractory Takayasu arteritis (TA) patients resistant to or dependent on glucocorticoids. METHODS Forty-four consecutive TA patients were enrolled, and the clinical characteristics and effectiveness of the immunosuppressive and biological agents in achieving and maintaining remission among glucocorticoid-resistant or glucocorticoid-dependent patients were investigated. RESULTS Fifteen patients showed favorable response to the initial glucocorticoid treatment, and 29 patients exhibited resistance to initial glucocorticoid treatment or relapsed with tapering glucocorticoid. Of the 29 patients, 5 responded to additional glucocorticoid treatment, and 22 of the remaining 24 glucocorticoid-resistant or glucocorticoid-dependent patients were prescribed immunosuppressive agents. Methotrexate was the most commonly used in these patients as the first-line treatment. In total, 10 patients maintained remission using immunosuppressive agents, with the effectiveness of each agent about 20%. The only significant difference between patients who were and were not able to achieve and maintain remission with immunosuppressive agents was the presence of the HLA-B52 allele (p<0.0001). Biological agents were administered to 6 patients refractory to immunosuppressive agents. All patients were administered tumor necrosis factor (TNF) inhibitors as the first-line treatment, and 3 patients maintained remission. Anti-interleukin-6 receptor antibody was administered to 2 patients who were resistant to the TNF inhibitors, and 1 patient achieved and maintained remission. CONCLUSION In our cohort, 64% of the glucocorticoid-resistant or glucocorticoid-dependent patients maintained remission through a combined treatment with glucocorticoid, immunosuppressive agents, and/or biological agents. The combined use of immunosuppressive and biological agents appears to be a promising treatment option for achieving and maintaining remission in refractory TA patients.

Aromatase knockout mice reveal an impact of estrogen on drug-induced alternation of murine electrocardiography parameters.

Our in vitro characterization showed that physiological concentrations of estrogen partially suppressed the I(Kr) channel current in guinea pig ventricular myocytes and the human ether-a-go-go-related gene (hERG) channel currents in CHO-K1 cells regardless of estrogen receptor signaling and revealed that the partially suppressed hERG currents enhanced the sensitivity to the hERG blocker E-4031. To obtain in vivo proof-of-concept data to support the effects of estrogen on cardiac electrophysiology, we here employed an aromatase knockout mouse as an in vivo estrogen-null model and compared the acute effects of E-4031 on cardiac electrophysiological parameters with those in wild-type mice (C57/BL6J) by recording surface electrocardiogram (ECG). The ablation of circulating estrogens blunted the effects of E-4031 on heart rate and QT interval in mice under a denervation condition. Our result provides in vivo proof of principle and demonstrates that endogenous estrogens increase the sensitivity of E-4031 to cardiac electrophysiology.

Novel Dielectric Coagulometer Identifies Hypercoagulability in Patients with a High CHADS2 Score without Atrial Fibrillation.

Background Recent reports showed that the CHADS2 score predicted the risk of strokes in patients without atrial fibrillation (AF). Although the hypercoagulability may contribute to the thrombogenesis, it has not been fully investigated due to a lack of a sensitive evaluation modality. Recently a novel dielectric blood coagulometry (DBCM) was invented for evaluating the coagulability by measuring the temporal change in whole blood dielectric permittivity. Objective We evaluated the utility of the DBCM for identifying the coagulability. Patients/Methods For fundamental experiments, 133 citrated blood samples were drawn from subjects with or without heparin administration. A DBCM analysis was performed to find the adequate coagulation index, and to delineate its measurement range by adding recombinant human tissue factor (TF) or heparin. Then the coagulability was assessed by DBCM and conventional coagulation assays in 84 subjects without AF, who were divided into 3 groups by their CHADS2 score. Another 17 patients who received warfarin were also assessed by DBCM to evaluate the effect of anticoagulants. Results and Conclusions We calculated the derivative of the dielectric permittivity change after recalcification, and extracted the end of acceleration time (EAT) as a novel index. The EAT showed a dose-dependent shortening with the addition of serial dilutions of TF (×10−2 to ×10−4), and a dose-dependent prolongation with the addition of heparin (0.05 to 0.15 U/ml). The EAT was significantly shorter in the higher CHADS2 score group (19.8 ± 4.8, 18.6 ± 3.1, and 16.3 ± 2.7 min in the CHADS2 = 0, 1, and ≥2 groups, respectively, p = 0.0065 by ANOVA). Patients receiving warfarin had a significantly more prolonged EAT than those without warfarin (18.6±4.2 vs. 25.8±7.3 min, p <0.001). DBCM detected the whole blood coagulability with a high sensitivity. Subjects with higher CHADS2 scores exhibited hypercoagulability without AF.

Dielectric permittivity change detects the process of blood coagulation: Comparative study of dielectric coagulometry with rotational thromboelastometry.

BACKGROUND Intravascular thrombus formation causes various cardiovascular diseases. To monitor coagulation is important for screening native status, prevention from bleeding and maintaining it within its therapeutic range. The prothrombin time and the activated partial thromboplastin time are widely used for assessment and recognized as the conventional methods. Prothrombin time methods employ enhancement of coagulation with thromboplastin. Since the laboratory data depend on the production lot and/or the manufacturer, the accurate methods are required for evaluation. Rotational thromboelastometry (ROTEM) is a method based on detection of the change in resistance to rotational movement during blood clotting, while dielectric blood coagulometry (DBCM) is a novel method for assessment of clotting by measuring the change of electrical permittivity. These methods are thus based on the technology for observation of different physical phenomena. The aim of this study was to compare parameters such as the clotting time obtained by ROTEM and DBCM to evaluate their clinical usefulness. METHODS AND RESULTS ROTEM and DBCM parameters were measured in 128 patients. The ROTEM clotting time showed a significant positive correlation with the DBCM coagulation time (R=0.707, p<0.001). Comparison of the DBCM coagulation time between patients with and without anticoagulant therapy (including novel oral anticoagulants) revealed a significant difference (43.8±11.9min in the anticoagulant group vs 29.4±8.3min in the control group, p<0.001). Evaluation of coagulation was equivalent with DBCM and ROTEM. CONCLUSIONS The present study suggested that DBCM, a novel method for measuring blood clotting, could provide the detail assessment for the status of anticoagulant therapy.

Clinical utility and functional analysis of variants in atrial fibrillation-associated locus 4q25

BACKGROUND Chromosome 4q25 has been repeatedly identified as atrial fibrillation (AF)-sensitive locus in multiple genome-wide association studies (GWAS) and is considered to hold some clues to AF pathogenesis. We aimed to investigate the clinical utilities in Japanese and to unveil the function of the 4q25 locus in affecting transcription of adjacent genes. METHODS We conducted AF GWAS in Japanese population (1382 AF cases and 1478 controls) and the replication panel (1666 AF cases and 1229 controls) with detailed clinical information which showed the acceleration of AF onset. Stepwise investigations with linkage disequilibrium analysis, histone code patterns, and reporter assay in the 4q25 locus were performed. RESULTS The AF GWAS confirmed a significant association of rs4611994 and rs1906617 in chromosome 4q25 with AF. In the clinical analysis, AF onset of the individuals with risk allele accelerated 2.5 years compared with those with protective allele (p=0.00012). Next, in the functional analysis, three single nucleotide polymorphisms (SNPs) in the variant group selected by linkage disequilibrium analysis were identified as candidates for the cis-regulatory element toward adjacent genes in chromatin immunoprecipitation assay. Among them, rs4611994 and rs72900144 regions showed higher effects on the transcriptional activity of luciferase gene in the risk alleles than those in the protective alleles (p<0.0001, p<0.005, respectively). CONCLUSIONS AF GWAS in Japanese confirmed the association with 4q25 locus and indicated that its SNP affected the acceleration of AF onset. The candidate regions of the causative SNPs, rs4611994 and rs72900144, could alter the adjacent gene expression level.

Chromosome 4q25 variants and recurrence after second-generation cryoballoon ablation in patients with paroxysmal atrial fibrillation

BACKGROUND Chromosome 4q25 single-nucleotide polymorphisms (SNPs) are associated with atrial fibrillation (AF) recurrence after radiofrequency catheter ablation, however the underlying mechanism is unknown. Pulmonary vein (PV) reconnections are common post-radiofrequency ablation. We explored the pre-procedural parameters, including AF susceptibility SNPs, predicting the response to PV isolation (PVI) using second-generation cryoballoons. METHODS One hundred fifty-seven paroxysmal AF patients undergoing PVI using second-generation cryoballoons and genetic testing were enrolled. The top 6 AF-associated Japanese ancestry SNPs were evaluated. Fourteen-day consecutive monitoring was performed to detect AF recurrences. RESULTS Early recurrence of AF (ERAF) was detected in 74(47.1%) patients, and the AF-free survival at 12-months after single procedures was 72.1%. Coxs proportional models determined that higher pro-BNP values (hazard ratio [HR]=1.001; 95% confidence interval [CI]=1.000-1.001; p=0.003) and the rs1906617 risk allele (HR=2.440; 95% CI=1.062-5.605; p=0.035) were independently associated with ERAFs, and the rs1906617 risk allele (HR=4.339; 95% CI=1.044-18.028; p=0.043) was the sole factor significantly associated with AF recurrence. Second procedures were performed in 41 patients a median of 6.0[5.0-9.5] months later, and 42/162(25.9%) PVs were reconnected. Reconnections were similarly observed in rs1906617 risk allele carriers and wild-type patients. Risk allele carriers at rs1906617 were more likely to have non-PV foci, but did not reach statistical significance (10/35 vs. 0/6, p=0.132). CONCLUSIONS AF risk alleles on chromosome 4q25 modulated the risk of AF recurrence after PVI using second-generation cryoballoons in patients with paroxysmal AF. Our study results suggested that non-PV foci might be the more likely mechanism of a high AF recurrence in chromosome 4q25 variant carriers.