Common Variant Near HEY2 Has a Protective Effect on Ventricular Fibrillation Occurrence in Brugada Syndrome by Regulating the Repolarization Current.

BACKGROUND: Risk stratification of Brugada syndrome (BrS) remains controversial and the majority of patients with BrS have no genetic explanation. We investigated relationships between genotypes of 3 single-nucleotide polymorphisms reported in a recent genome-wide association study and BrS phenotypes. METHODS AND RESULTS: SCN10A (rs10428132), SCN5A (rs11708996), and downstream from HEY2 (rs9388451) single-nucleotide polymorphisms were genotyped and compared between 95 Japanese patients with BrS and 1978 controls. Relationships between the single-nucleotide polymorphisms and clinical characteristics, 12-lead ECG findings, signal-averaged ECG findings, and electrophysiological parameters were also examined in patients with BrS. Both rs10428132 and rs9388451 were significantly associated with BrS (P=2.7×10(-14); odds ratio, 3.0; P=9.2×10(-4); odds ratio, 1.7, respectively). Interestingly, the HEY2 risk allele C was less frequent in BrS patients with ventricular fibrillation than in those without (59% versus 74%; P=4.1×10(-2); odds ratio, 0.5). A significant linear correlation was found between HEY2 genotypes and QTc interval (CC: 422±27 ms; CT: 408±21 ms; and TT: 381±27 ms; P= 4.0×10(-4)). The HEY2 mRNA expression level in the right ventricular specimens from patients with BrS (n=20) was significantly lower in patients with CC genotype than the other genotypes (P=0.04). Additionally, during 63±28 months follow-up periods after implantable cardioverter defibrillator implantation (n=90), Kaplan-Meier event-free survival curves revealed that the cumulative rate of ventricular fibrillation events was significantly lower in cases with HEY2 CC genotype (P=0.04). CONCLUSIONS: Our findings suggest that HEY2 CC genotype may be a favorable prognostic marker for BrS, protectively acting to prevent ventricular fibrillation presumably by regulating the repolarization current.

IFNL4/IL-28B haplotype structure and its impact on susceptibility to hepatitis C virus and treatment response in the Japanese population.

A new type III interferon, IFN lambda 4 (IFNL4), and its single-nucleotide polymorphism (SNP) ss469415590 causing a frame shift have been recently reported strongly to affect antiviral therapy for chronic hepatitis C virus (HCV) infection in African and Caucasian populations compared to previously reported IL-28B SNPs rs12979860 and rs8099917. To compare the predictability for treatment outcome among those polymorphisms, we estimated haplotype structure of IFNL4/IL-28B consisting of the three SNPs in 4630 Japanese chronic hepatitis C patients and 1122 healthy controls and then compared their impact on response to pegylated-IFN (PEG-IFN) plus ribavirin (RBV) combined therapy in 903 HCV-1b-infected patients. A total of five haplotypes were identified, although two major haplotypes accounted for >99 % of the variation. The SNPs were tightly linked but not in absolute linkage disequilibrium. We could not find any difference in the predictive impact of any of these three SNPs with regard to susceptibility to HCV and treatment response. However, patients with favourable rs8099917 TT, linked to unfavourable genotypes of ss469415590 and rs12979860, showed poor initial viral response compared with those with all favourable genotypes (P = 0.0022). These findings suggest that, in part, ss469415590 and rs12979860 may have better predictive impact on response to PEG-IFN plus RBV therapy in the Japanese population, especially in patients with any of the minor haplotypes consisting of these SNPs.

A nonsynonymous polymorphism in semaphorin 3A as a risk factor for human unexplained cardiac arrest with documented ventricular fibrillation.

Unexplained cardiac arrest (UCA) with documented ventricular fibrillation (VF) is a major cause of sudden cardiac death. Abnormal sympathetic innervations have been shown to be a trigger of ventricular fibrillation. Further, adequate expression of SEMA3A was reported to be critical for normal patterning of cardiac sympathetic innervation. We investigated the relevance of the semaphorin 3A (SEMA3A) gene located at chromosome 5 in the etiology of UCA. Eighty-three Japanese patients diagnosed with UCA and 2,958 healthy controls from two different geographic regions in Japan were enrolled. A nonsynonymous polymorphism (I334V, rs138694505A>G) in exon 10 of the SEMA3A gene identified through resequencing was significantly associated with UCA (combined P = 0.0004, OR 3.08, 95%CI 1.67-5.7). Overall, 15.7% of UCA patients carried the risk genotype G, whereas only 5.6% did in controls. In patients with SEMA3A(I334V), VF predominantly occurred at rest during the night. They showed sinus bradycardia, and their RR intervals on the 12-lead electrocardiography tended to be longer than those in patients without SEMA3A(I334V) (1031±111 ms versus 932±182 ms, P = 0.039). Immunofluorescence staining of cardiac biopsy specimens revealed that sympathetic nerves, which are absent in the subendocardial layer in normal hearts, extended to the subendocardial layer only in patients with SEMA3A(I334V). Functional analyses revealed that the axon-repelling and axon-collapsing activities of mutant SEMA3A(I334V) genes were significantly weaker than those of wild-type SEMA3A genes. A high incidence of SEMA3A(I334V) in UCA patients and inappropriate innervation patterning in their hearts implicate involvement of the SEMA3A gene in the pathogenesis of UCA.

Single-nucleotide polymorphisms in GALNT8 are associated with the response to interferon therapy for chronic hepatitis C.

New anti-hepatitis C virus (HCV) therapeutics developed recently are more effective and lead to improvements in sustained viral response. However, interferon (IFN) monotherapy is still used to a limited extent for fear of adverse effects. This study investigated host genetic factors affecting the IFN response in patients with chronic hepatitis C (CHC). Using a two-step design, a large-scale association screening including 1088 Japanese CHC patients treated with IFN was performed employing ~70 000 gene-based single-nucleotide polymorphisms (SNPs). Replication was tested in an independent Japanese cohort of 328 patients. Fine-mapping and functional analyses were also performed. Through two-step screening, it was found that rs2286580 in intron 6 of the gene encoding N-acetylgalactosaminyltransferase 8 (GALNT8) on chromosome 12 was significantly associated with a sustained viral response (combined P = 3.9×10(-6), odds ratio 1.52, 95 % confidence interval 1.29-1.82). The association was replicated in an additional cohort of 328 Japanese patients. In subgroup analysis, GALNT8 variants were associated with treatment outcome independently of HCV genotype. By contrast, the outcome of pegylated IFN and ribavirin combined therapy was not affected by the SNP. Fine-mapping analysis revealed that the association peak was at rs10849138 in intron 6 of GALNT8. Allele-specific transcription analysis demonstrated that GALNT8 expression was upregulated by an unfavourable allele of the variant. A luciferase reporter assay demonstrated that overexpression of GALNT8 attenuated IFN-α-induced gene transcription via the IFN-stimulated response element. These results suggest that GALNT8 variants contribute to the response to IFN therapy against CHC, providing a new insight into antiviral mechanisms of IFN.

Toward the establishment of a prediction system for the personalized treatment of chronic hepatitis C.

BACKGROUND: Although several direct-acting antivirals (DAAs) are now available, the therapy regimen for chronic hepatitis C will continue to include pegylated interferon and ribavirin for the foreseeable future. Despite their improved rate of sustained virological response (SVR), DAAs pose increased risks of side effects and selection for antiviral resistance. Not all patients require DAA to achieve SVR, whereas others are unlikely to respond even to triple therapy. Therefore, a personalized approach to candidate selection is necessary. METHODS: In this retrospective study, data from 640 Japanese patients who were treated for chronic hepatitis C genotype 1, 2, or 3 with pegylated interferon plus ribavirin combination therapy was compiled to identify robust pretreatment predictive factors for SVR. RESULTS: A logistic regression model for personalized therapy was developed based on age, viral genotype, initial viral load, aspartate aminotransferase/alanine aminotransferase ratio, α-fetoprotein levels, and IL28B single-nucleotide polymorphism genotype. The area under the receiver-operating characteristic curve (AUC) was 0.85. The mean AUC following 10 rounds of 10-fold cross validation was 0.82, with a true positive rate of 78.2%. CONCLUSIONS: A personalized approach to therapy may better inform treatment decisions and reduce incidence of side effects and antiviral resistance.

Prediction of response to peginterferon-alfa-2b plus ribavirin therapy in Japanese patients infected with hepatitis C virus genotype 1b.

Variation at the IL-28B locus was recently reported to be a significant predictive factor of viral response to pegylated-interferon plus ribavirin combination therapy against chronic hepatitis C. Predictive factors for the effect of therapy, including IL-28B polymorphism rs8099917 and viral and clinical factors were investigated. A total of 288 patients were enrolled who were chronically infected with hepatitis C virus (HCV) genotype 1b and treated with combination therapy. Among them, 87 patients completed 48 weeks of therapy without dose reduction or discontinuation. In multivariate regression analysis, the rs8099917 TT genotype was the only independent factor significantly associated with sustained viral response (P = 0.016, OR 61.5), whereas substitutions at amino acid 70 (aa 70) of the HCV core protein (P = 0.038, OR 5.9) and non-TT genotypes (P = 0.002, OR 17.2) were associated with nonvirological response. Both factors were also associated with viral dynamics during the initial stage of the therapy. Correlation analysis revealed that rs8099917 genotype was correlated with γ-glutamyl transpeptidase, hyaluronic acid, and HCV core aa 70. In conclusion, host (IL-28B polymorphism) and viral (aa 70) factors independently affect response to combination therapy.

IL-28B predicts response to chronic hepatitis C therapy--fine-mapping and replication study in Asian populations.

Type I interferon (IFN) is used for the treatment of chronic hepatitis C virus (HCV) infection. Despite advances in antiviral therapy, a large proportion of patients remain infected following current therapies. Through a genome-wide scan, we found two variants (rs8099917 and rs12979860) in the IL-28B locus that affect the outcome of PEG-IFN and ribavirin combination therapy, consistent with recent studies (P = 6.52×10(-8); odds ratio 2.46 and P = 8.63×10(-8), odds ratio 2.40, respectively). Significant associations were also observed in the case of IFN monotherapy for HCV genotypes 1b and 2a. With rs8099917, HCV genotype 1b patients had a significantly lower frequency of the favourable genotype (86.6 %) compared with healthy controls (91.7 %), and HCV genotype 2a patients had an intermediate frequency (89.9 %). Similar results were found for rs12979860. Fine-mapping analysis revealed that rs8099917 had the strongest association with treatment outcome and 14 others, including four novel single nucleotide polymorphisms, had comparable associations. Haplotype analysis revealed that none of the haplotypes showed stronger association than any single marker. Early non-responders who could not achieve 2 log viral decline during the first 12 weeks of treatment had higher odds ratios for these two variants. The favourable allele of rs8099917 is also associated with initial viral decline at 2 and 4 weeks following the start of therapy. Multivariate analysis of PEG-IFN and ribavirin-treated patients showed that rs8099917 genotype, viral load, fibrosis and age were significant predictors of response to therapy. Common variation at the IL-28B locus is predictive of various IFN-based therapies for HCV independent of regimen or HCV genotype.

ITPA polymorphism affects ribavirin-induced anemia and outcomes of therapy--a genome-wide study of Japanese HCV virus patients.

BACKGROUND & AIMS: Ribavirin-induced anemia is one of the major causes of discontinuation and dose reduction during anti-hepatitis C virus therapy. Factors influencing this anemia, especially host genetic factors, are poorly understood. In this study we investigated predictive factors in hepatitis C virus patients treated with combination therapy. METHODS: We performed a 2-step genome-wide screening followed by replication analysis and fine-mapping using a total of 923 Japanese hepatitis C virus 1b-infected patients treated with pegylated-interferon plus ribavirin. We also applied logistic regression analysis to search for possible independent associations of clinical parameters and genetic variants with treatment-induced hemoglobin (Hb) decline as well as treatment outcomes. RESULTS: We identified a variant, located upstream of the inosine triphosphate pyrophosphatase gene on chromosome 20p13 that is significantly associated with treatment-induced anemia (combined P = 6.0 × 10(-14)). Resequencing and fine-mapping revealed several single nucleotide polymorphisms (SNPs) strongly associated with Hb decline, including the nonsynonymous SNP rs1127354 (P = 3.5 × 10(-44)), which was recently reported for other ethnic groups. Another reported SNP, the splicing variant-related SNP rs7270101, was not polymorphic in the Japanese population. Stratified analysis based on rs1127354 genotype revealed that inosine triphosphate pyrophosphatase expression is not correlated with Hb decline, suggesting that rs1127354 is a direct causal variant in the Japanese population. Multivariate analysis demonstrated that age, baseline Hb, baseline platelet count, and rs1127354 were independently associated with severe anemia (Hb <10 g/dL). CONCLUSIONS: A missense substitution in inosine triphosphate pyrophosphatase gene affects ribavirin-induced anemia in hepatitis C virus-infected Japanese patients.

Monitoring of vasculogenesis-inhibiting activities in sewage effluents by using medaka embryos.

Environmental samples are known to be contaminated with complex chemicals such as estrogens, polycyclic aromatic hydrocarbons, and retinoids. These contaminants have potentially an adverse impact on survival of aquatic animals, because we found previously that medaka (Oryzias latipes) embryos are defective in the development of blood vessels and bones in the presence of these chemicals. Thus, it is important to test whether sewage effluents contain inhibitory activities against the embryonic development. To examine for such activity, medaka embryos were exposed for 48 h to extracts or freeze-evaporated concentrates of effluent samples collected from different municipal sewage treatment plants. We used the transgenic embryos that are hypersensitive to estrogens due to a high-level expression of estrogen receptor for detecting the total (sum of estrogenic and non-estrogenic) vessel-inhibiting activity. The embryos were specifically defective in blood-vessel formation in most effluent samples, showing the activities ranging from 3 to 30 ng of 17beta-estradiol equiv per liter. Detection limit of 17beta-estradiol was 10 ng per liter. For detection of the non-estrogenic vessel-inhibiting activity, we treated the transgenic embryos in the presence of an antiestrogen, tamoxifen, or used the wild-type embryos. The non-estrogenic activities were found in some (7 out of 18) effluents, ranging from half to all of the total activities. Our findings for the first time demonstrate the utility of the vascular assay for monitoring sewage effluents.

Retionic acid and its receptors are required for expression of aryl hydrocarbon receptor mRNA and embryonic development of blood vessel and bone in the medaka fish, Oryzias latipes.

Retinoic acid (RA), the active derivative of vitamin A, is essential for normal embryonic development of vertebrates because both the lack and excess of RA result in developmental malformations. We previously reported that aryl hydrocarbon receptor (AHR) is also required for vascular and bone formation by regulating cytochrome P450 expression. However, little is known about the roles of retinoic acid receptors (RAR) and retinoid X receptors (RXR) in the embryonic development of blood vessels and molecular cross-talk between RAR/RXR and AHR. We report for the first time that RA and RAR/RXR are required for expression of AHR mRNA and the embryonic development of blood vessel and bone. The embryonic organogenesis of medaka fish was specifically inhibited by an inhibitor of RA synthesis (diethylaminobenzaldehyde), antagonists of RAR (Ro41-5253) and RXR (Ro71-4595), agonist (beta-naphthoflavone) and antagonist (alpha-naphthoflavone) of AHR, and excess RA. These reagents are useful for future studies to elucidate molecular mechanisms for vascular and bone formation in the medaka embryogenesis. Our results also show that medaka embryos may be useful for screening inhibitors of vascular formation for anti-cancer drugs.