Keiichi Hirono

Mutation analysis of the G4.5 gene in patients with isolated left ventricular noncompaction.

Mutations in the gene G4.5, originally associated with Barth syndrome, have been reported to result in a wide spectrum of severe infantile X-linked cardiomyopathies. The purpose of this study was to investigate patients with isolated left ventricular noncompaction (LVNC) for disease-causing mutations in G4.5. In 27 patients including 10 families with isolated LVNC, mutation analysis of G4.5 was performed using single-strand DNA conformation polymorphism (SSCP) analysis and DNA sequencing. A novel splice acceptor site mutation of intron 8 of G4.5 was identified in a family with severe infantile X-linked LVNC without the usual findings of Barth syndrome. This mutation results in deletion of exon 9 from the mRNA, and is predicted to significantly disrupt the protein product. Genotype-phenotype correlation of G4.5 mutations in all 38 cases reported in the literature to date revealed that there was no correlation between location or type of mutation and either cardiac phenotype or disease severity. We suggest that males presenting with cardiomyopathy, particularly during infancy, even in the absence of the typical signs of Barth syndrome, should be evaluated for mutations in G4.5.

Neutrophils and Mononuclear Cells Express Vascular Endothelial Growth Factor in Acute Kawasaki Disease: Its Possible Role in Progression of Coronary Artery Lesions

Kawasaki disease (KD) is a syndrome of systemic vasculitis of unknown etiology that is complicated by coronary artery lesions (CAL), leading occasionally to cardiac ischemic sequelae. To examine whether vascular endothelial growth factor (VEGF) is responsible for CAL in KD, we determined serum VEGF levels by ELISA and peripheral blood mononuclear cell (PBMC) and neutrophil VEGF expression by immunoblot analysis. Significantly increased levels of VEGF were demonstrated in acute KD as well as in other vasculitis syndromes (p < 0.0001). In the 10 KD patients with CAL, serum VEGF levels were maximal approximately 2 wk postonset when CAL generally develops and were significantly higher than in 20 patients without CAL (mean, 474 and 241 pg/mL, respectively;p = 0.00015). During the same period, immunoblot analysis revealed maximal VEGF expression in PBMC, corresponding to serum VEGF levels in most patients and being particularly marked in patients with CAL (p < 0.01). Neutrophils expressed VEGF only in the early stage of acute KD and declined rapidly in the majority of KD patients regardless of the presence of CAL, showing a strikingly different expression pattern than that for PBMC. Predominant VEGF expression by PBMC was also demonstrated in patients with other vasculitis syndromes and only faintly in normal controls. The results suggest that VEGF is generated dynamically in KD, presumably reflecting its disease activity. Neutrophil-derived VEGF may play a role in regulating early vascular responses, whereas PBMC-derived VEGF may contribute to later vascular injury and remodeling.

Infliximab Reduces the Cytokine-Mediated Inflammation but Does Not Suppress Cellular Infiltration of the Vessel Wall in Refractory Kawasaki Disease

The aim of our study was to evaluate the efficacy of infliximab for the treatment of patients with refractory Kawasaki disease (KD) and investigate the dynamic changes of cytokines during infliximab treatment. We have performed a study of cytokine and proinflammatory molecule levels in 43 KD patients including 18 responders to IVIG, 14 nonresponders, and 11 patients treated with infliximab. We determined serum levels of soluble TNF receptor I (sTNFR I) and IL-6, as well as VEGF, damage associated molecular pattern (DAMP) molecules; myeloid-related protein (MRP)8/MRP14 and S100A12 sequentially. In eight patients, fever subsided immediately upon infliximab treatment. Four patients, who started infliximab after 12 d of illness, developed coronary artery lesions. Each of the cytokines was elevated before infliximab treatment in all patients. Although serum levels of proinflammatory cytokines decreased dramatically after infliximab treatment, DAMP molecules and VEGF and markers of local tissue damage were not suppressed. In contrast, in IVIG responders all cytokines decreased markedly after IVIG treatment. We show that infliximab is one of the adoptive therapies in refractory KD patients. Different behaviors of proinflammatory cytokines and DAMP molecules and VEGF after infliximab treatment suggest that infliximab is effective for suppression of cytokine-mediated inflammation, but could not completely block local vasculitis.

Effect of the VKORC1 Genotype on Warfarin Dose Requirements in Japanese Pediatric Patients

The primary aim of the present study was to evaluate the effect of the genotype of vitamin K epoxide reductase complex 1 (VKORC1) on warfarin dose requirements in Japanese pediatric patients. Forty-eight pediatric patients (0.42-19.25 years old) in whom stable anticoagulation was achieved by warfarin were enrolled in this study, and the polymorphic alleles of VKORC1 and CYP2C9 were determined for each subject. The relative impact of covariates on the anticoagulant effect of warfarin was evaluated by multiple regression analysis. It was found that VKORC1 genotype and age were major factors affecting the relationship between the weight-normalized warfarin dose and the therapeutic prothrombin time-international normalized ratio (PT-INR). Because only one patient had the CYP2C9*3 allele, we could not evaluate the effect of CYP2C9 polymorphisms on the anticoagulant effect of warfarin. In contrast, the anticoagulant effect of warfarin in patients with the VKORC1 1173CT or 1173CC genotype was 52.3% of that in patients with the 1173TT genotype. In addition, the anticoagulant effect of warfarin was shown to increase by 10.5% per year in Japanese pediatric patients. In conclusion, genotyping of VKORC1 will be useful in establishing individual anticoagulant therapy with warfarin, and it should be noted that a higher weight-normalized dose of warfarin is required in younger pediatric patients.

Identification of a novel TPM1 mutation in a family with left ventricular noncompaction and sudden death

Left ventricular noncompaction (LVNC) is a cardiomyopathy morphologically characterized by 2-layered myocardium, numerous prominent trabeculations, and deep intertrabecular recesses communicating with the left ventricular cavity. The purpose of this study was to investigate patients with LVNC for possible disease causing mutations. We screened 4 genes (TAZ, LDB3, DTNA and TPM1) in 51 patients with LVNC for mutations by polymerase chain reaction and direct DNA sequencing. A novel missense substitution in exon 1 of TPM1 (c.109A>G: p.Lys37Glu) was identified in three affected members of a family with isolated LVNC. The substitution brings about a change in amino acid charge at a highly conserved residue and could result in aberrant mRNA splicing. This variant was not identified in 200 normal control samples. Pathologic analysis of a right ventricular myocardial specimen from the probands maternal aunt revealed endocardial and subendocardial fibrosis with prominent elastin deposition, as well as the presence of adipose tissue between muscle layers, pathologic changes that are distinct from those seen in patients with HCM or DCM. Screening of the proband and her mother for variants in other sarcomeric protein-encoding candidate genes, MYH7, MYBPC3, TNNT2, TNNI3, ACTC, MYL2, and MYL3, did not identify any other non-synonymous variants or variants in splice donor-acceptor sequences that were potentially disease causing. We conclude TPM1 is a potential candidate disease-causing gene for isolated LVNC, especially in patients experiencing sudden death.

Pharmacokinetics of Bosentan in Routinely Treated Japanese Pediatric Patients with Pulmonary Arterial Hypertension

We evaluated the pharmacokinetics of routinely administered bosentan in 46 Japanese pediatric patients with pulmonary arterial hypertension. Plasma samples were taken twice at times corresponding to the peak and trough concentrations following repetitive oral administration. The population pharmacokinetic parameters of bosentan were estimated by use of the NONMEM program, in which a one-compartment model with repetitive bolus dosing was parameterized in terms of the oral clearance (CL/F) and elimination rate constant (k). Polymorphisms of CYP3A5, SLCO1B1, SLCO1B3, and SLCO2B1 had no significant effect on the disposition of bosentan. In addition, the pharmacokinetics of bosentan was not altered by heart failure or coadministration of sildenafil. In contrast, weight (WT)-normalized values of CL/F were correlated negatively with age (AGE). The final population mean values of CL/F and k were estimated to be 0.409 · (1 - 0.0377 · (AGE - 3.81)) · WT L/h and 0.175 h(-1), respectively.

Bosentan induces clinical and hemodynamic improvement in candidates for right-sided heart bypass surgery

OBJECTIVE To investigate the efficacy of bosentan in patients with single-ventricle physiology who were unable to undergo right-sided heart bypass surgery because of high pulmonary vascular resistance and pulmonary artery pressure. METHODS Eight patients with single-ventricle physiology (2 male and 6 female; aged 7 months to 5 years, median 1 year) were enrolled. Prior surgical interventions included pulmonary artery banding in 4 patients, Blalock-Taussig shunt operation in 2 patients, and bidirectional Glenn operation in 5 patients. Right-sided heart bypass surgery was contraindicated for all patients because of high pulmonary vascular resistance and pulmonary artery pressure. RESULTS Bosentan therapy successfully reduced pulmonary artery pressure and pulmonary vascular resistance in all patients. Mean pulmonary artery pressure at baseline and after bosentan therapy was 21.1 +/- 7.2 mm Hg and 11.9 +/- 4.1 mm Hg, respectively (P < .01). Mean pulmonary vascular resistance index at baseline and after bosentan therapy was 5.7 +/- 3.3 U/m(2) and 1.3 +/- 0.4 U/m(2), respectively (P < .01). Mean pulmonary vascular resistance/systemic vascular resistance at baseline and after bosentan therapy was 0.25 +/- 0.11 and 0.07 +/- 0.03, respectively (P < .01). All patients had improved clinical symptoms and underwent successful Fontan operations. CONCLUSION Bosentan induces mid-term clinical and hemodynamic improvement in patients with single-ventricle physiology and elevated pulmonary vascular resistance and pulmonary artery pressure. Bosentan therapy may increase the surgical options and improve outcomes in candidates for right-sided heart bypass surgery.

Postmortem genetic analysis of sudden unexplained death syndrome under 50 years of age: A next-generation sequencing study

BACKGROUND Recent studies on the genetic analysis of victims of sudden unexplained death syndrome (SUDS) have shown diagnostic potential. Previously, such analyses mainly targeted the major channelopathy-associated genes. OBJECTIVE The purpose of this study was to evaluate the utility of next-generation sequencing (NGS) in the postmortem diagnosis of SUDS. METHODS Our data are derived from 25 cases of SUDS (21 men and 4 women; age 19-50 years). A total of 70 genes were examined by NGS, and the pathogenicity of any detected rare variants with minor allele frequencies of <0.5% was evaluated using a widely used database and eight in silico algorithms. RESULTS Five known and 15 potentially pathogenic variants with a high in silico score were identified in 14 cases. In all, 6 channelopathy-related variants were identified in 5 cases, including 2 cases with history of arrhythmia; 11 cases had cardiomyopathy- or cardiac transcription factor-related variants. Three cases with desmosomal gene- or other cardiomyopathy-related variants showed possibly related pathologic changes. Three cases with RYR2 or TBX5 variants showed possible pathogenic fibrosis of the cardiac conduction system. Only 12 variants showed moderate or strong possible pathogenicity in SUDS cases compared with qualifying controls. CONCLUSION Hereditary heart diseases other than channelopathy may also be a significant cause of SUDS, even if clinical and pathologic findings do not show advanced disease. A combination of gene analysis using NGS and some predictive methods for detecting variants and careful pathologic evaluation may provide a reliable diagnosis of hereditary heart disease for potential SUDS cases.

Plasma concentrations of tadalafil in children with pulmonary arterial hypertension.

Background: There is no report documenting the plasma concentrations of tadalafil in children. This study was performed to evaluate the variability in the pharmacokinetics of tadalafil in children with pulmonary arterial hypertension (PAH) treated routinely with the drug. Methods: Plasma samples were taken twice (post- and predose) after repetitive oral administration, and the pharmacokinetic parameters (CL/F and V/F) in individual patients were estimated by the Bayesian method using the nonlinear mixed effects model. We also determined the unbound concentration of tadalafil using ultrafiltration. Results: Tadalafil was administered to 23 children aged between 0.25 and 17.4 years, with a mean age of 3.58 years. The mean (±SD) daily dose of tadalafil was 0.97 ± 0.41 mg/kg. Sixteen of the 23 children received bosentan concomitantly. The mean CL/F and V/F values of tadalafil were 0.149 L·h−1·kg−1 and 1.87 L/kg, respectively, which were higher than those reported in adults. No effects of age, bosentan, or the estimated glomerular filtration rate were observed on the CL/F value, indicating that other residual factors might account for the interindividual variability among children with PAH. The unbound tadalafil concentrations of the postdose samples ranged from 5.9 to 146 (46.9 ± 37.1) nmol/L, higher than the reported IC50 value of this phosphodiesterase-5 drug for humans (2–4 nmol/L, corresponding to 0.8–1.6 ng/mL). Conclusions: We demonstrated variability in the total and unbound plasma concentrations of tadalafil in children. However, all children received the empirical doses of the drug; a mean dose of 0.97 mg·kg−1·d−1 showed sufficient unbound concentrations needed for half-maximal inhibition of human phosphodiesterase-5 in vitro. These observations may provide information for the proper use of tadalafil to treat children with PAH.

A novel MYH7 gene mutation in a fetus with left ventricular noncompaction.

Left ventricular noncompaction (LVNC) is a recently defined cardiomyopathy characterized by a pattern of prominent trabecular meshwork and deep intertrabecular recesses. LVNC is rarely described in fetal life, and a small number of cases have been reported. We report the first fetal case, to our knowledge, of LVNC associated with a novel mutation in the MYH7 gene (c.1625A>C; p.Lys542Thr). This patient showed cardiomegaly on prenatal ultrasonographic examinations, with features indicating noncompaction of the myocardium apparent in the second trimester. This case highlights the importance of prenatal ultrasonography for the diagnosis of LVNC and suggests that abnormal myocardial development underlies the pathogenesis of LVNC.