Michio Yasunami

Replication of the association between a chromosome 9p21 polymorphism and coronary artery disease in Japanese and Korean populations

AbstractCoronary artery disease (CAD) has become a major health problem in many countries. Recent genome-wide association studies have identified the association between rs1333049 on chromosome 9p21 and susceptibility to CAD in Caucasoid populations. In this study, we evaluated the associations of rs1333049 with CAD in Japanese (604 patients and 1,151 controls) and Koreans (679 patients and 706 controls). We found a significant association in both Japanese [odds ratio (OR) = 1.30, 95% confidence interval (CI); 1.13–1.49, p = 0.00027, allele count model] and Koreans (OR = 1.19, 95% CI; 1.02–1.38, p = 0.025, allele count model). These observations demonstrated that chromosome 9p21 was the susceptibility locus for CAD also in East Asians.

Functional analysis of titin/connectin N2-B mutations found in cardiomyopathy

Hypertrophic cardiomyopathy and dilated cardiomyopathy are two major clinical phenotypes of “idiopathic” cardiomyopathy. Recent molecular genetic analyses have now revealed that “idiopathic” cardiomyopathy is caused by mutations in genes for sarcomere components. We have recently reported several mutations in titin/connectin gene found in patients with hypertrophic cardiomyopathy or dilated cardiomyopathy. A hypertrophic cardiomyopathy-associated titin/connectin mutation (Arg740Leu) was found to increase the binding to actinin, while other dilated cardiomyopathy-associated titin/connectin mutations (Ala743Val and Val54Met) decreased the binding to actinin and Tcap/telethonin, respectively. We also reported several other mutations in the N2-B region of titin/connectin found in hypertrophic cardiomyopathy and dilated cardiomyopathy. Since the N2-B region expresses only in the heart, it was speculated that functional alterations due to the mutations cause cardiomyopathies. In this study, we investigated the functional changes caused by the N2-B region mutations by using yeast-two-hybrid assays. It was revealed that a hypertrophic cardiomyopathy-associated mutation (Ser3799Tyr) increased the binding to FHL2 protein, whereas a dilated cardiomyopathy-associated mutation (Gln4053ter) decreased the binding. In addition, another TTN mutation (Arg25618Gln) at the is2 region was found in familial DCM. Because FHL2 protein is known to tether metabolic enzymes to N2-B and is2 regions of titin/connectin, these observations suggest that altered recruitment of metabolic enzymes to the sarcomere may play a role in the pathogenesis of cardiomyopathies.

Truncated KCNQ1 mutant, A178fs/105, forms hetero-multimer channel with wild-type causing a dominant-negative suppression due to trafficking defect.

We identified a novel mutation Ala178fs/105 missing S3–S6 and C‐terminus portions of KCNQ1 channel. Ala178fs/105‐KCNQ1 expressed in COS‐7 cells demonstrated no current expression. Co‐expression with wild‐type (WT) revealed a dominant‐negative effect, which suggests the formation of hetero‐multimer by mutant and WT. Confocal laser microscopy displayed intracellular retention of Ala178fs/105‐KCNQ1 protein. Co‐expression of the mutant and WT also increased intracellular retention of channel protein compared to WT alone. Our findings suggest a novel mechanism for LQT1 that the truncated S1–S2 KCNQ1 mutant forms hetero‐multimer and cause a dominant‐negative effect due to trafficking defect.

A novel link of HLA locus to the regulation of immunity and infection: NFKBIL1 regulates alternative splicing of human immune-related genes and influenza virus M gene

HLA locus contains immune-related genes and genetically regulates immune responses against both foreign- and self-antigens in humans. Inhibitor of κB-like protein (IκBL), encoded by HLA-linked NFKBIL1, is a protein of unknown function, while genetic variations in NFKBIL1 are known to associate with the susceptibility to inflammatory and/or autoimmune diseases. In this study, we found that IκBL suppressed exon exclusion in alternative splicing of human immune-related genes such as CD45. Yeast-two-hybrid screening and immunoprecipitation assay revealed molecular association of IκBL with CLK1, a serine/threonine and tyrosine kinase, which plays a role in the alternative splicing. Unexpectedly, we found that the regulation of alternative splicing in CD45 by IκBL was independent from the kinase activity of CLK1. On the other hand, it was demonstrated that an SR protein, ASF/SF2, bound both IκBL and CLK1 at the RNA-recognition motifs of ASF/SF2, implying a competition of IκBL and CLK1 on SR protein. In addition, IκBL was found to regulate the CLK1-dependent synthesis of M2 RNA, a splice variant of influenza A virus M gene. These observations suggest a functional involvement of IκBL in the regulation of alternative splicing in both human and viral genes, which is a novel link of HLA locus to the regulation of immunity and infection in humans.

A mutation in Wolfram syndrome type 1 gene in a Japanese family with autosomal dominant low-frequency sensorineural hearing loss

Conclusion. Our findings suggest that Wolfram syndrome type 1 gene (WFS1) mutation is an important cause of autosomal dominant low-frequency sensorineural hearing loss (LFSNHL) in Japan. Objective. DFNA6/14 is caused by a heterozygous mutation of WFS1 and is a common cause of autosomal dominant LFSNHL among populations in both Europe and the US. The purpose of this study was to investigate WFS1 mutations among Japanese patients whose phenotypes were consistent with those of DFNA6/14. Material and methods. Using audiometry and genetic analysis, we searched for WFS1 mutations in three unrelated Japanese patients with LFSNHL and a familial history of autosomal dominant hearing loss. Results. One patient carried a heterozygous G2700A mutation at codon 844 in exon 8, resulting in substitution of a threonine for an alanine (A844T). Genetic analysis of the available members of the patients family showed that the A844T mutation segregated with LFSNHL, but was not detected in any of 140 control chromosomes. It thus appears likely that the A844T mutation is causative for hearing loss in this group. Speech audiometry, self-recording audiometry and auditory brainstem responses showed the patient to have cochlear deafness without retrocochlear dysfunction. No mutation was found in the other two patients.

Megakaryoblastic leukemia factor-1 gene in the susceptibility to coronary artery disease

Coronary artery disease (CAD) is based on the atherosclerosis of coronary artery and may manifest with myocardial infarction or angina pectoris. Although it is widely accepted that genetic factors are linked to CAD and several disease-related genes have been reported, only a few could be replicated suggesting that there might be some other CAD-related genes. To identify novel susceptibility loci for CAD, we used microsatellite markers in the screening and found six different candidate CAD loci. Subsequent single nucleotide polymorphism (SNP) association studies revealed an association between CAD and megakaryoblastic leukemia factor-1 gene (MKL1). The association with a promoter SNP of MKL1, −184Cxa0>xa0T, was found in a Japanese population and the association was replicated in another Japanese population and a Korean population. Functional analysis of the MKL1 promoter SNP suggested that the higher MKL1 expression was associated with CAD. These findings suggest that MKL1 is involved in the pathogenesis of CAD.

Mutational Profiles and Molecular Etiologies of Hypertrophic Cardiomyopathy and Dilated Cardiomyopathy in Asian Populations

Idiopathic cardiomyopathy (ICM) had been defined as the cardiac disease of unknown etiology. There are two major clinical phenotypes of ICM; hypertrophic cardiomyopathy (HCM) and dilated cardiomyopathy (DCM). During the last decade, a considerable progress has been made for identifying gene mutations as the causes of HCM and DCM. We have searched for mutations in the known disease genes for ICM in Japanese and Korean patients with HCM or DCM, and identified a considerable number of disease-related mutations in a portion of HCM patients and a few DCM patients. In addition, our recent candidate gene approach has revealed that mutations in the titin gene cause both HCM and DCM, and functional analyses of the titin mutations have suggested that the HCM-related and DCM-related mutations would lead to stiff sarcomere and loose sarcomere, respectively. The notion that HCM is a disease of stiff sarcomere can also be supported by that most of the HCM-related mutations in the genes for sarcomeric proteins increase the calcium sensitivity of muscle contraction, i.e. cardiac muscles are contracted under relatively low calcium concentration. In other words, HCM-related sarcomere mutations may lead to relatively stiff sarcomere under physiological condition. On the other hand, most of the DCM-related mutations in Z-disc elements would manifest with loose sarcomere, which may lead to relatively inefficient transmission of power through the Z-disc, then, DCM at least in part can be considered as a disease of loose sarcomere. In this concept, cardiac hypertrophy in HCM can also be considered as compensatory response against the excess overload, and the decompensation could be occurred later, while DCM can be considered as to be with less compensatory hypertrophy and earlier decompensation than HCM.