Kimihiko Kato

Mitochondrial haplogroup N9a confers resistance against type 2 diabetes in Asians.

Because mitochondria play pivotal roles in both insulin secretion from the pancreatic beta cells and insulin resistance of skeletal muscles, we performed a large-scale association study to identify mitochondrial haplogroups that may confer resistance against or susceptibility to type 2 diabetes mellitus (T2DM). The study population comprised 2,906 unrelated Japanese individuals, including 1,289 patients with T2DM and 1,617 controls, and 1,365 unrelated Korean individuals, including 732 patients with T2DM and 633 controls. The genotypes for 25 polymorphisms in the coding region of the mitochondrial genome were determined, and the haplotypes were classified into 10 major haplogroups (i.e., F, B, A, N9a, M7a, M7b, G, D4a, D4b, and D5). Multivariate logistic-regression analysis with adjustment for age and sex revealed that the mitochondrial haplogroup N9a was significantly associated with resistance against T2DM (P=.0002) with an odds ratio of 0.55 (95% confidence interval 0.40-0.75). Even in the modern environment, which is often characterized by satiety and physical inactivity, this haplogroup might confer resistance against T2DM.

Genetic Risk for Ischemic and Hemorrhagic Stroke

Objective—We performed an association study to identify gene polymorphisms for assessing the genetic risk of ischemic or hemorrhagic stroke. Methods and Results—The study population comprised 3151 unrelated Japanese individuals: 1141 stroke patients (636 with atherothrombotic cerebral infarction, 282 with intracerebral hemorrhage, and 223 with subarachnoid hemorrhage) and 2010 controls. The genotypes for 202 polymorphisms of 152 genes were determined by suspension array technology. Multivariable logistic regression analysis with adjustment for conventional risk factors revealed that the –572G→C polymorphism of the interleukin-6 (IL-6) gene (IL6) was significantly (P<0.001) associated with both atherothrombotic cerebral infarction and intracerebral hemorrhage and that the –55C→T polymorphism of the uncoupling protein 3 gene (UCP3), the –863C→A polymorphism of the tumor necrosis factor (TNF) gene (TNF), and the G→A (Gly243Asp) polymorphism of the polycystic kidney disease 1–like gene (PKD1-like) were significantly associated with subarachnoid hemorrhage. Conclusions—IL6 genotype may be useful in assessing the genetic risk for atherothrombotic cerebral infarction and intracerebral hemorrhage, and genotypes for UCP3, TNF, and PKD1-like may be similarly beneficial in assessment of the risk for subarachnoid hemorrhage. Validation of our findings will require additional studies with independent subject panels.

Identification of CELSR1 as a susceptibility gene for ischemic stroke in Japanese individuals by a genome-wide association study

OBJECTIVEnWe have performed a genome-wide association study (GWAS) to identify genetic variants that confer susceptibility to ischemic stroke.nnnMETHODSnA total of 6341 individuals from three independent populations was examined. Subject panel A comprised 131 individuals with ischemic stroke and 135 controls; subject panel B comprised 790 individuals with ischemic stroke and 3435 controls; and subject panel C comprised 71 individuals with ischemic stroke and 1779 controls. A GWAS for ischemic stroke was performed in subject panel A with the use of the GeneChip Human Mapping 500K Array Set (Affymetrix).nnnRESULTSnThe relation of 100 single nucleotide polymorphisms (SNPs) selected by the GWAS to ischemic stroke was examined in 705 subjects with ischemic stroke and 3426 controls selected from subject panel B. Three SNPs (rs1671021 of LLGL2, rs9615362 of CELSR1, and rs753307 of RUVBL2) were significantly (P<0.05) associated with ischemic stroke. After DNA sequencing of linkage disequilibrium blocks containing these SNPs, three tag SNPs (rs6007897 of CELSR1, rs1671021 of LLGL2, and rs1062708 of RUVBL2) and a nonsynonymous SNP (rs4044210 of CELSR1) were examined for their relation to ischemic stroke in subject panels B and C. Both rs6007897 (A-->G, Thr2268Ala) and rs4044210 (A-->G, Ile2107Val) of CELSR1 as well as rs1671021 (T-->C, Phe479Leu) of LLGL2 were significantly associated with ischemic stroke in subject panel B. The rs6007897 and rs4044210 polymorphisms of CELSR1 were also significantly associated with ischemic stroke in subject panel C.nnnCONCLUSIONnCELSR1 is a susceptibility gene for ischemic stroke in Japanese individuals, although the functional relevance of the identified SNPs was not determined.

Women With Mitochondrial Haplogroup N9a Are Protected Against Metabolic Syndrome

To identify mitochondrial haplogroups that confer resistance against or susceptibility to metabolic syndrome, we performed a large-scale association study on 1,337 unrelated Japanese individuals, including 871 subjects with metabolic syndrome and 466 control subjects. Metabolic syndrome was diagnosed according to modified National Cholesterol Education Program Adult Treatment Panel III guidelines, using the cutoff point for obesity as a BMI of ≥25 kg/m2 instead of waist circumference. The genotypes for 25 polymorphisms in the coding region of the mitochondrial genome were determined, and the haplotypes were classified into 10 major haplogroups, i.e., F, B, A, N9a, M7a, M7b, G1, G2, D5, and D4. Multivariate logistic regression analysis revealed that the haplogroup N9a was significantly associated with resistance against metabolic syndrome in women with an odds ratio (OR) of 0.21 (95% CI 0.07–0.58, P = 0.0042). Women with haplogroups G1 and D5 tended to be resistant against metabolic syndrome with an OR of 0.22 (0.06–0.68, P = 0.0129) for G1 and with an OR of 0.32 (0.10–0.96, P = 0.0469) for D5, respectively. These results indicate that mitochondrial haplogroup N9a may be a protective factor against metabolic syndrome in Japanese women.

Mitochondrial haplogroup N9b is protective against myocardial infarction in Japanese males

Superoxide, which mitochondria mainly produce in vascular endothelial cells, plays an important role in the pathogenesis of atherosclerosis and coronary artery disease. Accordingly, mitochondrial functional differences are thought to be one of the most important factors for the risk of myocardial infarction among various individuals. In the present study, we surveyed mitochondrial haplogroups associated with myocardial infarction in Japanese subjects. The study population comprised 2,137 unrelated Japanese individuals, including 1,181 subjects with a first myocardial infarction (920 males, 261 females) and the control subjects (522 males, 434 females). Twenty-eight mitochondrial single nucleotide polymorphisms of 12 major mitochondrial haplogroups (A, B, D4, D5, F, G1, G2, M7a, M7b, M7c, N9a, and N9b) were determined by use of 28-plex PCR and fluorescent beads combined with sequence-specific oligonucleotide probes. After adjustment for age, sex, body mass index, and prevalence of smoking, hypertension, hypercholesterolemia, and type 2 diabetes, a significantly (Pxa0=xa00.0019) lower prevalence of haplogroup N9b was detected in subjects with myocardial infarction than in the controls. Especially, the prevalence of this haplogroup was significantly lower (Pxa0=xa00.0007) in the male subjects with the disease than in the male controls. In contrast, there were trends towards higher prevalence of the disease in haplogroup G1 for males (Pxa0<xa00.05). No significant haplogroup-related associations were detected for females. Our data suggest that haplogroup N9b confers resistance against myocardial infarction in Japanese males.

Genetic Factors for Ischemic and Hemorrhagic Stroke in Japanese Individuals

Background and Purpose— Although genetic epidemiologic studies have implicated several genetic variants as risk factors for ischemic or hemorrhagic stroke, the genetic determinants of these conditions remain largely unknown. We performed an association study to identify gene polymorphisms that confer susceptibility to atherothrombotic cerebral infarction, intracerebral hemorrhage, or subarachnoid hemorrhage. Methods— The study population comprised 3432 unrelated Japanese individuals: 1362 stroke patients (822 with atherothrombotic cerebral infarction, 333 with intracerebral hemorrhage, and 207 with subarachnoid hemorrhage) and 2070 controls. The genotypes for 50 polymorphisms of 38 candidate genes were determined by a method that combines the polymerase chain reaction and sequence-specific oligonucleotide probes with suspension array technology. Results— An initial &khgr;2 test (false discovery rate <0.05) and subsequent multivariable logistic-regression analysis with adjustment for conventional risk factors (P<0.05) revealed that the −14C→T polymorphism (rs1800977) of ABCA1, the A→C (rs3027898) and C→T (Ser532Leu, rs1059703) polymorphisms of IRAK1, and the G→C (Cys2229Ser) polymorphism (rs619203) of ROS1 were significantly associated with atherothrombotic cerebral infarction; that the −428G→A polymorphism (rs710968) of LIMK1 was significantly associated with intracerebral hemorrhage; and that the 13989A→G (Ile118Val) polymorphism (NC_000007.12) of CYP3A4 was significantly associated with subarachnoid hemorrhage. Conclusions— Genotypes for ABCA1, IRAK1, and ROS1 may prove useful for assessment of the genetic risk for atherothrombotic cerebral infarction, whereas those for LIMK1 and CYP3A4 may be similarly beneficial in assessment of the genetic risk for intracerebral hemorrhage and subarachnoid hemorrhage, respectively. Validation of these findings will require additional studies with independent subject panels.

Assessment of genetic risk for myocardial infarction

Although lifestyle and environmental factors influence the prevalence of myocardial infarction, genetic epidemiological studies have suggested that several genetic variants increase the risk for this condition. We have performed a large-scale association study to identify gene polymorphisms for reliable assessment of the genetic risk of myocardial infarction. The study population comprised 3,483 unrelated Japanese individuals (1,913 men; 1,570 women), including 1,192 subjects with myocardial infarction and 2,291 controls. The genotypes for 164 polymorphisms of 137 candidate genes were determined with an oligonucleotide ligation assay based on analysis of fluorescent microspheres with suspension array technology. Multivariable logistic regression analysis with adjustment for age, sex, body mass index, and the prevalence of smoking, hypertension, diabetes mellitus, and hypercholesterolemia revealed that the 677C-->T (Ala222Val) polymorphism of MTHFR, the 1595C-->G (Ser447Stop) polymorphism of LPL, and the -108/3G-->4G polymorphism of IPF1 were significantly associated with the prevalence of myocardial infarction. A stepwise forward selection procedure demonstrated that IPF1, MTHFR, and LPL genotypes significantly affected the prevalence of myocardial infarction. Combined genotype analysis of these polymorphisms yielded a maximum odds ratio of 2.54 for the combined genotype of TT for MTHFR, CC for LPL, and 3G3G for IPF1. The genotypes for MTHFR, LPL, and IPF1 may prove reliable for assessment of genetic risk for myocardial infarction. Determination of the combined genotype for these genes may contribute to primary, personalized prevention of this condition.

Association of genetic variants with myocardial infarction in Japanese individuals with metabolic syndrome

OBJECTIVEnThe purpose of the present study was to identify genetic variants that confer susceptibility to myocardial infarction (MI) in individuals with metabolic syndrome (MetS).nnnMETHODSnThe study population comprised 1887 Japanese individuals with MetS, including 773 subjects with MI and 1114 controls. The genotypes for 136 polymorphisms of 97 candidate genes were determined.nnnRESULTSnAn initial screen by the chi-square test revealed that seven polymorphisms were significantly (false discovery rate<0.05) associated with the prevalence of MI in individuals with MetS. Subsequent multivariable logistic regression analysis with adjustment for covariates revealed that the G-->A (Ser89Asn) polymorphism of UTS2 [odds ratio (OR), 1.90; 95% confidence interval (CI), 1.18-3.08], the 2445G-->A (Ala54Thr) polymorphism of FABP2 (OR, 1.72; 95% CI, 1.23-2.40), the -11377C-->G polymorphism of ADIPOQ (OR, 1.43; 95% CI, 1.15-1.79), the -231A-->G polymorphism of EDNRA (OR, 0.65; 95% CI, 0.48-0.89), and the -108/3G-->4G polymorphism of PDX1 (OR, 0.64; 95% CI, 0.48-0.87) were significantly (P<0.05) associated with MI. The variant alleles of UTS2, FABP2, and ADIPOQ were risk factors for MI, whereas the variant alleles of EDNRA and PDX1 were protective against this condition. A stepwise forward selection procedure demonstrated that UTS2, FABP2, ADIPOQ, EDNRA, and PDX1 genotypes were significant (P<0.05) and independent determinants of MI.nnnCONCLUSIONSnDetermination of genotypes for these polymorphisms of UTS2, FABP2, ADIPOQ, EDNRA, and PDX1 may prove informative for assessment of the genetic risk for MI in Japanese individuals with MetS.

Association of Genetic Variants with Chronic Kidney Disease in Japanese Individuals

BACKGROUND AND OBJECTIVESnAlthough genetic linkage analyses and association studies have implicated several loci and candidate genes in predisposition to chronic kidney disease (CKD), the genes that underlie genetic susceptibility to this condition have remained uncharacterized. The purpose of the present study was to identify genetic variants that confer susceptibility to CKD in Japanese individuals.nnnDESIGN, SETTING, PARTICIPANTS, & MEASUREMENTSnThe study population comprised 5217 Japanese individuals (2955 men, 2262 women), including 778 subjects (480 men, 298 women) with CKD [estimated GFR (eGFR), <50 ml min(-1) 1.73 m(-2)] and 4439 controls (2475 men, 1964 women; eGFR, > or =60 ml min(-1) 1.73 m(-2)). The genotypes for 40 polymorphisms of 32 candidate genes were determined.nnnRESULTSnThe chi-square test and multivariable logistic regression analysis with adjustment for covariates revealed that the -219G-->T polymorphism of APOE, the -519A-->G of MMP1, the -866G-->A of UCP2, the -1607/1G-->2G of MMP1, the A-->G (Lys45Glu) of MMP3, the G-->A (Ala163Thr) of AGTR1, the G-->A (Gly670Arg) of PECAM1, and the -55C-->T of UCP3 were significantly (false discovery rate <0.05) associated with CKD. Comparison of allele frequencies of these polymorphisms by the chi-square test between subgroups of CKD and control subjects individually matched for covariates revealed that the -519A-->G of MMP1 and the -866G-->A of UCP2 were significantly (P < 0.05) associated with CKD.nnnCONCLUSIONSnMMP1 and UCP2 may be susceptibility loci for CKD in Japanese individuals. Determination of genotypes for these polymorphisms may prove informative for prediction of genetic risk for CKD.

Identification of hypo- and hypermethylated genes related to atherosclerosis by a genome-wide analysis of DNA methylation

Epigenetic modification, particularly changes in DNA methylation at gene promoters, is implicated in the pathogenesis of atherosclerosis. However, the analysis of DNA methylation in atherosclerosis has been limited to a few selected candidate genes. In this study, we therefore performed a genome-wide analysis of DNA methylation in the atherosclerotic human aorta. A total of 48 post-mortem human aortic intima specimens were examined. To avoid the effects of interindividual variation, we performed intraindividual paired comparisons between atheromatous plaque lesions and corresponding plaque-free tissue for 24 subjects. Bisulfite-modified genomic DNA was analyzed for DNA methylation with a specific microarray (Illumina HumanMethylation450 BeadChip). We compensated for multiple comparisons by applying Bonferronis correction for statistical significance of association. DNA methylation was significantly (P<1.03x10⁻⁷) reduced at 15 CpG sites in 14 genes and increased at 30 CpG sites in 22 genes in atheromatous plaque compared with plaque-free intima. Three of the hypomethylated genes [Drosophila headcase (HECA), early B-cell factor 1 (EBF1) and nucleotide-binding oligomerization domain containing 2 (NOD2)] and three of the hypermethylated genes [human mitogen-activated protein kinase kinase kinase kinase 4 (MAP4K4), zinc finger E-box binding homeobox 1 (ZEB1) and FYN] were previously been implicated in atherosclerosis. The overexpression of HECA, EBF1 or NOD2 or the suppression of MAP4K4, ZEB1 or FYN expression in cultured HEK293 cells resulted in significant (P<4.80x10⁻⁷) changes in the expression of atherosclerosis-related genes, as determined with an expression microarray (Illumina HumanHT-12 v4 Expression BeadChip). Our findings suggested that HECA, EBF1 and NOD2 were significantly hypomethylated, whereas MAP4K4, ZEB1 and FYN were hypermethylated, in atheromatous plaque lesions compared with plaque-free intima. Epigenetic mechanisms may thus contribute to the pathogenesis of atherosclerosis.