RNF4-mediated SUMO-targeted ubiquitination relieves PARIS/ZNF746-mediated transcriptional repression.

The transcriptional repressor PARIS, which is a substrate of the ubiquitin E3 ligase parkin, represses the expression of the transcriptional co-activator, PGC-1α. However, little is known about how its repression activity is regulated. We have previously shown that PARIS is SUMOylated, and this SUMOylation plays an important role in regulating its transcriptional repression activity. In this study, we demonstrated that PARIS SUMOylation induced its ubiquitination and subsequent proteasomal degradation, which was mediated by the SUMO-targeted ubiquitin ligase RNF4. Reporter gene assays revealed that co-expression of SUMO3 and RNF4 relieved PARIS-mediated transcriptional repression. Conversely, the SUMO E3 ligase PIASy inhibited the RNF4-mediated ubiquitination of PARIS and blocked the RNF4-mediated relief of PARIS-mediated transcriptional repression. These results suggest that RNF4 regulates PARIS ubiquitination to control its transcriptional repression activity.

Evolutionary history of disease-susceptibility loci identified in longitudinal exome-wide association studies.

BACKGROUND: Our longitudinal exome-wide association studies previously detected various genetic determinants of complex disorders using ~26,000 single-nucleotide polymorphisms (SNPs) that passed quality control and longitudinal medical examination data (mean follow-up period, 5 years) in 4884-6022 Japanese subjects. We found that allele frequencies of several identified SNPs were remarkably different among four ethnic groups. Elucidating the evolutionary history of disease-susceptibility loci may help us uncover the pathogenesis of the related complex disorders. METHODS: In the present study, we conducted evolutionary analyses such as extended haplotype homozygosity, focusing on genomic regions containing disease-susceptibility loci and based on genotyping data of our previous studies and datasets from the 1000 Genomes Project. RESULTS: Our evolutionary analyses suggest that derived alleles of rs78338345 of GGA3, rs7656604 at 4q13.3, rs34902660 of SLC17A3, and six SNPs closely located at 12q24.1 associated with type 2 diabetes mellitus, obesity, dyslipidemia, and three complex disorders (hypertension, hyperuricemia, and dyslipidemia), respectively, rapidly expanded after the human dispersion from Africa (Out-of-Africa). Allele frequencies of GGA3 and six SNPs at 12q24.1 appeared to have remarkably changed in East Asians, whereas the derived alleles of rs34902660 of SLC17A3 and rs7656604 at 4q13.3 might have spread across Japanese and non-Africans, respectively, although we cannot completely exclude the possibility that allele frequencies of disease-associated loci may be affected by demographic events. CONCLUSION: Our findings indicate that derived allele frequencies of nine disease-associated SNPs (rs78338345 of GGA3, rs7656604 at 4q13.3, rs34902660 of SLC17A3, and six SNPs at 12q24.1) identified in the longitudinal exome-wide association studies largely increased in non-Africans after Out-of-Africa.

Two novel susceptibility loci for type 2 diabetes mellitus identified by longitudinal exome-wide association studies in a Japanese population.

Recent genome-wide association studies identified genetic variants that confer susceptibility to type 2 diabetes mellitus (T2DM). However, few longitudinal genome-wide association studies of this metabolic disorder have been reported to date. Therefore, we performed a longitudinal exome-wide association study of T2DM, using 24,579 single nucleotide polymorphisms (SNPs) and repeated measurements from 6022 Japanese individuals. The generalized estimating equation model was applied to test relations of SNPs to three T2DM-related parameters: prevalence of T2DM, fasting plasma glucose level, and blood glycosylated hemoglobin content. Three SNPs that passed quality control were significantly (P<2.26×10-7) associated with two of the three T2DM-related parameters in additive and recessive models. Of the three SNPs, rs6414624 in EVC and rs78338345 in GGA3 were novel susceptibility loci for T2DM. In the present study, the SNP of GGA3 was predicted to be a genetic variant whose minor allele frequency has recently increased in East Asia.

Identification of six novel susceptibility loci for dyslipidemia using longitudinal exome-wide association studies in a Japanese population.

Recent genome-wide association studies have identified various dyslipidemia-related genetic variants. However, most studies were conducted in a cross-sectional manner. We thus performed longitudinal exome-wide association studies of dyslipidemia in a Japanese population. We used ~244,000 genetic variants and clinical data of 6022 Japanese individuals who had undergone annual health checkups for several years. After quality control, the association of dyslipidemia-related phenotypes with 24,691 single nucleotide polymorphisms (SNPs) was tested using the generalized estimating equation model. In total, 82 SNPs were significantly (P < 2.03 × 10-6) associated with dyslipidemia phenotypes. Of these SNPs, four (rs74416240 of TCHP, rs925368 of GIT2, rs7969300 of ATXN2, and rs12231744 of NAA25) and two (rs34902660 of SLC17A3 and rs1042127 of CDSN) were identified as novel genetic determinants of hypo-HDL- and hyper-LDL-cholesterolemia, respectively. A replication study using the cross-sectional data of 8310 Japanese individuals showed the association of the six identified SNPs with dyslipidemia-related traits.

Identification of 12 novel loci that confer susceptibility to early-onset dyslipidemia.

The circulating concentrations of triglycerides, high density lipoprotein (HDL)­cholesterol, and low density lipoprotein (LDL)­cholesterol have a substantial genetic component, and the heritability of early­onset dyslipidemia might be expected to be higher compared with late­onset forms. In the present study, exome­wide association studies (EWASs) were performed for early­onset hypertriglyceridemia, hypo­HDL­cholesterolemia, and hyper­LDL­cholesterolemia, with the aim to identify genetic variants that confer susceptibility to these conditions in the Japanese population. A total of 8,073 individuals aged ≤65 years were enrolled in the study. The EWASs for hypertriglyceridemia (2,664 cases and 5,294 controls), hypo­HDL­cholesterolemia (974 cases and 7,085 controls), and hyper­LDL­cholesterolemia (2,911 cases and 5,111 controls) were performed with Illumina Human Exome­12 v1.2 DNA Analysis BeadChip or Infinium Exome­24 v1.0 BeadChip arrays. The association of allele frequencies for 31,198, 31,133, or 31,175 single nucleotide polymorphisms (SNPs) to hypertriglyceridemia, hypo­HDL­cholesterolemia, or hyper­LDL­cholesterolemia, respectively, was examined with Fisher's exact test. To compensate for multiple comparisons of genotypes with each of the three conditions, Bonferroni's correction was applied for statistical significance of association. The results demonstrated that 25, 28 and 65 SNPs were significantly associated with hypertriglyceridemia, hypo­HDL­cholesterolemia and hyper­LDL­cholesterolemia, respectively. Multivariable logistic regression analysis with adjustment for age and sex revealed that all 25, 28 and 65 of these SNPs were significantly associated with hypertriglyceridemia, hypo­HDL­cholesterolemia and hyper­LDL­cholesterolemia, respectively. Following examination of the association of the identified SNPs to serum concentrations of triglycerides, HDL­cholesterol, or LDL­cholesterol, linkage disequilibrium of the SNPs, and results of previous genome­wide association studies, we newly identified chromosomal region 19p12 as a susceptibility locus for hypertriglyceridemia, eight loci (MOB3C­TMOD4, LPGAT1, EHD3, COL6A3, ZNF860­CACNA1D, COL6A5, DCLRE1C, ZNF77) for hypo­HDL­cholesterolemia, and three loci (KIAA0319­FAM65B, UBD, LOC105375015) for hyper­LDL­cholesterolemia. The present study thus identified 12 novel loci that may confer susceptibility to early­onset dyslipidemia. Determination of genotypes for the SNPs at these loci may prove informative for assessment of genetic risk for hypertriglyceridemia, hypo­HDL­cholesterolemia, or hyper­LDL­cholesterolemia in the Japanese population.

Identification of 26 novel loci that confer susceptibility to early-onset coronary artery disease in a Japanese population.

Early-onset coronary artery disease (CAD) has a strong genetic component. Although genome-wide association studies have identified various genes and loci significantly associated with CAD mainly in European populations, genetic variants that contribute toward susceptibility to this condition in Japanese patients remain to be definitively identified. In the present study, exome-wide association studies (EWASs) were performed to identify genetic variants that confer susceptibility to early-onset CAD in Japanese. A total of 7,256 individuals aged ≤65 years were enrolled in the present study. EWAS were conducted on 1,482 patients with CAD and 5,774 healthy controls. Genotyping of single nucleotide polymorphisms (SNPs) was performed using Illumina Human Exome-12 DNA Analysis BeadChip or Infinium Exome-24 BeadChip arrays. The association between allele frequencies for 31,465 SNPs that passed quality control and CAD was examined using Fisher's exact test. To compensate for multiple comparisons of allele frequencies with CAD, a false discovery rate (FDR) of <0.05 was applied for statistically significant associations. The association between allele frequencies for 31,465 SNPs and CAD, as determined by Fisher's exact test, demonstrated that 170 SNPs were significantly (FDR <0.05) associated with CAD. Multivariable logistic regression analysis with adjustment for age, sex, and the prevalence of hypertension, diabetes mellitus and dyslipidemia revealed that 162 SNPs were significantly (P<0.05) associated with CAD. A stepwise forward selection procedure was performed to examine the effects of genotypes for the 162 SNPs on CAD. The 54 SNPs were significant (P<0.05) and independent [coefficient of determination (R2), 0.0008 to 0.0297] determinants of CAD. These SNPs together accounted for 15.5% of the cause of CAD. Following examination of results from previous genome-wide association studies and linkage disequilibrium of the identified SNPs, 21 genes (RNF2, YEATS2, USP45, ITGB8, TNS3, FAM170B-AS1, PRKG1, BTRC, MKI67, STIM1, OR52E4, KIAA1551, MON2, PLUT, LINC00354, TRPM1, ADAT1, KRT27, LIPE, GFY and EIF3L) and five chromosomal regions (2p13, 4q31.2, 5q12, 13q34 and 20q13.2) that were significantly associated with CAD were newly identified in the present study. Gene ontology analysis demonstrated that various biological functions were predicted in the 18 genes identified in the present study. The network analysis revealed that the 18 genes had potential direct or indirect interactions with the 30 genes previously revealed to be associated with CAD or with the 228 genes identified in previous genome-wide association studies. The present study newly identified 26 loci that confer susceptibility to CAD. Determination of genotypes for the SNPs at these loci may prove informative for assessment of the genetic risk for CAD in Japanese patients.

Identification of 13 novel susceptibility loci for early-onset myocardial infarction, hypertension, or chronic kidney disease.

Early­onset cardiovascular and renal diseases have a strong genetic component. In the present study, exome­wide association studies (EWASs) were performed to identify genetic variants that confer susceptibility to early­onset myocardial infarction (MI), hypertension, or chronic kidney disease (CKD) in Japanese individuals. A total of 8,093 individuals aged ≤65 years was enrolled in the study. The EWASs for MI, hypertension, and CKD were performed in 6,926 subjects (1,152 cases, 5,774 controls), 8,080 subjects (3,444 cases, 4,636 controls), and 2,556 subjects (1,051 cases, 1,505 controls), respectively. Genotyping of single nucleotide polymorphisms (SNPs) was performed with Illumina Human Exome­12 DNA Analysis BeadChip or Infinium Exome­24 BeadChip arrays. The associations of allele frequencies for 31,245, 31,276, or 31,514 SNPs that passed quality control to MI, hypertension, and CKD, respectively, was examined with Fisher's exact test. Bonferroni's correction for statistical significance of association was applied to compensate for multiple comparisons of genotypes with MI, hypertension, or CKD. The EWASs of allele frequencies revealed that 25, 11, and 11 SNPs were significantly associated with MI (P<1.60x10­6), hypertension (P<1.60x10­6), or CKD (P<1.59x10­6), respectively. Multivariable logistic regression analysis with adjustment for covariates showed that all 25, 11, and 11 SNPs were significantly associated with MI (P<0.0005), hypertension (P<0.0011), or CKD (P<0.0011), respectively. On examination of the results from previous genome­wide association studies and linkage disequilibrium of the identified SNPs, 11 loci (TMOD4, COL6A3, ADGRL3­CXCL8­MARCH1, OR52E4, TCHP­GIT2, CCDC63, 12q24.1, OAS3, PLCB2­VPS33B, GOSR2, ZNF77), six loci (MOB3C­TMOD4, COL6A3, COL6A5, CXCL8­MARCH1, NFKBIL1­6p21.3­NCR3, PLCB2­VPS33B), and seven loci (MOB3C­TMOD4, COL6A3, COL6A5, ADGRL3­CXCL8­MARCH1, MUC17, PLCB2­VPS33B, ZNF77) were identified as novel loci significantly associated with MI, hypertension, and CKD, respectively. Furthermore, six genes (TMOD4, COL6A3, CXCL8, MARCH1, PLCB2, VPS33B) were significantly associated with MI, hypertension and CKD; two genes (ADGRL3, ZNF77) with MI and CKD; and two genes (COL6A5, MOB3C) with hypertension and CKD. Therefore, 13 novel loci (MOB3C­TMOD4, COL6A3, ADGRL3­CXCL8­MARCH1, OR52E4, TCHP­GIT2, CCDC63, 12q24.1, OAS3, PLCB2­VPS33B, ZNF77, COL6A5, NFKBIL1­NCR3, MUC17) were identified that confer susceptibility to early­onset MI, hypertension, or CKD. The determination of genotypes for the SNPs at these loci may provide informative for assessment of the genetic risk for MI, hypertension, or CKD.

Six novel susceptibility loci for coronary artery disease and cerebral infarction identified by longitudinal exome-wide association studies in a Japanese population.

Coronary artery disease (CAD) and cerebral infarction (CI) remain major causes of morbidity and mortality in humans. Recent genome-wide association studies have identified various genetic variants associated with these diseases. However, these studies were commonly conducted in a cross-sectional manner. Therefore, the present research performed longitudinal exome-wide association studies for CAD and CI using data on ~244,000 genotyped variants and the clinical data of 6,026 Japanese individuals who had attended annual health checkups for several years (mean followed-up period, 5±3 years). Following quality controls, the significance [false discovery rate (FDR) of <0.05] of association of the diseases with 24,651 single nucleotide polymorphisms (SNPs) in 5,989 individuals for three inheritance models was tested using the generalized estimating equation model. SNPs that reached statistical significance were further screened against a threshold of approxdf (a scale of small effective sample size) of >30. The longitudinal exome-wide association studies revealed that three SNPs [rs4606855 of ADGRE3 (P=2.5×10-6; FDR=0.031; approxdf=71), rs3746414 of ZFP64 (P=5.9×10-6; FDR=0.048; approxdf=93) and rs7132908 of FAIM2 (P<2.0×10-16; FDR<4.9×10-12; approxdf=65)] were significantly associated with the prevalence of CAD. A different set of three SNPs [rs6580741 of FAM186A (P<2.0×10-16; FDR<4.9×10-12; approxdf=48), rs1324015 of LINC00400 (P<2.0×10-16; FDR<4.9×10-12; approxdf=49) and rs884205 of TNFRSF11A (P<2.0×10-16; FDR<4.9×10-12; approxdf=32)] was significantly associated with CI. The comparison of disease incidence with these SNPs demonstrated that all the minor alleles were associated with decreased susceptibility to CAD or CI. In conclusion, six novel SNPs were identified as susceptibility loci for CAD (rs4606855 of ADGRE3, rs3746414 of ZFP64, and rs7132908 of FAIM2) or CI (rs6580741 of FAM186A, rs1324015 of LINC00400, and rs884205 of TNFRSF11A).

Identification of nine novel loci related to hematological traits in a Japanese population.

Recent genome-wide association studies have identified various genetic variants associated with hematological traits. Although it is possible that quantitative data of hematological traits are varied among the years examined, conventional genome-wide association studies have been conducted in a cross-sectional manner that measures traits at a single point in time. To address this issue, we have traced blood profiles in 4,884 Japanese individuals who underwent annual health check-ups for several years. In the present study, longitudinal exome-wide association studies were conducted to identify genetic variants related to 13 hematological phenotypes. The generalized estimating equation model showed that a total of 67 single nucleotide polymorphisms (SNPs) were significantly [false discovery rate (FDR) of <0.01] associated with hematological phenotypes. Of the 67 SNPs, nine SNPs were identified as novel hematological markers: rs4686683 of SENP2 for red blood cell count (FDR = 0.008, P = 5.5 × 10-6); rs3917688 of SELP for mean corpuscular volume (FDR = 0.005, P = 2.4 × 10-6); rs3133745 of C8orf37-AS1 for white blood cell count (FDR = 0.003, P = 1.3 × 10-6); rs13121954 at 4q31.2 for basophil count (FDR = 0.007, P = 3.1 × 10-5); rs7584099 at 2q22.3 (FDR = 2.6 × 10-5, P = 8.8 × 10-8), rs1579219 of HCG17 (FDR = 0.003, P = 2.0 × 10-5), and rs10757049 of DENND4C (FDR = 0.008, P = 5.6 × 10-5) for eosinophil count; rs12338 of CTSB for neutrophil count (FDR = 0.007, P = 2.9 × 10-5); and rs395967 of OSMR-AS1 for monocyte count (FDR = 0.008, P = 3.2 × 10-5).

Identification of four genes as novel susceptibility loci for early-onset type 2 diabetes mellitus, metabolic syndrome, or hyperuricemia.

Given that early-onset type 2 diabetes mellitus (T2DM), metabolic syndrome (MetS), and hyperuricemia have been shown to have strong genetic components, the statistical power of a genetic association study may be increased by focusing on early-onset subjects with these conditions. Although genome-wide association studies have identified various genes and loci significantly associated with T2DM, MetS, and hyperuricemia, genetic variants that contribute to predisposition to these conditions in Japanese subjects remain to be identified definitively. We performed exome-wide association studies (EWASs) for early-onset T2DM, MetS, or hyperuricemia to identify genetic variants that confer susceptibility to these conditions. A total of 8,102 individuals aged ≤65 years were enrolled in the present study. The EWAS for T2DM was performed with 7,407 subjects (1,696 cases, 5,711 controls), that for MetS with 4,215 subjects (2,296 cases, 1,919 controls), and that for hyperuricemia with 7,919 subjects (1,365 cases, 6,554 controls). Single nucleotide polymorphisms (SNPs) were genotyped with Illumina Human Exome-12 DNA Analysis BeadChip or Infinium Exome-24 BeadChip arrays. The relationship of allele frequencies for 31,210, 31,521, or 31,142 SNPs that passed quality control for T2DM, MetS, or hyperuricemia, respectively, was examined with Fisher's exact test. To compensate for multiple comparisons of genotypes with T2DM, MetS, or hyperuricemia, we applied Bonferroni's correction for statistical significance of association. The EWAS of allele frequencies revealed that four, six, or nine SNPs were significantly associated with T2DM (P<1.60×10-6), MetS (P<1.59×10-6), or hyperuricemia (P<1.61×10-6), respectively. Multivariable logistic regression analysis with adjustment for age and sex revealed that three, six, or nine SNPs were significantly related to T2DM (P<0.0031), MetS (P<0.0021), or hyperuricemia (P<0.0014). After examination of the association of identified SNPs to T2DM-, MetS-, or hyperuricemia-related traits, linkage disequilibrium of the SNPs, and results of previous genome-wide association studies, newly identified ZNF860 and OR4F6 were the susceptibility loci for T2DM, OR52E4 and OR4F6 for MetS, and HERPUD2 for hyperuricemia. Given that OR4F6 was significantly associated with both T2DM and MetS, we newly identified four genes (ZNF860, OR4F6, OR52E4, HERPUD2) that confer susceptibility to early-onset T2DM, MetS, or hyperuricemia. Determination of genotypes for the SNPs in these genes may prove informative for assessment of the genetic risk for T2DM, MetS, or hyperuricemia.

Identification of nine genes as novel susceptibility loci for early-onset ischemic stroke, intracerebral hemorrhage, or subarachnoid hemorrhage.

Given that substantial genetic components have been shown in ischemic stroke, intracerebral hemorrhage (ICH), and subarachnoid hemorrhage (SAH), heritability may be higher in early-onset than late-onset individuals with these conditions. Although genome-wide association studies (GWASs) have identified various genes and loci significantly associated with ischemic stroke, ICH, or intracranial aneurysm mainly in European ancestry populations, genetic variants that contribute to susceptibility to these disorders remain to be identified definitively. We performed exome-wide association studies (EWASs) to identify genetic variants that confer susceptibility to ischemic stroke, ICH, or SAH in early-onset subjects with these conditions. A total of 6,649 individuals aged ≤65 years were examined. For the EWAS of ischemic or hemorrhagic stroke, 6,224 individuals (450 subjects with ischemic stroke, 5,774 controls) or 6,179 individuals (261 subjects with ICH, 176 subjects with SAH, 5,742 controls), respectively, were examined. EWASs were performed with the use of Illumina Human Exome-12 v1.2 DNA Analysis BeadChip or Infinium Exome-24 v1.0 BeadChip. To compensate for multiple comparisons of allele frequencies with ischemic stroke, ICH, or SAH, we applied a false discovery rate (FDR) of <0.05 for statistical significance of association. The association of allele frequencies of 31,245 single nucleotide polymorphisms (SNPs) that passed quality control to ischemic stroke was examined with Fisher's exact test, and 31 SNPs were significantly (FDR <0.05) associated with ischemic stroke. The association of allele frequencies of 31,253 or 30,970 SNPs to ICH or SAH, respectively, was examined with Fisher's exact test, and six or two SNPs were significantly associated with ICH or SAH, respectively. Multivariable logistic regression analysis with adjustment for age, sex, and the prevalence of hypertension and diabetes mellitus revealed that 12 SNPs were significantly [P<0.0004 (0.05/124)] related to ischemic stroke. Similar analysis with adjustment for age, sex, and the prevalence of hypertension revealed that six or two SNPs were significantly [P<0.0016 (0.05/32)] related to ICH or SAH, respectively. After examination of linkage disequilibrium of identified SNPs and results of previous GWASs, we identified HHIPL2, CTNNA3, LOC643770, UTP20, and TRIB3 as susceptibility loci for ischemic stroke, DNTTIP2 and FAM205A as susceptibility loci for ICH, and FAM160A1 and OR52E4 as such loci for SAH. Therefore, to the best of our knowledge, we have newly identified nine genes that confer susceptibility to early-onset ischemic stroke, ICH, or SAH. Determination of genotypes for the SNPs in these genes may prove informative for assessment of the genetic risk for ischemic stroke, ICH, or SAH in Japanese.

MicroRNAs as Potential Mediators for Cigarette Smoking Induced Atherosclerosis.

Smoking increases the risk of atherosclerosis-related events, such as myocardial infarction and ischemic stroke. Recent studies have examined the expression levels of altered microRNAs (miRNAs) in various diseases. The profiles of tissue miRNAs can be potentially used in diagnosis or prognosis. However, there are limited studies on miRNAs following exposure to cigarette smoke (CS). The present study was designed to dissect the effects and cellular/molecular mechanisms of CS-induced atherosclerogenesis. Apolipoprotein E knockout (ApoE KO) mice were exposed to CS for five days a week for two months at low (two puffs/min for 40 min/day) or high dose (two puffs/min for 120 min/day). We measured the area of atherosclerotic plaques in the aorta, representing the expression of miRNAs after the exposure period. Two-month exposure to the high dose of CS significantly increased the plaque area in aortic arch, and significantly upregulated the expression of atherosclerotic markers (VCAM-1, ICAM-1, MCP1, p22phox, and gp91phox). Exposure to the high dose of CS also significantly upregulated the miRNA-155 level in the aortic tissues of ApoE KO mice. Moreover, the expression level of miR-126 tended to be downregulated and that of miR-21 tended to be upregulated in ApoE KO mice exposed to the high dose of CS, albeit statistically insignificant. The results suggest that CS induces atherosclerosis through increased vascular inflammation and NADPH oxidase expression and also emphasize the importance of miRNAs in the pathogenesis of CS-induced atherosclerosis. Our findings provide evidence for miRNAs as potential mediators of inflammation and atherosclerosis induced by CS.

Identification of novel hyper- or hypomethylated CpG sites and genes associated with atherosclerotic plaque using an epigenome-wide association study.

DNA methylation is an important epigenetic modification that has been implicated in the pathogenesis of atherosclerosis. Although previous studies have identified various CpG sites and genes whose methylation is associated with atherosclerosis in populations with European or Mexican ancestry, the genome­wide pattern of DNA methylation in the atherosclerotic human aorta is yet to be elucidated in Japanese individuals. In the present study, a genome­wide analysis of DNA methylation at ~853,000 CpG sites was performed using 128 postmortem aortic intima specimens obtained from 64 Japanese patients. To avoid the effects of interindividual variation, intraindividual paired comparisons were performed between atheromatous plaque lesions and corresponding plaque­free tissue for each patient. Bisulfite­modified genomic DNA was analyzed using a specific microarray for DNA methylation. DNA methylation at each CpG site was calculated as the ß value, where ß = (intensity of the methylated allele)/(intensity of the methylated allele + intensity of the unmethylated allele + 100). Bonferroni's correction for statistical significance of association was applied to compensate for multiple comparisons. The methylation of 2,679 CpG sites differed significantly (P<5.86x10­8) between atheromatous plaque lesions and the corresponding plaque­free intima, with 2,272 and 407 CpG sites in atheromatous plaques being hyper­ or hypomethylated, respectively. A total of 5 hypermethylated CpG sites in atheromatous plaques were demonstrated to have a difference in ß value of >0.15 (plaque lesion­plaque­free intima) and 11 had a ß ratio of >1.50 (plaque/plaque­free intima). A further 15 and 17 hypomethylated CpG sites in atheromatous plaques were observed to have a difference in ß value of <­0.15 or a ß ratio of <0.67, respectively. According to these limits, a total of 16 novel genes that were significantly hyper­ or hypomethylated in atheromatous plaque lesions compared with the plaque­free intima were identified in the present study. The results of the present study suggest that the methylation of these genes may contribute to the pathogenesis of atherosclerosis in the Japanese population.

Identification of three genetic variants as novel susceptibility loci for body mass index in a Japanese population.

Recent genome-wide association studies have identified various obesity or metabolic syndrome (MetS) susceptibility loci. However, most studies were conducted in a cross-sectional manner. To address this gap, we performed a longitudinal exome-wide association study to identify susceptibility loci for obesity and MetS in a Japanese population. We traced clinical data of 6,022 Japanese subjects who had annual health check-ups for several years (mean follow-up period, 5 yr) and genotyped ~244,000 genetic variants. The association of single nucleotide polymorphisms (SNPs) with body mass index (BMI) or the prevalence of obesity and MetS was examined in a generalized estimating equation model. Our longitudinal exome-wide association studies detected 21 BMI- and five MetS-associated SNPs (false discovery rate, FDR <0.01). Among these SNPs, 16 have not been previously implicated as determinants of BMI or MetS. Cross-sectional data for obesity- and MetS-related phenotypes in 7,285 Japanese subjects were examined in a replication study. Among the 16 SNPs, three ( rs9491140 , rs145848316 , and rs7863248 ) were related to BMI in the replication cohort ( P < 0.05). In conclusion, three SNPs [ rs9491140 of NKAIN2 (FDR = 0.003, P = 1.9 × 10-5), rs145848316 of KMT2C (FDR = 0.007, P = 4.5 × 10-5), and rs7863248 of AGTPBP1 (FDR = 0.006, P = 4.2 × 10-5)] were newly identified as susceptibility loci for BMI.

Inverse Association Between Height-Increasing Alleles and Extreme Longevity in Japanese Women.

Growth hormone (GH)/insulin-like growth factor-1 (IGF-1)/insulin signaling is one of the most plausible biological pathways regulating aging and longevity. Previous studies have demonstrated that several single nucleotide polymorphisms (SNPs) in the GH/IGF-1/insulin signaling-associated genes influence both longevity and adult height, suggesting the possibility of a shared genetic architecture between longevity and height. We therefore examined the relationship between 30 height-associated SNPs and extreme longevity in a Japanese population consisting of 428 centenarians and 4,026 younger controls. We confirmed that height-increasing genetic scores (HGSs) constructed based on 30 SNPs were significantly associated with height in the controls (p = 6.95 × 10-23). HGS was significantly and inversely associated with extreme longevity in women (p = .011), but not in men, although no SNPs were significantly associated with extreme longevity after Bonferroni correction. The odds ratio for extreme longevity in the lowest HGS group (≤27) and the second lowest HGS group (28-30) relative to the highest HGS group (≥37) was 1.71 (p = .056) and 1.69 (p = .034), respectively, for women. In conclusion, the present study demonstrated an inverse association between height-increasing alleles with extreme longevity in Japanese women, providing novel insight into the genetic architecture of longevity and aging.

Identification of CDC42BPG as a novel susceptibility locus for hyperuricemia in a Japanese population.

Chronic kidney disease and hyperuricemia are serious global health problems. Recent genome-wide association studies have identified various genetic variants related to these disorders. However, most studies have been conducted in a cross-sectional manner. To identify novel susceptibility loci for chronic kidney disease or hyperuricemia, we performed longitudinal exome-wide association studies (EWASs), using ~ 244,000 genetic variants and clinical data of Japanese individuals who had undergone annual health checkups for several years. After establishing quality controls, the association of renal function-related traits in 5648 subjects (excluding patients with dialysis and population outliers) with 24,579 single nucleotide variants (SNVs) for three genetic models (P < 3.39 × 10- 7) was tested using generalized estimating equation models. The longitudinal EWASs revealed novel relations of five SNVs to renal function-related traits. Cross-sectional data for renal function-related traits in 7699 Japanese subjects were examined in a replication study. Among the five SNVs, rs55975541 in CDC42BPG was significantly (P < 4.90 × 10- 4) related to the serum concentration of uric acid in the replication cohort. We also examined the SNVs detected in our longitudinal EWASs with the information on P values in GKDGEN meta-analysis data. Four SNVs in SLC15A2 were significantly associated with the estimated glomerular filtration rate in European ancestry populations, although these SNVs were related to the serum concentration of uric acid with borderline significance in our longitudinal EWASs. Our findings indicate that CDC42BPG may be a novel susceptibility locus for hyperuricemia.

Longitudinal exome-wide association study to identify genetic susceptibility loci for hypertension in a Japanese population.

Genome-wide association studies have identified various genetic variants associated with complex disorders. However, these studies have commonly been conducted in a cross-sectional manner. Therefore, we performed a longitudinal exome-wide association study (EWAS) in a Japanese cohort. We aimed to identify genetic variants that confer susceptibility to hypertension using ~244 000 single-nucleotide variants (SNVs) and physiological data from 6026 Japanese individuals who underwent annual health check-ups for several years. After quality control, the association of hypertension with SNVs was tested using a generalized estimating equation model. Finally, our longitudinal EWAS detected seven hypertension-related SNVs that passed strict criteria. Among these variants, six SNVs were densely located at 12q24.1, and an East Asian-specific motif (haplotype) 'CAAAA' comprising five derived alleles was identified. Statistical analyses showed that the prevalence of hypertension in individuals with the East Asian-specific haplotype was significantly lower than that in individuals with the common haplotype 'TGGGT'. Furthermore, individuals with the East Asian haplotype may be less susceptible to the adverse effects of smoking on hypertension. The longitudinal EWAS for the recessive model showed that a novel SNV, rs11917356 of COL6A5, was significantly associated with systolic blood pressure, and the derived allele at the SNV may have spread throughout East Asia in recent evolutionary time.

Association of smoking with prevalence of common diseases and metabolic abnormalities in community-dwelling Japanese individuals.

Smoking is a significant risk factor for cardiovascular diseases (CVDs). Given that certain common pathologies, including hypertension, dyslipidemia and type 2 diabetes mellitus, are major risk factors for CVDs, the association of smoking with CVDs may be attributable, at least in part, to its effects on common diseases. The aim of the present study was to determine the association of smoking with the prevalence of common diseases and metabolic abnormalities in community-dwelling Japanese individuals. The study included 5,959 subjects (1,302 current smokers, 1,418 past smokers and 3,239 nonsmokers) recruited to the Inabe Health and Longevity Study, a longitudinal genetic epidemiological study of atherosclerotic, cardiovascular and metabolic diseases. Various metabolic parameters and prevalence of common diseases were compared between smokers and nonsmokers using multivariable regression or logistic regression analysis with adjustments for age. Analysis indicated significantly higher serum concentrations of triglycerides and lower concentrations of high-density lipoprotein (HDL)-cholesterol in current smokers compared with nonsmokers in men and women. Serum concentrations of creatinine and systolic blood pressure were significantly lower and estimated glomerular filtration rate was higher in male current smokers. In addition, body weight was higher in female current smokers. In multivariable logistic regression analysis, smoking was significantly associated with the prevalence of dyslipidemia [P=6.3×10-10; odds ratio (OR), 1.81], hypertriglyceridemia (P=2.3×10-20; OR, 2.39), hypo-HDL-cholesterolemia (P=2.0×10-9; OR, 2.14), metabolic syndrome (P=0.0003; OR, 1.61) and chronic kidney disease (P=4.4×10-15; OR, 0.54) in men, but not in women. The results indicated that smoking is significantly associated with various metabolic abnormalities and prevalence of common diseases in Japanese individuals, with certain sex differences, which may lead to accelerated development of CVDs.

Identification of five genetic variants as novel determinants of type 2 diabetes mellitus in Japanese by exome-wide association studies.

We performed exome-wide association studies to identify single nucleotide polymorphisms that either influence fasting plasma glucose level or blood hemoglobin A1c content or confer susceptibility to type 2 diabetes mellitus in Japanese. Exome-wide association studies were performed with the use of Illumina Human Exome-12 DNA Analysis or Infinium Exome-24 BeadChip arrays and with 11,729 or 8635 subjects for fasting plasma glucose level or blood hemoglobin A1c content, respectively, or with 14,023 subjects for type 2 diabetes mellitus (3573 cases, 10,450 controls). The relation of genotypes of 41,265 polymorphisms to fasting plasma glucose level or blood hemoglobin A1c content was examined by linear regression analysis. After Bonferroni's correction, 41 and 17 polymorphisms were significantly (P < 1.21 × 10-6) associated with fasting plasma glucose level or blood hemoglobin A1c content, respectively, with two polymorphisms (rs139421991, rs189305583) being associated with both. Examination of the relation of allele frequencies to type 2 diabetes mellitus with Fisher's exact test revealed that 87 polymorphisms were significantly (P < 1.21 × 10-6) associated with type 2 diabetes mellitus. Subsequent multivariable logistic regression analysis with adjustment for age and sex showed that four polymorphisms (rs138313632, rs76974938, rs139012426, rs147317864) were significantly (P < 1.44 × 10-4) associated with type 2 diabetes mellitus, with rs138313632 and rs139012426 also being associated with fasting plasma glucose and rs76974938 with blood hemoglobin A1c. Five polymorphisms-rs139421991 of CAT, rs189305583 of PDCL2, rs138313632 of RUFY1, rs139012426 of LOC100505549, and rs76974938 of C21orf59-may be novel determinants of type 2 diabetes mellitus.

Identification of TNFSF13, SPATC1L, SLC22A25 and SALL4 as novel susceptibility loci for atrial fibrillation by an exome­wide association study.

An exome­wide association study (EWAS) was performed to identify genetic variants, particularly low­frequency or rare coding variants with a moderate to large effect size, that confer susceptibility to atrial fibrillation in Japanese. The EWAS for atrial fibrillation was performed with 13,166 subjects (884 patients with atrial fibrillation and 12,282 controls) using an Illumina HumanExome­12 DNA Analysis BeadChip or Infinium Exome­24 BeadChip arrays. The association of atrial fibrillation with allele frequencies of 41,243 single nucleotide polymorphisms (SNPs) that passed quality control was examined with Fisher's exact test. Based on Bonferroni's correction, a P<1.21x10­6 was considered statistically significant. The EWAS for atrial fibrillation revealed that 122 SNPs were significantly associated with this condition. The association of the identified SNPs to atrial fibrillation was further examined by multivariable logistic regression analysis with adjustment for age, sex and the prevalence of hypertension. Eight SNPs were related (P<0.01) to atrial fibrillation, among which three polymorphisms, rs11552708 [G/A (G67R)]of TNF superfamily member 13 (TNFSF13; dominant model; P=9.36x10­9; odds ratio, 0.58), rs113710653 [C/T (E231 K)] of spermatogenesis and centriole associated 1 like (SPATC1L; dominant model; P=1.09x10­5; odds ratio, 3.27), and rs11231397 [G/C (R300T)] of solute carrier family 22 member 25 (SLC22A25; additive model; P=3.71x10­5; odds ratio, 1.77), were significantly (P<1.02x10­4) associated with this condition. The minor T allele of rs113710653 and the minor C allele of rs11231397 were risk factors for atrial fibrillation, whereas the minor A allele of rs11552708 was protective against this condition. In addition, rs77538589 [C/T (G117R)] of SALL4 exhibited a tendency to be associated with atrial fibrillation (dominant model; P=0.0002; odds ratio, 1.88), with the minor T allele representing a risk factor for this condition. TNFSF13, SPATC1L, SLC22A25 and SALL4 may thus be novel susceptibility loci for atrial fibrillation in the Japanese population.