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

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

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 genome‑wide pattern of DNA methylation in the atherosclerotic human aorta is yet to be elucidated in Japanese individuals. In the present study, a genome‑wide 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 plaque‑free tissue for each patient. Bisulfite‑modified genomic DNA was analyzed using a specific microarray for DNA methylation. DNA methylation at each CpG site was calculated as the β value, where β = (intensity of the methylated allele)/(intensity of the methylated allele + intensity of the unmethylated allele + 100). Bonferroni\u27s correction for statistical significance of association was applied to compensate for multiple comparisons. The methylation of 2,679 CpG sites differed significantly (P0.15 (plaque lesion‑plaque‑free intima) and 11 had a β ratio of >1.50 (plaque/plaque‑free intima). A further 15 and 17 hypomethylated CpG sites in atheromatous plaques were observed to have a difference in β value of <‑0.15 or a β 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 plaque‑free 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 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 similar to 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 x 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 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\u27s exact test. To compensate for multiple comparisons of genotypes with T2DM, MetS, or hyperuricemia, we applied Bonferroni\u27s correction for statistical significance of association. The EWAS of allele frequencies revealed that four, six, or nine SNPs were significantly associated with T2DM (P<1.60x10‑6), MetS (P<1.59x10‑6), or hyperuricemia (P<1.61x10‑6), 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 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 genome‑wide pattern of DNA methylation in the atherosclerotic human aorta is yet to be elucidated in Japanese individuals. In the present study, a genome‑wide 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 plaque‑free tissue for each patient. Bisulfite‑modified genomic DNA was analyzed using a specific microarray for DNA methylation. DNA methylation at each CpG site was calculated as the β value, where β = (intensity of the methylated allele)/(intensity of the methylated allele + intensity of the unmethylated allele + 100). Bonferroni\u27s correction for statistical significance of association was applied to compensate for multiple comparisons. The methylation of 2,679 CpG sites differed significantly (P0.15 (plaque lesion‑plaque‑free intima) and 11 had a β ratio of >1.50 (plaque/plaque‑free intima). A further 15 and 17 hypomethylated CpG sites in atheromatous plaques were observed to have a difference in β value of <‑0.15 or a β 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 plaque‑free 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 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)

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 30. The longitudinal exome‑wide association studies revealed that three SNPs [rs4606855 of ADGRE3 (P=2.5x10‑6; FDR=0.031; approxdf=71), rs3746414 of ZFP64 (P=5.9x10‑6; FDR=0.048; approxdf=93) and rs7132908 of FAIM2 (P<2.0x10‑16; FDR<4.9x10‑12; approxdf=65)] were significantly associated with the prevalence of CAD. A different set of three SNPs [rs6580741 of FAM186A (P<2.0x10‑16; FDR<4.9x10‑12; approxdf=48), rs1324015 of LINC00400 (P<2.0x10‑16; FDR<4.9x10‑12; approxdf=49) and rs884205 of TNFRSF11A (P<2.0x10‑16; FDR<4.9x10‑12; 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 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\u27s 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\u27s 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