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The genetic structure of the Asiatic black beat (Ursus thibetanus) in Japan was studied to understand the events that occurred during its evolution. The left domain of the mitochondrial control region (about 240 bp) was sequenced, defining 27 haplotypes that consisted of 23 haplotypes from 333 bears in Japan and 22 bears in the Asian continent. The network tree of the control region indicated that the Japanese population formed a distinct dade from the continental population. The phylogeographic analysis of the haplotypes indicated that the Shikoku and Ku Hanto populations had diverged during the initial phase from the ancestral population. After the 3 dominant haplotypes were rapidly distributed throughout Japan in the early stage of the population dispersal, the Japanese population diverged into eastern and western populations. Using the entire mitochondrial cytochrome b sequence, divergence time between the Japanese and the Continental populations suggested that the Japanese population might have colonized into Japan through the land bridge from the Korean Peninsula around 500 ka, which is consistent with paleontological evidence. Our finding that bears in western Japan exhibit lower genetic diversity and higher levels of genetic differentiation than bears in eastern Japan provides a vital contribution to conservation policy for these isolated populations. [ABSTRACT FROM AUTHOR]

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., (Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.)

Early‑onset cardiovascular and renal diseases have a strong genetic component. In the present study, exome‑wide association studies (EWASs) were performed to identify genetic variants that confer susceptibility to early‑onset 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 Exome‑12 DNA Analysis BeadChip or Infinium Exome‑24 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<1.60x10‑6), hypertension (P<1.60x10‑6), or CKD (P<1.59x10‑6), 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 genome‑wide association studies and linkage disequilibrium of the identified SNPs, 11 loci (TMOD4, COL6A3, ADGRL3‑CXCL8‑MARCH1, OR52E4, TCHP‑GIT2, CCDC63, 12q24.1, OAS3, PLCB2‑VPS33B, GOSR2, ZNF77), six loci (MOB3C‑TMOD4, COL6A3, COL6A5, CXCL8‑MARCH1, NFKBIL1‑6p21.3‑NCR3, PLCB2‑VPS33B), and seven loci (MOB3C‑TMOD4, COL6A3, COL6A5, ADGRL3‑CXCL8‑MARCH1, MUC17, PLCB2‑VPS33B, 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 (MOB3C‑TMOD4, COL6A3, ADGRL3‑CXCL8‑MARCH1, OR52E4, TCHP‑GIT2, CCDC63, 12q24.1, OAS3, PLCB2‑VPS33B, ZNF77, COL6A5, NFKBIL1‑NCR3, MUC17) were identified that confer susceptibility to early‑onset 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.

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 ).

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.

Background: The major histocompatibility complex (MHC) genes are one of the most important genetic systems in the vertebrate immune response. The diversity of MHC genes may directly influence the survival of individuals against infectious disease. However, there has been no investigation of MHC diversity in the Asiatic black bear (Ursus thibetanus). Here, we analyzed 270-bp nucleotide sequences of the entire exon 2 region of the MHC DQB gene by using 188 samples from the Japanese black bear (Ursus thibetanus japonicus) from 12 local populations., Results: Among 185 of 188 samples, we identified 44 MHC variants that encoded 31 different amino acid sequences (allotypes) and one putative pseudogene. The phylogenetic analysis suggests that MHC variants detected from the Japanese black bear are derived from the DQB locus. One of the 31 DQB allotypes, Urth-DQB*01, was found to be common to all local populations. Moreover, this allotype was shared between the black bear on the Asian continent and the Japanese black bear, suggesting that Urth-DQB*01 might have been maintained in the ancestral black bear population for at least 300,000 years. Our findings, from calculating the ratio of non-synonymous to synonymous substitutions, indicate that balancing selection has maintained genetic variation of peptide-binding residues at the DQB locus of the Japanese black bear. From examination of genotype frequencies among local populations, we observed a considerably lower level of observed heterozygosity than expected., Conclusions: The low level of observed heterozygosity suggests that genetic drift reduced DQB diversity in the Japanese black bear due to a bottleneck event at the population or species level. The decline of DQB diversity might have been accelerated by the loss of rare variants that have been maintained by negative frequency-dependent selection. Nevertheless, DQB diversity of the black bear appears to be relatively high compared with some other endangered mammalian species. This result suggests that the Japanese black bears may also retain more potential resistance against pathogens than other endangered mammalian species. To prevent further decline of potential resistance against pathogens, a conservation policy for the Japanese black bear should be designed to maintain MHC rare variants in each local population.

Genetic diversity estimation of the major histocompatibility complex (MHC) gene may be an important tool in the assessment of immune response ability against infectious disease. We were able to identify a near full-length expressed DQB sequence by RACE-PCR method from the Asiatic black bear, Ursus thibetanus in Japan. This is the first such full length expression in the Ursidae. The bear had at least one functional DQB locus. In phylogenetic tree analysis its DQB amino acid sequence formed a monophyletic group with DQB sequences from members of the order Carnivora and had a 90% nucleotide sequence similarity with the DQB allele of the California sea lion, Zalophus californianus. We compared the DQB amino acid composition of U. thibetanus with those of several other mammalian species including Homo sapiens. Amino acid residues known to be functionally important for human MHC genes, tended to be also conserved among other mammalian species while PBRs in the beta1 domain were heterogeneous among mammalian species. The DQB sequence obtained from the bear had not only no putative frameshifts or deletions but also no abnormal amino acid mutations such as had been observed in human DQB molecules. This suggests that the bear DQB sequence was an apparently functional DQB allele. As a preliminary study, we sequenced the exon 2 region of DQB alleles from genomic DNA, and succeeded to amplify the exon 2 of DQB loci. Our study will provide useful information for conservation genetics of the U. thibetanus as well as more generally regarding the mammalian MHC region.