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1.  Association between insulin resistance and plasma amino acid profile in non-diabetic Japanese subjects 
Elevation of the branched-chain amino acids (BCAAs), valine, leucine and isoleucine; and the aromatic amino acids, tyrosine and phenylalanine, has been observed in obesity-related insulin resistance. However, there have been few studies on Asians, who are generally less obese and less insulin-resistant than Caucasian or African-Americans. In the present study, we investigated the relationship between homeostasis model assessment of insulin resistance (HOMA-IR) and plasma amino acid concentration in non-diabetic Japanese participants.
Materials and Methods
A total of 94 healthy men and women were enrolled, and plasma amino acid concentration was measured by liquid chromatography/mass spectrometry after overnight fasting. The associations between HOMA-IR and 20 amino acid concentrations, and anthropometric and clinical parameters of lifestyle-related diseases were evaluated.
The mean age and body mass index were 40.1 ± 9.6 years and 22.7 ± 3.9, respectively. Significantly positive correlations were observed between HOMA-IR and valine, isoleucine, leucine, tyrosine, phenylalanine and total BCAA concentration. Compared with the HOMA-IR ≤ 1.6 group, the HOMA-IR > 1.6 group showed significantly exacerbated anthropometric and clinical parameters, and significantly elevated levels of valine, isoleucine, leucine, tyrosine, phenylalanine and BCAA.
The present study shows that the insulin resistance-related change in amino acid profile is also observed in non-diabetic Japanese subjects. These amino acids include BCAAs (valine, isoleucine and leucine) and aromatic amino acids (tyrosine and phenylalanine), in agreement with previous studies carried out using different ethnic groups with different degrees of obesity and insulin resistance.
PMCID: PMC4511300  PMID: 26221519
Amino acids; Homeostasis model assessment of insulin resistance; Insulin resistance
2.  VaDE: a manually curated database of reproducible associations between various traits and human genomic polymorphisms 
Nucleic Acids Research  2014;43(Database issue):D868-D872.
Genome-wide association studies (GWASs) have identified numerous single nucleotide polymorphisms (SNPs) associated with the development of common diseases. However, it is clear that genetic risk factors of common diseases are heterogeneous among human populations. Therefore, we developed a database of genomic polymorphisms that are reproducibly associated with disease susceptibilities, drug responses and other traits for each human population: ‘VarySysDB Disease Edition’ (VaDE; SNP-trait association data were obtained from the National Human Genome Research Institute GWAS (NHGRI GWAS) catalog and RAvariome, and we added detailed information of sample populations by curating original papers. In addition, we collected and curated original papers, and registered the detailed information of SNP-trait associations in VaDE. Then, we evaluated reproducibility of associations in each population by counting the number of significantly associated studies. VaDE provides literature-based SNP-trait association data and functional genomic region annotation for SNP functional research. SNP functional annotation data included experimental data of the ENCODE project, H-InvDB transcripts and the 1000 Genome Project. A user-friendly web interface was developed to assist quick search, easy download and fast swapping among viewers. We believe that our database will contribute to the future establishment of personalized medicine and increase our understanding of genetic factors underlying diseases.
PMCID: PMC4383886  PMID: 25361969
3.  RAvariome: a genetic risk variants database for rheumatoid arthritis based on assessment of reproducibility between or within human populations 
Rheumatoid arthritis (RA) is a common autoimmune inflammatory disease of the joints and is caused by both genetic and environmental factors. In the past six years, genome-wide association studies (GWASs) have identified many risk variants associated with RA. However, not all associations reported from GWASs are reproduced when tested in follow-up studies. To establish a reliable set of RA risk variants, we systematically classified common variants identified in GWASs by the degree of reproducibility among independent studies. We collected comprehensive genetic associations from 90 papers of GWASs and meta-analysis. The genetic variants were assessed according to the statistical significance and reproducibility between or within nine geographical populations. As a result, 82 and 19 single nucleotide polymorphisms (SNPs) were confirmed as intra- and inter-population-reproduced variants, respectively. Interestingly, majority of the intra-population-reproduced variants from European and East Asian populations were not common in two populations, but their nearby genes appeared to be the components of common pathways. Furthermore, a tool to predict the individual’s genetic risk of RA was developed to facilitate personalized medicine and preventive health care. For further clinical researches, the list of reliable genetic variants of RA and the genetic risk prediction tool are provided by open access database RAvariome.
Database URL:
PMCID: PMC3807080  PMID: 24158836
4.  PCDq: human protein complex database with quality index which summarizes different levels of evidences of protein complexes predicted from H-Invitational protein-protein interactions integrative dataset 
BMC Systems Biology  2012;6(Suppl 2):S7.
Proteins interact with other proteins or biomolecules in complexes to perform cellular functions. Existing protein-protein interaction (PPI) databases and protein complex databases for human proteins are not organized to provide protein complex information or facilitate the discovery of novel subunits. Data integration of PPIs focused specifically on protein complexes, subunits, and their functions. Predicted candidate complexes or subunits are also important for experimental biologists.
Based on integrated PPI data and literature, we have developed a human protein complex database with a complex quality index (PCDq), which includes both known and predicted complexes and subunits. We integrated six PPI data (BIND, DIP, MINT, HPRD, IntAct, and GNP_Y2H), and predicted human protein complexes by finding densely connected regions in the PPI networks. They were curated with the literature so that missing proteins were complemented and some complexes were merged, resulting in 1,264 complexes comprising 9,268 proteins with 32,198 PPIs. The evidence level of each subunit was assigned as a categorical variable. This indicated whether it was a known subunit, and a specific function was inferable from sequence or network analysis. To summarize the categories of all the subunits in a complex, we devised a complex quality index (CQI) and assigned it to each complex. We examined the proportion of consistency of Gene Ontology (GO) terms among protein subunits of a complex. Next, we compared the expression profiles of the corresponding genes and found that many proteins in larger complexes tend to be expressed cooperatively at the transcript level. The proportion of duplicated genes in a complex was evaluated. Finally, we identified 78 hypothetical proteins that were annotated as subunits of 82 complexes, which included known complexes. Of these hypothetical proteins, after our prediction had been made, four were reported to be actual subunits of the assigned protein complexes.
We constructed a new protein complex database PCDq including both predicted and curated human protein complexes. CQI is a useful source of experimentally confirmed information about protein complexes and subunits. The predicted protein complexes can provide functional clues about hypothetical proteins. PCDq is freely available at
PMCID: PMC3521179  PMID: 23282181
5.  H-InvDB in 2013: an omics study platform for human functional gene and transcript discovery 
Nucleic Acids Research  2012;41(Database issue):D915-D919.
H-InvDB ( is a comprehensive human gene database started in 2004. In the latest version, H-InvDB 8.0, a total of 244 709 human complementary DNA was mapped onto the hg19 reference genome and 43 829 gene loci, including nonprotein-coding ones, were identified. Of these loci, 35 631 were identified as potential protein-coding genes, and 22 898 of these were identical to known genes. In our analysis, 19 309 annotated genes were specific to H-InvDB and not found in RefSeq and Ensembl. In fact, 233 genes of the 19 309 turned out to have protein functions in this version of H-InvDB; they were annotated as unknown protein functions in the previous version. Furthermore, 11 genes were identified as known Mendelian disorder genes. It is advantageous that many biologically functional genes are hidden in the H-InvDB unique genes. As large-scale proteomic projects have been conducted to elucidate the functions of all human proteins, we have enhanced the proteomic information with an advanced protein view and new subdatabase of protein complexes (Protein Complex Database with quality index). We propose that H-InvDB is an important resource for finding novel candidate targets for medical care and drug development.
PMCID: PMC3531145  PMID: 23197657
6.  Prediction of Protein-Destabilizing Polymorphisms by Manual Curation with Protein Structure 
PLoS ONE  2012;7(11):e50445.
The relationship between sequence polymorphisms and human disease has been studied mostly in terms of effects of single nucleotide polymorphisms (SNPs) leading to single amino acid substitutions that change protein structure and function. However, less attention has been paid to more drastic sequence polymorphisms which cause premature termination of a protein’s sequence or large changes, insertions, or deletions in the sequence. We have analyzed a large set (n = 512) of insertions and deletions (indels) and single nucleotide polymorphisms causing premature termination of translation in disease-related genes. Prediction of protein-destabilization effects was performed by graphical presentation of the locations of polymorphisms in the protein structure, using the Genomes TO Protein (GTOP) database, and manual annotation with a set of specific criteria. Protein-destabilization was predicted for 44.4% of the nonsense SNPs, 32.4% of the frameshifting indels, and 9.1% of the non-frameshifting indels. A prediction of nonsense-mediated decay allowed to infer which truncated proteins would actually be translated as defective proteins. These cases included the proteins linked to diseases inherited dominantly, suggesting a relation between these diseases and toxic aggregation. Our approach would be useful in identifying potentially aggregation-inducing polymorphisms that may have pathological effects.
PMCID: PMC3506574  PMID: 23189203
7.  Reconstructing the Demographic History of the Human Lineage Using Whole-Genome Sequences from Human and Three Great Apes 
Genome Biology and Evolution  2012;4(11):1133-1145.
The demographic history of human would provide helpful information for identifying the evolutionary events that shaped the humanity but remains controversial even in the genomic era. To settle the controversies, we inferred the speciation times (T) and ancestral population sizes (N) in the lineage leading to human and great apes based on whole-genome alignment. A coalescence simulation determined the sizes of alignment blocks and intervals between them required to obtain recombination-free blocks with a high frequency. This simulation revealed that the size of the block strongly affects the parameter inference, indicating that recombination is an important factor for achieving optimum parameter inference. From the whole genome alignments (1.9 giga-bases) of human (H), chimpanzee (C), gorilla (G), and orangutan, 100-bp alignment blocks separated by ≥5-kb intervals were sampled and subjected to estimate τ = μT and θ = 4μgN using the Markov chain Monte Carlo method, where μ is the mutation rate and g is the generation time. Although the estimated τHC differed across chromosomes, τHC and τHCG were strongly correlated across chromosomes, indicating that variation in τ is subject to variation in μ, rather than T, and thus, all chromosomes share a single speciation time. Subsequently, we estimated Ts of the human lineage from chimpanzee, gorilla, and orangutan to be 6.0–7.6, 7.6–9.7, and 15–19 Ma, respectively, assuming variable μ across lineages and chromosomes. These speciation times were consistent with the fossil records. We conclude that the speciation times in our recombination-free analysis would be conclusive and the speciation between human and chimpanzee was a single event.
PMCID: PMC3752010  PMID: 22975719
human evolution; coalescence; speciation time; ancestral population size
8.  Comparative Genome Analysis of Three Eukaryotic Parasites with Differing Abilities To Transform Leukocytes Reveals Key Mediators of Theileria-Induced Leukocyte Transformation 
mBio  2012;3(5):e00204-12.
We sequenced the genome of Theileria orientalis, a tick-borne apicomplexan protozoan parasite of cattle. The focus of this study was a comparative genome analysis of T. orientalis relative to other highly pathogenic Theileria species, T. parva and T. annulata. T. parva and T. annulata induce transformation of infected cells of lymphocyte or macrophage/monocyte lineages; in contrast, T. orientalis does not induce uncontrolled proliferation of infected leukocytes and multiplies predominantly within infected erythrocytes. While synteny across homologous chromosomes of the three Theileria species was found to be well conserved overall, subtelomeric structures were found to differ substantially, as T. orientalis lacks the large tandemly arrayed subtelomere-encoded variable secreted protein-encoding gene family. Moreover, expansion of particular gene families by gene duplication was found in the genomes of the two transforming Theileria species, most notably, the TashAT/TpHN and Tar/Tpr gene families. Gene families that are present only in T. parva and T. annulata and not in T. orientalis, Babesia bovis, or Plasmodium were also identified. Identification of differences between the genome sequences of Theileria species with different abilities to transform and immortalize bovine leukocytes will provide insight into proteins and mechanisms that have evolved to induce and regulate this process. The T. orientalis genome database is available at
Cancer-like growth of leukocytes infected with malignant Theileria parasites is a unique cellular event, as it involves the transformation and immortalization of one eukaryotic cell by another. In this study, we sequenced the whole genome of a nontransforming Theileria species, Theileria orientalis, and compared it to the published sequences representative of two malignant, transforming species, T. parva and T. annulata. The genome-wide comparison of these parasite species highlights significant genetic diversity that may be associated with evolution of the mechanism(s) deployed by an intracellular eukaryotic parasite to transform its host cell.
PMCID: PMC3445966  PMID: 22951932
9.  Abundance of ultramicro inversions within local alignments between human and chimpanzee genomes 
Chromosomal inversion is one of the most important mechanisms of evolution. Recent studies of comparative genomics have revealed that chromosomal inversions are abundant in the human genome. While such previously characterized inversions are large enough to be identified as a single alignment or a string of local alignments, the impact of ultramicro inversions, which are such short that the local alignments completely cover them, on evolution is still uncertain.
In this study, we developed a method for identifying ultramicro inversions by scanning of local alignments. This technique achieved a high sensitivity and a very low rate of false positives. We identified 2,377 ultramicro inversions ranging from five to 125 bp within the orthologous alignments between the human and chimpanzee genomes. The false positive rate was estimated to be around 4%. Based on phylogenetic profiles using the primate outgroups, 479 ultramicro inversions were inferred to have specifically inverted in the human lineage. Ultramicro inversions exclusively involving adenine and thymine were the most frequent; 461 inversions (19.4%) of the total. Furthermore, the density of ultramicro inversions in chromosome Y and the neighborhoods of transposable elements was higher than average. Sixty-five ultramicro inversions were identified within the exons of human protein-coding genes.
We defined ultramicro inversions as the inverted regions equal to or smaller than 125 bp buried within local alignments. Our observations suggest that ultramicro inversions are abundant among the human and chimpanzee genomes, and that location of the inversions correlated with the genome structural instability. Some of the ultramicro inversions may contribute to gene evolution. Our inversion-identification method is also applicable in the fine-tuning of genome alignments by distinguishing ultramicro inversions from nucleotide substitutions and indels.
PMCID: PMC3227671  PMID: 22011259
10.  Towards BioDBcore: a community-defined information specification for biological databases 
The present article proposes the adoption of a community-defined, uniform, generic description of the core attributes of biological databases, BioDBCore. The goals of these attributes are to provide a general overview of the database landscape, to encourage consistency and interoperability between resources; and to promote the use of semantic and syntactic standards. BioDBCore will make it easier for users to evaluate the scope and relevance of available resources. This new resource will increase the collective impact of the information present in biological databases.
PMCID: PMC3017395  PMID: 21205783
11.  Towards BioDBcore: a community-defined information specification for biological databases 
Nucleic Acids Research  2010;39(Database issue):D7-D10.
The present article proposes the adoption of a community-defined, uniform, generic description of the core attributes of biological databases, BioDBCore. The goals of these attributes are to provide a general overview of the database landscape, to encourage consistency and interoperability between resources and to promote the use of semantic and syntactic standards. BioDBCore will make it easier for users to evaluate the scope and relevance of available resources. This new resource will increase the collective impact of the information present in biological databases.
PMCID: PMC3013734  PMID: 21097465
12.  CIPRO 2.5: Ciona intestinalis protein database, a unique integrated repository of large-scale omics data, bioinformatic analyses and curated annotation, with user rating and reviewing functionality 
Nucleic Acids Research  2010;39(Database issue):D807-D814.
The Ciona intestinalis protein database (CIPRO) is an integrated protein database for the tunicate species C. intestinalis. The database is unique in two respects: first, because of its phylogenetic position, Ciona is suitable model for understanding vertebrate evolution; and second, the database includes original large-scale transcriptomic and proteomic data. Ciona intestinalis has also been a favorite of developmental biologists. Therefore, large amounts of data exist on its development and morphology, along with a recent genome sequence and gene expression data. The CIPRO database is aimed at collecting those published data as well as providing unique information from unpublished experimental data, such as 3D expression profiling, 2D-PAGE and mass spectrometry-based large-scale analyses at various developmental stages, curated annotation data and various bioinformatic data, to facilitate research in diverse areas, including developmental, comparative and evolutionary biology. For medical and evolutionary research, homologs in humans and major model organisms are intentionally included. The current database is based on a recently developed KH model containing 36 034 unique sequences, but for higher usability it covers 89 683 all known and predicted proteins from all gene models for this species. Of these sequences, more than 10 000 proteins have been manually annotated. Furthermore, to establish a community-supported protein database, these annotations are open to evaluation by users through the CIPRO website. CIPRO 2.5 is freely accessible at
PMCID: PMC3013717  PMID: 21071393
14.  A comprehensive survey of human polymorphisms at conserved splice dinucleotides and its evolutionary relationship with alternative splicing 
Alternative splicing (AS) is a key molecular process that endows biological functions with diversity and complexity. Generally, functional redundancy leads to the generation of new functions through relaxation of selective pressure in evolution, as exemplified by duplicated genes. It is also known that alternatively spliced exons (ASEs) are subject to relaxed selective pressure. Within consensus sequences at the splice junctions, the most conserved sites are dinucleotides at both ends of introns (splice dinucleotides). However, a small number of single nucleotide polymorphisms (SNPs) occur at splice dinucleotides. An intriguing question relating to the evolution of AS diversity is whether mutations at splice dinucleotides are maintained as polymorphisms and produce diversity in splice patterns within the human population. We therefore surveyed validated SNPs in the database dbSNP located at splice dinucleotides of all human genes that are defined by the H-Invitational Database.
We found 212 validated SNPs at splice dinucleotides (sdSNPs); these were confirmed to be consistent with the GT-AG rule at either allele. Moreover, 53 of them were observed to neighbor ASEs (AE dinucleotides). No significant differences were observed between sdSNPs at AE dinucleotides and those at constitutive exons (CE dinucleotides) in SNP properties including average heterozygosity, SNP density, ratio of predicted alleles consistent with the GT-AG rule, and scores of splice sites formed with the predicted allele. We also found that the proportion of non-conserved exons was higher for exons with sdSNPs than for other exons.
sdSNPs are found at CE dinucleotides in addition to those at AE dinucleotides, suggesting two possibilities. First, sdSNPs at CE dinucleotides may be robust against sdSNPs because of unknown mechanisms. Second, similar to sdSNPs at AE dinucleotides, those at CE dinucleotides cause differences in AS patterns because of the arbitrariness in the classification of exons into alternative and constitutive type that varies according to the dataset. Taking into account the absence of differences in sdSNP properties between those at AE and CE dinucleotides, the increased proportion of non-conserved exons found in exons flanked by sdSNPs suggests the hypothesis that sdSNPs are maintained at the splice dinucleotides of newly generated exons at which negative selection pressure is relaxed.
PMCID: PMC2882926  PMID: 20433709
15.  H-DBAS: human-transcriptome database for alternative splicing: update 2010 
Nucleic Acids Research  2009;38(Database issue):D86-D90.
H-DBAS ( is a specialized database for human alternative splicing (AS) based on H-Invitational full-length cDNAs. In this update, for better annotations of AS events, we correlated RNA-Seq tag information to the AS exons and splice junctions. We generated a total of 148 376 598 RNA-Seq tags from RNAs extracted from cytoplasmic, nuclear and polysome fractions. Analysis of the RNA-Seq tags allowed us to identify 90 900 exons that are very likely to be used for protein synthesis. On the other hand, 254 AS junctions of human RefSeq transcripts are unique to nuclear RNA and may not have any translational consequences. We also present a new comparative genomics viewer so that users can empirically understand the evolutionary turnover of AS. With the unique experimental data closely connected with intensively curated cDNA information, H-DBAS provides a unique platform for the analysis of complex AS.
PMCID: PMC2808982  PMID: 19969536
16.  H-InvDB in 2009: extended database and data mining resources for human genes and transcripts 
Nucleic Acids Research  2009;38(Database issue):D626-D632.
We report the extended database and data mining resources newly released in the H-Invitational Database (H-InvDB; H-InvDB is a comprehensive annotation resource of human genes and transcripts, and consists of two main views and six sub-databases. The latest release of H-InvDB (release 6.2) provides the annotation for 219 765 human transcripts in 43 159 human gene clusters based on human full-length cDNAs and mRNAs. H-InvDB now provides several new annotation features, such as mapping of microarray probes, new gene models, relation to known ncRNAs and information from the Glycogene database. H-InvDB also provides useful data mining resources—‘Navigation search’, ‘H-InvDB Enrichment Analysis Tool (HEAT)’ and web service APIs. ‘Navigation search’ is an extended search system that enables complicated searches by combining 16 different search options. HEAT is a data mining tool for automatically identifying features specific to a given human gene set. HEAT searches for H-InvDB annotations that are significantly enriched in a user-defined gene set, as compared with the entire H-InvDB representative transcripts. H-InvDB now has web service APIs of SOAP and REST to allow the use of H-InvDB data in programs, providing the users extended data accessibility.
PMCID: PMC2808976  PMID: 19933760
17.  G-compass: a web-based comparative genome browser between human and other vertebrate genomes 
Bioinformatics  2009;25(24):3321-3322.
Summary: G-compass is designed for efficient comparative genome analysis between human and other vertebrate genomes. The current version of G-compass allows us to browse two corresponding genomic regions between human and another species in parallel. One-to-one evolutionarily conserved regions (i.e. orthologous regions) between species are highlighted along the genomes. Information such as locations of duplicated regions, copy number variations and mammalian ultra-conserved elements is also provided. These features of G-compass enable us to easily determine patterns of genomic rearrangements and changes in gene orders through evolutionary time. Since G-compass is a satellite database of H-InvDB, which is a comprehensive annotation resource for human genes and transcripts, users can easily refer to manually curated functional annotations and other abundant biological information for each human transcript. G-compass is expected to be a valuable tool for comparing human and model organisms and promoting the exchange of functional information.
Availability: G-compass is freely available at
PMCID: PMC2788932  PMID: 19846439
18.  Hyperlink Management System and ID Converter System: enabling maintenance-free hyperlinks among major biological databases 
Nucleic Acids Research  2009;37(Web Server issue):W17-W22.
Hyperlink Management System (HMS) is a system for automatically updating and maintaining hyperlinks among major public databases in the field of life science. We daily create corresponding tables of data IDs of major databases for human genes and proteins, and provide a CGI-program that returns correct and up-to-date URLs for showing data of various databases that correspond to user-specified IDs. The HMS can deal with various IDs: accession numbers of International Nucleotide Sequence Databases, HUGO Gene Symbols and IDs of UniProt, PDB, H-InvDB and others, and it can return URLs of various databases: H-InvDB, HUGO Gene Nomenclature Committee Database, NCBI Entrez Gene, UniProt, PDB and others. For example, 23 297 pages of Locus view of H-InvDB are reachable by using HUGO Gene Symbols through the HMS. Not only the CGI-program, the HMS provides a Web page for finding and opening URLs of these databases. Although hyperlinking is an effective way of relating biological data among different databases, updating hyperlinks has been a laborious work. The HMS fully automates the job, enabling maintenance-free hyperlinks. We also developed the ID Converter System (ICS) for simply converting data IDs by using corresponding tables in the HMS. The HMS and ICS are freely available at
PMCID: PMC2703917  PMID: 19454601
19.  VarySysDB: a human genetic polymorphism database based on all H-InvDB transcripts 
Nucleic Acids Research  2008;37(Database issue):D810-D815.
Creation of a vast variety of proteins is accomplished by genetic variation and a variety of alternative splicing transcripts. Currently, however, the abundant available data on genetic variation and the transcriptome are stored independently and in a dispersed fashion. In order to provide a research resource regarding the effects of human genetic polymorphism on various transcripts, we developed VarySysDB, a genetic polymorphism database based on 187 156 extensively annotated matured mRNA transcripts from 36 073 loci provided by H-InvDB. VarySysDB offers information encompassing published human genetic polymorphisms for each of these transcripts separately. This allows comparisons of effects derived from a polymorphism on different transcripts. The published information we analyzed includes single nucleotide polymorphisms and deletion–insertion polymorphisms from dbSNP, copy number variations from Database of Genomic Variants, short tandem repeats and single amino acid repeats from H-InvDB and linkage disequilibrium regions from D-HaploDB. The information can be searched and retrieved by features, functions and effects of polymorphisms, as well as by keywords. VarySysDB combines two kinds of viewers, GBrowse and Sequence View, to facilitate understanding of the positional relationship among polymorphisms, genome, transcripts, loci and functional domains. We expect that VarySysDB will yield useful information on polymorphisms affecting gene expression and phenotypes. VarySysDB is available at
PMCID: PMC2686441  PMID: 18953038
20.  Distribution and Effects of Nonsense Polymorphisms in Human Genes 
PLoS ONE  2008;3(10):e3393.
A great amount of data has been accumulated on genetic variations in the human genome, but we still do not know much about how the genetic variations affect gene function. In particular, little is known about the distribution of nonsense polymorphisms in human genes despite their drastic effects on gene products.
Methodology/Principal Findings
To detect polymorphisms affecting gene function, we analyzed all publicly available polymorphisms in a database for single nucleotide polymorphisms (dbSNP build 125) located in the exons of 36,712 known and predicted protein-coding genes that were defined in an annotation project of all human genes and transcripts (H-InvDB ver3.8). We found a total of 252,555 single nucleotide polymorphisms (SNPs) and 8,479 insertion and deletions in the representative transcripts in these genes. The SNPs located in ORFs include 40,484 synonymous and 53,754 nonsynonymous SNPs, and 1,258 SNPs that were predicted to be nonsense SNPs or read-through SNPs. We estimated the density of nonsense SNPs to be 0.85×10−3 per site, which is lower than that of nonsynonymous SNPs (2.1×10−3 per site). On average, nonsense SNPs were located 250 codons upstream of the original termination codon, with the substitution occurring most frequently at the first codon position. Of the nonsense SNPs, 581 were predicted to cause nonsense-mediated decay (NMD) of transcripts that would prevent translation. We found that nonsense SNPs causing NMD were more common in genes involving kinase activity and transport. The remaining 602 nonsense SNPs are predicted to produce truncated polypeptides, with an average truncation of 75 amino acids. In addition, 110 read-through SNPs at termination codons were detected.
Our comprehensive exploration of nonsense polymorphisms showed that nonsense SNPs exist at a lower density than nonsynonymous SNPs, suggesting that nonsense mutations have more severe effects than amino acid changes. The correspondence of nonsense SNPs to known pathological variants suggests that phenotypic effects of nonsense SNPs have been reported for only a small fraction of nonsense SNPs, and that nonsense SNPs causing NMD are more likely to be involved in phenotypic variations. These nonsense SNPs may include pathological variants that have not yet been reported. These data are available from Transcript View of H-InvDB and VarySysDB (
PMCID: PMC2561068  PMID: 18852891
21.  Low conservation and species-specific evolution of alternative splicing in humans and mice: comparative genomics analysis using well-annotated full-length cDNAs 
Nucleic Acids Research  2008;36(20):6386-6395.
Using full-length cDNA sequences, we compared alternative splicing (AS) in humans and mice. The alignment of the human and mouse genomes showed that 86% of 199 426 total exons in human AS variants were conserved in the mouse genome. Of the 20 392 total human AS variants, however, 59% consisted of all conserved exons. Comparing AS patterns between human and mouse transcripts revealed that only 431 transcripts from 189 loci were perfectly conserved AS variants. To exclude the possibility that the full-length human cDNAs used in the present study, especially those with retained introns, were cloning artefacts or prematurely spliced transcripts, we experimentally validated 34 such cases. Our results indicate that even retained-intron type transcripts are typically expressed in a highly controlled manner and interact with translating ribosomes. We found non-conserved AS exons to be predominantly outside the coding sequences (CDSs). This suggests that non-conserved exons in the CDSs of transcripts cause functional constraint. These findings should enhance our understanding of the relationship between AS and species specificity of human genes.
PMCID: PMC2582632  PMID: 18838389
22.  Two different classes of co-occurring motif pairs found by a novel visualization method in human promoter regions 
BMC Genomics  2008;9:112.
It is essential in modern biology to understand how transcriptional regulatory regions are composed of cis-elements, yet we have limited knowledge of, for example, the combinational uses of these elements and their positional distribution.
We predicted the positions of 228 known binding motifs for transcription factors in phylogenetically conserved regions within -2000 and +1000 bp of transcriptional start sites (TSSs) of human genes and visualized their correlated non-overlapping occurrences. In the 8,454 significantly correlated motif pairs, two major classes were observed: 248 pairs in Class 1 were mainly found around TSSs, whereas 4,020 Class 2 pairs appear at rather arbitrary distances from TSSs. These classes are distinct in a number of aspects. First, the positional distribution of the Class 1 constituent motifs shows a single peak near the TSSs, whereas Class 2 motifs show a relatively broad distribution. Second, genes that harbor the Class 1 pairs are more likely to be CpG-rich and to be expressed ubiquitously than those that harbor Class 2 pairs. Third, the 'hub' motifs, which are used in many different motif pairs, are different between the two classes. In addition, many of the transcription factors that correspond to the Class 2 hub motifs contain domains rich in specific amino acids; these domains may form disordered regions important for protein-protein interaction.
There exist at least two classes of motif pairs with respect to TSSs in human promoters, possibly reflecting compositional differences between promoters and enhancers. We anticipate that our visualization method may be useful for the further characterisation of promoters.
PMCID: PMC2292176  PMID: 18312685
23.  Evola: Ortholog database of all human genes in H-InvDB with manual curation of phylogenetic trees 
Nucleic Acids Research  2007;36(Database issue):D787-D792.
Orthologs are genes in different species that evolved from a common ancestral gene by speciation. Currently, with the rapid growth of transcriptome data of various species, more reliable orthology information is prerequisite for further studies. However, detection of orthologs could be erroneous if pairwise distance-based methods, such as reciprocal BLAST searches, are utilized. Thus, as a sub-database of H-InvDB, an integrated database of annotated human genes (, we constructed a fully curated database of evolutionary features of human genes, called ‘Evola’. In the process of the ortholog detection, computational analysis based on conserved genome synteny and transcript sequence similarity was followed by manual curation by researchers examining phylogenetic trees. In total, 18 968 human genes have orthologs among 11 vertebrates (chimpanzee, mouse, cow, chicken, zebrafish, etc.), either computationally detected or manually curated orthologs. Evola provides amino acid sequence alignments and phylogenetic trees of orthologs and homologs. In ‘dN/dS view’, natural selection on genes can be analyzed between human and other species. In ‘Locus maps’, all transcript variants and their exon/intron structures can be compared among orthologous gene loci. We expect the Evola to serve as a comprehensive and reliable database to be utilized in comparative analyses for obtaining new knowledge about human genes. Evola is available at
PMCID: PMC2238928  PMID: 17982176
24.  A genome-wide survey of changes in protein evolutionary rates across four closely related species of Saccharomyces sensu stricto group 
Changes in protein evolutionary rates among lineages have been frequently observed during periods of notable phenotypic evolution. It is also known that, following gene duplication and loss, the protein evolutionary rates of genes involved in such events changed because of changes in functional constraints acting on the genes. However, in the evolution of closely related species, excluding the aforementioned situations, the frequency of changes in protein evolutionary rates is still not clear at the genome-wide level. Here we examine the constancy of protein evolutionary rates in the evolution of four closely related species of the Saccharomyces sensu stricto group (S. cerevisiae, S. paradoxus, S. mikatae and S. bayanus).
For 2,610 unambiguously defined orthologous genes among the four species, we carried out likelihood ratio tests between constant-rate and variable-rate models and found 344 (13.2%) genes showing significant changes in the protein evolutionary rates in at least one lineage. Of all those genes which experienced rate changes, 139 and 49 genes showed accelerated and decelerated evolution, respectively. Most of the evolutionary rate changes could be attributed to changes in selective constraints acting on nonsynonymous sites, independently of species-specific gene duplication and loss. We estimated that the changes in protein evolutionary rates have appeared with a probability of 2.0 × 10-3 per gene per million years in the evolution of the Saccharomyces species. Furthermore, we found that the genes which experienced rate acceleration have lower expression levels and weaker codon usage bias than those which experienced rate deceleration.
Changes in protein evolutionary rates possibly occur frequently in the evolution of closely related Saccharomyces species. Selection for translational accuracy and efficiency may dominantly affect the variability of protein evolutionary rates.
PMCID: PMC1790889  PMID: 17257443
25.  H-DBAS: Alternative splicing database of completely sequenced and manually annotated full-length cDNAs based on H-Invitational 
Nucleic Acids Research  2006;35(Database issue):D104-D109.
The Human-transcriptome DataBase for Alternative Splicing (H-DBAS) is a specialized database of alternatively spliced human transcripts. In this database, each of the alternative splicing (AS) variants corresponds to a completely sequenced and carefully annotated human full-length cDNA, one of those collected for the H-Invitational human-transcriptome annotation meeting. H-DBAS contains 38 664 representative alternative splicing variants (RASVs) in 11 744 loci, in total. The data is retrievable by various features of AS, which were annotated according to manual annotations, such as by patterns of ASs, consequently invoked alternations in the encoded amino acids and affected protein motifs, GO terms, predicted subcellular localization signals and transmembrane domains. The database also records recently identified very complex patterns of AS, in which two distinct genes seemed to be bridged, nested or degenerated (multiple CDS): in all three cases, completely unrelated proteins are encoded by a single locus. By using AS Viewer, each AS event can be analyzed in the context of full-length cDNAs, enabling the user's empirical understanding of the relation between AS event and the consequent alternations in the encoded amino acid sequences together with various kinds of affected protein motifs. H-DBAS is accessible at .
PMCID: PMC1716722  PMID: 17130147

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