Identification of differentially methylated HpaII sites by NGS analysis of HpaII-digested libraries.

Differentially methylated regions (DMRs) have been widely explored as epigenetic biomarkers. Here, we developed a novel approach combining methylation-sensitive restriction enzyme (MSRE) and next-generation sequencing (NGS) to identify DMRs between chorionic villi (CV) and maternal blood cells (MBC). During NGS library preparation, adapter-ligated genomic DNA of CV and MBC were digested with the MSRE, HpaII, and PCR-amplified. As unmethylated HpaII sites were cleaved, the resulted library should contain only methylated HpaII sites. By sequencing both HpaII-digested CV and MBC libraries, 9 differentially methylated-HpaII sites on chromosome 21 which exhibited more than 50% methylation increase in CV were identified. These DMRs are epigenetic biomarkers to tell the difference between CV and MBC. Our approach will also be applicable to screen various tissue-specific epigenetic biomarkers.

VarySysDB: a human genetic polymorphism database based on all H-InvDB transcripts.

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 http://h-invitational.jp/varygene/.

A comprehensive survey of human polymorphisms at conserved splice dinucleotides and its evolutionary relationship with alternative splicing.

BACKGROUND: 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. RESULTS: 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. CONCLUSIONS: 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.

The H-Invitational Database (H-InvDB), a comprehensive annotation resource for human genes and transcripts.

Here we report the new features and improvements in our latest release of the H-Invitational Database (H-InvDB; http://www.h-invitational.jp/), a comprehensive annotation resource for human genes and transcripts. H-InvDB, originally developed as an integrated database of the human transcriptome based on extensive annotation of large sets of full-length cDNA (FLcDNA) clones, now provides annotation for 120 558 human mRNAs extracted from the International Nucleotide Sequence Databases (INSD), in addition to 54 978 human FLcDNAs, in the latest release H-InvDB_4.6. We mapped those human transcripts onto the human genome sequences (NCBI build 36.1) and determined 34 699 human gene clusters, which could define 34 057 (98.1%) protein-coding and 642 (1.9%) non-protein-coding loci; 858 (2.5%) transcribed loci overlapped with predicted pseudogenes. For all these transcripts and genes, we provide comprehensive annotation including gene structures, gene functions, alternative splicing variants, functional non-protein-coding RNAs, functional domains, predicted sub cellular localizations, metabolic pathways, predictions of protein 3D structure, mapping of SNPs and microsatellite repeat motifs, co-localization with orphan diseases, gene expression profiles, orthologous genes, protein-protein interactions (PPI) and annotation for gene families. The current H-InvDB annotation resources consist of two main views: Transcript view and Locus view and eight sub-databases: the DiseaseInfo Viewer, H-ANGEL, the Clustering Viewer, G-integra, the TOPO Viewer, Evola, the PPI view and the Gene family/group.

Publisher Correction: Massively parallel sequencing of cell-free DNA in plasma for detecting gynaecological tumour-associated copy number alteration.

A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has been fixed in the paper.

Massively parallel sequencing of cell-free DNA in plasma for detecting gynaecological tumour-associated copy number alteration.

The discovery of circulating tumour DNA molecules created a paradigm shift in tumour biomarkers as predictors of recurrence. Non-invasive prenatal testing (NIPT) to detect circulating cell-free foetal DNA in maternal plasma is increasingly recognised as a valuable substitute to perceive foetal copy number variation (CNV). This study aimed to determine whether the copy number detection in plasma samples using NIPT platform could be used as a prognostic biomarker in patients with gynaecological cancer. We conducted a prospective study using samples containing preoperative plasma from 100 women with gynaecological cancers. Samples were randomly rearranged and blindly sequenced using a low-coverage whole-genome sequencing plasma DNA, NIPT platform. The NIPT pipeline identified copy number alterations (CNAs) were counted in plasma as a gain or loss if they exceeded 10 Mb from the expected diploid coverage. Progression-free survival (PFS) and overall survival (OS) were analysed according to the presence of CNA in plasma using Kaplan-Meier analyses. The NIPT pipeline detected 19/100 cases of all gynaecological cancers, including 6/36 ovarian cancers, 3/11 cervical cancers, and 10/53 endometrial cancers. Patients with CNA in plasma had a significantly poorer prognosis in all stages concerning PFS and OS. Therefore, low-coverage sequencing NIPT platform could serve as a predictive marker of patient outcome.

Distribution and effects of nonsense polymorphisms in human genes.

BACKGROUND: 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 0.85x10(-3) per site, which is lower than that of nonsynonymous SNPs (2.1x10(-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. CONCLUSION/SIGNIFICANCE: 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 (http://h-invitational.jp/varygene/).

Cucurbitane glycosides from the fruits of Siraitia gros venorii and their inhibitory effects on Epstein-Barr virus activation.

Six new cucurbitane glycosides, mogroside II B (2), 11-deoxymogroside III (4), 7-oxomogroside II E (5), 7-oxomogroside V (6), 11-oxomogroside II A1 (7), and 11-oxomogroside IV A (8), and two known but new naturally occurring cucurbitane glycosides, mogroside II A1 (1) and mogroside III A2 (3), were isolated from an ethanol extract of the fruits of Siraitia grosvenorii. Upon evaluation of compounds 1-8 for inhibitory effects against the Epstein-Barr virus early antigen (EBV-EA) activation induced by 12-O-tetradecanoylphorbol-13-acetate (TPA), all compounds exhibited inhibitory effects with IC50 values of 346-400 mol ratio/32 pmol TPA. In addition, compounds 1-8 showed weak inhibitory effects on activation of (+/-)-(E)-methyl-2-[(E)-hydroxyimino]-5-nitro-6-methoxy-3-hexemide (NOR 1), a nitric oxide (NO) donor.