Ting-Fung Chan

Viral-Human Chimeric Transcript Predisposes Risk to Liver Cancer Development and Progression

The mutagenic effect of hepatitis B (HBV) integration in predisposing risk to hepatocellular carcinoma (HCC) remains elusive. In this study, we performed transcriptome sequencing of HBV-positive HCC cell lines and showed transcription of viral-human gene fusions from the site of genome integrations. We discovered tumor-promoting properties of a chimeric HBx-LINE1 that, intriguingly, functions as a hybrid RNA. HBx-LINE1 can be detected in 23.3% of HBV-associated HCC tumors and correlates with poorer patient survival. HBx-LINE1 transgenic mice showed heightened susceptibility to diethylnitrosamine-induced tumor formation. We further show that HBx-LINE1 expression affects β-catenin transactivity, which underlines a role in activating Wnt signaling. Thus, this study identifies a viral-human chimeric fusion transcript that functions like a long noncoding RNA to promote HCC.

Identification of a novel salt tolerance gene in wild soybean by whole-genome sequencing

Using a whole-genome-sequencing approach to explore germplasm resources can serve as an important strategy for crop improvement, especially in investigating wild accessions that may contain useful genetic resources that have been lost during the domestication process. Here we sequence and assemble a draft genome of wild soybean and construct a recombinant inbred population for genotyping-by-sequencing and phenotypic analyses to identify multiple QTLs relevant to traits of interest in agriculture. We use a combination of de novo sequencing data from this work and our previous germplasm re-sequencing data to identify a novel ion transporter gene, GmCHX1, and relate its sequence alterations to salt tolerance. Rapid gain-of-function tests show the protective effects of GmCHX1 towards salt stress. This combination of whole-genome de novo sequencing, high-density-marker QTL mapping by re-sequencing and functional analyses can serve as an effective strategy to unveil novel genomic information in wild soybean to facilitate crop improvement.

Whole-genome bisulfite sequencing of multiple individuals reveals complementary roles of promoter and gene body methylation in transcriptional regulation

BackgroundDNA methylation is an important type of epigenetic modification involved in gene regulation. Although strong DNA methylation at promoters is widely recognized to be associated with transcriptional repression, many aspects of DNA methylation remain not fully understood, including the quantitative relationships between DNA methylation and expression levels, and the individual roles of promoter and gene body methylation.ResultsHere we present an integrated analysis of whole-genome bisulfite sequencing and RNA sequencing data from human samples and cell lines. We find that while promoter methylation inversely correlates with gene expression as generally observed, the repressive effect is clear only on genes with a very high DNA methylation level. By means of statistical modeling, we find that DNA methylation is indicative of the expression class of a gene in general, but gene body methylation is a better indicator than promoter methylation. These findings are general in that a model constructed from a sample or cell line could accurately fit the unseen data from another. We further find that promoter and gene body methylation have minimal redundancy, and either one is sufficient to signify low expression. Finally, we obtain increased modeling power by integrating histone modification data with the DNA methylation data, showing that neither type of information fully subsumes the other.ConclusionOur results suggest that DNA methylation outside promoters also plays critical roles in gene regulation. Future studies on gene regulatory mechanisms and disease-associated differential methylation should pay more attention to DNA methylation at gene bodies and other non-promoter regions.

Rapid DNA mapping by fluorescent single molecule detection

DNA mapping is an important analytical tool in genomic sequencing, medical diagnostics and pathogen identification. Here we report an optical DNA mapping strategy based on direct imaging of individual DNA molecules and localization of multiple sequence motifs on the molecules. Individual genomic DNA molecules were labeled with fluorescent dyes at specific sequence motifs by the action of nicking endonuclease followed by the incorporation of dye terminators with DNA polymerase. The labeled DNA molecules were then stretched into linear form on a modified glass surface and imaged using total internal reflection fluorescence (TIRF) microscopy. By determining the positions of the fluorescent labels with respect to the DNA backbone, the distribution of the sequence motif recognized by the nicking endonuclease can be established with good accuracy, in a manner similar to reading a barcode. With this approach, we constructed a specific sequence motif map of lambda-DNA. We further demonstrated the capability of this approach to rapidly type a human adenovirus and several strains of human rhinovirus.

Hepatocellular carcinoma-derived exosomes promote motility of immortalized hepatocyte through transfer of oncogenic proteins and RNAs

Exosomes are increasingly recognized as important mediators of cell-cell communication in cancer progression through the horizontal transfer of RNAs and proteins to neighboring or distant cells. Hepatocellular carcinoma (HCC) is a highly malignant cancer, whose metastasis is largely influenced by the tumor microenvironment. The possible role of exosomes in the interactions between HCC tumor cell and its surrounding hepatic milieu are however largely unknown. In this study, we comprehensively characterized the exosomal RNA and proteome contents derived from three HCC cell lines (HKCI-C3, HKCI-8 and MHCC97L) and an immortalized hepatocyte line (MIHA) using Ion Torrent sequencing and mass spectrometry, respectively. RNA deep sequencing and proteomic analysis revealed exosomes derived from metastatic HCC cell lines carried a large number of protumorigenic RNAs and proteins, such as MET protooncogene, S100 family members and the caveolins. Of interest, we found that exosomes from motile HCC cell lines could significantly enhance the migratory and invasive abilities of non-motile MIHA cell. We further demonstrated that uptake of these shuttled molecules could trigger PI3K/AKT and MAPK signaling pathways in MIHA with increased secretion of active MMP-2 and MMP-9. Our study showed for the first time that HCC-derived exosomes could mobilize normal hepatocyte, which may have implication in facilitating the protrusive activity of HCC cells through liver parenchyma during the process of metastasis.

Mutation and polymorphism spectrum in osteogenesis imperfecta type II

Osteogenesis imperfecta (OI), also known as brittle bone disease, is a clinically and genetically heterogeneous disorder primarily characterized by susceptibility to fracture. Although OI generally results from mutations in the type I collagen genes, COL1A1 and COL1A2, the relationship between genotype and phenotype is not yet well understood. To provide additional data for genotype–phenotype analyses and to determine the proportion of mutations in the type I collagen genes among subjects with lethal forms of OI, we sequenced the coding and exon-flanking regions of COL1A1 and COL1A2 in a cohort of 63 subjects with OI type II, the perinatal lethal form of the disease. We identified 61 distinct heterozygous mutations in type I collagen, including five non-synonymous rare variants of unknown significance, of which 43 had not been seen previously. In addition, we found 60 SNPs in COL1A1, of which 17 were not reported previously, and 82 in COL1A2, of which 18 are novel. In three samples without collagen mutations, we found inactivating mutations in CRTAP and LEPRE1, suggesting a frequency of these recessive mutations of ∼5% in OI type II. A computational model that predicts the outcome of substitutions for glycine within the triple helical domain of collagen α1(I) chains predicted lethality with ∼90% accuracy. The results contribute to the understanding of the etiology of OI by providing data to evaluate and refine current models relating genotype to phenotype and by providing an unbiased indication of the relative frequency of mutations in OI-associated genes.

Genome-wide association study in a Chinese population identifies a susceptibility locus for type 2 diabetes at 7q32 near PAX4.

Aims/hypothesisMost genetic variants identified for type 2 diabetes have been discovered in European populations. We performed genome-wide association studies (GWAS) in a Chinese population with the aim of identifying novel variants for type 2 diabetes in Asians.MethodsWe performed a meta-analysis of three GWAS comprising 684 patients with type 2 diabetes and 955 controls of Southern Han Chinese descent. We followed up the top signals in two independent Southern Han Chinese cohorts (totalling 10,383 cases and 6,974 controls), and performed in silico replication in multiple populations.ResultsWe identified CDKN2A/B and four novel type 2 diabetes association signals with p < 1 × 10−5 from the meta-analysis. Thirteen variants within these four loci were followed up in two independent Chinese cohorts, and rs10229583 at 7q32 was found to be associated with type 2 diabetes in a combined analysis of 11,067 cases and 7,929 controls (pmeta = 2.6 × 10−8; OR [95% CI] 1.18 [1.11, 1.25]). In silico replication revealed consistent associations across multiethnic groups, including five East Asian populations (pmeta = 2.3 × 10−10) and a population of European descent (p = 8.6 × 10−3). The rs10229583 risk variant was associated with elevated fasting plasma glucose, impaired beta cell function in controls, and an earlier age at diagnosis for the cases. The novel variant lies within an islet-selective cluster of open regulatory elements. There was significant heterogeneity of effect between Han Chinese and individuals of European descent, Malaysians and Indians.Conclusions/interpretationOur study identifies rs10229583 near PAX4 as a novel locus for type 2 diabetes in Chinese and other populations and provides new insights into the pathogenesis of type 2 diabetes.

Deep sequencing of small RNA transcriptome reveals novel non-coding RNAs in hepatocellular carcinoma.

BACKGROUND & AIMS Small non-coding RNAs (ncRNA) are increasingly recognized to play important roles in tumorigenesis. With the advent of deep sequencing, efforts have been put forth to profile the miRNome in a number of human malignancies. However, information on ncRNA in hepatocellular carcinoma (HCC), especially the non-microRNA transcripts, is still lacking. METHODS Small RNA transcriptomes of two HCC cell lines (HKCI-4 and HKCI-8) and an immortalized hepatocyte line (MIHA) were examined using Illumina massively parallel sequencing. Dysregulated ncRNAs were verified in paired HCC tumors and non-tumoral livers (n=73) by quantitative reverse transcription-polymerase chain reaction. Clinicopathologic correlations and in vitro functional investigations were further carried out. RESULTS The combined bioinformatic and biological analyses showed the presence of ncRNAs and the involvement of a new PIWI-interacting RNA (piRNA), piR-Hep1, in liver tumorigenesis. piR-Hep1 was found to be upregulated in 46.6% of HCC tumors compared to the corresponding adjacent non-tumoral liver. Silencing of piR-Hep1 inhibited cell viability, motility, and invasiveness, with a concomitant reduction in the level of active AKT phosphorylation. In the analysis of miRNA, we showed for the first time, the abundant expression of miR-1323 in HCC and its distinct association in tumors arising from a cirrhotic background. Furthermore, miR-1323 overexpression in cirrhotic HCC correlated with poorer disease-free and overall survivals of patients (p<0.009). CONCLUSIONS Our study demonstrated the value of next-generation sequencing in dissecting the ncRNome in cancer. The comprehensive definition of transcriptome unveils virtually all types of ncRNAs and provides new insight into liver carcinogenetic events.

ViralFusionSeq: accurately discover viral integration events and reconstruct fusion transcripts at single-base resolution

Summary: Insertional mutagenesis from virus infection is an important pathogenic risk for the development of cancer. Despite the advent of high-throughput sequencing, discovery of viral integration sites and expressed viral fusion events are still limited. Here, we present ViralFusionSeq (VFS), which combines soft-clipping information, read-pair analysis and targeted de novo assembly to discover and annotate viral–human fusions. VFS was used in an RNA-Seq experiment, simulated DNA-Seq experiment and re-analysis of published DNA-Seq datasets. Our experiments demonstrated that VFS is both sensitive and highly accurate. Availability: VFS is distributed under GPL version 3 at http://hkbic.cuhk.edu.hk/software/viralfusionseq Contact: tf.chan@cuhk.edu.hk Supplementary information: Supplementary data are available at Bioinformatics Online

Genome-Wide Structural Variation Detection by Genome Mapping on Nanochannel Arrays.

Comprehensive whole-genome structural variation detection is challenging with current approaches. With diploid cells as DNA source and the presence of numerous repetitive elements, short-read DNA sequencing cannot be used to detect structural variation efficiently. In this report, we show that genome mapping with long, fluorescently labeled DNA molecules imaged on nanochannel arrays can be used for whole-genome structural variation detection without sequencing. While whole-genome haplotyping is not achieved, local phasing (across >150-kb regions) is routine, as molecules from the parental chromosomes are examined separately. In one experiment, we generated genome maps from a trio from the 1000 Genomes Project, compared the maps against that derived from the reference human genome, and identified structural variations that are >5 kb in size. We find that these individuals have many more structural variants than those published, including some with the potential of disrupting gene function or regulation.