Transcriptome and genome sequencing uncovers functional variation in humans

Summary Genome sequencing projects are discovering millions of genetic variants in humans, and interpretation of their functional effects is essential for understanding the genetic basis of variation in human traits. Here we report sequencing and deep analysis of mRNA and miRNA from lymphoblastoid cell lines of 462 individuals from the 1000 Genomes Project – the first uniformly processed RNA-seq data from multiple human populations with high-quality genome sequences. We discovered extremely widespread genetic variation affecting regulation of the majority of genes, with transcript structure and expression level variation being equally common but genetically largely independent. Our characterization of causal regulatory variation sheds light on cellular mechanisms of regulatory and loss-of-function variation, and allowed us to infer putative causal variants for dozens of disease-associated loci. Altogether, this study provides a deep understanding of the cellular mechanisms of transcriptome variation and of the landscape of functional variants in the human genome

XPC DEFICIENCY INCREASES RISK OF HEMATOLOGIC MALIGNANCIES THROUGH MUTATOR PHENOTYPE AND CHARACTERISTIC MUTATIONAL SIGNATURE

Recent studies demonstrated a dramatically increased risk of leukemia in patients with a rare genetic disorder, Xeroderma Pigmentosum group C (XP-C), characterized by constitutive deficiency of global genome nucleotide excision repair (GG-NER). The genetic mechanisms of non-skin cancers in XP-C patients remain unexplored. In this study, we analyze a unique collection of internal XP-C tumor genomes including 6 leukemias and 2 sarcomas. We observe a specific mutational pattern and an average of 25-fold increase of mutation rates in XP-C versus sporadic leukemia which we presume leads to its elevated incidence and early appearance. We describe a strong mutational asymmetry with respect to transcription and the direction of replication in XP-C tumors suggesting association of mutagenesis with bulky purine DNA lesions of probably endogenous origin. These findings suggest existence of a balance between formation and repair of bulky DNA lesions by GG-NER in human body cells which is disrupted in XP-C patients

Transcriptome and genome sequencing uncovers functional variation in humans

Genome sequencing projects are discovering millions of genetic variants in humans, and interpretation of their functional effects is essential for understanding the genetic basis of variation in human traits. Here we report sequencing and deep analysis of messenger RNA and microRNA from lymphoblastoid cell lines of 462 individuals from the 1000 Genomes Project--the first uniformly processed high-throughput RNA-sequencing data from multiple human populations with high-quality genome sequences. We discover extremely widespread genetic variation affecting the regulation of most genes, with transcript structure and expression level variation being equally common but genetically largely independent. Our characterization of causal regulatory variation sheds light on the cellular mechanisms of regulatory and loss-of-function variation, and allows us to infer putative causal variants for dozens of disease-associated loci. Altogether, this study provides a deep understanding of the cellular mechanisms of transcriptome variation and of the landscape of functional variants in the human genome

Comprehensive human virus screening using high-throughput sequencing with a user-friendly representation of bioinformatics analysis: a pilot study

High-throughput sequencing (HTS) provides the means to analyze clinical specimens in unprecedented molecular detail. While this technology has been successfully applied to virus discovery and other related areas of research, HTS methodology has yet to be exploited for use in a clinical setting for routine diagnostics. Here, a bioinformatics pipeline (ezVIR) was designed to process HTS data from any of the standard platforms and to evaluate the entire spectrum of known human viruses at once, providing results that are easy to interpret and customizable. The pipeline works by identifying the most likely viruses present in the specimen given the sequencing data. Additionally, ezVIR can generate optional reports for strain typing, can create genome coverage histograms, and can perform cross-contamination analysis for specimens prepared in series. In this pilot study, the pipeline was challenged using HTS data from 20 clinical specimens representative of those most often collected and analyzed in daily practice. The specimens (5 cerebrospinal fluid, 7 bronchoalveolar lavage fluid, 5 plasma, 2 serum, and 1 nasopharyngeal aspirate) were originally found to be positive for a diverse range of DNA or RNA viruses by routine molecular diagnostics. The ezVIR pipeline correctly identified 14 of 14 specimens containing viruses with genomes of <40,000 bp, and 4 of 6 specimens positive for large-genome viruses. Although further validation is needed to evaluate sensitivity and to define detection cutoffs, results obtained in this pilot study indicate that the overall detection success rate, coupled with the ease of interpreting the analysis reports, makes it worth considering using HTS for clinical diagnostics

Celiac disease T-cell epitopes from gamma-gliadins: immunoreactivity depends on the genome of origin, transcript frequency, and flanking protein variation

<p>Abstract</p> <p>Background</p> <p>Celiac disease (CD) is caused by an uncontrolled immune response to gluten, a heterogeneous mixture of wheat storage proteins. The CD-toxicity of these proteins and their derived peptides is depending on the presence of specific T-cell epitopes (9-mer peptides; CD epitopes) that mediate the stimulation of HLA-DQ2/8 restricted T-cells. Next to the thoroughly characterized major T-cell epitopes derived from the α-gliadin fraction of gluten, γ-gliadin peptides are also known to stimulate T-cells of celiac disease patients. To pinpoint CD-toxic γ-gliadins in hexaploid bread wheat, we examined the variation of T-cell epitopes involved in CD in γ-gliadin transcripts of developing bread wheat grains.</p> <p>Results</p> <p>A detailed analysis of the genetic variation present in γ-gliadin transcripts of bread wheat (<it>T. aestivum</it>, allo-hexaploid, carrying the A, B and D genome), together with genomic γ-gliadin sequences from ancestrally related diploid wheat species, enabled the assignment of sequence variants to one of the three genomic γ-gliadin loci, <it>Gli-A1</it>, <it>Gli-B1</it> or <it>Gli-D1</it>. Almost half of the γ-gliadin transcripts of bread wheat (49%) was assigned to locus <it>Gli-D1</it>. Transcripts from each locus differed in CD epitope content and composition. The <it>Gli-D1</it> transcripts contained the highest frequency of canonical CD epitope cores (on average 10.1 per transcript) followed by the <it>Gli-A1</it> transcripts (8.6) and the <it>Gli-B1</it> transcripts (5.4). The natural variants of the major CD epitope from γ-gliadins, DQ2-γ-I, showed variation in their capacity to induce <it>in vitro</it> proliferation of a DQ2-γ-I specific and HLA-DQ2 restricted T-cell clone.</p> <p>Conclusions</p> <p>Evaluating the CD epitopes derived from γ-gliadins in their natural context of flanking protein variation, genome specificity and transcript frequency is a significant step towards accurate quantification of the CD toxicity of bread wheat. This approach can be used to predict relative levels of CD toxicity of individual wheat cultivars directly from their transcripts (cDNAs).</p

Genomic mutation landscape of skin cancers from DNA repair-deficient xeroderma pigmentosum patients

Abstract Xeroderma pigmentosum (XP) is a genetic disorder caused by mutations in genes of the Nucleotide Excision Repair (NER) pathway (groups A-G) or in Translesion Synthesis DNA polymerase η (V). XP is associated with an increased skin cancer risk, reaching, for some groups, several thousand-fold compared to the general population. Here, we analyze 38 skin cancer genomes from five XP groups. We find that the activity of NER determines heterogeneity of the mutation rates across skin cancer genomes and that transcription-coupled NER extends beyond the gene boundaries reducing the intergenic mutation rate. Mutational profile in XP-V tumors and experiments with POLH knockout cell line reveal the role of polymerase η in the error-free bypass of (i) rare TpG and TpA DNA lesions, (ii) 3’ nucleotides in pyrimidine dimers, and (iii) TpT photodimers. Our study unravels the genetic basis of skin cancer risk in XP and provides insights into the mechanisms reducing UV-induced mutagenesis in the general population

Genomic mutation landscape of skin cancers from DNA repair-deficient xeroderma pigmentosum patients

Xeroderma pigmentosum (XP) is a genetic disorder caused by mutations in genes of the Nucleotide Excision Repair (NER) pathway (groups A-G) or in Translesion Synthesis DNA polymerase η (V). XP is associated with an increased skin cancer risk, reaching, for some groups, several thousand-fold compared to the general population. Here, we analyze 38 skin cancer genomes from five XP groups. We find that the activity of NER determines heterogeneity of the mutation rates across skin cancer genomes and that transcription-coupled NER extends beyond the gene boundaries reducing the intergenic mutation rate. Mutational profile in XP-V tumors and experiments with POLH knockout cell line reveal the role of polymerase η in the error-free bypass of (i) rare TpG and TpA DNA lesions, (ii) 3’ nucleotides in pyrimidine dimers, and (iii) TpT photodimers. Our study unravels the genetic basis of skin cancer risk in XP and provides insights into the mechanisms reducing UV-induced mutagenesis in the general population.</p

Comprehensive metagenomic analysis of glioblastoma reveals absence of known virus despite antiviral-like type I interferon gene response

Glioblastoma is a deadly malignant brain tumor and one of the most incurable forms of cancer in need of new therapeutic targets. As some cancers are known to be caused by a virus, the discovery of viruses could open the possibility to treat, and perhaps prevent, such a disease. Although an association with viruses such as cytomegalovirus or Simian virus 40 has been strongly suggested, involvement of these and other viruses in the initiation and/or propagation of glioblastoma remains vague, controversial and warrants elucidation. To exhaustively address the association of virus and glioblastoma, we developed and validated a robust metagenomic approach to analyze patient biopsies via high-throughput sequencing, a sensitive tool for virus screening. In addition to traditional clinical diagnostics, glioblastoma biopsies were deep-sequenced and analyzed with a multistage computational pipeline to identify known or potentially discover unknown viruses. In contrast to the studies reporting the presence of viral signatures in glioblastoma, no common or recurring active viruses were detected, despite finding an antiviral-like type I interferon response in some specimens. Our findings highlight a discrete and non-specific viral signature and uncharacterized short RNA sequences in glioblastoma. This study provides new insights into glioblastoma pathogenesis and defines a general methodology that can be used for high-resolution virus screening and discovery in human cancers

Passive and active DNA methylation and the interplay with genetic variation in gene regulation

DNA methylation is an essential epigenetic mark whose role in gene regulation and its dependency on genomic sequence and environment are not fully understood. In this study we provide novel insights into the mechanistic relationships between genetic variation, DNA methylation and transcriptome sequencing data in three different cell-types of the GenCord human population cohort. We find that the association between DNA methylation and gene expression variation among individuals are likely due to different mechanisms from those establishing methylation-expression patterns during differentiation. Furthermore, cell-type differential DNA methylation may delineate a platform in which local inter-individual changes may respond to or act in gene regulation. We show that unlike genetic regulatory variation, DNA methylation alone does not significantly drive allele specific expression. Finally, inferred mechanistic relationships using genetic variation as well as correlations with TF abundance reveal both a passive and active role of DNA methylation to regulatory interactions influencing gene expression. DOI:http://dx.doi.org/10.7554/eLife.00523.001