Exome sequencing of gastric adenocarcinoma identifies recurrent somatic mutations in cell adhesion and chromatin remodelling genes

Gastric cancer is a major cause of global cancer mortality. We surveyed the spectrum of somatic alterations in gastric cancer by sequencing the exomes of 15 gastric adenocarcinomas and their matched normal DNAs. Frequently mutated genes in the adenocarcinomas included TP53 (11/15 tumors), PIK3CA (3/15) and ARID1A (3/15). Cell adhesion was the most enriched biological pathway among the frequently mutated genes. A prevalence screening confirmed mutations in FAT4, a cadherin family gene, in 5% of gastric cancers (6/110) and FAT4 genomic deletions in 4% (3/83) of gastric tumors. Frequent mutations in chromatin remodeling genes (ARID1A, MLL3 and MLL) also occurred in 47% of the gastric cancers. We detected ARID1A mutations in 8% of tumors (9/110), which were associated with concurrent PIK3CA mutations and microsatellite instability. In functional assays, we observed both FAT4 and ARID1A to exert tumor-suppressor activity. Somatic inactivation of FAT4 and ARID1A may thus be key tumorigenic events in a subset of gastric cancers.

Transmission of two novel mutations in a pedigree with familial lecithin:cholesterol acyltransferase deficiency:structure-function relationships and studies in a compound heterozygous proband

Two novel mutations were identified in a compound heterozygous male with lecithin:cholesterol acyltransferase (LCAT) deficiency. Exon sequence determination of the LCAT gene of the proband revealed two novel heterozygous mutations in exons one (C110T) and six (C991T) that predict non-conservative amino acid substitutions (Thr13Met and Pro307Ser, respectively). To assess the distinct functional impact of the separate mutant alleles, studies were conducted in the proband's 3-generation pedigree. The compound heterozygous proband had negligible HDL and severely reduced apolipoprotein A-I, LCAT mass, LCAT activity, and cholesterol esterification rate (CER). The proband's mother and two sisters were heterozygous for the Pro307Ser mutation and had low HDL, markedly reduced LCAT activity and CER, and the propensity for significant reductions in LCAT protein mass. The proband's father and two daughters were heterozygous for the Thr13Met mutation and also displayed low HDL, reduced LCAT activity and CER, and more modest decrements in LCAT mass. Mean LCAT specific activity was severely impaired in the compound heterozygous proband and was reduced by 50% in individuals heterozygous for either mutation, compared to wild type family members. It is also shown that the two mutations impair both catalytic activity and expression of the circulating protein.

Within-host evolution of Burkholderia pseudomallei over a twelve-year chronic carriage infection

UNLABELLED: Burkholderia pseudomallei causes the potentially fatal disease melioidosis. It is generally accepted that B. pseudomallei is a noncommensal bacterium and that any culture-positive clinical specimen denotes disease requiring treatment. Over a 23-year study of melioidosis cases in Darwin, Australia, just one patient from 707 survivors has developed persistent asymptomatic B. pseudomallei carriage. To better understand the mechanisms behind this unique scenario, we performed whole-genome analysis of two strains isolated 139 months apart. During this period, B. pseudomallei underwent several adaptive changes. Of 23 point mutations, 78% were nonsynonymous and 43% were predicted to be deleterious to gene function, demonstrating a strong propensity for positive selection. Notably, a nonsense mutation inactivated the universal stress response sigma factor RpoS, with pleiotropic implications. The genome underwent substantial reduction, with four deletions in chromosome 2 resulting in the loss of 221 genes. The deleted loci included genes involved in secondary metabolism, environmental survival, and pathogenesis. Of 14 indels, 11 occurred in coding regions and 9 resulted in frameshift mutations that dramatically affected predicted gene products. Disproportionately, four indels affected lipopolysaccharide biosynthesis and modification. Finally, we identified a frameshift mutation in both P314 isolates within wcbR, an important component of the capsular polysaccharide I locus, suggesting virulence attenuation early in infection. Our study illustrates a unique clinical case that contrasts a high-consequence infectious agent with a long-term commensal infection and provides further insights into bacterial evolution within the human host.

IMPORTANCE: Some bacterial pathogens establish long-term infections that are difficult or impossible to eradicate with current treatments. Rapid advances in genome sequencing technologies provide a powerful tool for understanding bacterial persistence within the human host. Burkholderia pseudomallei is considered a highly pathogenic bacterium because infection is commonly fatal. Here, we document within-host evolution of B. pseudomallei in a unique case of human infection with ongoing chronic carriage. Genomic comparison of isolates obtained 139 months (11.5 years) apart showed a strong signal of adaptation within the human host, including inactivation of virulence and immunogenic factors, and deletion of pathways involved in environmental survival. Two global regulatory genes were mutated in the 139-month isolate, indicating extensive regulatory changes favoring bacterial persistence. Our study provides insights into B. pseudomallei pathogenesis and, more broadly, identifies parallel evolutionary mechanisms that underlie chronic persistence of all bacterial pathogens.

Next-generation sequencing: a change of paradigm in molecular diagnostic validation

Next-generation sequencing (NGS) is beginning to show its full potential for diagnostic and therapeutic applications. In particular, it is enunciating its capacity to contribute to a molecular taxonomy of cancer, to be used as a standard approach for diagnostic mutation detection, and to open new treatment options that are not exclusively organ-specific. If this is the case, how much validation is necessary and what should be the validation strategy, when bringing NGS into the diagnostic/clinical practice? This validation strategy should address key issues such as: what is the overall extent of the validation? Should essential indicators of test performance such as sensitivity of specificity be calculated for every target or sample type? Should bioinformatic interpretation approaches be validated with the same rigour? What is a competitive clinical turnaround time for a NGS-based test, and when does it become a cost-effective testing proposition? While we address these and other related topics in this commentary, we also suggest that a single set of international guidelines for the validation and use of NGS technology in routine diagnostics may allow us all to make a much more effective use of resources.

Characterization of nontypable Haemophilus influenzae isolates recovered from adult patients with underlying chronic lung disease reveals genotypic and phenotypic traits associated with persistent infection

Nontypable Haemophilus influenzae (NTHi) has emerged as an important opportunistic pathogen causing infection in adults suffering obstructive lung diseases. Existing evidence associates chronic infection by NTHi to the progression of the chronic respiratory disease, but specific features of NTHi associated with persistence have not been comprehensively addressed. To provide clues about adaptive strategies adopted by NTHi during persistent infection, we compared sequential persistent isolates with newly acquired isolates in sputa from six patients with chronic obstructive lung disease. Pulse field gel electrophoresis (PFGE) identified three patients with consecutive persistent strains and three with new strains. Phenotypic characterisation included infection of respiratory epithelial cells, bacterial self-aggregation, biofilm formation and resistance to antimicrobial peptides (AMP). Persistent isolates differed from new strains in showing low epithelial adhesion and inability to form biofilms when grown under continuous-flow culture conditions in microfermenters. Self-aggregation clustered the strains by patient, not by persistence. Increasing resistance to AMPs was observed for each series of persistent isolates; this was not associated with lipooligosaccharide decoration with phosphorylcholine or with lipid A acylation. Variation was further analyzed for the series of three persistent isolates recovered from patient 1. These isolates displayed comparable growth rate, natural transformation frequency and murine pulmonary infection. Genome sequencing of these three isolates revealed sequential acquisition of single-nucleotide variants in the AMP permease sapC, the heme acquisition systems hgpB, hgpC, hup and hxuC, the 3-deoxy-D-manno-octulosonic acid kinase kdkA, the long-chain fatty acid transporter ompP1, and the phosphoribosylamine glycine ligase purD. Collectively, we frame a range of pathogenic traits and a repertoire of genetic variants in the context of persistent infection by NTHi.

Independent genomewide screens identify the tumor suppressor VTRNA2-1 as a human epiallele responsive to periconceptional environment

Background: Interindividual epigenetic variation that occurs systemically must be established prior to gastrulation in the very early embryo and, because it is systemic, can be assessed in easily biopsiable tissues. We employ two independent genome-wide approaches to search for such variants.

Results: First, we screen for metastable epialleles by performing genomewide bisulfite sequencing in peripheral blood lymphocyte (PBL) and hair follicle DNA from two Caucasian adults. Second, we conduct a genomewide screen for genomic regions at which PBL DNA methylation is affected by season of conception in rural Gambia. Remarkably, both approaches identify the genomically imprinted VTRNA2-1 as a top environmentally responsive epiallele. We demonstrate systemic and stochastic interindividual variation in DNA methylation at the VTRNA2-1 differentially methylated region in healthy Caucasian and Asian adults and show, in rural Gambians, that periconceptional environment affects offspring VTRNA2-1 epigenotype, which is stable over at least 10 years. This unbiased screen also identifies over 100 additional candidate metastable epialleles, and shows that these are associated with cis genomic features including transposable elements.

Conclusions: The non-coding VTRNA2-1 transcript (also called nc886) is a putative tumor suppressor and modulator of innate immunity. Thus, these data indicating environmentally induced loss of imprinting at VTRNA2-1 constitute a plausible causal pathway linking early embryonic environment, epigenetic alteration, and human disease. More broadly, the list of candidate metastable epialleles provides a resource for future studies of epigenetic variation and human disease.

HES6 drives a critical AR transcriptional programme to induce castration-resistant prostate cancer through activation of an E2F1-mediated cell cycle network

Castrate-resistant prostate cancer (CRPC) is poorly characterized and heterogeneous and while the androgen receptor (AR) is of singular importance, other factors such as c-Myc and the E2F family also play a role in later stage disease. HES6 is a transcription co-factor associated with stem cell characteristics in neural tissue. Here we show that HES6 is up-regulated in aggressive human prostate cancer and drives castration-resistant tumour growth in the absence of ligand binding by enhancing the transcriptional activity of the AR, which is preferentially directed to a regulatory network enriched for transcription factors such as E2F1. In the clinical setting, we have uncovered a HES6-associated signature that predicts poor outcome in prostate cancer, which can be pharmacologically targeted by inhibition of PLK1 with restoration of sensitivity to castration. We have therefore shown for the first time the critical role of HES6 in the development of CRPC and identified its potential in patient-specific therapeutic strategies.

Exome sequencing of prostate cancer supports the hypothesis of independent tumour origins

BACKGROUND: Prostate cancer (PCa) is a clinically and pathologically heterogeneous disease. The rapid development of sequencing technology has the potential to deliver new biomarkers with emphasis on aggressive disease and to revolutionise personalised cancer treatment. However, a prostate harbouring cancer commonly contains multiple separate tumour foci, with the potential to aggravate tumour sampling. The level of intraprostatic tumour heterogeneity remains to be determined.

OBJECTIVE: To determine the level of intraprostatic tumour heterogeneity through genome-wide, high-resolution profiling of multiple tumour samples from the same individual.

DESIGN, SETTINGS, AND PARTICIPANTS: Multiple tumour samples were obtained from four individuals following radical prostatectomy. One individual (SWE-1) contained >70% cancer cells in all tumour samples, whereas the other three (SWE-2 to SWE-4) required the use of laser capture microdissection for tumour cell enrichment. Subsequently, DNA was extracted from all tissue samples, and exome sequencing was performed. All tumour foci of SWE-1 were also profiled using a high-resolution array for the identification of copy number alterations (CNA).

OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS: Shared somatic high-frequency single nucleotide variants (SNV) and CNAs were used to infer the level of intraprostatic tumour heterogeneity.

RESULTS AND LIMITATIONS: No high-frequency mutations, common for the three tumour samples of SWE-1, were identified. Ten randomly chosen positions were validated with Sanger sequencing in all foci, which verified the exome data. The high level of intraprostatic heterogeneity was consistent in all individuals. In total, three out of four individuals harboured tumours without an apparent common somatic denominator. Although we cannot exclude the presence of common structural rearrangements, a high-density array was used for the detection of deletions and amplifications in SWE-1, which agreed with the exome data.

CONCLUSIONS: We present evidence for the presence of somatically independent tumours within the same prostate. This finding will have implications for personalised cancer treatment and biomarker discovery.

New androgen receptor genomic targets show an interaction with the ETS1 transcription factor

The androgen receptor (AR) initiates important developmental and oncogenic transcriptional pathways. The AR is known to bind as a homodimer to 15-base pair bipartite palindromic androgen-response elements; however, few direct AR gene targets are known. To identify AR promoter targets, we used chromatin immunoprecipitation with on-chip detection of genomic fragments. We identified 1,532 potential AR-binding sites, including previously known AR gene targets. Many of the new AR target genes show altered expression in prostate cancer. Analysis of sequences underlying AR-binding sites showed that more than 50% of AR-binding sites did not contain the established 15 bp AR-binding element. Unbiased sequence analysis showed 6-bp motifs, which were significantly enriched and were bound directly by the AR in vitro. Binding sequences for the avian erythroblastosis virus E26 homologue (ETS) transcription factor family were also highly enriched, and we uncovered an interaction between the AR and ETS1 at a subset of AR promoter targets.

SNP discovery using Next Generation Transcriptomic Sequencing in Atlantic herring (Clupea harengus)

The introduction of Next Generation Sequencing (NGS) has revolutionised population genetics, providing studies of non-model species with unprecedented genomic coverage, allowing evolutionary biologists to address questions previously far beyond the reach of available resources. Furthermore, the simple mutation model of Single Nucleotide Polymorphisms (SNPs) permits cost-effective high-throughput genotyping in thousands of individuals simultaneously. Genomic resources are scarce for the Atlantic herring (Clupea harengus), a small pelagic species that sustains high revenue fisheries. This paper details the development of 578 SNPs using a combined NGS and high-throughput genotyping approach. Eight individuals covering the species distribution in the eastern Atlantic were bar-coded and multiplexed into a single cDNA library and sequenced using the 454 GS FLX platform. SNP discovery was performed by de novo sequence clustering and contig assembly, followed by the mapping of reads against consensus contig sequences. Selection of candidate SNPs for genotyping was conducted using an in silico approach. SNP validation and genotyping were performed simultaneously using an Illumina 1,536 GoldenGate assay. Although the conversion rate of candidate SNPs in the genotyping assay cannot be predicted in advance, this approach has the potential to maximise cost and time efficiencies by avoiding expensive and time-consuming laboratory stages of SNP validation. Additionally, the in silico approach leads to lower ascertainment bias in the resulting SNP panel as marker selection is based only on the ability to design primers and the predicted presence of intron-exon boundaries. Consequently SNPs with a wider spectrum of minor allele frequencies (MAFs) will be genotyped in the final panel. The genomic resources presented here represent a valuable multi-purpose resource for developing informative marker panels for population discrimination, microarray development and for population genomic studies in the wild.

Environmental selection on transcriptome-derived SNPs in a high gene flow marine fish, the Atlantic herring (Clupea harengus)

High gene flow is considered the norm for most marine organisms and is expected to limit their ability to adapt to local environments. Few studies have directly compared the patterns of differentiation at neutral and selected gene loci in marine organisms. We analysed a transcriptome-derived panel of 281 SNPs in Atlantic herring (Clupea harengus), a highly migratory small pelagic fish, for elucidating neutral and selected genetic variation among populations and to identify candidate genes for environmental adaptation. We analysed 607 individuals from 18 spawning locations in the northeast Atlantic, including two temperature clines (5-12 °C) and two salinity clines (5-35‰). By combining genome scan and landscape genetic analyses, four genetically distinct groups of herring were identified: Baltic Sea, Baltic-North Sea transition area, North Sea/British Isles and North Atlantic; notably, samples exhibited divergent clustering patterns for neutral and selected loci. We found statistically strong evidence for divergent selection at 16 outlier loci on a global scale, and significant correlations with temperature and salinity at nine loci. On regional scales, we identified two outlier loci with parallel patterns across temperature clines and five loci associated with temperature in the North Sea/North Atlantic. Likewise, we found seven replicated outliers, of which five were significantly associated with low salinity across both salinity clines. Our results reveal a complex pattern of varying spatial genetic variation among outlier loci, likely reflecting adaptations to local environments. In addition to disclosing the fine scale of local adaptation in a highly vagile species, our data emphasize the need to preserve functionally important biodiversity.

Novel tools for conservation genomics: comparing two high-throughput approaches for SNP discovery in the transcriptome of the European hake

The growing accessibility to genomic resources using next-generation sequencing (NGS) technologies has revolutionized the application of molecular genetic tools to ecology and evolutionary studies in non-model organisms. Here we present the case study of the European hake (Merluccius merluccius), one of the most important demersal resources of European fisheries. Two sequencing platforms, the Roche 454 FLX (454) and the Illumina Genome Analyzer (GAII), were used for Single Nucleotide Polymorphisms (SNPs) discovery in the hake muscle transcriptome. De novo transcriptome assembly into unique contigs, annotation, and in silico SNP detection were carried out in parallel for 454 and GAII sequence data. High-throughput genotyping using the Illumina GoldenGate assay was performed for validating 1,536 putative SNPs. Validation results were analysed to compare the performances of 454 and GAII methods and to evaluate the role of several variables (e.g. sequencing depth, intron-exon structure, sequence quality and annotation). Despite well-known differences in sequence length and throughput, the two approaches showed similar assay conversion rates (approximately 43%) and percentages of polymorphic loci (67.5% and 63.3% for GAII and 454, respectively). Both NGS platforms therefore demonstrated to be suitable for large scale identification of SNPs in transcribed regions of non-model species, although the lack of a reference genome profoundly affects the genotyping success rate. The overall efficiency, however, can be improved using strict quality and filtering criteria for SNP selection (sequence quality, intron-exon structure, target region score).

Application of SNPs for population genetics of nonmodel organisms:new opportunities and challenges

Recent improvements in the speed, cost and accuracy of next generation sequencing are revolutionizing the discovery of single nucleotide polymorphisms (SNPs). SNPs are increasingly being used as an addition to the molecular ecology toolkit in nonmodel organisms, but their efficient use remains challenging. Here, we discuss common issues when employing SNP markers, including the high numbers of markers typically employed, the effects of ascertainment bias and the inclusion of nonneutral loci in a marker panel. We provide a critique of considerations specifically associated with the application and population genetic analysis of SNPs in nonmodel taxa, focusing specifically on some of the most commonly applied methods.

Side effects of genome structural changes.

The first extensive catalog of structural human variation was recently released. It showed that large stretches of genomic DNA that vary considerably in copy number were extremely abundant. Thus it is conceivable that they play a major role in functional variation. Consistently, genomic insertions and deletions were shown to contribute to phenotypic differences by modifying not only the expression levels of genes within the aneuploid segments but also of normal copy-number neighboring genes. In this report, we review the possible mechanisms behind this latter effect.

Modeling sequencing errors by combining Hidden Markov models.

Among the largest resources for biological sequence data is the large amount of expressed sequence tags (ESTs) available in public and proprietary databases. ESTs provide information on transcripts but for technical reasons they often contain sequencing errors. Therefore, when analyzing EST sequences computationally, such errors must be taken into account. Earlier attempts to model error prone coding regions have shown good performance in detecting and predicting these while correcting sequencing errors using codon usage frequencies. In the research presented here, we improve the detection of translation start and stop sites by integrating a more complex mRNA model with codon usage bias based error correction into one hidden Markov model (HMM), thus generalizing this error correction approach to more complex HMMs. We show that our method maintains the performance in detecting coding sequences.