Anatomic position determines oncogenic specificity in melanoma.

Oncogenic alterations to DNA are not transforming in all cellular contexts1,2. This may be due to pre-existing transcriptional programmes in the cell of origin. Here we define anatomic position as a major determinant of why cells respond to specific oncogenes. Cutaneous melanoma arises throughout the body, whereas the acral subtype arises on the palms of the hands, soles of the feet or under the nails3. We sequenced the DNA of cutaneous and acral melanomas from a large cohort of human patients and found a specific enrichment for BRAF mutations in cutaneous melanoma and enrichment for CRKL amplifications in acral melanoma. We modelled these changes in transgenic zebrafish models and found that CRKL-driven tumours formed predominantly in the fins of the fish. The fins are the evolutionary precursors to tetrapod limbs, indicating that melanocytes in these acral locations may be uniquely susceptible to CRKL. RNA profiling of these fin and limb melanocytes, when compared with body melanocytes, revealed a positional identity gene programme typified by posterior HOX13 genes. This positional gene programme synergized with CRKL to amplify insulin-like growth factor (IGF) signalling and drive tumours at acral sites. Abrogation of this CRKL-driven programme eliminated the anatomic specificity of acral melanoma. These data suggest that the anatomic position of the cell of origin endows it with a unique transcriptional state that makes it susceptible to only certain oncogenic insults.

The origin and evolution of mutations in acute myeloid leukemia.

Most mutations in cancer genomes are thought to be acquired after the initiating event, which may cause genomic instability and drive clonal evolution. However, for acute myeloid leukemia (AML), normal karyotypes are common, and genomic instability is unusual. To better understand clonal evolution in AML, we sequenced the genomes of M3-AML samples with a known initiating event (PML-RARA) versus the genomes of normal karyotype M1-AML samples and the exomes of hematopoietic stem/progenitor cells (HSPCs) from healthy people. Collectively, the data suggest that most of the mutations found in AML genomes are actually random events that occurred in HSPCs before they acquired the initiating mutation; the mutational history of that cell is "captured" as the clone expands. In many cases, only one or two additional, cooperating mutations are needed to generate the malignant founding clone. Cells from the founding clone can acquire additional cooperating mutations, yielding subclones that can contribute to disease progression and/or relapse.

Clonal evolution in relapsed acute myeloid leukaemia revealed by whole-genome sequencing.

Most patients with acute myeloid leukaemia (AML) die from progressive disease after relapse, which is associated with clonal evolution at the cytogenetic level. To determine the mutational spectrum associated with relapse, we sequenced the primary tumour and relapse genomes from eight AML patients, and validated hundreds of somatic mutations using deep sequencing; this allowed us to define clonality and clonal evolution patterns precisely at relapse. In addition to discovering novel, recurrently mutated genes (for example, WAC, SMC3, DIS3, DDX41 and DAXX) in AML, we also found two major clonal evolution patterns during AML relapse: (1) the founding clone in the primary tumour gained mutations and evolved into the relapse clone, or (2) a subclone of the founding clone survived initial therapy, gained additional mutations and expanded at relapse. In all cases, chemotherapy failed to eradicate the founding clone. The comparison of relapse-specific versus primary tumour mutations in all eight cases revealed an increase in transversions, probably due to DNA damage caused by cytotoxic chemotherapy. These data demonstrate that AML relapse is associated with the addition of new mutations and clonal evolution, which is shaped, in part, by the chemotherapy that the patients receive to establish and maintain remissions.

Whole-genome analysis informs breast cancer response to aromatase inhibition.

To correlate the variable clinical features of oestrogen-receptor-positive breast cancer with somatic alterations, we studied pretreatment tumour biopsies accrued from patients in two studies of neoadjuvant aromatase inhibitor therapy by massively parallel sequencing and analysis. Eighteen significantly mutated genes were identified, including five genes (RUNX1, CBFB, MYH9, MLL3 and SF3B1) previously linked to haematopoietic disorders. Mutant MAP3K1 was associated with luminal A status, low-grade histology and low proliferation rates, whereas mutant TP53 was associated with the opposite pattern. Moreover, mutant GATA3 correlated with suppression of proliferation upon aromatase inhibitor treatment. Pathway analysis demonstrated that mutations in MAP2K4, a MAP3K1 substrate, produced similar perturbations as MAP3K1 loss. Distinct phenotypes in oestrogen-receptor-positive breast cancer are associated with specific patterns of somatic mutations that map into cellular pathways linked to tumour biology, but most recurrent mutations are relatively infrequent. Prospective clinical trials based on these findings will require comprehensive genome sequencing.

Genome Modeling System: A Knowledge Management Platform for Genomics.

In this work, we present the Genome Modeling System (GMS), an analysis information management system capable of executing automated genome analysis pipelines at a massive scale. The GMS framework provides detailed tracking of samples and data coupled with reliable and repeatable analysis pipelines. The GMS also serves as a platform for bioinformatics development, allowing a large team to collaborate on data analysis, or an individual researcher to leverage the work of others effectively within its data management system. Rather than separating ad-hoc analysis from rigorous, reproducible pipelines, the GMS promotes systematic integration between the two. As a demonstration of the GMS, we performed an integrated analysis of whole genome, exome and transcriptome sequencing data from a breast cancer cell line (HCC1395) and matched lymphoblastoid line (HCC1395BL). These data are available for users to test the software, complete tutorials and develop novel GMS pipeline configurations. The GMS is available at https://github.com/genome/gms.

Genome remodelling in a basal-like breast cancer metastasis and xenograft.

Massively parallel DNA sequencing technologies provide an unprecedented ability to screen entire genomes for genetic changes associated with tumour progression. Here we describe the genomic analyses of four DNA samples from an African-American patient with basal-like breast cancer: peripheral blood, the primary tumour, a brain metastasis and a xenograft derived from the primary tumour. The metastasis contained two de novo mutations and a large deletion not present in the primary tumour, and was significantly enriched for 20 shared mutations. The xenograft retained all primary tumour mutations and displayed a mutation enrichment pattern that resembled the metastasis. Two overlapping large deletions, encompassing CTNNA1, were present in all three tumour samples. The differential mutation frequencies and structural variation patterns in metastasis and xenograft compared with the primary tumour indicate that secondary tumours may arise from a minority of cells within the primary tumour.

CREST maps somatic structural variation in cancer genomes with base-pair resolution.

We developed 'clipping reveals structure' (CREST), an algorithm that uses next-generation sequencing reads with partial alignments to a reference genome to directly map structural variations at the nucleotide level of resolution. Application of CREST to whole-genome sequencing data from five pediatric T-lineage acute lymphoblastic leukemias (T-ALLs) and a human melanoma cell line, COLO-829, identified 160 somatic structural variations. Experimental validation exceeded 80%, demonstrating that CREST had a high predictive accuracy.

DNMT3A mutations in acute myeloid leukemia.

BACKGROUND: The genetic alterations responsible for an adverse outcome in most patients with acute myeloid leukemia (AML) are unknown. METHODS: Using massively parallel DNA sequencing, we identified a somatic mutation in DNMT3A, encoding a DNA methyltransferase, in the genome of cells from a patient with AML with a normal karyotype. We sequenced the exons of DNMT3A in 280 additional patients with de novo AML to define recurring mutations. RESULTS: A total of 62 of 281 patients (22.1%) had mutations in DNMT3A that were predicted to affect translation. We identified 18 different missense mutations, the most common of which was predicted to affect amino acid R882 (in 37 patients). We also identified six frameshift, six nonsense, and three splice-site mutations and a 1.5-Mbp deletion encompassing DNMT3A. These mutations were highly enriched in the group of patients with an intermediate-risk cytogenetic profile (56 of 166 patients, or 33.7%) but were absent in all 79 patients with a favorable-risk cytogenetic profile (P<0.001 for both comparisons). The median overall survival among patients with DNMT3A mutations was significantly shorter than that among patients without such mutations (12.3 months vs. 41.1 months, P<0.001). DNMT3A mutations were associated with adverse outcomes among patients with an intermediate-risk cytogenetic profile or FLT3 mutations, regardless of age, and were independently associated with a poor outcome in Cox proportional-hazards analysis. CONCLUSIONS: DNMT3A mutations are highly recurrent in patients with de novo AML with an intermediate-risk cytogenetic profile and are independently associated with a poor outcome. (Funded by the National Institutes of Health and others.).

SomaticSniper: identification of somatic point mutations in whole genome sequencing data.

MOTIVATION: The sequencing of tumors and their matched normals is frequently used to study the genetic composition of cancer. Despite this fact, there remains a dearth of available software tools designed to compare sequences in pairs of samples and identify sites that are likely to be unique to one sample. RESULTS: In this article, we describe the mathematical basis of our SomaticSniper software for comparing tumor and normal pairs. We estimate its sensitivity and precision, and present several common sources of error resulting in miscalls. AVAILABILITY AND IMPLEMENTATION: Binaries are freely available for download at http://gmt.genome.wustl.edu/somatic-sniper/current/, implemented in C and supported on Linux and Mac OS X. CONTACT: delarson@wustl.edu; lding@wustl.edu SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Recurring mutations found by sequencing an acute myeloid leukemia genome.

BACKGROUND: The full complement of DNA mutations that are responsible for the pathogenesis of acute myeloid leukemia (AML) is not yet known. METHODS: We used massively parallel DNA sequencing to obtain a very high level of coverage (approximately 98%) of a primary, cytogenetically normal, de novo genome for AML with minimal maturation (AML-M1) and a matched normal skin genome. RESULTS: We identified 12 acquired (somatic) mutations within the coding sequences of genes and 52 somatic point mutations in conserved or regulatory portions of the genome. All mutations appeared to be heterozygous and present in nearly all cells in the tumor sample. Four of the 64 mutations occurred in at least 1 additional AML sample in 188 samples that were tested. Mutations in NRAS and NPM1 had been identified previously in patients with AML, but two other mutations had not been identified. One of these mutations, in the IDH1 gene, was present in 15 of 187 additional AML genomes tested and was strongly associated with normal cytogenetic status; it was present in 13 of 80 cytogenetically normal samples (16%). The other was a nongenic mutation in a genomic region with regulatory potential and conservation in higher mammals; we detected it in one additional AML tumor. The AML genome that we sequenced contains approximately 750 point mutations, of which only a small fraction are likely to be relevant to pathogenesis. CONCLUSIONS: By comparing the sequences of tumor and skin genomes of a patient with AML-M1, we have identified recurring mutations that may be relevant for pathogenesis.

Genomic insights into the uncultivated marine Zetaproteobacteria at Loihi Seamount.

The Zetaproteobacteria are a candidate class of marine iron-oxidizing bacteria that are typically found in high iron environments such as hydrothermal vent sites. As much remains unknown about these organisms due to difficulties in cultivation, single-cell genomics was used to learn more about this elusive group at Loihi Seamount. Comparative genomics of 23 phylogenetically diverse single amplified genomes (SAGs) and two isolates indicate niche specialization among the Zetaproteobacteria may be largely due to oxygen tolerance and nitrogen transformation capabilities. Only Form II ribulose 1,5-bisphosphate carboxylase (RubisCO) genes were found in the SAGs, suggesting that some of the uncultivated Zetaproteobacteria may be adapted to low oxygen and/or high carbon dioxide concentrations. There is also genomic evidence of oxygen-tolerant cytochrome c oxidases and oxidative stress-related genes, indicating that others may be exposed to higher oxygen conditions. The Zetaproteobacteria also have the genomic potential for acquiring nitrogen from numerous sources including ammonium, nitrate, organic compounds, and nitrogen gas. Two types of molybdopterin oxidoreductase genes were found in the SAGs, indicating that those found in the isolates, thought to be involved in iron oxidation, are not consistent among all the Zetaproteobacteria. However, a novel cluster of redox-related genes was found to be conserved in 10 SAGs as well as in the isolates warranting further investigation. These results were used to isolate a novel iron-oxidizing Zetaproteobacteria. Physiological studies and genomic analysis of this isolate were able to support many of the findings from SAG analyses demonstrating the value of these data for designing future enrichment strategies.

Genomic and metabolic diversity of Marine Group I Thaumarchaeota in the mesopelagic of two subtropical gyres.

Marine Group I (MGI) Thaumarchaeota are one of the most abundant and cosmopolitan chemoautotrophs within the global dark ocean. To date, no representatives of this archaeal group retrieved from the dark ocean have been successfully cultured. We used single cell genomics to investigate the genomic and metabolic diversity of thaumarchaea within the mesopelagic of the subtropical North Pacific and South Atlantic Ocean. Phylogenetic and metagenomic recruitment analysis revealed that MGI single amplified genomes (SAGs) are genetically and biogeographically distinct from existing thaumarchaea cultures obtained from surface waters. Confirming prior studies, we found genes encoding proteins for aerobic ammonia oxidation and the hydrolysis of urea, which may be used for energy production, as well as genes involved in 3-hydroxypropionate/4-hydroxybutyrate and oxidative tricarboxylic acid pathways. A large proportion of protein sequences identified in MGI SAGs were absent in the marine cultures Cenarchaeum symbiosum and Nitrosopumilus maritimus, thus expanding the predicted protein space for this archaeal group. Identifiable genes located on genomic islands with low metagenome recruitment capacity were enriched in cellular defense functions, likely in response to viral infections or grazing. We show that MGI Thaumarchaeota in the dark ocean may have more flexibility in potential energy sources and adaptations to biotic interactions than the existing, surface-ocean cultures.

Retrotransposition of gene transcripts leads to structural variation in mammalian genomes.

BACKGROUND: Retroposed processed gene transcripts are an important source of material for new gene formation on evolutionary timescales. Most prior work on gene retrocopy discovery compared copies in reference genome assemblies to their source genes. Here, we explore gene retrocopy insertion polymorphisms (GRIPs) that are present in the germlines of individual humans, mice, and chimpanzees, and we identify novel gene retrocopy insertions in cancerous somatic tissues that are absent from patient-matched non-cancer genomes. RESULTS: Through analysis of whole-genome sequence data, we found evidence for 48 GRIPs in the genomes of one or more humans sequenced as part of the 1,000 Genomes Project and The Cancer Genome Atlas, but which were not in the human reference assembly. Similarly, we found evidence for 755 GRIPs at distinct locations in one or more of 17 inbred mouse strains but which were not in the mouse reference assembly, and 19 GRIPs across a cohort of 10 chimpanzee genomes, which were not in the chimpanzee reference genome assembly. Many of these insertions are new members of existing gene families whose source genes are highly and widely expressed, and the majority have detectable hallmarks of processed gene retrocopy formation. We estimate the rate of novel gene retrocopy insertions in humans and chimps at roughly one new gene retrocopy insertion for every 6,000 individuals. CONCLUSIONS: We find that gene retrocopy polymorphisms are a widespread phenomenon, present a multi-species analysis of these events, and provide a method for their ascertainment.

Effects of leptin and obesity on the upper airway function.

Obesity is associated with alterations in upper airway collapsibility during sleep. Obese, leptin-deficient mice demonstrate blunted ventilatory control, leading us to hypothesize that (1) obesity and leptin deficiency would predispose to worsening neuromechanical upper airway function and that (2) leptin replacement would acutely reverse neuromuscular defects in the absence of weight loss. In age-matched, anesthetized, spontaneously breathing C57BL/6J (BL6) and ob(-)/ob(-) mice, we characterized upper airway pressure-flow dynamics during ramp decreases in nasal pressure (P(N)) to determine the passive expiratory critical pressure (P(CRIT)) and active responses to reductions in P(N), including the percentage of ramps showing inspiratory flow limitation (IFL; frequency), the P(N) threshold at which IFL developed, maximum inspiratory airflow (Vi(max)), and genioglossus electromyographic (EMG(GG)) activity. Elevations in body weight were associated with progressive elevations in P(CRIT) (0.1 ± 0.02 cmH(2)O/g), independent of mouse strain. P(CRIT) was also elevated in ob(-)/ob(-) compared with BL6 mice (1.6 ± 0.1 cmH(2)O), independent of weight. Both obesity and leptin deficiency were associated with significantly higher IFL frequency and P(N) threshold and lower VI(max). Very obese ob(-)/ob(-) mice treated with leptin compared with nontreated mice showed a decrease in IFL frequency (from 63.5 ± 2.9 to 30.0 ± 8.6%) and P(N) threshold (from -0.8 ± 1.1 to -5.6 ± 0.8 cmH(2)O) and increase in VI(max) (from 354.1 ± 25.3 to 659.0 ± 71.8 µl/s). Nevertheless, passive P(CRIT) in leptin-treated mice did not differ significantly from that seen in nontreated ob(-)/ob(-) mice. The findings suggest that weight and leptin deficiency produced defects in upper airway neuromechanical control and that leptin reversed defects in active neuromuscular responses acutely without reducing mechanical loads.

Landscape of somatic retrotransposition in human cancers.

Transposable elements (TEs) are abundant in the human genome, and some are capable of generating new insertions through RNA intermediates. In cancer, the disruption of cellular mechanisms that normally suppress TE activity may facilitate mutagenic retrotranspositions. We performed single-nucleotide resolution analysis of TE insertions in 43 high-coverage whole-genome sequencing data sets from five cancer types. We identified 194 high-confidence somatic TE insertions, as well as thousands of polymorphic TE insertions in matched normal genomes. Somatic insertions were present in epithelial tumors but not in blood or brain cancers. Somatic L1 insertions tend to occur in genes that are commonly mutated in cancer, disrupt the expression of the target genes, and are biased toward regions of cancer-specific DNA hypomethylation, highlighting their potential impact in tumorigenesis.

Recurrent mutations in the U2AF1 splicing factor in myelodysplastic syndromes.

Myelodysplastic syndromes (MDS) are hematopoietic stem cell disorders that often progress to chemotherapy-resistant secondary acute myeloid leukemia (sAML). We used whole-genome sequencing to perform an unbiased comprehensive screen to discover the somatic mutations in a sample from an individual with sAML and genotyped the loci containing these mutations in the matched MDS sample. Here we show that a missense mutation affecting the serine at codon 34 (Ser34) in U2AF1 was recurrently present in 13 out of 150 (8.7%) subjects with de novo MDS, and we found suggestive evidence of an increased risk of progression to sAML associated with this mutation. U2AF1 is a U2 auxiliary factor protein that recognizes the AG splice acceptor dinucleotide at the 3' end of introns, and the alterations in U2AF1 are located in highly conserved zinc fingers of this protein. Mutant U2AF1 promotes enhanced splicing and exon skipping in reporter assays in vitro. This previously unidentified, recurrent mutation in U2AF1 implicates altered pre-mRNA splicing as a potential mechanism for MDS pathogenesis.