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Complete knowledge of the genetic variation in individual human genomes is a crucial foundation for understanding the etiology of disease. Genetic variation is typically characterized by sequencing individual genomes and comparing reads to a reference. Existing methods do an excellent job of detecting variants in approximately 90% of the human genome; however, calling variants in the remaining 10% of the genome (largely low-complexity sequence and segmental duplications) is challenging. To improve variant calling, we developed a new algorithm, DISCOVAR, and examined its performance on improved, low-cost sequence data. Using a newly created reference set of variants from the finished sequence of 103 randomly chosen fosmids, we find that some standard variant call sets miss up to 25% of variants. We show that the combination of new methods and improved data increases sensitivity by several fold, with the greatest impact in challenging regions of the human genome., National Human Genome Research Institute (U.S.) (Grant R01HG003474), National Human Genome Research Institute (U.S.) (Grant U54HG003067), National Institute of Allergy and Infectious Diseases (U.S.) (Contract HHSN272200900018C)

Evolution of Ebola virus over time The high rate of mortality in the current Ebola epidemic has made it difficult for researchers to collect samples of the virus and study its evolution. Gire et al. describe Ebola epidemiology on the basis of 99 whole-genome sequences, including samples from 78 affected individuals. The authors analyzed changes in the viral sequence and conclude that the current outbreak probably resulted from the spread of the virus from central Africa in the past decade. The outbreak started from a single transmission event from an unknown animal reservoir into the human population. Two viral lineages from Guinea then spread from person to person into Sierra Leone. Science , this issue p. 1369

Intermolecular RNA-RNA interactions are used by many noncoding RNAs (ncRNAs) to achieve their diverse functions. To aid in identifying these contacts, we developed a method based on RNA Antisense Purification to systematically map RNA-RNA interactions (RAP-RNA) and applied it to investigate two ncRNAs implicated in RNA processing: U1 snRNA, a component of the spliceosome, and Malat1, a lncRNA that localizes to nuclear speckles. U1 and Malat1 interact with nascent transcripts through distinct targeting mechanisms. Using differential crosslinking, we confirmed that U1 directly hybridizes to both 5’ splice sites and 5’-splice-site motifs throughout introns and found that Malat1 interacts with pre-mRNAs indirectly through protein intermediates. Interactions with nascent pre-mRNAs cause U1 and Malat1 to localize proximally to chromatin at active genes, demonstrating that ncRNAs can use RNA-RNA interactions to target specific premRNAs and genomic sites. RAP-RNA is sensitive to lower abundance RNAs as well, making it generally applicable for investigating ncRNAs., Hertz Foundation, American Society for Engineering Education. National Defense Science and Engineering Graduate Fellowship, Broad Institute of MIT and Harvard

Rabbits softly swept to domestication When people domesticate animals, they select for tameness and tolerance of humans. What else do they look for? To identify the selective pressures that led to rabbit domestication, Carneiro et al. sequenced a domestic rabbit genome and compared it to that of its wild brethren (see the Perspective by Lohmueller). Domestication did not involve a single gene changing, but rather many gene alleles changing in frequency between tame and domestic rabbits, known as a soft selective sweep. Many of these alleles have changes that may affect brain development, supporting the idea that tameness involves changes at multiple loci. Science , this issue p. 1074 ; see also p. 1000

N6-methyladenosine (m6A) is a common modification of mRNA with potential roles in fine-tuning the RNA life cycle. Here, we identify a dense network of proteins interacting with METTL3, a component of the methyltransferase complex, and show that three of them (WTAP, METTL14, and KIAA1429) are required for methylation. Monitoring m6A levels upon WTAP depletion allowed the definition of accurate and near single-nucleotide resolution methylation maps and their classification into WTAP-dependent and -independent sites. WTAP-dependent sites are located at internal positions in transcripts, topologically static across a variety of systems we surveyed, and inversely correlated with mRNA stability, consistent with a role in establishing “basal” degradation rates. WTAP-independent sites form at the first transcribed base as part of the cap structure and are present at thousands of sites, forming a previously unappreciated layer of transcriptome complexity. Our data shed light on the proteomic and transcriptional underpinnings of this RNA modification., National Human Genome Research Institute (U.S.). Centers of Excellence in Genomic Science (P50 HG006193), National Human Genome Research Institute (U.S.) (U54 HG003067), National Institutes of Health (U.S.) (Pioneer Award)

Ribosomal protein haploinsufficiency occurs in diverse human diseases including Diamond-Blackfan anemia (DBA)[superscript 1, 2], congenital asplenia[superscript 3] and T cell leukemia[superscript 4]. Yet, how mutations in genes encoding ubiquitously expressed proteins such as these result in cell-type– and tissue-specific defects remains unknown[superscript 5]. Here, we identify mutations in GATA1, encoding the critical hematopoietic transcription factor GATA-binding protein-1, that reduce levels of full-length GATA1 protein and cause DBA in rare instances. We show that ribosomal protein haploinsufficiency, the more common cause of DBA, can lead to decreased GATA1 mRNA translation, possibly resulting from a higher threshold for initiation of translation of this mRNA in comparison with other mRNAs. In primary hematopoietic cells from patients with mutations in RPS19, encoding ribosomal protein S19, the amplitude of a transcriptional signature of GATA1 target genes was globally and specifically reduced, indicating that the activity, but not the mRNA level, of GATA1 is decreased in patients with DBA associated with mutations affecting ribosomal proteins. Moreover, the defective hematopoiesis observed in patients with DBA associated with ribosomal protein haploinsufficiency could be partially overcome by increasing GATA1 protein levels. Our results provide a paradigm by which selective defects in translation due to mutations affecting ubiquitous ribosomal proteins can result in human disease., National Institutes of Health (U.S.) (Grant P01 HL32262), National Institutes of Health (U.S.) (Grant U54 HG003067-09)

Recent advances in genome engineering technologies based on the CRISPR-associated RNA-guided endonuclease Cas9 are enabling the systematic interrogation of mammalian genome function. Analogous to the search function in modern word processors, Cas9 can be guided to specific locations within complex genomes by a short RNA search string. Using this system, DNA sequences within the endogenous genome and their functional outputs are now easily edited or modulated in virtually any organism of choice. Cas9-mediated genetic perturbation is simple and scalable, empowering researchers to elucidate the functional organization of the genome at the systems level and establish causal linkages between genetic variations and biological phenotypes. In this Review, we describe the development and applications of Cas9 for a variety of research or translational applications while highlighting challenges as well as future directions. Derived from a remarkable microbial defense system, Cas9 is driving innovative applications from basic biology to biotechnology and medicine.

Translating whole-exome sequencing (WES) for prospective clinical use may have an impact on the care of patients with cancer; however, multiple innovations are necessary for clinical implementation. These include rapid and robust WES of DNA derived from formalin-fixed, paraffin-embedded tumor tissue, analytical output similar to data from frozen samples and clinical interpretation of WES data for prospective use. Here, we describe a prospective clinical WES platform for archival formalin-fixed, paraffin-embedded tumor samples. The platform employs computational methods for effective clinical analysis and interpretation of WES data. When applied retrospectively to 511 exomes, the interpretative framework revealed a 'long tail' of somatic alterations in clinically important genes. Prospective application of this approach identified clinically relevant alterations in 15 out of 16 patients. In one patient, previously undetected findings guided clinical trial enrollment, leading to an objective clinical response. Overall, this methodology may inform the widespread implementation of precision cancer medicine.

RNA, including long noncoding RNA (lncRNA), is known to be an abundant and important structural component of the nuclear matrix. However, the molecular identities, functional roles and localization dynamics of lncRNAs that influence nuclear architecture remain poorly understood. Here, we describe one lncRNA, Firre, that interacts with the nuclear-matrix factor hnRNPU through a 156-bp repeating sequence and localizes across an ~5-Mb domain on the X chromosome. We further observed Firre localization across five distinct trans-chromosomal loci, which reside in spatial proximity to the Firre genomic locus on the X chromosome. Both genetic deletion of the Firre locus and knockdown of hnRNPU resulted in loss of colocalization of these trans-chromosomal interacting loci. Thus, our data suggest a model in which lncRNAs such as Firre can interface with and modulate nuclear architecture across chromosomes.

Although a few cancer genes are mutated in a high proportion of tumours of a given type (.20%), most are mutated at intermediate frequencies (2–20%). To explore the feasibility of creating a comprehensive catalogue of cancer genes, we analysed somatic point mutations in exome sequences from 4,742 human cancers and their matched normal-tissue samples across 21 cancer types. We found that large-scale genomic analysis can identify nearly all known cancer genes in these tumour types. Our analysis also identified 33 genes that were not previously known to be significantly mutated in cancer, including genes related to proliferation, apoptosis, genome stability, chromatin regulation, immune evasion, RNA processing and protein homeostasis. Down-sampling analysis indicates that larger sample sizes will reveal many more genes mutated at clinically important frequencies. We estimate that near-saturation may be achieved with 600– 5,000 samples per tumour type, depending on background mutation frequency. The results may help to guide the next stage of cancer genomics. Comprehensive knowledge of the genes underlying human cancers is a critical foundation for cancer diagnostics, therapeutics, clinical-trial design and selection of rational combination therapies. It is now possible to use genomic analysis to identify cancer genes in an unbiased fashion, based on the presence of somatic mutations at a rate significantly higher than the expected background level. Systematic studies have revealed many new cancer genes, as well as new classes of cancer genes 1,2 . They have also made clear that, although some cancer genes are mutated at high frequencies, most cancer genes in most patients occur at intermediate frequencies (2–20%) or lower. Accordingly, a complete catalogue of mutations in this frequency class will be essential for recognizing dysregulated pathways and optimal targets for therapeutic intervention. However, recent work suggests major gaps in our knowledge of cancer genes of intermediate frequency. For example, a study of 183 lung adenocarcinomas 3 found that 15% of patients lacked even a single mutation affecting any of the 10 known hallmarks of cancer, and 38% had 3 or fewer such mutations. In this paper, we analysed somatic point mutations (substitutions and small insertion and deletions) in nearly 5,000 human cancers and their matched normal-tissue samples (‘tumour–normal pairs’) across 21 tumour types. The questions that we examine here are: first, whether large-scale genomic analysis across tumour types can reliably identify all known cancer genes; second, whether it will reveal many new candidate cancer genes; and third, how far we are from having a complete catalogue of cancer genes (at least those of intermediate frequency). We used rigorous statistical methods to enumerate candidate cancer genes and then carefully inspected each gene to identify those with strong biological connections to cancer and mutational patterns consistent with the expected function. The analysis reveals nearly all known cancer genes and revealed 33 novel candidates, including genes related to proliferation, apoptosis, genome stability, chromatin regulation, immune evasion, RNA processing and protein homeostasis. Importantly, the data show that the

Background Familial combined hypolipidemia, a Mendelian condition characterized by substantial reductions in all 3 major lipid fractions, is caused by mutations that inactivate the gene angiopoietin-like 3 (ANGPTL3). Whether ANGPTL3 deficiency reduces risk of coronary artery disease (CAD) is unknown. Objectives The study goal was to leverage 3 distinct lines of evidence—a family that included individuals with complete (compound heterozygote) ANGPTL3 deficiency, a population based-study of humans with partial (heterozygote) ANGPTL3 deficiency, and biomarker levels in patients with myocardial infarction (MI)—to test whether ANGPTL3 deficiency is associated with lower risk for CAD. Methods We assessed coronary atherosclerotic burden in 3 individuals with complete ANGPTL3 deficiency and 3 wild-type first-degree relatives using computed tomography angiography. In the population, ANGPTL3 loss-of-function (LOF) mutations were ascertained in up to 21,980 people with CAD and 158,200 control subjects. LOF mutations were defined as nonsense, frameshift, and splice-site variants, along with missense variants resulting in

Genetic screens help infer gene function in mammalian cells, but it has remained difficult to assay complex phenotypes—such as transcriptional profiles—at scale. Here, we develop Perturb-seq, combining single-cell RNA sequencing (RNA-seq) and clustered regularly interspaced short palindromic repeats (CRISPR)-based perturbations to perform many such assays in a pool. We demonstrate Perturb-seq by analyzing 200,000 cells in immune cells and cell lines, focusing on transcription factors regulating the response of dendritic cells to lipopolysaccharide (LPS). Perturb-seq accurately identifies individual gene targets, gene signatures, and cell states affected by individual perturbations and their genetic interactions. We posit new functions for regulators of differentiation, the anti-viral response, and mitochondrial function during immune activation. By decomposing many high content measurements into the effects of perturbations, their interactions, and diverse cell metadata, Perturb-seq dramatically increases the scope of pooled genomic assays. Keywords: single-cell RNA-seq; pooled screen; CRISPR; epistasis; genetic interactions

Genetic imputation is a cost-efficient way to improve the power and resolution of genome-wide association (GWA) studies. Current publicly accessible imputation reference panels accurately predict genotypes for common variants with minor allele frequency (MAF)≥5% and low-frequency variants (0.5≤MAF

Tiling screens using CRISPR-Cas technologies provide a powerful approach to map regulatory elements to phenotypes of interest, but computational methods that effectively model these experimental approaches for different CRISPR technologies are not readily available. Here we present CRISPR-SURF, a deconvolution framework to identify functional regulatory regions in the genome from data generated by CRISPR-Cas nuclease, CRISPR interference (CRISPRi), or CRISPR activation (CRISPRa) tiling screens. We validated CRISPR-SURF on previously published and new data, identifying both experimentally validated and new potential regulatory elements. With CRISPR tiling screens now being increasingly used to elucidate the regulatory architecture of the non-coding genome, CRISPRSURF provides a generalizable and accessible solution for the discovery of regulatory elements.

Objective: Angiosarcoma (AS) is a rare soft tissue sarcoma, with an incidence of 300 cases/yr and a 5-year DSS of 30%. The low incidence has impeded large-scale research efforts. To address this, we launched a patient-partnered genomics study which seeks to empower patients to accelerate research by remotely sharing their samples and clinical information. Methods: We developed a website (ASCproject.org) to allow remote acquisition of medical records (MR), saliva, blood, and archival tissue from patients in the US and Canada. Whole-exome sequencing (WES) of ~20,000 genes is performed on tumor and matched germline DNA. Transcriptome analysis is performed on tumor RNA. Ultra-low pass whole-genome sequencing (ULP-WGS) and in some cases WES is performed on cell free DNA (cfDNA) obtained from blood samples. Clinical data including information about demographics, diagnosis, treatments, and responses are obtained via patient-reported data (PRD) and through MR abstraction. The resulting clinically annotated genomic database is shared widely to identify genomic drivers and mechanisms of response and resistance to therapies. Results: Since launch on March 13 2017, 321 patients with AS have registered. The average age of patients is 56 yrs (range 22-89). Primary locations of AS were primary breast (24%), breast with prior radiation (20%), head/face/neck/scalp (HFNS) (21%), bone/limb (9%), abdominal (3%), heart (3%), lung (1%), liver (1%), lymph (0.5%), multiple locations (11%), and other locations (5%). 142 (48%) reported being disease free at the time of enrollment. To date, 153 saliva kits, 167 MRs, 43 blood samples, and 97 tissue samples have been obtained. WES analysis is complete for 14 samples.ULP-WGS is complete for 10 cfDNA samples, and WES on 4 cfDNA samples. Transcriptome sequencing is complete for 9 tumor samples. We identified several previously described genes known to be altered in AS, including recurrent alterations in KDR and TP53. Tumor mutational burden (TMB) and mutational signature activities were quantified for each tumor sample. All three of the AS from the HFNS in the initial cohort exhibited a high TMB (>150 mutations) and dominant UV light signature (COSMIC Signature 7). Based on this, we hypothesized that HFNS AS might respond well to immune checkpoint inhibitors. We identified through PRD 56 patients with HFNS AS who reported what medications they received. Of these, 2 reported receiving immune checkpoint inhibitors for the treatment of metastatic disease. Both patients had refractory metastatic HFNS AS and reported receiving off-label anti-PD1 therapy. Both had complete or near-complete responses following immunotherapy, and currently report having no evidence of disease. Clinical responses were confirmed through review of MRs. Sequencing is currently being performed on tumor samples from both patients. Conclusion: A patient-partnered approach enabled rapid identification and enrollment of over 300 patients with AS, an exceedingly rare cancer, in 15 months. We were able to obtain tumor, blood, saliva samples to perform genomic analyses, which were then merged with detailed clinical information. PRD, clinical, and genomic data generated from the first 12 patients and 14 samples have been released on cbioportal.org. Additional data will be released in six-month intervals. Initial results show high TMB and a UV signature in 3 out of 3 patients with HFNS AS. In addition, we identified 2 patients with HFNS AS who had extraordinary responses to immunotherapy. These findings suggest a common genomic basis for HFNS AS and could provide rationale for clinical interventions using checkpoint inhibitors for these AS. Analyses of additional samples are under way to further characterize mutational signatures in HFNS AS and implications for patient care. This study serves as proof of principle that patient-partnered genomics efforts can democratize cancer research for exceedingly rare cancers. Citation Format: Corrie Painter, Esha Jain, Michael Dunphy, Elana Anastasio, Mary McGillicuddy, Rachel Stoddard, Beena Thomas, Sara Balch, Kristin Anderka, Katie Larkin, Niall Lennon, Yen-Lin Chen, Andrew Zimmer, Esme O. Baker, Simone Maiwald, Jen Lapan, Jason L. Hornick, Chandrajit Raut, George Demetri, Eric S. Lander, Todd Golub. High mutation burden and response to immune checkpoint inhibitors in angiosarcomas of the scalp and face [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr B085.

SummaryWe describe the landscape of somatic genomic alterations based on multidimensional and comprehensive characterization of more than 500 glioblastoma tumors (GBMs). We identify several novel mutated genes as well as complex rearrangements of signature receptors, including EGFR and PDGFRA. TERT promoter mutations are shown to correlate with elevated mRNA expression, supporting a role in telomerase reactivation. Correlative analyses confirm that the survival advantage of the proneural subtype is conferred by the G-CIMP phenotype, and MGMT DNA methylation may be a predictive biomarker for treatment response only in classical subtype GBM. Integrative analysis of genomic and proteomic profiles challenges the notion of therapeutic inhibition of a pathway as an alternative to inhibition of the target itself. These data will facilitate the discovery of therapeutic and diagnostic target candidates, the validation of research and clinical observations and the generation of unanticipated hypotheses that can advance our molecular understanding of this lethal cancer.

M. tuberculosis is evolving antibiotic resistance, threatening attempts at tuberculosis epidemic control. Mechanisms of resistance, including genetic changes favored by selection in resistant isolates, are incompletely understood. Using 116 newly sequenced and 7 previously sequenced M. tuberculosis whole genomes, we identified genome-wide signatures of positive selection specific to the 47 drug-resistant strains. By searching for convergent evolution--the independent fixation of mutations in the same nucleotide position or gene--we recovered 100% of a set of known resistance markers. We also found evidence of positive selection in an additional 39 genomic regions in resistant isolates. These regions encode components in cell wall biosynthesis, transcriptional regulation and DNA repair pathways. Mutations in these regions could directly confer resistance or compensate for fitness costs associated with resistance. Functional genetic analysis of mutations in one gene, ponA1, demonstrated an in vitro growth advantage in the presence of the drug rifampicin.

Despite years of preclinical efforts and hundreds of clinical studies, therapeutic cancer vaccines with the routine ability to limit or eliminate tumor growth in humans have been elusive. With advances in genome sequencing, it is now possible to identify a new class of tumor-specific antigens derived from mutated proteins that are present only in the tumor. These “neoantigens” should provide highly specific targets for antitumor immunity. Although many challenges remain in producing and testing neoantigen-based vaccines customized for each patient, a neoantigen vaccine offers a promising new approach to induce highly focused antitumor T cells aimed at eradicating cancer cells. Cancer Immunol Res; 1(1); 11–15. ©2013 AACR.

Major international projects are underway that are aimed at creating a comprehensive catalogue of all the genes responsible for the initiation and progression of cancer. These studies involve the sequencing of matched tumour-normal samples followed by mathematical analysis to identify those genes in which mutations occur more frequently than expected by random chance. Here we describe a fundamental problem with cancer genome studies: as the sample size increases, the list of putatively significant genes produced by current analytical methods burgeons into the hundreds. The list includes many implausible genes (such as those encoding olfactory receptors and the muscle protein titin), suggesting extensive false-positive findings that overshadow true driver events. We show that this problem stems largely from mutational heterogeneity and provide a novel analytical methodology, MutSigCV, for resolving the problem. We apply MutSigCV to exome sequences from 3,083 tumour-normal pairs and discover extraordinary variation in mutation frequency and spectrum within cancer types, which sheds light on mutational processes and disease aetiology, and in mutation frequency across the genome, which is strongly correlated with DNA replication timing and also with transcriptional activity. By incorporating mutational heterogeneity into the analyses, MutSigCV is able to eliminate most of the apparent artefactual findings and enable the identification of genes truly associated with cancer.

Genetic changes underlying clear cell renal cell carcinoma (ccRCC) include alterations in genes controlling cellular oxygen sensing (for example, VHL) and the maintenance of chromatin states (for example, PBRM1). We surveyed more than 400 tumours using different genomic platforms and identified 19 significantly mutated genes. The PI(3)K/AKT pathway was recurrently mutated, suggesting this pathway as a potential therapeutic target. Widespread DNA hypomethylation was associated with mutation of the H3K36 methyltransferase SETD2, and integrative analysis suggested that mutations involving the SWI/SNF chromatin remodelling complex (PBRM1, ARID1A, SMARCA4) could have far-reaching effects on other pathways. Aggressive cancers demonstrated evidence of a metabolic shift, involving downregulation of genes involved in the TCA cycle, decreased AMPK and PTEN protein levels, upregulation of the pentose phosphate pathway and the glutamine transporter genes, increased acetyl-CoA carboxylase protein, and altered promoter methylation of miR-21 (also known as MIR21) and GRB10. Remodelling cellular metabolism thus constitutes a recurrent pattern in ccRCC that correlates with tumour stage and severity and offers new views on the opportunities for disease treatment.

We performed an integrated genomic, transcriptomic and proteomic characterization of 373 endometrial carcinomas using array- and sequencing-based technologies. Uterine serous tumours and ~25% of high-grade endometrioid tumours had extensive copy number alterations, few DNA methylation changes, low oestrogen receptor/progesterone receptor levels, and frequent TP53 mutations. Most endometrioid tumours had few copy number alterations or TP53 mutations, but frequent mutations in PTEN, CTNNB1, PIK3CA, ARID1A and KRAS and novel mutations in the SWI/SNF chromatin remodelling complex gene ARID5B. A subset of endometrioid tumours that we identified had a markedly increased transversion mutation frequency and newly identified hotspot mutations in POLE. Our results classified endometrial cancers into four categories: POLE ultramutated, microsatellite instability hypermutated, copy-number low, and copy-number high. Uterine serous carcinomas share genomic features with ovarian serous and basal-like breast carcinomas. We demonstrated that the genomic features of endometrial carcinomas permit a reclassification that may affect post-surgical adjuvant treatment for women with aggressive tumours., National Institutes of Health (U.S.) (Grant 5U24CA143799-04), National Institutes of Health (U.S.) (Grant 5U24CA143835-04), National Institutes of Health (U.S.) (Grant 5U24CA143840-04), National Institutes of Health (U.S.) (Grant 5U24CA143843-04), National Institutes of Health (U.S.) (Grant 5U24CA143845-04), National Institutes of Health (U.S.) (Grant 5U24CA143848-04), National Institutes of Health (U.S.) (Grant 5U24CA143858-04), National Institutes of Health (U.S.) (Grant 5U24CA143866-04), National Institutes of Health (U.S.) (Grant 5U24CA143867-04), National Institutes of Health (U.S.) (Grant 5U24CA143882-04), National Institutes of Health (U.S.) (Grant 5U24CA143883-04), National Institutes of Health (U.S.) (Grant 5U24CA144025-04), National Institutes of Health (U.S.) (Grant U54HG003067-11), National Institutes of Health (U.S.) (Grant U54HG003079-10), National Institutes of Health (U.S.) (Grant U54HG003273-10)

The incidence of esophageal adenocarcinoma (EAC) has risen 600% over the last 30 years. With a 5-year survival rate of ~15%, the identification of new therapeutic targets for EAC is greatly important. We analyze the mutation spectra from whole-exome sequencing of 149 EAC tumor-normal pairs, 15 of which have also been subjected to whole-genome sequencing. We identify a mutational signature defined by a high prevalence of A>C transversions at AA dinucleotides. Statistical analysis of exome data identified 26 significantly mutated genes. Of these genes, five (TP53, CDKN2A, SMAD4, ARID1A and PIK3CA) have previously been implicated in EAC. The new significantly mutated genes include chromatin-modifying factors and candidate contributors SPG20, TLR4, ELMO1 and DOCK2. Functional analyses of EAC-derived mutations in ELMO1 identifies increased cellular invasion. Therefore, we suggest the potential activation of the RAC1 pathway as a contributor to EAC tumorigenesis., National Human Genome Research Institute (U.S.) (Large Scale Sequencing Program Grant U54 HG003067)

A high-throughput screen (HTS) was conducted against stably propagated cancer stem cell (CSC)-enriched populations using a library of 300,718 compounds from the National Institutes of Health (NIH) Molecular Libraries Small Molecule Repository (MLSMR). A cinnamide analog displayed greater than 20-fold selective inhibition of the breast CSC-like cell line (HMLE_sh_Ecad) over the isogenic control cell line (HMLE_sh_eGFP). Herein, we report structure-activity relationships of this class of cinnamides for selective lethality towards CSC-enriched populations.

Detection of somatic point substitutions is a key step in characterizing the cancer genome. Mutations in cancer are rare (0.1–100/Mb) and often occur only in a subset of the sequenced cells, either due to contamination by normal cells or due to tumor heterogeneity. Consequently, mutation calling methods need to be both specific, avoiding false positives, and sensitive to detect clonal and sub-clonal mutations. The decreased sensitivity of existing methods for low allelic fraction mutations highlights the pressing need for improved and systematically evaluated mutation detection methods. Here we present MuTect, a method based on a Bayesian classifier designed to detect somatic mutations with very low allele-fractions, requiring only a few supporting reads, followed by a set of carefully tuned filters that ensure high specificity. We also describe novel benchmarking approaches, which use real sequencing data to evaluate the sensitivity and specificity as a function of sequencing depth, base quality and allelic fraction. Compared with other methods, MuTect has higher sensitivity with similar specificity, especially for mutations with allelic fractions as low as 0.1 and below, making MuTect particularly useful for studying cancer subclones and their evolution in standard exome and genome sequencing data.

Although genetic lesions responsible for some mendelian disorders can be rapidly discovered through massively parallel sequencing of whole genomes or exomes, not all diseases readily yield to such efforts. We describe the illustrative case of the simple mendelian disorder medullary cystic kidney disease type 1 (MCKD1), mapped more than a decade ago to a 2-Mb region on chromosome 1. Ultimately, only by cloning, capillary sequencing and de novo assembly did we find that each of six families with MCKD1 harbors an equivalent but apparently independently arising mutation in sequence markedly under-represented in massively parallel sequencing data: the insertion of a single cytosine in one copy (but a different copy in each family) of the repeat unit comprising the extremely long (~1.5–5 kb), GC-rich (>80%) coding variable-number tandem repeat (VNTR) sequence in the MUC1 gene encoding mucin 1. These results provide a cautionary tale about the challenges in identifying the genes responsible for mendelian, let alone more complex, disorders through massively parallel sequencing., National Institutes of Health (U.S.) (Intramural Research Program), National Human Genome Research Institute (U.S.), Charles University (program UNCE 204011), Charles University (program PRVOUK-P24/LF1/3), Czech Republic. Ministry of Education, Youth, and Sports (grant NT13116-4/2012), Czech Republic. Ministry of Health (grant NT13116-4/2012), Czech Republic. Ministry of Health (grant LH12015), National Institutes of Health (U.S.) (Harvard Digestive Diseases Center, grant DK34854)

SummaryClonal evolution is a key feature of cancer progression and relapse. We studied intratumoral heterogeneity in 149 chronic lymphocytic leukemia (CLL) cases by integrating whole-exome sequence and copy number to measure the fraction of cancer cells harboring each somatic mutation. We identified driver mutations as predominantly clonal (e.g., MYD88, trisomy 12, and del(13q)) or subclonal (e.g., SF3B1 and TP53), corresponding to earlier and later events in CLL evolution. We sampled leukemia cells from 18 patients at two time points. Ten of twelve CLL cases treated with chemotherapy (but only one of six without treatment) underwent clonal evolution, predominantly involving subclones with driver mutations (e.g., SF3B1 and TP53) that expanded over time. Furthermore, presence of a subclonal driver mutation was an independent risk factor for rapid disease progression. Our study thus uncovers patterns of clonal evolution in CLL, providing insights into its stepwise transformation, and links the presence of subclones with adverse clinical outcomes.

Neuroblastoma is a malignancy of the developing sympathetic nervous system that often presents with widespread metastatic disease, resulting in survival rates of less than 50%. To determine the spectrum of somatic mutation in high-risk neuroblastoma, we studied 240 affected individuals (cases) using a combination of whole-exome, genome and transcriptome sequencing as part of the Therapeutically Applicable Research to Generate Effective Treatments (TARGET) initiative. Here we report a low median exonic mutation frequency of 0.60 per Mb (0.48 nonsilent) and notably few recurrently mutated genes in these tumors. Genes with significant somatic mutation frequencies included ALK (9.2% of cases), PTPN11 (2.9%), ATRX (2.5%, and an additional 7.1% had focal deletions), MYCN (1.7%, causing a recurrent p.Pro44Leu alteration) and NRAS (0.83%). Rare, potentially pathogenic germline variants were significantly enriched in ALK, CHEK2, PINK1 and BARD1. The relative paucity of recurrent somatic mutations in neuroblastoma challenges current therapeutic strategies that rely on frequently altered oncogenic drivers., National Human Genome Research Institute (U.S.) (Grant U54HG003067), National Cancer Institute (U.S.) (Contract HHSN261200800001E)

Mammalian genomes are pervasively transcribed(1,2) to produce thousands of long noncoding RNAs (lncRNAs)(3,4). A few of these lncRNAs have been shown to recruit regulatory complexes through RNA-protein interactions to influence the expression of nearby genes(5–7), and it has been suggested that many other lncRNAs similarly act as local regulators(8,9). Such local functions could explain the observation that lncRNA expression is often correlated with the expression of nearby genes(2,10,11). However, such correlations have been challenging to dissect(12) and could alternatively result from processes that are not mediated by the lncRNA transcripts themselves. For example, some gene promoters have been proposed to have dual functions as enhancers(13–16), and the process of transcription per se has been proposed to contribute to gene regulation by recruiting activating factors or remodeling nucleosomes(10,17,18). Here we used genetic manipulations to dissect 12 genomic loci that produce lncRNAs and found that 5 of these loci influence the expression of a neighboring gene in cis. Surprisingly, none of these effects required the specific lncRNA transcripts themselves and instead involved general processes associated with their production, including enhancer-like activity of gene promoters, the process of transcription, and the splicing of the transcript. Importantly, such effects were not limited to lncRNA loci: we found that 4 of 6 protein-coding loci similarly influenced the expression of a neighbor. These results demonstrate that ‘crosstalk’ among neighboring genes is a prevalent phenomenon that can involve multiple mechanisms and cis regulatory signals, including a novel role for RNA splice sites. These mechanisms may explain the function and evolution of some genomic loci that produce lncRNAs and broadly contribute to the regulation of both coding and noncoding genes.

The genetic dependencies of human cancers widely vary. Here, we catalog this heterogeneity and use it to identify functional gene interactions and genotype-dependent liabilities in cancer. By using genome-wide CRISPR-based screens, we generate a gene essentiality dataset across 14 human acute myeloid leukemia (AML) cell lines. Sets of genes with correlated patterns of essentiality across the lines reveal new gene relationships, the essential substrates of enzymes, and the molecular functions of uncharacterized proteins. Comparisons of differentially essential genes between Ras-dependent and -independent lines uncover synthetic lethal partners of oncogenic Ras. Screens in both human AML and engineered mouse pro-B cells converge on a surprisingly small number of genes in the Ras processing and MAPK pathways and pinpoint PREX1 as an AML-specific activator of MAPK signaling. Our findings suggest general strategies for defining mammalian gene networks and synthetic lethal interactions by exploiting the natural genetic and epigenetic diversity of human cancer cells. Keywords: CRISPR; AML; synthetic lethality; gene networks; RAS; genetic screens, National Institutes of Health (U.S.) (Grant CA103866), National Institutes of Health (U.S.) (Grant F31CA189437)

Type 2 diabetes (T2D) affects Latinos at twice the rate seen in populations of European descent. We recently identified a risk haplotype spanning SLC16A11 that explains ∼20% of the increased T2D prevalence in Mexico. Here, through genetic fine-mapping, we define a set of tightly linked variants likely to contain the causal allele(s). We show that variants on the T2D-associated haplotype have two distinct effects: (1) decreasing SLC16A11 expression in liver and (2) disrupting a key interaction with basigin, thereby reducing cell-surface localization. Both independent mechanisms reduce SLC16A11 function and suggest SLC16A11 is the causal gene at this locus. To gain insight into how SLC16A11 disruption impacts T2D risk, we demonstrate that SLC16A11 is a proton-coupled monocarboxylate transporter and that genetic perturbation of SLC16A11 induces changes in fatty acid and lipid metabolism that are associated with increased T2D risk. Our findings suggest that increasing SLC16A11 function could be therapeutically beneficial for T2D. Video Abstract [Figure presented] Keywords: type 2 diabetes (T2D); genetics; disease mechanism; SLC16A11; MCT11; solute carrier (SLC); monocarboxylates; fatty acid metabolism; lipid metabolism; precision medicine

CRISPR screens illuminate enhancer function The noncoding regions around a gene that control the transcription of the protein-coding region are difficult to identify. Leveraging a CRISPR interference system (CRISPRi), Fulco et al. identified enhancer-promoter connections to map specific noncoding regions affecting gene regulation for the GATA1 and MYC loci (see the Perspective by Einstein and Yeo). Going forward, such CRISPRi-mapping can be used to evaluate promoter-enhancer screens functionally in an unbiased way. Science , this issue p. 769 ; see also p. 705

Cytokines are classically thought to stimulate downstream signaling pathways through monotonic activation of receptors. We describe a severe anemia resulting from a homozygous mutation (R150Q) in the cytokine erythropoietin (EPO). Surprisingly, the EPO R150Q mutant shows only a mild reduction in affinity for its receptor but has altered binding kinetics. The EPO mutant is less effective at stimulating erythroid cell proliferation and differentiation, even at maximally potent concentrations. While the EPO mutant can stimulate effectors such as STAT5 to a similar extent as the wild-type ligand, there is reduced JAK2-mediated phosphorylation of select downstream targets. This impairment in downstream signaling mechanistically arises from altered receptor dimerization dynamics due to extracellular binding changes. These results demonstrate how variation in a single cytokine can lead to biased downstream signaling and can thereby cause human disease. Moreover, we have defined a distinct treatable form of anemia through mutation identification and functional studies.

INTRODUCTION Almost all archaea and about half of bacteria possess clustered regularly interspaced short palindromic repeat (CRISPR)–CRISPR-associated genes (Cas) adaptive immune systems, which protect microbes against viruses and other foreign DNA. All functionally characterized CRISPR systems have been reported to target DNA, with some multicomponent type III systems also targeting RNA. The putative class 2 type VI system, which has not been functionally characterized, encompasses the single-effector protein C2c2, which contains two Higher Eukaryotes and Prokaryotes Nucleotide-binding (HEPN) domains commonly associated with ribonucleases (RNases), suggesting RNA-guided RNA-targeting function. RATIONALE Existing studies have only established a role for RNA interference, in addition to DNA interference, in the multicomponent type III-A and III-B systems. We investigated the possibility of C2c2-mediated RNA inference by heterologously expressing C2c2 locus from Leptotrichia shahii (LshC2c2) in the model system Escherichia coli. The ability of LshC2c2 to protect against MS2 single-stranded RNA (ssRNA) phage infection was assessed by using every possible spacer sequence against the phage genome. We next developed protocols to reconstitute purified recombinant LshC2c2 protein and test its biochemical activity when incubated with its mature CRISPR RNA (crRNA) and target ssRNA. We systematically evaluated the parameters necessary for cleavage. Last, to demonstrate the potential utility of the LshC2c2 complex for RNA targeting in living bacterial cells, we guided it to knockdown red fluorescent protein (RFP) mRNA in vivo. RESULTS This work demonstrates the RNA-guided RNase activity of the putative type VI CRISPR-effector LshC2c2. Heterologously expressed C2c2 can protect E. coli from MS2 phage, and by screening against the MS2 genome, we identified a H (non-G) protospacer flanking site (PFS) following the RNA target site, which was confirmed by targeting a complementary sequence in the β-lactamase transcript followed by a degenerate nucleotide sequence. Using purified LshC2c2 protein, we demonstrate that C2c2 and crRNA are sufficient in vitro to achieve RNA-guided, PFS-dependent RNA cleavage. This cleavage preferentially occurs at uracil residues in ssRNA regions and depends on conserved catalytic residues in the two HEPN domains. Mutation of these residues yields a catalytically inactive RNA-binding protein. The secondary structure of the crRNA direct repeat (DR) stem is required for LshC2c2 activity, and mutations in the 3′ region of the DR eliminate cleavage activity. Targeting is also sensitive to multiple or consecutive mismatches in the spacer:protospacer duplex. C2c2 targeting of RFP mRNA in vivo results in reduced fluorescence. The knockdown of the RFP mRNA by C2c2 slowed E. coli growth, and in agreement with this finding, in vitro cleavage of the target RNA results in “collateral,” nonspecific cleavage of other RNAs present in the reaction mix. CONCLUSION LshC2c2 is a RNA-guided RNase which requires the activity of its two HEPN domains, suggesting previously unidentified mechanisms of RNA targeting and degradation by CRISPR systems. Promiscuous RNase activity of C2c2 after activation by the target slows bacterial growth and suggests that C2c2 could protect bacteria from virus spread via programmed cell death and dormancy induction. A single-effector RNA targeting system has the potential to serve as a general chassis for molecular tools for visualizing, degrading, or binding RNA in a programmable, multiplexed fashion. C2c2 is an RNA-guided RNase that provides protection against RNA phage. CRISPR-C2c2 from L. shahii can be reconstituted in E. coli to mediate RNA-guided interference of the RNA phage MS2. Biochemical characterization of C2c2 reveals crRNA-guided RNA cleavage facilitated by the two HEPN nuclease domains. Binding of the target RNA by C2c2-crRNA also activates a nonspecific RNase activity, which may lead to promiscuous cleavage of RNAs without complementarity to the crRNA guide sequence.

DNA microarrays can be used to identify gene expression changes characteristic of human disease. This is challenging, however, when relevant differences are subtle at the level of individual genes. We introduce an analytical strategy, Gene Set Enrichment Analysis, designed to detect modest but coordinate changes in the expression of groups of functionally related genes. Using this approach, we identify a set of genes involved in oxidative phosphorylation whose expression is coordinately decreased in human diabetic muscle. Expression of these genes is high at sites of insulin-mediated glucose disposal, activated by PGC-1alpha and correlated with total-body aerobic capacity. Our results associate this gene set with clinically important variation in human metabolism and illustrate the value of pathway relationships in the analysis of genomic profiling experiments.

The evolution of the amniotic egg was one of the great evolutionary innovations in the history of life, freeing vertebrates from an obligatory connection to water and thus permitting the conquest of terrestrial environments. Among amniotes, genome sequences are available for mammals and birds, but not for non-avian reptiles. Here we report the genome sequence of the North American green anole lizard, Anolis carolinensis. We find that A. carolinensis microchromosomes are highly syntenic with chicken microchromosomes, yet do not exhibit the high GC and low repeat content that are characteristic of avian microchromosomes. Also, A. carolinensis mobile elements are very young and diverse—more so than in any other sequenced amniote genome. The GC content of this lizard genome is also unusual in its homogeneity, unlike the regionally variable GC content found in mammals and birds. We describe and assign sequence to the previously unknown A. carolinensis X chromosome. Comparative gene analysis shows that amniote egg proteins have evolved significantly more rapidly than other proteins. An anole phylogeny resolves basal branches to illuminate the history of their repeated adaptive radiations., National Science Foundation (U.S.) (NSF grant DEB-0920892), National Science Foundation (U.S.) (NSF grant DEB-0844624), National Human Genome Research Institute (U.S.)

The goal of the International HapMap Project is to determine the common patterns of DNA sequence variation in the human genome and to make this information freely available in the public domain. An international consortium is developing a map of these patterns across the genome by determining the genotypes of one million or more sequence variants, their frequencies and the degree of association between them, in DNA samples from populations with ancestry from parts of Africa, Asia and Europe. The HapMap will allow the discovery of sequence variants that affect common disease, will facilitate development of diagnostic tools, and will enhance our ability to choose targets for therapeutic intervention. © 2003 Nature Publishing Group.

Coronary heart disease (CHD) is a complex disorder constituting a major health problem in Western societies. To assess the genetic background of CHD, we performed a genomewide linkage scan in two study samples from the genetically isolated population of Finland. An initial study sample consisted of family material from the northeastern part of Finland, settled by a small number of founders approximately 300 years ago. A second study sample originated from the southwestern region of Finland, settled approximately 2,000 years ago. Families were ascertained through probands exhibiting premature CHD, defined as >50% stenosis of at least two coronary arteries at a young age, as verified by coronary angiography. Both study samples and the pooled data set provided evidence for linkage in two chromosomal regions. A region on chromosome 2q21.1-22 yielded two-point LOD scores of 3.2, 1.9, and 3.7, in the affected sib-pair (ASP) analyses of the northeastern, southwestern, and pooled study samples. The corresponding multipoint maximum-likelihood scores (MLSs) for these three study samples were 2.4, 1.3, and 3.0. In addition, a region on chromosome Xq23-26 resulted in two-point LOD scores of 1.9, 3.5, and 2.9 and in multipoint MLSs of 3.4, 3.1, and 2.5, respectively. In conclusion, this study identifies two loci likely to contribute to premature CHD: one on chromosome 2q21.1-22 and another on chromosome Xq23-26.

During interphase, the inactive X chromosome (Xi) is largely transcriptionally silent and adopts an unusual 3D configuration known as the “Barr body.” Despite the importance of X chromosome inactivation, little is known about this 3D conformation. We recently showed that in humans the Xi chromosome exhibits three structural features, two of which are not shared by other chromosomes. First, like the chromosomes of many species, Xi forms compartments. Second, Xi is partitioned into two huge intervals, called “superdomains,” such that pairs of loci in the same superdomain tend to colocalize. The boundary between the superdomains lies near DXZ4, a macrosatellite repeat whose Xi allele extensively binds the protein CCCTC-binding factor. Third, Xi exhibits extremely large loops, up to 77 megabases long, called “superloops.” DXZ4 lies at the anchor of several superloops. Here, we combine 3D mapping, microscopy, and genome editing to study the structure of Xi, focusing on the role of DXZ4. We show that superloops and superdomains are conserved across eutherian mammals. By analyzing ligation events involving three or more loci, we demonstrate that DXZ4 and other superloop anchors tend to colocate simultaneously. Finally, we show that deleting DXZ4 on Xi leads to the disappearance of superdomains and superloops, changes in compartmentalization patterns, and changes in the distribution of chromatin marks. Thus, DXZ4 is essential for proper Xi packaging.

The CRISPR-Cas adaptive immune system defends microbes against foreign genetic elements via DNA or RNA-DNA interference. We characterize the Class 2 type VI-A CRISPR-Caseffector C2c2 and demonstrate its RNA-guided RNase function. C2c2 from the bacterium Leptotrichia shahii provides interference against RNA phage.In vitro biochemical analysis show that C2c2 is guided by a single crRNA and can be programmed to cleave ssRNA targets carrying complementary protospacers. In bacteria, C2c2 can be programmed to knock down specific mRNAs. Cleavage is mediated by catalytic residues in the two conserved HEPN domains, mutations in which generate catalytically inactive RNA-binding proteins. These results broaden our understanding of CRISPR-Cas systems and suggest that C2c2 can be used to develop new RNA-targeting tools.

Resistance to the Bruton’s tyrosine kinase (BTK) inhibitor ibrutinib has been attributed solely to mutations in BTK and related pathway molecules. Using whole-exome and deep-targeted sequencing, we dissect evolution of ibrutinib resistance in serial samples from five chronic lymphocytic leukaemia patients. In two patients, we detect BTK-C481S mutation or multiple PLCG2 mutations. The other three patients exhibit an expansion of clones harbouring del(8p) with additional driver mutations (EP300, MLL2 and EIF2A), with one patient developing trans-differentiation into CD19-negative histiocytic sarcoma. Using droplet-microfluidic technology and growth kinetic analyses, we demonstrate the presence of ibrutinib-resistant subclones and estimate subclone size before treatment initiation. Haploinsufficiency of TRAIL-R, a consequence of del(8p), results in TRAIL insensitivity, which may contribute to ibrutinib resistance. These findings demonstrate that the ibrutinib therapy favours selection and expansion of rare subclones already present before ibrutinib treatment, and provide insight into the heterogeneity of genetic changes associated with ibrutinib resistance., University of Texas M.D. Anderson Cancer Center (Support Grant CA016672), National Science Foundation (U.S.) (DMR-1310266), Harvard University. Materials Research Science and Engineering Center (DMR-1420570), National Natural Science Foundation (China) (81372496)

Hi-C experiments explore the 3D structure of the genome, generating terabases of data to create high-resolution contact maps. Here, we introduce Juicer, an open-source tool for analyzing terabase-scale Hi-C datasets. Juicer allows users without a computational background to transform raw sequence data into normalized contact maps with one click. Juicer produces a hic file containing compressed contact matrices at many resolutions, facilitating visualization and analysis at multiple scales. Structural features, such as loops and domains, are automatically annotated. Juicer is available as open source software at http://aidenlab.org/juicer/., National Institutes of Health (U.S.) (New Innovator Award 1DP2OD008540), National Science Foundation (U.S.) Physics Frontier Center (Grant PHY-1427654), National Human Genome Research Institute (U.S.) (Grant HG006193), Robert A. Welch Foundation (Grant Q-1866), Cancer Prevention and Research Institute of Texas (Scholar Award R1304), NVIDIA Corporation (Research Center Award), IBM (University Challenge Award), Google (Research Award), Baylor College of Medicine (McNair Medical Institute Scholar Award), National Human Genome Research Institute (U.S.) (Grant HG003067), National Institutes of Health (U.S.) (4D Nucleome Grant U01HL130010)

Mammalian genomes are pervasively transcribed to produce thousands of spliced long noncoding RNAs (lncRNAs), whose functions remain poorly understood. Because recent evidence has implicated several specific lncRNA loci in the local regulation of gene expression, we sought to determine whether such local regulation is a property of many lncRNA loci. We used genetic manipulations to dissect 12 genomic loci that produce lncRNAs and found that 5 of these loci influence the expression of a neighboring gene in cis. Surprisingly, however, none of these effects required the specific lncRNA transcripts themselves and instead involved general processes associated with their production, including enhancer-like activity of gene promoters, the process of transcription, and the splicing of the transcript. Interestingly, such effects are not limited to lncRNA loci: we found similar effects on local gene expression at 4 of 6 protein-coding loci. These results demonstrate that ‘crosstalk’ among neighboring genes is a prevalent phenomenon that can involve multiple mechanisms and cis regulatory signals, including a novel role for RNA splicing. These mechanisms may explain the function and evolution of some genomic loci that produce lncRNAs.

We recently used in situ Hi-C to create kilobase-resolution 3D maps of mammalian genomes. Here, we combine these maps with new Hi-C, microscopy, and genome-editing experiments to study the physical structure of chromatin fibers, domains, and loops. We find that the observed contact domains are inconsistent with the equilibrium state for an ordinary condensed polymer. Combining Hi-C data and novel mathematical theorems, we show that contact domains are also not consistent with a fractal globule. Instead, we use physical simulations to study two models of genome folding. In one, intermonomer attraction during polymer condensation leads to formation of an anisotropic "tension globule." In the other, CCCTC-binding factor (CTCF) and cohesin act together to extrude unknotted loops during interphase. Both models are consistent with the observed contact domains and with the observation that contact domains tend to form inside loops. However, the extrusion model explains a far wider array of observations, such as why loops tend not to overlap and why the CTCF-binding motifs at pairs of loop anchors lie in the convergent orientation. Finally, we perform 13 genome-editing experiments examining the effect of altering CTCF-binding sites on chromatin folding. The convergent rule correctly predicts the affected loops in every case. Moreover, the extrusion model accurately predicts in silico the 3D maps resulting from each experiment using only the location of CTCF-binding sites in the WT. Thus, we show that it is possible to disrupt, restore, and move loops and domains using targeted mutations as small as a single base pair.

The gut microbial community is dynamic during the first 3 years of life, before stabilizing to an adult-like state. However, little is known about the impact of environmental factors on the developing human gut microbiome. We report a longitudinal study of the gut microbiome based on DNA sequence analysis of monthly stool samples and clinical information from 39 children, about half of whom received multiple courses of antibiotics during the first 3 years of life. Whereas the gut microbiome of most children born by vaginal delivery was dominated by Bacteroides species, the four children born by cesarean section and about 20% of vaginally born children lacked Bacteroides in the first 6 to 18 months of life. Longitudinal sampling, coupled with whole-genome shotgun sequencing, allowed detection of strain-level variation as well as the abundance of antibiotic resistance genes. The microbiota of antibiotic-treated children was less diverse in terms of both bacterial species and strains, with some species often dominated by single strains. In addition, we observed short-term composition changes between consecutive samples from children treated with antibiotics. Antibiotic resistance genes carried on microbial chromosomes showed a peak in abundance after antibiotic treatment followed by a sharp decline, whereas some genes carried on mobile elements persisted longer after antibiotic therapy ended. Our results highlight the value of high-density longitudinal sampling studies with high-resolution strain profiling for studying the establishment and response to perturbation of the infant gut microbiome., National Human Genome Research Institute (U.S.) (grant 2U54HG003067-10), Juvenile Diabetes Research Foundation International, National Institutes of Health (U.S.) (grant U54 DK102557), National Institutes of Health (U.S.) (grant R01 DK092405), National Institutes of Health (U.S.) (grant P30 DK043351), Leona M. and Harry B. Helmsley Charitable Trust, Crohn's and Colitis Foundation of America

Whole-genome sequencing using massively parallel sequencing technologies enables accurate detection of somatic rearrangements in cancer. Pinpointing large numbers of rearrangement breakpoints to base-pair resolution allows analysis of rearrangement microhomology and genomic location for every sample. Here we analyze 95 tumor genome sequences from breast, head and neck, colorectal, and prostate carcinomas, and from melanoma, multiple myeloma, and chronic lymphocytic leukemia. We discover three genomic factors that are significantly correlated with the distribution of rearrangements: replication time, transcription rate, and GC content. The correlation is complex, and different patterns are observed between tumor types, within tumor types, and even between different types of rearrangements. Mutations in the APC gene correlate with and, hence, potentially contribute to DNA breakage in late-replicating, low %GC, untranscribed regions of the genome. We show that somatic rearrangements display less microhomology than germline rearrangements, and that breakpoint loci are correlated with local hypermutability with a particular enrichment for transversions.

Genome-wide association studies (GWASs) have identified a genetic variant of moderate effect size at 6p21.1 associated with erythrocyte traits in humans. We show that this variant affects an erythroid-specific enhancer of CCND3. A Ccnd3 knockout mouse phenocopies these erythroid phenotypes, with a dramatic increase in erythrocyte size and a concomitant decrease in erythrocyte number. By examining human and mouse primary erythroid cells, we demonstrate that the CCND3 gene product cyclin D3 regulates the number of cell divisions that erythroid precursors undergo during terminal differentiation, thereby controlling erythrocyte size and number. We illustrate how cell type-specific specialization can occur for general cell cycle components—a finding resulting from the biological follow-up of unbiased human genetic studies., National Institutes of Health (U.S.) (Grant P01 HL32262), Amgen Inc.

Summary Despite recent insights into melanoma genetics, systematic surveys for driver mutations are challenged by an abundance of passenger mutations caused by carcinogenic UV light exposure. We developed a permutation-based framework to address this challenge, employing mutation data from intronic sequences to control for passenger mutational load on a per gene basis. Analysis of large-scale melanoma exome data by this approach discovered six novel melanoma genes ( PPP6C , RAC1 , SNX31 , TACC1 , STK19 , and ARID2 ), three of which— RAC1 , PPP6C , and STK19 —harbored recurrent and potentially targetable mutations. Integration with chromosomal copy number data contextualized the landscape of driver mutations, providing oncogenic insights in BRAF- and NRAS-driven melanoma as well as those without known NRAS / BRAF mutations. The landscape also clarified a mutational basis for RB and p53 pathway deregulation in this malignancy. Finally, the spectrum of driver mutations provided unequivocal genomic evidence for a direct mutagenic role of UV light in melanoma pathogenesis.

Breast carcinoma is the leading cause of cancer-related mortality in women worldwide with an estimated 1.38 million new cases and 458,000 deaths in 2008 alone1. This malignancy represents a heterogeneous group of tumours with characteristic molecular features, prognosis, and responses to available therapy2–4. Recurrent somatic alterations in breast cancer have been described including mutations and copy number alterations, notably ERBB2 amplifications, the first successful therapy target defined by a genomic aberration5. Prior DNA sequencing studies of breast cancer genomes have revealed additional candidate mutations and gene rearrangements 6–10. Here we report the whole-exome sequences of DNA from 103 human breast cancers of diverse subtypes from patients in Mexico and Vietnam compared to matched-normal DNA, together with whole-genome sequences of 22 breast cancer/normal pairs. Beyond confirming recurrent somatic mutations in PIK3CA11, TP536, AKT112, GATA313, and MAP3K110, we discovered recurrent mutations in the CBFB transcription factor gene and deletions of its partner RUNX1. Furthermore, we have identified a recurrent MAGI3-AKT3 fusion enriched in triple-negative breast cancer lacking estrogen and progesterone receptors and ERBB2 expression. The Magi3-Akt3 fusion leads to constitutive activation of Akt kinase, which is abolished by treatment with an ATP-competitive Akt small-molecule inhibitor.

Cataloged from PDF version of article. Generation of induced pluripotent stem cells (iPSCs) by somatic cell reprogramming involves global epigenetic remodelling. Whereas several proteins are known to regulate chromatin marks associated with the distinct epigenetic states of cells before and after reprogramming, the role of specific chromatin-modifying enzymes in reprogramming remains to be determined. To address how chromatin-modifying proteins influence reprogramming, we used short hairpin RNAs (shRNAs) to target genes in DNA and histone methylation pathways, and identified positive and negative modulators of iPSC generation. Whereas inhibition of the core components of the polycomb repressive complex 1 and 2, including the histone 3 lysine 27 methyltransferase EZH2, reduced reprogramming efficiency, suppression of SUV39H1, YY1 and DOT1L enhanced reprogramming. Specifically, inhibition of the H3K79 histone methyltransferase DOT1L by shRNA or a small molecule accelerated reprogramming, significantly increased the yield of iPSC colonies, and substituted for KLF4 and c-Myc (also known as MYC). Inhibition of DOT1L early in the reprogramming process is associated with a marked increase in two alternative factors, NANOG and LIN28, which play essential functional roles in the enhancement of reprogramming. Genome-wide analysis of H3K79me2 distribution revealed that fibroblast-specific genes associated with the epithelial to mesenchymal transition lose H3K79me2 in the initial phases of reprogramming. DOT1L inhibition facilitates the loss of this mark from genes that are fated to be repressed in the pluripotent state. These findings implicate specific chromatin-modifying enzymes as barriers to or facilitators of reprogramming, and demonstrate how modulation of chromatin-modifying enzymes can be exploited to more efficiently generate iPSCs with fewer exogenous transcription factors. © 2012 Macmillan Publishers Limited. All rights reserved.