Search

FLT3 fusions are associated with myeloid and lymphoid neoplasms with eosinophilia. We describe a patient presenting with clinicopathologic features of both chronic eosinophilic leukemia, not otherwise specified (CEL, NOS) and systemic mastocytosis (SM). The bone marrow demonstrated a myeloproliferative neoplasm with eosinophilia and aggregates of atypical mast cells. Cytogenetic analysis revealed a t(13;14)(q12;q32), which was subsequently molecularly characterized as a novel TRIP11-FLT3 rearrangement. A KIT D816V mutation was also identified. The patient rapidly transformed to T-lymphoblastic leukemia/lymphoma and expired shortly after diagnosis. This is the fifth FLT3 fusion gene described in the literature; the presence of both myeloid and lymphoid neoplasms implicates involvement of an early hematopoietic progenitor by rearranged FLT3. We suggest that leukemias and lymphomas with FLT3 fusion genes exhibit similar clinicopathologic features to, and should be included in, the WHO category of "Myeloid and lymphoid neoplasms with eosinophilia and abnormalities of PDGFRA, PDGFRB, or FGFR1, or with PCM1-JAK2."

The human and mouse antibody repertoires are formed by identical processes, but like all small animals, mice only have sufficient lymphocytes to express a small part of the potential antibody repertoire. In this study, we determined how the heavy chain repertoires of two mouse strains are generated. Analysis of IgM- and IgG-associated VDJ rearrangements generated by high-throughput sequencing confirmed the presence of 99 functional immunoglobulin heavy chain variable (IGHV) genes in the C57BL/6 genome, and inferred the presence of 164 IGHV genes in the BALB/c genome. Remarkably, only five IGHV sequences were common to both strains. Compared with humans, little N nucleotide addition was seen in the junctions of mouse VDJ genes. Germline human IgG-associated IGHV genes are rare, but many murine IgG-associated IGHV genes were unmutated. Together these results suggest that the expressed mouse repertoire is more germline-focused than the human repertoire. The apparently divergent germline repertoires of the mouse strains are discussed with reference to reports that inbred mouse strains carry blocks of genes derived from each of the three subspecies of the house mouse. We hypothesize that the germline genes of BALB/c and C57BL/6 mice may originally have evolved to generate distinct germline-focused antibody repertoires in the different mouse subspecies.

High-throughput DNA sequencing techniques have greatly accelerated the pace of research into the repertoires of antibody and T cell receptor gene rearrangements that confer antigen specificity to adaptive immune responses. Studies of aging-related changes in human B cell repertoires have benefited from the ability to detect and quantify thousands to millions of B cell clones in human samples, and study the mutational lineages and isotype switching relationships within each clonal lineage. Correlation of repertoire analysis with antibody gene data from antigen-specific B cells is poised to give much greater insight into clinically relevant B cell responses and memory storage. Here, we describe strategies for preparing and analyzing human antibody gene libraries for studying B cell repertoires.

We define a “threaded blockset,” which is a novel generalization of the classic notion of a multiple alignment. A new computer program called TBA (for “threaded blockset aligner”) builds a threaded blockset under the assumption that all matching segments occur in the same order and orientation in the given sequences; inversions and duplications are not addressed. TBA is designed to be appropriate for aligning many, but by no means all, megabase-sized regions of multiple mammalian genomes. The output of TBA can be projected onto any genome chosen as a reference, thus guaranteeing that different projections present consistent predictions of which genomic positions are orthologous. This capability is illustrated using a new visualization tool to view TBA-generated alignments of vertebrate Hox clusters from both the mammalian and fish perspectives. Experimental evaluation of alignment quality, using a program that simulates evolutionary change in genomic sequences, indicates that TBA is more accurate than earlier programs. To perform the dynamic-programming alignment step, TBA runs a stand-alone program called MULTIZ, which can be used to align highly rearranged or incompletely sequenced genomes. We describe our use of MULTIZ to produce the whole-genome multiple alignments at the Santa Cruz Genome Browser.

The University of California Santa Cruz (UCSC) Table Browser (http://genome.ucsc.edu/cgi-bin/hgText) provides text-based access to a large collection of genome assemblies and annotation data stored in the Genome Browser Database. A flexible alternative to the graphical-based Genome Browser, this tool offers an enhanced level of query support that includes restrictions based on field values, free-form SQL queries and combined queries on multiple tables. Output can be filtered to restrict the fields and lines returned, and may be organized into one of several formats, including a simple tab- delimited file that can be loaded into a spreadsheet or database as well as advanced formats that may be uploaded into the Genome Browser as custom annotation tracks. The Table Browser User's Guide located on the UCSC website provides instructions and detailed examples for constructing queries and configuring output.

The University of California Santa Cruz (UCSC) Genome Browser Database is an up to date source for genome sequence data integrated with a large collection of related annotations. The database is optimized to support fast interactive performance with the web-based UCSC Genome Browser, a tool built on top of the database for rapid visualization and querying of the data at many levels. The annotations for a given genome are displayed in the browser as a series of tracks aligned with the genomic sequence. Sequence data and annotations may also be viewed in a text-based tabular format or downloaded as tab-delimited flat files. The Genome Browser Database, browsing tools and downloadable data files can all be found on the UCSC Genome Bioinformatics website (http://genome.ucsc.edu), which also contains links to documentation and related technical information.

In order to identify novel somatic mutations associated with classic BCR/ABL1-negative myeloproliferative neoplasms, we performed high-coverage genome sequencing of DNA from peripheral blood granulocytes and cultured skin fibroblasts from a patient with MPL W515K-positive primary myelofibrosis. The primary myelofibrosis genome had a low somatic mutation rate, consistent with that observed in similar hematopoietic tumor genomes. Interfacing of whole-genome DNA sequence data with RNA expression data identified three somatic mutations of potential functional significance: i) a nonsense mutation in CARD6, implicated in modulation of NF-kappaB activation; ii) a 19-base pair deletion involving a potential regulatory region in the 5'-untranslated region of BRD2, implicated in transcriptional regulation and cell cycle control; and iii) a non-synonymous point mutation in KIAA0355, an uncharacterized protein. Additional mutations in three genes (CAP2, SOX30, and MFRP) were also evident, albeit with no support for expression at the RNA level. Re-sequencing of these six genes in 178 patients with polycythemia vera, essential thrombocythemia, and myelofibrosis did not identify recurrent somatic mutations in these genes. Finally, we describe methods for reducing false-positive variant calls in the analysis of hematologic malignancies with a low somatic mutation rate. This trial is registered with ClinicalTrials.gov (NCT01108159).

Background B cells expressing IgE contribute to immunity against parasites and venoms and are the source of antigen specificity in allergic patients, yet the developmental pathways producing these B cells in human subjects remain a subject of debate. Much of our knowledge of IgE lineage development derives from model studies in mice rather than from human subjects. Objective We evaluate models for isotype switching to IgE in human subjects using immunoglobulin heavy chain (IGH) mutational lineage data. Methods We analyzed IGH repertoires in 9 allergic and 24 healthy adults using high-throughput DNA sequencing of 15,843,270 IGH rearrangements to identify clonal lineages of B cells containing members expressing IgE. Somatic mutations in IGH inherited from common ancestors within the clonal lineage are used to infer the relationships between B cells. Results Data from 613,641 multi-isotype B-cell clonal lineages, of which 592 include an IgE member, are consistent with indirect switching to IgE from IgG- or IgA-expressing lineage members in human subjects. We also find that these inferred isotype switching frequencies are similar in healthy and allergic subjects. Conclusions We found evidence that secondary isotype switching of mutated IgG 1 -expressing B cells is the primary source of IgE in human subjects, with lesser contributions from precursors expressing other switched isotypes and rarely IgM or IgD, suggesting that IgE is derived from previously antigen-experienced B cells rather than naive B cells that typically express low-affinity unmutated antibodies. These data provide a basis from which to evaluate allergen-specific human antibody repertoires in healthy and diseased subjects.

The mission of the Encyclopedia of DNA Elements (ENCODE) Project is to enable the scientific and medical communities to interpret the human genome sequence and apply it to understand human biology and improve health. The ENCODE Consortium is integrating multiple technologies and approaches in a collective effort to discover and define the functional elements encoded in the human genome, including genes, transcripts, and transcriptional regulatory regions, together with their attendant chromatin states and DNA methylation patterns. In the process, standards to ensure high-quality data have been implemented, and novel algorithms have been developed to facilitate analysis. Data and derived results are made available through a freely accessible database. Here we provide an overview of the project and the resources it is generating and illustrate the application of ENCODE data to interpret the human genome., National Human Genome Research Institute (U.S.), National Institutes of Health (U.S.)

We construct several score functions for use in locating unusually conserved regions in a genomewide search of aligned DNA from two species. We test these functions on regions of the human genome aligned to the mouse genome. These score functions are derived from properties of neutrally evolving sites on the mouse and human genome and can be adjusted to the local background rate of conservation. The aim of these functions is to try to identify regions of the human genome that are conserved by evolutionary selection because they have an important function, rather than by chance. We use them to get a very rough estimate of the amount of DNA in the human genome that is under selection.

Levels of recombination vary among species, among chromosomes within species, and among regions within chromosomes in mammals. This heterogeneity may affect levels of diversity, efficiency of selection, and genome composition, as well as have practical consequences for the genetic mapping of traits. We compared the genetic maps to the genome sequence assemblies of rat, mouse, and human to estimate local recombination rates across these genomes. Humans have greater overall levels of recombination, as well as greater variance. In rat and mouse, the size of the chromosome and proximity to telomere have less effect on local recombination rate than in human. At the chromosome level, rat and mouse X chromosomes have the lowest recombination rates, whereas human chromosome X does not show the same pattern. In all species, local recombination rate is significantly correlated with several sequence variables, including GC%, CpG density, repetitive elements, and the neutral mutation rate, with some pronounced differences between species. Recombination rate in one species is not strongly correlated with the rate in another, when comparing homologous syntenic blocks of the genome. This comparative approach provides additional insight into the causes and consequences of genomic heterogeneity in recombination.

The rates at which human genomic DNA changes by neutral substitution and insertion of certain families of transposable elements covary in large, megabase-sized segments. We used the rat, mouse, and human genomic DNA sequences to examine these processes in more detail in comparisons over both shorter (rat–mouse) and longer (rodent–primate) times, and demonstrated the generality of the covariation. Different families of transposable elements show distinctive insertion preferences and patterns of variation with substitution rates. SINEs are more abundant in GC-rich DNA, but the regional GC preference for insertion (monitored in young SINEs) differs between rodents and humans. In contrast, insertions in the rodent genomes are predominantly LINEs, which prefer to insert into AT-rich DNA in all three mammals. The insertion frequency of repeats other than SINEs correlates strongly positively with the frequency of substitutions in all species. However, correlations with SINEs show the opposite effects. The correlations are explained only in part by the GC content, indicating that other factors also contribute to the inherent tendency of DNA segments to change over evolutionary time.

The laboratory rat (Rattus norvegicus) is an indispensable tool in experimental medicine and drug development, having made inestimable contributions to human health. We report here the genome sequence of the Brown Norway (BN) rat strain. The sequence represents a high-quality 'draft' covering over 90% of the genome. The BN rat sequence is the third complete mammalian genome to be deciphered, and three-way comparisons with the human and mouse genomes resolve details of mammalian evolution. This first comprehensive analysis includes genes and proteins and their relation to human disease, repeated sequences, comparative genome-wide studies of mammalian orthologous chromosomal regions and rearrangement breakpoints, reconstruction of ancestral karyotypes and the events leading to existing species, rates of variation, and lineage-specific and lineage-independent evolutionary events such as expansion of gene families, orthology relations and protein evolution.

We construct several score functions for use in locating unusually conserved regions in a genome-wide search of aligned DNA from two species. We test these functions on regions of the human genome aligned to the mouse genome. These score functions are derived from properties of neutrally evolving sites on the mouse and human genome, and can be adjusted to the local background rate of conservation. The aim of these functions is to try to identify regions of the human genome that are conserved by evolutionary selection, because they have an important function, rather than by chance. We use them to get a very rough estimate of the amount of DNA in the human genome that is under selection.

Six measures of evolutionary change in the human genome were studied, three derived from the aligned human and mouse genomes in conjunction with the Mouse Genome Sequencing Consortium, consisting of (1) nucleotide substitution per fourfold degenerate site in coding regions, (2) nucleotide substitution per site in relics of transposable elements active only before the human–mouse speciation, and (3) the nonaligning fraction of human DNA that is nonrepetitive or in ancestral repeats; and three derived from human genome data alone, consisting of (4) SNP density, (5) frequency of insertion of transposable elements, and (6) rate of recombination. Features 1 and 2 are measures of nucleotide substitutions at two classes of “neutral” sites, whereas 4 is a measure of recent mutations. Feature 3 is a measure dominated by deletions in mouse, whereas 5 represents insertions in human. It was found that all six vary significantly in megabase-sized regions genome-wide, and many vary together. This indicates that some regions of a genome change slowly by all processes that alter DNA, and others change faster. Regional variation in all processes is correlated with, but not completely accounted for, by GC content in human and the difference between GC content in human and mouse. [Supplemental material is available online atwww.genome.org andhttp://www.soe.ucsc.edu/research/compbio/covariation/.]

The sequence of the mouse genome is a key informational tool for understanding the contents of the human genome and a key experimental tool for biomedical research. Here, we report the results of an international collaboration to produce a high-quality draft sequence of the mouse genome. We also present an initial comparative analysis of the mouse and human genomes, describing some of the insights that can be gleaned from the two sequences. We discuss topics including the analysis of the evolutionary forces shaping the size, structure and sequence of the genomes; the conservation of large-scale synteny across most of the genomes; the much lower extent of sequence orthology covering less than half of the genomes; the proportions of the genomes under selection; the number of protein-coding genes; the expansion of gene families related to reproduction and immunity; the evolution of proteins; and the identification of intraspecies polymorphism.

As vertebrate genome sequences near completion and research refocuses to their analysis, the issue of effective genome annotation display becomes critical. A mature web tool for rapid and reliable display of any requested portion of the genome at any scale, together with several dozen aligned annotation tracks, is provided athttp://genome.ucsc.edu. This browser displays assembly contigs and gaps, mRNA and expressed sequence tag alignments, multiple gene predictions, cross-species homologies, single nucleotide polymorphisms, sequence-tagged sites, radiation hybrid data, transposon repeats, and more as a stack of coregistered tracks. Text and sequence-based searches provide quick and precise access to any region of specific interest. Secondary links from individual features lead to sequence details and supplementary off-site databases. One-half of the annotation tracks are computed at the University of California, Santa Cruz from publicly available sequence data; collaborators worldwide provide the rest. Users can stably add their own custom tracks to the browser for educational or research purposes. The conceptual and technical framework of the browser, its underlying MYSQL database, and overall use are described. The web site currently serves over 50,000 pages per day to over 3000 different users.

Abstract 2863 Background: JAK2 V617F is a recurrent, activating mutation in patients (pts) with BCR-ABL1-negative MPNs. Mutations in codon 515 of MPL occur in 1–5% and 5–10% of ET and PMF pts, respectively, and similar to JAK2 V617F, lead to constitutive JAK-STAT signaling. The acquisition of multiple mutational events affecting the JAK-STAT axis or components of the epigenetic machinery is common in MPNs and likely contributes to phenotypic diversity, including progression to acute myeloid leukemia. Mutated genes thus far implicated in MPN initiation and/or progression include TET2, CBL, SH2B3, ASXL1, DNMT3A, IDH1/2, IKZF1, EZH2, SRSF2, and TP53. In order to identify novel somatic mutations associated with classic BCR-ABL1-negative MPNs, we performed whole genome sequencing of DNA extracted from peripheral blood granulocytes and cultured skin fibroblasts from a patient with PMF and a known MPL W515K mutation. Methods: Whole genome sequencing (WGS) was undertaken in a 55 year-old man with untreated PMF four years after initial diagnosis. His DIPSS Plus risk group was low (score 0). His karyotype was normal, and molecular testing revealed wild-type JAK2 in addition to the MPL W515K mutation. WGS of purified granulocytes and paired cultured skin fibroblasts was performed using both Illumina HiSeq and Complete Genomics (CGI) platforms. The resulting data were analyzed using multiple independent aligners and variant callers. Amplicon-based targeted resequencing with the Illumina MiSeq platform was used to evaluate additional patient samples for recurrent mutations. Stanford institutional review board approval and informed pt consent was obtained for these analyses. Results: The PMF genome was sequenced to 88X (Illumina) and 128X (CGI) average fold coverage, and the cultured skin fibroblast genome was sequenced to 47X (Illumina) and 126X (CGI). The PMF genome had a low somatic mutation rate, consistent with that observed for other sequenced hematopoietic tumor genomes, with a low number of true somatic mutation calls. To definitively identify true mutations among various sequencing artefacts and germline variants, we use cultured skin fibroblasts which can be prepared with no contamination by neoplastic cells. In addition to re-identification of the MPL W515K mutation, this approach identified six additional somatic mutations that alter gene coding regions, splice sites, or known regulatory regions: a nonsense mutation in CARD6, implicated in modulation of NF-kappaB activation; a splice-site mutation in CAP2; three nonsynonymous point mutations in KIAA0355, SOX30, MFRP; and a 19-base pair (bp) deletion involving a regulatory region in the 5'-untranslated region (5'-UTR) of BRD2, a bromodomain-containing protein implicated in transcriptional regulation (Table). CARD6, BRD2, and KIAA0355, an uncharacterized protein, are expressed by the granulocytes derived from this patient, supporting a potential role in the development of PMF in this pt. Using massively parallel sequencing, we are currently examining the transcribed region of BRD2 and the coding region of the other five genes in additional pt samples. Analysis of the first 87 samples (MF=47, PV=20, ET=20) out of a cohort of 180 MPN pts has thus far not identified recurrent somatic mutations in these six genes. Conclusion: High-coverage genome sequencing of neoplastic and germline cells from a patient with MPL-mutated PMF identified six additional somatic mutations of potential functional significance. Work is ongoing to determine if somatic mutations in these genes are found in other patients with BCR-ABL1-negative MPNs, and their pathogenetic relevance to MPN biology. Disclosures: Snyder: Illumina: Consultancy; GenapSys: Membership on an entity's Board of Directors or advisory committees; Personalis: Consultancy, Founder Other.