Fusion Sequence Research Articles

Diversity of EML4-ALK fusion variants in non-small cell lung cancer

Objective To investigate the fusion sequence complexity of EML4-ALK in non-small cell lung cancer (NSCLC) patients, and the potential mutation in tyrosine kinase (TK) domain of ALK gene. Methods In routine practice，a novel echinoderm microtubule-associated protein-like4 and anaplastic lymphoma kinase (EML4-ALK) V3c variant was detected by rapid amplification of cDNA ends-polymerase chain reaction (RACE-PCR)-sequencing technology in a patient with NSCLC.The further consecutive 39 cases（total of 40 cases）were screened by use of reverse transcription（RT）-PCR for EML4-ALK fusion.Positive PCR products were purified and cloned into T vectors, transformed into DH5a germ cells and colony picked up and sequenced for sequence complexity analysis.Tyrosine kinase domain of ALK was amplified by RT-PCR and sequenced. Results Three out of 40 cases had EML4-ALK fusion.One case had six novel variants of EML4-ALK co-existing, termed as V3c(64.6%)，V3d(25.0%)，V3e(2.1%)，V3f(4.2%)，V3g(2.1%)and V3h(2.1%) variants, whereas without common V3a and V3b variants. In other two positive cases, one was V1 variant, another was concurrent V2, V3a and V3b variants.No mutations were detected in the TK domain of EML4-ALK in any case. Conclusions Several EML-ALK variants could co-exist in a given lung cancer tissue, which suggest that the diversity and sequence complexity of EML4-ALK fusion are exist.Attentions should be paid to screen all the variants in clinic to improve the pick-up rate.(Chin J Lab Med, 2012,35:593-597) Key words: Lung neoplasms; Carcinoma，non-small-cell lung; Oncogene proteins，fusion; Protein-tyrosine kinases; Polymerase chain reaction; Mutation

Seamless replacement of Autographa californica multiple nucleopolyhedrovirus gp64 with each of five novel type II alphabaculovirus fusion sequences generates pseudotyped virus that fails to transduce mammalian cells

Autographa californica multiple nucleopolyhedrovirus (AcMNPV), a member of the type I alphabaculoviruses, is able to transduce and deliver a functional gene to a range of non-host cells, including many mammalian lines and primary cells, a property mediated by the envelope fusion protein GP64. AcMNPV is non-cytopathic and inherently replication deficient in non-host cells. As such, AcMNPV represents a possible new class of gene therapy vector with potential future clinical utility. Whilst not a problem for in vitro gene delivery, the broad tropism displayed for non-host cells is less desirable in a gene therapy vector. The fusion protein F of type II alphabaculoviruses can substitute functionally for GP64, and such pseudotyped viruses display a severely impaired capacity for non-host-cell transduction. Thus, surface decoration of such an F-pseudotyped AcMNPV with cell-binding ligands may restore transduction competence and generate vectors with desirable cell-targeting characteristics. By seamlessly swapping the native gp64 coding sequence with each of five sequences encoding different F proteins, a set of F-pseudotyped AcMNPV was generated. This report details their relative abilities both to functionally replace GP64 in viral growth and to transduce human Saos-2 and HeLa cells. All five supported viable infections in insect cell cultures and one, the Mamestra configurata NPV (MacoNPV) F pseudotype, could be amplified to titres close to those of native AcMNPV. In contrast, none was able to transduce the Saos-2 and HeLa cell lines. The robust support provided by MacoNPV F in virus production makes the corresponding pseudotype a viable scaffold to display surface ligands to direct selective mammalian cell targeting.

Diagnostic Usefulness of Genomic Breakpoint Analysis of Various Gene Rearrangements in Acute Leukemias: A Perspective of Long Distance– or Long Distance Inverse-PCR–based Approaches

Diagnostic Usefulness of Genomic Breakpoint Analysis of Various Gene Rearrangements in Acute Leukemias: A Perspective of Long Distance– or Long Distance Inverse-PCR–based Approaches

Can silencing of transposons contribute to variation in effector gene expression inPhytophthora infestans?

Transposable elements are ubiquitous residents in eukaryotic genomes. Often considered to be genomic parasites, they can lead to dramatic changes in genome organization, gene expression, and gene evolution. The oomycete plant pathogen Phytophthora infestans has evolved a genome organization where core biology genes are predominantly located in genome regions that have relatively few resident transposons. In contrast, disease effector-encoding genes are most frequently located in rapidly evolving genomic regions that are rich in transposons. P. infestans, as a eukaryote, likely uses RNA silencing to minimize the activity of transposons. We have shown that fusion of a short interspersed element (SINE) to an effector gene in P. infestans leads to the silencing of both the introduced fusion and endogenous homologous sequences. This is also likely to occur naturally in the genome of P. infestans, as transcriptional inactivation of effectors is known to occur, and over half of the translocated “RXLR class” of effectors are located within 2 kb of transposon sequences in the P. infestans genome. In this commentary, we review the diverse transposon inventory of P. infestans, its control by RNA silencing, and consequences for expression modulation of nearby effector genes in this economically important plant pathogen.

Skeletal development inPan paniscuswith comparisons toPan troglodytes

Fusion of skeletal elements provides markers for timing of growth and is one component of a chimpanzee's physical development. Epiphyseal closure defines bone growth and signals a mature skeleton. Most of what we know about timing of development in chimpanzees derives from dental studies on Pan troglodytes. Much less is known about the sister species, Pan paniscus, with few in captivity and a wild range restricted to central Africa. Here, we report on the timing of skeletal fusion for female captive P. paniscus (n = 5) whose known ages range from 0.83 to age 11.68 years. Observations on the skeletons were made after the individuals were dissected and bones cleaned. Comparisons with 10 female captive P. troglodytes confirm a generally uniform pattern in the sequence of skeletal fusion in the two captive species. We also compared the P. paniscus to a sample of three unknown-aged female wild P. paniscus, and 10 female wild P. troglodytes of known age from the Taï National Park, Côte d'Ivoire. The sequence of teeth emergence to bone fusion is generally consistent between the two species, with slight variations in late juvenile and subadult stages. The direct-age comparisons show that skeletal growth in captive P. paniscus is accelerated compared with both captive and wild P. troglodytes populations. The skeletal data combined with dental stages have implications for estimating the life stage of immature skeletal materials of wild P. paniscus and for more broadly comparing the skeletal growth rates among captive and wild chimpanzees (Pan), Homo sapiens, and fossil hominins.

Non-MTC IGH-CCND1 Breakpoints in Mantle Cell Lymphoma Are Associated with CpG Sites and AID Hotspots

Abstract 398Most t(11;14)(q13;q32) breakpoints in mantle cell lymphoma (MCL) are scattered across a large genomic region centromeric to the CCND1 gene on chromosome 11q13. However, few t(11;14) breakpoints outside the major translocation cluster (MTC) have been sequenced. We report our analysis of 56 IGH-CCND1 fusion sequences from 32 non-MTC cases of MCL. Our analysis reveals remarkable breakpoint diversity at both CCND1 and IGH loci. The CCND1 breakpoints are located from 2 kb to 331 kb from the CCND1 gene, including 12 breakpoints (38%) in the 220kb region centromeric to the MTC, which is itself located 110 kb centromeric to the CCND1 gene. Twenty-one cases had a JH coding end breakpoint on the der(14) chromosome and a DH coding end breakpoint on the der(11). Two cases had der(14) and der(11) breakpoints derived from the coding and signal ends of the same JH or DH gene segment; one case had a JH/DH breakpoint on the der(14) and a VH breakpoint on the der(11); and one case had a breakpoint in the JH region located >100 bases from the nearest RSS motif, suggesting a RAG-independent break at IGH. No der(11) breakpoint could be amplified in eight cases. In sixteen cases (50%), one or both CCND1 breakpoints were within 4 bases of a CpG dinucleotide, a feature characteristic of translocation breakpoints in many human B cell lymphomas. Three CpG-associated “microclusters” were identified, i.e. breakpoints in two different tumors located at or near the same CpG site. Breakpoints in the sixteen (50%) “non-CpG” cases showed a significant association with AID hotspot motifs at the CCND1 locus. In addition, the non-CpG breakpoints were more likely to: 1) be located telomeric to the MTC, 2) involve 3’ JH and 5’ DH gene segments, and 3) be associated with immunoglobulin lambda light chain restriction; features that suggest occurrence at a late stage of pre-B cell maturation. In contrast, the CpG-associated breakpoints were: 1) more likely to be centromeric to the MTC, 2) not biased in their JH or DH segment usage, and 3) associated with kappa light chain restriction; features suggesting occurrence in an earlier pre-B cell or pro-B cell. Our results implicate AID in chromosomal breakage at both CpG and non-CpG sites within the CCND1 locus and suggest that AID and RAG collude to generate the chromosomal breaks underlying the t(11;14). Our findings also suggest that IGH-CCND1 rearrangements can occur at different stages of pre-B cell maturation. This study provides novel insights into the mechanism and developmental timing of the t(11;14) in human MCL, features that are likely to be relevant to a broad range of human lymphomas. Disclosures:Greisman:Signature Genomics, LLC: Patents & Royalties. Yi:Signature Genomics, LLC: Patents & Royalties.

What Is New? An Update of the MLL Recombinome Including the Three Novel Partner Genes ABI2, PDS5A, and TOP3A

Abstract 1351Chromosomal rearrangements of the MLL gene are associated with pediatric and adult de novo as well as therapy related acute myeloid leukemias, acute lymphoid leukemias, biphenotypic leukemias, and myelodysplastic syndromes. So far more than 70 MLL fusion partner genes have been characterized at the molecular level involving nearly all chromosomes. Though 11q23 rearrangements are associated with high-risk leukemias the clinical outcome of MLL rearrangements depends highly on the specific fusion partner involved. Nearly 40% of these partner genes have been identified at the Diagnostic Center of Acute Leukemia (DCAL) including the novel partner genes ABI2, PDS5A and TOP3A by analyzing more than 1400 MLL rearrangements. These rearrangements from 27 different countries include 52 different partner genes. This overview indicates all the specific introns of the translocation partner genes (TPGs) found to be involved in MLL translocations, their chromosomal locations and the type of genetic abnormality that is leading to the MLL fusion. More than 20% of all MLL rearrangements are complex ones. Within these complex rearrangements, the 5’ part of the MLL gene is generally fused in frame to one of the most frequent partner genes. But the 3’ part of the MLL gene is fused in many cases to a novel so-called “reciprocal MLL partner gene” like DENND4A or LRRTM4. To date more than 90 reciprocal MLL partner genes have been identified. In addition we present a recently analysed 4-way translocation where all four breakpoints could be identified by systematic breakpoint analysis using LDI-PCR. Also three novel “spliced MLL fusions”, a mechanism to generate functional chimeric fusion transcripts, involving MLLT4 (AF6), MYO1F, and CT45A2 have been identified at the DCAL. With these results, the number of genes involved in „spliced MLL fusions“ has increased from eight to eleven. Moreover we present the current breakpoint cluster region (bcr) for the 14 most frequent partner genes, namely AFF1 (AF4), MLLT3 (AF9), MLLT1 (ENL), MLLT10 (AF10), MLLT4 (AF6), ELL, EPS15, MLLT6 (AF17), SEPT6 (AF17), MLLT11 (AF1Q), SEPT9, AFF3 (LAF4), TET1, SEPT5 (PNUTL) and partial tandem duplications. For six patients, no partner gene could be identified at the molecular level and for 5 patients the identified fusion is out of frame. Unfortunetely all attemps to identify functional chimeric fusion transcripts by RACE and RT-PCR failed. These unusual MLL rearrangements probably represent a subclass of MLL abnormalities which have per se no or only a weak ability to transform hematopoeitic cells and are indentified only in the context of other hematopoeitic malignancies like the recently described MLL partner SACM1L. Finally, the determined patient-specific fusion sequences are succesfully used worldwide for minimal residual disease (MRD) monitoring to improve the treatment and outcome of acute leukemia patients. Disclosures:No relevant conflicts of interest to declare.

Influence of medium components on the expression of recombinant lipoproteins in Escherichia coli

Bacterial lipoproteins are crucial antigens for protective immunity against bacterial pathogens. Expression of exogenous lipoproteins in Escherichia coli at high levels is thought to be an extremely difficult endeavor because it frequently results in incomplete or absent lipid modification. Previously, we identified a fusion sequence (D1) from a Neisseria meningitidis lipoprotein that induced a non-lipidated protein, E3 (the domain III of the dengue virus envelope protein), to become lipidated. However, without optimizing the growth conditions, some of the D1-fusion proteins were not lipidated. Here, we report the influence of medium components on the expression of recombinant lipoproteins in E. coli. For high-level expression of mature lipoproteins in the C43 (DE3) strain, M9 medium was better than M63 and the rich medium. Furthermore, we analyzed the influence of other media factors (including nitrogen and carbon sources, phosphate, ferrous ions, calcium, magnesium, and pH) on the levels of lipoprotein expression. The results showed that excess nitrogen sources and phosphate in M9 medium could increase the amount of immature lipoproteins, and glucose was a better carbon source than glycerol for expressing mature lipoproteins. We also found that lipoproteins tended to be completely processed in the alkaline environment, even in the nutrient-rich medium. Additional constructs expressing different immunogens or lipid signal peptides as targets were also utilized, demonstrating that these targets could be expressed as completely mature lipoproteins in the M9 medium but not in the rich medium. Our results provide the useful information for expressing mature exogenous lipoproteins in E. coli.

Lipid composition modulates the interaction of peptides deriving from herpes simplex virus type I glycoproteins B and H with biomembranes

Lipid membranes play a key role in the viral life cycle. Enveloped viruses particularly require a sequence of fusion and fission events between the viral envelope and the target membranes for entry into the cell and egress from it. These processes are controlled by one or more viral glycoproteins that undergo conformational changes favoring the necessary micro- and mesoscopic lipid re-arrangements. Multiple regions from these glycoproteins are thought to interact with the membranes, according to a concerted mechanism, in order to generate the distortion necessary for fusion. In this work, we perform an EPR study on the role played by the membrane composition in tuning the interaction between lipid bilayers and two peptides, gH626–644 and gB632–650, that are highly fusogenic fragments of the gH and gB glycoproteins of herpes simplex virus. Our results show that both peptides interact with lipid bilayers, perturbing the local lipid packing. gH626–644 localizes close to the hydrophilic bilayer surface, while gB632–650 penetrates deeply into the membrane. Chain perturbation by the peptides increases in the presence of charged phospholipids. Finally, cholesterol does not alter the ability of gB632–650 to penetrate deeply in the membrane, whereas it limits penetration of the gH626–644 peptide to the more external layer. The different modes of interaction result in a higher fusogenic ability of gB632–650 towards cholesterol-enriched membranes, as demonstrated by lipid mixing assays. These results suggest that the mechanism of action of the gH and gB glycoproteins is modulated by the properties and composition of the phospholipid bilayer.

Identifying Protein Structural Classes by a Fusion Sequence Encoding Scheme

In this study, a novel sequence encoding scheme is introduced by fusing PseAA and PSSM. However, this sequence encoding scheme would correspond to a very high dimensional feature vector. A dimensionality reduction algorithm, the so-called NPE (Neighborhood Preserving Embedding) is introduced to extract the key features from the high-dimensional space. Finally, the K-NN (K-Nearest Neighbor) classifier is employed to identify the types of protein structures. Our jackknife test results thus obtained are quite encouraging, which indicate that the above methods are used effectively to deal with this complicated problem of predicting protein structural classes.

Clinical and molecular characterization of a transmitted reciprocal translocation t(1;12)(p32.1;q21.3) in a family co-segregating with mental retardation, language delay, and microcephaly

BackgroundChromosome translocation associated with neurodevelopmental disorders provides an opportunity to identify new disease-associated genes and gain new insight into their function. During chromosome analysis, we identified a reciprocal translocation between chromosomes 1p and 12q, t(1; 12)(p32.1; q21.3), co-segregating with microcephaly, language delay, and severe psychomotor retardation in a mother and her two affected boys.MethodsFluorescence in situ hybridization (FISH), long-range PCR, and direct sequencing were used to map the breakpoints on chromosomes 1p and 12q. A reporter gene assay was conducted in human neuroblastoma (SKNSH) and Chinese hamster ovary (CHO) cell lines to assess the functional implication of the fusion sequences between chromosomes 12 and 1.ResultsWe determined both breakpoints at the nucleotide level. Neither breakpoint disrupted any known gene directly. The breakpoint on chromosome 1p was located amid a gene-poor region of ~ 1.1 Mb, while the breakpoint on chromosome 12q was located ~ 3.4 kb downstream of the ALX1 gene, a homeobox gene. In the reporter gene assay, we discovered that the fusion sequences construct between chromosomes 12 and 1 had a ~ 1.5 to 2-fold increased reporter gene activity compared with the corresponding normal chromosome 12 sequences construct.ConclusionOur findings imply that the translocation may enhance the expression of the ALX1 gene via the position effect and result in the clinical symptoms of this family. Our findings may also expand the clinical phenotype spectrum of ALX1-related human diseases as loss of the ALX1 function was recently reported to result in abnormal craniofacial development.

Construction and identification of mutation of pgm gene attenuate the virulent of Brucella melitensis strain 16

Objective The construction of suicide plasmid vector could be used to make mutation of pgm gene which attenuates the virulent of Brucella melitensis strain 16, the research may lay a foundation for the development of novel live attenuated vaccines. Methods Sucrose sensitive gene as forward screening sign and fusion sequences of kanamycin resistance gene were constructed based on plasmid pucl9; pucS1.6K suicide plasmid vector was established by modifying pgm gene with fusion sequences of kanamycin resistance gene (insertion mutation); pgm gene mutation of Brucella melitensis strain 16 was obtained by electro transformation and mutation was confirmed by PCR amplification. Results The results showed that the identified Brucella melitensis strain 16 pgm gene was inactivated after insertion of kanamycin resistance gene, and the mutant pgm gene DNA fragment length was approximately 3525 bp, in line with expectations, Brucella pgm gene mutant melitensis strain 16 was successfully constructed. Conclusions The construction of suicide plasmid vector and precise mutation of Brucella melitensis strain 16 is successful, the study is not only provided an effective technology platform for constructing mutants of Brucella but also lays a foundation for the development of novel live attenuated vaccines. Key words: Brucella vaccine; pgm genes; Gene fusion; Mutagenesis, insertional

Determinants of Membrane Activity from Mutational Analysis of the HIV Fusion Peptide

We have synthesized a small library of 38 variants of the 23-residue fusion peptide domain found at the N-terminus of gp41 glycoprotein of HIV. This hydrophobic, glycine-rich sequence is critical for viral infectivity and is thought to be central in the membrane fusion of viral envelope with the host membrane. There has been extensive discussion regarding the origin of fusogenicity in this viral fusion sequence. Our library of fusion peptide variants was designed to address the biophysical importance of secondary structure, peptide flexibility, glycine content, and placement. We assayed each peptide for its ability to induce lipid mixing and membrane permeablization in synthetic vesicles. We find that the viral fusion peptide may be greatly simplified while retaining fusogenic function and minimizing membrane-permeablizing function; to the best of our knowledge, this is the first attempt to optimize fusogenic function of the HIV fusion peptide through sequence variation. Our data show that many flexible, linear, minimally hydrophobic peptides may achieve the biophysical function of fusion; glycine does not appear to be essential. These findings will be useful in the design of synthetic fusogens for cellular delivery.

DeFuse: An Algorithm for Gene Fusion Discovery in Tumor RNA-Seq Data

Gene fusions created by somatic genomic rearrangements are known to play an important role in the onset and development of some cancers, such as lymphomas and sarcomas. RNA-Seq (whole transcriptome shotgun sequencing) is proving to be a useful tool for the discovery of novel gene fusions in cancer transcriptomes. However, algorithmic methods for the discovery of gene fusions using RNA-Seq data remain underdeveloped. We have developed deFuse, a novel computational method for fusion discovery in tumor RNA-Seq data. Unlike existing methods that use only unique best-hit alignments and consider only fusion boundaries at the ends of known exons, deFuse considers all alignments and all possible locations for fusion boundaries. As a result, deFuse is able to identify fusion sequences with demonstrably better sensitivity than previous approaches. To increase the specificity of our approach, we curated a list of 60 true positive and 61 true negative fusion sequences (as confirmed by RT-PCR), and have trained an adaboost classifier on 11 novel features of the sequence data. The resulting classifier has an estimated value of 0.91 for the area under the ROC curve. We have used deFuse to discover gene fusions in 40 ovarian tumor samples, one ovarian cancer cell line, and three sarcoma samples. We report herein the first gene fusions discovered in ovarian cancer. We conclude that gene fusions are not infrequent events in ovarian cancer and that these events have the potential to substantially alter the expression patterns of the genes involved; gene fusions should therefore be considered in efforts to comprehensively characterize the mutational profiles of ovarian cancer transcriptomes.

Phylogenetic analysis of Austrian canine distemper virus strains from clinical samples from dogs and wild carnivores

Austrian field cases of canine distemper (14 dogs, one badger [Meles meles] and one stone marten [Martes foina]) from 2002 to 2007 were investigated and the case histories were summarised...

A DV protein based in domine II and III of E protein expressed in plants induce an effective immune response (67.7)

Abstract Dengue is the most important arboviral disease. Despite the efforts to develop an effective vaccine there is none available. In the virus the envelope protein is the most expose and it is the most immunity target. The E protein has 3 domains, the domain III (EIII) is involved in neutralizing Abs induction. Domain II (EII) contains a fusion peptide that may induce Abs with neutralizing activity. Recently Anti-DEC, has been considered as a potential adjuvant when fused with different viral proteins due to its use in targeting antigen to DCs. The goal of this work was to fuse genetically different sequences from domains II and III to heavy chain variable region of the Ab Anti-DEC-205 spaced by a flag sequence named OLLAS in order to improve the humoral response. The fusion sequences DEC-OLLAS-EII-EIII was cloned in the vector pICH31070. To express these DNA, the plants N. benthamiana were infiltrated and the protein expression was analyzed by W.B. at different days post-infiltration. The high expression was observed at day 8, then, the proteins were purify by preparative PAGE and refolded with L-arg buffer. Following groups of 8 mice were inoculated 3 times with 30μg of recombinant protein. The immunity of this protein was analyzed by ELISA, immobilizing DEN viral particles and important titer of antibodies were observed in the group of mice immunized with DEC-OLLAS-EIII-EII in contrast with the control group. Moreover neutralizing Abs was observed in the mentioned group

Bioinformatics and the allergy assessment of agricultural biotechnology products: Industry practices and recommendations

Bioinformatic tools are being increasingly utilized to evaluate the degree of similarity between a novel protein and known allergens within the context of a larger allergy safety assessment process. Importantly, bioinformatics is not a predictive analysis that can determine if a novel protein will ‘‘become” an allergen, but rather a tool to assess whether the protein is a known allergen or is potentially cross-reactive with an existing allergen. Bioinformatic tools are key components of the 2009 Codex Alimentarius Commission’s weight-of-evidence approach, which encompasses a variety of experimental approaches for an overall assessment of the allergenic potential of a novel protein. Bioinformatic search comparisons between novel protein sequences, as well as potential novel fusion sequences derived from the genome and transgene, and known allergens are required by all regulatory agencies that assess the safety of genetically modified (GM) products. The objective of this paper is to identify opportunities for consensus in the methods of applying bioinformatics and to outline differences that impact a consistent and reliable allergy safety assessment. The bioinformatic comparison process has some critical features, which are outlined in this paper. One of them is a curated, publicly available and well-managed database with known allergenic sequences. In this paper, the best practices, scientific value, and food safety implications of bioinformatic analyses, as they are applied to GM food crops are discussed. Recommendations for conducting bioinformatic analysis on novel food proteins for potential cross-reactivity to known allergens are also put forth.

Identification of Bcl-2/IgH fusion sequences using real-time PCR and chip-based microcapillary electrophoresis

The determination of polymerase chain reaction (PCR) amplification product sizes of the Bcl-2/IgH fusion gene from follicular lymphoma (FL) provides evidence of clonal identity. The present study describes detection of Bcl-2/IgH fusion gene clonality utilizing a small, simple microcapillary electrophoretic chip combined with a real-time PCR method. The microcapillary electrophoretic chip system effectively detects size differences among the Bcl-2/IgH fusion gene amplification products of FL from patient samples; something that is not possible using traditional gel electrophoresis. We also describe the potential of this system to utilize formalin-fixed, paraffin-embedded tissue samples sectioned on charged slides. The simple detection of Bcl-2/IgH fusion gene clonality using a microcapillary electrophoretic chip provides reliable information for monitoring minimal residual disease of FL, and can be an effective tool for use in clinical laboratories.

Phocine Distemper Virus in Seals, East Coast, United States, 2006

In 2006 and 2007, elevated numbers of deaths among seals, constituting an unusual mortality event, occurred off the coasts of Maine and Massachusetts, United States. We isolated a virus from seal tissue and confirmed it as phocine distemper virus (PDV). We compared the viral hemagglutinin, phosphoprotein, and fusion (F) and matrix (M) protein gene sequences with those of viruses from the 1988 and 2002 PDV epizootics. The virus showed highest similarity with a PDV 1988 Netherlands virus, which raises the possibility that the 2006 isolate from the United States might have emerged independently from 2002 PDVs and that multiple lineages of PDV might be circulating among enzootically infected North American seals. Evidence from comparison of sequences derived from different tissues suggested that mutations in the F and M genes occur in brain tissue that are not present in lung, liver, or blood, which suggests virus persistence in the central nervous system.

Targeted next-generation sequencing detects point mutations, insertions, deletions and balanced chromosomal rearrangements as well as identifies novel leukemia-specific fusion genes in a single procedure

DNA sequence enrichment from complex genomic samples using microarrays enables targeted next-generation sequencing (NGS). In this study, we combined 454 shotgun pyrosequencing with long oligonucleotide sequence capture arrays. We demonstrate the detection of mutations including point mutations, deletions and insertions in a cohort of 22 patients presenting with acute leukemias and myeloid neoplasms. Importantly, this one-step methodological procedure also allowed the detection of balanced chromosomal aberrations, including translocations and inversions. Moreover, the genomic representation of only one of the partner genes of a chimeric fusion on the capture platform also permitted identification of the novel fusion partner genes. Using acute myeloid leukemias harboring RUNX1 abnormalities as a model system, three novel chromosomal fusion sequences and KCNMA1 as a novel RUNX1 fusion partner gene were detected. This assay has the strong potential to become an important method for the comprehensive genetic characterization of particular leukemias and other malignancies harboring complex genomes.