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September 22, 2019

PHACTR1 splicing isoforms and eQTLs in atherosclerosis-relevant human cells.

Genome-wide association studies (GWAS) have identified a variant (rs9349379) at the phosphatase and actin regulator 1 (PHACTR1) locus that is associated with coronary artery disease (CAD). The same variant is also an expression quantitative trait locus (eQTL) for PHACTR1 in human coronary arteries (hCA). Here, we sought to characterize PHACTR1 splicing pattern in atherosclerosis-relevant human cells. We also explored how rs9349379 modulates the expression of the different PHACTR1 splicing isoforms.We combined rapid amplification of cDNA ends (RACE) with next-generation long-read DNA sequencing to discover all PHACTR1 transcripts in many human tissues and cell types. We measured PHACTR1 transcripts by qPCR to identify transcript-specific eQTLs.We confirmed a brain-specific long transcript, a short transcript expressed in monocytes and four intermediate transcripts that are different due to alternative splicing of two in-frame exons. In contrast to a previous report, we confirmed that the PHACTR1 protein is present in vascular smooth muscle cells. In 158 hCA from our collection and the GTEx dataset, rs9349379 was only associated with the expression levels of the intermediate PHACTR1 transcripts.Our comprehensive transcriptomic profiling of PHACTR1 indicates that this gene encodes six main transcripts. Five of them are expressed in hCA, where atherosclerotic plaques develop. In this tissue, genotypes at rs9349379 are associated with the expression of the intermediate transcripts, but not the immune-specific short transcript. This result suggests that rs9349379 may in part influence CAD by modulating the expression of intermediate PHACTR1 transcripts in endothelial or vascular smooth muscle cells found in hCA.

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September 22, 2019

Transcriptome sequencing reveals thousands of novel long non-coding RNAs in B cell lymphoma.

Gene profiling of diffuse large B cell lymphoma (DLBCL) has revealed broad gene expression deregulation compared to normal B cells. While many studies have interrogated well known and annotated genes in DLBCL, none have yet performed a systematic analysis to uncover novel unannotated long non-coding RNAs (lncRNA) in DLBCL. In this study we sought to uncover these lncRNAs by examining RNA-seq data from primary DLBCL tumors and performed supporting analysis to identify potential role of these lncRNAs in DLBCL.We performed a systematic analysis of novel lncRNAs from the poly-adenylated transcriptome of 116 primary DLBCL samples. RNA-seq data were processed using de novo transcript assembly pipeline to discover novel lncRNAs in DLBCL. Systematic functional, mutational, cross-species, and co-expression analyses using numerous bioinformatics tools and statistical analysis were performed to characterize these novel lncRNAs.We identified 2,632 novel, multi-exonic lncRNAs expressed in more than one tumor, two-thirds of which are not expressed in normal B cells. Long read single molecule sequencing supports the splicing structure of many of these lncRNAs. More than one-third of novel lncRNAs are differentially expressed between the two major DLBCL subtypes, ABC and GCB. Novel lncRNAs are enriched at DLBCL super-enhancers, with a fraction of them conserved between human and dog lymphomas. We see transposable elements (TE) overlap in the exonic regions; particularly significant in the last exon of the novel lncRNAs suggest potential usage of cryptic TE polyadenylation signals. We identified highly co-expressed protein coding genes for at least 88 % of the novel lncRNAs. Functional enrichment analysis of co-expressed genes predicts a potential function for about half of novel lncRNAs. Finally, systematic structural analysis of candidate point mutations (SNVs) suggests that such mutations frequently stabilize lncRNA structures instead of destabilizing them.Discovery of these 2,632 novel lncRNAs in DLBCL significantly expands the lymphoma transcriptome and our analysis identifies potential roles of these lncRNAs in lymphomagenesis and/or tumor maintenance. For further studies, these novel lncRNAs also provide an abundant source of new targets for antisense oligonucleotide pharmacology, including shared targets between human and dog lymphomas.

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September 22, 2019

Cell-type-specific splicing of Piezo2 regulates mechanotransduction.

Piezo2 is a mechanically activated ion channel required for touch discrimination, vibration detection, and proprioception. Here, we discovered that Piezo2 is extensively spliced, producing different Piezo2 isoforms with distinct properties. Sensory neurons from both mice and humans express a large repertoire of Piezo2 variants, whereas non-neuronal tissues express predominantly a single isoform. Notably, even within sensory ganglia, we demonstrate the splicing of Piezo2 to be cell type specific. Biophysical characterization revealed substantial differences in ion permeability, sensitivity to calcium modulation, and inactivation kinetics among Piezo2 splice variants. Together, our results describe, at the molecular level, a potential mechanism by which transduction is tuned, permitting the detection of a variety of mechanosensory stimuli. Published by Elsevier Inc.

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September 22, 2019

Isoform evolution in primates through independent combination of alternative RNA processing events.

Recent RNA-seq technology revealed thousands of splicing events that are under rapid evolution in primates, whereas the reliability of these events, as well as their combination on the isoform level, have not been adequately addressed due to its limited sequencing length. Here, we performed comparative transcriptome analyses in human and rhesus macaque cerebellum using single molecule long-read sequencing (Iso-seq) and matched RNA-seq. Besides 359 million RNA-seq reads, 4,165,527 Iso-seq reads were generated with a mean length of 14,875?bp, covering 11,466 human genes, and 10,159 macaque genes. With Iso-seq data, we substantially expanded the repertoire of alternative RNA processing events in primates, and found that intron retention and alternative polyadenylation are surprisingly more prevalent in primates than previously estimated. We then investigated the combinatorial mode of these alternative events at the whole-transcript level, and found that the combination of these events is largely independent along the transcript, leading to thousands of novel isoforms missed by current annotations. Notably, these novel isoforms are selectively constrained in general, and 1,119 isoforms have even higher expression than the previously annotated major isoforms in human, indicating that the complexity of the human transcriptome is still significantly underestimated. Comparative transcriptome analysis further revealed 502 genes encoding selectively constrained, lineage-specific isoforms in human but not in rhesus macaque, linking them to some lineage-specific functions. Overall, we propose that the independent combination of alternative RNA processing events has contributed to complex isoform evolution in primates, which provides a new foundation for the study of phenotypic difference among primates.© The Author 2017. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.

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September 22, 2019

A human-specific switch of alternatively spliced AFMID isoforms contributes to TP53 mutations and tumor recurrence in hepatocellular carcinoma.

Pre-mRNA splicing can contribute to the switch of cell identity that occurs in carcinogenesis. Here, we analyze a large collection of RNA-seq data sets and report that splicing changes in hepatocyte-specific enzymes, such as AFMID and KHK, are associated with HCC patients’ survival and relapse. The switch of AFMID isoforms is an early event in HCC development and is associated with driver mutations in TP53 and ARID1A The switch of AFMID isoforms is human-specific and not detectable in other species, including primates. Finally, we show that overexpression of the full-length AFMID isoform leads to a higher NAD+ level, lower DNA-damage response, and slower cell growth in HepG2 cells. The integrative analysis uncovered a mechanistic link between splicing switches, de novo NAD+ biosynthesis, driver mutations, and HCC recurrence.© 2018 Lin et al.; Published by Cold Spring Harbor Laboratory Press.

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September 22, 2019

Plasmodium knowlesi: a superb in vivo nonhuman primate model of antigenic variation in malaria.

Antigenic variation in malaria was discovered in Plasmodium knowlesi studies involving longitudinal infections of rhesus macaques (M. mulatta). The variant proteins, known as the P. knowlesi Schizont Infected Cell Agglutination (SICA) antigens and the P. falciparum Erythrocyte Membrane Protein 1 (PfEMP1) antigens, expressed by the SICAvar and var multigene families, respectively, have been studied for over 30 years. Expression of the SICA antigens in P. knowlesi requires a splenic component, and specific antibodies are necessary for variant antigen switch events in vivo. Outstanding questions revolve around the role of the spleen and the mechanisms by which the expression of these variant antigen families are regulated. Importantly, the longitudinal dynamics and molecular mechanisms that govern variant antigen expression can be studied with P. knowlesi infection of its mammalian and vector hosts. Synchronous infections can be initiated with established clones and studied at multi-omic levels, with the benefit of computational tools from systems biology that permit the integration of datasets and the design of explanatory, predictive mathematical models. Here we provide an historical account of this topic, while highlighting the potential for maximizing the use of P. knowlesi – macaque model systems and summarizing exciting new progress in this area of research.

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September 22, 2019

Genome characterization of oleaginous Aspergillus oryzae BCC7051: A potential fungal-based platform for lipid production.

The selected robust fungus, Aspergillus oryzae strain BCC7051 is of interest for biotechnological production of lipid-derived products due to its capability to accumulate high amount of intracellular lipids using various sugars and agro-industrial substrates. Here, we report the genome sequence of the oleaginous A. oryzae BCC7051. The obtained reads were de novo assembled into 25 scaffolds spanning of 38,550,958 bps with predicted 11,456 protein-coding genes. By synteny mapping, a large rearrangement was found in two scaffolds of A. oryzae BCC7051 as compared to the reference RIB40 strain. The genetic relationship between BCC7051 and other strains of A. oryzae in terms of aflatoxin production was investigated, indicating that the A. oryzae BCC7051 was categorized into group 2 nonaflatoxin-producing strain. Moreover, a comparative analysis of the structural genes focusing on the involvement in lipid metabolism among oleaginous yeast and fungi revealed the presence of multiple isoforms of metabolic enzymes responsible for fatty acid synthesis in BCC7051. The alternative routes of acetyl-CoA generation as oleaginous features and malate/citrate/pyruvate shuttle were also identified in this A. oryzae strain. The genome sequence generated in this work is a dedicated resource for expanding genome-wide study of microbial lipids at systems level, and developing the fungal-based platform for production of diversified lipids with commercial relevance.

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September 22, 2019

Stalking a lethal superbug by whole-genome sequencing and phylogenetics: Influence on unraveling a major hospital outbreak of carbapenem-resistant Klebsiella pneumoniae.

From July 2010-April 2013, Leipzig University Hospital experienced the largest outbreak of a Klebsiella pneumoniae carbapenemase 2 (KPC-2)-producing Klebsiella pneumoniae (KPC-2-Kp) strain observed in Germany to date. After termination of the outbreak, we aimed to reconstruct transmission pathways by phylogenetics based on whole-genome sequencing (WGS).One hundred seventeen KPC-2-Kp isolates from 89 outbreak patients, 5 environmental KPC-2-Kp isolates, and 24 K pneumoniae strains not linked to the outbreak underwent WGS. Phylogenetic analysis was performed blinded to clinical data and based on the genomic reads.A patient from Greece was confirmed as the source of the outbreak. Transmission pathways for 11 out of 89 patients (12.4%) were plausibly explained by descriptive epidemiology, applying strict definitions. Five of these and an additional 15 (ie, 20 out of 89 patients [22.5%]) were confirmed by phylogenetics. The rate of phylogenetically confirmed transmissions increased significantly from 8 out of 66 (12.1% for the time period before) to 12 out of 23 patients (52.2% for the time period after; P?<.001) after implementation of systematic screening for KPC-2-Kp (33,623 screening investigations within 11 months). Using descriptive epidemiology, systematic screening showed no significant effect (7 out of 66 [10.6%] vs 4 out of 23 [17.4%] patients; P?=?.465). The phylogenetic analysis supported the assumption that a contaminated positioning pillow served as a reservoir for the persistence of KPC-2-Kp.Effective phylogenetic identification of transmissions requires systematic microbiologic screening. Extensive screening and phylogenetic analysis based on WGS should be started as soon as possible in a bacterial outbreak situation. Copyright © 2018 Association for Professionals in Infection Control and Epidemiology, Inc. Published by Elsevier Inc. All rights reserved.

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September 22, 2019

Revisiting the contribution of gene duplication of blaOXA-23 in carbapenem-resistant Acinetobacter baumannii.

Gene duplication has been discovered for many antimicrobial resistance genes in bacterial genomes and has been considered a source of elevated antimicrobial resistance.1 The gene blaOXA-23is a major determinant in the emergence of carbapenem-resistant Acinetobacter baumannii (CRAB).2–4 We have previously reported the widespread duplication of blaOXA-23by surveying 113 clinical CRAB isolates in China.5 However, in these isolates the blaOXA-23 copy number did not correlate well with the MIC of imipenem. A similar phenomenon was also reported recently by Yoon et al.6 One reasonable explanation is that, in addition to gene duplica- tions, other mechanisms might also impact on the MIC, such as the presence of specific outer membrane proteins and/ortheover-expression of resistance–nodulation–division (RND)-type efflux pumps.7 Often, these mechanisms might vary in their performance when in different genomic contexts. Instead of making comparisons between clinical isolates, in this study we cultured A. baumannii under treatment with carbapenem, thus avoiding any interference induced in different genomic contexts. If an increase in the blaOXA-23 copy number or MIC were to occur within the same strain, the contribution of gene duplication to carbapenem resistance would be acknowledged.

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September 22, 2019

Somatic second hit mutation of RASA1 in vascular endothelial cells in capillary malformation-arteriovenous malformation.

Capillary malformation-arteriovenous malformation (CM-AVM) is an autosomal dominant vascular disorder that is associated with inherited inactivating mutations of the RASA1 gene in the majority of cases. Characteristically, patients exhibit one or more focal cutaneous CM that may occur alone or together with AVM, arteriovenous fistulas or lymphatic vessel abnormalities. The focal nature and varying presentation of lesions has led to the hypothesis that somatic “second hit” inactivating mutations of RASA1 are necessary for disease development. In this study, we examined CM from four different CM-AVM patients for the presence of somatically acquired RASA1 mutations. All four patients were shown to possess inactivating heterozygous germline RASA1 mutations. In one of the patients, a somatic inactivating RASA1 mutation (c.1534C > T, p.Arg512*) was additionally identified in CM lesion tissue. The somatic RASA1 mutation was detected within endothelial cells specifically and was in trans with the germline RASA1 mutation. Together with the germline RASA1 mutation (c.2125C > T, p.Arg709*) in the same patient, the endothelial cell somatic RASA1 mutation likely contributed to lesion development. These studies provide the first clear evidence of the second hit model of CM-AVM pathogenesis. Copyright © 2017 Elsevier Masson SAS. All rights reserved.

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September 22, 2019

Mutant JAK3 signaling is increased by loss of wild-type JAK3 or by acquisition of secondary JAK3 mutations in T-ALL.

The Janus kinase 3 (JAK3) tyrosine kinase is mutated in 10% to 16% of T-cell acute lymphoblastic leukemia (T-ALL) cases. JAK3 mutants induce constitutive JAK/STAT signaling and cause leukemia when expressed in the bone marrow cells of mice. Surprisingly, we observed that one third of JAK3-mutant T-ALL cases harbor 2 JAK3 mutations, some of which are monoallelic and others that are biallelic. Our data suggest that wild-type JAK3 competes with mutant JAK3 (M511I) for binding to the common ? chain and thereby suppresses its oncogenic potential. We demonstrate that JAK3 (M511I) can increase its limited oncogenic potential through the acquisition of an additional mutation in the mutant JAK3 allele. These double JAK3 mutants show increased STAT5 activation and increased potential to transform primary mouse pro-T cells to interleukin-7-independent growth and were not affected by wild-type JAK3 expression. These data extend our insight into the oncogenic properties of JAK3 mutations and provide an explanation of why progression of JAK3-mutant T-ALL cases can be associated with the accumulation of additional JAK3 mutations.© 2018 by The American Society of Hematology.

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September 22, 2019

MUMmer4: A fast and versatile genome alignment system.

The MUMmer system and the genome sequence aligner nucmer included within it are among the most widely used alignment packages in genomics. Since the last major release of MUMmer version 3 in 2004, it has been applied to many types of problems including aligning whole genome sequences, aligning reads to a reference genome, and comparing different assemblies of the same genome. Despite its broad utility, MUMmer3 has limitations that can make it difficult to use for large genomes and for the very large sequence data sets that are common today. In this paper we describe MUMmer4, a substantially improved version of MUMmer that addresses genome size constraints by changing the 32-bit suffix tree data structure at the core of MUMmer to a 48-bit suffix array, and that offers improved speed through parallel processing of input query sequences. With a theoretical limit on the input size of 141Tbp, MUMmer4 can now work with input sequences of any biologically realistic length. We show that as a result of these enhancements, the nucmer program in MUMmer4 is easily able to handle alignments of large genomes; we illustrate this with an alignment of the human and chimpanzee genomes, which allows us to compute that the two species are 98% identical across 96% of their length. With the enhancements described here, MUMmer4 can also be used to efficiently align reads to reference genomes, although it is less sensitive and accurate than the dedicated read aligners. The nucmer aligner in MUMmer4 can now be called from scripting languages such as Perl, Python and Ruby. These improvements make MUMer4 one the most versatile genome alignment packages available.

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September 22, 2019

Reduction of nonspecificity motifs in synthetic antibody libraries.

Successful antibody development requires both functional binding and desirable biophysical characteristics. In the current study, we analyze the causes of one hurdle to clinical development, off-target reactivity, or nonspecificity. We used a high-throughput nonspecificity assay to isolate panels of nonspecific antibodies from two synthetic single-chain variable fragment libraries expressed on the surface of yeast, identifying both individual amino acids and motifs within the complementarity-determining regions which contribute to the phenotype. We find enrichment of glycine, valine, and arginine as both individual amino acids and as a part of motifs, and additionally enrichment of motifs containing tryptophan. Insertion of any of these motifs into the complementarity-determining region H3 of a “clean” antibody increased its nonspecificity, with greatest increases in antibodies containing Trp or Val motifs. We next applied these rules to the creation of a synthetic diversity library based on natural frameworks with significantly decreased incorporation of such motifs and demonstrated its ability to isolate binders to a wide panel of antigens. This work both provides a greater understanding of the drivers of nonspecificity and provides design rules to increase efficiency in the isolation of antibodies with drug-like properties. Copyright © 2017 Elsevier Ltd. All rights reserved.

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September 22, 2019

High-quality assembly of Dermatophagoides pteronyssinus genome and transcriptome reveals a wide range of novel allergens.

House dust mites (HDM) are a predominant source of inhalant allergens that attribute to over 50% of worldwide allergy cases, while the full spectrum of HDM allergens remains unknown. Here we sequenced a high-quality genome of Dermatophagoides (D.) pteronyssinus to find known canonical allergens and allergen orthologs inferred from D. farinae genome.

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