Most patients with essential thrombocythemia or primary myelofibrosis that was not associated with a JAK2 or MPL alteration carried a somatic mutation in CALR. The clinical course in these patients was more indolent than that in patients with the JAK2 V617F mutation. (Funded by the MPN Research Foundation and Associazione Italiana per la Ricerca sul Cancro.).

BackgroundLong non-coding RNAs (lncRNAs) are increasingly implicated as gene regulators and may ultimately be more numerous than protein-coding genes in the human genome. Despite large numbers of reported lncRNAs, reference annotations are likely incomplete due to their lower and tighter tissue-specific expression compared to mRNAs. An unexplored factor potentially confounding lncRNA identification is inter-individual expression variability. Here, we characterize lncRNA natural expression variability in human primary granulocytes.ResultsWe annotate granulocyte lncRNAs and mRNAs in RNA-seq data from 10 healthy individuals, identifying multiple lncRNAs absent from reference annotations, and use this to investigate three known features (higher tissue-specificity, lower expression, and reduced splicing efficiency) of lncRNAs relative to mRNAs. Expression variability was examined in seven individuals sampled three times at 1- or more than 1-month intervals. We show that lncRNAs display significantly more inter-individual expression variability compared to mRNAs. We confirm this finding in two independent human datasets by analyzing multiple tissues from the GTEx project and lymphoblastoid cell lines from the GEUVADIS project. Using the latter dataset we also show that including more human donors into the transcriptome annotation pipeline allows identification of an increasing number of lncRNAs, but minimally affects mRNA gene number.ConclusionsA comprehensive annotation of lncRNAs is known to require an approach that is sensitive to low and tight tissue-specific expression. Here we show that increased inter-individual expression variability is an additional general lncRNA feature to consider when creating a comprehensive annotation of human lncRNAs or proposing their use as prognostic or disease markers.Electronic supplementary materialThe online version of this article (doi:10.1186/s13059-016-0873-8) contains supplementary material, which is available to authorized users.

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Studying the relationship between virus infection and cellular response is paradigmatic for our understanding of how perturbation changes biological systems. Immune response, in this context is a complex yet evolutionarily adapted and robust cellular change, and is experimentally amenable to molecular analysis. To visualize the full cellular response to virus infection, we performed temporal transcriptomics, proteomics, and phosphoproteomics analysis of vesicular stomatitis virus (VSV)-infected mouse macrophages. This enabled the understanding of how infection-induced changes in host gene and protein expression are coordinated with post-translational modifications by cells in time to best measure and control the infection process. The vast and complex molecular changes measured could be decomposed in a limited number of clusters within each category (transcripts, proteins, and protein phosphorylation) each with own kinetic parameter and characteristic pathways/processes, suggesting multiple regulatory options in the overall sensing and homeostatic program. Altogether, the data underscored a prevalent executive function to phosphorylation. Resolution of the molecular events affecting the RIG-I pathway, central to viral recognition, reveals that phosphorylation of the key innate immunity adaptor mitochondrial antiviral-signaling protein (MAVS) on S328/S330 is necessary for activation of type-I interferon and nuclear factor κ B (NFκB) pathways. To further understand the hierarchical relationships, we analyzed kinase–substrate relationships and found RAF1 and, to a lesser extent, ARAF to be inhibiting VSV replication and necessary for NFκB activation, and AKT2, but not AKT1, to be supporting VSV replication. Integrated analysis using the omics data revealed co-regulation of transmembrane transporters including SLC7A11, which was subsequently validated as a host factor in the VSV replication. The data sets are predicted to greatly empower future studies on the functional organization of the response of macrophages to viral challenges.

Myeloproliferative neoplasms (MPNs) repre sent a group of diseases that affect the myeloid lineage, characterized by the presence of an excess of terminally differentiated myeloid cells. Defects causing clonal hematopoiesis are a key factor in the emergence of these diseases. Throughout the years, a number of causative defects have been identified, predominantly affecting cytokine signaling and gene expression regulation. This review aims to provide an update on the current status of the MPN field in relation to identification of molecular defects involved in the disease and its clonal evolution.

The classical, BCR-ABL1 negative myeloproliferative neoplasms (MPN) are polycythemia vera (PV), essential thrombocythemia (ET) and primary myelofibrosis (PMF). The most common genetic alteration in MPN is the JAK2-V617F mutation detected in 95% of PV patients and in 50-60% of patients with ET or PMF. Mutations in exon 12 of JAK2 and in the thrombopoietin receptor gene MPL are found in an additional 5-10% of the cases. In recent years a number of other genes were shown to be affected in MPN. However, these mutations are not mutually exclusive with JAK2 and MPL mutations and are also found in other myeloid malignancies. A specific molecular marker for the remaining 40% of ET or PMF patients with wild type JAK2 and MPL is still unknown. We used whole-exome sequencing to identify novel mutations in PMF patients with wild type JAK2 and MPL. The analysis revealed recurrent somatic insertions and deletions in CALR encoding for calreticulin. All detected mutations resulted in a frameshift and clustered in the last exon (exon 9) of the gene. Following up on this finding we developed a PCR based assay to screen 1107 MPN patients for insertion/deletion mutations in exon 9 of CALR. No mutations were detected in PV. In ET and PMF CALR mutations were mutually exclusive with mutant JAK2 and mutant MPL. Of the patients with wild type JAK2 and MPL, 67% ET and 88% PMF had mutant CALR. We also tested 19 patients with wild type CALR-exon 9 for mutations in the other exons of the gene, but all were negative. Furthermore we did not find CALR-exon 9 mutations in 254 patients with de novo acute myeloid leukemia, 45 with chronic myeloid leukemia, 73 with myelodysplastic syndrome or 64 with chronic myelomonocytic leukemia. Out of 24 patients with refractory anemia with ringed sideroblasts associated with marked thrombocytosis (RARS-T), 3 patients carried CALR mutations. These patients were wild type for JAK2 and MPL. In total we detected 36 different types of mutations in CALR. A 52 bp deletion and a 5 bp insertion were the most prominent types found in 53% and 32% of all cases with mutant CALR. All 36 types of mutations result in a frameshift to the same alternative reading frame, generating a novel C-terminus of the mutated protein. The wild type C-terminal region of CALR contains a high-capacity calcium-binding domain and is highly negatively charged. As a consequence of the frameshift mutations the negatively charged amino acids are replaced by both neutral and positively charged amino acids. In addition, an endoplasmic reticulum retention signal present in the wild type protein is lost in the mutant variants. Expression in HEK cells showed that wild type CALR localizes in the endoplasmic reticulum, whereas this localization is less prominent in cells expressing mutant CALR. This observation is in line with the loss of the endoplasmic reticulum retention signal in the mutant protein. Overexpression of the most common CALR mutation (a 52 bp deletion) in interleukin-3 (IL-3) dependent Ba/F3 cells led to IL-3-independent growth and hypersensitivity to IL-3. Cells overexpressing the mutant were sensitive to the JAK-family kinase inhibitor SAR302503 and showed elevated STAT5 phosphorylation in absence of IL-3. This indicates that JAK-STAT signaling is involved in the observed cytokine independent growth of mutant CALR expressing Ba/F3 cells. ET and PMF patients with mutant CALR present with lower white blood cell counts (P<0.001 for ET, P=0.027 for PMF) and elevated platelet levels (P<0.001 in both entities) compared to patients with mutant JAK2. In both disease entities patients with mutant CALR show significantly better overall survival than patients with mutant JAK2 (P=0.043 in ET, P<0.001 in PMF). ET patients with mutant CALR had a lower risk of thrombosis in comparison to those with mutant JAK2 (P=0.003). Mutant CALR is a novel, specific molecular marker detected in the majority of MPN patients negative for JAK2 and MPL mutations. Use of this marker in the clinic is expected to improve diagnostic and therapeutic decision-making in MPN. Disclosures: No relevant conflicts of interest to declare.