JAK2 Exon 14 Skipping in Patients with Primary Myelofibrosis: A Minor Splice Variant Modulated by the JAK2-V617F Allele Burden

Background Primary myelofibrosis (PMF) is an acquired clonal disease of the hematopoietic stem cell compartment, characterized by bone marrow fibrosis, anemia, splenomegaly and extramedullary hematopoiesis. About 60% of patients with PMF harbor a somatic mutation of the JAK2 gene (JAK2-V617F) in their hematopoietic lineage. Recently, a splicing isoform of JAK2, lacking exon 14 (JAK2Δ14) was described in patients affected by myeloproliferative diseases. Materials and Methods By using a specific RT-qPCR method, we measured the ratio between the splicing isoform and the JAK2 full-length transcript (JAK2+14) in granulocytes, isolated from peripheral blood, of forty-four patients with PMF and nine healthy donors. Results We found that JAK2Δ14 was only slightly increased in patients and, at variance with published data, the splicing isoform was also detectable in healthy controls. We also found that, in patients bearing the JAK2-V617F mutation, the percentage of mutated alleles correlated with the observed increase in JAK2Δ14. Homozygosity for the mutation was also associated with a higher level of JAK2+14. Bioinformatic analysis indicates the possibility that the G>T transversion may interfere with the correct splicing of exon 14 by modifying a splicing regulatory sequence. Conclusions Increased levels of JAK2 full-length transcript and a small but significant increase in JAK2 exon 14 skipping, are associated with the JAK2-V617F allele burden in PMF granulocytes. Our data do not confirm a previous claim that the production of the JAK2Δ14 isoform is related to the pathogenesis of PMF.

[1]  E. Mansfield,et al.  Diagnosis of Down syndrome and other aneuploidies using quantitative polymerase chain reaction and small tandem repeat polymorphisms. , 1993, Human molecular genetics.

[2]  T. He,et al.  Erythropoietin-dependent Inhibition of Apoptosis Is Supported by Carboxyl-truncated Receptor Forms and Blocked by Dominant-negative Forms of Jak2 (*) , 1995, The Journal of Biological Chemistry.

[3]  U. Schumacher,et al.  Derivation of a new hematopoietic cell line with endothelial features from a patient with transformed myeloproliferative syndrome , 2000, Cancer.

[4]  U. Schumacher,et al.  Derivation of a new hematopoietic cell line with endothelial features from a patient with transformed myeloproliferative syndrome , 2000, Cancer.

[5]  G. Dreyfuss,et al.  Role of the Nonsense-Mediated Decay Factor hUpf3 in the Splicing-Dependent Exon-Exon Junction Complex , 2001, Science.

[6]  F. Speleman,et al.  Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes , 2002, Genome Biology.

[7]  Jo Vandesompele,et al.  Quantification of splice variants using real-time PCR , 2001, Nucleic Acids Res..

[8]  L. Maquat,et al.  Evidence for a Pioneer Round of mRNA Translation mRNAs Subject to Nonsense-Mediated Decay in Mammalian Cells Are Bound by CBP80 and CBP20 , 2001, Cell.

[9]  V. Cirigliano,et al.  X chromosome dosage by quantitative fluorescent PCR and rapid prenatal diagnosis of sex chromosome aneuploidies. , 2002, Molecular human reproduction.

[10]  A. Krainer,et al.  Listening to silence and understanding nonsense: exonic mutations that affect splicing , 2002, Nature Reviews Genetics.

[11]  J. Solassol,et al.  Rapid detection of common autosomal aneuploidies by quantitative fluorescent PCR on uncultured amniocytes , 2002, European Journal of Human Genetics.

[12]  Jinhua Wang,et al.  ESEfinder: a web resource to identify exonic splicing enhancers , 2003, Nucleic Acids Res..

[13]  Gene W. Yeo,et al.  Systematic Identification and Analysis of Exonic Splicing Silencers , 2004, Cell.

[14]  N. Bresolin,et al.  Silencer elements as possible inhibitors of pseudoexon splicing. , 2004, Nucleic acids research.

[15]  M. Hentze,et al.  Nonsense-mediated decay approaches the clinic , 2004, Nature Genetics.

[16]  L. Chasin,et al.  Computational definition of sequence motifs governing constitutive exon splicing. , 2004, Genes & development.

[17]  Mario Cazzola,et al.  A gain-of-function mutation of JAK2 in myeloproliferative disorders. , 2005, The New England journal of medicine.

[18]  Stefan N. Constantinescu,et al.  A unique clonal JAK2 mutation leading to constitutive signalling causes polycythaemia vera , 2005, Nature.

[19]  Sandra A. Moore,et al.  Activating mutation in the tyrosine kinase JAK2 in polycythemia vera, essential thrombocythemia, and myeloid metaplasia with myelofibrosis. , 2005, Cancer cell.

[20]  P. Campbell,et al.  Acquired mutation of the tyrosine kinase JAK2 in human myeloproliferative disorders , 2005, The Lancet.

[21]  Jun Kawai,et al.  A Simple Physical Model Predicts Small Exon Length Variations , 2006, PLoS genetics.

[22]  G. Ast,et al.  Comparative analysis identifies exonic splicing regulatory sequences--The complex definition of enhancers and silencers. , 2006, Molecular cell.

[23]  B. Frey,et al.  Quantitative microarray profiling provides evidence against widespread coupling of alternative splicing with nonsense-mediated mRNA decay to control gene expression. , 2006, Genes & development.

[24]  H. Drexler,et al.  JAK2 V617F tyrosine kinase mutation in cell lines derived from myeloproliferative disorders , 2006, Leukemia.

[25]  Tania Nolan,et al.  Quantification of mRNA using real-time RT-PCR , 2006, Nature Protocols.

[26]  François Girodon,et al.  The JAK2-V617F mutation is frequently present at diagnosis in patients with essential thrombocythemia and polycythemia vera. , 2006, Blood.

[27]  S. Brenner,et al.  Unproductive splicing of SR genes associated with highly conserved and ultraconserved DNA elements , 2007, Nature.

[28]  P. Duerksen-Hughes,et al.  Splicing and splice factor SRp55 participate in the response to DNA damage by changing isoform ratios of target genes. , 2008, Gene.

[29]  R. Kralovics Genetic complexity of myeloproliferative neoplasms , 2008, Leukemia.

[30]  Michael Q. Zhang,et al.  RNA landscape of evolution for optimal exon and intron discrimination , 2008, Proceedings of the National Academy of Sciences.

[31]  A. Tefferi,et al.  Classification and diagnosis of myeloproliferative neoplasms: The 2008 World Health Organization criteria and point-of-care diagnostic algorithms , 2008, Leukemia.

[32]  Andrew Collins,et al.  JAK2 haplotype is a major risk factor for the development of myeloproliferative neoplasms , 2009, Nature Genetics.

[33]  C. Béroud,et al.  Human Splicing Finder: an online bioinformatics tool to predict splicing signals , 2009, Nucleic acids research.

[34]  P. Campbell Somatic and germline genetics at the JAK2 locus , 2009, Nature Genetics.

[35]  Joseph K. Pickrell,et al.  Noisy Splicing Drives mRNA Isoform Diversity in Human Cells , 2010, PLoS genetics.

[36]  Xiuqiang Wang,et al.  JAK2 Exon 14 Deletion in Patients with Chronic Myeloproliferative Neoplasms , 2010, PloS one.

[37]  A. Tefferi Mutations galore in myeloproliferative neoplasms: Would the real Spartacus please stand up? , 2011, Leukemia.

[38]  N. Cross Genetic and epigenetic complexity in myeloproliferative neoplasms. , 2011, Hematology. American Society of Hematology. Education Program.

[39]  V. Spasovski,et al.  The influence of novel transcriptional regulatory element in intron 14 on the expression of Janus kinase 2 gene in myeloproliferative neoplasms , 2012, Journal of Applied Genetics.

[40]  A. Green,et al.  Molecular diagnosis of the myeloproliferative neoplasms: UK guidelines for the detection of JAK2 V617F and other relevant mutations , 2013, British journal of haematology.

[41]  C. Ghigna,et al.  HnRNP A1 controls a splicing regulatory circuit promoting mesenchymal-to-epithelial transition , 2013, Nucleic acids research.

[42]  A. Krainer,et al.  Splicing factor SRSF6 promotes hyperplasia of sensitized skin , 2014, Nature Structural &Molecular Biology.

[43]  R. Amann,et al.  Predictive Identification of Exonic Splicing Enhancers in Human Genes , 2022 .