Cristina Hurtado

LNK can also be mutated outside PH and SH2 domains in myeloproliferative neoplasms with and without V617FJAK2 mutation

LNK (SH2B3) is a member of a family of adaptor proteins that hare a proline-rich N-terminal dimerization domain, a pleckstrin omology (PH) domain, a Src homology 2 (SH2) domain, and a onserved C-terminal tyrosine residue [1]. It has been proposed s a putative tumor suppressor because it is a negative regulator f several cytokine receptors [2]. LNK−/− mice exhibit a MPN-like henotype with anomalies of hematopoiesis and abnormal accuulation of erythroid cells, megakaryocytes and B lymphocytes n different hematopoietic compartments [3]. In addition, some enome-wide association studies have found that one SNP in exon of this gene (rs3184504 and p.R262W) shows a significant assoiation with eosinophil counts (a common feature of some MPNs), everal blood parameters [4] and hypertension [5,6]. Recently, LNK mutations have been described in myeloprolifrative neoplasms, mainly in exon 2 that encodes part of the PH omain (encoded by exons 2–4) [7–9]. We have analysed the whole coding sequence of LNK in a cohort f 44 V617FJAK2 negative MPNs (four with polycythemia vera, 15 ith essential thrombocythemia, four with primary myelofibrosis, ne with chronic eosinophilic leukemia not otherwise specified, ve with MPNs unclassifiable and 15 with chronic myelomonoytic leukemia) [10] as well as 20 V617FJAK2 positive MPNs (8 V, 8 ET and 4 PMF) and 20 non-leukemic samples by dHPLC see Supplementary information), a highly sensitive, low-cost and edium-throughput method. The melting characteristics of exon (even when divided into fragments) made it impossible to scan or mutations by dHPLC analysis, so in this case samples were irectly sequenced. Results (Table 1) showed the presence of a eletion of 24 bp in exon 8 in a PMF V617FJAK2 negative sample c.1795 1818del and p.S480 E487del) and a c.1217T>C (p.F287S) hange in exon 4 of a PV V617FJAK2 positive sample (Fig. 1) also arboring a TET2 p.N1385S mutation. No changes were detected in NK exon 2. Analysis of exons 3, 4 and the coding part of the exon

CBL mutations in myeloproliferative neoplasms are also found in the gene's proline-rich domain and in patients with the V617FJAK2

Background Despite the discovery of the p.V617F in JAK2, the molecular pathogenesis of some chronic myeloproliferative neoplasms remains unclear. Although very rare, different studies have identified CBL (Cas-Br-Murine ecotropic retroviral transforming sequence) mutations in V617FJAK2-negative patients, mainly located in the RING finger domain. In order to determine the frequency of CBL mutations in these diseases, we studied different regions of all CBL family genes (CBL, CBLB and CBLC) in a selected group of patients with myeloproliferative neoplasms. We also included V617FJAK2-positive patients to check whether mutations in CBL and JAK2 are mutually exclusive events. Design and Methods Using denaturing high performance liquid chromatography, we screened for mutations in CBL, CBLB and CBLC in a group of 172 V617FJAK2-negative and 232 V617FJAK2-positive patients with myeloproliferative neoplasms not selected for loss of heterozygosity. The effect on cell proliferation of the mutations detected was analyzed on a 32D(FLT3) cell model. Results An initial screening of all coding exons of CBL, CBLB and CBLC in 44 V617FJAK2-negative samples revealed two new CBL mutations (p.C416W in the RING finger domain and p.A678V in the proline-rich domain). Analyses performed on 128 additional V617FJAK2-negative and 232 V617FJAK2-positive samples detected three CBL changes (p.T402HfsX29, p.P417R and p.S675C in two cases) in four V617FJAK2-positive patients. None of these mutations was found in 200 control samples. Cell proliferation assays showed that all of the mutations promoted hypersensitivity to interleukin-3 in 32D(FLT3) cells. Conclusions Although mutations described to date have been found in the RING finger domain and in the linker region of CBL, we found a similar frequency of mutations in the proline-rich domain. In addition, we found CBL mutations in both V617FJAK2-positive (4/232; 1.7%) and negative (2/172; 1.2%) patients and all of them promoted hypersensitivity to interleukin-3.

A new potential oncogenic mutation in the FERM domain of JAK2 in BCR/ABL1-negative and V617F-negative chronic myeloproliferative neoplasms revealed by a comprehensive screening of 17 tyrosine kinase coding genes

BCR/ABL1-negative chronic myeloproliferative neoplasms (CMPNs) are a heterogeneous group of clonal hematological malignancies. Over recent years, some genetic events in tyrosine kinase (TK) genes have been described as causal events of these diseases. To identify new genetic aberrations underlying these diseases, we used denaturing high performance liquid chromatography and fluorescence in situ hybridization (FISH) to analyze 17 genes from two receptor-TK families (III and IV) and from three cytoplasmic-TK families (Syk, Abl, and Jak) on samples from 44 BCR/ABL1-negative and JAK2(V617F)-negative CMPN patients with different clinical phenotypes. Although screening by FISH did not reveal novel chromosomal aberrations, several sequence changes were detected. None of them were frequent events, but we identified a new potential activating mutation in the FERM domain of JAK2(R340Q). None of the germline JAK2(V617F) single-nucleotide polymorphisms detected differed in distribution between patients and control subjects. In summary, data presented here show that these genes are not frequently mutated or rearranged in CMPNs, suggesting that molecular events causing these disorders must be located in other genes.

A meta-analysis of TET2 mutations shows a distinct distribution pattern in de novo acute myeloid leukemia and chronic myelomonocytic leukemia

Recurrent loss of heterozygosity (LOH) in 4q24 in patients with myeloid malignancies has led to the identifi cation of mutations in TET2 in diff erent myeloid neoplasms including myeloproliferative neoplasm (MPN), myelodysplastic syndrome (MDS), mixed myelodysplastic/myeloproliferative neoplasm (MDS/MPN) and acute myeloid leukemia (AML) [1 – 13]. Th eir impact on prognosis remains unresolved, and the mechanisms by which TET2 leads to transformation remain unclear. Based on its homology to TET1, it is possible that TET2 may play a role in epigenetic regulation [14] via the presence of catalytic domains in the two highly conserved regions of the gene. Mutations in these two regions could, by this mechanism, lead to inactivation of a specifi c tumor suppressor gene and/or activation of pro-proliferative pathways, although mutations in other areas of the gene could also result in a modifi cation of the protein. Studies using murine models of Tet2 inactivation show that inactivation of this gene establishes important roles for Tet2 in hematopoiesis and the development of myeloproliferative disease. Beacuse TET2 alterations in patients are commonly heterozygous, TET2 haploinsuffi ciency could be enough to promote defects in a hematopoietic progenitor and myeloproliferation [15]. We have collected data on all TET2 mutations published to date [1 – 13] (a total of 725 mutations in 3274 patients) (Table I) in order to analyze whether: (a) they are evenly distributed throughout the gene, (b) diff erent mutation types (missense, nonsense or frameshift) are more prevalent in diff erent regions of the gene and (c) distinct mutational patterns are found in diff erent types of myeloid neoplasms. To this end, we divided the sequence of the TET2 gene into four regions. Region B1 ( Box 1 ) and region B2 ( Box 2 ) are highly conserved in several species and in all three proteins of the TET family. Th e other two regions, which we denominated F1 and F2, are non-conserved regions (Figure 1). We calculated the frequency of mutations per amino acid in each of these regions (number of mutations divided by the number of amino acids

Low frequency of JAK2 exon 12 mutations in classic and atypical CMPDs.

The BCR-ABL negative chronic myeloproliferative disorders (CMPDs) are a group of stem cell clonal haematological malignancies characterised by abnormal proliferation and survival of one or more myeloid lineage cells. It has been proposed that they are caused by abnormalities in some signal transduction pathways mainly due to acquired somatic mutations, some of them in tyrosine kinase genes. One of the most prevalent mutations is V617F in the pseudokinase domain (JH2) coded by JAK2 exon 14. This mutation, reported in 2005, has been associated with nearly 95% of patients diagnosed of polycythaemia vera (PV) and near a half of the patients with essential thrombocythaemia (ET) and primary myelofibrosis (MF). However, the frequency of this mutation is below 20% for the remaining chronic myeloproliferative disorders and is absent in lymphoid neoplasms. JAK2V617F has been an important milestone in the knowledge of the molecular mechanisms leading to classic myeloproliferative disorders. However, there are still some patients with PV, ET and MF lacking V617F whose molecular defect is unknown. The disease in these cases could be due to other abnormalities in JAK2 or other related genes. In fact, in a few cases new point mutations have been reported, affecting also the JH2 domain, and one mutation affecting the JH1 domain [1] and reviewed in [2], some of them in other haematological malignancies. In addition, 10% of patients diagnosed of MF and some with ET show somatic activating mutations in the thrombopoietin receptor gene (MPL) inducing constitutive cytokine-independent activation of the JAK-STAT pathway.

CBL RING finger deletions are common in core-binding factor acute myeloid leukemias

Core-binding factor acute myeloid leukemia ( CBF -AML) is an acute myeloid neoplasm characterized by the presence of t(8;21)(q22;q22) [abbreviated as t(8;21)] or inv(16)(p13q22)/ t(16;16)(p13;q22) [inv(16)] that lead to fusion genes RUNX1/ RUNX1T1 and CBFB – MYH11 , respectively. Both chimeric transcripts are considered diagnostic markers of disease [1]. RUNX1 and CBFB code for subunits of the heterodimeric transcription factor core-binding factor (CBF). AML development is a multistep process that requires the cooperation of several genetic aberrations [2]. Class I mutations activate signal transduction pathways conferring a proliferative advantage, and they are mainly found in the receptor tyrosine kinase (RTK) genes FLT3 and KIT , but also in the GTPase-coding genes KRAS or NRAS . On the other hand, class II mutations aff ect transcription factors impairing differentiation, and are mainly found in CBF, C/EBP α and MLL. Both types of mutations would explain the accumulation of a large number of immature myeloid cells and loss of the capacity of diff erentiation into mature functional blood cells that characterize AML. In the last few years, the discovery of other aberrations in genes involved in epigenetic regulation has revealed an increasing complexity of cooperation of different abnormalities (for a review see [3]). Th e identifi cation of these mutations might have a signifi cant impact on the development of new therapeutic strategies and could explain the low rate of clinical responses with some single targeted therapies, such as FLT3 inhibitors [1]. In CBF -leukemias, class I activating mutations are mainly found in KIT (up to 40%), although some patients show the FLT3 D835Y change (up to 24% of CBFB – MYH11 and 7% of RUNX1/RUNX1T1 positive patients) and HRAS , NRAS and KRAS mutations. FLT3 internal tandem duplication (ITD) seems to be rare [1]. Several studies have identifi ed CBL mutations in myeloid neoplasms with frequencies ranging from 1 to 33%, mainly in patients without mutations in other molecules involved in signaling pathways such as FLT3 , JAK2 , NF1 , PTPN11 or RAS [4 – 7]. CBL ( Cas-Br-Murine ecotropic retroviral transforming sequence ) encodes an E3-ubiquitin ligase that acts as negative regulator of several RTKs [8], so mutations of this gene could be considered also as class I aberrations. Here, we have analyzed 26 samples of patients with primary AML for the presence of CBL mutations. All of them were classifi ed as CBF -leukemias, characterized by the presence of CBFB – MYH11 (in 12 cases) or RUNX1/RUNX1T1 (in 14 cases). Using denaturing high performance liquid

p.Y317H is a new JAK2 gain-of-function mutation affecting the FERM domain in a myelofibrosis patient with CALR mutation

p.V617F JAK2 mutation is the most frequent mutation in myeloproliferative neoplasms (MPNs), present in almost all patients with polycythaemia vera (PV), 50% of essential thrombocythemia (ET) and myelofibrosis (MF), and in some cases of atypical MPNs, myelodysplasia and AML. This mutation is located in exon 14 and affects the pseudokinase domain, resulting in the constitutive activation of kinase function. Other mutations have been found in this domain in PV, such as p.C616Y, p.C618R and p.D620E, or the somatic deletion of five amino acids (ΔIREED) that defines a distinct acute lymphoblastic leukemia (ALL) subgroup associated with trisomy 21. A region linking SH2 and JH2 domains (exon 12, residues 537 to 543) can be also mutated in some PV patients. During a comprehensive mutational screening of several tyrosine kinase genes, our group identified a new point mutation in JAK2 exon 8 (p.R340Q) in a MF patient. Subsequent analyses in additional samples from 443 MPN patients (174 p.V617F JAK2 negative and 269 p.V617F JAK2 positive), as well as 355 control samples without leukemia, led us to the identification of additional changes (p.Y317H and p.N337D) in the same exon. All of them were found in p.V617F JAK2 negative and MPL non-mutated MF cases, but none was recurrent. They affect the FERM domain of the protein, and they have not been described as SNPs in sequence databases except for p.N337D which was described as SNP (rs149683525) in 2014. In silico analysis with PolyPhen-2 showed that

A simple approach for classifying new mutations as somatic or germinal in DNA samples lacking paired tissue

When studying mutations in DNA samples, determining whether novel sequence changes are somatic mutations or germline polymorphisms can be difficult. Here we describe a novel and very simple approach for identification of somatic mutations and loss of heterozygosity (LoH) events in DNA samples where no matched tissue sample is available. Our method makes use of heterozygous polymorphisms that are located near the putative mutation to trace both germinal alleles.

A new KRT16 mutation associated with a phenotype of pachyonychia congenita

Pachyonychia congenita is a rare, autosomal dominant genetic disease characterized by painful palmoplantar keratoderma and hypertrophic nail dystrophy. This disorder is caused by mutations in any one of five cytoskeletal keratin proteins, K6a, K6b, K6c, K16 and K17. Here, we describe a new p.Leu421Pro (c.1262T>C) mutation in the highly conserved helix termination motif of K16 in a large Spanish family. Bioinformatic analyses as well as previous descriptions in the literature of homologous mutations in other keratin‐coding genes show that this mutation is probably causative of the disease.

Quantification of PDGFRA alternative transcripts improves the screening for X–PDGFRA fusions by reverse transcriptase-polymerase chain reaction

Hematological malignancies with eosinophilia are often associated with fusions in PDGFRA, PDGFRB, or FGFR1 genes. RT-PCR has proved to be useful for finding new PDGFRA gene fusions, but some studies have shown overexpression of the TK domain which cannot be explained by the existence of such aberrations. This fact could be related to the expression of alternative PDGFRA transcripts. We show that quantification of the expression of three different PDGFRA fragments discriminates between PDGFRA alternative transcripts and fusion genes, and we have tested this novel methodological approach in a group of eosinophilia cases. Our data show that alternative PDGFRA transcripts should be taken into account when screening for PDGFRA aberrations, such as gene fusions, by RT-PCR. Expression from an internal PDGFRA promoter seems to be a frequent event, in both normal and leukemic samples, and is probably related to physiological conditions, but it could have a role in other tumors. Even so, we show that our RQ-PCR methodology can discriminate expression of alternative transcripts from the presence of X–PDGFRA fusion genes.