A novel activating, germline JAK2 mutation, JAK2R564Q, causes familial essential thrombocytosis

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Key Points• Activating mutations outside exon 10 of MPL were identified in 10% (7 of 69) of triplenegative cases of ET and PMF.• JAK2-V625F and JAK2-F556V were identified in 2 triple-negative cases of ET and were shown to activate JAK-STAT5 signaling.Essential thrombocythemia (ET) and primary myelofibrosis (PMF) are chronic diseases characterized by clonal hematopoiesis and hyperproliferation of terminally differentiated myeloid cells. The disease is driven by somatic mutations in exon 9 of CALR or exon 10 of MPL or JAK2-V617F in >90% of the cases, whereas the remaining cases are termed "triple negative." We aimed to identify the disease-causing mutations in the triple-negative cases of ET and PMF by applying whole-exome sequencing (WES) on paired tumor and control samples from 8 patients. We found evidence of clonal hematopoiesis in 5 of 8 studied cases based on clonality analysis and presence of somatic genetic aberrations. WES identified somatic mutations in 3 of 8 cases. We did not detect any novel recurrent somatic mutations. In 3 patients with clonal hematopoiesis analyzed by WES, we identified a somatic MPL-S204P, a germline MPL-V285E mutation, and a germline JAK2-G571S variant. We performed Sanger sequencing of the entire coding region of MPL in 62, and of JAK2 in 49 additional triple-negative cases of ET or PMF. New somatic (T119I, S204F, E230G, Y591D) and 1 germline (R321W) MPL mutation were detected. All of the identified MPL mutations were gain-of-function when analyzed in functional assays. JAK2 variants were identified in 5 of 57 triple-negative cases analyzed by WES and Sanger sequencing combined. We could demonstrate that JAK2-V625F and JAK2-F556V are gain-of-function mutations. Our results suggest that triple-negative cases of ET and PMF do not represent a homogenous disease entity. Cases with polyclonal hematopoiesis might represent hereditary disorders. (Blood. 2016;127(3):325-332) IntroductionEssential thrombocythemia (ET), primary myelofibrosis (PMF), and polycythemia vera (PV) are the classical BCR-ABL-negative myeloproliferative neoplasms (MPNs). They are chronic diseases, characterized by clonal expansion of differentiated myeloid cells driven by somatic mutations. Most of the cases are sporadic, however, familial clustering has also been observed. In 95% of PV cases and 50% to 60% of ET and PMF cases the disease is driven by an acquired mutation in the JAK2 gene V617F.1-4 JAK2-V617F leads to the constitutive activation of the Janus kinase 2 (JAK2) and subsequently the JAK-signal transducer and activator of transcription (STAT) signaling pathway. The remaining cases of PV carry mutations in exon 12 of JAK2. Although JAK2 mutations in MPNs are acquired, in recent years several families with hereditary thrombocytosis (HT) have been described to have germline JAK2 mutations. [5][6][7] The second most commonly mutated gene in ET and PMF is CALR encoding calreticulin. Mutations in exon 9 of CALR have been described in 25% and 35% of patients with ET and PMF, respectively. 8,9 There are o...

“…[5][6][7] In all cases, they were weak gain-of-function mutations. Here, we reported 4 different JAK2 mutations in 5 triple-negative ET cases.…”

Section: Discussionmentioning

confidence: 99%

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confidence: 99%

Whole-exome sequencing identifies novel MPL and JAK2 mutations in triple-negative myeloproliferative neoplasms

Feenstra

Nivarthi

Gisslinger

et al. 2016

238

143

“…8 Likewise, 2 germ-line mutations, JAK2 V617I (different from the V617F substitution classically found in 60% of sporadic essential thrombocythemias [ETs]) and JAK2 R564Q (never observed in sporadic MPNs), have been reported. [9][10][11] Several mutations in the TPO gene that lead to increased TPO translation have also been described in families with a thrombocytosis. [12][13][14][15] Here, we report 2 families with hereditary thrombocytosis carrying so far unidentified heterozygous germ-line mutations in JAK2.…”

Section: Introductionmentioning

confidence: 99%

Germ-line JAK2 mutations in the kinase domain are responsible for hereditary thrombocytosis and are resistant to JAK2 and HSP90 inhibitors

Marty

Saint‐Martin

Pecquet

et al. 2014

Key Points• New germ-line mutations of JAK2 in the kinase domain were identified.• Specificity for MPL and resistance to JAK2 and HSP90 inhibitors was determined.The main molecular basis of essential thrombocythemia and hereditary thrombocytosis is acquired, and germ-line-activating mutations affect the thrombopoietin signaling axis. We have identified 2 families with hereditary thrombocytosis presenting novel heterozygous germ-line mutations of JAK2. One family carries the JAK2 R867Q mutation located in the kinase domain, whereas the other presents 2 JAK2 mutations, S755R/ R938Q, located in cis in both the pseudokinase and kinase domains. Expression of Janus kinase 2 (JAK2) R867Q and S755R/R938Q induced spontaneous growth of Ba/F3-thrombopoietin receptor (MPL) but not of Ba/F3-human receptor of erythropoietin cells. Interestingly, both Ba/F3-MPL cells expressing the mutants and platelets from patients displayed thrombopoietin-independent phosphorylation of signal transducer and activator of transcription 1. The JAK2 R867Q and S755R/R938Q proteins had significantly longer half-lives compared with JAK2 V617F. The longer half-lives correlated with increased binding to the heat shock protein 90 (HSP90) chaperone and with higher MPL cell-surface expression. Moreover, these mutants were less sensitive to JAK2 and HSP90 inhibitors than JAK2 V617F. Our results suggest that the mutations in the kinase domain of JAK2 may confer a weak activation of signaling specifically dependent on MPL while inducing a decreased sensitivity to clinically available JAK2 inhibitors. (Blood. 2014;123(9):1372-1383

“…25 Furthermore, additional preliminary reports have identified other germline JAK2 mutations in patients with myeloproliferative phenotype. [26][27][28] In the present studies, we further explored whether the JAK2V617I mutation is sufficient to induce hematopoietic disease as a sole genetic event. We have also characterized the hematopoietic stem and progenitor cell phenotype of patients carrying the germline JAK2V617I mutation to gain insights into the impact of an isolated JAK2 mutation on human hematopoiesis.…”

Section: Introductionmentioning

confidence: 99%

“…28 Furthermore, a number of additional preliminary reports have detected other germline JAK2 mutations in patients with thrombocytosis. 26,27 As additional germline JAK2 mutations are discovered, detailed characterization of the clinical, hematopoietic, and signaling phenotypes associated with specific mutations, as described in this study, will provide important and novel insights into the biology of distinct JAK2 mutations in MPN pathogenesis.…”

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confidence: 99%

Impact of isolated germline JAK2V617I mutation on human hematopoiesis

Mead

Chowdhury

Pecquet

et al. 2013

Key Points• Germline JAK2V617I mutation as a sole genetic event does not suppress hematopoietic stem cells.• JAK2V617I induces weaker constitutive activation than JAK2V617F but considerable cytokine hyperresponsiveness.The association between somatic JAK2 mutation and myeloproliferative neoplasms (MPNs) is now well established. However, because JAK2 mutations are associated with heterogeneous clinical phenotypes and often occur as secondary genetic events, some aspects of JAK2 mutation biology remain to be understood. We recently described a germline JAK2V617I mutation in a family with hereditary thrombocytosis and herein characterize the hematopoietic and signaling impact of JAK2V617I. Through targeted sequencing of MPN-associated mutations, exome sequencing, and clonality analysis, we demonstrate that JAK2V617I is likely to be the sole driver mutation in JAK2V617I-positive individuals with thrombocytosis. Phenotypic hematopoietic stem cells (HSCs) were increased in the blood and bone marrow of JAK2V617I-positive individuals and were sustained at higher levels than controls after xenotransplantation. In signaling and transcriptional assays, JAK2V617I demonstrated more activity than wild-type JAK2 but substantially less than JAK2V617F. After cytokine stimulation, JAK2V617I resulted in markedly increased downstream signaling compared with wild-type JAK2 and comparable with JAK2V617F. These findings demonstrate that JAK2V617I induces sufficient cytokine hyperresponsiveness in the absence of other molecular events to induce a homogeneous MPN-like phenotype. We also provide evidence that the JAK2V617I mutation may expand the HSC pool, providing insights into both JAK2 mutation biology and MPN disease pathogenesis. (Blood. 2013;121(20):4156-4165)