Milosevic Feenstra Jelena D, Nivarthi Harini, Gisslinger Heinz, Leroy Emilie, Rumi Elisa, Chachoua Ilyas, Bagienski Klaudia, Kubesova Blanka, Pietra Daniela, Gisslinger Bettina, Milanesi Chiara, Jäger Roland, Chen Doris, Berg Tiina, Schalling Martin, Schuster Michael, Bock Christoph, Constantinescu Stefan N, Cazzola Mario, Kralovics Robert
CeMM Research Center for Molecular Medicine of the Austrian Academy of Sciences, Vienna, Austria;
Department of Internal Medicine I, Division of Hematology and Blood Coagulation, Medical University of Vienna, Vienna, Austria;
Blood. 2016 Jan 21;127(3):325-32. doi: 10.1182/blood-2015-07-661835. Epub 2015 Sep 30.
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.
原发性血小板增多症（ET）和原发性骨髓纤维化（PMF）是慢性疾病，其特征为克隆性造血和终末分化髓系细胞的过度增殖。在超过90%的病例中，该疾病由CALR外显子9、MPL外显子10或JAK2-V617F的体细胞突变驱动，而其余病例则被称为“三阴性”。我们旨在通过对8例患者的肿瘤和对照样本进行全外显子测序（WES），来鉴定ET和PMF三阴性病例中的致病突变。基于克隆性分析和体细胞遗传畸变的存在，我们在8例研究病例中的5例中发现了克隆性造血的证据。WES在8例病例中的3例中鉴定出体细胞突变。我们未检测到任何新的复发性体细胞突变。在通过WES分析的3例克隆性造血患者中，我们鉴定出一个体细胞MPL-S204P、一个胚系MPL-V285E突变和一个胚系JAK2-G571S变异。我们对另外62例ET或PMF三阴性病例的MPL整个编码区以及49例JAK2进行了桑格测序。检测到新的体细胞（T119I、S204F、E230G、Y591D）和1个胚系（R321W）MPL突变。在功能分析中，所有鉴定出的MPL突变均为功能获得性突变。通过WES和桑格测序联合分析，在57例三阴性病例中的5例中鉴定出JAK2变异。我们能够证明JAK2-V625F和JAK2-F556V是功能获得性突变。我们的结果表明，ET和PMF的三阴性病例并不代表一个同质的疾病实体。多克隆造血的病例可能代表遗传性疾病。
