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標題: | 鈣網蛋白突變在骨髓增生性腫瘤的角色 The Roles of Calreticulin Mutations in Myeloproliferative Neoplasms |
作者: | Ken-Hong Lim 林建鴻 |
指導教授: | 郭遠燁(Yuan-Yeh Kuo) |
共同指導教授: | 周文堅(Wen-Chien Chou) |
關鍵字: | B細胞,鈣網蛋白,原發性血小板增多症,高分辨率融合分析,免疫,突變,斑馬魚, B cell,CALR,essential thrombocythemia,high-resolution melting analysis,immune,mutation,zebrafish, |
出版年 : | 2017 |
學位: | 博士 |
摘要: | 骨髓增生性腫瘤是克隆性造血幹細胞疾病,並且可以分類為“典型”和“非典型”骨髓增生性腫瘤。典型骨髓增生性腫瘤通常會在周邊的血液中表現出終端骨髓細胞的擴增,包括真性紅血血球增多症、原發性血小板增多症、原發性骨髓纖維化和慢性骨髓性白血病。近年來,在大約30%的JAK2/MPL-未突變的骨髓增生性腫瘤中發現鈣網蛋白(Calreticulin, CALR)突變。目前,鈣網蛋白突變已經成為診斷原發性血小板增多症和原發性骨髓纖維化的重要克隆標誌物。
這項研究的第一個目的是開發一種快速和敏感的篩選工具,用於檢測鈣網蛋白突變。我們使用CFX Connect即時系統成功開發了高分辨率熔解分析,以檢測原發性血小板增多症病人的鈣網蛋白第9外顯子突變。我們的高分辨率熔解分析系統在識別病人基因組DNA中的鈣網蛋白第1型和第2型突變體的最大敏感性為2.5%。我們的高分辨率熔解分析系統假陽性率為3%,並且無假陰性出現。 本研究的第二個目的是評估台灣原發性血小板增多症病人鈣網蛋白突變和JAK2 /CALR共突變的臨床和預後意義。我們在92例成年原發性血小板增多症病人中利用高分辨率熔解分析篩選了鈣網蛋白第9外顯子的改變,隨後並且進行了TA克隆。我們在21例(22.8%)病人中鑑定出典型的鈣網蛋白插入/刪除型突變。鈣網蛋白突變與年齡較輕(p = 0.025),血小板數較高(p <0.001)和較低的血紅素(p = 0.016)有相關。有趣的是,我們檢測到在59例JAK2突變的原發性血小板增多症病人中有13例(22%)的鈣網蛋白第9外顯子的改變。與這些具有JAK2 /CALR共突變的原發性血小板增多症病人有相關的因子包括年齡較大(p = 0.025),診斷後發生血栓事件較多(p = 0.048),診斷後主要動脈血栓事件較多(p = 0.022),且較多屬於血栓出血併發症高風險組病人(p = 0.023)。我們的研究顯示JAK2突變的原發性血小板增多症病人可以出現頻繁的鈣網蛋白第9外顯子的改變,並且界定出一群具有血栓事件風險增加的病人亞群。 本研究的第三個目的是要探討鈣網蛋白突變之原發性血小板增多症病人的B細胞免疫特徵。我們篩選了54例台灣成年原發性血小板增多症病人的鈣網蛋白突變並評估了其B細胞免疫特徵。在這54例原發性血小板增多症病人中有19例(35.2%)具有8種不同類型的鈣網蛋白笫9外顯子突變,其中包括4例(7.4%)同時伴有JAK2V617F共突變的病人。經過年齡、性別、追踨期和血液學參數校正後,我們進行的多變量分析證實,與健康成年人相比,活化的B細胞在JAK2突變,鈣網蛋白突變和三陰性原發性血小板增多症病人中都有增加的現象。因此,活化的B細胞增加在不同突變亞組的原發性血小板增多症病人中是一種普遍存在的現象。 本研究的第四個目的是使用斑馬魚動物模型研究鈣網蛋白突變的分子發病機制。我們確定了3種與人類鈣網蛋白直系同源的斑馬魚基因,稱為calr,calr3a和calr3b。CALR-del52和CALR-ins5突變體的表達使斑馬魚早期的造血幹/前驅細胞增加,並進而造成血小板球增多但不影響正常的血管生成。我們發現使用morpholino降低mpl但不是epor或csf3r可以顯著的減弱CALR突變體對血小板球增多的影響。此外,CALR突變體的表達也活化jak/stat信息傳遞路徑,而使用JAK抑製劑(ruxolitinib和fedratinib)可以抑制此活化現象。這些研究結果說明CALR突變體通過mpl依賴機制活化jak信息傳遞路徑導致斑馬魚致病性之血小板球生成。我們研究的結果也說明與突變型CALR腫瘤發生相關的訊息傳遞機制在人與斑馬魚之間是保守的。 The myeloproliferative neoplasms (MPNs) are clonal hematopoietic stem cell disorders and can be classified into 'classic' and 'atypical' MPNs. Classic MPNs usually exhibit terminal myeloid cell expansion in the peripheral blood and include polycythemia vera, essential thrombocythemia (ET), primary myelofibrosis and chronic myeloid leukemia. Calreticulin (CALR) mutations have recently been discovered in about 30% JAK2/MPL-unmutated myeloproliferative neoplasms (MPN), and have become an important clonal marker for the diagnosis of essential thrombocythemia (ET) and primary myelofibrosis. The first aim of this study is to develop a rapid and sensitive screening tool for the detection of CALR mutations. We successfully developed a high-resolution melting analysis (HRMA) with the CFX Connect real-time system to detect CALR exon 9 mutations in ET patients. The maximal sensitivity of our HRMA system in identifying both CALR type 1 and type 2 mutants from patients’ genomic DNA was 2.5%. Our HRMA has a false positive rate of 3% and no false negative. The second aim of this study is to evaluate the clinical and prognostic significance of CALR mutations and JAK2/CALR co-mutations in Taiwanese ET patients. We screened for CALR exon 9 alterations with HRMA followed by TA-cloning in 92 adult ET patients. We identified classic CALR indel mutations in 21 (22.8%) patients. CALR mutations were associated with younger age (p=0.025), higher platelet count (p<0.001) and lower hemoglobin level (p=0.016). Interestingly, we detected various CALR exon 9 alterations in 13 (22%) of 59 JAK2-mutated ET patients. JAK2-mutated ET patients with concomitant CALR alterations were associated with oldest age (p=0.025), higher thrombotic events after diagnosis (p=0.048), higher major arterial thrombotic events after diagnosis (p=0.022) and more patients being high risk group for thrombo-hemorrhagic complications (p=0.023). Frequent CALR exon 9 alterations in JAK2-mutated ET patients define a specific subgroup of patients with increased risk of thrombotic events. The third aim of this study is to determine the B cell immune profiles in CALR mutated ET patients. We screened for CALR mutations and evaluated B cell immune profiles in a cohort of 54 adult Taiwanese ET patients. 19 (35.2%) of 54 ET patients harbored 8 types of CALR exon 9 mutations including 4 (7.4%) patients with concomitant JAK2V617F mutations. Multivariate analysis adjusted for age, sex, follow-up period and hematological parameters confirmed that increased activated B cells were universally present in JAK2-mutated, CALR-mutated and triple-negative ET patients when compared to healthy adults. In conclusion, increased B cell activation is present in ET patients across different mutational subgroups. The fourth aim of this study is to investigate the molecular pathogenesis of CALR mutations using zebrafish animal models. We identified 3 zebrafish genes orthologous to human CALR, referred to as calr, calr3a and calr3b. Expression of the CALR-del52 and CALR-ins5 mutants caused an increase in the hematopoietic stem/progenitor cells followed by thrombocytosis without affecting normal angiogenesis. The expression of CALR mutants also perturbed early developmental hematopoiesis in zebrafish. Importantly, morpholino knockdown of mpl but not epor or csf3r could significantly attenuate the effects of mutant CALR. Furthermore, expression of mutant CALR caused jak-stat signaling activation in zebrafish that could be blocked by JAK inhibitors (ruxolitinib and fedratinib). These findings showed that mutant CALR activates jak-stat signaling through an mpl-dependent mechanism to mediate pathogenic thrombopoiesis in zebrafish, and illustrated that the signaling machinery related to mutant CALR tumorigenesis are conserved between human and zebrafish. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/2538 |
DOI: | 10.6342/NTU201700938 |
全文授權: | 同意授權(全球公開) |
顯示於系所單位: | 腫瘤醫學研究所 |
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