Department of Hematology Research, Instituto de Investigaciones Médicas Alfredo Lanari, University of Buenos Aires, National Council for Scientific and Technological Research (CONICET), Buenos Aires, Argentina.
Eur J Haematol. 2010 May;84(5):398-405. doi: 10.1111/j.1600-0609.2010.01421.x. Epub 2010 Jan 22.
To evaluate the frequency of MPL W515L, W515K and S505N mutations in essential thrombocythemia (ET) and primary myelofibrosis (PMF) and to determine whether MPLW515L leads to impaired Mpl expression, constitutive STAT3 and STAT5 activation and enhanced response to thrombopoietin (TPO).
Mutation detection was performed by allele-specific PCR and sequencing. Platelet Mpl expression was evaluated by flow cytometry, immunoblotting and real-time RT-PCR. Activation of STAT3 and STAT5 before and after stimulation with increasing concentrations of TPO was studied by immunoblotting. Plasma TPO was measured by ELISA.
MPLW515L was detected in 1 of 100 patients with ET and 1 of 11 with PMF. Platelets from the PMF patient showed 100% mutant allele, which was <50% in platelets from the ET patient, who also showed the mutation in granulocytes, monocytes and B cells. Mpl surface and total protein expression were normal, and TPO levels were mildly increased in the MPLW515L-positive ET patient, while MPL transcripts did not differ from controls in both MPLW515L-positive patients. Constitutive STAT3 and STAT5 phosphorylation was absent and dose response to TPO-induced phosphorylation was not enhanced.
The low frequency of MPL mutations in this cohort is in agreement with previous studies. The finding of normal Mpl levels in MPLW515L-positive platelets indicates this mutation does not lead to dysregulated Mpl expression, as frequently shown for myeloproliferative neoplasms. The lack of spontaneous STAT3 and STAT5 activation and the normal response to TPO is unexpected as MPLW515L leads to constitutive receptor activation and hypersensitivity to TPO in experimental models.
评估 MPL W515L、W515K 和 S505N 突变在原发性血小板增多症（ET）和原发性骨髓纤维化（PMF）中的频率，并确定 MPLW515L 是否导致 Mpl 表达受损、组成性 STAT3 和 STAT5 激活以及对血小板生成素（TPO）的反应增强。
通过等位基因特异性 PCR 和测序检测突变。通过流式细胞术、免疫印迹和实时 RT-PCR 评估血小板 Mpl 表达。通过免疫印迹研究刺激 TPO 浓度增加前后 STAT3 和 STAT5 的激活。通过 ELISA 测量血浆 TPO。
在 100 例 ET 患者和 11 例 PMF 患者中检测到 MPLW515L。PMF 患者的血小板显示 100%突变等位基因，而 ET 患者的血小板显示<50%，ET 患者的粒细胞、单核细胞和 B 细胞也显示突变。Mpl 表面和总蛋白表达正常，MPLW515L 阳性 ET 患者的 TPO 水平轻度升高，而在 MPLW515L 阳性患者中，MPL 转录物与对照无差异。无组成性 STAT3 和 STAT5 磷酸化，对 TPO 诱导的磷酸化的剂量反应未增强。
该队列中 MPL 突变的低频率与之前的研究一致。在 MPLW515L 阳性血小板中发现正常的 Mpl 水平表明该突变不会导致 Mpl 表达失调，正如在骨髓增生性肿瘤中经常观察到的那样。出乎意料的是，缺乏自发的 STAT3 和 STAT5 激活以及对 TPO 的正常反应，因为 MPLW515L 导致实验模型中受体的组成性激活和对 TPO 的超敏反应。
