Calreticulin (CALR) exon 9 frameshift mutations, commonly detected in essential thrombocythemia (ET) and primary myelofibrosis patients, activate signal transducer and activator of transcription (STAT) proteins in the presence of Myeloproliferative Leukemia Virus (MPL) and induce ET in vivo. Loss of the KDEL motif, an endoplasmic reticulum retention signal, and generation of many positively charged amino acids (AAs) in the mutated C-terminus are thought to be important for disease induction. To test this hypothesis, we generated mice harboring a Calr frameshift mutation using the CRISPR/Cas9 system. Deletion of 19-base pairs in exon 9 (c.1099-1117del), designated the del19 mutation, induced loss of the KDEL motif and generated many positively charged AAs, similar to human mutants. Calr del19 mice exhibited mild thrombocytosis, slightly increased megakaryocytes, and mild splenomegaly. In vitro experiments revealed that the murine CALR del19 mutant had a weaker ability to combine with murine MPL than the human CALR del52 mutant. Consequently, STAT5 activation was also very weak downstream of the murine mutant and murine MPL, and may be the reason for the mild disease severity. In summary, loss of the KDEL motif and positively charged AAs in the C-terminus of CALR is insufficient for MPL binding and ET development.

中文翻译：

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具有与人CALR突变同源的Carr突变的小鼠仅表现出轻度的血小板增多症。

钙网蛋白（CALR）外显子9移码突变通常在原发性血小板增多症（ET）和原发性骨髓纤维化患者中检测到，在骨髓增生性白血病病毒（MPL）存在的情况下激活信号转导和转录激活（STAT）蛋白，并在体内诱导ET。人们认为，KDEL基序，内质网保留信号的丢失以及在突变的C末端中产生许多带正电荷的氨基酸（AA）均对疾病的诱导很重要。为了验证这一假设，我们使用CRISPR / Cas9系统生成了带有Calr移码突变的小鼠。外显子9（c.1099-1117del）中19个碱基对的缺失，被指定为del19突变，诱导了KDEL基序的丧失，并产生了许多带正电荷的AA，类似于人类突变体。Calr del19小鼠表现出轻度的血小板增多症，巨核细胞略有增加，轻度脾肿大。体外实验表明，鼠CALR del19突变体与鼠MPL结合的能力比人CALR del52突变体弱。因此，STAT5激活在鼠类突变体和鼠类MPL的下游也非常弱，这可能是轻度疾病严重性的原因。总之，在CALR的C末端失去KDEL基序和带正电荷的AA对于MPL结合和ET发育是不足的。