Currently, the triad of true erythrocytosis (PV), essential thrombocythemia (ET), and primary myelofibrosis (PMF) is referred to as Philadelphia chromosome-negative myeloproliferative neoplasms (MPN). The discovery of the JAK2V617F gene mutation a decade ago can be considered a milestone in the molecular diagnosis of MPN, and has also facilitated basic research in MPN genetics and biology, reclassification of the disease, as well as development of novel therapeutic agents and clinical applications. I. JAK2 V617F gene mutation Research has confirmed that this gene mutation exists in 95% of PV patients and 50%-60% of ET and PMF patients; other myeloid tumors such as acute myeloid leukemia (AML) and MDS can also have low-frequency mutations, especially ring-associated rhabdomyoloblastic anemia with thrombocytosis (RARS-T). This molecular marker has become an important diagnostic basis for PV, ET, and PMF in the WHO classification. In cases that do not meet the WHO diagnostic criteria, the presence of the JAK2V617F mutation suggests a potential risk of MPN or an inert subclinical form of MPN.Studies have found that there are 2 types of JAK2 V617F mutations, pure and heterozygous.ET is almost always heterozygous, whereas 28% of patients with PV and 14% of patients with PMF are pure.The JAK2 V617F allele loading The JAK2 V617F allele load is differently associated with different phenotypes of MPN and has prognostic significance: PV patients with a high JAK2V617F mutation load are more likely to develop myelofibrosis and thrombosis, increased leukocyte counts and hemoglobin levels, decreased platelets, and splenomegaly; in ET patients, the level of JAK2V617F correlates positively with leukocyte counts and negatively with platelet counts. Second, JAK2 exon 12 mutation JAK2V617F gene mutation is present in about 97% of PV cases. JAK2 exon 12 mutation should be further tested in a few mutation-negative cases, in which there is marked erythroid hyperplasia and decreased levels of erythropoietin (EPO), with no megakaryocyte or granulocyte hyperplasia. patients with ET and PMF are basically free of JAK2 exon 12 mutation. Studies have shown that JAK2 exon 12 mutations can be substitutions, deletions, insertions, or duplications of the gene. Clinically, patients with JAK2 exon 12 mutations are younger, approximately 40% of patients are under 50 years of age, and tend to exhibit pure erythroid lineage hyperplasia (higher hemoglobin levels and blood cell volume), although a small percentage of patients have triple lineage hyperplasia. Since JAK2 exon 12 mutations tend to be expressed at low levels, it is particularly important to choose an appropriate and highly sensitive assay. Third, MPL exon 10 mutation One year after the discovery of the JAK2V617F mutation, myeloproliferative leukemia viral oncogene (MPL) mutations were found in patients with ET and PMF who were negative for the mutation, but not in patients with PV.The MPL gene, located on chromosome 1p34, encodes the thrombopoietin receptor and affects the proliferation and differentiation of megakaryocytes. Acquired mutations in codon W515 constitutively activate the thrombopoietin receptor through cytokine activation of the downstream JAK-STAT pathway. The two most common mutations are W515L and W515K, which are present in 15% of JAK2 V617F-negative MPN patients, of which 5% of ET and more than 10% of PMF patients can have this mutation. In addition, MPL mutations can occur simultaneously with JAK2 V617F mutations. MPL exon 10 mutations are often clustered around amino acid 515 in the transmembrane region. Reproducible disease-causing mutations typically include the W515L and W515K mutations, as well as the rare W515A, W515R, and W515S mutations.The clinical phenotype of patients with MPL exon 10 mutations is often characterized by relatively low hemoglobin levels. Compared with ET patients positive for the JAK2V617F mutation, ET patients with the MPL mutation had higher platelet counts, marked megakaryocytosis, and higher serum EPO levels.The W515K mutation had a higher allelic mutation load than patients with the W515L mutation, suggesting that the mechanisms of the two variants may be different. Screening for MPL mutations is strongly recommended for patients with suspected ET or PMF without JAK2 and CALR mutations. IV. CALR exon 9 mutations The CALR gene encodes calreticulin, a calcium-binding molecular chaperone associated with the endoplasmic reticulum. CALR mutations associated with MPN are insertions, deletions, or both in exon 9 and are usually heterozygous mutations. CALR mutations have been found to be present in 60% to 80% of JAK2 and MPL mutation-negative patients with ET and MPF, and occasionally in patients with MDS-RARS-T, rarely in patients with PV, and rarely concurrently with JAK2V617F or BCR-ABL mutations. A retrospective analysis of ET and PMF found that patients with CALR mutations exhibited higher platelet counts, low hemoglobin levels, and higher overall survival than patients with JAK2 mutations, MPL mutations, or triple negatives. Relative to JAK2-negative patients, ET patients with CALR mutations had fewer thrombotic and cardiovascular events and a higher incidence of myelofibrosis, and patients with CALR-positive PMF had a lower incidence of erythrocyte transfusion dependence and higher survival.The significance of quantitative monitoring of the CALR mutation is unclear, and the observation that patients with interferon therapy have a reduced CALR allele load may provide guidance on disease monitoring.