Reasoning
PS3 is met at strong strength. Multiple independent, variant-specific functional studies provide concordant evidence across recombinant protein, engineered cell, patient-derived cell, hematopoietic colony, and murine systems. JAK2 V617F shows approximately threefold higher catalytic activity than wild-type JAK2 JH1-JH2 (493±19 versus 150±9 relative activity per pmol protein) in a recombinant kinase assay; constitutive phosphorylation of STAT5, STAT3, STAT1, ERK1/2, and p38 is observed in patient-derived HEL cells; cytokine-independent proliferation/survival and increased STAT5 phosphorylation are demonstrated in BaF3 and UT-7/TPO cells with appropriate cytokine-dependent controls; and expression in a mouse transplantation model produces erythrocytosis. These orthogonal assays directly test the specified variant and include wild-type, cytokine-stimulation, cytokine-withdrawal, inhibitor, or disease-model comparisons, supporting a reproducible gain-of-function/damaging effect on JAK2 function.
Rule
Well-established in vitro or in vivo functional studies supportive of a damaging effect on the gene or gene product.
Evidence considered
- Analysis of Jak2 catalytic function by peptide microarrays (PMID:21533163): recombinant JAK2 JH1-JH2 V617F had approximately threefold higher catalytic activity than JH1-JH2 wild type, 493±19 versus 150±9 relative activity per pmol protein; the assay used wild-type, V617F, and JH1 constructs and 144 peptide substrates.
- SOCS-mediated downregulation of mutant Jak2 (PMID:19543316): patient-derived HEL cells naturally carrying V617F showed constitutive phosphorylation of STAT5, STAT3, STAT1, ERK1/2, and p38; inducible HEK models demonstrated constitutive signaling and SOCS-dependent regulation of the mutant.
- Acquired mutation of the tyrosine kinase JAK2 (PMID:15781101): the exact Val617Phe variant was found in erythroid and granulocyte-macrophage colonies and in all examined erythropoietin-independent erythroid colonies, with dysregulated kinase activity in a cohort including 90 controls.
- A gain-of-function mutation of JAK2 (PMID:15858187): BaF3 and UT-7/TPO cell assays showed V617F-associated cytokine hypersensitivity, proliferation, survival, and STAT5 phosphorylation, including growth and survival without interleukin-3.
- A unique clonal JAK2 mutation (PMID:15793561): variant-specific cell assays showed STAT5 activation without erythropoietin, constitutive JAK2/STAT5 signaling, growth-factor-independent growth, and erythropoietin hypersensitivity; a mouse bone-marrow transplantation model produced erythrocytosis.
- JAK2/IDH-mutant-driven myeloproliferative neoplasm (PMID:29355841): conditional murine Jak2V617F models and patient-derived samples demonstrated disease-promoting effects and impaired hematopoietic differentiation in vivo; this is supportive functional context, although the exact cDNA notation was not stated in the paper.
Sources