Abstract
Janus kinase 2 (JAK2) mutations are key drivers of Philadelphia chromosome-negative myeloproliferative neoplasms (MPNs), with somatic JAK2 V617F mutation, present in ~99% of cases, being the most prevalent. However, the studies of rare and novel JAK2 variants may reveal previously unrecognized pathophysiological mechanisms of myelopoiesis. Here, we describe the functional impact of a novel germline frameshift mutation, JAK2 R989fs, found in a 24-year-old woman with a clinical phenotype resembling an atypical MPN. The patient, after suffering cerebral stroke, was found to have unexplained thrombocytosis (platelets 557 × 10³/µL; range 150-450 × 10³/µL), borderline hematocrit (45.4%; range 36-46%), and normal hemoglobin (14.5 g/dL; range 12-15,5 g/dL) with normal leukocytes and differential with decreased transferrin saturation at 17%, while taking iron supplementation. Her erythropoietin (EPO) was low-normal range at 7 and 8 mU/mL (normal range 4-27 mU/mL); however, her thrombopoietin was increased at 158 pg/ml (~2x level of upper normal range).
The JAK2 R989fs variant, a 2-nucleotide deletion at positions c.2967_2968 (exon 22) with allelic burden at 46,9%, was identified through next generation sequencing and confirmed to be heterozygous in granulocyte DNA; its germline origin was confirmed in her fibroblasts. The same mutation was found to be inherited from her mother and was also identified in her older sister, who had hemoglobin: 14.8 g/dL, hematocrit: 45.3%, platelets: 411 × 10³/µL; however, possible iron deficiency was not excluded. Her brother was found not to carry the mutation and had normal blood counts. Her mother, maternal great grandmother and maternal uncle had a history of repeated thrombosis, but their blood counts were not available. The JAK2 R989fs mutation results in a premature stop codon, predicting a truncated protein of 990 amino acids (~114 kDa), lacking the distal C-terminal portion of the kinase domain. The expression of the truncated JAK protein was confirmed by Western blot analysis of HEK293 cells transfected with expression plasmid bearing JAK2 R989fs mutation.
To investigate the structural consequences of the R989fs truncation, we performed AlphaFold3 modeling of both wild-type and R989fs-mutant JAK2 proteins. The truncation occurs at Val990, within the JH1 kinase domain, leading to the loss of key regulatory motifs including the DFG motif (Asp994–Phe996–Gly997), activation loop tyrosines (Tyr1007 and Tyr1008), and portions of the C-lobe required for regulatory inhibition and substrate specificity. Despite this, the truncated kinase retains the ATP-binding VAIK (Lys882) and HRD (His974–Asp976) motifs, and the core kinase cavity, suggesting retained catalytic potential. Structural comparison showed a more compact and potentially more stable conformation, possibly due to loss of flexible regulatory regions. Notably, the absence of the activation loop may result in constitutive activity, broader substrate specificity, and reduced sensitivity to competitive inhibitors.
Her early peripheral erythroid progenitors (BFU-Es) grew independently of EPO (hallmark of polycythemia vera (PV)) comparable to two JAK2 V617F positive PV controls and one JAK2 V617F positive essential thrombocythemia control. In an in vitro liquid erythroid expansion of mononuclear cells differentiated into erythroid progenitors (PMID:17976518), she had increased proportion of CD71⁺ and CD235a⁺ erythroid cells, compared to controls, but comparable to JAK2 V617F-positive cells from PV, suggesting accelerated differentiation of JAK2 R989fs mutant erythroid cells. JAK2 R989fs erythroid progenitors had greater proliferative capacity than PV JAK2 V617F-positive cells, while apoptosis rates remained unaltered. In in vitro erythroid expansion, phospho-STAT5 (p-STAT5) levels were elevated on day 16 of erythroid expansion although not surpassing the levels observed in the JAK2 V617F-positive control. She achieved complete hematologic remission with ropeginterferon alpha (100 µg administered every two weeks), and p-STAT5 levels in her erythroid cells were normalized.
We report that the novel germline JAK2 R989fs truncated variant encodes a constitutively active JAK2 kinase that enhances in vitro proliferation and differentiation of erythroid progenitors, with increased STAT5 signaling. Additional studies are in progress to define the variant's role in megakaryocytic and myeloid differentiation, along with ongoing molecular analyses.
