Novel germline JAK2R715T mutation causing PV-like erythrocytosis in 3 generations. Amelioration by Ropeg-Interferon.

Polycythemia vera (PV) is a clonal disorder arising from the acquired somatic mutations of the JAK2 gene, including JAK2V617F or several others in exon 12. A 38-year-old female had a stroke at age 32 and found to have elevated hemoglobin, normal leukocytes, normal platelets, and tested negative for JAK2V617F and exon 12 mutations. Next generation sequencing revealed a novel mutation: JAK2R715T in the pseudokinase domain (JH2) at 47.5%. Its presence in her nail DNA confirmed a germline origin. Her mother and her son similarly had erythrocytosis and a JAK2R715T mutation. Computer modeling indicated gain-of-function JAK2 activity. The propositus and her mother had polyclonal myelopoiesis, ruling out another somatic mutation-derived clonal hematopoiesis. Some erythroid progenitors of all three generations grew without erythropoietin, a hallmark of PV. The in vitro reporter assay confirmed increased activity of the JAK2R715T kinase. Similar to PV, the JAK2R715T native cells have increased STAT5 phosphorylation, augmented transcripts of prothrombotic and inflammatory genes, and decreased KLF2 transcripts. The propositus was not controlled by hydroxyurea, and JAK2 inhibitors were not tolerated; however, Ropeginterferon-alfa-2b (Ropeg-IFN-α) induced a remission. Ropeg-IFN-α treatment also reduced JAK2 activity in the propositus, her mother and JAK2V617F PV subjects. We report dominantly inherited erythrocytosis secondary to a novel germline JAK2R715T gain-of-function mutation with many but not all comparable molecular features to JAK2V617F PV. We also document a previously unreported inhibitory mechanism of JAK2 signaling by Ropeg-IFN-α.

Polycythemia vera is not initiated by JAK2V617F mutation.

OBJECTIVE: The somatic JAK2(V617F) mutation is seen in most polycythemia vera (PV) patients; however, it is not clear if JAK2(V617F) is the PV-initiating mutation. METHODS: In order to examine this issue, we developed a novel real-time quantitative allele-specific PCR, in which allelic discrimination is enhanced by the synergistic effect of a mismatch in the -1 position, and a locked nucleic acid (LNA) nucleoside at the -2 position. RESULTS: Determination of allelic frequencies was reproducible (SD = 1.5%) and sensitive--0.1% mutant allele detected in 40 ng of DNA. The JAK2(V617F) frequency in clonal granulocytes from 3 PV females was less than 50% (27.5 +/- 11) and in 7 females greater than 50% (75 +/- 10.5). We also found that wild-type JAK2 BFU-E colonies from PV patients can grow without erythropoietin. The identification of the primary genetic lesion resulting in PV is essential for the development of novel therapeutic strategies. CONCLUSION: Our studies correlating the frequency of JAK2(V617F) mutant allele and clonality, as well as the presence of homozygous wild-type JAK2 erythropoietin-independent erythroid colonies, provide compelling evidence that the JAK2(V617F) is not the PV-initiating mutation. This supports a model wherein the JAK2(V617F) mutation arises as a secondary genetic event. Furthermore, our results indicate that an undefined molecular lesion, preceding JAK2(V617F), is responsible for clonal hematopoiesis in PV. We conclude that development of therapeutic strategies that target the JAK2(V617F) clonal cells may not be sufficient for eradication of PV.

Mutations of von Hippel-Lindau tumor-suppressor gene and congenital polycythemia.

The von Hippel-Lindau (pVHL) protein plays an important role in hypoxia sensing. It binds to the hydroxylated hypoxia-inducible factor 1 alpha (HIF-1 alpha) and serves as a recognition component of an E3-ubiquitin ligase complex. In hypoxia or secondary to a mutated VHL gene, the nondegraded HIF-1 alpha forms a heterodimer with HIF-beta and leads to increased transcription of hypoxia-inducible genes, including erythropoietin (EPO). The autosomal dominant cancer-predisposition von Hippel-Lindau (VHL) syndrome is due to inheritance of a single mutated allele of VHL. In contrast, we recently showed that homozygous germline 598C-->T VHL mutation leads to Chuvash polycythemia (CP). We subsequently found VHL mutations in three unrelated individuals unaffected with CP, one of whom was compound heterozygous for the 598C-->T mutation and another VHL mutation. We now report seven additional polycythemic patients with VHL mutations in both alleles. Two Danish siblings and another American boy were homozygous for the VHL 598C-->T mutation. Three unrelated white Americans were compound heterozygotes for 598C-->T and another VHL mutation, 562C-->G in two and 574C-->T in the third. Additionally, a Croatian boy was homozygous for a 571C-->G VHL mutation, the first example of homozygous VHL germline mutation causing polycythemia, other than the VHL 598C-->T mutation. We have not observed VHL syndrome-associated tumors in polycythemic subjects or their heterozygous relatives; however, this will need to be evaluated by longitudinal studies. Over all, we found that up to half of the consecutive patients with apparent congenital polycythemia and increased serum Epo we have examined have mutations of both VHL alleles. Those findings, along with reports of CP, underscore that VHL mutations are the most frequent cause of congenital polycythemia and define a new class of polycythemic disorder, polycythemias due to augmented hypoxia sensing.

Discrimination of polycythemias and thrombocytoses by novel, simple, accurate clonality assays and comparison with PRV-1 expression and BFU-E response to erythropoietin.

Essential thrombocythemia (ET) and polycythemia vera (PV) are clonal myeloproliferative disorders that are often difficult to distinguish from other causes of elevated blood cell counts. Assays that could reliably detect clonal hematopoiesis would therefore be extremely valuable for diagnosis. We previously reported 3 X-chromosome transcription-based clonality assays (TCAs) involving the G6PD, IDS, and MPP1 genes, which together were informative in about 65% of female subjects. To increase our ability to detect clonality, we developed simple TCA for detecting the transcripts of 2 additional X-chromosome genes: Bruton tyrosine kinase (BTK) and 4-and-a-half LIM domain 1 (FHL1). The combination of TCA established the presence or absence of clonal hematopoiesis in about 90% of female subjects. We show that both genes are subject to X-chromosome inactivation and are polymorphic in all major US ethnic groups. The 5 TCAs were used to examine clonality in 46 female patients along with assays for erythropoietin-independent erythroid colonies (EECs) and granulocyte PRV-1 mRNA levels to discriminate polycythemias and thrombocytoses. Of these, all 19 patients with familial polycythemia or thrombocytosis had polyclonal hematopoiesis, whereas 22 of 26 patients with clinical evidence of myeloproliferative disorder and 1 patient with clinically obscure polycythemia were clonal. Interestingly, interferon alpha therapy in 2 patients with PV was associated with reversion of clonal to polyclonal hematopoiesis. EECs were observed in 14 of 14 patients with PV and 4 of 12 with ET, and increased granulocyte PRV-1 mRNA levels were found in 9 of 13 patients with PV and 2 of 12 with ET. Thus, these novel clonality assays are useful in the diagnosis and follow-up of polycythemic conditions and disorders with increased platelet levels.