Familial Polycythemia Research Articles

Familial Polycythemia Associated with JAK2 Mutation Previously Known As Variant of Unknown Significance

Introduction Mutations in the Janus Kinase 2 gene (JAK2) are commonly encountered in patients with myeloproliferative neoplasms (MPN) including polycythemia vera (PV) and essential thrombocythemia. JAK2 mutations particularly V617F is associated with PV in 98% cases. Gain of function mutations in the JAK2 exon 12 are reported to be associated with isolated erythropoiesis. Atypical mutations in JAK2 including (c.3323A>G, p.N1108S) were reported in patients with MPN who tested negative for the established driver mutations including exon 14 JAK 2, exon 9 CALR and exon 10 MPL genes. Primary familial congenital polycythemia (PFCP) is a rare disease inherited in an autosomal dominant fashion caused by hypersensitivity of EPO receptor. In such cases, an EPO receptor mutation is only found in 12-15% of affected patients. Due to associated increased risk of thrombosis, bleeding, leukemic involvement and myelofibrosis in PV, bone marrow evaluation is essential in this patient population. In patients with polycythemia, the goals of treatment are to maintain appropriate level of hemoglobin/hematocrit and to reduce severity of symptoms. We present a family affected by polycythemia and leukocytosis associated with an atypical heterozygous variant of JAK2 [c.3323A>G (p.Asn1108Ser)] currently reported as benign or variant of unknown significance (VUS) in literature. Methods Case series, retrospective electronic medical record review with institutional board review exemption Results We report a case series of three Caucasian family members affected with JAK2 VUS mutation (mother and two sons). The proband is a 17-year-old male who presented to an outside facility at the age of 14 with complaints of fatigue, headache, and intense pruritis after hot showers. Initial workup was significant for marked erythrocytosis and reticulocytosis without evidence of abnormal cells on peripheral film. Bone marrow evaluation showed trilineage hematopoiesis with no morphological features of a myeloproliferative neoplasm. Genetic testing was negative for JAK2 V617F and exon 12 mutations. However, whole exome sequencing (WES) revealed a heterozygous mutation of JAK2 gene, c.3323A>G (p.Asn1108Ser). Additional investigation on proband's younger brother, who complained of similar symptoms, also revealed the same JAK2 variant. The proband's mother reported non-specific symptoms including headache, flushing, and occasional blurry vision that she previously attributed to hypertension. Her labs were significant for leukocytosis, monocytosis and thrombocytosis in the presence of normal red blood cell count and WES showed the same mutation. Bone marrow evaluation of the brother and the mother showed normal trilineage hematopoiesis and was negative for malignancy (Table 1). Proband's father is reportedly negative for the aforesaid mutation and is clinically asymptomatic. The proband and his brother are currently managed with phlebotomy every 3 to 4 weeks to maintain hematocrit below 45%, leading to improvement in symptoms. Cytoreductive therapies have been discussed but not initiated. Proband's mother is managed by an adult hematologist. Scheduled phlebotomies with close surveillance is recommended for clinical practice along with interval research sequencing. Conclusion Our case series reports a family with clinical features and laboratories suggestive of a familial form of polycythemia, WES detected the presence of an atypical JAK2 mutation: c.3323A>G, (p.Asn1108Ser). The phenotypical and clinical significance of this mutation has been considered non canonical per reports and classified as likely benign by reputable commercial genetic testing laboratories. However, other reports identify this JAK2 variant as having a significant increased prevalence in patients with MPNs and AML compared to the general population. It may result in a gain of function of the JAK2 protein and subsequent development of MPNs. Enrollment in research sequencing protocols is encouraged in such patients to closely follow the pathologic potential and spectrum of genetic variants rarely reported in literature. Additional research to determine the pathogenicity of this genetic variant is highly warranted.

Hypoxia-inducible factor underlies von Hippel-Lindau disease stigmata.

von Hippel-Lindau (VHL) disease is a rare hereditary cancer syndrome that causes a predisposition to renal clear-cell carcinoma, hemangioblastoma, pheochromocytoma, and autosomal-recessive familial polycythemia. pVHL is the substrate conferring subunit of an E3 ubiquitin ligase complex that binds to the three hypoxia-inducible factor alpha subunits (HIF1-3α) for polyubiquitylation under conditions of normoxia, targeting them for immediate degradation by the proteasome. Certain mutations in pVHL have been determined to be causative of VHL disease through the disruption of HIFα degradation. However, it remains a focus of investigation and debate whether the disruption of HIFα degradation alone is sufficient to explain the complex genotype-phenotype relationship of VHL disease or whether the other lesser or yet characterized substrates and functions of pVHL impact the development of the VHL disease stigmata; the elucidation of which would have a significant ramification to the direction of research efforts and future management and care of VHL patients and for those manifesting sporadic counterparts of VHL disease. Here, we examine the current literature including the other emergent pseudohypoxic diseases and propose that the VHL disease-phenotypic spectrum could be explained solely by the varied disruption of HIFα signaling upon the loss or mutation in pVHL.

Selective Proliferation of Gene-Modified Human Erythroid Cells with Truncated Erythropoietin Receptors

Hematopoietic stem cell gene therapy for hemoglobin disorders, such as sickle cell disease, has stringent requirements for high-level gene marking with robust β-globin expression in erythroid cells for widespread successful clinical application. In vivo selection of gene-modified cells could allow for enhanced therapeutic effects. Erythropoietin (Epo) is a key cytokine for erythroid cell proliferation and differentiation. Various mutations in a C-terminal negative regulatory region of Epo receptor (EpoR) were reported in familial polycythemia. We hypothesized that coexpression of EpoR activated by C-terminal deletions could confer a selective advantage among gene-modified erythroid cells, thus raising the curative potential of therapeutic globin vectors.We generated a selectable truncated human EpoR (thEpoR) by deleting a 40 amino acid C-terminus, and prepared lentiviral vectors encoding both thEpoR and GFP (thEpoR/GFP) connected by internal ribosome entry site (IRES) controlled by murine stem cell virus promoter (Mp). K562 cells, Epo-dependent immortalized erythrocyte HUDEP-2 cells, and human CD34+ cell-derived erythroid cells (Epo-based differentiation) were transduced with thEpoR/GFP vector, and GFP-positive percentages (%GFP) were evaluated for 1-2 weeks. We observed increasing %GFP over 1 week for thEpoR/GFP-transduced HUDEP-2 cells at various Epo doses (3, 1, 0.3, and 0.1U/ml, p<0.01 in all doses) but not in the GFP only control (Figure). Additionally, there was no increase of %GFP for both thEpoR/GFP and GFP only vectors in Epo-independent K562 cells. Two weeks after erythroid differentiation of transduced CD34+ cells (2U/ml Epo), we observed higher glycophorin A-positive erythroid cell percentages (%GPA; 81±0 vs 50±3%, p<0.01), higher GFP and GPA-double positive percentages (%GFP+GPA+; 63±0 vs 44±3%, p<0.01), and larger cell expansion (2.8±0.6 vs 1.6±1.0 fold, p<0.01) in thEpoR/GFP transduction, as compared to GFP only control. These data demonstrate that thEpoR enhances erythroid cell expansion under Epo exposure in human erythroid cells. Accelerated erythroid differentiation can be induced by thEpoR in human CD34+ cells.Since Epo-based negative feedback among erythroid cells is required to maintain normal hematopoiesis, we switched from the constitutive Mp to the erythroid specific Ankyrin-1 (ANKp) or EpoR promoter with or without a WPRE enhancer. After erythroid differentiation of transduced CD34+ cells with the erythroid specific thEpoR/GFP vectors, we observed higher %GPA (81-85% vs 69±4%, p<0.01) in all thEpoR/GFP vectors, and higher %GFP+GPA+ (32±3 and 45±0 vs 22±1%, p<0.01) in thEpoR/GFP vectors with Mp and ANKp-WPRE, as compared to GFP only (Mp). Interestingly, higher γ-globin production in thEpoR/GFP transduction (12±0 vs 8±0%, p<0.01) was detected by HPLC, as compared to GFP only and no transduction controls. These data demonstrate that a thEpoR-based selective advantage in human erythroid cells can be controlled by erythroid specific promoters, and possibly induce γ-globin production.We then evaluated the ANKp-WPRE-derived thEpoR/GFP utilizing either an IRES or a 2A sequence to coexpress a GFP reporter (thEpoR-IRES-GFP, thEpoR-2A-GFP, and GFP-2A-thEpoR), since lower GFP intensity was observed in all thEpoR-IRES-GFP vectors (17-24 vs 95±5, p<0.01) than GFP only vector (Mp). In transduced CD34+ cell-derived erythroid cells, we observed higher %GFP+GPA+ in the GFP-2A-thEpoR vector (50±2 vs 37±3%, p<0.01), and greater cell expansion in thEpoR-IRES-GFP and GFP-2A-thEpoR vectors (100±7 and 88±6 vs 62±5 fold, p<0.05), as compared to GFP only vector with ANKp-WPRE. Lower GFP intensity was observed in all thEpoR/GFP vectors (14-34 vs 106±5, p<0.01) than the GFP only control, while the highest GFP intensity was obtained in GFP-2A-thEpoR (34±0). These data suggest that ANKp-WPRE with GFP-2A-thEpoR is optimal for erythroid specific expression, which should allow for both selection and detection of GFP signals in gene-modified erythroid cells in vitro and in vivo animal models.In summary, we developed erythroid specific thEpoR-expressing vectors, allowing for larger expansion and accelerated differentiation in human erythroid cells. While farther in vivo studies are desirable, this system should be beneficial for providing a selective advantage for gene-modified erythroid cells in gene therapy strategies for hemoglobin disorders. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Sustained fetal hemoglobin induction in vivo is achieved by BCL11A interference and coexpressed truncated erythropoietin receptor.

Hematopoietic stem cell gene therapy for hemoglobin disorders, including sickle cell disease, requires high-efficiency lentiviral gene transfer and robust therapeutic globin expression in erythroid cells. Erythropoietin is a key cytokine for erythroid proliferation and differentiation (erythropoiesis), and truncated human erythropoietin receptors (thEpoR) have been reported in familial polycythemia. We reasoned that coexpression of thEpoR could enhance the phenotypic effect of a therapeutic vector in erythroid cells in xenograft mouse and autologous nonhuman primate transplantation models. We generated thEpoR by deleting 40 amino acids from the carboxyl terminus, allowing for erythropoietin-dependent enhanced erythropoiesis of gene-modified cells. We then designed lentiviral vectors encoding both thEpoR and B cell lymphoma/leukemia 11A (BCL11A)-targeting microRNA-adapted short hairpin RNA (shmiR BCL11A) driven by an erythroid-specific promoter. thEpoR expression enhanced erythropoiesis among gene-modified cells in vitro. We then transplanted lentiviral vector gene-modified CD34+ cells with erythroid-specific expression of both thEpoR and shmiR BCL11A and compared to cells modified with shmiR BCL11A only. We found that thEpoR enhanced shmiR BCL11A-based fetal hemoglobin (HbF) induction in both xenograft mice and rhesus macaques, whereas HbF induction with shmiR BCL11A only was robust, yet transient. thEpoR/shmiR BCL11A coexpression allowed for sustained HbF induction at 20 to 25% in rhesus macaques for 4 to 8 months. In summary, we developed erythroid-specific thEpoR/shmiR BCL11A-expressing vectors, enhancing HbF induction in xenograft mice and rhesus macaques. The sustained HbF induction achieved by addition of thEpoR and shmiR BCL11A may represent a viable gene therapy strategy for hemoglobin disorders.

Familial Polycythemia Likely Due to Novel Hemoglobin Variant Hemoglobin Hyden

Adult hemoglobin (HbA) is made up of two pairs of globin chains. Some rare mutations of the globin chains can result in high affinity towards hemoglobin molecule thus changing the equilibrium of normal oxygen loading in lungs and the delivery of same to the tissues. Because of change in the affinity to the oxygen these mutations can result in erythrocytosis (polycythemia). Here we discuss a case of Familial Polycythemia likely due to novel hemoglobin variant.42-year Caucasian male presents to the clinic with high hemoglobin for several years but otherwise denies any symptoms of headache, vision changes, chest pain. He had history of multiple phlebotomies. His past medical history is significant for Polycythemia, PICC line associated clot, Type2 Diabetes, Hypertension, Epidural abscess. Social history is significant for smokeless tobacco but otherwise non-smoker, non-alcoholic, not an IVDA and doesn't use testosterone. Family history is significant for his sister, her two 14year old daughters and multiple other family members with elevated hemoglobin and undergoes phlebotomies, maternal grandfather died of cancer in his 30s.Physical examination is only significant for BMI of 32.46 otherwise no skin discoloration or cyanosis. Laboratory data WBC 9.4 with normal differential, hemoglobin 18.2, hematocrit 55.4, platelet count 180,000, peripheral blood smear was within normal limits, negative for JAK-2, normal erythropoietin, negative for hemochromatosis, Oxygen dissociation p50 of 19 which is low indicating left shifted dissociation curve, hemoglobin electrophoresis HbA: 61.2%, HbA2: 3%, HbF: 0%, Beta variant: 35.8%. In order to further characterize beta variant Bidirectional sequence analysis for Molecular alterations was performed and the following alterations were detected Gene: HBB, DNA change: Codon 39, heterozygous CAG>CCG Protein change: P.G1n39Pro. [glutamine (Q) to proline (P)]. HGVS: c.119A>C, p.Q40P, Classification: Likely deleterious variant. (GenBank accession number NM_000518.4).This is a previously unreported beta chain hemoglobin variant present. This hemoglobin variant is named as Hemoglobin Hyden based on the place where this is found in Hyden, Kentucky. There have been four variants reported at codon 40 of the beta globin gene, which is an external contact site between beta globin and alpha-2 globin. One variant, Hb vassa, is associated with mild hemolytic anemia. The three other variants (Hb Alabama, Hb Tianshui and Hb San Bruno) are not associated with clinical or hematological abnormalities. In our opinion p.Q40P is likely a cause of erythrocytosis as the same mutation is found in atleast three other patients with polycythemia who are unrelated but all of them presenting from same county. In order to further establish the causality it may be beneficial to test first degree relatives in this family in order to determine whether the p.Q40P alteration tracks with disease and is not present in unaffected individuals.Hemoglobin threshold for phlebotomy to lower the risk of thrombosis and cardiovascular events is yet to be defined. DisclosuresNo relevant conflicts of interest to declare.

113 - Identification of candidate predisposing factors in familial polycythemia vera with exome sequencing

113 - Identification of candidate predisposing factors in familial polycythemia vera with exome sequencing

Relative polycythemia in acute pancreatitis: a series of case report

Polycythemia is an unusual manifestation of acute pancreatitis. When polycythemia does occur, it is almost invariably due to intravascular fluid depletion, contraction of Plasma volume, Gaisbock’s Syndrome, Primary congenital and familial polycythemia, polycythemia vera, Hypoxia as in high altitude, pulmonary cyanotic congenital heart diseases, hypoventilation syndromes like sleep apnea, Smoker’s Polycythemia, carbonmonoxide intoxication caused by industrial exposure, Erythropoietin secreting tumors, post renal transplantation erythrocytosis and congenital polycythemias. This case series report presents four patients with acute pancreatitis and relative polycythemia due to reduction in plasma volume as a result of intravascular volume depletion, as demonstrated by Haemoglobin and Hematocrit during acute pancreatitis and the significance of this association is discussed. Among the case series, the case 1 was relative polycythemia with acute pancreatitis presented as pseudocyst, whereas the case 2, 3 and 4 presented as relative polycythemia with necrotizing pancreatitis. The clinical course of the patient suggested that the relative polycythemia developed subsequent to the clinical onset of acute pancreatitis. It is believed that this represents the first recorded case series in the English Literature of acute pancreatitis with Relative Polycythemia due to reduction in plasma volume as a result of intravascular volume depletion. Four Patients presented with Relative polycythemia during acute pancreatitis was found.

Retrospective outcomes in patients with BCR-ABL positive Acute Lymphoid Leukemia without Allogeneic stem cell transplantation at the Houston Methodist

S176 Introduction: Ph-like acute lymphoblastic leukemia (ALL) is characterized by a variety of rearrangements and other genetic alterations activating cytokine receptor and kinase signaling and poor outcome. We recently identified recurring rearrangements of the erythropoietin receptor (EPOR) into the immunoglobulin heavy (IGH) or kappa (IGK) chain loci. The goal of this study was to examine the role of these alterations in kinase signaling and lymphoid transformation.Methods:Wild-type or EPOR rearranged alleles were expressed in interleukin-3 (IL-3)-dependent mouse hematopoietic Ba/F3 cells and interleukin-7 (IL-7)-dependent pre-B cells harboring alterations of Arf and/or the dominant negative IKZF1 allele IK6 observed in EPOR-rearranged ALL. Proliferation and signaling were examined in the absence or presence of EPO. EPOR expression and signaling in cell lines and primary leukemic cells were examined by immunofluorescence, flow cytometry and immunoblotting. Luciferase-marked xenografts of human EPORrearranged ALL were established in NOD-SCID-IL2R gamma (NSG) null mice, and signaling, EPO-dependent proliferation and sensitivity to the JAK inhibitor ruxolitinib were assessed ex vivo and in vivo. Results: Eight cases out 154 Ph-like ALL (5.2%) harbored rearrangements of EPOR into either the IGH or IGK loci with two consequences: i) inversion and insertion of EPOR 5’ untranscribed region into the the promoter and enhancer region of IGH/IGK; ii) truncation of the last coding exon of EPOR, resulting in overexpression of a C-terminal truncated receptor that retains the phosphorylation site required for STAT5 activation, but lacking multiple residues required for negative EPOR regulation. Notably, the location of the truncation sites is similar to those arising from inherited mutations in primary familial congenital polycythemia. The truncated alleles were expressed at higher levels than wild-type EPOR in IL-3-dependent Ba/F3 and IL-7-dependent Arf-/mouse pre-B cells, and sustained cell proliferation and increased STAT5 phosphorylation following stimulation with exogenous EPO. Xenograft leukemic cells from EPOR-rearranged cases exhibited high levels of mutant EPOR on the cell surface, constitutive pSTAT5 and noteworthy ex vivo and in vivo sensitivity to the JAK2 inhibitor ruxolitinib. Conclusions: Rearrangements of truncated EPOR into the IGH/IGK chain loci define a new subgroup of Phlike ALL and unexpectedly implicate EPOR signaling as an important factor influencing B-lymphoid malignancies. Finally, these findings suggest that aberrant EPOR signaling is amenable to JAK-STAT5 inhibition in cases harboring these rearrangements.

Polycythemias

Polycythemia, also called erythrocytosis, is an increase in the number of circulating red blood cells per volume of blood, as reflected by an elevated hematocrit or hemoglobin level. The three major categories of polycythemia are relative polycythemia, secondary polycythemia, and polycythemia vera. Included in this chapter are discussions on initial evaluation; familial polycythemia; and polycythemia caused by renal and hepatic disorders, drug use, and appropriate increases in erythropoietin production. A flowchart depicts an approach to the evaluation of a patient with polycythemia. Other figures depict the oxygen-hemoglobin dissociation curve characteristic of various polycythemias and a gene mutation associated with familial polycythemia. This review contains 3 highly rendered figures and 27 references.

A Novel EPO Gene Mutation In a Family With Autosomal Dominant Polycythemia

Familial polycythemia/erythrocytosis (ECYT by OMIM), characterized by an absolute increase in red cell mass, is a heterogeneous group of disorders that can be attributed to either intrinsic erythroid progenitor defects, resulting in their hyperproliferation (primary polycythemias), or from circulating extracellular factors such as erythropoietin (EPO) stimulating erythropoiesis (secondary polycythemias). Known primary familial causes include gain-of-function EPO receptor gene mutations. Secondary polycythemia causes include gene mutations that increase hemoglobin oxygen affinity (a, b, and g globins and 2, 3 bisphosphoglycerate mutase, and cytochrome b5 reductase). Several mutations result in alterations of the hypoxia-sensing pathway, which is a primary regulator of EPO production, and familial polycythemia. These include gain-of-function mutations of the HIF-2-a gene (HIF2A), and loss-of-function mutations of two negative regulators of HIFs (EGLN1/PHD2) and von Hippel-Lindau (VHL) genes. Some of these mutations exhibit overlapping features of both primary and secondary polycythemias, such as Chuvash polycythemia. No mutations of the EPO gene have yet been reported.Here we report a 5-generation Caucasian family with autosomal dominant polycythemia. The propositus (III-11) was initially seen 28 years ago at the age of 2 years old. He had moderately increased EPO levels, no splenomegaly, and normal leukocyte and platelet numbers. He was not hypoxic and his hemoglobin oxygen dissociation (P50) was normal. His 21 family members agreed to be tested and 10 were polycythemic, while 11 were normal. Later linkage analyses of 18 re-consented relatives examined the association of a polycythemic phenotype with polymorphisms of HIF2A, HIF1A, EPOR, PHD2, and VHL genes, ruling these out as the cause. Exome sequencing of 5 affected individuals revealed a novel nucleotide change in chromosome 7 at -136nt upstream of the EPO gene (NG_021471 -136 G>A) from the ATG initiation site. This variant has not been reported in any publicly available genome databases and none of the 8 unrelated Caucasian controls have this variant. To determine the distribution of this variant and its segregation, we screened 7 affected and 8 unaffected relatives; all 8 unaffected samples were negative for this EPO variant, while all 7 affected individuals were heterozygous for this variant in the 5'UTR of the EPO gene.The effect(s) of this 5'UTR variant on EPO gene transcription is being examined by Luciferase assay, as well as in an expression assay using HEK293 EPO producing cells. In brief, constructs containing the -136nt variant having either the G (wild-type) or A (mutant) nucleotide of the EPO gene have been made and inserted into the pGL3 vector for the luciferase assay. EPO transcript quantitation studies will be carried out using human EPO cDNA clones with or without the -136nt variant transfected into HEK293 cell line and analyzed under two different conditions, ambient and at 5% oxygen. EPO expression levels under these two different conditions will be determined at multiple time points.This is the first report of an EPO gene mutation associated with familial polycythemia; its functional impact is being studied. Disclosures:No relevant conflicts of interest to declare.

Novel compound VHL heterozygosity (VHL T124A/L188V) associated with congenital polycythaemia

Novel compound <scp>VHL</scp> heterozygosity (<i><scp>VHL</scp></i> T124A/L188V) associated with congenital polycythaemia

Physiopathology, Etiologic Factors, Diagnosis, and Course of Polycythemia Vera as Related to Therapy According to William Dameshek, 1940-1950

According to Dameshek, true polycythemia (polycythemia vera: PV) is a chronic myeloproliferative disorder of the total bone marrow without any evidence of invasiveness, in which erythrocytosis, leukocytosis, and thrombocytosis are all simultaneously present. A possible hereditary or transmitted tendency may be present, but actual familial polycythemia is rare. As to the etiology, Dameshek proposed 2 highly speculative possibilities in 1950: the presence of excessive bone marrow stimulation by an unknown factor or factors, and a lack or a diminution in the normal inhibitory factor or factors. Dameshek’s hypothesis was confirmed in 2005 by Vainchenker in France by the discovery of the acquired JAK2V617F mutation as the cause of 3 phenotypes of classical myeloproliferative neoplasms: essential thrombocythemia, PV, and myelofibrosis. The JAK2V617F mutation induces a loss of inhibitory activity of the JH2 pseudokinase part on the JH1 kinase part of Janus kinase 2 (JAK2). This leads to enhanced activity of the normal JH1 kinase activity of JAK2, which makes the mutated hematopoietic stem cells hypersensitive to the hematopoietic growth factors thrombopoietin, erythropoietin, insulin-like growth factor-1, stem cell factor, and granulocyte colony-stimulating factor, resulting in trilinear myeloproliferation. In retrospect, the situation observed by Dameshek where all “stops” to blood production in the bone marrow are pulled in PV is caused by the JAK2V617F mutation.Dameshek considered PV patients as fundamentally normal and therefore the treatment should be as physiologic as possible. For this reason, a systematic phlebotomy/iron deficiency method of treatment was recommended; the use of radioactive phosphorus is reserved for refractory cases and cases of major thrombosis. If the patient lives long enough and does not succumb to the effects of thrombosis or other complications, the marrow will gradually show signs of diminished activity. The blood smear shows nucleated red cells, increased polychromatophilia, and immature granulocytes of various types. With increasing reduction of erythropoietic tissue, myelofibrosis becomes more of an organized mass of fibrous tissue. There is prominent extramedullary hematopoiesis in the spleen, which becomes extraordinarily large and in some cases occupies almost the entire abdominal cavity. The enlarged spleen is made up largely of metaplastic marrow tissue in primary myeloid metaplasia of the spleen.Conflict of interest:None declared.

Abstract 1109: c-Cbl ubiquitination of p85 is essential for EpoR downregulation.

Abstract Impaired deactivation of growth factor receptors is strongly associated with carcinogenesis. Among these receptors erythropoietin receptor (EpoR) is the main regulator for red blood cell production. EpoR−/− mice die of anemia, whereas constitutive EpoR signaling is oncogenic. Binding of Epo to the EpoR activates cytosolic JAK2 kinase, which in turn phosphorylates tyrosine residues in the EpoR cytoplasmic domain, to recruit and activate signaling proteins for erythropoiesis. Following this activation, EpoR is internalized and degraded to terminate the signaling. We have shown that Epo induces clathrin-dependent EpoR internalization and p85 regulatory subunit of PI3 Kinase (PI3K) plays a key role in this process. Interestingly, this mechanism does not require PI3K activity. One disease associated with this down-regulation mechanism is Primary familial and congenital polycythemia (PFCP). PFCP patients harbor truncated EpoRs, have increased red blood cell mass and their erythroid progenitors are hypersensitive to Epo. These truncated EpoR lack the binding site for p85 and restoring p85 binding site rescues EpoR internalization and corrects Epo hypersensitivity. However, the exact mechanism of how p85 aids in EpoR internalization is unclear. We observed that Epo induces p85 mono-ubiquitination. To test if ubiquitination of p85 regulates EpoR internalization, we generated p85 truncation mutants to identify the domains required for binding to EpoR. We found that the C-terminal SH2 domain and p110 binding domain of p85 interact with the EpoR. c-Cbl-/- mice were reported to have erythroid hyperplasia and c-Cbl was shown to bind p85. We hypothesized that upon stimulation c-Cbl ubiquitinates p85, which recruits endocytic proteins such as Epsin for EpoR down-regulation. In an in vitro kinase assay, c-Cbl can be directly phosphorylated by wild-type but not kinase-deficient JAK2. In addition, Y731 of c-Cbl became phosphorylated upon Epo stimulation which is in the pYXXM p85 binding motif. Importantly, Epo induced EpoR internalization was abolished in cells knock down of c-Cbl by siRNA or in mouse embryonic fibroblasts (MEFs) isolated from c-Cbl knockout animals. Two ligase-deficient c-Cbl mutants,ΔY368 and ΔY371 both had a dominant negative effect on the internalization of EpoR. Together, our results show that c-Cbl, and its ligase activity, play a crucial role in Epo-induced EpoR internalization. Secondly, we observed that upon Epo stimulation EpoR co-localizes with Epsin1 on the plasma membrane. We examined if PFCP EpoR recruits Epsin1 upon Epo stimulation. Consistent with our hypothesis that Epsin1 is recruited to the EpoR by ubiquitinated p85, PFCP EpoR does not co-localize with Epsin1 on the plasma membrane upon Epo stimulation. Studying molecular players and mechanisms in EpoR down-regulation will shed light onto our understanding of hematological malignancies. Citation Format: Gamze B. Bulut, Lily Huang. c-Cbl ubiquitination of p85 is essential for EpoR downregulation. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1109. doi:10.1158/1538-7445.AM2013-1109

Clinical utility gene card for: familial polycythaemia vera

1.5 Mutational spectrum Somatic JAK2 exon 14 G1849T/V617F (80–100% of all familial and sporadic PV patients).1–4 Multiple somatic JAK2 exon 12 (non-V617F) mutations (o10% of familial and sporadic PV patients).5–8 Germ-line JAK2 polymorphism rs10974944.9–12 Multiple somatic TET2 exons 3–12 mutations (10–15% of V617Fpositive familial and sporadic PV patients).12–14 Germ-line EGLN1 G471C/G147H (JAK2 V617F-positive familial PV case).15 Recurrent somatic cytogenetic aberrations in 20–30% of familial PV and sporadic PV (þ 8, þ 9, del(9p), del(13q) and del(20q)).16,17

Autosomal dominant inheritance with variable penetrance in primary familial and congenital polycythemia: A family tree

Primary familial and congenital polycythemia is a rare congenital disorder with only one case ever reported from Indian Subcontinent. Here, we are reporting an entire family inflicted with primary familial and congenital polycythemia, first ever of its kind from Indian subcontinent. We are of firm belief that our report would create awareness among medical fraternity in India about this under reported disorder. Keywords: Primary familial and congenital polycythemia, erythropoietin, autosomal dominant, variable penetrance, sporadic, erythropoietin receptor, Chuvash polycythemia.

Benign Familial Polycythaemia in a Young Male

Polycythaemia has been reported rarely as a familial condition. There is evidence to suggest transmission as a Mendelian dominant trait, but recessive inheritance has also been described. We present here a case of benign familial polycythaemia in a 25-year-old male with similar presentation in his family members. Our patient presented with reddish discolouration of the eyes, early satiety , weight loss and itching at intervals, for four years. An additional examination revealed red beefy tongue and Grade III clubbing. The importance of presenting this case lies in the fact that the prognosis appears to be good in these patients, but regular observation is necessary as Kiladjian and colleagues have mentioned that there is a risk of leukaemia, thrombosis and myelofibrosis in these patients later on, as the idiopathic erythrocytosis group contains a certain number of polycythaemia patients.

Genetic Investigation by Shared Genomic Segment and Linkage Study of a Unique Family with Primary Familial and Congenital Polycythemia

AbstractAbstract 4783 Introduction:Polycythemia/Erythrocytosis may be congenital and/or familial, or acquired. The first recognized familial polycythemias are associated with mutations in the globins gene resulting in increased affinity of Hb to oxygen, which could also be caused by congenital deficiency of 2, 3 DPG. Subsequently gain-of-function of erythropoietin receptor gene mutations have been shown to cause familial dominantly inherited polycythemia. More recently, mutation in the genes involved in hypoxia sensing pathway have been described as causes of congenital polycythemia; these include recessively inherited VHL (Chuvash polycythemia), and dominantly inherited HIF2a and PHD2 mutations. Yet unexplained familial polycythemias still exist. Here we investigated a family with dominant polycythemia present in five generations wherein all known mutations in polycythemia-causing genes were excluded through linkage analyses and direct sequencing. Familial clustering of the disease provided us an opportunity to identify a novel genetic cause modulating erythropoiesis. We utilized a shared genomic segment (SGS) and linkage analysis approach, to provide evidence of chromosomal candidate regions for further efforts to localize the causative gene. Methods:A five generation family pedigree consisting of 26 phenotyped members (10 affected/16 unaffected) with 16 subjects (5 affected and 11 unaffected) genotyped on an Affymetrix v5.0 SNP platform. A conventional two-point linkage analysis did not identify a potential causative genomic region. Multipoint linkage analysis was subsequently performed because this allows for optimal use of the genetic SNP data to infer inheritance patterns and leads to lower false positive rates than two-point linkage methods. In order to identify potential causative regions for harboring the polycythemia-causing defect, we also performed shared genomic segment (SGS) analysis that recognizes a region identical by descent must also be shared by identical by state. We analyzed 409,426 reference dbSNPs with a 94% call rate for genotyped subjects to determine runs of loci at which affected individuals share a common allele. Parametric multipoint linkage analysis used a pruned set of 8,394 non-monomorphic markers with minimum spacing of 100 kb, minimum heterozygosity of 0.3 and maximum pairwise r2 of 0.16 based on HapMap CEU data. We used a Markov chain Monte Carlo linkage method (MCLINK) which allows for fully informative multilocus linkage analysis on extended pedigrees. Results:SGS identified five runs of shared loci that achieved chromosome-wide significance among all five affected subjects (chromosome, run length): 18, 1980 SNPs; 14, 1292 SNPs and 1095 SNPs; 16, 1060 SNPs and 20, 500 SNPs. The SGS regions on chr. 14 (1292 and 1095 run lengths out of 13,149 total SNPs) begins at (NCBI build 36.1) 55,956,582,765bp (rs17254216) through 73,408,302bp (rs1778222) and contain regions of 80 and 79 consecutive SNPs at which all cases share homozygously from a homozygous shared genomic segment analysis (HGS). Multipoint linkage identified a peak LOD score of 3.41 on chr. 16 at 78,033,240bp (rs4888031) achieving genomewide significance which is contained within an observed SGS region of interest (77,507,467bp to 81,049,285bp). [Display omitted] Conclusion:We submit that genetic epidemiological analyses (SGS and Linkage) augment likelihood of identifying the disease-causing genomic regions that are good candidates for the underlying familial susceptibility variant(s). We observed an 8Mb region of interest on chromosome 14 which may contain a segment of loss-of-heterozygosity, and a 3.9Mb region on chromosome 16 both are now being analyzed by sequencing studies. Disclosures:No relevant conflicts of interest to declare.

Clinical and Biological Features, Treatment and Long-Term Outcome of 65 Children with Ph- Myeloprolipherative Disorders (MPD).

Abstract 1889Poster Board I-912 Introduction:MPD rarely occur in children and published data of pediatric patients (pts) are limited. In adults, current diagnostic criteria are those of the WHO2008 and the therapeutic options are supported by controlled studies. From our experience, we suggested that the biological markers show a clinical relevance in Ph- MPD children and may influence their management. Patients and methods:We retrospectively analyzed 65 pts aged ≤20 years (yrs) at diagnosis (dx) with Ph-negative MPD, observed between December 1981 and March 2009. Dx was performed according to the PVSG or WHO criteria. Since 2002, 52/65 pts were studied for JAK2 mutations, polycytemia rubra vera-1 (PRV-1) RNA expression, thrombopoietin (TPO) and its receptor (c-MPL) mutations, erythropoietin receptor (EPO-r) gene mutations, spontaneous endogenous erythroid colony (EECs) growth, clonality on female pts. Results:Thirty-nine females and 26 males with a median age at dx of 14 yrs were classified as follow: 34 (52%) essential thrombocythemia (ET), 16 (24.5%) familial thrombocythemia (FT), 11 (17%) polycythemia vera (PV), 3 (4.5%) familial polycythemia (FP) and 1 idiopathic myelofibrosis (IM). Clinical and biological data, treatment and outcome are reported. Pts with familial disease were younger than those with the sporadic form (p<.05). Hematocrit (Hct) values were higher in ET than in FT pts (p .0047). Fifty pts underwent a bone marrow aspirate and biopsy (15 FT and FP children were excluded). No chromosome abnormalities were found. Overt fibrosis was present in 1 pt. Symptoms at dx were recorded in 21/64 pts (33%) and splenomegaly was present in 13/64 (20%), more frequently in sporadic disease pts. The JAK2V617F mutation was found in 47.5% of ET and 27% of PV. Clonal myelopoiesis was present in 7/11 (63.5%) ET and 2/3 PV females. None of the FT and FP pts showed JAK2V617F mutations or clonality. EECs grew in 58% of ET, in 42% of FT and in 40% of PV. PRV-1 RNA overexpression was found in 29/44 pts (66%). No EECs growth or EPO-r gene mutation was found in FP pts. The median EPO level was 5 mU/ml (1.1–16.4) in polycythemic pts. MPLS505A mutations were detected in15/16 FT pts and a novel HIF2A mutation in 1 FP pt. Treatment was modified during follow-up (f-up): 15 children did not undergo any treatment and 16 pts received more than one cytoreductive drugs. Phlebothomies were carried out in 9 polycythemic pts. Overall, 26 pts were treated with different cytoreductive drugs (hydroxyurea, interferone-alpha, anagrelide and pipobroman). Because of the persistently higher platelet (PLT) count, ET children needed more cytoreductive drugs than FT pts (p .0006). Anti-platelet agents were used in 40 pts and stopped in 30. No hemorrhagic events were recorded; 3 pts (5%) developed thromboses. Eight pts had 11 pregnancies (4 abortions: 2 spontaneous). Five pts showed progressive splenomegaly, 2 (1 PV and 1 ET, untreated) of them developed IM. Two pts developed a cancer. All pts are alive after a median f-up of 10 yrs. Conclusions:This represents the largest reported series of pediatric MPD. A broad familial work-up combined to molecular analyses allowed us to characterize the MPD disorders in our pediatric population, to plan suitable treatments and to better manage their disease.ET (34 pts)FT (16 pts)PV (11 pts)FP (3 pts)IM (1 pt)M/F10/247/98/31/2FAge at Dx (yrs)15.53 (5.24–19.75)11.91 (0.22–17.45)16.47 (3.68–18.8)11.18 (11.18–14.16)19WBC × 109/L9,88 (5,62–22,22)8,43 (5,17–16,06)6,73 (4,86–28,67)6,76 (5,4–6,77)13,35Hct %41.65 (29–53)36.15 (32.3–42)53.9 (50–72.5)53.20 (42.4–54.5)31PLT × 109/L1109 (633–2800)990,5 (611–2950)217 (166–945)207 (202–271)995Symptoms12/33 (36%)4/16 (25%)5/11 (45%)0/3noSplenomegaly8/34 (23.5%)2/15 (13%)3/11 (27%)0/3noAnti-platelet agents27/34 (79.4%)9/16 (56%)4/11 (36%)0/3noCytoreductive drugs23/34 (68%)2/16 (12.5%)1/11 (9%)0/3noEECs pos11/19 (58%)5/12 (42%)4/10 (40%)0/3noPRV-1 RNA overexpr14/19 (74%)6/12 (50%)6/10 (60%)3/3 (100%)yesJAK2 V617F mut10/21 (47.5%)0/133/11 (27%)0/3noMonoclonality7/11 (63.5%)0/72/3 (66%)0/2yesMPLS505A mut0/1515/16 (94%)–––Thromboses1/34 (3%)2/16 (12.5%)0/110/3noPregnancies92–––F-up (yrs)10.02 (0.37–27.28)11.02 (0.86–26.96)9 (0.25–17.14)2 (2–9)12 Disclosures:No relevant conflicts of interest to declare.

Molecular and Clinical Features of the Myeloproliferative Neoplasm Associated with JAK2 Exon 12 Mutations: a European Multicenter Study.

Abstract 3904Poster Board III-840While about 95% of patients with polycythemia vera carry the unique V617F mutation in JAK2 exon 14, several mutations in exon 12 have been described in the minority of JAK2 (V617F)-negative subjects. The initial study [N Engl J Med 2007 Feb 1;356(5):459-68] led to the conclusion that JAK2 exon 12 mutations define a distinctive myeloproliferative syndrome that affects patients who currently receive a diagnosis of polycythemia vera or idiopathic erythrocytosis. Very recent studies suggest that the 'GGCC' haplotype of JAK2 confers susceptibility to the somatic acquisition of both JAK2 (V617F) and exon 12 mutations [Nat Genet 2009 Apr;41(4):450-4, Leukemia 2009 May 14, Epub ahead of print]. Indeed, we reported pedigrees with familial polycythemia vera in which there were both JAK2 (V617F)-positive and JAK2 exon 12 mutation-positive siblings [Blood 2008 Feb 1;111(3):1686-9]. The myeloproliferative neoplasm associated with JAK2 exon 12 mutations is a rare disorder, and only small groups of patients have been reported so far by various investigators. We therefore started a collaborative study in Europe with the aim of collecting about 100 patients with this condition in order to define the molecular and clinical features of this myeloproliferative neoplasm. An ad hoc database was developed for data collection and management. As of August 1, 2009, 77 patients with the required clinical and hematologic data at diagnosis have been recruited (median follow-up 3.2 years, range 0-27 years), while complete follow-up information was available for 57 of these patients. Various approaches were employed for the detection of JAK2 exon 12 mutations, including genomic DNA sequencing, allele-specific PCR assays, and high resolution melting. Overall, 16 different exon 12 mutations were identified. The most frequent mutation were N542-E543del (26 patients), K539L (12 patients), R541-E543delinsK (6 patients), and F537-K539delinsL (6 patients); the remaining mutations occurred less frequently. With respect to the clinical phenotype at presentation, the Kruskal-Wallis test did not reveal any significant difference between the above most frequent mutations. Median age at diagnosis was 53 years (range 15-92), and the male/female ratio was 43/34. Mean hemoglobin level was 19.3 ± 2.2 g/dL, mean WBC count 8.5 ± 3.2 × 109/L, and mean PLT count 334 ± 197 × 109/L. Overall, 48 out of 77 (62%) patients presented with isolated erythrocytosis, 12 (16%) with erythrocytosis and leukocytosis (WBC count > 10 × 109/L), 8 (10%) with erythrocytosis and thrombocytosis (PLT count > 400 × 109/L), and 8 (10%) displayed a full myeloproliferative pattern (erythrocytosis, leukocytosis and thrombocytosis). Serum erythropoietin level was below the lower normal limit in 46 out of 58 (79%) patients. Twenty-one of 25 (84%) patients had endogenous erythroid colonies. During follow-up, two patients had deep venous thrombosis, two progressed to post-polycythemia vera myelofibrosis (diagnosed according to the IWG-MRT criteria) and two developed a myelodysplastic syndrome. In conclusion, the available data indicate that the myeloproliferative neoplasm associated with JAK2 exon 12 mutations is mainly associated with isolated erythrocytosis at clinical onset, but also suggest that the subsequent clinical course may be similar to that of JAK2 (V617F)-positive polycythemia vera, at least in a portion of patients. Disclosures:No relevant conflicts of interest to declare.

β-Trcp mediates ubiquitination and degradation of the erythropoietin receptor and controls cell proliferation

AbstractControl of intensity and duration of erythropoietin (Epo) signaling is necessary to tightly regulate red blood cell production. We have recently shown that the ubiquitin/proteasome system plays a major role in the control of Epo-R signaling. Indeed, after Epo stimulation, Epo-R is ubiquitinated and its intracellular part is degraded by the proteasome, preventing further signal transduction. The remaining part of the receptor and associated Epo are internalized and degraded by the lysosomes. We show that β-Trcp is responsible for Epo-R ubiquitination and degradation. After Epo stimulation, β-Trcp binds to the Epo-R. This binding, like Epo-R ubiquitination, requires Jak2 activation. The Epo-R contains a typical DSG binding sequence for β-Trcp that is highly conserved among species. Interestingly, this sequence is located in a region of the Epo-R that is deleted in patients with familial polycythemia. Mutation of the serine residue of this motif to alanine (Epo-RS462A) abolished β-Trcp binding, Epo-R ubiquitination, and degradation. Epo-RS462A activation was prolonged and BaF3 cells expressing this receptor are hypersensitive to Epo, suggesting that part of the hypersensitivity to Epo in familial polycythemia could be the result of the lack of β-Trcp recruitment to the Epo-R.