Hereditary Erythrocytosis Research Articles

Two Novel Genetic Variants Involved in the Oxygen Sensing Pathway in JAK2-unmutated Erythrocytosis.

JAK2-unmutated erythrocytosis or non-polycythemia vera erythrocytosis is a rare condition comprising both acquired and hereditary forms. Although acquired erythrocytosis has been well-studied, hereditary erythrocytosis remains poorly studied. Genetic alterations associated with hereditary erythrocytosis include mutations in erythropoietin receptor and erythropoietin (EPO), altered oxygen affinity mutations, and variants associated with the oxygen-sensing pathway. We established a molecular diagnostic approach based on these genes and retrospectively evaluated. Peripheral blood from 56 erythrocytosis patients, lacking JAK2 mutation, were screened for oxygen-sensing pathway abnormalities. Two novel mutations were identified in the EGLN1 gene: NM_022051.2:c.712G > C (p.Gly238Arg) and NM_022051.2:c.122A > C (p.Tyr41Ser) in two patients separately. Notably, both reported heterozygous mutations were absent in the population database. Predictions using multiple computer software indicated that these two missense mutations were harmful and induced a highly conserved amino acid change in EGLN1. Patients with the two mutations exhibited normal serum EPO levels and high hemoglobin and hematocrit levels. Additionally, three other variants of genes were identified in the oxygen-sensing pathway, including endothelial PAS domain protein 1 (EPAS1) rs184760160(2/56), and EGLN1 rs186996510(2/56), rs555121182(2/56). These variants were categorized as benign or likely benign. Our findings provide a framework for etiological research and highlight the importance of screening for genetic mutations associated with erythrocytosis in clinical practice.

Characterization of genetic variants in the EGLN1/PHD2 gene identified in a European collection of patients with erythrocytosis.

Hereditary erythrocytosis is a rare hematologic disorder characterized by an excess of red blood cell production. Here we describe a European collaborative study involving a collection of 2,160 patients with erythrocytosis sequenced in ten different laboratories. We focused our study on the EGLN1 gene and identified 39 germline missense variants including one gene deletion in 47 probands. EGLN1 encodes the PHD2 prolyl 4-hydroxylase, a major inhibitor of hypoxia-inducible factor. We performed a comprehensive study to evaluate the causal role of the identified PHD2 variants: (i) in silico studies of localization, conservation, and deleterious effects; (ii) analysis of hematologic parameters of carriers identified in the UK Biobank; (iii) functional studies of the protein activity and stability; and (iv) a comprehensive study of PHD2 splicing. Altogether, these studies allowed the classification of 16 pathogenic or likely pathogenic mutants in a total of 48 patients and relatives. The in silico studies extended to the variants described in the literature showed that a minority of PHD2 variants can be classified as pathogenic (36/96), without any differences from the variants of unknown significance regarding the severity of the developed disease (hematologic parameters and complications). Here, we demonstrated the great value of federating laboratories working on such rare disorders in order to implement the criteria required for genetic classification, a strategy that should be extended to all hereditary hematologic diseases.

JAK2 unmutated erythrocytosis: 2023 Update on diagnosis and management.

JAK2 unmutated or non-polycythemia vera (PV) erythrocytosis encompasses a heterogenous spectrum of hereditary and acquired entities. Foremost in the evaluation of erythrocytosis is the exclusion of PV through JAK2 (inclusive of exons 12-15) mutation screening. Initial assessment should also include gathering of previous records on hematocrit (Hct) and hemoglobin (Hgb) levels, in order to streamline the diagnostic process by first distinguishing longstanding from acquired erythrocytosis; subsequent subcategorization is facilitated by serum erythropoietin (Epo) measurement, germline mutation screening, and review of historical data, including comorbid conditions and medication list. Hereditary erythrocytosis constitutes the main culprit in the context of longstanding erythrocytosis, especially when associated with a positive family history. In this regard, a subnormal serum Epo level suggests EPO receptor mutation. Otherwise, considerations include those associated with decreased (high oxygen affinity Hgb variants, 2,3-bisphosphoglycerate deficiency, PIEZO1 mutations, methemoglobinemia) or normal oxygen tension at 50% Hgb saturation (P50). The latter include germline oxygen sensing pathway (HIF2A-PHD2-VHL) and other rare mutations. Acquired erythrocytosis commonly results from central (e.g., cardiopulmonary disease, high-altitude habitat) or peripheral (e.g., renal artery stenosis) hypoxia. Other noteworthy conditions associated with acquired erythrocytosis include Epo-producing tumors (e.g., renal cell carcinoma, cerebral hemangioblastoma) and drugs (e.g., testosterone, erythropoiesis stimulating agents, sodium-glucose cotransporter-2 inhibitors). Idiopathic erythrocytosis is an ill-defined terminology that presumes the existence of an increased Hgb/Hct level without an identifiable etiology. Such classification often lacks accounting for normal outliers and is marred by truncated diagnostic evaluation. Current consensus treatment guidelines are not supported by hard evidence and their value is further undermined by limited phenotypic characterization and unfounded concerns for thrombosis. We are of the opinion that cytoreductive therapy and indiscriminate use of phlebotomy should be avoided in the treatment of non-clonal erythrocytosis. However, it is reasonable to consider therapeutic phlebotomy if one were to demonstrate value in symptom control, with frequency determined by symptoms rather than Hct level. In addition, cardiovascular risk optimization and low dose aspirin is often advised. Advances in molecular hematology might result in better characterization of "idiopathic erythrocytosis" and expansion of the repertoire for germline mutations in hereditary erythrocytosis. Prospective controlled studies are needed to clarify potential pathology from JAK2 unmutated erythrocytosis, as well as to document the therapeutic value of phlebotomy.

Comprehensive in silico and functional studies for classification of EPAS1/HIF2A genetic variants identified in patients with erythrocytosis.

Gain-of-function mutations in the EPAS1/HIF2A gene have been identified in patients with hereditary erythrocytosis that can be associated with the development of paraganglioma, pheochromocytoma and somatostatinoma. In the present study, we describe a unique European collection of 41 patients and 28 relatives diagnosed with an erythrocytosis associated with a germline genetic variant in EPAS1. In addition we identified two infants with severe erythrocytosis associated with a mosaic mutation present in less than 2% of the blood, one of whom later developed a paraganglioma. The aim of this study was to determine the causal role of these genetic variants, to establish pathogenicity, and to identify potential candidates eligible for the new hypoxia-inducible factor-2 α (HIF-2α) inhibitor treatment. Pathogenicity was predicted with in silico tools and the impact of 13 HIF-2b variants has been studied by using canonical and real-time reporter luciferase assays. These functional assays consisted of a novel edited vector containing an expanded region of the erythropoietin promoter combined with distal regulatory elements which substantially enhanced the HIF-2α-dependent induction. Altogether, our studies allowed the classification of 11 mutations as pathogenic in 17 patients and 23 relatives. We described four new mutations (D525G, L526F, G527K, A530S) close to the key proline P531, which broadens the spectrum of mutations involved in erythrocytosis. Notably, we identified patients with only erythrocytosis associated with germline mutations A530S and Y532C previously identified at somatic state in tumors, thereby raising the complexity of the genotype/phenotype correlations. Altogether, this study allows accurate clinical follow-up of patients and opens the possibility of benefiting from HIF-2α inhibitor treatment, so far the only targeted treatment in hypoxia-related erythrocytosis disease.

Clinical and molecular diagnosis of hereditary and idiopathic erythrocytosis

Clinical and molecular diagnosis of hereditary and idiopathic erythrocytosis

Heritable disorders of oxygen sensing.

Hypoxia-inducible factors (HIFs) activate gene transcription in response to reduced O2 availability and play critical roles in development, physiology, and disease pathogenesis. Mutations that dysregulate HIF activity are the genetic basis for tumor predisposition in the von Hippel-Lindau syndrome and excess red blood cell production in hereditary erythrocytosis.

Heritable disorders of oxygen sensing.

Hypoxia-inducible factors (HIFs) activate gene transcription in response to reduced O2 availability and play critical roles in development, physiology, and disease pathogenesis. Mutations that dysregulate HIF activity are the genetic basis for tumor predisposition in the von Hippel-Lindau syndrome and excess red blood cell production in hereditary erythrocytosis.

Clinical Features of Idiopathic Erythrocytosis in a Racially Diverse Population

Background: Idiopathic erythrocytosis (IE) is an entity characterized by a persistently elevated hemoglobin, variable erythropoietin (EPO) level, absence of janus kinase 2 (JAK2) mutations suggestive of polycythemia vera (PV) and no identifiable secondary cause. Previous studies have compared IE to PV, showing a lower incidence of venous thrombosis and leukemic transformation in IE but similar incidence of arterial events. PV is known to be associated with constitutional symptoms and splenomegaly, while hereditary erythrocytosis can be associated with recurrent headaches and fatigue. A comprehensive assessment of clinical features including symptoms in IE has not been performed. Methods: Patients signed informed consent to participate in an observational study approved by the Institutional Review Board. Enrollment criteria included: age 18 years or older; hemoglobin level greater than 16 g/dL on two occasions at least 3 months apart or greater than 15 g/dL if undergoing phlebotomy; negative testing for JAK2 mutations; and negative work up for secondary causes of erythrocytosis. Baseline assessment included history and physical exam, vital signs, pulse oximetry, and body mass index. Baseline laboratory exams included a complete blood cell count, complete metabolic panel, C-reactive protein, iron panel, ferritin, hemoglobin A1C, erythropoietin level. Abdominal ultrasound was performed to evaluate for splenomegaly. The Myeloproliferative Symptom Assessment Form (MPN-SAF) was used to assess for the presence and severity of a broad range of symptoms that may be expected to occur in patients with IE. The MPN-SAF was administered at baseline and every 6 months thereafter. Results: 35 patients had data available for analysis. Patient characteristics are shown in Table 1. The most prevalent co-morbid conditions were those known to be associated with cardiovascular disease risk and metabolic syndrome, including hepatic steatosis identified on abdominal ultrasound in 63% of patients. Three (8.6%) patients had a history of venous or arterial thrombosis. Two (5.8%) patients had a history of lymphoma (NK/T-cell and Hodgkin). Three patients (8.6%) had a first-degree relative with a myeloproliferative neoplasm (chronic myelomonocytic leukemia, essential thrombocytosis and polycythemia vera) and one patient had a son with IE and history of stroke. 16 (46%) patients were taking aspirin and 11 (31%) had undergone phlebotomy within 3 months of study enrollment. Patients reported the following symptoms on the MPN-SAF at baseline: fatigue (77%), early satiety (57%), difficulty sleeping (57%), numbness/tingling (51%), headaches (49%), concentration problems (40%), itching (40%), bone pain (37%), night sweats (37%), depression (37%), abdominal pain (37%), abdominal discomfort (37%), inactivity (37%), problems with sexual desire/function (34%), dizziness/lightheadedness (31%), cough (26%), fever (17%), and unintentional weight loss (17%). Fatigue carried the highest average symptom intensity (3.77, SD 3.17). Discussion: In this study, we describe the clinical features associated with IE in a multiracial cohort. Patients with IE are frequently symptomatic and have a high incidence of hepatic steatosis by ultrasound. Disclosures No relevant conflicts of interest to declare.

The Genomics and Genetics of Oxygen Homeostasis.

Human survival is dependent upon the continuous delivery of O2 to each cell in the body in sufficient amounts to meet metabolic requirements, primarily for ATP generation by oxidative phosphorylation. Hypoxia-inducible factors (HIFs) regulate the transcription of thousands of genes to balance O2 supply and demand. The HIFs are negatively regulated by O2-dependent hydrox-ylation and ubiquitination by prolyl hydroxylase domain (PHD) proteins and the von Hippel-Lindau (VHL) protein. Germline mutations in the genes encoding VHL, HIF-2α, and PHD2 cause hereditary erythrocytosis, which is characterized by polycythemia and pulmonary hypertension and is caused by increased HIF activity. Evolutionary adaptation to life at high altitude is associated with unique genetic variants in the genes encoding HIF-2α and PHD2 that blunt the erythropoietic and pulmonary vascular responses to hypoxia.

Characterization of Pathogenic Variants Associated with Hereditary Thrombocytopenias in Families from the Czech Republic

Introduction Inherited thrombocytopenias (IT) are a heterogeneous group of 33 different forms of monogenic disorders caused by molecular defects affecting 40 genes at least. The pathogenic germline variants play an important role in the development and maintenance of hematopoietic system (megakaryopoesis and thrombopoesis). These changes lead to disruption of these processes and are presented as the thrombocytopenia phenotype (low platelet count, blood-examination). However, patients are occasionally misdiagnosed with the immune thrombocytopenia and unsuccessfully treated with steroid therapy and splenectomy. In some patients, accurate diagnosis of IT can only be established based on the results of molecular genetic testing. Furthermore, it has also been shown that some hematological conditions with Mendelian type of hereditability precede the development of hematooncological disease. Patients and Methods DNA samples from peripheral blood or buccal swabs of four unrelated families were isolated. The whole exome sequencing (WES) was performed using the NextSeq 500 Illumina instrument with adequate chemistry and sequencing libraries were prepared according to the SeqCap EZ Human Exome Probes v3 protocol. The generated data were processed using in-house bioinformatics pipelines. The detected pathogenic variants were confirmed by Sanger sequencing. Moreover, the novel variant was analyzed in silico using analytical procedures including protein modelling, too. Germline DNA analysis was performed on all available samples and somatic DNA analysis was done for the oncological patient. Within each family, the obtained pathogenic variants were compared between the individuals with IT phenotype and their disease-free relatives. Results The pathogenic variants were characterized in four families with different forms of IT. Moreover, the additional genetic variants were detected in three of them which predispose to the development of hematological malignancies. In the first family, a novel heterozygous variant c.320C>T; p.(Thr107Met) in TUBB1 gene is probably responsible for essential thrombocytopenia disease because all rare TUBB1 variants until now have been detected in patients with macrothrombocytopenia. The known pathogenic variant c.1402G>T; p.(Val468Phe) in JAK2 gene (10.9% frequency) was identified in a family member suffering from the myeloproliferative disease. In the second family, heterozygous pathogenic variants c.3076C>T; p.(Arg1026Trp) in ITGA2B gene and c.3188G>A; p.(Arg1063His) in JAK2 gene were detected, associated with platelet-type bleeding disorders and hereditary erythrocytosis with megakaryocytic atypia and predisposition for hematological malignancy, respectively. It is known that stomach tumor occurred in patient´s family before. In the third family, heterozygous pathogenic variant c.3493C>T; p.(Arg1165Cys) in MYH9 gene was identified in a patient with macrothrombocytopenia. This variant was associated with Sebastian syndrome, macrothrombocytopenia and granulocyte inclusions and predisposition to kidney failure, hearing loss, and cataracts. In the fourth family, ANKRD26-related thrombocytopenia with predisposition to myeloid malignancy was probably identified in a patient with detected heterogeneous known variant c.-140C>G in 5´ UTR of ANKRD26 gene. Moreover, the novel c.682C>T; p.(Arg228Trp) variant in SYTL3 gene with uncertain significance was detected in this patient. Conclusions The pathogenic variants were detected in unrelated affected families with macrothrombocytopenia, platelet-type bleeding disorders and hereditary erythrocytosis with megakaryocytic atypia, Sebastian syndrome, and ANKRD26-related thrombocytopenia. Moreover, the genetic variants predispose to myeloid malignancy were identified. Molecular genetic testing helped the clinicians to determine the correct diagnosis in these patients. This study was supported by Ministry of Health of the Czech Republic (grant No 16-29447A), and TA CR (TE02000058). Disclosures No relevant conflicts of interest to declare.

Algorithmic evaluation of hereditary erythrocytosis: Pathways and caveats.

Multiple algorithms have been published for the evaluation of hereditary erythrocytosis (HE). Typical entry points begin after excluding the more common acquired conditions through investigations of clinical history and assessment of cardiac, pulmonary, or vascular system disorders. Prior exclusion of JAK2 mutations, particularly the common JAK2 V617F mutation, is indicated in adults but less so in pediatric populations. Key decision trees are based on serum erythropoietin levels and p50 results. Recent data reveal some overlap in clinical presentation and laboratory findings in erythrocytosis. Caveats to consider when using algorithmic approaches are discussed.

JAK2 R1063H Variant Enhances V617F Constitutive Signaling and Favors Development of Essential Thrombocythemia with Increased Hemoglobin and Neutrophils

The clinical consequences of the highly prevalent JAK2 V617F mutation in Ph-negative myeloproliferative neoplasms (MPNs) are well studied. However, the impact on the MPN phenotype of co-occurring JAK2 variants is less well characterized. In this study, we focused on JAK2 R1063H, a variant previously reported in a pediatric case of hereditary erythrocytosis (Kapralova et al, Blood 2016) and in 3 out of 93 polycythemia vera patients that were JAK2 V617F-positive (Levine et al, Cancer Cell 2005). It has been described as a very rare polymorphism in population (frequency 0.004377).In order to gain insight into the clinical and functional relevance of coexisting JAK2 mutations, we tested a cohort of 390 JAK2 V617F-positive MPN patients for JAK2 R1063H and identified 14 carriers of both mutations. From the clinical perspective, the double-positive patients exhibited predominantly an essential thrombocythemia phenotype that was accompanied by significantly higher neutrophil granulocyte counts and hemoglobin values, when compared to those harboring only JAK2 V617F mutation. By employing targeted NGS for genes known to be involved in myeloid malignancies we found that the mutational profile and the number of additional somatic mutations in double-mutant patients seemed to be comparable to previous studies of JAK2 V617F-positive MPNs.Next, we used digital droplet PCR (ddPCR) for JAK2 R1063H and JAK2 V617F allele burden assessment. Quantification of JAK2 R1063H allele indicated that in 8 patients the variant was heterozygous, likely inherited (the percentage of the mutant allele being around 50%). In 3 patients with a high JAK2 V617F allelic burden a nearly homozygous status for R1063H was identified (a fractional abundance >80%) suggesting that one R1063H allele was inherited and the second one was acquired by uniparental disomy, as a result of mitotic recombination. In 3 other patients, the JAK2 R1063H mutation was most likely acquired due to the low percentage of the mutant allele (between 20.7% and 31.5%). For cis/trans configuration analysis of JAK2 V617F and R1063H mutations, we performed single-colony sequencing of subcloned JAK2 cDNA from 9 out of 14 patients. A cis configuration of the mutations was detected in 7 and a trans configuration in 2 cases. In one patient, the cis configuration of JAK2 mutations combined with results obtained by ddPCR (the allele burdens for R1063H and V617F were 31.5% and 52.8%, respectively) suggested that R1063H was acquired, following V617F acquisition.To assess the effect on JAK2 signaling of coexisting JAK2 V617F and R1063H mutations in cis or trans, we created human cDNA JAK2 mutants (V617F, R1063H and V617F/R1063H). STAT5 transcriptional activity of the JAK2 WT and JAK2 mutants in the presence of myeloid dimeric cytokine receptors (EPOR, given the higher hemoglobin levels, G-CSFR, given the observed neutrophilia in patients carrying JAK2 V617F and R1063H, and TPOR) measured in JAK2-deficient γ-2A cells by dual luciferase assay revealed a significantly higher constitutive activity of JAK2 V617F/R1063H (cis mutant) compared to that of JAK2 V617F, in both homozygous and heterozygous configurations and with either of the dimeric myeloid cytokine receptors. In the trans configuration of JAK2 V617F and JAK2 R1063H, we did not observe a significant difference compared to combination of JAK2 V617F and JAK2 WT. Western blot analysis assessing activated phosphorylated forms of JAK2, STAT5 and ERK1/2 demonstrated a higher level of constitutive activation of JAK2 and STAT5 generated by the double V617F/R1063H mutant versus JAK2 V617F and similar effect was shown also for ERK1/2 signaling.Our results suggest that either an acquisition of V617F in cis on a germline R1063H allele or a gain of additional JAK2 R1063H on JAK2 V617F will lead to an increase in downstream signaling by the driver JAK2 V617F. The oncogenic signaling of JAK2 V617F enhanced by the R1063H mutation has a clinical effect, leading to a significantly increased number of neutrophilic granulocytes and hemoglobin values, in agreement with higher levels of signaling via EPOR and G-CSFR.The work was supported by projects COP A1.1.4. ID: P_37_798, Contract 149/26.10.2016, (MySMIS2014+: 106774), MyeloAL, WELBIO F 44/8/5-MCF/UIG-10955, ARC: N° 16/21-073, by Ludwig Institute for Cancer Research, Salus Sanguinis Foundation, Fondation Les avions de Sébastien, Belgium and by GACR 17-05988S and MSMT LTAUSA17142. DisclosuresNo relevant conflicts of interest to declare.

Genotype-Phenotype Correlation of Hereditary Erythrocytosis Mutations, a single center experience.

Hereditary erythrocytosis is associated with high oxygen affinity hemoglobin variants (HOAs), 2,3-bisphosphoglycerate deficiency and abnormalities in EPOR and the oxygen-sensing pathway proteins PHD, HIF2α, and VHL. Our laboratory has 40 years of experience with hemoglobin disorder testing and we have characterized HOAs using varied protein and molecular techniques including functional assessment by p50 analysis. In addition, we have more recently commenced adding the assessment of clinically relevant regions of the VHL, BPGM, EPOR, EGLN1 (PHD2), and EPAS1 (HIF2A) genes in a more comprehensive hereditary erythrocytosis panel of tests. Review of our experience confirms a wide spectrum of alterations associated with erythrocytosis which we have correlated with phenotypic and clinical features. Through generic hemoglobinopathy testing we have identified 762 patients with 81 distinct HOA Hb variants (61 β, 20 α), including 12 that were first identified by our laboratory. Of the 1192 cases received for an evaluation specific for hereditary erythrocytosis, approximately 12% had reportable alterations: 85 pathogenic/likely pathogenic mutations and 58 variants of unknown significance. Many have not been previously reported. Correlation with clinical and phenotypic data supports an algorithmic approach to guide economical evaluation; although, testing is expanded if the suspected causes are negative or of uncertain significance. Clinical features are similar and range from asymptomatic to recurrent headaches, fatigue, restless legs, chest pain, exertional dyspnea and thrombotic episodes. Many patients were chronically phlebotomized with reported relief of symptoms. This article is protected by copyright. All rights reserved.

Cooperation of germ line JAK2 mutations E846D and R1063H in hereditary erythrocytosis with megakaryocytic atypia

AbstractThe role of somatic JAK2 mutations in clonal myeloproliferative neoplasms (MPNs) is well established. Recently, germ line JAK2 mutations were associated with polyclonal hereditary thrombocytosis and triple-negative MPNs. We studied a patient who inherited 2 heterozygous JAK2 mutations, E846D from the mother and R1063H from the father, and exhibited erythrocytosis and megakaryocytic atypia but normal platelet number. Culture of erythroid progenitors from the patient and his parents revealed hypersensitivity to erythropoietin (EPO). Using cellular models, we show that both E846D and R1063H variants lead to constitutive signaling (albeit much weaker than JAK2 V617F), and both weakly hyperactivate JAK2/STAT5 signaling only in the specific context of the EPO receptor (EPOR). JAK2 E846D exhibited slightly stronger effects than JAK2 R1063H and caused prolonged EPO-induced phosphorylation of JAK2/STAT5 via EPOR. We propose that JAK2 E846D predominantly contributes to erythrocytosis, but is not sufficient for the full pathological phenotype to develop. JAK2 R1063H, with very weak effect on JAK2/STAT5 signaling, is necessary to augment JAK2 activity caused by E846D above a threshold level leading to erythrocytosis with megakaryocyte abnormalities. Both mutations were detected in the germ line of rare polycythemia vera, as well as certain leukemia patients, suggesting that they might predispose to hematological malignancy.

47-Year-Old Man With Pruritus

47-year-old man with a medical history of headaches and seasonal allergies presented to the primary care clinic in late fall for evaluation of diffuse pruritus of 6 weeks’ duration that had occurred after initiating a new diet. The diet mostly restricted foods like dairy, soy, nuts, grains, and processed foods, but he stated that he had substantially increased his consumption of coconut and various spices including turmeric. The itching was present over most of his body but most prominent on the inner thighs and most notable after a hot shower. He provided picturesfromrecent episodes thatillustrated linear excoriations on the thighs. When he showered in cooler water, the diffuse pruritus was still present but minimally improved compared with a hot shower. He had been using the same body soap for years but tried a new soap without relief from the itching. In addition, he changed laundry detergent from one he had used for years with no relief. He had no occupational or habitual exposures that are suspected to contribute to pruritus. Overthe-counter hydrocortisone cream applied to his thighs provided minimal relief. His weight had been stable, and he reported no constitutional symptoms. On physical examination, the patient was a

Spectrum of Mutations Associated with Hereditary Erythrocytosis

▪Background: Mechanisms of hereditary erythrocytosis have been elucidated recently. These include high oxygen affinity (HOA) hemoglobin (Hb) variants, bisphosphoglycerate mutase (BPGM) deficiency, abnormalities in the erythropoietin receptor (EPOR) and oxygen-sensing pathway (OSP) proteins prolyl hydroxylase domain-2 (PHD2), hypoxia-inducible factor-2 alpha subunit (HIF2A), and von Hippel-Lindau (VHL). We present our experience with these disorders.Methods: Evaluation of erythrocytosis patients' blood samples for HOA Hb variants by protein or DNA sequencing methods has been performed in our laboratory for over 30 years. Testing has included alkaline/acid electrophoresis, isoelectric focusing, capillary electrophoresis, high performance liquid chromatography, mass spectrometry and Sanger sequencing. BPGM deficiency was determined by enzyme activity assay and/or genetic testing. Since 2012 we have evaluated the EPOR, EGLN1 (PHD2), EPAS1(HIF2A), and VHL genes. Reflexive evaluations, which include testing for p50 and pertinent Sanger sequencing of HBB, HBA1, HBA2, EPOR (exon 8), EGLN1 (exons 1-5), EPAS1 (exon 12) and VHL, have been performed on a subset of cases. Genetic results were correlated with clinical and phenotypic data.Results: Retrospective database review identified 62 confirmed HOA Hb variants (48β, 14α) with clinical erythrocytosis (Table 1). Multiple Hb variants were silent on at least one protein method but showed decreased p50 values. One homozygous BPGM mutation case was identified. Of the 394 cases evaluated for EPOR and OSP abnormalities, 39 cases (10%) contained genetic alterations. Of these, 11 were known pathogenic mutations and 28 were novel alterations including 4 pathogenic, 12 likely pathogenic and 12 variants of unknown significance (VUS). Eighteen EGLN1, 10 EPAS1, and 7 EPOR alterations were detected, all heterozygous. Four VHL mutation cases were identified: 3 homozygous/compound heterozygous known mutations and one novel heterozygous VUS (p.R200Q). All EPOR mutations resulted in a premature stop codon. Most of the EPAS1 alterations were located near proline 531, although two predicted splice site disturbances were found. The EGLN1 alterations were novel, variable, and affect the enzyme's catalytic domain. All 4 VHL cases involved the R200 amino acid position in at least one allele. Serum Epo levels varied: decreased in EPOR (5/5); normal in PHD2 (11/11) and most HIF2A (6/7) and VHL (3/4) cases; increased in 1 HIF2A and 1 VHL case. p50 was normal in EPOR and OSP cases (31/31 tested).Conclusion: A spectrum of abnormalities was identified. Salient points include: 1) Diagnostically, a high index of suspicion for HOA Hb variants is required as they can give false negative results by routine protein detection methods. p50 and Epo levels were useful for triage. 2) Phenotypically, serum Epo levels were low in EPOR and normal in PHD2 and HIF2A cases. Unexpectedly, the two classic Chuvash polycythemia VHL cases were associated with normal Epo levels. 3) Mechanistically, the alterations were truncating in EPOR, variable and scattered in EGLN1 and clustered near proline 531 in EPAS1. VHL mutations were rare and consistently affected the R200 amino acid position in at least one allele. Novel alterations were frequent, especially in EGLN1. 4) Clinically, many patients were asymptomatic but a subset demonstrated recurrent headaches and one had chest pain. Clotting complications included cerebrovascular accident in one EPAS1 and EGLN1 case each and portal vein thrombosis in one EGLN1 case.Table 1Clinically significant high oxygen affinity Hb variants at Mayo ClinicnBetaAlpha>30MalmoTarrant10 - 20Bethesda, San Diego, Andrew-Minneapolis, M-Saskatoon, Olympia, Syracuse, AbruzzoDallas5 - 10Ty Gard, Ypsilanti, Alberta, Coimbra, North ChicagoJ-Cape Town2 - 4Brigham, Kempsey, Little Rock, Puttelange, Wood, Brisbane, Cowtown, Creteil, Linkoping, Osler, Potomac, Providence, Ranier, Sparta, Vanderbilt, Heathrow, Helsinki, Hiroshima, Homozygous HPFH, McKees-Rocks, Pierre Benite, ReginaChesapeake, Ethiopia, Chiapas, Columbia Missouri, Burlington, Legnano1Alcorn County*, Bologna-St. Orsola, Bunbury, Cambridge-MA, Cardarelli, Chandigarh, Johnstown, Nantes, Nebraska, Olomouc, Palmerston North, Pitie-Salpetriere, Saint-Jacques, South MilwaukeeLinwood, Longview*, Milledgeville, Sarasota Springs, Voorhees*novel DisclosuresPardanani:Stemline: Research Funding.

Hereditary gene mutations in Korean patients with isolated erythrocytosis

Most cases of erythrocytosis occur secondary to chronic tissue hypoxia or as a clonal disease such as polycythemia vera with somatic mutations in the Janus kinase 2 (JAK2) gene. Rarely, erythrocytosis is caused by hereditary gene mutations. This study investigated hereditary gene mutations in 38 unrelated Korean patients with isolated erythrocytosis without (1) JAK2 mutation and (2) secondary causes of erythrocytosis other than smoking history. Direct sequencing analyses were performed on six genes associated with hereditary erythrocytosis [HBB, exon 2 and exon 3 of HBA2, VHL, EGLN1 (previously PHD2), exon 12 of EPAS1 (previously HIF2A), and exons 5-8 of EPOR]. As a result, mutations were detected in five patients (three never smokers and two current smokers) out of 38 patients (13.2%). The mutations detected in those five patients were EPOR:p.W439*, EPOR:p.G212C, HBB:p.H98Q (or conventionally H97Q, Hb Malmö [β 97(FG4) His > Gln]), HBB:p.V138M (V137M), and EGLN1:p.L279Tfs43*, all in heterozygous state. No patient had mutations in HBA2, VHL, or in EPAS1. This study indicates that workup for hereditary gene mutations is needed for isolated erythrocytosis with or without smoking history.

Oxygen Sensing, Hypoxia-Inducible Factors, and Disease Pathophysiology

Hypoxia-inducible factors (HIFs) are transcriptional activators that function as master regulators of oxygen homeostasis, which is disrupted in disorders affecting the circulatory system and in cancer. The role of HIFs in these diseases has been elucidated by clinical studies and by analyses of mouse models. HIFs play a protective role in the pathophysiology of myocardial ischemia due to coronary artery disease, limb ischemia due to peripheral arterial disease, pressure-overload heart failure, wound healing, and chronic rejection of organ transplants. In contrast, HIFs contribute to the pathogenesis of pulmonary arterial hypertension, systemic hypertension associated with sleep apnea, ocular neovascularization, hereditary erythrocytosis, and cancer.

Inherited predisposition to myeloproliferative neoplasms

Myeloproliferative neoplasms (MPNs) are haematological disorders characterized by an overproduction of mature myeloid cells with a tendency to transform to acute myeloid leukaemia. Clonal proliferation of myeloid progenitor cells is driven by somatically acquired mutations, most notably JAK2 V617F, but there are important features relating to pathogenesis and phenotypic diversity that cannot be explained by acquired mutations alone. In this review we consider what is currently known about the role that inherited factors play in the development and biology of both sporadic and familial forms of MPN. Although most MPN cases appear to be sporadic, familial predisposition has been recognized for many years in a subset of cases and epidemiological studies have indicated the presence of common susceptibility alleles. Currently the JAK2 46/1 haplotype (also referred to as 'GGCC') is the strongest known predisposition factor for sporadic MPNs carrying a JAK2 V617F mutation, explaining a large proportion of the heritability of this disorder. Less is known about what genetic variants predispose to MPNs that lack JAK2 V617F, but there have been recent reports of interesting associations in biologically plausible candidates, and more loci are set to emerge with the application of systematic genome-wide association methodologies. Several highly penetrant predisposition variants that affect erythropoietin signalling, thrombopoietin signalling or oxygen sensing have been characterized in families with nonclonal hereditary erythrocytosis or thrombocytosis, but much less is known about familial predisposition to true clonal MPN. The heterogeneous pattern of inheritance and presumed genetic heterogeneity in these families makes analysis difficult, but whole exome or genome sequencing should provide novel insights into these elusive disorders.

First Achievements of MPN&MPNr-EuroNet (COST Action BM0902), a New European Network Dedicated to the Diagnosis of Myeloproliferative Neoplasms and Hereditary Erythrocytosis and Thrombocytosis

Abstract 2809 Background:The MPN&MPNr-EuroNet network, created in November 2009, is supported by the European program COST (CoOperation in Science and Technology). It is open to all colleagues active in the fields of myeloprolifeative neoplasms (MPN) and related hereditary diseases (MPNr: hereditary erythrocytosis and thrombocytosis). AIMS:To facilitate, improve and innovate in the diagnosis of MPN and hereditary erythrocytosis and thrombocytosis in Europe. Methods:MPN&MPNr-EuroNet has formed 4 working groups (WG): WG 1 focuses on JAK2 -mutated MPN; WG 2 is dedicated to thrombocythemia and myelofibroses without mutation of JAK2 and includes subgroups specialized in hereditary thrombocytosis (HT) and in histopathology; WG 3 is dedicated to hereditary erythrocytosis (HE); WG 4 is responsible for scientific cooperation and the diffusion of scientific knowledge. Results:During the first 18 months of MPN&MPNr-EuroNet activity, 77 colleagues from 19 countries (16 European countries plus Israel, Turkey, and the USA), joined the network and participated in the four WG, resulting in the achievements listed below.WG 1: 1) determination of the best JAK2 V617F assays, a joint MPN&MPNr-EuroNet/European LeukemiaNet project; 2) on-going study of MPN cases with low JAK2 V617F burden; 3) on-going study of MPN cases with multiple JAK2 mutation.WG 2: 1) list of laboratories responsible for the diagnosis of MPL and THPO mutations in Europe; 2) first international quality test of the detection of MPL mutations; 3) on-going study of new THPO and MPL mutations in HT cases; 4) on-going study of the histopathology of MPN without JAK2 mutation.WG 3: 1) list of laboratories responsible for the diagnosis of HE in Europe; 2) consensus on a diagnostic algorithm for the diagnosis of HE; 3) close interaction with COST Action TD0901 (HypoxiaNet) to facilitate the discovery of new genes of interest for the diagnosis of HE; 4) exchange of positive control samples for the main mutations responsible for HE; 5) study of idiopathic erythrocytosis.WG 4: 1) MPN&MPNr-EuroNet website: www.mpneuronet.eu; 2) organization of bi-annual meetings (5th meeting: March 7–9, 2012, Belfast, United Kingdom); 3) organization of annual training schools: a May training school dedicated to the molecular detection of JAK2 and MPL mutations (in Nîmes, France), and an October training school dedicated to hereditary erythrocytosis (in Coimbra, Portugal); 4) financial support for short term scientific missions for exchange and collaborative studies between participating institutions. Conclusion:MPN&MPNr-EuroNet will enable European researchers, biologists and clinicians to define new diagnostic tools and exchange technologies. MPN&MPNr-EuroNet is open to all interested physicians and scientists and we invite new members, including those from outside Europe, to join. Scholarships are available to finance participation in meetings and training schools, and to facilitate exchanges between participating institutions. For detailed information on all MPN&MPNr-EuroNet activities, see www.mpneuronet.eu. Disclosures:Schnittger:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Vannucchi:Novartis: Honoraria.