Idiopathic Erythrocytosis Research Articles

Impact of HFE mutations on thrombotic risk in patients with idiopathic erythrocytosis: A single‐centre study

Impact of <i>HFE</i> mutations on thrombotic risk in patients with idiopathic erythrocytosis: A single‐centre study

TNF-α is a predictive marker in distinguishing myeloproliferative neoplasm and idiopathic erythrocytosis/thrombocytosis: development and validation of a non-invasive diagnostic model.

Philadelphia-chromosome negative myeloproliferative neoplasms (MPN) exhibit phenotypic similarities with JAK/STAT-unmutated idiopathic erythrocytosis and thrombocytosis (IE/IT). We aimed to develop a clinical diagnostic model to discern MPN and IE/IT. A retrospective study was performed on 77 MPN patients and 32 IE/IT patients in our center from January 2018 to December 2023. We investigated the role of hemogram, cytokine and spleen size in differentiating MPN and IE/IT among newly onset erythrocytosis and thrombocytosis patients. Independent influencing factors were integrated into a nomogram for individualized risk prediction. The calibration and discrimination ability of the model were evaluated by concordance index (C-index), calibration curve. MPN had significantly higher TNF-α level than IE/IT, and the TNF-α level is correlated with MF-grade. Multivariable analyses revealed that TNF-α, PLT count, age, size of spleen were independent diagnostic factors in differentiating MPN and IE/IT. Nomograms integrated the above 4 factors for differentiating MPN and IE/IT was internally validated and had good performance, the C-index of the model is 0.979. The elevation of serum TNF-α in MPN patients is of diagnostic significance and is correlated with the severity of myelofibrosis. The nomogram incorporating TNF-α with age, PLT count and spleen size presents a noteworthy tool in the preliminary discrimination of MPN patients and those with idiopathic erythrocytosis or thrombocytosis. This highlights the potential of cytokines as biomarkers in hematologic disorders.

Coexistence of Multiple Gene Variants in Some Patients with Erythrocytoses.

Erythrocytosis is a relatively common condition; however, a large proportion of these patients (70%) remain without a clear etiologic explanation. We set up a targeted NGS panel for patients with erythrocytosis, and 118 sporadic patients with idiopathic erythrocytosis were studied. In 40 (34%) patients, no variant was found, while in 78 (66%), we identified at least one germinal variant; 55 patients (70.5%) had 1 altered gene, 18 (23%) had 2 alterations, and 5 (6.4%) had 3. An altered HFE gene was observed in 51 cases (57.1%), EGLN1 in 18 (22.6%) and EPAS1, EPOR, JAK2, and TFR2 variants in 7.7%, 10.3%, 11.5%, and 14.1% patients, respectively. In 23 patients (19.45%), more than 1 putative variant was found in multiple genes. Genetic variants in patients with erythrocytosis were detected in about 2/3 of our cohort. An NGS panel including more candidate genes should reduce the number of cases diagnosed as "idiopathic" erythrocytosis in which a cause cannot yet be identified. It is known that HFE variants are common in idiopathic erythrocytosis. TFR2 alterations support the existence of a relationship between genes involved in iron metabolism and impaired erythropoiesis. Some novel multiple variants were identified. Erythrocytosis appears to be often multigenic.

Idiopathic erythrocytosis: A diagnostic and management challenge with emerging areas for exploration.

Despite published algorithms for approaching the work-up of erythrocytosis, a significant proportion of patients are left with uncertainty as to its aetiology and prognosis. The term 'idiopathic erythrocytosis' (IE) is applied when known primary and secondary aetiologies have been ruled out. However, the assignment of secondary aetiologies is not always straightforward or evidence based, which can lead to misdiagnosis and heterogeneity in cohort studies. Furthermore, new studies have identified germline or somatic mutations that may affect prognosis. Epidemiological and cohort data are inconsistent as to whether IE increases the risk for complications such as arterial and venous thromboembolism, clonal transformation or comorbid conditions. Randomized trials assessing the role of phlebotomy for long-term management of IE have not been performed, so treatment remains a vexing problem for clinicians. Standardization of terminology and testing strategies, including comprehensive genetic screening in clinical research, are key to refining our understanding of IE.

Idiopathic erythrocytosis: a germline disease?

Polycythemia Vera (PV) is typically caused by V617F or exon 12 JAK2 mutations. Little is known about Polycythemia cases where no JAK2 variants can be detected, and no other causes identified. This condition is defined as idiopathic erythrocytosis (IE). We evaluated clinical-laboratory parameters of a cohort of 56 IE patients and we determined their molecular profile at diagnosis with paired blood/buccal-DNA exome-sequencing coupled with a high-depth targeted OncoPanel to identify a possible underling germline or somatic cause. We demonstrated that most of our cohort (40/56: 71.4%) showed no evidence of clonal hematopoiesis, suggesting that IE is, in large part, a germline disorder. We identified 20 low mutation burden somatic variants (Variant allelic fraction, VAF, < 10%) in only 14 (25%) patients, principally involving DNMT3A and TET2. Only 2 patients presented high mutation burden somatic variants, involving DNMT3A, TET2, ASXL1 and WT1. We identified recurrent germline variants in 42 (75%) patients occurring mainly in JAK/STAT, Hypoxia and Iron metabolism pathways, among them: JAK3-V722I and HIF1A-P582S; a high fraction of patients (48.2%) resulted also mutated in homeostatic iron regulatory gene HFE-H63D or C282Y. By generating cellular models, we showed that JAK3-V722I causes activation of the JAK-STAT5 axis and upregulation of EPAS1/HIF2A, while HIF1A-P582S causes suppression of hepcidin mRNA synthesis, suggesting a major role for these variants in the onset of IE.Graphical abstract

Utility of next-generation sequencing in identifying congenital erythrocytosis in patients with idiopathic erythrocytosis.

Congenital erythrocytosis (CE) is increasingly recognized as the cause of erythrocytosis in patients in whom polycythemia vera and secondary acquired causes have been excluded. The aim of our study was to determine possible genetic background in patients with idiopathic erythrocytosis. 40 patients with idiopathic erythrocytosis, referred to our institution in a 5-year period, were analyzed. We collected data on erythropoietin (Epo) levels, hemoglobin (Hgb), hematocrit (Hct), erythrocyte count, age, gender, past thrombotic events, concomitant diseases, and smoking status. CE was tested using next-generation sequencing (NGS), in the majority of patients also measurement of P50 and Hgb electrophoresis were performed. Patients with signs of iron overload were tested for genetic variants in the HFE gene. The median patient age at analysis was 46.5 years (range 22-73), with 37 out of 40 being males (93 %). The median Hgb, Hct and red blood cells count were 180 g/L, 0.51, 5.985 x 1012/L in men and 171 g/L, 0.50 and 5.68 x 1012/L in women, respectively. Epo levels were decreased in three, increased in one patient and within the normal range in the rest (median 7.55 mIU/mL; range 2.90-19.50). Eight patients (20 %) smoked. 32 (80 %) were treated with low-dose aspirin, and 20 (50 %) underwent at least one phlebotomy. Thromboembolic events were recorded in 2 patients (5 %). P50 was measured in 20 out of 40 patients, and it was above 24 mm Hg (3.12 kPa) in all of them. Hemoglobin electrophoresis was performed in 73 % of patients, with no abnormal Hgb detected. Variants in the HFE gene were found in 8 out of 40 patients (20 %), but in only one patient the results were associated with an increased risk for hemochromatosis. Although no pathogenic variants for CE were detected by NGS, two variants of uncertain significance, namely EGLN1 (NM_022051.2):c.1072C>T (p.(Pro358Ser)) and EGLN1 (NM_022051.2):c.1124A>G (p.(Glu375Gly)) were identified as strong etiologic candidates. CE is an extremely rare condition. Genetic testing is advised in young individuals with a long-standing persistent erythrocytosis, possibly with a family history and after exclusion of more frequent secondary causes and polycytemia vera.

Targeted NGS analysis reveals a complex genetic background of idiopathic erythrocytosis in a large Venetian family

Targeted NGS analysis reveals a complex genetic background of idiopathic erythrocytosis in a large Venetian family

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.

Sphingomyelin and Phosphatidylserine Derived Compounds Found into RBCs of Two Unrelated Individuals with Idiopathic Erythrocytosis with Altered Hemoglobin (Hb) Function Properties and No Hemoglobinopathies - Preliminary Results

Sphingomyelin and phosphatidylserine derived compounds were found into Red Blood Cells (RBCs) of two unrelated individuals with idiopathic erythrocytosis, whom were referred to the Laboratory of Hemoglobinopathies - State University of Campinas (State of São Paulo, Brazil) to investigate the presence of hemoglobin (Hb) variants with high oxygen affinity. Both unrelated individuals were 5-year-old age Caucasian Brazilian boys with the following counts: RBC (x 10/6mm3) 6.15 and 7.69, Hb (g/dL) 20.1 and 20, Htc (%) 54 and 58.7, respectively. Most common causes of erythrocytosis, including polycythemia vera (PV) and other secondary erythrocytosis; JAK2, VHL and EPOR mutational screen were previously excluded in the Centro Infantil Boldrini (Campinas, State of São Paulo, Brazil), where the children are followed. No hemoglobin or globin variants were detected by standard methods: alkaline electrophoresis in cellulose acetate (pH 8.9), acidic electrophoresis in agar gel (pH 5.8), cation-exchange HPLC (Variant II, Bio-Rad) and RP-HPLC (Alliance, Waters). Direct genome sequencing of HBA and HBB performed in automated sequencer (ABI PRISM™ 377 DNA Automated Sequencer, Applied BioSystems) after DNA amplification obtained from peripheral leukocytes (QIAamp DNA Blood, Qiagen) did not reveal any mutation. Familial study also did not show any alteration in blood count, hemoglobin or even globin gene mutation in their relatives. Deletional and non-deletional alpha thalassemia was also excluded by Multiplex-gap-PCR and restriction enzyme reaction (Hph and NcoI). Despite of normal results for Hb characterization, functional tests performed with Hb purified by exclusion (Sephadex G-25, Sigma-Aldrich) and ion exchange chromatography (Amberlite MB-3, Sigma-Aldrich) in Hepes buffer indicated some alteration. O2 affinity was evaluated by determining P50 with stripped lysates ([Hb]=70 μM/Heme) in pHs of 6.5, 7.0, 7.5, 8.0 and 8.5, for Bohr effect calculations. Heme-heme cooperativity was determined by Hill coefficient (n) and the activity of allosteric effector in the Hb-O2 ligation was also tested, using inositol hexaphosphate ([IHP]=1mM), a mimetic compound of the intraerythrocytic 2,3-BPG. P50 was slightly decreased (both patients, stripped Hb samples) at extreme acidic and alkaline pH (pH 6.5 and 8.5) leading to altered Bohr effect, in comparison to healthy and erythrocytosis control (caused by cardiopathy, with no Hb variant). P50 was also found decreased at pH 8.5 in the presence of IHP. Heme-heme cooperativity was very decreased at pHs 6.5, 7.0, 8.0 and 8.5, in stripped state as well as in the presence of IHP. In order to investigate the unexpected results mass spectrometry of the purified intraerythrocytic content was performed. RBC lysate was separated from stroma by ultracentrifugation (14.000rpm) and purified by molecular exclusion filtration (Amicon filter - Millipore, 10KDa cutoff) and ion exchange chromatography (Amberlite MB-3, Sigma-Aldrich) for desalinization. Quadrupole Mass spectrometry (MS) with 0.8 ppm MS and MS/MS accuracy (Impact High DefinitionTM, Bruker), preceded by UPLC (Ultra Pressure Liquid Chromatography) system (Shimadzu Nexera) with C18 Polymer RP-column (Supelco apHera C18 Polymer 150 x 2 mm, 5um) was used to separate and identify and compare the compounds to control sample (secondary erythrocytosis with no Hb function alteration). No alteration in peptide, hemoglobin or heme were found. Four small compounds, two derived of sphingomyelin and two derived of phosphatidylserine were identified in both patient samples (C39H79N2O6P, C42H80NO8P, C42H82NO8P, C44H84NO8P) - Figure 1. Sphingomyelin and phosphatidylserine are RBC membrane compounds rich in PO4-, which theoretically could bind b1b2 allosteric 2,3-BPG site in Hb. There is no evidence that these molecules were effectively bound to Hb except that were found in purified Hb samples with some function alteration, particularly with decreased heme-heme cooperativity. Some evidence suggests that analog compounds could be found in higher concentrations in PV or secondary erythrocytosis. To our knowledge it is the first-time that alteration in Hb function was observed in samples with these molecules. These preliminary results should be further validated and at least 10 other similar cases should be better characterized. (Financial support: Fapesp, Faepex-Unicamp, Educorp-Unicamp). Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Clinical Features and Outcomes of Polycythemia Vera Patients with Different JAK2 V617F Mutational Status and Allele Burden

Polycythemia vera (PV) is the most common myeloproliferative neoplasms (MPNs) characterized by the overproduction of morphologically and functionally normal blood cells. Whereas, the clinical usefulness of JAK2 V617F allele burden is still under investigation. We retrospectively reviewed the clinical parameters and haematologic information of 276 patients with PV, including 247 JAK2 mutated and 29 JAK2 unmutated patients, and 38 patients with idiopathic erythrocytosis in the First Affiliated Hospital of Zhejiang University School of Medicine and the Fourth Affiliated Hospital of Zhejiang University School of Medicine(ChiCTR2200061208). JAK2 V617F allele mutational burden was performed in 183 PV patients by RQ-PCR. The expression of WT1 gene was performed in 28 PV patients .JAK2 mutated PV exhibited higher leukocyte counts[12.4(5.0-36.8)×109/L vs 7.1(4.7-17.1)×109/L, P<0.001], neutrophil counts[11.3(3.9-26.6)×109/L vs 4.4(2.2-6.3)×109/L, P<0.001], monocyte counts[0.7(0.3-2.2)×109/L vs 0.6(0.3-0.9)×109/L, P=0.004], eosinophil counts[0.26(0.02-0.96)×109/L vs 0.12(0.04-0.28)×109/L, P<0.001], basophil counts[0.13(0.01-0.62)×109/L vs 0.05(0.02-0.06)×109/L, P<0.001], red blood cell distribution width values[15.2(12.3-23.1)fL vs 12.4(11.7-15.5)fL, P<0.001], platelet counts[616(153-1436)×109/L vs 230(158-375)×109/L, P<0.001], lactate dehydrogenase values[320(144-547) U/L vs 196(117-261) U/L, P<0.001], onset ages[61(26-89) years vs 52(17-72) years, P=0.003], a higher frequency of splenomegaly(41.3% vs 6.9%, P<0.001), lower body mass index[22.91(15.57-32.58) kg/m2 vs 24.83(19.62-34.90) kg/m2, P=0.019] , lymphocyte counts[1.6(0.8-8.7)×109/L vs 2.2(1.3-3.9)×109/L, P=0.015], mean corpuscular hemoglobin values[30.0(22.2-38.6)pg vs 31.5(28.9-36.8)pg, P=0.008], mean corpuscular hemoglobin concentration values[327(294-362)g/L vs 345(324-361)g/L, P<0.001] and serum cholesterol level[3.81(1.84-6.44) mmol/L vs 5.04(3.67-6.41) mmol/L, P=0.001] than JAK2 unmutated patients. PV patients exhibited higher leukocyte counts[13.8(5.0-36.8)×109/L vs 7.5(3.8-11.3)×109/L, P<0.001], neutrophil counts[10.2(2.2-26.6)×109/L vs 4.8(2.1-7.5)×109/L, P<0.001], monocyte counts[0.7(0.3-2.2)×109/L vs 0.5(0.1-1.0)×109/L, P=0.001], eosinophil counts[0.23(0.02-0.96)×109/L vs 0.12(0.03-0.58)×109/L, P=0.001], basophil counts[0.09(0.01-0.62)×109/L vs 0.04(0-0.12)×109/L, P<0.001], red blood cell distribution width values[14.3(11.7-23.1)fL vs 12.5(11.7-15.4)fL, P<0.001], platelet counts[493(153-1436)×109/L vs 233(137-428)×109/L, P<0.001], lactate dehydrogenase values[294(117-547) U/L vs 203(134-301) U/L, P<0.001], onset ages[60(17-89) years vs 45 (20-83) years, P<0.001], a higher frequency of splenomegaly(37.7% vs 7.9%, P<0.001), lower body mass index[23.11(15.57-34.90) kg/m2 vs 25.97(18.40-39.18) kg/m2, P=0.001], mean corpuscular hemoglobin concentration values[332(294-362)g/L vs 342(314-377)g/L, P<0.001] and serum cholesterol level[4.12(1.84-6.44) mmol/L vs 5.00(2.78-7.83) mmol/L, P=0.001] than idiopathic erythrocytosis patients. Among the 276 PV patients, 183 were measured with JAK2 V617F allele burden. The analysis of spearman rank correlation showed that JAK2 V617F allele burdens were positively correlated with white blood cell counts(r=0.5516, P<0.0001), neutrophil count(r=0.8358, P<0.0001), lactate dehydrogenase levels(r=0.6915, P<0.0001). Moreover, the expression level of WT1 gene was positively correlated with JAK2 V617F allele burden(r=0.6653, P=0.0001), white blood cell counts(r=0.4276, P=0.0184), neutrophil count(r=0.4238, P=0.0220), platelet counts(r=0.3914, P=0.0324) and lactate dehydrogenase levels(r=0.6500, P=0.0001). In conclusion, the JAK2 V617F allele burden and the expression level of WT1 gene were correlated with hematologic parameters and clinical outcomes of PV patients. These two tests are recommended for all patients with PV, irrespective of hemoglobin level. The research was supported by the Key R&D Program of Zhejiang, No. 2022C03137; Public Technology Application Research Program of Zhejiang, China, No. LGF21H080003; the Key Project of Jinhua Science and Technology Plan, China, No. 2020-3-011;the 2019-2022 Key Medical Discipline (Hematology) Fund of Jinhua, China. Correspondence: Jian Huang househuang@zju.edu.cn. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Effects of idiopathic erythrocytosis on the left ventricular diastolic functions and the spectrum of genetic mutations: A case control study.

Background:We have aimed at exposing left ventricular diastolic functions and the presence of known genetic mutations for familial erythrocytosis, in patients who exhibit idiopathic erythrocytosis.Methods:Sixty-four patients with idiopathic erythrocytosis (mean age, 46.4 ± 2.7 years) and 30 age-matched healthy subjects were prospectively evaluated. The regions of interest of the erythropoietin receptor, hemoglobin beta-globin, von Hippel-Lindau, hypoxia-inducible factor 2 alpha, and Egl-9 family hypoxia-inducible factor genes were amplified by PCR. Left ventricular (LV) mass was measured by M-mode and 2-dimensional echocardiography. LV diastolic functions were assessed by conventional echocardiography and tissue Doppler imaging.Results:As a result of genetic analyses, genetic mutations for familial erythrocytosis were detected in 5 patients. It has been observed in our study that the risk of cardiovascular disorders is higher in patients. Interventricular septum thickness, left atrial diameter, and some diastolic function parameters such as deceleration time and isovolumetric relaxation time have been found to be significantly higher in idiopathic erythrocytosis group than in the controls.Conclusion:This study has shown that LV diastolic functions were impaired in patients with idiopathic erythrocytosis. In this patient group with increased risk of cardiovascular disorders, the frequent genetic mutations have been detected in 5 patients only. Therefore, further clinical investigations are needed as novel genetic mutations may be discovered in patients with idiopathic erythrocytosis because of cardiovascular risk.

PB2050: EVALUATION OF NON-CLONAL ERYTHROCYTOSIS: SINGLE CENTRE EXPERIENCE

Background: Secondary erythrocytosis has a broad differential diagnosis that includes hypoxic lung disease, cyanotic congenital heart disease, kidney disease, medications and erythropoietin-producing malignant disorders. Aetiology remains unknown in some patients, they have the so-called idiopathic erythrocytosis (IE). Among the latter, patients with inherited/congenital erythrocytosis can be found, especially if they have a positive family history. Aims: The aim of our study was to analyse the characteristics of non-clonal erythrocytosis in patients referred to our Haematology department in a 5-year period (2016 – 2021) and to compare the IE group to the group with known causes. Methods: We included all individuals who were Janus kinase 2 (JAK2) mutation negative and had erythrocytosis. Data was retrospectively collected with the help of the hospital database and statistically analysed with SPSS. Results: The study included 250 patients, 176 (70%) men and 74 (30%) women, with mean age 58.8 years (21 – 94 years). Their average haemoglobin values within a 1-year time period were 182.5 g/L for men and 169 g/L for women. Average haematocrit levels were 0.53 for men and 0.50 for women. Elevated erythropoietin (EPO) levels were found in 10.5% patients, 83.4% had normal and 6.1% decreased values. Symptoms were reported by 48% of the patients, mostly itchiness (16.4%), sweating (12%) and other (30%) such as tinnitus, myalgia, joint pain and snoring. The most probable reasons for secondary erythrocytosis were smoking/chronic obstructive pulmonary disease (COPD) (32%), IE (29%), dehydration (7%), nephrological aetiology (6%), obesity (4%), sleep apnoea (4%), asthma (4%), infection (4%), heart or pulmonary disease hypoxia (4%), possible hemochromatosis (3%), medications (2%) and high altitude/training (1%). Patients with IE had an average age of 57.2 years (24 – 83 years), 57 (78%) were men and 8 (11%) had a positive family history. EPO levels in this group were elevated in 7.8%, normal in 88.2% and decreased in 3.9%. Past thrombotic events were reported by 23.3% of individuals with IE and 20.3% of patients with known secondary cause. There was no statistical difference in age, gender, EPO, haemoglobin levels and past thrombotic events between the IE group and group with known causes. Patients with IE significantly differed from the group with known causes in haematocrit levels (p=0.0219) with lower levels in IE patients. Summary/Conclusion: Among 250 patients with non-clonal erythrocytosis the majority had acquired aetiology. The most common cause was hypoxia caused by smoking or COPD. Patients with IE significantly differed from the group with known causes in haematocrit levels, while there were no significant differences between groups in age, gender, EPO levels, haemoglobin levels and past thrombotic events.

P1012: A NUMBER OF CONGENITAL ERYTHROCYTOSIS ARE MULTIGENIC

Background: Imbalance in erythropoiesis and oxygen homeostasis could lead to the onset of Erythrocytosis, a clinical condition characterized by persistently raised hemoglobin (Hb) and hematocrit (Ht) levels. Nowadays, a high number of patients are classified as “Idiopathic Erythrocytosis” (IE) meaning that it is not possible to identify the cause of hematocrit increase. Recently, HFE mutations has been found to be often present in idiopathic erythrocytic patients. The HFE mutations could facilitate the increase in red cell mass, and this indicates the possible involvement of Iron Metabolism in causes of IE. Aims: To identify the possible mutations of IE patients using our gene panel for Next Generation Sequencing (NGS). Methods: In 118 patients with IE, regularly followed in our surgery, we used a gene panel for NGS composed by fifteen genes: JAK2, EGLN1 (PHD2), EPO-R, FTL, FTH, ASXL1, HFE, HFE2, TFR2, HAMP, SLC40A1, SLC11A2, VHL, BPMG, EPAS1 (HIF-2α). We analyzed all the exons parts of these genes. The approach used for the library preparation was a multiplexing PCR and data were analyzed by bioinformatics tools. In all patients, the mutations found were confirmed with Sanger Sequencing. Results: In 78 out of the 118 patients (66%) evaluated, we found one 1 mutated gene in 55 patients (7 EGLN1, 1 VHL, 34 HFE, 6 TFR2, 3 JAK2 and 4 EPO-R), 2 mutated genes in 18 patients (1 EGLN1/JAK2, 4 EGLN1/HFE, 2 EPAS1/HFE, 1 EPAS1/JAK2, 1 EPAS1/EGLN1, 2 EPO-R/HFE, 2 HFE/JAK2, 1 JAK2/TFR2, 2 with TFR2/EGLN1, 1 VHL/HFE, 1 TFR2/HFE) and 3 mutated genes in 5 patients (1 EGLN1/EPO-R/HFE, 1 VHL/HFE/EGLN1, 1 EPAS1/EPO-R/HFE, 1 EPAS1/HFE/EGLN1 and HFE/TFR2/JAK2). All the mutations found were germline. Interestingly, 21 out of the 27 (78%) patients carrying HFE mutation displayed 2 or more mutations. Summary/Conclusion: The use of NGS panels in various clinical conditions has frequently demonstrated that multiple mutations coexist in various patients and that the monogenic diseases are quite rare. This study is the first at our best knowledge that gives evidence of more than one mutated gene in erythrocytotic patients, and this is the results of the use of an appropriate NGS panel. The data here reported suggest that we can confirm that HFE mutations are common in IE patients, in some cases associated with other mutations, but the present data ask some questions: HFE mutations alone can induce erythrocytosis? or other genetic imbalances are needed? The presence of multiple mutations could lead to a greater severity of the disease? A significant number of IE patients displayed TFR2 mutations, giving more relevance to the hypothesis of the iron metabolism implication in IE genesis. Finally, the presence of 11 patients carrying germlines JAK2 mutations is surprising, and we suppose it will be necessary to strictly watch these patients in the suspicion of a future appearance of a polycythemia vera. The present study demonstrates the need to extend the biomolecular knowledge in patients with idiopathic erythrocytosis.

Clinical and molecular diagnosis of hereditary and idiopathic erythrocytosis

Clinical and molecular diagnosis of hereditary and idiopathic erythrocytosis

Importance of Sequencing HBA1, HBA2 and HBB Genes to Confirm the Diagnosis of High Oxygen Affinity Hemoglobin.

High oxygen affinity hemoglobin (HOAH) is the main cause of constitutional erythrocytosis. Mutations in the genes coding the alpha and beta globin chains (HBA1, HBA2 and HBB) strengthen the binding of oxygen to hemoglobin (Hb), bringing about tissue hypoxia and a secondary erythrocytosis. The diagnosis of HOAH is based upon the identification of a mutation in HBA1, HBA2 or HBB in specialized laboratories. Phenotypic studies of Hb are also useful, but electrophoretic analysis can be normal in 1/3 of cases. The establishment of the dissociation curve of Hb can be used as another screening test, a shift to the left indicating an increased affinity for Hb. The direct measurement of venous P50 using a Hemox Analyzer is of great importance, but due to specific analytic conditions, it is only available in a few specialized laboratories. Alternatively, an estimated measurement of the P50 can be obtained in most of the blood gas analyzers on venous blood. The aim of our study was therefore to determine whether a normal venous P50 value could rule out HOAH. We sequenced the HBB, HBA1 and HBA2 genes of 75 patients with idiopathic erythrocytosis. Patients had previously undergone an exhaustive medical check-up after which the venous P50 value was defined as normal. Surprisingly, sequencing detected HOAH in three patients (Hb Olympia in two patients, and Hb St Nazaire in another). A careful retrospective examination of their medical files revealed that (i) one of the P50 samples was arterial; (ii) there was some air in another sample; and (iii) the P50 measurement was not actually done in one of the patients. Our study shows that in real life conditions, due to pre-analytical contingencies, a venous P50 value that is classified as being normal may not be sufficient to rule out a diagnosis of HOAH. Therefore, we recommend the systematic sequencing of the HBB, HBA1 and HBA2 genes in the exploration of idiopathic erythrocytosis.

Single Centre Analysis of JAK2 V617F and Exon 12 Negative Idiopathic Erythrocytosis

▪IntroductionThe molecular categorisation of myeloproliferative neoplasms (MPNs) has changed the landscape of diagnosis and treatment. Polycythaemia vera (PV) is characterised by red blood cell proliferation and JAK2 V617F or Exon 12 mutations in up to 98% of patients 1. However, some patients without such mutations have an arduous diagnostic course with varying management and prognostic outcomes 2. We present our experience in managing this challenging cohort and aim to illuminate a potential diagnostic pathway for patients.MethodWe searched electronic records of patients attending haematology clinics over the last 20 years at University College Hospital with a prior diagnosis of PV / erythrocytosis (presenting with raised Haemoglobin (Hb) &/ Haematocrit (Hct)) with no evidence of JAK2 exon 12 or 14 mutations on bone marrow or peripheral blood molecular analysis (multiplex PCR sensitivity 0.1%). We reviewed their diagnostic workup, which included full blood count & where available, bone marrow myeloid (Illumina TruSight Myeloid) & erythroid next generation sequencing (NGS) panels.Results37 patients with JAK2 V617F & Exon 12 mutation negative erythrocytosis were identified. Patients were categorised in to 3 groups 2; idiopathic erythrocytosis (IE), secondary polycythaemia (SP) & high affinity haemoglobinopathies (HAH); patient characteristics are summarised in Table 1. The median age of IE & HAH was younger, their presenting Hb/Hct levels was higher compared to SP, with a male predominance. Constitutional symptoms were only reported in the IE cohort. Erythropoietin (EPO) was elevated in HAH & IE patients but within normal range in SP. Thrombotic events occurred in all cohorts, most frequently in IE. Splenomegaly was reported in 4/21 IE, 1/13 SP, but was not a feature in HAH patients. When performed, IE red cell mass (RCM) studies were raised but within normal range in SP patients. Table 2 details IE cohort erythroid mutations. Myeloid NGS only identified MPL and BCOR mutations of pathogenic significance and multiple single nucleotide polymorphisms of no known significance. No abnormalities were demonstrated in 15% of SP patients that underwent bone marrow myeloid mutational analysis. Venesections (VS) were instigated in 95% of the IE cohort, antiplatelets (AP) in 52%, anticoagulation (AC) in 14% and cytoreductive therapy (CT) in 19% due to intolerance/failure of VS. VS programme was instigated in 46% of SP patients, AP in 7% and AC in 47%.DiscussionThe median age of our IE cohort was 48 yrs with a 19% incidence of thrombosis. Where performed, bone marrow histology demonstrated hypercellularity but was not consistent with MPN diagnostic criteria. Myeloid NGS panel mutations such as BCOR may represent clonal haematopoieis of indeterminate potential. Heterozygous VHL C598T & C376A mutations, in keeping with Chuvash polycythaemia, were demonstrated. Mutations in EGLN1 &BPGM,were detected in our patients, however there was an absence of correlating haematological parameters or family history to support a diagnosis of congenital erythrocytosis (2). Variants of unknown significance were also detected in SH2B3, BMP6 &EGLN3 gene duplication. SP patients were older (median age 68 years) and where performed had normal RCM and no myeloid mutations identified.The initial approach adopted at our centre for diagnosing and managing JAK2 V617F & Exon 12 negative erythocytosis begins with clinical evaluation for secondary causes. This is followed by assessment of EPO level, RCM study, extended molecular mutational analysis involving screening for high affinity haemoglobins, congenital erythrocytosis and bone marrow histology. Where patients are symptomatic or considered at high risk for thrombosis, we venesect to a personalised target and patients are offered AP and/or AC. CT is instigated where VS is not tolerated or ineffective, however this approach is not corroborated in the literature. Our experience highlights the clinical heterogeneity of JAK2 negative erythrocytosis and the need to develop a robust and systematic diagnostic and treatment algorithm with further clarification of the role of molecular profiling.1. William W, Kralovics R. Genetic basis and molecular pathophysiology of classical myeloproliferative neoplasms. Blood. 2017 Feb; 129(6):667-6792. McMullin MF. Idiopathic erythrocytosis: a disappearing entity. Hematology Am Soc Educ Program. 2009; 2009(1):629-635 [Display omitted] DisclosuresSekhar: Novartis: Consultancy, Research Funding.

Idiopathic Erythrocytosis: Vascular Complications

Idiopathic Erythrocytosis (IE) is a diagnosis of exclusion once primary and secondary causes have been eliminated; yet IE remains an enigmatic disorder. Published series from decades ago (Modan & Modan J. Hematol [1968] 14:375; Pearson & Wetherly-Mein, Clin.Lab Hematol. [1974] 1:189) have reported high rates of vascular complications (46%) and advise phlebotomy to keep the hematocrit to less than 45%. A More recent series (McMullin B.J. Hematol. [2005] 130:174) advise no need for phlebotomy in patients with no co-morbidities such as vascular disease, diabetes, or hypertension. In an attempt to determine some practice guidelines, we undertook a retrospective analysis of patients identified with IE within our practice and to quantify the rates of vascular complications, if any.From January 1, 2016 to December 31. 2020 99 patients were referred because of erythrocytosis. Twenty of these 99 patients had polycythemia rubra vera and 77 had secondary erythrocytosis ( 64 males, 13 females, median age 61 years). Of these 77 patients with secondary erythrocytosis, 40 had obstructive sleep apnea, 10 men abused testosterone, and 1 patient each was either post splenectomy or post kidney transplant, 1 patient had a renal cell carcinoma, and 1 patient had familial erythrocytosis. Twenty patients were identified as having IE and all 20 did not have a JAK2 mutation. There 13 males and 7 females with a median age of 63 years (range 31-76). The median hemoglobin was 17.2 gm/dl and the median hematocrit was 51.2 (range 15.0 to 18.4 and 49% to 55% respectively). The median erythropoietin level was 9.9 mu/ml (range 5-38.5). Two of the 20 patients underwent therapeutic phlebotomy and developed no vascular complications. Eighteen patients were simply followed with phlebotomy despite having comorbidities of hypertension, increased BMI, type II diabetes, and none developed vascular complications. IE remain an enigmatic disorder that requires uniform diagnostic criteria as well as uniform practice guidelines; however, given our retrospective review, we do not believe that therapeutic phlebotomy is justified. DisclosuresNo relevant conflicts of interest to declare.

Molecular Pathways Involved in the Development of Congenital Erythrocytosis.

Patients with idiopathic erythrocytosis are directed to targeted genetic testing including nine genes involved in oxygen sensing pathway in kidneys, erythropoietin signal transduction in pre-erythrocytes and hemoglobin-oxygen affinity regulation in mature erythrocytes. However, in more than 60% of cases the genetic cause remains undiagnosed, suggesting that other genes and mechanisms must be involved in the disease development. This review aims to explore additional molecular mechanisms in recognized erythrocytosis pathways and propose new pathways associated with this rare hematological disorder. For this purpose, a comprehensive review of the literature was performed and different in silico tools were used. We identified genes involved in several mechanisms and molecular pathways, including mRNA transcriptional regulation, post-translational modifications, membrane transport, regulation of signal transduction, glucose metabolism and iron homeostasis, which have the potential to influence the main erythrocytosis-associated pathways. We provide valuable theoretical information for deeper insight into possible mechanisms of disease development. This information can be also helpful to improve the current diagnostic solutions for patients with idiopathic erythrocytosis.

Genetic Background of Congenital Erythrocytosis.

True erythrocytosis is present when the red cell mass is greater than 125% of predicted sex and body mass, which is reflected by elevated hemoglobin and hematocrit. Erythrocytosis can be primary or secondary and congenital or acquired. Congenital defects are often found in those diagnosed at a young age and with a family history of erythrocytosis. Primary congenital defects mainly include mutations in the Erythropoietin receptor gene but SH2B3 has also been implicated. Secondary congenital erythrocytosis can arise through a variety of genetic mechanisms, including mutations in the genes in the oxygen sensing pathway, with high oxygen affinity hemoglobin variants and mutations in other genes such as BPMG, where ultimately the production of erythropoietin is increased, resulting in erythrocytosis. Recently, mutations in PIEZ01 have been associated with erythrocytosis. In many cases, a genetic variant cannot be identified, leaving a group of patients with the label idiopathic erythrocytosis who should be the subject of future investigations. The clinical course in congenital erythrocytosis is hard to evaluate as these are rare cases. However, some of these patients may well present at a young age and with sometimes catastrophic thromboembolic events. There is little evidence to guide the management of congenital erythrocytosis but the use of venesection and low dose aspirin should be considered.

Diagnosis and management of non-clonal erythrocytosis remains challenging: a single centre clinical experience

Erythrocytosis has a diverse background. While polycythaemia vera has well defined criteria, the diagnostic approach and management of other types of erythrocytosis are more challenging. The aim of study was to retrospectively analyse the aetiology and management of non-clonal erythrocytosis patients referred to a haematology outpatient clinic in an 8-year period using a 3-step algorithm. The first step was inclusion of patients with Hb > 185 g/L and/or Hct > 0.52 in men and Hb > 165 g/L and/or Hct > 0.48 in women on two visits ≥ two months apart, thus confirming true erythrocytosis. Secondly, polycythaemia vera was excluded and secondary causes of erythrocytosis (SE) identified. Thirdly, idiopathic erythrocytosis patients (IE) were referred to next-generation sequencing for possible genetic background evaluation. Of the 116 patients, 75 (65%) are men and 41 (35%) women, with non-clonal erythrocytosis 34/116 (29%) had SE, 15/116 (13%) IE and 67/116 (58%) stayed incompletely characterized (ICE). Patients with SE were significantly older and had significantly higher Hb and Hct compared to patients with IE. Most frequently, SE was attributed to obstructive sleep apnoea and smoking. Phlebotomies were performed in 56, 53 and 40% of patients in the SE, IE, and ICE group, respectively. Approx. 70% of patients in each group received aspirin. Thrombotic events were registered in 12, 20 and 15% of SE, IE and ICE patients, respectively. Congenital erythrocytosis type 4 (ECYT4) was diagnosed in one patient. The study demonstrates real-life management of non-clonal erythrocytosis which could be optimized using a 3-step diagnostic algorithm.