Unexplained Erythrocytosis Research Articles

206 A patient with unexplained erythrocytosis. Is there an SGLT2 inhibitor (SGLT2i) on the medication list?

206 A patient with unexplained erythrocytosis. Is there an SGLT2 inhibitor (SGLT2i) on the medication list?

First Observation of Hemoglobin Ypsilanti in Saudi Arabia: A High Oxygen Affinity Hemoglobin Presenting with Extreme Erythrocytosis

Abstract High oxygen affinity hemoglobin (HOAH) variants are rare hemoglobinopathies sometimes associated with congenital erythrocytosis. Patients with HOAH are usually asymptomatic and are incidentally discovered when unexplained erythrocytosis is observed in the complete blood count. Here, we report the first patient with hemoglobin (Hb) Ypsilanti from high-altitude Southern Saudi Arabia, who presented with chronic extreme erythrocytosis (polycythemia) with an Hb concentration of 23 g/dL and hematocrit of 70%. The patient lacked any finding suggestive of secondary erythrocytosis, and the evaluation for acquired polycythemia vera was negative. Hb protein analysis and genetic testing confirmed the presence of a high-affinity Hb variant. Living at high altitude contributed to the presentation of extreme erythrocytosis. Besides highlighting important clinical and diagnostic characteristics, this case illustrates two important diagnostics caveats: high-affinity Hb can be misdiagnosed as another more common qualitative hemoglobinopathy such as Hb S or Hb D carrier state, and it can interfere with HbA1C analysis.

Whole Exome Sequencing Reveals Novel Variants in Unexplained Erythrocytosis.

Erythrocytosis is characterized by an increase in red cells in peripheral blood. Polycythemia vera, the commonest primary erythrocytosis, results from pathogenic variants in JAK2 in ∼98% of cases. Although some variants have been reported in JAK2-negative polycythemia, the causal genetic variants remain unidentified in ∼80% of cases. To discover genetic variants in unexplained erythrocytosis, we performed whole exome sequencing in 27 patients with JAK2-negative polycythemia after excluding the presence of any mutations in genes previously associated with erythrocytosis (EPOR, VHL, PHD2, EPAS1, HBA, and HBB). We found that the majority of patients (25/27) had variants in genes involved in epigenetic processes, including TET2 and ASXL1 or in genes related to hematopoietic signaling such as MPL and GFIB. Based on computational analysis, we believe that variants identified in 11 patients in this study could be pathogenic although functional studies will be required for confirmation. To our knowledge, this is the largest study reporting novel variants in individuals with unexplained erythrocytosis. Our results suggest that genes involved in epigenetic processes and hematopoietic signaling pathways are likely associated with unexplained erythrocytosis in individuals lacking JAK2 mutations. With very few previous studies targeting JAK2-negative polycythemia patients to identify underlying variants, this study opens a new avenue in evaluating and managing JAK2-negative polycythemia.

Erythrocytosis associated with IgA nephropathy.

SummaryBackgroundErythrocytosis is a hematological disorder usually related to hematopoietic stem cell somatic mutations. However, unexplained erythrocytosis remains frequent. In this study, we evaluated the involvement of IgA1, a regulator of erythropoiesis also implicated in IgA nephropathy (IgAN) pathophysiology, in unexplained polycythemia/erythrocytosis (PE) of IgAN patients.MethodsIgAN-PE patients’ serum was collected, analyzed and used to study IgA1 effect on proliferation and differentiation of erythroid progenitors. Hematological parameters of transgenic mice for human alpha1 heavy chain were studied. Multicentric observational cohorts of chronic kidney disease (CKD) patients, including both native kidney diseases and renal transplants, were studied to analyze patient hemoglobin levels.FindingsWe retrospectively identified 6 patients with IgAN and unexplained PE. In large CKD cohorts, IgAN was associated with PE in 3.5% of patients (p<0.001 compared to other nephropathies). IgAN was an independent factor associated with higher hemoglobin levels (13.1g/dL vs 12.2 g/dL, p=0.01). During post-transplant anemia, anemia recovery was faster in IgAN patients. Elevated polymeric/monomeric IgA1 ratio as well as high Gd-IgA1 rate were observed in circulating IgA1 of the 6 IgAN-PE patients as compared with control or IgAN patients without PE. IgA1 from these patients increased the sensitivity of erythroid progenitors to Epo. In mice, we also observed an elevation of hematocrit in alpha1 knock-in mice compared to wild type controls.InterpretationThese data identify a new etiology of erythrocytosis and demonstrate the role of pIgA1 in human erythropoiesis. This syndrome of IgA-related erythrocytosis should be investigated in case of unexplained erythrocytosis and renal disease.FundingThis work was supported by INSERM (French national institute for health and medical research), Labex GRex and Imagine Institute (Paris, France)

Identification of Variants Associated With Rare Hematological Disorder Erythrocytosis Using Targeted Next-Generation Sequencing Analysis.

An erythrocytosis is present when the red blood cell mass is increased, demonstrated as elevated hemoglobin and hematocrit in the laboratory evaluation. Congenital predispositions for erythrocytosis are rare, with germline variants in several genes involved in oxygen sensing (VHL, EGLN1, and EPAS1), signaling for hematopoietic cell maturation (EPOR and EPO), and oxygen transfer (HBB, HBA1, HBA2, and BPGM) that were already associated with the eight congenital types (ECYT1–8). Screening for variants in known congenital erythrocytosis genes with classical sequencing approach gives a correct diagnosis for only up to one-third of the patients. The genetic background of erythrocytosis is more heterogeneous, and additional genes involved in erythropoiesis and iron metabolism could have a putative effect on the development of erythrocytosis. This study aimed to detect variants in patients with yet unexplained erythrocytosis using the next-generation sequencing (NGS) approach, targeting genes associated with erythrocytosis and increased iron uptake and implementing the diagnostics of congenital erythrocytosis in Slovenia. Selected 25 patients with high hemoglobin, high hematocrit, and no acquired causes were screened for variants in the 39 candidate genes. We identified one pathogenic variant in EPAS1 gene and three novel variants with yet unknown significance in genes EPAS1, JAK2, and SH2B3. Interestingly, a high proportion of patients were heterozygous carriers for two variants in HFE gene, otherwise pathogenic for the condition of iron overload. The association between the HFE variants and the development of erythrocytosis is not clearly understood. With a targeted NGS approach, we determined an actual genetic cause for the erythrocytosis in one patient and contributed to better management of the disease for the patient and his family. The effect of variants of unknown significance on the enhanced production of red blood cells needs to be further explored with functional analysis. This study is of great significance for the improvement of diagnosis of Slovenian patients with unexplained erythrocytosis and future research on the etiology of this rare hematological disorder.

Haemoglobin Olympia {β Codon 20 (B2) G→A, Val→Met}: A Silent Haemoglobin Variant

High oxygen affinity haemoglobin variants are rare and often underdiagnosed in persistent erythrocytosis with no apparent aetiology. Here the author present a 29-year-old Indian male patient with a long-standing history of erythrocytosis which was incidentally detected. The proband had a prothrombotic family history of cerebral vessel stroke in his paternal grandfather at a young age and unexplained erythrocytosis in his father and brother. A review of his haemograms showed persistent high haemoglobin values. Routine tests did not reveal any specific aetiology and haemoglobin electrophoresis by High-Performance Liquid Chromatography (HPLC) showed absence of any abnormal peak or unstable haemoglobin. DNA sequencing of the β globin gene revealed heterozygosity for codon 20 {GTG→ATG, Valine (Val)→ Methionine (Met)} mutation confirming the presence of an electrophoretically silent Hb variant - Haemoglobin Olympia in him and his extended family members. This case study emphasises importance of this rare entity of high oxygen affinity haemoglobin variant as a differential diagnosis while screening for erythrocytosis. This is the first case report of Haemoglobin Olympia from India reported in the literature.

Prevalence of Unexplained Erythrocytosis and Thrombocytosis - an Nhanes Analysis

Introduction Polycythemia vera (PV) and essential thrombocythemia (ET) are myeloproliferative neoplasms characterized by erythrocytosis and thrombocytosis, with an estimated prevalence of 22 and 24 per 100,000, respectively in the United States (Ma X et al AJH 2008). However, the prevalence of undiagnosed disease is unknown. We examined the National Health and Nutritional Examination Survey (NHANES) database for unexplained erythrocytosis (UE) and thrombocytosis (UT). Methods NHANES is a cross-sectional, nationally representative, population-based survey administered by the Centers for Disease Control and Prevention that utilizes a combination of interviews, physical examinations, and laboratory testing. In the UE group, participants were included if they had a hemoglobin &amp;gt;18.5 g/dL in men or &amp;gt;16.5 g/dL in women or a hematocrit &amp;gt;55.5% in men and &amp;gt;49.5% in women. Participants were excluded if they required oxygen supplementation, had a reported history or pulmonary function test evidence of obstructive lung disease, were receiving exogenous steroids or smoked cigarettes, or had a history of blood, kidney, liver, or uterine cancer. The UT group included participants with a platelet count greater than 450 x 10^9/L, and excluded those with laboratory evidence of iron deficiency, an elevated LDH suggesting inflammation, or reported a current infection, history of cancer, or rheumatologic conditions. Given the availability of necessary eligibility data, NHANES was queried from 2007-2008 for the UE group and 1999-2002 for the UT group. Weighted statistics are presented. Results Of the 3348 participants evaluable for UE, 16 (0.38%; 383.0 per 100,000 population) had erythrocytosis of which 4 were unexplained (0.04% of the overall population; 40.8 per 100,000 population). Reasons for erythrocytosis are noted in Table 1. UE participants had a mean hemoglobin of 18.1g/dL (standard error [SE] 0.6) and hematocrit of 50.7% (SE 1.5). Table 2 shows the estimated prevalence of UE by age, gender, and race. The average age was 58.8 years (SE 2.8), which was significantly older than the general population without erythrocytosis (42.8 years [SE 0.5], p&amp;lt;0.0001). All UE participants in the sample were United States (US) born and had an educational level below a college degree. The mean family poverty income ratio was 2.4 (SE 0.6), which was lower, although not significantly different than the NHANES population without erythrocytosis (mean 3.2 [SE 0.1], p=0.2113). Of the 7403 participants evaluable for thrombocytosis, 108 participants (1.47%; 1,474.6 per 100,000 population) had thrombocytosis of which 33 (0.49% of the overall population; 494.2 per 100,000) were UT. Reasons for thrombocytosis are shown in Table 1. The mean platelet count of the UT group was 494.5 x 10^9/L (SE 9.1). The average age was 42.6 years (SE 3.5) which was not significantly different than the general population without thrombocytosis (45.1 years [SE 0.3], p=0.4582), but those with UT were significantly more likely to be female (76.4% vs. 50.5%, p=0.0117). Table 2 shows the estimated prevalence of UT by age, gender, and race group. The weighted percentage of US born was 86.7% in the UT group, with 63.1% having an educational level below a college degree. The mean family poverty income ratio was 3.1 (SE 0.4), which was not significantly different than the NHANES population without thrombocytosis (mean 3.0 [SE 0.1], p=0.8974). Conclusions Although this study is limited by inability to determine World Health Organization (WHO) diagnosis of ET and PV, this NHANES analysis suggests that the prevalence of UE and UT may be higher than commonly appreciated, with an estimated prevalence of 40.8 and 494.2 per 100,000, respectively. This is almost certainly an overestimate as a result of methodologic limitations of an NHANES analysis, including the fact that estimates are based on a small number of participants with UE or UT obtained at a static time point. It is also important to note that not all patients UE and UT fulfill WHO criteria for PV and ET. However, our data do suggest that PV and ET may be under-evaluated in the general population. While it is possible that certain factors may predispose some patients to having undiagnosed disease, the number of participants with unexplained conditions were too small to analyze this in more detail. Disclosures Mascarenhas: Celgene, Prelude, Galecto, Promedior, Geron, Constellation, and Incyte: Consultancy; Incyte, Kartos, Roche, Promedior, Merck, Merus, Arog, CTI Biopharma, Janssen, and PharmaEssentia: Other: Research funding (institution).

Identifying Myeloid Mutations By NGS in Patients with Unexplained Erythrocytosis

Introduction: Unexplained erythrocytosis is a common reason for consultation in hematology. Identification of JAK2V617F mutation has facilitated the diagnosis of Polycythemia Vera (PV), but a proportion of patients without clear secondary causes for erythrocytosis remain undiagnosed or presumptively diagnosed with an either Exon 12 mutation or JAK2 negative PV. Since 2005, our institution has been performing JAK2V617F testing by PCR. In 2018 we switched to an NGS panel which includes JAK2/exon 12 and 40 other genes implicated in myeloid malignancies. We reviewed all previously diagnosed PV who had NGS myeloid panel performed to determine whether patients with a clinical diagnosis of JAK2 negative PV had other myeloid mutations that might explain their erythrocytosis and alter their management. Methods: We identified all cases with clinically suspected PV or confirmed JAK2 mutated PV who went on to have had NGS testing performed between January 2018 and February 2019 at London Health Sciences Centre, a tertiary care center servicing a population of approximately 2.5 million in Ontario, Canada. The Oncomine Myeloid NGS panel (Thermo-Fisher, MA, USA) examines DNA sequence variants in 40 genes (17 full genes and 23 hotspot genes) along with an RNA-based panel of 29 fusion driver genes and their over 600 fusion partners. Diagnosis was based on the WHO 2016 Classification of Tumours of Haematopoietic and Lymphoid Tissues. The clinical impact was assessed from retrospective review of electronic medical record to determine whether there was a diagnostic or management impact. Results: A total of 143 patients followed for PV or unexplained erythrocytosis had NGS testing during the study period. Of those, 137/143 (95.8%) patients had previous JAK2V617F PCR tested and 48/137 (35%) were identified with JAK2V617F mutation. Of the 48 patients with previous JAK2V617F PCR detected, NGS confirmed JAK2 mutation in 40/48 (83.3%) with additional non-JAK2 mutations in 17/40 (42.5%) patients. Of note 8/48 (16.7%) patients previously detected JAK2V617F by PCR had undetectable JAK2 mutation when repeat testing was performed by NGS. Of those 89/137 (65%) patients with previous JAK2V617F PCR negative result, NGS revealed JAK2 exon 12 mutation in 3/89 (3.4%) patients and JAK2V617F/JAK2L611V mutations in 1/89 (1.1%) patient resulting in diagnosis as PV, whereas non-JAK2 mutations in 6/89 (6.7%) patients. No MPL or CALR positive cases were identified in this cohort. Remaining 79/89 (88.8%) had no mutations identified (Figure1) and in this group, 13/79 (16.5%) patients were discharged from hematology clinic, 7/79 (8.9%) had therapies such as phlebotomy, aspirin or hydroxyurea stopped or reduced, whereas 2/79 (2.5%) patients had further evaluation or testing for unexplained erythrocytosis. (Table 1) Conclusions: In the unexplained erythrocytosis JAK2V617F PCR negative group, JAK2exon 12 mutation was identified in 3.4% in keeping with known incidence of this mutation. Some previously positive PCR JAK2V617F mutation were not identified by NGS panel (16%) which may reflect changes in clone size either with time or therapy or inherent differences in assay sensitivity (2.5% mutational alleles for NGS versus 0.1% for PCR). Lack of identifiable myeloid mutation and clonal hematopoiesis by NGS testing influenced clinical management. Specifically, mutation negative patients were more likely assigned to non-MPN group and called secondary erythrocytosis which resulted in reducing interventions. Non-JAK2 mutations occurred in more than 1/3 of previously identified JAK2 positive PCR tested PV. The clinical impact of most these mutations is uncertain and requires longer follow up. Disclosures No relevant conflicts of interest to declare.

Genetic basis of unexplained erythrocytosis in Indian patients.

To evaluate the spectrum of genetic defects in Indian patients with unexplained erythrocytosis. Fifteen families (18 patients) with unexplained erythrocytosis were enrolled after excluding polycythemia vera and secondary erythrocytosis. Focused Sanger sequencing from genomic DNA was performed for EPOR (exon 8), VHL (exons 2-3), EGLN1 (exons 2-5), EPAS1 (exon 12), and all exons of HBB, HBA1, and HBA2 genes. Eleven of the 18 patients (including two pairs of brothers) had Chuvash polycythemia, that is, homozygosity for VHL:c.598C>T (p.Arg200Trp). Three patients (two of whom were brothers) had HBB mutations associated with increased oxygen-affinity hemoglobin-one had a heterozygous Hb McKees Rocks HBB:c.438T>A (p.Tyr146*), and two brothers showed heterozygous Hb Rainier HBB:c.437A>G (p.Tyr146Cys). No pathogenic variants were found in the remaining four cases. A gene-by-gene Sanger sequencing approach could determine a genetic basis for erythrocytosis in 11 of the 15 (73%) Indian families, with homozygous VHL:c.598C>T (p.Arg200Trp) being the commonest pathogenic variant. This first study from the Indian subcontinent provides a rationale for analyzing this variant in patients with suspected congenital erythrocytosis from this region. Rare first occurrences of Hb McKees Rocks and Hb Rainier in Indians are also being reported.

Diagnosis and Management of Anabolic Androgenic Steroid Use.

The lifetime prevalence of anabolic androgenic steroid (AAS) use is estimated at 1% to 5% worldwide. AAS use occurs primarily male elite athletes and men who want a muscular appearance. The evidence for effective, safe management of AAS cessation and withdrawal is weak. Key studies were extracted from PubMed (1990-2018) and Google Scholar with reference searches from relevant retrieved articles. The proven adverse effects of AASs include suppression of the gonadal axis and infertility, hirsutism and defeminization in women, and erythrocytosis. Alkylated AASs that are taken orally may cause hepatopathy. There is an association between high-dosage AAS use and increased risk of cardiovascular disease. Clues for AAS use include very low serum high-density cholesterol and sex hormone-binding globulin concentrations and unexplained erythrocytosis. For elite athletes, the biological passport (monitoring of blood or urinary androgen and androgen precursor concentrations after determining the athlete's baseline) is useful for detecting AAS use. For nonelite athletes, the best method to confirm AAS use is to inquire in a nonjudgmental manner. Cessation of chronic AAS use is associated with a withdrawal syndrome of anxiety and depression. Men who use AASs <1 year typically recover normal hypothalamic-pituitary-testicular axis function within 1 year after cessation. Men who have infertility due to high-dosage AAS use ≥1 year might benefit from short-term treatment with clomiphene or human chorionic gonadotropin.

Hb Tarrant [α126(H9)Asp→Asn; HBA2: c.379G > A (or HBA1)] in a Chinese Family as a Cause of Familial Erythrocytosis

Hb Tarrant [α126(H9)Asp→Asn; HBA2: c.379G > A (or HBA1)], is a rare high oxygen affinity hemoglobin (Hb) variant that causes erythrocytosis, previously described in a few Mexican–American families. Here we report the first Chinese family with this Hb variant presenting with unexplained familial erythrocytosis. No evidence of hemolysis was seen. A locally adapted approach to the diagnostic process in clinical laboratories is discussed. Molecular analysis has an important role in confirmation of the diagnosis. Proper identification of this rare but clinically significant Hb variant is helpful for family counseling and will help to guide appropriate management of absolute erythrocytosis.

The TEMPI Syndrome: Telangiectasias, Elevated Erythropoietin and Erythrocytosis, Monoclonal Gammopathy, Perinephric Fluid Collections, and Intrapulmonary Shunting

Abstract 1037▪▪This icon denotes a clinically relevant abstractWe report a rare and novel syndrome which we have termed TEMPI: telangiectasias, elevated erythropoietin and erythrocytosis, monoclonal gammopathy, perinephric fluid collections, and intrapulmonary shunting. In 2010, the index case of a man with unexplained erythrocytosis and perinephric fluid collections was published in the Case Records of the Massachusetts General Hospital in the New England Journal of Medicine. In the case report, we appealed to readers to share insights and similar cases. As a result, two strikingly similar cases were identified: a woman in Antwerp, Belgium and a woman in Los Angeles, California. The patients share five characteristics constituting the TEMPI syndrome.The three patients had all been healthy until their 3rd or 4th decades when they began to manifest symptoms. Telangiectasias developed spontaneously and were most prominent over the trunk, arms, hands, face, and oropharynx; there was no evidence of liver dysfunction. Erythrocytosis was present, with the presenting hematocrit ranging between 58–64% (hemoglobin 19–22 g/dL). The serum erythropoietin level was elevated at diagnosis, and gradually rose to values greater than 5000 mU/ml. Extensive imaging did not reveal a source of erythropoietin production, and evaluations incluing hemoglobin electrophoresis, hemoglobin oxygen affinity, and gene sequencing were negative. An IgG-kappa monoclonal gammopathy was identified, at similar concentrations of approximately 0.7g/dL, without hypercalcemia or lytic bone lesions, and the bone marrow biopsies revealed fewer than 10% plasma cells, consistent with a diagnosis of monoclonal gammopathy of undetermined significance (MGUS). The progressive accumulation of bilateral perinephric fluid collections is a striking and unique feature of the syndrome. There were no intraparenchymal renal cysts; the fluid was localized between the kidney and the renal capsule. Sampling of the fluid revealed a clear, aseptic, serous fluid with low protein, few leukocytes, and no cholesterol or triglycerides. Microscopic intrapulmonary shunting was identified, and slowly progressed with worsening hypoxia and shortness of breath. There was no evidence of pulmonary hypertension on echocardiogram or right heart catheterization. All three patients have suffered spontaneous venous thromboses, and two of the patients suffered spontaneous intracranial hemorrhages without identifiable arterio-venous malformations on MRI/MRA or cerebral angiography.The pathophysiology underlying the TEMPI syndrome is unknown. The similar characteristics shared by patients of different ages, genders, and geographies suggest an acquired rather than an inherited disorder. The presence of an IgG-kappa paraprotein in all three patients is suggests that the paraprotein might be involved in the pathophysiology of this disease. One of the patients underwent empiric treatment with the proteasome inhibitor bortezomib, with dramatic improvement in her symptoms after six cycles of therapy. The telangiectasias resolved, the level of serum erythropoietin normalized, the monoclonal gammopathy disappeared, the perinephric fluid collections resolved, and the degree of intrapulmonary shunting decreased to the point that she was able to discontinue her supplemental oxygen. Based on her clinical improvement, the decision was made to treat the other patients similarly. We will report in detail the characteristics of this novel syndrome as well as the treatment results. The response of one patient to the treatment of her MGUS suggests that the monoclonal gammopathy is involved in this syndrome by a mechanism that remains to be defined. Disclosures:Off Label Use: In this presentation, the authors used bortezomib as an empiric treatment for an undiagnosed medical condition.

A novel mutation of the erythropoietin receptor gene associated with primary familial and congenital polycythaemia

Primary familial and congenital polycythaemia (PFCP) is a rare form of inherited erythrocytosis caused by heterozygous mutations in the erythropoietin receptor gene (EPOR). We present a novel mutation in the EPOR in a 15-year-old male who was referred to our clinic for investigation of a persistently elevated haemoglobin level. A significant family history of unexplained erythrocytosis spanning four generations of the patient's family was established. The family history was also significant for an apparent increased rate of cerebrovascular disease in individuals with erythrocytosis. The mutation detected in our patient resides in exon 8 of EPOR, similar to all other EPOR mutations responsible for PFCP. These mutations result in truncation of the cytoplasmic domain of the receptor and impair down-regulation of signalling via the erythropoietin receptor (EPOR). Clinical manifestations in published cases have varied widely and there is a paucity of firm recommendations regarding the management of affected patients. Given the strong family history of complications attributable to erythrocytosis we have recommended venesection with a haematocrit target of ≤0.45 for our patient.

LNK Mutation Studies In Chronic- and Blast-Phase Myeloproliferative Neoplasms and JAK2 Mutation-Negative Erythrocytosis

AbstractAbstract 4105 Background:JAK2 mutations are found in approximately 99% of patients with polycythemia vera (PV) and 60% of those with essential thrombocythemia (ET) or primary myelofibrosis (PMF). It is currently assumed that other mutations relevant to JAK signaling contribute to the pathogenesis of JAK2 mutation-negative myeloproliferative neoplasms (MPN). The same might hold true for some cases of “idiopathic erythrocytosis” associated with subnormal serum erythropoietin level (sEpo). LNK is a plasma membrane-bound adaptor protein whose function includes inhibition of wild-type and mutant JAK2 phosphorylation. LNK exon 2 mutations were recently described in two patients with JAK2V617F-negative ET or PMF. Both mutations involved the LNK pleckstrin homology (PH) domain; one was a 5 base-pair deletion and missense mutation leading to a premature stop codon (603_607delGCGCT; 613C>G) and the other a missense mutation (622G>C; glutamic acid to glutamine substitution; E208Q). Objectives:i) To estimate the prevalence of LNK mutations in chronic and blast phase MPNii) To determine if LNK mutations are mutually exclusive of other MPN-associated mutationsiii) To test the hypothesis that LNK mutations might contribute to the pathogenesis of JAK2 mutation-negative PV or otherwise unexplained erythrocytosis Methods:LNK mutation analysis was performed on bone marrow or blood cells using modified primers for amplifying the SH2 and PH domains, using previously published methods (Oh et al. Blood First Edition Paper, prepublished online April 19, 2010; DOI 10.1182/blood-2010-02-270108). Results:LNK mutation studies were performed in 172 patients; paired chronic-blast phase samples were analyzed in 26 cases. Diagnoses in the 172 study patients were as follows: 78 chronic-phase MPN (39 JAK2V617F-positive PV, 25 PMF and 14 ET) enriched for TET2, IDH, JAK2V617F, or MPL mutations, 61 blast-phase MPN (41 blast-phase PMF, 11 blast-phase PV and 9 blast-phase ET), 25 JAK2V617F-negative PV and 8 “unexplained erythrocytosis” associated with subnormal sEpo and negative for Epo receptor (EPOR) mutations.Ten novel heterozygous LNK mutations, all but one affecting exon 2 in the PH domain, were identified: 6 missense mutations involving codons 215, 220, 223, 229 and 234, one nonsense mutation involving codon 208 (622G>T leading to glutamic acid to a stop codon substitution; E208X), one synonymous mutation involving codon 208, and 2 deletion mutations involving exon 2 (685-691_delGGCCCCG) or exon 5 (955_delA). LNK mutations were most frequent in blast-phase MPN, occurring in 6 (9.8%) of the 61 informative patients; chronic-phase sample analysis in 4 of these revealed the same mutation in only one case. Mutant LNK was detected in chronic-phase samples only in 2 additional patients with blast-phase MPN. Among the 8 LNK-mutated blast-phase MPN cases, 7 had blast-phase PMF. The clinical phenotype in the remaining 2 LNK-mutated patients was that of an isolated erythrocytosis without overt features of PV. JAK2V617F was documented in 3 and IDHR140Q in 1 LNK-mutated patients. Conclusions:LNK mutations i) target an exon 2 ‘hot spot’ in the PH domain spanning residues E208-D234, ii) might be most prevalent in blast-phase PMF iii) are not mutually exclusive of JAK2 or IDH mutations and iv) might be part of the “missing link” in the pathogenesis of JAK2 mutation-negative “idiopathic” erythrocytosis. Disclosures:No relevant conflicts of interest to declare.

Novel exon 12 mutations in the HIF2A gene associated with erythrocytosis

AbstractErythrocytosis can arise from deregulation of the erythropoietin (Epo) axis resulting from defects in the oxygen-sensing pathway. Epo synthesis is controlled by the hypoxia inducible factor (HIF) complex, composed of an α and a β subunit. There are 2 main α subunits, HIF-1α and HIF-2α. Recently, a HIF-2α Gly537Trp mutation was identified in a family with erythrocytosis. This raises the possibility of HIF2A mutations being associated with other cases of erythrocytosis. We now report a subsequent analysis of HIF2A in a cohort of 75 erythrocytosis patients and identify 4 additional patients with novel heterozygous Met535Val and Gly537Arg mutations. All patients presented at a young age with elevated serum Epo. Mutations at Gly-537 account for 4 of 5 HIF2A mutations associated with erythrocytosis. These findings support the importance of HIF-2α in human Epo regulation and warrant investigation of HIF2A in patients with unexplained erythrocytosis.

Prevalence and clinicopathologic correlates of JAK2 exon 12 mutations in JAK2V617F-negative polycythemia vera

After accounting for misdiagnosis and treatment effect, allele-specific (AS)-PCR detects the JAK2V617F mutation in >95% of polycythemia vera (PV) patients. Using database inquiry, we identified 6 of a total 220 cases with PV that were JAK2V617F-negative (prevalence=3%). Of these, five cases ( approximately 80%) were found to harbor one of the two JAK2 exon 12 mutations (F537-K539delinsL or N542-E543del) in bone marrow (BM) and/or peripheral blood cells. Similar screening of six additional cases - three each with idiopathic erythrocytosis (IE) or otherwise unexplained erythrocytosis (UE) - did not reveal either JAK2V617F or JAK2 exon 12 mutations. We found JAK2 exon 12 mutations in PV cases to be readily detected by both DNA sequencing and AS-PCR, regardless of whether BM or peripheral blood cells were used as the source for DNA. Although erythroid hyperplasia was the predominant histologic feature on BM examination, megakaryocyte abnormalities and reticulin fibrosis were noted in most PV patients harboring exon 12 mutations. However, similar BM morphologic changes can also be seen in some JAK2V617F-positive PV cases; therefore, distinct genotype-phenotype association cannot be established.

Polycythemia as the first manifestation of Cushing’s disease

A 39-yr-old man presented to our hospital with unexplained erythrocytosis and hypertension. His follow-up for erythrocytosis had begun 2 yr earlier in another hospital and he had been diagnosed with polycythemia rubra vera. On admission to our hospital he was hypertensive (165/95 mmHg) and, except for the presence of moon-like face and facial plethora, his physical examination was normal. His hemoglobin concentration was 19.2 g/dl, and hematocrit was 58.9% with an increased red blood cell mass of 58 ml/kg as measured by radioisotope (Cr51). Blood film, other hematological indices except for elevated leukocyte alkaline phosphatase score, arterial gas analysis, and examination of aspirated bone marrow were all normal. An abdominal ultrasonography showed no evidence of splenomegaly. A diagnosis of probable secondary erythrocytosis was made. Early-morning serum cortisol and 24-h urinary free cortisol concentration as well as serum ACTH were high. Serum cortisol was not suppressed by low-dose dexamethasone, but suppressed by high-dose dexamethasone. Pituitary magnetic resonance imaging showed no lesion. After inferior petrosal sinus sampling suggesting right-central ACTH secretion, the patient underwent transnasal-transsphenoidal pituitary adenomectomy. Both hypercortisolemia and erythrocytosis regressed completely after the adenomectomy. After the operation, the patient's hemoglobin concentration and hematocrit decreased steadily, and 1 month post-adenomectomy his hemoglobin is 14.9 g/dl and hematocrit 44.8%. Thus, Cushing's syndrome should be a routine part of evaluation of unexplained polycythemia.

Minimizing Error in the Determination of P50

The measurement of P 50 in whole blood, defined as the oxygen tension corresponding to 50% oxygen saturation, gives a quantitative measure of hemoglobin oxygen affinity. The determination of P 50 is useful for screening for hemoglobin variants in cases of unexplained anemia or erythrocytosis, both because it is easily performed and because many hemoglobin variants with abnormal oxygen affinity are “silent”, i.e., not detectable with conventional electrophoretic techniques (1). The determination of P 50 previously required obtaining several points on the oxyhemoglobin dissociation curve. The procedure was seldom performed because of the labor and specialized apparatus required. However, it was suggested >25 years ago that P 50 could be accurately calculated from single measurements of oxygen tension ( P o2) and the corresponding oxygen saturation ( S o2) (2). Since then, an improved mathematical model of the oxyhemoglobin dissociation curve (3) has been incorporated into a guideline for the so-called “single-point” determination of P 50 (4) that has been tested (5)(6) and subsequently approved by the IFCC. The single-point approach is based on the fact that a plot of log P o2 vs log[ S o2/(1 − S o2)], the so-called Hill plot, is linear over a fairly wide interval. Therefore, if one knows both the slope and one point on the line, any other point on the line can be determined. There have been different recommendations for the interval within which the single-point method is valid. Some studies have suggested that it can be used for oxygen saturations as low as 20% (5). The original IFCC guideline (4) specified saturations in the interval between 40% and 80%, and a subsequent IFCC document on definitions of quantities used in blood pH and gas analysis (7) extended the upper limit to 90% saturation. …

Electrospray Mass Spectrometry: An Efficient Method to Detect Silent Hemoglobin Variants Causing Erythrocytosis

Erythrocytosis, which is characterized by high hematocrit (packed cell volume), is a common condition causing complications attributable to hyperviscosity of blood. According to modern nomenclature, patients can be subdivided into one group of “apparent erythrocytosis” and one of “absolute erythrocytosis” (1). The absolute-erythrocytosis group contains both genetic disorders, acquired conditions such as polycythemia, and secondary reactions attributable to chronic hypoxemia or renal disease. The occurrence of hemoglobin (Hb) variants with increased oxygen affinity is a well-known cause of hereditary erythrocytosis, but in recent years interest has also been given to mutations in the erythropoietin receptor gene (2). Theoretically, functional studies of oxygen saturation curves would be the ideal method to screen for Hb variants that cause erythrocytosis, but such methods are not generally available and there are diverging opinions whether P 50 measurements of stored or shipped samples can be used (3)(4). Of the nearly 200 Hb variants with increased oxygen affinity described, fewer than one-half have been found in conjunction with significant erythrocytosis (3). Several of the variants are attributable to amino acid substitutions that do not affect the net charge, i.e., the substitutions are “silent” when investigation is performed by conventional electrophoretic techniques. In contrast, almost all Hb variants in this group can readily be found by mass spectrometry exploiting the change in molecular weight of the variant globin. Using electrospray mass spectrometry (ESMS), we have screened 70 consecutive samples from patients with unexplained erythrocytosis that had showed normal Hb pattern on isoelectric focusing (IEF) (5) and HPLC (6)(7) analysis. The investigation was approved by the Ethics Committee at Huddinge University Hospital. In three cases, we detected β-globin variants. This report illustrates how the information from electrophoretic methods and the knowledge of the wild-type globin gene sequence can facilitate the interpretation of mass spectral …

The detection of occult renal tumors in children by elevated hematocrit on routine complete blood count: A report of two cases

Wilms' tumor is a renal neoplasm usually found in young children and is rarely seen in teenagers. The production of erythropoietin by these tumors may result in secondary erythrocytosis, which should be reflected in complete blood counts (CBC). A search of the literature for reports of occult Wilms' tumors initially suspected on the basis of erythrocytosis was unrewarding. Two teenagers are reported in whom unexplained erythrocytosis was the initial indication of a Wilms' tumor. In both cases, a previous CBC showed elevations in hemoglobin and hematocrit that might have led to an earlier diagnosis. We conclude that unexplained erythrocytosis should sound the alert for further diagnostic studies to evaluate the possibility of occult renal neoplasia in older children.