Heterozygosity for bisphosphoglycerate mutase deficiency expressing clinically as congenital erythrocytosis: A case series and literature review.

Erythrocytosis is associated with increased red blood cell mass and can be either congenital or acquired. Congenital secondary causes are rare and include germline variants increasing haemoglobin (Hb)-oxygen affinity (e.g., Hb or bisphosphoglycerate mutase (BPGM) variants) or affecting oxygen-sensing pathway proteins. Here, we describe five adults from three kindreds with erythrocytosis associated with heterozygosity for BPGM variants, including one novel. Functional analyses showed partial BPGM deficiency, reduced 2,3-bisphosphoglycerate levels and/or increased Hb-oxygen affinity. We also review currently known BPGM variants. This study contributes to raising awareness of BPGM variants, and in particular that heterozygosity for BPGM deficiency may already manifest clinically.

A Gain-of-Function Mutation in EPO in Familial Erythrocytosis.

Familial erythrocytosis with elevated erythropoietin levels is frequently caused by mutations in genes that regulate oxygen-dependent transcription of the gene encoding erythropoietin ( EPO). We identified a mutation in EPO that cosegregated with disease with a logarithm of the odds (LOD) score of 3.3 in a family with autosomal dominant erythrocytosis. This mutation, a single-nucleotide deletion (c.32delG), introduces a frameshift in exon 2 that interrupts translation of the main EPO messenger RNA (mRNA) transcript but initiates excess production of erythropoietin from what is normally a noncoding EPO mRNA transcribed from an alternative promoter located in intron 1. (Funded by the Gebert Rüf Foundation and others.).

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.

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.

Congenital erythrocytosis.

Erythrocytosis, or increased red cell mass, may be labeled as primary or secondary, depending on whether the molecular defect is intrinsic to the red blood cells/their precursors or extrinsic to them, the latter being typically associated with elevated erythropoietin (EPO) levels. Inherited/congenital erythrocytosis (CE) of both primary and secondary types is increasingly recognized as the cause in many patients in whom acquired, especially neoplastic causes have been excluded. During the past two decades, the underlying molecular mechanisms of CE are increasingly getting unraveled. Gain-in-function mutations in the erythropoietin receptor gene were among the first to be characterized in a disorder termed primary familial and congenital polycythemia. Another set of mutations affect the components of the oxygen-sensing pathway. Under normoxic conditions, the hypoxia-inducible factor (HIF), upon hydroxylation by the prolyl-4-hydroxylase domain protein 2 (PHD2) enzyme, is degraded by the von Hippel-Lindau protein. In hypoxic conditions, failure of prolyl hydroxylation leads to stabilization of HIF and activation of the EPO gene. CE has been found to be caused by loss-of-function mutations in VHL and PHD2/EGLN1 as well as gain-of-function mutations in HIF-2α (EPAS1), all resulting in constitutive activation of EPO signaling. Apart from these, globin gene mutations leading to formation of high oxygen affinity hemoglobins also cause CE. Rarely, bisphosphoglycerate mutate mutations, affecting the 2,3-bisphosphoglycerate levels, can increase the oxygen affinity of hemoglobin and cause CE. This narrative review examines the current mutational spectrum of CE and the distinctive pathogenetic mechanisms that give rise to this increasingly recognized condition in various parts of the world.

Genetic analysis of 39 erythrocytosis and hereditary hemochromatosis-associated genes in the Slovenian family with idiopathic erythrocytosis.

BACKGROUND: Erythrocytosis is a condition with an excessive number of erythrocytes, accompanied by an elevated haemoglobin and/or haematocrit value. Congenital erythrocytosis has a diverse genetic background with several genes involved in erythropoiesis. In clinical practice, nine genes are usually examined, but in approximately 70% of patients, no causative mutation can be identified. In this study, we screened 39 genes, aiming to identify potential disease-driving variants in the family with erythrocytosis of unknown cause. PATIENTS AND METHODS: Two affected family members with elevated haemoglobin and/or haematocrit and negative for acquired causes and one healthy relative from the same family were selected for molecular-genetic analysis of 24 erythrocytosis and 15 hereditary haemochromatosis-associated genes with targeted NGS. The identified variants were further analysed for pathogenicity using various bioinformatic tools and review of the literature. RESULTS: Of the 12 identified variants, two heterozygous variants, the missense variant c.471G>C (NM_022051.2) (p.(Gln157His)) in the EGLN1 gene and the intron variant c.2572-13A>G (NM_004972.3) in the JAK2 gene, were classified as low-frequency variants in European population. None of the two variants were present in a healthy family member. Variant c.2572-13A>G has potential impact on splicing by one prediction tool. CONCLUSION: For the first time, we included 39 genes in the erythrocytosis clinical panel and identified two potential disease-driving variants in the Slovene family studied. Based on the reported functional in vitro studies combined with our bioinformatics analysis, we suggest further functional analysis of variant in the JAK2 gene and evaluation of a cumulative effect of both variants.

Novel mutations in the EPO-R, VHL and EPAS1 genes in the Congenital Erythrocytosis patients.

Congenital erythrocytosis (CE) can be classified as primary and secondary and 82 consecutive patients of erythrocytosis who were JAK-2 mutation negative, were further investigated. The genomic DNA was extracted from all the patients and the EPO-R, VHL, EGLN1 and EPAS1 genes were PCR amplified and sequenced. The sequence analysis showed (28/82) 34.14% patients had mutations. Among them, (19/28) 67.86% patients had mutations in exon 8 of EPO-R gene, of which six were novel missense mutations, p.(Gly418Ala), p.(Gly390Ala), p.(Ala411Thr), p.(Gly475Val), p.(Glu490Asp), p.(Glu362Gln) and three were novel frameshift mutations, p.(Glu336*), p.(Pro327Hisfs*68), p.(Gly479Alafs*37). All these EPO-R patients were heterozygotes and were forming endogenous erythrocyte colonies (EEC). Some patients (8/28) 28.57% had mutations in VHL gene, out of which 3 novel homozygous missense mutations in exon 1 of VHL gene, p.Gly80Asp, p.Gln107Glu and p.Gln113Glu, were identified. In addition, (1/28) 3.5% patients had one reported heterozygous missense mutation in exon 12 of EPAS1 gene p.Gly537Arg and one novel frameshift mutation p.(Ala553Glyfs*58). Further, in silico analysis indicated most of the mutations, probably, were damaging the protein structures, causing the CE in these patients. In this study the mutations in EPO-R and EPAS1 genes were identified for the first time in India.

Pregnancy with Chuvash Polycythaemia and Other Congenital Erythrocytosis.

This original report describes the management of a pregnant woman with congenital erythrocytosis (Chuvash polycythaemia) and reviews the scarce data available in the literature. Therapy consisted of low-dose aspirin and phlebotomies to maintain haematocrit <50% while monitoring iron stores to avoid severe deficiency detrimental to the foetus. Despite normal initial foetal growth, the pregnancy was complicated by preterm birth due to chorioamnionitis. The placenta showed no signs of thrombotic events. The published reports cover 13 pregnancies in 8 patients, showing 1 first-trimester miscarriage, 5 infants with intrauterine growth restriction and/or preterm birth and 1 maternal thrombotic event. These cases were managed with phlebotomies, low-dose aspirin and/or low-molecular-weight heparin, although inconsistently.

Genetic basis of unexplained erythrocytosis in Indian patients.

OBJECTIVE: To evaluate the spectrum of genetic defects in Indian patients with unexplained erythrocytosis. METHODS: 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. RESULTS: 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. CONCLUSION: 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.

Genetic variability of hypoxia-inducible factor alpha (HIFA) genes in familial erythrocytosis: Analysis of the literature and genome databases.

Familial erythrocytosis (FE) is a congenital disorder, defined by elevated red blood cell number, hemoglobin, and hematocrit. Among eight types of FE, type 4 is caused by variants in the EPAS1 gene. Two other hypoxia-inducible factor alpha (HIFA) subunits, HIF1A and HIF3A, have not yet been associated with medical history of FE, but have potential role in the development of erythrocytosis. To improve diagnosis, it is crucial to identify new variants in genes involved in erythrocyte production. Published literature and data from genome browsers were used to obtain HIFA sequence variants associated with erythrocytosis and to locate them on protein sequence and regulatory sites. We retrieved 24 variants from the literature: 2 in HIF1A, 20 in EPAS1 and 2 in HIF3A gene. Sixteen out of 20 variants in the EPAS1 gene are positioned in a conserved region of 13 amino acids within exon 12, next to regulatory post-translational modification and binding sites, suggesting that EPAS1 has an important role in erythropoiesis. The role of HIF1A and HIF3A in the development of erythrocytosis should be further investigated.

Role of fetal intertwin difference in middle cerebral artery peak systolic velocity in predicting neonatal twin anemia-polycythemia sequence.

OBJECTIVES: To evaluate the relationship between the fetal intertwin difference in middle cerebral artery peak systolic velocity (MCA-PSV) and intertwin difference in hemoglobin (Hb) concentration at birth in monochorionic diamniotic (MCDA) twin pregnancies in order to assess its potential role in the prediction of twin anemia-polycythemia sequence (TAPS). METHODS: This was a retrospective cohort study of MCDA twin pregnancies delivered between January 2012 and January 2018. All pregnancies with measurements of MCA-PSV within 7 days prior to delivery and in which neonatal Hb concentration was available were included. The correlation between fetal intertwin difference in MCA-PSV, expressed in multiples of the median (MoM), and neonatal intertwin difference in Hb concentration was investigated. Receiver-operating characteristics (ROC) curve analysis was used to assess the performance of fetal intertwin difference in MCA-PSV for predicting intertwin difference in Hb > 90th centile at birth. RESULTS: A total of 154 out of 256 MC twin pregnancies fulfilled the inclusion criteria. Fetal intertwin difference in MCA-PSV MoM correlated positively with neonatal intertwin difference in Hb concentration (r = 0.79; P < 0.001). The 90th centile for intertwin difference in Hb was 7.25 g/dL. There were 15 (9.7%) cases with a Hb difference ≥ 7.25 g/dL at birth. ROC curve analysis showed a high accuracy of fetal intertwin MCA-PSV MoM difference for the prediction of neonatal intertwin Hb difference ≥ 7.25 g/dL at birth (area under the ROC curve, 0.976 (95% CI, 0.935-0.993); P = 0.012). The optimal cut-off for intertwin MCA-PSV MoM difference was 0.373, with a sensitivity of 93.3% (95% CI, 68.1-99.8%) and a specificity of 95.7% (95% CI, 90.8-98.4%). The positive predictive value was 70% (95% CI, 45.7-88.1%) and the negative predictive value was 99.3% (95% CI, 95.9-100%). CONCLUSION: Our findings show that fetal intertwin MCA-PSV MoM difference is a good predictor of neonatal intertwin Hb concentration difference > 90th centile and potentially of TAPS. Copyright © 2018 ISUOG. Published by John Wiley & Sons Ltd.

Improved prediction of twin anemia-polycythemia sequence by delta middle cerebral artery peak systolic velocity: new antenatal classification system.

OBJECTIVES: To investigate the diagnostic accuracy of delta middle cerebral artery peak systolic velocity (MCA-PSV) > 0.5 multiples of the median (MoM) and compare its predictive value with that of the current MCA-PSV cut-off values of > 1.5 MoM in the donor and < 1.0 MoM in the recipient, for the diagnosis of twin anemia-polycythemia sequence (TAPS) in monochorionic twin pregnancy. METHODS: This was a retrospective consecutive cohort study comprising all uncomplicated monochorionic twin pregnancies and twin pregnancies with a postnatal diagnosis of TAPS managed between 2003 and 2017 in the Dutch national referral center for fetal therapy. Cases with incomplete MCA-PSV Doppler measurements 1 week prior to delivery or with incomplete hemoglobin measurements within 1 day after birth were excluded. The postnatal diagnosis of TAPS was based on an intertwin hemoglobin difference > 8 g/dL and at least one of the following: reticulocyte count ratio > 1.7 or presence of minuscule anastomoses on the placental surface. We compared the predictive accuracy of the current diagnostic method using MCA-PSV cut-off values of > 1.5 MoM in the donor and < 1.0 MoM in the recipient with that of a new method based on intertwin difference in MCA-PSV > 0.5 MoM for prediction of TAPS. RESULTS: In total, 45 uncomplicated and 35 TAPS monochorionic twin pregnancies were analyzed. The sensitivity and specificity of the cut-off MCA-PSV values (donor > 1.5 MoM, recipient < 1.0 MoM) to predict TAPS was 46% (95% CI, 30-62%) and 100% (95% CI, 92-100%), respectively; positive predictive value was 100% (95% CI, 81-100%) and negative predictive value 70% (95% CI, 58-80%). Delta MCA-PSV showed a sensitivity of 83% (95% CI, 67-92%) and a specificity of 100% (95% CI, 92-100%); the positive and negative predictive values were 100% (95% CI, 88-100%) and 88% (95% CI, 77-94%), respectively. Of the 35 cases with TAPS diagnosed postnatally, 13 twin pairs showed a delta MCA-PSV > 0.5 MoM but did not fulfill the cut-off MCA-PSV criteria. Of these 13 TAPS twins, nine donors and four recipients had normal MCA-PSV values. There was a high correlation between delta MCA-PSV and intertwin difference in hemoglobin level (R = 0.725, P < 0.01). CONCLUSION: Delta MCA-PSV > 0.5 MoM has a greater diagnostic accuracy for predicting TAPS compared to the current MCA-PSV cut-off criteria. We therefore propose a new antenatal classification system for TAPS. In monochorionic twin pregnancies with delta MCA-PSV > 0.5 MoM on Doppler ultrasound, but normal MCA-PSV values in the donor or recipient, obstetricians should be aware of the therapeutic implications and neonatal morbidities associated with TAPS. © 2018 The Authors. Ultrasound in Obstetrics & Gynecology published by John Wiley & Sons Ltd on behalf of the International Society of Ultrasound in Obstetrics and Gynecology.

A Kindred with a ß-Globin Base Substitution [ß89(F5)Ser→Arg (AGT>AGG); HBB: c.270T>G] Resulting in Hemoglobin Vanderbilt.

High oxygen affinity hemoglobins (Hbs), characterized by a decreased ability to release oxygen to the tissues and a left-shifted oxygen dissociation curve, are a rare cause of secondary erythrocytosis. Here, we report a base substitution in the ß-globin gene at codon 89 (AGT>AGG) in a kindred with familial erythrocytosis resulting in Hb Vanderbilt, a high oxygen affinity variant.

First Description of Hb San Diego (HBB: c.328G>A) in a Chinese Family with Congenital Erythrocytosis.

Congenital erythrocytosis is a rare and hereditary disorder of red blood cell (RBC) production that can be caused by high oxygen affinity hemoglobin (Hb) variants. We applied a genetic approach including whole exome sequencing and Sanger sequencing. We identified a heterozygous ß-globin gene (Hb San Diego or HBB: c.328G>A) in exon 3 as a causative germline mutation in a Chinese family with congenital erythrocytosis. We concluded that in erythrocytosis with a dominant inheritance and normal or inappropriately high erythropoietin (EPO) levels, the high oxygen affinity Hb variants should be considered. In addition, as a tool for identification of mutations in congenital erythrocytosis, whole exome sequencing improves diagnostic accuracy and provides the opportunity for discovery of novel variants.

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

The 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.

Whole-exome sequencing identifies novel candidate predisposition genes for familial polycythemia vera.

BACKGROUND: Polycythemia vera (PV), characterized by massive production of erythrocytes, is one of the myeloproliferative neoplasms. Most patients carry a somatic gain-of-function mutation in JAK2, c.1849G > T (p.Val617Phe), leading to constitutive activation of JAK-STAT signaling pathway. Familial clustering is also observed occasionally, but high-penetrance predisposition genes to PV have remained unidentified. RESULTS: We studied the predisposition to PV by exome sequencing (three cases) in a Finnish PV family with four patients. The 12 shared variants (maximum allowed minor allele frequency <0.001 in Finnish population in ExAC database) predicted damaging in silico and absent in an additional control set of over 500 Finns were further validated by Sanger sequencing in a fourth affected family member. Three novel predisposition candidate variants were identified: c.1254C > G (p.Phe418Leu) in ZXDC, c.1931C > G (p.Pro644Arg) in ATN1, and c.701G > A (p.Arg234Gln) in LRRC3. We also observed a rare, predicted benign germline variant c.2912C > G (p.Ala971Gly) in BCORL1 in all four patients. Somatic mutations in BCORL1 have been reported in myeloid malignancies. We further screened the variants in eight PV patients in six other Finnish families, but no other carriers were found. CONCLUSIONS: Exome sequencing provides a powerful tool for the identification of novel variants, and understanding the familial predisposition of diseases. This is the first report on Finnish familial PV cases, and we identified three novel candidate variants that may predispose to the disease.

The von Hippel-Lindau Chuvash mutation in mice alters cardiac substrate and high-energy phosphate metabolism.

Hypoxia-inducible factor (HIF) appears to function as a global master regulator of cellular and systemic responses to hypoxia. HIF pathway manipulation is of therapeutic interest; however, global systemic upregulation of HIF may have as yet unknown effects on multiple processes. We used a mouse model of Chuvash polycythemia (CP), a rare genetic disorder that modestly increases expression of HIF target genes in normoxia, to understand what these effects might be within the heart. An integrated in and ex vivo approach was employed. Compared with wild-type controls, CP mice had evidence (using in vivo magnetic resonance imaging) of pulmonary hypertension, right ventricular hypertrophy, and increased left ventricular ejection fraction. Glycolytic flux (measured using [(3)H]glucose) in the isolated contracting perfused CP heart was 1.8-fold higher. Net lactate efflux was 1.5-fold higher. Furthermore, in vivo (13)C-magnetic resonance spectroscopy (MRS) of hyperpolarized [(13)C1]pyruvate revealed a twofold increase in real-time flux through lactate dehydrogenase in the CP hearts and a 1.6-fold increase through pyruvate dehydrogenase. (31)P-MRS of perfused CP hearts under increased workload (isoproterenol infusion) demonstrated increased depletion of phosphocreatine relative to ATP. Intriguingly, no changes in cardiac gene expression were detected. In summary, a modest systemic dysregulation of the HIF pathway resulted in clear alterations in cardiac metabolism and energetics. However, in contrast to studies generating high HIF levels within the heart, the CP mice showed neither the predicted changes in gene expression nor any degree of LV impairment. We conclude that the effects of manipulating HIF on the heart are dose dependent.