A study of 36 unrelated cases with pure erythrocytosis revealed three new mutations in the erythropoietin receptor gene

Thirty-six unrelated cases with erythrocytosis of unknown originwere investigated. Exons 5–8 of the erythropoietin receptorgene (EPOR), the von Hippel-Lindau gene, and the prolyl hydroxylasedomain protein 2 gene (PHD2) were screened by direct DNA sequencing.The Janus kinase 2 mutation, JAK2 (Val617Phe), was screenedby allele specific PCR. In this study, three new mutations ofEPOR causing deletions in exon 8 were found: the first led directlyto a stop codon [g.5957_5958delTT (p.Phe424X)], the second toa stop codon after one residue [g.5828_5829delCC (p.Pro381GlnfsX1)]and the third to a stop codon following a frameshift sequenceof 23 residues [g.5971delC (p.Leu429TrpfsX23)]. One patienthad a previously reported EPOR mutation [g.6146A>G (p.Asn487Ser)]and another, a silent one (g.5799G>A). All were heterozygotes.In addition, 2 patients were positive for JAK2 (Val617Phe),and 2 reported elsewhere, were mutated in the PHD2 gene [c.606delG(p.Met202IlefsX71).

Key words: polycythemia, erythrocytosis, erythropoietin receptor, von Hippel-Lindau, JAK2, prolyl hydroxylase domain protein 2.

Introduction

TOP

Introduction

Polycythemia refers to a group of disorders with increased hematocritand hemoglobin levels, and increased red cell mass. Most polycythemiacases are acquired, secondary to a chronic tissue hypoxia, orto a primary bone marrow disease, such as polycythemia vera(PV). In this latter case activating mutations in the Januskinase 2 (JAK2) have been found: JAK2 (Val617Phe)¹ is the mostfrequent, however, 10 others have been recently reported inexon 12.^2–3 Congenital polycythemia can result from variousetiologies such as hemoglobins with high oxygen affinity, deficienciesin 2,3-diphosphoglycerate mutase,⁴ mutations of the EPOR, andof genes coding for factors involved in the oxygen sensing pathway⁵[Von Hippel-Lindau (VHL), prolyl hydroxylase domain 2 (PHD2)].^6–8

Percy recently reviewed the mutations in the EPOR associatedwith erythrocytosis⁹ (an absolute increase in red cell massand hematocrit without elevation of the megakaryocytic or granulocyticlineages). This subgroup of abnormalities is usually characterizedby low to normal plasma erythropoietin levels, and hypersensitivityof erythroid progenitors to exogenous erythropoietin in vitro.¹⁰The patients are usually clinically asymptomatic or presentingwith mild symptoms, however, this condition could still contributeto cardiovascular problems.¹¹ We investigated 36 unrelated caseswith erythrocytosis of unidentified origin. In all samples wescreened for EPOR abnormalities, focusing on the exons encodingfor the cytoplasmic region (exons 7–8) which interactswith kinases, and the transmembrane region (exon 6) which isimportant for proper receptor activation.¹² All these patientshave already been investigated for PHD2 mutations:⁸ two sibsand four patients with missense mutations were found. In addition,we searched for JAK2 (Val617Phe) and VHL mutations. This reportfocuses on the mutations found in EPOR.

Design and Methods

TOP

Design and Methods

Patients
The patients were referred to our laboratory for molecular diagnosisof erythrocytosis. Polycythemia vera or causes of secondaryerythrocytosis were eliminated. All patients had initially elevatedhemoglobin and hematocrit levels. None of them carried hemoglobinwith increased oxygen affinity. In 10 cases a familial historyof erythrocytosis was found, and a total of 44 samples wereanalyzed corresponding to 36 unrelated cases. The hematologicdata of the patients are summarized in Table 1.

View this table:
[in this window]
[in a new window]
[Download PPT slide]
Table 1. Hematologic and clinical presentation of the 36 cases including 44 patients with presumed pure erythrocytosis.

Erythroid colony formation assays
Bone marrow or peripheral blood mononuclear cells were used,and the assays performed according to standard procedures.¹³

Molecular biology studies
All patients gave their signed informed consents. The studywas approved by the local ethics committee and performed inaccordance with the World Medical Association Declaration ofHelsinki. Genomic DNA was prepared from peripheral blood byphenol chloroform procedure. Screening for JAK2 (Val617Phe)mutation was performed by allele-specific polymerase chain reaction(PCR), as previously described.¹⁴ Exons 5–8 of EPOR genewere amplified by PCR using Ampli Taq^® DNA polymerase (Roche,New Jersey, USA), in a Gene Amp^® PCR system 2700 (AppliedBiosystems, Foster city, CA, USA). Details and primer sequencesare available upon request. The PCR products were purified.Sequencing reactions were carried out using Big Dye^® sequencingkit, (Applied Biosystems) and analyzed on the ABI Prism^®3100 Genetic analyzer (Applied Biosystems) according to themanufacturer’s protocol. The three exons of VHL gene wereanalyzed according to a similar procedure (details for primersequences and PCR conditions are available upon request).

Results and Discussion

TOP

Results and Discussion

In this study, three different mutations of EPOR, and one frameshiftmutation of PHD2 appeared to be responsible for the observederythrocytosis. In addition, 2 patients had the JAK2 (Val617Phe)mutation. The first EPOR mutation resulted in a stop codon atposition 424 (p.Phe424X), predicting a protein shortened by85 residues. It was found, over 3 generations, in 4 membersof a family (Figure 1A). Erythropoietin level in the third generationwas low. Erythroid colony formation assays showed hypersensitivityof erythroid progenitors to exogenous erythropoietin (Figure 1B).Electrophoresis of the PCR product showed a heteroduplex. Sequencingof the PCR revealed a heterozygous deletion of two thyminesat positions 5957 and 5958 in exon 8 (g.5957_5958delTT) (Figure 1C).

View larger version (32K):
[in this window]
[in a new window]
[Download PPT slide]
Figure 1. Experimental data concerning the first (A, B, C), the second (D), and the third (E) EPOR mutations. (A) Pedigree of the 3-generation family. (B) Erythroid colony formation assays in one member of the family (PG6) and in 2 normal controls (T1 and T2). The vertical axis indicates the number of BFU-E-derived colonies per 2.5x10⁵ peripheral blood mononuclear cells. The final EPO concentration added to the cultures is indicated in the horizontal axis. It shows in vitro hypersensitivity of BFU-E to erythropoietin at low concentrations. (C) DNA sequencing showing g.5957_5958delTT. (D) DNA sequencing showing g.5828_5829delCC. (E) DNA sequencing showing g.5971delC.

The second EPOR mutation was a two nucleotides deletion encodingat position 381 for a glutamine followed by a stop. This wasobserved in a 47-year-old woman (Hb: 18.5g/dl, Hct: 56%). Oneof her cousins was erythrocytosic as well, with a low EPO level.Both patients were treated by venesection. Electrophoresis ofthe PCR product revealed a heteroduplex. Sequencing showed aheterozygous deletion of cytosines 5828 and 5829 in exon 8 (g.5828_5829delCC)(Figure 1D), replacing codon 382 by a stop codon. This led toa 127 amino acid truncation (p.Pro381GlnfsX1), which is thelargest reported to date in the EPOR molecule.

The third EPOR mutation led to a 57 amino acid truncation. Thiscase of familial erythrocytosis, observed in a 31-year-old mother,and in her two daughters (9 and 11 years old), was discoveredwhen the youngest was hospitalized at 9 years old for a convulsiveepisode associated with fever. Because erythrocytosis was observed(Hb 20.2 g/dL, Hct 58%) a complete blood count was carried outfor the sister and the mother, revealing Hb levels of 18.8 and20.6 g/dl, and Hct 54.8 and 60.2% respectively. The probandhad an EPO level of less than 5 mUI/mL, (normal range: 5–24mUI/mL). Electrophoresis of the PCR products showed no specialfeature, but DNA sequencing revealed a heterozygous deletionof a cytosine at position 5971 in exon 8 (g.5971delC) (Figure 1E).This caused a frameshift at position 429, predicting an introductionof 23 amino acids followed by a premature stop codon (p.Leu429TrpfsX23).The three mutations associated with familial erythrocytosisdescribed above result in truncation in the distal region ofthe protein, involving the loss of 6 or 7 functionally importanttyrosines. In addition, a previously described mutation¹⁵ wasfound in a 62-year-old man with erythrocytosis (Hb 17.4 g/dl,Hct 54%, normal serum EPO level), treated by venesection. Sequencingof the PCR product of exon 8 showed a heterozygous A>G mutationof nt 6146 resulting in an aspargine to serine substitutionat position 487 [g.6146A>G (p.Asn487Ser)]. However, the patienthad a chronic respiratory failure with an arterial SaO2 of 86%.This raised the question of whether this base change might bea polymorphism or whether it has some relation with the erythrocytosis.The in vitro studies in murin Ba/F3 cell line could not demonstrateif this mutation had biological consequences.¹⁵ We found thelast EPOR mutation in a 6-year-old erythrocytosic child (Hb19 g/dL), with an elevated serum EPO level (30.8 mUI/mL) andno familial history of erythrocytosis. Sequencing of the PCRproduct of exon 8 showed a heterozygous guanine to adenine substitutionat nucleotide 5799 (g.5799G>A), which does not modify theencoded amino acid, and thus is unlikely to be the cause oferythrocytosis.

This work emphasizes the negative growth-regulatory role ofthe distal region of the EPOR molecule in erythropoiesis. AfterEPO binding and conformational change of EPOR, JAK2 triggersthe signaling cascade by autophosphorylation and phosphorylationof EPOR on tyrosine residues, which become docking sites forpositive and negative regulators. The former group includes:signal transducers and activators of transcription (STAT5a/b),p85 ${alpha}$ regulatory subunit of PI-3Kinase, and Lyn tyrosine kinase.¹⁶Among the negative regulatory signals are protein tyrosine phosphatase(SHP-1), CIS (cytokine inducible Src homology-2 containing proteins)or SOCS (suppressors of cytokin signaling), and Lnk.¹⁷ Accordingto residue positions in the human EPOR, it has been suggestedthat SHP1 interacts with P-Tyr454,¹⁸ CIS3, also referred toas SOCS3, interacts with P-Tyr426, P-Tyr454 and P-Tyr456,^19–20down regulating cytokine signaling in each case. To date, includingthis work, 16 mutations affecting the intracellular domain ofEPOR have been described.⁹ The percentage of familial and congenitalpolycythemias found, in this study, to be associated with EPORmutations in exon 8 is similar to that described in the literature(<15%). This suggests that mutations in other regions ofthe EPOR, or in other genes, could be responsible for the unresolvedcases.²¹ Mutations affecting the negative or the positive regulatorsof EPOR signaling cascade are candidates for further exploration.In this study, screening for JAK2 (Val617Phe) mutation was foundpositive in 2 patients, which were reclassified as PV (Table 2).This observation is in agreement with two reports that JAK2(Val617Phe) is found at a low incidence in patients with idiopathicerythrocytosis,^22,23 and has to be screened for. The 2 patientsdid not fulfill the criteria of PV since both had normal leukocyteand platelet counts, no splenomegaly, and negative endogenouserythroid colony (EEC) assay. This is also in agreement withthe reported patients’ characteristics.^22,23 For the secondpatient, the relative percentage of the JAK2 (Val617Phe) allelecould be quantified on granulocytes (stored before cytotoxictreatment) and was found to be quite low, about 5%. This mayargue for a false negative result of the EEC assay in this patient,because if only few hematopoietic progenitors harbored the JAK2(Val617Phe) allele conferring EPO hypersensitivity, the numberof endogenous colonies might be low and difficult to detect.In contrast to other studies reporting that mutations in theVHL gene constitute around 10%²⁴ of all cases with idiopathicerythrocytosis, the screening for VHL mutations was negativein all our patients who presented with variable serum EPO levelsand a wide age range. The PHD2 mutation found in the two sibs[c.606delG (p.Met202IlefsX71)] led to a protein truncated byits 154 C-terminal amino acids. Further studies are in progressto verify biological consequences of the point mutations.

View this table:
[in this window]
[in a new window]
[Download PPT slide]
Table 2. Main hematologic characteristics of the two patients found positive for the JAK2(Val617Phe).

The screening for exon 12 mutations in JAK2 must still be performedon new samples of the DNA extracted from granulocytes, or fromthat of the endogenous erythroid colonies.³ In conclusion, ourfindings add to the spectrum of the molecular defects identifiedso far reflecting the heterogeneity of the erythrocytosis. However,in the majority of the patients, the genetic defect(s) remainelusive and require further research.

Acknowledgments

we thank Dr. W. Vainchenker for scientific discussion, the referringphysicians (Dr. S. Ansoborlo, Dr. J.J. Kiladjian, and Dr. J.Roussi)for providing us with the clinical data, and Mrs. I. Teyssandierfor technical assistance.

Footnotes

Authorship and Disclosures

MA-S performed PCR and sequencing experiments, collected theclinical data and wrote the manuscript EM contributed to theexperimental work; BCG performed VHL sequencing; HW wrote themanuscript and discussed the results; CP designed the study,performed sequencing experiments and wrote the manuscript; VUdesigned the study, performed EPOR sequencing, JAK2 V617F screening,and in vitro cultures, and contributed to writing the manuscript.CP and VU contributed equally to this work. The authors reportedno potential conflicts of interest.

Received for publication September 17, 2007. Revision received February 8, 2008. Accepted for publication March 3, 2008.

References

TOP