chapter8202-219).pdf · chapter8 familialandacquired polycythaemia 8.1 familialpolycythaemia...

* CHAPTER 8

Familial and acquiredpolycythaemia

* 8.1Familial polycythaemia

Josef Prchal, Victor Gordeuk

IRON2009_CAP.8.1(202-219):EBMT2008 4-12-2009 16:22 Pagina 202

1. IntroductionOur understanding of the regulation of erythropoiesis has improved in recent years.The elucidation of the pathophysiology of congenital polycythaemias (erythrocytoses)provides new information regarding the molecular control of red cell production andleads to precise diagnosis. In this chapter, we review the pathophysiology oferythropoiesis and those clinical congenital disorders wherein the molecular basishas been defined. These can be divided into three categories.1. Familial polycythaemia may be due to an intrinsic red cell precursor defect thatleads to increased erythropoiesis despite low erythropoietin (Epo) levels. Thisis called primary familial polycythaemia and is often due to a gain-of-functionmutation in the erythropoietin receptor gene.

2. Familial polycythaemia may be due to inherited defects that cause tissue hypoxiaand a secondary increase in erythropoietin concentrations. Examples includehaemoglobins with high oxygen affinity, defects that lead tomethaemoglobinaemia or deficiency of 2,3-BPG.

3. Familial polycythaemia may be due to inherited defects that lead to increasederythropoietin concentrations in the setting of normal tissue oxygenation. Theseconditions of abnormal hypoxia sensing may result from mutations in the vonHippel-Lindau (VHL) gene, the gene for proline hydroxylase domain protein 2 orthe gene for hypoxia inducible factor-2α.

Once thought to be benign, it is now clear that many forms of familial polycythaemiaare complicated by thrombotic and other non-erythroid manifestations and increasedmortality. Optimal therapy has not been determined. While polycythaemia vera (PV)is the most frequent primary polycythaemic disorder and is acquired, about 5% ofPV patients have relatives with PV or with other myeloproliferative disorders. Thissuggests that these families inherit a yet-to-be identified gene(s) that increasesthe acquisition of PV somatic mutation(s).

2. Regulation of erythropoiesis as it relates to familial polycythaemia

2.1 Stages of erythropoiesisThe production of erythrocytes, erythropoiesis, is a tightly regulated process, butall aspects of its regulation are not fully elucidated. Much remains to be learnedfrom uncovering the molecular basis of congenital and acquired mutations that disruptthe control of erythropoiesis. The process of erythropoiesis can be viewed ashaving three stages. In the initial stage, pluripotent haematopoietic progenitorstransform to committed erythroid precursors. In the second stage, erythroidprogenitors proliferate by a process that is largely regulated by Epo and is madeeffective by the appearance of its receptor (EpoR). Expression of EpoR peaks on the

DISORDERS OF ERYTHROPOIESIS, ERYTHROCYTES AND IRON METABOLISM203

CHAPTER 8.1 • Familial polycythaemia


THE HANDBOOK 2009 EDITION204

surface of early erythroblasts during this stage and then declines. In the third orterminal stage, late erythroid precursors undergo enucleation and removal ofnucleotides and other remnants of organelles that may be toxic to mature circulatingerythrocytes. Defects in erythropoiesis that lead to polycythaemia invariably occurat the second stage of erythropoiesis.

2.2 Alterations in regulation that lead to polycythaemiaIn this review we will concentrate only on those defects that have been demonstratedto lead to, or potentially could lead to, polycythaemia, i.e. increased red cell mass.Polycythaemia is synonymous with the term “erythrocytosis”, and since no consensuson usage has been reached, we will refer to individual entities by the conventionalusage associated with them.Polycythaemias can be classified as being appropriate or inappropriate (Table 1A).

Table 1: Classification of polycythaemia by two approaches

B. Classification according to whether or not erythropoietin is increased

Congenital Acquired

Primary (erythropoietin not increased; defect intrinsic to erythroid progenitor cells)

Primary and familial congenital polycythaemia Polycythaemia vera

Secondary (erythropoietin increased)

Congenital heart disease with right to leftshuntingMethaemoglobinaemiaHaemoglobins with high affinity for oxygenLow erythrocyte 2,3-biphosphoglycerate levelsCongenital polycythaemias due to abnormalhypoxia sensing

Acquired cardiac and pulmonary disorderscharacterised by hypoxiaExposure to cobalt or cyanateSmoking and increased carboxyhaemoglobinEctopic secretion of erythropoietin by tumours

A. Classification according to appropriateness (tissue oxygenation)

Congenital Acquired

Appropriate polycythaemias (tissue hypoxia present)

Haemoglobins with high affinity for oxygen

MethaemoglobinaemiaLow erythrocyte 2,3-biphosphoglycerate levels

Cardiac and pulmonary disorders characterisedby hypoxiaExposure to cobaltSmoking and increased carboxyhaemoglobin

Inappropriate polycythaemias (tissue hypoxia absent)

Primary and familial congenital polycythaemiaCongenital polycythaemias due to abnormalhypoxia sensing

Polycythaemia veraEctopic secretion of erythropoietin by tumours



Appropriate polycythaemias (1) are defined by physiologically intact upregulation ofEpo production to meet tissue oxygen demand, which then drives augmentederythropoiesis. These disorders may be inherited or acquired conditions that lead totissue hypoxia. Inappropriate polycythaemias (1) are caused by inappropriateincreases of erythropoietin signalling such as seen in congenital or acquired primarypolycythaemias, in polycythaemia of disordered hypoxia sensing, in ectopic productionof erythropoietin by tumours. Dysregulation of angiotensin II signalling in renaltransplant patients might also be considered an inappropriate polycythaemia (2, 3).Polycythaemias can also be classified as being primary or secondary (Table 1B).Primary polycythaemias are characterised by a defect within the progenitors thatleads to Epo hypersensitivity or independence, which drives the polycythaemiaphenotype. These disorders are characterised by a low Epo level and can be dividedinto two categories:1. Congenital gain-of-function mutations of the EPOR gene lead to primary familial andcongenital polycythaemia (PFCP) wherein the Epo signal is augmented by the mutation.2. In PV, a somatic (acquired) mutation located downstream of the physiologicalstage of EpoR activation, i.e. the JAK2V617F gain-of-function mutation, contributesto the pathogenesis.Secondary polycythaemias are largely Epo-driven disorders. There are however otherstimulators of erythropoiesis including cobalt, angiotensin II, and insulin-likegrowth factor 1 (IGF-1) (2). Some congenital defects lead to dysregulation of Epoproduction and polycythaemia due to inappropriately high Epo levels. Some of thesecongenital disorders have features of both primary and secondary polycythaemia(i.e. Chuvash polycythaemia).

2.3 Regulation of erythropoietin productionEpo is the principal regulator of erythropoietin signalling as evidenced by itsmodulation of erythroid proliferation, prevention of apoptosis, and promotion oferythroid differentiation (reviewed by Krantz (4)). Regulation of Epo production canbe mediated by hypoxia or be independent of hypoxia.

2.3.1 Molecular basis of hypoxia sensingHypoxia inducible factor transcription factorsHypoxia influences erythropoiesis, energy metabolism, vasculogenesis, iron metabolism,tumour promotion and other processes (reviewed by Hirota (5) and (6)). Thetranscription factors, hypoxia inducible factor (HIF)-1 and HIF-2, are induced in hypoxiccells and bind to a cis-acting nucleotide sequence referred to as the hypoxia-responsive element (HRE), first identified in the 3’-flanking region of the human EPOgene (7). Many hypoxia-inducible genes are directly regulated by HIFs. Dr. Semenza’s



group showed that ~ 3% of all genes expressed in endothelial tissue are HIF-1 regulated(8). HIFs are heterodimeric transcription factors composed of an HIF-α subunit andan HIF-β subunit (Figure 1). The HIF-β subunit belongs to the basic helix-loop-helix(bHLH)-containing PER-ARNT-SIM (PAS)-domain family of transcription factors. Onlythe HIF-1α and HIF-2α subunits are regulated by hypoxia and exist exclusively inHIF. Thus HIF-α is the key subunit determining the hypoxia-modulated amount andactivity of HIF and the transcription of hypoxia-inducible genes.

HIF-1αThe half-life of HIF-1α in the cell is only minutes under normoxic conditions, as theprotein is rapidly degraded by the VHL protein ubiquitin-proteasome pathway (9).


O2-independentpathway

Transcriptionalregulation

O2-dependentpathway

HSP90

[O2]

[O2]

[O2]

UbiUbi

E2

E2VHL

RACK1RACK1

HIF-1aHIF-1a

HIF-1a

HIF-1b Elongin C

Elongin C

26S Proteasome

HIF-1a regulation

p300

HIF-1α

Figure 1: Hypoxic and normoxic control of HIF-1a

Overview of HIF-1a regulation. The left panel depicts the oxygen dependent pathway. In the middle ofthe cartoon, in the presence of oxygen, HIF-1a is proline-hydroxylated, which allows interaction withelongins C and E2, and VHL protein leading to ubiquitinisation and destruction of HIF-1a in theproteasome. The left portion of the cartoon depicts the lack of HIF-1a degradation in hypoxia, withformation of HIF-1a and HIF-1b dimer, resulting in transcriptional regulation of HIF controlled genes.The right panel depicts the oxygen independent pathway wherein RACK1 interacting with elongins C andE2 promote ubiquitinisation and destruction of HIF-1a in the proteasome.



This is initiated by posttranslational hydroxylation of proline at position 564 of theHIF-1α molecule by one of several iron-containing proline hydroxylases. Proline 564hydroxylation facilitates the binding of HIF-1α to VHL protein and subsequentdegradation. Under hypoxic conditions, proline 564 of HIF-1α is not hydroxylated.The unmodified protein escapes VHL-binding, ubiquitination, and degradation, butrather is translocated to the cell nucleus where it dimerises with HIF-1β to form HIF-1 and then activates transcription through binding to specific HREs. Anotherregulatory step involves O2-dependent hydroxylation of asparagine at position 803of HIF-1α, which requires the enzyme FIH-1 (factor inhibiting HIF-1). Hydroxylationof asparagine 803 during normoxia blocks the binding of two other transcriptionfactors, p300 and CBP, to HIF-1 resulting in inhibition of HIF-1 mediated genetranscription. Under hypoxic conditions, asparagine 803 of HIF-1α is not hydroxylated,p300 and CBP can bind to the HIF-1 dimer, and transcriptional activation of HIF-1target genes occurs. In summary, the targeting and subsequent polyubiquitinationof HIF1α requires pVHL, iron, O2 and proline hydroxylase activity, and, as depictedin Figure 1, this complex constitutes the oxygen sensor (10, 11).

HIF-2αHIF-1α and HIF-2α exhibit high sequence homology but have different mRNAexpression patterns. HIF-1α is expressed ubiquitously whereas HIF-2α expression isrestricted to certain tissues (5, 12). Both HIF-1α and HIF-2α are regulated throughidentical mechanism by hypoxia and form a dimer with the same HIF-1β subunit.The kidneys are the main site of Epo production, i.e. renal interstitial cells, and HIF-1 is the principal regulator of EPO transcription in this organ (5). In other tissues,such as the liver (14), which generates ~ 20% of circulating Epo, and the brain (13),EPO gene transcription is HIF-2-regulated (12). The discovery of an iron-responsiveelement in the 5′ untranslated region of HIF2α reveals a novel regulatory linkbetween iron availability and HIF-2α expression (15) that may impact control oferythropoiesis. Thus, when iron supply is limited, HIF-2α would decrease, and wheniron is abundant, hepatic HIF-2α would increase, enhancing liver-synthesised Epoproduction and promoting erythropoiesis (16).

Hypoxia independent regulation of HIFO2-dependent regulation of the HIF-1α subunit is mediated by prolyl hydroxylases,VHL protein, and a proteosomal complex as described above. A regulatory pathwayof HIF that is independent of hypoxia has also been described. This mechanism involvesthe receptor of activated protein kinase C (RACK1) as an HIF-1α-interacting proteinthat promotes prolyl hydroxylase/VHL-independent proteasomal degradation of HIF-



1α. RACK1 competes with heat shock protein 90 (HSP90) for binding to the PAS-Adomain of HIF-1α. HIF-1α degradation is abolished by RACK1 loss-of-function.RACK1 binds to the proteasomal subunit, elongin-C, and promotes ubiquitination ofHIF-1α (Figure 1). Thus, RACK1 is an essential component of an O2/PHD/VHL-independent mechanism for regulating HIF-1α (17).

3. Primary familial polycythaemia (low Epo levels and normal tissueoxygenation)Primary familial and congenital polycythaemia (PFCP) is the only recognised typeof primary familial polycythaemia. It is characterised by an autosomal dominant modeof inheritance, and less frequently, by the occurrence of sporadic cases (18-21). Thiscan be contrasted with Chuvash polycythaemia where the inheritance is autosomalrecessive. The clinical features of PFCP include absence of predisposition to thedevelopment of acute leukaemia or other myeloproliferative disorders, absence ofsplenomegaly, normal white blood cell and platelet counts, low plasma Epo levels,normal haemoglobin-oxygen dissociation curve (indicated by a normal P50) andhypersensitivity of erythroid progenitors to exogenous erythropoietin in vitro (20-25). PFCP is generally thought to be a benign condition, but it has been reportedto be associated with a predisposition to cardiovascular problems, such ashypertension, coronary artery disease, and cerebrovascular events, and these eventsare not clearly related to an elevated haematocrit (26-28). Association withcardiovascular disease has not been described in all series.The distal cytoplasmic region of the EPOR, in association with SHP-1, is required fordown-regulation of Epo-mediated activation of JAK2/STAT5 proteins (29, 30). Thus far,more than 10 mutations of the Epo receptor gene (EPOR) have been convincingly linkedwith PFCP (20-25), 3155. In general, these mutations result in truncation of the EPORcytoplasmic carboxyl terminal leading to loss of its negative regulatory domain,resulting in a gain-of function of EPOR. Additional missense EPORmutations have beendescribed that are not clearly linked to PFCP or other disease phenotypes (21, 55).Absence of polycythaemic phenotype in some patients with EPORmutation is suggestiveof a role played by gene modifiers or epigenetic factors in phenotypic penetrance (32).

4. Secondary familial polycythaemia (elevated Epo levels due to tissuehypoxia)

4.1 High oxygen affinity haemoglobin mutantsAffinity of haemoglobin for oxygen is expressed as the P50, which is the partialpressure of oxygen in blood at which 50% of the haemoglobin is saturated with




oxygen. An abnormally low P50 reflects an increased affinity of haemoglobin foroxygen. During oxygenation and deoxygenation, there is considerable movement alongthe α1/β2 interface (33). Several haemoglobin mutants have substitutions affectingthis interface. Other haemoglobin variants have amino acid substitutions involvingthe C-terminal residues of the β chain or of the 2, 3 BPG binding sites. All thesesubstitutions can affect the cooperative nature of oxygen binding with haem, thechange from T to R state and vice versa, and in turn can change the affinity ofhaemoglobin for oxygen. The majority of mutations affecting oxygen affinity resultin high affinity haemoglobin variants that lead to relative tissue hypoxia andcompensatory secondary appropriate polycythaemia. There are 91 haemoglobinvariants, listed on the globin server, known to be associated with high affinity foroxygen (http://globin.bx.psu.edu/hbvar/menu.html; last accessed December 24,2008). Thus an autosomal dominant polycythaemia with normal to elevated Epo levelsis suggestive of a mutant haemoglobin with high affinity for oxygen (33). The therapyof this compensatory polycythaemia by phlebotomies is ill advised.Haemoglobin F binds oxygen normally but in the cell has high oxygen affinity dueto reduced binding of 2,3-BPG (see below).

4.2 2, 3-BPG deficiencyCongenitally low erythrocyte 2, 3 biphosphoglycerate (2, 3 BPG) level can occur becauseof deficiency of the red cell enzyme 2, 3-BPG mutase (34, 35). This is an extremelyrare autosomal recessive condition. This disorder should be suspected in the case ofisolated polycythaemia (without any feature of myeloproliferative disorders such asprogressive increase of RBC mass, high platelet and granulocyte counts andsplenomegaly), absence of a family history and low P50 (signifying high oxygenaffinity). A haemoglobin mutation needs to be ruled out first. In these cases, the redcells will have high oxygen affinity; however, unlike high affinity haemoglobin, theoxygen affinity of the haemolysate is normal and the level of 2, 3-BPG is very low.

4.3 Congenital methaemoglobinaemiasThere are three types of hereditary methaemoglobinaemias (reviewed by Gregg & Prchal(36)). Two are inherited as autosomal recessive traits: cytochrome b5R deficiency andcytochrome b5 deficiency. The third type is an autosomal dominant disorder, haemoglobinM (Hb M) disease in which there is a mutation of one of the globin genes. Allcongenital methaemoglobinaemias are associated with suboptimal delivery of oxygenper red cell and this may result in compensatory secondary appropriate polycythaemia.The resulting polycythaemia is typically mild and its treatment is not advisable as itwould only decrease tissue oxygen delivery and lead to tissue hypoxia.



5. Disorders of hypoxia sensing (elevated Epo levels and normal tissueoxygenation)

5.1 Chuvash polycythaemiaThis disorder was the first hereditary condition of augmented hypoxia sensing tobe recognised. Chuvash polycythaemia, the only known endemic polycythaemia, isan autosomal recessive hereditary polycythaemia. The Chuvash people reside in themid-Volga River region in Russia where Chuvash polycythaemia affects hundreds ofpeople, making it the most common congenital polycythaemia (37). Outside ofChuvashia, this form of familial polycythaemia has also been found sporadically indiverse ethnic and racial groups (38-40), and a high prevalence of this disorder wasreported in the Italian island of Ischia (41). In a study of five Chuvash families withpolycythaemia, a mutation of VHL (598CÆT) was found in the affected individuals(42). This mutation impairs the interaction of pVHL with HIF-1α, thus reducing therate of ubiquitin-mediated destruction of HIF-1α. As a result, the level of the HIF-1 increases and leads to increased expression of target genes including endothelin-1, EPO, plasminogen activator inhibitor (PAI), transferrin, transferrin receptor andVEGF (42-45). The VHL598CÆT mutation also impairs the interaction of pVHL withHIF-2α (46), and an animal model of the Chuvash polycythaemia mutation indicatesthat it may be increased levels of HIF-2α rather than HIF-1α that mediate theincreased Epo levels and polycythaemia in this condition (47). Chuvash polycythaemiais characterised by an intact response to hypoxia despite increased basal expressionof a broad range of hypoxia-regulated genes in normoxia. Unexpectedly, theerythroid progenitors are hypersensitive to Epo; thus Chuvash polycythaemia sharesfeatures of primary as well as secondary polycythaemia.Chuvash polycythaemia is a unique VHL syndrome characterised by homozygousgermline mutation of VHL leading to predisposition to development of varicose veins,benign vertebral haemangiomas, low systemic blood pressures, pulmonary hypertension,thrombosis, bleeding, cerebral vascular events, and increased mortality (Table 2).Peripheral blood profiling indicates lower white blood cell and platelet counts thancontrols, lower CD4 counts, elevated levels of both pro-inflammatory and anti-inflammatory cytokines, and altered plasma thiol levels with elevated homocysteineand glutathione and low cysteine concentrations (44, 45, 48-50). Despite increasedexpression of HIF-1α and VEGF in normoxia, Chuvash polycythaemia patients do notdisplay a predisposition to tumour formation. Imaging studies of 33 Chuvashpolycythaemia patients revealed unsuspected cerebral ischaemic lesions in 45% butno tumours characteristic of VHL syndrome (44). It is not known whether therapy withaspirin or phlebotomy in patients with Chuvash polycythaemia prevents complications.



MolecularHomozygous VHL598CÆTDecreased capture of HIF-α by VHL proteinDecreased degradation of HIF-α by the ubiquitin-proteasome pathwayIncreased HIF-α under normoxic conditionsAltered transcription of multiple HIF-regulated genes

Increased plasma concentrations of products of HIF-regulated genesEndothelin-1ErythropoietinPlasminogen activator inhibitor-1Vascular endothelial growth factorTransferrinTransferrin receptor

Physical findingsRuddy complexionVaricose veins, increased prevalenceHeart murmur, increased prevalenceClubbing of digits, increased prevalenceLower systemic blood pressureHigher pulmonary artery pressure on echocardiogramBenign vertebral haemangiomas on MRICerebral ischaemic lesions on MRI

Complete blood countPolycythaemia (erythrocytosis)Lower white blood cell countLower platelet count

Table 2: Characteristics of Chuvash polycythaemia



Immunologic profileIncreased plasma concentrations of Th-1 cytokines (GM-CSF, IFN-γ, IL-2 and IL-12, TNF-α)Increased plasma concentrations of Th-2 cytokines (IL-4, IL-5, IL-10, IL-13)Normal ratio of Th-1 to Th-2 cytokinesLower CD4 counts

Thiol metabolismIncreased plasma concentrations of homocysteine, glutathione and cysteinylglycineReduced plasma concentration of cysteine

Clinical courseNo increase in incidence of renal cell carcinoma, pheochromocytoma or haemangioblastomaIncreased peptic ulcersIncreased major thrombosesIncreased haemorrhageIncreased cerebrovascular accidentsIncreased mortality


Homozygosity for VHL 598CÆT occurs sporadically in Caucasians in the United Statesand Europe and in people of Southeast Asian (Indian subcontinent) ancestry (38-41). Thrombotic complications have been reported in some of these individuals.To address the question of whether the VHL 598CÆT substitution occurred in a singlefounder or resulted from recurrent mutational events, haplotype analysis wasperformed on subjects bearing the VHL 598CÆT mutation and normal unrelatedindividuals from Chuvash, Asian, Caucasian, Hispanic and African-American ethnicgroups (51). These studies indicated that in most individuals, the VHL 598CÆTmutation arose in a single ancestor between 12,000 and 51,000 years ago. However,a polycythaemic family in Turkey had a different haplotype indicating that the VHL598CÆT mutation in this family occurred independently (52).In contrast, autosomal dominant mutations of the VHL gene cause VHL syndrome(53). Heterozygotes for dominant VHL mutations are at increased risk of developinghaemangioblastomas, renal cell carcinoma, pheochromocytoma, pancreatic endocrinetumours, and endolymphatic sac tumours when they acquire a somatic mutationin the normal VHL allele. Some patients with VHL syndrome also develop acquiredpolycythaemia (53). Increased expression of HIF-1 and possibly VEGF may underliethe VHL tumour predisposition syndrome and the development of haemangioblastomaand renal cell carcinoma. However, the absence of a predisposition to tumourigenesisin Chuvash polycythaemia patients implies that deregulation of HIF-1 and VEGFmay not be sufficient to cause predisposition towards tumour formation in VHLsyndrome.Most heterozygotes for VHL598CÆT do not have polycythaemia. However, anEnglish patient has been described who was a heterozygote for VHL 598CÆT (40),although the inheritance of a deletion of a VHL allele or a null VHL allele in a transposition was not excluded in this patient. Subsequently, two VHL heterozygouspatients with polycythaemia were described in whom a null VHL allele was morerigorously excluded (52, 54); one of these patients also had ataxia-telangiectasia(54). Some patients with congenital polycythaemia have proven to be compoundheterozygotes for the Chuvash mutation, VHL 598CÆT, and other VHL mutationsincluding 562CÆG, 574CÆT, 388CÆG, and 311GÆT (39, 52, 54).

5.2 Non-Chuvash germline VHL mutationsNon-Chuvash germline VHL mutations also cause polycythaemia. A Croatian boy washomozygous for VHL 571CÆG, the first example of a homozygous VHL germlinemutation other than VHL 598CÆT causing polycythaemia (39). Additionally, aPortuguese girl was a compound heterozygote for VHL 562CÆG and VHL 253CÆT(54, 55). A few cases of congenital polycythaemia with mutations of only one non-Chuvash polycythaemia VHL allele have been described. Two Ukrainian children with



polycythaemia were heterozygotes for VHL 376GÆT, but the father with the samemutation was not polycythaemic (38). Peripheral blood erythroid progenitors fromthe children and father were hyper-responsive to recombinant Epo in in vitroclonogenic assays in a way similar to that seen in Chuvash polycythaemia patients.

5.3 Proline hydroxylase deficiencyA family with a proline hydroxylase domain protein 2 (PHD2) mutation (950CÆG)was recently described in which heterozygotes for this mutation have mild orborderline polycythaemia (56). Since then, four additional patients with unexplainedpolycythaemia who are heterozygote carriers of different mutations in PHD2 havebeen reported (two frameshift mutations, 606delG and 840_841insA, both locatedin exon 1, and two nonsense mutations, 1112GÆA and 1129CÆT, in exon 3) (57,58). One of these patients had a major thrombotic event (thrombosis of thesagittal sinus) (58). These observations underscore the importance of defective HIFsignalling in the genesis of polycythaemic disorders.

5.4 HIF-2a gain-of-function mutationsRecently, a family was described in which members with polycythaemia were heterozygouscarriers of a HIF-2α Gly537Trp mutation which served to stabilise the HIF-2α protein(59). The same group subsequently reported four additional polycythaemic patientswith heterozygous Met535Val or Gly537Arg mutations in the HIF-2α gene (60). Thepatients tend to present at a young age with elevated serum Epo. These findings supportthe importance of the proline hydroxylase, HIF-2α, VHL axis in human Epo regulationand in the pathogenesis of familial anaemias due to abnormal hypoxia sensing.

5.5 Congenital polycythaemias of yet unidentified defect with elevated orinappropriately normal levels of EpoNot all patients with congenital polycythaemias with normal or elevated Epo levels dohave VHL, PHD2 or HIF-2αmutations, and the molecular basis of polycythaemia in thesecases remains to be elucidated. It is not clear why in some families, the polycythaemiais dominantly inherited (61), in others recessively, and in some it is sporadic and, whyin families with the same mutation the phenotype can be different (55, 62). Lesionsin genes linked to hypoxia-independent regulation of HIF as well as oxygen-dependentgene regulation pathways are leading candidates for mutation screening in polycythaemicpatients with normal or elevated Epo without VHL, PHD2 or HIF-2α mutations.

6. Familial polycythaemia vera and other myeloproliferative disordersThere is growing evidence that the acquired primary polycythaemic disorder





polycythaemia vera, which is caused by somatic mutation(s), often has a familialpredisposition (63); in some families other myeloproliferative disorders are alsopresent (64-65). It is estimated that about 5% of PV patients have relatives withPV or with other myeloproliferative disorders (65). This suggests the existence ofgerm-line mutations that either facilitate the somatic mutation(s) or contribute tosomatic acquired mutations to result in familiar clustering of myeloproliferativedisorders. The molecular basis of these interactions yet remains to be defined. Thissuggests that these families inherit a yet-to-be identified gene(s) that increasesthe acquisition of PV somatic mutation(s).

AcknowledgementsSupported in part by grant nos. 2 R25 HL003679-09 (VG) and 1 R01 HL079912-03 (VG) fromNHLBI, by Howard University GCRC grant no 2MOI RR10284-11 (VG) from NCRR, NIH, Bethesda,MD and R01HL50077-14 (JP) and 1P01CA108671-03 (JP).

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Multiple Choice Questionnaire

To find the correct answer, go to http://www.esh.org/iron-handbook2009answers.htm

1. Mutation of which of the following genes has not been associatedwith congenital polycythaemia?a) VHL. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .b) HIF-2 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .c) EPOR . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

d) EPO. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

2. Chuvash polycythaemia is caused by homozygosity for the 598CÆÆTmutation in the VHL gene. Which of the following is a recognisedmanifestation of Chuvash polycythaemia?a) Benign vertebral hemangiomas . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . b) Pheochromocytoma . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . c) Hypertension . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

d) CNS cerebellar haemangioblastoma . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

3. Which of these polycythaemias is caused by a somatic mutation(s)?a) Primary familial congenital polycythaemia . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

b) HIF-2 mutation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

c) Polycythaemia vera . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

d) Chuvash polycythaemia . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

4. Which of these congenital polycythaemic states is associated withlow erythropoietin level and augmented erythropoietin signaling?a) Primary familial congenital polycythaemia

(due to EPO receptor mutations) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . b) Chuvash polycythaemia (598CÆT VHL mutation) . . . . . . . . . . . . . . . . . . . . . . . . . . . c) HIF-2 mutation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

d) Proline hydroxylase mutation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

5. Which of these polycythaemia states is inherited as an autosomal dominant disorder?



a) Polycythaemia vera . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

b) Chuvash polycythaemia . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

c) Mutations of globin gene causing high haemoglobin oxygen affinity . . . . . . .

d) Cytochrome b5 reductase mutations causing methaemoglobinaemia and polycythaemia . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .




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