Von Hippel-Lindau-dependent polycythemia is endemic on the island of Ischia: Identification of a novel cluster

Abstract

Chuvash polycythemia (MIM 263400) is an autosomal recessive disorder characterized by a high hemoglobin level, relatively high serum erythropoietin, and early death. It results from a Von Hippel-Lindau (VHL) gene mutation (C598T) that causes increased HIF-1alpha activity and erythrocyte production in the face of normoxia. This polycythemia is endemic in Chuvashia, whereas its worldwide frequency is very low. We investigated the incidence of the Chuvash-type VHL mutation in Campania (South Italy) and identified 14 affected subjects (5 families). Twelve live on the island of Ischia (Bay of Naples). From analysis of the mutated allele, we found that the disease was more frequent on Ischia (0.070) than in Chuvashia (0.057). The haplotype of all patients matched that identified in the Chuvash cluster, thereby supporting the single-founder hypothesis. We also found that nonaffected heterozygotes had increased HIF-1alpha activity, which might confer a biochemical advantage for mutation maintenance. In conclusion, we have identified the first large cluster of Chuvash erythrocytosis outside Chuvashia, which suggests that this familial polycythemia might be endemic in other regions of the world.

Full text

HEMATOPOIESIS
Von Hippel-Lindau–dependent polycythemia is endemic on the island of Ischia:
identification of a novel cluster
Silverio Perrotta, Bruno Nobili, Marcella Ferraro, Carmela Migliaccio, Adriana Borriello, Valeria Cucciolla, Vincenzo Martinelli,
Francesca Rossi, Francesca Punzo, Paola Cirillo, Giuseppe Parisi, Vincenzo Zappia, Bruno Rotoli, and Fulvio Della Ragione
Chuvash polycythemia (MIM 263400) is
an autosomal recessive disorder charac-
terized by a high hemoglobin level, rela-
tively high serum erythropoietin, and early
death. It results from a Von Hippel-Lindau
(VHL) gene mutation (C598T) that causes
increased HIF-1␣activity and erythrocyte
production in the face of normoxia. This
polycythemia is endemic in Chuvashia,
whereas its worldwide frequency is very
low. We investigated the incidence of the
Chuvash-type VHL mutation in Campania
(South Italy) and identified 14 affected
subjects (5 families). Twelve live on the
island of Ischia (Bay of Naples). From
analysis of the mutated allele, we found
that the disease was more frequent on
Ischia (0.070) than in Chuvashia (0.057).
The haplotype of all patients matched
that identified in the Chuvash cluster,
thereby supporting the single-founder hy-
pothesis. We also found that nonaffected
heterozygotes had increased HIF-1␣activ-
ity, which might confer a biochemical
advantage for mutation maintenance. In
conclusion, we have identified the first
large cluster of Chuvash erythrocytosis
outside Chuvashia, which suggests that
this familial polycythemia might be en-
demic in other regions of the world.
(Blood. 2006;107:514-519)
©2006 by The American Society of Hematology
Introduction
The Chuvash variant of familial polycythemia was first described
in more than 100 individuals from about 80 families living in the
mid-Volga River region of European Russia.1The disease is
characterized by a high hemoglobin level, increased plasma
erythropoietin (Epo) level, varicose veins, vertebral hemangiomas,
low blood pressure, and an elevated serum concentration of
vascular endothelial growth factor (VEGF).2Patients affected by
Chuvash polycythemia die early, mainly as a result of cerebral
vascular events or peripheral thrombosis. These injuries seem to be
linked to mechanisms other than blood hyperviscosity or serum
Epo content.2Indeed, the prevalence of low blood pressure in
patients with Chuvash polycythemia contrasts with the hyperten-
sion frequently associated with polycythemia vera and other
familial polycythemias resulting from excess Epo.
Genome-wide screening and candidate gene characterization
demonstrated that the Arg200Trp mutation (C598T) of the Von
Hippel-Lindau (VHL) gene causes the Chuvash form of polycythe-
mia.3Thereafter, the mutation was detected in homozygosity in
patients with sporadic or familial congenital erythrocytosis from
diverse ethnic groups.4-8 However, 19 homozygotes have been
identified among the more than 150 known cases of non-Chuvash
familial erythrocytosis.9Furthermore, 8 other VHL mutations
(Arg79Cys, Gly104Val, Asp126Tyr, Val130Leu, Gly144Arg,
Tyr175Cys, Leu188Val, His191Asp, Pro192Ala) were detected in
either homozygotes or compound heterozygotes.4,5,7,8,10 These
mutations were detected in a total of 10 cases, which indicates
that the C598T transition is the major cause of VHL-related
erythrocytosis.
The C598T mutation likely originated from a single founder
event because the VHL haplotype in non-Chuvash patients is
identical to that in polycythemic patients from Chuvashia.6,11 The
C598T allele is very rare outside the Chuvash population. In fact,
its frequency in Chuvashia is about 0.057,3whereas the worldwide
frequency of the Chuvash-associated haplotype is about 0.001 377.11
The different haplotype recently identified in a patient of Turkish
ancestry probably represents an independent mutational event.7
Together with other proteins (elongin B, C, Rbx1, and Cul2),
the VHL protein participates in the hypoxia-sensing pathway,
where it binds the proline-hydroxylated form of the hypoxia-
inducible factor-1␣(HIF-1␣), thereby committing the transcription
factor to polyubiquitination and proteasomal degradation.12-15
Under normoxic conditions, HIF-1␣is hydroxylated and rapidly
degraded, thereby resulting in down-regulation of the transcription
of HIF-1␣–regulated genes.13,16 Conversely, the C598T mutation
impairs VHL function and causes an increase in the HIF-1
complex, which in turn could cause overexpression of its target
genes.3HIF-1␣regulates such important genes as EPO,VEGF,
SDF1,GLUT1, triosephosphate isomerase 1 (TP1) transferrin, and
the transferrin receptor.3,17,18 Although an increased serum level of
Epo is considered the major cause of polycythemia,1other HIF-1␣–
modulated genes might be involved in the pathogenesis of
erythrocytosis.
From the Department of Pediatrics, Second University of Naples, Italy; the
Department of Biochemistry and Biophysics “F. Cedrangolo,” Second
University of Naples, Italy; the Division of Hematology, Federico II University,
Naples, Italy; and the Divisione di Pediatria, Ospedale Anna Rizzoli, Lacco
Ameno d’Ischia, Naples, Italy.
Submitted June 20, 2005; accepted September 5, 2005. Prepublished online as
Blood First Edition Paper, October 6, 2005; DOI 10.1182/blood-2005-06-2422.
Supported by grants from “Progetto di Ricerca di Ateneo” Seconda Universita`
di Napoli, Fondo per gli Investimenti della Ricerca di Base (FIRB), Progetti di
Rilevante Interesse Nazionale (PRIN), Ministero dell’Istruzione dell’Universita`
e della Ricerca (MIUR), and Associazione Italiana per la Ricerca sul Cancro
(AIRC).
An Inside Blood analysis of this article appears at the front of this issue.
Reprints: Silverio Perrotta, Department of Pediatrics, Second University of Naples,
Via Luigi De Crecchio, 4, Naples, Italy; e-mail: silverio.perrotta@unina2.it
The publication costs of this article were defrayed in part by page charge
payment. Therefore, and solely to indicate this fact, this article is hereby
marked ‘‘advertisement’’ in accordance with 18 U.S.C. section 1734.
© 2006 by The American Society of Hematology
514 BLOOD, 15 JANUARY 2006 䡠VOLUME 107, NUMBER 2
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In this study, we investigated the frequency of the C598T VHL
mutation in the Campania Region of South Italy. Unexpectedly, we
discovered a cluster of the disease on the island of Ischia (Bay of
Naples), which has a population of about 55 000. This is the first
region other than Chuvashia where this congenital polycythemia
is endemic.
Patients, materials, and methods
Patients
Twenty-two patients from 13 families with suspected Chuvash-like congeni-
tal polycythemia were included in the study. Diagnostic criteria included (1)
persistent elevated hemoglobin level (⬎180 g/L [18 g/dL] in males, ⬎165
g/L [16.5 g/dL] in females, or ⬎2 SD above the median of the sex- and
age-specific normal range in children); (2) absence of splenomegaly; (3)
normal leukocyte and platelet counts; (4) normal hemoglobin oxygen
affinity; (5) high or inappropriately high serum Epo level,1,3,9,19 and (6)
absence of known causes of secondary erythrocytosis. The median age of
patients at diagnosis was 19 years (range, 1-34 years). Some patients had
undergone sporadic or regular phlebotomy treatment. Eleven patients were
members of 2 unrelated families: 8 from family A and 3 from family B. The
other 11 subjects reported no affected relatives. All cases were recruited
through the Department of Pediatrics (Second University of Naples) and the
Division of Hematology (Federico II University of Naples). The study was
approved by the Institutional Review Board of the Second University of
Naples and performed in accordance with the World Medical Association
Declaration of Helsinki of 1975, as revised in 2000. Written informed
consent for molecular genetic analysis, data analysis, and publication was
obtained from all participants.
Family A. Family A includes 8 polycythemic patients: a mother (P13),
her 2 sons (P15 and P16), her brother (P12), her father (P05), her uncle
(P04), and 2 cousins (P07 and P08) (Figure 1A). All lived on the island of
Ischia. No patient had a history or evidence of thrombotic complications or
cancer. There is no record of consanguinity in the family. Erythrocytosis
was discovered in the mother when she was 10 years old, at which time the
hemoglobin (Hb) was 165 g/L (16.5 g/dL) and packed cell volume (PCV)
was .54 (54%). The serum Epo concentration was 35 IU/L (mIU/mL)
(normal range, 11-30 IU/L [mIU/mL]) before phlebotomy therapy was
initiated. Her husband was not affected by erythrocytosis. Polycythemia
was diagnosed in her 2 sons shortly after birth, and they began a therapeutic
phlebotomy program to maintain their hematocrit level below .45 (45%).
Their Epo levels were 55 and 99 IU/L (mIU/mL) before phlebotomies.
Family B. Family B includes 3 polycythemic subjects. A 9-year-old
girl from Ischia (P22) (Figure 1B) was diagnosed with erythrocytosis at the
age of 3 months. Hb was 210 g/L (21.0 g/dL) and PCV was .59 (59%),
whereas O2P50 was normal. The Epo concentration was 41 IU/L
(mIU/mL). Polycythemia was diagnosed in her mother (P19) and uncle
(P21) when they were 10 and 12 years old, respectively. They had elevated
levels of Hb (197 g/L [19.7 g/dL] and 225 g/L [22.5 g/dL], respectively) and
Epo (34 IU/L [mIU/mL] and 75 IU/L [mIU/mL], respectively). There is no
record of consanguinity in the family. The patients do not have a history of
cerebrovascular complications or cancer.
Detection of gene mutations
Genomic DNA was extracted from peripheral blood leukocytes with the
Flexigene DNA Kit (Qiagen GmbH, Hilden, Germany). To search for VHL
mutations, we sequenced all 3 VHL exons and their intron-exon boundaries.
Polymerase chain reaction (PCR) was performed essentially as reported in
Ang et al,3and the reaction products were purified using a QIAquick Gel
Extraction Kit (Quiagen GmbH). The products were sequenced using the
ABI 310 DNA Sequencer and the ABI PRISM Dye Terminator Cycle
Sequencing Reaction Kit (Applied Biosystems, Milan, Italy), according to
the manufacturer’s instructions. The coding regions of the elongin B and
elongin C genes and the HIF-1␣sequences spanning the oxygen-dependent
degradation/pVHL interaction domain (residues 417-698, exons 10-12)
were screened for DNA sequence variations by PCR amplification and
DNA sequencing using the oligonucleotide primers and PCR conditions
described in Clifford et al.20 DNA sequencing of PCR products was carried
out as reported.
Mutation screening for the C598T base change
The C598T mutation abolishes a Fnu4HI restriction endonuclease recogni-
tion site. Thus, to screen for the mutation, we digested 14 ␮L PCR-
amplified exon 3 product with 0.5 U Fnu4HI (New England Biolabs,
Hitchin, Herts, United Kingdom) for 3 hours at 37°C. The digested products
were visualized by electrophoresis on 2% agarose.
Haplotype analysis
We used 8 single-nucleotide polymorphisms that span the VHL gene
(rs1056286, rs722509, rs779805 A⬎G, rs779808, rs1678607, 1149A⬎G,
rs696356, rs378630), which are known to be highly informative in the
Chuvash population,11 to characterize polycythemic patients. The PCR
products were sequenced as reported in “Detection of gene mutations.”
Reverse transcription polymerase chain reaction
We isolated RNA from 5 ⫻106Epstein-Barr virus (EBV)–transformed
lymphoblastoid cells using the Trizol Reagent Kit (Invitrogen, Carlsbad,
CA). Total RNAwas prepared from reticulocytes as described elsewhere.21
cDNA was synthesized using the Superscript II Kit (Invitrogen) with
random hexamers and 1.5 ␮g total RNA. PCR amplification of the whole
VHL coding region was carried out with primers cVHL1-3F1 5⬘-
CAGCTCCGCCCCGCGTCCGAC-3⬘(located at the 5⬘-untranslated re-
gion) and cVHL1-3R1 5⬘-AAGGAAGGAACCAGTCCTGT-3⬘(located at
the 3⬘-untranslated region). PCR conditions were as reported in Cario et al.7
The reaction products were analyzed by agarose electrophoresis. cDNA was
amplified with a primer located in the second VHL exon (cVHL2-3F1
5⬘-CTCTTCAGAGATGCAGGGACAC-3⬘) and the cVHL1-3R1 primer
in separate experiments. The reaction product (377 bp) was digested
with 0.5 U Fnu4HI as described in “Mutation screening for the C598T
base change.”
The expression of the EPO,VEGF,SDF1, and TP1 genes was evaluated
by reverse transcriptase (RT)–PCR with cDNAs prepared as described in
“Detection of gene mutations.” The primers and conditions were as follows:
Figure 1. Pedigrees of 2 families affected by VHL-dependent polycythemia
living on the island of Ischia. The Pcode denotes individuals from whom DNA
samples were obtained. Filled symbols denote polycythemic subjects who are
homozygous for the C598T mutation; half-filled symbols, heterozygous subjects.
VHL-DEPENDENT POLYCYTHEMIAIS ENDEMIC IN ISCHIA 515BLOOD, 15 JANUARY 2006 䡠VOLUME 107, NUMBER 2
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EPO (5⬘-CGCGCCCGCTCTGCTCCGACACC-3⬘[forward] and 5⬘-
GGAGCGACAGCAGGGACAGGAGA-3⬘[reverse] for 32 cycles, each
consisting in steps at 95°C for 45 seconds, 56°C for 45 seconds, and 68°C
for 45 seconds), VEGF (5⬘-TCGGGCCTCCGAAACCATGA-3⬘[forward]
and 5⬘-CTCCTCCTTCTGCCATGGGT-3⬘[reverse] for 32 cycles, each
consisting in steps at 95°C for 45 seconds, 56°C for 45 seconds, and 68°C
for 45 seconds), SDF-1 (5⬘-GTGTCACTGGCGACACGTAG-3⬘[forward]
and 5⬘-TCCCATCCCACAGAGAGAAG-3⬘[reverse] for 32 cycles, each
consisting in steps at 95°C for 45 seconds, 58°C for 45 seconds, and 68°C
for 45 seconds), TP1 (5⬘-GTGAAGGACTGGAGCAAGGT-3⬘[forward]
and 5⬘-GGGCTCATTGTTTGGCATTG-3⬘[reverse] for 28 cycles, each
consisting in steps at 95°C for 45 seconds, 58°C for 45 seconds, and 68°C
for 45 seconds). The PCR products were analyzed by electrophoresis on
1.8% agarose gel.
Before amplification with each specific primer pair, an aliquot of the
cDNA preparation was amplified using primers for ␤-actin to determine the
integrity of the generated cDNA (BD Biosciences Clontech, San Jose, CA).
Moreover, we used 5 different cDNA concentrations to ensure that signals
were proportional to input mRNA. Each experiment was performed at least
in triplicate and, in several cases, in quadruplicate. The expression of
␤-actin, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), EPO, and
SDF-1 was also evaluated by real-time PCR as reported elsewhere.17,22,23
Human kidney RNA (BD Biosciences Clontech) served as a positive
control for EPO expression.
Preparation of lymphoblastoid cell lines
We established EBV-transformed lymphoblastoid cell lines from the
peripheral blood of 8 subjects: 2 homozygotes for the C598T mutation (P12
and P22), 2 polycythemic heterozygotes (P24 and P25), 2 nonpolycythemic
heterozygotes (P09 and P11), and 2 control subjects. The EBV lines were
produced with 0.2-nm filtered culture medium of the B-95.8 EBV-
producing marmoset line24 and PHA-M. Cells (107) were pelleted and
resuspended in 1 mL B-95.8 cell line supernatant. This preparation was
incubated in a conical tube at 37°C and occasionally resuspended. After
60 to 90 minutes, the cells were pelleted and resuspended in RPMI 1640
supplemented with L-glutamine (2 mM), penicillin/streptomycin (100 ␮g/
mL), gentamycin (100 ␮g/mL), and 10% heat-inactivated fetal bovine
serum in the presence of 5 ␮g/mL PHA-M. Half the medium was replaced
every 3 to 4 days. Outgrowth of EBV-transformed cells was evident after
4 to 6 weeks.
HIF-1␣functional analysis and immunoblotting
HIF-1␣transcription factor activity was determined with the TransAM
HIF-1␣Kit (Active Motif, Carlsbad, CA) according to the manufacturer’s
instructions. Briefly, in this enzyme-linked immunoadsorbent assay (ELISA)
method, an oligonucleotide containing the hypoxia-response element is
immobilized on a 96-well plate. HIF dimers in nuclear extracts specifically
bind to the oligonucleotide and are identified by means of an anti–HIF-1␣
antibody. The procedures used for immunoblotting are reported else-
where.25 The antibodies directed against aldolase and GAPDH were from
Santa Cruz Biotechnology (Santa Cruz, CA); the antisera against HIF-1␣is
contained in the kit.
Results
The VHL gene C598T mutation in the Campania Region
We studied 22 patients from 13 families putatively affected by
Chuvash-like erythrocytosis. We studied only patients whose
hemoglobin-adjusted Epo serum concentration was higher than
normal under nonphlebotomized conditions.3,9 All subjects live in
the Campania region of southern Italy, which has a population of
about 5 million. All declared that their family had resided in
Campania for several generations. No patient is of Chuvash origin.
We first evaluated the occurrence of the C598T mutation by
sequencing the 3 VHL exons (Figure 2A). We verified the results by
amplifying the third VHL exon and digesting the PCR product with
Fnu4HI (Figure 2B). Because the mutation abolishes the Fnu4HI
restriction site, the amplified product containing C598T was not
digested (Figure 2B). Identical results were obtained with the 2
approaches, thereby validating the second method in the screening
of multiple DNA samples.
We identified the mutation in 14 patients (5 families) of the 22
examined (Figure 2B). Twelve patients were homozygotes for the
C598T mutation and 2 were heterozygotes. A similar screening of
putative Chuvash-like polycythemic patients living in other Italian
regions revealed no patient with the C598T mutation (data not shown).
All the 12 homozygote patients (3 families) live on Ischia.
Although the 2 heterozygotes do not live on Ischia, their parents
who carried the VHL mutation live on the Naples coastline directly
facing the island. No mutations were found in the 3 VHL exons
of the other 8 polycythemic patients who did not carry the
C598T mutation.
VHL mutation in heterozygotes
The 2 patients with a heterozygous C598T mutation were a
4-year-old boy (P24) and a 30-year-old man (P25). Erythrocytosis
was discovered at the age of 2 years (P24) and 10 years (P25). Their
present hemoglobin levels are 170 g/L (17 g/dL) and 205 g/L (20.5
g/dL), PCV of .54 (54%) and .65 (65%), and serum Epo 33 IU/L
(mIU/mL) and 54 IU/L (mIU/mL), respectively. Thus far, they have
no hyperviscosity symptoms or thromboembolic complications.
These 2 patients (and their parents) declared that no other family
member is affected by erythrocytosis. There was no consanguinity
in the parents. In both the cases, the C598T mutation was inherited
from the father, but neither father had any clinical or laboratory
signs of polycythemia.
Subjects heterozygous for the C598T mutation do not usually
manifest erythrocytosis. However, 2 independent cases of polycy-
themic patients heterozygous for the C598T mutation have been
Figure 2. Detection of the C598T mutation in DNA samples from 2 heterozy-
gotes and 12 homozygotes. (A) Examples of sequences of the relevant region of
the VHL gene in a control subject, in a heterozygote (P24), and in a homozygote
(P04) patient. (B) FnU4HI digested the 268-bp PCR product of the wild-type VHL
allele into 187- and 81-bp bands. Conversely, the C598T mutation resulted in an
uncut 268-bp band. Patients P24 and P25 were heterozygotes for the mutation,
whereas the other patients are homozygotes. MW indicates molecular weight
standards; Heter, heterozygotes.
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reported.6,7,8,10 We next carried out a series of analyses to look for
other genetic aberrations (eg, mutations, deletions, and silencings)
that could affect the apparently wild-type VHL allele.
First, we sequenced the VHL gene promoter and did not find any
mutations. However, this finding does not exclude abnormalities
that may affect the expression of the wild-type VHL allele (eg,
epigenetic events) or result in altered transcripts. Therefore, we
analyzed the VHL transcripts. We retrotranscribed total RNA from
EBV-transformed B lymphoblasts of the 2 patients and amplified
the cDNA using primers localized in the 5⬘- and 3⬘-UTR of the
VHL mRNA. This experiment revealed 2 full-length VHL tran-
scripts, as previously reported.26 One transcript includes all 3 ex-
ons, and a smaller transcript results from splicing-induced skipping
of the second exon. The 2 amplified products were purified and
digested with Fnu4HI, and the assay mixtures were separated by
electrophoresis on agarose gel. Both the wild-type and the mutated
VHL allele expressed the 2 transcripts (data not shown). We also
amplified the cDNAs using a forward primer localized in the
second exon and a reverse primer at the 3⬘-UTR of the mRNA. In
this case, only 1 amplified product was obtained. The PCR product
was digested with Fnu4HI. Although these results are semiquantita-
tive, they suggest that the 2 alleles are transcribed with a similar
efficiency (Figure 3A).
Because the 2 heterozygote polycythemic patients have high
Epo serum levels, it is conceivable that genetic alterations of
components of the oxygen-sensing pathway other than VHL may
contribute to erythrocytosis. Consequently, we looked for muta-
tions in the HIF1A gene and in 2 genes (ie, elongin B and
elongin C) that encode other components of the E3 complex that
ubiquitinates HIF-1␣. No mutations were found (data not shown).
VHL gene mutation is endemic on Ischia
The 12 patients, homozygous for the Chuvash-like mutation, come
from 3 families that live on Ischia (Figures 1-2). Greater than 50%
of the patients are affected by hypotension and varicose veins. No
cancer or other symptoms of Chuvash polycythemia were ob-
served. In addition to families A and B (corresponding to 11
patients), the other homozygous subject (P23) was a 35-year-old
man with a long-standing history of erythrocytosis. He is the only
member of his family affected by polycythemia, and at present he
has a hemoglobin level of 210 g/L (21 g/dL), PCV of .64 (64%),
and a serum Epo level of 42 IU/L (mIU/mL). His heterozygous
parents have no history of consanguinity.
Our 2 affected families included 6 homozygotes (P08, P12, P13,
P15, P16, and P22) who had one homozygous parent (P04, P05,
P13, and P19) (Figures 1-2). Because the other parent was an
obligate heterozygote (P03, P06, P14, and P20) and parental
consanguinity was denied, we inferred a high frequency of the
C598T mutation on Ischia. In this population, we determined that
the mutation occurred with a frequency of 0.0703 (9 heterozygotes
in 64 healthy subjects). None of the investigated subjects belonged
to families that included homozygote patients. Conversely, we
found no VHL mutations in 100 healthy subjects (200 chromo-
somes) from other Italian areas. The haplotype pattern in our 12
patients (data not shown) was identical to that previously reported
in patients with Chuvash polycythemia.11
The data obtained in this novel VHL-dependent polycythemic
cluster suggest that heterozygotes may have some selective advan-
tages that favor the spread and maintenance of the mutated allele.
To address this issue, we determined HIF-1␣activity in EBV-
transformed B lymphoblasts from healthy subjects, heterozygotes
and homozygotes. As shown in Figure 4A, HIF-1␣activity was
3-fold higher in homozygotes compared with controls. Surpris-
ingly, activity was also increased in heterozygous subjects. These
results were confirmed by a sensitive immunoblotting procedure
(Figure 4B). Using RT-PCR, we also measured the expression of
the EPO,VEGF,SDF1 (Figure 5A) and TP1 (data not shown)
Figure 3. VHL expression in lymphoblastoid cells from a control subject (Con),
2 heterozygotes (P24 and P25), and a homozygote (P22). (A) Total RNA from each
cell line was retrotranscribed to cDNA, which was amplified as reported in “Patients,
materials, and methods,” and the product (377 bp) was digested with FnU4HI. The
normal allele yielded 2 fragments of 245 bp and 132 bp. The C598T mutation
abolished the restriction site and resulted in an uncut 377-bp band. The 2
heterozygote patients (P24 and P25) expressed the allele in roughly similar amounts.
Conversely, the homozygote for the VHL mutation shows a single undigested band.
(B) The expression of the actin gene served as control.
Figure 4. HIF-1␣activity in lymphoblastoid cell lines. (A) Samples from 2 control
subjects (Con), 2 healthy heterozygotes (P09 and P11), and 2 homozygotes (P12 and
P22) were cultured, and nuclear extracts were assayed for HIF-1␣activity with the
TransAM HIF-1 kit (Active Motif) and (B) with immunoblotting with the antibody in the
TransAM HIF-1 kit. Error bars indicate 2 SDs.
Figure 5. HIF-1␣–dependent expression in lymphoblastoid cell lines and
reticulocytes. (A) Total RNA from lymphoblastoid cells of 2 control subjects (Con)
and 2 homozygote patients (P12 and P22). Kidney total RNA (KR) was used as a
positive control. Expression of the actin gene served as a control. (B) Total RNA from
the reticulocytes of a control and a homozygote patient (P12) were used as starting
material. (C) Immunoblotting analysis of aldolase and GAPDH in cellular extracts of 2
control subjects and 2 homozygotes (P12 and P22). See “Patients, materials, and
methods” for further details.
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genes, which are targets of activated HIF-1␣.27 In particular, SDF1
has recently been demonstrated to be modulated by HIF-1␣.17
Because the SDF-1 protein is involved in angiogenesis, it could
play a role in lowering blood pressure in homozygous and
heterozygous subjects with Chuvash polycythemia. As shown in
Figure 5A, VEGF expression was increased in homozygotes (by
more than 3-fold as assessed by gel scanning). In contrast, EPO and
SDF1 (Figure 5A) and TP1 (data not shown) gene transcription was
identical in all samples.
We also evaluated the expression of SDF1, VEGF, and EPO in
reticulocytes from a healthy subject and from a C598T homozy-
gote. Only VEGF transcription was up-regulated, whereas EPO
and SDF1 expression was unchanged, thereby confirming the
findings obtained in lymphoblastoid cells (Figure 5B). Quantitative
PCR confirmed the data on EPO and SDF1 expression. Immuno-
blotting experiments with 2 other HIF-1␣targets—aldolase and
GAPDH—revealed no variations (Figure 5C).
Discussion
This study demonstrates that Chuvash polycythemia is frequent in
Campania and is endemic on the island of Ischia. This is the only
cluster known besides the original Chuvash cluster. Our observa-
tion supports the notion that this erythrocytosis variant is spread
throughout the world and demonstrates that it might be very
frequent in some areas. A study carried out in other Italian regions
(data not shown) suggests that the Ischia cluster is unique in Italy.
Moreover, the finding that our 12 patients have the same haplotype
as the Chuvash patients11 supports the single-founder hypothesis.
Although there is no proof of direct contact between the
Chuvash and the inhabitants of Ischia, there is evidence, albeit
weak, of a link between the 2 populations that might account for the
high incidence of the disease on the island. The Chuvash derive
from the Huns, who are thought to derive from the Middle East
populations of the Sumerians and Scythians. The Huns also
interacted with the Hungars and Vandals.28 Thus, the C598T VHL
allele may have reached Ischia consequent to (1) the Vandals’
invasion of Ischia that started from a Carthaginian harbor at the
time of Attila the Hun, (2) the Hungars’ conquest of central and
south Italy, and (3) the pillaging by Turks of Ischia and
surrounding areas.29
Irrespective of the route of transmission, we cannot explain the
high incidence of the C598T mutation in Ischia and not in other
Italian regions with a similar history. It is conceivable that a
founder effect in a small isolated population within an island under
social and environmental conditions that retard outbreeding may
have led to the emergence of the mutation. Another possibility is
that the altered gene might convey advantages in terms of iron
metabolism, erythropoiesis, and embryonic development.2,3 For
instance, improved erythropoiesis could compensate for iron
deficiency consequent to a fish-based diet. Moreover, a slight
increase of HIF-1␣–regulated cytokines might be useful in such
conditions as preeclampsia.
Our data on HIF target gene transcription in Chuvash polycythe-
mia differ from those of a previous study3in 2 aspects. First, EPO
expression was not up-regulated in our EBV-transformed B-
lymphocytes, which might reflect the low expression of EPO in
these cells. Second, and more intriguing, is the observation that of
the 5 genes expressed in lymphoblastoid cells, namely VEGF,
SDF1,TPI, aldolase, and GAPDH,30,31 only VEGF appears to be
up-regulated in the Chuvash-like polycythemic lymphoid cells. It is
probable that, in an identical genetic background, different HIF-1␣
levels are required to express specific genes or sets of genes, which
would explain, at least in part, the distinct phenotypes observed in
subjects with different VHL mutations.
The mechanism underlying VHL-dependent polycythemia in
patients with only one altered allele is not clear. In this context, it is
noteworthy that we also found polycythemic patients who fulfilled
the Chuvash-like erythrocytosis criteria9and had high serum Epo,
but who had no VHL mutations (S.P. and F.D.R., manuscript
submitted). This raises the possibility of alterations at other steps of
the HIF-1␣–related pathway.
A clinical aspect of this study is that in regions, such as
Chuvashia and Ischia, congenital polycythemia should be consid-
ered a “frequent” nonbenign hematologic disease. Awareness of
this frequency may lead to early diagnosis and hence better patient
management. Finally, because it is not strictly confined to Chu-
vashia and not solely a result of the C598T mutation, we suggest
that “VHL-dependent polycythemia” would be a more accurate
term for this condition.
Acknowledgments
We thank Drs Marco Picardi, Rosanna Ciancia, and Fara Petruz-
ziello for their invaluable assistance regarding the clinical aspects
of the research, and Stefania Arciello for her technical help. We
also thank Jean Gilder for text editing.
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VHL-DEPENDENT POLYCYTHEMIAIS ENDEMIC IN ISCHIA 519BLOOD, 15 JANUARY 2006 䡠VOLUME 107, NUMBER 2
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October 6, 2005 originally published onlinedoi:10.1182/blood-2005-06-2422
2006 107: 514-519
Vincenzo Zappia, Bruno Rotoli and Fulvio Della Ragione
Cucciolla, Vincenzo Martinelli, Francesca Rossi, Francesca Punzo, Paola Cirillo, Giuseppe Parisi,
Silverio Perrotta, Bruno Nobili, Marcella Ferraro, Carmela Migliaccio, Adriana Borriello, Valeria
Ischia: identification of a novel cluster
dependent polycythemia is endemic on the island of−Von Hippel-Lindau
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