The heterozygote advantage of the Chuvash polycythemia VHLR200W mutation may be protection against anemia

Article (PDF Available)inHaematologica 96(9):1371-4 · May 2011with17 Reads
DOI: 10.3324/haematol.2011.045609 · Source: PubMed
Abstract
The germ-line loss-of-function VHL(R200W) mutation is common in Chuvashia, Russia and occurs in other parts of the world. VHL(R200W) homozygotes have elevated hypoxia inducible factor (HIF)-1 and HIF-2 levels, increased hemoglobin concentration, propensity to thrombosis and early mortality. Because the mutation persists from an ancient origin, we hypothesized that there is a heterozygote advantage. Thirty-four VHL(R200W) heterozygotes and 44 controls over 35 years of age from Chuvashia, Russia were studied. Anemia was defined as hemoglobin less than 130 g/L in men and less than 120 g/L in women. Mild anemia was present in 15% of VHL(R200W) heterozygotes and 34% of controls without a mutated VHL allele. By multivariate logistic regression, the odds of anemia were reduced an estimated 5.6-fold in the VHL(R200W) heterozygotes compared to controls (95% confidence interval 1.4-22.7; P=0.017). In conclusion, heterozygosity for VHL(R200W) may provide protection from anemia; such protection could explain the persistence of this mutation.
haematologica | 2011; 96(9)
BRIEF REPORTS
1371
Introduction
The R200W mutation of the von Hippel Lindau gene (VHL)
is present on the same haplotype in almost all persons of het-
erogeneous racial and ethnic backgrounds, indicating that the
mutation may have originated in a founder prior to diver-
gence of human races.
1
There is only one reported exception
to this genetic linkage.
2
Homozygosity for VHL
R200W
is respon-
sible for Chuvash polycythemia, the first recognized congen-
ital disorder of augmented hypoxia sensing.
3
Chuvash poly-
cythemia is common in the Chuvash Republic of the Russian
Federation
4
and on the Italian island of Ischia;
5
the condition
also occurs in other parts of the world.
2,6
Chuvash poly-
cythemia is characterized by increased levels of HIF-1α and
HIF-2α under ambient oxygen conditions
3,7
and upregulation
of a number of the target genes of HIF.
3,8,9
Clinical manifesta-
tions include lower systemic blood pressure, higher pul-
monary artery pressure and other changes in pulmonary vas-
cular physiology, varicose veins, vertebral and hepatic
hemangiomas, lower white blood cell and platelet counts,
increased serum concentrations of inflammatory cytokines,
changes in plasma thiol concentrations, arterial and venous
thrombosis, major bleeding episodes, cerebral vascular events
and premature mortality. In contrast to von Hippel Lindau
cancer predisposition disease, malignant tumors typical of
this syndrome have not been found, and no increased risk of
cancer has been demonstrated.
8-13
Given a negative selection for VHL
R200W
homozygotes, the
mutation should be associated with some type of heterozy-
gote advantage, albeit slight. However, the effect of heterozy-
gosity for VHL
R200W
is not known. Only rare cases of poly-
cythemia associated with VHL
R200W
heterozygosity have been
reported.
2,6,14
In one study that included 9 Chuvash VHL
R200W
heterozygotes and 77 Chuvash participants with normal VHL
alleles, the VHL
R200W
heterozygotes had significantly lower
systemic blood pressures and higher serum PAI-1 concentra-
tions. In addition, although no VHL
R200W
heterozygote was
polycythemic, the mean hemoglobin concentration was high-
er by 4 g/L but this difference was not statistically significant.
8
The present study was conducted to prospectively deter-
mine if heterozygotes for VHL
R200W
have discernable physio-
logical and clinical differences from individuals without a
mutated VHL allele, and if such differences exist, to consider
whether they may represent a heterozygote advantage. We
previously searched for a possible protection of VHL
R200W
het-
erozygotes from eclampsia, a major cause of maternal mor-
The heterozygote advantage of the Chuvash polycythemia VHL
R200W
mutation
may be protection against anemia
Galina Y. Miasnikova,
1
Adelina I. Sergueeva,
2
Mehdi Nouraie,
3
Xiaomei Niu,
3
Daniel J. Okhotin,
4
Lydia A. Polyakova,
1
T
omas Ganz,
5
J
osef T. Prchal,
6
a
nd Victor R. Gordeuk
3
1
Chuvash Republic Clinical Hospital No. 1, Cheboksary Russia;
2
Cheboksary Children’s Hospital, Cheboksary Russia;
3
Center for
Sickle Cell Disease and Department of Medicine, Howard University, Washington, DC;
4
Russian Research Services, Camas, WA
USA;
5
Dept. of Medicine, David Geffen School of Medicine at UCLA, Los Angeles, USA; and
6
Division of Hematology, University of
Utah and VAH, Salt Lake City, UT, USA
Funding: supported in part by grant n. UH1-HL03679-05 from the National Heart, Lung and Blood Institute (VRG) and the Office of Research on Minority
Health, by a grant from Amgen, and by NIH grants n. 1 R01 HL079912-01 (VRG) and R01 HL50077-14 (JTP).
Manuscript received on April 7, 2011. Revised version arrived on May 11, 2011. Manuscript accepted May 16, 2011.
Correspondence: Victor R. Gordeuk, M.D., Center for Sickle Cell Disease, Howard University, 2041 Georgia Ave. NW, Washington, DC 20060 USA. Phone:
international +1.202.8651941. Fax: international +1.202.2326719. E-mail: vgordeuk@howard.edu
The germ-line loss-of-function VHL
R200W
mutation is common
in Chuvashia, Russia and occurs in other parts of the world.
VHL
R200W
homozygotes have elevated hypoxia inducible factor
(HIF)-1 and HIF-2 levels, increased hemoglobin concentration,
propensity to thrombosis and early mortality. Because the
mutation persists from an ancient origin, we hypothesized
that there is a heterozygote advantage. Thirty-four VHL
R200W
heterozygotes and 44 controls over 35 years of age from
Chuvashia, Russia were studied. Anemia was defined as
hemoglobin less than 130 g/L in men and less than 120 g/L in
women. Mild anemia was present in 15% of VHL
R200W
het-
erozygotes and 34% of controls without a mutated VHL
allele. By multivariate logistic regression, the odds of anemia
were reduced an estimated 5.6-fold in the VHL
R200W
heterozy-
gotes compared to controls (95% confidence interval 1.4-22.7;
P=0.017). In conclusion, heterozygosity for VHL
R200W
may pro-
vide protection from anemia; such protection could explain
the persistence of this mutation.
Key words: heterozygosity, VHL
R200W
, mutation, protection,
anemia.
Citation: Miasnikova GY, Sergueeva AI, Nouraie M, Niu X,
Okhotin DJ, Polyakova LA, Ganz T, Prchal JT, and Gordeuk
VR. The heterozygote advantage of the Chuvash polycythemia
VHL
R200W
mutation may be protection against anemia.
Haematologica 2011;96(9):1371-1374.
doi:10.3324/haematol.2011.045609
©2011 Ferrata Storti Foundation. This is an open-access paper.
ABSTRACT
haematologica | 2011; 96(9)
bidity and mortality that is related to dysregulation of HIF-
1-regulated VEGF, but this was not validated (Gordeuk et
al., unpublished data, 2011). In this study, we examined
the effect of VHL
R200W
heterozygosity on anemia in an oth-
e
rwise unselected Chuvash sample. We postulated that
the identification of any VHL
R
200W
heterozygote advantage
could point to potential benefits of chronic augmentation
of HIFs by pharmacological agents, such as inhibitors of
PHDs.
1
5,16
Design and Methods
Study objective
T
he study objective was to compare clinical and molecular char-
acteristics of a group of VHL
R200W
heterozygotes over 35 years of
age and a similar number of controls without a mutated VHL allele
over 35 years of age in the Chuvash Republic of the Russian
Federation. Budgetary restraints confined the study to less than 80
research participants.
Research protocol
The Howard University Institutional Review Board approved
the research and all participants provided written informed con-
sent. The study was carried out in the Chuvash Autonomous
Republic of the Russian Federation, which is located about 650
kilometers southeast of Moscow along the Volga River. VHL
R200W
heterozygotes over 35 years of age were identified by studying
first-degree family members of patients with Chuvash poly-
cythemia. In addition, unaffected, unrelated controls, also of
Chuvash ethnicity, and of similar age and sex distribution were
enrolled from the community in the same geographical area of
Chuvashia without previous knowledge of their health status. The
study participants, who were in their usual state of health, were
characterized by medical history, physical examination including
blood pressure and body weight, and laboratory tests of the
peripheral blood.
Laboratory procedures
Complete blood count was performed by an automated analyz-
er (Sysmex XT 2000i, Sysmex Corporation, Kobe, Hyogo, Japan).
Serum ferritin concentration was determined by enzyme
immunoassay (Ramco Laboratories Inc., Stafford, TX, USA).
Plasma concentrations of VEGF and serum concentrations of ery-
thropoietin and soluble transferrin receptor were determined by
enzyme linked immunosorbent assay (ELISA) (R& D Systems,
Minneapolis, MN, USA). Plasma PAI-1 concentration was also
determined by ELISA (Innovative Research, Inc., Novi, MI, USA).
Serum hepcidin concentration was measured by competitive
ELISA as previously described.
17
Genotyping for VHL
R200W
was per-
formed by PCR as previously described.
8
Statistics
The primary study comparison was between VHL
R200W
heterozy-
gotes and genotypically normal subjects. Analysis of continuous
variables was made by the Student’s t-test (after normal transfor-
mation) or by analysis of variance with adjustment for significant
covariates. Analysis of categorical variables was by Pearson’s χ
2
test or by logistic regression with adjustment for other significant
variables. Skewed continuous variables were log-transformed to
approximate a normal distribution. Analyses were performed
with Stata 10.1 (StatCorp., College Station, TX, USA).
Role of the funding source
Amgen helped in the design of the study but Dr. Gordeuk had
full responsibility for implementing and conducting the study, col-
lecting, managing and interpreting the data, and writing the man-
uscript.
Results and Discussion
The clinical characteristics of the study participants are
summarized in Table 1 according to VHL genotype. The
mean value for MCV was lower among the VHL
R200W
het-
erozygotes (P=0.033) and the white blood cell counts were
higher (P=0.036). The serum ferritin concentrations were
similar in VHL
R200W
heterozygotes and controls. The mean
(standard deviation [SD]) hemoglobin concentration was
134 (14) g/L in the VHL
R200W
heterozygotes and 128 (16) g/L
in the controls without a mutated VHL allele (P=0.10). The
study sample size of 78 has a power of 0.50 to detect the
observed 6 g/L difference in hemoglobin concentration
between VHL
R200W
heterozygotes and controls at a signifi-
cance level of P<0.05. A sample size of 170 would have a
power of 0.8 to detect such a difference and a sample size
of 230 would have a power of 0.9.
Using the World Health Organization definition of
hemoglobin (less than 130 g/L in males and less than 120
g/L in females),
18
20 (26%) of the 77 study participants
were anemic (Table 2) and the prevalence by gender was
15 (32%) of 47 females and 5 (17%) of 30 males. The ane-
mia was mild and associated with decreases in the MCHC
(P=0.002), ferritin concentration (P=0.007) and hepcidin
concentration (P=0.009), and an increase in the erythro-
poietin concentration (P=0.06; Table 3), suggesting that
the anemia may have been predominantly related to iron
deficiency.
The prevalence of anemia was 15% in the VHL
R200W
het-
erozygotes and 34% in the controls (P=0.061 by Pearson’s
χ
2
test). In a logistic regression analysis that adjusted for
Table 1. Clinical characteristics of study participants according to VHL
genotype.*
VHL
R200W
Controls with P
heterozygotes normal VHL
alleles
N = 34 N = 44
Age (years) 53 (12) 53 (12) 0.9
Female gender 19 (56%) 29 (66%) 0.4
History of smoking 6 (18%)
1
5 (11%) 0.4
History of alcohol consumption 8 (24%)
1
9 (20%) 0.7
History of bleeding in the past year 3 (9%)
1
3 (7%) 0.7
History of thrombosis 4 (12%)
1
5 (11%) 0.9
BMI (kg/m
2
) 26.1 (4.3)
2
25.7 (5.2) 0.4
Systolic blood pressure (mm Hg) 136 (28)
1
134 (24) 0.9
Diastolic blood pressure (mm Hg) 87 (14)
1
85 (11) 0.8
Mean blood pressure (mm Hg) 103 (18)
1
101 (15) 0.8
Hemoglobin (g/L) 134 (14) 128 (16) 0.10
Mean corpuscular volume (fL) 88 (4.5) 90 (6.1) 0.026
White blood cells (¥10
9
/L ) 7.6 (1.7) 6.9 (1.5) 0.036
Platelets (¥10
9
/L) 238 (69.5) 258 (56.0) 0.09
Ferritin (mg/L)** 62 (24-159) 56 (21-147) 0.6
*Results in mean (SD) or n. (%). Comparison by Student’s t-test or Pearson’s χ
2
test.
**Geometric mean and SD range.
1
n=33;
2
n=32
G
.Y. Miasnikova
et al.
1372
Heterozygosity for VHL
R200W
and anemia
haematologica | 2011; 96(9)
1373
age, gender, and an interaction between age and gender,
the estimated odds of anemia were 5.6-fold lower in the
VHL
R200W
heterozygotes than the controls (95% confidence
interval 1.4-22.7; P=0.017). Among the 5 VHL
R200W
het-
erozygotes with anemia, one 39-year old woman had a
serum ferritin concentration of 6 mg/L and hepcidin of 2.5
ng/mL, indicating iron deficiency, and 3 women and one
man had unexplained anemia. Among the 15 controls
with anemia, six women 53 years of age or under had
serum ferritin concentration less than 20 mg/L and hep-
cidin less than 21 ng/mL indicating iron deficiency. Two
men in the control group had changes consistent with
alcohol effect (macrocytosis and a history of alcohol con-
sumption), one man and 4 women under 60 years of age
had unexplained anemia, and one man and 2 women over
65 years of age had otherwise unexplained anemia (com-
monly referred to as ‘unexplained anemia of the elderly’).
Thus, this study suggests that, in contrast to the deleteri-
ous effects of homozygosity for VHL
R200W
in the form of
Chuvash polycythemia, heterozygosity for VHL
R200W
may
lead to protection from anemia. Based on our data, this
protective mechanism may apply to iron deficiency ane-
mia and to anemia in general; whether it is pertinent to
anemia of inflammation or anemia of the elderly in partic-
ular cannot be answered in this relatively small study.
However, these questions could be addressed in a larger
study in humans and in experiments utilizing a mouse
model of Chuvash polycythemia.
7
It seems possible that the observed lower risk for ane-
mia in VHL
R200W
heterozygotes may be due to a mild
increase in HIF activity in normoxia. HIF transcription fac-
tors are known to up-regulate erythropoietin, plasmino-
gen activator inhibitor-1, transferrin receptor, and vascular
endothelial growth factor and down-regulate hepcidin.
19,20
Concentrations of these products as adjusted for signifi-
cant covariates are presented in Table 3. There were high-
er transferrin receptor concentrations (P=0.026), and a
trend to higher vascular endothelial growth factor (P=0.14)
and erythropoietin (P=0.18) concentrations among the
VHL
R
200W
heterozygotes. Hepcidin concentrations did not
differ by VHL genotype. Although there were no signifi-
cant differences in the circulating erythropoietin concen-
trations between VHL
R200W
heterozygotes and VHL wild-
type controls in this study, there is evidence for direct
stimulation of erythropoiesis by HIFs independent of ery-
thropoietin concentration, as VHL
R200W
homozygote ery-
throid progenitors have a heightened response to erythro-
poietin.
3,21
Evidence from a mouse model suggests that, in
addition to promoting erythropoietin and transferrin
receptor expression and down-regulating hepcidin expres-
sion, HIF likely also stimulates erythropoiesis by an iron-
dependent mechanism yet to be defined.
22
A limitation to this study is the small sample size. To
confirm the present findings and identify other possibly
subtle benefits of the heterozygous state for an autoso-
mal recessive disease, study of a larger cohort would be
required. Another potential drawback is that the overall
prevalence of anemia in this study seems high.
Information on the hematologic status of adults in Russia
in general or Chuvashia in particular is scarce. In our pre-
viously published study from Chuvashia,
8
the prevalence
of anemia among adult controls from the community
was similar to the present study (24% overall, 34% in
women, 14% in men) (V. Gordeuk, unpublished observa-
tions, 2011).
In conclusion, a level of protection from anemia might
explain a heterozygote advantage for the VHL
R200W
allele,
despite the high mortality of the homozygotes.
Furthermore, the data presented here suggest that mild
increases of HIF-1 and HIF-2 signaling in normoxia do not
compromise erythropoietic activity under normal physio-
logical conditions to a degree that would noticeably
change the hemoglobin concentration in healthy Chuvash
heterozygotes. However, when a pathological insult
increases demand on erythropoiesis, augmented signaling
of HIFs in heterozygotes may increase erythropoiesis to a
discernible degree, resulting in either prevention or
improvement of anemia. Thus, the cumulative effect of
VHL
R200W
heterozygosity may provide a degree of protec-
Table 2. Clinical characteristics of study participants according to the
presence or absence of anemia (hemoglobin <130 g/dL men, <120
g/dL women).*
A
nemia No anemia P
N
= 20 N = 57
Age (years) 53 (12) 53 (12) 0.8
Female sex 15 (75%) 32 (56%) 0.14
VHL
R200W
heterozygote 5 (25%) 28 (49%) 0.061
H
istory of alcohol consumption 4 (20%) 13 (23%) 0.8
History of bleeding in the past year 3 (15%) 3 (5%) 0.16
BMI (kg/m
2
) 24.2 (3.3) 26.5 (5.1) 0.040
WBC (¥10
9
/L) 6.7 (1.2) 7.4 (1.8) 0.07
Hemoglobin (g/L) 112 (10) 138 (11) ---
MCV (fL) 89 (7.3) 89 (4.9) 0.4
MCHC (g/dL) 32.1 (1.3) 33.2 (1.9) 0.002
RDW 14.0 (3.1) 13.8 (3.9) 0.9
Platelets (¥10
9
/L) 270 (77) 242 (56) 0.18
Ferritin (mg/L )** 33 (12-92) 71 (30-167)
1
0.007
Transferrin receptor (mMol/L)** 18.4 (12.3-2.0)
2
20.0 (15.8-25.4)
3
0.06
Erythropoietin (IU/L)** 12 (7-19) 9 (6-15) 0.06
Hepcidin (ng/mL)** 25 (9-78) 58 (22-153) 0.009
*Results in mean (SD) or n. (%). Comparison by the Student’s t-test or Pearson’s χ
2
test.
*
*Geometric mean and SD range.
1
n
=56;
2
n
=16;
3
n
=49.
Table 3. Products of HIF-regulated genes in study participants accord-
ing to VHL genotype.*
VHL
R200W
VHL wildtype P
heterozygotes controls
N = 34 N = 44
Erythropoietin (IU/L)** 11 (10-12) 10 (9-10) 0.18
Hepcidin (ng/mL)*** 46 (41-53) 48 (42-55) 0.8
Plasminogen activator 3.1 (21-4.5)
1
2.0 (1.4-2.9)
1
0.41
inhibitor-1 (ng/mL)
Transferrin receptor 20.2 (19.5-20.9)
1
18.1 (17.5-18.7)
1
0.026
(nMol/L)****
Vascular endothelial 17.5 (14.9-205)
1
12.3 (10.4-14.6)
1
0.14
growth factor (ng/mL)
*
Geometric mean (SE range) from ANOVA. **Adjusted for anemia. Two outliers exclud-
ed. ***Adjusted for ferritin and anemia. ****Adjusted for ferritin and anemia. Four out-
l
iers excluded.
1
n
=33
tion from anemia that might afford an advantage for main-
taining or even increasing the frequency of this genetic
polymorphism in humans. Furthermore, our findings sug-
gest that raising levels of HIF-α by pharmacological inhibi-
t
ion of PHDs in humans might be a safe means to increase
erythropoietin levels for the correction and prevention of
anemia and/or to produce other potentially beneficial
aspects of the hypoxic response.
15
A
uthorship and Disclosures
The information provided by the authors about contributions from
persons listed as authors and in acknowledgments is available with
t
he full text of this paper at www.haematologica.org.
Financial and other disclosures provided by the authors using the
ICMJE (www.icmje.org) Uniform Format for Disclosure of
Competing Interests are also available at www.haematologica.org.
G
.Y. Miasnikova
et al.
1374
haematologica | 2011; 96(9)
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