AbstractThe germ-line loss-of-function VHLR200W mutation is common in Chuvashia, Russia and occurs in other parts of the world. VHLR200W 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 VHLR200W 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 VHLR200W 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 VHLR200W heterozygotes compared to controls (95% confidence interval 1.4–22.7; P=0.017). In conclusion, heterozygosity for VHLR200W may provide protection from anemia; such protection could explain the persistence of this mutation.
The R200W mutation of the von Hippel Lindau gene (VHL) is present on the same haplotype in almost all persons of heterogeneous racial and ethnic backgrounds, indicating that the mutation may have originated in a founder prior to divergence of human races.1 There is only one reported exception to this genetic linkage.2 Homozygosity for VHLR200W is responsible for Chuvash polycythemia, the first recognized congenital disorder of augmented hypoxia sensing.3 Chuvash polycythemia is common in the Chuvash Republic of the Russian Federation4 and on the Italian island of Ischia;5 the condition also occurs in other parts of the world.2,6 Chuvash polycythemia is characterized by increased levels of HIF-1α and HIF-2α under ambient oxygen conditions3,7 and upregulation of a number of the target genes of HIF.3,8,9 Clinical manifestations include lower systemic blood pressure, higher pulmonary artery pressure and other changes in pulmonary vascular 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 VHLR200W homozygotes, the mutation should be associated with some type of heterozygote advantage, albeit slight. However, the effect of heterozygosity for VHLR200W is not known. Only rare cases of polycythemia associated with VHLR200W heterozygosity have been reported.2,6,14 In one study that included 9 Chuvash VHLR200W heterozygotes and 77 Chuvash participants with normal VHL alleles, the VHLR200W heterozygotes had significantly lower systemic blood pressures and higher serum PAI-1 concentrations. In addition, although no VHLR200W heterozygote was polycythemic, the mean hemoglobin concentration was higher by 4 g/L but this difference was not statistically significant.8
The present study was conducted to prospectively determine if heterozygotes for VHLR200W have discernable physiological 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 VHLR200W heterozygotes from eclampsia, a major cause of maternal morbidity 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 VHLR200W heterozygosity on anemia in an otherwise unselected Chuvash sample. We postulated that the identification of any VHLR200W heterozygote advantage could point to potential benefits of chronic augmentation of HIFs by pharmacological agents, such as inhibitors of PHDs.15,16
Design and Methods
The study objective was to compare clinical and molecular characteristics of a group of VHLR200W 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.
The Howard University Institutional Review Board approved the research and all participants provided written informed consent. 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. VHLR200W heterozygotes over 35 years of age were identified by studying first-degree family members of patients with Chuvash polycythemia. 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.
Complete blood count was performed by an automated analyzer (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 erythropoietin 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 VHLR200W was performed by PCR as previously described.8
The primary study comparison was between VHLR200W heterozygotes and genotypically normal subjects. Analysis of continuous variables was made by the Student’s t-test (after normal transformation) or by analysis of variance with adjustment for significant covariates. Analysis of categorical variables was by Pearson’s χ2 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, collecting, managing and interpreting the data, and writing the manuscript.
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 VHLR200W heterozygotes (P=0.033) and the white blood cell counts were higher (P=0.036). The serum ferritin concentrations were similar in VHLR200W heterozygotes and controls. The mean (standard deviation [SD]) hemoglobin concentration was 134 (14) g/L in the VHLR200W 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 VHLR200W heterozygotes and controls at a significance 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 anemia 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 erythropoietin 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 VHLR200W heterozygotes and 34% in the controls (P=0.061 by Pearson’s χ test). In a logistic regression analysis that adjusted for age, gender, and an interaction between age and gender, the estimated odds of anemia were 5.6-fold lower in the VHLR200W heterozygotes than the controls (95% confidence interval 1.4–22.7; P=0.017). Among the 5 VHLR200W heterozygotes with anemia, one 39-year old woman had a serum ferritin concentration of 6 μg/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 μg/L and hepcidin 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 consumption), 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 (commonly referred to as ‘unexplained anemia of the elderly’). Thus, this study suggests that, in contrast to the deleterious effects of homozygosity for VHLR200W in the form of Chuvash polycythemia, heterozygosity for VHLR200W may lead to protection from anemia. Based on our data, this protective mechanism may apply to iron deficiency anemia and to anemia in general; whether it is pertinent to anemia of inflammation or anemia of the elderly in particular 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 anemia in VHLR200W heterozygotes may be due to a mild increase in HIF activity in normoxia. HIF transcription factors are known to up-regulate erythropoietin, plasminogen activator inhibitor-1, transferrin receptor, and vascular endothelial growth factor and down-regulate hepcidin.19,20 Concentrations of these products as adjusted for significant covariates are presented in Table 3. There were higher 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 VHLR200W heterozygotes. Hepcidin concentrations did not differ by VHL genotype. Although there were no significant differences in the circulating erythropoietin concentrations between VHLR200W heterozygotes and VHL wild-type controls in this study, there is evidence for direct stimulation of erythropoiesis by HIFs independent of erythropoietin concentration, as VHLR200W homozygote erythroid progenitors have a heightened response to erythropoietin.3,21 Evidence from a mouse model suggests that, in addition to promoting erythropoietin and transferrin receptor expression and down-regulating hepcidin expression, 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 autosomal 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 previously 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 observations, 2011).
In conclusion, a level of protection from anemia might explain a heterozygote advantage for the VHLR200W 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 physiological 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 VHLR200W heterozygosity may provide a degree of protection from anemia that might afford an advantage for maintaining or even increasing the frequency of this genetic polymorphism in humans. Furthermore, our findings suggest that raising levels of HIF-α by pharmacological inhibition 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
- 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).
- Authorship and Disclosures The information provided by the authors about contributions from persons listed as authors and in acknowledgments is available with the 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.
- Received April 7, 2011.
- Revision received May 11, 2011.
- Accepted May 16, 2011.
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