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Prepublished online August 29, 2011; doi:10.1182/blood-2011-03-345512
Chuvash polycythemia VHLR200W mutation is associated with downregulation of hepcidin expression Victor R. Gordeuk, Galina Y. Miasnikova, Adelina I. Sergueeva, Xiaomei Niu, Mehdi Nouraie, Daniel J. Okhotin, Lydia A. Polyakova, Tatiana Ammosova, Sergei Nekhai, Tomas Ganz and Josef T. Prchal
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Blood First Edition Paper, prepublished online August 29, 2011; DOI 10.1182/blood-2011-03-345512 From bloodjournal.hematologylibrary.org by guest on November 8, 2012. For personal use only.
Chuvash polycythemia VHLR200W mutation is associated with down-regulation of hepcidin expression
Victor R. Gordeuk1, Galina Y. Miasnikova2, Adelina I. Sergueeva3, Xiaomei Niu1, Mehdi Nouraie1, Daniel J. Okhotin4, Lydia A. Polyakova2, Tatiana Ammosova1, Sergei Nekhai1, Tomas Ganz5, Josef T. Prchal6
1
Center for Sickle Cell Disease and Department of Medicine, Howard University, Washington,
DC 20060 USA 2
Chuvash Republic Clinical Hospital No. 1, Cheboksary 428022 Russia
3
Cheboksary Children’s Hospital, Cheboksary 428022 Russia
4
Russian Research Services, Camas, WA 98607 USA
5
Dept. of Medicine, David Geffen School of Medicine at UCLA, Los Angeles
6
Division of Hematology, University of Utah and VAH, Salt Lake City, UT
Correspondence to: Victor R. Gordeuk, M.D., Center for Sickle Cell Disease, Howard University, 2041 Georgia Ave. NW, Washington, DC 20060 USA Phone 202-865-1941; Fax 202-232-6719;
[email protected]
1 Copyright © 2011 American Society of Hematology
From bloodjournal.hematologylibrary.org by guest on November 8, 2012. For personal use only.
Abstract Hypoxia is known to reduce the expression of hepcidin, the master regulator of iron metabolism. However, it is not clear whether this response is primarily related to increased erythropoiesis driven by hypoxically stimulated erythropoietin or to a more direct effect of hypoxia on hepcidin expression. The germ-line loss-of-function VHLR200W mutation is common in Chuvashia, Russia and also occurs elsewhere. VHLR200W homozygotes have elevated hypoxia inducible factor (HIF)-1α and HIF-2α levels, increased red cell mass, propensity to thrombosis and early mortality. Ninety VHLR200W homozygotes and 52 controls with normal VHL alleles from Chuvashia, Russia were studied under basal circumstances. In univariate analyses, serum hepcidin concentration correlated positively with serum ferritin concentration and negatively with homozygosity for VHLR200W. After adjustment for serum erythropoietin and ferritin concentrations by multiple linear regression, the geometric mean (95% confidence interval of mean) hepcidin concentration was 8.1 (6.3-10.5) ng/ml in VHLR200W homozygotes versus 26.9 (18.6-38.0) ng/ml in controls (P 20 years of age, were recruited from the community, and were in their usual state of health. The study participants were characterized by medical history, physical examination including blood pressure and body weight and laboratory tests of the peripheral blood. Serum samples were collected from 2004 to 2008 and they were stored at -70 degrees C. Collection and storage of samples from Chuvash polycythemia patients and controls was identical. Samples were transported in dry shippers with liquid nitrogen or on dry ice. They were analyzed for hepcidin in 2010, and had undergone two or less freeze-thaw cycles. About half of the patients with Chuvash polycythemia had been treated with phlebotomy within the year before the date of the study. The other half had never undergone phlebotomy or had received phlebotomy more than one year before the date of the study.
Laboratory procedures. The 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). Serum concentration of erythropoietin was determined by enzyme linked immunosorbent assay (ELISA) (R& D Systems, Minneapolis, MN). Serum hepcidin was measured by competitive ELISA as described previously.34 Serum albumin, total protein, and iron concentrations and total iron binding capacity were determined by Quest Diagnostics (Auburn Hills, MI) using spectrophotometric methodology. The globulin fraction, the albumin to globulin ratio and the transferrin saturation were calculated.
5
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Statistics. The primary study comparison was between VHLR200W homozygotes and genotypically normal subjects with regard to serum hepcidin concentration using multiple linear regression. For hepcidin concentrations below the detection limit, we assigned a value of 2.6 ng/ml, which is half-way between the limit of detection of 5.2 ng/ml and 0. Skewed continuous variables were log-transformed to approximate a normal distribution. Generalized linear models were also applied to validate the linear regression. The clinical characteristics of the VHLR200W homozygotes and controls were assessed with the student t-test or Pearson chi square. Bivariate relationships of various measurements with hepcidin were performed with Spearman correlation. Analyses were performed by Stat 10.0 (StataCorp, College Station, TX).
Results Clinical characteristics according VHL genotype. The clinical characteristics of the study participants are summarized in Table 1. Mean ages were 43 years in 90 VHLR200W homozygotes compared to 49 years in 52 controls (P = 0.008). Females made up slightly more than half of both cohorts. Histories of alcohol consumption, substantial bleeding in the past year, thrombosis and systemic hypertension were not different in VHLR200W homozygotes compared to controls. Body mass index was lower in VHLR200W homozygotes than controls (P = 0.004) and history of smoking was higher (P = 0.024).
Complete blood count, iron measures, erythropoietin and hepcidin concentrations according to VHL genotype (Table 2). After adjustment for gender by multiple linear regression, the geometric mean (95 % CI of mean) hemoglobin concentration was 17.3 (16.8 17.8) g/L in the VHLR200W homozygotes and 12.8 (12.3 - 13.3) g/L in controls without mutated VHL alleles (P
