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1.  The Hypoxic Response Contributes to Altered Gene Expression and Pre-Capillary Pulmonary Hypertension in Patients with Sickle Cell Disease 
Circulation  2014;129(16):1650-1658.
We postulated that the hypoxic response in sickle cell disease (SCD) contributes to altered gene expression and pulmonary hypertension, a complication associated with early mortality.
Methods and Results
To identify genes regulated by the hypoxic response and not other effects of chronic anemia, we compared expression variation in peripheral blood mononuclear cells from 13 SCD subjects with hemoglobin SS genotype and 15 Chuvash polycythemia subjects (VHLR200W homozygotes with constitutive up-regulation of hypoxia inducible factors in the absence of anemia or hypoxia). At 5% false discovery rate, 1040 genes exhibited >1.15 fold change in both conditions; 297 were up-regulated and 743 down-regulated including MAPK8 encoding a mitogen-activated protein kinase important for apoptosis, T-cell differentiation and inflammatory responses. Association mapping with a focus on local regulatory polymorphisms in 61 SCD patients identified expression quantitative trait loci (eQTL) for 103 of these hypoxia response genes. In a University of Illinois SCD cohort the A allele of a MAPK8 eQTL, rs10857560, was associated with pre-capillary pulmonary hypertension defined as mean pulmonary artery pressure ≥25 and pulmonary capillary wedge pressure ≤15 mm Hg at right heart catheterization (allele frequency=0.66; OR=13.8, P=0.00036, n=238). This association was confirmed in an independent Walk-PHaSST cohort (allele frequency=0.65; OR=11.3, P=0.0025, n=519). The homozygous AA genotype of rs10857560 was associated with decreased MAPK8 expression and present in all 14 identified pre-capillary pulmonary hypertension cases among the combined 757 patients.
Our study demonstrates a prominent hypoxic transcription component in SCD and a MAPK8 eQTL associated with pre-capillary pulmonary hypertension.
PMCID: PMC4287376  PMID: 24515990
sickle cell disease; MAPK8; hypoxic response; expression quantitative trait loci; association mapping; pre-capillary pulmonary hypertension
2.  Iron deficiency modifies gene expression variation induced by augmented hypoxia sensing 
Blood cells, molecules & diseases  2013;52(1):10.1016/j.bcmd.2013.07.016.
In congenital Chuvash polycythemia (CP), VHLR200W homozygosity leads to elevated hypoxia inducible factor (HIF) levels at normoxia. CP is often treated by phlebotomy resulting in iron deficiency, permitting us to examine the separate and synergistic effects of iron deficiency and HIF signaling on gene expression. We compared peripheral blood mononuclear cell gene expression profiles of eight VHLR200W homozygotes with 17 wildtype individuals with normal iron status and found 812 up-regulated and 2120 down-regulated genes at false discovery rate 0.05. Among differential genes we identified three major gene regulation modules involving induction of innate immune responses, alteration of carbohydrate and lipid metabolism, and down-regulation of cell proliferation, stress-induced apoptosis and T-cell activation. These observations suggest molecular mechanisms for previous observations in CP of lower blood sugar without increased insulin and low oncogenic potential. Studies including 16 additional VHLR200W homozygotes with low ferritin indicated that iron deficiency enhanced the induction effect of VHLR200W for 50 genes including hemoglobin synthesis loci but suppressed the effect for 107 genes enriched for HIF-2 targets. This pattern is consistent with potentiation of HIF-1α protein stability by iron deficiency but a trend for down-regulation of HIF-2α translation by iron deficiency overriding an increase in HIF-2α protein stability.
PMCID: PMC3852195  PMID: 23993337
3.  Novel Compound VHL Heterozygosity (VHL T124A/L188V) Associated With Congenital Polycythaemia 
British journal of haematology  2013;162(6):851-853.
PMCID: PMC3759558  PMID: 23772956
polycythaemia; erythrocytosis; mutations; molecular haematology; genetics
4.  Differential Sensitivity to JAK Inhibitory Drugs by Isogenic Human Erythroblasts and Hematopoietic Progenitors Generated from Patient-Specific Induced Pluripotent Stem Cells 
Stem cells (Dayton, Ohio)  2014;32(1):269-278.
Disease-specific induced pluripotent stem cells (iPSCs) provide an unprecedented opportunity to establish novel disease models and accelerate drug development using distinct tissue target cells generated from isogenic iPSC lines with and without disease-causing mutations. To realize the potential of iPSCs in modeling acquired diseases which are usually heterogeneous, we have generated multiple iPSC lines including two lines that are JAK2-wild-type and four lines homozygous for JAK2-V617F somatic mutation from a single polycythemia vera (PV) patient blood. In vitro differentiation of the same patient-derived iPSC lines have demonstrated the differential contributions of their parental hematopoietic clones to the abnormal erythropoiesis including the formation of endogenous erythroid colonies. This iPSC approach thus may provide unique and valuable insights into the genetic events responsible for disease development. To examine the potential of iPSCs in drug testing, we generated isogenic hematopoietic progenitors and erythroblasts from the same iPSC lines derived from PV patients and normal donors. Their response to three clinical JAK inhibitors, INCB018424 (Ruxolitinib), TG101348 (SAR302503), and the more recent CYT387 was evaluated. All three drugs similarly inhibited erythropoiesis from normal and PV iPSC lines containing the wild-type JAK2 genotype, as well as those containing a homozygous or heterozygous JAK2-V617F activating mutation that showed increased erythropoiesis without a JAK inhibitor. However, the JAK inhibitors had less inhibitory effect on the self-renewal of CD341 hematopoietic progenitors. The iPSC-mediated disease modeling thus underlies the ineffectiveness of the current JAK inhibitors and provides a modeling system to develop better targeted therapies for the JAK2 mutated hematopoiesis.
PMCID: PMC4096297  PMID: 24105986
Induced pluripotent stem cells; Hematopoietic progenitor cells; Erythropoiesis; Preclinical drug evaluation; Hematopoietic malignancies
5.  New Syndrome of Paraganglioma and Somatostatinoma Associated With Polycythemia 
Journal of Clinical Oncology  2013;31(13):1690-1698.
The occurrence of ≥ two distinct types of tumors, one of them paraganglioma (PGL), is unusual in an individual patient, except in hereditary cancer syndromes.
Patients and Methods
Four unrelated patients were investigated, with thorough clinical evaluation. Plasma and tissue catecholamines and metanephrines were measured by high-performance liquid chromatography. Anatomic and functional imaging were performed for tumor visualization. Germline and tumor tissue DNA were analyzed for hypoxia-inducible factor 2 alpha (HIF2A) mutations. The prolyl hydroxylation and stability of the mutant HIF2α protein, transcriptional activity of mutant HIF2A, and expression of hypoxia-related genes were also investigated. Immunohistochemical staining for HIF1/2α was performed on formalin-fixed, paraffin-embedded tumor tissue.
Patients were found to have polycythemia, multiple PGLs, and duodenal somatostatinomas by imaging or biochemistry with somatic gain-of-function HIF2A mutations. Each patient carried an identical unique mutation in both types of tumors but not in germline DNA. The HIF2A mutations in these patients were clustered adjacent to an oxygen-sensing proline residue, affecting HIF2α interaction with the prolyl hydroxylase domain 2–containing protein, decreasing the hydroxylation of HIF2α, and reducing HIF2α affinity for the von Hippel–Lindau protein and its degradation. An increase in the half-life of HIF2α was associated with upregulation of the hypoxia-related genes EPO, VEGFA, GLUT1, and END1 in tumors.
Our findings indicate the existence of a new syndrome with multiple PGLs and somatostatinomas associated with polycythemia. This new syndrome results from somatic gain-of-function HIF2A mutations, which cause an upregulation of hypoxia-related genes, including EPO and genes important in cancer biology.
PMCID: PMC3807138  PMID: 23509317
6.  A Novel EPAS1/HIF2A germline mutation in a Congenital Polycythemia with Paraganglioma 
Congenital polycythemias have diverse etiologies, including mutations in the hypoxia sensing pathway. These include HIF2A at exon 12, VHL gene (Chuvash polycythemia), and PHD2 mutations, which in one family was also associated with recurrent pheochromocytoma/paraganglioma (PHEO/PGL). Over the past two decades, we have studied seven unrelated patients with sporadic congenital polycythemia who subsequently developed PHEO/PGL with, until now, no discernible molecular basis. We now report a polycythemic patient with a novel germline HIF2AF374Y (exon 9) mutation, inherited from his mother, who developed PHEO/PGL.
We show that this is a gain-of-function mutation and demonstrate no loss-of-heterozygosity or additional somatic mutation of HIF2A in the tumor, indicating HIF2AF374Y may be predisposing rather than causative of PHEO/PGL. This report, in view of 2 other concomitantly reported PHEO/PGL patients with somatic mutations of HIF2A and polycythemia, underscores the PHEO/PGL promoting potential of mutations of HIF2A that alone are not sufficient for PHEO/PGL development.
PMCID: PMC3570726  PMID: 23090011
HIF2A; Paraganglioma; Familial erythrocytosis
7.  Concomitant JAK2 V617F-positive Polycythemia Vera and B-Cell Chronic Lymphocytic Leukemia in Three Patients Originating from Two Separate Hematopoietic Stem Cells 
American journal of hematology  2012;88(2):157-158.
The simultaneous occurrence of polycythemia vera (PV) and chronic lymphocytic leukemia (CLL) is a rare event that offers a possibility to study their common origin. PV originates from self-renewing hematopoietic stem cells (HSC) with both lymphoid and myeloid potential(1–3). It has been reported that CLL also originates from self-renewing HSC with a potential for both lymphoid and myeloid differentiation(4, 5). We report 3 females with concomitant CLL and PV whose X-chromosome inactivation patterns of the neoplastic cells revealed that granulocytes/platelets and B-lymphocytes used different X-chromosome alleles. These data indicate that both PV and CLL have arisen independently and from different HSC.
PMCID: PMC3556177  PMID: 23280542
PV; CLL; JAK2 V617F; clonality
8.  An Unconventional Antigen Translated by a Novel Internal Ribosome Entry Site Elicits Antitumor Humoral Immune Reactions1 
Self-tumor Ags that elicit antitumor immune responses in responses to IFN-α stimulation remain poorly defined. We screened a human testis cDNA library with sera from three polycythemia vera patients who responded to IFN-α and identified a novel Ag, MPD6. MPD6 belongs to the group of cryptic Ags without conventional genomic structure and is encoded by a cryptic open reading frame located in the 3′-untranslated region of myotrophin mRNA. MPD6 elicits IgG Ab responses in a subset of polycythemia vera patients, as well as patients with chronic myelogenous leukemia and prostate cancer, suggesting that it is broadly immunogenic. The expression of myotrophin-MPD6 transcripts was upregulated in some tumor cells, but only slightly increased in K562 cells in response to IFN-α treatment. By using bicistronic reporter constructs, we showed that the translation of MPD6 was mediated by a novel internal ribosome entry site (IRES) upstream of the MPD6 reading frame. Furthermore, the MPD6-IRES-mediated translation, but not myotrophin-MPD6 transcription, was significantly upregulated in response to IFN-α stimulation. These findings demonstrate that a novel IRES-mediated mechanism may be responsible for the translation of unconventional self-Ag MPD6 in responsive to IFN-α stimulation. The eliciting antitumor immune response against unconventional Ag MPD6 in patients with myeloproliferative diseases suggests MPD6 as a potential target of novel immunotherapy.
PMCID: PMC3902139  PMID: 16982933
9.  Decreased serum glucose and glycosylated hemoglobin levels in patients with Chuvash polycythemia: a role for HIF in glucose metabolism 
In Chuvash polycythemia, a homozygous 598C>T mutation in the von Hippel-Lindau gene (VHL) leads to an R200W substitution in VHL protein, impaired degradation of α-subunits of hypoxia inducible factor (HIF)-1 and HIF-2, and augmented hypoxic responses during normoxia. Chronic hypoxia of high altitude is associated with decreased serum glucose and insulin concentrations. Other investigators reported that HIF-1 promotes cellular glucose uptake by increased expression of GLUT1 and increased glycolysis by increased expression of enzymes such as PDK. On the other hand, inactivation of Vhl in murine liver leads to hypoglycemia associated with a HIF-2-related decrease in the expression of the gluconeogenic enzymes genes Pepck, G6pc, and Glut2. We therefore hypothesized that glucose concentrations are decreased in individuals with Chuvash polycythemia. We found that 88 Chuvash VHLR200W homozygotes had lower random glucose and glycosylated hemoglobin A1c levels than 52 Chuvash subjects with wildtype VHL alleles. Serum metabolomics revealed higher glycerol and citrate levels in the VHLR200W homozygotes. We expanded these observations in VHLR200W homozygote mice and found that they had lower fasting glucose values and lower glucose excursions than wild-type control mice but no change in fasting insulin concentrations. Hepatic expression of Glut2 and G6pc but not Pdk2 was decreased and skeletal muscle expression of Glut1, Pdk1 and Pdk4 was increased. These results suggest that both decreased hepatic gluconeogenesis and increased skeletal uptake and glycolysis contribute to the decreased glucose concentrations. Further study is needed to determine whether pharmacologically manipulating HIF expression might be beneficial for treatment of diabetic patients.
PMCID: PMC3537876  PMID: 23015148
VHL; hypoxia inducible factors; glucose; insulin; glycolysis; gluconeogenesis
10.  Molecular basis of polycythemic disorders due to aberrant hypoxia sensing and its relevance to acute leukemia 
The author of this lecture has been especially honored to be selected to deliver the Ernest Beutler Memorial Lecture at the Acute Leukemia Forum 2012 and to write this overview. Ernest Beutler was the pivotal influence in my introduction to academic life, and his contribution to hematology in the last 5 decades was unsurpassed. Taking a cue from Ernie’s example, I have elected in the keynote speech and this brief treatise, to start with an unconventional introduction and to expand on some discoveries made in my laboratory. Then I will extend these findings to the focus of the Acute Leukemia Forum to address potentially new approaches to therapies of acute leukemias. Somatic and germline mutations of acute leukemias are unfortunately caused by arrays of somatic and germline mutations. Simultaneous targeting of so many mutations makes it not possible to efficiently target all for cure. Albeit we should be aware that we should not in the near future ignore targeted therapy of those functionally important genetic and epigenetic events that are either initiating or contributing to aggressivity of acute leukemia, as these may be ameliorated by targeted intervention against one, or even a few together, of these defined molecular lesions. Yet, leukemic cells, like other cancer cells, have the unique metabolic feature to generate energy, referred as the Warburg effect, which can potentially be targeted to suppress or even eradicate cancer.
PMCID: PMC3513693  PMID: 23200548
Acute Leukemia Forum; Beutler; cancer cell metabolism; glycolysis; hypoxia-inducible factor; HIF; HIF-1; HIF-2; HIF-3; IDH1; IDH2; isocitrate dehydrogenase; leukemia; polycythemia; tumorigenesis; von Hippel-Lindau; Warburg
11.  Interim analysis of safety and efficacy of ruxolitinib in patients with myelofibrosis and low platelet counts 
Ruxolitinib, a Janus kinase 1 and 2 inhibitor, demonstrated improvements in spleen volume, symptoms, and survival over placebo and best available therapy in intermediate-2 or high-risk myelofibrosis patients with baseline platelet counts ≥100 × 109/L in phase III studies. The most common adverse events were dose-dependent anemia and thrombocytopenia, which were anticipated because thrombopoietin and erythropoietin signal through JAK2. These events were manageable, rarely leading to treatment discontinuation. Because approximately one-quarter of MF patients have platelet counts <100 × 109/L consequent to their disease, ruxolitinib was evaluated in this subset of patients using lower initial doses. Interim results of a phase II study of ruxolitinib in myelofibrosis patients with baseline platelet counts of 50-100 × 109/L are reported.
Ruxolitinib was initiated at a dose of 5 mg twice daily (BID), and doses could be increased by 5 mg once daily every 4 weeks to 10 mg BID if platelet counts remained adequate. Additional dosage increases required evidence of suboptimal efficacy. Assessments included measurement of spleen volume by MRI, MF symptoms by MF Symptom Assessment Form v2.0 Total Symptom Score [TSS]), Patient Global Impression of Change (PGIC); EORTC QLQ-C30, and safety/tolerability.
By week 24, 62% of patients achieved stable doses ≥10 mg BID. Median reductions in spleen volume and TSS were 24.2% and 43.8%, respectively. Thrombocytopenia necessitating dose reductions and dose interruptions occurred in 12 and 8 patients, respectively, and occurred mainly in patients with baseline platelet counts ≤75 × 109/L. Seven patients experienced platelet count increases ≥15 × 109/L. Mean hemoglobin levels remained stable over the treatment period. Two patients discontinued for adverse events: 1 for grade 4 retroperitoneal hemorrhage secondary to multiple and suspected pre-existing renal artery aneurysms and 1 for grade 4 thrombocytopenia.
Results suggest that a low starting dose of ruxolitinib with escalation to 10 mg BID may be appropriate in myelofibrosis patients with low platelet counts.
Trial registration NCT01348490.
PMCID: PMC4176265  PMID: 24283202
Janus kinase inhibitor; Myelofibrosis; Phase II; Platelet count; Ruxolitinib; Spleen volume; Total symptom score
12.  Could Hypoxia increase the prevalence of thrombotic complications in Polycythemia Vera? 
Thromboses represent a major cause of morbidity and mortality in Polycythemia Vera (PV) but the contributing mechanisms are not fully described.
Patients and methods
To evaluate whether environmental conditions such as altitude/hypoxia could impact thromboses history, we retrospectively analyzed thrombosis history in 71 PV patients living at an elevation of 5,000 feet or more in the SLC area (SLC) and 166 PV patients living near sea level in the Baltimore area (BLM). The SLC cohort was older with a longer disease duration. No significant differences in type of anticoagulation therapy or prothrombotic factors were present between the two cohorts. After adjusting for age, sex and disease duration, SLC patients experienced an estimated 3.9-fold increase in the odds of a history of thromboses compared to BLM patients (95% confidence interval 1.8-7.6; p = 0.0004). A history of cardiovascular event was present in 58% of the SLC patients compared to 27% of the BLM patients (p<0.0001). Before diagnosis thromboses occurred in 18% and 4% of the SLC and BLM groups respectively (p =0.003). No correlation between JAK2V617F allele burden and thrombosis was observed in this study.
This retrospective study suggests that even moderate hypoxia associated with 5,000 feet elevation should be considered as independent prothrombotic risk factor. This observation needs to be confirmed by prospective studies.
PMCID: PMC3796441  PMID: 23392352
13.  Metabolic insight into mechanisms of high-altitude adaptation in Tibetans 
Molecular genetics and metabolism  2012;106(2):244-247.
Recent studies have identified genes involved in high-altitude adaptation in Tibetans. Genetic variants/haplotypes within regions containing three of these genes (EPAS1, EGLN1, and PPARA) are associated with relatively decreased hemoglobin levels observed in Tibetans at high altitude, providing corroborative evidence for genetic adaptation to this extreme environment. The mechanisms that afford adaptation to high-altitude hypoxia, however, remain unclear. Considering the strong metabolic demands imposed by hypoxia, we hypothesized that a shift in fuel preference to glucose oxidation and glycolysis at the expense of fatty acid oxidation would improve adaptation to decreased oxygen availability. Correlations between serum free fatty acids and lactate concentrations in Tibetan groups living at high altitude and putatively selected haplotypes provide insight into this hypothesis. An EPAS1 haplotype that exhibits a signal of positive selection is significantly associated with increased lactate concentration, the product of anaerobic glycolysis. Furthermore, the putatively advantageous PPARA haplotype is correlated with serum free fatty acid levels, suggesting a possible decrease in the activity of fatty acid oxidation. Although further studies are required to assess the molecular mechanisms underlying these patterns, these associations suggest that genetic adaptation to high altitude involves alteration in energy utilization pathways.
PMCID: PMC3437309  PMID: 22503288
14.  Genetic Adaptation to Extreme Hypoxia. Study of High-Altitude Pulmonary Edema in a Three Generations of Chinese Han Family 
Blood cells, molecules & diseases  2009;43(3):221-225.
Organismal response to hypoxia is essential for critical regulation of erythropoiesis, other physiological functions and survival. There is an evidence of individual variation in response to hypoxia as some but not all of the affected individuals develop polycythemia, and or pulmonary and cerebral edema. A significant population difference in response to hypoxia exist as many Tibetans, Ethiopian and Andean natives developed an adaptive mechanisms to extreme hypoxia. A proportion of any non-adapted individuals exposed to high altitude develop pulmonary edema (HAPE), pulmonary hypertension, cerebral edema and extreme polycythemia. The isolation of causative gene(s) responsible for HAPE and other extreme hypoxia complications would provide a rational basis for specific targeted therapy of HAPE, allow its targeted prevention for at-risk populations, and clarification of its, and pathophysiology of other hypoxic maladaptations. As today, the only suggested linkage in unrelated individual with HAPE has been with endothelial nitric oxide synthase (eNOS) gene. Here we describe a family with multiple members affected with HAPE in three generations. Families with multiple affected members with HAPE have not been described. We first ruled out linkage of HAPE with eNOS gene. We then performed analysis of the whole genome using high-density SNP arrays (Affymetrix v5.0) and assuming a single gene causation of HAPE ruled out a linkage with 34 other candidate genes. Only HIF2A haplotype was shared by individuals who exhibit the HAPE phenotype, and the work on their possible causative role in HAPE is in progress. Clearly a small size of our family does not provide sufficient power for a conclusive analysis of linkage; we hope that collaboration with other investigators referring us more HAPE patients in effort to increase sample size would lead to identification of gene(s) responsible for HAPE and possibly other maladaptive hypoxic complications.
PMCID: PMC3506116  PMID: 19481479
Hypoxia; Erythropoietin; High Altitude Pulmonary Edema (HAPE); Endothelial Nitric Oxide (eNOS); Hypoxia Inducible Factor (HIF)
15.  Somatic HIF2α gain-of-function mutations in paraganglioma and somatostatinoma associated with polycythemia 
The New England journal of medicine  2012;367(10):922-930.
Hypoxia inducible factors (HIFs) are transcription factors controlling energy, iron metabolism, erythropoiesis, and development, and, when dysregulated, contribute to tumorigenesis, cancer progression, and invasion. However, HIFα mutations have not previously been identified in any cancer. Here we report two novel somatic gain-of-function HIF2α mutations in two patients, one presenting with a paraganglioma and a second with both paraganglioma and somatostatinoma. Both mutations were shown to confer increased HIF2α activity and protein half-life. While germline mutations of regulators of HIFα, including VHL and EGLN1, have been reported in pheochromocytomas/paragangliomas, this is the first report of a somatic gain-of-function mutation in HIF.
PMCID: PMC3432945  PMID: 22931260
HIF2α; paraganglioma; somatostatinoma; polycythemia
16.  Molecular Basis of Two Novel Mutations Found in Type I Methemoglobinemia 
Blood cells, molecules & diseases  2011;46(4):277-281.
Congenital methemoglobinemia due to NADH-cytochrome b5 reductase 3 (CYB5R3) deficiency is an autosomal recessive disorder that occurs sporadically worldwide, although endemic clusters of this disorder have been identified in certain ethnic groups. It is present as two distinct phenotypes, type I and type II. Type I methemoglobinemia is characterized by CYB5R3 enzyme deficiency restricted to erythrocytes and is associated with benign cyanosis. The less frequent type II methemoglobinemia is associated with generalized CYB5R3 deficiency affecting all cells and is lethal in early infancy. Here we describe the molecular basis of type I methemoglobinemia due to CYB5R3 deficiency in four patients from three distinct ethnic backgrounds, Asian Indian, Mexican and Greek.
The CYB5R3 gene of three probands with type I methemoglobinemia and their relatives were sequenced revealing several putative causative mutations; in one subject multiple mutations were present. Two novel mutations, S54R and F157C, were identified and the previously described A179T, V253M mutations were also identified. All these point mutations mapped to the NADH binding domain and or the FAD binding domain. Each has the potential to sterically hinder cofactor binding causing instability of the CYB5R3 protein. Wild type CYB5R3 as well as two of these novel mutations, S54R and F157C, were amplified, cloned, and purified recombinant peptide obtained. Kinetic and thermodynamic studies of these proteins show that the above mutations lead to decreased thermal stability.
PMCID: PMC3075332  PMID: 21349748
methemoglobinemia; NADH-cytochrome b5 reductase 3; gene mutation; cyanosis; protein structure
17.  Allogeneic haematopoietic cell transplantation for myelofibrosis in 30 patients 60–78 years of age 
British journal of haematology  2011;153(1):76-82.
We analysed the results of haematopoietic cell transplantation (HCT) in 30 patients aged 60–78 (median 65) years, with primary myelofibrosis or myelofibrosis evolving from antecedent polycythaemia vera or essential thrombocythaemia. Donors were human leucocyte antigen (HLA)-identical siblings (N = 15) or unrelated individuals (N = 15). Various conditioning regimens were used, ranging from very low intensity (fludarabine plus 2 Gy total body irradiation) to high dose (busulfan plus cyclophosphamide). Stem cell sources were granulocyte colony-stimulating factor mobilized peripheral blood progenitor cells in 29 patients and marrow in one patient. Sustained engraftment was documented in 27 of 30 patients. Day -100 mortality was 13%. With a median follow-up of 22 (range 0.5–69) months, 3-year overall survival and progression-free survival were 45% and 40%, respectively. Currently, 13 patients are surviving. Seven patients died with disease progression at 0.5–22 months, and 10 patients died from other causes at 1.5–37.5 months after HCT. While the selection of older patients for transplantation was probably biased, the present results are encouraging. Motivated older patients with myelofibrosis without substantial comorbid conditions should be offered the option of allogeneic HCT.
PMCID: PMC3236079  PMID: 21323890
myelofibrosis, older patients; haematopoietic cell transplantation
18.  Quantification of Fibrosis and Osteosclerosis in Myeloproliferative Neoplasms: A Computer-Assisted Image Study 
Leukemia research  2010;34(7):871-876.
Evaluation of bone marrow fibrosis and osteosclerosis in myeloproliferative neoplasms (MPN) is subject to interobserver inconsistency. Performance data for currently utilized fibrosis grading systems are lacking, and classification scales for osteosclerosis do not exist. Digital imaging can serve as a quantification method for fibrosis and osteosclerosis. We used digital imaging techniques for trabecular area assessment and reticulin-fiber quantification. Patients with all Philadelphia negative MPN subtypes had higher trabecular volume than controls (p ≤0.0015). Results suggest that the degree of osteosclerosis helps differentiate primary myelofibrosis from other MPN. Numerical quantification of fibrosis highly correlated with subjective scores, and interobserver correlation was satisfactory. Digital imaging provides accurate quantification for osteosclerosis and fibrosis.
PMCID: PMC2875327  PMID: 20122729
bone marrow fibrosis; digital imaging; myeloproliferative neoplasms; osteosclerosis
19.  Association of G6PD202A,376G with lower haemoglobin concentration but not increased haemolysis in patients with sickle cell anaemia 
British journal of haematology  2010;150(2):218-225.
The genetic bases of the highly variable degrees of anaemia and haemolysis in persons with Hb SS are not fully known, but several studies have indicated that G6PD deficiency is not a factor. The G6PD202A and G6PD376G alleles and α-thalassaemia were determined by molecular genetic testing in 261 children and adolescents with Hb SS in a multicentre study. G6PD202A,376G (G6PD A-) was defined as hemizygosity for both alleles in males and homozygosity in females. Among the participants 41% were receiving hydroxycarbamide.
The prevalence of G6PD202A,376G was 13.6% in males and 3.3% in females with an overall prevalence of 8.7%. G6PD202A,376G was associated with a 10 g/l decrease in haemoglobin concentration (P=0.008) but not with increased haemolysis as measured by lactate dehydrogenase, bilirubin, aspartate-aminotransferase, reticulocyte count or a haemolytic component derived from these markers (P>0.09). Similar results were found within a sub-group of children who were not receiving hydroxycarbamide. By comparison, single and double α-globin deletions were associated with progressively higher haemoglobin concentrations (P=0.005 for trend), progressively lower values for haemolytic component (P=0.007), and increased severe pain episodes (P<0.001).
In conclusion, G6PD202A,376G may be associated with lower haemoglobin concentration in sickle cell anaemia by a mechanism other than increased haemolysis.
PMCID: PMC2906678  PMID: 20507315
sickle cell anaemia; G6PD; haemolysis; alpha-thalassaemia; haemoglobin concentration
20.  Increased size of solid organs in patients with Chuvash polycythemia and in mice with altered expression of HIF-1α and HIF-2α 
Chuvash polycythemia, the first hereditary disease associated with dysregulated oxygen-sensing to be recognized, is characterized by a homozygous germ-line loss-of-function mutation of the VHL gene (VHLR200W) resulting in elevated hypoxia inducible factor (HIF)-1α and HIF-2α levels, increased red cell mass and propensity to thrombosis. Organ volume is determined by the size and number of cells, and the underlying molecular control mechanisms are not fully elucidated. Work from several groups has demonstrated that the proliferation of cells is regulated in opposite directions by HIF-1α and HIF-2α. HIF-1α inhibits cell proliferation by displacing MYC from the promoter of the gene encoding the cyclin-dependent kinase inhibitor, p21Cip1, thereby inducing its expression. In contrast, HIF-2α promotes MYC activity and cell proliferation. Here we report that the volumes of liver, spleen, and kidneys relative to body mass were larger in 30 individuals with Chuvash polycythemia than in 30 matched Chuvash controls. In Hif1a+/− mice, which are heterozygous for a null (knockout) allele at the locus encoding HIF-1α, hepatic HIF-2α mRNA was increased (2-fold) and the mass of the liver was increased, compared with wild-type littermates, without significant difference in cell volume. Hepatic p21Cip1 mRNA levels were 9.5-fold lower in Hif1a+/− mice compared with wild-type littermates. These data suggest that, in addition to increased red cell mass, the sizes of liver, spleen, and kidneys are increased in Chuvash polycythemia. At least in the liver, this phenotype may result from increased HIF-2α and decreased p21Cip1 levels leading to increased hepatocyte proliferation.
PMCID: PMC2866078  PMID: 20140661
Polycythemia; VHL; Hypoxia inducible factor; Organ size; p21Cip1; Genetics; Gene expression; Hematology; HIF; Hypoxia; Mouse models
21.  Erythropoietin Receptor Signaling Regulates Both Erythropoiesis and Megakaryopoiesis In Vivo 
Transgenic expression of a gain-of-function truncated mouse erythropoietin receptor gene (EpoR) leads to expansion of the HSC pool in response to human erythropoietin (Epo). We have re-examined this observation using a knock-in mouse model, wherein the mouse EpoR gene was replaced in its proper genetic locus by a single copy of either a wild type human or a polycythemia-inducing truncated human EPOR gene. Bone marrow cells obtained from knock-in mice were transplanted together with competitor bone marrow cells in a model that allows tracking of erythroid, platelet, and leukocyte contributions by each genotype. Secondary transplants were also performed. Stem/progenitor cells were identified phenotypically and isolated for colony-forming assays to evaluate cytokine responsiveness by cells with the wild type human or truncated human EPOR gene. Augmented Epo signaling increased erythroid repopulation post-transplant as expected, but had no effect on short-term or long-term leukocyte repopulation. However, the wild type human EPOR knock-in mouse showed decreases in both erythroid and platelet repopulation compared to marrow cells from the mutant human EPOR knock-in mouse or normal B6 animals. These results provide evidence supporting a role for Epo signaling in megakaryopoiesis in vivo and suggest a role for Epo signaling early in hematopoietic development.
PMCID: PMC2818228  PMID: 19836979
erythropoietin; hematopoietic stem cells; progenitor cells; erythrocytes; platelets
22.  EPO Receptor Gain-of-Function Causes Hereditary Polycythemia, Alters CD34+ Cell Differentiation and Increases Circulating Endothelial Precursors 
PLoS ONE  2010;5(8):e12015.
Gain-of-function of erythropoietin receptor (EPOR) mutations represent the major cause of primary hereditary polycythemia. EPOR is also found in non-erythroid tissues, although its physiological role is still undefined.
Methodology/Principal Findings
We describe a family with polycythemia due to a heterozygous mutation of the EPOR gene that causes a G→T change at nucleotide 1251 of exon 8. The novel EPOR G1251T mutation results in the replacement of a glutamate residue by a stop codon at amino acid 393. Differently from polycythemia vera, EPOR G1251T CD34+ cells proliferate and differentiate towards the erythroid phenotype in the presence of minimal amounts of EPO. Moreover, the affected individuals show a 20-fold increase of circulating endothelial precursors. The analysis of erythroid precursor membranes demonstrates a heretofore undescribed accumulation of the truncated EPOR, probably due to the absence of residues involved in the EPO-dependent receptor internalization and degradation. Mutated receptor expression in EPOR-negative cells results in EPOR and Stat5 phosphorylation. Moreover, patient erythroid precursors present an increased activation of EPOR and its effectors, including Stat5 and Erk1/2 pathway.
Our data provide an unanticipated mechanism for autosomal dominant inherited polycythemia due to a heterozygous EPOR mutation and suggest a regulatory role of EPO/EPOR pathway in human circulating endothelial precursors homeostasis.
PMCID: PMC2916842  PMID: 20700488
23.  Erythropoiesis in Polycythemia Vera is Hyper-Proliferative and has Accelerated Maturation 
Polycythemia vera (PV) is an acquired myeloproliferative clonal disorder, characterized by augmented erythropoiesis. To better define PV pathogenesis, we performed an in vitro erythroid expansion from peripheral blood mononuclear cells of controls and PV patients and evaluated the cells for proliferation, apoptosis, erythroid differentiation, and morphology at the defined time points. PV erythroid progenitors exhibited increased proliferation at days 9~14 and accelerated maturation at days 7~14, with a larger S-phase population (40%) than controls (20%) at day 11; however, the proportion of apoptotic cells was comparable to controls. Previously, we have identified PV-specific dysregulation of several microRNAs (i.e. miR-150, 451, 222, 155, 378). We had analyzed expression profiles of selected target genes of these microRNAs based on in silico prediction and their known function pertinent to the observed PV-specific erythropoiesis differences. p27, cMYB and EPOR showed differential expression in PV erythroid progenitors at the specific stages of erythroid differentiation. In this study, we identified accelerated maturation and hyper-proliferation at early stages of PV erythropoiesis. We speculate that aberrant expression of p27, c-MYB, and EPOR may contribute to these abnormal features in PV erythropoiesis.
PMCID: PMC2693444  PMID: 19264517
polycythemia vera; in vitro erythroid expansion; erythroid maturation; erythropoiesis; miRNA in polycythemia vera; p27; c-MYB; erythropoietin receptor genes
24.  Elevated homocysteine, glutathione and cysteinylglycine concentrations in patients homozygous for the Chuvash polycythemia VHL mutation 
Haematologica  2008;93(2):279-282.
In Chuvash polycythemia, homozygous von Hippel-Lindau (VHL) 598C>T leads to increased hypoxia inducible factor-1α and 2α, thromboses and lower systemic blood pressures. Circulating homocysteine, glutathione, γ-glutamyltransferase and cysteinylglycine concentrations were higher in 34 VHL598C>T homozygotes than in 37 normal controls and cysteine was lower. Multivariate analysis showed elevated homocysteine independently associated with higher mean systemic blood pressures and elevated glutathione was associated with lower pressures to a similar degree. Among VHL598C>T homozygotes, homocysteine was elevated with low and normal folate concentrations, consistent with a possible defect in the remethylation pathway. The elevated glutathione and γ-glutamyltranserase levels correlated positively with cysteinylglycine, consistent with possible upregulation of a glutathione synthetic enzyme and γ-glutamyltransferase. Cysteinylglycine correlated inversely with cysteine, consistent with possible reduced cysteinyldipeptidase activity. We conclude that up-regulated hypoxia-sensing may influence multiple steps in thiol metabolism. The effects of the resultant elevated levels of homocysteine and glutathione on systemic blood pressure may largely balance each other out.
PMCID: PMC2852873  PMID: 18223282
VHL; polycythemia; homocysteine; folate; glutathione
25.  Angiogenic and Inflammatory Markers of Cardiopulmonary Changes in Children and Adolescents with Sickle Cell Disease 
PLoS ONE  2009;4(11):e7956.
Pulmonary hypertension and left ventricular diastolic dysfunction are complications of sickle cell disease. Pulmonary hypertension is associated with hemolysis and hypoxia, but other unidentified factors are likely involved in pathogenesis as well.
Design and Methods
Plasma concentrations of three angiogenic markers (fibroblast growth factor, platelet derived growth factor–BB [PDGF-BB], vascular endothelial growth factor [VEGF]) and seven inflammatory markers implicated in pulmonary hypertension in other settings were determined by Bio-Plex suspension array in 237 children and adolescents with sickle cell disease at steady state and 43 controls. Tricuspid regurgitation velocity (which reflects systolic pulmonary artery pressure), mitral valve E/Edti ratio (which reflects left ventricular diastolic dysfunction), and a hemolytic component derived from four markers of hemolysis and hemoglobin oxygen saturation were also determined.
Plasma concentrations of interleukin-8, interleukin-10 and VEGF were elevated in the patients with sickle cell disease compared to controls (P≤0.003). By logistic regression, greater values for PDGF-BB (P = 0.009), interleukin-6 (P = 0.019) and the hemolytic component (P = 0.026) were independently associated with increased odds of elevated tricuspid regurgitation velocity while higher VEGF concentrations were associated with decreased odds (P = 0.005) among the patients with sickle cell disease. These findings, which are consistent with reports that PDGF-BB stimulates and VEGF inhibits vascular smooth muscle cell proliferation, did not apply to E/Etdi.
Circulating concentrations of angiogenic and pro-Inflammatory markers are altered in sickle cell disease children and adolescents with elevated tricuspid regurgitation velocity, a subgroup that may be at risk for developing worsening pulmonary hypertension. Further studies to understand the molecular changes in these children are indicated.
PMCID: PMC2776981  PMID: 19956689

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