Publications by authors named "Melanie J Percy"

54 Publications

Tibetan , an allele with loss-of-function properties.

Proc Natl Acad Sci U S A 2020 06 15;117(22):12230-12238. Epub 2020 May 15.

Department of Pathology and Laboratory Medicine, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104;

Tibetans have adapted to the chronic hypoxia of high altitude and display a distinctive suite of physiologic adaptations, including augmented hypoxic ventilatory response and resistance to pulmonary hypertension. Genome-wide studies have consistently identified compelling genetic signatures of natural selection in two genes of the Hypoxia Inducible Factor pathway, and The product of the former induces the degradation of the product of the latter. Key issues regarding Tibetan are whether it is a gain-of-function or loss-of-function allele, and how it might contribute to high-altitude adaptation. Tibetan PHD2 possesses two amino acid changes, D4E and C127S. We previously showed that in vitro, Tibetan PHD2 is defective in its interaction with p23, a cochaperone of the HSP90 pathway, and we proposed that Tibetan is a loss-of-function allele. Here, we report that additional PHD2 mutations at or near Asp-4 or Cys-127 impair interaction with p23 in vitro. We find that mice with the Tibetan allele display augmented hypoxic ventilatory response, supporting this loss-of-function proposal. This is phenocopied by mice with a mutation in that abrogates the PHD2:p23 interaction. haploinsufficiency, but not the Tibetan allele, ameliorates hypoxia-induced increases in right ventricular systolic pressure. The Tibetan allele is not associated with hemoglobin levels in mice. We propose that Tibetans possess genetic alterations that both activate and inhibit selective outputs of the HIF pathway to facilitate successful adaptation to the chronic hypoxia of high altitude.
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http://dx.doi.org/10.1073/pnas.1920546117DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7275716PMC
June 2020

International external quality assurance of JAK2 V617F quantification.

Ann Hematol 2019 May 8;98(5):1111-1118. Epub 2018 Dec 8.

Department of Hematology and Department of Pathology, Molecular Unit, Herlev Hospital, University of Copenhagen, Herlev Ringvej 75, DK-2730, Herlev, Denmark.

External quality assurance (EQA) programs are vital to ensure high quality and standardized results in molecular diagnostics. It is important that EQA for quantitative analysis takes into account the variation in methodology. Results cannot be expected to be more accurate than limits of the technology used, and it is essential to recognize factors causing substantial outlier results. The present study aimed to identify parameters of specific importance for JAK2 V617F quantification by quantitative PCR, using different starting materials, assays, and technical platforms. Sixteen samples were issued to participating laboratories in two EQA rounds. In the first round, 19 laboratories from 11 European countries analyzing JAK2 V617F as part of their routine diagnostics returned results from in-house assays. In the second round, 25 laboratories from 17 countries participated. Despite variations in starting material, assay set-up and instrumentation the laboratories were generally well aligned in the EQA program. However, EQA based on a single technology appears to be a valuable tool to achieve standardization of the quantification of JAK2 V617F allelic burden.
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http://dx.doi.org/10.1007/s00277-018-3570-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6469832PMC
May 2019

A novel splicing site IRP1 somatic mutation in a patient with pheochromocytoma and JAK2 positive polycythemia vera: a case report.

BMC Cancer 2018 03 13;18(1):286. Epub 2018 Mar 13.

Section on Medical Neuroendocrinology, Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD, 20892, USA.

Background: The role of the hypoxia signaling pathway in the pathogenesis of pheochromocytoma/paraganglioma (PPGL)-polycythemia syndrome has been elucidated. Novel somatic mutations in hypoxia-inducible factor type 2A (HIF2A) and germline mutations in prolyl hydroxylase type 1 and type 2 (PHD1 and PHD2) have been identified to cause upregulation of the hypoxia signaling pathway and its target genes including erythropoietin (EPO) and its receptor (EPOR). However, in a minority of patients presenting with this syndrome, the genetics and molecular pathogenesis remain unexplained. The aim of the present study was to uncover novel genetic causes of PPGL-polycythemia syndrome.

Case Presentation: A female presented with a history of JAK2 positive PV, diagnosed in 2007, and right adrenal pheochromocytoma diagnosed and resected in 2011. Her polycythemia symptoms and hematocrit levels continued to worsen from 2007 to 2011, with an increased frequency of phlebotomies. Postoperatively, until early 2013, her hematocrit levels remained normalized. Following this, the hematocrit levels ranged between 46.4 and 48.9% [35-45%]. Tumor tissue from the patient was further tested for mutations in genes related to upregulation of the hypoxia signaling pathway including iron regulatory protein 1 (IRP1), which is a known regulator of HIF-2α mRNA translation. Functional studies were performed to investigate the consequences of these mutations, especially their effect on the HIF signaling pathway and EPO. Indel mutations (c.267-1_267delGGinsTA) were discovered at the exon 3 splicing site of IRP1. Minigene construct and splicing site analysis showed that the mutation led to a new splicing site and a frameshift mutation of IRP1, which caused a truncated protein. Fluorescence in situ hybridization analysis demonstrated heterozygous IRP1 deletions in tumor cells. Immunohistochemistry results confirmed the truncated IRP1 and overexpressed HIF-2α, EPO and EPOR in tumor cells.

Conclusions: This is the first report which provides direct molecular genetic evidence of association between a somatic IRP1 loss-of-function mutation and PHEO and secondary polycythemia. In patients diagnosed with PHEO/PGL and polycythemia with negative genetic testing for mutations in HIF2A, PHD1/2, and VHL, IRP1 should be considered as a candidate gene.
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http://dx.doi.org/10.1186/s12885-018-4127-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5850917PMC
March 2018

Molecular diagnostics of myeloproliferative neoplasms.

Eur J Haematol 2015 Oct 18;95(4):270-9. Epub 2015 May 18.

Laboratoire d'Hématologie, Centre Hospitalier Universitaire de Nantes, Nantes, France.

Since the discovery of the JAK2 V617F mutation in the majority of the myeloproliferative neoplasms (MPN) of polycythemia vera, essential thrombocythemia and primary myelofibrosis ten years ago, further MPN-specific mutational events, notably in JAK2 exon 12, MPL exon 10 and CALR exon 9 have been identified. These discoveries have been rapidly incorporated into evolving molecular diagnostic algorithms. Whilst many of these mutations appear to have prognostic implications, establishing MPN diagnosis is of immediate clinical importance with selection, implementation and the continual evaluation of the appropriate laboratory methodology to achieve this diagnosis similarly vital. The advantages and limitations of these approaches in identifying and quantitating the common MPN-associated mutations are considered herein with particular regard to their clinical utility. The evolution of molecular diagnostic applications and platforms has occurred in parallel with the discovery of MPN-associated mutations, and it therefore appears likely that emerging technologies such as next-generation sequencing and digital PCR will in the future play an increasing role in the molecular diagnosis of MPN.
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http://dx.doi.org/10.1111/ejh.12578DOI Listing
October 2015

DEK oncogene expression during normal hematopoiesis and in Acute Myeloid Leukemia (AML).

Blood Cells Mol Dis 2015 Jan 12;54(1):123-31. Epub 2014 Aug 12.

Centre for Cancer Research and Cell Biology (CCRCB), Queen's University Belfast, Belfast, United Kingdom; Haematology Department, Belfast City Hospital, Belfast Health and Social Care Trust, Belfast, United Kingdom. Electronic address:

DEK is important in regulating cellular processes including proliferation, differentiation and maintenance of stem cell phenotype. The translocation t(6;9) in Acute Myeloid Leukemia (AML), which fuses DEK with NUP214, confers a poor prognosis and a higher risk of relapse. The over-expression of DEK in AML has been reported, but different studies have shown diminished levels in pediatric and promyelocytic leukemias. This study has characterized DEK expression, in silico, using a large multi-center cohort of leukemic and normal control cases. Overall, DEK was under-expressed in AML compared to normal bone marrow (NBM). Studying specific subtypes of AML confirmed either no significant change or a significant reduction in DEK expression compared to NBM. Importantly, the similarity of DEK expression between AML and NBM was confirmed using immunohistochemistry analysis of tissue mircorarrays. In addition, stratification of AML patients based on median DEK expression levels indicated that DEK showed no effect on the overall survival of patients. DEK expression during normal hematopoiesis did reveal a relationship with specific cell types implicating a distinct function during myeloid differentiation. Whilst DEK may play a potential role in hematopoiesis, it remains to be established whether it is important for leukemagenesis, except when involved in the t(6;9) translocation.
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http://dx.doi.org/10.1016/j.bcmd.2014.07.009DOI Listing
January 2015

Molecular heterogeneity of familial myeloproliferative neoplasms revealed by analysis of the commonly acquired JAK2, CALR and MPL mutations.

Fam Cancer 2014 Dec;13(4):659-63

Cancer Molecular Diagnostics, Central Pathology Laboratory, St. James's Hospital, Dublin 8, Ireland,

The myeloproliferative neoplasms (MPN) are clonal, hematological malignancies that include polycythemia vera, essential thrombocythemia and primary myelofibrosis. While most cases of MPN are sporadic in nature, a familial pattern of inheritance is well recognised. The phenotype and status of the commonly acquired JAK2 V617F, CALR exon 9 and MPL W515L/K mutations in affected individuals from a consecutive series of ten familial MPN (FMPN) kindred are described. Affected individuals display the classical MPN phenotypes together with one kindred identified suggestive of hereditary thrombocytosis. In affected patients the JAK2 V617F mutation is the most commonly acquired followed by CALR exon nine mutations with no MPL W515L/K mutations detected. The JAK2 V617F and CALR exon 9 mutations appear to occur at approximately the same frequency in FMPN as in the sporadic forms of these diseases. The familial nature of MPN may often be overlooked and accordingly more common than previously considered. Characterisation of these FMPN kindred may allow for the investigation of molecular events that contribute to this inheritance.
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http://dx.doi.org/10.1007/s10689-014-9743-2DOI Listing
December 2014

The role of PHD2 mutations in the pathogenesis of erythrocytosis.

Hypoxia (Auckl) 2014 1;2:71-90. Epub 2014 Jul 1.

Department of Pathology and Laboratory Medicine, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA.

The transcription of the erythropoietin () gene is tightly regulated by the hypoxia response pathway to maintain oxygen homeostasis. Elevations in serum EPO level may be reflected in an augmentation in the red cell mass, thereby causing erythrocytosis. Studies on erythrocytosis have provided insights into the function of the oxygen-sensing pathway and the critical proteins involved in the regulation of transcription. The α subunits of the hypoxia-inducible transcription factor are hydroxylated by three prolyl hydroxylase domain (PHD) enzymes, which belong to the iron and 2-oxoglutarate-dependent oxygenase superfamily. Sequence analysis of the genes encoding the PHDs in patients with erythrocytosis has revealed heterozygous germline mutations only occurring in Egl nine homolog 1 (, also known as ), the gene that encodes PHD2. To date, 24 different mutations comprising missense, frameshift, and nonsense mutations have been described. The phenotypes associated with the patients carrying these mutations are fairly homogeneous and typically limited to erythrocytosis with normal to elevated EPO. However, exceptions exist; for example, there is one case with development of concurrent paraganglioma (PHD2-H374R). Analysis of the erythrocytosis-associated PHD2 missense mutations has shown heterogeneous results. Structural studies reveal that mutations can affect different domains of PHD2. Some are close to the hypoxia-inducible transcription factor α/2-oxoglutarate or the iron binding sites for PHD2. In silico studies demonstrate that the mutations do not always affect fully conserved residues. In vitro and in cellulo studies showed varying effects of the mutations, ranging from mild effects to severe loss of function. The exact mechanism of a potential tumor-suppressor role for PHD2 still needs to be elucidated. A knockin mouse model expressing the first reported PHD2-P317R mutation recapitulates the phenotype observed in humans (erythrocytosis with inappropriately normal serum EPO levels) and demonstrates that haploinsufficiency and partial deregulation of PHD2 is sufficient to cause erythrocytosis.
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http://dx.doi.org/10.2147/HP.S54455DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5045058PMC
July 2014

Polycythaemia-inducing mutations in the erythropoietin receptor (EPOR): mechanism and function as elucidated by epidermal growth factor receptor-EPOR chimeras.

Br J Haematol 2014 May 18;165(4):519-28. Epub 2014 Feb 18.

Department of Cell and Developmental Biology, Sackler Faculty of Medicine, Tel-Aviv University, Tel Aviv, Israel.

Primary familial and congenital polycythaemia (PFCP) is a disease characterized by increased red blood cell mass, and can be associated with mutations in the intracellular region of the erythropoietin (EPO) receptor (EPOR). Here we explore the mechanisms by which EPOR mutations induce PFCP, using an experimental system based on chimeric receptors between epidermal growth factor receptor (EGFR) and EPOR. The design of the chimeras enabled EPOR signalling to be triggered by EGF binding. Using this system we analysed three novel EPOR mutations discovered in PFCP patients: a deletion mutation (Del1377-1411), a nonsense mutation (C1370A) and a missense mutation (G1445A). Three different chimeras, bearing these mutations in the cytosolic, EPOR region were generated; Hence, the differences in the chimera-related effects are specifically attributed to the mutations. The results show that the different mutations affect various aspects related to the signalling and metabolism of the chimeric receptors. These include slower degradation rate, higher levels of glycan-mature chimeric receptors, increased sensitivity to low levels of EGF (replacing EPO in this system) and extended signalling cascades. This study provides a novel experimental system to study polycythaemia-inducing mutations in the EPOR, and sheds new light on underlying mechanisms of EPOR over-activation in PFCP patients.
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http://dx.doi.org/10.1111/bjh.12782DOI Listing
May 2014

Hypoxia-inducible factor 2α regulates key neutrophil functions in humans, mice, and zebrafish.

Blood 2014 Jan 6;123(3):366-76. Epub 2013 Nov 6.

Department of Infection and Immunity and.

Neutrophil lifespan and function are regulated by hypoxia via components of the hypoxia inducible factor (HIF)/von Hippel Lindau/hydroxylase pathway, including specific roles for HIF-1α and prolyl hydroxylase-3. HIF-2α has both distinct and overlapping biological roles with HIF-1α and has not previously been studied in the context of neutrophil biology. We investigated the role of HIF-2α in regulating key neutrophil functions. Human and murine peripheral blood neutrophils expressed HIF-2α, with expression up-regulated by acute and chronic inflammatory stimuli and in disease-associated inflammatory neutrophil. HIF2A gain-of-function mutations resulted in a reduction in neutrophil apoptosis both ex vivo, through the study of patient cells, and in vivo in a zebrafish tail injury model. In contrast, HIF-2α-deficient murine inflammatory neutrophils displayed increased sensitivity to nitrosative stress induced apoptosis ex vivo and increased neutrophil apoptosis in vivo, resulting in a reduction in neutrophilic inflammation and reduced tissue injury. Expression of HIF-2α was temporally dissociated from HIF-1α in vivo and predominated in the resolution phase of inflammation. These data support a critical and selective role for HIF-2α in persistence of neutrophilic inflammation and provide a platform to dissect the therapeutic utility of targeting HIF-2α in chronic inflammatory diseases.
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http://dx.doi.org/10.1182/blood-2013-05-500207DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3894493PMC
January 2014

A knock-in mouse model of human PHD2 gene-associated erythrocytosis establishes a haploinsufficiency mechanism.

J Biol Chem 2013 Nov 11;288(47):33571-33584. Epub 2013 Oct 11.

Department of Pathology and Laboratory Medicine, University of Pennsylvania, Philadelphia, Pennsylvania 19104. Electronic address:

The central pathway for controlling red cell mass is the PHD (prolyl hydroxylase domain protein):hypoxia-inducible factor (HIF) pathway. HIF, which is negatively regulated by PHD, activates numerous genes, including ones involved in erythropoiesis, such as the ERYTHROPOIETIN (EPO) gene. Recent studies have implicated PHD2 as the key PHD isoform regulating red cell mass. Studies of humans have identified erythrocytosis-associated, heterozygous point mutations in the PHD2 gene. A key question concerns the mechanism by which human mutations lead to phenotypes. In the present report, we generated and characterized a mouse line in which a P294R knock-in mutation has been introduced into the mouse Phd2 locus to model the first reported human PHD2 mutation (P317R). Phd2(P294R/+) mice display a degree of erythrocytosis equivalent to that seen in Phd2(+/-) mice. The Phd2(P294R/+)-associated erythrocytosis is reversed in a Hif2a(+/-), but not a Hif1a(+/-) background. Additional studies using various conditional knock-outs of Phd2 reveal that erythrocytosis can be induced by homozygous and heterozygous knock-out of Phd2 in renal cortical interstitial cells using a Pax3-Cre transgene or by homozygous knock-out of Phd2 in hematopoietic progenitors driven by a Vav1-Cre transgene. These studies formally prove that a missense mutation in PHD2 is the cause of the erythrocytosis, show that this occurs through haploinsufficiency, and point to multifactorial control of red cell mass by PHD2.
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http://dx.doi.org/10.1074/jbc.M113.482364DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3837105PMC
November 2013

Genetic basis of congenital erythrocytosis: mutation update and online databases.

Hum Mutat 2014 Jan 22;35(1):15-26. Epub 2013 Oct 22.

Department of Hematology, Centro Hospitalar e Universitário de Coimbra, Coimbra, Portugal.

Congenital erythrocytosis (CE), or congenital polycythemia, represents a rare and heterogeneous clinical entity. It is caused by deregulated red blood cell production where erythrocyte overproduction results in elevated hemoglobin and hematocrit levels. Primary congenital familial erythrocytosis is associated with low erythropoietin (Epo) levels and results from mutations in the Epo receptor gene (EPOR). Secondary CE arises from conditions causing tissue hypoxia and results in increased Epo production. These include hemoglobin variants with increased affinity for oxygen (HBB, HBA mutations), decreased production of 2,3-bisphosphoglycerate due to BPGM mutations, or mutations in the genes involved in the hypoxia sensing pathway (VHL, EPAS1, and EGLN1). Depending on the affected gene, CE can be inherited either in an autosomal dominant or recessive mode, with sporadic cases arising de novo. Despite recent important discoveries in the molecular pathogenesis of CE, the molecular causes remain to be identified in about 70% of the patients. With the objective of collecting all the published and unpublished cases of CE the COST action MPN&MPNr-Euronet developed a comprehensive Internet-based database focusing on the registration of clinical history, hematological, biochemical, and molecular data (http://www.erythrocytosis.org/). In addition, unreported mutations are also curated in the corresponding Leiden Open Variation Database.
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http://dx.doi.org/10.1002/humu.22448DOI Listing
January 2014

Erythrocytosis in children and adolescents-classification, characterization, and consensus recommendations for the diagnostic approach.

Pediatr Blood Cancer 2013 Nov 14;60(11):1734-8. Epub 2013 Jun 14.

Department of Pediatrics and Adolescent Medicine, University Medical Center Ulm, Ulm, Germany.

During recent years, the increasing knowledge of genetic and physiological changes in polycythemia vera (PV) and of different types of congenital erythrocytosis has led to fundamental changes in recommendations for the diagnostic approach to patients with erythrocytosis. Although widely accepted for adult patients this approach may not be appropriate with regard to children and adolescents affected by erythrocytosis. The "congenital erythrocytosis" working group established within the framework of the MPN&MPNr-EuroNet (COST action BM0902) addressed this question in a consensus finding process and developed a specific algorithm for the diagnosis of erythrocytosis in childhood and adolescence which is presented here.
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http://dx.doi.org/10.1002/pbc.24625DOI Listing
November 2013

Erythrocytosis and pulmonary hypertension in a mouse model of human HIF2A gain of function mutation.

J Biol Chem 2013 Jun 2;288(24):17134-44. Epub 2013 May 2.

Department of Pathology and Laboratory Medicine, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania 19104, USA.

The central pathway for oxygen-dependent control of red cell mass is the prolyl hydroxylase domain protein (PHD):hypoxia inducible factor (HIF) pathway. PHD site specifically prolyl hydroxylates the transcription factor HIF-α, thereby targeting the latter for degradation. Under hypoxia, this modification is attenuated, allowing stabilized HIF-α to activate target genes, including that for erythropoietin (EPO). Studies employing genetically modified mice point to Hif-2α, one of two main Hif-α isoforms, as being the critical regulator of Epo in the adult mouse. More recently, erythrocytosis patients with heterozygous point mutations in the HIF2A gene have been identified; whether these mutations were polymorphisms unrelated to the phenotype could not be ruled out. In the present report, we characterize a mouse line bearing a G536W missense mutation in the Hif2a gene that corresponds to the first such human mutation identified (G537W). We obtained mice bearing both heterozygous and homozygous mutations at this locus. We find that these mice display, in a mutation dose-dependent manner, erythrocytosis and pulmonary hypertension with a high degree of penetrance. These findings firmly establish missense mutations in HIF-2α as a cause of erythrocytosis, highlight the importance of this HIF-α isoform in erythropoiesis, and point to physiologic consequences of HIF-2α dysregulation.
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http://dx.doi.org/10.1074/jbc.M112.444059DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3682519PMC
June 2013

Methemoglobin reductase deficiency: novel mutation is associated with a disease phenotype of intermediate severity.

J Pediatr Hematol Oncol 2012 Aug;34(6):457-60

Department of Haematology, Belfast City Hospital, Belfast, Northern Ireland.

Background: Cytochrome b5 reductase (CB5R) deficiency is a recessively inherited autosomal disorder that is either benign (type I) or associated with severe neurological problems (type II). Specific mutations in the CYB5R gene are not exclusive to each type.

Observation: Two cyanotic children with developmental delay but with slow progression were investigated for CB5R deficiency. A novel mutation, p.Arg58Pro, was independently detected in both cases.

Conclusions: The clinical variability and severity of the disease reflect the combined effects of impaired function of the 2 mutant enzymes. As illustrated by these 2 cases, inheritance of p.Arg58Pro with either p.Gly76Ser or pLeu188del causes a clinical condition more severe than type I and less severe than the type II cases reported to date.
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http://dx.doi.org/10.1097/MPH.0b013e318257a492DOI Listing
August 2012

A nonsynonymous LNK polymorphism associated with idiopathic erythrocytosis.

Am J Hematol 2011 Nov 22;86(11):962-4. Epub 2011 Aug 22.

Department of Haematology, Centre for Cancer Research and Cell Biology, Queen’s University Belfast, Northern Ireland, United Kingdom.

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http://dx.doi.org/10.1002/ajh.22154DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3674579PMC
November 2011

Potential prognostic marker ubiquitin carboxyl-terminal hydrolase-L1 does not predict patient survival in non-small cell lung carcinoma.

J Exp Clin Cancer Res 2011 Aug 30;30:79. Epub 2011 Aug 30.

Department of Haematology, Centre for Cancer Research and Cell Biology, Queen's University Belfast, 97 Lisburn Road, Belfast, Northern Ireland, UK, BT9 7BL.

Background: Ubiquitin Carboxyl-Terminal Hydrolase-L1 (UCH-L1) is a deubiquitinating enzyme that is highly expressed throughout the central and peripheral nervous system and in cells of the diffuse neuroendocrine system. Aberrant function of UCH-L1 has been associated with neurological disorders such as Parkinson's disease and Alzheimer's disease. Moreover, UCH-L1 exhibits a variable expression pattern in cancer, acting either as a tumour suppressor or promoter, depending on the type of cancer. In non-small cell lung carcinoma primary tumour samples, UCH-L1 is highly expressed and is associated with an advanced tumour stage. This suggests UCH-L1 may be involved in oncogenic transformation and tumour invasion in NSCLC. However, the functional significance of UCH-L1 in the progression of NSCLC is unclear. The aim of this study was to investigate the role of UCH-L1 using NSCLC cell line models and to determine if it is clinically relevant as a prognostic marker for advanced stage disease.

Methods: UCH-L1 expression in NSCLC cell lines H838 and H157 was modulated by siRNA-knockdown, and the phenotypic changes were assessed by flow cytometry, haematoxylin & eosin (H&E) staining and poly (ADP-ribose) polymerase (PARP) cleavage. Metastatic potential was measured by the presence of phosphorylated myosin light chain (MLC2). Tumour microarrays were examined immunohistochemically for UCH-L1 expression. Kaplan-Meier curves were generated using UCH-L1 expression levels and patient survival data extracted from Gene Expression Omnibus data files.

Results: Expression of UCH-L1 was decreased by siRNA in both cell lines, resulting in increased cell death in H838 adenocarcinoma cells but not in the H157 squamous cell line. However, metastatic potential was reduced in H157 cells. Immunohistochemical staining of UCH-L1 in patient tumours confirmed it was preferentially expressed in squamous cell carcinoma rather than adenocarcinoma. However the Kaplan-Meier curves generated showed no correlation between UCH-L1 expression levels and patient outcome.

Conclusions: Although UCH-L1 appears to be involved in carcinogenic processes in NSCLC cell lines, the absence of correlation with patient survival indicates that caution is required in the use of UCH-L1 as a potential prognostic marker for advanced stage and metastasis in lung carcinoma.
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http://dx.doi.org/10.1186/1756-9966-30-79DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3180428PMC
August 2011

A novel base change leading to Hb Vanderbilt [β89(F5)Ser→Arg, AGT>AGA].

Hemoglobin 2011 ;35(4):428-9

Department of Haematology, University College Hospital, Galway, Republic of Ireland.

We describe a high oxygen affinity hemoglobin (Hb) variant (Hb Vanderbilt) as a result of a heterozygous novel base change from T to A at codon 89 (AGT>AGA) leading to an amino acid change from serine to arginine.
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http://dx.doi.org/10.3109/03630269.2011.594137DOI Listing
December 2011

Cardiopulmonary function in two human disorders of the hypoxia-inducible factor (HIF) pathway: von Hippel-Lindau disease and HIF-2alpha gain-of-function mutation.

FASEB J 2011 Jun 9;25(6):2001-11. Epub 2011 Mar 9.

Department of Physiology, Anatomy and Genetics, University of Oxford, Parks Rd., Oxford, OX1 3PT, UK.

The hypoxia-inducible factors (HIFs; isoforms HIF-1α, HIF-2α, HIF-3α) mediate many responses to hypoxia. Their regulation is principally by oxygen-dependent degradation, which is initiated by hydroxylation of specific proline residues followed by binding of von Hippel-Lindau (VHL) protein. Chuvash polycythemia is a disorder with elevated HIF. It arises through germline homozygosity for hypomorphic VHL alleles and has a phenotype of hematological, cardiopulmonary, and metabolic abnormalities. This study explores the phenotype of two other HIF pathway diseases: classic VHL disease and HIF-2α gain-of-function mutation. No cardiopulmonary abnormalities were detected in classic VHL disease. HIF-2α gain-of-function mutations were associated with pulmonary hypertension, increased cardiac output, increased heart rate, and increased pulmonary ventilation relative to metabolism. Comparison of the HIF-2α gain-of-function responses with data from studies of Chuvash polycythemia suggested that other aspects of the Chuvash phenotype were diminished or absent. In classic VHL disease, patients are germline heterozygous for mutations in VHL, and the present results suggest that a single wild-type allele for VHL is sufficient to maintain normal cardiopulmonary function. The HIF-2α gain-of-function phenotype may be more limited than the Chuvash phenotype either because HIF-1α is not elevated in the former condition, or because other HIF-independent functions of VHL are perturbed in Chuvash polycythemia.
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http://dx.doi.org/10.1096/fj.10-177378DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3159892PMC
June 2011

The HIF pathway and erythrocytosis.

Annu Rev Pathol 2011 ;6:165-92

Department of Pathology and Laboratory Medicine, University of Pennsylvania School of Medicine, Philadelphia, 19104, USA.

Because of the central role that red blood cells play in the delivery of oxygen to tissues of the body, red blood cell mass must be controlled at precise levels. The glycoprotein hormone erythropoietin (EPO) regulates red blood cell mass. EPO transcription, in turn, is regulated by a distinctive oxygen-sensing mechanism. In this pathway, prolyl hydroxylase domain protein (PHD) site-specifically hydroxylates the α-subunit of the transcription factor hypoxia-inducible factor α (HIF-α), thereby targeting the latter for degradation by the von Hippel-Lindau tumor-suppressor protein (VHL). Under hypoxic conditions, this posttranslational modification of HIF-α is inhibited, which stabilizes it and promotes the transcriptional activation of genes, including that for EPO. Rare patients with erythrocytosis have mutations in the genes encoding for PHD2, HIF-2α, and VHL, which implicates these proteins as critical to the proper control of red blood cell mass in humans.
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http://dx.doi.org/10.1146/annurev-pathol-011110-130321DOI Listing
April 2011

Regulation of human metabolism by hypoxia-inducible factor.

Proc Natl Acad Sci U S A 2010 Jul 28;107(28):12722-7. Epub 2010 Jun 28.

Department of Physiology, Anatomy and Genetics, University of Oxford, Oxford OX1 3PT, United Kingdom.

The hypoxia-inducible factor (HIF) family of transcription factors directs a coordinated cellular response to hypoxia that includes the transcriptional regulation of a number of metabolic enzymes. Chuvash polycythemia (CP) is an autosomal recessive human disorder in which the regulatory degradation of HIF is impaired, resulting in elevated levels of HIF at normal oxygen tensions. Apart from the polycythemia, CP patients have marked abnormalities of cardiopulmonary function. No studies of integrated metabolic function have been reported. Here we describe the response of these patients to a series of metabolic stresses: exercise of a large muscle mass on a cycle ergometer, exercise of a small muscle mass (calf muscle) which allowed noninvasive in vivo assessments of muscle metabolism using (31)P magnetic resonance spectroscopy, and a standard meal tolerance test. During exercise, CP patients had early and marked phosphocreatine depletion and acidosis in skeletal muscle, greater accumulation of lactate in blood, and reduced maximum exercise capacities. Muscle biopsy specimens from CP patients showed elevated levels of transcript for pyruvate dehydrogenase kinase, phosphofructokinase, and muscle pyruvate kinase. In cell culture, a range of experimental manipulations have been used to study the effects of HIF on cellular metabolism. However, these approaches provide no potential to investigate integrated responses at the level of the whole organism. Although CP is relatively subtle disorder, our study now reveals a striking regulatory role for HIF on metabolism during exercise in humans. These findings have significant implications for the development of therapeutic approaches targeting the HIF pathway.
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http://dx.doi.org/10.1073/pnas.1002339107DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2906567PMC
July 2010

Erythropoietin-induced activation of the JAK2/STAT5, PI3K/Akt, and Ras/ERK pathways promotes malignant cell behavior in a modified breast cancer cell line.

Mol Cancer Res 2010 Apr 30;8(4):615-26. Epub 2010 Mar 30.

Centre for Cancer Research and Cell Biology, Queen's University Belfast, Belfast, Northern Ireland, United Kingdom.

Erythropoietin (Epo), the major regulator of erythropoiesis, and its cognate receptor (EpoR) are also expressed in nonerythroid tissues, including tumors. Clinical studies have highlighted the potential adverse effects of erythropoiesis-stimulating agents when used to treat cancer-related anemia. We assessed the ability of EpoR to enhance tumor growth and invasiveness following Epo stimulation. A benign noninvasive rat mammary cell line, Rama 37, was used as a model system. Cell signaling and malignant cell behavior were compared between parental Rama 37 cells, which express few or no endogenous EpoRs, and a modified cell line stably transfected with human EpoR (Rama 37-28). The incubation of Rama 37-28 cells with pharmacologic levels of Epo led to the rapid and sustained increases in phosphorylation of signal transducers and activators of transcription 5, Akt, and extracellular signal-regulated kinase. The activation of these signaling pathways significantly increased invasion, migration, adhesion, and colony formation. The Epo-induced invasion capacity of Rama 37-28 cells was reduced by the small interfering RNA-mediated knockdown of EpoR mRNA levels and by inhibitors of the phosphoinositide 3-kinase/Akt and Ras/extracellular signal-regulated kinase signaling pathways with adhesion also reduced by Janus-activated kinase 2/signal transducers and activators of transcription 5 inhibition. These data show that Epo induces phenotypic changes in the behavior of breast cancer cell lines and establishes links between individual cell signaling pathways and the potential for cancer spread.
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http://dx.doi.org/10.1158/1541-7786.MCR-09-0264DOI Listing
April 2010

The JAK2 46/1 haplotype predisposes to MPL-mutated myeloproliferative neoplasms.

Blood 2010 Jun 19;115(22):4517-23. Epub 2010 Mar 19.

Wessex Regional Genetics Laboratory, Salisbury District Hospital, Salisbury, United Kingdom.

The 46/1 JAK2 haplotype predisposes to V617F-positive myeloproliferative neoplasms, but the underlying mechanism is obscure. We analyzed essential thrombocythemia patients entered into the PT-1 studies and, as expected, found that 46/1 was overrepresented in V617F-positive cases (n = 404) versus controls (n = 1492, P = 3.9 x 10(-11)). The 46/1 haplotype was also overrepresented in cases without V617F (n = 347, P = .009), with an excess seen for both MPL exon 10 mutated and V617F, MPL exon 10 nonmutated cases. Analysis of further MPL-positive, V617F-negative cases confirmed an excess of 46/1 (n = 176, P = .002), but no association between MPL mutations and MPL haplotype was seen. An excess of 46/1 was also seen in JAK2 exon 12 mutated cases (n = 69, P = .002), and these mutations preferentially arose on the 46/1 chromosome (P = .029). No association between 46/1 and clinical or laboratory features was seen in the PT-1 cohort either with or without V617F. The excess of 46/1 in JAK2 exon 12 cases is compatible with both the "hypermutability" and "fertile ground" hypotheses, but the excess in MPL-mutated cases argues against the former. No difference in sequence, splicing, or expression of JAK2 was found on 46/1 compared with other haplotypes, suggesting that any functional difference of JAK2 on 46/1, if it exists, must be relatively subtle.
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http://dx.doi.org/10.1182/blood-2009-08-236448DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3145114PMC
June 2010

Erythrocytosis associated with a novel missense mutation in the HIF2A gene.

Haematologica 2010 May 8;95(5):829-32. Epub 2009 Dec 8.

Laboratory for Red Blood Cell Research, Department of Clinical Chemistry and Haematology, University Medical Center Utrecht, Utrecht, the Netherlands.

The ERYTHROPOIETIN (EPO) gene is regulated by the transcription factor Hypoxia Inducible Factor-alpha (HIF-alpha). In this pathway, Prolyl Hydroxylase Domain protein 2 (PHD2) hydroxylates two prolyl residues in HIF-alpha, which in turn promotes HIF-alpha degradation by the von Hippel Lindau (VHL) protein. Evidence that HIF-2alpha is the important isoform for EPO regulation in humans comes from the recent observation that mutations in the HIF2A gene are associated with cases of erythrocytosis. We report here a new erythrocytosis-associated mutation, p.Asp539Glu, in the HIF2A gene. Similar to all reported cases, the affected residue is in close vicinity and C-terminal to the primary hydroxylation site in HIF-2alpha, Pro531. This mutation, however, is notable in producing a rather subtle amino acid substitution. Nonetheless, we find that this mutation compromises binding of HIF-2alpha to both PHD2 and VHL, and we propose that this mutation is the cause of erythrocytosis in this individual.
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http://dx.doi.org/10.3324/haematol.2009.017582DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2864390PMC
May 2010

A new polycythaemia vera-associated SOCS3 SH2 mutant (SOCS3F136L) cannot regulate erythropoietin responses.

Br J Haematol 2009 Nov 5;147(4):450-8. Epub 2009 Sep 5.

Centre for Infection and Immunity, Queen's University, Belfast, Northern Ireland.

Recently several different JAK2 exon12 mutations have been identified in V617F negative polycythaemia vera (PV) or idiopathic erythrocytosis (IE) patients. The patients present with erythrocytosis, ligand-independent cell growth and low serum erythropoietin (EPO) levels. Within this group, a deletion of amino acids 542-543 (N542-E543del) of JAK2 is most prevalent. We have previously shown that in the presence of JAK2(V617F), suppressor of cytokine signalling 3 (SOCS3) is unable to negatively regulate EPO signalling and proliferation of V617F-expressing cells. Here we report a PV patient heterozygous for the somatic JAK2(N542-E543del) mutation and a previously unreported germline mutation within the SH2 domain of SOCS3 (F136L). Interestingly, the SOCS3(F136L) mutation was detected in a Japanese myeloproliferative disorder patient cohort at double the frequency of healthy controls. Cells expressing SOCS3(F136L) had markedly elevated EPO-induced proliferation and extended EPO-induced JAK2 phosphorylation. Additionally, compared to wild-type SOCS3, mutant SOCS3 had an extended half-life in the presence of JAK2 and JAK2(N542-E543del). Our findings suggest that this loss-of-function SOCS3 mutation may have contributed to disease onset by causing deregulated JAK2 signalling in the presence of a constitutively active JAK2(N542-E543del) mutant.
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http://dx.doi.org/10.1111/j.1365-2141.2009.07860.xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2838228PMC
November 2009

A novel L218P mutation in NADH-cytochrome b5 reductase associated with type I recessive congenital methemoglobinemia.

Pediatr Hematol Oncol 2009 Jul-Aug;26(5):381-5

Department of Pediatric Hematology, Dr. Sami Ulus Children's Health Education and Research Hospital, Ankara, Turkey.

The presence of central cyanosis that is unrelated to cardiopulmonary causes alerts clinicians to a possible diagnosis of methemoglobinemia. Congenital methemoglobinemia due to deficiency of nicotinamide-adenine dinucleotide (NADH)-cytochrome b5 reductase (cb(5)r) is an autosomal recessive disorder characterized by life long cyanosis. Here we report a six-year old boy who presented with central cyanosis and upon examination revealed a methemoglobin level of 19.0%. Sequencing the CYB5R3 gene identified a homozygous T-->C transition at base c.653, which changed codon 218 from leucine to proline (L218P) in cb(5)r protein. Treatment with ascorbic acid relieved the cyanosis and returned methemoglobin levels to normal.
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http://dx.doi.org/10.1080/08880010902979233DOI Listing
September 2009

Erythrocytosis-associated HIF-2alpha mutations demonstrate a critical role for residues C-terminal to the hydroxylacceptor proline.

J Biol Chem 2009 Apr 10;284(14):9050-8. Epub 2009 Feb 10.

Department of Pathology and Laboratory Medicine, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104, USA.

A classic physiologic response to hypoxia in humans is the up-regulation of the ERYTHROPOIETIN (EPO) gene, which is the central regulator of red blood cell mass. The EPO gene, in turn, is activated by hypoxia inducible factor (HIF). HIF is a transcription factor consisting of an alpha subunit (HIF-alpha) and a beta subunit (HIF-beta). Under normoxic conditions, prolyl hydroxylase domain protein (PHD, also known as HIF prolyl hydroxylase and egg laying-defective nine protein) site specifically hydroxylates HIF-alpha in a conserved LXXLAP motif (where underlining indicates the hydroxylacceptor proline). This provides a recognition motif for the von Hippel Lindau protein, a component of an E3 ubiquitin ligase complex that targets hydroxylated HIF-alpha for degradation. Under hypoxic conditions, this inherently oxygen-dependent modification is arrested, thereby stabilizing HIF-alpha and allowing it to activate the EPO gene. We previously identified and characterized an erythrocytosis-associated HIF2A mutation, G537W. More recently, we reported two additional erythrocytosis-associated HIF2A mutations, G537R and M535V. Here, we describe the functional characterization of these two mutants as well as a third novel erythrocytosis-associated mutation, P534L. These mutations affect residues C-terminal to the LXXLAP motif. We find that all result in impaired degradation and thus aberrant stabilization of HIF-2alpha. However, each exhibits a distinct profile with respect to their effects on PHD2 binding and von Hippel Lindau interaction. These findings reinforce the importance of HIF-2alpha in human EPO regulation, demonstrate heterogeneity of functional defects arising from these mutations, and point to a critical role for residues C-terminal to the LXXLAP motif in HIF-alpha.
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http://dx.doi.org/10.1074/jbc.M808737200DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2666553PMC
April 2009

Genetic origins and clinical phenotype of familial and acquired erythrocytosis and thrombocytosis.

Am J Hematol 2009 Jan;84(1):46-54

Department of Haematology, Belfast City Hospital, Belfast, Northern Ireland, UK.

Familial and acquired erythrocytosis and thrombocytosis are characterized by myeloid lineage hyperproliferation, which is either single or multi-lineage in origin. The single lineage disorders exhibit Mendelian inheritance with polyclonal hematopoiesis and often arise from a single genetic defect. In contrast, the multi-lineage disorders exhibit complex patterns of inheritance with multi-genetic origins and clonal hematopoiesis. They have the potential to acquire JAK2 somatic mutations, but this is not the primary event. Identification of the disease-causing genes will enable better classification of familial and acquired erythrocytosis and thrombocytosis. Furthermore, it will provide an insight into the mechanisms regulating myeloid cell proliferation.
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http://dx.doi.org/10.1002/ajh.21313DOI Listing
January 2009
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