M.J. Percy

Establishing optimal quantitative-polymerase chain reaction assays for routine diagnosis and tracking of minimal residual disease in JAK2-V617F-associated myeloproliferative neoplasms: a joint European LeukemiaNet/MPN&MPNr-EuroNet (COST action BM0902) study.

Reliable detection of JAK2-V617F is critical for accurate diagnosis of myeloproliferative neoplasms (MPNs); in addition, sensitive mutation-specific assays can be applied to monitor disease response. However, there has been no consistent approach to JAK2-V617F detection, with assays varying markedly in performance, affecting clinical utility. Therefore, we established a network of 12 laboratories from seven countries to systematically evaluate nine different DNA-based quantitative PCR (qPCR) assays, including those in widespread clinical use. Seven quality control rounds involving over 21 500 qPCR reactions were undertaken using centrally distributed cell line dilutions and plasmid controls. The two best-performing assays were tested on normal blood samples (n=100) to evaluate assay specificity, followed by analysis of serial samples from 28 patients transplanted for JAK2-V617F-positive disease. The most sensitive assay, which performed consistently across a range of qPCR platforms, predicted outcome following transplant, with the mutant allele detected a median of 22 weeks (range 6–85 weeks) before relapse. Four of seven patients achieved molecular remission following donor lymphocyte infusion, indicative of a graft vs MPN effect. This study has established a robust, reliable assay for sensitive JAK2-V617F detection, suitable for assessing response in clinical trials, predicting outcome and guiding management of patients undergoing allogeneic transplant.

Genetic basis of Congenital Erythrocytosis mutation update and online databases

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.

Recessive congenital methaemoglobinaemia: functional characterization of the novel D239G mutation in the NADH-binding lobe of cytochrome b5 reductase

Type I recessive congenital methaemoglobinaemia (RCM), caused by the reduced form of nicotinamide adenine dinucleotide (NADH)‐cytochrome b5 reductase (cytb5r) deficiency, manifests clinically as cyanosis without neurological dysfunction. Two mutations, E255‐ and G291D, have been identified in the NADH‐binding lobe of cytb5r in previously reported patients, and we have detected a further novel mutation, D239G, in this lobe in two unrelated Irish families. Although one family belongs to the genetically isolated Traveller Community, which separated from the general Irish population during the 1845–48 famine, the D239G mutation was present on the same haplotype in both families. Three known cytb5r mutations were also identified, including the R159‐ mutation, which causes loss of the entire NADH‐binding lobe and had previously been reported in an individual with type II RCM. Characterization of the three NADH‐binding lobe mutants using a heterologous expression system revealed that all three variants retained stoichiometric levels of flavin adenine dinucleotide with spectroscopic and thermodynamic properties comparable with those of native cytb5r. In contrast to the E255‐ and G291D variants, the novel D239G mutation had no adverse impact on protein thermostability. The D239G mutation perturbed substrate binding, causing both decreased specificity for NADH and increased specificity for NADPH. Thus cytb5r deficient patients who are heterozygous for an NADH‐binding lobe mutation can exhibit the clinically less severe type I phenotype, even in association with heterozygous deletion of the NADH‐binding lobe.

Comparison of diagnostic criteria for polycythaemia vera

Abstract Three sets of diagnostic criteria for polycythaemia vera (PY); the Polycythaemia Vera Study Group (PVSG) criteria (1975), the British Committee for Standards in Haematology (BCSH) criteria (1996) and the World Health Organisation (WHO) criteria (2001) have been described. We compared the ability of each set of criteria to accurately diagnose PV and differentiate it from secondary, apparent and idiopathic erythrocytosis. A cohort was drawn from a clinical database of erythrocytosis patients currently attending the Belfast City Hospital and the relevant information from the time of diagnosis for each patient was assessed according to each set of criteria, with the BCSH criteria used as a comparator. Sufficient data was available on 71 patients: 46 PV, 8 idiopathic, 8 apparent and 9 secondary erythrocytosis. The BCSH criteria classified 34 of 46 patients (73.9%) as PV and the WHO criteria had a sensitivity and specificity of 100% for classifying PV. For idiopathic and apparent erythrocytosis, the specificity of the WHO criteria, compared to the BCSH criteria, declined to 66.7 and 87.5%, respectively. The PVSG criteria were limited by the unavailability of required data for some patients resulting in a sensitivity and specificity of 50% for PV and specificity of 100% for all other groups. The Janus kinase 2 (JAK2) V617F mutation was present in 34 (85.3%) PV, 2 (50%) IE, 1 (50%) apparent and no secondary erythrocytosis cases. We concluded that the BCSH criteria were the most accurate diagnostic criteria for PV as they had an acceptable level of sensitivity and could differentiate between PV and other erythrocytoses.

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

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.

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

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.

The prevalence of CALR mutations in a cohort of patients with myeloproliferative neoplasms.

To investigate the prevalence of calreticulin (CALR) mutations in JAK2‐ and MPL‐non‐mutated patients with suspected myeloproliferative neoplasm (MPN) from a large MPN clinic and confirm a diagnosis of MPN.

A novel mutation, Ile289Thr, in the ALAS2 gene in a family with pyridoxine responsive sideroblastic anaemia

X-linked sideroblastic anaemia (XLSA; OMIM 301 300) is characterised by accumulation of inorganic iron in erythroblast mitochondria, visualised on staining as distinctive perinuclear rings. It arises from a deficiency of the erythroid specific isoenzyme of δ-aminolaevulinate synthase (ALAS2; E.C. 2.3.1.3.7), caused mainly by mutations affecting the catalytic or substrate-binding domains.1,2 ALAS2 uses pyridoxal-5-phosphate as a cofactor to catalyse the first, rate-limiting step of erythroid haem synthesis and pyridoxine treatment can alleviate anaemia in many cases, although the response is variable and affected by factors such as mutation, age and iron load.3,4 …