Joanne Marsden

Definition of subtypes of essential thrombocythaemia and relation to polycythaemia vera based on JAK2 V617F mutation status: a prospective study

BACKGROUND An acquired V617F mutation in JAK2 occurs in most patients with polycythaemia vera, but is seen in only half those with essential thrombocythaemia and idiopathic myelofibrosis. We aimed to assess whether patients with the mutation are biologically distinct from those without, and why the same mutation is associated with different disease phenotypes. METHODS Two sensitive PCR-based methods were used to assess the JAK2 mutation status of 806 patients with essential thrombocythaemia, including 776 from the Medical Research Councils Primary Thrombocythaemia trial (MRC PT-1) and two other prospective studies. Laboratory and clinical features, response to treatment, and clinical events were compared for V617F-positive and V617F-negative patients with essential thrombocythaemia. FINDINGS Mutation-positive patients had multiple features resembling polycythaemia vera, with significantly increased haemoglobin (mean increase 9.6 g/L, 95% CI 7.6-11.6 g/L; p<0.0001), neutrophil counts (1.1x10(9)/L, 0.7-1.5x10(9)/L; p<0.0001), bone marrow erythropoiesis and granulopoiesis, more venous thromboses, and a higher rate of polycythaemic transformation than those without the mutation. Mutation-positive patients had lower serum erythropoietin (mean decrease 13.8 U/L; 95% CI, 10.8-16.9 U/L; p<0.0001) and ferritin (n=182; median 58 vs 91 mug/L; p=0.01) concentrations than did mutation-negative patients. Mutation-negative patients did, nonetheless, show many clinical and laboratory features that were characteristic of a myeloproliferative disorder. V617F-positive individuals were more sensitive to therapy with hydroxyurea, but not anagrelide, than those without the JAK2 mutation. INTERPRETATION Our results suggest that JAK2 V617F-positive essential thrombocythaemia and polycythaemia vera form a biological continuum, with the degree of erythrocytosis determined by physiological or genetic modifiers.

C-Terminal Deletions in the ALAS2 Gene Lead to Gain of Function and Cause X-linked Dominant Protoporphyria without Anemia or Iron Overload

All reported mutations in ALAS2, which encodes the rate-regulating enzyme of erythroid heme biosynthesis, cause X-linked sideroblastic anemia. We describe eight families with ALAS2 deletions, either c.1706-1709 delAGTG (p.E569GfsX24) or c.1699-1700 delAT (p.M567EfsX2), resulting in frameshifts that lead to replacement or deletion of the 19-20 C-terminal residues of the enzyme. Prokaryotic expression studies show that both mutations markedly increase ALAS2 activity. These gain-of-function mutations cause a previously unrecognized form of porphyria, X-linked dominant protoporphyria, characterized biochemically by a high proportion of zinc-protoporphyrin in erythrocytes, in which a mismatch between protoporphyrin production and the heme requirement of differentiating erythroid cells leads to overproduction of protoporphyrin in amounts sufficient to cause photosensitivity and liver disease.

Erythropoietin depletion and anaemia in diabetes mellitus.

Aims To discover whether Type 1 diabetic patients with autonomic neuropathy might be anaemic and erythropoietin (EPO)‐depleted.

Erythropoietin response to hypoxia in patients with diabetic autonomic neuropathy and non‐diabetic chronic renal failure

Aims An erythropoietin (EPO)‐deficient anaemia is recognized in Type 1 diabetic patients with early nephropathy and symptomatic autonomic neuropathy (DN). The aim of this study was to determine whether the EPO response to hypoxia was deficient in order to clarify the mechanisms involved in this process.

Autonomic function and serum erythropoietin levels in chronic fatigue syndrome

OBJECTIVE Given previous findings, we wished to investigate whether there was evidence of autonomic dysfunction in patients with chronic fatigue syndrome, and whether this could be related to reduced erythropoietin levels and altered red blood cell indices. METHODS We assessed autonomic function and analysed blood parameters (including erythropoietin) in 22 patients with chronic fatigue syndrome who were medication-free and without comorbid depression or anxiety. Results were compared to 23 iron-deficiency anaemia patients and 18 healthy individuals. RESULTS Autonomic testing in patients with chronic fatigue syndrome yielded a significantly greater increase in heart rate together with a more pronounced systolic blood pressure fall on standing compared to healthy individuals. Heart rate beat-to-beat variation on deep breathing and responses to the Valsalva manoeuvre were normal. Two of 22 patients with chronic fatigue had mild normochromic normocytic anaemia with normal ferritin, vitamin B12 and folate levels. Serum erythropoietin levels were within reference range. CONCLUSION Some autonomic dysfunction is present in chronic fatigue syndrome (CFS) patients; the explanation remains uncertain, but could relate to cardiovascular deconditioning. There were no major haematological, biochemical or immunological abnormalities in these patients.

Successful control of hyperparathyroidism in patients on continuous ambulatory peritoneal dialysis using magnesium carbonate and calcium carbonate as phosphate binders.

To avoid the use of aluminium as a phosphate binder, patients on CAPD who were stable were dialysed against a peritoneal dialysis fluid which was magnesium free. A mixture of calcium and magnesium carbonate was used as a phosphate binder over a period in excess of 1 year. Vitamin D analogues were used in the majority. Results show satisfactory control of hyperparathyroidism with mean parathyroid hormone concentration for the group of 121 pg/ml (normal < 100 pg/ml), calcium concentration of 2.41 mmol/l, magnesium 0.97 mmol/l, phosphate 1.36 mmol/l and aluminium 0.35 mmol/l (normal < 0.2 mumol/l). These results were as good as and better in some respects than a minority using calcium carbonate alone or remaining on aluminium hydroxide, the latter remaining on Mg-containing CAPD fluid.

Erythropoietin deficiency and anaemia in multiple system atrophy.

Serum erythropoietin (EPO) levels are partially controlled by the sympathetic outflow to the kidney. We have studied whether patients with multiple system atrophy (MSA), known to be associated with dysautonomia, are EPO‐deficient. Eighteen MSA patients were studied along with 32 idiopathic Parkinsons disease (PD) patients, 23 controls with iron‐deficiency anaemia, and 18 healthy individuals. Serum creatinine was normal in all groups. Mean haemoglobin (Hb) concentration in MSA patients was 13.7 ± 1.7 g/dL. Four MSA patients had unexplained anaemia (minimum Hb: 10.5 g/dL) and abnormal autonomic function tests including significant postural hypotension, whereas none of the PD patients was anaemic. Serum EPO levels were suppressed in relation to anaemia in MSA patients compared to elevated EPO levels in iron‐deficiency anaemia patients (difference of regression lines P < 0.001), indicating EPO deficiency in the anaemic MSA patients. Serum EPO levels in PD patients were within normal range. A subset of MSA patients has anaemia and postural hypotension, which may be associated with EPO deficiency. This may have therapeutic implications.

European Specialist Porphyria Laboratories: Diagnostic Strategies, Analytical Quality, Clinical Interpretation, and Reporting as Assessed by an External Quality Assurance Program

BACKGROUND The porphyrias are a group of rare metabolic disorders whose diagnosis depends on identification of specific patterns of porphyrin precursor and porphyrin accumulation in urine, blood, and feces. Diagnostic tests for porphyria are performed by specialized laboratories in many countries. Data regarding the analytical and diagnostic performance of these laboratories are scarce. METHODS We distributed 5 sets of multispecimen samples from different porphyria patients accompanied by clinical case histories to 18-21 European specialist porphyria laboratories/centers as part of a European Porphyria Network organized external analytical and postanalytical quality assessment (EQA) program. The laboratories stated which analyses they would normally have performed given the case histories and reported results of all porphyria-related analyses available, interpretative comments, and diagnoses. RESULTS Reported diagnostic strategies initially showed considerable diversity, but the number of laboratories applying adequate diagnostic strategies increased during the study period. We found an average interlaboratory CV of 50% (range 12%-152%) for analytes in absolute concentrations. Result normalization by forming ratios to the upper reference limits did not reduce this variation. Sixty-five percent of reported results were within biological variation-based analytical quality specifications. Clinical interpretation of the obtained analytical results was accurate, and most laboratories established the correct diagnosis in all distributions. CONCLUSIONS Based on a case-based EQA scheme, variations were apparent in analytical and diagnostic performance between European specialist porphyria laboratories. Our findings reinforce the use of EQA schemes as an essential tool to assess both analytical and diagnostic processes and thereby to improve patient care in rare diseases.

Erythropoietin - measurement and clinical applications

Erythropoietin (EPO) is an endogenous hormone produced primarily by the kidney which controls the production of erythrocytes. The main stimulus to production is low tissue oxygen (hypoxia) and EPO triggers the formation of red blood cells by binding to a receptor on erythroid progenitor target cells. Alteration in the EPO regulatory system produces a change in circulating EPO in a variety of disease states, such as renal anaemia and polycythaemia. The availability of recombinant EPO in the 1980s transformed the treatment of anaemia, particularly anaemia of end stage renal disease, and led to the development of more sensitive and specific assays for the measurement of EPO. There are more widespread uses for EPO and preliminary studies indicate that EPO may be useful as a neuroprotective agent by reducing inflammation near the site of injury. The use of EPO to boost endurance in athletes has attracted unwanted publicity, although analytical techniques are now available that can differentiate between endogenous and recombinant EPO. Different types of erythropoietic agents have been developed with a longer plasma half-life and the ability to maintain haemoglobin levels for longer periods and reduce the need for frequent dosing with EPO.

Urinary excretion of porphyrins, porphobilinogen and δ-aminolaevulinic acid following an attack of acute intermittent porphyria

Background and objectives The porphyrias are a group of rare, mainly inherited, diseases caused by a deficiency of one of the enzymes of the haem biosynthesis pathway. The biochemical hallmark of an acute attack is an increase in urine porphobilinogen (PBG), together with an increase in urinary excretion of δ-aminolaevulinic acid (ALA) and total urine porphyrins (TUP). In patients with acute intermittent porphyria (AIP) the concentrations of the porphyrin precursors are thought to remain elevated for many years following an acute attack, although this has not been well documented. Methods We measured urine ALA, PBG and TUP excretion in 20 patients with AIP following an attack of acute porphyria over a time period of 3 months to 23 years after their last documented acute attack. Results We showed that urinary concentrations of all metabolites remain elevated for many years. The urinary half life of TUP was 5.3 years, ALA 7.7 years and PBG 10.6 years. Even after 20 years, PBG concentrations remained elevated above the normal range. Conclusions Our study highlights the difficulties of using urinary analysis for diagnosing recurrent attacks, and also raises important questions about the pathophysiology of the condition.