Angel F. Remacha

Matriptase-2 mutations in iron-refractory iron deficiency anemia patients provide new insights into protease activation mechanisms

Mutations leading to abrogation of matriptase-2 proteolytic activity in humans are associated with an iron-refractory iron deficiency anemia (IRIDA) due to elevated hepcidin levels. Here we describe two novel heterozygous mutations within the matriptase-2 (TMPRSS6) gene of monozygotic twin girls exhibiting an IRIDA phenotype. The first is the frameshift mutation (P686fs) caused by the insertion of the four nucleotides CCCC in exon 16 (2172_2173insCCCC) that is predicted to terminate translation before the catalytic serine. The second mutation is the di-nucleotide substitution c.467C>A and c.468C>T in exon 3 that causes the missense mutation A118D in the SEA domain of the extracellular stem region of matriptase-2. Functional analysis of both variant matriptase-2 proteases has revealed that they lead to ineffective suppression of hepcidin transcription. We also demonstrate that the A118D SEA domain mutation causes an intra-molecular structural imbalance that impairs matriptase-2 activation. Collectively, these results extend the pattern of TMPRSS6 mutations associated with IRIDA and functionally demonstrate that mutations affecting protease regions other than the catalytic domain may have a profound impact in the regulatory role of matriptase-2 during iron deficiency.

Impact of ABO incompatibility on allogeneic peripheral blood progenitor cell transplantation after reduced intensity conditioning.

BACKGROUND:  Most studies indicate that ABO incompatibility has no effect on the clinical outcome after allogeneic peripheral blood progenitor cell (PBPC) transplantation (allo‐PBPCT). However, it carries additional risks of hemolytic reactions, delayed red blood cell (RBC) engraftment, and pure red cell aplasia (PRCA). Data on these events after reduced intensity conditioning (RIC) regimens are limited, but recent studies have suggested a higher transplant‐related mortality (TRM) and morbidity in this setting.

VITAMIN B-12 ABNORMALITIES IN HIV-INFECTED PATIENTS

Abstract: A prospective study of 60 consecutively admitted patients with HIV infection was performed to document the prevalence, etiology and manifestations of low serum vitamin B‐12 in such patients. Low serum B‐12 levels were found in 10 patients (16.7%). In 6, vitamin B‐12 absorption was impaired and hog intrinsic factor addition did not improve it. Patients with low vitamin B‐12 levels showed lower hemoglobin, leukocytes, lymphocytes, CD4 lymphocytes and CD4/CD8 lymphocyte ratio than HIV patients with physiological serum vitamin B‐12 levels. However, bone marrow megaloblastosis was found in only 3 low vitamin B‐12 patients and the deoxyuridine suppression test was pathological in only 1 case. In 7 patients, parented treatment was begun with variable response despite serum vitamin B‐12 correction. In conclusion, low serum vitamin B‐12 is often found in HIV‐infected patients and it could be related to malabsorption, but clear megaloblastic abnormalities and treatment response could not be demonstrated. A decreased concentration of the serum binders due to disturbances in the leukocytes and related immunocompetent cell may play an additional role.

Serum soluble transferrin receptor concentrations are increased in central obesity. Results from a screening programme for hereditary hemochromatosis in men with hyperferritinemia.

BACKGROUND A decrease in the serum concentrations of the soluble transferrin receptor (sTfR) is considered a good index of tissue iron. Because obesity is associated with hyperferritinemia and this is considered a sign of iron overload, a decrease in sTfR would be expected for the obese. We evaluated whether obese men with hyperferritinemia, detected in a genetic screening programme for hereditary hemochromatosis (HH), have lower serum concentrations of sTfR than their non-obese counterparts. METHODS 75 men (age: 55.4+/-12.4 years) with hyperferritinemia (serum ferritin--SF > 200 microg/L) and no known conditions of iron overload were evaluated for body mass index (BMI), waist circumference (WC), blood pressure, traditional indices of iron status, sTfR, fasting plasma glucose, lipid profile, insulin resistance (HOMA-IR), highly-sensitive C-reactive protein, hepatic enzymes and HFE gene mutations of HH. RESULTS sTfR correlated with BMI (r=0.289; p=0.014) and with WC (r=0.420; p<0.001). Thirty-two subjects were obese (BM > or = 30 kg/m(2)) and had a significantly higher sTfR (2.95 (2.22-3.28) vs 2.28 (1.88-2.91) mg/L; p=0.013), hemoglobin (157+/-12 vs 152+/-11 gr/L; p=0.049) and HOMA-IR (1.38 (1.04-2.69) vs 1.02 (0.60-1.55) mg/L; p=0.009) than the non-obese. WC explained separately more variability of the sTfR than BMI (r(2)=0.177; p=0.002 and r=0.077; p=0.042, respectively), after adjusting for potential confounders. CONCLUSION An increase in serum concentrations of sTfR is associated with central obesity in men with hyperferritinemia.

Mutations in HAMP and HJV genes and their impact on expression of clinical hemochromatosis in a cohort of 100 Spanish patients homozygous for the C282Y mutation of HFE gene

Most hereditary hemochromatosis (HH) patients are homozygous for the C282Y mutation of the HFE gene. Nevertheless, penetrance of the disease is very variable. In some patients, penetrance can be mediated by concomitant mutations in other iron master genes. We evaluated the clinical impact of hepcidin (HAMP) and hemojuvelin mutations in a cohort of 100 Spanish patients homozygous for the C282Y mutation of the HFE gene. HAMP and hemojuvelin mutations were evaluated in all patients by bidirectional direct cycle sequencing. Phenotype–genotype interactions were evaluated. A heterozygous mutation of the HAMP gene (G71D) was found in only one out of 100 cases. Following, we performed a study of several members of that family, and we observed several members had a digenic inheritance of the C282Y mutation of the HFE gene and the G71D mutation of the HAMP gene. This mutation in the HAMP gene did not modify the phenotype of the individuals who were homozygous for the C282Y mutation. One other patient presented a new polymorphism in the hemojuvelin gene, without consequences in iron load or clinical course of the disease. In conclusion, HAMP and hemojuvelin mutations are rare among Spanish HH patients, and their impact in this population is not significant.

Does the SLC40A1 gene modify HFE-related haemochromatosis phenotypes?

Most hereditary haemochromatosis patients are homozygous for the C282Y mutation of the HFE gene. However, the phenotypic expression and clinical aggressiveness of the disease differs considerably from patient to patient. The main objective of this work was to study the role of variants in the SLC40A1 gene in the severity of iron overload and his clinical consequences in 100 Spanish probands homozygous for the C282Y mutation of the HFE gene. We performed automated sequencing of the coding regions, including intron–exon junctions of the SLC40A1 gene. We studied the association between polymorphisms in the SLC40A1 gene and median values of iron removed, taking into account statistical corrections for multiple comparisons. No pathogenic mutations in the SLC40A1 were detected. Five known single nucleotide polymorphisms (SNPs) were identified, and two of them were associated with phenotypic characteristics. IVS1-24 C>G was associated with the amount of iron removed and presence of liver disease: Of the 83 patients finally studied for this SNP, the amount of iron removed was above the median in 36 of 56 (64.3%) for C/C, in nine of 23(39.1%) for C/G and in zero of four (0%) for G/G patients (P = 0.01). Liver damage was observed in 34 of 56 patients (60.7%) for C/C, in eight of 23 (34.8%) for C/G and in zero of four (0%) for G/G (P = 0.01). Both associations remained significant at multivariate analysis (P = 0.011 and P = 0.023, respectively). IVS1-24 C>G on the ferroportin gene seems to be a genetic modifier for clinical aggressiveness of HFE1 haemochromatosis.

Genotype–phenotype correlation in a Spanish population homozygous for the H63D mutation of the HFE gene

Hereditary hemochromatosis (HH) is an autosomal recessive disorder of iron metabolism with excessive cellular iron levels resulting in tissue damage [1–3]. In 1996, Feder et al. showed that in most HH patients, the HFE gene is mutated in position 282 (replacement of cysteine by tyrosine, C282Y) [4]. In Spain, 85–90% of HH patients showed this mutation [5, 6]. However, a second mutation, the replacement of histidine in position 63 by aspartic acid (H63D), is much more frequent. In our area, the Northeast of Spain, the H63D mutation was present in 37% of the general population, and 4% were homozygous [7]. In a murine model, the contribution of the H63D homozygosity (DD genotype) was demonstrated. However, the iron overload (IO) was below that of the C282Y homozygosity (YY genotype) [8]. Moreover, data in humans support that the DD genotype could result in IO [9–11]. Given the high prevalence of this mutation in our population, we evaluated the genotype–phenotype correlation in homozygotes for the H63D mutation. Two studies were conducted in two Spanish populations. First, the correlation between the HFE mutations and the amount of iron in the liver was evaluated in 78 cases (7 cases were DD homozygotes) [12]. Liver biopsy was carried out because of a biochemical IO [serum transferrin saturation >50% and/or serum ferritin >350 μg/l]. Hemochromatosis was considered when the hepatic iron index (HII) exceeded 1.9 μmol/g of dry weight multiplied by the age in years, μmol/g.year [1–3]. In 30 out of 78 cases, HII exceeded 1.9 μmol/g.year, including three cases (10%) homozygous for the H63D mutation (HII=4.2, 3.9, and 3.8 μmol/g.year). Moreover, in one patient homozygous for the H63D mutation, the HII was 1.8 μmol/g.year Therefore, four out of the seven DD homozygotes showed moderate liver IOs (57%). C282Y homozygotes showed the highest levels of HII. DD homozygotes had higher levels of HII than wild-type patients and H63D heterozygotes (Table 1). The second study was conducted between 1998 and 2004 in 55 consecutive patients with the DD genotype. All patients came from our university hospital and were studied for all the known secondary causes of biochemical IO [13, 14]. Data were not available in one case, which was excluded from the study. Hepcidin (HAMP) gene mutations were investigated given that these mutations could modify the severity of the IO [15]. Transferrin saturation and serum ferritin were evaluated by automated methodologies (Hitachi 911 and Elecsys, Roche). Moreover, total iron removed by phlebotomy was calculated in grams. Mutations of the HFE gene (C282Y, H63D, and S65C) were analyzed with LightCycler equipment [6, 7, 14]. The HAMP gene was evaluated using single strand conformational polymorphism and direct sequencing [15]. The patients were 36 men and 18 women, with a median age was 51 years. They were chosen for the study because of a biochemical IO (41 cases) or a family study (13 cases). Forty-five patients (83%) showed a biochemical IO pattern. An underlying cause of biochemical IO was observed in 35 A. F. Remacha (*) . M. P. Sarda . M. J. Barcelo . V. Bach . A. Altes . M. Baiget . C. Guarner . I. Blesa Hematology Department, Hospital de Sant Pau, Avda Padre Claret 167, Barcelona, 0825, Spain e-mail: aremacha@hsp.santpau.es

Intermittent hypoxia exposure in a hypobaric chamber and erythropoietin abuse interpretation

Abstract The aim of this study was to assess the effect of intermittent hypoxia exposure on direct and indirect methods used to evaluate recombinant human erythropoietin (rhEPO) misuse. Sixteen male triathletes were randomly assigned to either the intermittent hypoxia exposure group (experimental group) or the control normoxic group (control group). The members of the experimental group were exposed to simulated altitude (from 4000 to 5500 m) in a hypobaric chamber for 3 h per day, 5 days a week, for 4 weeks. Blood and urine samples were collected before and after the first and the final exposures, and again 2 weeks after the final exposure. While serum EPO significantly increased after the first [from a mean 8.3 IU · l−1 (s = 3.2) to 16.6 IU · l−1 (s = 4.7)] and final exposures [from 4.6 IU · l−1 (s = 1.4) to 24.8 IU · l−1 (s = 9.3)], haemoglobin, percentage of reticulocytes, and soluble transferrin receptor were not elevated. Second-generation ON/OFF models (indirect rhEPO misuse detection) were insensitive to intermittent hypoxia exposure. The distribution of the urinary EPO isoelectric profiles (direct rhEPO misuse detection) was altered after intermittent hypoxia exposure with a slight shift towards more basic isoforms. However, those shifts never resulted in misinterpretation of results. The intermittent hypoxia exposure protocol studied did not produce any false-positive result for indirect or direct detection of rhEPO misuse in spite of the changes in EPO serum concentrations and urinary EPO isoelectric profiles, respectively.

The relationship between iron overload and clinical characteristics in a Spanish cohort of 100 C282Y homozygous hemochromatosis patients

We studied the relationship between iron removed by venesection, sex, age, and clinical characteristics in a group of 100 Spanish probands with hereditary hemochromatosis (HH), all C282Y homozygous in the HFE gene. Iron overload was higher in men than in women (P < 0.0001) and increased with age (P = 0.02). Forty-four patients presented with liver disease (28 had fibrosis–cirrhosis of the liver), 24 with diabetes, 18 with arthropathy, and 13/73 men with impotence. No clinical consequences of hemochromatosis were observed in 43 patients. The number of clinical complications was higher in men (P = 0.01) and increased with age (P = 0.006) and with the amount of iron removed (P < 0.0001). The amount of iron removed was significantly higher by univariate analysis in patients with liver disease (P < 0.0001), diabetes (P = 0.007), arthropathy (P = 0.006), and impotence (P = 0.003) than in patients without these complications. In the multivariant analysis, only liver disease maintained a significant relationship with the amount of iron removed (P < 0.0001). Diabetes and arthropathy were closely related with previous liver disease, and impotence appeared mainly in hemochromatosic men with diabetes and alcoholism.

Guidelines on haemovigilance of post-transfusional iron overload.

Currently, haematologists working both in haemotherapy and clinical haematology have no guidelines for the management and treatment of patients with post-transfusional iron overload. To address this issue, the Spanish Society of Haematology and Haemotherapy (SEHH) and the Spanish Society of Blood Transfusion (SETS) selected several of their members to develop “Guidelines on Haemovigilance of Post-Transfusional Iron Overload”. The first step consisted in a systematic MEDLINE search that included articles published from 1980 to October 2010 using the search term limits “iron overload”, “iron chelation therapy”, “thalassemia”, “myelodysplastic syndromes”, and “magnetic resonance imaging”. Then, in a face-to-face working meeting held in November 2009, the contents and sections of the guidelines were established, the group coordinators and members were selected, the working method to be followed was defined, and the deadlines were set. The members and group coordinators consisted of a team of experts appointed by the SEHH and the SETS, who were to work in one of five working groups. Overall, each of the five working groups consisted of three to five members (one member could work in more than one working group), and one group coordinator who was responsible for establishing a dialogue between members, and setting deadlines. The purpose of the five working groups was to: (i) define the importance of monitoring iron overload in patients with chronic transfusion dependence; (ii) characterise the target population, i.e. which patients should be monitored and how this monitoring should be carried out; (iii) describe the procedures for monitoring transfusional haemosiderosis, including assessment of ferritin levels, transferrin saturation index, units of packed red blood cell units transfused, and general measures for the care of patients; (iv) define which information mechanisms and alert systems should be put into practice, what information must be recorded in a patient’s transfusion history and how this information can be made accessible to treating physicians and generate a computerised warning system for patients at risk of transfusional haemosiderosis based on the patient’s transfusion history; and (v) describe an action protocol in the case of iron overload (Figure 1). Figure 1 Process of elaboration of this guideline. Once each working group had developed the contents of the five main topics, the information was first sent to the appropriate group coordinator to be revised and approved, and then to the rest of the authors for their approval. Before June 2010, two additional face-to-face working meetings and several videoconferences were held in order to unify concepts and issue final recommendations. The contents of these guidelines are, therefore, divided into five different sections. The purpose is to provide the best recommendations from currently available scientific evidence on the issues raised, thus filling the void in the field of post-transfusional iron overload. Because the clinical benefit of many strategies discussed herein is widely accepted, despite not being supported by high-quality randomised trials, the group of experts agreed that the use of levels of evidence was not compulsory in the present guidelines.