Genetic contribution to iron status: SNPs related to iron deficiency anaemia and fine mapping of CACNA2D3 calcium channel subunit.

Numerous studies associate genetic markers with iron- and erythrocyte-related parameters, but few relate them to iron-clinical phenotypes. Novel SNP rs1375515, located in a subunit of the calcium channel gene CACNA2D3, is associated with a higher risk of anaemia. The aim of this study is to further investigate the association of this SNP with iron-related parameters and iron-clinical phenotypes, and to explore the potential role of calcium channel subunit region in iron regulation. Furthermore, we aim to replicate the association of other SNPs reported previously in our population. We tested 45 SNPs selected via systematic review and fine mapping of CACNA2D3 region, with haematological and biochemical traits in 358 women of reproductive age. Multivariate analyses include back-step logistic regression and decision trees. The results replicate the association of SNPs with iron-related traits, and also confirm the protective effect of both A allele of rs1800562 (HFE) and G allele of rs4895441 (HBS1L-MYB). The risk of developing anaemia is increased in reproductive age women carriers of A allele of rs1868505 (CACNA2D3) and/or T allele of rs13194491 (HIST1H2BJ). Association of SNPs from fine mapping with ferritin and serum iron suggests that calcium channels could be a potential pathway for iron uptake in physiological conditions.

Evolution of iron overload in patients with low-risk myelodysplastic syndrome: iron chelation therapy and organ complications.

This study aimed to evaluate the evolution of iron overload, assessed by serum ferritin (SF), in transfusion-dependent lower risk patients with myelodysplastic syndrome (MDS), as well as to describe the occurrence of organ complications, and to analyze its relationship with iron chelation therapy. This observational retrospective study was conducted from March 2010 to March 2011 in 47 Spanish hospitals. A total of 263 patients with lower risk MDS (International Prognostic Scoring System [IPSS] low/intermediate-1 risk or Spanish Prognostic Index [SPI] 0-1 risk), transfusion-dependent, and who had received ≥10 packed red blood cells (PRBC) were included. At MDS diagnosis, patients received a mean of 2.8 ± 3.9 PRBC/month, and 8.7% of patients showed SF ≥1000 µg/L. Over the course of the disease, patients received a mean of 83.4 ± 83.3 PRBC, and 36.1% of patients presented SF ≥2500 µg/L. Cardiac, hepatic, endocrine, or arthropathy complications appeared/worsened in 20.2, 11.4, 9.9, and 3.8% of patients, respectively. According to investigator, iron overload was a main cause of hepatic (70.0%) and endocrine (26.9%) complications. A total of 96 (36.5%) patients received iron chelation therapy for ≥6 months, being deferasirox the most frequent first chelation treatment (71.9%). Chelation-treated patients showed longer overall survival (p < 0.001), leukemia-free survival (p = 0.007), and cardiac event-free survival (p = 0.017) than non-chelated patients. In multivariable analyses, age (p = 0.011), IPSS (p < 0.001), and chelation treatment (p = 0.015) were predictors for overall survival; IPSS (p = 0.014) and transfusion frequency (p = 0.001) for leukemia-free survival; and chelation treatment (p = 0.040) and Sorror comorbidity index (p = 0.039) for cardiac event-free survival. In conclusion, these results confirm the potential survival benefit of iron chelation therapy and provide additional evidence on the deleterious effect of iron overload in lower risk MDS patients.

Combined cobalamin and iron deficiency anemia: a diagnostic approach using a model based on age and homocysteine assessment.

Macrocytosis, the hallmark of cobalamin/folate deficiency anemia, is frequently absent. Clinicians have to be aware of coexisting conditions that can mask the macrocytosis expression of megaloblastic anemia, especially iron deficiency. The objective of this work was to investigate the degree of overlap between iron deficiency anemia (IDA) and cobalamin deficiency and to develop a predictive model for differentiating IDA from combined deficiency. A prospective case and control study was carried out to investigate vitamin B12 and folate status in iron deficiency anemia. A total of 658 patients were recruited, 41 of whom (6.2 %) were excluded. The remaining 617 subjects consisted of 130 controls and 487 with IDA. Low vitamin B12 (LB12) was considered when serum vitamin B12 was ≤200 pmol/L. High serum homocysteine (Hcy) was defined by Hcy >17 µM/L. A multivariate analysis (including a logistic regression) was performed to develop a diagnostic model. Low vitamin B12 levels were found in 17.8 % of IDA subjects. Ten out of 11 subjects (91 %) with IDA and serum vitamin B12 (B12) ≤100 pmol/L showed vitamin B12 deficiency. Moreover, vitamin B12 deficiency was demonstrated in 48 % of cases with IDA and B12 between 101 and 150 pmol/L and in 40 % with IDA and B12 between 151 and 200 pmol/, respectively. As a result of multivariate logistic analysis, neutrophil counts and age predicted subjects with vitamin B12 ≤200 and Hcy >17 µmol/L, [Formula: see text]. Using the age of 60 as a cutoff, sensitivity was 91 % (39 out of the 43 patients with vitamin B12 deficiency and IDA were identified). In summary, low vitamin B12 was found in 18 % of patients with IDA. Vitamin B12 deficiency was demonstrated in many patients with LB12 and IDA. Age over 60 years was used to separate patients with combined deficiency (sensitivity 91 %). Therefore, for a diagnostic purpose, serum vitamin B12 should be evaluated in IDA patients over 60 years. This diagnostic model needs to be validated in a different population.

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.

Iron overload and chelation therapy in patients with low-risk myelodysplastic syndromes with transfusion requirements.

The main objective of the study was to analyze the incidence of iron overload (IO) and its management in transfusion-dependent patients with low-risk myelodysplastic syndrome (MDS) before the license of deferasirox. This observational, cross-sectional, and multicenter study was conducted from January to May 2007 in 81 Spanish hospitals. Eligible patients had a low or intermediate-1 risk score and had to have received at least ten units of packed red blood cell (PRBC). Of the 549 patients analyzed, 75% had received more than 20 PRBC units since diagnosis; 14% had IO at diagnosis and 58% at last follow-up. Thirty-eight percent of patients received chelation therapy; of those, 92% were treated with desferrioxamine. Ferritin levels at the start of chelation therapy were higher than 1,000 microg/L in 76% and over 2,500 microg/L in 24% of patients. Of the 202 patients who received some form of chelation therapy, ferritin levels increased from a mean +/- SD of 1,986 +/- 1,398 to 2,480 +/- 1,648 microg/L at last follow-up in 86% (p < 0.001). In the remaining 29 patients treated with a minimally effective therapy, ferritin levels did not increase. Of these, only 11 patients received such therapy lasting more than 12 months. In conclusion, most low-risk transfusion-dependent MDS patients develop IO, but only a minority receives a minimally effective and timely iron chelation therapy.

Iron Deficiency in Menstruating Adult Women: Much More than Anemia.

Background: Iron deficiency anemia (IDA) is highly prevalent in women of child-bearing age. However, their nonhematological symptoms have been overlooked. This study aims to analyze the nonhematological features and symptoms of IDA in a group of women of reproductive age and the changes occurred during iron therapy. Materials and Methods: IDA women underwent dietary, physical activity, menstrual blood loss, and cognitive function assessment at baseline. Hematological and biochemical parameters were analyzed. Executive attention was tested by the flanker task and working memory by the 2-back task. Oral iron therapy (ferrous sulfate) was given to 35 women for 8 weeks and the changes in iron status, biochemical markers, cognitive function, and nonhematological symptoms were evaluated. Results: Patients presented nonhematological symptoms: pica, 32.4%; cheilitis, 20.6%; restless legs syndrome (RLS), 20.6%; diffuse hair loss, 55.9%; and ungual alterations, 38.2%. Two or more symptoms were present in 58.8% of women. Serum iron and working memory were correlated at baseline. Multivariate analyses show associations (odds ratio [OR], 95% confidence interval [CI]) between pica and reaction time in the working memory test (OR 2.14, 95% CI 1.19-3.87, p = 0.012); RLS with total serum protein (OR 0.08, 95% CI 0.06-0.92, p = 0.043); and cheilitis with mean corpuscular hemoglobin (OR 0.388, 95% CI 0.189-0.799, p = 0.01). Pica, cheilitis, and RLS completely resolved with iron therapy, and ungual alterations and hair loss improved in 92.3% and 84.2% of women, respectively. Better performance in executive attention and working memory was observed after iron therapy. Conclusions: More attention should be given to the nonhematological manifestations of IDA to improve the quality of life of menstruating women.

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.

Tumour cell lines HT-29 and FaDu produce proinflammatory cytokines and activate neutrophils in vitro: possible applications for neutrophil-based antitumour treatment.

There is evidence that polymorphonuclear neutrophils (PMNs) can exert severe antineoplastic effects. Cross-talk between tumour cells and endothelial cells (ECs) is necessary for the accumulation of PMN around a tumour. This work reports the ability of two PMN-sensitive, human, permanent cell lines-colorectal adenocarcinoma (HT-29) and pharyngeal squamous-cell carcinoma (FaDu) cells-to act as inflammatory foci. PMNs were cytotoxic to both lines, the adhesion of the PMNs to the tumour cells being important in this effect. The tumour cells released appreciable amounts of IL-8 and GROalpha, and induced the transmigration of PMN through human microvascular-EC monolayers. Conditioning media associated with both lines induced the adhesion of PMN and the surface expression of ICAM-1 in microvascular-EC. In addition, FaDu-conditioning-medium strongly induced the production of proinflammatory cytokines by microvascular-EC. These results support the idea that tumour cells might normally induce a potent acute inflammatory response, leading to their own destruction.

MYH9 Associated nephropathy. / Nefropatía asociada a mutación del gen MYH9.

MYH9 related diseases are caused by mutations in the MYH9 gene and constitute a rare group of genetic entities. Its inheritance follows an autosomal dominant pattern. The MYH9 gene, encodes the nonmuscle myosin heavy chain IIA, expressed in different tissues and especially in podocytes and mesangial cells. The disorder is characterized by the presence of macrothrombocytopenia, leukocyte inclusions and a variable risk of developing renal failure, hearing loss and early-onset cataracts. We describe the case of a 27-year-old Caucasian woman, diagnosed initially with idiopathic thrombocytopenic purpura. After a detailed family history and the appearance of renal involvement and hearing loss, genetic testing allowed to make the diagnosis of nephropathy associated with MYH9 mutation. This case is an example of the delayed diagnosis of uncommon diseases and highlights the usefulness genetic testing. A review of the disease is provided.

Ultrastructural, cytogenetic, and molecular findings in mast cell leukemia: Case report.

We report a de novo aleukemic form of MCL with a complex monosomic karyotype with LOH for multiple chromosomes and TP53 mutation. Additionally, whereas D816V KIT was not found, the c-Kit transmembrane domain p.M541L variant was detected which is the most common SNP of KIT gene in humans with controversial pathogenic role. In these cases, it is crucial to perform a rapid broad molecular study for an accurate diagnosis which could help to initiate targeted therapy.

The V617F mutation of JAK2 is very uncommon in patients with thrombosis.

Given that many cases of thrombosis do not have a clear cause, a myeloproliferative disease could be involved. We investigated the V617F mutation of the JAK2 gene in 295 patients with thrombosis. Only one case was positive. Therefore, the study of this mutation is not necessary in all patients with idiopathic thrombosis.

The relationship between transferrin saturation and erythropoiesis during stem cell transplantation.

Ninety-seven percent of 81 patients had a transferrin saturation (TS) level >80% from day 0 of their stem cell transplant. This phenomenon was inversely related with reticulocyte count changes (p<0.0001). The time with a TS > 80% was predicted by reticulocyte recovery (p=0.031) in multivariate analysis. The kinetics of TS is a direct consequence of erythropoietic activity during stem cell transplantation.

Occurrence of the JAK2 V617F mutation in the WHO provisional entity: myelodysplastic/myeloproliferative disease, unclassifiable-refractory anemia with ringed sideroblasts associated with marked thrombocytosis.

The JAK2/V617F mutation has been noted in essential thrombocytemia. We investigated 19 cases with refractory anemia with ringed sideroblasts (RARS), including three RARS with thrombocytosis (RARS-T). Only the RARS-T patients showed this mutation. More cases need to be analyzed to determine the prevalence of the JAK2/V617F mutation in RARS-T.

[Treatment of type 1 hereditary haemochromatosis with oral magnesium]. / Tratamiento de la hemocromatosis hereditaria tipo 1 con magnesio oral.

BACKGROUND AND OBJECTIVE: An essential step in the pathogenesis of hereditary hemochromatosis seems to be the increased expression of a duodenal divalent cation transporter (DMT1) responsible for absorption of non-heminic iron2+. The objective of the present study was to ascertain whether the competitive blockade of DMT1 by the administration of high doses of oral Mg2+ reduces iron absorption in patients homozygous for the C282Y mutation. PATIENTS AND METHOD: Iron absorption was evaluated by a low dose iron absorption test in 15 patients before and after treatment with oral magnesium (809.6 mg every 8 hours) for two weeks. RESULTS: We did not observe secondary effects or significant differences in iron absorption before or after magnesium treatment (14.7 micromol/L; 95% confidence interval [CI], 9.8-19.6 vs 14.9 micromol/L; 95% CI, 8.5-21.4, P = 0.7). CONCLUSIONS: Treatment with oral magnesium does not reduce iron absorption in homozygous C282Y patients. This treatment can not be used in these subjects.

[Evaluation of V617F mutation of JAK2 in negative chromosome Philadelphia chronic myeloproliferative disorders]. / Valoración de la mutación V617F del gen JAK2 en síndromes mieloproliferativos crónicos con cromosoma Filadelfia negativo.

BACKGROUND AND OBJECTIVE: Polycythemia vera (PV) and essential thrombocytemia (ET) are chronic myeloproliferative diseases (MPD) characterized by overactive hemopoiesis. A single point mutation of JAK2 (Val617Phe) has been detected in PV, ET and myelofibrosis (MF). The aim of this work was to investigate the JAK2 mutation in patients with MPD and to compare the results to those of the endogenous formation of BFU-E erythroid colonies (EEC). Finally, different sources of hematopoietic cells to obtain DNA were evaluated. PATIENTS AND METHOD: In this work 146 patents were studied (81 MPD: 27 PV, 28 ET, 11 MF and 15 with myeloid chronic leukemia). Moreover, 28 patients showed secondary polycythemias or reactive thrombocytosis, 8 MPD/myelodysplastic syndromes and 29 other disorders. In 54 patients, EEC were also evaluated. Peripheral blood cells were used as source of DNA in 122 patients, bone marrow in 33, cells from BFU-E in 14 and cells from EEC in 24 patients. Their DNA samples were analyzed using an allele-specific polimerase chain reaction methodology. RESULTS: The JAK2 mutation was present in 96% of PV patients, 59% of ET and 63.6% of MF. None of the remaining patients showed this mutation. Diagnostic agreement was excellent between EEC and the mutation (kappa index = 0.93; 97% positive agreement and 95% negative agreement). DNA was obtained in 119 out of 122 samples from peripheral blood, in all patients with bone marrow, and in 50% of patients with BFU-E or EEC. In 7 cases, samples from different cell sources were studied. Their results were identical. CONCLUSIONS: The V617F mutation of JAK2 is present in most of PV patients and half of those with MF or ET. There is an excellent concordance with the EEC results.

Influence of cobalamin deficiency compared with that of cobalamin absorption on serum holo-transcobalamin II.

BACKGROUND: Cobalamin attached to transcobalamin II (TC II), known as holo-TC II, is the active cobalamin fraction taken up by tissues. Holo-TC II is also the form in which absorbed cobalamin enters the circulation from the ileum. Therefore, holo-TC II has been proposed variously as a marker of cobalamin adequacy, cobalamin absorption, or both, including even its advocacy as a surrogate Schilling test. Such claims carry conflicting diagnostic implications because metabolic adequacy and absorption are not identical. OBJECTIVE: The objective was to examine metabolic and absorptive influences on holo-TC II. DESIGN: Treated patients with pernicious anemia (PA), who have abnormal absorption but a normal metabolic status, were chosen as the model to differentiate between the effects of the 2 cobalamin-related characteristics. Serum holo-TC II and indexes of cobalamin metabolism in 23 treated patients were compared with those of 6 untreated PA patients (abnormal absorption and metabolic status) and 33 control subjects (normal absorption and metabolic status). RESULTS: Holo-TC II, which correlated directly with cobalamin and inversely with homocysteine, was significantly higher in treated PA patients in metabolic remission than in untreated PA patients (74 +/- 59 compared with 9 +/- 6 pmol/L) and was significantly lower than in control subjects (105 +/- 58 pmol/L), although the latter difference was small and the values overlapped greatly. CONCLUSIONS: Metabolic cobalamin status is a major determinant of serum holo-TC II. Absorption status may have mild influence as well, although other explanations remain possible. Serum holo-TC II cannot be used clinically to diagnose cobalamin malabsorption because of overlap with normal values. The influences on holo-TC II are complex and require careful analysis.