miR-146a rs2431697 identifies myeloproliferative neoplasm patients with higher secondary myelofibrosis progression risk.

Myelofibrosis (MF) occurs as part of the natural history of polycythemia vera (PV) and essential thrombocythemia (ET), and remarkably shortens survival. Although JAK2V617F and CALR allele burden are the main transformation risk factors, inflammation plays a critical role by driving clonal expansion toward end-stage disease. NF-κB is a key mediator of inflammation-induced carcinogenesis. Here, we explored the involvement of miR-146a, a brake in NF-κB signaling, in MPN susceptibility and progression. rs2910164 and rs2431697, that affect miR-146a expression, were analyzed in 967 MPN (320 PV/333 ET/314 MF) patients and 600 controls. We found that rs2431697 TT genotype was associated with MF, particularly with post-PV/ET MF (HR = 1.5; p < 0.05). Among 232 PV/ET patients (follow-up time=8.5 years), 18 (7.8%) progressed to MF, being MF-free-survival shorter for rs2431697 TT than CC + CT patients (p = 0.01). Multivariate analysis identified TT genotype as independent predictor of MF progression. In addition, TT (vs. CC + CT) patients showed increased plasma inflammatory cytokines. Finally, miR-146a-/- mice showed significantly higher Stat3 activity with aging, parallel to the development of the MF-like phenotype. In conclusion, we demonstrated that rs2431697 TT genotype is an early predictor of MF progression independent of the JAK2V617F allele burden. Low levels of miR-146a contribute to the MF phenotype by increasing Stat3 signaling.

Detection of new pathogenic mutations in patients with congenital haemolytic anaemia using next-generation sequencing.

INTRODUCTION: Congenital haemolytic anaemia (CHA) refers to a group of genetically heterogeneous disorders, mainly caused by changes in genes encoding globin chains, cytoskeletal proteins and red cell enzymes, in which accurate diagnosis can be challenging with conventional techniques. METHODS: To set-up a comprehensive assay for detecting mutations that could improve aetiological diagnosis, we designed a custom panel for sequencing coding regions from 40 genes known to be involved in the pathogenesis of CHA, using the Ion Torrent™ (Thermo Fisher Scientific, S.L. Waltham, MA, USA) Personal Genome Machine (PGM) Sequencer. A control group of 16 samples with previously known mutations and a test group of 10 patients with unknown mutations were included for assay validation and application, respectively. RESULTS: In the test group, we identified pathogenic mutations in all cases: four patients had novel mutations in genes related to membrane defects (SPTB, ANK1, SLC4A1 and EPB41), four were homozygous or compound heterozygous for mutations in genes related to enzyme deficiencies (GPI, TPI1 and GSS), one had a mutation in the HBB gene and another presented a homozygous mutation in the ADAMTS13 gene. CONCLUSIONS: Ion PGM sequencing with our custom panel is a highly efficient way to detect mutations causing haemolytic anaemia, including new variations. It is a high-throughput detection method that is ready for application in clinical laboratories.

Darbepoetin alfa for anemia in patients with low or intermediate-1 risk myelodysplastic syndromes and positive predictive factors of response.

BACKGROUND: Current guidelines support the use of erythropoiesis-stimulating agents for the treatment of anemia associated with low-risk myelodysplastic syndromes (MDS). DESIGN AND METHODS: Single-arm, open-label, multi-center, phase 2 trial that evaluated the efficacy and safety of darbepoetin alfa (DA) in patients with low or intermediate-risk MDS, hemoglobin <100 g/L, erythropoietin (EPO) levels <500 IU/L and transfusion requirements <2 units/month over the preceding 2 months. Erythroid response (major [MaR] or minor [MiR]) and fatigue (Functional Assessment of Cancer Therapy-Fatigue [FACT-F]) were evaluated at 8, 16 and 24 weeks. DA was initiated at 300 µg weekly. For patients who did not achieve MaR by 8 weeks, filgrastim 300 µg weekly was added. CLINICAL TRIAL REGISTRATION: clinicaltrials.gov identifier: NCT01039350. RESULTS: Forty-four patients (72.7% transfusion independent) were included. Median age was 76.0 years (range 41.3-92.4), 54.5% were male, and 90.9% presented ECOG Status (0-1). Eighteen patients received filgrastim. An erythroid response was achieved by 31 of 44 patients (70.5%) at week 8 (47.7% MaR, 22.7% MiR), 31 of 44 patients (70.5%) at week 16 (61.4% MaR, 9.1% MiR), and 32 of 44 patients (72.7%) at week 24 (61.3% MaR, 11.4% MiR). Mean (95% CI) change in FACT-F at week 24 was 3.61 (0.72 to 6.51). Baseline EPO levels <100 IU/L were a predictive factor of response. DA was well tolerated. Four mild (two iron deficiencies, flu syndrome and headache) and one fatal (thromboembolic event) adverse events were considered related to darbepoetin alfa. CONCLUSIONS: A fixed dose of 300 µg of darbepoetin alfa weekly (with or without filgrastim) seems to be an effective and safe treatment for anemic patients with low or intermediate-risk MDS, low transfusion burden and EPO levels <500 IU/L. Results may not be extrapolable to unselected MDS patients.

Hb Johnstown [beta 109 (G11) Val-->Leu]: second case described and associated for the first time with beta(0)-thalassemia in two Spanish families.

Hb Johnstown, a high oxygen affinity hemoglobin, was identified in four members from two unrelated Spanish families with erythrocytosis and left-shifted hemoglobin-oxygen dissociation curve. This hemoglobin variant, electrophoretically silent, was analyzed by reverse-phase high-performance liquid chromatography, and the mutation was characterized at the DNA level by beta gene sequencing. In one of these families, two members are affected with Hb Johnstown in association with beta(0)-thalassemia. In these cases the erythrocytosis and low values for P(50) due to Hb Johnstown remain in spite of the beta-thalassemia.

Erythropoietin plus granulocyte colony-stimulating factor in the treatment of myelodysplastic syndromes. Identification of a subgroup of responders. The Spanish Erythropathology Group.

BACKGROUND AND OBJECTIVE: Anemia leading to transfusion is probably the most important problem in patients with myelodysplastic syndromes (MDS). Human recombinant erythropoietin (rHuEpo) and granulocyte colony-stimulating factor (G-CSF) have been used to treat patients with anemia of MDS, but fewer than 50% respond. The aim of this work was to evaluate the benefit of rHuEpo +/- G-CSF treatment and to isolate the response predictive variables in a group of selected patients with MDS. DESIGN AND METHODS: A non-randomized multicenter trial was carried out in 32 patients with MDS. The inclusion criteria were age >= 18 years, refractory anemia (RA) or refractory anemia with ringed sideroblasts, Hb <= 100 g/L or receiving transfusions and serum erythropoietin <= 250 U/L. These patients were treated with subcutaneous rHuEpo (300 U/kg) three times a week for 8 weeks. In the case of partial response (PR) or no response (NR) subcutaneosly administered G-CSF (1 microg/kg) three times a week was added to the rHuEpo for 8 more weeks. If the patient achieved complete response (CR) or PR in the second phase, he was included in a follow-up phase of 24 weeks in which the dose of growth factors was tapered down. Several variables, including the score published by the Scandinavian-American group, were used as possible predictive variables. RESULTS: An erythroid response was observed in 16 patients (50%); in 12 it was a CR and in 4 it was a PR. During the period of rHuEpo administration, 7 CR and 4 PR (34.4%) were documented. Of the 14 patients in whom G-CSF was added to rHuEpo, 7 (50%) responded (3 CR and 4 PR). No major side-effects associated with growth factors were observed. The multivariate analysis showed that of the different variables evaluated only the Scandinavian-American response score was significant with a relative probability of response of 11.8 (95% confident intervals: 2.5-53) when this score was > +1 (77% of cases responded). In contrast, when this score was <= 1 only 15 % of the cases responded. INTERPRETATION AND CONCLUSIONS: Use of the Scandinavian-American response score is to be recommended in a patient-oriented approach to treating MDS cases with the Epo and G-CSF. Treatment with rHuEpo and G-CSF is safe, its main drawback being its cost. However, a long-term study evaluating the regimen's cost-benefit ratio is warranted.

Rh-deficiency of the regulator type caused by splicing mutations in the human RH50 gene.

The Rh polypeptides and the glycoproteins Rh50, CD47, LW, and glycophorin B, which interact in the red blood cell membrane to form a multisubunit complex, are lacking or are severely reduced in the Rh-deficiency syndrome. We previously reported that in several Rhnull patients the RH50 gene was altered at the coding sequence level, resulting in either a single amino acid substitution or the synthesis of a truncated polypeptide. In the present report, we have detected two mutations in the intronic region of the RH50 gene that identify a new molecular mechanism involved in Rh-deficiency. The first mutation affected the invariant G residue of the 3' acceptor splice-site of intron 6, causing the skipping of the downstream exon and the premature termination of translation. The second mutation occurred at the first base of the 5' donor splice-site of intron 1. Both these mutations were found in homozygote state. RNase protection assays demonstrated that the Rh50 mRNA level was strongly reduced or undetectable in the 3' and 5' splice mutants, respectively. The different mutations affecting the RH50 gene are indicative of an heterogeneous mutational pattern, which further supports the hypothesis that the lack of the Rh50 protein may prevent the assembly or transport of the Rh membrane complex to the red blood cell surface.

Red blood cell phenotypes in alpha-thalassemias in the Spanish population.

BACKGROUND AND OBJECTIVE: alpha-thalassemia is very common on all thalassemic geographical regions. The present work aimed at analyzing the relationship between the degree of microcytosis and hematological parameters and the type of alpha-thalassemic mutation. DESIGN AND METHODS: Five hundred and thirty-six subjects with 4 kinds of alpha-thalassemia were examined using established techniques that determined all hematological parameters, and globin synthesis and molecular biological techniques to study the DNA of globin genes by Southern blotting. RESULTS: Adult carriers of alpha (+)-thalassemia (-alpha/alpha alpha) present very few hematological alterations. In a statistical comparison with normal individuals (alpha alpha/alpha alpha), significant differences were found between the hemocytometric data and the MCV and MCH of heterozygous alpha + thalassemia and the heterozygous alpha zero or homozygous alpha + genotype. Hb H disease was detected in 15 patients, presenting a severe degree of anemia, a significant increase in RDW and globin chain synthesis with an alpha/beta ratio of 0.5 +/- 0.1. INTERPRETATION AND CONCLUSIONS: These data provide reference values for geographical areas where alpha + thalassemia is common. These hematocytometric data, together with hemoglobin analysis, could be useful as a future reference data for new patients diagnosed with alpha-thalassemia.

[Identification of 5 variants of the hemoglobin beta chain using DNA analysis (amplification, sequencing and restriction enzymes)]. / Identificación de 5 variantes de Hb de cadena beta mediante análisis del ADN (amplificación, secuenciación y enzimas de restricción).

PURPOSE: To confirm the conventional techniques for studying structural haemoglobinopathies and to show off the simplicity and efficacy of new methods based on the study of DNA. PATIENTS AND METHODS: Peripheral blood samples of 17 patients with 5 haemoglobin variants detected by conventional and shown off by means of sequencing according to Sanger's method, plus PCR-RFLP, were studied. RESULTS: Five structural haemoglobin variants were found, which distributed as follows: 7 cases of Hb Complutense (beta 127 Gln-->Glu), 1 Hb D-Punjab (beta 121 Glu-->Gln), 3 Hb Hofu (beta 126 Val-->Glu), 3 Hb J-Baltimore (beta 16 Gly-->Asp) and 3 Hb San Diego (beta 109 Val-->Met). CONCLUSIONS: These results allow us to stress the simplicity and usefulness of DNA analysis (sequencing , amplification and enzymatic digestion) to identify haemoglobin variants as opposed to laborious analysis of the primary structure by means of HPLC peptide separation.

[Cutaneous infiltration in multiple myeloma]. / Infiltración cutánea en el mieloma múltiple.

A 44 year old woman with multiple myeloma and cutaneous involvement is presented. She had a painless tumor over the malar region. Microscopic study showed a cutaneous infiltration of plasma cells. Survival from diagnosis lasted 5 months. Over this period, multiple and progressive cutaneous nodules appeared, having the same structural characteristics.

[Hormonal contraceptive therapy and thromboembolic disease]. / Terapéutica anticonceptiva hormonal y enfermedad tromboembólica.

A group of 55 patients with rheumatoid arthritis (RA) and 25 healthy persons is studied. In peripheral blood, the number of E rosettes is similar in both groups but in patients with RA who are not on steroid therapy, these is a decreased number of the absolute value of T lymphocytes. There is a positive correlation between quantitative cellular immunity and different biologic and clinical parameters.

PIP: Through a careful review of the published literature this document presents the most recent knowledge on the action of estrogens on the mechanism of blood coagulation. It now seems clear that oral contraceptives (OCs) cause an increase in the activity of platelet factor 3, and also an increase in platelet aggregation; at the same time coagulation is activated through decreased levels of antighrombin 3, resulting in a concentration of soluble fibrin fragments. Alteration of fibrinolysis is very possible, although the literature is contradictory on this point. Estrogens can also cause thrombotic accidents by alteration of the vascular walls, and by alteration of the lipid metabolism. According to different authors, the risk of thrombotic accident in OC users is greater than in nonusers. The risk greatly increases in smokers. It is possible that thrombotic risk can be decreased with the use of OCs with a lower estrogen content.