Showing papers in "Blood Cells Molecules and Diseases in 2010"

Hematologically important mutations: The autosomal recessive forms of chronic granulomatous disease (second update)

Abstract: Chronic granulomatous Disease (CGD) is an immunodeficiency disorder affecting about 1 in 250,000 individuals. The disease is caused by mutations in the genes encoding the components of the leukocyte NADPH oxidase. This enzyme produces superoxide, which is essential in the process of intracellular pathogen killing by phagocytic leukocytes. Four of the five genes involved in CGD are autosomal; these are CYBA, encoding p22-phox, NCF2, encoding p67-phox, NCF1, encoding p47-phox, and NCF4, encoding p40-phox. This article lists all mutations identified in these genes in the autosomal forms of CGD. Moreover, polymorphisms in these genes are also given, which should facilitate the recognition of future disease-causing mutations.

The cancer stem cell selective inhibitor salinomycin is a p-glycoprotein inhibitor.

Abstract: Salinomycin, a polyether antibiotic acting as a highly selective potassium ionophore and widely used as an anticoccidial drug, was recently shown to act as a specific inhibitor of cancer stem cells. In the present study we report that salinomycin acts as a potent inhibitor of multidrug resistance gp170, as evidenced through drug efflux assays in MDR cancer cell lines overexpressing P-gp (CEM-VBL 10 and CEM-VBL 100; A2780/ADR). Conformational P-gp assay provided evidence that the inhibitory effect of salinomycin on P-gp function could be mediated by the induction of a conformational change of the ATP transporter. Treatment of the MDR cell lines with salinomycin restored a normal drug sensitivity of these cells. The observation that salinomycin is a MDR-1 inhibitor may have important implications for the understanding of the mechanisms through which this drug impairs the viability of cancer stem cells. Interestingly, nigericin and abamectin, two additional drugs identified as cancer stem cells inhibitors, also act as potent gp170 inhibitors.

MicroRNAs expression signatures are associated with lineage and survival in acute leukemias.

Abstract: MicroRNAs (miRNAs) are small ( approximately 22 nucleotide) non-coding RNAs whose altered expression has been associated with various types of cancers, including leukemia. In the present study, we conducted a quantitative PCR (qPCR) analysis of expression of 23 human precursor miRNAs in bone marrow specimens of 85 Chinese primary leukemia patients, including 53 acute myeloid leukemia (AML) and 32 acute lymphoblastic leukemia (ALL) cases. We show that 16 miRNAs were differentially expressed between AMLs and ALLs. Of them, eight were previously reported (i.e., miR-23a, miR-27a/b, miR-128a, miR-128b, miR-221, miR-222, miR-223, and let-7b) and eight were newly identified (i.e., miR-17, miR-20a, miR-29a/c, miR-29b, miR-146a, miR-150, miR-155, and miR-196b). More importantly, through correlating miRNA expression signatures with outcome of patients, we further show that expression signatures of a group of miRNAs are associated with overall survival of patients. Of them, three (i.e., miR-146a, miR-181a/c, and miR-221), five (i.e., miR-25, miR-26a, miR-29b, miR-146a, and miR-196b), and three (i.e., miR-26a, miR-29b, and miR-146a) miRNAs are significantly associated with overall survival (P<0.05) of the 32 ALL, 53 AML, and 40 non-M3 AML patients, respectively. Particularly, the expression signature of miR-146a is significantly inversely correlated with overall survival of both ALL and AML patients. The prognostic significance of miR-146a in AML has been confirmed further in an independent study of 61 Chinese new AML patient samples. We also identified 622 putative target genes of miR-146a that are predicted by at least three out of the five major prediction programs (i.e., TragetScan, PicTar, miRanda, miRBase Targets, and PITA). Through gene ontology analysis, we found that these genes were particularly enriched (2- to 9-fold higher than expected by chance) in the GO categories of "negative regulation of biology processes," "negative regulation of cellular processes," "apoptosis," and "cell cycle," which may be related to the association of miR-146a with poor survival.

Self inhibition of phagocytosis: the affinity of ‘Marker of Self’ CD47 for SIRPα dictates potency of inhibition but only at low expression levels

Abstract: Phagocytes engulf foreign cells but not 'self' in part because self cells express CD47 as a ligand for signal regulatory protein SIRPalpha, which inhibits phagocytosis. Motivated by reports of upregulation of CD47 on both normal and cancerous stem cells [1: Jaiswal et al., 2009] and also by polymorphisms in SIRPalpha [2: Takenaka et al., 2007], we show here that inhibition of engulfment correlates with affinity of CD47 for SIRPalpha - but only at low levels of CD47. One common human polymorph of SIRPalpha is studied and binds more strongly to human-CD47 than to mouse-CD47 (K(d) approximately 0.12 microM and 6.9 microM, respectively) and does not bind sheep red blood cells (RBCs) - which are well-established targets of human macrophages; in comparison, a common mouse polymorph of SIRPalpha binds with similar affinity to human and mouse CD47 (K(d) approximately 0.22 microM). Using immunoglobulin (IgG)-opsonized particles with varying levels of either human- or mouse-CD47, the effective inhibition constants K(i) for blocking phagocytosis are then determined with both human- and mouse-derived macrophages. Only human phagocytes show significant differences in man versus mouse K(i)'s and only at CD47 levels below normal densities for RBCs. While phospho-signaling through human-SIRPalpha shows similar trends, consistent again with the affinity differences, saturating levels of CD47 (>K(i)) can signal and inhibit phagocytosis regardless of man versus mouse. Quantitative analyses here prompt more complete characterizations of both CD47 levels and SIRPalpha polymorphisms when attempting to study in vivo effects of these key proteins in innate immunity.

Runx2 in normal tissues and cancer cells: A developing story.

Abstract: The Runx transcription factors are essential for mammalian development, most notably in the haematopoietic and osteogenic lineages. Runx1 and its binding partner, CBFbeta, are frequently targeted in acute leukaemia but evidence is accumulating that all three Runx genes may have a role to play in a wider range of cancers, either as tumour promoters or tumour suppressors. Whilst Runx2 is renowned for its role as a master regulator of bone development we discuss here its expression pattern and putative functions beyond this lineage. Furthermore, we review the evidence that RUNX2 promotes neoplastic development in haematopoietic lineages and in advanced mammary and prostate cancer.

Force majeure: therapeutic measures in response to restricted supply of imiglucerase (Cerezyme) for patients with Gaucher disease.

Abstract: Gaucher disease is the first lysosomal disorder for which clinically effective enzyme replacement therapy has been introduced. Lifelong treatment with imiglucerase, the recombinant glucocerebrosidase manufactured by the Genzyme Corporation (MA, USA), is administered intravenously - usually at biweekly intervals. An acute shortage of imiglucerase (to 20% of prior global supply) has occurred as a result of viral contamination of the production facility; production was halted, and a full supply of imiglucerase is not anticipated until January 2010. An urgent meeting of physicians, researchers, and patients was convened through the agency of the European Working Group for Gaucher Disease; this was instigated by patients internationally represented by the European Gaucher Alliance. Here we present a position statement based on the findings of the group, with key recommendations about identification and monitoring of at-risk patients threatened by the abrupt withdrawal of treatment, the equitable distribution of residual imiglucerase - and access to alternative treatments including those that have completed phase III clinical trials but have not yet been licensed.

Band 3 multiprotein complexes in the red cell membrane; of mice and men.

Abstract: The bicarbonate/chloride exchanger band 3 (Anion Exchanger 1, AE1) is the most abundant protein in the erythrocyte membrane, it has an important role in gas exchange and functions as a point of attachment for the cytoskeletons maintaining the mechanistic and osmotic properties of the erythrocyte. Band 3 is found in three distinct protein complexes within the erythrocyte membrane: an ankyrin-dependent tetrameric band 3 complex, a dimeric band 3 complex bound to the protein 4.1-GPC junctional complex and as freely diffusing dimeric band 3 complexes. Much if not all of our present knowledge of these protein complexes is derived from mouse knockout model systems and human variant blood samples. This review will explore what is known about the band 3 complexes of mice and humans, focussing on the observed species differences and their potential functional consequences.

Isolated microvesicles from peripheral blood and body fluids as observed by scanning electron microscope

Abstract: Microvesicles are sub-micron structures shed from the cell membrane in a final step of the budding process After being released into the microenvironment they are free to move and carry signaling molecules to distant cells, thereby they represent a communication system within the body Since all cells shed microvesicles, it can be expected that they will be found in different body fluids The potential diagnostic value of microvesicles has been suggested, however, a standardized protocol for isolation has not yet been agreed upon It is unclear what is the content of the isolates and whether the isolated microvesicles were present in vivo or—have they been created within the isolation procedure To present evidence in this direction, in this work we focus on the visualization of the material obtained by the microvesicle isolation procedure We present scanning electronic microscope images of microvesicles isolated from blood, ascites, pleural fluid, cerebrospinal fluid, postoperative drainage fluid and chyloid fluid acquired from human and animal patients Vesicular structures sized from 1 µm downto 50 nm are present in isolates of all considered body fluids, however, the populations differ in size and shape reflecting also the composition of the corresponding sediments Isolates of microvesicles contain numerous cells which indicates that methods of isolation and determination of the number of microvesicles in the peripheral blood are to be elaborated and improved

Polymorphisms and mutations of human TMPRSS6 in iron deficiency anemia

Abstract: Male subjects with iron deficiency from the general population were examined for polymorphisms or sporadic mutations in TMPRSS6 to identify genetic risk factors for iron deficiency anemia. Three uncommon non-synonymous polymorphisms were identified, G228D, R446W, and V795I (allele frequencies 0.0074, 0.023 and 0.0074 respectively), of which the R446W polymorphism appeared to be overrepresented in the anemic population. In addition, three children with iron refractory iron deficiency anemia, and one sibling with iron responsive iron deficiency anemia were also examined for polymorphisms or sporadic mutations in TMPRSS6. Two children (family 1) were compound heterozygotes for a L674F mutation and a previously described splicing defect predicted to cause skipping of exon 13 (IVS13+1 G>A). One child from the second family was homozygous for a deletion (497T) causing a frameshift (L166X+36) and premature termination. The sibling and mother from the second family were compound heterozygotes for the L166X mutation and the uncommon R446W polymorphism. Although in vitro expression studies demonstrated that the R446W isoform was biologically similar to wildtype Tmprss6, clinical data indicate that the R446W produces a milder disease when carried in trans with severe mutation in Tmprss6. The four children carrying mutations in TMPRSS6 all exhibited inappropriately high urinary hepcidin levels for the degree of iron deficiency.

The impact of MYC expression in lymphoma biology: beyond Burkitt lymphoma.

Abstract: Although classically described as the driving oncogene in Burkitt lymphoma (BL), abnormalities of MYC have been recognized in other non-Hodgkin lymphomas as well. For example, MYC is overexpressed in approximately 10% of diffuse large B-cell lymphomas (DLBCL), conferring an adverse prognosis with chemoresistance and shortened survival; only approximately 30% of patients achieve long-term survival despite modern therapies. In contrast to BL, MYC aberrations in DLBCL are usually associated with multiple cytogenetic abnormalities and other genetic lesions, such as concurrent BCL2 translocations. Patients with so-called "double-hit" lymphomas have a worse outcome with few survivors beyond 6 months. It is unclear why MYC translocations are diagnostic in BL but prognostic in other lymphomas; different mechanisms underlying MYC abnormalities and a unique target set of genes may explain some of the variance. Furthermore, MYC possesses nontranscriptional functions other than transcriptional controls on genes regulating cell growth and may also influence the lymphoma microenvironment. Here we summarize current knowledge regarding MYC in lymphomas other than Burkitt lymphoma, with an emphasis on transcriptional, epigenetic, clinical, and microenvironmental consequences.

Binding patterns of BCL11A in the globin and GATA1 loci and characterization of the BCL11A fetal hemoglobin locus

Abstract: BCL11A is a major regulator of fetal hemoglobin production. Reduced levels of BCL11A have been shown to delay switching from fetal to adult hemoglobin, suggesting that it acts as a stage-specific repressor of gamma globin expression. We have carried out a survey of BCL11A binding in the globin, BCL11A and GATA1 loci by ChIP-on-chip analysis in primary human erythroid cells. We found strong occupancy in both alpha and beta globin upstream regulatory regions as well as in regions involved in switching and hereditary persistence of fetal hemoglobin. Genetic studies have identified a restricted 14kb region in BCL11A intron 2 as being highly associated with HbF levels. Strong GATA-1 binding and acetylated histone H3 was found in this area, which could be indicative of a regulatory element, changes in which might be responsible for the overall regulation of BCL11A. We also observed BCL11A and GATA-1 binding in a known auto-regulatory promoter element of the GATA1 locus.

Altered phosphorylation of cytoskeleton proteins in sickle red blood cells: the role of protein kinase C, Rac GTPases, and reactive oxygen species.

Abstract: The small Rho GTPases Rac1 and Rac2 regulate actin structures and mediate reactive oxygen species (ROS) production via NADPH oxidase in a variety of cells. We have demonstrated that deficiency of Rac1 and Rac2 GTPases in mice disrupts the normal hexagonal organization of the RBC cytoskeleton and reduces erythrocyte deformability. This is associated with increased phosphorylation of adducin at Ser-724, (corresponding to Ser-726 in human erythrocytes), a domain target of protein kinase C (PKC). PKC phosphorylates adducin and leads to decreased F-actin capping and dissociation of spectrin from actin, implicating a significant role of such phosphorylation in cytoskeletal remodeling. We evaluated adducin phosphorylation in erythrocytes from patients with sickle cell disease and found it consistently increased at Ser-726. In addition, ROS concentration is elevated in sickle erythrocytes by 150-250% compared to erythrocytes from normal control individuals. Here, we review previous studies demonstrating that altered phosphorylation of erythrocyte cytoskeletal proteins and increased ROS production result in disruption of cytoskeleton stability in healthy and sickle cell erythrocytes. We discuss in particular the known and potential roles of protein kinase C and the Rac GTPases in these two processes.

The XmnI Gγ polymorphism influences hemoglobin F synthesis contrary to BCL11A and HBS1L-MYB SNPs in a cohort of 57 β-thalassemia intermedia patients

Abstract: The HbF level is a quantitative trait influenced by many loci inside or outside the β-globin gene cluster. The aim of this study was to analyze in 57 β-thalassemia intermedia patients with very various genotypes the effects on fetal hemoglobin levels of SNPs lying in three genes or chromosome regions which include the XmnI Gγ polymorphism at position − 158 of the HBG2 promoter (rs7482144), two SNPs located in the BCL11A region (rs4671393 and rs11886868) and three SNPs located in the HBS1L-MYB region (rs28384513, rs9399137 and rs4895441). Our study shows a strong correlation between the XmnI Gγ polymorphism and the fetal hemoglobin expression in this patient population (p = 0.002). Unfortunately, although recent studies clearly showed a role of SNPs in BCL11A and a HBS1L-MYB region on either clinical expression or fetal hemoglobin levels of β-hemoglobinopathies such as sickle cell disease and β-thalassemia, SNPs in BCL11A and the HBS1L-MYB region did not show statistically significant correlations with fetal hemoglobin levels. This suggests that the BCL11A and HBS1L-MYB loci have a minor effect on HbF level compared to the XmnI QTL in β-thalassemia intermedia patients.

Runx family genes, niche, and stem cell quiescence.

Abstract: In multicellular organisms, terminally differentiated cells of most tissues are short-lived and therefore require constant replenishment from rapidly dividing stem cells for homeostasis and tissue repair. For the stem cells to last throughout the lifetime of the organism, however, a small subset of stem cells, which are maintained in a hibernation-like state known as stem cell quiescence, is required. Such dormant stem cells reside in the niche and are activated into proliferation only when necessary. A multitude of factors are required for the maintenance of stem cell quiescence and niche. In particular, the Runx family genes have been implicated in stem cell quiescence in various organisms and tissues. In this review, we discuss the maintenance of stem cell quiescence in various tissues, mainly in the context of the Runx family genes, and with special focus on the hematopoietic system.

The aryl hydrocarbon receptor: Regulation of hematopoiesis and involvement in the progression of blood diseases

Abstract: The aryl hydrocarbon receptor (AhR) is a basic helix–loop–helix protein that belongs to the superfamily of environment-sensing PAS (Per–ARNT–Sim) proteins. A large number of ligands have been described to bind AhR and promote its nuclear translocation. In the nucleus, the AhR and its dimerization partner the AhR nuclear translocator (ARNT) form a DNA-binding complex that acts as a transcriptional regulator. Animal and human data suggest that, beyond its mediating responses to xenobiotic and/or unknown endogenous ligands, the AhR has a role, although as yet undefined, in the regulation of cell cycle and inflammation. The AhR also appears to regulate the hematopoietic and immune systems during development and adult life in a cell-specific manner. While accidental exposure to xenobiotic AhR ligands has been associated with leukemia in humans, the specific mechanisms of AhR involvement are still not completely understood. However, recent data are consistent with a functional role of the AhR in the maintenance of hematopoietic stem and/or progenitor cells (HSCs/HPCs). Studies highlighting AhR regulation of HSCs/HPCs provide a rational framework to understand their biology, a role of the AhR in hematopoietic diseases, and a means to develop interventions for these diseases.

Increased survival and reversion of iron-induced cardiac disease in patients with thalassemia major receiving intensive combined chelation therapy as compared to desferoxamine alone.

Abstract: Myocardial iron overload is the leading cause of death in patients with β-thalassemia major. An intensification monotherapy with deferoxamine (DFO) as well as a combination therapy with DFO and deferiprone (DFP) reduces myocardial iron and improves cardiac function. However, the prognosis for thalassemia major patients with established cardiac disease switched from DFO monotherapy to combined DFP/DFO chelation is unknown. Twenty-eight thalassemia major patients with cardiac disease were enrolled in a prospective study lasting 42 ± 6 months. Fifteen (9 high-ferritin and 6 low-ferritin) were placed on DFP/DFO (DFP, 75 mg/kg t.i.d.; DFO, 40–50 mg/kg over 8–12 h at night 5–7 days/week), while 13 (5 high- and 8 low-ferritin) received DFO alone. No cardiac events were observed among high-ferritin patients on combination therapy, whereas 4 cardiac events ( p = 0.0049), including three deaths, occurred in high-ferritin patients on DFO monotherapy. These findings demonstrate that in thalassemia major patients with well-established cardiac disease combined iron-chelation therapy with DFP/DFO is superior to DFO monotherapy.

Serum hepcidin but not prohepcidin may be an effective marker for anemia of inflammation (AI).

Abstract: Anemia in cancer patients can result from a complex interaction of numerous factors including iron deficiency, inflammation, toxicity related to therapy and effect of cancer on the marrow. Determining effective anemia treatment can therefore be complex, requiring a combination of diagnostic tests. Research on iron metabolism has highlighted the importance of hepcidin and its potential role in development of anemia of inflammation (AI). Hepcidin is a peptide that controls iron flow, is induced by inflammation and is speculated to cause the sequestration of iron in patients with inflammation. In the present study, serum hepcidin concentration determined by LC-MS/MS was shown to correlate with inflammatory markers in patients with anemia of cancer (AoC). In the absence of a widely-available serum hepcidin detection assay, detection of prohepcidin using a commercial assay has been used for several years as a surrogate for measuring serum hepcidin concentration. Analysis of prohepcidin concentration did not reveal any correlation with hepcidin or with inflammatory markers in patient samples and our data suggest that prohepcidin may not be stable in serum. Algorithms to sub-classify AoC patients showed that hepcidin was strongly associated with the population subset with inflammation and without iron deficiency. Serum hepcidin concentrations may therefore be a good predictor of AI, useful in diagnosis of anemia etiology and in treatment determination.

Novel large deletions in the human alpha-globin gene cluster: Clarifying the HS-40 long-range regulatory role in the native chromosome environment.

Abstract: Globin genes, which encode the protein subunits of hemoglobin (Hb), are organized in two different gene clusters and present a coordinated and differential pattern of expression during development. Concerning the human α-globin gene cluster (located at chromosome region 16p13.3), four upstream highly conserved elements known as multispecies conserved sequences (MCS-R1-4) or DNase I hypersensitive sites (HSs) are implicated in the long-range regulation of downstream gene expression. However, only the absence of the MCS-R2 site (HS-40) has proven to drastically downregulate the expression of those genes, and consequently, it has been regarded as the major and crucial distal regulatory element. In this study, Multiplex Ligation-dependent Probe Amplification was used to screen for deletions in the telomeric region of the short arm of chromosome 16, in an attempt to explain the α-thalassemia or the HbH disease present in a group of Portuguese patients. We report four novel and five uncommon deletions that remove the α-globin distal regulatory elements and/or the complete α-globin gene cluster. Interestingly, one of them occurred de novo and removes all HSs except HS-10, while other eliminates only the HS-40 site, the latter being replaced by the insertion of a 39 nucleotide orphan sequence. Our results demonstrate that HS-10 alone does not significantly enhance the α-globin gene expression. The absence of HS-40 in homozygosity, found in a patient with Hb H disease, strongly downregulates the expression of α-globin genes but it is not associated with a complete absence of α-globin chain production. The study of naturally occurring deletions in this region is of great interest to understand the role of each upstream regulatory element in the native human erythroid environment.

Vivo-Morpholino knockdown of αIIb: A novel approach to inhibit thrombocyte function in adult zebrafish

Abstract: Knockdown of protein function by antisense oligonucleotides has been used to understand the protein function not only in development but also in human diseases. Recently, Vivo-Morpholinos, chemically modified morpholinos which penetrate the cells, have been used in adult experimental animal models to alter the splicing and thereby change the protein expression. Until now, there have been no such studies using Vivo-Morpholinos to evaluate hemostatic function in adult animals. We injected αIIb Vivo-Morpholinos intravenously into adult zebrafish. Thrombocyte function was assayed by time to aggregation assay of the citrated blood, annexin V binding to thrombocytes, and gill bleeding. The thrombocyte functional inhibition occurred in 24 h after αIIb Vivo-Morpholinos injection and reached a maximum in 48 h. However, in 72 h, the inhibition was no longer observed. Reduction of annexin V binding to thrombocytes and increased gill bleeding were observed 48 h after αIIb Vivo-Morpholino injections. The action of the αIIb Vivo-Morpholino was demonstrated by the presence of an alternatively spliced αIIb mRNA and the reduction of αIIb in thrombocytes of fish treated with αIIb Vivo-Morpholino. These results provide the first proof of principle that thrombocyte function can be inhibited by thrombocyte-specific Vivo-Morpholinos in adult zebrafish and presents an approach to knockdown thrombocyte-specific genes to conduct biochemical studies in thrombocytes. This study also provides the first antisense antithrombotic approach to inhibit thrombocyte function in adult zebrafish.

Plasma asymmetric dimethylarginine concentrations in sickle cell disease are related to the hemolytic phenotype

Abstract: Asymmetric dimethylarginine (ADMA) is associated with pulmonary hypertension (PHT) in sickle cell disease (SCD). We studied the relationship of ADMA to other SCD-related complications. Plasma ADMA and associated parameters were determined in 52 HbSS/HbS beta(0)-thalassemia and 24 HbSC/HbS beta(+)-thalassemia patients. As expected ADMA levels were higher in HbSS/HbS beta(0)-thalassemia patients with PHT (p = 0.018), but also in those with other hemolysis-associated complications such as leg ulcers (p = 0.012), cholelithiasis (p = 0.008) and priapism (p = 0.02) compared with counterparts without these complications. ADMA levels did not differ between patients with and without other disease related complications such as retinopathy and avascular osteonecrosis. Higher ADMA concentrations therefore seem to be associated to the hemolytic phenotype of SCD. (c) 2010 Elsevier Inc. All rights reserved.

Alpha-thalassemia is associated with a decreased occurrence and a delayed age-at-onset of albuminuria in sickle cell anemia patients.

Abstract: The aim of this study was to identify possible risk factors for albuminuria, an early marker of sickle cell anemia (SCA) glomerulopathy, in a cohort of 189 SCA adult patients followed at the Sickle Cell Center of Guadeloupe, a French Caribbean island. Biological parameters obtained at baseline, α-globin gene status, and βS haplotypes were compared in patients stratified accordingly to graded albuminuria. Abnormal albumin excretion rate was detected in half of the studied adult patients and macroalbuminuria occurred in 21.6%. Graded albuminuria was associated with advanced age (p = 0.006), systolic blood pressure (p = 0.031), and worsened anemia, i.e. low hemoglobin rate (p

Strong association between a new marker of hemolysis and glomerulopathy in sickle cell anemia.

Abstract: To perform a precise evaluation of the hemolytic status of patients with sickle cell anemia (SCA), advanced red blood cell parameters provided by the last generation analyzers were investigated in a series of SCA patients. The search for precise markers of hemolysis was performed to identify if patients so exposed develop organic complications related to a postulated hemolysis-linked endothelial dysfunction. Red blood cell survival was evaluated by the ratio between mature red blood cell (RBC) and reticulocyte (RET) hemoglobin (RBC-Hb/RET-Hb). In comparison with serum lactate dehydrogenase (LDH) and total bilirubin, the log (RBC-Hb/RET-Hb) was identified as the most discriminant hematological parameter to evaluate hemolysis. Furthermore, by combining this parameter with LDH, we defined a composite variable, which we called CVar, that is highly correlated with albuminuria and might constitute a powerful new marker of risk for this complication.

Thalassemia in Western Australia: 11 novel deletions characterized by Multiplex Ligation-dependent Probe Amplification

Abstract: The number of immigrants in Western Australia from many different areas where hemoglobinopathies are endemic has increased dramatically since the 1970s. Therefore, many different thalassemia mutations have been introduced in the country, which add a technological diagnostic problem to the serious burden of hemoglobinopathy management and to public health care. Recently, we have developed a rapid and simple technique based on Multiplex Ligation-dependent Probe Amplification to detect deletions causing alpha-and beta-thalassemia, deltabeta-thalassemia and Hereditary Persistence of Fetal Hemoglobin. A screening for (unknown) deletions was performed in a cohort of patients of different ethnic backgrounds preselected for their thalassemia phenotype, in which common deletions and point mutations were excluded. Out of 37 cases suspected to carry a deletion, 27 were found to carry 17 different deletion types of which 6 causing alpha-thalassemia and 5 causing beta-thalassemia were novel. For 3 of the deletions, we have been able to characterize the exact breakpoint sequences by long-range PCR and direct sequencing. These results show that MLPA is a suitable technology to detect unknown and uncommon deletions. These could represent a diagnostic problem when offering prevention to couples at risk presenting with unclear phenotypes and might result in a serious fetal problem when the deletion involves embryonic genes.

Altered hepatic BMP signaling pathway in human HFE hemochromatosis

Abstract: Human hemochromatosis (HC) has been associated with the common C282Y polymorphism of HFE or rare pathogenic mutations of TfR2, HJV, FPN and HAMP. All forms of human HC seem to be caused by low or inadequate levels of hepcidin, the iron hormone. We and others have recently shown that Hfe(-/-) mice exhibit an impairment in the bone morphogenetic protein (BMP) signaling pathway controlling hepcidin. However, all data indicating the central role of BMPs in hepcidin regulation and an impaired BMP/SMAD signaling in HC have been collected in mice. In this study we investigated whether also in humans the expression of BMP signaling targets, SMAD7 and Id1, are associated with liver iron concentration (LIC) and whether such regulation is disrupted in HFE-HC. We correlated the mRNA expression, assessed by RT-PCR, of HAMP, SMAD7 and Id1 with LIC in liver biopsies from patients with normal iron status, HFE-HC or non-HC hepatic iron overload. We found that in human liver, not only HAMP, but also SMAD7 and Id1 mRNA significantly correlate with the extent of hepatic iron burden. However, this correlation is lost in patients with HFE-HC, but maintained in subjects with non-hemochromatotic iron overload. These data indicate that in human HFE-HC a disrupted BMP/SMAD signaling in the liver is key in the pathogenesis of the disease.

Zebrafish von Willebrand factor.

Abstract: von Willebrand factor (vWF) is a large protein involved in primary hemostasis A dysfunction in this protein or an insufficient production of the protein leads to improper platelet adhesion/aggregation, resulting in a bleeding phenotype known as von Willebrand disease (vWD) To gain a better understanding of vWF interactions in vivo, the use of zebrafish as a model is ideal because of the transparency of the embryos and larvae In this article, we examined the presence and function of vWF in hemostasis of zebrafish utilizing a variety of molecular methods Using RT-PCR and antibody staining, we have shown that vWF mRNA is present in thrombocytes Through antibody staining, we demonstrated vWF is synthesized in blood vessels The role of zebrafish vWF in hemostasis was established through knockdown methods using vWF morpholino (vWF MO) antisense oligonucleotides Embryos injected with vWF MO at the one to four cell stages resulted in a bleeding phenotype Injection of embryos with vWF MO also caused an increase in time to occlusion within arteries in larvae upon laser induced injury We then used vWF-specific Vivo-morpholinos (VMO) to induce vWF knockdown in adult zebrafish by targeting the exon homologous to the human exon 28 of the vWF gene The reduced ristocetin-mediated agglutination of thrombocytes in a plate tilting assay, using blood from adult zebrafish injected with VMO, provided evidence that vWF is involved in the hemostatic process We also administered desmopressin acetate to larvae and adults which resulted in enhanced aggregation/agglutination of thrombocytes Zebrafish genome database analysis revealed the presence of GPIbβ gene It also revealed the exon of zebrafish vWF gene corresponding to exon 28 of human vWF gene is highly similar to the exon 28 of human vWF gene, except that it has an insertion that leads to a translated peptide sequence that separates the two A domains coded by this exon This exon is also conserved in other fishes In summary, we established that zebrafish vWF has a role similar to that of vWF found in humans, thus, making zebrafish a useful model for studying the cell biology of vWF in vivo

Backtracking RAS mutations in high hyperdiploid childhood acute lymphoblastic leukemia

Abstract: High hyperdiploidy is the single largest subtype of childhood acute lymphoblastic leukemia (ALL) and is defined by the presence of 51–68 chromosomes in a karyotype. The 5 or more extra chromosomes characterizing this subtype are known to occur in a single mitotic event, prenatally. We screened for RAS mutations among 517 acute childhood leukemias (including 437 lymphocytic, of which 393 were B-cell subtypes) and found mutations in 30% of high hyperdiploids compared to only 10% of leukemias of other subtypes ( P KRAS mutations occurred before birth using a PCR-restriction enzyme-mediated Taqman quantitative PCR reaction, and found no evidence for prenatal KRAS mutations in 14 patients tested. While RAS mutations were previously associated with prior chemical exposures in childhood and adult leukemias, in this study RAS -mutated cases were not significantly associated with parental smoking when compared to study controls. IGH rearrangements were backtracked in three RAS -positive patients (which were negative for KRAS mutation at birth) and found to be evident before birth, confirming a prenatal origin for the leukemia clone. We posit a natural history for hyperdiploid leukemia in which prenatal mitotic catastrophe is followed by a postnatal RAS mutation to produce the leukemic cell phenotype.

Deoxygenation affects tyrosine phosphoproteome of red cell membrane from patients with sickle cell disease.

Abstract: Sickle cell disease (SCD) is a worldwide distributed hereditary red cell disorder related to the production of a defective form of hemoglobin, hemoglobin S (HbS). One of the hallmarks of SCD is the presence of dense, dehydrate highly adhesive sickle red blood cells (RBCs) that result from persistent membrane damage associated with HbS polymerization, abnormal activation of membrane cation transports and generation of distorted and rigid red cells with membrane perturbation and cytoskeleton dysfunction. Although modulation of phosphorylation state of the proteins from membrane and cytoskeleton networks has been proposed to participate in red cell homeostasis, much still remains to be investigated in normal and diseased red cells. Here, we report that tyrosine (Tyr-) phosphoproteome of sickle red cells was different from normal controls and was affected by deoxygenation. We found proteins, p55 and band 4.1, from the junctional complex, differently Tyr-phosphorylated in SCD RBCs compared to normal RBCs under normoxia and modulated by deoxygenation, while band 4.2 was similarly Tyr-phosphorylated in both conditions. In SCD RBCs we identified the phosphopeptides for protein 4.1R located in the protein FERM domain (Tyr-13) and for α-spectrin located near or in a linker region (Tyr-422 and Tyr-1498) involving protein areas crucial for their functions in the context of red cell membrane properties, suggesting that Tyr-phosphorylation may be part of the events involved in maintaining membrane mechanical stability in SCD red cells.

Promoter methylation of DAPK1, E-cadherin and thrombospondin-1 in de novo and therapy-related myeloid neoplasms.

Abstract: DNA methylation is one of the major epigenetic changes in human cancers, leading to silencing of tumor suppressor genes, with a pathogenetic role in tumor development and progression in myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML). Methylation of key promoter regions, induced by cytotoxic therapy together with complex genetic changes, is important in the biology of therapy-related myeloid neoplasms (t-MN). We were interested in the characterization of the methylation pattern of AML and MDS de novo and therapy-related. We studied 385 patients (179 females, 206 males), of a median age of 66 years (range 16-98 years). There were 105 MDS, 208 de novo AML and 72 t-MN (45 MDS and 27 AML). Using a methylation-specific PCR, we studied the promoter methylation status of E-cadherin (CDH1), TSP1 and DAP-Kinase 1. These genes have been shown to be involved in the malignant transformation, interfering with angiogenesis, interaction with micro-environment, apoptosis and xenobiotic detoxification. We found no associations between promoter hypermethylation and gender or age at the time of initial diagnosis. In patients with MDS, there were no associations between hypermethylation and clinical characteristics, including IPSS score, WHO classification and cytogenetics. DAPK1 was more frequently methylated in t-MDS/AML when compared to de novo MDS and AML (39% vs 15.3% and 24.4%, p=0.0001), while methylation of CDH1 was similar in t-MDS/AML and AML (51% and 53.4%), but less frequent in de novo MDS (29%) (p=0.003). In the t-MDS/AML group, we found that the methylation pattern appeared to be related to the primary tumor, with DAPK1 more frequently methylated in patients with a previous lymphoproliferative disease (75% vs 32%, p=0.006). On the other hand, methylation of CDH1 was associated to radiotherapy for the primary malignancy (84.5% vs 38%, p=0.003). TSP1 hypermethylation was rare and not characteristic of t-MDS/AML. In 177 patients studied for concurrent methylation of several promoters, t-MN and AML de novo were significantly more frequently hypermethylated in 2 or more promoter regions than de novo MDS (20% vs 12.4%, p<0.001). Chemotherapy and individual genetic predisposition have a role in t-MDS/AML development, the identification of specific epigenetic modifications may explain complexity and genomic instability of these diseases and give the basis for targeted-therapy. The significant association with previous malignancy subtypes may underlie a likely susceptibility to methylation of specific targets and a role for constitutional epimutations as predisposing factors for the development of therapy-related myeloid neoplasm.

Hemoglobin Q-Thailand related disorders: origin, molecular, hematological and diagnostic aspects.

Abstract: We describe the molecular and hematological profiles of thalassemia syndromes caused by interactions of hemoglobin (Hb) Q-Thailand [α74(EF3) Asp-His] and various hemoglobinopathies found in 52 unrelated adult Thai subjects. Ten genotypes including several previously undescribed conditions were observed, which were classified into 4 groups. Group I included 26 Hb Q-Thailand heterozygotes and a homozygotous subject. Group II included subjects with Hb Q-Thailand and other α-thalassemia alleles in trans including 1 compound Hb Q-Thailand/α(+)-thalassemia (-α(3.7)), 2 Hb Q-Thailand/Hb Constant Spring disease and 6 Hb H/Q-Thailand disease. The average levels of Hb Q-Thailand were found to be 29.8%, 82.3%, 34.7%, 49.2-49.3% and 79.4%, respectively. Both Hbs Bart's and H were observed in addition to Hb Q-Thailand in all 6 cases with Hb Q-H disease but not in a homozygous Hb Q-Thailand. Group III included 7 double heterozygotes for Hb Q-Thailand/Hb E, 3 Hb Q-Thailand/Hb E/α(+)-thalassemia (-α(3.7)), 3 heterozygous Hb Q-Thailand/homozygous Hb E and 1 triple heterozygote for Hb Q-Thailand/Hb Constant Spring/Hb E. In this group, Hbs E (α(A)(2)β(E)(2)), Q-Thailand (α(QT)(2)β(A)(2)) and QE (α(QT)(2)β(E)(2)) were observed on both HPLC and capillary electrophoresis. The Hb QE, rather than Hb Q-Thailand, was detected in all 3 cases with heterozygous Hb Q-Thailand and homozygous Hb E. The remaining two cases in group 4 were double heterozygotes for Hb Q-Thailand and β(0)-thalassemia in which Hb Q-Thailand, elevated Hb A(2) (α(A)(2)δ(2)), and Hb QA(2) (α(QT)(2)δ(2)) were detected. DNA analysis identified the Hb Q-Thailand mutation (α74: GAC-CAC) and the linked (-α(4.2)) in all cases. Analysis of α-globin gene haplotype provided the first evidence of a single origin of this Hb variant in Thai population.

A common SNP near BMP2 is associated with severity of the iron burden in HFE p.C282Y homozygous patients: a follow-up study.

Abstract: BACKGROUND AND OBJECTIVES: It is now generally admitted that penetrance of the common HFE p.C282Y/p.C282Y genotype is incomplete, and identification of modifier genes is the concern of a growing number of research projects. We recently identified a significant association between pretherapeutic serum ferritin level and the common rs235756 single nucleotide polymorphism (SNP) of the BMP2 gene region. Our results further suggested an interactive effect between the BMP2 rs235756 SNP and the rs16827043 SNP in HJV, with a small additive effect of the rs4901474 SNP in BMP4. DESIGN AND METHODS: The present study has been designed as a replication study in an independent cohort of 450 HFE p.C282Y homozygous patients from a nearby French region (Brittany). Information on individual alcohol consumption and amount of iron removed by phlebotomy being available for a substantial part of this cohort, additional analyses were conducted. RESULTS: Only the use of the Amount of Iron Removed by phlebotomy (AIR) as marker of iron burden has provided positive results. Indeed, a significant association was detected between rs235756 and AIR adjusted for sex and age, with a mean AIR increasing with the number of BMP2 T alleles in the genotype groups. The effect of rs235657 was not strong enough to detect effects of gene combinations. Still, the trend in two-locus genotype risks involving BMP2 and HJV for AIR was concordant with the specific interactive effect described in the initial study. INTERPRETATION AND CONCLUSIONS: Although we failed to replicate results of the initial study, we argue that, altogether, our results help to consider genes involved in the regulation of hepcidin synthesis as potential modifiers of the p.C282Y/pC282Y genotype expression and especially BMP2.