Β-thalassemia Intermedia Patients Research Articles

Increased levels of circulating platelet microparticles as a risk of hypercoagulable state in β-thalassemia intermedia patients

Introduction Thromboembolism is a frequent complication in thalassemia. Platelet microparticles (PMPs) may have a role in the hypercoagulability in thalassemia. Objective To determine the levels of PMPs in β-thalassemia intermedia (β-TI) patients and whether increased levels of PMPs in these patients are correlated with coagulation parameters or not. Patients and methods Sixty-three β-TI patients, classified into 46 nonsplenectomized (NS) patients, 17 splenectomized (S) patients, and 20 age-matched and sex-matched volunteers as controls. For all, full medical history, through clinical examination, and laboratory investigations: complete blood count, prothrombin time, prothrombin concentration, activated partial thromboplastin time, FVIII, FXI, fibrinogen, D-dimer, and PMPs were detected by using flow-cytometry approach. Results PMPs were significantly higher in S and NS β-TI patients than controls (P<0.001, P=0.002), respectively. FVIII and D-dimer were significantly higher in S patients than controls (P=0.004, 0.048), respectively. Fibrinogen level was significantly lower in both NS and S β-TI patients than in controls (P<0.001). A significant positive correlation between PMPs and platelet count in S patients (r=0.549, P=0.023). Conclusion Thrombotic risk in β-TI patients is related to increased circulating PMPs and platelet count. Splenectomy was considered a risk factor of thrombosis in our study.

Membranoproliferative glomerulonephritis in β-thalassemia intermedia; a case report

Introduction: β-thalassemia intermedia reduces the body’s ability to produce adult hemoglobin and causes anemia. In contrast to β-thalassemia major, β-thalassemia intermedia patients do not require lifelong transfusion and are often independent of blood transfusion until young age. Moreover, chronic hypoxia and iron overload may cause tubular and glomerular dysfunction in patients with thalassemia. Case Presentation: We report a 21-year-old female with β-thalassemia intermedia (β-TI) presenting with generalized edema and proteinuria and showed membranoproliferative glomerulonephritis (MPGN) after renal biopsy. Conclusion: The possibility of occurrence of MPGN in patients with thalassemia should be considered. To our knowledge, it is the first case of thalassemia that was reported with MPGN and, more investigation is required to assess the association of thalassemia and MPGN.

Characterization of Iron Complexes in the Red Blood Cells of β-thalassaemia patients using 57Fe Mössbauer Spectroscopy

Red Blood Cells (RBCs) samples from healthy volunteers, β-thalassaemia major and β-thalassaemia intermedia patients were studied by means of 57Fe Mössbauer Spectroscopy (MS). The MS spectra from the RBCs of the thalassaemia patients showed the existence of significant amounts of ferritin-like iron, and in particular in the 82% of the β-thalassaemia intermedia ones. Also, β-thalassaemia intermedia patients were found to have higher Liver Iron Concentration (LIC) when compared to the β-thalassaemia major patients, as assessed by MRI exams, even though they had on an average about half the serum ferritin levels.

First description of the rs45496295 polymorphism of the C/EBPE gene in β-thalassemia intermedia patients

The C/EBPE gene, located in 14q11.2, encodes for a B/zip-type transcription factor. The C/EBPɛ is involved in terminal differentiation and functional maturity of granulocyte progenitor cells and in cell apoptosis during myeloid differentiation. A C/EBPE gene has recently been described as a candidate gene involved in clinical variability of β-thalassemia (β-thal). In this study, the C/EBPE gene was sequenced in 146 subjects divided into the severe type of β-thal major (β-TM) and moderate type of β-thal intermedia (β-TI), and a control group. The analysis identified the rs45496295 (C > T) polymorphism in the heterozygous state in 73.9% β-TI patients, which was not the case in the β-TM patients or in the control group. Thus, the T allele is consequently associated with the β-TI group (p = 10−3). According to the Human Splicing Finder (version 3.0, Marseille, France), the presence of the rs45496295 polymorphism leads the creation of a new intronic exotic splicing enhancer (ESE) site. Moreover, the T allele of rs45496295 is associated with a lower transfusion regimen (p = 10−3) and a higher pretransfusion hemoglobin (Hb) rate (p = .006). The comparison of several factors concerning T allele carriers and non-carriers showed that the T allele does not act on the Hb F rate. The T allele of rs45496295, associated with moderate type of β-thal, seems to modify the C/EBPɛ action, thereby preventing the hemolysis.

Genetic Variants at BCL11A and HBS1L-MYB loci Influence Hb F Levels in Chinese Zhuang β-Thalassemia Intermedia Patients

Increased Hb F levels can ameliorate the symptoms of β-thalassemia (β-thal). Due to the genetic heterogenicity of β-thal, the relationship between genetic variants in modifier genes and Hb F level has been studied in different populations. The Chinese Zhuang has the second largest population in China and has 6.78% prevalence of β-thal. However, the effects of these single nucleotide polymorphism (SNP) variants on the Hb F levels of β-thal intermedia (β-TI) patients in this population have not been reported. To explore the association between modifier loci (β-globin gene cluster, HBS1L-MYB intergenic region and BCL11A) and Hb F levels in Chinese Zhuang β-TI patients, 96 unrelated β-TI patients (50 males and 46 females) with different Hb F levels were recruited and genotyped by mass spectrometry. A total of 13 SNPs were confirmed to be in a significant relationship with Hb F levels in this population. Of these, high-risk genotypes of six Hb F-associated SNPs, rs9376090, rs7776054, rs9399137, rs9389268, rs9402685 in the HBS1L-MYB intergenic region and rs189984760 in the BCL11A locus, showed association with high Hb F levels, especially for SNPs in linkage disequilibrium. One novel Hb F-associated SNP, rs189984760, was identified in our study. Our findings will be of valuable reference for correlation between modifier genes and Hb F in Chinese Zhuang populations and may lead to better understand the modifying mechanisms for β-thal.

Can hydroxyurea serve as a free radical scavenger and reduce iron overload in β-thalassemia patients?

In this study, we hypothesize that hydroxyurea could provide an additional benefit as a free radical scavenger and/or iron chelator in β-thalassemia patients with iron overload. Twenty-one β-thalassemia intermedia patients who presented between 3 and 17 years but later required regular blood transfusions were enrolled for hydroxyurea therapy for a year. Fourteen patients responded to the therapy with hemoglobin levels maintained above 7.5 g/dl without transfusions. Hydroxyurea was discontinued after 6 months in seven patients who did not respond to the therapy and had to be continued on regular blood transfusions. We observed a statistically significant decrease in serum ferritin levels from 4194 ± 4850 ng/ml to 2129 ± 2380 ng/ml among the responders and from 2955 ± 2909 ng/ml to 2040 ± 2432 ng/ml among the non-responders and statistically significant decrease in labile iron pool from 18678.7 ± 10067.4 mean fluorescence intensity (MFI) to 14888.5 ± 5284.0 MFI among responders and from 17986.3 ± 9079.8 MFI to 15634.8 ± 8976.9 MFI among the non-responders after therapy. Phosphatidylserine externalization also showed a statistically significant decrease from 44.2 ± 22.2 MFI to 16.6 ± 6.7 MFI among the responders and from 46.9 ± 33.1 MFI to 39.8 ± 7.4 MFI among the non-responders along with a statistically significant decrease in the levels of reactive oxygen species from 72.8 ± 35.5 MFI to 29.0 ± 8.3 MFI among the responders and from 80.9 ± 41.4 MFI to 40.5 ± 15.8 MFI among the non-responders after therapy. A statistically significant increase in reduced glutathione levels was also observed from 430.8 ± 201.1 MFI to 715.5 ± 292.4 MFI among the responders and from 359.6 ± 165.6 MFI to 450.3 ± 279.5 MFI among the non-responders after therapy. This suggests the possible additional role of hydroxyurea as a free radical scavenger and/or iron chelator but requires a larger study for substantiation.

Decreased expression of CD47 and CD55 surface molecules on density-based subsets of red cells of β-thalassaemia intermedia patients compared to red cells of healthy blood donors

Decreased expression of CD47 and CD55 surface molecules on density-based subsets of red cells of β-thalassaemia intermedia patients compared to red cells of healthy blood donors

Clinical and Haematological Effects of Hydroxyurea in β-Thalassemia Intermedia Patients.

It is well known that hydroxyurea (HU) impacts on clinical and haematologic indices in thalassemia. We aimed to evaluate the effect of hydroxyurea on clinical and haematological improvement in children with thalassemia intermedia. After the patients' enrollment in the study their data such as transfusion, hospitalization, spleen size, visit, total Hb, HbF levels, MCV and MCH were compared before and after treatment with HU 10 mg/kg/day/for one year. In patients with thalassemia intermedia, HU significantly diminished the rate of transfusion, hospitalization, spleen size and significantly increased Hb MCH, HbF and MCV. Moreover HU was well tolerated in our patients and we got no remarkable adverse effect. We divulged hydroxyurea 10 mg/kg/day during one year. This significantly increased HbF, total haemoglobin, MCV, MCH, without any remarkable adverse events.

Priapism, an Emerging Complication in β-Thalassemia Intermedia Patients

The increase in survival rate of β-thalassemia (β-thal) patients allowed for the appearance and manifestation of several complications in almost every organ system. Priapism in β-thal patients is rarely reported in the literature. We herein report and investigate the occurrence of two cases of priapism in two young patients with β-thal intermedia (β-TI). The potential mechanisms are due to either a cellular mechanism involving a thrombus obstructing the efferent venules of the corpora cavernosa leading to priapism, or a recently elucidated functional mechanism that causes alteration of nitric oxide (NO) response of the penis, ultimately causing priapism. This should incite clinicians for a close follow-up and monitoring of high risk patients who are susceptible to developing priapism.

Iron overload in β-thalassemia intermedia

The aim is to overview recent evidence on consequences, assessment, and management of iron overload in transfusion-independent patients with β-thalassemia intermedia. Despite their transfusion-independence, β-thalassemia intermedia patients can still accumulate iron due to increased intestinal absorption. Recent observational studies show that iron burden in this group of patients can ultimately reach considerably high thresholds, and leads to a variety of serious morbidities involving the liver, endocrine glands, and arguably the vascular system. The diagnosis of iron overload in this patient population can follow established guidelines from β-thalassemia major patients, although with careful interpretation of spot serum ferritin levels. Data from a recent randomized clinical trial demonstrated the efficacy and safety of iron chelation therapy in decreasing liver iron concentration in this group of patients, which may ultimately help in reducing morbidity risk. Iron overload in transfusion-independent patients with β-thalassemia intermedia deserves careful attention, and prompt diagnosis and management are recommended.

KLF10 Gene Expression is Associated with High Fetal Hemoglobin Levels and with Response to Hydroxyurea Treatment in β-Hemoglobinopathy Patients

In humans, fetal hemoglobin (HbF) production is controlled by many intricate mechanisms that, to date, remain only partly understood. Pharmacogenomic analysis of the effects of hydroxyurea (HU) on HbF production was undertaken in a collection of Hellenic β-thalassemia and sickle cell disease (SCD) compound heterozygotes and a collection of healthy and KLF1-haploinsufficient Maltese adults, to identify genomic signatures that follow high HbF patterns. KLF10 emerged as a top candidate. Moreover, genotype analysis of β-thalassemia major and intermedia patients and an independent cohort of β-thalassemia/SCD compound heterozygous patients that do or do not respond to HU treatment showed that the homozygous mutant state of a tagSNP in the KLF10 3'UTR is not present in β-thalassemia intermedia patients and is underrepresented in β-thalassemia/SCD compound heterozygous patients that respond well to HU treatment. These data suggest that KLF10 may constitute a pharmacogenomic marker to discriminate between response and nonresponse to HU treatment.

α-Haemoglobin stabilising protein expression is influenced by mean cell haemoglobin and HbF levels in HbE/β-thalassaemia individuals

The alpha haemoglobin stabilising protein (AHSP) acts as a molecular chaperone for α-globin by stabilising nascent α-globin before transferring it to waiting free β-globin chains. Binding of AHSP to α-globin renders α-globin chemically inert whereby preventing it from precipitating and forming reactive oxygen species byproducts. The AHSP has been actively studied in the recent years, particularly in its relation to β-thalassaemia. Studies have shown that AHSP is a modifier in β-thalassaemia mice models. However, this relationship is less established in humans. Studies by some groups showed no correlation between the AHSP haplotypes and the severity of β-thalassaemia, whereas others have shown that certain AHSP haplotype could modify the phenotype of β-thalassaemia intermedia patients. We investigated the expression of AHSP in relation to selected demographic data, full blood count, HPLC results, HbE/β-thalassaemia genotype, Xmn-1 Gγ polymorphism, α-globin, β-globin and γ-globin expression. We found that AHSP expression was significantly correlated to mean cell haemoglobin level, HbF %, α-globin, β-globin and excess α-globin expression. We concluded that AHSP could be a secondary compensatory mechanism in red blood cells to counterbalance the excess α-globin chains in HbE/β-thalassaemia individuals.

Clinicopathological and Radiological Study of Egyptian β-Thalassemia Intermedia and β-Thalassemia Major Patients: Relation to Complications and Response to Therapy

The clinico epidemiological characteristics, frequency of complications, and response to various therapeutic modalities in 80 Egyptian β-thalassemia intermedia (β-TI) patients were compared with 70 β-thalassemia major (β-TM) patients. β-Thalassemia intermedia patients had a higher incidence of left atrium dilatation, right ventricular dilatation and pulmonary hypertension, whereas, β-TM patients showed a higher incidence of left ventricular (LV) dilatation, restrictive LV filling and impaired LV contractility, with an overall higher incidence of heart disease (p <0.001). Short stature, delayed puberty, osteoporosis, bone fractures, diabetes mellitus and viral hepatitis was frequently observed in β-TM patients compared with β-TI patients (p <0.05). Administration of hydroxyurea (HU) alone was associated with significant improvement in hematological parameters and quality of life for β-TI patients. In conclusion, the risk of complications still burdens the life of Egyptian thalassemia patients and their frequency varies between β-TI and β-TM. We provide evidence that calls for the use of HU in β-TI patients.

Early Echocardiographic Findings in β-Thalassemia Intermedia Patients Using Standard and Tissue Doppler Methods

Heart complications are among the serious problems of patients with β-thalassemia intermedia. This study aimed to evaluate myocardial function in these patients. Clinical parameters and both standard Doppler and pulsed Doppler tissue imaging parameters were compared in 51 β-thalassemia intermedia patients (mean age, 17.05 ± 5.8 years) and 20 normal subjects (mean age, 17.81 ± 7.35 years, p = 0.98). In 11 patients (21.5%), pulmonary artery hypertension was detected. M-mode echocardiographic findings such as ejection fraction and fractional shortening did not show statistically significant changes (p > 0.005). Pulsed Doppler showed a significant difference in the early (E) to late diastolic (A) velocity ratio of the tricuspid and mitral valve between the patients and the control subjects (p < 0.05). In the pulsed tissue Doppler study, the peak systolic velocity of the septum (Ss), the peak atrial velocity of the septum (Aas), the peak systolic velocity of the tricuspid annulus (St), the peak early diastolic velocity of the tricuspid annulus (Eat), and the peak late diastolic velocity of the tricuspid annulus (Aat) were increased significantly (p < 0.05). The pulse tissue Doppler of the lateral mitral annulus did not change significantly (p > 0.005). The peak systolic velocity of the posterior wall and the peak late diastolic velocity of the anterior wall changed significantly (p < 0.05). This study showed that β-thalassemia intermedia patients with normal M-mode and two-dimensional echocardiography had statistically significant changes in pulsed Doppler and pulsed tissue Doppler imaging.

Down Regulation of Hepcidin and Haemojuvelin Expression in the Hepatocyte Cell-Line HepG2 Induced by Thalassaemic Sera.

β-thalassaemia represents a group of diseases, in which ineffective erythropoiesis is accompanied by iron overload. In a mouse model of β-thalassaemia we observed that the liver expresses relatively low levels of hepcidin, which is a key factor in the regulation of iron absorption by the gut and of iron recycling by the reticuloendothelial system. We hypothesized that despite the overt iron overload, a putative plasma factor found in β-thalassaemia might suppress liver hepcidin expression. We therefore compared sera from β-thalassaemia and haemochromatosis (C282Y mutation) patients with those of healthy individuals in terms of their capacity to evoke changes in expression of key genes of iron metabolism in human HepG2 hepatoma cells. Sera from β-thalassaemia major patients evoked a major decrease in hepcidin (HAMP) and lipocalin2 (oncogene 24p3) (LCN2) expression, as well as a moderate decrease in haemojuvelin (HFE2) expression, compared to sera from healthy individuals. Significant correlation was found between the degree of downregulation of HAMP and HFE2 evoked by b-thalassaemia major sera (r=0.852, p<0.0009). Decreased HAMP expression was also found in HepG2 cells treated with sera collected from β-thalassaemia intermedia patients. In contrast, the majority of sera from hereditary haemochromatosis patients evoked an increase in HAMP expression, which correlated with their transferrin saturation (r=0.765, p<0.0099). Our results suggest that in β-thalassaemia, serum factors might override the potential effect of iron overload on HAMP expression, thereby providing an explanation for the failure to arrest excessive intestinal iron absorption.

Pulmonary hypertension in thalassemia: Association with platelet activation and hypercoagulable state

The pathogenesis of pulmonary hypertension (PAH), a serious complication in thalassemia, is not well understood. Thromboembolism has been postulated as one of the causative factors; however, there are currently limited specific data on its role. To examine whether increased platelet activation and hypercoagulability are linked to PAH, 25 beta-thalassemia major and beta-thalassemia intermedia patients were evaluated with Doppler echocardiograms for estimation of pulmonary artery pressure and with laboratory assays for indications of a prothrombotic state. The association of clinical variables and abnormal coagulation assays with PAH was determined. PAH was identified in 17 (68%) patients; mean pulmonary artery systolic pressure was 39.8 +/- 5.4 mm Hg. PAH was significantly associated with prior splenectomy, older age, and evidence for chronic hemolysis, diagnosed in both transfused (n = 10) and nontransfused (n = 7) patients. Increased platelet activation, measured by P-selectin, was significantly associated with PAH (P = 0.001). Increased thrombin-antithrombin III level was more prevalent in the presence of PAH, but increased fibrinolysis or low protein C levels were not. This study underscores the role of platelet activation in the development of PAH and stresses its occurrence even among patients who are regularly transfused, especially those who are older and have had splenectomies.

The Effect of Hydroxyurea on the Coagulation System in Sickle Cell Anemia and β-Thalassemia Intermedia Patients: A Preliminary Study

The Effect of Hydroxyurea on the Coagulation System in Sickle Cell Anemia and β-Thalassemia Intermedia Patients: A Preliminary Study

The Effect of Hydroxyurea on the Coagulation System in Sickle Cell Anemia and β-Thalassemia Intermedia Patients: A Preliminary Study

Hydroxyurea (HU) has been shown to reduce the frequency of vaso-occlusive manifestations in both adults and children with sickle cell disease (SCD), and the induction of hemoglobin F (HbF) is thought to be the underlying mechanism responsible for clinical improvement in some patients. However, there exists no good correlation between the amount of HbF increase and clinical response. Recent studies suggest that increased activity of the coagulation system may be important in the pathogenesis of vascular occlusion in sickle cell disease. To analyze the effect of HU on the coagulation system in children, the authors studied the levels of some coagulation factors and natural inhibitors. Eleven children who had been treated with HU because of SCD (5 patients), sickle- g -thalassemia (3 patients), and g -thalassemia intermedia (3 patients) were enrolled in the study. Levels of the coagulation factors II, V, VII, VIII, IX, X, XI, and XII, and of protein C and protein S, prothrombin times, activated partial thromboplastine times, thrombin times, and reptilase times were measured before the treatment and at the 5th or 6th months of HU therapy when the patients were in a steady-state condition. There was a decrease in all of the coagulation factors except for FIX and FXII and in inhibitors such as protein C and protein S. However, statistically significant decreases were observed only in factor VIII and protein C levels. The rates of decrease were 54.8 and 12.5% ( p = .015 and p = .018) in FVIII and protein C, respectively. This result shows that HU has significant effects on the coagulation and natural inhibitory systems.

Hypocholesterolemia in children and adolescents with β-thalassemia intermedia

Objectives: To conduct a prospective study to evaluate the lipid profile in children and adolescents with β-thalassemia intermedia and major, and to examine the contribution of different factors to hypocholesterolemia observed in these patients. Study design: Demographic, clinical, and laboratory data were prospectively obtained from patients with β-thalassemia intermedia (n = 9) and major (n = 47). Lipid profiles were also determined in a control group of healthy children (n = 18). Lipid values of β-thalassemics and controls were compared and the relationships between lipid levels and different covariates were determined. Results: β-thalassemia intermedia patients had significantly lower total cholesterol (TC), high-density lipoprotein cholesterol (HDL-C) and low-density lipoprotein cholesterol (LDL-C) compared with β-thalassemia major and controls (P <.001). With regression analysis, serum lipid levels (TC, HDL-C, and triglycerides) correlated with diagnosis (β-thalassemia major or intermedia) but not with age, sex, hemoglobin, or ferritin. LDL-C was influenced by both diagnosis and ferritin levels. Conclusions: Children and adolescents with β-thalassemia intermedia have significantly lower cholesterol levels than patients with β-thalassemia major. This is related to their disorder and not influenced by age, sex, hemoglobin, or ferritin levels. In these patients, needless investigations for hypolipidemia should be avoided. (J Pediatr 2002;141:543-7)

β-THALASSEMIA INTERMEDIA FROM SOUTHERN IRAN: IVS-II-1 (G→A) IS THE PREVALENT THALASSEMIA INTERMEDIA ALLELE

The preliminary results of a pilot study are reported, intended as an initiation of a research plan, focused on the prevention of β-thalassemia in Iran. The aims of this study are: (i) to improve the knowledge of the molecular background of β-thalassemia intermedia in Southern Iran; (ii) to verify the role of the −158 Gγ (C→T) (Xmn I) polymorphism as a modulating factor in thalassemia intermedia; (iii) to test the validity of the multiplex and single mutation specific amplification refractory mutation system in analyzing the molecular defects causing β-thalassemia in multiethnic populations; and (iv) to develop suitable strategies for the application of prevention protocols in Iran. To accomplish the task we have selected 87 β-thalassemia intermedia patients and adapted the DNA methodology to detect the following 11 frequent mutations in Iran: codon 5 (−CT); frameshift codons (FSC) 8/9 (+G); codon 30 (G→C); IVS-I-1 (G→A); IVS-I-5 (G→C); IVS-I-6 (T→C); IVS-I-110 (G→A); codons 36/37 (−T); codon 44 (−C); IVS-II-1 (G→A); IVS-II-745 (C→G). Because of the multiethnicity of the population we have also included the Indian IVS-I (25 bp deletion) and the Mediterranean IVS-I-130 (G→C) and codon 39 (C→T) mutations. Forty-eight patients were randomly studied for the Xmn I polymorphism together with 50 healthy volunteers as a control group. The molecular analysis conducted in Iran, identified only 31% of the alleles that were presumed to be thalassemic, revealing either a strategic or a technical insufficiency of the chosen method. However, the mutations with the highest prevalence in the country (IVS-II-1, IVS-I-110, IVS-I-1 and FSC 8/9) were found. As expected the IVS-II-1 defect, being the most frequent in south Iran, was present at the highest rate (24%). The Xmn I polymorphism was found in association with this prevalent mutation and was detected in the homozygous state in 87.5% of the patients homozygous for the IVS-II-1 (G→A) mutation. The overall positivity for Xmn I was found in 40.6% of the thalassemic alleles vs. 14% in the non-thalassemic, confirming the hypothesis of an older event, antecedent to the IVS-II-1 mutation. In trying to assess a more suitable molecular detection method we intend to continue this study in collaboration with the European centers involved, applying more effective technologies and better defining the molecular spectrum of β-thalassemia in the sub-populations. We also intend to verify the effect of α-thalassemia in the genotype/phenotype correlation of β-thalassemia intermedia.