Beta-thalassemia Intermedia Patients Research Articles

A survey of intracranial blood flow velocity in thalassemia intermedia in Khuzestan Province, Iran

Background: Beta-thalassemia intermedia (BTI) is a type of hemoglobinopathy with an increased risk of cerebrovascular accidents, and transcranial cerebral Doppler ultrasonography (TCD) through determining the mean cerebral blood flow velocity (CBFV) can serve to predict the risk of a developing stroke. This study aims to compare patients with beta-thalassemia intermedia and healthy individuals in terms of the cerebral blood flow velocity.
 Materials and Methods: This research was a case control study on 35 BTI patients and 25 healthy subjects. The patients were categorized into three age groups including 7-10, 11-15 and 16-20 years old. The mean CBFVs were compared between the two groups. The factors of age, gender, serum ferritin level, hemoglobin level, spleen size, thrombocytosis, and thalassemia genotypes were evaluated for their effects on CBFVs.
 Results: Mean CBFVs were significantly higher in all the intracranial arteries of BTI patients compared to normal subjects (p-value < 0.05). The hemoglobin levels showed a negative correlation between the left and right vertebral arteries of BTI patients in terms of blood flow velocity (p-value < 0.05). The mean CBFVs in the left vertebral and basilar arteries were negatively correlated to age in BTI. There was no correlation among ferritin level, thrombocytosis, splenomegaly, splenectomy, XmnI polymorphism, and cerebral blood flow velocity in the BTI patients group (p-value > 0.05).
 Conclusion: This study showed that cerebral blood flow velocities of BTI patients were higher than normal control group. In addition, CBFVs were not affected by factors such as gender, serum ferritin, platelet count, size of spleen and XmnI genotype, however, there was negative correlation between age and hemoglobin level with CBFVs.

Usefulness of Eosin-5’-Maleimide (EMA) Dye for the Diagnosis of Hereditary Spherocytosis

Abstract Background the normal biconcave discoid shape of the red blood cell (RBC) is maintained by cytoskeletal proteins underneath the membrane. This protein network and its link with the lipid bilayer are essential for RBCs to remain flexible and carry out their physiological functions. A number of disorders including hereditary spherocytosis (HS) and hereditary elliptocytosis (HE) are caused by decreased expression or mutation of cytoskeletal proteins. Aim of the Work to evaluate the usefulness eosin-5-maleamide (EMA) dye binding flow cytometric test in the diagnosis of HS and assess the sensitivity and specificity of this test to distinguish HS patients from patients having anemia due to other causes. Subjects and Methods this study was conducted on 52 patients with chronic anemia, 21 diagnosed as HS (group I) and 31 patients with anemia due to other causes (group II). The patients were 28 males and 23 females with a male to female ratio 1.2:1, their ages ranged between 3 to 12.5 years old attending at the Pediatric Hematology Clinics, Ain Shams University Hospitals. Normal control group (group III) was included in the study. Results median fluorescence intensity (MFI) had shown significantly lower values in HS patients (group I) (3.78±0.48) than (iron deficiency anemia) IDA patients (group IIa) (5.56±0.35), auto immune hemolytic anemia (AIHA) patients (group IIb) (5.7±0.27), beta thalassemia intermedia (BTI) patients (group IIc) (6.01±0.63) and control group (group III) (5.3±0.43). The % reduction in EMA fluorescence proved to be significantly higher in HS patient (group I) (29.88±8.27) than in IDA patients (group IIa) (1.13±6.18), AIHA patients (-6.14±10.44) and BTI patients (group IIc) (-5.1±10.07). Multi regression analysis had been used to check the most sensitive predictors for diagnosis of HS, both EMA MFI and the % reduction in EMA fluorescence proved to be the best sensitive predictive values for discriminating HS patients, however the higher F-ratio in EMA MFI (F = 25.222) than in % reduction in EMA fluorescence intensity (F = 19.866) showed that the MFI is even better than % reduction in fluorescence in diagnosis of HS. Conclusion the present study highlighted the role of flow cytometric EMA binding test in differentiating HS patients from patients having anemia due to other causes.

Global analysis of erythroid cells redox status reveals the involvement of Prdx1 and Prdx2 in the severity of beta thalassemia.

β-thalassemia is a worldwide distributed monogenic red cell disorder, characterized by an absent or reduced beta globin chain synthesis. The unbalance of alpha-gamma chain and the presence of pathological free iron promote severe oxidative damage, playing crucial a role in erythrocyte hemolysis, exacerbating ineffective erythropoiesis and decreasing the lifespan of red blood cells (RBC). Catalase, glutathione peroxidase and peroxiredoxins act together to protect RBCs from hydrogen peroxide insult. Among them, peroxiredoxins stand out for their overall abundance and reactivity. In RBCs, Prdx2 is the third most abundant protein, although Prdxs 1 and 6 isoforms are also found in lower amounts. Despite the importance of these enzymes, Prdx1 and Prdx2 may have their peroxidase activity inactivated by hyperoxidation at high hydroperoxide concentrations, which also promotes the molecular chaperone activity of these proteins. Some studies have demonstrated the importance of Prdx1 and Prdx2 for the development and maintenance of erythrocytes in hemolytic anemia. Now, we performed a global analysis comparatively evaluating the expression profile of several antioxidant enzymes and their physiological reducing agents in patients with beta thalassemia intermedia (BTI) and healthy individuals. Furthermore, increased levels of ROS were observed not only in RBC, but also in neutrophils and mononuclear cells of BTI patients. The level of transcripts and the protein content of Prx1 were increased in reticulocyte and RBCs of BTI patients and the protein content was also found to be higher when compared to beta thalassemia major (BTM), suggesting that this peroxidase could cooperate with Prx2 in the removal of H2O2. Furthermore, Prdx2 production is highly increased in RBCs of BTM patients that present high amounts of hyperoxidized species. A significant increase in the content of Trx1, Srx1 and Sod1 in RBCs of BTI patients suggested protective roles for these enzymes in BTI patients. Finally, the upregulation of Nrf2 and Keap1 transcription factors found in BTI patients may be involved in the regulation of the antioxidant enzymes analyzed in this work.

Evaluation of Endocrine Complications in Beta-Thalassemia Intermedia Patients: A Cross Sectional Multi-Center Study

The diagnosis of beta thalassemia intermedia (BTI) is mainly based on the severity of clinical phenotype. It is associated with a wide range of specific complications including extramedullary hematopoiesis, leg ulcers, gallstones, hypercoagulable state, pulmonary hypertension (PHT), endocrine disorders and osteoporosis. The commonest endocrine complication in beta thalassemia major (TM) patients is hypogonadism followed by hypothyroidism and diabetes but the data on endocrine disorders in BTI patients are scarce. The aim of this study is to determine the prevalence of endocrine complications in a large series of BTI patients.Methods: In this multi-countries cross-sectional study, all BTI patients registered at 12 thalassemic treatment centers in Iran (2 Centers), Italy (2 Centers), Greece, Turkey (2 centers), Oman, Qatar, Jordan, Cyprus and United Kingdom were enrolled during 2017. Non transfusion- dependent beta-thalassemia patients or those who received blood transfusion 3-4 times or less annually, were evaluated. Required information was collected from medical records using a designed questionnaire. Demographic data, clinical characteristics and laboratory data included age, sex, splenectomy, type of treatment (blood transfusion, iron chelation, hydroxyurea), hormonal assays, bone mineral density, calcium -phosphorous metabolism, serum ferritin, liver function tests, fetal and total hemoglobin levels, platelet and nucleated red blood cell counts, were collected.Results: A total of 721 BTI patients were enrolled in the survey from 9 countries. The most prevalent disease-related complications were osteoporosis (21.6%) and hypogonadism (12.6%) followed by: central hypothyroidism (8.3%), non-insulin-dependent diabetes mellitus (7.8%), primary hypothyroidism (5.5%), insulin-dependent diabetes mellitus (4.2%), hypoparathyroidism (2.2%), growth hormone deficiency (1.1%), adrenal mass (1%) and thyroid cancer (0.5%).Conclusion: This study evaluated the largest cohort of BTI patients with endocrine disorders. Although BTI patients are non-transfusion dependent or only occasionally transfused, iron-overload due to increased intestinal iron absorption and enhanced bone marrow activity cause endocrine disorders and osteoporosis. This study demonstrate that although endocrine complications are less common in patients with BTI compared to data reported in literature in TM patients , a regular monitoring with timely diagnosis and proper management underscoring on osteoporosis and gonadal disorders are crucial to prevent endocrine complications in these patients. DisclosuresNo relevant conflicts of interest to declare.

A comparison of heart function and arrhythmia in clinically asymptomatic patients with beta thalassemia intermedia and beta thalassemia major

ABSTRACTBackground: The goal of this study was to compare heart function and arrhythmia in clinically asymptomatic patients with beta thalassemia intermedia and beta thalassemia major.Method: In this cross-sectional study, 60 patients with beta thalassemia major and 60 patients with beta thalassemia intermedia who had clinically no symptoms of arrhythmia and clinically normal heart function were evaluated using 24-hour ambulatory electrocardiogram monitoring and echocardiography. For data analysis SPSS ver.20 software was used. A P-value of less than 0.05 was considered statistically significant.Results: The mean age of the beta thalassemia intermedia patients was 24.18 ± 7.9 years and the mean age in beta thalassemia major was 24.38 ± 7.7 years (P>0.05). Premature atrial contractions (PACs) were observed in 14 (23.3%) patients with beta thalassemia intermedia and in 22 (36.6%) beta thalassemia major patients. Premature ventricular contractions (PVCs) were detected in 8 (13.3%) patients in the beta thalassemia intermediate group and 16 (26.6) patients in the beta thalassemia major group, respectively. The left ventricular diastolic dimension, end-diastolic volume, and stroke volume were significantly higher in beta thalassemia intermedia group (P<0.05). Pulmonary acceleration time as an indicator of pulmonary pressure was lower in beta thalassemia intermedia group.Conclusion: Both atrial and ventricular arrhythmias were more common in the beta thalassemia major group. Higher end-diastolic volume and stroke volume were detected in the beta thalassemia intermedia group. Pulmonary acceleration time was lower in the beta thalassemia intermedia group, which can be an indicator of higher pulmonary pressure.

Comparative study of radiographic and laboratory findings between Beta thalassemia major and Beta thalassemia intermedia patients with and without treatment by hydroxyurea.

Background:In patients with thalassemia, chronic anemia causes bone marrow expansion and consequently skeletal manifestation in spine, skull, face and rib bones.Objectives:We aimed to compare chest radiographic findings and facial bone deformity in patients with thalassemia major (TM) and intermedia.Patients and Methods:In this cross sectional study, 86 consecutive thalassemia patients referring to the Thalassemia clinic in Shiraz, Southern Iran were evaluated during 2012. Patients were divided into three groups including TM and thalassemia intermedia (TI) with and without taking hydroxyurea (HU). Findings ofchest radiography (trabeculation, rib widening and paraspinal masses) as well as facial bone deformity were evaluated by an expert radiologist. Besides, laboratory findings were measured regarding hemoglobin, ferritin, NRBC and platelet count.Results:All radiologic findings were significantly higher in patients with TI compared to TM (P< 0.05). In patients with TI, only trabeculation was observed with a higher frequency in patients with HU compared to those without HU (68% vs. 27.3%, P= 0.008). In the regression model, from all variables evaluated, only NRBC showed a significant correlation with trabeculation (Exp B = 1.014, CI: 1.004-1.025, P = 0.008) and age showed a significant correlation with paravertebral mass (Exp B = 1.147, CI: 1.03-1.27, P = 0.013).Conclusions:In patients with TM, bone widening, trabeculation, paraspinal masses and facial bone deformity were lower than patients with TI, whichcan be related to effectiveness of therapy with blood transfusion irrespective of its adverse effects in TM patients.

Frequency of cholelithiasis in patients with Beta-thalassemia intermedia with and without hydroxyurea.

Background:Recent studies regarding the effect of hydroxyurea (HU) in thalassemia have revealed favorable effects on the reduction of ineffective erythropoiesis.Objectives:The aim of the current study was to evaluate whether or not HU can have an effect on the gallstone formation rate in patients with beta-thalassemia intermedia (BTI).Patients and Methods:In this case control cross-sectional study, from a total of 250 transfusion-independent BTI patients, 51 patients who were taking HU, participated in the study. Patients were registered in the Thalassemia clinic of Shiraz University of Medical Sciences, Shiraz, which is a referral center located in southern Iran, during 2011-2012. Mean dose of HU consumption in the case group was 10 ± 2.5 mg/kg/day (range of 8-15 mg/kg/day), with a mean duration of consumption of 7.5 ± 3.8 years (range 1-14 years). In addition, 41 age- and sex-matched BTI patients who did not use HU were randomly selected as a control group. All patients underwent abdominal ultrasound by a radiologist for evaluation of gallstones.Results:Mean age of the participants was 21.4 ± 6.5 years (10-40 years). There was no statistically significant difference regarding the frequency of cholelithiasis between the two groups of patients (P = 0.822). Our study showed significantly lower hemoglobin levels and a higher percentage of nucleated red blood cells in the HU group compared with the control group (P = 0.001 and P = 0.005, respectively).Conclusions:It seems that taking HU for long periods can reduce hemolysis and bone marrow suppression, and that decreases the rate of cholelithiasis. We believe that if these patients had not been treated with HU, we would probably have observed a significantly higher frequency of cholelithiasis, due to more hemolysis compared with patients not taking HU. Further studies with larger sample sizes are suggested.

A killer revealed: 10-year experience with beta-thalassemia intermedia

ObjectivesPatients with beta-thalassemia intermedia tend to present later in life with milder anemia than beta-thalassemia major patients. The incidence of mortality and its causes in this patient population remains unknown. We aim to reveal the incidence and most common causes of death in this population.Methods and resultsWe reviewed the charts of all of the beta-thalassemia intermedia patients who had been followed at the Chronic Care Center in Hazmieh, Lebanon during a 10-year period. A total of 18 patients out of 127 had died during the follow-up period giving a cumulative 10-year mortality incidence of 14%. The most common causes of cardiac deaths were due to renal and cardiac causes.DiscussionMost causes of death have been linked to the high levels of iron coupled with anemia present in this patient population. Many of deaths could be prevented by adequate treatment.ConclusionLarger studies with more comprehensive data capture on risk factors of mortality in this patient population are called for.

Comparative study of hypogonadism in beta-thalassemia intermedia patients with and without hydroxyurea

BackgroundWe aimed at evaluating the effect of long-term use of hydroxyurea (HU) on gonad function in patients with beta-thalassemia intermedia (beta-TI) in Iran.MethodsSeventy-five beta-TI patients aged ≥11 years, mean serum ferritin level of <1000 ng/dL in the recent 5 years, and taking HU with a dose of 8–15 mg per kg body weight per day for at least 5 years were randomly selected during 2010 in southern Iran. Thirty-one beta-TI patients without HU were considered as a control group. Serum levels of prolactin, luteinizing hormone, follicle-stimulating hormone, testosterone, and estradiol were measured. An expert endocrinologist examined all participants and reviewed their hormonal profile.ResultsThe mean age of the participants was 22.7 ± 5.1 years (age range of 12–41 years). Overall, 10 patients with hypogonadotropic hypogonadism were detected including seven patients (9.3%) with HU and three patients (9.7%) without HU. The difference between the two groups was not statistically significant (P = 0.605).ConclusionIt appears that long-term use of HU at a dose of 8–15 mg per kg body weight per day has no effect on gonad function in beta-TI patients. However, further prospective studies and large clinical trials are needed to confirm this subject.

HQK-1001 Is Well Tolerated and Augments Hemoglobin F and Hemoglobin Levels In Patients with Beta Thalassemia Intermedia

AbstractAbstract 4280Beta thalassemia intermedia syndromes are serious conditions for which there is no satisfactory therapy to correct the underlying globin chain imbalance. Some agents that induce fetal globin gene expression have ameliorated anemia in thalassemia patients by reducing the imbalance in alpha: non-alpha globin synthesis, but none have been broadly accepted or are currently approved by regulatory authorities. HQK-1001 is an oral agent that targets the fetal globin gene promoter, thereby increasing fetal hemoglobin (HbF) expression. It has been well tolerated in single dose and multiple dose escalation clinical studies in healthy volunteers. We now report the results of a randomized, double blind, placebo-controlled, multiple ascending dose Phase I/II trial in 21 adult patients with beta thalassemia intermedia (BTI), including 14 with HbE/ß0 thalassemia and 7 with ß+/ß0 thalassemia (including 12 different beta globin gene mutations). Study medication was taken as a single daily dose for 8 weeks. Four ascending dose levels (10, 20, 30, and 40 mg/kg/day) were sequentially evaluated in 4 dose level cohorts after the preceding dose and schedule were determined safe by an independent and unblinded Safety Monitoring Committee. HQK-1001 was well-tolerated. Adverse events in treated subjects included headache, upper respiratory infection and nausea, but the rates of such events were not markedly different than those observed in the placebo-treated subjects. The 20 mg/kg dose was associated with a 10% mean increase above baseline in HbF, (p< 0.001). Total hemoglobin (Hgb) increased by a mean of 1.1 gram/dL in 3 of 6 treated BTI patients with Mediterranean mutations. F-cells increased over the study period with maximal increases often observed 2 weeks following therapy. Doses higher than 20 mg/kg were not associated with the same magnitude of pharmacodynamic effects. These observations indicate that HQK-1001 is well-tolerated at doses associated with favorable pharmacodynamic effects on Hgb and HbF. These findings with brief treatment provide a rationale for conducting larger and longer studies in BTI patients. Disclosures:Fuchareon:HemaQuest Pharmaceuticals, Inc: Honoraria, Research Funding. Inati:HemaQuest Pharmaceuticals, Inc: Honoraria, Research Funding. Boosalis:HemaQuest Pharmaceuticals, Inc: Equity Ownership, Research Funding. Thein:HemaQuest Pharmaceuticals, Inc: Research Funding. Wallis:HemaQuest Pharmaceuticals: Consultancy, Equity Ownership. Bobbitt:HemaQuest Pharmaceuticals, Inc: Employment, Equity Ownership, Patents & Royalties. Thomson:HemaQuest Pharmaceuticals: Employment, Equity Ownership. Johnson:HemaQuest Pharmaceuticals: Employment, Equity Ownership. Berenson:HemaQuest Pharmaceuticals, Inc: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties. Perrine:HemaQuest Pharmaceuticals, Inc: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties, Research Funding.

Prevalence of thrombotic risk factors among β-thalassemia patients from Western Iran

There is evidence for increased risk of thrombosis in patients with beta-thalassemia intermedia and beta-thalassemia major. The present study investigated the prevalence of thromboembolic risk factors of prothrombin G20210A, factor V Leiden G1691A and methylentetrahydrofolate reductase (MTHFR) C677T, as well as the hematological and clinical profiles in beta-thalassemia major and intermedia patients from Western Iran. Patients consisted of 158 beta-thalassemia patients, 151 beta-thalassemia major and 7 beta-thalassemia intermedia patients, including 82 males and 76 females aged 13.6 +/- 6.3 years. The control group were 180 healthy blood donors and school students, consisting of 103 males and 77 females aged 16.8 +/- 2.1. Genotyping was done by PCR-RFLP using Mnl I, Hind III and Hinf I for factor V Leiden and prothrombin G20210A and MTHFR, respectively. The prevalence of prothrombin G20210A variant in patients and healthy individuals were 1.3 and 3.3%, respectively. Factor V Leiden G1691A was insignificantly higher in beta-thalassemia patients (prevalence 5.7% and allele frequency 3.2%) compared to healthy individuals (2.8%). This mutation was found in eight beta-thalassemia major (5.3%) and one beta-thalassemia intermedia (14.3%) patients. The prevalence of MTHFR C677T polymorphism was slightly higher in patients (50%) compared to healthy individuals (48.3%). Around 71% of beta-thalassemia intermedia and 38.4% of beta-thalassemia major patients had undergone splenectomy. In beta-thalassemia major patients, 5.3% had insulin dependent diabetes mellitus (IDDM) and 6.6% had HCV antibodies. All patients with IDDM were splenectomized and in one of them the prothrombin G20210A variant was found. Two patients, a 7-year-old boy with beta-thalassemia intermedia receiving regularly blood transfusion and a beta-thalassemia major patient (a 22-year-old splenectomized female), were found to be homozygous for MTHFR 677TT and heterozygous for factor V Leiden G1691A. Double heterozygosity for factor V Leiden G1691A and MTHFR C677T and also homozygous factor V Leiden 1691AA were found in two beta-thalassemia major patients. No thromboembolic event has been recorded in the files of patients. The results of present study establish the prevalence of biological risk factors of thrombosis in beta-thalassemia patients from Western Iran. It seems that thrombophilic mutations may not be associated with thrombotic events in thalassemic patients, which needs to be confirmed by the study of larger sample sizes.

Increased γ‐globin gene expression in β‐thalassemia intermedia patients correlates with a mutation in 3′HS1

We report a novel set of genetic markers in the DNaseI hypersensitive sites comprising the human beta-globin locus chromatin hub (CH), namely HS-111 and 3'HS1. The HS-111 (-21 G>A) and 3'HS1 (+179 C>T) transitions form CH haplotypes, which occur at different frequencies in beta-thalassemia intermedia and major patients and normal (nonthalassemic) individuals. We also show that the 3'HS1 (+179 C>T) variation results in a GATA-1 binding site and correlates with increased fetal hemoglobin production in beta-thalassemia intermedia patients. In contrast, the HS-111 (+126 G>A) transition, found in three normal chromosomes, is simply a rare polymorphism. We conclude that the CH haplotypes are useful genetic determinants for beta-thalassemia major and intermedia patients, while the 3'HS1 (+179 C>T) mutation may have functional consequences in gamma-globin genes expression.

Circulating Microparticles in Lebanese Patients with beta-Thalassemia Intermedia.

Introduction: Beta-thalassemia is a genetic disease with a prevalence that varies in the world reaching 3% in Lebanon. One-third of these patients have beta-thalassemia intermedia. Patients with thalassemia intermedia have complications not present in those with thalassemia major, mainly leg ulcers, extramedullary hematopoiesis and thrombosis. The beta-thalassemia intermedia patients not receiving transfusions and who are splectomized, are at higher risk of thrombosis as well as the other complications. Microparticles (MP) are shed submicrometric plasma membrane fragments (~0.1–1μm) harboring negatively charged phosphatidylserine (PS) in their extracellular membrane leaflet. They mainly stem from apoptotic or activated cells, and generally present a procoagulant potential. Increased levels of MP were described in disorders with major vascular and thrombotic symptoms such as myocardial infarction, diabetes, renal failure or sickle cell disease.Methods: In this study we determined circulating MP levels in platelet-free plasma samples of patients and healthy donors using a solid-phase capture assay on insolubilized streptavidin. The presence of circulating MP was first evaluated using annexin V-coupled biotin that binds the negatively charged PS in MP. Independent sample t-test was conducted to examine significant difference between patients and controls. Thirty-two patients with beta-thalassemia intermedia and 17 healthy controls were included. P-values less than 0.05 were considered to be statistically significant.Results: Fifty percent of the patients and 81.3% of the control group were males. The average age of the patients was 26.5 ± 9.5 years. Around 77% of the patients had splenectomy. Shed membrane MP were elevated to near statistical significance in platelet-free plasma of patients with beta-thalassemia intermedia but not in control individuals (7.3 ± 5.7 vs 4.7 ± 2.9 nmol/L PS equivalent, p=0.09). The cell origin of MP was further investigated after capture onto specific biotinylated antibodies used instead of annexin V. MP of red blood cell origins were elevated, as expected in patients with beta-thalassemia intermedia vs controls (6.05 ± 3.8 vs 2.54 ± 3.8 nmol/L PS equivalent, p<0.01 for patients and controls, respectively). Interestingly, both platelet and endothelial-derived MP were elevated in patients vs controls (of platelet origin: 3.02 ± 2.06 vs 1.91 ± 1.15 nmol/L PS equivalent, p=0.05; and of endothelial origin: 1.58 ± 0.87 vs 0.41 ± 0.3 nmol/L PS equivalent, p<0.01). Leukocyte and neutrophil-derived MP were also elevated in patients vs control (p=<0.01).Conclusion: These results indicate that circulating membrane MP are increased in beta-thalassemia intermedia and testify to increased platelet, endothelium and leukocyte activation, in probable association with the increased thrombotic risk. Further investigations are required to assess the significance of these enhanced markers of cell activation, with respect to a possible deleterious potential towards vascular or blood cell functions.

Downregulation of hepcidin and haemojuvelin expression in the hepatocyte cell‐line HepG2 induced by thalassaemic sera

Beta-thalassaemia represents a group of diseases, in which ineffective erythropoiesis is accompanied by iron overload. In a mouse model of beta-thalassaemia, we observed that the liver expressed 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. It was hypothesised that, despite the overt iron overload, a putative plasma factor found in beta-thalassaemia might suppress liver hepcidin expression. Sera from beta-thalassaemia and haemochromatosis (C282Y mutation) patients were compared with those of healthy individuals regarding their capacity to induce changes the expression of key genes of iron metabolism in human HepG2 hepatoma cells. Sera from beta-thalassaemia major patients induced a major decrease in hepcidin (HAMP) and lipocalin2 (oncogene 24p3) (LCN2) expression, as well as a moderate decrease in haemojuvelin (HFE2) expression, compared with sera from healthy individuals. A significant correlation was found between the degree of downregulation of HAMP and HFE2 induced by beta-thalassaemia major sera (r = 0.852, P < 0.0009). Decreased HAMP expression was also found in HepG2 cells treated with sera from beta-thalassaemia intermedia patients. In contrast, the majority of sera from hereditary haemochromatosis patients induced an increase in HAMP expression, which correlated with transferrin (Tf) saturation (r = 0.765, P < 0.0099). Our results suggest that, in beta-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 in these patients.

Clinical phenotype of triplicated α-globin genes and heterozygosity for β0-thalassemia in Chinese subjects

The presence of extra copies of alpha-globin gene has been shown to worsen the degree of anemia in beta-thalassemia heterozygotes. We describe the clinical phenotype of eight Chinese subjects with heterozygosity for both triplicated alpha-globin gene and a beta0-thalassemia allele. They were identified through genotyping of beta-thalassemia intermedia and major patients, and through community-based thalassemia screening program in Hong Kong. Standard molecular techniques were used in the determination of genotype. All subjects in this series showed five copies of alpha-globin genes (alphaalphaalpha/alphaalpha) in association with a beta0-thalassemia allele. Although genotypically identical, six subjects showed a beta-thalassemia intermedia phenotype while two were clinically indistinguishable from beta-thalassemia minor, implying the presence of genetic modifying factors that remained undefined. Triplication of alpha-globin gene and heterozygosity for beta0-thalassemia accounted for 15% of beta-thalassemia intermedia patients at our locality and was associated with a mild clinical phenotype. This genotype was not found among beta-thalassemia major patients. They presented in adulthood and were usually not transfusion dependent. When compared with simple beta-thalassemia heterozygotes, they showed obvious red cell abnormalities (hypochromasia, anisopoikilocytosis, circulating normoblasts), lower hemoglobin (Hb) and higher HbF levels. The presence of triplicated alpha-globin genes should always be considered in apparent beta-thalassemia carriers who were more symptomatic than expected, so that unnecessary investigations for the cause of anemia could be avoided. Finally, triplication of alpha-globin genes should be looked for in families with children affected by beta-thalassemia intermedia in which only one parent showed a picture of beta-thalassemia on Hb analysis.

Nucleotide changes in the gamma-globin promoter and the (AT)xNy(AT)z polymorphic sequence of beta LCRHS-2 region associated with altered levels of HbF.

We have studied 31 beta-thalassaemia intermedia, 30 beta-thalassaemia major patients and 50 normal individuals from Turkey, determining the relationship between the nucleotide variations in beta-globin gene cluster, the altered levels of foetal haemoglobin and the relative ratios of beta- and gamma mRNAs. We have found in beta-thalassaemia intermedia patients with high foetal haemoglobin expression that the three nucleotide variations in the 5' sequences of the gamma globin genes, A-->G at G gamma - 1396, the T-->C at A gamma - 228, and the GA-->AG at A gamma - 603/4, are linked to haplotype II in haplotypic homozygotes and the (AT)8N14(AT)7 motif in beta LCR. Conversely, the three single nucleotide substitutions in the 5' sequences of gamma globin genes, the G-->A at G gamma - 1225, the A-->G at A gamma + 25 and the C-->G at A gamma - 369, which have a strong linkage with haplotype I, V or VI in haplotypic homozygotes and the (AT)10N12(AT)12 and the (AT)9N12(AT)12 motifs in HS-2 of beta LCR are all associated with low foetal haemoglobin levels. The number of nucleotide changes in beta-globin gene cluster implied in our study are not the primary cause of the differences in haemoglobin F levels. They perhaps may contribute to the variations in the clinical severity observed among beta thalassaemia intermedia and major patients with other yet unknown gene conversions.

The interaction of anti 3.7 type quadruplicated alpha-globin genes and heterozygous beta-thalassemia.

Human alpha-globin gene mapping was carried out using a variety of restriction endonucleases (Bgl II, Bam HI, Hind III, Eco RI, Hpa I, Pvu II and Rsa I) on members of a family from El Salvador and a female from Hawaii, of Chinese descent, whose hematological and clinical parameters were those of beta-thalassemia intermedia. Southern blot DNA analysis showed that the beta-thalassemia intermedia patients from the above two families had the same anti 3.7 type quadruplicated alpha-genes on the one chromosome, and that they had the alpha genotype alpha 2, alpha 1 alpha 2, alpha 1 alpha 2, alpha 1/alpha 2, alpha 1. The alpha/beta globin synthesis ratios of the three affected Salvadoran patients were around 2.5, and the affected Hawaiian patient was 2.9. These ratios strongly suggest that the additional alpha-genes in the anti 3.7 type rearrangement are biologically active, thus accounting for the severity of the heterozygous beta-thalassemia observed among these patients.