Globin Chain Synthesis Research Articles

Electrocardiographic Presentation, Cardiac Arrhythmias, and Their Management in β-Thalassemia Major Patients.

Beta-thalassemia major (β-TM) is a genetic hemoglobin disorder characterized by an absent synthesis of globin chains that are essential for hemoglobin formation, causing chronic hemolytic anemia. Clinical management of thalassemia major consists in regular long-life red blood cell transfusions and iron chelation therapy to remove iron introduced in excess with transfusions. Iron deposition in combination with inflammatory and immunogenic factors is involved in the pathophysiology of cardiac dysfunction in these patients. Heart failure and arrhythmias, caused by myocardial siderosis, are the most important life-limiting complications of iron overload in beta-thalassemia patients. Cardiac complications are responsible for 71% of global death in the beta-thalassemia major patients. The aim of this review was to describe the most frequent electrocardiographic abnormalities and arrhythmias observed in β-TM patients, analyzing their prognostic impact and current treatment strategies.

Revisiting a Complex Rearrangement Involving a 619 Base Pairs Deletion, 6 Nucleotide Insertion Followed by a A > G Substitution Causing β°-Thalassemia

One of the prevalent inherited blood disorders is thalassemia syndrome that characterized by reduction (β+) or absence (β0) of β globin chain synthesis. The β globin (HBB) gene map in the short arm of chromosome 11 and most of the mutations in this gene are single nucleotide substitutions, insertions or deletions of nucleotides. Nucleotide sequence analysis of a partially deleted β-globin gene from an Iranian carrier of β-thalassemia displayed a complex rearrangement involving a 619 base pairs (bp) deletion. This rearrangement had originally been named as the 619bp deletion and later on as the 619bp deletion with a 7bp insertion. In our study, using by single chain sequencing, we have shown that the actual rearrangement involves a 619bp deletion, a 6bp insertion followed by a G>A substitution deleting the exon 3 of the β-globin gene. This clarification has to be inserted into the relevant databases as some of them still site the original 619bp deletion with wrong breakpoints.

THE IMPORTANCE OF IRP AS REGULATORS OF IRON METABOLISM IN β THALASSEMIA PATIENTS

Thalassemia is a hereditary blood disease. Beta (s)-thalassemia which caused by a point mutation on s-globin gene localized on short arm of chromosome 11 as a cluster, is an autosomal recessive and also one of the most common genetic diseases in worldwide. In other words, this disease is characterized by malfunctions during the globin chain synthesis of hemoglobin synthesis process. Unbalanced globin chain synthesis is the major cause of low level hemoglobin production leading to anemia. Current theraphy for this disease includes regular blood transfusions and iron chelation. Excessive hemolysis, increased intestinal iron absorption as well as frequent blood transfusions during theraphy results in chronically increased iron load and consequently oxidative stress. Iron homeostasis is provided by iron regulatory proteins (IRPs) in body. Therefore, variants of these proteins lead to differences in iron uptake and the storage. In this study, the role of IRP-1 and IRP-2 in iron uptake and the effect of oxidative stress consequent upon the iron toxicity in thalassemia individuals will be examined in detail. Keywords: IRP-1, IRP-2, gene polymorphism, oxidative stress and thalassemia.

Vertebra Plana due to Thalassemia: A Case Report

Beta thalassemia syndromes are a group of hereditary blood disorders causing a reduction of hemoglobin (Hb) content of red blood cells due to reduced or absent β globin chain synthesis. Beta thalassemia major emerges between 6th and 24th months of life and is characterized by growth retardation, pallor, jaundice, muscular weakness, genu valgum, hepatosplenomegaly, foot ulcers, mass lesions due to extramedullary hematopoiesis, and skeletal changes secondary to bone marrow expansion [1]. A normal growth rate can be achieved for 10 to 12 years provided that hemoglobin concentration is kept between 9.5 and 10.5 g/dl by regular blood transfusions [2]. In patients undergoing transfusions certain complications secondary to excessive iron loading may occur and include delayed sexual maturation, cardiomyopathy, endocrine gland dysfunction, hypersplenism, chronic hepatitis, HIV infection, venous thrombosis, and osteoporosis [1,3]. Vertebra plana is a radiological diagnosis defined as vertebral flattenLnJ or straightening with preserved intervertebral disc height. It arises most commonly due to eosinophilic granuloma while metastatic disease, multiple myeloma, lymphoma, leukemia, Ewing’s sarcoma, Gaucher disease, tuberculosis, and aneurysmal bone cysts are other causes [4]. НalassemLa major, on the other hand, is not a frequent cause of vertebra plana. НLs paper discusses the etiopathogenesis and management approach of osteoporosis-induced vertebra plana of L4 vertebra in a case with thalassemia major.

Prevalence of Beta-Thalassemia Trait Among Students of the University College of Science and Technology-Palestine

Thalassemias are a group of recessively inherited genetic disorders mostly common in the Mediterranean, the equatorial and near equatorial regions of Africa and Asia. Large number of mutations cause abnormal globin gene expression and result in complete absence or reduction of globin chain synthesis which lead to thalassemia. β-thalassemia is the result of deficient or absent synthesis of β-globin chains, leading to excess α chains. This study was conducted in order to determine the prevalence of β-thalassemia trait among students of University College of Science and Technology (UCST) in Khan Younis, Gaza Strip-Palestine. Allele Specific PCR (ASPCR) was used to determine the intervening sequence IVSI-6 (T →C) and IVSI-110 (G →A) mutations. The study population consisted of 348 subjects recruited from the UCST (144 males: 41% and 204 females: 59%). Blood samples were collected in EDTA tube for CBC. Mentzer index was calculated for all samples. Blood film was done and stained using Giemza stain. DNA was isolated from 12 samples that had normal RBCs and low MCV and whose Mentzer index was >13. These samples were subjected to Allele Specific PCR in order to detect IVSI-6 (T →C) and IVSI-110 (G →A) mutations. The hemoglobin level in females was found to be about 20% less than the level recorded in males (11.40±1.01 vs 14.30±0.79 g/dl). The results also revealed that there were significant differences in all measured CBC parameters and indices between males and females except that for WBC, RBC, MCHC and PLT. IVSI-6 (T →C) mutation was detected only in two samples and both were heterozygous. IVSI-110 (G →A) mutation was not detected in this study. The present results showed that the case of β-thalassemia carrier have normal RBC, MCH, Hb and normal Mentzer index which could be missed in routine screening test.

The LSD1 Inhibitor RN-1 Recapitulates the Fetal Pattern of Hemoglobin Synthesis in Anemic and Normal, Non-Anemic Baboons

Increased fetal hemoglobin (HbF) levels lessen the severity of symptoms and increase the lifespan of patients with sickle cell disease (SCD). Hydroxyurea (HU), the only drug approved for the treatment of sickle cell disease, increases HbF levels, reduces pain crises, and increases the lifespan of patients. Because HU is not effective in a large proportion of patients, new pharmacological agents that increase HbF levels have long been sought. Recent studies identifying LSD-1 as a repressor of γ-globin expression led to experiments demonstrating that the LSD-1 inhibitor RN-1 increased γ-globin expression in SCD mice. As the arrangement and developmental stage-specific expression pattern of the β-like globin genes is highly conserved between man and baboon, the baboon remains the best predictor of the activity of HbF-inducing agents in man. Therefore, experiments were performed to test the effect of RN-1 on HbF in anemic baboons. Anemia (Hct=20) was induced by repeated phlebotomies for 14 days prior to administration of RN-1. Five anemic baboons were treated with varying doses of RN-1(0.125-2.5 mg/kg/d; 5d; sc). Dose-dependent increases in HbF, F cells, F retics, and γ-globin chain synthesis in reticulocytes were associated with decreased neutrophils and increased monocytes. Globin chain synthesis analysis following RN-1 treatment showed that the 5' Iγ and 3' Vγ-globin genes were expressed in the ratio characteristic of fetal development (5'Iγ/3'Vγ>2) rather than that induced in adults by erythropoietic stress (5'Iγ/3'Vγ<0.5) or decitabine treatment where changes in the Iγ/Vγ ratio are observed but the fetal ratio is not attained. RT-PCR analysis of pre-and post-treatment BM showed that RN-1 increased γ-globin mRNA nearly 5 fold (p<0.05) with no effect on ε-globin mRNA. ChIP analysis of pre- and post-treatment BM erythroid cells showed increased levels of pol II, H3K9ac, H3K4me2 and H3K4me3 associated with the γ-globin but not with the ε- or β-globin promoter and IVS II regions consistent with increased γ-globin transcription. Levels of H3K9me2 were increased at the β-globin promoter and IVSII regions. Bisulfite sequence analysis showed a small decrease in level of DNA methylation of the γ-globin promoter in post-treatment (0.68+0.07% total cytosine) compared to pre-treatment (0.79+0.05%; p<0.05). To investigate whether induction of γ-globin expression was dependent on erythropoietic stress, four normal, non-anemic baboons were treated with varying doses and schedules of RN-1 (0.2-0.5mg/kg/d; 5d/wk; 1-10wks; sc). The effect of RN-1 was measured by analysis of F cells and globin chain synthesis in peripheral blood. The results (Table) show that RN-1 increases γ-globin expression and F cells in normal, non-anemic baboons in a dose-dependent manner. Analysis of globin chain synthesis showed predominant synthesis of the Iγ-globin chain with an Iγ/Vγ ratio exceeding the fetal ratio in all individuals.Effect of RN-1 in Normal, Non-Anemic Baboons TableAnimalRN-1 Dose (mg/kg/d)ScheduleGlobin chain synthesis (γ/γ+β)Iγ/Vγ chain synthesisF cells (%)ANC (/μl)Plt(X103/μl)Mono(%)PrePostPrePostNadirNadirPeak85490.55d0.030.714.63NDND191016921.685490.255d0.010.264.47NDND191021914.586980.255d/wk/2 wks0.010.2020.12.09.21210687.986960.2-0.255d/wk/10 wksND0.106.633.526.81440646.1Neutropenia following RN-1 treatment of anemic baboons (mean ANC nadir=746±156) was reduced in non-anemic animals (2 of 4 baboons >1500; mean ANC nadir=1617±350; p<0.02). Increased monocytes and decreased platelet counts were observed. Differences between anemic and non-anemic baboons may reflect perturbation of hematopoietic differentiation by phlebotomy. We conclude that RN-1 is a powerful in vivo inducer of HbF in both anemic and non-anemic baboons that preferentially increases synthesis of the 5' Iγ-globin gene and recapitulates the fetal pattern of hemoglobin synthesis. Our data predicts that RN-1 treatment will induce clinically relevant levels of HbF in SCD patients, although careful titration of dose may be required to minimize effects on hematopoiesis. DisclosuresNo relevant conflicts of interest to declare.

Therapeutic efficacy of different iron chelators in Egyptian children with Beta Thalassemia with iron overload.

Beta thalassemia is a hereditary blood disorder characterized by reduced or absent beta chains of hemoglobin resulting in imbalanced globin chain synthesis with early destruction of RBCs and anemia. Patients with thalassemia major become transfusion- dependent with subsequent iron overload. Effective iron chelation therapy remains the main target of management of thalassemia major. 'The aim of this work was to compare the efficacy of different iron chelating agents' in the treatment of ' iron overload in children with beta thalassemia major'. 'The current study was conducted on 120 children with beta thalassemia major with serum ferritin level of more than 1000 ng/ml who were divided into 4 groups': Group A: 30 patients were treated with 8 hours intravenous infusion of Desferrioxamine, '40 mg/kg/day, 6 days per week for 6 months'. Group B: 30 patients were treated with subcutaneous infusion of Desferrioxamine, 40 mg/kg/day, 6 days per week 8-12 hours per day at night using Desferal pump for 6 months. Group C: 30 patients were treated with oral Deferiprone 75 mg/kg/day in three divided doses daily for 6 months. Group D: 30 patients were treated with oral Deferasirox 30 mg/kg/day in single dose on empty stomach daily for 6 months. 'For all patients laboratory investigations were carried out including complete blood count (CBC), measurement of serum ferritin, serum iron, TIBC (total iron binding capacity), liver enzymes and kidney functions'. There were significant reductions in 'serum ferritin and serum iron' after treatment in all studied groups with the highest reduction in group A, group B, group D and group C but without statistically significant differences between the four studied groups before and after chelation therapy. 'There were no significant differences in' the mean values of the parameters of CBC, liver enzymes and kidney functions between the studied groups before and after chelation therapy. From this study we concluded that there was significant reduction in serum ferritin and serum iron after chelation therapy in studied groups with the highest reduction in group A (IV Desferrioxamine), group B (SC Desferrioxamine), group D (oral Deferasirox) and group C (oral Deferiprone) with no statistically significant differences between the studied groups of patients before and after 6 months of regular chelation.

Coexistence of Malaria and Thalassemia in Malaria Endemic Areas of Thailand.

Hemoglobinopathy and malaria are commonly found worldwide particularly in malaria endemic areas. Thalassemia, the alteration of globin chain synthesis, has been reported to confer resistance against malaria. The prevalence of thalassemia was investigated in 101 malaria patients with Plasmodium falciparum and Plasmodium vivax along the Thai-Myanmar border to examine protective effect of thalassemia against severe malaria. Hemoglobin typing was performed using low pressure liquid chromatography (LPLC) and α-thalassemia was confirmed by multiplex PCR. Five types of thalassemia were observed in malaria patients. The 2 major types of thalassemia were Hb E (18.8%) and α-thalassemia-2 (11.9%). There was no association between thalassemia hemoglobinopathy and malaria parasitemia, an indicator of malaria disease severity. Thalassemia had no significant association with P. vivax infection, but the parasitemia in patients with coexistence of P. vivax and thalassemia was about 2-3 times lower than those with coexistence of P. falciparum and thalassemia and malaria without thalassemia. Furthermore, the parasitemia of P. vivax in patients with coexistence of Hb E showed lower value than coexistence with other types of thalassemia and malaria without coexistence. Parasitemia, hemoglobin, and hematocrit values in patients with coexistence of thalassemia other than Hb E were significantly lower than those without coexistence of thalassemia. Furthermore, parasitemia with coexistence of Hb E were 2 times lower than those with coexistence of thalassemia other than Hb E. In conclusion, the results may, at least in part, support the protective effect of thalassemia on the development of hyperparasitemia and severe anemia in malaria patients.

Prenatal Diagnosis of Hemoglobinopathies: From Fetoscopy to Coelocentesis

Prenatal diagnosis of hemoglobinopathies involves the study of fetal material from blood, amniocytes, trophoblast coelomatic cells and fetal DNA in maternal circulation. Its first application dates back to the 70s and it involves globin chain synthesis analysis on fetal blood. In the 1980s molecular analysis was introduced as well as amniocentesis and chorionic villi sampling under high-resolution ultrasound imaging. The application of direct sequencing and polymerase chain reactionbased methodologies improved the DNA analysis procedures and reduced the sampling age for invasive prenatal diagnosis from 18 to 16–11 weeks allowing fetal genotyping within the first trimester of pregnancy. In the last years, fetal material obtained at 7–8 weeks of gestation by coelocentesis and isolation of fetal cells has provided new platforms on which to develop diagnostic capabilities while non-invasive technologies using fetal DNA in maternal circulation are starting to develop.

α:Non–α and Gγ:Aγ globin chain ratios in thalassemia intermedia patients treated with hydroxyurea

To elucidate the possible ways by which hydroxyurea molecules affect globin chain (α or β-like) synthesis. A total of 23 thalassemia intermedia patients (13 male and 10 female) aged between 5 and 26 years were treated for five months with 15 mg/(kg·day) of hydroxyurea. Hemoglobins electrophoresis and globin chain electrophoresis was performed on each sample at different time points before and during the treatment. Fetal hemoglobin increased significantly in most patients and average episodes of transfusion decreased. Both Gγ and Aγ-globin chains increased significantly and α-globin:Nonα-globin chain as well as Gγ-globin:Aγ globin chains ratios decreased. Improvement in α:non-α ratio and consequent decrease of free α-globin chain might be the cause of beneficial effects of hydroxyurea therapy. Two patients who felt better didn't show significant increase in their fetal hemoglobin level, and this is in contradiction with the hypothesis claiming that the HbF level increase is the cause of such therapeutic effect. In spite of the unclear mechanism of action of this drug, hydroxyurea therapy had noticeable impacts on thalassemia intermedia and also sickle cell disease and even patients suffering from thalassemia major.

Seroprevalence of Hepatitis B, Hepatitis C and Human Immunodeficiency Virus Among Multitransfused Thalassaemic Children in Dhaka, Bangladesh

Background: Thalassaemia is a congenital hemolytic disease caused by defective globin chain synthesis of haemoglobin and largely treated by repeated blood transfusions. Transfusion-transmitted infections still make a great challenge in the management of patients with thalassaemia major. The most important worldwide transfusion transmitted infections (TTI) are hepatitis B virus (HBV), hepatitis C virus (HCV) and human immunodeficiency virus (HIV). Despite concern about a possible increase in the incidence of these infections there are no recent data about the prevalence of HBV, HCV and HIV from Bangladesh. Objectives: To evaluate the prevalence of hepatitis B, hepatitis C and human immunodeficiency virus in multi-transfused thalassaemia patients (MTP), to identify the possible risk factors and to evaluate the effect of compulsory screening of blood to prevent these infections. Methodology: This cross-sectional study was conducted during 2011 to 2012 on 100 consecutive multi-transfused thalassaemic patients who were interviewed using a structured questionnaire and tested for serological markers of hepatitis B virus (HBsAg), hepatitis C virus (Anti-HCV) and human immunodeficiency virus (Anti-HIV 1+2). Results: The overall prevalence of HCV, HBV, HIV and co-infection among (MTP) were 31%, 3%, 0% and 1%, respectively. Children who developed infection had a higher incidence of receiving transfusion from professional donors or unknown donors than the non-infected ones. Infected children had a higher frequency of receiving transfusions without screening and receiving more number of transfusions than their counterpart. Other non-transfusion related (NTR) risk factors such as surgical operation, dental procedures, needle stick injury were significantly higher in patients who acquired transfusion transmitted infections (TTI). Conclusions: HCV infection was the most prevalent transfusion transmitted infection (TTI) among multi-transfused thalassaemia patients (MTP) and remains a major health problem for these patients. Children who received transfusion from professional donors and received unscreened blood had more chance of getting infection with transfusion transmitted infection. DOI: http://dx.doi.org/10.3329/bjch.v37i3.18618 Bangladesh J Child Health 2013; Vol.37(3): 146-153

English

BACKGROUND: Beta-thalassemia syndromes are a group of hereditary blood disorders characterized by reduced or absent beta globin chain synthesis, resulting in transfusion dependent severe anemia, leading to iron overload, resulting in hypothyroidism as the most common endocrine problem. OBJECTIVE: There is lack of information about frequency of hypothyroidism in thalassemic patients in central part of India, so this was undertaken to determine the frequency of hypothyroidism in patients suffering from homozygous s-thalassemia and to study its correlation with serum ferritin and transfusion index. METHOD: This descriptive study included 60 diagnosed thalassemia major patients aged 2-18 years. Demographic data as well as history of blood transfusion, from which transfusion index was estimated, was taken. Serum total T3, T4 and TSH by sandwich ELISA method using Eliscan kit, serum ferritin level by sandwich ELISA method using Accu-bind kit were measured from fasting blood sample. Hypothyroidism was defined by a TSH level >6.4μIU/ml. RESULTS: Study of the thyroid panel among all 60 patients showed a mean TSH level of 4.65±2.41 μIU/ml. Mean total T3 and total T4 levels were 1.35±0.48 ng/ml and 7.4±1.93 μg/dl respectively. Mean serum ferritin level was 557.25±198.66 ng/dl. Hypothyroidism was detected in 14 (23.33%) out of 60 β thalassemia patients. Out of these, compensated hypothyroid (normal T3 and T4 with raised TSH) was seen in 9 patients (15%) and decompensated hypothyroid (Decrease T3 or T4 and Raised TSH) was seen in 5 patients (8.33%). There was significant positive correlation of TSH levels with serum ferritin levels, age and transfusion index. However total T3 and total T4 did not show any correlation with serum ferritin levels, age or transfusion index. CONCLUSION: Hypothyroidism, are more common in second decade of life. Early recognition and hence prevention of these complications will definitely help to improve both the longevity & quality of life of these patients.

Correlation between Plasma Interleukin-3, the α/β Globin Ratio, and Globin mRNA Stability.

Background. Globin chain synthesis (GCS) analysis is used in the diagnosis of thalassemia. However, the wide reference range limits its use as a decisive diagnostic tool. It has been shown that α and β globin mRNA increase through stimulation of cells by interleukin-3 (IL-3). Therefore, this study investigates the relationship between plasma IL-3 and the β/α globin ratio. Methods. Blood samples were collected from 32 healthy participants on two occasions one month apart. GCS analysis, real-time PCR, and ELISA tests were conducted to determine the β/α globin ratio, globin mRNA expression and stability rate, and IL-3 levels. Results. On the basis of IL-3 levels, the participants were divided in two groups. One group included participants who showed a significant increase in IL-3 as indicated by a significant rise in mean values of α, β, and γ globin mRNA, α and β globin, RBC, and hemoglobin. The other group included participants who showed no difference in IL-3 levels with no significant variations in the above-mentioned parameters. Conclusion. The results of this study indicate that IL-3 has an equivalent positive effect on α and β globin chain synthesis. Therefore, IL-3 levels do not explain the wide reference range of the α/β globin ratio.

Saudi Guidelines on the Diagnosis and Treatment of Pulmonary Hypertension: Pulmonary hypertension associated with hemolytic anemia.

Hereditary hemoglobin disorders affecting the globin chain synthesis namely thalassemia syndromes and sickle cell disease (SCD) are the most common genetic disorders in human. Around 7% of the world population carries genes for these disorders, mainly the Mediterranean Basin, Middle and Far East, and Sub-Saharan Africa. An estimated 30 million people worldwide are living with sickle cell disease, while 60-80 million carry beta thalassemia trait. About 400,000 children are born with severe hemoglobinopathies each year.Cardiovascular complications of hemoglobinopathies include left and right ventricular (RV) dysfunction, arrhythmias, pericarditis, myocarditis, valvular heart disease, myocardial ischemia, and notably pulmonary hypertension (PH).Because of a unique pathophysiology, pulmonary hypertension associated with hemolytic disorders was moved from WHO group I to group V PH diseases. Treatment strategies are also unique and include blood transfusion, iron chelation, hydroxyurea, and oxygen therapy. The role of PH-specific agents has not been established.

Screening of Thalassaemia Carriers and Its Limitations

The α and β-thalassaemias (thal) are common genetic disorders of globin chain synthesis where the carriers have deficiency of α or β globin chain respectively. In resource limited settings, the naked eye single tube red cell osmotic fragility (NESTROFT) has been used for screening of thalassaemia. It has low specificity compared with its sensitivity. Screening of carriers encompasses automated/semi-automated techniques and manual methods for presumptive diagnosis of thalassaemia. The current strategy in screening involves a step by step process simplified into the following acronym, BHES. B, refers to blood counts and a film; H, haemoglobin (Hb) subtyping; E, Hb electrophoresis; and S, stability (H-inclusion and Sickle cell test). Blood counts and red blood cell indices are now available by automated instrumentation where a cut-off value of mean corpuscular Hb (MCH)<27 pg necessitates further investigation for thalassaemia or iron deficiency. The distribution of normal Hb subtypes (HbA, HbA2 and HbF) is done commonly by high performance liquid chromatography (HPLC) or capillary zone electrophoresis (CZE). The thalassaemia trait phenotype is indicated by a near normal Hb, microcytic and hypochromic red cell indices. In classical β-thalassaemia trait, the cut-off value of HbA2 value is 4% and above. However, milder mutations can result in HbA2 values between 3.2 to 3.9%. In alpha thalassaemia trait the HbA2 falls within the normal range. In a carrier of α0-thalassaemia, occasional or few H-inclusions suggests its presence. However, absence of H-inclusions does not rule out its presence. Recently, it has been shown that the current screening method of thalassaemia carriers has its limitations. Mild mutations may show normal or near normal red cell indices and be missed in the screening process. Carriers with these mutations can cause thalassaemia intermedia. In addition there are mutations that have more profound effects resulting in more severe outcome but have carriers with normal red cell indices. In α-thalassaemia intermedia, HbH disease, those with nondeletional mutations are clinically more severe. These non-deletion mutations include Hb Constant Spring, Hb Pakse and Hb Adana. Carriers with these non-deletional α-thal mutations have normal red cell indices. Cases of hydrops foetalis have been reported with a homozygous state of Hb Adana and also in α0 with Hb Adana. Coinheritance of α or β-thal also effects the red cell indices making them near normal. Thalassaemia intermedia and major have serious implications and prevention requires strategies to detect couples at risk. Mutational spectrum of the globin genes is updated continuously with improved diagnostic technology. Clinical programs preventing thalassaemia are presently based upon prospective screening and prenatal diagnosis. In order for prevention to be effective, there is a need to be aware that mutations can exist in thalassaemia carriers where the red cell indices are normal.

A Novel β Globin Gene Mutation, Reported As An α Chain Hemoglobin Variant By High Performance Liquid Chromatography (HPLC) & As Sickle Cell Trait On New Born Screening: A case Report

IntroductionThere are 827 variants of β thalassemia reported to the registry of human hemoglobin variants and thalassemias registry 1. Genetic mutations of β thalassemia are very diverse but can be broadly divided in to non deletion forms and deletion forms. The new mutation is a frame shift insertion in exon 2 of the β globin gene. To the best of our knowledge this mutation has never been described before and presents as a mild form β thalassemia intermedia. ObjectiveTo describe the phenotypic presentation of the new β globin variant, due to insertion of 9 nucleotides (AAAGTGCTC) between nucleotides c.207 and c.208. Case reportA 22 months-old Hispanic boy who was referred for evaluation of persistent anemia. The new born screening for the child was positive for sickle cell trait. Initial hemoglobin (Hb) was 8.8, hematocrit was 27 and MCV was 65.5, which were decreased. RDW was 25.2, which was increased. Hemoglobin evaluation by acid and alkaline electrophoresis and HPLC revealed a HbS 0%, HbF 6.5% HbA 78.4%, HbA2 4.9% and a Hb variant 10.2%. The interpretation of the results was an α globin variant suggestive of Hb Montefiore and β thalassemia trait. Alpha thalassemia PCR for the 7 most common deletions in the α globin chain was negative. Subsequent α globin gene sequencing revealed no α globin gene mutations. On physical exam there were no bony changes or hepatosplenomegaly.Family HistoryThe mother is 29-year-old with HbAA. The father is 39-years-old with the same mutation on beta globin gene analysis. Hb electrophoresis also suggested an α and β globin mutation. Alpha thalassemia PCR was negative and he had a normal α globin gene copy number. At age 19 he had a splenectomy secondary to splenomegaly and hypersplenism. He is consistently anemic with Hb< 9 and MCV < 70.The child has a paternal half brother, who is 21 years old and has similar problems as father. He had a splenectomy at the age of 17 after admission for abdominal pain. The patient has a 5-year-old sibling, who is normal with HbAA and another paternal half sibling reported as no anemia. DiscussionThough β thalassemia intermedia is most commonly homozygous or compound heterozygous, less frequently it can be due to single locus mutation. DNA sequencing of the α globin gene of the index case was completely normal and there was normal copy number of the α globin gene in the father. No Hb S was found on Hb electrophoresis.The new mutation adds 9 base pairs to exon 2 and 3 amino acids (Lys-Val-Leu) between amino acid 68 and 69 of the protein. This elongates the beta chain which can lead to instability and precipitation of Hb as well as hemolysis and anemia2. Further studies like short time incubation and pulse chase globin chain synthesis experiments are needed to know the stability of the β globin protein3. In addition, an increase of α globin gene copy number can also be a reason for increasing the severity of β thalassemia trait2. However the α gene copy was normal in the father. ConclusionWe present a case of a child with a false positive abnormal newborn screen suggestive of sickle cell trait, as well as a Hb electrophoresis suggestive of an alpha globin mutation. As the father and paternal brother have had a splenectomy in their teen years with noted hepatosplenomegaly, suggestive of increased hemolysis, and the anemia is more severe than usual for β thalassemia trait, this suggests that phenotypically the c.199_207dup variant presents as a mild β thalassemia intermedia. In addition, there does not seem to be any bony abnormalities associated with marrow hyperplasia. As both our patients are heterozygous for this novel mutation with normal α globin gene copy number and alpha globin sequencing, we suspect that elongation of the β globin produces an unstable hemoglobin with a mild β thalassemia intermedia phenotype2.

VASCULAR ENDOTHELIAL GROWTH FACTOR IN CHILDREN WITH THALASSEMIA MAJOR PDF

BackgroundThe β-Thalassemia syndromes are the most common hereditary chronic hemolytic anemia due to impaired globin chain synthesis. Vascular endothelial growth factor (VEGF) plays several roles in angiogenesis which is a crucial process in the pathogenesis of several inflammatory, autoimmune and malignant diseases. Endothelial damage and inflammation make a significant contribution to the pathophysiology of β-thalassemia.Purpose: The aim of the study was to assess serum VEGF level in children with beta-thalassemia major as a marker of angiogenesis.MethodsA total of 50 children entered the study, 40 patients with thalassemia major and 10 healthy controls. We used enzyme-linked immunosorbent assay for quantitative evaluation of VEGF.ResultsVEGF level was significantly higher in patients with β-thalassemia major than healthy controls (p=0.001). VEGF level was also higher in splenectomised thalassemic patients than non splenectomised ones (p=0.001). There were a positive correlation between VEGF and chelation starting age (p=0.008), and a negative correlation between VEGF and frequency of blood transfusion (p=0.002).ConclusionThalassemia patients, especially splenectomized, have elevated serum levels of VEGF. Early chelation and regular blood transfusion help to decrease serum VEGF and the risk of angiogenesis.

Extramedullary Haematopoiesis Causing Spinal Cord Compression in a Thalassemia Intermedia Patient

The thalassemias are a heterogenous group of inherited disorders of hemoglobin (Hb) synthesis characterized by a lack of or decreased /defective synthesis of globin chains. The name Mediterranean anemia, which Whipple introduced, is misleading because the condition can be found in any part of the world. In ?-thalassemia, ?-globin chain synthesis is reduced, whereas in ?-thalassemia, ?-globin chain synthesis is defective. Their clinical severity varies widely, ranging from asymptomatic forms to severe or even fatal entities. Like most anemias, erythropoietin production and erythropoiesis is increased in thalassemia causing erythroid marrow hyperplasia as well as extramedullary haematopoiesis which is a common compensatory mechanism for chronic anemia found in patients with hemoglobinopathies such as thalassemia, sickle cell anemia, and hereditary spherocytosis. These patients are usually asymptomatic. Extramedullary hematopoiesis (EMH) usually manifests in the liver, spleen, kidneys and as paravertebral masses. Extramedullary hematopoietic tissue occurring within the spinal canal and causing cord compression is an unusual but well-described entity. Total surgical excision is usually not feasible because of the diffuse spread of extramedullary hematopoietic tissue and the possibility of recurrence, but acute neurological impairment does require emergency surgery. Extramedullary hematopoiesis is radiosensitive so radiation therapy is widely accepted as a very effective mode of therapy. Repeated blood transfusion and cytostatic agents are also recommended for treatment.We report a case of a 35-year-old man with underlying beta thalassemia intermedia who developed pain in lower back and progressive paresthesia and weakness of both lower limbs due to multifocal extramedullary epidural hematopoietic tissue occurring within the spinal canal with compression over the cord. Nepalese Journal of Radiology; Vol. 2; Issue 2; July-Dec. 2012; 81-85 DOI: http://dx.doi.org/10.3126/njr.v2i2.7692

Thalassemia – Increasingly Frequent Cause of Microcytic Anemias in Children in Poland

AbstractAbstract 5184Thalassemias are a group of genetically determined hemolytic anemias related to disturbed synthesis of globin chains, and are classified as quantitative hemoglobinopathies.We would like to present our experience in diagnosis of thalassemia in children as an increasingly frequent cause of microcytic anemias as well as to point out that it is the cause of microcytic anemia in Poland.Between 2005–2012 we established the diagnosis of thalassemia minor in 94 children of Polish origin, referred to the Department of Pediatrics, Hematology and Oncology, Medical University of Warsaw. At admission all patients presented microcytosis, erythrocytosis, hypochromia and normal or elevated iron status. Most of them had long-lasting uneffective iron therapy in anamnesis. Their age ranged from 3 to 17 years. The group consisted of 40 girls and 54 boys.Routinely employed diagnostic methods, including blood morphology, determination of iron and ferritin, electrophoretic separation of hemoglobins, determination of HgbA2 and HgbF, as applied in all cases, often turn out to be insufficient to diagnose thalassemia, substantiating the need to use modern molecular biological techniques.Thalassemia alfa was diagnosed in 9 children in whom we detected deletion α 3, 7 using molecular biological techniques.In 2 children coexistence of both thalassemia alfa and beta was established. Both children presented macrocytosis and normal range of haemoglobin and red blood cells count. The molecular biological techniques used in both cases identified deletion α 3, 7 characteristic for alfa thalassemia, and Hb Koln (Codon GTC>ATG) and Del 4, 2 (Taq1) characteristic for beta thalassemia.In the remaining 83 patients we made the diagnosis of thalassemia beta. The diagnosis was possible after the evaluation of Hgb A2 and F levels. In 24 cases we managed to performed molecular biological techniques, identifying mutation IVS I-6 (T>C) as the most frequent.Generally associated with the Mediterranean Basin, thalassemia is, however, more and more often diagnosed in Poland. This is due to the international human traffic. Growing availability of diagnostic tests, like molecular biological techniques, especially useful in equivocal cases, is also important.The amplification of diagnostic possibilities by genetic tests in the future may be useful in detection of genetic mutations responsible for thalassemia and characteristic for Polish population. Disclosures:No relevant conflicts of interest to declare.

Mitochondria Superoxide Is an Early Trigger of Apoptosis in an Ex Vivo Model of Human Beta Thalassemia.

AbstractAbstract 2131Apoptotic cell death is a central feature of beta-thalassemia and other anemias caused by ineffective erythropoiesis. In this study, markers of apoptosis were examined using an ex vivo model of human beta-thalassemia to explore the cause(s) and timing of erythroblast cell death. Beta-globin gene and protein expression were knocked down by lentiviral transduction of beta-globin shRNA (beta-KD) in adult human CD34+ cells. Beta-globin mRNA (Control=4.0E+07 ± 1.4E+06 copies/ng, vs. beta-KD=2.5E+06 ± 1.6E+06 copies/ng, p=0.014) and protein were reduced in beta-KD cells by 90% compared to controls while alpha globin expression was maintained. The effects of imbalanced globin chain synthesis were therefore studied according to the stage of erythroblast maturation. Erythroid progenitor cell commitment and proliferation occur over the first two weeks in culture. During the third week of culture (days 14–21), the proerythroblasts undergo terminal differentiation with the characteristic loss of CD71 from the cell surface.Prior to culture day 14, phenotypic analyses demonstrated low levels of apoptosis in beta-KD and control cultures. On culture day 14, a small but significant increase in active caspase 3 was detected in the beta-KD cells compared to controls (beta-KD=4.0±1.0%, Control=0.7±0.3%, p=0.02) suggesting that apoptosis was initiated during the early stages of terminal maturation. Increases in annexin V staining did not achieve statistical significance on day 14 beta-KD cells (beta-KD=13.1±3.9%, Control=7.6±2.2%, p=0.16). By culture day 18, when orthochromic normoblasts are the most prevalent population in control cultures, a large population of apoptotic cells was detected in the beta-KD. The beta-KD erythroblasts demonstrated further increases in active caspase 3 (beta-KD=11.4±2.2% vs. Control=1.1±0.1%, p=0.014), as well as significant increases in surface annexin V (beta-KD=75.8±3.3%, vs. Control=35.9±12.7%, p=0.024). Western analysis of culture day 18 beta-KD membranes demonstrated a marked increase in alpha-chains compared with culture day 14.Since cleaved caspase 3 was increased in beta-KD cells near the beginning of their terminal maturation and prior to the accumulation of alpha chains in the cell membranes, other triggers of apoptosis were investigated. Mitochondrial superoxide is a reactive species that can be generated by iron or other mitochondrial toxins. Increased levels of mitochondrial superoxide cause apoptosis. The cultured erythroblasts were stained with MitoSOX, a cell-permeable dye that specifically detects mitochondrial superoxide. On culture day 11, mitochondrial superoxide was barely detectable in beta-KD and control cell populations (beta-KD=5.2±3.3% vs. Control=3.6±2.9%, p=0.051). Thereafter, the superoxide detection was increased significantly in beta-KD cells on culture day 14 (beta-KD=54.2±6.7% vs. Control=9.1±2.9%, p=0.003), and culture day 18 (beta-KD=81.1±3.2% vs. Control=34.6±3.8%, p=0.007).Oxidation of cellular membranes by hemichromes and free alpha chains damages thalassemic erythrocytes and their precursors. These data suggest that imbalanced globin chain synthesis also triggers apoptosis during the early stages of terminal differentiation by increasing superoxide formation in the mitochondria. Disclosures:No relevant conflicts of interest to declare.