Transient neonatal hemolytic anemia due to the novel gamma globin gene mutation HBG2:C.290T>C, p.Leu97Pro (hemoglobin Wareham).

Unstable gamma globin variants can cause transient neonatal hemolytic anemia. We have identified a novel variant in a newborn who presented with jaundice and anemia requiring phototherapy and red blood cell transfusion. The patient was found to be heterozygous for the mutation HGB2:c.290T>C, p.Leu97Pro, which we have termed hemoglobin (Hb) Wareham. This substitution is expected to generate an unstable hemoglobin with increased oxygen affinity based on the homologous mutation previously described in the beta globin gene, which is termed as Hb Debrousse. The patient fully recovered by 9 months of age as expected with the transition from fetal to adult hemoglobin.

Antioxidant-Rich Nutraceutical as a Therapeutic Strategy for Sickle Cell Disease.

Sickle cell disease (SCD) is a genetically inherited disease in which the "SS" individual possesses two copies of the abnormal beta-globin gene. This disease is one of the most dominant genetic diseases in the world. SCD is marked by the propensity of red cell hemoglobin to polymerize and distort the red cell from a biconcave disk shape into a sickle shape, resulting in a typical vaso-occlusive episode and accelerated hemolysis. Plants are rich sources of bioactive compounds that are promising anti-sickling agents to scavenge free radicals, thereby ensuring oxidative balance. The current review highlights the potential therapeutic benefits of antioxidant-rich nutraceutical in the treatment and management of sickle cell disease. The anti-sickling potential of nutraceutical is attributed to the presence of antioxidant bioactive chemicals such as alkaloids, polyphenols, vitamins, and minerals, which acts as scavengers of free radicals that prevent oxidative damage of the hemoglobin and prevent hemolysis, facilitating longer erythrocyte lifespan. The challenges of current therapies for SCD and future directions are also discussed.KEY TEACHING POINTSSickle cell disease is a genetically inherited disease in which SS individuals possess two copies of the abnormal beta-globin gene.Oxidative stress contributes to the pathophysiology of secondary dysfunction in sickle cell patients.Antioxidants can play a vital role in maintaining a balance between oxidant and antioxidant defense systems.Nutraceutical rich in antioxidants such as alkaloids, polyphenols, vitamins, and minerals is potential therapeutic agents for sickle cell disease.An antioxidant-rich nutraceutical may act to reduce vaso-occlusive crises.

Current status of beta-thalassemia and its treatment strategies.

BACKGROUND: Thalassemia is an inherited hematological disorder categorized by a decrease or absence of one or more of the globin chains synthesis. Beta-thalassemia is caused by one or more mutations in the beta-globin gene. The absence or reduced amount of beta-globin chains causes ineffective erythropoiesis which leads to anemia. METHODS: Beta-thalassemia has been further divided into three main forms: thalassemia major, intermedia, and minor/silent carrier. A more severe form among these is thalassemia major in which individuals depend upon blood transfusion for survival. The high level of iron deposition occurs due to regular blood transfusion therapy. RESULTS: Overloaded iron raises the synthesis of reactive oxygen species (ROS) that are noxious and prompting the injury to the hepatic, endocrine, and vascular system. Thalassemia can be analyzed and diagnosed via prenatal testing (genetic testing of amniotic fluid), blood smear, complete blood count, and DNA analysis (genetic testing). Treatment of thalassemia intermediate is symptomatic; however; it can also be accomplished by folic supplementation and splenectomy. CONCLUSION: Thalassemia major can be cured through regular transfusion of blood, transplantation of bone marrow, iron chelation management, hematopoietic stem cell transplantation, stimulation of fetal hemoglobin production, and gene therapy.

Curcuminoids supplementation ameliorates iron overload, oxidative stress, hypercoagulability, and inflammation in non-transfusion-dependent ß-thalassemia/Hb E patients.

Curcuminoids, polyphenol compounds in turmeric, possess several pharmacological properties including antioxidant, iron-chelating, and anti-inflammatory activities. Effects of curcuminoids in thalassemia patients have been explored in a limited number of studies using different doses of curcuminoids. The present study aims to evaluate the effects of 24-week curcuminoids supplementation at the dosage of 500 and 1000 mg/day on iron overload, oxidative stress, hypercoagulability, and inflammation in non-transfused ß-thalassemia/Hb E patients. In general, both curcuminoids dosages significantly lowered the levels of oxidative stress, hypercoagulability, and inflammatory markers in the patients. In contrast, reductions in iron parameter levels were more remarkable in the 1000 mg/day group. Subgroup analysis revealed that a marker of hypercoagulability was significantly decreased only in patients with baseline ferritin ≤ 1000 ng/ml independently of curcuminoids dosage. Moreover, the alleviation of iron loading parameters was more remarkable in patients with baseline ferritin > 1000 ng/ml who receive 1000 mg/day curcuminoids. On the other hand, the responses of oxidative stress markers were higher with 500 mg/day curcuminoids regardless of baseline ferritin levels. Our study suggests that baseline ferritin levels should be considered in the supplementation of curcuminoids and the appropriate curcuminoids dosage might differ according to the required therapeutic effect. Thai Clinical Trials Registry (TCTR): TCTR20200731003; July 31, 2020 "retrospectively registered".

Discovery of Novel Fetal Hemoglobin Inducers through Small Chemical Library Screening.

The screening of chemical libraries based on cellular biosensors is a useful approach to identify new hits for novel therapeutic targets involved in rare genetic pathologies, such as ß-thalassemia and sickle cell disease. In particular, pharmacologically mediated stimulation of human γ-globin gene expression, and increase of fetal hemoglobin (HbF) production, have been suggested as potential therapeutic strategies for these hemoglobinopathies. In this article, we screened a small chemical library, constituted of 150 compounds, using the cellular biosensor K562.GR, carrying enhanced green fluorescence protein (EGFP) and red fluorescence protein (RFP) genes under the control of the human γ-globin and ß-globin gene promoters, respectively. Then the identified compounds were analyzed as HbF inducers on primary cell cultures, obtained from ß-thalassemia patients, confirming their activity as HbF inducers, and suggesting these molecules as lead compounds for further chemical and biological investigations.

Beta-Thalassemia Intermedia: A Single Thalassemia Center Experience from Northeastern Iraq.

OBJECTIVE: To determine the molecular characterization and disease-associated complications of beta-thalassemia intermedia (ß-TI) patients in Sulaymaniyah province, northeastern Iraq. METHODS: A total of 159 ß-TI) patients in Sulaymaniyah province, northeastern Iraq. ß-TI) patients in Sulaymaniyah province, northeastern Iraq. RESULTS: Nineteen different ß-globin gene mutations arranged in 37 various genotypes were determined. The most frequent were IVS-II-I (G>A) (47.2%), followed by IVS-I-6 (T>C) (23.3%) and IVS-I-110 (G>A) (5%). Among disease-related morbidities documented, bone disease amounted to 53% (facial deformity and osteoporosis), followed by endocrinopathies 17.6% (growth retardation and subclinical hypothyroidism), cholelithiasis 13.8%, pulmonary hypertension 11.3%, and abnormal liver function test 7.5%, whereas venous thrombosis, extramedullary hemopoiesis, and leg ulcer were less frequently observed. Age ≥ 35 and female sex were risk factors for cholelithiasis, while age was an independent risk for hypothyroidism and female sex was associated with increased risk for osteoporosis. Mean serum ferritin of ≥1000 µg/L was associated with an increased risk of osteoporosis, whereas chelation therapy was protective for a multitude of other complications. Transfusion, on the other hand, increased the risk of osteoporosis, yet it was protective for cholelithiasis and hypothyroidism. Moreover, splenectomy was protective for cholelithiasis, although it was an independent risk for hypothyroidism. Finally, hydroxyurea was associated with an increased risk of osteoporosis, while it was protective for cholelithiasis. Discussion and Conclusion. ß +-thalassemia mutation had contributed to 41.25 of families with a less severe ß-thalassemia phenotype in the northeastern part of Iraq, justifying the need to investigate the contribution of genetic modifiers in ameliorating disease severity. In addition, the substantial number of ß-TI patients developed disease-related morbidities, which necessitates the need for more appropriate clinical management with earlier intervention.ß-TI) patients in Sulaymaniyah province, northeastern Iraq. µg/L was associated with an increased risk of osteoporosis, whereas chelation therapy was protective for a multitude of other complications. Transfusion, on the other hand, increased the risk of osteoporosis, yet it was protective for cholelithiasis and hypothyroidism. Moreover, splenectomy was protective for cholelithiasis, although it was an independent risk for hypothyroidism. Finally, hydroxyurea was associated with an increased risk of osteoporosis, while it was protective for cholelithiasis. Discussion and Conclusion. ß +-thalassemia mutation had contributed to 41.25 of families with a less severe ß-thalassemia phenotype in the northeastern part of Iraq, justifying the need to investigate the contribution of genetic modifiers in ameliorating disease severity. In addition, the substantial number of ß-TI patients developed disease-related morbidities, which necessitates the need for more appropriate clinical management with earlier intervention.Discussion and Conclusion. ß +-thalassemia mutation had contributed to 41.25 of families with a less severe ß-thalassemia phenotype in the northeastern part of Iraq, justifying the need to investigate the contribution of genetic modifiers in ameliorating disease severity. In addition, the substantial number of ß-TI patients developed disease-related morbidities, which necessitates the need for more appropriate clinical management with earlier intervention.ß-TI) patients in Sulaymaniyah province, northeastern Iraq. ß-TI) patients in Sulaymaniyah province, northeastern Iraq.

Oral ferroportin inhibitor ameliorates ineffective erythropoiesis in a model of ß-thalassemia.

ß-Thalassemia is a genetic anemia caused by partial or complete loss of ß-globin synthesis, leading to ineffective erythropoiesis and RBCs with a short life span. Currently, there is no efficacious oral medication modifying anemia for patients with ß-thalassemia. The inappropriately low levels of the iron regulatory hormone hepcidin enable excessive iron absorption by ferroportin, the unique cellular iron exporter in mammals, leading to organ iron overload and associated morbidities. Correction of unbalanced iron absorption and recycling by induction of hepcidin synthesis or treatment with hepcidin mimetics ameliorates ß-thalassemia. However, hepcidin modulation or replacement strategies currently in clinical development all require parenteral drug administration. We identified oral ferroportin inhibitors by screening a library of small molecular weight compounds for modulators of ferroportin internalization. Restricting iron availability by VIT-2763, the first clinical stage oral ferroportin inhibitor, ameliorated anemia and the dysregulated iron homeostasis in the Hbbth3/+ mouse model of ß-thalassemia intermedia. VIT-2763 not only improved erythropoiesis but also corrected the proportions of myeloid precursors in spleens of Hbbth3/+ mice. VIT-2763 is currently being developed as an oral drug targeting ferroportin for the treatment of ß-thalassemia.

Production and Transduction of a Human Recombinant ß-Globin Chain into Proerythroid K-562 Cells To Replace Missing Endogenous ß-Globin.

Protein replacement therapy (PRT) has been applied to treat severe monogenetic/metabolic disorders characterized by a protein deficiency. In disorders where an intracellular protein is missing, PRT is not easily feasible due to the inability of proteins to cross the cell membrane. Instead, gene therapy has been applied, although still with limited success. ß-Thalassemias are severe congenital hemoglobinopathies, characterized by deficiency or reduced production of the adult ß-globin chain. The resulting imbalance of α-/ß-globin chains of adult hemoglobin (α2ß2) leads to precipitation of unpaired α-globin chains and, eventually, to defective erythropoiesis. Since protein transduction domain (PTD) technology has emerged as a promising therapeutic approach, we produced a human recombinant ß-globin chain in fusion with the TAT peptide and successfully transduced it into human proerythroid K-562 cells, deficient in mature ß-globin chain. Notably, the produced human recombinant ß-globin chain without the TAT peptide, used as internal negative control, failed to be transduced into K-562 cells under similar conditions. In silico studies complemented by SDS-PAGE, Western blotting, co-immunoprecipitation and LC-MS/MS analysis indicated that the transduced recombinant fusion TAT-ß-globin protein interacts with the endogenous native α-like globins to form hemoglobin α2ß2-like tetramers to a limited extent. Our findings provide evidence that recombinant TAT-ß-globin is transmissible into proerythroid K-562 cells and can be potentially considered as an alternative protein therapeutic approach for ß-thalassemias.

Emerging Therapies.

At present, the only definitive cure for ß-thalassemia is a bone marrow transplant (BMT); however, HLA-blood-matched donors are scarcely available. Current therapies undergoing clinical investigation with most potential for therapeutic benefit are the ß-globin gene transfer of patient-specific hematopoietic stem cells followed by autologous BMT. Other emerging therapies deliver exogenous regulators of several key modulators of erythropoiesis or iron homeostasis. This review focuses on current approaches for the treatment of hemoglobinopathies caused by disruptions of ß-globin.

2,000 Year old ß-thalassemia case in Sardinia suggests malaria was endemic by the Roman period.

OBJECTIVES: The island of Sardinia has one of the highest incidence rates of ß-thalassemia in Europe due to its long history of endemic malaria, which, according to historical records, was introduced around 2,600 years ago by the Punics and only became endemic around the Middle Ages. In particular, the cod39 mutation is responsible for more than 95% of all ß-thalassemia cases observed on the island. Debates surround the origin of the mutation. Some argue that its presence in the Western Mediterranean reflects the migration of people away from Sardinia, others that it reflects the colonization of the island by the Punics who might have carried the disease allele. The aim of this study was to investigate ß-globin mutations, including cod39, using ancient DNA (aDNA) analysis, to better understand the history and origin of ß-thalassemia and malaria in Sardinia. MATERIALS AND METHODS: PCR analysis followed by sequencing were used to investigate the presence of ß-thalassemia mutations in 19 individuals from three different Roman and Punic necropolises in Sardinia. RESULTS: The cod39 mutation was identified in one male individual buried in a necropolis from the Punic/Roman period. Further analyses have shown that his mitochondrial DNA (mtDNA) and Y-chromosome haplogroups were U5a and I2a1a1, respectively, indicating the individual was probably of Sardinian origin. CONCLUSIONS: This is the earliest documented case of ß-thalassemia in Sardinia to date. The presence of such a pathogenic mutation and its persistence until present day indicates that malaria was likely endemic on the island by the Roman period, earlier than the historical sources suggest.

Iron distribution and histopathological study of the effects of deferoxamine and deferiprone in the kidneys of iron overloaded ß-thalassemic mice.

Renal glomerular and tubular dysfunctions have been reported with high prevalence in ß-thalassemia. Iron toxicity is implicated in the kidney damage, which may be reversed by iron chelation therapy. To mimic heavy iron overload and evaluate the efficacy of iron chelators in the patients, iron dextran (180mg iron/mouse) was intraperitoneally (i.p.) injected in heterozygous ß-globin knockout mice ((mußth-3/+), BKO) and wild type mice (C57BL/6J, WT) over a period of 2 weeks, followed by daily i.p. injection of deferoxamine (DFO) or deferiprone (L1) for 1 week. In BKO mice, iron preferentially accumulated in the proximal tubule with a grading score of 0-1 and increased to grade 3 after iron loading. In contrast, iron mainly deposited in the glomerulus and interstitial space in iron overloaded WT mice. Increased levels of kidney lipid peroxidation, glomerular and medullar damage and fibrosis in iron overloaded mice were reversed by treatment with iron chelators. L1 showed higher efficacy than DFO in reduction of glomerular iron, which was supported by a significantly decreased the amount of glomerular damage. Notably, DFO and L1 demonstrated a distinct pattern of iron distribution in the proximal tubule of BKO mice. In conclusion, chelation therapy has beneficial effects in iron-overloaded kidneys. However, the defect of kidney iron metabolism in thalassemia may be a determining factor of the treatment outcome in individual patients.

Non-Invasive DNA Sampling for Molecular Analysis of Beta-Thalassemia: Amiable Alternative Sampling Methods with Accurate Results for Pediatric Patients.

BACKGROUND: Beta-thalassemia is the most common genetic disorder in Malaysia. Confirmation of the ß-globin gene mutations involved in thalassemia is usually carried out by molecular analysis of DNA extracted from leukocytes in whole blood. Molecular analysis is generally carried out when affected children are around 1 - 2 years as clinical symptoms are expressed during this period. Blood taking at this age can be distressing for the child. High yield and pure DNA extracted from non-invasive sampling methods can serve as alternative samples in molecular studies for genetic diseases especially in pediatric cases. METHODS: In this study, mouthwash, saliva, and buccal cytobrush samples were collected from ß-thalassemia major patients who had previously been characterized using DNA extracted from peripheral blood. DNA was extracted from mouthwash, saliva, and buccal cytobrush samples using the conventional inexpensive phenol-chloroform method and was measured by spectrophotometry for yield and purity. Molecular characterization of ß-globin gene mutations was carried out using the amplification refractory mutation system (ARMS). RESULTS: DNA extracted from mouthwash, saliva, and buccal cytobrush samples produced high concentration and pure DNA. The purified DNA was successfully amplified using ARMS. Results of the ß-globin gene mutations using DNA from the three non-invasive samples were in 100% concordance with results from DNA extracted from peripheral blood. CONCLUSIONS: The conventional in-house developed methods for non-invasive sample collection and DNA extraction from these samples are effective and negate the use of more expensive commercial kits. In conclusion, DNA extracted from mouthwash, saliva, and buccal cytobrush samples provided sufficiently high amounts of pure DNA suitable for molecular analysis of ß-thalassemia.

Effects of Iron Chelators on Pulmonary Iron Overload and Oxidative Stress in ß-Thalassemic Mice.

AIM: To evaluate the effect of iron chelators on iron-related pulmonary pathology and oxidative stress in an animal model of ß-thalassemia. METHODS: Pulmonary iron overload was induced in heterozygous ß-globin knockout mice (mußth-3/+, BKO). Over a period of 2 weeks, 180 mg of iron/mouse was loaded by intraperitoneal injection of iron dextran, and subsequently treated daily via intraperitoneal with either deferoxamine (DF) or deferiprone (L1) at an equimolar concentration of iron binding (0.2 and 0.6 µmol/g body weight, respectively) for 7 days. RESULTS: Iron loading resulted in iron deposition in peribronchial regions, septa and also in alveolar macrophages with a grading score of 3. This iron burden resulted in lung epithelial injuries, fibrosis and corresponded with increased lipid peroxidation and decreased tissue catalase activity. Treatment with DF or L1 resulted in a reduction of iron-laden alveolar macrophages and decreased oxidative stress and tissue damage, showing the iron mobilizing ability of both compounds. CONCLUSION: Iron chelation therapy, with DF and L1, may protect against pulmonary damage by sequestering catalytic iron and improving oxidative status. It may be beneficial in the prevention of pulmonary complications in thalassemia.

DNA damage: beta zero versus beta plus thalassemia.

BACKGROUND: ß thalassemia results in an increase in the α to non-α chain ratio. Iron released from unpaired α chains in RBCs and that ensuing from regular transfusions is the major cause of cellular damage. The use of iron chelators to counter the iron overload is accompanied by side-effects. The extent of iron toxicity could vary from one patient to another and could help in determining the optimal chelator dose for each patient. AIM: To observe the pro-oxidant/antioxidant disturbance and the extent of DNA damage in ß thalassemia patients with different ß globin gene anomalies. METHODS: The formation of Reactive Oxygen Species (ROS ) was observed by incubation of cell suspensions with 2',7', dichlorofluorescin-diacetate (DCFH DA) and DNA damage was demonstrated by single cell gel electrophoresis. Heinz bodies were observed by staining blood smears. SUBJECTS: The study group comprised 50 regularly transfused beta thalassemia patients and 40 non thalassemic controls. RESULTS: While Heinz bodies and nucleated RBCs were seen in all the patients, oxidation of DCFH and DNA damage were seen to be associated with the ß globin gene defect. DNA damage was found to be greater in ß(0) homozygotes as compared to the ß(+) homozygotes, and was maximum in patients presenting with the 619 base pair deletion. CONCLUSION: In the present study, iron toxicity, as indicated by DNA damage, has been seen to vary in the patients. Thus, monitoring of the dose of iron chelators, according to the type of mutation in the beta globin gene, may help improve the compliance of beta thalassemics to chelation therapy and prevent side-effects in patients with beta plus mutations.

Hepcidin is suppressed by erythropoiesis in hemoglobin E ß-thalassemia and ß-thalassemia trait.

Hemoglobin E (HbE) ß-thalassemia is the most common severe thalassemia syndrome across Asia, and millions of people are carriers. Clinical heterogeneity in HbE ß-thalassemia is incompletely explained by genotype, and the interaction of phenotypic variation with hepcidin is unknown. The effect of thalassemia carriage on hepcidin is also unknown, but it could be relevant for iron supplementation programs aimed at combating anemia. In 62 of 69 Sri Lankan patients with HbE ß-thalassemia with moderate or severe phenotype, hepcidin was suppressed, and overall hepcidin inversely correlated with iron accumulation. On segregating by phenotype, there were no differences in hepcidin, erythropoiesis, or hemoglobin between severe or moderate disease, but multiple linear regression showed that erythropoiesis inversely correlated with hepcidin only in severe phenotypes. In moderate disease, no independent predictors of hepcidin were identifiable; nevertheless, the low hepcidin levels indicate a significant risk for iron overload. In a population survey of Sri Lankan schoolchildren, ß-thalassemia (but not HbE) trait was associated with increased erythropoiesis and mildly suppressed hepcidin, suggesting an enhanced propensity to accumulate iron. In summary, the influence of erythropoiesis on hepcidin suppression associates with phenotypic disease variation and pathogenesis in HbE ß-thalassemia and indicates that the epidemiology of ß-thalassemia trait requires consideration when planning public health iron interventions.

A cell-based high-throughput screen for novel chemical inducers of fetal hemoglobin for treatment of hemoglobinopathies.

Decades of research have established that the most effective treatment for sickle cell disease (SCD) is increased fetal hemoglobin (HbF). Identification of a drug specific for inducing γ-globin expression in pediatric and adult patients, with minimal off-target effects, continues to be an elusive goal. One hurdle has been an assay amenable to a high-throughput screen (HTS) of chemicals that displays a robust γ-globin off-on switch to identify potential lead compounds. Assay systems developed in our labs to understand the mechanisms underlying the γ- to ß-globin gene expression switch during development has allowed us to generate a cell-based assay that was adapted for a HTS of 121,035 compounds. Using chemical inducer of dimerization (CID)-dependent bone marrow cells (BMCs) derived from human γ-globin promoter-firefly luciferase ß-globin promoter-Renilla luciferase ß-globin yeast artificial chromosome (γ-luc ß-luc ß-YAC) transgenic mice, we were able to identify 232 lead chemical compounds that induced γ-globin 2-fold or higher, with minimal or no ß-globin induction, minimal cytotoxicity and that did not directly influence the luciferase enzyme. Secondary assays in CID-dependent wild-type ß-YAC BMCs and human primary erythroid progenitor cells confirmed the induction profiles of seven of the 232 hits that were cherry-picked for further analysis.

Prevalence of the ß(S) gene among scheduled castes, scheduled tribes and other backward class groups in Central India.

Sickle cell disease is an inherited disorder of the blood, and characterized by vasoocclusive crises (VOC), risks for pneumococcal infections and organ toxicities, is associated with morbidity and premature mortality. India, with a population of 1.2 billion individuals, is estimated to be home to over 50.0% of the world's patients with sickle cell disease. The ß(S) gene [ß6(A3)Glu→Val; HBB: c.20A>T] has the highest prevalence in three socio-economically disadvantaged ethnic categories: the Scheduled Castes (SC), the Scheduled Tribes (ST), and Other Backward Class (OBC) groups in India. The tradition of endogamy practiced by the ethnic groups in India provides the rationale for the screening of individual populations to better understand the distribution of the ß(S) gene, guide counseling and awareness programs and aid development of public policy. We undertook a study to describe the prevalence of the ß(S) gene in these ethnic groups in the district of Nagpur, Maharashtra in Central India. Through community screening and subsequent targeted screening of high risk individuals, 35,636 individuals were screened, of whom 5466 were found to have sickle cell trait and 1010 were identified with sickle cell disease. Community screening revealed a sickle cell trait prevalence of 13.0% in the SC, 12.0% in the ST and 3.4% in the OBC population. This study describes the prevalence of the ß(S) gene within these groups in Central India determined by large scale community screening. This program has uncovered previously undiagnosed cases, provided detailed information to guide population-based disease counseling, prevention and comprehensive care programs.

Recent trends for novel options in experimental biological therapy of ß-thalassemia.

INTRODUCTION: ß-thalassemias are caused by nearly 300 mutations of the ß-globin gene, leading to low or absent production of adult hemoglobin. Achievements have been recently obtained on innovative therapeutic strategies for ß-thalassemias, based on studies focusing on the transcriptional regulation of the γ-globin genes, epigenetic mechanisms governing erythroid differentiation, gene therapy and genetic correction of the mutations. AREAS COVERED: The objective of this review is to describe recently published approaches (the review covers the years 2011 - 2014) useful for the development of novel therapeutic strategies for the treatment of ß-thalassemia. EXPERT OPINION: Modification of ß-globin gene expression in ß-thalassemia cells was achieved by gene therapy (eventually in combination with induction of fetal hemoglobin [HbF]) and correction of the mutated ß-globin gene. Based on recent areas of progress in understanding the control of γ-globin gene expression, novel strategies for inducing HbF have been proposed. Furthermore, the identification of microRNAs involved in erythroid differentiation and HbF production opens novel options for developing therapeutic approaches for ß-thalassemia and sickle-cell anemia.

Iron distribution and histopathological characterization of the liver and heart of ß-thalassemic mice with parenteral iron overload: Effects of deferoxamine and deferiprone.

The liver and heart are the major target organs for iron accumulation and iron toxicity in ß-thalassemia. To mimic the phenomenon of heavy iron overload resulting from repeated blood transfusions, a total of 180 mg of iron dextran was intraperitoneally injected into C57BL/6J mice (WT) and heterozygous ß-globin knockout mice ((mu)ß(th-3/+), BKO). The effects of deferiprone and deferoxamine in this model were investigated. The iron was distributed homogenously throughout the 4 liver lobes (left, caudate, right and median) and was present in hepatocytes, Kupffer cells and the sinusoidal space. Iron accumulation in phagocytic macrophages, recruitment of hepatic lymphocytes and nucleus membrane degeneration were observed as a result of iron overload in the WT and BKO mice. However, the expansion of hepatic extramedullary hematopoiesis was observed only in the BKO mice with iron overload. In the heart, the iron accumulated in the cardiac interstitium and myocytes, and moderate hypertrophy of the myocardial fibers and cardiac myocyte degeneration were observed. Although the total liver iron was not significantly altered by iron chelation therapy, image analysis demonstrated a difference in the efficacies of two iron chelators. The major site of chelation was the extracellular compartment, but treatment with deferiprone also resulted in intracellular iron chelation. Interestingly, iron chelators reversed the pathological changes resulting from iron overload in WT and BKO mice despite being used for only a short treatment period. We suggest that some of these effects may be secondary to the anti-inflammatory activity of the chelators.

Development of single-strand conformational polymorphism to screen for mutations in hotspot regions of beta-globin gene of beta-thalassemia patients of Sri Lanka.

Beta-thalassemia is prevalent in Sri Lanka and imposes a heavy economic and social burden in the country due to the patients' life-long need for regular blood transfusion and treatment with iron chelation therapy. Thus, there is a need to develop a rapid, reliable and effective population-based presymptomatic and prenatal screening method for beta-thalassemia. Single-strand conformational polymorphism (SSCP) technique was developed as an adjunct for the previously developed allele-specific PCR (ASP) technique to screen the presence of mutations in beta-globin gene. A hotspot region of beta-globin gene containing 98% of known beta-thalassemia mutations was amplified from 24 clinically diagnosed beta-thalassemia patients and two normal individuals. Two overlapping amplicons of 238 bp and 268 bp were subjected to SSCP analysis. The SSCP banding patterns of these two fragments from beta-thalassemia patients were different from the corresponding regions of normal individuals. Sequence analysis of these regions revealed the presence of 4 mutations in the form of deletion and substitution that have not been reported previously from Sri Lanka. Therefore, the SSCP protocol developed in this study together with ASP should provide an appropriate screening approach for presymptomatic and parental diagnosis of beta-thalassemia in the Sri Lankan population.