Thalassemia — From Genotype to Phenotype

Thalassemia encompasses serious diseases with complex pathophysiology that is difficult to explain since it is considered a group of defects with similar clinical effects, still not a single disorder

Genetic basis of hereditary persistence of fetal haemoglobin

This thesis investigated the extent to which genetic factors underlie the variations observed in fetal haemoglobin (HbF) levels. This is important as it is known that an elevated HbF level can ameliorate the symptoms of many of the haemoglobinopathies. The frequency and range of hereditary persistence of fetal haemoglobin (HPFH) in the UK population was determined which was otherwise unknown. All 4 categories of deletion mutations were identified and demonstrated the high frequency of deletional HPFH in UK patients. Four potentially novel deletions were identified and 2 fully characterised. One novel deletion was the first reported case of a large b0-thalassaemia deletion mutation in the Afghan population. Mutations in the g-globin gene promoters were identified as a frequent (21% of patients) cause of non-deletion HPFH in the UK. The majority of mutations being in white British individuals with elevated HbF levels only, probably arisen independently through genetic drift. The strongest association with the three polymorphisms and HbF expression was seen in b-thalassaemia trait subjects with the XmnI-HBG2 polymorphism. The SNPs in BCL11A and HBS1L-MYB failed to show statistical correlations with HbF. Heterozygosity for ten novel mutations in the KLF1 gene were indentified in patients with a high HbF indicating that a single altered KLF1 allele can elevate HbF. The identification of a KLF1 mutation in an individual with a particularly mild form of sickle cell disease could provide in-vivo evidence that controlled reduction of KLF1 expression could be an effective treatment for sickle cell disease. Finally, the finding that Asian Indian newborns undergo haemoglobin switching earlier than other ethnic groups was investigated. Birth weight, gestation and chromosomal abnormalities were not responsible and the frequency of the 4.9kb g-thalassaemia deletion mutation was determined to be low

Novel Therapy Approaches in β-Thalassemia Syndromes — A Role of Genetic Modifiers

The β-thalassemia syndromes are heterogeneous autosomal recessive hereditary disorders, caused by alterations in the HBB gene and characterized by absent or reduced β-globin chain synthesis. The β-thalassemia phenotypes are variable, ranging from severe, transfusion-dependent thalassemia major to mild, asymptomatic thalassemia trait. This interpatient clinical variability has swayed researchers toward identifying genetic modifiers for these disorders. Primary modifiers refer to type of alterations affecting β-globin gene. Secondary modifiers include variations in genes affecting α/β-globin chain equilibrium, such as genes involved in the γ-globin gene expression and genes affecting the amount and stability of α-globin chains. Tertiary modifiers are gene variations affecting the phenotype with regard to the complications caused by β-thalassemia syndromes. A role of secondary genetic modifiers in ameliorating the clinical phenotype has been observed. Secondary genetic modifiers are the most common targets for modern therapy and could be located within α- and γ-globin genes or outside globin gene cluster. The most potent secondary modifier genes are γ-globin genes. Production of fetal hemoglobin (HbF) trough adulthood ameliorates the severity of β-thalassemia phenotype. Large family and genome-wide association studies have shown that regions outside of the β-globin gene cluster are also implicated in γ-globin gene expression regulation. HBS1-MYB intragenic region and BCL11A gene have been particularly studied. Variants within these loci, along with γ-globin gene variants, account for approximately 50% of the HbF level variation, suggesting that additional factors are involved (transcription regulators (KLF1), regulators of α-globin chain stability (AHSP), epigenetic regulators (FoP)). Until recently a definitive cure for β-thalassemia could be achieved with bone marrow transplantation. However, it is available for less than 30% of the patients and bears a significant risk of morbidity and mortality. Alternative strategies, such as gene therapy and development of induced pluripotent stem cells (iPSCs) have been explored. The targets for gene therapy are hematopoietic stem cells, which are harvested from patient bone marrow or peripheral blood, purified by immunoselection, transduced by “therapeutic gene” aimed at correcting the effect of defective β-globin gene, and returned to the patient. Various types of vectors have been considered for gene transfer, including non viral (tRNK and ribozymes) and viral (retroviral and lentiviral vectors). In the past few years, iPSCs emerged as an interesting candidate for gene transfer. The feature that makes these cells appealing in the field of gene therapy is their susceptibility to gene correction by homologous recombination. Therapy protocols based on molecular basis of β-thalassemia are the best example of novel approaches in disease treatment

Modifiers of γ-Globin Gene Expression and Treatment of β-Thalassemia

Beta thalassemia (β-thalassemia) is an autosomal recessive genetic disease with many genes involved. It is a heterogeneous disorder caused by variations in the inactivation mechanism of the Beta-globin (β-globin) genes. Despite seemingly similar genotypes, the patients with Beta-thalassemia have a remarkable variability in anaemia, growth development, and hepatospleenomegaly and transfusion requirements. The genetic factors may differ in each race or ethnic group for therapy and prevention. Despite remarkable successes in the treatment of Beta-thalassemia in the past decades, it is still the leading cause of death and premature disability in developed and developing countries. Possible factors that influence the severity of anaemia in thalassemia may be inherited or non-inherited. The inherited factors include the type of β-thalassemia, coinheritance of alpha thalassemia (α-thalassemia) and factors that stimulate fetal hemoglobin (HbF) production. In this chapter, respective contributions of known modifiers and also the pharmaceutical agents currently in use and under clinical trials for regulating the globin gene expression will be discussed

The Hellenic type of nondeletional hereditary persistence of fetal hemoglobin results from a novel mutation (g.-109G>T) in the HBG2 gene promoter

Nondeletional hereditary persistence of fetal hemoglobin (nd-HPFH), a rare hereditary condition resulting in elevated levels of fetal hemoglobin (Hb F) in adults, is associated with promoter mutations in the human fetal globin (HBG1 and HBG2) genes. In this paper, we report a novel type of nd-HPFH due to a HBG2 gene promoter mutation (HBG2:g.-109G>T). This mutation, located at the 3′ end of the HBG2 distal CCAAT box, was initially identified in an adult female subject of Central Greek origin and results in elevated Hb F levels (4.1%) and significantly increased Gγ-globin chain production (79.2%). Family studies and DNA analysis revealed that the HBG2:g.-109G>T mutation is also found in the family members in compound heterozygosity with the HBG2:g.-158C>T single nucleotide polymorphism or the silent HBB:g.-101C>T β-thalassemia mutation, resulting in the latter case in significantly elevated Hb F levels (14.3%). Electrophoretic mobility shift analysis revealed that the HBG2:g.-109G>T mutation abolishes a transcription factor binding site, consistent with previous observations using DNA footprinting analysis, suggesting that guanine at position HBG2/1:g.-109 is critical for NF-E3 binding. These data suggest that the HBG2:g-109G>T mutation has a functional role in increasing HBG2 transcription and is responsible for the HPFH phenotype observed in our index cases

Recent advances in β-thalassemias

β-thalassemias are heterogeneous hereditary anemias characterized by a reduced output of β-globin chains. The disease is most frequent in the temperate regions of the world, where it represents an important health problem. In the last decades, several programs, aimed at controlling the birth rate of thalassemia newborns by screening and prenatal diagnosis of populations with high risk of β-thalassemia, have been successful accomplished. Bone marrow transplantation has offered a definitive cure for the fraction of patients with available donors. In the same time, steady improvements were made in the traditional clinical management of β-thalassemia patients. The introduction of the oral iron chelators deferiprone that preferentially chelates hearth iron and the development of novel NMR diagnostic methods has led to reduced morbility, increased survival and improved quality of life. More recently, major advances have being made in the discovery of critical modifier genes, such as Myb and especially BCL11A (B cell lymphoma 11A), a master regulator of HbF (fetal hemoglobin) and hemoglobin switching. Polimorphysms of BCL11A, Myb and γ-globin genes account for most of the variability in the clinical phenotypes in β-thalassemia and sickle cell anemia patients. Finally, the year 2010 has brought in the first successful experiment of gene therapy in a β-thalassemia patient, opening up the perspective of a generalized cure for all β- thalassemia patients