Beta thalassemia syndromes: New insights

Abstract

Beta thalassemia (β-thalassemia) syndromes are a heterogeneous group of inherited hemoglobinopathies caused by molecular defects in the beta-globin gene that lead to the impaired synthesis of beta-globin chains of the hemoglobin. The hallmarks of the disease include ineffective erythropoiesis, chronic hemolytic anemia, and iron overload. Clinical presentation ranges from asymptomatic carriers to severe anemia requiring lifelong blood transfusions with subsequent devastating complications. The management of patients with severe β-thalassemia represents a global health problem, particularly in low-income countries. Until recently, management strategies were limited to regular transfusions and iron chelation therapy, with allogeneic hematopoietic stem cell transplantation available only for a subset of patients. Better understanding of the underlying pathophysiological mechanisms of β-thalassemia syndromes and associated clinical phenotypes has paved the way for novel therapeutic options, including pharmacologic enhancers of effective erythropoiesis and gene therapy.

Key Words: Beta thalassemia; Hemoglobin; Molecular defects; Ineffective erythropoiesis; Hemolysis; Transfusion; Iron chelation; Novel therapies

Core Tip: Beta thalassemia syndromes are among the most common monogenic disorders worldwide, characterized by impaired hemoglobin synthesis that leads to ineffective erythropoiesis, chronic hemolysis, iron overload, and subsequent complications. The clinical manifestations are diverse, as a result of the underlying beta-globin gene variants and the coinheritance of modifying factors. Recent advances in the understanding of the underlying molecular and cellular mechanisms have facilitated the development of novel therapeutic strategies. The purpose of this article is to briefly describe new insights in the pathophysiology of this old disease, and discuss revolutionary changes in the treatment landscape for severe forms of beta thalassemia.

INTRODUCTION

Beta thalassemia (β-thalassemia) syndromes are a heterogenous group of autosomal recessive anemias characterized by reduced or absent synthesis of β-globin (HBB) chains of the hemoglobin (Hb)[1,2]. The disorder represents a global health burden. It is estimated that about 1.5% of the world population (80 million people to 90 million people) are carriers of β-thalassemia[3], with > 40000 affected individuals born each year[4]. The highest prevalence is in the traditional malaria-endemic “thalassemia belt”, which extends from sub-Saharan Africa, the Mediterranean, the Middle East to Southeast Asia, reflecting the relative resistance of carriers against Plasmodium falciparum and the higher frequency of consanguineous marriages[5]. However, due to population migrations from high-prevalence areas, β-thalassemia is now encountered worldwide[6]. As carriers for α-thalassemia and sickle cell disease (SCD) are similarly protected against Plasmodium falciparum, the regions where β-thalassemia is prevalent overlap with those of α-thalassemia and SCD. Consequently, coinheritance of two or more Hb variants is not unusual in these regions[7].

PATHOPHYSIOLOGY

β-thalassemia syndromes are recessive disorders caused by pathogenic variants in the HBB gene, resulting in reduced or absent β-globin chain production and an imbalance of α-subunit and β-subunit that form Hb tetramer. An excess of free α-globin chains triggers the most important pathogenic events of the disease: Ineffective erythropoiesis, chronic anemia/hemolysis, and iron overload[8]. Unpaired α-chains precipitate in erythroid precursors, causing their premature destruction. Besides, they accumulate on erythroid membranes and are oxidized into methemoglobin and insoluble hemichromes[9,10]. Free iron catalyzes the formation of reactive oxygen species. An oxidative stress environment contributes to reduced differentiation and apoptosis of erythroblasts[11]. A continuous state of chronic stress erythropoiesis arises, and the expanded pool of erythroid progenitors is unable to generate red blood cells (RBCs). Biochemical signaling from marrow expansion involving the bone morphogenetic protein pathway inhibits hepcidin production, thereby leading to increased iron absorption and iron overload[12,13]. An activated intestinal hypoxia-inducible factor 2 alpha leads to the upregulation of genes responsible for duodenal iron absorption and contributes also to iron overload[10]. Macrophages in the bone marrow are activated by iron overload, and selectively phagocyte apoptotic erythroblasts, thereby contributing to ineffective erythropoiesis. Increased levels of several inflammatory cytokines have also been reported[14]. Chronic anemia and hypoxia cause increased erythropoietin production, resulting in the increased proliferation and accumulation of abnormal erythroid progenitors. Chronic hemolysis in the peripheral blood, and the binding of immunoglobulins and complement trigger the sequestration of RBCs in the spleen, resulting in hypersplenism, which eventually aggravates the severity of the anemia[15]. The type of HBB mutation and the degree of globin chain imbalance are the primary determinants of the clinical phenotype. However, patients with the same HBB genotype can show tremendous phenotypic diversity, and a set of genetic modifiers at different levels have been identified[16]. Secondary modifiers of β-thalassemia are genetic variants that alter the relative imbalance of α-globin and HBB chains. Co-inheritance of an α-thalassemia trait and the hereditary persistence of HbF are associated with a milder phenotype. Tertiary modifiers include genetic and environmental factors that alter the clinical complications of the disease[17]. The best delineated genetic variants mapping outside the HBB cluster are those affecting bilirubin metabolism, iron homeostasis, bone disease, and cardiac disease[18,19].

DIAGNOSIS

The diagnosis of β-thalassemia should be considered in any individual with microcytic hypochromic anemia and the absence of iron deficiency[9,20]. A peripheral blood smear reveals microcytosis, hypochromia, anisocytosis, poikilocytosis, and nucleated RBCs[20]. The reticulocyte count is normal or slightly elevated. Hb electrophoresis with decreased or completely absent HbA, increased HbA2 and often HbF, is an important tool to establish the diagnosis and to recognize carriers and patients with an intermediate and severe phenotype. High-performance liquid chromatography and capillary electrophoresis are two standard techniques used for quantifying HbA2 and detecting other hemoglobinopathies that may interact with β-thalassemia[21,22]. The use of DNA genotyping has become increasingly important to obtain an accurate diagnosis, predict disease severity, provide therapeutic targets, and guide management[7,9].

MOLECULAR GENETICS

HBB is located on chromosome 11 at position p15.5. HBB maps clustered with four other functional globin genes: The embryonal HBE gene, the HBG1 and HBG2 genes, and the adult HBD, as well as the ψβ-pseudogene[23]. The genes are arranged along the chromosome in the order of their developmental expression to produce different Hb tetramers[24]. More than 400 HBB causative variants have been described in patients with β-thalassemia[19,25]. In contrast to α-thalassemia, which is mainly caused by larger deletions involving HBA1 and HBA2 genes, most HBB variants are point mutations, including mainly single nucleotide substitutions, or some indels (insertions or deletions) leading to frameshift[1,7,19,26]. Multiple stages of HBB expression can be affected by the vast spectrum of non-deletional variants, from transcription, through RNA processing, to RNA translation and stability[19,27,28]. The prevalence of variants differs by region. Worldwide, only 20 HBB causing variants account for more than 80% of β-thalassemia cases, because of the geographical clustering of populations with a few common mutations[29]. Larger deletions are rare in β-thalassemia, and are classified into deletions confined to HBB, deletions extending to other genes, and deletions of the locus control region[26]. HBB alterations are broadly classified according to the extent to which β-globin chain synthesis is reduced to three categories: (1) β⁰ (complete absence of β-globin); (2) β⁺ (β-globin is produced, but less than normal); and (3) β⁺⁺ or silent (a mild reduction in β-globin synthesis)[27]. Table 1 shows the main types of HBB variants and their association with the severity of β-thalassemia.

Table 1 Beta thalassemia variants.

Type of β-globin gene variant		Subtype of β-globin gene variant	Beta-thalassemia severity type
Point mutations	Trancriptional variants	Promoter regulatory elements; 5’ untranslated regions	β+ or β++
	Variants involving RNA processing	Splice junction; consensus splice sites; cryptic splice sites; RNA cleavage - poly A signal; others in 3’ untranslated regions	β0, β+ or β++
	Variants involving RNA translation	Initiation codon; nonsense codons; frameshift	β0
Deletions			β0

CLASSIFICATION

β-thalassemia are traditionally classified into three main forms based on the severity of the clinical phenotype: Major (also known as Mediterranean anemia or Cooley anemia), intermedia, and minor (β-thalassemia carrier or trait). β-thalassemia minor is a heterozygous state with one unaffected HBB and one affected, either β⁺ or β⁰. Homozygosity or compound heterozygosity with β⁺ or β⁰ causes thalassemia intermedia and major[30]. Over the past decade, there has been a transition to classification based on blood transfusion requirements into transfusion-dependent thalassemia (TDT) or non-TDT (NTDT)[9]. Patients with TDT require regular lifelong blood transfusions, starting before the age of 2 years. Patients with NTDT may need blood transfusion occasionally or for a limited period, e.g., during surgery or pregnancy. Transfusion requirements should be re-evaluated intermittently, and patients may shift clinically between TDT or NTDT over time.

CLINICAL MANIFESTATIONS

The complex pathophysiology of β-thalassemia leads to diverse clinical manifestations, ranging from asymptomatic individuals to severe fatal anemias in utero or early childhood[31]. Clinical spectra vary depending on the underlying HBB molecular defect and the coinheritance of other genetic modifiers. β-thalassemia carriers (β-thalassemia trait) have an absence of symptoms and hematological abnormalities (so called silent carriers) or microcytosis/mild microcytic hypochromic anemia. In pregnant women carriers, significant anemia (Hb < 7 g/dL) may develop, usually by the third trimester[15]. The carriers are mostly identified as part of family screening or a population survey, or incidentally during intercurrent illness[32]. Patients with β-thalassemia major typically present between 6 months and 24 months of age when Hb production is switched off from fetal to adult. Clinical presentation includes extreme pallor due to severe anemia, failure to thrive, and hepatosplenomegaly. Feeding problems, jaundice, irritability or somnolence, diarrhea, gallstones, and recurrent fever may occur[15,20,30]. Untreated or inadequately treated children suffer from growth retardation and bone deformities due to bone marrow expansion. Frontal bossing, larger cheekbones, and maxillary hypertrophy are common skeletal findings (“thalassemic facies”)[1]. Progressive hepatosplenomegaly may lead to pancytopenia[32]. Long-term complications associated with iron overload include dilated cardiomyopathy, liver disease, and endocrinopathies (hypogonadism, hypothyroidism, diabetes mellitus)[20,31]. Genetic modifiers, mainly affecting bilirubin metabolism, iron metabolism, bone disease, and cardiac abnormalities, can influence the clinical course and response to therapy[33]. The presence of TA sequence polymorphism in the gene promoter of uridine diphosphate-glucuronosyltransferase family 1 member A1 responsible for Gilbert syndrome may increase the predisposition to cholelithiasis[34,35]. SP1-binding site polymorphism in the regulatory region of the collagen type I alpha 1 gene is strongly associated with osteoporosis in patients with β-thalassemia major[36,37]. Besides, variants in vitamin D receptor, collagen type I alpha 2, and transforming growth factor beta-1 genes can modify bone mass in thalassemia patients[7]. The coinheritance of the homeostatic iron regulator C282Y variant, which causes the common type of hereditary hemochromatosis, may increase iron overload[38,39]. Furthermore, the presence of apolipoprotein E ε4 is a risk factor for left ventricular heart failure as the main cause of death in thalassemic patients[40]. Glutathione S-transferase M1 gene polymorphisms have been associated with cardiac iron deposition in patients with β-thalassemia major[41,42]. Conversely, the β-thalassemia trait may contribute to overall better cardiovascular health through a favorable lipidemic and blood pressure profile[43,44].

Many children who are adequately transfused (with the maintenance of Hb level > 9 g/dL) and are compliant with iron chelation therapy, have normal growth and pubertal development, and become sexually mature[32]. β-thalassemia intermedia belongs to NTDT, with mild to moderate hemolytic anemia maintaining Hb level ranging from 7 g/dL to 10 g/dL without transfusion support, and encompassing a wide spectrum of clinical phenotypes between those of thalassemia minor and major[45]. Mildly affected patients can be asymptomatic until adulthood. Clinical presentation in more severe forms of β-thalassemia intermedia typically occurs at 2 years to 4 years of age, and manifestations can include pallor, jaundice, hepatosplenomegaly, and skeletal changes. Growth and development are generally better than in patients with thalassemia major. However, compared to TDT, patients with β-thalassemia intermedia are at greater risk of cholelithiasis, thromboembolic complications (particularly splenectomized patients), and pulmonary hypertension. Extramedullary hematopoiesis primarily affects the spleen, liver, lymph nodes, and vertebrae, and may cause neurological symptoms[32]. Pulmonary hypertension is considered a primary cause of heart failure[46]. Many patients with β-thalassemia intermedia will require transfusions at some point in their lives. When their transfusion requirements reach > 8 units per year, they are reclassified as TDT or β-thalassemia major[15].

TREATMENT

No specific treatment is required for β-thalassemia carriers, but they should receive genetic counselling. The treatment of patients with thalassemia major and intermedia should be individualized[47]. Conventional modalities for the management of β-thalassemia include blood transfusions, iron chelation therapy, splenectomy, and hematopoietic stem-cell transplantation (HSCT) for a subgroup of patients[48]. Recent advances in the understanding of the pathophysiology of β-thalassemia have led to the development of new therapeutic strategies.

Conventional management

In TDT, regular RBC transfusions, usually every 2 weeks to 5 weeks, to maintain the pretransfusion Hb level > 9 g/dL to 10.5 g/dL, are needed to correct anemia, suppress ineffective erythropoiesis, and inhibit increased gastrointestinal iron absorption[1,17]. Iron chelation therapy is also required to manage the iron overload as an inevitable complication of regular transfusions. Chelation is initiated in childhood after the first 10-20 transfusions, when ferritin levels exceed 1000 ng/mL[49]. Three iron chelators are currently available: Deferoxamine, which is administered parenterally, and the oral alternatives deferiprone and deferasirox. Chelation is usually started with an oral chelator but switched to deferoxamine if it fails[45]. Combined chelation therapy (deferoxamine and deferiprone, or deferasirox and deferiprone) is a safe and effective option in patients with severe iron overload[50,51]. Splenectomy is considered for thalassemic patients with a progressive increase in blood requirements and significant increases in iron stores despite iron chelation therapy, hypersplenism with a worsening of anemia, leukopenia, and thrombocytopenia, and splenomegaly with a risk of splenic rupture[32,52]. The main complications of splenectomy are overwhelming infections, and an increased risk of thromboembolic events and pulmonary hypertension[1]. Allogenic HSCT is an established curative and most widely used therapy for TDT, with the overall survival reaching almost 90%[32]. The best clinical outcomes are reported when HSCT is performed from matched sibling donors and in those aged less than 14 years[53]. The main risks of HSCT include the toxicity of intensive conditioning regimens and graft-versus-host disease. The progressive availability of stem cell sources from unrelated donors or umbilical cord blood has made HSCT a feasible option for an increasing number of TDT patients lacking an identical sibling donor[54].

Novel therapies

Innovative therapies in β-thalassemia can be classified into three major categories based on their pathological target: Addressing ineffective erythropoiesis, modulating iron metabolism, and altering globin gene expression and globin chain imbalance[55]. Luspatercept and sotatercept are recombinant fusion proteins that bind ligands of the transforming growth factor beta superfamily and promote late-stage erythroid maturation[48,56]. Janus kinase-2 inhibitors (fedratinib, ruxolitinib) have been shown to prevent proliferation of thalassemic erythroid cells and reduce the spleen volume[17,57]. Small allosteric activators of RBC pyruvate kinase (mitapivat), which increase adenosine triphosphate production, have shown a promising role in the treatment of the late phase of ineffective erythropoiesis[58].

Molecules targeting iron dysregulation include minihepcidins that restrict iron absorption, ferroportin inhibitors (vamifeport), apotransferrin that upregulates hepcidin and downregulates transferrin receptor 1, and transmembrane protease serine 6 that increases the hepatic synthesis of hepcidin[48,55]. Gene therapy aims to restore the RBC function and ameliorate anemia by repairing altered genes[59]. Gene manipulation strategies for modifying patient autologous hematopoietic stem cells include gene addition using lentiviral vectors (β-like globin gene replacement, HbF induction) and gene editing[60]. Gene editing involves the correction of β-thalassemic alterations using endonuclease: Zinc finger nucleases, transcription activator-like effector nucleases, and clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9[59].

CRISPR-Cas technology represents the optimal tool for developing therapeutic methods since the CRISPR-Cas9 system has some major advantages, including high genome editing efficiency, low cytotoxicity and transient expression when delivered as ribonucleoproteins, and the possibility of multiplexing, which enables the simultaneous targeting of multiple loci[61]. Two main CRISPR-based approaches are known for the treatment of β-thalassemia. The first approach is the repair of the mutated form of the HBB gene. This approach is challenging, given the large number of variants that can cause β-thalassemia, and is limited to the correction of known HBB gene mutations. The second approach is based on the reactivation of HbF production by targeting γ-globin gene expression and leading to the amelioration of the clinical manifestations of β-thalassemia. Previous studies attempted to induce γ-globin gene expression in bone marrow-derived adult CD34⁺ hematopoietic stem and progenitor cells (HSPC) by delivering target site specific SaCas9 to remove part of the HBB locus including HBB and HBD genes, and repair it by non-homologous end joining[62]. After differentiation into erythroid cells in vitro, HSPC demonstrated a significantly increased expression of the γ-globin gene compared to cells without these 12.9 kb deletions. Therefore, these cells have demonstrated a potential new approach to autologous transplantation therapy for the treatment of homozygous β-thalassemia[62].

Although remarkable advances in gene therapy and gene editing have been achieved over the years, there are still many drawbacks. One is the fact that viral vector insertion in the HSPC genome is an uncontrolled process that can lead to polyclonal reconstitution[63]. On the other hand, the main obstacles of gene editing are off-target activity and chromosomal rearrangement events[64]. Despite all the difficulties, the United States Food and Drug Administration approved two innovative treatments for SCD in December 2023. The first one, lovotibeglogene autotemcel, is a cell-based gene therapy. Using a lentiviral vector, genetic modification is introduced into the blood stem cells of a patient with SCD, enabling them to produce Hb similar to HbA instead of HbS[65]. The second agent, exagamglogene autotemcel, is the first United States Food and Drug Administration approved treatment to utilize CRISPR-Cas technology. Using the genome editing approach, the erythroid-specific enhancer region of the BCL11A gene in HSPC is targeted, resulting in diminished BCL11A expression and subsequent increased production of γ-globin[66]. Both drugs are approved for the treatment of patients older than 12 years with SCD and a history of vaso-occlusive events. Both medications are derived from the patient’s own genetically modified blood stem cells, which are administered back to the patient in a single, one-time infusion as part of an autologous HSCT. Exagamglogene autotemcel was also approved by the European Medicines Agency in 2024 as the first CRISPR-Cas9 medicine for β-thalassemia and SCD[67]. It is to be noted that, while both gene therapy and gene editing have the potential to prevent years of long-term morbidity with a single treatment for β-thalassemia, their cost exceeds the available healthcare funding in most countries worldwide. Available therapies for β-TDT are listed in Table 2. A number of new agents and technologies are emerging, and the efficacy and potential toxicity of novel therapies need to be further tested to establish optimal management.

Table 2 Available therapy for transfusion-dependent beta thalassemia.

Treatment	Mode of action
Red blood cell transfusion	Correction of anemia
Deferoxamine	Iron chelation
Deferiprone
Deferasirox
Hematopoietic stem cell transplantation	Replacement with healthy hematopoietic stem cells
Luspatercept	Late-stage erythroid maturation
Exagamglogene autotemcel and lovotibeglogene autotemcel	Autologous genome-modifying/editing based therapy

CONCLUSION

β-thalassemia syndromes are a heterogeneous group of inherited disorders caused by alterations in HBB that result in ineffective hematopoiesis, chronic hemolytic anemia, and iron overload leading to end-organ damage. The increasing understanding of the molecular basis of multiple mechanisms involved in the pathophysiology of the disease has led to the development of novel therapeutic modalities beyond transfusion and iron chelation, with a promising impact on the reduction of the disease burden, an improvement in the long-term clinical outcome, and the quality of life of patients with β-thalassemia.