Thalassemia refers to a group of hereditary quantitative hemoglobinopathies, blood disorders that derive from a quantitative reduction of the synthesis of the globin chains of hemoglobin. On the basis of the genetic defect, they are divided into alpha and beta-thalassemia and are both inherited in an autosomal recessive manner. Thalassemias have wide phenotypic variability, from asymptomatic individuals up to very poor prognosis. Clinically, the phenotype is related to altered erythropoiesis, anemia, hemolysis and in the case of multiple transfusions, accumulation of iron in organs such as liver and spleen. Diagnosis is mainly clinical, based on blood tests, and is confirmed with DNA analysis. Treatment is closely related to clinical severity, however, blood transfusion or use of iron chelators or hematopoietic cell transplantation is frequent.
Learn more about hemoglobin
Hemoglobin is a tetrameric molecule (made up of 4 subunits), whose task is to bind and transport oxygen in the blood. Each subunit, called “globin chain” or “globin” coordinates within it a heme group, which contains an iron atom capable of oxidizing and binding oxygen and/or carbon dioxide. Within the tetramer, the subunits are equal to 2 to 2.
During the development of an individual, the composition of the globin subunits varies over time: in the fetal phase, hemoglobin (HbF) is composed of two α chains and two γ chains (α2γ2); gradually, HbF is replaced by the form HbA1 (α2β2) which becomes predominant in adulthood. In adults, a small part of hemoglobin is instead made up of the HbA2 form (α2δ2).
Detailed clinical description
Alpha thalassemia is due to a poor or a lack of synthesis of the α globin subunit. Clinically, it is characterized by microcytic and hypochromic anemia, inefficient erythropoiesis and hemolysis, which can cause splenomegaly. Additionally, patients may experience fatigue, weakness, irritability, and dizziness. Patients with thalassemia often require continuous transfusions, which cause iron overload in certain organs such as the liver, which can enlarge and cause hepatomegaly. However, the clinical picture is highly variable and closely related to the number of deleted or mutated alleles. Indeed, the α chains are encoded by two genes, for each of which each individual inherits two alleles.
Subjects in whom only one of the alleles is deleted are healthy carriers and do not show clinical signs. Subjects in which two alleles are deleted (in cis or in trans) are carriers of the thalassemia tract, show marked microcytosis, but hemoglobin levels are practically normal and they may be clinically asymptomatic. Patients in whom 3 out of 4 alleles do not function show a moderate to severe clinical picture, which is called HbH disease. Finally, in patients in whom all the alleles are not functional there is Bart’s disease (hydrops fetalis), a disorder with prenatal onset that is incompatible with life.
Beta-thalassemia is due to poor or lack of synthesis of the β globin subunit. The beta-thalassemia phenotype is divided into β-thalassemia minor, β-thalassemia intermediate and β-thalassemia major, depending on the severity of the symptoms. Individuals with β-thalassemia minor are asymptomatic or have mild anemia. The phenotype of β-thalassemia major is the most serious, which occurs from childhood and often leads to infant death. Patients with this condition experience severe microcytic anemia associated with fatigue, weakness, shortness of breath and migraine. Often there are jaundice, hepato- and spleno-megalia, accumulation of iron in the organs, expansion of the bone marrow causing skeletal deformities. Intermediate beta-thalassemia is characterized by a variable phenotype, with mild to moderate anemia associated in some cases with other typical symptoms. Also in this case, the phenotypic manifestation is closely linked to the underlying genetic defect: subjects who have only one mutated allele have β-thalassemia minor, those with both mutated alleles can have the intermediate or major form depending on the effect of the mutation on the protein.
Thalassemias are more frequent in those regions of the world where malaria is endemic such as Southeast Asia, the Indian subcontinent and Africa and in some coastal areas of the Mediterranean, while it remains rare in North America, Europe, Australia. and in South Korea. About 7% of the world’s population is estimated to be carrier of thalassemia.
The genes that code for globins are grouped into two gene clusters: the α-globin cluster, which is located on the short arm of chromosome 16 and the β globin cluster, which is located on the short arm of chromosome 11.
The α-globin cluster contains, under the control of a locus control region (LCR) that determines its alternative expression, the ζ2 gene involved in the formation of embryonic hemoglobin, four non-functional pseudogenes and the two genes for α-globins, α1 and α2. Genes α1 and α2 derive from an ancestral event of tandem duplication and therefore have a highly homologous and repeated sequence.
The β globin cluster contains, under the control of a LCR, the genes encoding the globins δ, β, γ, ε and a non-functioning pseudogene.
The pathogenetic mechanism is based on the imbalance of the synthesis between the α chains and the β chains of hemoglobin. Chains that excess tend to aggregate, precipitate and cause the destruction of red blood cell precursors, as well as sequester properly functioning chains through a negative dominance mechanism.
Alpha-thalassemia is an autosomal recessive inherited disease, mainly due to the deletion of one or more alleles of the HBA1 and HBA2 genes, due to the non-homologous recombination process that can occur because of the high sequence homology of the two genes. In clinical practice, α-thalassemia is classified as α0-thalassemia, when both alleles of a gene are deleted (-/-) and α+-thalassemia when only one of the alleles is deleted (α/-). In some cases, the α+ genotype can be caused by sequence mutations (and not deletions) such as SNV or small insertions/deletions: most of these variants fall into the HBA2 gene.
Beta-thalassemia is an autosomal recessive disease caused by mutations in the HBB gene. Unlike α-thalassemia, in β-thalassemia the mutations are not mainly deletions (which may still exist, but very rarely), but SNV or small insertions/deletions. On the basis of the residual amount of synthesis of the β subunit, we can distinguish three types of alleles: β0, associated with the absence of synthesis; β+, associated with a reduced synthesis; β++, associated with a minimal reduction in synthesis (also known as silent). Nonsense, frameshift and splice mutations usually cause a β0 allele, while mutations affecting regulatory sites, UTRs, and transcription may instead produce β+ or β++ alleles.
Nowadays, there are several techniques that allow confirming molecularly the diagnosis of thalassemia. These techniques allow identifying both large deletions/duplications and sequence mutations. Regarding large deletions/duplications, the two main techniques used are MLPA and GAP PCR, which is used to search for deletions whose breakpoint is known, such as the common deletions of 3.7kb and 4.2Kb (α3.7 e α4.2). For sequence variants, it is possible to use allele-specific PCR or reverse dot-blot to identify the most common known mutations, while for the identification of unknown variants Sanger sequencing or Next-Generation Sequencing (NGS) remain the methods more reliable. However, particular attention must be paid to the high sequence homology of HBA1 and HBA2 which can prevent the correct localization of the mutation.
The differential diagnosis of α-thalassemia includes:
- Alpha-thalassemia/mental retardation syndrome: an X-linked syndrome due to mutations in the ATRX gene, characterized by developmental delay, characteristic facies, genitourinary abnormalities and alpha thalassemia.
- Hydrops fetalis whose cause can depend on numerous factors such as chromosomal abnormalities, infections or other genetic diseases.
- Hemolytic anemias.
The differential diagnosis of β-thalassemia includes:
- Sideroblastic anemia and congenital dyserythropoietic anemia
- Tumor disorders such as myelomonocytic leukemia and aplastic anemia
- X-linked thrombocytopenia with beta-thalassemia, an X-linked recessive disease characterized by thrombocytopenia, haemolytic anemia, splenomegaly and alterations in globin synthesis due to mutations in the GATA1 gene.
Genetic testing strategy
The suspicion of thalassemia is mainly clinical and supported by hematological tests on red blood cells. Molecular testing is used both to confirm the diagnosis and to identify asymptomatic at-risk relatives or to aid prenatal diagnosis. For molecular confirmation, it is recommended to proceed with the analysis of a Next-Generation Sequencing gene panel that includes all the genes associated with this disorder. Breda Genetics offers the analysis of genes for thalassemias through multi-gene panels based on exome (EXOME 60MB) or whole genome (FULL GENOME) sequencing. Furthermore, in the event of a negative result, it is possible to proceed with the analysis of large deletions/duplications on the genes of interest through methods such as MLPA.
Beta-Thalassemia OMIM # 603902
Alpha-Thalassemia OMIM # 604131
Munkongdee T, Chen P, Winichagoon P, Fucharoen S, Paiboonsukwong K. Update in Laboratory Diagnosis of Thalassemia. Front Mol Biosci. 2020 May 27;7:74. doi: 10.3389/fmolb.2020.00074. PMID: 32671092;
Gilad O, Shemer OS, Dgany O, Krasnov T, Nevo M, Noy-Lotan S, Rabinowicz R, Amitai N, Ben-Dor S, Yaniv I, Yacobovich J, Tamary H. Molecular diagnosis of α-thalassemia in a multiethnic population. Eur J Haematol. 2017 Jun;98(6):553-562. doi: 10.1111/ejh.12866. Epub 2017 Apr 6. PMID: 28160324.
Lee JS, Cho SI, Park SS, Seong MW. Molecular basis and diagnosis of thalassemia. Blood Res. 2021 Apr 30;56(S1):S39-S43. doi: 10.5045/br.2021.2020332. PMID: 33935034; PMCID: PMC8093999.