Test Diagnosis of Thalassemia/Thalassemia is an autosomal recessive hereditary chronic hemolytic anemia due to a partial or complete deficiency in the synthesis of α-globin chains (α-thal) or β-globin chains (β-thal) which compose the major adult hemoglobin (HbA), a tetramer of α 2β 2. It is caused by one or more of several hundred mutations in the corresponding genes. The unpaired globin chains are unstable; they precipitate intracellularly, resulting in hemolysis, premature destruction (by apoptosis) of red blood cell (RBC) precursors in the bone marrow, and a short life-span of mature RBCs in the circulation.
Beta-thal is classified into three main subgroups based on their clinical expression: major, intermedia, and minor. β-thal major presents itself within the first 2 years of life with severe anemia, poor growth, and skeletal abnormalities and requires regular, lifelong blood transfusions. β-thal intermedia requires only periodic blood transfusions, while β-thal minor does not require specific treatment. Alpha-thal presents with moderate anemia when there is a significant lack of synthesis of α-globin chains (HbH disease).
Another Name
The term thalassemia is derived from the Greek, Thalassa (sea) and haima (blood). Beta-thalassemia includes three main forms:
- Thalassemia Major variably referred to as Cooley’s Anemia and Mediterranean Anemia, Thalassemia Intermedia and Thalassemia Minor also called beta-thalassemia carrier, beta-thalassemia trait or heterozygous beta-thalassemia.
- Apart from the rare dominant forms, subjects with thalassemia major are homozygotes or compound heterozygotes for beta0 or beta+ genes, subjects with thalassemia intermedia are mostly homozygotes or compound heterozygotes and subjects with thalassemia minor are mostly heterozygotes.
Types of Thalassemia
Thalassemia consists of a group of disorders that may range from a barely detectable abnormality of the blood to severe or fatal anemia. Adult hemoglobin is composed of two alpha (α) and two betas (β) polypeptide chains. There are two copies of the hemoglobin alpha gene (HBA1 and HBA2), which each encode an α-chain, and both genes are located on chromosome 16. The hemoglobin beta gene (HBB) encodes the β-chain and is located on chromosome 11.
In α-thalassemia, there is a deficient synthesis of α-chains. The resulting excess of β-chains binds oxygen poorly, leading to a low concentration of oxygen in tissues (hypoxemia). Similarly, in β-thalassemia, there is a lack of β-chains. However, excess α-chains can form insoluble aggregates inside red blood cells. These aggregates cause the death of red blood cells and their precursors, causing very severe anemia. The spleen becomes enlarged as it removes damaged red blood cells from the circulation.
Alpha-thalassemias
The α-thalassemias involve the genes HBA1[rx] and HBA2,[rx] inherited in a Mendelian recessive fashion. Two gene loci and so four alleles exist. It is also connected to the deletion of the 16p chromosome. α Thalassemias result in decreased alpha-globin production, therefore fewer alpha-globin chains are produced, resulting in an excess of β chains in adults and excess γ chains in newborns. The excess β chains form unstable tetramers (called hemoglobin H or HbH of 4 beta chains), which have abnormal oxygen dissociation curves. Alpha thalassemias often are found in people from Southeast Asia, the Middle East, China, and in those of African descent.[rx]
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Alpha-thalassemia syndromes
| Syndrome | Number of alpha-globin genes affected | Clinical features | Hemoglobin pattern |
| Silent carrier (α+) | 1 | No or minimal anemia | 1%–2% Hb Barts (γ4) |
| Thalassemia trait (α+) | 2 | Mild anemia Hypochromic microcytic | 5%–10% Hb Barts (γ4) |
| HbH disease (α0 + α+) | 3 | Moderate anemia Hypochromic microcytic RBC inclusion bodies | 10%–30% HbH (β4) |
| Hydrops fetalis (α0) | 4 | Death in utero or at birth Severe anemia | 97% Hb Barts (γ4) 3% HbH (β4) |
| of missing genes | Types of alpha thalassemia[rx] | Symptoms |
|---|---|---|
| 1 | Silent carrier | No symptoms |
| 2 | Alpha thalassemia trait | Minor anemia |
| 3 | Hemoglobin H disease | Mild to moderate anemia may lead to normal life |
| 4 | Hydrops fetalis | Fetal death usually occurs at birth |
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Beta-thalassemia
Typical beta-thalassemia carriers are identified by analysis of RBC indices, which shows microcytosis (low MCV) and reduced content of Hb per red cell (low MCH), and by qualitative and quantitative Hb analysis, which displays the increase of HbA2.
Beta-thalassemia associated with other Hb anomalies
The interaction of HbE and beta-thalassemia results in thalassemia phenotypes ranging from a condition indistinguishable from thalassemia major to a mild form of thalassemia intermedia. Depending on the severity of symptoms three categories may be identified:
- Mild HbE/beta-thalassemia – It is observed in about 15% of all cases in Southeast Asia. This group of patients maintains Hb levels between 9 and 12 g/dl and usually does not develop clinically significant problems. No treatment is required.
- Moderately severe HbE/beta-thalassemia – The majority of HbE/beta-thalassemia cases fall into this category. The Hb levels remain at 6-7 g/dl and the clinical symptoms are similar to thalassemia intermedia. Transfusions are not required unless infections precipitate further anemia. Iron overload may occur.
- Severe HbE/beta-thalassemia – The Hb level can be as low as 4-5 g/dl. Patients in this group manifest symptoms similar to thalassemia major and are treated as thalassemia major patients.
Beta thalassemias are due to mutations in the HBB gene on chromosome 11,[rx] also inherited in an autosomal, recessive fashion. The severity of the disease depends on the nature of the mutation and on the presence of mutations in one or both alleles.
- β thalassemia major – (Mediterranean anemia or Cooley anemia) is caused by a βo/βo genotype. No functional β chains are produced, and thus no hemoglobin A can be assembled. This is the most severe form of β-thalassemia;
- β thalassemia intermedia – is caused by a β+/βo or β+/β+ genotype. In this form, some hemoglobin A is produced;
- β thalassemia minor – is caused by a β/βo or β/β+ genotype. Only one of the two β globin alleles contains a mutation, so β chain production is not terribly compromised and patients may be relatively asymptomatic.
Most thalassemias are inherited as recessive traits. Beta-thalassemias can be classified into
– Beta-thalassemia
- Thalassemia major
- Thalassemia intermedia
- Thalassemia minor
Beta-thalassemia with associated Hb anomalies
- HbC/Beta-thalassemia
- HbE/Beta-thalassemia
- HbS/Beta-thalassemia (a clinical condition more similar to sickle cell disease than to thalassemia major or intermedia)
- Hereditary persistence of fetal Hb and beta-thalassemia & Autosomal dominant forms
Beta-thalassemia associated with other manifestations
- Beta-thalassemia-trichothiodystrophy
- X-linked thrombocytopenia with thalassemia
Delta-thalassemia
As well as alpha and beta chains present in hemoglobin, about 3% of adult hemoglobin is made of alpha and delta chains. Just as with beta-thalassemia, mutations that affect the ability of this gene to produce delta chains can occur.
Combination Hemoglobinopathies
Thalassemia can coexist with other hemoglobinopathies. The most common of these are:
- Hemoglobin E/thalassemia – common in Cambodia, Thailand, and parts of India, it is clinically similar to β thalassemia major or thalassemia intermedia.
- Hemoglobin S/thalassemia – common in African and Mediterranean populations, is clinically similar to sickle-cell anemia, with the additional feature of splenomegaly.[citation needed]
- Hemoglobin C/thalassemia – common in the Mediterranean and African populations, hemoglobin C/βo thalassemia causes a moderately severe hemolytic anemia with splenomegaly; hemoglobin C/β+ thalassemia produces a milder disease.
- Hemoglobin D/thalassemia – common in the northwestern parts of India and Pakistan (Punjab region).
Causes of Thalassemia
Alpha thalassemia
Alpha thalassemia is caused by a deletion or mutation in one or more of the four alpha-globin gene copies. The mutation causes a decrease in the production of alpha-globin. The more genes that are affected, the less alpha globin is produced by the body. The four different types of alpha thalassemia are classified according to the number of genes affected and include:
- Silent Carrier State (1 gene affected) – People who have a mutation(s) in only one alpha-globin gene are silent carriers. They usually have normal hemoglobin levels and red cell indices but can pass on the affected gene to their children. These individuals have no signs or symptoms and are usually identified only after having a child with thalassemia. The only way to identify a silent carrier is by DNA analysis.
- Alpha Thalassemia Trait (2 genes affected) – People who have alpha thalassemia trait have red blood cells (RBCs) that are smaller (microcytic) and paler (hypochromic) than normal, have a decreased MCV (mean corpuscular volume, a measurement of the average size of a single RBC), and have a mild chronic anemia. They generally do not have other signs and sometimes may lack symptoms. This form of anemia does not respond to iron supplements. Diagnosis of alpha thalassemia trait is usually done by exclusion of other causes of microcytic anemia. Confirmatory testing by DNA analysis is available but is not routinely done.
- Hemoglobin H Disease (3 genes affected) – With this condition, the large decrease in alpha-globin chain production causes an excess of beta chains, which then come together into groups of 4 beta chains, known as Hemoglobin H, which is visible inside red blood cells on a specially stained blood smear. Hb H disease can cause moderate to severe anemia and serious health problems such as an enlarged spleen, bone deformities, and fatigue. The signs and symptoms associated with Hb H disease vary widely. Some individuals are asymptomatic while others have severe anemia, requiring regular medical care. Hemoglobin H disease is found most often in individuals of Southeast Asian or Mediterranean descent.
- Alpha Thalassemia Major (also called hydrops fetalis, 4 genes affected) – This is the most severe form of alpha thalassemia. In this condition, no alpha globin is produced, therefore, no normal hemoglobin is produced. Fetuses affected by alpha thalassemia major become anemic early during the pregnancy. They retain excess fluids (hydropic) and frequently have enlarged hearts and livers. This diagnosis is frequently made in the last months of pregnancy when a fetal ultrasound indicates a hydropic fetus. There are also risks for the pregnant mother. About 80% of the time, the mother will have “toxemia” (protein in the urine, high blood pressure, swollen ankles and feet) and can develop severe postpartum bleeding (hemorrhage). Fetuses with alpha thalassemia major are usually miscarried, stillborn, or die shortly after birth. In very rare cases, children with alpha thalassemia have survived through in utero blood transfusions and extensive medical care.
Beta thalassemia is caused by mutations in one or both of the beta-globin genes. There have been more than 250 mutations identified, but only about 20 are the most common. The severity of the anemia caused by beta thalassemia depends on which mutations are present and whether there is decreased beta-globin production (called beta+ thalassemia) or if production is completely absent (called beta0 thalassemia). The different types of beta-thalassemia include:
- Beta Thalassemia Trait or Beta Thalassemia Minor – Individuals with this condition have one normal gene and one with a mutation, causing a mild decrease in beta-globin production. They usually have no health problems other than abnormally small red blood cells and possibly mild anemia that will not respond to iron supplements. An individual’s children can inherit this gene.
- Thalassemia Intermedia – In this condition, an affected person has two abnormal genes, causing moderate to a severe decrease in beta-globin production. These individuals may develop symptoms later than those with thalassemia major (see below) and often with milder symptoms. They rarely require treatment with blood transfusion. The severity of the anemia and health problems experienced depends on the mutation types present. The dividing line between thalassemia intermedia and thalassemia major is the degree of anemia and the number and frequency of blood transfusions required. Those with thalassemia intermedia may need occasional transfusions but do not require them on a regular basis.
- Thalassemia Major or Cooley’s Anemia – This is the most severe form of beta-thalassemia. These individuals have two abnormal genes that cause either a severe decrease or complete lack of beta-globin production, preventing the production of significant amounts of normal hemoglobin (Hb A). This condition usually appears within the first two years of life and causes life-threatening anemia, poor growth, and skeletal abnormalities during infancy. This anemia requires lifelong regular blood transfusions and considerable ongoing medical care. Over time, these frequent transfusions lead to excessive amounts of iron in the body. Left untreated, this excess iron can deposit in the liver, heart, and other organs and can lead to premature death from organ failure. Therefore, individuals undergoing transfusion may need chelation therapy to reduce iron overload.
Other forms of thalassemia occur when a gene for beta-thalassemia is inherited in combination with a gene for a hemoglobin variant. The most important of these are:
- Hb E-beta thalassemia – Hb E is one of the most common hemoglobin variants. It is found predominantly in people of Southeast Asian and African descent. If a person inherits one Hb E gene and one beta-thalassemia gene, the combination produces Hb E-beta thalassemia, which causes moderately severe anemia similar to beta-thalassemia intermedia.
- Hb S-beta thalassemia or sickle cell-beta thalassemia – Hb S is one of the most well known of the hemoglobin variants. Inheritance of one Hb S gene and one beta-thalassemia gene results in Hb S-beta thalassemia. The severity of the condition depends on the amount of beta-globin produced by the beta gene. If no beta globin is produced, the clinical picture is similar to sickle cell disease but with even worse baseline anemia. The American College of Medical Genetics advises screening all newborns for hemoglobin S/beta-thalassemia as well as sickle cell anemia. It is also required in all 50 states.
Others Causes
Thalassemia has an autosomal recessive pattern of inheritance. Both α- and β-thalassemias are often inherited in an autosomal recessive manner. Cases of dominantly inherited α- and β-thalassemias have been reported, the first of which was in an Irish family with two deletions of 4 and 11 bp in exon 3 interrupted by an insertion of 5 bp in the β-globin gene. For the autosomal recessive forms of the disease, both parents must be carriers for a child to be affected. If both parents carry a hemoglobinopathy trait, the risk is 25% for each pregnancy for an affected child.
Symptoms of Thalassemia
The right hand of a person with severe anemia compared to the left hand of one without
- Iron overload – People with thalassemia can get an overload of iron in their bodies, either from the disease itself or from frequent blood transfusions. Too much iron can result in damage to the heart, liver, and endocrine system, which includes glands that produce hormones that regulate processes throughout the body. The damage is characterized by excessive deposits of iron. Without adequate iron chelation therapy, almost all patients with beta-thalassemia accumulate potentially fatal iron levels.[rx]
- Infection – People with thalassemia have an increased risk of infection. This is especially true if the spleen has been removed.[rx]
- Bone deformities – Thalassemia can make the bone marrow expand, which causes bones to widen. This can result in abnormal bone structure, especially in the face and skull. Bone marrow expansion also makes bones thin and brittle, increasing the risk of broken bones.[rx]
- Enlarged spleen – The spleen aids in fighting infection and filters unwanted material, such as old or damaged blood cells. Thalassemia is often accompanied by the destruction of a large number of red blood cells and the task of removing these cells causes the spleen to enlarge. Splenomegaly can make anemia worse, and it can reduce the life of transfused red blood cells. Severe enlargement of the spleen may necessitate its removal.[rx]
- Slowed growth rates – anemia can cause a child’s growth to slow. Puberty also may be delayed in children with thalassemia.[rx]
- Heart problems – Diseases, such as congestive heart failure and abnormal heart rhythms, may be associated with severe thalassemia.[rx]
- Fatigue
- Weakness
- Pale or yellowish skin
- Facial bone deformities
- Slow growth
- Abdominal swelling
- Dark urine
- Dizziness
- Shortness of breath
- A fast heartbeat
- Headache
- Difficulty concentrating
- Pale skin
- Chest pain
- Cold hands and feet
- Shortness of breath
- Leg cramps
- Poor feeding
- Delayed growth
- Greater susceptibility to infections
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Facial features and oral manifestations of thalassemia
| Features | Cause | |
|---|---|---|
| Facial features | Class II malocclusion | The maxillary protrusion, mandibular atrophy |
| Maxillary protrusion [rx, rx] | Early fusion of occipital sutures, hyperplasia of anterior maxillofacial structures | |
| Lateral displacement of orbits [rx] | Marrow overgrowth in maxillary bone | |
| Chipmunk facies [rx, rx] | Malar prominence, saddle nose, frontal bossing | |
| Brodie syndrome [rx] | Mandibular arch is telescoped within the maxillary arch | |
| Pneumatisation of paranasal sinuses | Hyperplasia of marrow in frontal, temporal and facial bones [rx, rx] | |
| Oral manifestations | Malocclusion | The maxillary protrusion, increased overjet, anterior open bite |
| High caries index [rx] | Poor oral hygiene, less phosphorus, and IgA in saliva | |
| Mucosal pallor, atrophic glossitis | Decreased hemoglobin levels | |
| Severe gingivitis | If splenectomy did [rx] | |
| Inflammation of salivary glands | Iron deposits | |
| Dark-colored gingiva | High ferritin levels in the blood | |
| Thin mandibular cortex [rx] | Marrow proliferation, expansion of medulla | |
| Multiple diastemas | ||
| Roots-short and spike-shaped, taurodontism [rx] | ||
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Diagnosis of Thalassemia
- Genetic counseling and prenatal diagnosis – Prevention of beta-thalassemia is based on carrier identification, genetic counseling and prenatal diagnosis [rx]. Carrier detection has been previously described. Genetic counseling provides information for individuals and at-risk couples (i.e. both carriers) regarding the mode of inheritance, the genetic risk of having affected children and the natural history of the disease including the available treatment and therapies under investigation.
- Clinical Diagnosis – Thalassemia major is usually suspected in an infant younger than two years of age with severe microcytic anemia, mild jaundice, and hepatosplenomegaly. Thalassemia intermedia presents at a later age with similar but milder clinical findings. Carriers are usually asymptomatic but sometimes may have mild anemia.
- Hematologic Diagnosis – RBC indices show microcytic anemia. Thalassemia major is characterized by reduced Hb level (<7 g/dl), mean corpuscular volume (MCV) > 50 < 70 fl and mean corpuscular Hb (MCH) > 12< 20 pg. Thalassemia intermedia is characterized by Hb level between 7 and 10 g/dl, MCV between 50 and 80 fl and MCH between 16 and 24 pg. Thalassemia minor is characterized by reduced MCV and MCH, with increased Hb A2 level [rx].
- Stimulating HbF production – Individuals with HPFH demonstrate that preventing or reversing the switch from fetal to adult hemoglobin would provide efficacious therapy for thalassemia and various other hemoglobinopathies. It has been observed that some patients recovering from cytotoxic therapy have reactivated HbF synthesis. Several therapeutic agents such as erythropoietin, hydroxyurea, cytarabine, and butyrate analogs have produced an increase in HbF synthesis in the thalassemic patients by stimulating the HbF-producing progenitor cell population [rx, rx].
- Bone marrow transplantation – To date, over 1000 bone marrow transplants have been performed in thalassemic patients at medical centers of excellence [rx]. Disease-free survival of 80% to 90% was achieved in patients who had adequate iron chelation and did not suffer from liver failure or hepatomegaly. In patients without these good prognostic features, disease-free survival rates dropped to 50%.
- Somatic gene therapy – Human globin genes have been transferred into mouse cells [rx]. Successful application of gene transfer for the treatment of thalassemia is experimental and will require that the newly introduced genes do not alter the growth properties of the bone marrow cells by the recombinant retroviral genome [rx]. Design of vectors that ensure adequate production of globin messenger RNA to correct the deficiency in thalassemic red cells requires more experimentation.
Peripheral blood smear
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Affected individuals demonstrate the red blood cell (RBC) morphologic changes of microcytosis, hypochromia, anisocytosis, poikilocytosis (spiculated tear-drop and elongated cells), and nucleated red blood cells (i.e., erythroblasts). The number of erythroblasts is related to the degree of anemia and is markedly increased following splenectomy.
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Carriers demonstrate reduced mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH) [rx], and RBC morphologic changes that are less severe than in affected individuals. Erythroblasts are normally not seen.
Qualitative and quantitative hemoglobin analysis
By cellulose acetate electrophoresis and DE-52 micro chromatography or HPLC identifies the amount and type of hemoglobin present. The following hemoglobin (Hb) types are most relevant to β-thalassemia:
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Hemoglobin A (HbA) – two globin alpha chains and two globin beta chains (α2β2)
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Hemoglobin F (HbF) – two globin alpha chains and two globin gamma chains (α2γ2)
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Hemoglobin A2 (HbA2) – two globin alpha chains and two globin delta chains (α2δ2)
- The Hb pattern in beta-thalassemia varies according to beta-thalassemia type. In beta0 thalassemia, homozygotes HbA is absent and HbF constitutes the 92-95% of the total Hb. In beta+ thalassemia homozygotes and beta+/beta0 genetic compounds, HbA levels are between 10 and 30% and HbF between 70-90%. HbA2 is variable in beta-thalassemia homozygotes and it is enhanced in beta-thalassemia minor.
- Hb electrophoresis and HPLC – also detect other hemoglobinopathies (S, C, E, Arab, Lepore) that may interact with beta-thalassemia.
Peripheral blood smear
- Affected individuals show RBC morphologic changes [microcytosis, hypochromia, anisocytosis, poikilocytosis (spiculated tear-drop and elongated cells)], and nucleated RBC (i.e., erythroblasts). The number of erythroblasts is related to the degree of anemia and is markedly increased after splenectomy.
- Carriers have less severe RBC morphologic changes than affected individuals. Erythroblasts are normally not seen.
Molecular Genetic Analysis
- The prevalence of a limited number of mutations in each population has greatly facilitated molecular genetic testing.
- Commonly occurring mutations of the beta-globin gene are detected by PCR-based procedures [rx]. The most commonly used methods are reverse dot blot analysis or primer-specific amplification, with a set of probes or primers complementary to the most common mutations in the population from which the affected individual originated.
- If targeted mutation analysis fails to detect the mutation, beta-globin gene sequence analysis can be used to detect mutations in the beta-globin gene.
Pitfalls in carrier identification by hematologic testing are
- Coinheritance of alpha-thalassemia – which may normalize the RBC indices. However, in alpha/beta double heterozygotes, the HbA2 concentration remains in the beta-thalassemia carrier range and thus is diagnostic. Therefore, HbA2 determination should always be performed for beta-thalassemia carrier identification.
- Coinheritance of delta-thalassemia – which may reduce to normal the increased Hb A2 levels typical of the beta-thalassemia carrier state. Double heterozygosity for delta- and beta-thalassemia can be distinguished from the most common alpha-thalassemia carrier state by globin chain synthesis or globin gene analysis.
- Silent mutations – i.e., very mild mutations associated with the consistent residual output of Hb beta chains and with normal RBC indices and normal or borderline HbA2. The above-reported groups of carriers are referred to as atypical carriers.
When the hematologic analysis is abnormal, molecular genetic testing of the beta-globin gene is performed to identify the disease-causing mutation [rx].
Differential Diagnosis
Few conditions share similarities with homozygous beta-thalassemia:
- The genetically-determined sideroblastic anemias are easily differentiated because of ring sideroblasts in the bone marrow and variably elevated serum concentration of erythrocyte protoporphyrin. Most sideroblastic anemias are associated with defects in the heme biosynthetic pathway, especially delta-aminolevulinic acid synthase.
- Congenital dyserythropoietic anemias do not have high HbF and do have other distinctive features, such as multinuclearity of the red blood cell precursors.
- A few acquired conditions associated with high HbF (juvenile chronic myelomonocytic leukemia with normal karyotype, aplastic anemia both congenital and acquired during the recovery phase) may be mistaken for beta-thalassemia, even though they have very characteristic clinical and hematological features.
Treatment of Thalassemia
Thalassemia major
Regular transfusions correct the anemia, suppress erythropoiesis, and inhibit increased gastrointestinal absorption of iron.
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Before starting the transfusions, the following are absolutely necessary:
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Hepatitis B vaccination
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Extensive red blood cell antigen typing, including Rh, Kell, Kidd, and Duffy and serum immunoglobulin determination – the latter of which detects individuals with IgA deficiency, who need special (repeatedly washed) blood unit preparation before each transfusion
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The transfusion regimen is designed to obtain a pre-transfusion Hb concentration of 95-100 g/L.
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Transfusions are usually given every two to three weeks.
Thalassemia intermedia
Treatment of individuals with thalassemia intermedia is symptomatic and based on splenectomy and folic acid supplementation.
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Treatment of extramedullary erythropoietic masses is based on radiotherapy, transfusions, or, in selected cases, hydroxyurea (with a protocol similar to that used for sickle cell disease).Hydroxyurea also increases globin gamma chains and may have other undefined effects.
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Individuals with thalassemia intermedia may develop iron overload from increased gastrointestinal absorption of iron or from occasional transfusions; chelation therapy with deferasirox has been demonstrated to be safe and effective in persons age ten years or older with a liver iron concentration ≥5 mg Fe/g dry weight or serum ferritin ≥800 ng/mL (thresholds after which the risk of serious iron-related morbidity is increased) [rx].
Bone marrow transplantation
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Bone marrow transplantation (BMT) from an HLA-identical sib represents an alternative to traditional transfusion and chelation therapy. If BMT is successful, iron overload may be reduced by repeated phlebotomy, thus eliminating the need for iron chelation.
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The outcome of BMT is related to the pretransplantation clinical conditions, specifically the presence of hepatomegaly, the extent of liver fibrosis, and the magnitude of iron accumulation. In children who lack the above risk factors, disease-free survival is higher than 90%. Adults with beta-thalassemia are at increased risk for transplant-related toxicity due to an advanced phase of the disease and have a two-year overall survival of 80% and a two-year event-free survival of 76% with current treatment protocol [rx].
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BMT from unrelated donors has been carried out on a limited number of individuals with β-thalassemia. Provided that selection of the donor is based on stringent criteria of HLA compatibility and that individuals have limited iron overload, results are comparable to those obtained when the donor is a compatible sib [rx].
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Severe acute graft-vs-host disease (GVHD) may occur in 9% of individuals, with a lower risk observed in those with an HLA-matched sib donor.
Cord blood transplantation
- Cord blood transplantation from a related donor offers a good probability of a successful cure and is associated with a low risk for GVHD [rx]. For couples who have already had a child with thalassemia and who undertake prenatal diagnosis in a subsequent pregnancy, prenatal identification of HLA compatibility between the affected child and an unaffected fetus allows the collection of placental blood at delivery and the option of cord blood transplantation to cure the affected child [rx].
Transfusional Iron Overload
- The most common secondary complications are those related to transfusional iron overload, which can be prevented by adequate iron chelation.
Assessment of iron overload
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Serum ferritin concentration – In clinical practice, the effectiveness of chelators is monitored by routine determination of serum ferritin concentration. However, serum ferritin concentration is not always reliable for evaluating iron burden because it is influenced by other factors, the most important being the extent of liver damage.
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Liver biopsy – Determination of liver iron concentration in a liver biopsy specimen shows a high correlation with total body iron accumulation and is the gold standard for evaluation of liver iron overload. However, (1) liver biopsy is an invasive technique involving the possibility (though low) of complications; (2) liver iron content can be affected by hepatic fibrosis, which commonly occurs in individuals with iron overload and hepatitis C virus infection; and (3) irregular iron distribution in the liver can lead to false-negative results [rx].
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Magnetic biosusceptometry (SQUID) – which gives a reliable measurement of hepatic iron concentration, is another option [rx]; however, magnetic susceptometry is presently available only in a limited number of centers worldwide.
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MRI techniques – for assessing iron loading in the liver and heart are commonly used [rx, rx]. T2 and T2parameters have been validated for liver iron concentration. Cardiac T2 is reproducible, is applicable between different scanners, correlates with cardiac function, and relates to tissue iron concentration [rx, rx]. Clinical utility of T2 in monitoring individuals with siderotic cardiomyopathy has been demonstrated. In one study, 12 human hearts from transfusion-dependent affected individuals after either death or transplantation for end-stage heart failure underwent cardiovascular magnetic resonance R2 (the reciprocal of T2) measurement. Tissue iron concentration was measured in multiple samples of each heart with inductively coupled plasma atomic emission spectroscopy, providing calibration in humans for cardiovascular magnetic resonance R2 against myocardial iron concentration and detailing the iron distribution throughout the heart in iron overload [rx].
Chelation therapy
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Desferrioxamine B (DFO) – The first chelator introduced clinically was desferrioxamine B (DFO) administered five to seven days a week by 12-hour continuous subcutaneous infusion via a portable pump. The recommended dosage depends on the individual’s age and serum ferritin concentration. Young children start with 20-30 mg/kg/day, increasing up to 40 mg/kg/day after age five to six years. The maximum dose is 50 mg/kg/day after growth is completed. The dose may be reduced if serum ferritin concentration is low. By maintaining the total body iron stores below critical values (i.e., hepatic iron concentration <7.0 mg per gram of dry weight liver tissue), desferrioxamine B therapy prevents the secondary effects of iron overload, resulting in a consistent decrease in morbidity and mortality [rx].
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Ascorbate repletion (daily dose ≤100-150 mg) increases the amount of iron removed after DFO administration.
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Side effects of DFO chelation therapy are more common in the presence of relatively low iron burden and include ocular and auditory toxicity, growth restriction, and, rarely, renal impairment and interstitial pneumonitis. DFO administration also increases susceptibility to Yersinia infections. The major drawback of DFO chelation therapy is low compliance resulting from complications of administration.
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Deferiprone – a bidentate oral chelator, is administered in a dose of 75-100 mg/kg/day. The main side effects of deferiprone therapy include arthropathy, gastrointestinal symptoms, and, above all, neutropenia and agranulocytosis [rx] that demand close monitoring. The effect of deferiprone on liver iron concentration may vary among the individuals treated. However, results from independent studies suggest that deferiprone is more cardioprotective than desferrioxamine: compared to those being treated with DFO, individuals being treated with deferiprone have better myocardial MRI pattern and less probability of developing (or worsening pre-existing) cardiac disease [rx, rx]. These retrospective observations have been confirmed in a prospective study [rx].
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Deferasirox – was developed as a once-daily oral monotherapy for the treatment of transfusional iron overload. It is effective in adults and children and has a defined safety profile that is clinically manageable with appropriate monitoring. The most common treatment-related adverse events are gastrointestinal disorders, skin rash, and a mild, non-progressive increase in serum creatinine concentration [rx]. Cases of renal failure, hepatic failure, cytopenias, and gastrointestinal hemorrhage have been reported in the post-marketing phase. Provided adequate doses are given, there is a good response to deferasirox across the full range of baseline liver iron concentration values. Prospective data demonstrate the efficacy of deferasirox in improving myocardial T2* and in maintaining a normal left ventricle ejection fraction [rx, rx]. Deferasirox has not been evaluated in formal trials for affected individuals with symptomatic heart failure or low left-ventricle ejection fraction.
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Combination therapies – Strategies of chelation using a combination of desferrioxamine and deferiprone have been effective in individuals with severe iron overload. Retrospective, prospective, and randomized clinical studies have shown that combined iron chelation with desferrioxamine and deferiprone rapidly reduces myocardial siderosis, improves cardiac and endocrine function, reduces liver iron and serum ferritin concentration, reduces cardiac mortality, and improves survival; toxicity is manageable
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Removal of excess iron – Repeated bleeding (phlebotomy) is used to remove excess iron in patients with normal Hb levels, such as in patients with hereditary hemochromatosis, where IO is caused by mutations in the iron homeostasis system [rx]. Patients after hematopoietic stem cell (HSC) transplantation (HSCT) who had IO prior to transplantation due to multiple transfusions may also benefit from this treatment [rx]. Three iron chelators are currently in clinical use. Deferoxamine, the first to be used clinically, is given by a slow, continuous, subcutaneous, overnight infusion through a portable pump. While its side effects are minimal, its mode of administration results in low compliance [rx]. Deferasirox, the first effective oral chelator, is given at 20–30 mg/kg once/day.
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Iron overload – Normally, 1–2 mg of iron is absorbed from the diet per day, with an equivalent amount lost by the turnover of gastrointestinal tract epithelial cells. The body has no mechanism for disposing of excess iron [rx]; therefore, in thal and other transfusion-dependent anemias, IO may accumulate in a relatively short time (transfusional IO). An RBC transfusion requirement of two units (200–250 mg of iron per unit) per month will result in over 20 g of excess body iron in 4 years [rx].
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Dyserythropoiesis – The chronic anemia and its associated hypoxia in thal stimulate increased production of RBCs (chronic stress erythropoiesis). This is mediated by overproduction of erythropoietin (EPO), the main erythropoietic stimulating hormone, and other factors, such as members of the transforming growth factor (TGF)-β and activin receptor-II (ActR-II) trap ligands [rx]. Binding of EPO to its surface receptor on erythroid precursors activates transduction pathways, including Jak2/Stat5, which inhibit apoptosis and induce proliferation and differentiation of the developing cells.
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Activin receptor-II trap ligands – Luspatercept and Sotatercept, compounds that bind to trap ligands and GDF-11, developed in animal models, are currently in clinical trials [rx]. They prevent activins binding to ActR-II and the activation of the Smad 4-dependent signaling pathway, improving erythroid maturation and RBC production. A phase 3, multicenter, multinational study with luspatercept is ongoing in β-thal and HbE/β-thal subjects, with encouraging preliminary results [rx].
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JAK2 inhibitors – The EPO signaling of erythropoiesis involves Jak2 phosphorylation. Beta-thal mice have elevated EPO levels associated with increased Jak2 phosphorylation, resulting in ineffective erythropoiesis and extramedullary hematopoiesis [rx]. Jak2 inhibitors effectively reduce splenomegaly in such mice. Several Jak2 inhibitors have been developed with beneficial results in patients with myelofibrosis and Jak2-related polycythemia vera [rx]. Jak2 inhibitors could be also beneficial for non-transfusion-dependent thal patients with splenomegaly [rx].
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Induction of the Hsp70 chaperone machinery – The heat shock protein 70 (Hsp70) is a molecular chaperone needed for normal termination of erythropoiesis [rx]. It is predominant in the late erythroid precursors when it is translocated to the nucleus and protects the globin transcription factor-1 (GATA-1), the principal transcriptional factor for erythropoiesis, from caspase-3 cleavage [rx]. In β-thal major, HSP70 is sequestrated in the cytoplasm, leaving GATA-1 unprotected from cleavage, resulting in end-stage maturation arrest and apoptosis [rx]. Exportins, such as XPO1, are factors that control the nucleocytoplasmic trafficking of proteins and RNAs.
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Stimulating HbF production – Individuals with HPFH demonstrate that preventing or reversing the switch from fetal to adult hemoglobin would provide efficacious therapy for thalassemia and various other hemoglobinopathies. It has been observed that some patients recovering from cytotoxic therapy have reactivated HbF synthesis. Several therapeutic agents such as erythropoietin, hydroxyurea, cytarabine, and butyrate analogs have produced an increase in HbF synthesis in the thalassemic patients by stimulating the HbF-producing progenitor cell population [rx, rx].
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Bone marrow transplantation – To date, over 1000 bone marrow transplants have been performed in thalassemic patients at medical centers of excellence [rx]. Disease-free survival of 80% to 90% was achieved in patients who had adequate iron chelation and did not suffer from liver failure or hepatomegaly. In patients without these good prognostic features, disease-free survival rates dropped to 50%.
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Somatic gene therapy – Human globin genes have been transferred into mouse cells [rx]. Successful application of gene transfer for the treatment of thalassemia is experimental and will require that the newly introduced genes do not alter the growth properties of the bone marrow cells by the recombinant retroviral genome [rx]. Design of vectors that ensure adequate production of globin messenger RNA to correct the deficiency in thalassemic red cells requires more experimentation.
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Allogeneic hematopoietic stem cell transplantation – Allogeneic HSCT (allo-HSCT) is currently the only definitive cure for transfusion-dependent young patients before the development of IO-related tissue damage [rx]. β-thal major patients with good risk features have a >90% chance of a successful outcome, but allo-HSCT in high-risk patients is challenging because of graft rejection and transplant-related mortality. Novel modified or reduced-intensity conditioning regimens are being evaluated in an attempt to improve the outcome in such patients with favorable results.
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Gene therapy – Gene therapy involves in vivo genetic manipulation of the autologous HSCs, which are then transplanted to the patient for reconstitution. This approach has focused on two areas. (A) Increasing the production of γ-globin by the addition of its gene, overexpression of its endogenous activating transcription factors, and silencing of its repressors, as discussed above. (B) Increasing the production of β-globin by the addition of a normal gene or correction of the mutated gene.
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Gene modification approach – The patient’s HSCs are subjected to gene editing ex vivo and then returned to the patient for reconstitution. Increased production of γ-globin has been accomplished using lentiviral vectors that express a zinc finger protein which interacts with the promoter of the γ-globin gene or by carrying microRNAs that silence its repressors. Two potent transcriptional repressors of γ-globin, BCL11A, and ZBTB7A, have been identified. They act with additional trans-acting epigenetic repressive complexes, lineage-defining factors, and developmental programs to silence the γ-globin genes by working on cis-acting sequences at the globin gene loci. Inhibition of these repressors could reactivate γ-globin production in adult patients.
- Stimulation of HbF production – During prenatal life in humans, the major Hb is fetal Hb (HbF), a tetramer of α- and γ-globin (α 2γ 2) which is replaced during the first year of life by HbA (α 2β 2) (Hb switching). Thus, the clinical features of β-hemoglobinopathies, including β-thal, are not apparent at birth; only as HbF levels wane are the symptoms manifested. Patients with β-thal produce high but variable levels of HbF compared to normal individuals. High levels of HbF ameliorate the severity of the disease, mainly by reducing the surplus of α-globin chains.
- HbF induction – HbF induction is an attempt to reactivate fetal globin gene transcription.[rx] Efforts involve trying to disrupt the fetal globin gene promoter.[rx]
Complications
Complications of iron overload include the following
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In children, growth restriction and failure of sexual maturation
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In adults, the involvement of the heart (dilated cardiomyopathy), liver (fibrosis and cirrhosis), and endocrine glands (resulting in diabetes mellitus and insufficiency of the parathyroid, thyroid, pituitary, and, less commonly, adrenal glands)
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Hypersplenism
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Chronic hepatitis (resulting from infection with the viruses that cause hepatitis B and/or hepatitis C)
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Cirrhosis (from iron overload and chronic hepatitis)
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HIV infection
References
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