Thalassemias

Diagnosis

Indications for Testing

• Anemia or isolated microcytosis
• Fetus or infant with hydrops fetalis

Laboratory Testing

• CBC with differential and serum iron studies to determine if anemia represents thalassemia or is caused by iron deficiency
  • Expected red blood cell indices are listed in Table 1 for both types of thalassemias (from GeneTests.org)
• Globin chain synthesis assay
• Hemoglobin evaluation by HPLC (high performance liquid chromatography) or electrophoresis – hemoglobin patterns listed in Table 2 for both types of thalassemias (from GeneTests.org) 
• Molecular testing to confirm α or β thalassemia
  • β thalassemia – HBB gene sequencing
  • α thalassemia – HBA1 and HBA2 molecular analysis
    • Initial testing – deletion testing for HBA1 and HBA2 by polymerase chain reaction to identify 7 common α globin gene deletions
    • Second-line testing – gene sequencing for HBA1 and HBA2 when deletion testing has detected the inactivation of 2 or less globin genes

Differential Diagnosis

• β thalassemia
  • Sideroblastic anemia
  • Congenital dyserythropoietic anemia (CDA)
  • Juvenile chronic myelomonocytic leukemia
• α thalassemia
  • Severe (hydrops)
    • Parvovirus
    • Rh incompatibility
    • Turner syndrome
  • Mild (anemia)
    • Iron deficiency anemia

Screening

Clinical Background

Thalassemias are a group of common, inherited hemoglobin disorders that result in the unbalanced synthesis of β and α globin chains. Most forms are not associated with significant hemolysis, although some are (notably hemoglobin H disease).

Epidemiology

• Prevalence – estimated 5-7% of the population worldwide carries clinically significant hemoglobin mutations
  • β thalassemia is most commonly found in the populations of Southern Europe, Southeast Asia, Africa, and India
  • α thalassemia is widespread in Africa, Mediterranean populations, the Middle East, and Southeast Asia
    • Carrier frequencies of α thalassemia in commonly affected populations
      • African, African American – ~1:3
      • Middle Eastern, Southeast Asian – 1:20
      • Mediterranean – 1:30-50

Inheritance

• Usually autosomal recessive; infrequently dominantly inherited

Pathophysiology

• Hemoglobin, a tetramer of two α and two β or β-like (δ and γ) globin chains found in red blood cells, is stable only as a tetramer; free globin chains, a hallmark of thalassemias, have variable toxicity
• Adult hemoglobin consists primarily of hemoglobin A (α₂ β₂) plus small amounts of hemoglobin A₂ (α₂ δ₂ <2-3%) and hemoglobin F (α₂ γ₂ <1%)
• Symptoms of thalassemia result from inadequate hemoglobin production and accumulation of free globin subunits that are toxic to erythroid precursors (free α chains are relatively more toxic than free β chains)
  • Genetic mutations in the globin genes (α or β) result in decreased or absent production of that globin chain and relative excess of the other
• Disease named according to the defective or absent globin unit
  • Two main types – β thalassemia and α thalassemia
  • Rare forms of thalassemia (δβ-, γ, δ, ε) may produce hematological or clinical symptoms in the heterozygous form
  • Most forms of thalassemias have no clinical significance
• The α globin subunit is synthesized by the α₁ (HBA1) and α₂ (HBA2) genes on chromosome 16
  • Normal individuals have four functioning α globin genes (αα/αα)
  • 95% of α thalassemia is caused by HBA1 and HBA2 gene deletions; nondeletion or regulatory region mutations are rare
  • For prenatal counseling, it is important to know whether the nonfunctioning α globin genes lie on the same chromosome (cis) or on opposite chromosomes (trans)
    • When both parents carry a cis deletion of HBA1 and HBA2 (––/αα), the risk for hydrops fetalis associated with Hb Bart in their offspring may be 1:4 (homozygosity for certain cis deletions results in embryonic lethality)
• The β globin subunits are synthesized by the ε, γ (2 copies), δ and β genes on chromosome 11
  • β thalassemia is often caused by single nucleotide substitutions; far less commonly, β thalassemia is caused by small insertions or deletions or other sequence variations of the β globin gene HBB
• Over 200 known mutations are categorized into 2 classes
  • β zero (β⁰)
    • No β globin chain synthesis from the affected allele
  • β plus (β⁺)
    • Decreased β globin chain synthesis from the affected allele
• Deletions involving the β globin gene are rare
  • Indian – partial deletion of β globin gene (β⁰ mutation)
  • δβ thalassemia – deletion of the entire β gene and a majority of the δ gene; deletion is only partially compensated by increased γ globin production (β⁺ mutation)
  • Hereditary persistence of fetal hemoglobin (pancellular) – large deletions within the globin gene cluster which alter normal hemoglobin switching and result in increased γ globin production (raised HbF)
  • Hb Lepore – large deletion resulting in the fusion of the δ and β globin genes (β⁺ mutation)

Thalassemia Types

• α thalassemia

  Epidemiology Incidence – the most common inherited disorder of hemoglobin worldwide Ethnicity α globin mutations commonly observed in the following populations – Mediterranean (1:30–50), Southeast Asian (1:20), African, Middle Eastern, and African American (~1:3) Hb Bart hydrops fetalis and HbH syndromes occur more often in Southeast Asian, Asian-Indian, and Mediterranean populations than African populations due to the rarity of cis deletions (−−/αα) in Africa Genetics Autosomal recessive inheritance Normally, individuals have four functioning α globin genes (αα/αα); two genes, α-1 (HBA1) and α-2 (HBA2), are present on each copy of chromosome 16 Clinical Presentation Clinically significant forms of α thalassemia Hb Bart (γ4) hydrops fetalis syndrome Loss of function of all four α globin genes (−−/−−) Clinical findings include fetal generalized edema, ascites, pleural and pericardial effusions, and severe hypochromic anemia, as well as fetal or perinatal death Maternal complications during pregnancy are common and include preeclampsia, polyhydramnios or oligohydramnios, antepartum hemorrhage, premature delivery Hemoglobin H (HbH) disease Generally occurs due to loss of function of three α globin genes (−−/−α) Commonly seen  in some populations (Malaysia, Thailand, Vietnam) due to a combination of α thalassemia and hemoglobin Constant Spring (HbCS) Clinical findings include moderate microcytic hypochromic anemia, hemolysis with Heinz bodies, splenomegaly, rare extramedullary hematopoiesis, and propensity of acute hemolysis after oxidative stress, drug therapy, or infection Presence of HbH (β4), or in a neonate presence of Hb Bart (γ4) Carrier states for α thalassemia α thalassemia trait Loss of function of two α globin genes (–α/−α) or (−−/αα) Mild microcytic anemia may be present; normal hemoglobin electrophoresis Often misdiagnosed as iron deficiency Prenatal counseling – critical to define whether non-functioning α globin genes lay on the same chromosome (cis) or opposite chromosomes (trans) When both parents carry a cis deletion of HBA1 and HBA2 (−−/αα), the risk for hydrops fetalis associated with Hb Bart in their offspring may be 1:4 (homozygosity for certain cis deletions results in embryo death) Typically asymptomatic; mild anemia and mild microcytosis Normal hemoglobin electrophoresis, often misdiagnosed as iron deficiency α thalassemia silent carrier Loss of function of a single α globin gene (−α/αα) Typically asymptomatic; borderline anemia or mild microcytosis may be present, but may have normal red cell indexes Normal hemoglobin electrophoresis, often misdiagnosed as iron deficiency

  β thalassemia (Cooley Anemia)

  Epidemiology Ethnicity – β thalassemias are most commonly observed in individuals from Southern Europe, Northern Africa, and India; it is extremely rare among African Americans Genetics Autosomal recessive >500 β globin gene (HBB) mutations have been described Clinical Presentation Asymptomatic at birth – neonates have not yet switched from fetal to adult hemoglobin (γ genes to β genes) Certain HBB deletions impair the developmental switch from fetal to adult Hb, resulting in hereditary persistence of fetal Hb, which incompletely compensates for the absent HbA in mutation carriers Types β thalassemia major – homozygous or a compound heterozygote for two β0 mutations Clinical presentation Associated with severe anemia and hepatosplenomegaly Symptoms typically first appear at 6-24 months Early mortality if patient does not receive chronic recurrent transfusions or bone marrow transplantation Growth retardation, failure to thrive Pallor Hepatosplenomegaly Jaundice (HbF is protective in first 6 months) Symptoms in older patients – leg ulcers, extramedullary hematopoiesis, thrombophilia, pulmonary arterial hypertension, endocrine dysfunction, osteoporosis Resulting complications are a major cause of morbidity and mortality Iron overload from transfusions compounded by increased iron absorption leading to cardiac and liver failure is the main cause of death Treatment Affected individuals are transfusion dependent Regular transfusions with chelation to prevent iron overload prolongs life expectancy Bone marrow or cord blood transplantation is the only currently available curative option; gene therapy strategies are in development β thalassemia intermedia – milder presentation β+ homozygote, β0/β+ compound heterozygote, or β0 in combination with a thalassemic hemoglobinopathy (eg, HbE, Hb Lepore) Clinical presentation Variable presentation – may be nearly as severe as β thalassemia major Pallor, jaundice, cholelithiasis, liver and spleen enlargement, moderate/severe skeletal changes, leg ulcers, extramedullary masses of hyperplastic erythroid marrow Often associated with iron overload due to increased intestinal absorption of iron caused by ineffective erythropoiesis, even in subjects never transfused Treatment Patients may require transfusions Splenectomy controversial β thalassemia minor Heterozygous for β0 or β+ mutation Clinical presentation Clinically asymptomatic with minor hematologic anomalies such as reduced mean corpuscular volume (MCV) and elevated HbA2

Indications for Laboratory Testing

• Tests generally appear in the order most useful for common clinical situations
• Click on number for test-specific information in the ARUP Laboratory Test Directory

Diagnostic Algorithm

  Hemolytic Anemias Testing Algorithm