Thalassemias

Diagnosis

Indications for Testing

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

Screening

  • Indications for carrier screening – family history of α or β thalassemia, patients belonging to high-risk ethnic groups, reproductive partners of known thalassemia carriers, microcytosis, and no identified iron deficiency
    • Patients from high-risk ethnic groups – initial screening by CBC with RBC indices
    • Patients of African descent – initial screening should also include HPLC to detect sickle cell carriers
  • Microcytosis – (MCV <80 fL) and hypochromia (MCH <27 pg) in the absence of iron deficiency suggest carrier status
    • Genetic counseling is recommended for couples when both partners are carriers for the same type of thalassemia

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 A22 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 Barts 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)
      • Can be beneficial in patients with sickle cell disease or β thalassemia
      • Testing is important to distinguish from other etiologies such as δβ thalassemia
    • Hb Lepore – large deletion resulting in the fusion of the δ and β globin genes (β+ mutation)
      • May be inherited with β thalassemia trait

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 Barts 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 Barts (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 Barts (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 Barts 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+ 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
Test Name and Number Recommended Use Limitations Follow Up
CBC with Platelet Count and Automated Differential 0040003
Method: Automated Cell Count/Differential

Initial test for evaluation of hemoglobinopathy

   
Iron, Plasma or Serum 0020037
Method: Quantitative Spectrophotometry

Initial test for evaluating microcytic hypochromic anemia to rule out iron deficiency as cause of anemia

   
Hemoglobin Evaluation Reflexive Cascade 2005792
Method: High Performance Liquid Chromatography/Electrophoresis/RBC Solubility/Polymerase Chain Reaction/Fluorescence Resonance Energy Transfer/Sequencing

Optimal test for the initial and confirmatory diagnosis of any suspected hemoglobinopathy or thalassemia

Not recommended for routine carrier screening in healthy adults for  purposes of reproductive decision making

Cascade reflex testing may include electrophoresis, solubility testing, and/or molecular analyses of the globin genes

A faculty hematopathologist personally directs and interprets each stage of testing to completion

A comprehensive report is provided

Do not use for the follow-up of an individual with a known diagnosis

Sensitivity/specificity – varies, depending on test components

Cascade may not detect all Hb variants, including rare HBA1/2 deletions, regulatory region mutations, or mutations involving the delta or gamma genes

Diagnostic errors can occur due to rare sequence variations

 
Hemoglobin Evaluation with Reflex to Electrophoresis and/or RBC Solubility 0050610
Method: High Performance Liquid Chromatography/Electrophoresis/RBC Solubility

Effective test for screening and follow up of individuals  with hemoglobinopathies

Sensitivity/specificity – varies, depending on test components

May not detect all hemoglobin variants

Diagnostic errors can occur due to rare sequence variations

 
Beta Globin (HBB) Sequencing and Deletion/Duplication 2010117
Method: Polymerase Chain Reaction/Sequencing/Multiplex Ligation-dependent Probe Amplification

Preferred test for molecular confirmation of β thalassemia or a hemoglobinopathy involving the β-globin gene

Clinical sensitivity – 99% (~97% by sequencing and ~2% by deletion analysis) for β thalassemia and hemoglobinopathies associated with the HBB gene

Analytical sensitivity – 99%

Diagnostic errors may occur due to rare sequence variations

Breakpoints of large deletions will not be determined

Precise clinical phenotype associated with a particular deletion may not be known (eg, HPFH vs δ-β thalassemia)

Intragenic deletions in the β-globulin cluster genes, other than HBB, may not be detected

Does not assess for point mutations within the coding or regulatory regions of the HBD, HBG1, HBG2, and HBE1 genes

 
Beta Globin (HBB) Gene Sequencing 0050578
Method: Polymerase Chain Reaction/Sequencing

Molecular confirmation of suspected structural hemoglobinopathy or β thalassemia

Mutations tested – complete protein coding sequence with exon/intron boundaries, proximal promoter, 5’ and 3’ untranslated regions, and intronic mutations IVS-II-654, IVS-II-705 and IVS-II-745"

Clinical sensitivity – ~97%, for β thalassemia and hemoglobinopathies associated with the HBB gene

Analytical sensitivity – 99%

Diagnostic errors may occur due to rare sequence variations

Breakpoints of large deletions will not be determined

Precise clinical phenotype associated with a particular deletion may not be known (eg, HPFH vs δ-β thalassemia)

Intragenic deletions in the β-globulin cluster genes, other than HBB, may not be detected

Does not assess for point mutations within the coding or regulatory regions of the HBD, HBG1, HBG2, and HBE1 genes

 
Alpha Thalassemia (HBA1 and HBA2) 7 Deletions 0051495
Method: Polymerase Chain Reaction/Gel Electrophoresis

First-tier genetic test for confirmation of suspected α thalassemia

Detect the 7 most common α globin gene deletions of HBA1 and HBA2 (-α3.7, -α4.2, -(α)20.5, --SEA, --MED, --THAI, --FIL)

Clinical sensitivity – up to 90%, depending on ethnicity

Analytical sensitivity/specificity – 99%

Rare α-globin gene deletions, nondeletional mutations, gene duplications, and mutations of the regulatory region will not be detected

α-globin gene duplications will not be detected

Diagnostic errors can occur due to rare sequence variations

 
Beta Globin (HBB) Sequencing, Fetal 0050388
Method: Polymerase Chain Reaction/Sequencing

Confirmatory genetic test on fetal specimens for the prenatal detection of structural hemoglobinopathies and β thalassemia

Parental β globin mutations must be provided prior to fetal testing

Mutations in the β globin gene exons, intron/exon borders, proximal promoter, 5' and 3' UTRs, intronic mutations IVS-II 654f, IVS-II 705, and IVS-II 745 and the Asian Indian 619 bp deletion will be detected

Large HBB gene deletions and duplications other than 619del will not be detected

Rare diagnostic errors can occur due to primer site mutations

 
Hemoglobin (Hb) A2 and F by Column 0050613
Method: High Performance Liquid Chromatography

Confirm previously abnormal values

   
Beta Globin (HBB) HbS, HbC, and HbE Mutations 0051421
Method: Polymerase Chain Reaction/Fluorescence Resonance Energy Transfer

Confirm suspected HbS, HbC, or HbE mutations

Clinical sensitivity

  • Sickle cell disease – >70%
  • Other hemoglobinopathies –vary by ethnicity

Analytical sensitivity – 99%

Diagnostic errors can occur due to rare sequence variations

Detects only the 3 most common missense variants in the β-globulin gene

Other β- and α-globulin variants are not identified

 
Beta Globin (HBB) HbS, HbC, and HbE Mutations, Fetal 0051422
Method: Polymerase Chain Reaction/Fluorescence Resonance Energy Transfer

Genetic test on fetal specimens for prenatal detection of HbS, HbC, and HbE mutations

Prenatal diagnosis when both parents are known carriers of HbS, HbC, or HbE

Clinical sensitivity

  • Sickle cell disease – >70%
  • Other hemoglobinopathies –varies by ethnicity

Analytical sensitivity – 99%

Diagnostic errors can occur due to rare sequence variations

Detects only the 3 most common missense variants in the β-globulin gene

Other β- and α-globulin variants are not identified

 
Hereditary Persistence of Fetal Hemoglobin (HPFH) 8 Mutations 2005408
Method: Polymerase Chain Reaction/Electrophoresis

Detects 8 common deletions associated with HPFH – HPFH-1-7; and SEA-HPFH

Clinical sensitivity/specificity - unknown

Only the 8 targeted deletions associated with HPFH will be detected

Point mutations or rare deletions that cause HPFH or delta/beta thalassemia will not be identified

Other genetic modifiers of HbF levels will not be assessed

This test is unable to differentiate homozygosity for an HPFH deletion from compound heterozygosity for an HPFH deletion and a rare globin cluster deletion

Diagnostic errors can occur due to rare sequence variations

 
Hemoglobin Lepore (HBD/HBB Fusion) 3 Mutations 2004686
Method: Qualitative Polymerase Chain Reaction/Qualitative Electrophoresis

Molecular confirmation of a suspected Hb Lepore variant identified by hemoglobin evaluation

Carrier screening for individuals with family history of Hb Lepore

Detects 3 common mutations – Hb Lepore-Washington-Boston; Hb Lepore-Baltimore; Hb Lepore-Hollandia

Clinical sensitivity/specificity – unknown

Analytical sensitivity/specificity – 99%

Diagnostic errors may occur due to rare sequence variation

Negative result does not exclude β thalassemia, as other β-globulin gene mutations are not identified by this assay

 
Additional Tests Available
 
Click the plus sign to expand the table of additional tests.
Test Name and NumberComments
Beta Globin (HBB) Deletion/Duplication 2010113
Method: Multiplex Ligation-dependent Probe Amplification

Second-tier test detects large deletions of the β-globin gene cluster associated with β thalassemia or hereditary persistence of fetal hemoglobin (HPFH)

Preferred initial test is the combined sequencing and deletion/duplication test

Alpha Globin (HBA1 and HBA2) Sequencing 2001582
Method: Polymerase Chain Reaction/Sequencing

Second-tier genetic test for detection of α thalassemia

Confirm a clinical diagnosis of α thalassemia when HBA1 `and HBA2 deletion testing has detected the inactivation of 2 or fewer α-globin genes

Confirm the identity of a variant detected through hemoglobin evaluation that may be pathogenic or benign