Thalassemias

  • Diagnosis
  • Algorithms
  • Screening
  • Background
  • Lab Tests
  • References
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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 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

  • 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

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 may be associated with significant hemolysis such as hemoglobin H disease.

Epidemiology

  • Prevalence – estimated 5-7% of the population worldwide carries clinically significant hemoglobin mutations

Inheritance

  • Autosomal recessive
  • Autosomal dominant – unusual

Pathophysiology

  • Hemoglobin, a tetramer of two α and two β or β-like (δ and γ) globin chains found in red blood cells
    • 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

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.

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

Limitations 

Cascade may not detect all Hb variants

Regulatory region mutations and sequence variants in genes other than HBB, HBA1, and HBA2 will not be detected

The phase of identified mutations may not be determined

Specific breakpoints of large deletions/duplications will not be determined, and it may not be possible to distinguish mutations of similar size

Individuals carrying both a deletion and duplication within the α-globin gene cluster may appear to have a normal number of α-globin gene copies

Sequencing of both HBA1 and HBA2 genes may not be possible in individuals harboring large α-globin deletions on both alleles

Rare syndromic or acquired forms of α thalassemia associated with ATRX gene mutations will not be detected

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

Limitations 

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

Limitations 

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-globin 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 Globin (HBA1 and HBA2) Deletion/Duplication 2011622
Method: Multiplex Ligation-dependent Probe Amplification

Limitations 

Breakpoints of large deletions/duplications will not be determined; therefore, it may not be possible to distinguish mutations of similar size

Assay does not assess for nondeletional mutations within the coding or regulatory regions of the α-globin cluster genes

Individuals carrying both a deletion and duplication within the α-globin gene cluster may appear to have a normal number of α-globin gene copies

Rare syndromic or acquired forms of α thalassemia associated with ATRX mutations will not be detected

Diagnostic errors can occur due to rare sequence variations

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

Limitations 

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

Rare syndromic or acquired forms of α thalassemia will not be detected

Alpha Globin (HBA1 and HBA2) Sequencing and Deletion/Duplication 2011708
Method: Polymerase Chain Reaction/Sequencing./Multiplex Ligation-dependent Probe Amplification.

Limitations 

Diagnostic errors can occur due to rare sequence variations

Deletion/Duplications

  • Breakpoints of large deletions/duplications will not be determined; therefore, it may not be possible to distinguish mutations of similar size
  • Assay does not assess for nondeletion mutations within the coding or regulatory regions of the α-globin genes
  • Individuals carrying both a deletion and triplication within the α-globin gene cluster may appear to have a normal number of α-globin gene copies
  • Rare syndromic or acquired forms of α thalassemia associated with ATRX mutations will not be detected

Sequencing

  • The phase of most identified mutations may not be determined
  • Sequencing of both HBA1 and HBA2 may not be possible in individuals harboring large α-globin deletions on both alleles
  • Rare syndromes associated with α thalassemia, such as ATR-X and ATR-16, will not be detected

Hemoglobin (Hb) A[2] and F by Column 0050613
Method: High Performance Liquid Chromatography

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

Limitations 

Diagnostic errors can occur due to rare sequence variations

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

Other β- and α-globin variants are not identified

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

Limitations 

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

Rare diagnostic errors can occur due to primer-site mutations

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

Limitations 

Diagnostic errors can occur due to rare sequence variations

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

Other β- and α-globin variants are not identified

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

Limitations 

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

Limitations 

Diagnostic errors may occur due to rare sequence variation

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

Guidelines

ACOG Committee on Obstetrics. ACOG Practice Bulletin No. 78: hemoglobinopathies in pregnancy. Obstet Gynecol. 2007; 109(1): 229-37. PubMed

Trent RJ, Webster B, Bowden DK, Gilbert A, Holl J, Lindeman R, Lammi A, Rowell J, Hinchcliffe M, Colley A, Wilson M, Saleh M, Blackwell J, Petrou V. Complex phenotypes in the haemoglobinopathies: recommendations on screening and DNA testing. Pathology. 2006; 38(6): 507-19. PubMed

General References

Cao A, Galanello R. Beta-thalassemia. Genet Med. 2010; 12(2): 61-76. PubMed

Fucharoen S, Weatherall DJ. The hemoglobin E thalassemias. Cold Spring Harb Perspect Med. 2012; 2(8): PubMed

Galanello R, Origa R. Beta-thalassemia. Orphanet J Rare Dis. 2010; 5: 11. PubMed

Harteveld CL, Higgs DR. Alpha-thalassaemia. Orphanet J Rare Dis. 2010; 5: 13. PubMed

Higgs DR, Engel JDouglas, Stamatoyannopoulos G. Thalassaemia. Lancet. 2012; 379(9813): 373-83. PubMed

Hoppe CC. Prenatal and newborn screening for hemoglobinopathies. Int J Lab Hematol. 2013; 35(3): 297-305. PubMed

Lo YM Dennis, Chiu RW K. Noninvasive approaches to prenatal diagnosis of hemoglobinopathies using fetal DNA in maternal plasma. Hematol Oncol Clin North Am. 2010; 24(6): 1179-86. PubMed

Olivieri NF, Muraca GM, O'Donnell A, Premawardhena A, Fisher C, Weatherall DJ. Studies in haemoglobin E beta-thalassaemia. Br J Haematol. 2008; 141(3): 388-97. PubMed

Origa R, Moi P, Galanello R, Cao A. Alpha-Thalassemia. In: Pagon RA, Adam MP, Ardinger HH, et al, editors. GeneReviews, University of Washington, 1993-2015. Seattle, WA [Last updated Nov 2013; Accessed: Mar 2016]

Peters M, Heijboer H, Smiers F, Giordano PC. Diagnosis and management of thalassaemia. BMJ. 2012; 344: e228. PubMed

Piel FB, Weatherall DJ. The α-thalassemias. N Engl J Med. 2014; 371(20): 1908-16. PubMed

Traeger-Synodinos J, Harteveld CL. Advances in technologies for screening and diagnosis of hemoglobinopathies. Biomark Med. 2014; 8(1): 119-31. PubMed

Waye JS, Eng B. Diagnostic testing for α-globin gene disorders in a heterogeneous North American population. Int J Lab Hematol. 2013; 35(3): 306-13. PubMed

References from the ARUP Institute for Clinical and Experimental Pathology®

Agarwal AM, Nussenzveig RH, Hoke C, Lorey TS, Greene DN. Identification of one or two α-globin gene deletions by isoelectric focusing electrophoresis. Am J Clin Pathol. 2013; 140(3): 301-5. PubMed

Greene DN, Vaughn CP, Crews BO, Agarwal AM. Advances in detection of hemoglobinopathies Clin Chim Acta. 2015; 439: 50-7. PubMed

Lanikova L, Kucerova J, Indrak K, Divoka M, Issa J, Papayannopoulou T, Prchal JT, Divoky V. β-Thalassemia due to intronic LINE-1 insertion in the β-globin gene (HBB): molecular mechanisms underlying reduced transcript levels of the β-globin(L1) allele. Hum Mutat. 2013; 34(10): 1361-5. PubMed

Nussenzveig RH, Vanhille DL, Hussey D, Reading S, Agarwal AM. Development of a rapid multiplex PCR assay for identification of the three common Hemoglobin-Lepore variants (Boston-Washington, Baltimore, and Hollandia) and identification of a new Lepore variant. Am J Hematol. 2012; 87(10): E74-5. PubMed

Reading S, Sirdah MM, Tarazi IS, Prchal JT. Detection of nine Mediterranean β-thalassemia mutations in Palestinians using three restriction enzyme digest panels: a reliable method for developing countries. Hemoglobin. 2014; 38(1): 39-43. PubMed

Medical Reviewers

Last Update: August 2016