Thalassemias

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
- Red blood cell indices (from GeneTests.org)
  - α thalassemia (refer to table 1)
  - β thalassemia (refer to table 1)
Globin chain synthesis assay
Hemoglobin evaluation by HPLC (high performance liquid chromatography) or electrophoresis – hemoglobin patterns (from GeneTests.org)
- α thalassemia (refer to table 2)
- β thalassemia (refer to table 2)
Molecular testing to confirm α or β thalassemia
- β thalassemia – HBB gene sequencing
- α thalassemia – HBA1 and HBA2 molecular analysis
  - Deletion testing for HBA1 and HBA2 by polymerase chain reaction to identify 7 common α globin gene deletions
  - 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

Indications for carrier screening – family history of α or β thalassemia, patients belonging to high-risk ethnic groups, reproductive partners of known thalassemia carriers
- 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

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; non-deletion or regulatory region mutations are rare
- For prenatal counseling, it is important to know whether the non-functioning 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 occurs 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 pre-eclampsia, polyhydramnios or oligohydramnios, antepartum hemorrhage, and 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

Tests

Tests generally appear in the order most useful for common clinical situations

Test name: CBC with Platelet Count & Automated Differential

ARUP #: 0040003

Methodology: Automated Cell Count with Flow Cell Differential

Use:

Rule out other causes of anemia

Test name: Iron, Plasma or Serum

ARUP #: 0020037

Methodology: Quantitative Spectrophotometry

Use:

Rule out iron deficiency as cause of anemia

Test name: Beta Globin (HBB) Gene Sequencing

ARUP #: 0050578

Methodology: Polymerase Chain Reaction/Sequencing

Use:

Confirm diagnosis of hemoglobinopathies and beta thalassemias

Use when a definitive diagnosis cannot be made by HPLC or gel electrophoresis

Mutations in the beta 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 619bp deletion will be detected

Limitations:

Large gene deletions (other than 619del) or mutations in the distal regulatory elements, (eg, locus control region) will not be detected

Test name: Beta Globin (HBB) Sequencing, Fetal

ARUP #: 0050388

Methodology: Polymerase Chain Reaction/Sequencing

Use:

Confirm diagnosis of hemoglobinopathies and beta thalassemias in an at-risk pregnancy

Parental beta globin mutations must be provided prior to fetal testing

Mutations in the beta 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

Limitations:

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

Rare diagnostic errors can occur due to primer site mutations

Test name: Alpha Thalassemia (HBA1 & HBA2) 7 Deletions

ARUP #: 0051495

Methodology: Polymerase Chain Reaction/Gel Electrophoresis

Use:

Detect the 7 most common alpha globin gene deletions [-alpha3.7, -alpha4.2, -(alpha)20.5, --SEA, --MED, --THAI, --FIL]; clinical sensitivity varies by ethnicity and may be as high as 90%

Limitations:

Rare alpha globin gene deletions, non-deletion mutations, gene duplications, and mutations of the regulatory region will not be detected

Test name: Hemoglobin Evaluation with Reflex to Electrophoresis and/or RBC Solubility

ARUP #: 0050610

Methodology: High Performance Liquid Chromatography/Electrophoresis/RBC Solubility

Use:

Detect common thalassemic hemoglobinopathies (eg, HbCS)

Test name: Alpha Globin (HBA1 and HBA2) Sequencing

ARUP #: 2001582

Methodology: Polymerase Chain Reaction/Sequencing

Use:

Up to 10% clinical sensitivity; most HBA1 and/or HBA2 gene mutations are large deletions not detectable by sequencing

Contact genetic counselor before submitting

Limitations:

Rare diagnostic errors can occur due to primer site mutations

Large deletions/duplications and some mutations of the regulatory regions will not be detected

Phase of identified mutations may not be determined

Rare syndromes associated with alpha thalassemia such as ATR-X and ATR-16 will not be detected

Test name: Hemoglobin (Hb) A₂ & F by Column

ARUP #: 0050613

Methodology: High Performance Liquid Chromatography

Use:

Confirm previously abnormal values

Test name: Hemoglobin Bart

ARUP #: 0050528

Methodology: High Performance Liquid Chromatography

Use:

Detect alpha thalassemia in infants <3 months old

Determine etiology of hydrops fetalis

Detect levels of Hb Bart and Hb H

Test name: Beta Globin (HBB) HbS, HbC, & HbE Mutations

ARUP #: 0051421

Methodology: Polymerase Chain Reaction/Fluorescence Resonance Energy Transfer

Use:

Detect 3 common beta globin mutations: HbS, HbC and HbE

Limitations:

Only beta globin mutations causing HbS, HbC and HbE will be detected

Test name: Beta Globin (HBB) HbS, HbC, and HbE Mutations, Fetal

ARUP #: 0051422

Methodology: Polymerase Chain Reaction/Fluorescence Resonance Energy Transfer

Use:

Confirm a diagnosis of hemoglobinopathies and beta thalassemias in an at-risk pregnancy

Parental beta globin mutations must be provided before fetal testing

Limitations:

Only beta globin mutations causing HbS, HbC and HbE will be detected

Test name: Hereditary Persistence of Fetal Hemoglobin (HPFH) 8 Mutations

ARUP #: 2005408

Methodology: Polymerase Chain Reaction/Electrophoresis

Use:

Detect 8 common beta globin gene cluster deletions resulting in HPFH

Limitations:

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

Test name: Hemoglobin Lepore (HBD/HBB Fusion) 3 Mutations

ARUP #: 2004686

Methodology: Qualitative Polymerase Chain Reaction/Qualitative Electrophoresis

Use:

Detect Hb Lepore resulting from rearrangements of the delta and beta globin genes; carrier screening for individuals with family history of Hb Lepore

Limitations:

Only the three common Hb Lepore mutations will be detected

Rare diagnostic errors may occur due to primer-site mutations

Additional Tests Available

Test name: Hemoglobin Evaluation Reflexive Cascade

ARUP #: 2005792

Methodology: Refer to individual components

Comments:

References

Guidelines

General

Cao A, Galanello R. Beta-thalassemia. Genet Med. 2010; 12 (2) :61-76.PubMed
El Rassi F, Cappellini MD, Inati A, Taher A. Beta-thalassemia intermedia: an overview. Pediatr Ann. 2008; 37 (5) :322-328.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, Weatherall DJ. The alpha thalassaemias. Cell Mol Life Sci. 2009; 66 (7) :1154-1162.PubMed
Kutlar F. Diagnostic approach to hemoglobinopathies. Hemoglobin. 2007; 31 (2) :243-250.PubMed
Muncie HL Jr, Campbell J. Alpha and beta thalassemia. Am Fam Physician. 2009; 80 (4) :339-344.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) :388-397.PubMed
Wajcman H, Riou J. Globin chain analysis: an important tool in phenotype study of hemoglobin disorders. Clin Biochem. 2009; 42 (18) :1802-1806.PubMed
Wajcman H, Traeger-Synodinos J, Papassotiriou I, Giordano PC, Harteveld CL, Baudin-Creuza V, Old J. Unstable and thalassemic alpha chain hemoglobin variants: a cause of Hb H disease and thalassemia intermedia. Hemoglobin. 2008; 32 (4) :327-349.PubMed
Weatherall DJ. The Thalassemias: Disorders of Globin Synthesis. In Kaushansky, Lichtman, Seligson, Kipps, and Prchal, eds. Williams Hematology, 8th ed. New York: McGraw-Hill, 2010. pp. 675-707.

Medical Reviewers

Comprehensive Review: April 2011
Last Update: November 2011