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FeedbackMultiplex Ligation-Dependent Probe Amplification (MLPA)/Sequencing
- Preferred first-tier genetic test for confirmation of suspected α thalassemia or α thalassemia trait
- Detects common, rare, and novel deletions or duplications in the α-globin gene cluster and its HS-40 regulatory region
- Multiplex ligation-dependent probe amplification (MLPA) used to detect Hb Constant Spring (HbCS) (HBA2 c.427T>C; p.Ter143Gln); targeted Sanger sequencing is performed to assess HbCS copy number in absence of a concurrent HBA2 deletion.
Multiplex Ligation-Dependent Probe Amplification (MLPA)
- First-tier genetic test for confirmation of suspected α thalassemia or α thalassemia trait
- Detects common, rare, and novel deletions or duplications in the α-globin gene cluster (HBZ, HBM, HBA1, HBA2, HBQ1) and its HS-40 regulatory region
Multiplex Ligation-Dependent Probe Amplification (MLPA)/Sequencing/Polymerase Chain Reaction (PCR)
- Comprehensive genetic test for detection of α thalassemia or α thalassemia trait
- Detect deletional and nondeletional variants in HBA1 and HBA2
Multiplex Ligation-Dependent Probe Amplification (MLPA)/Sequencing
- Diagnostic testing for α thalassemia in fetus with suggestive clinical findings or at risk for α thalassemia due to familial HBA1/HBA2 deletions or HbCS variant
- Detects common, rare, and novel deletions or duplications in the α-globin gene cluster and its HS-40 regulatory region
- MLPA is used to detect HbCS (HBA2 c.427T>C; p.Ter143Gln); targeted Sanger sequencing is performed to assess HbCS copy number in absence of a concurrent HBA2 deletion.
Alpha (α) thalassemia is the most common inherited disorder of hemoglobin (Hb) worldwide and is caused by HBA1 and HBA2 gene variants. Decreased or absent synthesis of the hemoglobin (Hb) α chain results in clinical presentations ranging from asymptomatic silent carriers to severe anemia and fetal lethality. The two clinically significant forms of α thalassemia are Hb Bart hydrops fetalis syndrome and hemoglobin H (HbH) disease. Alpha thalassemia is found more often in certain ethnicities, including African, African American, Mediterranean, Middle Eastern, and Southeast Asian.
Disease Overview
Prevalence and/or Incidence
- Most common inherited disorder of Hb worldwide
- Carrier frequencies in high-risk populations:
- African, African American: 1/3
- Middle Eastern, Southeast Asian: 1/20
- Mediterranean: 1/30-50
- Hb Bart hydrops fetalis syndrome and HbH disease are more frequent in Southeast Asian, Asian Indian, and Mediterranean populations than in African populations.
Symptoms
| Phenotype | Associated Symptoms |
|---|---|
|
α Thalassemia silent carrier |
Typically asymptomatic, though borderline anemia or mild microcytosis may be present Often misdiagnosed as iron deficiency Normal Hb electrophoresis |
|
α Thalassemia trait |
Mild microcytic anemia may be present Often misdiagnosed as iron deficiency Normal Hb electrophoresis |
|
HbH disease |
Moderate to severe form of α thalassemia Moderate microcytic hypochromic anemia Hemolysis with Heinz bodies Splenomegaly Rare extramedullary hematopoiesis Propensity for acute hemolysis after oxidative stress, drug therapy, or infection |
|
Hb Bart hydrops fetalis syndrome |
Most severe form of α thalassemia Risk for fetus
Maternal complications during pregnancy
|
Pathophysiology
Typically, individuals have four functioning α-globin genes (αα/αα). Two genes, HBA1 and HBA2, are present on each copy of chromosome 16 and α-globin chains function as subunits of fetal Hb (HbF: α2γ2) and adult Hb (HbA: α2β2). The number of α-globin genes deleted or inactivated correlates with different α thalassemia phenotypes. Genotype/phenotype correlations in α thalassemia are complex and may be influenced by coinheritance of other Hb variants or α-globin gene duplications.
| Phenotype | Genotype(s) |
|---|---|
|
α Thalassemia silent carrier |
-α/αα |
|
α Thalassemia trait |
-α/-α --/αα |
|
HbH disease |
--/-α |
|
Hb Bart hydrops fetalis syndrome |
--/-- |
Genetics
Genes
HBA1 and HBA2
Inheritance
Autosomal recessive
Variants
- HBA1 and HBA2 large gene deletions account for approximately 90% of pathogenic α-thalassemia variants.
- -α3.7 and -α4.2 deletions result in the deletion of a single gene.
- -(α)20.5, --SEA, --MED-I, --FIL, and --THAI deletions result in the deletion of both HBA1 and HBA2 genes from the same chromosome
- Sequence variants and regulatory region variants occur mainly in HBA2 and account for up to 15% of causative variants.
- Nondeletional variants include:
- Sequence variants that inactivate the gene
- Small insertions/deletions
- Variants that result in unstable α-globin protein (eg, Hb Constant Spring or HBcS)
- Nondeletional α-globin variants may be pathogenic or benign.
- Both may result in an abnormal protein detectable by Hb evaluation.
- Pathogenic nondeletional variants often have a more severe effect than single gene deletions.
- Nondeletional variants include:
- Alpha-globin gene duplication results in three or more active α-globin genes on a single chromosome.
- Typically benign
- May alter expected clinical phenotypes and hematological features when coinherited with beta (β) thalassemia
Test Interpretation
Alpha Globin (HBA1 and HBA2) Sequencing and Deletion/Duplication
Sensitivity/Specificity
- Analytical sensitivity/specificity: 99% for both duplication/deletion analysis and sequencing
- Clinical sensitivity: most pathogenic HBA1 and/or HBA2 gene variants are large deletions not detectable by sequencing
Results
| Result | Variant(s) Detected | Interpretation |
|---|---|---|
|
Positive |
Genotype (-a/aa)
|
Silent carrier |
|
Genotype (-a/-a) or (--/aa)
|
α Thalassemia trait |
|
|
1 pathogenic sequence variant
|
Silent carrier or α thalassemia trait |
|
|
Genotype (--/-a) |
HbH disease
|
|
|
Genotype (--/--)
|
Hb Bart hydrops fetalis syndrome |
|
|
Genotype (aa/aaa) |
Extra functional alpha globin gene copy is present |
|
|
Negative |
No pathogenic variants detected |
Greatly decreased probability that the individual is affected with, or a carrier of, α thalassemia |
|
Inconclusive |
Variant(s) of uncertain clinical significance identified |
Unknown clinical significance |
Limitations
- Diagnostic errors can occur due to rare sequence variations.
- Sequence analysis will not detect all regulatory region variants or variants in α-globin cluster genes other than HBA1 and HBA2.
- Sequencing of both HBA1 and HBA2 may not be possible in individuals harboring large α-globin deletions on both alleles.
- This assay is unable to sequence HBA2-HBA1 fusion genes; thus, HBA1 or HBA2 sequence variants occurring in cis with a 3.7 kb deletion or other HBA2-HBA1 hybrid gene will not be detected.
- It may not be possible to determine the phase of identified sequence variants.
- Specific breakpoints of large deletions/duplications will not be determined; therefore, it may not be possible to distinguish deletion variants 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.
- Rare syndromic or acquired forms of α thalassemia associated with ATRX variants will not be detected.
References
-
GeneReviews Alpha-Thalassemia - Thalassemia
Origa R, Moi P. Alpha-thalassemia. In: Adam MP, Ardinger HH, Pagon RA, et al, eds. GeneReviews. University of Washington, Seattle. Last update Oct 2020; accessed Jan 2021.