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FeedbackMassively Parallel Sequencing
- Use to confirm carrier status or diagnosis of β thalassemia or β globinopathy in an individual with clinical findings or family history of β thalassemia or hemoglobinopathy
- Use to identify or confirm abnormal hemoglobin variant(s) detected by HPLC or Hb electrophoresis
Massively Parallel Sequencing
Use for molecular confirmation of β thalassemia or β globinopathy on fetal samples
Test Description
Massively parallel sequencing of all coding exons, exon-intron junctions, 5' proximal promoter and untranslated region, 3' polyadenylation signal, and intronic variants c.93-21G>A (IVS-I-110), c.316-197C>T (IVS-II-654), c.316-146T>G (IVS-II-705), and c.316-106C>G (IVS-II-745) of the HBB gene
If a familial sequence variant has been previously identified, targeted sequencing for that variant may be appropriate; refer to the Laboratory Test Directory for additional information.
Multiplex Ligation-Dependent Probe Amplification (MLPA) / Capillary Electrophoresis
Use to identify HBB gene deletions associated with β thalassemia, elevated hemoglobin F (such as in hereditary persistence of fetal hemoglobin [HPFH] or δβ thalassemia), or to confirm gene fusion hemoglobin variants (e.g., hemoglobin Lepore).
Variants in the beta (β)-globin gene (HBB) can result in anemia, β thalassemia, or sickling disorders of varying severity. Initial testing includes biochemical assessment for abnormal hemoglobin (Hb) variants using high-performance liquid chromatography (HPLC) and electrophoresis. A diagnosis is confirmed using molecular analysis of the HBB gene.
Disease Overview
Associated Phenotypes
| Phenotype | Characteristics |
|---|---|
| Thalassemia: decrease in protein produced | β thalassemia major
β thalassemia intermedia
β thalassemia minor (trait)
|
| Hemoglobinopathy: structurally abnormal protein | Sickling disorders:
Microcytic or hemolytic anemia Cyanosis (reduced oxygen-affinity HbS) Erythrocytosis (increased oxygen-affinity HbS) No clinical effect |
| HPFH | Persistent HbF production resulting from variants of the β-globin gene cluster that alter normal Hb switching Clinically benign condition |
| HbA2, hemoglobin, alpha 2; HbF, fetal hemoglobin; HPFH, hereditary persistence of fetal hemoglobin; MCV, mean corpuscular volume | |
Etiology
β thalassemia and certain hemoglobinopathies are caused by pathogenic germline variants within the HBB gene or variants involving the β globin gene cluster and its regulatory elements.
Prevalence and/or Incidence
- Approximately 5% of the world’s population carries clinically important Hb variants.
- 300,000 individuals with a severe hemoglobinopathy are born annually.
- β thalassemias are most commonly observed in individuals from southern Europe, northern Africa, and India.
Genetics
Gene
HBB (NM_000518)
Inheritance
Autosomal recessive (typically)
Structure/Function
- Major adult Hb (HbA) is composed of two β-globin chains and two alpha (α)-globin chains.
- Typically, adults have two functional β-globin genes (HBB) and four functional α-globin genes (two copies each of HBA1 and HBA2).
- β-globin chains with different variants may interact to alleviate or exacerbate the effects of the individual variants.
- Variants in the HBB gene can result in formation of a structurally abnormal protein or decrease the amount of protein produced.
- Certain HBB deletions impair the developmental switch from HbF to HbA, resulting in HPFH.
Test Interpretation
Clinical Sensitivity
This test is 99% sensitive for β thalassemia and hemoglobinopathies associated with the HBB gene (≥90% for large deletions using multiplex ligation probe amplification [MLPA]).
Analytic Sensitivity for Next Generation Sequencing
| Variant Class | Analytic Sensitivity (PPA) Estimatea (%) and 95% Credibility Region (%) | Analytic Specificity (NPA) |
|---|---|---|
| SNVs | >99 (96.9-99.4) | >99.9 |
| Deletions 1-10 bpb | 93.8 (84.3-98.2) | >99.9 |
| Insertions 1-10 bpb | 94.8 (86.8-98.5) | >99.9 |
aGenes included on this test are a subset of a larger methods-based validation from which the PPA values are derived. bVariants greater than 10 bp may be detected, but the analytic sensitivity may be reduced. bp, base pairs; NPA, negative percent agreement; PPA, positive percent agreement; SNVs, single nucleotide variants | ||
Results
| Result | Variant(s) Detected | Clinical Interpretation |
|---|---|---|
| Heterozygous | One pathogenic variant detected | Carrier of a structurally abnormal Hb or β thalassemia, depending on the specific variant identified |
| Homozygous or compound heterozygous | Two pathogenic variants detected (either the same variant or two different variants) | Variably affected, depending on the specific variant(s) identified |
| Negative | No pathogenic variants detected | Significantly decreases possibility of β thalassemia or β globinopathy Clinically benign structural variants predicted to produce an abnormal electrophoresis/HPLC result will be reported |
Limitations
- A negative result does not exclude a diagnosis of β thalassemia.
- Diagnostic errors can occur due to rare sequence variations.
- Interpretation of this test result may be impacted if this patient has had an allogeneic stem cell transplantation.
- Certain gene therapies may impact the performance of this test and interpretation of results; the presence or absence of variants, zygosity, and HBB gene copy number may not be determined in such cases.
- The following will not be evaluated:
- Variants outside the HBB coding regions and intron-exon boundaries by massively parallel sequencing (MPS)
- Regulatory region variants upstream of c.-250 and deep intronic variants other than: c.93-21G>A (IVS-I-110), c.316-197C>T (IVS-II-654), c.316-146T>G (IVS-II-705), and c.316-106C>G (IVS-II-745)
- Noncoding transcripts
- Large exonic deletions/duplications/inversions by MPS
- Sequence variants by MLPA
- The following may not be detected:
- Deletions/duplications/insertions of any size by MPS
- Single exon deletions/duplications based on the breakpoints of the rearrangement by MLPA
- Intragenic deletions other than HBB in the β globin cluster genes by MLPA
- Low-level mosaic or somatic variants
- Certain other variants, due to technical limitations in the presence of pseudogenes or repetitive/homologous regions