Beta Globin (HBB) Sequencing and Deletion/Duplication

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.

Indications for Ordering

  • Confirm carrier status or diagnosis of β thalassemia or β globinopathy in individual with clinical findings or family history of β thalassemia or hemoglobinopathy
  • Identify or confirm abnormal Hb variant(s) detected by HPLC or Hb electrophoresis
Test Description
  • Polymerase chain reaction (PCR) amplification and bidirectional sequencing of HBB coding region, intron/exon boundaries, proximal promoter and untranslated regions, and deep intronic variants (IVS-II-654, IVS-II-705, IVS-II-745)
  • Multiplex ligation-dependent probe amplification (MLPA) of the β-globin gene cluster (HBB, HBD, HBG1, HBG2, HBE1) and its locus control region
Beta Globin (HBB) Gene Sequencing 0050578
Method: Polymerase Chain Reaction/ Sequencing

Molecular confirmation of a suspected structural hemoglobinopathy or β thalassemia

Indications for Ordering
  • Confirm carrier status or diagnosis of β thalassemia or β globinopathy in individual with clinical findings or family history of β thalassemia or hemoglobinopathy
  • Identify or confirm abnormal Hb variant(s) detected by HPLC or Hb electrophoresis
Test Description

Polymerase chain reaction (PCR) amplification and bidirectional sequencing of HBB coding region, intron/exon boundaries, proximal promoter and untranslated regions, and deep intronic variants (IVS-II-654, IVS-II-705, IVS-II-745)

Beta Globin (HBB) Deletion/Duplication 2010113
Method: Multiplex Ligation-dependent Probe Amplification

Detects large deletions of the β-globin gene cluster associated with β thalassemia or HPFH

Indications for Ordering
  • Confirm carrier status or diagnosis of β thalassemia or β globinopathy in individual with clinical findings or family history of β thalassemia or hemoglobinopathy
  • Assess for deletional HPFH in individuals with elevated Hb F
Test Description

Multiplex ligation-dependent probe amplification (MLPA) of the β-globin gene cluster (HBB, HBD, HBG1, HBG2, HBE1) and its locus control region

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 known hemoglobinopathies

Variants in the beta (β)-globin gene (HBB) can result in anemia, β thalassemia or sickling disorders of varying severity. Typical testing strategy is as follows:

  • Initial testing: screen for abnormal hemoglobin (Hb) variants using high-performance liquid chromatography (HPLC) and electrophoresis
  • Secondary testing: molecular analysis to identify or confirm abnormal Hb variant(s) detected by HPLC or Hb electrophoresis

Disease Overview

Prevalence

  • ~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

Symptoms

Phenotypes caused by HBB variants
Phenotype Characteristics

Thalassemia: decrease in protein produced

β thalassemia minor (trait)

  • Usually clinically asymptomatic, mild anemia may be present
  • Minor hematologic anomalies, including reduced MCV and elevated HbA2

β thalassemia major

  • Associated with severe microcytic anemia and hepatosplenomegaly
  • Affected individuals are transfusion dependent

β thalassemia intermedia

  • Milder clinical presentation than β thalassemia major

Hemoglobinopathy: structurally abnormal protein

 

Sickling disorders

  • Sickle cell anemia (HbSS)
  • Hb S-C disease

Microcytic or hemolytic anemia

Cyanosis

  • Reduced oxygen-affinity Hbs

Erythrocytosis

  • Increased oxygen-affinity Hbs

No clinical effect

Hereditary persistence of fetal Hb (HPFH)

Persistent HbF production resulting from variants of β-globin gene cluster that alter normal Hb switching

Clinically benign condition

MCV, mean corpuscular volume

Genetics

Gene

HBB

Inheritance

Autosomal recessive (typically)

Structure/Function

  • Major adult Hb (HbA): composed of two β-globin chains and two α-globin chains
  • Normal 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 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 fetal to adult Hb, resulting in hereditary persistence of fetal Hb

Variants

>500 β-globin variants

Test Interpretation

Sensitivity/Specificity

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

Results

  • Pathogenic HBB gene variant(s) detected
    • Heterozygous
      • Carrier of a structurally abnormal Hb or β thalassemia, depending on the specific variant identified
    • Homozygous or compound heterozygous
      • Variably affected, depending on the specific variant(s) identified
  • No pathogenic HBB gene 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

  • Diagnostic errors can occur due to rare sequence variations
  • Breakpoints of large deletions and duplications will not be determined
  • Precise clinical phenotype associated with a particular deletion may not be known (eg, HPFH vs. delta-beta [δ-β] thalassemia)
  • Intragenic deletions in the β-globin cluster genes, other than HBB, may not be detected
  • Does not assess for sequence variants within the coding or regulatory regions of the HBD, HBG1, HBG2, and HBE1 genes

Last Update: August 2019