Hemoglobinopathies

Diagnosis

Indications for Testing

Laboratory Testing

  • Initial testing – CBC with peripheral smear demonstrating any of the following
    • Polychromasia, spherocytes, schistocytes, sickle cells, Heinz bodies, basophilic stippling; however, the lack of any of these cells does not rule out hemolytic anemia
  • For thalassemia testing, see Thalassemias topic
  • Many hemoglobinopathies can be diagnosed using electrophoretic or high-performance liquid chromatography (HPLC) techniques, but some are missed
    • Reflexive cascade is preferred for initial screening
  • Recommended screening tests for specific phenotypes
    • Hemoglobin instability test (isopropanol or heat instability test) for unstable hemoglobins causing hemolytic anemia
    • Determination of heme oxygen dissociation curve (P50) for those with polycythemia and decreased oxygen affinity hemoglobin
      • Testing for congenital polycythemia (VHL mutation)
      • Spectrophotometric techniques for M-hemoglobins
  • P50 testing for polycythemia syndrome 
    • Some mutant hemoglobins cannot be readily identified by electrophoretic or HPLC techniques
    • Confirmation of suspected hemoglobin variants can be obtained by DNA analysis

Screening

  • Newborn screening for sickle cell disease is mandated in all states in the U.S.
  • Carrier screening is recommended for individuals belonging to high-risk ethnic groups
    • Initial screening – HPLC or thin layer isoelectric focusing, per U.S. Preventive Services Task Force

Clinical Background

Hemoglobinopathies are a group of common, inherited disorders of hemoglobin, resulting in either the synthesis of structurally abnormal globin subunits or a reduced synthesis of structurally normal globin subunits (thalassemias).

Epidemiology

  • Incidence
    • Hemoglobinopathies are among the most common monogenic diseases, with ~5% of the world’s population carrying a hemoglobin mutation
    • >300,000 children affected with severe hemoglobinopathy worldwide  

Pathophysiology

  • Hemoglobin, a tetramer of two α and two β or β-like (δ and γ) globin chains found in red blood cells, combines reversibly with oxygen and is the medium by which oxygen is transported within the body
  • Synthesis of globins in adults is controlled by 5 genes: α, β, γa, γg and δ
    • Primary hemoglobin (Hb) in normal adults is HbA, with small amounts of HbA2 and HbF
    • Hemoglobinopathies are the result of mutations of the globin genes
    • Hemoglobin can also be modified by environmental factors or inherited mutations that do not affect the globin chain
      • Examples include methemoglobin, carboxyhemoglobin, and S-nitrosohemoglobin (also referred to as dyshemoglobinemia)
    • >400 hemoglobin variants have been identified as resulting from α, β, γ or δ globin gene mutations
    • Identification of the hemoglobin variant leads to correct diagnosis, improved treatment, and accurate genetic counseling
  • Hemoglobin variants may produce different phenotypes
    • Benign – clinically and hematologically insignificant
    • Sickling disease – associated with several genotypes
    • Hemolytic anemias – unstable hemoglobins
    • Methemoglobinemia (M-hemoglobins) – associated with heme in oxidized ferric (Fe +3) rather than normal ferrous (Fe +2) state, which renders methemoglobin unable to deliver oxygen and results in cyanosis
      • Methemoglobin can also be acquired or inherited from cytochrome b5 reductase mutations
    • Polycythemia – associated with increased oxygen hemoglobin affinity (ie, decreased p50)
    • Anemia – associated with decreased oxygen hemoglobin affinity (ie, increased p50)
    • Thalassemia phenotype (imbalance of α and β/β-like chains, ie, HbE, Hb Constant Spring, Hb Lepore) – shares features of both hemoglobinopathies and thalassemias

Types of hemoglobinopathies

  • Sickle cell disease

    Epidemiology

    • Incidence
      • 1/200-600 African Americans
      • HbS causes 60-70% of sickle cell disease in U.S. (1/2,000)
    • Ethnicity
      • HbS occurs most commonly in sub-Saharan Africans and some ethnic and racial groups in Southern Europe (Greece, Turkey and Sicily), India, and the Middle East
      • HbC common in West Africa
      • HbE common in Southeast Asia

    Genetics

    • Autosomal recessive
    • Sickle cell anemia is characterized by homozygosity for HbS
    • Sickle cell trait (Hb AS) is the asymptomatic carrier state for sickle cell disease
      • Carrier frequency of HbS is ~10% in African Americans
    • Sickle cell disease refers to the inheritance of HbS in combination with another β-globin mutation (such as HbE, HbC, Hb Lepore, or β thalassemia) to produce a sickling phenotype
      • Common genotypes associated with sickle cell disease
      • HbSS – sickle cell anemia
      • HbS/C
      • HbS/β0 and HbS/β+ – sickle cell β thalassemia
      • HbS/E
      • HbS/Lepore
      • HbS/D Punjab
      • HbS/O Arab
    • Parental screening for sickle cell trait and interacting hemoglobinopathies can aid in genetic counseling

    Clinical Presentation

    • Asymptomatic at birth – neonate has not yet switched from fetal to adult hemoglobin (γ genes to β genes)
    • Severity of disease varies and is influenced by genotype
    • Acute, painful, vaso-occlusive crises (long bones, chest, and back) are common
    • Complications – spleen fibrosis and acute splenic sequestration, cerebrovascular accident, kidney failure, lungs (acute chest syndrome and pulmonary hypertension), central nevous system and bone infarcts, tissue ischemia, priapism, gallstone disease, increased risk of invasive bacterial infections, aplastic crisis
    • Milder forms present with hemolytic anemia
    Congenital methemoglobinemia

    Epidemiology

    • Incidence – not common; type II is sporadic
    • Ethnicity – type I is endemic in Asthabascan and Navajo native Americans, Yakuts, and Siberian natives; sporadic in other races

    Genetics

    • Autosomal dominant disorder
      • Mutations of α-globin genes; lifelong phenotype of cyanosis  
      • Mutations of β-globin genes; phenotype of cyanosis after 2-6 months of age
      • Mutations of γ-globin genes; phenotype of cyanosis during first 2-6 months of age
    • Autosomal recessive disorder
      • Homozygous or compound heterozygous mutations of cytochrome b5 reductase
      • Type I – affects mature red blood cells only
      • Type II – affects all cell types

    Clinical Presentation

    • Type I – anemia, gray skin, cyanosis; no reduced life expectancy
      • Tolerate levels of M-hemoglobins up to 40%
    • Type II – manifests as intellectual diversity and developmental delay; reduced life expectancy
    Polycythemia or anemia (associated with hemoglobin variants with high oxygen affinity)

    Epidemiology

    • Incidence – rare cause of congenital polycythemia

    Genetics

    • Autosomal dominant; de novo mutations have been reported
    • More than 100 high-affinity hemoglobin variants have been described; low-affinity variants are less common
    • Mutations resulting in hemoglobins with altered oxygen affinity may occur in either the α or β-globin genes
    • Mutations of the α-globin gene resulting in hemoglobins with altered oxygen affinity have a milder clinical effect than similar mutations of the β-globin gene due to the number of α gene copies
    • Homozygotes for β-globin gene mutations resulting in hemoglobins with altered oxygen affinity may be more severely affected than heterozygotes
    • Coinheritance of a thalassemia allele may alter clinical expression

    Clinical Presentation

    • High-oxygen-affinity variants
      • Associated with
        • Benign hereditary polycythemia
        • Increased hematocrit
        • Increased blood hemoglobin concentration
          • Normal leukocyte and platelet counts
          • Lack of splenomegaly
      • Characterized by
        • Decreased p50 value – partial pressure of oxygen where hemoglobin is 50% oxygenated
          • Sigmoidal oxygen dissociation curve (ODC) shifted to the left
        • Rare endemic familial congenital polycythemia variance associated with mutation of VHL gene
    • Low-oxygen-affinity variants
      • Associated with anemia and possible cyanosis

Indications for Laboratory Testing

  • 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
Test Name and Number Recommended Use Limitations Follow Up
CBC with Platelet Count and Automated Differential 0040003
Method: Automated Cell Count/Differential
Initial test for evaluation of hemoglobinopathy    
Hemoglobin Evaluation Reflexive Cascade 2005792
Method: High Performance Liquid Chromatography/Electrophoresis/RBC Solubility/Polymerase Chain Reaction/Fluorescence Resonance Energy Transfer/Sequencing

Optimal test for the initial and confirmatory diagnosis of any suspected hemoglobinopathy or thalassemia

Not recommended for routine carrier screening in healthy adults for  purposes of reproductive decision making

Cascade reflex testing may include electrophoresis, solubility testing, and/or molecular analyses of the globin genes

A faculty hematopathologist personally directs and interprets each stage of testing to completion

A comprehensive report is provided

Do not use for the follow-up of an individual with a known diagnosis

Sensitivity/specificity – varies, depending on test components

Cascade may not detect all Hb variants, including rare HBA1/2 deletions, regulatory region mutations, or mutations involving the delta or gamma genes

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

Effective test for screening and follow up of individuals  with hemoglobinopathies

Sensitivity/specificity – varies, depending on test components

May not detect all hemoglobin variants

Diagnostic errors can occur due to rare sequence variations

 
Hemoglobin S, Evaluation with Reflex to RBC Solubility 0050520
Method: High Performance Liquid Chromatography

Determines presence of hemoglobin S

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

Confirm suspected HbS, HbC, or HbE mutations

Clinical sensitivity – 

  • Sickle cell disease – >70%
  • Other hemoglobinopathies –vary by ethnicity

Analytical sensitivity – 99%

Diagnostic errors can occur due to rare sequence variations

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

Other β- and α-globulin variants are not identified

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

Genetic test on fetal specimens for prenatal detection of HbS, HbC, and HbE mutations

Prenatal diagnosis when both parents are known carriers of HbS, HbC, or HbE

Clinical sensitivity –

  • Sickle cell disease – >70%
  • Other hemoglobinopathies –varies by ethnicity

Analytical sensitivity – 99%

Diagnostic errors can occur due to rare sequence variations

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

Other β- and α-globulin variants are not identified

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

First-tier genetic test for confirmation of suspected α thalassemia

Detects the 7 most common α-globin gene deletions of HBA1 and HBA2 (-α3.7, -α4.2, -(α)20.5, --SEA, --MED, --THAI, --FIL)

Clinical sensitivity – up to 90%, depending on ethnicity

Analytical sensitivity/specificity – 99%

Rare α-globin gene deletions, nondeletional mutations, 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

 
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

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

Analytical sensitivity – 99%

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-globulin 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

 
Beta Globin (HBB) Gene Sequencing 0050578
Method: Polymerase Chain Reaction/Sequencing

Molecular confirmation of suspected structural hemoglobinopathy or β thalassemia

Mutations tested – complete protein coding sequence with exon/intron boundaries, proximal promoter, 5’ and 3’ untranslated regions, and intronic mutations IVS-II-654, IVS-II-705 and IVS-II-745

Clinical sensitivity – 97% for β thalassemia and hemoglobinopathies associated with the HBB gene

Analytical sensitivity – 99%

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-globulin 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

 
Oxygen Dissociation (P50) by Hemoximetry 2002984
Method: Spectrophotometry/Clark Electrode

Suspected hemoglobin (Hb) variant with altered oxygen affinity

  • Cyanotic individuals with or without anemia
  • Family history of hemoglobinopathy with polycythemia

A simultaneously drawn control specimen from a healthy, nonsmoking individual who is not biologically related to the patient is required for meaningful interpretation

Specimens must be analyzed within 48 hours of collection; P50 value decreases with time

Will not distinguish between the possible causes for decreased P50 value, which include 2,3-DPG deficiency; high-oxygen-affinity Hb variants; methemoglobinemia; carboxyhemoglobinemia

Decreased P50 values in chronic smokers (carboxyhemoglobinemia) should be interpreted with caution; Hb has higher affinity for carbonmonoxy than for oxygen

P50 values should be correlated with age; HbF may produce a decreased P50 level

 
von Hippel-Lindau (VHL) Sequencing 2002970
Method: Polymerase Chain Reaction/Sequencing

Determine cause of congenital polycythemia in symptomatic individuals    

Large deletions and duplications, deep intronic mutations, and regulatory region mutations are not detected

Diagnostic errors may occur due to rare sequence variation

Polycythemia due to causes other than VHL gene mutations will not be detected

 
Hemoglobin Lepore (HBD/HBB Fusion) 3 Mutations 2004686
Method: Qualitative Polymerase Chain Reaction/Qualitative Electrophoresis

Molecular confirmation of suspected Hb Lepore variant identified by hemoglobin evaluation

Carrier screening for individuals with family history of Hb Lepore

Detects 3 common mutations – Hb Lepore-Washington-Boston; Hb Lepore-Baltimore; Hb Lepore-Hollandia

Clinical sensitivity/specificity – unknown

Analytical sensitivity/specificity – 99%

Diagnostic errors may occur due to rare sequence variation

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

 
Additional Tests Available
 
Click the plus sign to expand the table of additional tests.
Test Name and NumberComments
Beta Globin (HBB) Deletion/Duplication 2010113
Method: Multiplex Ligation-dependent Probe Amplification

Second-tier test detects large deletions of the β-globin gene cluster associated with β thalassemia or hereditary persistence of fetal hemoglobin (HPFH)

Preferred initial test is the combined sequencing and deletion/duplication test

Alpha Globin (HBA1 and HBA2) Sequencing 2001582
Method: Polymerase Chain Reaction/Sequencing

Second-tier genetic test for detection of α thalassemia

Confirm a clinical diagnosis of α thalassemia when HBA1 and HBA2 deletion testing has detected the inactivation of 2 or fewer α-globin genes

Confirm the identity of a variant detected through hemoglobin evaluation that may be pathogenic or benign

Methemoglobin Reductase, Blood 2011015
Method: Quantitative Spectrophotometry

Confirm cases of heterozygous or homozygous methemoglobin reductase deficiency