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
    • Reflextive 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

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 to detect hemoglobin variants

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

   
Hemoglobin Evaluation with Reflex to Electrophoresis and/or RBC Solubility 0050610
Method: High Performance Liquid Chromatography/Electrophoresis/RBC Solubility

Acceptable test for the initial confirmatory diagnosis of any suspected hemoglobinopathy

Some mutations are electrophoretically silent; false positives may occur for hemoglobin S RBC solubility  
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 – ~97%, dependent on ethnicity

Analytical sensitivity – 99%

Clinical sensitivity -

Sickle cell disease >70%

Other hemoglobinopathies - varies by ethnicity

Mutations other than HbC (c.19G>A), HbS (c.20A>T) and HbE (c.79G>A) will not be detected

Clinical sensitivity – >70% for sickle cell disease; other hemoglobinopathies vary depending on individual’s ethnicity

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

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

Clinical sensitivity – ~97%, dependent on ethnicity

Analytical sensitivity – 99%

Mutations other than HbC (c.19G>A), HbS (c.20A>T) and HbE (c.79G>A) will not be detected

Clinical sensitivity – >70% for sickle cell disease; other hemoglobinopathies vary depending on individual’s ethnicity

 
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 (-α3.7, -α4.2, -(α)20.5, --SEA, --MED, --THAI, --FIL); clinical sensitivity varies by ethnicity and may be as high as 90%

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

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

First-tier genetic test for confirmation of suspected structural hemoglobinopathy or β thalassemia

  • Confirm high-performance liquid chromatography (HPLC) or gel electrophoresis results that suggests carrier status or diagnosis of a β thalassemia or β globinopathy
  • Diagnostic testing in individuals with clinical findings of β thalassemia or a hemoglobinopathy
  • Carrier testing for individuals with a family history of β thalassemia or a hemoglobinopathy
  • To confirm a specific HBB mutation in parents prior to prenatal diagnosis
  • Prenatal diagnosis

Mutations in the β globin gene exons, intron/exon borders, proximal promoter, 5’ and 3’ UTRs, and Asian Indian 619bp deletion will be detected

Clinical sensitivity – ~97%, dependent on ethnicity

Analytical sensitivity – 99%

Rare diagnostic errors may occur due to primer-site mutations

Large β globin deletions or fusion genes will not be detected

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

Second-tier genetic test for detection of α thalassemia when HBA1 and HBA2 deletion testing has detected the inactivation of 2 or fewer α globin genes

Contact genetic counselor before submitting

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 α thalassemia such as ATR-X and ATR-16 will not be detected

Test is not able to identify sequence variants in  alpha globin gene in cis with the common 3.7 kb deletion; therefore, sequencing is not possible in individuals homozygous for the 3.7 kb deletion

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

Indicated for patients with familial polycythemia or patients with isolated polycythemia who lack a JAK2 mutation and do not exhibit clinical findings (ie, leukocytosis, thrombocytosis, splenomegaly) associated with polycythemia vera

   
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

Rare diagnostic errors may occur due to primer-site mutations

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 (g.63632_71046del)
  • Hb Lepore-Baltimore (g.63564_70978del)
  • Hb Lepore-Hollandia (g.63290_70702del)

Clinical sensitivity/specificity – unknown

Analytical sensitivity/specificity – 99%

Rare diagnostic errors may occur due to primer-site mutations