Hemoglobinopathies

Indications for Testing

Hemolytic anemia; family history of hemoglobinopathy

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 may be missed
- 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

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

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, γ^a, γ^g 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 chains; examples include methemoglobin, carboxyhemoglobin, and S-nitrosohemoglobin (also referred to as dyshemoglobinemia)
>400 hemoglobin variants have been identified 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/or 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

Sickle Cell Disease

Epidemiology

Incidence
- 1/200-600 African Americans
- HbS causes 60-70% of sickle cell disease in U.S. (1/2000)
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; life-long 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 disability and development 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 more mild 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

Tests generally appear in the order most useful for common clinical situations

Test name: CBC with Platelet Count & Automated Differential

ARUP #: 0040003

Methodology: Automated Cell Count with Flow Cell Differential

Use: Initial screening

Test name: Hemoglobin Evaluation Reflexive Cascade

ARUP #: 2005792

Methodology: Refer to individual components

Use:

Detect hemoglobin variants from a single sample

Limitations:

This cascade may not detect all hemoglobin variants, including large HbB deletions, rare HbA1/2 deletions, regulatory region mutations, or mutations involving the

Test name: Hemoglobin Evaluation with Reflex to Electrophoresis and/or RBC Solubility

ARUP #: 0050610

Methodology: High Performance Liquid Chromatography/Electrophoresis/RBC Solubility

Use:

Rapidly distinguishes many hemoglobin variants when an abnormal hemoglobin is suspected; determines and confirms the presence of hemoglobin S

Limitations: Some mutations are electrophoretically silent; false positives may occur for hemoglobin S RBC solubility

Test name: Hemoglobin S, Evaluation with Reflex to RBC Solubility

ARUP #: 0050520

Methodology: High Performance Liquid Chromatography

Use:

Determines presence of hemoglobin S

Test name: Beta Globin (HBB) HbS, HbC, and HbE Mutations, Fetal

ARUP #: 0051422

Methodology: Polymerase Chain Reaction/Fluorescence Resonance Energy Transfer

Use:

Fetal testing when both parental variants have been identified as HbC (c.19G>A), HbS (c.20A>T) or HbE (c.79G>A)

Limitations:

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

Test name: Oxygen Dissociation (P50) by Hemoximetry

ARUP #: 2002984

Methodology: Spectrophotometry/Clark Electrode

Use:

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

Test name: von Hippel-Lindau (VHL) Sequencing

ARUP #: 2002970

Methodology: Polymerase Chain Reaction/Sequencing

Use:

Determine cause of congenital polycythemia in symptomatic individuals

Limitations:

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

Test name: Hemoglobin Lepore (HBD/HBB Fusion) 3 Mutations

ARUP #: 2004686

Methodology: Qualitative Polymerase Chain Reaction/Qualitative Electrophoresis

Use:

Detect Hb Lepore resulting from rearrangements of the delta and beta globin genes; carrier screening for individuals with family history of Hb Lepore

Limitations:

Only the three common Hb Lepore mutations will be detected

Rare diagnostic errors may occur due to primer-site mutations

Additional Tests Available

Test name: Beta Globin (HBB) HbS, HbC, & HbE Mutations

ARUP #: 0051421

Methodology: Polymerase Chain Reaction/Fluorescence Resonance Energy Transfer

Comments:

Molecular confirmation of HbC (c.19G>A), HbS (c.20A>T) and HbE (c.79G>A) variants

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

Test name: Beta Globin (HBB) Gene Sequencing

ARUP #: 0050578

Methodology: Polymerase Chain Reaction/Sequencing

Comments:

Confirm the diagnosis of hemoglobinopathies and beta thalassemias

Use when a definitive diagnosis cannot be made by HPLC or gel electrophoresis

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

Large beta globin deletions or fusion genes will not be detected

Test name: Alpha Thalassemia (HBA1 & HBA2) 7 Deletions

ARUP #: 0051495

Methodology: Polymerase Chain Reaction/Gel Electrophoresis

Comments:

Detect the 7 most common alpha globin gene deletions (-alpha3.7, -alpha4.2, -(alpha)20.5, --SEA, --MED, --THAI, --FIL); clinical sensitivity varies by ethnicity and may be as high as 90%

Test name: Alpha Globin (HBA1 and HBA2) Sequencing

ARUP #: 2001582

Methodology: Polymerase Chain Reaction/Sequencing

Comments:

Detect rare alpha globin mutations

Contact genetic counselor before submitting

Phase of identified mutations may not be determined

Rare syndromes associated with alpha thalassemia such as ATR-X and ATR-16 will not be detected