Hereditary Hemolytic Anemia Panel, Sequencing

Hereditary Hemolytic Anemia Panel Sequencing 2012052
Method: Massively Parallel Sequencing

Indications for testing:

Determine etiology, elicit inheritance pattern, and assess recurrence risk in individuals with:

  • Unexplained hemolytic anemia
  • Unexplained hyperbilirubinemia in neonates
  • Family history of unexplained hemolytic anemia
  • Pregnancy with hydrops fetalis of unknown etiology
Familial Mutation, Targeted Sequencing 2001961
Method: Polymerase Chain Reaction/Sequencing
  • Recommended test for a known familial sequence variant previously identified in a family member.
  • A copy of the family member’s test result documenting the familial variant is required.

Hereditary hemolytic anemia (HHA) is characterized by premature red blood cell (RBC) destruction and anemia due to intrinsic RBC defects, and encompasses a diverse group of heterogeneous disorders. Genetic testing is indicated when initial test results do not explain clinical presentation or mode of inheritance.

Disease Overview

Clinical presentation is highly variable and may include anemia, fatigue, gallstones, hyperbilirubinemia/jaundice, pallor, scleral icterus, and splenomegaly. Laboratory findings include decreased hemoglobin concentration, hematocrit, and RBC count. Blood smear abnormalities such as spherocytes, acanthocytes, schistocytes, bite cells, stomatocytes, polychromasia, and target cells may be present.

Associated Disorders

RBC Membrane Disorders

Disorders characterized by weakened RBC membrane, resulting in irregular shape, increased fragility, and hemolysis:

  • Hereditary spherocytosis (HS) – ANK1, EPB42, SLC4A1, SPTA1, and SPTB
    • The most common RBC membrane disorder and is characterized by spherically shaped RBCs with decreased deformability.
    • Approximately 75% of HS is autosomal dominant and 25% is autosomal recessive or de novo.
  • Hereditary elliptocytosis and hereditary pyropoikilocytosis (HE/HPP) are related disorders with a wide spectrum of clinical phenotypes.
    • HE is characterized by elliptically shaped RBCs.
    • Marked anisopoikilocytosis with elliptocytes, microspherocytes, and bizarrely shaped RBCs are characteristically seen in HPP.
    • HE is autosomal dominant and HPP is autosomal recessive. Variants in SPTA1, SPTB, and, less commonly, EPB41 are causative.
    • Individuals with HE are generally asymptomatic but can have mild compensated hemolytic anemia.
    • HPP patients usually present with moderate to severe anemia.
  • Dehydrated hereditary stomatocytosis (xerocytosis) is characterized by decreased intracellular potassium content, loss of cell water, increased cytoplasmic viscosity, and increased mean cell hemoglobin concentration.
    • Inheritance is autosomal dominant and the most commonly involved gene is PIEZO1.
    • Splenectomy should be avoided in patients with some forms of hereditary stomatocytosis, as it may predispose the patient to life-threatening thrombotic events.

RBC Enzymopathies

More than 20 recognized disorders caused by deficiencies of enzymes involved with glycolysis, hexose monophosphate shunt, glutathione metabolism, and nucleotide metabolism 

  • Common forms:
    • G6PD deficiency (G6PD)
    • Pyruvate kinase deficiency (PK)
    • Pyrimidine 5’-nucleotidase (NT5C3A)
  • Associated findings:
    • Usually normocytic normochromic hemolytic anemia with no specific abnormalities of RBC morphology
    • Severity of hemolysis variable and may be a result of an external stressor (eg, infection, administration of drugs, or ingestion of some foods)
    • Nonhematological manifestations may include:
      • Myopathy
      • Neurological dysfunction
      • Intellectual disability

Hemoglobinopathies

Quantitative defect in biosynthesis of one type of hemoglobin (Hb) chain or a structurally abnormal Hb

  • Alpha or beta thalassemia results from a quantitative defect in the synthesis of either the alpha- or beta-globin chain.
    • The unpaired subunits precipitate, bind to the RBC membrane, and lead to hemolysis.
  • Structural Hb variants result from a structurally abnormal Hb that may polymerize, precipitate, or crystalize within the RBC, leading to membrane changes and hemolysis.
  • Next generation sequencing (NGS) analysis is complex in these disorders due to the high level of gene homology, copy number variations, and duplication within the alpha and beta globin operons. These disorders are not included as part of the current testing panel.

Prevalence

HHA disorders –1/500-1,100

  • Hereditary spherocytosis – 1/2,000 northern Europeans
  • Hereditary elliptocytosis/HPP – 1,2000-1,4000 worldwide
  • G6PD deficiency – 400 million worldwide
    • Varies by ethnicity – 7/10 Kurdish Jewish males, 1/6-10 African American males, 1/7-9 Arabic males, 1/6-16 Southeast Asian males
  • Pyruvate kinase deficiency – 1/20,000 Europeans

Inheritance

Dependent on gene – autosomal recessive, autosomal dominant, or X-linked

Test Description

See Genes Tested table for genes included in the panel.

Clinical Sensitivity

Variable and dependent on phenotype/condition

Testing Strategy

See ARUP Consult’s Hemolytic Anemias Testing Algorithm.

  • Molecular testing for hemolytic anemia is indicated when initial test results do not explain clinical presentation or mode of inheritance.

Limitations

  • A negative result does not exclude a heritable form of hemolytic anemia.
  • Diagnostic errors can occur due to rare sequence variations.
  • Interpretation of this test result may be impacted if this individual has had an allogeneic stem cell transplantation or recently received a blood transfusion.
  • The following will not be evaluated:
    • Variants outside the coding regions and intron-exon boundaries of the targeted genes
    • Regulatory region variants and deep intronic variants
    • Large deletions/duplications
    • Genes of the alpha- and beta-globin clusters
    • Noncoding transcripts
  • The following may not be detected:
    • Deletions/duplications/insertions of any size by massively parallel sequencing
    • Some variants due to technical limitations in the presence of pseudogenes, repetitive, or homologous regions
    • Low-level somatic variants

Analytical Sensitivity

Variant Class

Analytical Sensitivity (PPA) Estimatea (%)

Analytical Sensitivity (PPA) 95% Credibility Regiona (%)

SNVs

99.2

96.9-99.4

Deletions 1-10 bp

93.8

84.3-98.2

Deletions 11-44 bp

100

87.8-100

Insertions 1-10 bp

94.8

86.8-98.5

Insertions 11-23 bp

100

62.1-100

aGenes included on this test are a subset of a larger methods-based validation from which the PPA values are derived.

bp, base pairs; PPA, positive percent agreement; SNVs, single nucleotide variants

Genes Tested

Gene MIM Number Disorder Inheritance

AK1

103000

Hemolytic anemia due to adenylate kinase deficiency 

AR

ALDOA

103850

Glycogen storage disease XII

AR

ANK1

612641

Spherocytosis, type 1 

AD

CDAN1

607465

Anemia, congenital dyserythropoietic, type IA

AR

CYB5R3

613213

Methemoglobinemia due to deficiency of methemoglobin reductase

AR

EPB41

130500

Elliptocytosis 1

AD

Elliptocytosis 1

AR

EPB42

177070

Spherocytosis, type 5 

AR

G6PD

305900

Nonspherocytic hemolytic anemia due to G6PD deficiency

XL

GCLC

606857

Hemolytic anemia due to gamma-glutamylcysteine synthetase deficiency 

AR

GPI

172400

Nonspherocytic hemolytic anemia due to glucose phosphate isomerase deficiency

AR

GSR

138300

Hemolytic anemia due to glutathione reductase deficiency

AR

GSS

601002

Hemolytic anemia due to glutathione synthetase deficiency

AR

HK1

142600

Nonspherocytic Hemolytic Anemia due to Hexokinase Deficiency

AR

NT5C3A

606224

Hemolytic Anemia due to uridine 5-prime monophosphate hydrolase deficiency

AR

PFKM

610681

Glycogen storage disease VII

AR

PGK1

311800

Phosphoglycerate kinase 1 deficiency

XL

PIEZO1

611184

Dehydrated hereditary stomatocytosis with or without pseudohyperkalemia and/or perinatal edema

AD

PKLR

609712

Pyruvate kinase deficiency

AR

SEC23B

610512

Congenital dyserythropoietic anemia type II

AR

SLC4A1

109270

Spherocytosis, type 4
Ovalocytosis, southeast Asian type
Cryohydrocytosis

AD

SLCO1B1

604843

Hyperbilirubinemia, rotor type

AR (digenic)

SLCO1B3

605495

Hyperbilirubinemia, rotor type

AR (digenic)

SPTA1

182860

Elliptocytosis 2

AD

Pyropoikilocytosis
Spherocytosis, type 3 

AR

SPTB

182870

Spherocytosis, type 2
Elliptocytosis 3

AD

TPI1

190450

Hemolytic anemia due to triosephosphate isomerase deficiency

AR

UGT1A1

191740

Gilbert syndrome
Crigler-Najjar syndrome, types I and II
Hyperbilirubinemia, transient familial neonatal

AR

UGT1A6

606431

 

 

UGT1A7

606432

 

 

AD, autosomal dominant; AR, autosomal recessive; XL, X-linked
References 

Bolton-Maggs PH, Langer JC, Iolascon A, Tittensor P, King M, General Haematology Task Force of the British Committee for Standards in Haematology. Guidelines for the diagnosis and management of hereditary spherocytosis--2011 update Br J Haematol. 2012; 156(1): 37-49. PubMed

Gallagher PG. Abnormalities of the erythrocyte membrane Pediatr Clin North Am. 2013; 60(6): 1349-62. PubMed

Koralkova P, van Solinge WW, van Wijk R. Rare hereditary red blood cell enzymopathies associated with hemolytic anemia - pathophysiology, clinical aspects, and laboratory diagnosis Int J Lab Hematol. 2014; 36(3): 388-97. PubMed

Last Update: February 2019