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Test Code WGSEQ Gamma Globin Full Gene Sequencing, Varies

Important Note

EPIC/Beaker order code is LAB1230225

Useful For

An adjunct in the interpretation of hemoglobin electrophoresis results

 

Evaluation for suspected gamma variants or nondeletional hereditary persistence of fetal hemoglobin (HPFH)

 

Assess for unstable gamma chain variants (there are occasionally newborns who are jaundiced at birth, often requiring phototherapy, in which all other tests for causes of hemolysis are unrevealing)

Genetics Test Information

The beta-like hemoglobins include the epsilon, gamma, beta, and delta globins, whose genes are present on chromosome 11 in a linked cluster (ie, the beta globin complex). The gamma genes, HBG1 (Ay) and HBG2 (Gy), contain 3 exonic coding regions and 2 intronic intervening sequences (IVS). The genes produce gamma globin chains that form tetramers with alpha globin chains to create fetal hemoglobin (Hb F). HBG1 and HBG2 differ only in which amino acid is located at position 136 (alanine or glycine). The resultant proteins are named A-gamma and G-gamma, respectively. Although G-gamma is predominant at birth, this gradually reverses during the first year of life to become the normal adult G-gamma/A-gamma ratio, which is 2:3. Some people maintain an increased G-gamma:A-gamma ratio throughout life, which has been linked to certain polymorphisms in either gene. Additionally, some alterations in the promoter regions of the gamma globin genes are known to cause a form of hereditary persistence of fetal hemoglobin (HPFH), which is characterized by a significant but harmless elevation of Hb F into adulthood. If coinherited with sickle cell disease, HPFH has a strong modulating effect on the condition and appears to protect against some, but not all, of its complications. Some gamma mutations result in gamma chain hemoglobin variants, most of which are clinically insignificant; however, an incompletely studied subset causes neonatal disorders, such as hemolytic anemia, cyanosis, and methemoglobinemia.

Method Name

Polymerase Chain Reaction (PCR) Amplification/Sanger Sequence Analysis

Reporting Name

Gamma Globin Full Gene Sequencing

Specimen Type

Varies


Shipping Instructions


Specimens must arrive within 30 days (720 hours) of collection.



Necessary Information


A complete patient history is strongly encouraged.



Specimen Required


Submit only 1 of the following specimens:

 

Specimen Type: Peripheral blood

Container/Tube:

Preferred: Lavender top (EDTA)

Acceptable: Yellow top (ACD)

Specimen Volume: 4 mL

Collection Instructions:

1. Invert several times to mix blood.

2. Send specimen in the original tube.

Specimen Stability: Refrigerate

 

Specimen Type: Extracted DNA from whole blood

Container/Tube: 1.5-2mL tube with indication of volume and concentration of DNA

Specimen Volume: Entire specimen

Collection Instructions:

1. Label specimen as extracted DNA from blood.

2. Write the DNA concentration on the specimen tube.

Specimen Stability: Frozen (preferred)/Refrigerate/Ambient


Specimen Minimum Volume

Blood: 1 mL; Extracted DNA: 50 mcL at 50 ng/mcL concentration

Specimen Stability Information

Specimen Type Temperature Time
Varies Varies 30 days

Reject Due To

Hemolysis

Gross OK; Mild OK

Lipemia

NA

Icterus

NA

Other

Sodium heparin, bone marrow, paraffin-embedded tissue, frozen tissue, paraffin-embedded bone marrow aspirate clot, methanol-acetic acid (MAA)-fixed pellets, moderately to severely clotted

Clinical Information

Hemoglobin F (Hb F) is the dominant hemoglobin at birth but is gradually replaced by adult hemoglobin (Hb A) during the year after birth (normal value ≤1% of total hemoglobin after age 2). Increased Hb F levels may continue after the neonatal period and into adulthood for various reasons. Genetic causes include deletional and nondeletional forms of hereditary persistence of fetal hemoglobin (HPFH) and delta-beta thalassemia mutations. Over 100 mutations have been described in the gamma genes and, if detectable, the protein expression will vary over time according to the overall Hb F expression. Gamma globin mutations can manifest either as a quantitative (gamma thalassemia or nondeletional HPFH) or a qualitative (gamma variant) abnormality. Nondeletional HPFH mutations frequently modulate the expected severity of sickling disorders due to the inhibitory properties of Hb F on sickle formation. Many gamma chain variants are benign, although some, such as unstable, high- and low-oxygen affinity, or M hemoglobin variants, cause hemolytic anemia/hyperbililrubinemia, erythrocytosis, cyanosis, and methemoglobinemia, respectively. The percentages of gamma variants will vary according to if they are present on the HBG1 or HBG2 genes, as these genes are differentially expressed depending on the age of the patient. Symptoms due to gamma variants are expected to decrease along with the normal decrease in Hb F and therefore most resolve after the first 6 months of life.

Reference Values

An interpretive report will be provided.

Interpretation

An interpretive report will be provided and will include specimen information, assay information, and whether the specimen was positive for any mutations in the gene. If positive, the mutation will be correlated with clinical significance, if known.

Cautions

This test cannot be used in isolation to confirm or exclude hemoglobin conditions. Large deletions, crossover events, as well as other mutations may not be detected. This test is used in conjunction with adequately studied protein analysis results.

 

If multiple mutations are identified, gamma globin gene sequencing is not able to distinguish between mutations that are found in the same allele (in cis) and mutations found on different alleles (in trans). This limitation of sequencing may complicate diagnosis or classification and has implications for inheritance and genetic counseling. To resolve these cases, molecular results must be correlated with electrophoretic and protein data and/or family studies.

Clinical Reference

1. Crowley MA, Mollan TL, Abdulmalik OY, et al: A hemoglobin variant associated with neonatal cyanosis and anemia. N Engl J Med 2011;364:1837-1843

2. Cui J, Baysdorfer C, Azimi M, et al: Identification of three novel Hb F variants: Hb F-Hayward [Gy(NA1)Gly->Asp, GGT>GAT], Hb F-Chori-I [AyT16(A13)Gly->Asp, GGC>GAC] and Hb F-Chori-II [AyI29(B11)Gly->Glu, GGA>GAA]. Hemoglobin 2012;36:305-309

3. Akinsheye I, Alsultan A, Solovieff N, et al: Fetal hemoglobin in sickle cell anemia. Blood 2011;118:19-27

4. Disorders of Hemoglobin Genetics, Pathophysiology, and Clinical Management. Second edition. Edited by M Steinberg, B Forget, D Higgs, D Weatherall. New York, Cambridge University Press, 2009

5. Molecular Hematology. Third edition. Edited by D Provan, J Gribben. Malden, Massachusets, Blackwell Publishing, 2010

6. Color Atlas of Hemoglobin Disorders: A Compendium Based on Proficiency Testing. Edited by JD Hoyer, SH Kroft. Northfield, IL. College of American Pathologists, 2003

7. Merchant S, Oliveira JL, Hoyer JD, Viswanatha DS: Chapter 24. Molecular diagnosis in hematopathology. In Hematopathology: A Volume in the Series: Foundations in Diagnostic Pathology. Second edition. Edited by J Goldblum. E Hsi. Churchill Livingstone. 2012

Method Description

Total genomic DNA is extracted from the sample and the full gamma globin genes are amplified by PCR in separate reactions followed by Sanger sequencing. Review of the sequence data is performed using a combination of automated calls and manual inspection.(Unpublished Mayo method)

Day(s) and Time(s) Performed

Monday through Friday

Analytic Time

4 days

Specimen Retention Time

Whole Specimen: 14 days. DNA stored for 3 months

Performing Laboratory

Mayo Medical Laboratories in Rochester

Test Classification

This test was developed and its performance characteristics determined by Mayo Clinic in a manner consistent with CLIA requirements. This test has not been cleared or approved by the U.S. Food and Drug Administration.

CPT Code Information

81479-Unlisted molecular

LOINC Code Information

Test ID Test Order Name Order LOINC Value
WGSEQ Gamma Globin Full Gene Sequencing In Process

 

Result ID Test Result Name Result LOINC Value
46952 Gamma Globin Gene Sequencing Result In Process
46953 Gamma Globin Interpretation In Process

Forms

1. Thalassemia/Hemoglobinopathy Patient Information (T358) in Special Instructions

2. New York Clients-Informed consent is required. Please document on the request form or electronic order that a copy is on file. An Informed Consent for Genetic Testing (T576) in Special Instructions.