Alpha Hemoglobin DNA Sequence

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General Information

Lab Name
Alpha Hemoglobin DNA Sequence
Lab Code
HASEQ
Epic Ordering
ALPHA HEMOGLOBIN DNA SEQUENCE
Description

There are currently over 800 hemoglobin variants catalogued, of which approximately 300 are due to mutations in the alpha-globin gene. In addition, approximately 5% of alpha-thalassemia is caused by point mutations. This test, which sequences the coding regions and introns of both the alpha-globin 1 (HBA1) and alpha-globin 2 (HBA2) genes in both directions, identifies hemoglobin variants that are not easily diagnosed by electrophoresis/HPLC and can determine the cause of non-deletional alpha-thalassemia.

Indications for testing include:

  • Identification of hemoglobin variants detected by electrophoresis or HPLC
  • Differential diagnosis of microcytic anemia
  • Evaluation of nondeletional Hemoglobin H disease
  • Evaluation of a relative of an individual with a known alpha-globin mutation
  • Prenatal diagnosis of nondeletional alpha-thalassemia in pregnancies at risk for Hb H hydrops fetalis syndrome

NOTE: For outside patients please provide additional clinical and family history. Please include the following available laboratory data: CBC, Hb electrophoresis, and iron studies.

Notes: To order testing for a relative of an individual with a known alpha-globin mutation, see the Laboratory Medicine Online Test Guide for Alpha Hemoglobin Sequencing, Relative [HAREL]. If testing for deletional alpha-thalassemia is indicated, order “Alpha Thalassemia DNA Screen”.

References
  • Huisman, T. H. J., Carver, M. F. H., and Efremov, G. D. (1996). A Syllabus of Hemoglobin Variants (Augusta, GA: The Sickle Cell Anemia Foundation).
  • Higgs DR, et al. A review of the molecular genetics of the human alpha-globin gene cluster. Blood 1989, 73:1081-104. 2649166 .
  • Skogerboe KJ, et al. Screening for alpha-thalassemia. Correlation of hemoglobin H inclusion bodies with DNA-determined genotype. Arch Pathol Lab Med 1992, 116:1012-8. 1329692
  • Molchanova TP, Pobedimskaya DD, and Postnikov YuV. A simplified procedure for sequencing amplified DNA containing the alpha 2- or alpha 1-globin gene. Hemoglobin 1994, 18:251-5. 7928384
  • Old JM. Screening and genetic diagnosis of haemoglobin disorders. Blood Rev 2003, 17:43-53. 12490210
  • Chui DH, Fucharoen S, and Chan V. Hemoglobin H disease: not necessarily a benign disorder. Blood 2003, 101:791-800. 12393486
  • Chan V, et al. Molecular defects in Hb H hydrops fetalis. Br J Haematol 1997, 96:224-8. 9029003
  • Globin Gene Server: http://globin.cse.psu.edu/
Forms & Requisitions

Genetics Requisition

Synonyms
Alpha Thalassemia, Constant Spring, Globin, Hemoglobin Bart's, Hemoglobin H, Hemoglobinopathy, Hydrops fetalis
Components

Interpretation

Method

DNA sequencing of the coding regions and introns of both the alpha1 (HBA1) and alpha 2 globin (HBA2) genes in both directions to detect point mutations causing alpha globin variants and some types of alpha thalassemia.

This test detects alpha globin variants and non-deletional alpha thalassemia mutations (e.g. Hb Constant Spring). The coding regions and introns of both HBA1 and HBA2 genes are sequenced in bidirectionally. The reference mRNA sequences are HBA1 (NM_000558.3) and HBA2 (NM_000517.4) with nucleotide 1 corresponding to the A of the AUG initiation codon (nucleotide 38 of both reference sequences) and codon 1 corresponding to the valine encoded by GTG (nucleotides 41-43 of both reference sequences). The sensitivity of this test for detecting nucleotide substitutions, small insertions and deletions and in the alpha 1 (HBA1) and alpha 2 (HBA2) genes is theoretically >98%. This test will not detect mutations that lie outside of the sequenced regions, nor large HBA1 and HBA2 gene deletions (e.g. –a3.7, – –SEA). This test was developed and its performance characteristics determined by the Department of Laboratory Medicine at the University of Washington.

Reference Range
See individual components
Ref. Range Notes

No mutation detected.

References
  • Huisman, T. H. J., Carver, M. F. H., and Efremov, G. D. (1996). A Syllabus of Hemoglobin Variants (Augusta, GA: The Sickle Cell Anemia Foundation).
  • Higgs DR, et al. A review of the molecular genetics of the human alpha-globin gene cluster. Blood 1989, 73:1081-104. 2649166 .
  • Skogerboe KJ, et al. Screening for alpha-thalassemia. Correlation of hemoglobin H inclusion bodies with DNA-determined genotype. Arch Pathol Lab Med 1992, 116:1012-8. 1329692
  • Molchanova TP, Pobedimskaya DD, and Postnikov YuV. A simplified procedure for sequencing amplified DNA containing the alpha 2- or alpha 1-globin gene. Hemoglobin 1994, 18:251-5. 7928384
  • Old JM. Screening and genetic diagnosis of haemoglobin disorders. Blood Rev 2003, 17:43-53. 12490210
  • Chui DH, Fucharoen S, and Chan V. Hemoglobin H disease: not necessarily a benign disorder. Blood 2003, 101:791-800. 12393486
  • Chan V, et al. Molecular defects in Hb H hydrops fetalis. Br J Haematol 1997, 96:224-8. 9029003
  • Globin Gene Server: http://globin.cse.psu.edu/
Guidelines

Ordering & Collection

Specimen Type
Blood/Cultured amniocytes or chorionic villus cells/Extracted DNA from blood, chorionic villi, and amniocyte. Direct chorionic villi, amniocyte, or amniotic fluid testing require Genetics Director approval. Please call the lab at 206-598-7021
Collection

Acceptable:

  1. Whole blood:5 mL lavender top (EDTA) tube or yellow (ACD) top tube or 2 mL microtainer lavender top tube

  2. Extracted DNA from blood, chorionic villi, and amniocytes: 500 ng (concentration >10 ng/uL)

  3. Cultured amniocytes/chorionic villi: MCC is required for testing fetal samples. See MCC OLTG.

  4. Also acceptable, but requires the Genetics Director's approval and a backup culture. Direct chorionic villi and/or TISSUE: Send 20mg of tissue in a sterile tube or RPMI culture media

    *NOTE: If a fetal sample (cultured amniocytes or chorionic villi) was received, add MCC to the order. Prenatal testing requires concomitant testing for maternal cell contamination (see Online Test Guide, MCC for ordering and specimen requirements). See Special Instructions.

Unacceptable: Heparin green top tubes, buccal swab

Forms & Requisitions

Genetics Requisition

Handling Instructions

SPS specimen handling:

  1. Whole blood sample: store in the refrigerator

  2. Cultured amniocytes/chorionic villi: store at room temperature. Call the Genetics lab upon receipt (206)598-7021.

  3. Extracted DNA: store in the refrigerator
Quantity
requested: Entire specimen
minimum: Blood: 1 mL. If volume is less than 1mL, do not cancel. Send to Genetics lab. Confluent cultured cells: One (1) T25 flask. Extracted DNA: 250 ng

Processing

Processing

If fetal tissue (cultured amniocytes or chorionic villi) was received for prenatal testing, consultation with the laboratory is required. Please notify the Genetics lab about prenatal studies via email at geneticshelp@uw.edu or call 206-598-7021.

For clients outside of UW, please include the most recent CBC and Hb electrophoresis result/s (if available), and/or any relevant clinical history.

Performance

LIS Dept Code
Genetics (GEN)
Performing Location(s)
UW-MT Genetics

Attention: Genetics Lab
Clinical lab, Room NW220
University of Washington Medical Center
1959 NE Pacific Street
Seattle, WA 98195

Tel: 206-598–6429 M–F (7:30 AM–4:00 PM)
Fax: 206-616-4584
Lab email: cgateam@uw.edu

Tel (EXOME only): 206-543-0459

Faculty
Jillian Buchan, PhD, FACMG
Runjun Kumar, MD, PhD
Regina Kwon, MD, MPH
Christina Lockwood, PhD, DABCC, DABMGG
Abbye McEwen, MD, PhD
Colin Pritchard, MD, PhD
Vera Paulson, MD, PhD
Eric Konnick, MD, MS
He Fang, PhD
Frequency
Performed weekly. Results within 3 weeks.
Available STAT?
No

Billing & Coding

CPT codes
81259
Billing Comments

For pricing information, contact Client Support Services 206-520-4600 or 800-713-5198.

LOINC
60540-2
Interfaced Order Code
UOW2161