Alpha Hemoglobin DNA Sequence

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”.

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.

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

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

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.