Beta Hemoglobin DNA Sequencing

There are currently over 800 hemoglobin variants catalogued, of which approximately 500 are due to mutations in the beta-globin gene. In addition, approximately 95% of beta-thalassemia is caused by point mutations. This test, which sequences the coding regions and introns of the beta-globin gene (HBB) in both directions, identifies hemoglobin variants that are not easily diagnosed by electrophoresis/HPLC and can determine the cause of beta-thalassemia.

Indications for testing include:

• Identification of hemoglobin variants detected by electrophoresis or HPLC
• Differential diagnosis of microcytic anemia
• Molecular characterization of beta-thalassemia mutations
• Testing of a relative of an individual with a known beta-globin mutation
• Prenatal diagnosis of potentially severe beta-thalassemia
• Characterization of the sickle-cell mutation

Notes: To order testing for a relative of an individual with a known beta-globin mutation, see the Laboratory Medicine Online Test Guide for Beta Hemoglobin Sequencing, Relative [HBREL]

DNA sequencing of the coding regions, introns, and flanking sequences of the hemoglobin beta (HBB) gene in both directions to detect point mutations causing beta globin variants and beta thalassemia.

This test sequences bidirectionally a 2 kb contiguous region of the HBB (beta globin) gene from approximately bases 61941 to 63927 of Genbank U01317, corresponding to bases minus 195 to plus 1792 relative to the start of transcription. Patient sequences are compared against Genbank U01317 as genomic reference sequence to detect mutations. Mutations are reported at the genomic level, with base 1 corresponding to the start of transcription. Mutations that change an amino acid are reported at the protein level according to the sequence of the mature protein without its initiator methionine, and may also be described by the traditional name of the hemoglobin variant.

Correspondence between this mutation nomenclature system and the HGVS nomenclature system may be found at the web site http://globin.cse.psu.edu.

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.