Beta-Thalassemia, Beta Globin (HBB) Gene Sequencing

Gene sequencing test is done to analyze mutations in the HBB gene on chromosome 11 by amplification through PCR followed by Sanger sequencing. Some hemoglobin disorders will not be detected, such as large deletional alterations and crossover events. In testing algorithm this is a second tier test usually done to confirm abnormal Hb electrophoresis and red blood cell indices result.
In addition to thalassemia the following hemoglobins HbS/ HbE/ HbD/ HbC are also detected.
It is also done to evaluate chronic hemolytic anemia of unknown etiology
It is also done as a part of preconception screening in persons with family history of thalassemia; particularly spouses of carriers with borderline HbA2 values (≥3.3%)

The HBB gene on chromosome 11 is examined using PCR amplification and Sanger sequencing. HBB test is performed for the following reasons:

• Confirmation of thalassemia or a structural hemoglobinopathy involving the globin gene at the molecular level.
• Detect beta-thalassemia sequence variants, like + 0.
• Detect haemoglobin sequence variants like HbS, HbE, HbD, and HbC.
• Detect other point mutations linked to HbE, HbD, HbS, and HbC.

Gene sequencing test detects 30 common mutations in human beta globin gene (HBB) 

Beta-Thalassemia, Beta Globin (HBB) test detects haemoglobin variants and the common beta thalassemia sequence variants, including beta plus and beta zero thalassemias.

Gene sequencing test requires a blood sample. A tourniquet (elastic) band is placed tightly on the upper arm. The patient is then asked to make a fist. This helps in the build-up of blood filling the veins. The skin is disinfected before needle insertion and the blood sample is collected in vacutainer
Clinical history, referring clinicians contact number, HPLC Report, CBC findings, clinical/family history and TRF are mandatory

β0 is indicated by the following mutations :
619 bp deletion Codon 5(-CT)
Codon 8 (-AA) Codon 8/9(+G)
Codon 15(GG-AG) Codon 16(-C)
Codon 30(G-C) Codon 30(G-A)
Codon 39(C-T) Codon 41/42(-TTCT)
Codon 47/48 (+ATCT) Codon 88(+T)
IVS-I-1(G-A) IVS-I-1(G-T)
IVS-I-130(G-A) IVS-I-130(G-C)
IVS-I-25 (25 bp del)
β+ is indicated by the following mutations:
-88(C-T) Cap +1(A-C)
IVSI-5(G-C) (β+Severe) IVSI-110(G-A)
IVSI-128(T-G) IVS-II-654(C-T)(β+Severe)
IVS-II-745 (C-G) IVS II -837 (T-G)(β+/ β0)
Hemoglobin variants by the following mutations:
Hb S Codon 6(A-T); Hb C Codon 6(G-A); Hb E Codon 26(G-A); Hb D Punjab Codon 121(G-C); Hb D Iran Codon 22(G-C)
Patients with β-thalassemia major typically present in early childhood with severe anemia,
hepatosplenomegaly, and failure to thrive and are at risk for a shortened life expectancy; repeated transfusions are necessary
Individuals with thalassemia intermedia present later in life and have milder anemia that requires transfusions only intermittently
Carriers of β-thalassemia, on the other hand, have a single gene having β0/β+ mutation with the other gene being normal. Clinically, they have a mild microcytic hypochromic anemia with normal life expectancy

HBB test is recommended for individuals with:

• Clinical findings of anaemia and splenomegaly.
• Family history of thalassemia or hemoglobinopathy.
• Persistent haemolytic anaemia of unknown origin.
• Abnormal results on haemoglobin evaluation by electrophoresis or high-performance liquid chromatography (HPLC).
• Abnormal results on the complete blood count (CBC) test.
• Abnormal DNA tests for alpha and beta globins.

The test can be performed for anyone at any time to check for thalassemia or to determine if you are a carrier of thalassemia and are at risk of passing it on to your child. Pregnant mothers can take this test to check if they are carriers of the thalassemia gene. If the mother is found to be a carrier, the test is also performed on the father. 

No special preparation is required before taking the test.

The following mutations represent β0 (absence of production of beta globin)-

• Deletion of 619 bp Codon 5 (-CT).
• Codon 8 (-AA) (+G) codon 8/9.
• Codon 15 (GG-AG) Codon 16 (-C).
• Codon 30 (G-C) Codon 30 (G-A).
• Codon 39 (C-T) Codon 41/42 (-TTCT).
• (ATCT) Codon 47/48 88(+T) codon.
• IVS-I-1(G-A) IVS-I-1(G-T).
• IVS-I-130(G-A) IVS-I-130(G-C).
• IVS-I-25 (25 bp del) (25 bp del).

The following mutations suggest β+ (decreased but not absent production of beta globin)-

• -88(C-T) Cap +1(A-C) (A-C)
• The IVSI-5 (G-C) (+Severe) IVSI-110(G-A)
• IVSI-128(T-G) IVS-II-654(C-T)(β+Severe)
• IVS-II-745 (C-G) (C-G) IVS II -837 (T-G) (β+/ β0)

The following mutations suggest hemoglobin variants -

• Hb S Codon 6(A-T).
• Hb C Codon 6(G-A).
• Hb E Codon 26(G-A).
• Hb D Punjab Codon.
• 121(G-C).
• Hb D Iran Codon 22(G-C).

The doctor may order HPLC with reflex to electrophoresis and/or RBC solubility test, and molecular tests to identify haemoglobin variants

• Beta Globin
• Beta Thalassemia
• Thalassemia: Beta
• β-Thalassemia: HBB (Full Gene Sequencing)
• Beta Thalassemia HBB
• Beta-globin gene (HBB) sequencing