Gene Sequencing Test

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%)

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

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