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Hematology and Blood Disorders | Volume 2
Page 28
July 25-26, 2019 | Amsterdam, Netherlands
OF EXCELLENCE
IN INTERNATIONAL
MEETINGS
alliedacademies.comYEARS
Global Hematology 2019
4
th
International Conference on
HEMATOLOGY AND
BONE MARROW TRANSPLANTATION
SCREENING OF Β-GLOBIN GENE (HBB) FOR RARE MUTATIONS IN Β-THALASSEMIA
PATIENTS INCLUDED HAEMOGLOBIN S D-PUNJAB USING SANGER SEQUENCING
Zeeshan Ansar, Asghar Nasir, Azra Samreen, Kahkashan Imam and Tariq Moatter
Aga Khan University, Pakistan
Objective:
β-thalassemia is an autosomal recessive disorder which results in the formation of abnormal hae-
moglobin due to a variety of different mutations found in the HBB gene. These mutations render patients inca-
pable of producing correct form of haemoglobin. The aim of this study was to identify HBB gene mutations in
β-thalassemic patients, not included in the common-mutation panel of ARMS PCR, by sequencing HBB coding,
intronic and promoter.
Method:
A total of 10 samples previously tested for HBB gene mutations by ARMS PCR common-panel (i.e. IVS
1-1, IVS 1-5, Codon 8/9, Codon 41/42 and 619bp deletion) were analyzed by Sanger sequencing. Two healthy
subjects were included as negative controls. Genomic DNA was isolated and HBB gene was amplified. Column
purified amplified products were utilized for bidirectional cycle sequencing (Big Dye Terminator, ABI, USA).
Results:
In the present study, a total of 10 samples were analyzed. Four were males and six females. The Mean
of the patients was three years. All patients were diagnosed as β-thalassemia major based on their family his-
tory, clinical and laboratory findings. On average, patients were receiving transfusions every second week.
Seven rare mutations in HBB gene were detected including point mutations. The mutations spanned in the
promoter region HBB:c.138C>A (-88 C>A), exon1 HBB:c.17_18delCT (Codon5 –CT), HBB:c.47G>A (Codon15
G>A), HBB:c.92G>C (Codon30 G>C), HBB:c.50A>C (CAP+1 A>C), exon2 HBB:c.118C>T (Codon39) and intron2
HBB:c.315+1G>A (IVS II-I G>A) and a heterozygous change at codon 6 (GAG—>GTG) and also a heterozygous
mutation at codon 121 (GAA—>CAA) . All control subjects showed normal HBB gene sequence. In addition, a
polymorphism T>C in codon3 at position HBB: c.59 was detected in majority of the patients and controls.
Conclusion:
Although ARMS PCR is a fast and convenient method for detection of common mutations in the
HBB gene, a small subset of patients may be missed because of rare mutations, which would require other
means for diagnosis. Sanger sequencing is an accurate and robust technique to manage such patients.
Hematol Blood Disord 2019, Volume 2