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Comparison Between Three Molecular Diagnostics for the Identification of Heterozygous Hemoglobin E.

BACKGROUND AND OBJECTIVES
Hemoglobin E is a variant hemoglobin caused due to the base substitution G→A at codon 26 in the β-globin-coding gene that is followed by the alteration of glutamic acid (GAG) to lysine (AAG). Various types of molecular analysis methods such as tetra-primer amplification refractory mutation system (T-ARMS-PCR), Tm-shift real-time polymerase chain reaction (Tm-shift qPCR) and high-resolution melting analysis (HRMA) are commonly used to detect several mutations in the β-globin-coding gene. This study was conducted to compare the detection result of Cd 26 (G→A) mutation in the β-globin-coding gene of heterozygous HbE between the above-mentioned methods.


MATERIALS AND METHODS
DNA samples were isolated from blood archive of heterozygous HbE and analyzed for the detection of the mutation using HRMA and Tm-shift on a real-time PCR instrument, whereas T-ARMS analysis was performed on a conventional PCR equipment. High resolution melt v3.1 software and Bio-Rad CFX Manager software were used to analyze the result of HRMA and Tm-shift qPCR, whereas the T-ARMS-PCR result was analyzed by observing the number and size of DNA bands on gel electrophoresis.


RESULTS
Among 21 samples, the Cd 26 mutation was detected in numbers 18, 19 and 21 by HRMA, Tm-shift qPCR and T-ARMS-PCR. DNA Sequencing confirmed Cd 26 mutation on 5 ambiguous samples and revealed two homozygous mutation.


CONCLUSION
The Cd 26 (G→A) mutation was detected in proportions 100, 91 and 86% by T-ARMS-PCR, Tm-shift qPCR and HRMA, respectively.