Comparison between three molecular diagnostics for the identification of heterozygous hemoglobin E

Abstract

Background and Objectives: Hemoglobin E is a variant hemoglobin caused due to the base substitution G6A at codon 26 in the $-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 $-globin-coding gene. This study was conducted to compare the detection result of Cd 26 (G6A) mutation in the $-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 (G6A) mutation was detected in proportions 100, 91 and 86% by T-ARMS-PCR, Tm-shift qPCR and HRMA, respectively.