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

Area of Science:

• Molecular Biology
• Genetics
• Biochemistry

Background:

• Hemoglobin E (HbE) is a prevalent hemoglobin variant caused by a specific G→A base substitution at codon 26 of the beta-globin gene.
• This mutation results in the alteration of glutamic acid to lysine, impacting hemoglobin function.
• Accurate detection of the HbE mutation is crucial for genetic counseling and clinical management.

Purpose of the Study:

• To compare the diagnostic performance of three molecular methods for detecting the codon 26 G→A mutation associated with HbE.
• To evaluate the efficacy of 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) in identifying heterozygous HbE.

Main Methods:

• DNA samples from individuals with heterozygous HbE were analyzed using HRMA and Tm-shift qPCR on a real-time PCR instrument.
• T-ARMS analysis was performed using conventional PCR equipment.
• Results were analyzed using specialized software for HRMA and Tm-shift qPCR, and gel electrophoresis for T-ARMS-PCR.

Main Results:

• T-ARMS-PCR successfully detected the codon 26 mutation in 100% of the analyzed samples.
• Tm-shift qPCR detected the mutation in 91% of samples, while HRMA detected it in 86% of samples.
• DNA sequencing confirmed the mutation in ambiguous samples and identified two homozygous cases.

Conclusions:

• T-ARMS-PCR demonstrated the highest sensitivity and accuracy for detecting the HbE codon 26 mutation among the tested methods.
• Tm-shift qPCR and HRMA are viable alternatives but showed slightly lower detection rates in this study.
• The findings support the use of T-ARMS-PCR as a reliable method for HbE mutation screening.