Abstract

The aim of the present study was to determine the phenotype and genotype frequency of the most common β-globin mutations among the thalassemia major (TM) patients, using high-resolution melting (HRM) curve analysis as a novel, reliable and rapid scanning method. In this descriptive-analytical study, real time polymerase chain reaction (PCR) was applied besides HRM assay in the same reaction tube for mutation detection by melting curve behavior analysis of EvaGreen™ dye. The phenotypic and genotypic frequency of β-globin mutations between 120 patients including IVS-II-1(G-A), IVS-I-110 (G-A), IVS-I-5(G-C), FSC 8/9, FSC 36/37, Codon 30, IVS-I-6 (T-C) and IVS-I-1(G-A) was successfully detected. TM patients showed significant genotype heterogeneity. The clinical outcomes of our TM patients were mostly explained by the genotypes linked to the β⁰ type of β- tahalssemia. Genotypic analysis showed 70 patients with homozygous mutations TM (40 β⁰/ β⁰ and 30 β⁺/ β⁺) and 15 with compound heterozygous TM (14 β⁺/ β⁰ and 1 β⁰/Hb Variant). HRM method as a simpler and cost effective way, accurately characterized the molecular basis of the TM patients.

Key words: β-Globin, β-thalassemia major, real time polymerase chain reaction, high-resolution melting analysis, melting curve, direct sequencing, single nucleotide polymorphism.

Abbreviation

HBB, Hemoglobin beta; HRM, High-Resolution Melting; Hb, Hemoglobin; Tm, Temperature melting; PCR, Polymerase Chain Reaction; SNP, Single Nucleotide Polymorphism; ARMS, Amplification Refractory Mutation System, ARMS; IVS, Intervening sequence; FSC, Frame shift; Cd, codon; TM, Tahalssemia Major