Rapid Identification of Common β-Thalassemia Mutations in the Chinese Population Using Duplex or Triplex Amplicon Genotyping by High-Resolution Melting Analysis

Abstract

β-Thalassemia is one of the most prevalent inherited diseases in China. To date, over 20 β-thalassemia mutations have been identified in the Chinese population, and four mutations [CD41-42 (-4 bp), IVS-2-654C→T, CD17A→T, and -28A→G] account for approximately 90% of the cases. Therefore, the exploration of simple, reliable, and rapid approaches for molecular detection of these common mutations is important for prevention and early diagnosis of the disease. High-resolution melting (HRM) analysis is a new technique for mutation detection that has the advantages of rapidity, accuracy, and convenience. Building on one-amplicon genotyping by HRM analysis, we developed duplex and triplex amplicon genotyping to simultaneously identify these common β-thalassemia mutations in patients or carriers. Two or three sets of primers were combined to conduct duplex or triplex amplicon genotyping, which distinguished a variety of genotypes by HRM based on the melting curve shapes. Seventy-one DNA samples from β-thalassemia traits or patients were analyzed using the described approaches and 65 were identified to carry the 4 common β-thalassemia alleles including 56 heterozygous mutations [23 for CD41-42 (-4 bp), 18 for IVS-2-654C→T, 11 for CD17A→T, and 4 for -28A→G], 3 homozygous mutations for IVS-2-654C→T, and 6 compound heterozygous mutations [CD41-42 (-4 bp)/IVS-2-654C→T (4 cases), -28A→G/CD17A→T (1 case), IVS-2-654C→T/CD17A→T (1 case)]. The whole procedure for mutation detection was completed within only half an hour. The results derived from HRM analysis were fully in accordance with sequencing. We suggest this rapid and accurate method for molecular screening to detect the common β-thalassemia mutations in the Chinese population as well as in other ethnic groups and nationalities in which the above four β-thalassemia alleles are prevalent.