The value of single-molecule real-time technology in the diagnosis of rare thalassemia variants and analysis of phenotype–genotype correlation

To compare single-molecule real-time technology (SMRT) and conventional genetic diagnostic technology of rare types of thalassemia mutations, and to analyze the molecular characteristics and phenotypes of rare thalassemia gene variants, we used 434 cases with positive hematology screening as the cohort, then used SMRT technology and conventional gene diagnosis technology [(Gap-PCR, multiple ligation probe amplification technology (MLPA), PCR-reverse dot blot (RDB)] for thalassemia gene screening. Among the 434 enrolled cases, conventional technology identified 318 patients with variants (73.27%) and 116 patients without variants (26.73%), SMRT identified 361 patients with variants (83.18%), and 73 patients without variants (16.82%). The positive detection rate of SMRT was 9.91% higher than conventional technology. Combination of the two methods identified 485 positive alleles among 49 types of variant. The genotypes of 354 cases were concordant between the two methods, while 80 cases were discordant. Among the 80 cases, 76 cases had variants only identified in SMRT method, 3 cases had variants only identified in conventional method, and 1 false positive result by the traditional PCR detection technology. Except the three variants in HS40 and HBG1-HBG2 loci, which was beyond the design of SMRT method in this study, all the other discordant variants identified by SMRT were validated by further Sanger sequencing or MLPA. The hematological phenotypic parameters of 80 discordant cases were also analyzed. SMRT technology increased the positive detection rate of thalassemia genes, and detected rare thalassemia cases with variable phenotypes, which had great significance for clinical thalassemia gene screening.