Molecular spectrum and prevalence of thalassemia investigated by third-generation sequencing in the Dongguan region of Guangdong Province, Southern China

Background: PCR, Sanger sequencing and NGS are often employed for carrier screening of thalassemia but all of these methods have limitations. In this study, we evaluated a new third-generation sequencing-based approach termed comprehensive analysis of thalassemia alleles (CATSA) to explore the prevalence of thalassemia in the Dongguan region of southern China.Methods: 19,932 subjects were recruited for thalassemia screening and hemoglobin testing was performed for each of them. Routine PCR was performed for all the hemoglobin testing-positive subjects and CATSA was conducted for randomly selected subjects from hemoglobin testing-positive and negative subjects.Results: In the 2716 subjects tested both by PCR and CATSA, 2569 had the same results and 147 had discordant results between the two methods. Sanger sequencing, specially designed PCR and MLPA confirmed the results of CATSA were all correct. In total, CATSA correctly detected 787 subjects with variants while routine PCR correctly detected 640 subjects with variants. CATSA yielded a 5.42% (147 of 2716) increment compared with routine PCR. In the 447 hemoglobin testing-negative subjects, CATSA identified pathogenic variants in 12 subjects. Moreover, CATSA identified a novel deletion (chr16:171262-202032) in the alpha-globin gene cluster. As a result, the deduced carrier frequency of alpha-thalassemia,beta-thalassemia and alpha-/beta-thalassemia was 5.62%, 3.85% and 0.93%, respectively.Conclusions: Our study demonstrated CATSA was a more comprehensive and precise approach than the routine PCR in a large scale of samples, which is highly beneficial for carrier screening of thalassemia. It provided a broader molecular spectrum of hemoglobinopathies and a better basis for a control program in Dongguan region.