High accuracy of single‐molecule real‐time sequencing in detecting a rare α‐globin fusion gene in carrier screening population

Abstract

Introduction The α‐globin fusion gene between the HBA2 and HBAP1 genes becomes clinically important in thalassemia screening because this fusion gene can cause severe hemoglobin (Hb) H disease when combining with α0‐thalassemia (α0‐thal). Due to its uncommon rearrangement in the α gene cluster without dosage changes, this fusion gene is undetectable by common molecular testing approaches used for α‐thal diagnosis. Methods In this study, we used the single‐molecule real‐time (SMRT) sequencing technique to detect this fusion gene in 23 carriers identified by next‐generation sequencing (NGS) among 16,504 screened individuals. Five primers for α and β thalassemia were utilized. Results According to the NGS results, the 23 carriers include 14 pure heterozygotes, eight compound heterozygotes with common α‐thal alleles, and one homozygote. By using SMRT, the fusion mutant was successfully detected in all 23 carriers. Furthermore, SMRT corrected the diagnosis in two “pure” heterozygotes: one was compound heterozygote with anti‐3.7 triplication, and the other was homozygote. Conclusion Our results indicate that SMRT is a superior method compared to NGS in detecting the α fusion gene, attributing to its efficient, accurate, and one‐step properties.

Third generation sequencing transforms the way of the screening and diagnosis of thalassemia: a mini-review

“…Compared to NGS, recent studies with larger sample sizes indicate that the detection rate of TGS is higher, especially for deletion variants and heterozygotes ( 16 , 17 , 23 ). This is due to the significant advantage of TGS in read length, which is much longer than that of NGS, making it easier to detect deletion variants in thalassemia genes ( 23 ).…”