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

Introduction: The alpha-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 alpha(0)-thalassemia (alpha(0)-thal). Due to its uncommon rearrangement in the a gene cluster without dosage changes, this fusion gene is undetectable by common molecular testing approaches used for alpha-that 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 alpha and beta thalassemia were utilized. Results: According to the NGS results, the 23 carriers include 14 pure heterozygotes, eight compound heterozygotes with common alpha-that 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 alpha fusion gene, attributing to its efficient, accurate, and onestep properties.