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.