Single-Molecule Real-Time Sequencing Reveals Hidden Diversity and Corrects Misdiagnosis in α-Thalassemia.

Abstract

Objectives: Conventional methods for α-thalassemia gene diagnosis, such as Gap-PCR, are primarily designed for common deletions. However, their limitations in detecting complex rearrangements that maintain the total copy number of α-globin genes can lead to misdiagnosis, as exemplified by the misinterpretation of "-α^3.7 heterozygous" carriers. This study aimed to evaluate the application of single-molecule real-time (SMRT) sequencing in addressing these diagnostic challenges.

Materials and methods: From January 2023 to April 2025, 9180 individuals from Dongguan were enrolled. Peripheral blood samples underwent routine thalassemia genetic testing. Samples identified as -α^3.7/αα by Gap-PCR were further analyzed using multiplex ligation-dependent probe amplification (MLPA) and SMRT sequencing.

Results: SMRT sequencing demonstrated high precision in distinguishing genotypes that are difficult to differentiate by traditional methods. It accurately identified benign variants like HKαα and pathogenic triplications such as ααα^anti4.2, which is crucial for assessing the risk of offspring developing intermediate β-thalassemia. Furthermore, the technology successfully revealed "hidden" balanced complex rearrangements (e.g., -α^3.7/ααα^anti3.7) within the blind spots of conventional techniques. Notably, a specific genotype, -α^3.7/αα12, was reported for the first time in the Chinese population. Additionally, the sequencing process concurrently enabled the detection of point variants, achieving a comprehensive "one-stop" assessment.

Conclusion: This study demonstrates that SMRT sequencing technology, leveraging its advantages of long reads, single-molecule resolution, and haplotype phasing, can significantly improve the diagnostic accuracy for complex α-thalassemia genotypes. It effectively corrects misdiagnoses and omissions associated with conventional methods, thereby providing a reliable basis for precise genetic counseling and risk assessment.