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

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.

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.

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.

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.