Enhancing Thalassemia Diagnosis: Advantages of Third-Generation Sequencing.

Background: This study aimed to evaluate the efficacy of third-generation sequencing (TGS) and a thalassemia (Thal) gene diagnostic kit in identifying Thal gene mutations.

Methods: Blood samples (n = 119) with positive hematology screening results were tested using polymerase chain reaction (PCR)-based methods and TGS on the PacBio-Sequel-II-platform, respectively.

Results: Out of the 119 cases, 106 cases showed fully consistent results between the two methods, with TGS identified HBA1/2 and HBB gene mutations in 82 individuals. Notably, TGS exhibited a 5.04% higher detection rate compared to PCR-based methods (68.91% vs. 63.87%). For HBA1/2 mutations, TGS accurately detected three types of rare HBA1/2 mutations (--THAI, HBA2:c.34A>C, and HBA1:c.354_355insATC), two types of rare HBA compound mutations (ɑWSɑ/ɑCap+23(C>G)ɑ and -ɑ3.7/ɑIVS-Ⅱ-34ɑ), and three rare triplicates of α-globin variants (ɑɑ/ɑɑɑanti3.7, --SEA/HKɑɑ, and ɑɑ/ɑɑɑanti4.2). For the HBB gene, TGS detected two rare HBB mutations, namely HBB:c.316-45G>C and HBB:c.170G>A. For these 13 cases of rare thalassemia gene mutations, most patients exhibited varying degrees of microcytic hypochromia. However, patients with mutation in HBA2:c.34A>C, HBA1:c.354_355insATC, and HBB:c.170G>A did not exhibit typical results in blood routine tests but had abnormal hemoglobin composition. Additionally, TGS confirmed the cis/trans configuration of 2 allelic gene mutations in one step.

Conclusions: Compared to traditional genotyping methods, TGS increased the detection rate of positive HB gene mutations and identified rare Thal cases with variable phenotypes. For Thal screening, it is recommended to perform both blood routine tests and hemoglobin electrophoresis, combined with TGS, to minimize the risk of missed or incorrect diagnoses in clinical practice. Although TGS is currently more expensive than other methods, it pro-vides a comprehensive approach for Thal screening and clinical diagnosis, particularly for rare Thal variants. As sequencing throughput increases and costs decrease, TGS can be widely applied in the screening of genetic diseases.