Single-molecule real-time (SMRT) sequencing technology shows advantages in the clinical diagnosis of complex thalassemia

Abstract

Thalassemia is a hereditary hemolytic anemia caused by pathogenic variants in the α- or β-globin genes, resulting in imbalanced globin chain synthesis. Clinical manifestations vary from asymptomatic carrier states to severe transfusion-dependent anemia. Current routine diagnostic methods in China, gap polymerase chain reaction (gap-PCR) and reverse dot blot hybridization (RDB), detect only 24 common variants, leading to frequent genotype-phenotype mismatches and undiagnosed cases. In this study, we employed multiplex ligation-dependent probe amplification (MLPA), Sanger sequencing, and SMRT sequencing to genetically screen 30 cases of complex thalassemia, aiming to clarify the underlying genetic causes. SMRT sequencing successfully resolved 28/30 cases (93.3%), with genotypes confirmed by MLPA and Sanger sequencing. Notably, three novel deletions were identified: a 32.8-kb (-- 32.8 , chr16:149,860–182,697) and a 5.9-kb (chr16:169,916–175,863) deletion in the α-globin gene cluster, and an 8.3-kb deletion (chr11:5,224,209–5,232,469) in the β-globin gene cluster. In addition to detecting rare single-nucleotide variants (SNVs) and large deletions, SMRT sequencing additionally identified complex structural variants that are often missed or difficult to characterize using MLPA and Sanger sequencing. Our findings demonstrate that SMRT sequencing offers superior resolution and diagnostic power for complex thalassemia and is a valuable tool for improving molecular diagnosis in challenging cases.