Detection of rare thalassemia mutations using long-read single-molecule real-time sequencing

Gap- polymerase chain reaction (PCR), reverse dot-blot assay (RDB), real-time PCR based multicolor melting curve analysis (MMCA assay), multiplex ligation-dependent probe amplification (MLPA) and Sanger sequencing are conventional methods to diagnose thalassemia but all of them have limitations. In this study, we applied single-molecule real-time (SMRT) sequencing following multiplex long-range PCR to uncover rare mutations in nine patients and their family members. The patients with different results between Gap-PCR and MMCA assay or with phenotype not matching genotype were included. Using SMRT sequencing, we first identified the carriers with alpha alpha alpha(anti3.7)/HK alpha alpha, -alpha(762bp)alpha/alpha alpha (chr16:172,648-173,409), alpha alpha(fusion)/alpha(QS)alpha (in a trans configuration), two cases with novel gene rearrangements and another case with a novel 341 bp insertion in alpha-globin gene cluster, respectively. One carrier with -(SEA)/alpha alpha alpha(anti4.2), and two carriers with the coexistence of globin variant and an alpha-globin gene duplication were also found. Most importantly, we could determine two defects in alpha-globin gene cluster being a cis or trans configuration in a single test. Our results showed that SMRT has great advantages in detection of alpha-globin gene triplication, rare deletions and determination of a cis or trans configuration. SMRT is a comprehensive and one-step method for thalassemia screening and diagnosis, especially for detection of rare thalassemia mutations.

Automated summary

In this study, we utilized single-molecule real-time (SMRT) sequencing to uncover rare thalassemia mutations in nine patients and their family members. The results demonstrate the importance of SMRT sequencing in thalassemia screening and diagnosis, particularly for detecting rare mutations.