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

Area of Science:

• Genetics
• Molecular Biology
• Hematology

Background:

• Conventional thalassemia diagnostic methods like Gap-PCR and MMCA assay have limitations in detecting rare mutations.
• Accurate diagnosis of alpha-globin gene cluster variations is crucial for understanding thalassemia phenotypes.

Purpose of the Study:

• To evaluate the efficacy of single-molecule real-time (SMRT) sequencing combined with multiplex long-range PCR for identifying rare alpha-globin gene mutations.
• To determine the configuration (cis or trans) of alpha-globin gene defects in a single test.

Main Methods:

• Applied SMRT sequencing following multiplex long-range PCR on nine patients and their family members.
• Included patients with discordant results between Gap-PCR and MMCA assay or phenotype-genotype mismatch.
• Utilized SMRT sequencing to identify various rare mutations, gene rearrangements, insertions, and duplications.

Main Results:

• Identified carriers with rare alpha-globin gene mutations including αααanti3.7/HKαα, -α762bpα/αα, and ααfusion/αQSα in trans configuration.
• Discovered novel gene rearrangements, a 341 bp insertion, and alpha-globin gene duplications.
• Successfully determined the cis or trans configuration of alpha-globin gene cluster defects in a single test.

Conclusions:

• SMRT sequencing demonstrates significant advantages in detecting alpha-globin gene triplications, rare deletions, and determining cis/trans configurations.
• SMRT sequencing provides a comprehensive, one-step method for thalassemia screening and diagnosis, particularly for rare mutations.