Multiplex Quantitative Real-Time Polymerase Chain Reaction and High-Resolution Melting Analysis for Identification of a Couple At-Risk of Having a Newborn with Severe Thalassemia

Abstract

Many polymerase chain reaction (PCR)-based techniques have been used for routine diagnosis of α- and β-thalassemias. However, most require a multi step of post-PCR processes that are time-consuming and labor-intensive procedures. This study reported the successful use of multiplex quantitative real-time PCR (qPCR), with high-resolution melting (HRM) analysis for diagnosis of two common deletional α⁰-thalassemia (α⁰-thal) and 15 common β-thalassemia (β-thal) mutations, in order to identify a couple at-risk of having a newborn with severe thalassemia in the northern region of Thailand. With this approach, 22 (7.2%) of 306 couples were diagnosed as being at-risk for having a child with severe thalassemia, including three homozygous α⁰-thal, five homozygous β-thal and 14 Hb E (HBB: c.79G>A)/β⁰-thal disease. Our findings indicated that multiplex qPCR with HRM is applicable for routine molecular diagnosis in order to identify a couple at-risk of having a newborn with severe thalassemia, especially in an endemic region.

Keywords:

• β-Thalassemia major (β-TM)
• Hb Bart’s hydrops fetalis
• Hb E/β-thal disease
• high-resolution melting (HRM) analysis
• thalassemia

Acknowledgements

The authors would like to thank medical technicians and nurses at the 15 district hospitals for their help and assistance.

Author contributions

C. Ruengdit and M. Punyamung performed all the experiments. C. Ruengdit, M. Punyamung, P. Khamphikham, P. Pongpunyayuen, N. Intasai and S. Pornprasert analyzed data and interpreted the results. C. Ruengdit, P. Khamphikham and S. Pornprasert wrote the manuscript. S. Pornprasert conceptualized the research idea. All authors have read and approved the final manuscript.

Disclosure statement

The authors report no conflicts of interest. The authors alone are responsible for the content and writing of this article.

Data availability

The data that support the findings of this study are available from the corresponding author upon reasonable request.