Detection of Hb H Disease Genotypes Common in Northern Thailand by Quantitative Real-Time Polymerase Chain Reaction and High Resolution Melting Analyses

Abstract

We used quantitative real-time polymerase chain reaction (qPCR) and high resolution melting (HRM) analyses for the detection of the common α-thalassemia (α-thal) genotypes in 40 northern Thai Hb H (β4) patients. The α⁰-thal [– –^SEA (Southeast Asian) deletion] was detected by a multiplex gap real-time PCR. To determine the α⁺-thal, three primer pairs were designed. The A-primer pair was used to amplify the 3' terminal DNA sequences of the HBA2 gene, and the B-primer pair amplified the 5' flanking region of the HBA1 gene. The C-primer pair amplified the 3' terminal DNA sequences of the HBA1 gene and was used as an internal control. The –α^4.2 (leftward) and –α^3.7 (rightward) deletions were determined by monitoring the absence of PCR product(s). The Hb H patients who had a negative PCR result for the A-primer pair but positive for the B- and C-primer pairs carried the –α^4.2 deletion, while the –α^3.7 deletion carriers were negative for the A- and B-primer pairs. In the case of Hb H with Hb Constant Spring (Hb CS, α142, Term→Gln; HBA2: c.427T>C), all primer pairs were positive, HRM analysis of the PCR product of the A-primer pair was introduced to analyze the Hb CS gene. It can distinguish clearly between normal and Hb CS genotypes. The applied methods for the determination of the α⁰- and α⁺-thal genotypes revealed the results to be as accurate as conventional gap-PCR and the direct DNA sequencing methods but resulted in a much simpler and faster procedure.

• Hb H (β4) disease
• Quantitative real-time polymerase chain reaction (qPCR)
• High resolution melting (HRM) analysis

ACKNOWLEDGMENTS

We thank Professor Dr. Jurgen Horst, University Münster, Münster, Germany, for reading the manuscript and helpful discussions. We also thank the medical technicians at the Thalassaemia Laboratory, Chiang Mai University Hospital, Chiang Mai, Thailand, for their assistance.

Declaration of Interest: This study was supported by a grant from the Faculty of Medical Science and the Faculty of Science, Naresuan University, Phitsanulok, Thailand. The authors report no conflicts of interest. The authors alone are responsible for the content and writing of this article.