Diagnosis of α⁰-thalassemia Chiang Rai (‐‐^CR) deletion by melt curve analysis in Northern Thailand

Abstract

A large novel 44.6 kb deletion named α⁰-thalassemia Chiang Rai (--^CR) was first described in the individuals with uncommon Hb Bart’s hydrops fetalis and HbH disease. This study aimed to develop a real-time gap PCR and melt curve analysis for the detection of --^CR and investigate its frequency in northern Thailand. Among 4,952 blood samples, the assay was performed in 525 samples with a mean corpuscular volume (MCV) < 80 fL, HbA₂ < 3.5%, HbA₂+E < 25%, and negative for common deletional α⁰-thalassemia --^SEA and --^THAI. The developed method showed T_m values of 85.8 ± 0.0 °C and 91.5 ± 0.1 °C, which were specific for --^CR and wild-type alleles, respectively. Nine (0.18% of 4,952 or 1.71% of 525) were positive for --^CR, in which two were HbH disease and the rest were heterozygous for --^CR. This study demonstrated the success of real-time gap PCR with melt curve analysis for --^CR diagnosis. Additionally, the prevalence of --^CR in the northern Thai population was comparable to --^THAI. Thus, this study implies the importance of --^CR in northern Thailand. Moreover, the developed real-time gap PCR with melt curve analysis is simple and highly accurate, and may be considered as an additional tool for routine α⁰-thalassemia --^CR diagnosis in this region.

Keywords:

• α⁰-thalassemia
• Hb Bart’s hydrops fetalis
• melt curve analysis
• Chiang Rai deletion
• northern Thailand

Ethical approval

This study was approved by the Ethics Committee of the Faculty of Associated Medical Sciences, Chiang Mai University, Chiang Mai, Thailand (AMSEC-63EM-015).

Author contributions

C.R., P.K., and S.P. designed the research. C.R., P.K. and M.P. performed DNA extraction. C.R. and M.P. performed Hb analysis. C.R., P.K., M.P., and P.P. performed real-time gap PCR and melt curve analysis. C.R., P.K., P.P., and S.P. interpreted the results. P.K. and S.P. wrote the first manuscript draft. S.P. conceptualized the idea of this research project. All authors have read and approved the final version of the manuscript for publication.

Disclosure statement

No potential conflict of interest was reported by the author(s).

Data availability statement

Data supporting the findings of this study are available from the corresponding author on reasonable request.

Additional information

Funding

C.R. was supported by grants from the Teaching Assistant and Research Assistant (TA/RA) scholarships, Graduate School, Chiang Mai University, Chiang Mai, Thailand. P.P. was supported by grants from Faculty of Associated Medical Sciences, Chiang Mai University, Chiang Mai, Thailand.