The α -thalassemia 44.6 kb or Chiang Rai (– ) deletion has been reported in northern Thailand and is capable of causing hemoglobin (Hb) H disease and a lethal α-thalassemia genotype, Hb Bart’s hydrops fetalis, in this region. However, there are no current data regarding the frequency of — nationwide due to a lack of effective diagnostic assay. Therefore, this study aimed to develop a reliable platform for simultaneous genotyping of — and two common α -thalassemias in Thailand (– and — ) and investigate the frequency of — across Thailand.
Multiplex gap-PCR assay and five renewable plasmid DNA controls for — , — , — , α2-globin (HBA2), and β-actin (ACTB) were newly developed and validated with reference methods. The developed assay was further tested on 1046 unrelated individuals with a reduced mean corpuscular volume (MCV) of less than 75 fl for investigating genotypic and allelic spectrum of — .
Our developed assay showed 100% concordance with reference methods. The results were valid and reproducible throughout hundreds of reactions. Comparison of the genotypic and allelic spectra revealed that heterozygous — (– /αα) and — alleles were dominant with the frequency of 22.85% (239/1046) and 13.34% (279/2092), respectively. Of these, — and — were relatively rare in this population and comparable to each other with the allelic frequency of 0.14% (3/2092).
This study successfully established a reliable molecular diagnostic platform for genotyping of — , — , and — in a single reaction. Additionally, we demonstrated the frequency of — in Thailand for the first time and provided knowledge basis for the planning of severe α-thalassemia prevention and control programs in Thailand, where thalassemia is endemic.

© 2023 John Wiley & Sons Ltd/University College London.