Development of molecular diagnostic platform for alpha(0)-thalassemia 44.6 kb (Chiang Rai, --(CR)) deletion in individuals with microcytic red blood cells across Thailand

Introduction: The alpha(0)-thalassemia 44.6 kb or Chiang Rai (--(CR)) deletion has been reported in northern Thailand and is capable of causing hemoglobin (Hb) H disease and a lethal alpha-thalassemia genotype, Hb Bart's hydrops fetalis, in this region. However, there are no current data regarding the frequency of --(CR) nationwide due to a lack of effective diagnostic assay. Therefore, this study aimed to develop a reliable platform for simultaneous genotyping of --(CR) and two common alpha(0)-thalassemias in Thailand (--(SEA) and --(THAI)) and investigate the frequency of --(CR) across Thailand.Methods: Multiplex gap-PCR assay and five renewable plasmid DNA controls for --(CR), --(SEA), --(THAI), alpha 2-globin (HBA2), and beta-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 --(CR).Results: 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 --(SEA) (--(SEA)/alpha alpha) and --(SEA) alleles were dominant with the frequency of 22.85% (239/1046) and 13.34% (279/2092), respectively. Of these, --(THAI) and --(CR) were relatively rare in this population and comparable to each other with the allelic frequency of 0.14% (3/2092).Conclusion: This study successfully established a reliable molecular diagnostic platform for genotyping of --(CR), --(SEA), and --(THAI) in a single reaction. Additionally, we demonstrated the frequency of --(CR) in Thailand for the first time and provided knowledge basis for the planning of severe alpha-thalassemia prevention and control programs in Thailand, where thalassemia is endemic.