Development of molecular diagnostic platform for α0-thalassemia 44.6 kb (Chiang Rai, --CR) deletion in individuals with microcytic red blood cells across Thailand

Abstract

Introduction

The α⁰-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 α-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 α⁰-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, 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 --^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/αα) 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 α-thalassemia prevention and control programs in Thailand, where thalassemia is endemic.