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

AbstractIntroductionThe 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.MethodsMultiplex 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.ResultsOur 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).ConclusionThis 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.