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

Pinyaphat Khamphikham; Oravee Hanmanoviriya; Somsakul Pop Wongpalee; Thongperm Munkongdee; Kittiphong Paiboonsukwong; Yupin Jopang; Chaowanee Wangchauy; Charan Sancharernsook; Nathawat Jinorose; Sakorn Pornprasert

doi:10.1111/ahg.12496

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

Ann Hum Genet. 2023 May;87(3):137-145. doi: 10.1111/ahg.12496. Epub 2023 Jan 29.

Affiliations

¹ Division of Clinical Microscopy, Department of Medical Technology, Faculty of Associated Medical Sciences, Chiang Mai University, Chiang Mai, Thailand.
² Hematology and Health Technology Research Center, Department of Medical Technology, Faculty of Associated Medical Sciences, Chiang Mai University, Chiang Mai, Thailand.
³ Department of Microbiology, Faculty of Medicine, Chiang Mai University, Chiang Mai, Thailand.
⁴ Thalassemia Research Center, Institute of Molecular Biosciences, Mahidol University, Nakhon Pathom, Thailand.
⁵ Regional Health Promotion Center 9 Nakhon Ratchasima, Department of Health, Ministry of Public Health, Nakhon Ratchasima, Thailand.
⁶ Hematology Unit, Department of Pathology, Faculty of Medicine, Prince of Songkla University, Songkhla, Thailand.
⁷ Department of Medical Technology, Sunpasitthiprasong Hospital, Ubon Ratchathani, Thailand.
⁸ Department of Medical Technology, Phayao Hospital, Phayao, Thailand.

PMID: 36709419
DOI: 10.1111/ahg.12496

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.

Keywords: Bart's hydrops fetalis; Chiang Rai deletion; microcytic anemia; severe thalassemia; α-thalassemia.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Erythrocytes
Female
Humans
Hydrops Fetalis / genetics
Pathology, Molecular
Thailand
alpha-Thalassemia* / diagnosis
alpha-Thalassemia* / genetics