Rapid Identification of β-Thalassemia, Hb E, and High Hb F Determinants Using a High-Resolution Melting Analysis: Application in Prenatal Diagnosis in Southern Thailand

β-thalassemia (thal), hemoglobin (Hb) E, and high Hb F determinants, which are caused by mutations in the β-globin gene cluster, are common genetic disorders in Thailand and Southeast Asia. Prenatal diagnosis is essential for couples at risk to identify severe forms, including homozygous β-thal and Hb E/β-thal. Conventional methods, including reverse dot-blot hybridization and gap-polymerase chain reaction (PCR) for genotyping of point and large deletion mutations, require post-PCR steps, which are time-consuming and costly. This study aimed to develop a rapid and efficient method using monoplex high-resolution melting (HRM) analysis for genotyping of Hb E and 11 β-thal mutations; multiplex HRM analysis for identifying six deletional mutations, including two β⁰-thal mutations (3.5 and 45 kb deletion); and a novel method for detecting four high Hb F determinants, namely, δβ⁰-thal (12.5 kb deletion), HPFH6, Indian inv-del (^Aγδβ)⁰-thal, and Thai del-inv-ins (^Aγδβ)⁰-thal. The developed assays were validated using 182 blinded fetal DNA samples with 41 β-thal genotypes. Different HRM patterns were observed among wild-type, heterozygote, homozygote, and compound heterozygote genotypes. Six deletional mutations showed specific melt curves. This technique demonstrated 100% concordance with conventional methods. The assay showed 100% sensitivity, specificity, and positive and negative predictive values within the limit of detection at DNA concentrations of 8.0 ng/reaction. Finally, this developed assay was efficient in identifying both point mutations and large deletion, convenient, rapid, and cost-effective and did not require post-PCR steps. Thus, this technique has potential for application in prenatal diagnosis of thal and can inform prevention and control programs.