Detection of Common α-Hemoglobin Variants in Thailand by Using Real-Time PCR with High Resolution Melting Analysis.

α-Hemoglobin (Hb) variants are common genetic mutations worldwide. The appropriate techniques must be followed to scan and identify these variants, particularly in routine investigations for thalassemia and hemoglobinopathies in countries with high prevalence of α and β-thalassemia. High resolution melting (HRM) analysis is a popular and effective technique for identifying genetic variations with rapid output results. This study designed four newly developed primer pairs that had full coverage of the HBA genes for detection of α-Hb variants using real-time PCR with HRM analysis. Forty-one blood samples were collected from individuals with known or suspected α-Hb variants. The results demonstrated clearly distinguished melting patterns of nine α-Hb variants including Hb Constant Spring, Hb Q-Thailand, Hb Pakse', Hb Hekinan, Hb Nakhon Ratchasima, Hb Siam, Hb Thailand, Hb Queens, and Hb Quong Sze compared with the wild-type sample pattern. All mutations were confirmed by DNA nucleotide sequencing. This study presents the first case report of the combination of Hb Shaare Zedek co-inherited with Hb Hekinan in a Thai patient. Interactions between these two Hb variants displayed a high level of Hb F (23.5%) on an HPLC Hb chromatogram and a mild symptom phenotype with low mean corpuscular volume (71.3 fL) and mean corpuscular hemoglobin (21.8 pg) in the proband. Overall, HRM analysis is a suitable, rapid, and powerful technique for the identification of gene mutations, and for the diagnosis of common and rare α-Hb variants to prevent and control thalassemia in Thailand.