Association of Hb A₂ Variants with Several Forms of α- and β-Thalassemia in Thailand

In this study, Hb A₂ variants and their association with α- and β-thalassemia (α- and β-thal) were analyzed. We performed molecular analyses to identify α-thal [- -^SEA (Southeast Asian), - -^THAI (Thai), -α^3.7 (rightward) and -α^4.2 (leftward)] deletions, and Hb Constant Spring (Hb CS; HBA2: c.427T>C), Hb A₂-Melbourne (HBD: c.130G>A), Hb A₂' (HBD: c.49G>C), Hb A₂-Lampang (HBD: c.142G>A). β⁰-Thalassemia mutations included codon 17 (A>T) (HBB: c.52A>T), codons 41/42 (-TCTT) (HBB: c.126_129delCTTT), codons 71/72 (+A) (HBB: c.216_217insA) and IVS-I-1 (G>T) (HBB: c.92+1G>T) in 23 samples which had a Hb A₂ variant peak in zone 1 of the capillary electrophoresis (CE) electropherogram. Results showed that 20 patients (87.0%) carried Hb A₂-Melbourne with seven different genotypes for α- and β-thal, two (8.7%) carried Hb A₂' and one (4.3%) carried Hb A₂-Lampang. All three samples doubly heterozygous for Hb A₂-Melbourne/β⁰-thal had Hb A₂ levels lower than 4.0%, while summation of Hb A₂ and Hb A₂-Melbourne ranged from 4.9-5.3%, reaching the accepted range (4.0-10.0%) for β-thal trait. Hb A₂-Melbourne is the most common δ-globin variant in the Thai population. Hb A₂ variant and Hb A₂ levels must be combined in order to diagnose carriers of β-thal. β-Globin haplotype analysis showed an association with a single β-globin haplotype [+ - - - - + +] of Hb A₂-Melbourne, Hb A₂' and Hb A₂-Lampang, indicating that they were of the same origin. We developed a multiplex allele-specific polymerase chain reaction (ASPCR) for simultaneous detection of these three Hb A₂ variants.