Molecular analysis
Genomic DNA was extracted from peripheral blood leukocytes using a standard protocol of phenol-chloroform extraction. Alpha-globin genotyping was performed by a single-tube multiplex gap polymerase chain reaction (Gap-PCR) for detecting seven common α-globin deletions (–SEA, –THAI, -(α)20.5, –FIL, –MED3.7, -α4.2) and a single-tube multiplex amplification refractory mutation system (ARMS-PCR) for screening six common non-deletional α-globin mutations in Thailand: initiation codon (ATGA-G), codon 30 (ΔGAG), codon 59 (GGCGAC), codon 125 (CTGCCG) or Hb QuangSze, termination codon (TAACAA) or Hb Constant Spring, and a termination codon (TAATAT) or Hb Paksé.31 Beta-globin genotyping was performed by ARMS-PCR for detecting 16 common beta-globin mutations (-28, CD8/9, CD17, CD19, CD26 (Hb E), CD26 G>T (stop codon), CD27/28, IVSI-I, IVSI-5, CD35, CD41, CD41/42, CD43, CD71/72, CD95, and IVSII-654).32 A single-tube multiplex Gap-PCR and enzymatic amplification was used for common beta-globin gene deletions (3.48 kb, 619 bp, Filipino (β)°, SEA HPFH (β)°, ChineseGγ( Aγδβ)°, Thai (δβ)°, Hb Lepore, HPFH-6 Gγ( Aγδβ)°, Siriraj-thalGγ( Aγδβ)°, Asian Indian type A, and Asian Indian type B).33,34 Hemoglobin E testing was studied by restriction fragment length polymorphism (RFLP)-PCR utilizing Mnl I restriction enzyme.35