title: Scanning for α-Hemoglobin Variants by High-Resolution Melting Analysis.
abstract: BACKGROUND
  Definitive detection of hemoglobin (Hb) variants requires DNA sequencing. High-resolution melting (HRM) analysis of polymerase chain reaction (PCR) amplicons was applied to detect and discriminate among uncommon α-Hb variants found in Thailand.
  
  
  METHODS
  Uncommon suspected α-Hb variants observed in Hb typing were identified by sequencing of DNA from whole blood samples. Three pairs of PCR primers covering the mutation regions in the three α-globin exons then were used for PCR coupled with difference in HRM analysis to subtract out the concomitant melting profile of the normal allele in the heterozygous state.
  
  
  RESULTS
  DNA sequencing identified six heterozygous α-Hb variants, namely, Hb G-Waimanalo (HBA2: exon 2, codon 64 G>A), Hb J-Buda (HBA1: exon 2, codon 61 G>T), Hb Kurosaki (HBA2: exon 1; codon 7 A>G), Hb O-Indonesia (HBA1: exon 3 codon 116 G>A), Hb Q-India (HBA1:exon 2, codon 64 G>C), and Hb Q-Thailand (HBA1: exon 2 codon 74 G>C). Difference HRM analysis showed one temperature melting profile using exon 1 primer pair, four different profiles with exon 2 primer pair, and one profile with exon 3 primer pair.
  
  
  CONCLUSIONS
  PCR-HRM analysis was effective in detecting and discriminating among single point mutations causing six uncommon α-Hb variants in heterozygous individuals. The method can be applied for routine screening due to its simplicity and relatively low cost.
authors.affiliation: Department of Pathology, Faculty of Medicine, Ramathibodi Hospital, Mahidol University, Bangkok, Thailand.
authors.forename: Walaiporn
authors.initials: W
authors.lastname: Yimniam
authors.affiliation: Department of Pathology, Faculty of Medicine, Ramathibodi Hospital, Mahidol University, Bangkok, Thailand. sumalee.jin@mahidol.ac.th.
authors.forename: Sumalee
authors.initials: S
authors.lastname: Jindadamrongwech
mesh_terms: D001483:Base Sequence; D014644:Genetic Variation; D006801:Humans; D009691:Nucleic Acid Denaturation / Q000235:genetics*; D016133:Polymerase Chain Reaction / Q000379:methods*; D017422:Sequence Analysis, DNA; D013785:Thailand; D055542:alpha-Globins / Q000235:genetics*
chemical_list: D055542:alpha-Globins
keywords: DNA sequencing; hemoglobin typing; hemoglobin variant; high-resolution melting (HRM) analysis; quantitative PCR