title: High resolution melting analysis to genotype the most common variants in the HFE gene.
abstract: BACKGROUND
  High resolution melting (HRM) analysis of PCR amplicons was recently introduced as a closed-tube, rapid, and inexpensive method of genotyping. This study evaluated this system as an option for detecting the three most common mutations in the HFE gene (C282Y, H63D, S65C), accounting for the main form of hereditary haemochromatosis.
  
  
  METHODS
  Ninety samples, previously screened by direct sequencing, and 27 controls were used. The analysis were performed on the Rotor Gene Q, using the commercial HRM mix containing the Eva Green dye (Qiagen). Specific primers allowed the amplification of the regions of interest in the HFE gene. Following amplification, a HRM analysis was conducted to detect DNA variants. The thermal denaturation profiles of the samples were compared with those of the controls.
  
  
  RESULTS
  One hundred percent of heterozygous and homozygous samples were readily identified. Heterozygotes were easily identified because heteroduplexes altered the shape of the melting curves, but significant differences were also present in the melting curves of the homozygous carries compared with those of the wild-type subjects.
  
  
  CONCLUSIONS
  HRM analysis is an appealing technology for HFE gene screening. It is a robust technique that can be widely adopted in diagnostic laboratories to facilitate gene mutation screening.
authors.affiliation: Dipartimento di Medicina Chirurgia e Odontoiatria, Università degli Studi di Milano, Milan, Italy.
authors.forename: Roberta V
authors.initials: RV
authors.lastname: Marotta
authors.forename: Olivia
authors.initials: O
authors.lastname: Turri
authors.forename: Antonella
authors.initials: A
authors.lastname: Morandi
authors.forename: Manuela
authors.initials: M
authors.lastname: Murano
authors.forename: Gianlodovico Melzi
authors.initials: GM
authors.lastname: d'Eril
authors.forename: Maria Luisa
authors.initials: ML
authors.lastname: Biondi
mesh_terms: D004252:DNA Mutational Analysis / Q000191:economics / Q000379:methods*; D060005:Genotyping Techniques / Q000191:economics / Q000379:methods*; D000071020:Hemochromatosis Protein; D015395:Histocompatibility Antigens Class I / Q000235:genetics*; D006801:Humans; D008565:Membrane Proteins / Q000235:genetics*; D009154:Mutation / Q000235:genetics*; D009691:Nucleic Acid Denaturation; D016133:Polymerase Chain Reaction; D013997:Time Factors; D044366:Transition Temperature
chemical_list: C103673:HFE protein, human; D000071020:Hemochromatosis Protein; D015395:Histocompatibility Antigens Class I; D008565:Membrane Proteins