title: Genotyping of the hemochromatosis HFE p.H63D and p.C282Y mutations by high-resolution melting with the Rotor-Gene 6000® instrument.
abstract: BACKGROUND
  The genotyping of HFE p.C282Y and p.H63D mutations is one of the most requested molecular analyses in the laboratorial routine. In this scenario, the main aim was to develop a genotyping assay that has advantages compared to other methods.
  
  
  METHODS
  Genotypes for the HFE p.C282Y (c.G845A; rs1800562) and p.H63D (c.C187G, rs1799945) mutations were assessed by polymerase chain reaction (PCR) followed by high resolution melting (HRM) analysis with the Rotor-Gene 6000(®) instrument. Validation studies were conducted in samples bi-directionally sequenced.
  
  
  RESULTS
  The melting assay was developed in a unique procedure and to ensure the result in approximately 112 min (31 min for sample preparation and 81 min for the PCR-HRM step). Genotypes for the HFE p.C282Y mutation were easily distinguished in the region of 80-86°C. For the HFE p.H63D, genotypes were also easily distinguished in the region of 76-82°C, but using the addition of known wild-type genotype DNA in all unknown samples plus a reaction without addition. In validation, genotypes were 100% concordant between methods.
  
  
  CONCLUSIONS
  Our genotyping assay with the Rotor-Gene 6000(®) instrument applies to the laboratorial routine with several advantages, especially in large-scale demand. The main advantages were the non-dependence on gel electrophoresis and on mutagenic reagents for visualization of fragments, reduction of the chances for contamination due to sample preparation, the lack of use of probe-based methods and cost-effectiveness.
authors.affiliation: Laboratory of Genetics and Molecular Cardiology, Heart Institute (InCor), University of Sao Paulo Medical School, SP, Brazil.
authors.forename: Paulo Caleb Junior Lima
authors.initials: PC
authors.lastname: Santos
authors.forename: Renata Alonso Gadi
authors.initials: RA
authors.lastname: Soares
authors.forename: Jose Eduardo
authors.initials: JE
authors.lastname: Krieger
authors.forename: Elvira Maria
authors.initials: EM
authors.lastname: Guerra-Shinohara
authors.forename: Alexandre Costa
authors.initials: AC
authors.lastname: Pereira
mesh_terms: D060005:Genotyping Techniques / Q000295:instrumentation*; D006432:Hemochromatosis / Q000175:diagnosis / Q000235:genetics*; D000071020:Hemochromatosis Protein; D015395:Histocompatibility Antigens Class I / Q000235:genetics*; D006801:Humans; D008565:Membrane Proteins / Q000235:genetics*; D009154:Mutation; D016133:Polymerase Chain Reaction; D012015:Reference Standards
chemical_list: C103673:HFE protein, human; D000071020:Hemochromatosis Protein; D015395:Histocompatibility Antigens Class I; D008565:Membrane Proteins