title: Linked linear amplification for simultaneous analysis of the two most common hemochromatosis mutations.
abstract: BACKGROUND
  Two mutations in HFE, G845A (amino acid substitution C282Y) and C187G (H63D), are associated with hereditary hemochromatosis. We developed and validated a novel method, linked linear amplification (LLA), for detection of these two mutations.
  
  
  METHODS
  Two segments of HFE were amplified by a multiplex LLA reaction that generated biotinylated LLA products. Aliquots of the multiplex LLA reaction were captured in microwells by hybridization to immobilized allele-specific oligonucleotides (ASOs). One wild-type and one mutant ASO represented the DNA sequence at each of the two mutation sites. Hybridization was detected by a streptavidin-horseradish peroxidase-based colorimetric method. Genotypes obtained by LLA and PCR-restriction fragment length polymorphism (PCR-RFLP) methods for 320 individuals were compared.
  
  
  RESULTS
  The amplified samples included the following genotypes as determined by PCR-RFLP: wild-type 282 and 63 codons (n = 105), C282Y homozygous mutant (n = 54), C282Y heterozygous (n = 52), H63D homozygous mutant (n = 17), H63D heterozygous (n = 59), and compound H63D and C282Y heterozygous mutant (n = 33). There was complete concordance between the results obtained by LLA and those obtained by PCR-RFLP analysis. The presence of another HFE mutation, A193T (encoding S65C), did not interfere with genotyping at codon 63.
  
  
  CONCLUSIONS
  LLA provides a reliable method to detect the common mutations in HFE that cause hereditary hemochromatosis.
authors.affiliation: Department of Pathology, University of Michigan, Ann Arbor, MI 48109, USA. kille001@umn.edu
authors.forename: Anthony A
authors.initials: AA
authors.lastname: Killeen
authors.forename: John W
authors.initials: JW
authors.lastname: Breneman
authors.forename: Arlene R
authors.initials: AR
authors.lastname: Carillo
authors.forename: Luis A
authors.initials: LA
authors.lastname: Ugozzoli
authors.forename: Jimmie D
authors.initials: JD
authors.lastname: Lowery
authors.forename: Jason
authors.initials: J
authors.lastname: Liu
authors.forename: Craig S
authors.initials: CS
authors.lastname: Hixson
mesh_terms: D019943:Amino Acid Substitution; D003062:Codon; D005838:Genotype; D006432:Hemochromatosis / Q000235:genetics*; D000071020:Hemochromatosis Protein; D015395:Histocompatibility Antigens Class I / Q000235:genetics*; D006801:Humans; D008565:Membrane Proteins / Q000235:genetics*; D009154:Mutation; D021141:Nucleic Acid Amplification Techniques / Q000379:methods; D012150:Polymorphism, Restriction Fragment Length; D015203:Reproducibility of Results
chemical_list: D003062:Codon; C103673:HFE protein, human; D000071020:Hemochromatosis Protein; D015395:Histocompatibility Antigens Class I; D008565:Membrane Proteins