title: Simultaneous detection of HFE C282Y, H63D and S65C mutations associated with type 1 haemochromatosis using a multiplex luminex bead assay.
abstract: Type 1 hereditary haemochromatosis (HH) is a common genetic disorder in Caucasoids resulting from mutations in the HFE gene. Routine diagnostic testing for type 1 HH involves genotyping for two of these described HFE mutations, C282Y and H63D. In some cases typing of a third mutation, S65C is also performed. Several techniques have been reported for HFE genotyping and these include polymerase chain reaction (PCR)-sequence-specific primers (SSP), PCR-restriction fragment length polymorphism (RFLP), PCR-sequence-specific oligonucleotide probe (SSOP), real-time PCR followed by melting curve analysis and TaqMan assay. The aim of this study was to develop an alternative method to both conventional PCR and real-time PCR/TaqMan assay to detect all three HFE mutations in a single assay using Luminex technology. DNA controls of known genotypes (n = 109) were used to evaluate this approach. These controls were selected to represent the three possible genotypes (wild type, mutant, heterozygous) for each mutation. Subsequently, blind DNA samples (n = 100) were used to validate this method. This new assay was then compared with current techniques (in-house PCR-SSP and TaqMan assay). Comparison of genotypes obtained with the Luminex method with those previously reported by both in-house PCR-SSP and TaqMan assay showed 100% concordance for both DNA controls and blind DNA samples and no discrepancies were observed. Allelic frequency for C282Y, H63D and S65C mutations were 22%, 16% and 2%, respectively. We report here a high-throughput, accurate and robust multiplex luminex bead assay for routine clinical testing of C282Y, H63D and S65C mutations in the HFE gene.
authors: Histocompatibility and Immunogenetics Laboratory, National Health Service Blood and Transplant (NHSBT), Colindale Centre, London, UK. cristina.navarrete@nhsbt.nhs.uk
authors: S P
authors: SP
authors: Cardoso
authors: R
authors: R
authors: Patel
authors: C
authors: C
authors: Brown
authors: C
authors: C
authors: Navarrete
mesh_terms: D004247:DNA / Q000235:genetics; D005838:Genotype; D006432:Hemochromatosis / Q000235:genetics*; D000071020:Hemochromatosis Protein; D015395:Histocompatibility Antigens Class I / Q000235:genetics*; D006801:Humans; D007118:Immunoassay; D008565:Membrane Proteins / Q000235:genetics*; D009154:Mutation / Q000235:genetics*; D016133:Polymerase Chain Reaction; D012150:Polymorphism, Restriction Fragment Length
chemical_list: C103673:HFE protein, human; D000071020:Hemochromatosis Protein; D015395:Histocompatibility Antigens Class I; D008565:Membrane Proteins; D004247:DNA