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Simultaneous detection of HFE C282Y, H63D and S65C mutations associated with type 1 haemochromatosis using a multiplex luminex bead assay

Cardoso, SP; Patel, R; Brown, C; Navarrete, C; (2011) Simultaneous detection of HFE C282Y, H63D and S65C mutations associated with type 1 haemochromatosis using a multiplex luminex bead assay. TISSUE ANTIGENS , 78 (3) 171 - 177. 10.1111/j.1399-0039.2011.01736.x.

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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.

Type:Article
Title:Simultaneous detection of HFE C282Y, H63D and S65C mutations associated with type 1 haemochromatosis using a multiplex luminex bead assay
DOI:10.1111/j.1399-0039.2011.01736.x
Keywords:allele-specific primer extension, haemochromatosis, HFE, iron overload, Luminex, multiplex, mutations, REAL-TIME PCR, HEREDITARY HEMOCHROMATOSIS, BLOOD-DONORS, GENETIC HEMOCHROMATOSIS, POPULATION, OVERLOAD, DISEASE, PREVALENCE, DIAGNOSIS
UCL classification:UCL > School of Life and Medical Sciences > Faculty of Medical Sciences > Surgery and Interventional Science (Division of)

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