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.

Analytical Validation of a Personalized Medicine APOL1 Genotyping Assay for Nondiabetic Chronic Kidney Disease Risk Assessment

“…Our group has previously used this technology to develop multiplexed Ashkenazi Jewish carrier screening panels, 15 and novel genotyping assays with this open platform have also been reported for hereditary hemochromatosis, 16 HLA typing, 17 human papillomavirus detection, 18 and other applications. 19 To the best of our knowledge, this APOL1 G1/G2 genotyping assay represents the first disease risk prediction genetic test validated for clinical use with this technology.…”

Multiplex Allele-Specific PCR for Simultaneous Detection of H63D and C282Y HFE Mutations in Hereditary Hemochromatosis

“…Numerous assays have been developed to screen for the C282Y and H63D mutations (10)(11)(12)(13)(14)(15). Here, we report on a novel inhouse-developed multiplex allele-specific PCR assay that allows rapid and inexpensive genotyping of the C282Y and H63D mutations.…”