Cardoso S P, Patel R, Brown C, Navarrete C
Histocompatibility and Immunogenetics Laboratory, National Health Service Blood and Transplant (NHSBT), Colindale Centre, London, UK.
Tissue Antigens. 2011 Sep;78(3):171-7. doi: 10.1111/j.1399-0039.2011.01736.x. Epub 2011 Jul 8.
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.
1型遗传性血色素沉着症（HH）是白种人中常见的一种遗传性疾病，由HFE基因突变引起。1型HH的常规诊断检测包括对上述两种HFE突变（C282Y和H63D）进行基因分型。在某些情况下，也会对第三种突变S65C进行分型。已有多种技术用于HFE基因分型，包括聚合酶链反应（PCR）-序列特异性引物（SSP）、PCR-限制性片段长度多态性（RFLP）、PCR-序列特异性寡核苷酸探针（SSOP）、实时PCR结合熔解曲线分析和TaqMan检测法。本研究的目的是开发一种替代传统PCR和实时PCR/TaqMan检测法的方法，利用Luminex技术在单一检测中检测所有三种HFE突变。使用已知基因型的DNA对照（n = 109）来评估这种方法。这些对照被选来代表每种突变的三种可能基因型（野生型、突变型、杂合子）。随后，使用盲法DNA样本（n = 100）来验证该方法。然后将这种新检测法与当前技术（内部PCR-SSP和TaqMan检测法）进行比较。将Luminex方法获得的基因型与之前内部PCR-SSP和TaqMan检测法报告的基因型进行比较，结果显示DNA对照和盲法DNA样本的一致性均为100%，未观察到差异。C282Y、H63D和S65C突变的等位基因频率分别为22%、16%和2%。我们在此报告一种用于HFE基因中C282Y、H63D和S65C突变常规临床检测的高通量、准确且稳健的多重Luminex微珠检测法。
