Sample-to-SNP kit: A reliable,easy and fast tool for the detection of HFE p.H63D and p.C282Y variations associated to hereditary hemochromatosis |
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Authors: | Peter B. Nielsen Maja S. Petersen Viviana Ystaas Rolf V. Andersen Karin M. Hansen Vibeke Blaabjerg Mette Refstrup |
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Affiliation: | 1. Department of Clinical Biochemistry, Division of Molecular Genetic Diagnostics, Copenhagen University Hospital, DK-2100, Copenhagen, Denmark;2. Department of Immunology, Copenhagen University Hospital, DK-2100, Copenhagen, Denmark;3. Department of Pathology, Copenhagen University Hospital, DK-2100, Copenhagen, Denmark;4. Professionshøjskolen Metropol, University College, DK-2200, Copenhagen, Denmark |
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Abstract: | Classical hereditary hemochromatosis involves the HFE-gene and diagnostic analysis of the DNA variants HFE p.C282Y (c.845G > A; rs1800562) and HFE p.H63D (c.187C > G; rs1799945). The affected protein alters the iron homeostasis resulting in iron overload in various tissues. The aim of this study was to validate the TaqMan-based Sample-to-SNP protocol for the analysis of the HFE-p.C282Y and p.H63D variants with regard to accuracy, usefulness and reproducibility compared to an existing SNP protocol. The Sample-to-SNP protocol uses an approach where the DNA template is made accessible from a cell lysate followed by TaqMan analysis. Besides the HFE-SNPs other eight SNPs were used as well. These SNPs were: Coagulation factor II-gene F2 c.20210G > A, Coagulation factor V-gene F5 p.R506Q (c.1517G > A; rs121917732), Mitochondria SNP: mt7028 G > A, Mitochondria SNP: mt12308 A > G, Proprotein convertase subtilisin/kexin type 9-gene PCSK9 p.R46L (c.137G > T), Plutathione S-transferase pi 1-gene GSTP1 p.I105V (c313A > G; rs1695), LXR g.-171 A > G, ZNF202 g.-118 G > T. In conclusion the Sample-to-SNP kit proved to be an accurate, reliable, robust, easy to use and rapid TaqMan-based SNP detection protocol, which could be quickly implemented in a routine diagnostic or research facility. |
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Keywords: | EDTA, ethylene diamine tetraacetic acid HH, hereditary hemochromatosis MGB, minor groove binder NTC, non template control PCR, polymerase chain reaction SNP, single nucleotide polymorphism HFE, hemochromatosis F2, coagulation factor II F5, coagulation factor V GSTP1, glutathione S-transferase pi 1 HAMP, hepcidin antimicrobial peptide HJV, hemojuvelin LXR, liver X receptor PCSK9, proprotein convertase subtilisin/kexin type 9 SLC40A1, solute carrier family 40 member 1 TFR2, transferrin receptor 2 ZNF202, zinc finger protein 202 |
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