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

Department of Clinical Biochemistry, Division of Molecular Genetic Diagnostics, Copenhagen University Hospital, DK-2100, Copenhagen, Denmark.
Gene (Impact Factor: 2.14). 06/2012; 507(1):79-84. DOI: 10.1016/j.gene.2012.06.020
Source: PubMed


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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    • "Genotyping of P450 2A6 (P450 2A6*1A, 2A6*1B, and 2A6*4) was carried out by conventional PCR amplification (Fig. 1) as described previously [2] [3] [4]. Blood samples were obtained with puncture needles, lysed, and stabilized [5]. Blood samples from healthy non-smoking Japanese volunteers or DNA fractions extracted separately by the standard protocol from the volunteers' buccal cells were used for real-time detection of P450 2A6 wild-type and whole-gene deletion [1]. "
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    ABSTRACT: This data article contains a supplementary figure and validation data relating to the research article entitled “Genotyping of wild-type cytochrome P450 2A6 and whole-gene deletion using human blood samples and a multiplex real-time polymerase chain reaction method with dual-labeled probes” (Shimizu et al., Clinica Chimica Acta 441, 71-74, 2015), which presents a multiplex real-time polymerase chain reaction method with dual-labeled probes for human P450 2A6 wild-type and whole-gene deletion. Real-time methods have dramatically improved the speed of complex genetic diagnostics compared to conventional assays based on restriction enzyme digestion. Here, we show the basic assay validation data by single and multiplex determinations in comparison with commercial TaqMan copy number assays for P450 2A6.
    Full-text · Article · Oct 2015
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    ABSTRACT: Genetic polymorphisms of human cytochrome P450 2A6 (CYP2A6) are one of the determinants of smoking behavior and/or tobacco-related lung cancer risk in male Japanese smokers. To help identify those at high risk, we developed a multiplex real-time polymerase chain reaction (PCR)-based genotyping method with dual-labeled probes to detect wild-type and whole-gene deletion of CYP2A6 directly from blood samples without DNA isolation. We validated the new real-time PCR method that uses dual-labeled probes by utilizing 116 genomic DNA samples that had been genotyped previously and 33 blood samples. The new method could discriminate CYP2A6 from highly homologous CYP2A7 and CYP2A13 genes and could also determine CYP2A6*1 (wild type) and CYP2A6*4 (whole-gene deletion) alleles in perfect accordance with previous analysis data. Amplification curve profiles were obtained by multiplex real-time PCR assay with CYP2A6*1 and CYP2A6*4 primer sets and dual-labeled probes using one-drop blood samples previously genotyped for CYP2A6*1/*1, CYP2A6*1/*4, and CYP2A6*4/*4. A real-time multiplex PCR assay for genotyping wild-type CYP2A6 and whole-gene deletion was developed with dual-labeled probes. The new method achieved 100% agreement with data from the conventional PCR method for 116 genomic DNA samples and samples from 33 volunteers, thereby establishing its validity. Copyright © 2014. Published by Elsevier B.V.
    No preview · Article · Dec 2014 · Clinica Chimica Acta