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High resolution melting analysis to genotype the most common variants in the HFE gene

  • Roberta V. Marotta , Olivia Turri , Antonella Morandi , Manuela Murano , Gianlodovico Melzi d'Eril and Maria Luisa Biondi EMAIL logo
Published/Copyright: June 1, 2011
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Clinical Chemistry and Laboratory Medicine
From the journal Clinical Chemistry and Laboratory Medicine Volume 49 Issue 9

Abstract

Background: High resolution melting (HRM) analysis of PCR amplicons was recently introduced as a closed-tube, rapid, and inexpensive method of genotyping. This study evaluated this system as an option for detecting the three most common mutations in the HFE gene (C282Y, H63D, S65C), accounting for the main form of hereditary haemochromatosis.

Methods: Ninety samples, previously screened by direct sequencing, and 27 controls were used. The analysis were performed on the Rotor Gene Q, using the commercial HRM mix containing the Eva Green dye (Qiagen). Specific primers allowed the amplification of the regions of interest in the HFE gene. Following amplification, a HRM analysis was conducted to detect DNA variants. The thermal denaturation profiles of the samples were compared with those of the controls.

Results: One hundred percent of heterozygous and homozygous samples were readily identified. Heterozygotes were easily identified because heteroduplexes altered the shape of the melting curves, but significant differences were also present in the melting curves of the homozygous carries compared with those of the wild-type subjects.

Conclusions: HRM analysis is an appealing technology for HFE gene screening. It is a robust technique that can be widely adopted in diagnostic laboratories to facilitate gene mutation screening.

Keywords: haemochromatosis; HFE gene; high resolution melting
Corresponding author: Maria Luisa Biondi, U.O. Diagnostica Molecolare Infettivologica, Azienda Ospedaliera San Paolo, Via A. Di Rudinì 8 20142 Milano, Italy Phone: +39 02/81844314, Fax: +39 02/81844027
Received: 2010-12-7
Accepted: 2011-4-27
Published Online: 2011-06-1
Published in Print: 2011-09-01

©2011 by Walter de Gruyter Berlin Boston

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https://doi.org/10.1515/CCLM.2011.237
Keywords for this article
haemochromatosis; HFE gene; high resolution melting

Articles in the same Issue

  1. Editorial
  2. Athlete's biological passport: to test or not to test?
  3. Review
  4. Prognostic value of cystatin C in acute coronary syndromes: enhancer of atherosclerosis and promising therapeutic target
  5. Opinion Papers
  6. The function of oxalic acid in the human metabolism
  7. Current limitations of the Athlete's Biological Passport use in sports
  8. Limits and pitfalls of Athlete's Biological Passport
  9. The Athlete Biological Passport from the perspective of an anti-doping organization
  10. Perspectives
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  12. Guidelines and Recommendations
  13. IFCC primary reference procedures for the measurement of catalytic activity concentrations of enzymes at 37 °C. Part 9: Reference procedure for the measurement of catalytic concentration of alkaline phosphatase
  14. Genetics and Molecular Diagnostics
  15. A template for mutational data analysis of the CFTR gene
  16. High resolution melting analysis to genotype the most common variants in the HFE gene
  17. General Clinical Chemistry and Laboratory Medicine
  18. Bayesian analysis of an international ELISA comparability study
  19. Inflammatory markers in preeclamptic patients
  20. Pre-analytical effects of different lithium heparin plasma separation tubes in the routine clinical chemistry laboratory
  21. Pre-acquisition system assessment of the Sysmex® Coagulation System CS-2100i and comparison with end-user verification; a model for the regional introduction of new analysers and methods
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  33. N-terminal pro-B-type natriuretic peptide in early and advanced phases of obesity
  34. Vaspin plasma concentrations and mRNA expressions in patients with stable and unstable angina pectoris
  35. Prospective study of first stroke in relation to plasma homocysteine and MTHFR 677C>T and 1298A>C genotypes and haplotypes – evidence for an association with hemorrhagic stroke
  36. Letters to the Editor
  37. Evaluation of the analytical performance of the Beckman Coulter AU680 automated analytical system based on quality specifications for allowable performance derived from biological variation
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  40. Modified phosphatidylserine-dependent antiprothrombin ELISA enables identification of patients negative for other antiphospholipid antibodies and also detects low avidity antibodies
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