Quantitative PCR high-resolution melting (qPCR-HRM) curve analysis, a new approach to simultaneously screen point mutations and large rearrangements: application to MLH1 germline mutations in Lynch syndrome

Authors

  • Etienne Rouleau,

    Corresponding author
    1. Centre René Huguenin, Laboratoire d'Oncogénétique, Institut Nationale de la Santé et de la Recherche Médicale (INSERM) U735,Saint-Cloud, France
    • Centre René Huguenin, Laboratoire d'Oncogénétique, INSERM U735, 35 rue Dailly, 92210 Saint Cloud, France
    Search for more papers by this author
  • Cédrick Lefol,

    1. Centre René Huguenin, Laboratoire d'Oncogénétique, Institut Nationale de la Santé et de la Recherche Médicale (INSERM) U735,Saint-Cloud, France
    Search for more papers by this author
  • Violaine Bourdon,

    1. Institut Paoli-Calmettes, Département d'Oncologie Génétique Prévention et Dépistage, INSERM CIC P 9502, Université Aix Marseille II, Marseille, France
    Search for more papers by this author
  • Florence Coulet,

    1. Groupe Hospitalier Pitié-Salpêtrière, Université Pierre et Marie Curie-Paris 6, Laboratoire d'Oncogénétique et Angiogénétique Moléculaire, Paris, France
    Search for more papers by this author
  • Tetsuro Noguchi,

    1. Institut Paoli-Calmettes, Département d'Oncologie Génétique Prévention et Dépistage, INSERM CIC P 9502, Université Aix Marseille II, Marseille, France
    Search for more papers by this author
  • Florent Soubrier,

    1. Groupe Hospitalier Pitié-Salpêtrière, Université Pierre et Marie Curie-Paris 6, Laboratoire d'Oncogénétique et Angiogénétique Moléculaire, Paris, France
    Search for more papers by this author
  • Ivan Bièche,

    1. Centre René Huguenin, Laboratoire d'Oncogénétique, Institut Nationale de la Santé et de la Recherche Médicale (INSERM) U735,Saint-Cloud, France
    Search for more papers by this author
  • Sylviane Olschwang,

    1. Centre de Recherches en Cancérologie de Marseille, INSERM U891, Institut Paoli-Calmettes, Marseille, France
    Search for more papers by this author
  • Hagay Sobol,

    1. Institut Paoli-Calmettes, Département d'Oncologie Génétique Prévention et Dépistage, INSERM CIC P 9502, Université Aix Marseille II, Marseille, France
    Search for more papers by this author
  • Rosette Lidereau

    1. Centre René Huguenin, Laboratoire d'Oncogénétique, Institut Nationale de la Santé et de la Recherche Médicale (INSERM) U735,Saint-Cloud, France
    Search for more papers by this author

Abstract

Several techniques have been developed to screen mismatch repair (MMR) genes for deleterious mutations. Until now, two different techniques were required to screen for both point mutations and large rearrangements. For the first time, we propose a new approach, called “quantitative PCR (qPCR) high-resolution melting (HRM) curve analysis (qPCR-HRM),” which combines qPCR and HRM to obtain a rapid and cost-effective method suitable for testing a large series of samples. We designed PCR amplicons to scan the MLH1 gene using qPCR HRM. Seventy-six patients were fully scanned in replicate, including 14 wild-type patients and 62 patients with known mutations (57 point mutations and five rearrangements). To validate the detected mutations, we used sequencing and/or hybridization on a dedicated MLH1 array–comparative genomic hybridization (array-CGH). All point mutations and rearrangements detected by denaturing high-performance liquid chromatography (dHPLC)+multiplex ligation-dependent probe amplification (MLPA) were successfully detected by qPCR HRM. Three large rearrangements were characterized with the dedicated MLH1 array-CGH. One variant was detected with qPCR HRM in a wild-type patient and was located within the reverse primer. One variant was not detected with qPCR HRM or with dHPLC due to its proximity to a T-stretch. With qPCR HRM, prescreening for point mutations and large rearrangements are performed in one tube and in one step with a single machine, without the need for any automated sequencer in the prescreening process. In replicate, its reagent cost, sensitivity, and specificity are comparable to those of dHPLC+MLPA techniques. However, qPCR HRM outperformed the other techniques in terms of its rapidity and amount of data provided. Hum Mutat 0, 1–9, 2009. © 2009 Wiley-Liss, Inc.

Ancillary