title: 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.
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.
authors.affiliation: Centre René Huguenin, Laboratoire d'Oncogénétique, Institut Nationale de la Santé et de la Recherche Médicale (INSERM) U735, Saint-Cloud, France. rouleau@crh1.org
authors.forename: Etienne
authors.initials: E
authors.lastname: Rouleau
authors.forename: Cédrick
authors.initials: C
authors.lastname: Lefol
authors.forename: Violaine
authors.initials: V
authors.lastname: Bourdon
authors.forename: Florence
authors.initials: F
authors.lastname: Coulet
authors.forename: Tetsuro
authors.initials: T
authors.lastname: Noguchi
authors.forename: Florent
authors.initials: F
authors.lastname: Soubrier
authors.forename: Ivan
authors.initials: I
authors.lastname: Bièche
authors.forename: Sylviane
authors.initials: S
authors.lastname: Olschwang
authors.forename: Hagay
authors.initials: H
authors.lastname: Sobol
authors.forename: Rosette
authors.initials: R
authors.lastname: Lidereau
mesh_terms: D048868:Adaptor Proteins, Signal Transducing / Q000235:genetics*; D003123:Colorectal Neoplasms, Hereditary Nonpolyposis / Q000235:genetics*; D004252:DNA Mutational Analysis / Q000379:methods*; D017931:DNA Primers / Q000378:metabolism; D005091:Exons / Q000235:genetics; D015321:Gene Rearrangement; D018095:Germ-Line Mutation / Q000235:genetics*; D006801:Humans; D000070957:MutL Protein Homolog 1; D009687:Nuclear Proteins / Q000235:genetics*; D009691:Nucleic Acid Denaturation; D009693:Nucleic Acid Hybridization; D017354:Point Mutation / Q000235:genetics*; D016133:Polymerase Chain Reaction / Q000379:methods*; D012015:Reference Standards
chemical_list: D048868:Adaptor Proteins, Signal Transducing; D017931:DNA Primers; C501486:MLH1 protein, human; D009687:Nuclear Proteins; D000070957:MutL Protein Homolog 1