title: A one-step prescreening for point mutations and large rearrangement in BRCA1 and BRCA2 genes using quantitative polymerase chain reaction and high-resolution melting curve analysis.
abstract: High-resolution melting (HRM) of DNA is a versatile method for mutation scanning that monitors the fluorescence of double-strand DNA with saturating dye. Performing HRM on a real-time thermocycler enables semiquantitative analysis (quantitative polymerase chain reaction, qPCR) to be associated to HRM analysis for detection of both large gene rearrangements and point mutations (qPCR-HRM). We evaluated this method of mutation screening for the two major breast and ovarian cancer susceptibility genes BRCA1 and BRCA2. Screening of these two genes is time-consuming and must include exploration of large rearrangements that represent 5% to 15% of the alterations observed in these genes. To assess the reliability of the HRM technology, 201 known nucleotide variations scattered over all amplicons were tested. The sensitivity of qPCR was evaluated by analyzing seven large rearrangements. All previously identified variants tested were detected by qPCR-HRM. A retrospective study was done with 45 patients: qPCR-HRM allowed all the variants previously tested by denaturing high-performance liquid chromatography to be identified. qPCR analysis showed three cases of allele dropout (due to a 104-bp deletion, SNP primer mismatch, and an Alu insertion). A prospective study was done with 165 patients allowing 22 deleterious mutations, 16 unclassified variants, and 2 rearrangements to be detected. qPCR-HRM is a simple, sensitive, and fast method that does not require modified PCR primers. Thus, this method allows in one step the detection of point mutation, gene rearrangements, and prevention of missing a mutation due to primer mismatch.
authors.affiliation: Laboratoire d'Oncogénétique et Angiogénétique moléculaire, Groupe Hospitalier Pitié-Salpêtrière, Assistance Publique-Hopitaux de Paris, Université Pierre et Marie Curie, Paris, France. florence.coulet@psl.aphp.fr
authors.forename: Florence
authors.initials: F
authors.lastname: Coulet
authors.forename: Filipe
authors.initials: F
authors.lastname: Pires
authors.forename: Etienne
authors.initials: E
authors.lastname: Rouleau
authors.forename: Cedrick
authors.initials: C
authors.lastname: Lefol
authors.forename: Sabrina
authors.initials: S
authors.lastname: Martin
authors.forename: Chrystelle
authors.initials: C
authors.lastname: Colas
authors.forename: Odile
authors.initials: O
authors.lastname: Cohen-Haguenauer
authors.forename: Irina
authors.initials: I
authors.lastname: Giurgea
authors.forename: Anne
authors.initials: A
authors.lastname: Fajac
authors.forename: Catherine
authors.initials: C
authors.lastname: Noguès
authors.forename: Liliane
authors.initials: L
authors.lastname: Demange
authors.forename: Agnès
authors.initials: A
authors.lastname: Hardouin
authors.forename: Rosette
authors.initials: R
authors.lastname: Lidereau
authors.forename: Florent
authors.initials: F
authors.lastname: Soubrier
mesh_terms: D000483:Alleles; D001943:Breast Neoplasms / Q000235:genetics; D002851:Chromatography, High Pressure Liquid; D055028:Comparative Genomic Hybridization; D003199:Computer Systems; D017931:DNA Primers; D005260:Female; D005470:Fluorometry / Q000379:methods; D019398:Genes, BRCA1; D024522:Genes, BRCA2; D005820:Genetic Testing / Q000379:methods*; D006801:Humans; D016254:Mutagenesis, Insertional; D009691:Nucleic Acid Denaturation; D017354:Point Mutation; D016133:Polymerase Chain Reaction / Q000379:methods*; D011446:Prospective Studies; D012189:Retrospective Studies; D012680:Sensitivity and Specificity; D017422:Sequence Analysis, DNA
chemical_list: D017931:DNA Primers