title: DHPLC/SURVEYOR nuclease: a sensitive, rapid and affordable method to analyze BRCA1 and BRCA2 mutations in breast cancer families.
abstract: Hereditary breast cancer accounts for about 10% of all breast cancers and BRCA1 and BRCA2 genes have been identified as validated susceptibility genes for this pathology. Testing for BRCA gene mutations is usually based on a pre-screening approach, such as the partial denaturation DHPLC method, and capillary direct sequencing. However, this approach is time consuming due to the large size of BRCA1 and BRCA2 genes. Recently, a new low cost and time saving DHPLC protocol has been developed to analyze gene mutations by using SURVEYOR(®) Nuclease digestion and DHPLC analysis. A subset of 90 patients, enrolled in the Genetic Counseling Program of the National Cancer Centre of Bari (Italy), was performed to validate this approach. Previous retrospective analysis showed that 9/90 patients (10%) were mutated in BRCA1 and BRCA2 genes and these data were confirmed by the present approach. DNA samples underwent touchdown PCR and, subsequently, SURVEYOR(®) nuclease digestion. BRCA1 and BRCA2 amplicons were divided into groups depending on amplicon size to allow multiamplicon digestion. The product of this reaction were analyzed on Transgenomic WAVE Nucleic Acid High Sensitivity Fragment Analysis System. The operator who performed the DHPLC surveyor approach did not know the sequencing results at that time. The SURVEYOR(®) Nuclease DHPLC approach was able to detect all alterations with a sensitivity of 95%. Furthermore, in order to save time and reagents, a multiamplicon setting preparation was validated.
authors.affiliation: National Cancer Centre Giovanni Paolo II, Molecular Genetics Laboratory, v. Orazio Flacco 65, 70124 Bari, Italy.
authors.forename: Brunella
authors.initials: B
authors.lastname: Pilato
authors.forename: Simona
authors.initials: S
authors.lastname: De Summa
authors.forename: Katia
authors.initials: K
authors.lastname: Danza
authors.forename: Stavros
authors.initials: S
authors.lastname: Papadimitriou
authors.forename: Paolo
authors.initials: P
authors.lastname: Zaccagna
authors.forename: Angelo
authors.initials: A
authors.lastname: Paradiso
authors.forename: Stefania
authors.initials: S
authors.lastname: Tommasi
mesh_terms: D019313:BRCA1 Protein / Q000235:genetics*; D024682:BRCA2 Protein / Q000235:genetics*; D001483:Base Sequence; D001943:Breast Neoplasms / Q000235:genetics*; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D004252:DNA Mutational Analysis / Q000191:economics* / Q000379:methods*; D004273:DNA, Neoplasm / Q000235:genetics; D003851:Deoxyribonucleases / Q000378:metabolism*; D005190:Family; D005260:Female; D005820:Genetic Testing / Q000191:economics* / Q000379:methods*; D061325:Hereditary Breast and Ovarian Cancer Syndrome / Q000235:genetics; D006801:Humans; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics; D009691:Nucleic Acid Denaturation / Q000235:genetics*; D016133:Polymerase Chain Reaction; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity
chemical_list: D019313:BRCA1 Protein; D024682:BRCA2 Protein; D004273:DNA, Neoplasm; D003851:Deoxyribonucleases
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