Twenty-three percent of first diagnosed breast cancer patients resulted to be candidate for BRCA genetic test and, among these, BRCA1 and BRCA2 mutations occur in almost 20% patients. BRCA testing is complicated by the large gene region and the extent of gene variations. Elective methods to evaluate BRCA known and unknown alterations is a prescreening by dHPLC plus sequencing of altered regions even if other approaches, chip-based have been set-up. Our aim was to provide a WAVE®-HS System-based methodology to perform BRCA1/BRCA2 analysis with Surveyor Nuclease enzyme using a Multi-Amplicon approach in order to reduce the overall time needed to screen the genes and the costs. Fourteen patients and 29 controls with specific BRCA alterations already evidenced by standard methods have been used for blind analysis by the new approach. DNA fragments including exons 8, 11, 20 and 24 of BRCA1 and 4,10, 11, 12, 17, 18, 25 and 27 of BRCA2 were amplified by polymerase chain reaction using DNA samples extracted from blood. Denaturation followed by gradual annealing of the amplified fragments formed a heteroduplex with a base-mismatch if a mutation existed because the DNA samples contained wild-type DNA derived from non-malignant cells. After cleavage by Surveyor nuclease, a new single-strand-specific endonuclease that cleaves heteroduplex DNA at a base-mismatch site in both DNA strands, mutations were simply detected by dHPLC WAVE Nucleic Acid High Sensitivity Fragment Analysis System (Transgenomic) followed by sequencing of variant gene segments. At the moment, only 3 amplicons can be contemporarily analyzed with a sensitivity of 90% and a specificity of 100% for pathological samples, and 100% of sensitivity and specificity for healthy controls. We also showed that this enzyme does not demonstrate any preference regarding mismatch cleavage at specific positions. In conclusion, the surveyor nuclease method, used with a multi-amplicon approach, was comparable to direct sequencing for detecting BRCA mutations, achieving high sensitivity with lower cost, providing an important tool for genetic analysis of complex genes.
Rapid screening by Surveyor Nuclease-based mutation detection for BRCA1 and BRCA2 genes / B., Pilato; M., Iorio; M., Martinucci; S., Papadimitriou; P., Zaccagna; Salvatore, Francesco; A., Paradiso; Frisso, Giulia; S., Tommasi. - (2009). (Intervento presentato al convegno Hereditary breast and ovarian cancer (HBOC): risks and challenges tenutosi a Bari nel 10-12 settembre 2009).
Rapid screening by Surveyor Nuclease-based mutation detection for BRCA1 and BRCA2 genes
SALVATORE, FRANCESCO;FRISSO, GIULIA;
2009
Abstract
Twenty-three percent of first diagnosed breast cancer patients resulted to be candidate for BRCA genetic test and, among these, BRCA1 and BRCA2 mutations occur in almost 20% patients. BRCA testing is complicated by the large gene region and the extent of gene variations. Elective methods to evaluate BRCA known and unknown alterations is a prescreening by dHPLC plus sequencing of altered regions even if other approaches, chip-based have been set-up. Our aim was to provide a WAVE®-HS System-based methodology to perform BRCA1/BRCA2 analysis with Surveyor Nuclease enzyme using a Multi-Amplicon approach in order to reduce the overall time needed to screen the genes and the costs. Fourteen patients and 29 controls with specific BRCA alterations already evidenced by standard methods have been used for blind analysis by the new approach. DNA fragments including exons 8, 11, 20 and 24 of BRCA1 and 4,10, 11, 12, 17, 18, 25 and 27 of BRCA2 were amplified by polymerase chain reaction using DNA samples extracted from blood. Denaturation followed by gradual annealing of the amplified fragments formed a heteroduplex with a base-mismatch if a mutation existed because the DNA samples contained wild-type DNA derived from non-malignant cells. After cleavage by Surveyor nuclease, a new single-strand-specific endonuclease that cleaves heteroduplex DNA at a base-mismatch site in both DNA strands, mutations were simply detected by dHPLC WAVE Nucleic Acid High Sensitivity Fragment Analysis System (Transgenomic) followed by sequencing of variant gene segments. At the moment, only 3 amplicons can be contemporarily analyzed with a sensitivity of 90% and a specificity of 100% for pathological samples, and 100% of sensitivity and specificity for healthy controls. We also showed that this enzyme does not demonstrate any preference regarding mismatch cleavage at specific positions. In conclusion, the surveyor nuclease method, used with a multi-amplicon approach, was comparable to direct sequencing for detecting BRCA mutations, achieving high sensitivity with lower cost, providing an important tool for genetic analysis of complex genes.I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
