1 / 17

Breast cancer and the BRCA genes

Validation of BRCA2 mutation scanning using the LightScanner system for high resolution melt analysis Lewis Darnell Nottingham Regional Molecular Genetics Service. Breast cancer and the BRCA genes.

edna
Download Presentation

Breast cancer and the BRCA genes

An Image/Link below is provided (as is) to download presentation Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author. Content is provided to you AS IS for your information and personal use only. Download presentation by click this link. While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server. During download, if you can't get a presentation, the file might be deleted by the publisher.

E N D

Presentation Transcript

  1. Validation of BRCA2 mutation scanning using the LightScanner system for high resolution melt analysisLewis Darnell Nottingham Regional Molecular Genetics Service

  2. Breast cancer and the BRCA genes • Breast cancer accounts for 22% of all cancers among women giving an annual worldwide incidence of approximately 1,000,000 cases. • 5%-10% of breast cancers are hereditary with 30%-50% due to mutations in either BRCA1 or BRCA2. • Mutations in BRCA1 or 2 confera high lifetime risk of both breast and ovarian cancers.

  3. BRCA2 • BRCA2 covers approximately 70Kb of genomic DNA of 13q12. • 27 exons make up an approximately 11Kb mRNA, coding for a 3,418 amino acid protein. • BRCA2 is involved in double-strand break DNA repair. • Pathogenic mutations: • Point mutations (nonsense, missense and splicing alterations) • Small deletions, duplications and insertions. • Large, whole exon deletions and duplications.

  4. Current mutation scanning strategy • Currently both BRCA1 and BRCA2 are screened concurrently: • Two week sample collection • Multiplex ligation-dependent probe amplification (MLPA) for deletion/duplication detection • Conformation sensitive capillary electrophoresis (CSCE) pre-screen. 33 and 46 amplicons for BRCA1 and BRCA2 • Bi-directional sequencing of any patient amplicons that do not match the wild-type (WT) or polymorphic controls • In the last year the results for all BRCA1/2 mutation scans were reported within the 40-day reporting time. • However, a high failure rate for CSCE increases the cost of the screen and can put pressure on reporting times.

  5. High Resolution Melt analysis • High Resolution Melt (HRM) analysis offers rapid and low cost detection of sequence variants. • The DNA region of interest is amplified and heteroduplexed in the presence of LCGreen. • LCGreen binds dsDNA to saturation and will emit a fluorescent signal when incorporated. • Sequence variants alter the melting profile of a heteroduplexed sample. • Fluorescence change, under controlled melting, is analysed using the LightScanner (Idaho Technologies).

  6. BRCA2 primers and validation criteria • 47 primer pairs designed by Idaho Technologies. • Cover the entire coding region of BRCA2 and at least 15 base pairs either side of coding exons. • Primers covering sites of known frequency single nucleotide polymorphisms (SNPs) were modified to contain inosine or degenerate bases where applicable. • Validation criteria: • No false-negative results • Fewer than 25% false positives

  7. Initial amplicon Validation • Each amplicon was tested using a panel of 20 wild type (WT) controls and samples containing all available amplicon specific variants. • Using the standard protocol supplied with the primer sequences, 25 of the 47 amplicons met the validation criteria. • Further optimisation was carried out with the remaining 22 amplicons.

  8. WT Variant False-positive Further optimisation • Primer concentration: • Reduced from 0.25μM to 0.20μM • Number of false-positives reduced • 7 amplicons needed lower primer concentrations to meet the validation criteria • Further reductions decrease the amplification Amplicon 11-3: 0.25μM 0.20μM

  9. WT Variant False-positive Further optimisation • PCR cycles: • Cycles increased from 35 to 40 • Results improved for all amplicons trialled • Amplification more consistent between samples • Reduced false positive rate and increased variant detection Amplicon 11-12: 35 cycles 40 cycles

  10. Further optimisation • Genomic DNA clean up: • Using CA reagent, supplied by Clent Life Science, prior to sample dilution • Reduced variation between samples of the same genotype Amplicon 10-3: Untreated DNA CA reagent cleaned DNA WT c.1365A>G c.1359delA False-positive

  11. Amplicons that have not met the validation criteria • Amplicons 03 and 10-3 both failed to detect a variant: • c.68-7T>A and c.1359delA respectively • Both variants lie within mononucleotide tracts close to the end of their respective fragments • Other variants have been detected closer to the end of a fragment • Variants have also been detected in mononucleotide tracts E.g. c.8940insA in amplicon 22

  12. Amplicons that have not met the validation criteria • Amplicons 10-1b, 10-2 and 11-1 all have problems with common polymorphisms: • 10-1b – common SNPs under both primers • 10-2 – common internal polymorphism, poor results • 11-1 – three common polymorphisms in fragment • These five amplicons would be sequenced directly using alternative primers where appropriate.

  13. Results after optimisation • After amplicon optimisation 42 of 47 amplicons met the validation criteria. • 140 of 142 variants detected. Sensitivity: 98.6%. • Not including results from amplicons 10-1b, 10-2 and 11-1.

  14. Summary of validation results • Even amplification and low variation between samples are essential. • All amplicons are now run with the 40-cycle PCR. • Seven amplicons will be amplified using 0.2μM primers. • All samples will be cleaned up prior to dilution using CA reagent. • Polymorphisms can make analysis difficult. • WT and polymorphism controls will need to be run with amplicons 11-5, 11-6, 14-2 and 17 to differentiate WT and polymorphic groups. Amplicon 17: WT c.7806-14T>C Het c.7806-14T>C Hom c.7822C>G c.7927G>A

  15. Advantages of HRM as a pre-screen in the Nottingham laboratory • Rapid: • The 42 amplicons for 10 patients can be amplified, analysed and scored in two days by one person • CSCE would take five days to do the same screen using a 16-capillary ABI 3130xl. • Low cost: • HRM is approximately 35% cheaper than CSCE for consumables • Quick setup and analysis reduce the cost further • Reduced sequencing: • Very low failure rate • Fewer samples to sequence than CSCE, including the five amplicons needing direct sequencing • Increased sequencing capacity for other applications

  16. Future work • Direct comparison to CSCE: • HRM analysis for BRCA2 currently running concurrently with CSCE using the same patient samples • Will provide more conclusive sensitivity and specificity values • Further validate HRM as a diagnostic tool • Amplicons requiring further work: • Trial high sensitivity mastermix for amplicons 03 and 10-3 • Potentially redesign primers for 10-1b, 10-2 and 11-1

  17. Acknowledgements • Nottingham Molecular Genetics Service • Rachael Tredwell • Jo Field • Gareth Cross • All staff • Clent Life Science • David Harris • Idaho Technologies • Jason McKinney

More Related