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Validation of BRCA2 mutation scanning using the LightScanner system for high resolution melt analysis Lewis Darnell Nottingham Regional Molecular Genetics.

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Presentation on theme: "Validation of BRCA2 mutation scanning using the LightScanner system for high resolution melt analysis Lewis Darnell Nottingham Regional Molecular Genetics."— Presentation transcript:

1 Validation of BRCA2 mutation scanning using the LightScanner system for high resolution melt analysis Lewis 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 confer a high lifetime risk of both breast and ovarian cancers. GeneCancer typeLifetime risk (%) BRCA1Breast65-85 Ovarian39-63 BRCA2Breast45-84 Ovarian11-27

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 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 WT Variant False-positive

9 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 WT Variant False-positive

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. Mutation typeNumber of variants detectedNumber variants not detected Nucleotide change1061 Nucleotide deletion301 Nucleotide duplication30 Nucleotide insertion10 Total1402

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

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