Analysis of Norwegian BRCA mutations using Sequenom MALDI TOF MS

1. Analysis of Norwegian BRCA mutations using Sequenom MALDI TOF MS Ann Curtis on behalf of James Eden Institute of Human Genetics Newcastle University

2. Challenges to mutation analysis of BRCA1 and BRCA2 • ~430,000 new cases per year in Europe • ~5% with mutations in BRCA1 or BRCA2 • BRCA1 and BRCA2 are large genes • >3000 distinct BRCA1 and BRCA2 mutations and polymorphisms reported on BIC • Current BRCA mutation testing - sequencing of entire coding regions – expensive and time consuming

3. Targeted mutation analysis of BRCA1 and BRCA2 • Almost no founder mutations or hot spots • Geographical/ethnic differences in BRCA1 and BRCA2 mutation frequencies • Migration between populations complicates mutation screening and produces unfeasibly large numbers of mutations

4. Familial breast cancer in Norway • Frequent but unevenly distributed • Reduced population caused by Bubonic plague 25 generations ago and then rapid expansion • Total of ~70 BRCA1 and BRCA2 mutations • 4 of these make up 68% of BRCA1 mutation carriers • Newcastle Molecular Genetics laboratory: 102 BRCA1/BRCA2 mutations • Only 13 of these mutations common to both populations • Feasible number and population-specific nature of Norwegian BRCA mutations makes Norway a candidate for country-wide targeted BRCA mutation detection

5. Norwegian BRCA collaboration Professor John Burn Dr Pål Møller Institute of Human Genetics Department of Medical Genetics Newcastle University Norwegian Radium Hospital, Oslo Aim: • To develop a BRCA1 and BRCA2 assay that will identify >95% of familial breast cancer in the Norwegian population 2 techniques in parallel can detect all 70 Norwegian mutations: 1. SEQUENOMTM MALDI TOF mass spectrometer 62 mutations (deletions, substitutions, insertions) 2. Multiplex Ligation-dependent Probe Amplification (MLPA) 8 mutations (large exonic changes)

6. SEQUENOMTM for mutation testing of BRCA1 and BRCA2 • Able to study up to 30 mutations simultaneously • Cheap, simple preparation of samples • Rapid data analysis (1hr per 384 DNA samples) • Minimal data interpretation – automated software for calling mutations + confidence score

7. SEQUENOM iPLEX reaction for mutation detection and genotyping Wild type allele (T) Mutant allele (C) ‘Extension’ primer of specific mass anneals immediately upstream of mutation. Extension Primer (5500Da) Extension Primer (5500Da) C T +Polymerase enzyme +ddATP/ddCTP/ ddTTP/ddGTP extended Primer (5800Da) extended Primer (6100Da) If supplied with all 4 ddNTPs, the primer is extended by one nucleotide generating a product of specific mass. G A T C

8. SEQUENOMTM MALDI TOF MASS SPECTROMETERMatrix Assisted Laser Desorption/Ionisation Time of Flight mass spectrometry Detector Flight path Time of flight Laser Sequenom chip (matrix)

9. The masses of the 2 extension products are distinguished by the mass spectrometer, allowing the patient to be genotyped for the mutation. 7000Da 7000Da 5500Da 7000Da 5500Da 5500Da Homozygous WT (TT) Heterozygote (TC) Homozygous mut (CC)

10. Power of SEQUENOM iPLEX for mutation detection • Step 1:Multiplex PCR using up to 30 sets of primers per reaction Each of the 30 PCR products contains a mutation site • Step 2:iPLEX reaction. 30 iPLEXes can be analysed simultaneously on the Mass Spectrometer • Each SEQUENOM chip holds 384 DNA samples • 384 plate of 30plex PCR can be transferred to a chip • 30 x 384 = 11,520 mutations to be genotyped in 1 run

11. Norwegian mutation assay – design and strategy • 62/70 Norwegian BRCA mutations can be studied by Sequenom • Complications of high multiplex PCRs: Strongly working PCRs out-compete weaker ones • Strategy: To amplify each multiplex in turn, redesigning the failing (weak) assays into the next multiplex • 1 assay failed primer design (BRCA2.7462delA). Proximal SNP prevented extension primer binding. Use of degenerate primer overcame problem • 1 assay will not pool into 4 plexes 1 – 4 (BRCA2.4075delGT). Not economical to run as 1-plex • Final design: 60/62 BRCA mutations for Sequenom analysis pooled into 4 multiplexes: MP1. 26-plex MP2. 20-plex MP3. 12-plex MP4. 3-plex All multiplexes gave clean results on wild type DNA

12. Validation using mutation control DNA • Able to validate test for 55/61 mutations using positive control DNA sent from Norway • No DNA sent for: • BRCA1.185insA • BRCA1.1048delA • BRCA1.1675delA • BRCA1.2594delC • BRCA1.5002T>C • BRCA1.4418delA • Mutation nomenclature was a nightmare • All 55 positive controls tested on Sequenom for the 61 functional Sequenom assays • PCRs performed in duplicate, all at 56°C annealing temp, 35 cycles • Expected to detect 1 mutation in each positive control, negative results for all other mutations

13. Validation results • 50/55 positive controls: Correct mutation detected by Sequenom in both replicates. No other mutation detected within same sample • 1/55 positive controls: Correct mutation detected but 1 of other 61 mutations detected also BRCA2.IVS23-2 A>G – also detected BRCA1.C5002T • 1/55 positive controls: Correct mutation not detected. 1 of other 61 mutations detected. BRCA1.IVS22-25 T>A – Detected in this sample: BRCA1.185insA (?mislabelling, no +ve control for this mutation) • 3/55 positive controls: Correct mutation not detected. No other mutations detected BRCA1.5382insC BRCA1.3171ins5 BRCA1.576_577ins21 Confusing nomenclature makes insertion sequences difficult to pinpoint. Are we looking in the right place?

14. BRCA2_T7786C Mutation correctly detected in mutant sample Mutation absent in all other samples

15. BRCA2.IVS23-2 A>GBRCA1.IVS22-25 T>A Mutation correctly detected in mutant sample Mutation not detected in mutant sample Different mutation found in same sample BRCA1.185insA Other mutation detected in same sample BRCA1.C5002T

16. Summary 70 Norwegian mutations 8 MLPA 62 Sequenom 1 failed assay design 61 mutations – wild type sequence detected 55 mutation controls for validaion 51 mutant sequences detected 3 fails (all insertions) 1 mislabelling (different mutation detected)

17. Norwegian BRCA mutation list

18. What next • Confirm location of 3 insertions mutations by DNA sequencing (failed assays). Redesign extension primers • Confirm presence of BRCA1.185insA in ?mislabelled sample • Organise delivery of the 6 untested positive controls. 3 of these mutations are found in Newcastle families so we have samples already: BRCA1.185insA, BRCA.1048delA, BRCA1.2594del • Pooling BRCA2.4075delGT into Multiplex 4 and attempting a 4-plex • Blind study: Will the Sequenom pick up the correct mutations?

19. Conclusion Overall very optimistic • 51/62 working assays • Confident that difficulties associated with 10 of remaining 11 will be solved • Cheap (£1.06 per sample) • Fast (1 day to prepare reactions, analyse data next day) • High throughput - 11,500 genotypes per chip

20. ...and finally • Application to other • Populations • Diseases • Genes

21. We raised £281.23 for Everyman cancer charity Acknowledgements Pat Bond Anna Jeffery Smith Jonathan Coxhead Jane Cooper Joytika Attari Rob Brown John Burn Bernard Keavney Pål Møller