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 BurnDr 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. SEQUENOM TM MALDI TOF mass spectrometer 62 mutations (deletions, substitutions, insertions) 2. Multiplex Ligation-dependent Probe Amplification (MLPA) 8 mutations (large exonic changes)

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

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

9. 5500Da 7000Da5500Da 7000Da 5500Da 7000Da Homozygous WT (TT)Homozygous mut (CC)Heterozygote (TC) The masses of the 2 extension products are distinguished by the mass spectrometer, allowing the patient to be genotyped for the mutation.

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: 1. BRCA1.185insA 2. BRCA1.1048delA 3. BRCA1.1675delA 4. BRCA1.2594delC 5. BRCA1.5002T>C 6. 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. Mutation correctly detected in mutant sample Mutation absent in all other samples BRCA2_T7786C

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

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 detected3 fails (all insertions) 1 mislabelling (different mutation detected)

17. MUTATIONEXON 1BRCA1 del exons 1-13- 2BRCA1 del exons 18-24- 3BRCA1 del exons 3-16- 4BRCA1 del exons 5-7- 5BRCA1 del exons 8-13- 6BRCA1 dup exon 13- 7BRCA1.2677ins35611 8BRCA2 del exon 3- 9BRCA1.120A>G2 10BRCA1.185insA2 11BRCA1.187delAG2 12BRCA1.458ins217 13BRCA1.505delG7 14BRCA1.816delGT11 15BRCA1.913delCT11 16BRCA1.967T>A11 17BRCA1.1048delA11 18BRCA1.1135insA11 19BRCA1.1177G>A11 20BRCA1.1191delC11 21BRCA1.1569G>T11 22BRCA1.1675delA11 23BRCA1.1806C>T11 MUTATIONEXON 25BRCA1.2557insG11 26BRCA1.2594delC11 27BRCA1.2988C>T11 28BRCA1.3109insAA11 29BRCA1.3124delA11 30BRCA1.3171ins511 31BRCA1.3203del1111 32BRCA1.3297G>T11 33BRCA1.3347delAG11 34BRCA1.3438G>T11 35BRCA1.3450delCAAG11 36BRCA1.3726C>T11 37BRCA1.4056C>T11 38BRCA1.4085delA11 39BRCA1.4154delA11 40BRCA1.4184del411 41BRCA1.4418delA13 42BRCA1.4731C>T15 43BRCA1.4808C>G16 44BRCA1.4864delA16 45BRCA1.5002T>C16 46BRCA1.5166G>T17 47BRCA1.IVS17-2A>CIVS17 MUTATIONEXON 49BRCA1.5382insC18 50BRCA1.5630G>A24 51BRCA1.5653delA24 52BRCA2.IVS2-7T>AIVS2 53BRCA2.999delTCAAA9 54BRCA2.1886T>G10 55BRCA2.2024del510 56BRCA2.2275delTCTC11 57BRCA2.3036delACAA11 58BRCA2.3824delACTG11 59BRCA2.4075delGT11 60BRCA2.4088delA11 61BRCA2.5445delTTTAAGT11 62BRCA2.5805delT11 63BRCA2.6287delAACA11 64BRCA2.6312del511 65BRCA2 6839_6840insC11 66BRCA2.7462delA14 67BRCA2.7786C>T15 68BRCA2.IVS23-2A>GIVS23 69BRCA2.9481insA24 70BRCA2.9751G>T26 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. Acknowledgements Pat Bond Anna Jeffery Smith Jonathan Coxhead Jane Cooper Joytika Attari Rob Brown John Burn Bernard Keavney Pål Møller We raised £281.23 for Everyman cancer charity