Analysis of Norwegian BRCA mutations using Sequenom MALDI

TOF MS

Ann Curtis on behalf of

James EdenInstitute of Human Genetics

Newcastle University

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

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

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

Norwegian BRCA collaborationProfessor John Burn Dr Pål Møller Institute of Human Genetics Department of Medical GeneticsNewcastle 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 spectrometer62 mutations (deletions, substitutions, insertions)

2. Multiplex Ligation-dependent Probe Amplification (MLPA) 8 mutations (large exonic changes)

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

SEQUENOM iPLEX reaction for mutation detection and genotyping

extended Primer (6100Da)

T

C

C

T

Mutant allele (C)

Extension Primer (5500Da) 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.

SEQUENOMTM MALDI TOF MASS SPECTROMETERMatrix Assisted Laser Desorption/Ionisation Time of Flight mass

spectrometry

Laser

Flight path

Detector

Time of flight

Sequenom chip

(matrix)

5500Da 7000Da 5500Da 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.

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

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-plexMP2. 20-plexMP3. 12-plexMP4. 3-plex

All multiplexes gave clean results on wild type DNA

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. BRCA1.185insA2. BRCA1.1048delA3. BRCA1.1675delA4. BRCA1.2594delC5. BRCA1.5002T>C6. 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

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

BRCA1.5382insCBRCA1.3171ins5 BRCA1.576_577ins21

Confusing nomenclature makes insertion sequences difficult to pinpoint. Are we looking in the right place?

Mutation correctly detected in mutant sample

Mutation absent in all other samples

BRCA2_T7786C

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

Summary70 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)

MUTATIONEXO

N

1BRCA1 del exons 1-13 -

2BRCA1 del exons 18-24 -

3BRCA1 del exons 3-16 -

4 BRCA1 del exons 5-7 -

5BRCA1 del exons 8-13 -

6 BRCA1 dup exon 13 -

7 BRCA1.2677ins356 11

8 BRCA2 del exon 3 -

9 BRCA1.120A>G 2

10 BRCA1.185insA 2

11 BRCA1.187delAG 2

12 BRCA1.458ins21 7

13 BRCA1.505delG 7

14 BRCA1.816delGT 11

15 BRCA1.913delCT 11

16 BRCA1.967T>A 11

17 BRCA1.1048delA 11

18 BRCA1.1135insA 11

19 BRCA1.1177G>A 11

20 BRCA1.1191delC 11

21 BRCA1.1569G>T 11

22 BRCA1.1675delA 11

23 BRCA1.1806C>T 11

MUTATIONEXO

N

25 BRCA1.2557insG 11

26 BRCA1.2594delC 11

27 BRCA1.2988C>T 11

28 BRCA1.3109insAA 11

29 BRCA1.3124delA 11

30 BRCA1.3171ins5 11

31 BRCA1.3203del11 11

32 BRCA1.3297G>T 11

33 BRCA1.3347delAG 11

34 BRCA1.3438G>T 11

35BRCA1.3450delCAAG 11

36 BRCA1.3726C>T 11

37 BRCA1.4056C>T 11

38 BRCA1.4085delA 11

39 BRCA1.4154delA 11

40 BRCA1.4184del4 11

41 BRCA1.4418delA 13

42 BRCA1.4731C>T 15

43 BRCA1.4808C>G 16

44 BRCA1.4864delA 16

45 BRCA1.5002T>C 16

46 BRCA1.5166G>T 17

47 BRCA1.IVS17-2A>C IVS17

MUTATIONEXO

N

49 BRCA1.5382insC 18

50 BRCA1.5630G>A 24

51 BRCA1.5653delA 24

52 BRCA2.IVS2-7T>A IVS2

53 BRCA2.999delTCAAA 9

54 BRCA2.1886T>G 10

55 BRCA2.2024del5 10

56 BRCA2.2275delTCTC 11

57 BRCA2.3036delACAA 11

58 BRCA2.3824delACTG 11

59 BRCA2.4075delGT 11

60 BRCA2.4088delA 11

61BRCA2.5445delTTTAAGT 11

62 BRCA2.5805delT 11

63 BRCA2.6287delAACA 11

64 BRCA2.6312del5 11

65 BRCA2 6839_6840insC 11

66 BRCA2.7462delA 14

67 BRCA2.7786C>T 15

68 BRCA2.IVS23-2A>G IVS23

69 BRCA2.9481insA 24

70 BRCA2.9751G>T 26No

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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?

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

...and finally

Application to other

Populations

Diseases

Genes

Acknowledgements

Pat BondAnna Jeffery Smith Jonathan CoxheadJane CooperJoytika AttariRob BrownJohn BurnBernard KeavneyPål Møller

We raised £281.23 for Everyman cancer charity