Analysis of Norwegian BRCA mutations
using Sequenom MALDI TOF MS
Ann Curtis on behalf of
James Eden
Institute 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 collaboration
Professor John Burn
Institute of Human Genetics
Newcastle University
Dr Pål Møller
Department of Medical Genetics
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)
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
Wild type allele (T)
Mutant allele (C)
Extension Primer (5500Da)
Extension Primer (5500Da)
T
C
+Polymerase enzyme
+ddATP/ddCTP/
ddTTP/ddGTP
extended Primer (5800Da)
A
T
extended Primer (6100Da)
G
C
‘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 SPECTROMETER
Matrix Assisted Laser Desorption/Ionisation Time of Flight mass spectrometry
Detector
Flight
path
Time of flight
Laser
Sequenom chip
(matrix)
5500Da
7000Da
Homozygous WT (TT)
5500Da
7000Da
Heterozygote (TC)
5500Da
7000Da
The masses of the 2
extension products
are distinguished by
the mass
spectrometer,
allowing the patient
to be genotyped for
the mutation.
Homozygous mut (CC)
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-plex
All multiplexes gave clean results on wild type DNA
MP2. 20-plex
MP3. 12-plex
MP4. 3-plex
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.
2.
3.
4.
5.
6.
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
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?
BRCA2_T7786C
Mutation correctly
detected in mutant
sample
Mutation absent in
all other samples
BRCA2.IVS23-2 A>G
Mutation correctly
detected in
mutant sample
Other mutation
detected in same
sample
BRCA1.C5002T
BRCA1.IVS22-25 T>A
Mutation not
detected in mutant
sample
Different mutation
found in same
sample
BRCA1.185insA
Summary
70 Norwegian mutations
62 Sequenom
8 MLPA
1 failed assay design
61 mutations – wild type sequence detected
55 mutation controls for validaion
51 mutant sequences detected
detected)
3 fails (all insertions)
1 mislabelling (different
mutation
Norwegian BRCA mutation list
MUTATION
EXON
MUTATION
EXON
MUTATION
EXON
1
BRCA1 del exons 1-13
-
25
BRCA1.2557insG
11
49
BRCA1.5382insC
18
2
BRCA1 del exons 18-24
-
26
BRCA1.2594delC
11
50
BRCA1.5630G>A
24
3
BRCA1 del exons 3-16
-
27
BRCA1.2988C>T
11
51
BRCA1.5653delA
24
4
BRCA1 del exons 5-7
-
28
BRCA1.3109insAA
11
52
BRCA2.IVS2-7T>A
5
BRCA1 del exons 8-13
-
29
BRCA1.3124delA
11
53
BRCA2.999delTCAAA
6
BRCA1 dup exon 13
-
30
BRCA1.3171ins5
11
54
BRCA2.1886T>G
10
7
BRCA1.2677ins356
11
31
BRCA1.3203del11
11
55
BRCA2.2024del5
10
8
BRCA2 del exon 3
-
32
BRCA1.3297G>T
11
56
BRCA2.2275delTCTC
11
9
BRCA1.120A>G
2
33
BRCA1.3347delAG
11
57
BRCA2.3036delACAA
11
10
BRCA1.185insA
2
34
BRCA1.3438G>T
11
58
BRCA2.3824delACTG
11
11
BRCA1.187delAG
2
35
BRCA1.3450delCAAG
11
59
BRCA2.4075delGT
11
12
BRCA1.458ins21
7
36
BRCA1.3726C>T
11
60
BRCA2.4088delA
11
13
BRCA1.505delG
7
37
BRCA1.4056C>T
11
61
BRCA2.5445delTTTAAGT
11
14
BRCA1.816delGT
11
38
BRCA1.4085delA
11
62
BRCA2.5805delT
11
15
BRCA1.913delCT
11
39
BRCA1.4154delA
11
63
BRCA2.6287delAACA
11
16
BRCA1.967T>A
11
40
BRCA1.4184del4
11
64
BRCA2.6312del5
11
17
BRCA1.1048delA
11
41
BRCA1.4418delA
13
65
BRCA2 6839_6840insC
11
18
BRCA1.1135insA
11
42
BRCA1.4731C>T
15
66
BRCA2.7462delA
14
19
BRCA1.1177G>A
11
43
BRCA1.4808C>G
16
67
BRCA2.7786C>T
15
20
BRCA1.1191delC
11
44
BRCA1.4864delA
16
68
BRCA2.IVS23-2A>G
21
BRCA1.1569G>T
11
45
BRCA1.5002T>C
16
69
BRCA2.9481insA
24
22
BRCA1.1675delA
11
46
BRCA1.5166G>T
17
70
BRCA2.9751G>T
26
23
BRCA1.1806C>T
11
47
BRCA1.IVS17-2A>C
IVS17
IVS2
9
IVS23
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 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