Validation of BRCA2 mutation scanning using the LightScanner system for high resolution melt analysis Lewis Darnell Nottingham Regional Molecular Genetics Service.
Download ReportTranscript Validation of BRCA2 mutation scanning using the LightScanner system for high resolution melt analysis Lewis Darnell Nottingham Regional Molecular Genetics Service.
Slide 1
Validation of BRCA2 mutation scanning
using the LightScanner system for high
resolution melt analysis
Lewis Darnell
Nottingham Regional Molecular Genetics Service
Slide 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.
Gene
Cancer type
Lifetime risk (%)
BRCA1
Breast
65-85
Ovarian
39-63
Breast
45-84
Ovarian
11-27
BRCA2
Slide 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.
Slide 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) prescreen. 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.
Slide 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).
Slide 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
Slide 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.
Slide 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
Slide 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
Slide 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
WT
CA reagent cleaned DNA
c.1365A>G
c.1359delA
False-positive
Slide 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
Slide 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.
Slide 13
Results after optimisation
After amplicon optimisation 42 of 47 amplicons met the
validation criteria.
Mutation type
Number of variants detected
Number variants not detected
Nucleotide change
106
1
Nucleotide deletion
30
1
Nucleotide duplication
3
0
Nucleotide insertion
1
0
Total
140
2
140 of 142 variants detected. Sensitivity: 98.6%.
Not including results from amplicons 10-1b, 10-2 and 11-1.
Slide 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 115, 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
Slide 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 16capillary 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
Slide 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
Slide 17
Acknowledgements
Nottingham Molecular Genetics Service
•
•
•
•
Rachael Tredwell
Jo Field
Gareth Cross
All staff
Clent Life Science
• David Harris
Idaho Technologies
• Jason McKinney
Validation of BRCA2 mutation scanning
using the LightScanner system for high
resolution melt analysis
Lewis Darnell
Nottingham Regional Molecular Genetics Service
Slide 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.
Gene
Cancer type
Lifetime risk (%)
BRCA1
Breast
65-85
Ovarian
39-63
Breast
45-84
Ovarian
11-27
BRCA2
Slide 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.
Slide 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) prescreen. 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.
Slide 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).
Slide 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
Slide 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.
Slide 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
Slide 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
Slide 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
WT
CA reagent cleaned DNA
c.1365A>G
c.1359delA
False-positive
Slide 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
Slide 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.
Slide 13
Results after optimisation
After amplicon optimisation 42 of 47 amplicons met the
validation criteria.
Mutation type
Number of variants detected
Number variants not detected
Nucleotide change
106
1
Nucleotide deletion
30
1
Nucleotide duplication
3
0
Nucleotide insertion
1
0
Total
140
2
140 of 142 variants detected. Sensitivity: 98.6%.
Not including results from amplicons 10-1b, 10-2 and 11-1.
Slide 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 115, 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
Slide 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 16capillary 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
Slide 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
Slide 17
Acknowledgements
Nottingham Molecular Genetics Service
•
•
•
•
Rachael Tredwell
Jo Field
Gareth Cross
All staff
Clent Life Science
• David Harris
Idaho Technologies
• Jason McKinney