Development of a BRCA2 screening service –
Introduction of high resolution MELT analysis
A Grade Trainee Project
Nick Camm
Yorkshire Regional Genetics Service
Overview
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BRCA2 screening strategy
High resolution MELT analysis background
Workup of High resolution MELT analysis
Results from initial testing
Potential for screening
Conclusions
BRCA2 Screening
• BRCA2 has 27 exons and spans ~70Kb encoding a
protein of 3418 amino acids.
• Current Screening Strategy in Leeds
– Bi-directional sequencing of 30 amplicons
– Protein truncation test for exon 11
• Proposed to reduce time and cost of screening by
introducing high resolution MELT analysis
High Resolution MELT (HRM)
Analysis
• HRM analysis is a rapid, closed tube
method used for the detection of
sequence variants
• Uses a double stranded DNA
saturating dye (LCGreen) incorporated
at the PCR stage
• PCR products are melted and the
fluorescence measured
• Sequence variants are identified via
melting curve analysis using the Light
scanner (Idaho Technologies)
Which Amplicons to choose for
HRM?
• Useful to have majority of samples with the same
genotype
– Reduces the need to confirm variants by alternative method
– Amplicons with one or more common SNPs could
complicate analysis
• Assessed SNP content and frequency for BRCA2
– Ensembl database
– Analysing sequence data for 95 patients from target
population
SNPs Present
30 Amplicons
20 Amplicons
Too large
12 Amplicons
HRM Analysis Workup
• 12 BRCA2 Amplicons suitable
for HRM analysis
• Optimise PCR using
Lightscanner Master mix
• Presence of LCGreen raised
the optimal annealing
temperature ~7°C to 62°C
• Products from temperature
gradient PCRs were analysed
• Screened a small panel of
genomic samples of known
sequence to test the ability to
detect variants
62°C
HRM analysis
• Analysis of PCR products was
carried out using the
LightScanner® instrument from
Idaho Technology
• Melt curves were obtained
between 62 -95ºC
• A normalisation process is carried
out on the raw curves
• The data can then be grouped
according to similarity
• Viewing the data as a difference
plot enables the differences in
variant samples to be clearly
seen
HRM analysis
• Analysis of PCR products was
carried out using the
LightScanner® instrument from
Idaho Technology
• Melt curves were obtained
between 62 -95ºC
• A normalisation process is carried
out on the raw curves
• The data can then be grouped
according to similarity
• Viewing the data as a difference
plot enables the differences in
variant samples to be clearly
seen
HRM analysis
• Analysis of PCR products was
carried out using the
LightScanner® instrument from
Idaho Technology
• Melt curves were obtained
between 62 -95ºC
• A normalisation process is carried
out on the raw curves
• The data can then be grouped
according to similarity
• Viewing the data as a difference
plot enables the differences in
variant samples to be clearly
seen
Results
• All variants clearly
distinguishable from wild-type
samples
• Wild type samples group tightly
together
Exon 21 - 14 wild type samples, 1
variant sample (c.8668C>A)
• Poor quality DNA samples can
appear to be variants
• Sub-optimal PCR conditions
prevent useful analysis
• Different sequence changes can
give similar melt curves
Exon 24 – 14 wild type samples, 1
variant sample (c.9117G>A)
Results
• All variants clearly
distinguishable from wild-type
samples
• Wild type samples group tightly
together
Exon 9 - 15 wild type samples
• Poor quality DNA samples can
appear to be variants
• Sub-optimal PCR conditions
prevent useful analysis
• Different sequence changes can
give similar melt curves
Exon 15 - 15 wild type samples
Results
• All variants clearly
distinguishable from wild-type
samples
• Wild type samples group tightly
together
• Poor quality DNA samples can
appear to be variants
• Sub-optimal PCR conditions
prevent useful analysis
• Different sequence changes can
give similar melt curves
Exon 5&6 - 15 wild type samples
Sample was nanodropped
and found to have
absorbance ratio’s indicative
of a poor quality sample.
Results
• All variants clearly
distinguishable from wild-type
samples
• Wild type samples group tightly
together
Exon 26
• Poor quality DNA samples can
appear to be variants
• Sub-optimal PCR conditions
prevent useful analysis
Exon 26 - 15 wild type samples
• Different sequence changes can
give similar melt curves
Results
• All variants clearly
distinguishable from wild-type
samples
• Wild type samples group tightly
together
• Poor quality DNA samples can
appear to be variants
• Sub-optimal PCR conditions
prevent useful analysis
• Different sequence changes can
give similar melt curves
Exon 23 - 13 wild type samples + 2
variant samples
Variants c.9038C>T and c.9117G>A
Gave indistinguishable melt profiles
Blind screening
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A panel of 45 genomic samples
were analysed blind for the 11
optimised amplicons
results compared to those
obtained by direct sequencing
Melt curves
analysed
Variants
detected
False
positives
451
3
4
False
Sensitivity Specificity
negatives
0
100%
4 samples were discounted from the analysis either due to poor
quality or lack of sequencing results
99.11%
Conclusions
• HRM analysis could be used as a reliable mutation scanning
method for ~1/3 of BRCA2 amplicons screened in Leeds
• Up to 2/3 of BRCA2 could be screened by HRM analysis
• HRM is quicker and cheaper than sequencing analysis
– Time to process a 96 well plate
• Sequencing ~11 hours
• HRM analysis ~3 hours
– Approximately 1/3 of the cost of bi-directional sequencing
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Good quality genomic DNA sample is essential
Fully optimised PCR is required
Sequencing of any variants must be carried out to confirm result
HRM analysis could potentially be used as a mutation scanning
technique for many genes currently screened by sequencing
Acknowledgements
• Yorkshire Regional DNA Laboratory
– Ruth Charlton
– Rachel Robinson
– Teresa Patrick
• Cancer Research UK (Leeds)
– Claire Taylor
– Graham Taylor
• Wessex National Genetics Reference laboratory
– Helen White