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BRCA screening by HRM on the LightCycler 480


BRCA screening by HRM on the LightCycler? 480

Tom Janssens
Center for Human Genetics Laboratory for Molecular Diagnostics University Hospital Leuven, Belgium

HRM

r />What is HRM?
High resolution melting Mutation scanning Based on difference in melting patterns between homo- and heteroduplexes

DNA with heterozygote SNP

PCR

C G

+
C G T A C A T G

T A

Denaturation reannealing + Intercalating fluorescent dye

C G T A C A T G

C G T A

Increasing temperature

ESHG conference Barcelona, Spain

BRCA screening by HRM on the LightCycler 480

June 1st 2008

HRM

Applications
Mutation scanning (screening for unknown sequence variations) Genotyping (detection of known SNPs using unlabeled probes) Mutation scanning + genotyping (combination) Methylation analysis
ESHG conference Barcelona, Spain BRCA screening by HRM on the LightCycler 480 June 1st 2008

BRCA1 and BRCA2 screening
Very challenging genes
BRCA1: 22 coding exons

BRCA2: 26 coding exons

Lower than average GC content No mutation hot spots (insertions, deletions, duplications) Increasing number of clinical samples 2 strategies: direct sequencing or 2-tier strategy
ESHG conference Barcelona, Spain BRCA screening by HRM on the LightCycler 480 June 1st 2008

DHPLC vs HRM

Good performance …
high sensitivity (>95%) high specificity (>98%)

Cheaper system No expensive consumables “maintenance-free” Closed-tube system No separation of duplexes 10-15min melt

… but some limitations
Throughput Maintenance

Serial system Separation of duplexes multiple Tm

Daily Weekly Monthly

Much faster !!!

ESHG conference Barcelona, Spain

BRCA screening by HRM on the LightCycler 480

June 1st 2008

LC480 validation study: Setup
Extensive EuroGentest validation
Collaboration (Leuven, Leiden, Geneva) Many samples – different types of sequence variations Inter-laboratory parameters

3 phases
PCR optimization Mutation detection optimization Blind study

Aim
A complete primer set for BRCA1 and BRCA2 screening Power to determine a sensitivity of at least 99% Easy implementation in a diagnostic setting
ESHG conference Barcelona, Spain BRCA screening by HRM on the LightCycler 480 June 1st 2008

PCR optimization
Primers:
Based on existing primer sets for dHPLC or sequencing SNPcheck (NGRL Manchester) Fragment length (~ 200 – 400bp) M13 tags universal primers streamlined sequencing
BRCA1: 42 amplicons BRCA2: 61 amplicons

PCR
Criteria
- sigmoid shape of real-time curve - Cp < 28 - specific bands on 2% agarose - < 3 melting domains

Starting conditions
- 10 ?l reactions - 1x HRM master (Roche Applied Science) - 2 or 3 mM magnesium - 250 nM primers - 20 ng template DNA - same annealing temperature if possible - 40 cycles
June 1st 2008

ESHG conference Barcelona, Spain

BRCA screening by HRM on the LightCycler 480

PCR optimization: starting conditions

BRCA2 exon 2 Ta: 60°C 2mM Mg

S B

ESHG conference Barcelona, Spain

BRCA screening by HRM on the LightCycler 480

June 1st 2008

PCR optimization: starting conditions
No sigmoid shape Cp > 28 Low signal

BRCA2 exon 3 Ta: 60°C 2mM Mg

B S

ESHG conference Barcelona, Spain

BRCA screening by HRM on the LightCycler 480

June 1st 2008

PCR optimization: fine-tuning

BRCA2 exon 3 Ta: 57°C 3mM Mg Critical parameters Annealing temperature Magnesium concentration

S B

ESHG conference Barcelona, Spain

BRCA screening by HRM on the LightCycler 480

June 1st 2008

PCR optimization: redesign amplicon
BRCA2 exon 7, 3mM Mg, 60°C annealing No sigmoid Cp > 28 Low signal 3 MDs!

BRCA2 exon 7 Ta: 60°C 3mM Mg Smeared sample Redesign primer!
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S B 3 MDs!

BRCA screening by HRM on the LightCycler 480

June 1st 2008

PCR optimization: Summary
103 amplicons
BRCA1: 42 BRCA2: 61

fixed annealing temperatures
BRCA1: 4 blocks of at least 6 amplicons (59°C, 62°C, 63°C, 65°C) BRCA2: all at 57°C

all amplicons met required criteria

ESHG conference Barcelona, Spain

BRCA screening by HRM on the LightCycler 480

June 1st 2008

Mutation detection optimization
Gene scanning software (v1.3 v1.5)

Critical parameters for mutation detection
Pre- and post normalization interval settings Sensitivity setting
ESHG conference Barcelona, Spain BRCA screening by HRM on the LightCycler 480

Mutation detection optimization
Setup
Set of ~ 300 DNA samples with known sequence variations
Types of sequence variations

substitutions

64% 8%

insertions/dup lications deletions delins

1%

26%

Determine optimal settings for each amplicon
Normalization strategy: 1°C intervals right before and after melt region Sensitivity: range without false negatives (and false positives)

ESHG conference Barcelona, Spain

BRCA screening by HRM on the LightCycler 480

Mutation detection optimization: Example
BRCA2 exon 9 Sensitivity range = 0.63 – 1.00 OK

Automatic grouping function
IVS9+1G>A

Sample ID

c.714dupA

wt c.708T>C

ESHG conference Barcelona, Spain

BRCA screening by HRM on the LightCycler 480

June 1st 2008

Mutation detection optimization: Summary
All analyses performed on software v1.3 Detection rates:
BRCA1: 94% BRCA2: 97%

Fixed normalization intervals Variable sensitivity ranges

ESHG conference Barcelona, Spain

BRCA screening by HRM on the LightCycler 480

June 1st 2008

Blind study
Aim
determine sensitivity and specificity of HRM ~ 300 positive control samples (~ 150 per gene) Power to reach a sensitivity of at least 99% (CI: 95%) At least as many wild type samples to have equal power for specificity

Setup
Random distribution of samples (11 per amplicon + 1 ntc) Random distribution of amplicons over 3 labs inter-lab variability Blind and automatic analysis Pre-defined and optimized normalization and sensitivity settings Upgrade from Software 1.3 to 1.5

ESHG conference Barcelona, Spain

BRCA screening by HRM on the LightCycler 480

June 1st 2008

Blind study: Results
Normalization settings
Poor reproducibility between laboratories Shift in normalization intervals

Lab1

Lab2

Sensitivity settings
Fixed at 0.60 for most amplicons with software v1.5 some amplicons higher sensitivity setting

Preliminary sensitivity
BRCA1: > 97% BRCA2: > 99%
ESHG conference Barcelona, Spain

Preliminary specificity
BRCA1: > 92% BRCA2: > 77%
(88% with v1.3) (82% with v1.3)

(96% with v1.3) (96% with v1.3)

BRCA screening by HRM on the LightCycler 480

June 1st 2008

Back to theory …
Tm varies by: GC-content amplicon length

Tm is influenced by: Salt concentration MgCl2 concentration dye concentration

Highest Stability

Lowest Stability

G:C > A:T > G:G > G:T = G:A >T:T = A:A > T:C > A:C > C:C
BRCA genes are very challenging because of size and GC content!
ESHG conference Barcelona, Spain BRCA screening by HRM on the LightCycler 480 June 1st 2008

Conclusions
New software package v1.5:
Better performance More user-friendly

Normalization settings are not reproducible between labs
Use automatic software settings

Specificity needs improvement High sensitivity close to 99%...

adjust sensitivity setting

… however still 4 false negatives out of 296 tested samples
Current primer set not finalized yet for diagnostic purpose
ESHG conference Barcelona, Spain BRCA screening by HRM on the LightCycler 480 June 1st 2008

Future work / actions
Tackle false negative issues
Primer design Secondary structures GC content Literature: touchdown PCR

increased specificity?

Validation report Standard Operating Procedure (SOP) for practical use in diagnostic laboratories Poster at ESHG: P08.39
“Interlaboratory validation of High Resolution Melting (HRM) for BRCA1 and BRCA2 on the LightCycler? 480”
ESHG conference Barcelona, Spain BRCA screening by HRM on the LightCycler 480 June 1st 2008

Acknowledgements
University of Leuven (Belgium) Gert Matthijs Geneviève Michils Tina Overloop Geneva university hospital (Switzerland) Philippe Maillet Rapha?le Buser Leiden university hospital (The Netherlands) Bert Bakker Nienke van der Stoep Chantal van Paridon Roche Applied Science Penzberg (Germany) Udo Eichenlaub Reinhard Beck Gudrun Tellmann Oliver Geulen

ESHG conference Barcelona, Spain

BRCA screening by HRM on the LightCycler 480

June 1st 2008


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