Development of a BRCA2 screening service – Introduction of high resolution MELT analysis A Grade...

Development of a BRCA2 screening service –

Introduction of high resolution MELT analysis

A Grade Trainee Project

Nick Camm

Yorkshire Regional Genetics Service

Overview

• 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

30 Amplicons 12 Amplicons20 Amplicons

SNPs Present Too large

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

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 24 – 14 wild type samples, 1 variant sample (c.9117G>A)

Exon 21 - 14 wild type samples, 1 variant sample (c.8668C>A)

Results






Exon 15 - 15 wild type samples


Results






Exon 5&6 - 15 wild type samples

Sample was nanodropped and found to have absorbance ratio’s indicative of a poor quality sample.

Results







Exon 26

Results






Exon 23 - 13 wild type samples + 2 variant samples

Variants c.9038C>T and c.9117G>A Gave indistinguishable melt profiles

Blind screening

• 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

False negatives

Sensitivity Specificity

451 3 4 0 100% 99.11%

4 samples were discounted from the analysis either due to poor quality or lack of sequencing results

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

• 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

Development of a BRCA2 screening service – Introduction of high resolution MELT analysis A Grade...

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