1. Development of a BRCA2 screening service – Introduction of high resolution MELT analysis A Grade Trainee Project Nick Camm Yorkshire Regional Genetics Service

2. Overview BRCA2 screening strategy High resolution MELT analysis background Workup of High resolution MELT analysis Results from initial testing Potential for screening Conclusions

3. 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

4. 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)

5. 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 Amplicons12 Amplicons20 Amplicons SNPs PresentToo large

6. 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

7. 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

8. 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

9. 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

10. 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)

11. 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 15 - 15 wild type samples Exon 9 - 15 wild type samples

12. 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.

13. 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 26 - 15 wild type samples Exon 26

14. 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

15. 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 SensitivitySpecificity 451340100%99.11% 4 samples were discounted from the analysis either due to poor quality or lack of sequencing results

16. 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

17. 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