1. Mutation scanning in Marfan syndrome using High Resolution Melt analysis Kate Sergeant, Northern Genetics Service, Newcastle upon Tyne

2. Marfan syndrome Autosomal dominant, 1 in 5 000 – 1 in 10 000 Autosomal dominant, 1 in 5 000 – 1 in 10 000 Connective tissue disorder Connective tissue disorder Affects ocular, skeletal & Affects ocular, skeletal & cardiovascular systems – risk of sudden death cardiovascular systems – risk of sudden death FBN1 chr 15, 65 exons FBN1 chr 15, 65 exons 350 kDa extracellular matrix protein Fibrillin 350 kDa extracellular matrix protein Fibrillin

3. FBN1 mutations Over 600 reported mutations (UMD-FBN1) Over 600 reported mutations (UMD-FBN1) Most mutations are unique Most mutations are unique Most pathogenic mutations are missense affecting cysteine residues Most pathogenic mutations are missense affecting cysteine residues Mutation analysis of FBN1 exons detects ~80% Mutation analysis of FBN1 exons detects ~80% Identifying a mutation gives a definitive diagnosis – cardiological screening to those at risk Identifying a mutation gives a definitive diagnosis – cardiological screening to those at risk

4. Aims Set up an assay for mutation scanning in FBN1 Set up an assay for mutation scanning in FBN1 Using the LightScanner High Resolution Melt (HRM) analysis system for heteroduplex detection Using the LightScanner High Resolution Melt (HRM) analysis system for heteroduplex detection Validate this method using positive controls Validate this method using positive controls Test Marfan syndrome patients for FBN1 mutations Test Marfan syndrome patients for FBN1 mutations

5. + LightScanner HRM system

6. HRM analysis Temperature Fluorescence variant Temperature  Fluorescence

7. FBN1 assay design

8. Validation with positive controls

9. Results Exon 2 c.247+1G>A het Exon 43 c.5297-2A>G het Exon 57_2 c.7204+63C>A het Exon 29 c.3609_3610ins13 het

10. Results c.7852G>A63 c.4038C>G32 c.7204+63C>A57 c.3963A>G31 c.6888G>A56 c.3609_3610ins1329 c.6817A>G55 c.3511T>C28 ( ) c.6617-21A>T54 c.2684_2689del622 c.6594C>T53 c.2559C>A21 c.5816G>A47 c.2023_2026delTTTG16 ( ) c.5672-63G>T46 c.1875T>C15  c.5671+28dupT45 c.1793insTT14 c.5297-2A>G43 c.1122delT9 c.4942+3_4942+9del739 c.772C>T7 c.4588C>T37 c.718C>T6 c.4408T>C35 c.443-35A>G5 c.4270C>G34 c.306T>C3 c.4139G>A33 c.247+1G>A2 Identified? Nucleotide changeExon Identified? Nucleotide changeExon

11. Exons 46 and 54 – false negatives? c.5672-63G>T het wild type Exon 46 c.5672-63G>T het wild type ?

12. Exon 45 – false negative c.5671+28dupT het Exon 45

13. Exon 45 – larger sample number c.5671+28dupT het

14. False positives 22 false positives were encountered 22 false positives were encountered Problem with archived DNA and different extraction methods Problem with archived DNA and different extraction methods Reduce this by Reduce this by Standardising extraction methods Standardising extraction methods Dilute DNA samples in a common buffer Dilute DNA samples in a common buffer Double reaction volume Double reaction volume

15. Summary of validation 28 positive controls tested 28 positive controls tested 1 “true” false negative 1 “true” false negative 22 false positives 22 false positives  Sensitivity ~ 96%  Specificity ~ 94%

16. Patient panel 6 patients tested so far 6 patients tested so far Correctly identified 12 SNPs Correctly identified 12 SNPs Reduced number of false positives Reduced number of false positives  Specificity ~98%

17. Conclusions + Sensitive + Quick + Low cost  False positives  Different DNA samples  Some user variability Suitable scanning technique for a large gene

18. Acknowledgements All in the Newcastle laboratory All in the Newcastle laboratory David Bourn David Bourn Claire Healey, Val Wilson & Danny Routledge Claire Healey, Val Wilson & Danny Routledge Salisbury laboratory – Catharina Yearwood Salisbury laboratory – Catharina Yearwood