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Mutation scanning in Marfan syndrome using High Resolution Melt analysis

Mutation scanning in Marfan syndrome using High Resolution Melt analysis. Kate Sergeant, Northern Genetics Service, Newcastle upon Tyne. Marfan syndrome. Autosomal dominant, 1 in 5 000 – 1 in 10 000 Connective tissue disorder Affects ocular, skeletal &

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Mutation scanning in Marfan syndrome using High Resolution Melt analysis

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  1. Mutation scanning inMarfan syndrome usingHigh Resolution Melt analysis Kate Sergeant, Northern Genetics Service, Newcastle upon Tyne

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

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

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

  5. LightScanner HRM system +

  6. HRM analysis variant Fluorescence D Fluorescence variant Temperature Temperature

  7. FBN1 assay design

  8. Exon Nucleotide change Exon Nucleotide change 2 c.247+1G>A 33 c.4139G>A 3 c.306T>C 34 c.4270C>G 5 c.443-35A>G 35 c.4408T>C 6 c.718C>T 37 c.4588C>T 7 c.772C>T 39 c.4942+3_4942+9del7 9 c.1122delT 43 c.5297-2A>G 14 c.1793insTT 45 c.5671+28dupT 15 c.1875T>C 46 c.5672-63G>T 16 c.2023_2026delTTTG 47 c.5816G>A 21 c.2559C>A 53 c.6594C>T 22 c.2684_2689del6 54 c.6617-21A>T 28 c.3511T>C 55 c.6817A>G 29 c.3609_3610ins13 56 c.6888G>A 31 c.3963A>G 57 c.7204+63C>A 32 c.4038C>G 63 c.7852G>A Validation with positive controls

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

  10. Results Exon Nucleotide change Identified? Exon Nucleotide change Identified? 2 c.247+1G>A  33 c.4139G>A  3 c.306T>C  34 c.4270C>G  5 c.443-35A>G  35 c.4408T>C  6 c.718C>T  37 c.4588C>T  7 c.772C>T  39 c.4942+3_4942+9del7  9 c.1122delT  43 c.5297-2A>G  14 c.1793insTT  45 c.5671+28dupT  15 c.1875T>C  46 c.5672-63G>T () 16 c.2023_2026delTTTG  47 c.5816G>A  21 c.2559C>A  53 c.6594C>T  22 c.2684_2689del6  54 c.6617-21A>T () 28 c.3511T>C  55 c.6817A>G  29 c.3609_3610ins13  56 c.6888G>A  31 c.3963A>G  57 c.7204+63C>A  32 c.4038C>G  63 c.7852G>A 

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

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

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

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

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

  16. Patient panel • 6 patients tested so far • Correctly identified 12 SNPs • 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 • David Bourn • Claire Healey, Val Wilson & Danny Routledge • Salisbury laboratory – Catharina Yearwood

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