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 & cardiovascular systems – risk of sudden death 

FBN1

chr 15, 65 exons  350 kDa extracellular matrix protein Fibrillin

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

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

+ LightScanner HRM system

HRM analysis variant

Temperature

variant

Temperature

FBN1 assay design

Validation with positive controls

7 9 14 15 16 21 22 28 29 31 32 3 5 6

Exon

2

Nucleotide change

c.247+1G>A c.306T>C c.443-35A>G c.718C>T c.772C>T c.1122delT

c.1793insTT

c.1875T>C c.2023_2026delTTTG

c.2559C>A c.2684_2689del6

c.3511T>C c.3609_3610ins13

c.3963A>G c.4038C>G 39 43 45 46 47 53 54 55 56 57 63

Exon

33 34 35 37

Nucleotide change

c.4139G>A c.4270C>G c.4408T>C c.4588C>T c.4942+3_4942+9del7 c.5297-2A>G c.5671+28dupT c.5672-63G>T c.5816G>A c.6594C>T c.6617-21A>T c.6817A>G c.6888G>A c.7204+63C>A c.7852G>A

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

28 29 31 32 15 16 21 22 6 7 9 14 3 5

Exon

2

Nucleotide change

c.247+1G>A c.306T>C c.443-35A>G c.718C>T c.772C>T c.1122delT

c.1793insTT

c.1875T>C c.2023_2026delTTTG

c.2559C>A c.2684_2689del6

c.3511T>C c.3609_3610ins13

c.3963A>G c.4038C>G

Results Identified?

               55 56 57 63 46 47 53 54 37 39 43 45

Exon

33 34 35

Nucleotide change

c.4139G>A c.4270C>G c.4408T>C c.4588C>T c.4942+3_4942+9del7 c.5297-2A>G c.5671+28dupT c.5672-63G>T c.5816G>A c.6594C>T c.6617-21A>T c.6817A>G c.6888G>A c.7204+63C>A c.7852G>A

Identified?

   (  )         (  )  

Exons 46 and 54 – false negatives?

Exon 46 c.5672-63G>T het wild type c.5672-63G>T het wild type ?

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

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

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

Summary of validation

 28 positive controls tested  1 “true” false negative  22 false positives  Sensitivity ~ 96%  Specificity ~ 94%

Patient panel

 6 patients tested so far  Correctly identified 12 SNPs  Reduced number of false positives  Specificity ~98%

Conclusions

+ + + Sensitive Quick Low cost    False positives Different DNA samples Some user variability 

Suitable scanning technique for a large gene

Acknowledgements

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