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
+
variant
Temperature
D Fluorescence
Fluorescence
HRM analysis
variant
Temperature
FBN1 assay design
Validation with positive controls
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
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
Results
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

Identified?
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 – false negative
Exon 45
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