Mutation scanning inMutation scanning inMarfan syndrome usingMarfan syndrome usingHigh Resolution Melt High Resolution Melt

analysisanalysis

Kate Sergeant,Kate Sergeant, Northern Genetics Service, Northern Genetics Service, Newcastle upon TyneNewcastle upon Tyne

Marfan syndromeMarfan syndrome

Autosomal dominant, 1 in 5 000 – 1 in 10 000Autosomal dominant, 1 in 5 000 – 1 in 10 000

Connective tissue disorderConnective tissue disorder Affects ocular, skeletal &Affects ocular, skeletal &

cardiovascular systems – risk of sudden deathcardiovascular systems – risk of sudden death FBN1FBN1 chr 15, 65 exons chr 15, 65 exons 350 kDa extracellular matrix protein Fibrillin350 kDa extracellular matrix protein Fibrillin

FBN1FBN1 mutations mutations Over 600 reported mutations (UMD-FBN1)Over 600 reported mutations (UMD-FBN1) Most mutations are uniqueMost mutations are unique Most pathogenic mutations are missense Most pathogenic mutations are missense

affecting cysteine residuesaffecting cysteine residues Mutation analysis of Mutation analysis of FBN1FBN1 exons detects ~80% exons detects ~80% Identifying a mutation gives a definitive Identifying a mutation gives a definitive

diagnosis – cardiological screening to those at diagnosis – cardiological screening to those at riskrisk

AimsAims

Set up an assay for mutation scanning in Set up an assay for mutation scanning in FBN1FBN1 Using the LightScanner High Resolution Melt Using the LightScanner High Resolution Melt

(HRM) analysis system for heteroduplex (HRM) analysis system for heteroduplex detectiondetection

Validate this method using positive Validate this method using positive controlscontrols

Test Marfan syndrome patients for Test Marfan syndrome patients for FBN1FBN1 mutationsmutations

++

LightScanner HRM systemLightScanner HRM system

HRM analysisHRM analysis

Temperature

Flu

ores

cenc

e

variant

variant

Temperature

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luor

esce

nce

FBN1FBN1 assay design assay design

Validation with positive Validation with positive controlscontrols

c.7852G>A63c.4038C>G32

c.7204+63C>A57c.3963A>G31

c.6888G>A56c.3609_3610ins1329

c.6817A>G55c.3511T>C28

c.6617-21A>T54c.2684_2689del622

c.6594C>T53c.2559C>A21

c.5816G>A47c.2023_2026delTTTG16

c.5672-63G>T46c.1875T>C15

c.5671+28dupT45c.1793insTT14

c.5297-2A>G43c.1122delT9

c.4942+3_4942+9del739c.772C>T7

c.4588C>T37c.718C>T6

c.4408T>C35c.443-35A>G5

c.4270C>G34c.306T>C3

c.4139G>A33c.247+1G>A2

Nucleotide changeExonNucleotide changeExon

ResultsResultsExon 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

ResultsResults

c.7852G>A63c.4038C>G32

c.7204+63C>A57c.3963A>G31

c.6888G>A56c.3609_3610ins1329

c.6817A>G55c.3511T>C28

()c.6617-21A>T54c.2684_2689del622

c.6594C>T53c.2559C>A21

c.5816G>A47c.2023_2026delTTTG16

()c.5672-63G>T46c.1875T>C15

c.5671+28dupT45c.1793insTT14

c.5297-2A>G43c.1122delT9

c.4942+3_4942+9del739c.772C>T7

c.4588C>T37c.718C>T6

c.4408T>C35c.443-35A>G5

c.4270C>G34c.306T>C3

c.4139G>A33c.247+1G>A2

Identified?Nucleotide changeExonIdentified?Nucleotide changeExon

Exons 46 and 54 – false Exons 46 and 54 – false negatives?negatives?

c.5672-63G>T het

wild type

Exon 46

c.5672-63G>T het

wild type

?

Exon 45 – false negativeExon 45 – false negative

c.5671+28dupT het

Exon 45

Exon 45 – larger sample Exon 45 – larger sample numbernumber

c.5671+28dupT het

False positivesFalse positives

22 false positives were encountered22 false positives were encountered Problem with archived DNA and different Problem with archived DNA and different

extraction methodsextraction methods Reduce this byReduce this by

Standardising extraction methodsStandardising extraction methods Dilute DNA samples in a common bufferDilute DNA samples in a common buffer Double reaction volumeDouble reaction volume

Summary of validationSummary of validation

28 positive controls tested28 positive controls tested 1 “true” false negative1 “true” false negative 22 false positives22 false positives

Sensitivity ~ 96%Sensitivity ~ 96% Specificity ~ 94%Specificity ~ 94%

Patient panelPatient panel

6 patients tested so far6 patients tested so far Correctly identified 12 SNPsCorrectly identified 12 SNPs Reduced number of false positivesReduced number of false positives

Specificity ~98%Specificity ~98%

ConclusionsConclusions

+ SensitiveSensitive+ QuickQuick+ Low costLow cost

False positivesFalse positives Different DNA samplesDifferent DNA samples Some user variabilitySome user variability

Suitable scanning technique for a large gene

AcknowledgementsAcknowledgements

All in the Newcastle laboratoryAll in the Newcastle laboratory David BournDavid Bourn Claire Healey, Val Wilson & Danny RoutledgeClaire Healey, Val Wilson & Danny Routledge

Salisbury laboratory – Catharina YearwoodSalisbury laboratory – Catharina Yearwood