mutation scanning in marfan syndrome using high resolution melt analysis
Post on 06-Jan-2016
88 Views
Preview:
DESCRIPTION
TRANSCRIPT
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
F
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
top related