Using the Reveal Genetic Mutation Discovery System ( Discovery

Using the Reveal Genetic Mutation Discovery System (
Discovery System ( Spectrumedix
Spectrumedix ) for Mutation Screening in a Diagnostic Laboratory JK Campbell
JK Campbell Ø Ø Overview of Spectrumedix System Ø Ø Evaluation using mutation detection reference reagents Ø Ø Summary of current use at Guy’s
Summary of current use at Guy’s Reveal Genetic Mutation Discovery System (( Spectrumedix
Spectrumedix ) Ø Ø
Temperature gradient capillary electrophoresis (TGCE) l l
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Fragments with different melting properties can be analysed together Multiple DNA fragments can be analysed together Uses ethidium bromide to detect DNA l l
No need for fluorescent primers 96 Capillaries Ø Ø Specific software to compare peak traces and identify mutations Ø Ø Can also be used for genotyping and sequencing
sequencing Ø Ø
Reveal Genetic Mutation Discovery System Final Temp Tm amplicon B and C p
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Tm amplicon A T
Initial Temp em
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Time A Time B mutation start normal mutation normal mutation Time A normal mutation normal Time B Revelation Mutation Detection Software Raw data Normalised data Mutation Report
Revelation Mutation Detection Software
Revelation Mutation Detection Software Revelation Mutation Detection Software
Revelation Mutation Detection Software Revelation Mutation Detection Software
Revelation Mutation Detection Software Evaluation using mutation detection reference reagents Aims Ø Ø To determine sensitivity and specificity Ø Ø To determine the best temperature range for analysis Ø Ø To determine any correlation between type of mutation and position with ease of detection
mutation and position with ease of detection Study Design § §
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Generic mutation detection reference reagents supplied by The Na
Generic mutation detection reference reagents supplied by The Na tional Genetics Reference Laboratory (Wessex). 4 plasmid based fragments produced with an average GC content of 20, 40, 60 or 80%. Each of these sequences has been mutated to produce all possible base substitutions at three positions within the amplicon
amplicon . Mutation created
Mutation created Sequence generated Heteroduplex produced Heteroduplex produced A > C nnnC Gnnn nnn CGnnn C:T & G:A A > T nnnT Gnnn nnn TGnnn T:T & A:A G > A nnnAA nnn nnnA Annn A:C & G:T G > C nnnAC nnn nnnA Cnnn C:C & G:G …nnnAGnnn... Position 1 (P1) …nnnAGnnn... Position 2 (P2) …nnnAGnnn... Position 3 (P3) Results – Sensitivity and Specificity TGCE CSCE
CSCE True positive 84 89 True negative 54 73 False positive 26 6 False negative 4 7 Fails 8 1 Sensitivity 95% 93% Specificity 68% 92% Results – Best Analysis Temperature Ø Ø
Temperature range can be set by user l l
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No major differences in mutation detection at different temperature ranges, however l l
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35
35 ­­ 45 o C 50
50 ­­ 60 o C (default) 55
55 ­­ 65 o C 64
64 ­­ 67 o C – recommended for high GC fragments 20% GC fragments degraded at all temps except 35
20% GC fragments degraded at all temps except 35 ­­ 45 o C 40% GC fragments degraded at 64
40% GC fragments degraded at 64 ­­ 67 o C Several mutations produced only subtle traces at 35
Several mutations produced only subtle traces at 35 ­­ 45 o C Overall conclusion was that a single temperature range (50
(50 ­­ 60 o C) was sufficient to detect all mutations
C) was sufficient to detect all mutations Results ­ Correlations Ø Ø
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Heteroduplex traces were classified according to how clear they were: l l
1 – mutation missed l l
2 – very subtle (
very subtle ( eg wider) l l
3 – subtle (
subtle ( eg small shoulder) l l
4 – Obvious l l
5 – Amazing (
Amazing ( eg 3 or 4 separate peaks) Data was stratified according to position and type of mutation to look for correlations.
mutation to look for correlations. strength of heteroduplex Position of Mutation 5.0 amazing
4.0 obvious TGCE CSCE 3.0 subtle 2.0 very subtle 1.0 missed 0.0 P1 P2 Position of mutation P3 Type of Mutation 5.0 amazing
4.0 obvious Strength of heteroduplex TGCE CSCE 3.0 subtle 2.0 very subtle 1.0 missed 0.0 A>C A>T G>A Type of mutation G>C Strength of heteroduplex GC Content of Fragment 5.0 amazing
4.0 obvious TGCE CSCE 3.0 subtle 2.0 very subtle 1.0 missed 0.0 20 40 60 GC conte nt of fragment 80 Summary of mutation screening using the Spectrumedix at Guy’s Disease
Gene(s) Congenital Muscular Dystrophy POMT1, POMT2, POMGnT1, LARGE, Fukutin LAMA2 HSP Alports HBOC DMD/BMD Spastin (SPG4) COL4A5 BRCA1 + BRCA2 Dystrophin 82 80 M # fragments 80 65 single/ multiplex M S S M M Analysis temperature 50­60 50­60 50­60 55­60 50­60 and 35­45 sensitivity 100% (4/4) 100% (9/9) 95.8% (23/24) 90% (27/30) 97.7 (43/44) sequencing Exons sequenced 16 51 (+1 control) 14.4% (8.6% HD, 5.8% fails) 1 1 2 Summary Ø Ø TGCE is being used successfully at Guy’s for mutation scanning Ø Ø Sensitivity is comparable to other techniques Ø Ø TGCE has several advantages: l l
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Analysis of fragments with different melt properties simultaneously Analysis of multiplex data No need for fluorescent primers Analysis software with mutation calling
Analysis software with mutation calling Acknowledgements Ø Ø Helen White Ø Ø Judith Pagan Ø Ø Rachael Mein Ø Ø Caroline Godfrey Ø Ø David Moore Ø Ø Annabel Whibley
Annabel Whibley