Nick Parkin Trainee Clinical Scientist Wessex Regional Genetics Laboratory
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Transcript of Nick Parkin Trainee Clinical Scientist Wessex Regional Genetics Laboratory
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Nick Parkin
Trainee Clinical Scientist
Wessex Regional Genetics Laboratory
A false negative result for Myotonic Dystrophy type 2 using quadruplet
primed PCR
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Myotonic dystrophy type 2 (DM2)/ Proximal Myotonic Myopathy (PROMM)
• Autosomal dominant, multi-systemic degenerative myopathy characterized by;
• Progressive muscle weakness,
• Myotonia
• Cataracts
• Cardiac abnormalities
• Frontal balding
• Infertility
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ZNF9 (CNBP1 )
• CCTG expansion in intron 1
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ZNF9 (CNBP1 )
• CCTG repeat normal range <75 repeats (including up to 3 interspersions)
(TG)n(TCTG)n(CCTG)n
• CCTG expansion in intron 1
• Complicated polymorphic repeat region
• CCTG affected range 75-11,000 repeats
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CL3N58D
TGn TCTGn CCTGn CL3N58D FCL3N58D R
Repeat primerRepeat primer
CCTG
Tail specific primerTail specific primer
CCTG
Tail specific primer Repeat primerTail specific primerTail specific primerTail specific primer
CCTG
Tail specific primer Repeat primerTail specific primer
CCTGCCTGCCTG
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ZNF9 Southern blotting
• Somatic mosaicism makes large expansions hard to detect
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• CL3N58D primers (Day et al 2003)
• 50 presumed normal, 3 known controls (2 negative and 1 positive)
• Known controls from were patients from Wessex previously tested at another centre
• 1 of the previously reported negatives tested positive
DM2 set up in Wessex
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Normal vs. expansion image
DM2 set up in Wessex
Normal
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DM2 set up in Wessex
Positive
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• 2 other family members available
• Also tested positive
• Samples sent out to other testing laboratories on UKGTN
• 2 centres found all 3 negative
• 2 centres (not including my study) found all 3 positive
DM2 set up in Wessex
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Inter-centre audit of DM2 testing strategies
• Information on other laboratory’s testing acquired and comparedCentre TP-PCR P1 TP-PCR P3 TP-PCR P4
Wessex GGCCTTATAACCATGCAAATG(FAM) TACGCATCCGAGTTTGAGACG TACGCATCCGAGTTTGAGACGCAGGCAGGCAGGCAGGCAGG
1 GCCTAGGGGACAAAGTGAGA (6-FAM) TACGCATCCCAGTTTGAGACG TACGCATCCCAGTTTGAGACGCCTGCCTGCCTGCCTG
TACGCATCCCAGTTTGAGACGBCTGBCTGBCTGBCTG
2 GCCTAGGGGACAAAGTGAGA(FAM) TACGCATCCCAGTTTGAGACG TACGCATCCCAGTTTGAGACGCCTGCCTGCCTGCCTG
3 GCC TAG GGG ACA AAG TGA GA TAC GCA TCC CAG TTT GAG ACG 3’ (DYE 4 labelled) TAC GCA TCC CAG TTT GAG ACG CCT GCC TGC CTG CCT G
4GCC TAGG GGA CAA AGT GAG A
(FAM) TAC GCA TCC CAG TTT GA GAC G TAC GCA TCC CAG TTT GAG ACG CCT GCC TGC CTG CCT G
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Inter-centre audit of DM2 testing strategies
• Information on other laboratory’s testing acquired and compared
• No major differences seen except for the use of the common primer (My testing strategy used the opposite primer to all of the other centres)
Taq Mix Conditions Cycles Denatured? Run On
denaturation annealing elongation
Epi-centre Fail safe Premix J 94°C for 1 min 51°C for 2 min 72°C for 2 min x30 No 3100
Immolase 4.0µl 5.5M betaine 94°C for 1 min 60°C for 1 min 72°C for 2 min x35 Yes-95°C 3100/3130
Immolase 4.0µl 5.5M betaine 94°C for 1 min 58°C for 1 min 72°C for 2 min x35 Yes-95°C 3100/3130
Amplitaq Gold 1µl DMSO 94ºC 1min 55ºC* 1min 72ºC 2min x32 Yes-95°C 3100
Megamix 1µl DMSO 95ºC 30s 60ºC 30s 72ºC 1min x31 Yes-95°C 3100
Fast start Taq 8µl 5U/µl betaine 94°C for 1 min 58°C for 1 min 72°C for 2 min x35 No CEQ8000
FastStart PCR kit CG rich 5µl 94°C for 1 min 60°C for 1 min 72°C for 2 min x35 Yes-95°C 3100
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Data from another of the testing centres
• Normal
• Affected family from Wessex
• +ve control
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TGn TCTGn CCTGn CL3N58D FCL3N58D R
CCTGCCTGCCTGCCTGCCTGCCTGCCTG TCTG TCTCACTTTGTCCCCTAGGC
Sequencing of primer sites
Sequencing Primer
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• Normal • Mutation (c.120-801T>C)
• Mutation not found in 80 normals, more being processed
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TGn TCTGn CCTGn CL3N58D FCL3N58D R
Location of Mutation
CCTGCCTGCCTGCCTGCCTGCCTGCCTG TCTG YCTCACTTTGTCCCCTAGGC
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Conclusions
• Testing is left short due to overlapping sites
• Primers need to be re-designed
• This is difficult due to the high sequence homology upstream
• Not many viable options
TGn TCTGn CCTGn CL3N58D FCL3N58D R
• Re-designed PCR primers to miss mutation
• New QP-PCR primers that no longer overlap
• Optimisation and validation are ongoing
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Acknowledgements
WRGL:
Oliver Wood
Sophie Marks
Phillipa Duncan
Esta Cross
David Robinson
James Macpherson
John Harvey
Plus:
All the scientists that were involved from all the other centres