Nick Parkin

Trainee Clinical Scientist

Wessex Regional Genetics Laboratory

A false negative result for Myotonic Dystrophy type 2 using quadruplet

primed PCR

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

ZNF9 (CNBP1 )

• CCTG expansion in intron 1

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

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

ZNF9 Southern blotting

• Somatic mosaicism makes large expansions hard to detect

• 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

Normal vs. expansion image

DM2 set up in Wessex

Normal

DM2 set up in Wessex

Positive

• 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

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

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

Data from another of the testing centres

• Normal

• Affected family from Wessex

• +ve control

TGn TCTGn CCTGn CL3N58D FCL3N58D R

CCTGCCTGCCTGCCTGCCTGCCTGCCTG TCTG TCTCACTTTGTCCCCTAGGC

Sequencing of primer sites

Sequencing Primer

• Normal • Mutation (c.120-801T>C)

• Mutation not found in 80 normals, more being processed

TGn TCTGn CCTGn CL3N58D FCL3N58D R

Location of Mutation

CCTGCCTGCCTGCCTGCCTGCCTGCCTG TCTG YCTCACTTTGTCCCCTAGGC

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

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