Nick Parkin Trainee Clinical Scientist Wessex Regional Genetics Laboratory

1. A false negative result for Myotonic Dystrophy type 2 using quadruplet primed PCR Nick Parkin Trainee Clinical Scientist Wessex Regional Genetics Laboratory

2. 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

3. ZNF9 (CNBP1 ) CCTG expansion in intron 1

4. ZNF9 (CNBP1 ) CCTG expansion in intron 1 Complicated polymorphic repeat region (TG)n(TCTG)n(CCTG)n CCTG repeat normal range <75 repeats (including up to 3 interspersions) CCTG affected range 75-11,000 repeats

5. Tail specific primer Tail specific primer Tail specific primer Tail specific primer Tail specific primer Tail specific primer Tail specific primer Tail specific primer Repeat primer Repeat primer Repeat primer Repeat primer TGn TCTGn CCTGn CCTG CCTG CCTG CCTG CCTG CCTG CL3N58D CL3N58D R CL3N58D F

6. ZNF9 Southern blotting Somatic mosaicism makes large expansions hard to detect

7. DM2 set up in Wessex 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

8. DM2 set up in Wessex Normal vs. expansion image Normal

9. DM2 set up in Wessex Positive

10. DM2 set up in Wessex 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

11. Inter-centre audit of DM2 testing strategies Information on other laboratory’s testing acquired and compared

12. 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)

13. Data from another of the testing centres Normal Affected family from Wessex +ve control

14. CCTGCCTGCCTGCCTGCCTGCCTGCCTG TCTG TCTCACTTTGTCCCCTAGGC Sequencing of primer sites Sequencing Primer CL3N58D R TGn TCTGn CCTGn CL3N58D F

15. Mutation (c.120-801T>C) Normal Mutation not found in 80 normals, more being processed

16. CCTGCCTGCCTGCCTGCCTGCCTGCCTG TCTG YCTCACTTTGTCCCCTAGGC Location of Mutation CL3N58D R TGn TCTGn CCTGn CL3N58D F

17. CL3N58D R TGn TCTGn CCTGn CL3N58D F 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 Re-designed PCR primers to miss mutation New QP-PCR primers that no longer overlap Optimisation and validation are ongoing

18. 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