1. High density array comparative genomic hybridisation (aCGH) for dosage analysis and rapid breakpoint mapping in Duchenne Muscular Dystrophy (DMD) Victoria Cloke CMGS Spring Conference April 2010

2. Overview High density dystrophin gene aCGH platform Validation Application to specialised testing –Complex mutations –Therapeutic exon skipping trials

3. Cy3 labelled patient DNA Cy5 labelled control DNA Principle of aCGH

4. High density dystrophin array 4x44K format array designed by Madhuri Hegde’s group at Emory University 16,248 unique probes for the dystrophin gene region plus their reverse compliments Exons Introns 10bp 60bp 100bp

5. High density aCGH Validation +1 0 Dystrophin gene Stage 1: Normal control vs Normal control

6. +7 +6 +5 +4 +3 +2 +1 0 -1 -2 -3 -4 -5 -6 -7 Dystrophin exons 9 87 6 5 4 32 32.58743Mb 32.719041Mb32.850653Mb32.982265Mb High density aCGH Validation CNV in intron 2 Deletion dystrophin exons 3-7 Stage 2: Known exonic deletions and duplications Hemizygous male deletion

7. Heterozygous deletion dystrophin exon 45 High density aCGH Validation Stage 2: Known exonic deletions and duplications Heterozygous female deletions and duplications

8. High density aCGH Validation Stage 2: Known exonic deletions and duplications Heterozygous female deletions and duplications Heterozygous duplication dystrophin exons 49-50

9. High density aCGH Validation Stage 3: Inversion samples Deletions in dystrophin introns 44 and 45 Inversion Exon 45 c.6438+96064_6614+1540

10. IL1RAPL1 High density aCGH Validation Dystrophin Deletion dystrophin Exon 52 -2 -3 +6 +5 +4 +3 +2 +1 0 -4 -5 -6 Stage 3: Inversion samples Inversion Exon 53 –> 79 3’ deletion including 11 genes +7 -7

11. High density aCGH Validation Stage 3: Inversion samples Inversion Ex62 c.9164-10300_c.9224+12600 Intron 62 deletion

12. Applications of high density dystrophin aCGH Finding mutations in MLPA and point mutation negative patients Dystrophin Exon 44

13. Applications of dystrophin high density aCGH Difficulties in exon skipping for duplications —Orientation —Structure —Position of breakpoints Dystrophin aCGH study of 25 duplications —Structure of duplications —Rapid breakpoint mapping —Understanding how dystrophin duplications arise Informing a exon skipping trial targeting duplications

14. Duplication of dystrophin exons 17-45 Duplication aCGH results 31.692239Mb 31.698543Mb31.704848Mb31.711153Mb +2 +1 -1 -2 +2 +1 -1 -2 0 Dystrophin exons51 Duplication of dystrophin exon 51 +1

15. Duplication breakpoint mapping Exon 51

16. Breakpoint sequencing results Ease of breakpoint mapping –15/25 breakpoints (60%) needed just one round of PCR and sequencing 20/25 (80%) central breakpoints amplified and sequenced Tandem orientation

17. Breakpoint sequencing results Intron 7 Duplication sequence Intron 2 Intron 30 Intron 17 Duplication sequence Intron 1 Intron 4 Duplication sequence Microhomology 1-4 nucleotides 14/20 (70%) Small insertion 1-4 nucleotides 4/20 (20%) Clean breakpoint 2/20 (10%)

18. Genomic DNA: Duplication Exons 3-37 Breakpoint close to exon 37 RNA level: Duplication Exons 3-36 Duplication study Exon 37 13bp Exon 36Exon 3Exon 36Exon 3Exon 4-37 Exon 38-> Exon 1-2Exon 3-35 Comparison with RNA results

19. Duplication study Non-allelic homologous recombination (NAHR) –Lack of homology between breakpoints 34% - 48% (mean 42%) sequence identity No shared repetitive element homology Non-homologous recombination (NHR) –Simple tandem structure –Non-recurrent breakpoints –Microhomology and insertions  DNA repair mechanism such as non-homologous endjoining (NHEJ)  Replication based mechanism such as fork stalling and template switching (FoSTeS) Understanding the mechanism of duplications E.g.

20. Array CGH vs MLPA Array CGH as a specialist test –Solving difficult cases –Rapid breakpoint mapping e.g. to Inform therapeutic strategies Conclusions High Density aCGHMLPA Consumable Cost£178£40 Information provided44K probesLimited to ~1 probe/exon ReliabilityMultiple 60nucleotide probes/aberration Risk of false positives FlexibilityCustom designLimited kit availability

21. Acknowledgements Dr Steve Abbs Dr Michael Yau Jo McCauley Dr Joo Wook Ahn Prof Francesco Muntoni Jihee Kim Dr Madhuri Hegde Ephrem Chin