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

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

  3. Principleof aCGH Cy5 labelled control DNA Cy3 labelled patient DNA

  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 60bp 100bp 10bp Exons Introns

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

  6. 32.58743Mb 32.719041Mb 32.850653Mb 32.982265Mb +7 +6 +5 +4 +3 +2 +1 0 - 1 - 2 - 3 - 4 - 5 - 6 - 7 Dystrophin exons 9 8 7 6 5 4 3 2 CNV in intron 2 High density aCGH Validation Stage 2: Known exonic deletions and duplications Hemizygous male deletion Deletion dystrophin exons 3-7

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

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

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

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

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

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

  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. 31.692239Mb 31.698543Mb 31.704848Mb 31.711153Mb +2 +2 +1 +1 +1 0 - - 1 1 - - 2 2 Dystrophin exons 51 Duplication of dystrophin exon 51 Duplication aCGH results Duplication of dystrophin exons 17-45

  15. Duplication breakpoint mapping Exon 51 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 1 Microhomology 1-4 nucleotides 14/20 (70%) Duplication sequence Intron 4 Intron 30 Small insertion 1-4 nucleotides 4/20 (20%) Duplication sequence Intron 17 Intron 7 Clean breakpoint 2/20 (10%) Duplication sequence Intron 2

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

  19. Duplication study Understanding the mechanism of duplications • 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) E.g.

  20. Conclusions • Array CGH vs MLPA • Array CGH as a specialist test • Solving difficult cases • Rapid breakpoint mapping e.g. to Inform therapeutic strategies

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