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Overview

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. Overview. High density dystrophin gene aCGH platform Validation

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Overview

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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

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