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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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slide1

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

overview
Overview
  • High density dystrophin gene aCGH platform
  • Validation
  • Application to specialised testing
    • Complex mutations
    • Therapeutic exon skipping trials
slide3

Principleof aCGH

Cy5 labelled control DNA

Cy3 labelled patient DNA

high density dystrophin array
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

high density acgh validation
High density aCGH Validation

Stage 1: Normal control vs Normal control

+1

0

-1

Dystrophin gene

high density acgh validation1

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

high density acgh validation2
High density aCGH Validation

Stage 2: Known exonic deletions and duplications

Heterozygous female deletions and duplications

Heterozygous deletion dystrophin exon 45

high density acgh validation3
High density aCGH Validation

Stage 2: Known exonic deletions and duplications

Heterozygous female deletions and duplications

Heterozygous duplication dystrophin exons 49-50

high density acgh validation4

Inversion Exon 45 c.6438+96064_6614+1540

Deletions in dystrophin introns 44 and 45

High density aCGH Validation

Stage 3: Inversion samples

high density acgh validation5
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

high density acgh validation6
High density aCGH Validation

Stage 3: Inversion samples

Inversion Ex62 c.9164-10300_c.9224+12600

Intron 62 deletion

applications of high density dystrophin acgh

Dystrophin

Exon 44

Applications of high density dystrophin aCGH

Finding mutations in MLPA and point mutation negative patients

applications of dystrophin high density acgh
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

duplication acgh results

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

breakpoint sequencing results
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
breakpoint sequencing results1
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

duplication study

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

duplication study1
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.

conclusions
Conclusions
  • Array CGH vs MLPA
  • Array CGH as a specialist test
    • Solving difficult cases
    • Rapid breakpoint mapping e.g. to Inform therapeutic strategies
acknowledgements
Acknowledgements
  • Dr Steve Abbs
  • Dr Michael Yau
  • Jo McCauley
  • Dr Joo Wook Ahn
  • Prof Francesco Muntoni
  • Jihee Kim
  • Dr Madhuri Hegde
  • Ephrem Chin