Download High density array comparative genomic hybridisation (aCGH)

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
• Application to specialised testing
– Complex mutations
– Therapeutic exon skipping trials
Principle of aCGH
Cy3 labelled
patient DNA
Cy5 labelled
control DNA
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
Stage 1: Normal control vs Normal control
+1
0
-1
Dystrophin
gene
High density aCGH Validation
Stage 2: Known exonic deletions and duplications
Hemizygous male deletion
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
98
7 6 5
4 3
Deletion dystrophin
exons 3-7
2
CNV in intron 2
High density aCGH Validation
Stage 2: Known exonic deletions and duplications
Heterozygous female deletions and duplications
31.77632Mb
31.936032Mb
32.095744Mb
32.255457Mb
+2
+1
0
-1
-2
Dystrophin exons
48
31.645784Mb
47 46
45
44
31.71414Mb
31.78250Mb
+2
+1
0
-1
Heterozygous
deletion dystrophin
exon 45
43
42
31.85086Mb
31.77632Mb
31.936032Mb
32.095744Mb
32.255457Mb
High density aCGH Validation
+2
+1
0
Stage 2: Known exonic deletions and duplications
-1
Heterozygous female deletions and duplications
-2
Dystrophin exons
48
47 46
31.77632Mb
31.645784Mb
45
44
31.936032Mb
31.71414Mb
43
32.095744Mb
31.78250Mb
42
32.255457Mb
31.85086Mb
+2
+2
+1
+1
0
0
-1
-1
-2
-2
Dystrophin exons
48
Dystrophin exons
52
31.645784Mb
47 46
45
51
31.71414Mb
50
49
44
31.78250Mb
+2
+1
0
-1
Heterozygous duplication
dystrophin exons 49-50
48
43
42
31.85086Mb
High density aCGH Validation
Stage 3: Inversion samples
Inversion Exon 45 c.6438+96064_6614+1540
31.827229Mb
31.935944Mb
32. 044659Mb
32.153374Mb
+7
+6
+5
+4
+3
+2
+1
0
-1
-2
-3
-4
-5
-6
-7
Dystrophin exons
47 46
45
Deletions in dystrophin introns 44 and 45
44
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
3’ deletion including
11 genes
Deletion
dystrophin
Exon 52
High density aCGH Validation
Stage 3: Inversion samples
Inversion Ex62 c.9164-10300_c.9224+12600
31.183465Mb
31.217965Mb
31.252466Mb
31.286967Mb
+7
+6
+5
+4
+3
+2
+1
0
-1
-2
-3
-4
-5
-6
-7
Dystrophin exons
63
62
Intron 62 deletion
61
Applications of high density
dystrophin aCGH
Finding mutations in MLPA and point mutation negative patients
Dystrophin
Exon 44
Applications of dystrophin high density
aCGH
Informing a exon skipping trial targeting duplications
• 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
Duplication aCGH results
31.692239Mb
31.741906Mb
31.698543Mb
32.04018Mb
31.704848Mb
32.33846Mb
31.711153Mb
32.63674Mb
+2+2
+1+1
00
-1
-1
-2
-2
Dystrophinexons
exons
Dystrophin
50 - 46
45
51
44 - 17
Duplication of
of dystrophin
dystrophin exons
exon 51
Duplication
17-45
16 - 8
5
4
Duplication breakpoint mapping
45
Exon
5
651 5
5Exon
651
6
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 results
32775529 atgggaggcatgtatactatagaacagaaattttagtattctactcagaagc
gtattcagaaacttaaatagccatataatacatattttggagcatgtcagaa
Intron 1
<<<<<<<< |||||||||||||||||||||||||||
|| | || |
|||
|
Microhomology atgggaggcatgtatactatagaacagggatctaggttgtgtgctccttatg
|||||||||||||||||||||||||||| | ||
|||
|
Duplication sequence
1-4 <<<<<<<<
nucleotides
|gtattcagaaacttaaatagccatataagaattaactgtttgcacaatctac
|
|
|||||||||||||||||||||||||||
||
|
|
||
||||||||||||||||||||||||||||
32850655
aaccctattgtgacctgcgcatgcgagggatctaggttgtgtgctccttatg
14/20
(70%)
Intron 4
tctaacacatgcaattttaagaggataagaattaactgtttgcacaatctac
Intron 30
32323093
catgaagatgataaatacttatgttaatatgattaggagatggaattaatta
Small insertion
<<<<<<<< ||||||||||||||||||||||||
|
|
|
| |
1-4 nucleotides
catgaagatgataaatacttatgtattttaaaaaccggctttgttaagcaat
Duplication sequence
4/20
(20%)
<<<<<<<<
| ||
|
||||||||||||||||||||||||
32462155 gtattttgttatgtaagtgctagctgactaaaaaccggctttgttaagcaat
Intron 17
Clean breakpoint
2/20 (10%)
attaaggatctgtgtgagattctgctggatggtgtagattttcttgaaaa
Intron 7
|||||||||||||||||||||||||
|
| |
| ||
attaaggatctgtgtgagattctgcataaattcagcttatgttaaggaag
Duplication sequence
| |
||
|||||||||||||||||||||||||
Intron 2
ggcttttgtgttacattgaacagagataaattcagcttatgttaaggaag
Duplication study
Comparison with RNA results
• Genomic DNA: Duplication Exons 3-37
• Breakpoint close to exon 37
Exon 36
Exon 37
Exon 3
13bp
• RNA level: Duplication Exons 3-36
Exon 1-2
Exon 3-35
Exon 4-37
Exon 38->
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
E.g.  DNA repair mechanism such as non-homologous endjoining
(NHEJ)
 Replication based mechanism such as fork stalling and template
switching (FoSTeS)
Conclusions
• Array CGH vs MLPA
High Density aCGH
MLPA
Consumable Cost
£178
£40
Information provided
44K probes
Limited to ~1 probe/exon
Reliability
Multiple 60nucleotide
probes/aberration
Risk of false positives
Flexibility
Custom design
Limited kit availability
• Array CGH as a specialist test
– Solving difficult cases
– Rapid breakpoint mapping e.g. to Inform therapeutic
strategies
Acknowledgements
•
•
•
•
Dr Steve Abbs
Dr Michael Yau
Jo McCauley
Dr Joo Wook Ahn
• Prof Francesco Muntoni
• Jihee Kim
• Dr Madhuri Hegde
• Ephrem Chin