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

Cy3 labelled patient DNA Cy5 labelled control DNA Principle of aCGH

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

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

Dystrophin exons Mb Mb Mb Mb High density aCGH Validation CNV in intron 2 Deletion dystrophin exons 3-7 Stage 2: Known exonic deletions and duplications Hemizygous male deletion

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

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 Validation Stage 3: Inversion samples Deletions in dystrophin introns 44 and 45 Inversion Exon 45 c _

IL1RAPL1 High density aCGH Validation Dystrophin Deletion dystrophin Exon Stage 3: Inversion samples Inversion Exon 53 –> 79 3’ deletion including 11 genes +7 -7

High density aCGH Validation Stage 3: Inversion samples Inversion Ex62 c _c Intron 62 deletion

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

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 of dystrophin exons Duplication aCGH results Mb Mb Mb Mb Dystrophin exons51 Duplication of dystrophin exon 51 +1

Duplication breakpoint mapping Exon 51

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 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%)

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

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.

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

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