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Array CGH

Array CGH. Louise McClelland 10 th November 2010. Introduction. Background Technical details Applications Interpreting CNV. Key terms. Array comparative genomic hybridisation ( aCGH ) Copy number variants (CNV) Catch all term for any copy number change Copy number polymorphisms (CNP)

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Array CGH

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  1. Array CGH Louise McClelland 10th November 2010

  2. Introduction • Background • Technical details • Applications • Interpreting CNV

  3. Key terms • Array comparative genomic hybridisation (aCGH) • Copy number variants (CNV) • Catch all term for any copy number change • Copy number polymorphisms (CNP) • A CNV likely to be benign • BAC array • Oligoarray • Microdeletion • Microduplication • Frontline testing

  4. Array Comparative Genome Hybridisation (aCGH) • Technique for identifying loss or gain of subchromosomal band regions (microdeletions and duplications) • Compares differences between the patient DNA and a reference DNA • Based on the cytogenetic technique CGH • CGH the target ‘reference’ DNA is a metaphase chromosome • aCGH target reference DNA is normal genomic DNA in specific locations on a microarray • The 2003 Genetics white paper funding was used to establish many UK aCGH services

  5. Technique • Patient DNA is fragmented, fluorescently labelled then hybridised to reference (“normal”) DNA which has an alternative fluorescent label

  6. BAC vs Oligo • BAC – Large insert clones: ~100-200Kb • Oligo – Synthetic “in silico” probes; ~60-80 “mer” • BAC • Data is generally more “transparent” than oligoarrays • Accurate copy number determination from single data point • Oligos • Advantages • Higher theoretical resolution • Cheaper to produce • More flexible (more amenable to “multiple format design”, more options for probe selection) • Disadvantages • Smoothing algorithms required • Single data point can not be used for copy no. • Increased manipulation of data can introduce artefact

  7. aCGH Resolution Karyotyping 3-10 Mb Sequencing /MLPA 1bp 6Kb 5Mb >10Mb • Balance required between sensitivity and specificity

  8. Platforms • BlueGnome • Agilent • Roche Nimblegen

  9. Hybridisation options • Dye swap • Hybridise patient DNA to reference ‘normal’ DNA • Repeat in opposite colours • 2 arrays required • Loop • 3 patient samples labelled to each of 2 colours • Each sample hybridised against the other 2 • Only 1 array per patient • Patient/Patient • Hybridised patient to another phenotypically mismatched patient • The dye ratios inform ownership of any imbalances detected • This method is employed by the Guys lab

  10. Analysis: Normalisation • Spatial ratio bias algorithm • Removes bias arising from hybridisation and scanning • e.g. scanner bias causing one side of array to be brighter • GC bias algorithm • Data tends to be noisy towards GC rich regions • Different clones corrected by GC bias algorithm differently depending on GC content

  11. Analysis: Exclusion • Individual replicates with a substantially different log2 ratio to the other replicates • Low intensity spots with an inadequate signal to noise ratio • Spots with debris or uneven signal across spot surface

  12. Analysis: Quality control • 95% of clones must have worked to pass QC • For a copy number change to be significant it must exceed 3 SD from the mean

  13. Abnormal aCGH results • Due to the volume of data generated a high number of uncertain results may be obtained • An abnormal result should always be validated using an alternative method; • FISH • Gives positional information • Resolution can inhibit use in duplications (two fluorescent signals may appear as one because close together) • MLPA • Mental retardation kits P064-MR1, P094-MR2 • Broad subtelomere kits P069, P036, P070 • Centromere kits P181, P182 • Microdeletion syndrome kits P245, P297 • Reference kits P200, P300 (to be used with bespoke MLPA probes) • QF-PCR

  14. Applications in a diagnostic setting • Currently used in combination with karyotyping for selective; • Constitutional postnatal cases • Fetal pathology cases • Leukaemia cases • Used for more widespread referrals • Prenatal testing of abnormal ultrasound cases (being validated) • Prognostics and residual disease monitoring in cancers

  15. aCGH: Frontline testing • For mental retardation (MR), autistic spectrum (ASD), multiple congenital abnormalities (MCA) • These referrals represent the largest referral number to cytogenetics • Advantages over conventional testing • Higher sensitivity than karyotyping • 15-20% compared to 3% if Down syndrome is excluded • Better resolution that FISH for reciprocal duplications of known microdeletions e.g. • 7q11 Williams Beuren • 17p11.2 Potocki-Lupski • Disadvantages • Increased unknown clinical significance CNV • Will not detect: • Truly balanced translocations • Low level mosaicism (BAC arrays can detect 10%, Oligos 20-30%)

  16. Recent publications • April 2010 – Ahn et al. • Guys lab • Their experience of using aCGH as a frontline test (since May 2008) • May 2010 – Miller et al. • Reviewed 33 studies of DD/ID, ASD, MCA, 21, 698 patients • Both papers suggest using oligoarrays

  17. Guys experience (Ahn et al., 2010) • Oligoarrays as first line test 1169 patients and 22% abnormality rate • 89% of abnormalities wouldn’t have been detected by karyotyping • 14% of imbalances detected fell within known sites of recurrent microdeletion/dups • Low parental sample received for follow up (40% of those requested) • Reduced costs by; • Patient/patient hybridisation method • Normal arrays – only 1 analysis was required • Risk of reciprocal imbalance in the two patients minimal (but don’t get a lot of clinical info) • Batching and robotics, streamlined IT systems • Offer aCGH at same cost as karyotype analysis • Karyotyping to remain for; • Turners, trisomy 13,18, 21, Kleinfelter • Distinguish free trisomy from translocateion associated trisomy • Multiple miscarriage – for balanced translocations

  18. Deciphering developmental disorders (DDD) project • 12,000 UK children with abnormal development • Recruited from all over the UK • Aim to transform clinical practice for children with abnormal development aCGH CNV Normal Exome sequencing Investigate and report back to Regional labs for confirmation work Sequencing variant http://decipher.sanger.ac.uk/ddd

  19. Interpreting CNVs • Pathogenic deletions more common than duplications – perhaps because duplications are harder to validate • Consider size of imbalance • Most pathogenic CNV >1 Mb and de novo (Miller et al., 2010) • Has a del/dup of any of the region been seen before • DECIPHER • Can publish data in DECIPHER if consent is given • DECIPHER v.5 Haploinsufficiency LOD score (Huang et al .,2010 ) • Help prioritize genes for investigation • Database of Genomic Variation (DGV) • Comprehensive info regarding benign and unlikely to be pathogenic CNVs • Ensembl search for genes in region • Any likely candidate genes for clinical features • Consider neighboring genes and regulatory elements • Literature search • ID overlapping del/dup? • Similar clinical features • Do literature search for each gene in / around region • Trio studies • Is the variant de novo • Exclusion of balanced parental rearrangements by FISH is recommended for apparently de novo abnormalities • If inherited, does the parent show any features

  20. Frequency 1 in 5000 Most common microdeletion syndrome in Ahn et al., 2010 (15 cases, 0.62%) http://decipher.sanger.ac.uk/syndromes

  21. “Our ability to discover genetic variation is running ahead of our ability to interpret them” Huang et al., 2010 Conclusions • More unknown clinical significance variants; reporting and interpreting challenges  • This will improve as more data published more CNP will be confirmed etc • Change to infrastructure of diagnostic genetics labs • Reduce referral numbers for karyotyping, fragile X, PWS (will become a reflex tests) • More integration between molecular and cyto as karyotyping reduced • Increased demand on high spec hardware and IT storage solutions

  22. References • ACC BPG for constitutional aCGH analysis • Ahn et al., Molecular Cytogenetics, 3, 9, 2010 • Huang et al., PLOS, 6(10), e1001154, 2010 • Miller et al., Am J Hum Genet, 86, 749-764, 2010 • Oostlander et al., Clin Genet, 66, 488-495, 2004 • Sagoo et al., Genet in Med, 11(3), 139-146, 2009 • www.mrc-holland.com • http://decipher.sanger.ac.uk/ddd • http://decipher.sanger.ac.uk/syndromes • www.cambridgebluegnome.com/ • www.genomics.agilent.com/ • www.nimblegen.com/products/cgh/human.html

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