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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

Array CGH

Louise McClelland

10th November 2010


  • Background

  • Technical details

  • Applications

  • Interpreting CNV

Key terms
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

Array comparative genome hybridisation acgh
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


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

Bac vs oligo
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




Karyotyping 3-10 Mb

Sequencing /MLPA





  • Balance required between sensitivity and specificity


  • BlueGnome

  • Agilent

  • Roche Nimblegen

Hybridisation options
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

Analysis normalisation
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

Analysis exclusion
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

Analysis quality control
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

Abnormal acgh results
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

Applications in a diagnostic setting
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

Acgh frontline testing
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%)

Recent publications
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

Guys experience ahn et al 2010
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

Deciphering developmental disorders ddd project
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




Exome sequencing

Investigate and report back to Regional labs for confirmation work

Sequencing variant

Interpreting cnvs
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


      • 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

Frequency 1 in 5000

Most common microdeletion syndrome in Ahn et al., 2010 (15 cases, 0.62%)


“Our ability to discover genetic variation is running ahead of our ability to interpret them”

Huang et al., 2010


  • 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

References ahead of our ability to interpret them”

  • 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