Mutation Detection Session FRCPath Preparation Course 9/11/09

1. Outline the methodology behind array CGH, concentrating on the systems in routine diagnostic use. Describe the advantages and disadvantages of the technology and give examples on how you could go about confirming the significance of a change Mutation Detection Session FRCPath Preparation Course 9/11/09

2. http://www.aist.go.jp/aist_e/aist_today/2003_07/hot_line/hot_line_20.htmlhttp://www.aist.go.jp/aist_e/aist_today/2003_07/hot_line/hot_line_20.html http://www.sanger.ac.uk/HGP/Cytogenetics/ • Comparatative Genomic Hybridization (CGH) • Developed to investigate solid tumours as they usually give poor preparations of metaphase spreads. • DNA is extracted from the test sample and a normal reference sample. • Test and reference DNA are differentially labelled with two different fluorochromes; • test is usually green (Cy3) and reference is red (Cy5). • Labelled samples are applied to target metaphase chromosomes and compete for complementary hybridisation sites. • Ratios of test to reference fluorescence along the chromosome are quantified using digital imaging analysis. • Gains and losses are identified: Gains are chromosomal regions of increased fluorescence ratios (green) and losses are chromosomal regions of reduced ratios (red). • Necessary to karyotype in order to map the areas of imbalance. Resolution is limited to that of metaphase chromosomes • (5-10Mb).

3. Array Comparative Genomic Hybridisation (aCGH) Metaphase chromosomes are replaced as the target by large numbers of mapped clones that are spotted onto a standard glass slide. Improved resolution for screening of genomic gains and losses. Test and normal reference are differentially labelled and co-hybridised to the microarray – competitive binding to complementary clones. The array is imaged and the relative fluorescence intensities are calculated for each mapped clone, and plotted relative to each clone’s position in the genome. The resulting copy number ratio reflects the DNA copy number difference. http://www.nature.com/scitable/content/Diagram-of-the-microarray-based-comparative-genomic-41020

4. aCGH probes Technology has advanced rapidly in last decade Platforms aim for increasing number of targets & shorter sequence length of probes • Large insert clones: BAC, PAC (~75-200kb) • Small insert clones: Cosmids (~30-40kb), Fosmids (~40-50kb) • cDNA clones (0.5-2kb) • PCR products (100bp-1.5kb) • Oligonucleotides (25-85 mer) Resolution of the array depends on the size, the distance between the probes and signal to noise ratio Probes mainly used in arrays: BACs and Oligonucleotides BACS are more stable that other types of cloning vectors cDNAs: high resolution but not good for CNV due to uneven distribution of genes PCR products: high resolution but poor signal to noise ratio and expensive to construct.

5. Backbone Backbone Types of aCGH Targeted arrays: coverage of specific areas of the genome that is of interest Whole Genome arrays: whole genome coverage (tiling BAC or high density oligo) A well designed diagnostic array will achieve the maximum detection of clinically relevant chromosomal imbalances and minimizing the number of confirmatory FISH tests needed. Most diagnostic array designs have a ‘backbone’ of probes evenly distributed across the genome (excluding highly repetitive regions such as heterochromatin) plus higher density coverage at regions of known clinical significance. Increased Coverage WS Region

6. BAC arrays vs Oligonucleotide arrays vs SNP arrays BAC arrays: Comprehensive coverage of the genome with robust low noise hybridisations Problems with noise from repeat sequences in the clones are suppressed by competitive hybridisation with Cot-1 DNA Limitation on the number of clones that can be spotted on slide CNV shorter than BAC will be missed Lowest screening resolution of BAC arrays is approx. 1Mb Oligo arrays: Highest potential resolution and density Oligos prepared from repeat free segments of DNA reduce problems of non-specific hybridisation due to repeat sequences in the genome (can reduce amount of Cot-1 DNA used) Allow more precise localization of the CNV Better than BAC arrays at detecting smaller CNV Poorer signal to noise ratios compared to BAC arrays

7. BAC arrays vs Oligonucleotide arrays vs SNP arrays SNP arrays: Two sets of probes present: SNP probes and non-SNP probes Each SNP is at both sense and antisense strand. The probe intensities that correspond to the two possible alleles of the SNP reveal which of the expected genotypes is present (AA or AB or BB), and also used for measuring copy number. Additional non-SNP probes also measure copy number. These probes are chosen to behave in a linear manner to dosage. Apart from CNV can additionally loss of heterozygosity and copy neutral UPD. Not strictly ‘comparative’ genomic hybridisation; only the test sample is hybridised to the array; CNV is identified by comparison to separate control set. Poorer signal to noise ratio as compared to BAC arrays.

8. Representational Oligonucleotide Microarray Analysis (ROMA) • Reduction of complexity of genomic DNA used for hybridisation. • Technique can be used in oligonucleotide and SNP arrays to improve signal to noise ratio. • DNA digestion  Fragments ligated to adapters  Amplification using universal primers  Labelling  Hybridisation • Only smaller restriction fragments (up to ~1.2kb) become amplified (i.e. reduction of representation of DNA). • Complexity depends on the enzyme used in digestion (enzyme that cuts infrequently few small fragments  low complexity and vice versa Disadvantages: Complexity reduction may lead to differential representation of parts of the genome, which may be interpreted erroneously as copy number changes Different individuals may have different restriction digestion patterns, and it is possible that some individual probe ratios may be related to restriction fragment size differences rather than to true copy-number changes.

9. Commercial platforms: • BAC arrays: BlueGnome CytoChip v3 • Disease focused design; increased coverage in >100 regions associated with disease. • >5000 replicated BAC clones to enable investigation of 143 OMIM genes at 100 Kb resolution, subtelomeres at 250 Kb and screening of genomic backbone at 0.5Mb resolution. • CytoChips are manufactured with clones from the widely validated Roswell Park Cancer Institute BAC library (RP nomenclature). • Each clone on the CytoChip may be ordered as a labelled FISH probe to directly validate the physical location of results and to investigate parental samples. • 8 arrays per kit (can do 8 samples or 4 if dye-swap) Oligonucleotide arrays: Agilent Technologies, NimbleGen Systems Inc. • Agilent arrays: • Preconfigured (44K, 105A, 244A) and custom made 60mer oligos probes printed onto derivatized glass slides using a non-contact industrial inject printing process. • NimbleGen Systems Inc: • Preconfigured genome wide, CNV, chromosome specific and custom made • Probes are synthesised directly onto the silica surface using light directed photochemistry. • Chromosome/CNV arrays (50-75mer), whole genome arrays (60mer). • Whole genome (2.1M, 3x720K, 12x125K).

10. SNP arrays: Agilent Technologies, NimbleGen Systems Inc. • Affymetrix: • 25-mer SNP based oligonucleotides directly synthesized on the array surface using a process called photolithography. • Requires genome complexity reduction (procedure similar to ROMA) • Only the test genome is labelled and hybridised • Control set for evaluation of CNV is available from Affymetrix in the form of array data obtained from healthy HapMap individuals. • 100K, 500K, 5.0, 6.0 • Illumina Inc. BeadChip arrays: • BeadArray technology: silica beads self assemble in microwells on silica slides. Each bead is covered with hundreds of thousands of copies of a specific 50-mer SNP based oligonucleotide probe. • Genomic DNA WGA  Fragmentation Two step allele detection • 1) Hybridisation of unlabelled DNA to array stopping one base before the interrogated marker. 2) Enzymatic single-base extension to incorporate a labelled nucleotide • Dual-colour florescent staining allows the labelled nucleotide to be detected by Illumina’s imagining system • Signal intensity then compared to a control set for evaluation of copy number changes. • HumanCytoChip12, Human660W-Quad, Human 1M-Duo, HumanOmni1-quad

11. Considerations when choosing an array • Design • Cost • Appropriateness for intended use • Consider the number of probes, the chromosomal location and sensitivity of • detection • - Interprobe distance (=genome size/number of probes) • - Expected resolution (=average interprobe distance x 5) • Level of noise in the experiment • - Inversely proportional to the length of the probe (SNP > oligo > BAC) • - Another contribution to noise in SNP arrays comes from comparing the data • with a control set and not from co-hybridisation with a reference • - Effect on resolution; there must be quality metrics to estimate the confidence • of the data. • Computing power and storage • Additional experimental cost • - Reagents, additional equipment (e.g. hybridisation chambers, scanners)

12. aCGH advantages • No requirement for prior knowledge of the chromosome imbalance involved. • No requirement for culturing. • Can use very little starting DNA (or WGA if necessary). • Can detect virtually all the unbalanced abnormalities. Resolution is only limited to the size of the clone target used and the distance between them • In one experiment can perform the equivalent of multiple FISH or MLPA tests. Equal sensitivity for deletions and duplications. • Better that FISH in detecting duplications (~5-10Mb) • Better than conventional cytogenetics in detecting trisomy mosaicism and marker chromosomes. • Can be automated for high throughput applications. -> Automation can decrease hands on time, improve reproducibility, drive cost down. • aCGH disadvantages • Cannot detect rearrangements that do not involve genomic imbalances (balanced chromosome translocations, inversions, ploidy (whole genome copy number changes) • Does not provide information about the structural arrangements of the chromosome segments involved • Can detect CNV of uncertain significance – will need follow up studies and may remain unresolved. • Currently expensive (costs will go down in the future)

13. Confirmation of aCGH results • FISH (metaphase or interphase) • - detection of duplications may be problematic • - can use same BAC as in array; if using oligoarray chose BACs overlapping change • Customised MLPA • qPCR. • Mircosatellite genotyping. • Long range PCR. • Another aCGH. • In true CGH platforms, dye-swap experiments can confirm a result • In SNP arrays, genotype information can serve as confirmation of the copy number results (e.g. if microdeletion is present all the SNPs in that region will be hemizygous) • -Confirmation is first carried out on the proband; also serves as evidence that the new test is going to work on the parental samples (if required to be tested). • Important to categorise CNVs as pathogenic, benigh or of unknown clinical significance.

14. Factors influencing the risk assessment of a CNV • Nature of CNV: • Duplications better tolerated than deletions • Single copy deletions have been observed in healthy individuals. • Single copy deletions may still be pathogenic if the other copy has a point mutation. • CNVs that exist as both deletions and duplications in a population are more difficult to interpret (for example a duplicated CNV in a healthy individual may be haploinsufficient in affected individuals). • ** Not all inherited copy number changes are benign; a parent with the same alteration as the proband may be phenotypically normal or have a milder phenotype (e.g. 1q21.1 and 15q13.3 microdeletions.

15. Check the CNV databases: • Database of Genomic Variants (http://projects.tcag.ca/variation); CNVs detected in normal individuals by Toronto Center of Applied Genomics, also periodically updated with published results. • http://www.sanger.ac.uk/humgen/cnv • http://humanparalogy.gs.washington.edu/build36/build36htm • Databases that provide clinical information with CNV data: • Database of Chromosomal Imbalance and Phenotype in Humans using Ensembl Resources (DESIPHER) • Chromosome Abnormality Database (CAD) • Mendelian Cytogenetics Network Online Database • - European Cytogenetics Association Register of Unbalanced Chromosome Abberations (ECARUCA) • Important to see how many times a CNV has been reported. • Validation. • Breakpoints.

16. Array CGH in the Diagnostic Lab • Improved resolution for the detection of copy number change • Confirmation of cytogenetically visible abnormality • Elucidation of cytogenetically visible copy number change • Detection of Microscopically Invisible Copy Number Change • Main clinical referrals: MR, dev. delay, ASD (studies have reported 10-15% overall rate of detection in MR cases) • Increasingly becoming the front line test • Other uses: • Prenatal diagnosis – issues to address: • Choice of array; targeted clinically relevant array. • Referrals; application to all prenatal specimens or limited to abn. scans with normal karyotype. • Processing times and costs • confirmation of results, processing of parental samples, • Counselling • Will not detect balanced changes • May detect other disorders, not just trisomies; important for counselling • Presence of unknown CNV may complicate interpretation increase patient anxiety

17. PGD/IVF applications: screening of blastomeres or eggs • Screening of egg polar bodies- if polar body has abnormal chromosome number so does the egg. • Has been used in private fertility clinic in Nottingham, in women who have suffered recurrent miscarriages, to chose eggs used for fertilization. • Baby Oliver was born earlier this year after his mother had her eggs screened (apparently had 13 previous unsuccessful IVF cycles). 5/20 treated cases have also conceived. The test costs £1,950, on top of the £3,000 or so for IVF. It is not available on the NHS or at other private clinics. Wordsworth et al Genomic Med. 2007 September; 1(1-2): 35–45. Diagnosing idiopathic learning disability: a cost-effectiveness analysis of microarray technology in the National Health Service of the United Kingdom aCGH more expensive on single test comparison: the average cost of aCGH was £442 and the average cost of karyotyping was £117 (based on data from four centres) On context of follow up, aCGH more cost effective due to long term savings regardless of result. Earlier diagnoses save costs of additional diagnostic tests. Negative results are cost-effective in minimising follow-up test choice In a hypothetical cohort of 100 idiopathic learning disability children, aCGH was found to cost less per diagnosis (£3,118) than a karyotyping and multi-telomere FISH approach (£4,957).

18. Research applications (pm session) Cancer genetics: identification of tumour suppressor genes Identification of genome architecture and copy number variation Identification of new syndromes: e.g. 17q21.31 microdeletion, CHARGE syndrome References: Carter (2007). Methods and strategies for analyzing copy number variation using DNA microarrays Nature Genetics 39, S16 - S21 Theisen  (2008). Microarray-based comparative genomic hybridization (aCGH). Nature Education 1(1) Pergament (2007). Controversies and challenges of array comparative genomic hybridisation in prenatal genetic diagnosis. Genet. Med. Vol 9, No 9, 596-598 Stankiewicz and Beaudet 2007. Use of array CGH in the evaluation of dysmoprhology, malformations, developmental delay and idiopathic mental retardation. Current Opinion in Genetics and Development, 17: 182-192 Shaikh 2007. Oligonucleotide arrays for high resolution analysis of copy number alteration in mental retardation/multiple congenital anomalies. Genetics in Medicine Vol. 9, No 9: 617-625