FRCPath Part 1 Self Help Session

1. FRCPath Part 1 Self Help Session Mutation Detection Helene Schlecht

2. What methods are available to reliably detect copy number variants responsible for human genetic disease?

3. Keywords • CNV • G-banding • CGH (arrayCGH) • SNP arrays • Next generation sequencing – paired end mapping/read depth • FISH • qPCR • Paralogue Ratio Testing • MLPA

4. Copy Number Variants • Defined as stretches of DNA (over 1000bp) found at variable copy number. • If present in >1% population – Copy Number Polymorphism. • Types of CNVs: deletions, duplications, deletions/duplications, multi-allelic and complex. • Heritable/de novo; Germline/somatic

6. Copy Number Variants - Formation 3 main pathways: • Non Allelic Homologous Recombination (NAHR) • Non Homologous End Joining (NHEJ) • Fork Stalling and Template Switching (FoSTeS/MMBIR)

7. Copy Number Variants – human disease • Copy Number Variation known about very early on in the form of Down Syndrome and other syndromes associated with visible deletions or duplications. • Also normal variation seen in size of heterochromatin at centromeres of 1, 9, 16 and long arm Y, the short arm of acrocentric (13, 14, 15, 21, 22) and at fragile sites • All reflect varying copy number of tandem repeated sequence. • 2006 1st CNV map showing about 12% genome is covered by CNVs (Redon et al. 2006).

8. Copy Number Variants • Deletions in 22q11.2 region cause DiGeorge/velocardiofacial syndrome. • Deletions in 15q11-q13 cause Prader-Willi syndrome • 3.7Mb del on short arm of chromosome 17 responsible for 70% cases of Smith-Magenis Syndrome. • EGFR copy number higher than normal in non small cell lung carcinoma.

11. Techniques • Genome wide: • G Banding • CGH • SNP arrays • Next Generation Sequencing

12. Techniques – G-banding • Metaphase chromosomes digested with trypsin then stained with Giemsa. • Gives: • dark G bands – AT rich, gene poor; • pale G negative bands – GC rich, gene rich.

13. Techniques – G-banding • Advantages – straight forward technique. • Disadvantage – resolution, looking at isolated population of cells. • Used in – Karyotyping

14. Techniques – CGH • DNA from subject tissue and normal control tissue labelled with different fluorescent tags. • Hybridised to normal metaphase chromosomes. • Look at colour ratios.

15. Techniques – Array CGH • Metaphase chromosomes replaced with arrays of clones mapped to human genome and spotted onto a solid support. • Probes to most known genes and non-coding regions of the genome. • Resolution determined by size and density of sequences on array. • Range from 200kb (BAC arrays) to 25bp (oligo arrays)

17. Techniques – CGH • Advantages • CGH: better resolution (than G-banding), automated detection. • Arrays: No culturing required, more cost effective, • Arrays: better sensitivity then FISH for duplications, equal deletions. • Arrays: can be automated • Oligo arrays: Better resolution, and more accurate definition of CNV boundaries (vs BAC). • BAC arrays: Higher signal to noise ratio.

18. Techniques – CGH • Disadvantages • CGH: resolution still low. • Arrays: Cannot detect balanced rearrangments – no information on structural arrangement. • Arrays: expensive • BAC arrays: Low resolution – 50kb. • Oligo arrays: Poor signal to noise ratio which causes experimental variability between studies; more expensive (vs BAC). • Used in – dev. delay

19. Techniques – SNP arrays • 1000’s of single hybridisation assays are simultaneously done under the same conditions. • Probes are fixed to solid surface in a high-density grid. • Test DNA, fluorescently labelled, is added. • Amount of target DNA bound by each probe reflects quantity in the sample.

20. Techniques – SNP arraysAffymetrix chip technology

21. Techniques – SNP arrays • Illumina Bead Array technology.

22. Techniques – SNP arrays • Advantages – High resolution • Disadvantages – DNA quality, Cost, Not technically simply, Analysis more complex • Used in – dev. delay screen

23. Techniques – Next gen • Alignment of DNA sequences from different sources. • Next Generation assemblies of complete genome of an individual allows for more robust, reliable genome comparisons. • Computational analysis: • Read Depth • Based on Paired end mapping

24. Techniques – Paired end mapping • Generation of library of fragments (300-500bp). • Subject to deep sequencing to give paired-end spans. • Compared to reference genome. Use PEM and RD in combination

25. (i) Genomic DNA sheared to yield DNA fragments ~3 kb; (ii) biotinylated hairpin adapters were ligated to the fragment ends; (iii) fragments circularized; (iv) and randomly sheared; (v) linker (+) fragments were isolated; (vi) the library was subjected to 454 sequencing (13). (vii) Paired ends were analyzed computationally to determine (viii) the distribution of “paired-end spans” (shown for a single 454 sequencing pool).0 Variation in the Human Genome Paired-End Mapping Reveals Extensive Structural Science 318, 420 (2007); Jan O. Korbel, et al.

26. Techniques – Next generation sequencing • Advantages – can identify inversions and provide resolution of CNV boundaries down to single nucleotide level, cost. • Disadvantages –PEM cannot detect CNVs larger than fragment size; cannot reliably identify variants in complex genomic regions. • Used in – Mental retardation screens

27. Techniques – Locus specific • FISH • Real-time PCR • Paralogue Ratio Testing • MLPA

28. Techniques – Fluorescence in situ hybridisation (FISH) • Fluorescent probes that bind specifically to regions of the genome. • Fluorescent microscopy used for analyses. • Probes tagged with fluorophores (by nick translation or PCR using tagged oligos). • Interphase or metaphase chromosome preparation.

29. Techniques – FISH • Advantages – well established, inexpensive • Disadvantages – labour intensive, resolution, need specific probes. • Used in - Prader-Willi/AS syndrome, 22q13 del syndrome, known microdeletion syndromes

30. Techniques – Real-time PCR • PCR theoretically amplifies DNA exponentially. When the PCR is in the log phase the amount of product present should be proportional to the amount of template. • A Taqman probe labelled with two fluorophores, one reporter dye and the other a quencher. • PCR reaction and the taq polymerase displaces the TaqMan probe from the template. This separates the quencher from the reporter allowing it to fluoresce. The fluorescence is measured and is quantified as the reaction continues.

31. Techniques – Real-time PCR • Advantages – Technically simple, relatively high throughput. • Disadvantages – Not suitable for simultaneous amplification of many targets, does not give a precise measurement of copy number. • Used in – confirmations?

32. Techniques – Paralogue Ratio Test • Simultaneous amplification of target element and an unlinked reference locus. • Use same set of primers in a single reaction. • Target and reference locus distinguished by size or restriction digest. • Quantified and compared by capillary electrophoresis.

33. Techniques – Paralogue Ratio Test • Advantages – Accurate, relatively high throughput, decreases experimental variability, detect genes that vary in number over a high range. • Disadvantages – Targeted only, assay design/optimisation. • Used in - DEFB4 (varies between two and seven copies).

34. Techniques – MLPA

35. Techniques – MLPA • Advantages – Technically simple, sensitive • Disadvantages – Mainly for locus specific, not high throughput. • Used in – NF1,

37. Databases • Normal – www.1000genomes.org • Normal – projects.tcag.ca.variation • Pathogenic – decipher.sanger.ac.uk

38. References • Redon et al 2006. Nature 444:444-454 (First CNV map) • Fanciulli et al 2010. Clin Genet. 77:201-213 (Good recent CNV review) • Armour et al. 2006. Nucleic Acids Res 35:e19 (PRT) • Choy et al 2010. BJOG 117:391-398 (review) • Korbel et al. 2007. Science318, 420 (PEM)