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Microarrays/CNVs

Microarrays/CNVs. FRCPath PartII Study Day Birmingham 7 January 2011 Thalia Antoniadi. Exceptional healthcare, personally delivered. Array CGH.

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Microarrays/CNVs

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  1. Microarrays/CNVs FRCPath PartII Study Day Birmingham 7 January 2011 Thalia Antoniadi Exceptional healthcare, personally delivered

  2. Array CGH • Copy Number Variants (CNVs) are associated with a significant proportion of cases with developmental delay, phenotypes with dysmorphic features, mental retardation, autistic spectrum disorders • Karyotyping (3-5Mb resolution) • FISH detect sub-microscopic • Quantitative PCR copy number variations • MLPA ‘targeted’ methods • Microarray-based Comparative Genomic Hybridization (array CGH) is a technique to scan the genome for gains and losses of chromosomal material • Array-based whole genome investigation enables the genome wide detection of large and sub-microscopic imbalances Exceptional healthcare, personally delivered

  3. Overview Exceptional healthcare, personally delivered

  4. Types of Arrays • Microarray Comparative Genomic Hybridization (CGH) to look for genomic gains and losses • SNP arrays • To study disease susceptibility/ dosage effect • Expression microarrays • Determining the level, or volume, at which a certain gene/path of genes is expressed • The immobilized DNA is cDNA derived from the mRNA of known genes • The control and sample DNA hybridized to the chip is cDNA derived from the mRNA of normal and diseased tissue, respectively. If a gene is overexpressed in a certain disease state, then more sample cDNA, as compared to control cDNA, will hybridize to the spot representing that expressed gene. Exceptional healthcare, personally delivered

  5. BAC Oligo Types of Array targets • There are two types of array targets: • Bacterial Artificial Chromosome arrays (BACs) - contain DNA isolated from large insert clones that range in size from 150 – 200 kb - they are very sensitive and results can be validated with FISH using the BAC DNA as a probe. - the production of BAC DNA is labour-intensive, and resolution is relatively limited • Oligonucleotide arrays - offer greater genome coverage - oligonucleotides 10-60 bases - they are preferable to BACs and have extremely high resolution ___ ___ ______ ||||| ||||| |||||| |||| |||||||||| ||||||||| |||||||||||||||||| |||||| ||||||||||||| ||||| ||||||||||||||| |||| ||| ||||||||||||| ||||||||||||||| |||| ||||||||||||||| ||||||||||||||||||||||||||||| |||| ||||||||| |||||||||||| ||||||||||| Exceptional healthcare, personally delivered

  6. Design Approaches • There are also two approaches when it comes to designing arrays: targeted and whole genome/tiling. • Targeted arrays contain specific regions of the genome, such as those responsible for known microdeletion/microduplication syndromes and do not have probes that span the whole genome. • Whole genome/tiling path arrays give full genome coverage and for clinical testing have usually a resolution of 50kb to 1Mb between adjacent probes. The enhanced coverage has led to the identification of an additional 5% of abnormalities when compared to a targeted array. Exceptional healthcare, personally delivered

  7. Average diagnostic array: oligos 60 bases/ 60,000 oligos per array / x8 www.ngrl.org.uk/Wessex/downoads Technology Assessment Report July 2010 Exceptional healthcare, personally delivered

  8. Examples of scanned images Exceptional healthcare, personally delivered

  9. dup(7)(q21.13q22.1) Exceptional healthcare, personally delivered

  10. Array CGH process Exceptional healthcare, personally delivered

  11. www.ngrl.org.uk/Wessex/downoads Technology Assessment Report July 2010 Exceptional healthcare, personally delivered

  12. Allows for investigation of the whole genome and has a very high resolution Detects mosaicism to a level of 10-30% abnormal cells Limitation of aCGH: it does not detect balanced rearrangements An audit of a 10 year period of karyotype results (Guy’s): 4/36,663 balanced rearrangements in postnatal samples: 0.0001% Complexity: CNVs have been found in phenotypically normal people and can occur at a high frequency in the general population Array CGH Exceptional healthcare, personally delivered

  13. Copy number variations (CNVs) • A significant amount of human variation is due to structural changes of the genome rather than to base-pair changes in the DNA. • This variation accounts for roughly 12% of human genomic DNA and may range from 1kb to several megabases in size. • Copy number variations are alterations of genomic DNA that correspond to relatively large regions of the genome that have been deleted or amplified on certain chromosomes. • CNVs may either be inherited or caused by a de novo event. Exceptional healthcare, personally delivered

  14. Copy number variations (CNVs) • CNVs can be caused by genomic rearrangements such as deletions, duplications, inversions, and translocations. • Rearrangements of the human genome can be categorised in to two major groups on the basis of breakpoint analysis: • Recurrent rearrangements • Non recurrent rearrangements • Recurrent rearrangements Research on recurrent rearrangements with breakpoint clustering at Low Copy Repeats and Segmental Duplications enabled the elucidation of the mechanism: Non Allelic Homologous Recombination (NAHR) Factors such as size, orientation, percentage similarity and the distance between the copies influence the susceptibility of LCRs to genomic rearrangement. Exceptional healthcare, personally delivered

  15. Copy number variations (CNVs) Non-recurrent rearrangements - Non Homologous End Joining (NHEJ) Genomic disorders caused by non-recurrent rearrangements have uncovered a new replication-based human genomic rearrangement mechanism: - Fork Stalling and Template Switching (FoSTeS) The FoSTeS mechanism has been generalised and the molecular details refined, including through genetic and genomic observations on chromosomal rearrangements in other model organisms, and resulted in a model operative in all life forms: Microhomology-Mediated Break-Induced Replication(MMBIR) MMBIR can explain complex rearrangements such as duplication-triplication-duplication. Exceptional healthcare, personally delivered

  16. Interpretation • Database of Genomic Variants (DGV) • a comprehensive summary of structural variation in the human genome -DNA segments >1kb • http://projects.tcag.ca/variation/ • Imbalances of regions represented in DGV> NOT reported • DECIPHER • a tool for collating information about very rare chromosomal disorders • http://decipher.sanger.ac.uk • The DECIPHER database of sub-microscopic chromosomal imbalance collects clinical information about chromosomal microdeletions/duplications/insertions, translocations and inversions Exceptional healthcare, personally delivered

  17. Interpretation • CNVs outside areas of known syndromic imbalance can be reported as preliminary findings • Parental bloods should be requested to establish inheritance pattern • Assessment of pathogenicity of a CNV (benign or pathogenic) • The imbalance is previously described • De novo origin: more likely to be pathogenic • CNV size: larger >more likely to be pathogenic • CNV overlaps genomic coordinates for an known imbalance syndrome • CNV contains morbid OMIM genes/ is gene rich • CNV is an amplification (greater than 1 copy gain) • CNV is a homozygous deletion • Inherited: phenotypic assessment of parent is required • CNV present in affected parent or relative Exceptional healthcare, personally delivered

  18. Clinical utility of arrays • Postnatal diagnosis In patients with idiopathic developmental delay, mental retardation or MR/congenital abnormalities, aCGH produces a higher clinical yield (20-25%) than karyotype and FISH/MLPA (5-10%) The benefits of this significant increase in diagnostic power include: - proper referrals to specialists, allowing for appropriate therapeutic interventions and screening - less future diagnostic procedures for the patient - a diagnosis may also relieve anxiety for families and allow for planning of future pregnancies Exceptional healthcare, personally delivered

  19. Clinical utility of arrays Prenatal diagnosis Most is carried out in the context of maternal age and/or increased risk of Down Syndrome based on ultrasound/serum markers aCGH will detect any CNV, also of unknown pathogenicity Develop arrays with a limited number of well-known targets Infertility and recurrent miscarriages Chromosomal translocations are a significant cause of infertility Recurrent miscarriages are often due to balanced translocations Exceptional healthcare, personally delivered

  20. DD/LD: aCGH in diagnosis, today. • UKGTN second report, November 2010 “use of aCGH as a first line test for postnatal referrals with developmental delay/learning disabilities with dysmorphic features and/or congenital anomalies • aCGH testing superior to standard karyotyping • UKGTN recommends it should be funded throughout NHS immediately Exceptional healthcare, personally delivered

  21. Consensus statement: chromosomal microarray is a first-tier clinical diagnostic test for individuals with developmental disabilities or congenital anomalies.Miller DT et al. • Chromosomal microarray offers a much higher diagnostic yield (15-20%) for genetic testing for individuals with unexplained developmental delay/intellectual disability or multiple congenital anomalies than a G-banded karyotype (~3%, excluding Down syndrome and other common syndromes) • Truly balanced rearrangements and low level mosaicism are generally not detectable but these are relative infrequent causes of abnormal phenotypes in this population (<1%) Exceptional healthcare, personally delivered

  22. Molecular Cytogenetics 2010, 3:9Validation and implementation of aCGH as a first line test in place of postnatal karyotyping for genome imbalanceJoo Wook Ahn et al. (Guy’s &St Thomas’) 24% of patients tested had CNVs potentially pathogenic At least 89% of the abnormalities detected by first line testing would not have been detected by standard karyotype analysis ‘patient/patient’ hybridization: instead of reference they used another patient’s DNA > reduced the cost -limitation: phenotypically mismatched patients Exceptional healthcare, personally delivered

  23. Am J Hum Genet 86, 765-772, May 14, 2010Value for money? Array Genomic Hybridization for Diagnostic Testing for Genetic Causes of Intellectual Disability.Reiger et al. • A decision analytic model was used to synthesize the economic and clinical outcomes accrued to a hypothetical cohort of patients with idiopathic intellectual disability (ID) undergoing conventional or AGH testing strategy • Pooled data from several published reports examining AGH for idiopathic ID • Assumed that AGH would identify each of the diagnoses obtained by karyotyping • Cost analysis included lab tests, clinic visits, resource utilization etc Exceptional healthcare, personally delivered

  24. Am J Hum Genet 86, 765-772, May 14, 2010 --- Exceptional healthcare, personally delivered

  25. Am J Hum Genet 86, 765-772, May 14, 2010 The results suggested that AGH testing after a karyotype for each individual, versus the use of AGH as a first line diagnostic test, resulted in costs that exceeded benefits overall AGH cost will reduce in time Effectiveness was not measured in Quality Adjusted Life Years (QALY) because of the age of the patients Biased cohort as it included patients between 5-10yrs old The AGH platforms compared were not the same Exceptional healthcare, personally delivered

  26. DECIPHERing Developmental Disorders (DDD)

  27. References • The DNA replication FoSTes/MMBIR mechanism can generate genomic, genic, and exonic cpmplex rearrangements in humans Zhang F et al. Nat Genet 2009 • Validation and implementation of aCGH as a first line test in place of post natal karyotyping for genome imbalance Zoo Wook Ahn et al, Molecular Cytogenetics 2010, 3:9 • Guidelines for molecular karyotyping in constitutional genetic diagnosis Vermeesch JR et al. Eur J Hum Genet 2007 15, 1105-14 • Value for money? Array Genomic Hybridization for Diagnostic Testing for Genetic Causes of Intellectual Disability Reiger et al. Am J Hum Genet 86, 765-772, May 14, 2010 • Consensus statement: chromosomal microarray is a first-tier clinical diagnostic test for individuals with developmental disabilities or congenital anomalies.Miller DT et al. Am J Hum Genet 86: 749-764, May 14, 2010 Exceptional healthcare, personally delivered

  28. Rare deletions at 16p13.11 predispose to a diverse spectrum of sporadic epilepsy syndromes.Heinzen EL et al. • Genome-wide screen to identify CNV in 3812 patients with a diverse spectrum of epilespy syndromes (schizophrenia, mental retardation, idiopathic generalised epilepsy)- 1299 controls • Large deletions >100kb in 23 patients; no control had a deletion >16kb • 12 genomic regions harboring >2Mb deletions I patients • Haploinsufficiency as a contributor to the pathogenicity.---complementary info- Exceptional healthcare, personally delivered

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