Pooling expertise in genetic testing: an innovative approach to NHS ...

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Pooling expertise in genetic testing: an innovative approach to NHS cytogenetics The field of cytogenetics has undergone significant change over the past few years. The rapid development of microarrays has meant that traditional techniques are now taking a subsidiary role in the detection of chromosomal abnormalities to array comparative genome hybridisation for constitutional disorders.

Karyotyping, the staining of metaphase chromosomes to produce distinctive banding patterns, has historically been an important technology for cytogeneticists to view gross genetic differences. However, the requirement to grow cells in culture, and the ability to only detect aberrations >5 Mb, are both limitations to this methodology. Fluorescence in situ hybridisation (FISH) techniques, which use fluorescently labelled DNA probes to identify sequences of interest on the chromosome, have the advantage that they can provide higher resolution and can detect microdeletions, as well as balanced and unbalanced translocation events. Although FISH can be performed on uncultured cells, the number of probes that can be used simultaneously on the DNA is limited. Microarrays are now a preferred choice for a variety of reasons. First, the higher resolution permits an unparalleled view of the genome and a better insight into the mechanisms behind certain genetic diseases. Thanks to microarray technology, researchers have a clearer idea of the role copy number variation (CNV) plays in the 136

pathogenesis of a number of diseases. Copy number variation is defined as the duplication/deletion of large segments of DNA, including one or more genes. They have been implicated in a number of genetic disorders including constitutional diseases and syndromes with aspects of developmental delay (eg 17q21.31 microdeletion syndrome1–6). Second, research published by the International Standards for Cytogenomic Arrays (ISCA) consortium, a group of clinical cytogenetics and molecular genetics laboratories committed to improving quality of patient care related to clinical genetic testing,7 showed that a higher diagnostic yield (15–20%) is produced with microarrays than with traditional karyotyping (3%). As a result, the benefits of a faster and more reliable genetic diagnosis means that adequate medical care and genetic counselling can be given to patients. Third, although microarray testing facilities are reasonably expensive to set up, the cost per diagnosis can be less than the cost of a G-banded karyotype plus a customised FISH test. As a result of the above, the ISCA consortium7 and the American College of

Medical Genetics (ACMG)8 have recently recommended that cytogenetic microarrays should replace G-banded karyotypes as the ‘first tier’ technology to study individuals with unexplained developmental and intellectual disabilities or multiple congenital anomalies. Oxford Gene Technology (OGT) has worked in collaboration with the ISCA consortium to design a range of microarray formats that focus on disease- and syndrome-related regions of the genome. The CytoSure ISCA aCGH arrays provide probes that cover the whole genome, allowing enhanced detection,9 and are able to identify genetic aberrations with high signal-to-noise ratios.

Development of aCGH microarrays Existing single nucleotide polymorphism (SNP) genotyping platforms can be used to identify CNVs. However, one of the major limitations of this technique is that SNP platforms provide reasonably low-quality data on CNVs as they are not fully optimised for this purpose. In order to understand further the complexities of CNVs and their role in disease, there is a call for new technical methods that do not rely on SNP detection protocols.10 One such method uses aCGH and provides direct comparison of an unknown sample with a known reference. In this technique, reference and test samples are compared using dual-colour fluorescence and the intensity of the dyes highlights the differences in copy number between the samples. Such aCGH arrays use long oligonucleotide probes covering the whole genome and can detect both known and de novo CNVs. In a recent comparison of microarray platforms, the technology used to make OGT’s PATHOLOGY IN PRACTICE NOVEMBER 2010

CHROMOSOME RUNNING TESTING HEAD (Sanger Institute, UK). Finally, the ‘databasing’ facility enables storage of data for easy reference at a later date and permits comparison of current results with previously recorded ‘legacy’ data.

Diagnostic power of a fully integrated service Oxford Gene Technology has developed a high-throughput processing and Male sample analysis service (CytoSure Services) which can screen more than 2000 samples per week in its state-of-the-art genomics laboratory, without the costs associated with in-house processing and set up. The company’s expertise has been certified by the British Standards Institute (BSI) to comply with BS EN ISO 9001:2008, demonstrating OGT’s Fig 1. CytoSure Interpret software showing isodisomy of chromosome 8 (isodisomy is where both commitment to delivering high-quality chromosomes in a complement arise from a single duplicated chromosome from one parent, as products and services by meeting opposed to heterodisomy where the chromosome complement is received as a heterologous pair from one parent). The red and green bars indicate copy number (CN) calls. The allele distribution is customer requirements and taking steps approximately 50% on the normal chromosomes. continually to improve its products and services. Building on this, OGT has recently received certification to ISO/IEC CytoSure arrays emerged as the best unaltered, so any reference DNA can be 27001:2005, an international information aCGH platform and showed superior used, and the DNA does not need to be security management standard, which signal-to-noise ratios than traditional cut with restriction enzymes. In addition, provides clear evidence of robust SNP-based CNV platforms.11 near-identical labelling and hybridisation information security management to meet all customer needs, by protecting conditions to standard aCGH are The new range of CytoSure ISCA the integrity and confidentiality of utilised, meaning that this additional aCGH arrays, currently marketed for information and data. Oxford Gene UPD functionality can be integrated research use only, features a 60-mer Technology is one of only 453 rapidly and easily into existing probe design and has been designed workflows. Used in conjunction with the companies in the UK to achieve the level following multiple rounds of of data and information management optimisation. In addition, the microarray updated version of OGT’s CytoSure required to comply with ISO 27001. Interpret Software (Fig 1), clear platform’s two-colour labelling system To date, research groups such as the identification of regions of loss of allows ‘on array’ sample comparison, ensuring higher sensitivity and accuracy heterozygosity (LOH) is possible and the Wellcome Trust Case Control data processing tools allow these areas Consortium have used OGT’s data plus greater reproducibility than other processing and analysis capability. competitive platforms.11,12 The reliability of genetic diversity to be further investigated. In combination, the special As the world’s largest CNV study, over of the arrays to detect genetic aberrations and their high signal-to-noise array design and extensive data analysis two billion high-quality data points were generated from more than 20,000 capability provided by OGT permit ratio has ensured that they are already segmental and whole chromosome UPD samples in just 20 weeks.13 The study used at a number of key facilities in the UK. Until recently, however, aCGH-based to be detected. demonstrated the capability of OGT to processes could not distinguish whether provide high-quality data to a tight alleles came from the same or different deadline. In addition, research Interpreting the data parents. This is important for diagnosing CytoSure Interpret Software, which scientists are currently using OGT’s diseases where two copies of a full CytoSure product range to identify accompanies OGT’s range of aCGH chromosome or part of a chromosome genetic aberrations causing a range of arrays, is an easy-to-use package that are received from one parent (a developmental disorders (eg DiGeorge facilitates the analysis of aCGH data. phenomenon known as uniparental Standardised data analysis is possible, and Williams-Beuren). disomy [UPD]). This is especially alongside customised, user-defined important in the diagnosis of recessive analysis, which allows the software to Improving patient diagnosis conditions. However, a new method has be optimised for individual laboratory through collaboration been developed very recently by OGT needs. Reproducible results are The current economic climate and the that embraces all the benefits of the obtained due to the ‘automatic rapid advancement of technology aCGH technology and includes the aberration detection’ function, which warrants a new approach to healthcare ability to detect UPD. greatly reduces subjectivity in analysis. service delivery. In order to improve The CytoSure ISCA UPD 4x180K array Direct weblinks to external databases detection of genetic disorders and was designed after multiple rounds of and resources put aberrations into reduce testing costs, OGT would like to SNP probe selection and validation, context and there is full integration take its expertise a step further by targeting over 6000 SNPs with evenly with existing data and publicly collaborating with genetic testing distributed probes. The combined ISCA- available cytogenetics databases, laboratories in the UK. UPD array has been developed to including the Database of Genomic As OGT has the consumables and the ensure that the aCGH protocol is largely Variants (DGV Canada) and DECIPHER analytical tools in place, it proposes an PATHOLOGY IN PRACTICE NOVEMBER 2010

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RUNNING HEADTESTING CHROMOSOME of obesity due to deletions on chromosome 16p11.2. Nature 2010; 463: 671–5. Sample purification and quality control MONDAY 5 Koolen DA, Sharp AJ, Hurst JA et al. • Sample purification in you lab, optimised in consultation with OGT Clinical and molecular delineation of Your • Secure shipping to OGT the 17q21.31 microdeletion lab • DNA quality checked and quality control metrics reported syndrome. J Med Genet 2008; • Sample concentration normalised 45(11): 710–20. • Samples transferred to 96-well plates 6 Slavotinek AM. Novel microdeletion Array processing (over 40 sample quality control checks performed) syndromes detected by chromosome • Samples logged into LIMS microarrays. Hum Genet 2008; • Labelling reactions 124(1): 1–17 OGT • Hybridisation 7 Miller DT, Adam MP, Aradhya S et al. • Washing, screening and quality control Consensus statement: chromosomal • Data transferred to you microarray is a first-tier clinical diagnostic test for individuals with Data interpretation developmental disabilities or • Data returned for analysis in your laboratory, initially using OGT’s congenital anomalies. Am J Hum Your industry leading CytoSure Interpret lab Genet 2010; 86 (5): 749–64. • Software will be provided with full training 8 Manning M, Hudgins L; for the FRIDAY Professional Practice and Guidelines Committee. Array-based technology Your reliable results and recommendations for utilization in medical genetics practice for Fig 2. Schematic of the CytoSure service. detection of chromosomal abnormalities. Genet Med 2010; Oct 18. ePub ahead of print outsourcing of the processing and data microarray systems to include ISCA (www.acmg.net/StaticContent/PPG/C generation of clinical samples in order endorsement and the addition of UPD MA_2010.pdf). to broaden access to high-resolution detection features. In addition, recent cytogenetic methodologies and thus guidance from the ISCA consortium and 9 Baldwin EL, Lee Jy, Blake DM et al. improve patient diagnoses. Under this ACMG supports the use of cytogenetic Enhanced detection of clinically outsourcing service, OGT would also microarrays as the first-tier technology relevant genomic imbalances using a bear the responsibility of keeping its to study individuals with unexplained targeted plus whole genome technology up to date, as the methods developmental and intellectual oligonucleotide microarray. Genet to detect genetic diseases are finedisabilities or multiple congenital Med 2008; 10 (6): 415–29. tuned and increases in throughput are anomalies. 10 Ionita-Laza J, Rogers AJ, Lange C, developed over time. The expertise OGT has in microarray Raby BA, Lee C. Genetic association The proposed solution of robust design has been combined with analysis of copy-number variation sample analysis, using CytoSure ISCA CytoSure Services to produce fast (CNV) in human disease aCGH arrays, alongside a streamlined, turnaround of data and reliable results. pathogenesis. Genomics 2009; 93 easy-to-use data analysis package, The service has resulted in a method of (1): 22–6. would provide a variety of benefits for identifying a broad range of genetic 11 Curtis C, Lynch AG, Dunning MJ et al. the NHS including proven data quality, syndromes without additional The pitfalls of platform comparison: fast turnaround times, no technical investment in equipment or personnel DNA copy number array technologies failures, repeat testing or downtime, training. Working in collaboration with assessed. BMC Genomics 2009; 10: and no need for new equipment or the NHS, OGT can provide a faster, more 588–610. costly service contracts. This leaves reliable and more cost-effective genetic 12 Sparkes N. Array comparative P more time for data interpretation and testing service. genomic hybridisation (aCGH) follow-up, which, given the current and delivers superior performance in the increasing numbers of genetic tests detection and quantification of CNVs. References required each year, is extensive. Oxford Gene Technology 1 Fanciulli M, Norsworthy PJ, Petretto E (www.ogt.co.uk/documents/Technical et al. FCGR3B copy number variation paperCNVplatformcomparison.pdf). is associated with susceptibility to Refined service systemic, but not organ-specific, Owing to recent advances in the field of 13 Conrad DF, Pinto D, Redon R et al. autoimmunity. Nat Genet 2007; 39: cytogenetics, testing for chromosomal Origins and functional impact of copy 721–3. abnormalities has become vastly more number variation in the human detailed and accurate. This is largely a genome. Nature 2010; 464: 704–12. 2 Sebat J, Lakshmi B, Malhotra D et al. result of the shift from traditional Strong association of de novo copy methods of analysing the genome, such number mutations with autism. as karyotyping and FISH, towards the Science 2007; 316: 445–9. Further details are available from: use of microarrays, in order to 3 Walsh T, McClellan JM, McCarthy SE Oxford Gene Technology understand the role that structural et al. Rare structural variants disrupt Begbroke Science Park changes to the DNA (eg CNV) play in the multiple genes in Sandy Lane, Yarnton pathology of disease. neurodevelopmental pathways in Oxford OX5 1PF Oxford Gene Technology has been a schizophrenia. Science 2008; 320: Tel: +44 (0)1865 856800 key player in the development of aCGH 539–43. Email: [email protected] microarrays. In particular, it has been 4 Walters RG, Jacquemont S, Valsesia Web: www.ogt.co.uk responsible for the refinement of these A. et al. A new highly penetrant form

Simple and integrated workflow

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PATHOLOGY IN PRACTICE NOVEMBER 2010