Use of Affymetrix Arrays in the Diagnosis of Gene Copy‐Number Variation

Farah R. Zahir1, Marco A. Marra1

1 University of British Columbia, Department of Medical Genetics, Vancouver, British Columbia
Publication Name:  Current Protocols in Human Genetics
Unit Number:  Unit 8.13
DOI:  10.1002/0471142905.hg0813s85
Online Posting Date:  April, 2015
GO TO THE FULL TEXT: PDF or HTML at Wiley Online Library


Diagnosing constitutional pathogenic copy number variants (CNVs) requires detecting submicroscopic segmental chromosomal imbalances. The Affymetrix GeneChip mapping array was one of the initial microarray platforms used to measure duplication and deletion of genetic material in DNA samples. Unlike oligonucleotide microarrays from NimbleGen and Agilent, developed around the same time to infer copy number status for the DNA sequence covered by the probe, the Affymetrix GeneChip system used 25‐mer oligonucleotide probes designed to interrogate SNPs. Thus, it was possible to use the Affymetrix ‘SNP chips’ to both identify SNPs and to identify copy number status. Affymetrix now offers the CytoScan microarray platforms, which are optimized for copy‐number analyses, and provides accompanying software. They also offer several other microarray platforms suitable for copy‐number analyses. Here we discuss the application of the CytoScan high‐density (HD) platform for the detection of genomic imbalance. We provide an overview of the sequence of computational analyses involved in identifying pathogenic CNVs and highlight important parameters for consideration in assessing the pathogenicity of a detected CNV. © 2015 by John Wiley & Sons, Inc.

Keywords: CMA; chromosome aberrations; software; CytoScan; Affymetrix

PDF or HTML at Wiley Online Library

Table of Contents

  • Introduction
  • Overview of Informatics Steps in Analyzing Microarray Data
  • Background Information
  • Summary
  • Figures
PDF or HTML at Wiley Online Library


PDF or HTML at Wiley Online Library



Literature Cited

Literature Cited
  Ambros, I.M., Brunner, C., Abbasi, R., Frech, C., and Ambros, P.F. 2014. Ultra‐high density SNParray in neuroblastoma molecular diagnostics. Front. Oncol. 4:202.
  Anaka, M., Hudson, C., Lo, P.H., Do, H., Caballero, O.L., Davis, I.D., Dobrovic, A., Cebon, J., and Behren, A. 2013. Intratumoral genetic heterogeneity in metastatic melanoma is accompanied by variation in malignant behaviors. BMC Med. Genomics 6:40.
  Asadollahi, R., Oneda, B., Joset, P., Azzarello‐Burri, S., Bartholdi, D., Steindl, K., Vincent, M., Cobilanschi, J., Sticht, H., Baldinger, R., Reissmann, R., Sudholt, I., Thiel, C.T., Ekici, A.B., Reis, A., Bijlsma, E.K., Andrieux, J., Dieux, A., FitzPatrick, D., Ritter, S., Baumer, A., Latal, B., Plecko, B., Jenni, O.G., and Rauch, A. 2014. The clinical significance of small copy number variants in neurodevelopmental disorders. J. Med. Genet. 51:677‐688.
  Berkel, S., Marshall, C.R., Weiss, B., Howe, J., Roeth, R., Moog, U., Endris, V., Roberts, W., Szatmari, P., Pinto, D., Bonin, M., Riess, A., Engels, H., Sprengel, R., Scherer, S.W., and Rappold, G.A. 2010. Mutations in the SHANK2 synaptic scaffolding gene in autism spectrum disorder and mental retardation. Nat. Genet. 42:489‐491.
  Brand, H., Pillalamarri, V., Collins, R.L., Eggert, S., O'Dushlaine, C., Braaten, E.B., Stone, M.R., Chambert, K., Doty, N.D., Hanscom, C., Rosenfeld, J.A., Ditmars, H., Blais, J., Mills, R., Lee, C., Gusella, J.F., McCarroll, S., Smoller, J.W., Talkowski, M.E., and Doyle, A.E. 2014. Cryptic and complex chromosomal aberrations in early‐onset neuropsychiatric disorders. Am. J. Hum. Genet. 95:454‐461.
  Campbell, I.M., Yuan, B., Robberecht, C., Pfundt, R., Szafranski, P., McEntagart, M.E., Nagamani, S.C., Erez, A., Bartnik, M., Wisniowiecka‐Kowalnik, B., Plunkett, K.S., Pursley, A.N., Kang, S.H., Bi, W., Lalani, S.R., Bacino, C.A., Vast, M., Marks, K., Patton, M., Olofsson, P., Patel, A., Veltman, J.A., Cheung, S.W., Shaw, C.A., Vissers, L.E., Vermeesch, J.R., Lupski, J.R., and Stankiewicz, P. 2014. Parental somatic mosaicism is underrecognized and influences recurrence risk of genomic disorders. Am. J. Hum. Genet. 95:173‐182.
  Carvalho, C.M., Ramocki, M.B., Pehlivan, D., Franco, L.M., Gonzaga‐Jauregui, C., Fang, P., McCall, A., Pivnick, E.K., Hines‐Dowell, S., Seaver, L.H., Friehling, L., Lee, S., Smith, R., Del Gaudio, D., Withers, M., Liu, P., Cheung, S.W., Belmont, J.W., Zoghbi, H.Y., Hastings, P.J., and Lupski, J.R. 2011. Inverted genomic segments and complex triplication rearrangements are mediated by inverted repeats in the human genome. Nat. Genet. 43:1074‐1081.
  Darvishi, K. 2010. Application of nexus copy number software for CNV detection and analysis. Curr. Protoc. Hum. Genet. 65:4.14.1‐4.14.28.
  Fiegler, H. and Carter, N.P. 2004. Genomic array technology. Meth. Cell Biol. 75:769‐785.
  Friedman, J., Adam, S., Arbour, L., Armstrong, L., Baross, A., Birch, P., Boerkoel, C., Chan, S., Chai, D., Delaney, A.D., Flibotte, S., Gibson, W.T., Langlois, S., Lemyre, E., Li, H.I., MacLeod, P., Mathers, J., Michaud, J.L., McGillivray, B.C., Patel, M.S., Qian, H., Rouleau, G.A., Van Allen, M.I., Yong, S.L., Zahir, F.R., Eydoux, P., and Marra, M.A. 2009. Detection of pathogenic copy number variants in children with idiopathic intellectual disability using 500 K SNP array genomic hybridization. BMC. Genomics 10:526.
  Hu, W.F., Chahrour, M.H., and Walsh, C.A. 2014. The diverse genetic landscape of neurodevelopmental disorders. Annu. Rev. Genomics Hum. Genet. 15:195‐213.
  Hurles, M.E., Dermitzakis, E.T., and Tyler‐Smith, C. 2008. The functional impact of structural variation in humans. Trends Genet. 24:238‐245.
  Iafrate, A.J., Feuk, L., Rivera, M.N., Listewnik, M.L., Donahoe, P.K., Qi, Y., Scherer, S.W., and Lee, C. 2004. Detection of large‐scale variation in the human genome. Nat. Genet. 36:949‐951.
  Kearney, H.M., South, S.T., Wolff, D.J., Lamb, A., Hamosh, A., and Rao, K.W., and Working Group of the American College of Medical, G. 2011. American College of Medical Genetics recommendations for the design and performance expectations for clinical genomic copy number microarrays intended for use in the postnatal setting for detection of constitutional abnormalities. Genet. Med. 13:676‐679.
  Liu, P., Carvalho, C.M., Hastings, P.J., and Lupski, J.R. 2012. Mechanisms for recurrent and complex human genomic rearrangements. Curr. Opin. Genet. Dev. 22:211‐220.
  Liu, P., Erez, A., Nagamani, S.C., Dhar, S.U., Kolodziejska, K.E., Dharmadhikari, A.V., Cooper, M.L., Wiszniewska, J., Zhang, F., Withers, M.A., Bacino, C.A., Campos‐Acevedo, L.D., Delgado, M.R., Freedenberg, D., Garnica, A., Grebe, T.A., Hernandez‐Almaguer, D., Immken, L., Lalani, S.R., McLean, S.D., Northrup, H., Scaglia, F., Strathearn, L., Trapane, P., Kang, S.H., Patel, A., Cheung, S.W., Hastings, P.J., Stankiewicz, P., Lupski, J.R., and Bi, W. 2011. Chromosome catastrophes involve replication mechanisms generating complex genomic rearrangements. Cell 146:889‐903.
  Mason‐Suares, H., Kim, W., Grimmett, L., Williams, E.S., Horner, V.L., Kunig, D., Goldlust, I.S., Wu, B.L., Shen, Y., Miller, D.T., Martin, C.L., and Rudd, M.K. 2013. Density matters: Comparison of array platforms for detection of copy‐number variation and copy‐neutral abnormalities. Genet. Med. 15:706‐712.
  Medvedev, P., Stanciu, M., and Brudno, M. 2009. Computational methods for discovering structural variation with next‐generation sequencing. Nat. Methods 6:S13‐20.
  Menten, B., Buysse, K., Zahir, F., Hellemans, J., Hamilton, S.J., Costa, T., Fagerstrom, C., Anadiotis, G., Kingsbury, D., McGillivray, B.C., Marra, M.A., Friedman, J.M., Speleman, F., and Mortier, G. 2007. Osteopoikilosis, short stature and mental retardation as key features of a new microdeletion syndrome on 12q14. J. Med. Genet. 44:264‐268.
  Miller, D. T., Shen, Y., and Wu, B.‐L. 2012. Oligonucleotide microarrays for clinical diagnosis of copy number variation and zygosity status. Curr. Protoc. Hum. Genet. 74:8.12.1‐8.12.17.
  Mills, R.E., Walter, K., Stewart, C., Handsaker, R.E., Chen, K., Alkan, C., Abyzov, A., Yoon, S.C., Ye, K., Cheetham, R.K., Chinwalla, A., Conrad, D.F., Fu, Y., Grubert, F., Hajirasouliha, I., Hormozdiari, F., Iakoucheva, L.M., Iqbal, Z., Kang, S., Kidd, J.M., Konkel, M.K., Korn, J., Khurana, E., Kural, D., Lam, H.Y., Leng, J., Li, R., Li, Y., Lin, C.Y., Luo, R., Mu, X.J., Nemesh, J., Peckham, H.E., Rausch, T., Scally, A., Shi, X., Stromberg, M.P., Stutz, A.M., Urban, A.E., Walker, J.A., Wu, J., Zhang, Y., Zhang, Z.D., Batzer, M.A., Ding, L., Marth, G.T., McVean, G., Sebat, J., Snyder, M., Wang, J., Ye, K., Eichler, E.E., Gerstein, M.B., Hurles, M.E., Lee, C., McCarroll, S.A., Korbel, J.O., and Genomes, P. 2011. Mapping copy number variation by population‐scale genome sequencing. Nature 470:59‐65.
  Molenaar, J.J., Koster, J., Zwijnenburg, D.A., van Sluis, P., Valentijn, L.J., van der Ploeg, I., Hamdi, M., van Nes, J., Westerman, B.A., van Arkel, J., Ebus, M.E., Haneveld, F., Lakeman, A., Schild, L., Molenaar, P., Stroeken, P., van Noesel, M.M., Ora, I., Santo, E.E., Caron, H.N., Westerhout, E.M., and Versteeg, R. 2012. Sequencing of neuroblastoma identifies chromothripsis and defects in neuritogenesis genes. Nature 483:589‐593.
  Pavlistova, L., Zemanova, Z., Sarova, I., Lhotska, H., Berkova, A., Spicka, I., and Michalova, K. 2014. Change in ploidy status from hyperdiploid to near‐tetraploid in multiple myeloma associated with bortezomib/lenalidomide resistance. Cancer Genet. 207:326‐331.
  Poduri, A., Evrony, G.D., Cai, X., and Walsh, C.A. 2013. Somatic mutation, genomic variation, and neurological disease. Science 341:1237758.
  Pugh, T.J., Delaney, A.D., Farnoud, N., Flibotte, S., Griffith, M., Li, H.I., Qian, H., Farinha, P., Gascoyne, R.D., and Marra, M.A. 2008. Impact of whole genome amplication on analysis of copy number variants. Nucleic Acids Res. 36:e80. doi: 10.1093/nar/gkn378.
  Ramachandran, D., Mulle, J.G., Locke, A.E., Bean, L.J., Rosser, T.C., Bose, P., Dooley, K.J., Cua, C.L., Capone, G.T., Reeves, R.H., Maslen, C.L., Cutler, D.J., Sherman, S.L., and Zwick, M.E. 2014. Contribution of copy‐number variation to down syndrome‐associated atrioventricular septal defects. Genet. Med. doi: 10.1038/gim.2014.144. [Epub ahead of print].
  Redon, R., Ishikawa, S., Fitch, K.R., Feuk, L., Perry, G.H., Andrews, T.D., Fiegler, H., Shapero, M.H., Carson, A.R., Chen, W., Cho, E.K., Dallaire, S., Freeman, J.L., Gonzalez, J.R., Gratacos, M., Huang, J., Kalaitzopoulos, D., Komura, D., MacDonald, J.R., Marshall, C.R., Mei, R., Montgomery, L., Nishimura, K., Okamura, K., Shen, F., Somerville, M.J., Tchinda, J., Valsesia, A., Woodwark, C., Yang, F., Zhang, J., Zerjal, T., Zhang, J., Armengol, L., Conrad, D.F., Estivill, X., Tyler‐Smith, C., Carter, N.P., Aburatani, H., Lee, C., Jones, K.W., Scherer, S.W., and Hurles, M.E. 2006. Global variation in copy number in the human genome. Nature 444:444‐454.
  Sdano, M.R., Vanzo, R.J., Martin, M.M., Baldwin, E.E., South, S.T., Rope, A.F., Allen, W.P., and Kearney, H. 2014. Clinical utility of chromosomal microarray analysis of DNA from buccal cells: Detection of mosaicism in three patients. J. Genet. Couns. 23:922‐927.
  Sebat, J., Lakshmi, B., Troge, J., Alexander, J., Young, J., Lundin, P., Maner, S., Massa, H., Walker, M., Chi, M., Navin, N., Lucito, R., Healy, J., Hicks, J., Ye, K., Reiner, A., Gilliam, T.C., Trask, B., Patterson, N., Zetterberg, A., and Wigler, M. 2004. Large‐scale copy number polymorphism in the human genome. Science 305:525‐528.
  Shaw‐Smith, C., Redon, R., Rickman, L., Rio, M., Willatt, L., Fiegler, H., Firth, H., Sanlaville, D., Winter, R., Colleaux, L., Bobrow, M., and Carter, N.P. 2004. Microarray based comparative genomic hybridisation (array‐CGH) detects submicroscopic chromosomal deletions and duplications in patients with learning disability/mental retardation and dysmorphic features. J. Med. Genet. 41:241‐248.
  South, S.T., Lee, C., Lamb, A.N., Higgins, A.W., and Kearney, H.M., and Working Group for the American College of Medical, G., and Genomics Laboratory Quality Assurance, C. 2013. ACMG Standards and Guidelines for constitutional cytogenomic microarray analysis, including postnatal and prenatal applications: Revision 2013. Genet. Med. 15:901‐909.
  Stankiewicz, P. and Lupski, J.R. 2010. Structural variation in the human genome and its role in disease. Annu. Rev. Med. 61:437‐455.
  Tan, R., Wang, Y., Kleinstein, S.E., Liu, Y., Zhu, X., Guo, H., Jiang, Q., Allen, A.S., and Zhu, M. 2014. An evaluation of copy number variation detection tools from whole‐exome sequencing data. Hum. Mutat. 35:899‐907.
  Tucker, T., Zahir, F.R., Griffith, M., Delaney, A., Chai, D., Tsang, E., Lemyre, E., Dobrzeniecka, S., Marra, M., Eydoux, P., Langlois, S., Hamdan, F.F., Michaud, J.L., and Friedman, J.M. 2014. Single‐exon‐resolution targeted chromosomal microarray analysis of known and candidate intellectual disability genes. Eur. J. Hum. Genet. 22:792‐800.
  Tucker, T., Montpetit, A., Chai, D., Chan, S., Chenier, S., Coe, B.P., Delaney, A., Eydoux, P., Lam, W.L., Langlois, S., Lemyre, E., Marra, M., Qian, H., Rouleau, G.A., Vincent, D., Michaud, J.L., and Friedman, J.M. 2011. Comparison of genome‐wide array genomic hybridization platforms for the detection of copy number variants in idiopathic mental retardation. BMC. Med. Genomics 4:25.
  Verdin, H., D'Haene, B., Beysen, D., Novikova, Y., Menten, B., Sante, T., Lapunzina, P., Nevado, J., Carvalho, C.M., Lupski, J.R., and De Baere, E. 2013. Microhomology‐mediated mechanisms underlie non‐recurrent disease‐causing microdeletions of the FOXL2 gene or its regulatory domain. PLoS Genet. 9:e1003358.
  Wang, Y., Cottman, M., and Schiffman, J.D. 2012. Molecular inversion probes: A novel microarray technology and its application in cancer research. Can. Gen. 205:341‐355.
  Webb, B.D., Scharf, R.J., Spear, E.A., Edelmann, L.J., and Stroustrup, A. 2014. Evaluation of the Affymetrix CytoScan Dx Assay for developmental delay. Expert Rev. Mol. Diagn. 1‐8.
  Yu, Y.P., Michalopoulos, A., Ding, Y., Tseng, G., and Luo, J.H. 2014. High fidelity copy number analysis of formalin‐fixed and paraffin‐embedded tissues using Affymetrix Cytoscan HD chip. PloS one 9:e92820.
  Zahir, F. and Friedman, J.M. 2007. The impact of array genomic hybridization on mental retardation research: A review of current technologies and their clinical utility. Clin Genet 72:271‐287.
  Zahir, F.R., Baross, A., Delaney, A.D., Eydoux, P., Fernandes, N.D., Pugh, T., Marra, M.A., and Friedman, J.M. 2008. A patient with vertebral, cognitive and behavioural abnormalities and a de novo deletion of NRXN1alpha. J. Med. Genet. 45:239‐243.
  Zahir, F., Firth, H.V., Baross, A., Delaney, A.D., Eydoux, P., Gibson, W.T., Langlois, S., Martin, H., Willatt, L., Marra, M.A., and Friedman, J.M. 2007. Novel deletions of 14q11.2 associated with developmental delay, cognitive impairment and similar minor anomalies in three children. J Med Genet 44:556‐561.
  Zhang, F., Khajavi, M., Connolly, A.M., Towne, C.F., Batish, S.D., and Lupski, J.R. 2009. The DNA replication FoSTeS/MMBIR mechanism can generate genomic, genic and exonic complex rearrangements in humans. Nat. Genet. 41:849‐853.
  Zhang, F., Seeman, P., Liu, P., Weterman, M.A., Gonzaga‐Jauregui, C., Towne, C.F., Batish, S.D., De Vriendt, E., De Jonghe, P., Rautenstrauss, B., Krause, K.H., Khajavi, M., Posadka, J., Vandenberghe, A., Palau, F., Van Maldergem, L., Baas, F., Timmerman, V., and Lupski, J.R. 2010. Mechanisms for nonrecurrent genomic rearrangements associated with CMT1A or HNPP: Rare CNVs as a cause for missing heritability. Am. J. Hum. Genet. 86:892‐903.
Internet Resources
  Affymetrix Web site containing information about the microarrays, wet lab protocols for preparing DNA, procedures for processing and scanning chips, and annotation files for analyses.
  Web site for the Database of Genomic Variants (DGV; also known as the Toronto Database). Lists known copy number polymorphisms from the literature.
  Online Mendelian Inheritance of Man database: A manually curated database of comprehensive gene and genetic‐region centric information.
  Database of Chromosomal Imbalance and Phenotype in Humans using Ensembl Resources: A repository of pathogenic CNVs and clinical information. A validated user name and password is used to access clinical data.
  International Collaboration for Clinical Genomics:Database of CNVs detected by clinical and accredited molecular testing laboratories worldwide.
PDF or HTML at Wiley Online Library