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Chapter 15
Array-Based Comparative Genomic Hybridization (aCGH)
Chengsheng Zhang , Eliza Cerveira , Mallory Romanovitch , and Qihui Zhu
Abstract
Copy number variations (CNVs) in the genomes have been suggested to play important roles in human evolution, genetic diversity, and disease susceptibility. A number of assays have been developed for the detection of CNVs, including fl uorescent in situ hybridization (FISH), array-based comparative genomic hybridization (aCGH), PCR-based assays, and next-generation sequencing (NGS). In this chapter, we describe a microarray method that has been used for the detection of genome-wide CNVs, loss of hetero- zygosity (LOH), and uniparental disomy (UPD) associated with constitutional and neoplastic disorders.
Key words Microarray , Array-based comparative genomic hybridization (aCGH) , Copy number variation (CNV) , Single nucleotide polymorphism (SNP) , Loss of heterozygosity (LOH) , Uniparental disomy (UPD)
1 Introduction
Structural variations ( SVs ) in the human genome are present in many forms, including single nucleotide polymorphisms ( SNPs ), small insertion/deletions (Indels), inversions, translocations, repetitive sequences, and copy number variations (CNVs) [ 1 – 3 ]. CNVs , which encompass more total nucleotides than SNPs in the human genomes, have been suggested to play important roles in human evolution, genetic diversity, and disease susceptibility [ 4 – 12 ]. In addition, CNVs have been shown to be associated with a variety of constitutional and neoplastic disorders. For instance, a number of microdeletion/micro- duplication syndromes (e.g., Williams-Beuren syndrome, Angelman Syndrome, DiGeorge syndrome, Pallister Killian syndrome, and Potocki-Lupski syndrome) result from copy number gain or loss in the genome [ 13 ]. Cancer is a genetic disease, which is driven by genetic changes in the oncogenes, tumor suppressor genes, and DNA repair genes, as well as the chromosomal instability [ 14 ]. Numerous studies from the Cancer Genome Atlas ( TCGA ) Consortium and other research groups have indicated that CNVs may also play signifi - cant roles in the process of tumorigenesis [ 15 – 17 ].
A number of assays have been developed for detection of CNVs in the human genomes, including fl uorescence in situ hybridiza- tion (FISH), array-based comparative genomic hybridization (aCGH), PCR-based assays, and next-generation sequencing [ 1 – 3 , 13 ]. The aCGH has many advantages over the conventional cyto- genetic techniques, such as genome‐wide coverage, high resolu- tion, and amenable to automation. However, it also has a number of limitations. For instance, it is unable to detect the balanced chromosomal rearrangements (e.g., inversions and translocations).
In early CGH experiments, the DNA targets were analyzed with the metaphase chromosome spreads [ 18 – 20 ]. This technology later evolved so that the DNA targets are hybridized to immobile cDNA fragments or bacterial artifi cial chromosomes (BACs) on a microarray surface [ 21 – 25 ]. The current advanced technology measures copy number differences using oligonucleotide microar- rays from Agilent, Affymetrix, and Illumina [ 26 – 28 ]. Some of the aCGH arrays label the test and normal reference DNA samples with different fl uorescent dyes (e.g., Cy5 and Cy3) respectively, and co-hybridized to the microarray [ 28 ]. The fl uorescence inten- sity ratio of the two labeled dyes at each DNA fragment refl ects the copy number ratio of that DNA sequence in the test DNA com- pared to the reference DNA [ 28 ]. Others are not required to be hybridized simultaneously with the test DNA sample [ 29 ]. The test sample data is compared to the data of normal reference sam- ples in the database developed by the manufacturers through bio- informatics analysis. Regions of DNA that have been deleted or amplifi ed are seen as changes in the ratio of test data against the normal control data along the target chromosomes. The SNP arrays are able to detect CNVs , loss of heterozygosity (LOH) , and uniparental disomy ( UPD ) [ 29 ].
In this chapter, we describe an aCGH assay using the CytoScan HD platform from Affymetrix [ 29 ]. This array contains roughly 2.7 million probes throughout the human genome and offers the highest resolution coverage for constitutional and cancer genes. It covers all 36,000 RefSeq genes including 12,000 OMIM , all ISCA constitutional regions, and Sanger cancer genes. The effective res- olution is 12 kilobase pairs (kb) on OMIM genes, 22 kb on other RefSeq genes, 10 kb on ISCA and cancer genes, and 50 kb on the backbone. It provides a genome-wide approach for detection of CNVs (gains or losses), LOH, regions identical-by-descent, and UPD on a single array. This array platform has been widely used in research and clinical laboratories for studies of constitutional and neoplastic disorders [ 30 – 38 ]. In 2010, the American College of Medical Genetics and Genomics (ACMGG) recommended employing chromosomal microarray as a fi rst-tier test for patients with unexplained developmental or intellectual disability, autism spectrum disorders, and congenital anomalies [ 39 ]. In 2014, Affymetrix received the clearance from the U.S. Food and Drug
Administration (FDA) to market its CytoScan Dx assay for pediat- ric patients with developmental delay and/or intellectual disability [ 40 ]. However, for DNA samples extracted from the formalin- fi xed, paraffi n-embedded ( FFPE ) tissue specimens, we are using the Affymetrix OncoScan CNV FFPE array platform, which has been developed and optimized specifi cally for FFPE DNA samples [ 41 – 43 ].
2 Materials