Genome-wide Mapping of Copy Number Variations Using SNP Arrays

Abstract

The availability of high-density single nucleotide polymorphism (SNP) microarrays in recent years has proven to be a great step forward in the context of global analysis of genomic abnormalities in disease. SNP arrays offer great robustness, high resolution and the possibility to detect a variety of different genomic copy number variations such as submicroscopic deletions, amplifications, loss of heterozygosity and uniparental disomy. Moreover, they can be used to perform genome wide association studies. Therefore, SNP arrays harbor several advancements over traditional molecular methods to analyze genomic aberrations, such as cytogenetic analyses, fluorescence in situ hybridization or comparative genomic hybridization methods. Until now, SNP arrays have exclusively been used in experimental research and have enabled seminal new discoveries in many fields by identifying common genomic lesions underlying specific diseases, especially cancer. However, it is foreseeable that SNP arrays will also take up a position in routine diagnostic processes in the future. This review focuses on technical principles of the SNP array technology and their utilization to detect submicroscopic genomic and polymorphic markers associated with disease.

Fig. 1

Principles of Affymetrix SNP array technology. Genomic DNA is digested by restriction enzymes to form fragments of varying lengths. These are subjected to ligation with adapters to enable a one-primer PCR to produce fragments of selected size (200-1,100 bp). Subsequently, these are labeled with a fluorochrome and hybridized to the microarray. There, DNA fragments containing a SNP specifically bind to their allele-specific perfect match probes. The hybridized and washed array is then scanned by a laser, picking up fluorescent signals in dependency of quantitative binding. The raw data can be calculated into intensity data giving information about the DNA copy number, and determination of the SNP alleles provides information about the genotype.

Fig. 2

Genomic alterations detectable by SNP arrays, visualized with the CNAG (Copy Number Analyser for GeneChip®) software and allele-specific copy number determination. A Deletions on chromosome 9 in an ALL sample: Hemizygous deletions are characterized by reduction of the copy number value from 2 to 1. This is evidenced by a downward deviation of the copy number signals of single SNPs (single dots in the upper line), the averaged copy number signal (continuous line in the second panel from above) and deletion of one allele as shown by downward deviation of one of the allele-specific copy number signals (two separate lines in the lower panel). In homozygous deletions, both alleles are deleted, and the copy number value is reduced to 0. B Duplications and amplifications on chromosome 8q: Duplications are identified by an upward deviation of the copy number value to 3 (for duplications) or >3 for amplification of genomic material. C aUPD on chromosome 17q. aUPD is displayed by divergence of the allele-specific copy number signals in the lower panel. These indicate that one parental allele is duplicated while the other one is deleted. Throughout this region, the copy number value remains = 2, but a LOH is evidenced by the drastic loss of heterozygous SNP calls (vertical lines below the cytoband), the remaining ones are erroneous SNP calls caused by contamination of the tumor sample with normal DNA.

Fig. 3

Identification of target genes by identifying commonly affected genomic regions with the CNAG software. Shown is an integral view of chromosome 9 of a collection of ALL samples. Deletions are shown by horizontal lines below the cytoband, each line represents one sample. While many samples harbor big deletions, containing hundreds of potential target genes of the genomic lesion, overlapping the data from many samples leads to a reduction of size of the common region. This is impressively shown in ALL for hemi- and homozygous deletions pinpointing the tumor suppressor genes CDKN2A/CDKN2B or for the transcription factor Pax5.