Although copy number analysis is a powerful tool for studying genomic aberrations, it lacks the capacity to detect genotypic changes which are copy-neutral. If we consider loss of heterozygosity (LOH), this event may be caused by a hemizygous deletion in which one allele is lost and the other allele remains present (Figure 1,  middle panel). That type of LOH can be recognized not only by SNP-genotyping, but by copy-number analysis as well. However, an allele may lost initially, but the subsequent amplification of the remaining copy creates a copy-neutral LOH (Figure 1, right panel), also known as uniparental disomy. Different mechanisms have been described to create copy-neutral LOH in meiosis and mitosis; the common feature is that copy-neutral LOH can only be detected when copy number is studied in combination with SNP genotype. Please note that irrespective of the preservation of total number of copies, the biological effect is still important as recessive mutations would no longer be masked by their dominant normal counterparts.

Figure 1. Possible mechanisms of LOH and their impact on copy number. Left panel: heterozygous SNP; numbers indicate the number of copies of each allele (“normal” or most common allele = green, “mutant” = red). Middle panel: hemizygous deletion leading to the loss of normal allele. Right panel: duplication of the ”mutant” allele. The situation in the middle panel changes the gene copy number, while the situation in the right panel is copy-number neutral

A solution for detection of copy-neutral events is the integration of copy number workflow with LOH or the use of allelic imbalance (AI) under the Allele-Specific Copy Number (AsCN) workflow (advantages of AI over LOH are discussed below). With this approach, the copy number data are supplemented with SNP genotyping data (currently available with Affymetrix® and Illumina®) to label the genomic regions in the following fashion: amplification without LOH/AI, amplification with LOH/AI, deletion without LOH/AI, deletion with LOH/AI, copy-neutral LOH/AI (Figure 2). The last category, copy-neutral LOH/AI, is the added value of the workflow integration

A further consideration is that the correct interpretation of currently available algorithms for LOH has been proven complex and difficult because cancer cells frequently deviate from the diploid state, and tumor specimens often contain a significant proportion of normal cells. For instance, it has been shown that as the proportion of tumor cells in a sample decreases and approaches 50% or less, the capacity to detect the LOH diminishes (Yamamoto et al., Am J Hum Gen 2007). Moreover, the genotyping algorithms fail to call a heterozygote SNP accordingly in a situation when only one of two alleles gets amplified (e.g. 3×A and 1×B): a false positive LOH call can be the consequence.

Figure 2. Integration of copy number workflow with loss of heterozygosity (LOH) or allelic imbalance (AI) under allele-specific copy number (AsCN) workflows enables the identification of copy-neutral events

AsCN analysis, on the other hand, is a method that enables reliable detection of allelic imbalance in tumor samples even in the presence of large proportions of normal cells. Unlike LOH, it does not require a large set of normal reference samples. For a heterozygous SNP (only those are informative), a balance is expected between the two alleles (1×A and 1×B, or 1:1 ratio). The AsCN algorithm provides an estimated number of copies of each allele and therefore enables the detection of allelic imbalance even in cases when alleles are amplified or deleted (e.g. 3×A and 1×B). Moreover, LOH can be considered a special case of AI (e.g., 1×A, B allele deleted) (Figure 3). Therefore, due to its improved robustness, AsCN may be a preferred application in tumor-focused applications.

Figure 3. Loss of heterozygosity (LOH) as a special case of allelic imbalance. The situation on the left represents a normal heterozygous SNP, with one copy of each allele

References

Diskin SJ, Li M, Hou C, Yang S, Glessner J, Hakonarson H, Bucan M, Maris JM, Wang K. Adjustment of genomic waves in signal intensities from whole-genome SNP genotyping platforms. Nucleic Acids Res. 2008 Nov;36(19):e126.

Ramakrishna M, Williams LH, Boyle SE, Bearfoot JL, Sridhar A, Speed TP, Gorringe KL, Campbell IG. Identification of candidate growth promoting genes in ovarian cancer through integrated copy number and expression analysis. PLoS One. 2010 Apr 8;5(4):e9983.

Yamamoto G, Nannya Y, Kato M, Sanada M, Levine RL, Kawamata N, Hangaishi A, Kurokawa M, Chiba S, Gilliland DG, Koeffler HP, Ogawa S. Highly sensitive method for genomewide detection of allelic composition in nonpaired, primary tumor specimens by use of Affymetrix single-nucleotide-polymorphism genotyping microarrays. Am J Hum Genet. 2007 Jul;81(1):114-26.

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