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Tutorials : Nov 1, 2010 (Vol. 30, No. 19)

Improving LOH Detection on CGH Microarrays

Technology Strives to Bypass a Number of Limitations of Current Products
  • Anniek De Witte
  • ,
  • Jeanene Swanson

Due to its higher resolution, oligo array CGH has recently made strong inroads in the cytogenetic lab. It’s not surprising. With an increased sensitivity of more than 1,000-fold, microarrays can detect much smaller genetic aberrations—microduplications and microdeletions—that may be linked to abnormal phenotypes and diseases than can karyotype analysis.

As basic research studies revealing the role of varied chromosomal aberrations in susceptibility to diseases like autism, mental retardation, and developmental delays continue to amass, cytogenetic researchers are beginning to understand the need to detect more and smaller changes.

One problem, however, with array CGH microarrays is that they only detect copy-number changes, since they measure total copies of alleles present in a sample. Copy-neutral changes such as loss of heterozygosity (LOH), uniparental disomy (UPD), and consanguinity, as well as balanced translocations, go undetected. 

During cell division, recombination between chromosomes might occur, resulting in material exchange with maintenance of the copy number. One of the resulting possibilities is UPD, where a person inherits two copies of a chromosome pair from one parent and no copy from the other parent. If the chromosomes involved are imprinted such that the genes on these chromosomes are monoallelically active (i.e., only the maternal or paternal allele of the pair is expressed), the resulting phenotype may be abnormal. UPD in tumor cells is often referred to as acquired UPD or copy-neutral LOH (cnLOH). Copy-neutral LOH is common in both hematologic and solid tumors.

Being able to detect copy-neutral and copy-number changes simultaneously would go a long way toward improving upon current efforts to create detailed genomic profiles of disease susceptibility and disease states.

Agilent Technologies’  new SurePrint G3 CGH+SNP microarray was designed to overcome the limitations of current array CGH microarrays. It allows cytogenetic researchers to detect both copy-number and copy-neutral chromosomal changes using one microarray. The same labeling and hybridization assay can be used, and an algorithm has been incorporated into Agilent Genomic Workbench 6.5 software to report both copy-number and copy-neutral changes.

The basis of Agilent’s new array is the inclusion of both CGH and SNP probes. Like with Agilent’s SurePrint CGH microarrays, the CGH probes measure the total number of alleles in a chromosomal region. The SNP probes, however, make it possible to measure copy-neutral, allele-specific changes.

Using a restriction fragment enzymatic digestion assay, SNPs located in the enzymes’ recognition site can be genotyped. Regions of copy-neutral LOH or UPD are then located by identifying genomic regions with a scarcity of heterozygous SNP calls.

Protocol

The sample-preparation and hybridization protocol is exactly the same as that for CGH-only microarrays. We digested genomic DNA from an unknown test sample and a control sample with known genotype with AluI and RsaI. Then we labeled the test sample with Cy5 dye and the control sample with Cy3 dye and hybridized both samples to the same array. After washing, we scanned the slides at 3 micron resolution on Agilent’s High Resolution C Scanner and extracted and analyzed the images using Agilent Genomic Workbench 6.5 software.

Methodology

Our method is similar to restriction fragment length polymorphism (RFLP). In RFLP analysis a genomic DNA sample is digested by restriction enzymes and the resulting fragments are separated by gel electrophoresis. Instead of using gel electrophoresis we use a microarray.

Restriction enzymes cut molecules of DNA at specific recognition sites, so cutting with a particular enzyme should always produce the same size and number of fragments. However, when a site is polymorphic, or existing as two alleles, the restriction enzyme will not recognize one allele and will not cut there, leading to a length polymorphism.

The ~60,000 SNP probes on the CGH+SNP microarray span variant AluI or RsaI restriction enzyme recognition sites and measure the copy number of the uncut allele at those loci (Figure 1). We measure the total copy number of the region encompassing the SNP site by neighboring CGH probes. The copy number of the cut allele can then be inferred from the total copy number and the copy number of the uncut allele.

A SNP copy-number call is made from the log ratio of the test sample (Cy5 signal) versus a genotyped internal reference (Cy3 signal). The copy number of each SNP in the reference sample is known. In order to determine the allele-specific copy number of the test sample, the log ratios of the SNP probes are adjusted by the copy number of the reference sample.

The reference-adjusted log ratios fall into three categories corresponding to the copy numbers of the uncut alleles in the sample, which correspond to the three possible diploid genotypes for the SNPs: AA, AB, or BB. Regions of copy-neutral LOH or UPD are then located by identifying genomic regions with a statistically significant scarcity of heterozygous SNP calls.

Results/Conclusions

With high-quality DNA samples, the SNP call rate is greater than 95% with greater than 99% accuracy, and the presence of SNP probes does not affect the performance of CGH probes. The number and quality of copy number aberrations detected on the SurePrint G3 CGH+SNP microarrays is comparable to detection using SurePrint G3 CGH-only microarrays with the added benefit of simultaneous identification of copy-neutral aberrations.

For a normal diploid region of the genome, one expects the 0, 1, and 2 SNP copy numbers of the uncut allele to be randomly distributed. In a diploid genome carrying a copy-neutral LOH or UPD aberration, the SNP probes will only report alleles that are homozygously cut and uncut (0 and 2 uncut alleles) and, therefore, only two states are found. Figure 2 shows an example of UPD observed in an individual known to have genomic aberrations associated with Angelman syndrome.

In conclusion, the SurePrint G3 CGH+SNP microarray affords the precision of SNP detection to measure copy-neutral genomic changes with ~5–10 Mb resolution as well as the reproducibility of Agilent’s CGH platform—all on a single microarray. As a result, researchers no longer need to choose between high-resolution, high-quality CGH data and the detection of LOH/UPD or alternatively run two separate experiments.