In addition to the surprising revelation that our chromosomes harbor fewer genes than originally predicted, the Human Genome Project also unveiled that all individuals share approximately 99.9% of their DNA.
The remaining 0.1% of the genome subsequently enjoyed enhanced scrutiny, as it promised to shed light on interindividual genomic variation, which became one of the most fascinating biomedical topics in recent years.
While single nucleotide polymorphisms (SNPs) were initially the focus of many studies, subsequent discoveries unveiled an additional source of genomic variability, which became known as copy-number variation (CNV). Array comparative genomic hybridization (array CGH) helped characterize copy-number variants and advanced research in several clinically relevant biomedical areas, but the quantitative characterization of copy-number variation remains a technically challenging endeavor.
“We have been using CGH arrays for many years, and they allow the detection of genome-wide copy-number changes, but one thing they cannot detect are copy-neutral aberrations,” says Anniek De Witte, technical marketing manager at Agilent Technologies.
One of the technical shortcomings of array CGH is that it can only measure the total number of allele copies in a sample and, therefore, it is only informative about copy-number changes. Acquired uniparental disomy or copy-neutral loss of heterozygosity, a previously underappreciated modification in which a pair of chromosomes or chromosomal segments is inherited from only one parent, that is increasingly being described in hematologic and solid tumors, would not be detected by this approach.
“The only way to detect these aberrations was by adding SNPs to the arrays, to make the distinction whether two chromosomes are different from each other or not, and this is why we incorporated SNP probes into our CGH arrays,” De Witte explains.
This approach helped investigators unveil significant diversity in several chronic lymphocytic leukemia tumor samples that they examined. In addition, this platform, combined with novel algorithms, is able to determine the fraction of aneuploid cells in cancer samples down to 10% for copy-number changes and down to 20% for copy-neutral changes.
“We wanted to address certain technical questions related to the performance of array CGH on small clinical samples, and the use of ultrahigh-resolution platforms in such samples,” says Mark Basik, M.D., assistant professor in surgery and oncology at McGill University and attending surgical oncologist at the Jewish General Hospital in Montréal.
Dr. Basik and colleagues proposed to test whether the density of array CGH allowed smaller DNA lesions, such as copy-number gains or losses, that could not be seen otherwise, to be visualized.
“The answer is yes,” reveals Dr. Basik. In a recent study that used MCF7 cell lines, investigators in Dr. Basik’s group discovered that the 1M CGH array has a remarkable capacity to unveil micro-copy number alterations, defined as alterations affecting chromosomal regions smaller than 1 Mb.
“More recently, we expanded this approach to clinical samples and found very similar results,” adds Dr. Basik. At the same time, Dr. Basik and colleagues visualized many intragenic breaks and, subsequently, by comparing the data with results from the original study that sequenced the same cell line, they identified approximately half of the gene fusion events that had been defined in that study. “Array CGH could become a valuable screening tool to look for gene fusion events,” reveals Dr. Basik.
An additional question that Dr. Basik and colleagues addressed revolves around the use of whole-genome amplification prior to performing high-resolution arrays. With clinical research increasingly relying on biopsies, which generally provide a very limited amount of biological material, whole-genome amplification is often required prior to the analysis of chromosomal variants.
A comparison between the 244K and the 1M CGH Agilent array platforms revealed that whole-genome amplification introduces many amplification artifacts, such as small deletions, that are much more detectable with the 1M platform than with the 244K platform. “This was surprising, and suggested that the use of whole-genome amplification with very high-resolution platforms is problematic and, in fact, algorithms used to automatically detect aberrations will pick up many artifacts and classify them as real aberrations,” cautions Dr. Basik.