Early efforts to examine genomic changes in the clinical setting relied on cytogenetic techniques such as chromosome karyotyping, a widely used approach to examine chromosomes and identify changes that could be responsible for a specific phenotype. This technique is sometimes inadequate, however, due to its crude limit of resolution, five to ten million bases, after which chromosome changes can be difficult to detect under a microscope.
An approach that has emerged over recent years, array-based comparative genomic hybridization, or array CGH, allows genome-wide scans to detect chromosomal rearrangements at a greatly improved resolution, down to hundreds of bases, or more than one thousand-fold better than karyotyping.
In array CGH, the control or reference genome and the patient’s genome are differentially fluorescently labeled and competitively hybridized to several hundreds or thousands of DNA targets. “The high resolution has become a very powerful aspect of this technology, and many times when we cannot detect any abnormalities at the chromosome level, we discover a lot of changes at the genome level by array CGH,” explains Jun Gu, M.D., Ph.D., instructor/education coordinator in the cytogenetic technology program at University of Texas and M.D. Anderson Cancer Center.
At the Association of Genetic Technologists’ annual meeting to be held next month in Phoenix, Dr. Gu will provide an update on advances at the frontline of array CGH. He will also offer a case study describing applications that are suitable for this type of analysis.
“Array CGH has emerged as a high-resolution whole-genome approach that could potentially help us classify genetic diseases with greater precision. This is the beginning of a new era, and many exciting discoveries are in front of us.”
As part of their ongoing efforts to find de novo genomic changes linked to autism, Simon G. Gregory, Ph.D., associate professor of medical genetics at Duke University, together with collaborators, used array CGH to examine the genomic profiles of 119 children with idiopathic autism.
This approach unveiled over 100 copy number variants, from which the investigators focused on a 0.7-Mb deletion on the short arm of chromosome 3, a region containing five genes. One of these genes, OXTR, encodes the oxytocin receptor that, having previously been implicated in autism, emerged as an interesting candidate to pursue.
The authors found that one child’s mother also carried the deletion, and she had self-reported obsessive-compulsive disorder symptoms, a condition previously linked to the same gene. However, to the investigators’ initial surprise, the child’s brother, despite being affected with autism, did not harbor this deletion. “We were briefly disappointed, until we decided to look at the methylation profile of this receptor,” recalls Dr. Gregory.
Previous reports documented that the OXTR gene is regulated by its DNA methylation status. By performing disulphide sequencing, Dr. Gregory and colleagues identified five CpG dinucleotides that exhibited differential methylation patterns among family members.
At three of these sites, the affected brother showed extensive methylation, an epigenetic modification correlated with decreased gene expression. Furthermore, on small groups of patients with autism, the authors showed that several of these dinucleotides were hypermethylated in peripheral blood mononuclear cells and in the temporal lobe cortex, as compared to unaffected individuals, a finding that they are proposing to further explore on larger patient groups.
Another research effort that Dr. Gregory is conducting in collaboration with medical oncologist Andrew J. Armstrong, M.D., ScM, proposes to profile copy number changes in primary and secondary malignant prostate tumor cells.
Cells that originate from primary tumors often metastasize to distant sites in various organs and establish secondary tumors that contribute to the lethal behavior of cancer; however, the factors that shape this process, and the specific affinity that primary prostate cancer cells have for certain secondary dissemination sites, are insufficiently understood.
“We are trying to identify the circulating tumor cells, pool them, and use unbiased genomic amplification to conduct copy number profiling and unveil potential copy number variations that would make primary tumor cells more likely to metastasize or increase their affinity for specific secondary tissues,” explains Dr. Gregory.
In addition, as part of a project seeking to identify factors associated with tumor behavior, Dr. Gregory and collaborators are using array CGH to explore correlations between copy number variation and the progression of oligodendroglyomas.
In addition to the research world, array CGH is also becoming commonplace in the clinical community and is increasingly replacing G-banded karyotyping as the gold standard for cytogenetic analysis.