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Aug 1, 2011 (Vol. 31, No. 14) , Jul 1, 2011 (Vol. 31, No. 13)

aCGH Opens Up Novel Avenues of Study

Technique that Augments Karotyping Now Being Used in Prenatal Testing, Cancer, and Autism

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    Oxford Gene Technology’s high-throughput genomic services laboratory reportedly processes over 2,000 aCGH samples per week.

    Array-based comparative genomic hybridization (aCGH) has become a standard method for detecting copy-number variations (CNVs) and other genetic defects associated with genetic diseases. Once primarily the domain of postnatal testing for developmental disorders, the technique is branching into prenatal (and preconception) testing and cancer.

    aCGH involves labeling test and reference genomes with differently colored dyes, mixing them, then hybridizing the samples to a microarray of probes covering a genomic area of interest. The scope may range from a few hundred genes to the entire genome.

    aCGH differs from standard CGH in its resolution. Because conventional CGH hybridizes samples directly to chromosomes, resolution is limited to five megabases or so. Arrays employ not whole chromosomes but probes consisting of 50 to 75 bases. The densest commercially available arrays today consist of between two and three million probes. More common are 180,000-spot slides, which provide whole-genome analysis at a resolution of 5–10 kilobases.

    “The advantage of microarrays is that they allow viewing of the entire genome as well as regions of special interest,” said James Clough, vp for clinical and genomic solutions at Oxford Gene Technology (OGT).

    Founded by Edwin Southern, Ph.D., the Oxford professor of Southern blot fame who also invented microarrays, OGT began to provide microarray services about seven years ago. “It soon became clear that array CGH had a role to play in the investigation of constitutional disorders,” added Clough.

    At the time karyotyping was king, and oligonucleotide arrays were thought to be too noisy. But eventually, researchers found clinical relevance beneath signals originally ascribed to noise. But what eventually won over researchers was the clinical yield: just 5% to 8% for karyotyping, up to 17% on BAC arrays (an early embodiment of oligo arrays), and up to 22% for aCGH.

    The most pronounced limitation of aCGH is the inability of the native technique to detect balanced translocations in which precisely the same quantity of genetic material switches from one chromosome to another, and vice versa. Probes can only detect the relative amount of material corresponding to a genetic region, but not its actual location. Balanced translocations are associated with many solid and hematopoietic cancers.

    Another limitation is that aCGH cannot distinguish triploidy, tetraploidy, or any other abnormality where every chromosome in the cell is detected in multiple copies, generally termed polyploidy.

    “These rearrangements can only be detected using conventional chromosome testing techniques like karyotyping,” said Gary Harton, head of molecular research at Reprogenetics. “However, aCGH can be used to detect the inheritance of unbalanced translocations in embryos from couples that carry these chromosome rearrangements.”

    At the “Association of Genetic Technologies” annual meeting, PerkinElmer described a work-around for balanced translocations, thereby opening up new areas of investigation for aCGH.

    PerkinElmer's approach uses gene amplification of known translocations followed by aCGH analysis. It involves polymerase and primers for the affected chromosomes.

    For example, the Philadelphia translocation in chronic myeloid leukemia involves chromosomes 9 and 22. As the primer for chromosome 9 and polymerase amplify the gene, they encounter the sequence from chromosome 22 and amplify that as well. Since only the primer for chromosome 9 is present, one would not expect an amplified signal for chromosome 22. Separately, the primer for the breakpoint on chromosome 22 amplifies the genetic material from chromosome 9.

    “Now, with that amplified pool in hand, when we look at the array we see chromosome 22 has a signal where you wouldn't expect one,” said Christopher Williams, global market segment leader for oncology.

    Further analysis would reveal where the DNA broke apart, which holds significance for both prognosis and treatment.

    PerkinElmer is offering a service based on this approach and a new product, OncoChip, which targets more than 1,800 loci associated with hematologic cancers. Onco- Chip provides baseline coverage of one oligo probe every 35 kb, and one probe every 500–5,000 bases in targeted regions.

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