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OMICS Array CGH Continued from page 1 analysis and interpretation," says Kenneth J. Craddock, M.D., a cytogeneticist at Toronto General Hospital. While copy-number abnormalities can be found in nearly all medical conditions, their signifcance must be explored individually, particularly because some variants simply refect normal inter-individual variation, while others may be of unknown signifcance. "At this time, the fndings for which we know the signifcance are a minority, and this constitutes another huge challenge in the feld," Dr. Craddock says. With the generation of more complex datasets, the availability of computing power is becoming increasingly important in analyzing and understanding the signifcance of structural changes in the chromosome. "Support from informaticians and computer programmers is not very prominent in genetics labs that are testing for various diseases, including cancer, but with the newly emerging technologies, these aspects will need to be addressed," Dr. Craddock says. Importantly, this support will be crucial in helping differentiate physiological structural variants from the ones that are pathologically signifcant. "This will also help connect the data to existing databases and increase automation, something for which most hospitals currently do not have the necessary resources," he adds. "While we have a great capacity to sequence genomes in a high-throughput manner at lower costs, we still lack accurate computational algorithms to detect copy-number variations from sequencing data," according to Santhosh Girirajan, Ph.D, assistant professor of biochemistry and molecular biology at Penn State University. Dr. Girirajan and his colleagues used array CGH to visualize chromosomal rearrangements in several developmental disorders. For a recent effort, they examined a cohort of more than 2,300 children who had copy-number variants associated with intellectual disability, fnding that harboring two large copy-number variants of unknown signifcance is associated with an over eightfold higher likelihood of developmental delay. This fnding suggested that multiple copynumber variants may interact with one another to shape the clinical presentation in complex diseases, and explains previous reports of phenotypic heterogeneity, when dissimilar clinical presentations were described in individuals harboring identical chromosomal abnormalities. More recently, Dr. Girirajan and colleagues examined the global load of chromosomal deletions and duplications in autism, a heterogeneous disorder that, based on 2013 estimates by the Centers for Disease Control and Prevention, affects one in 50 schoolchildren in the U.S. This study revealed an approximately sevenfold increase in duplications and a twofold increase in deletions in children with autism, and pointed toward the relationship between an increased genomic load of copynumber variations, particularly duplications, and the risk to develop this condition. "This 26 | TreeView representation of cDNAs that exhibit great ape or human LS aCGH signatures are presented. Order of genes within each lineage is based on the average log2 fluorescence ratios (ordered highest to lowest) of the respective species. The dataset used for this figure was not collapsed by UniGene cluster to minimize the chance that significant LS cDNAs would be missed. Fluorescence ratios are depicted using a pseudocolor scale (indicated). University of Colorado Denver/Andrew Fortna, et al. PLoS Biol. 2004 July;2(7):e207. While array CGH found broad applicability in clinical and research laboratories, the statistical models of experimental design and data analysis remain an area that received relatively limited attention. also points toward the need to understand the impact of deletions or duplications of chromosomal regions harboring tens of genes, as opposed to the more simple genetic mutations that we often talk about in human genetics," Dr. Girirajan says. "CGH, a very well understood and accepted test, provides a cost-effective way to fnd copy-number variants, but interpreting the results and fguring out what the variants mean, is currently the major challenge," says Robert L. Nussbaum, M.D., professor of medicine and chief of the division of medical genetics at the University of California, San Francisco. Complex Disorders One of the challenges stems from the fact that some copy-number variations, even the large ones, might not have a particular impact on the phenotype. On the other hand, some symptomatic patients were shown to harbor copy-number variants that are present in parents who might not be affected by August 2013 | GENengnews.com | Genetic Engineering & Biotechnology News the disorder. "That might suggest either that the copynumber variation does not have anything to do with the condition, or that there is an additional factor that interacts with it, such as a mutation, which may be in another gene or on another chromosome," Dr. Nussbaum says. The challenges are more pronounced when testing is performed in a prenatal setting, due to the signifcant diffculties in predicting the severity of a specifc disease whose onset may be years or decades in the future. Array CGH is reshaping many biomedical areas. One of these is preimplantation genetic diagnosis, which entails a small biopsy that is performed to remove one of the 6-8 cells of a three-day embryo formed after in vitro fertilization, prior to its implantation into the uterus. This widely performed procedure revolutionized assisted reproductive technologies but, like any medical procedure, is not devoid of risks. "For the frst time, we were able to obtain DNA from the embryo and perform genetic analyses without removing cells from the blastomere," says Simone Palini, Ph.D., senior clinical embryologist of the research group directed by Carlo Bulletti, M.D., in the physiopathology of reproduction unit at Cervesi General Hospital in Cattolica, Italy. Together with Luca Galluzzi, Ph.D., professor of recombinant and molecular biotechnology, and Mauro Magnani, Ph.D., professor in biochemistry in the department of biomolecular science at the University of Urbino, the investigators demonstrated that fuid removed from blastocel cavity of a fve- day-old human blastocyst can provide suffcient DNA to perform genetic analyses. Genomic DNAv was found in approximately 90% of the blastocyst fuid samples that were collected during the vitrifcation procedure, as part of the cryopreservation of the embryos obtained by in vitro fertilization. "In this approach, the embryologist places the needle only 7 microns inside the blastocyst, which is 250–300 micrometers in diameter, and preimplanatation genetic testing is performed without disturbing the embryo, in a procedure that is comparable to the intracytoplasmic sperm injection," says Dr. Palini. The 0.3–0.5 nanoliters of fuid that are retrieved during this process contain a median of 9.9 pg DNA, which can be used to perform various types of tests. "For most labs, a thermocycler is like a coffee machine, and virtually all labs can afford to perform PCR and look for mutations in specifc genes of interest," say Dr. Palini and Dr. Galluzzi. In a proof-of-principle experiment, Dr. Bulletti and Dr. Magnani's groups used the DNA isolated from the blastocyst fuid for whole-genome amplifcation and array CGH analyses, illustrating the possibility to detect several aneuploidies and to determine the sex of the embryos. "CGH, a very important tool for reproductive medicine, will most likely see further improvements in the future, and it assumes an important role in preventive medicine, even if more studies should be performed to validate the protocol," Dr. Palini says. Evolutionary Applications Gene duplication is a major mechanism behind evolutionary change. With this in mind, Dr. Sikela in collaboration with Dr. Jonathan Pollack at Stanford University, focused on fnding genes that have been highly duplicated along specifc primate lineages in several species, including humans. "We used gene-based array CGH genomewide to identify genes that are important for human and primate evolution," says James M. Sikela, Ph.D., professor of biochemistry and molecular genetics at the University of Colorado Denver. This marked the frst time genome-wide array CGH analysis was applied to perform a cross-species comparison of humans and other primates. "In the analysis that we performed, cDNAs corresponding to virtually all human genes had been deposited on the arrays, so this was a cDNA array, as compared to the typical oligonucleotide array. This had the advantage that we could survey the copy number of virtually all human genes in each experiment," Dr. Sikela explains. This strategy revealed that approximately 140 genes were specifcally changed in the human lineage, represented and were present in more or fewer copies in humans as compared with nonhuman primates. One of the strongest signals was from a gene that encoded multiple copies of DUF1220, a protein of unknown function. Further analysis revealed that DUF1220 was present in many more copies in humans as compared to the genome of any other