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Aug 1, 2013 (Vol. 33, No. 14)

Diving Deep with Array CGH

  • While single-nucleotide polymorphisms were an initial focus, the subsequent discovery of copy-number variation unveiled a new dimension of genomic diversity, and made apparent the need to develop more refined technologies to capture chromosomal rearrangements.

    Comparative genomic hybridization (CGH) provided the opportunity to detect chromosomal imbalances in a high-throughput manner, at the genome-wide scale, and with higher resolution than with previously available methods, such as G-banding and fluorescence in situ hybridization. At the same time, the wealth of CGH data generated gave rise to several challenges.

    “Identifying the genomic changes that are significant is a challenging aspect of CGH array data 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 significance must be explored individually, particularly because some variants simply reflect normal inter-individual variation, while others may be of unknown significance. “At this time, the findings for which we know the significance are a minority, and this constitutes another huge challenge in the field,” Dr. Craddock says.

    With the generation of more complex datasets, the availability of computing power is becoming increasingly important in analyzing and understanding the significance 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 significant. “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, finding that harboring two large copy-number variants of unknown significance is associated with an over eightfold higher likelihood of developmental delay. This finding suggested that multiple copy-number 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 copy-number variations, particularly duplications, and the risk to develop this condition. “This 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 find copy-number variants, but interpreting the results and figuring 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

    Click Image To Enlarge +
    Day 5 blastocyst. The holding pipette and the injection pipette are shown. After aspiration, the fluid is stored in an Eppendorf tube and immediately analyzed. Blastocoele fluid content seems to be useful for pre-implantation genetic diagnosis and to detect viability of the embryo without removing embryonic cells. [Cervesi Hospital Cattolica]

    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 the disorder.

    “That might suggest either that the copy-number 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 significant difficulties in predicting the severity of a specific 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 first 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 fluid removed from blastocel cavity of a five-day-old human blastocyst can provide sufficient DNA to perform genetic analyses.

    Genomic DNAv was found in approximately 90% of the blastocyst fluid samples that were collected during the vitrification 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 fluid 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 specific 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 fluid for whole-genome amplification 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.

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