Molecular karyotyping

Array comparative genomic hybridization (array CGH), also called molecular karyotyping, uses microarray technology (high density DNA probes on a solid surface) to detect in parallel whether any of several thousands of chromosomal fragments are deleted or duplicated in a genomic DNA sample. Array CGH can thus identify chromosomal abnormalities in patients with constitutional or acquired disorders and in embryos.
From our own experience in developing this highly effective molecular technique and rolling it out in a clinical setting, we can confidently assert that, within the next five to ten years, array CGH will replace conventional karyotyping, the most widely used genetic diagnostic technique for prenatal and postnatal genome analysis. Indeed, whereas the resolution of conventional karyotyping is limited to 5 Mb, the resolution of molecular karyotyping is only limited by the number and the size of the targets on the array. In addition, molecular karyotyping reduces analysis time from three weeks to two days, is amenable to automation, and makes data analysis more objective. As shown in this proposal, it is now becoming possible to obtain a molecular karyotype from minute amounts of DNA. The consortium members are at the cutting edge of array CGH development to answer genetic questions and at the forefront of introducing this novel technology in genetic diagnostic practice.
Furthermore, array CGH is leading to a paradigm shift in genetic diagnosis and research. During the last 20 years, since the development of molecular genetics, a major focus has been on the identification of nucleotide variations in both constitutional and acquired diseases. This focus has successfully led to the identification of the genetic causes of most of the common and rare Mendelian inherited diseases. In contrast to SNPs and small changes in the genetic code, array CGH is now revealing a hitherto unexpected source of variation, large scale copy number variation (CNPs; Copy Number Polymorphism). Segments ranging from 5 to 500 kb can be hemizygous or present in three or even four copies in different individuals. These CNPs often carry genes. Data is now accumulating that this variation may be a major cause of phenotypic variation and may better explain disease susceptibility variation, pharmacogenomic variation, and hitherto unexplained disorders.
This research proposal aims to push the technical limits of this novel technology. By advancing the potential of molecular karyotyping, both by improving technology and data analysis methods, novel applications in research as well as novel possibilities for genetic diagnosis will be achieved. First, we will develop genome wide analysis of genomic imbalances from a single cell. This will allow us to answer long standing questions about early embryogenesis and tumor development. Second, we will push back the limits of the technology to detect low grade mosaicisms, which are genetic imbalances in only a fraction of a mixture of cells. This in turn will allow us to investigate foetal DNA in the maternal plasma as well as improved analysis of tumors, lymphomas, and leukemias and probing potentially undetected causes of recurrent miscarriages and subfertility. Third, we will investigate the role of epigenetic phenomena in X chromosomal anomalies, constitutional anomalies, and cancer. Finally, we will maximize the effectiveness of these advances in array CGH by developing novel data interpretation methods. Statistical approaches, data storage and vizualisation tools developed in each of these domains wil cross fertilize the advances in the other domains.
These advances will not only enable to answer new basic questions, but change human genetic diagnosis. Single cell array CGH analysis will improve preimplantation genetic diagnosis and possibly the diagnosis of certain types of tumors. Mosaicism detection would influence and change the fields of prenatal and postnatal diagnosis of constitutional and acquired disorders. It is well known that epigenetic processes play an important role in both constitutional and acquired diseases. The potential of arrays to get a genome wide overview of epigenetic changes might be tremendous. Especially for the diagnosis of several types of cancers, the lack of a genome wide screening tool was limiting the diagnostic potential. Finally, we foresee that the huge amount of novel (biological) information coming from these analyses will require bioinformatics approaches to aid clinicians and laboratory directors in decision making and help researchers in understanding their findings. Since each application might use the different proposed technological fields, data storage, visualization and analysis tools will be recuperated among applications.