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In the early years of scientific investigations in the field of heredity, the methods used to obtain data were considered genetical but once the physical basis of genetic conditions were recognized several studies were performed using methods of both cytology and genetics, using the data collected by genetic procedures along with observations made using cytological techniques. This dual approach to problems of heredity was coined the term cytogenetics. Cytogenetics has allowed to understand the genetic basis of diseases as well as a powerful tool to diagnose genetic conditions. Some the traditional cytogenetics techniques are chromosome banding and fluorescent in situ hybridization (FISH). In chromosome banding the chromosomes are treated with different chemicals to stain them and interpretations are made from how the chromosome stains, based on the dyes used they could be G-banding, Q-banding, C-banding or R-banding.
In FISH, a nucleic acid probe is conjugated with a fluorescent dye, this is then used to detect the complementary sequences on the metaphase chromosomes or in interphase nuclei. After hybridization, the location of the bound probes is detected and analyzed by using fluorescence microscopy and digital imaging technology. The above mentioned traditional techniques are powerful tools and upon which the foundation have been built but they have their limitations, for instance, chromosome banding is unable to detect deletions less than 5mb i. e. microdeletions and in FISH the appropriate probes needs to selected in order to identify chromosomal aberrations. To overcome the shortcomings there have been several advancements in the field and these technological leaps have helped improve our ability to investigate and define cellular processes at a molecular level which has benefiting both scientists and clinicians. Few of the new FISH-based technologies are reverse FISH, multiplex FISH(M-FISH), spectral karyotyping (SKY), comparative genomic hybridization (CGH) analysis and matrix or microarray – CGH (M-CGH). Instead of discussing all the modern cytogenetics methods and their impact on identifying basis of diseases and clinical diagnostic services, in this essay the focus will be on comparative genomic hybridization and its implications.
Cytogenetic technologies have had a major impact on the field of medicine and in particular reproductive medicine and oncology, they have enabled us to understand and analyze the frequencies at which chromosomal aberrations occur during gametogenesis, embryonic development and tumor development. We are now able to better understand and detect the genetic abnormalities that are associated with tumor origins, tumor progression, spontaneous abortions and congenital anomalies. CGH technology allows us to identify and map genomic regions for any chromosomal losses or gains in a single experiment without even having any knowledge on the chromosomal abnormality in question. CGH can produce a map of DNA sequence copy number changes as a function of chromosomal location throughout the entire genome. There are two ways of performing CGH, either using the direct method or the indirect method, for the direct method we use fluorochromes whereas for the indirect method we use haptens, using haptens could be more beneficial in some cases since they are both cost-effective and more flexible. In the event there is a need for a reference standard for data analysis, CGH is performed with different labeled normal DNA. In order to perform a more detailed analysis, CGH is coupled with sensitive monochrome cooled-charge coupled device (CCD) camera and an automated image analysis software. Regions of gain or loss of DNA will be represented as ratios of the two fluorochrome intensity on the chromosome that are being studied, in the case of chromosomal duplication or gene amplification in tumor DNA there would be increased green-to-red ratios whereas in chromosomal deletions there would be a decreased ratio.
Microarray-based comparative genomic hybridization (array CGH) is able to combine the advantages of molecular diagnostics along with the traditional cytogenetic techniques and furthering the field of cytogenetics. Studies performed on individuals with developmental delay and dysmorphic features have shown that array CGH has the ability to chromosomal aberrations such as duplications, deletions, amplifications and aneuploidies. In case of individuals with normal results before cytogenetic testing the detection rates of chromosome aberrations was 5-17%, copy number variants (CNVs) can also be identified. Array CGH is a powerful tool with potential to being a diagnostic tool for identifying visible and submicroscopic chromosome anomalies in mental retardation and other development disabilities.
Tumor genetics Cancer can be aptly described as a disease resultant of genomic instability and given that CGH is designed to identify segmental genomic alterations, it is an appropriate tool to study the genetic basis of cancer and the chromosomal anomalies that are associated with it. Advances in array CGH technologies have made it possible to examine chromosomal regions with extraordinary detail and has revolutionized our understanding of the tumor genome. Several array-based technologies that are being developed to further improve the resolution of CGH will enable researches to identify and analyze that is genomic regions responsible for cancer proliferation and facilitate in rapid gene discovery.
Take this case for instance, the p53 tumor suppressor gene has been explored as a target for gene therapy in ovarian cancer, the idea is to HER2/neu/erB2 for antibody-mediated therapy (Herceptin) and E1 A-mediated gene therapy. In the study conducted by Kudoh et al. , they were able to show that the increased presence of copy number at 1q21 and 13q13 correlated to the lack of response to the chemotherapy regimen of doxorubicin, cisplatin and cyclophosphamide, so it is possible to identify and characterize the genes driving the copy number abnormalities using CGH which could lead to new therapeutic targets in ovarian cancer and possibly disturb tumor cell growth or even change the sensitivity to chemotherapy.
Chromosome specific microarraysIt is also possible to design microarray that are specific to a single chromosome or a chromosome arm. For instance, using a chromosome 20 array with 22 cosmid, P1 phage artificial chromosome (PAC) and bacterial artificial chromosome (BAC) clones as intervals markers covering chromosome 20 at 3 Mb resolution, by using this array to study breast cancer it detected SeGAs in multiple regions suggesting that by using a higher density array it would be possible to gain more insight into the complex chromosomal alterations in cancer genomes than previously thought. This is yet another instance where CGH has helped identify novel techniques to understand the basis of a genetic condition. Genome-wide approach CGH microarrays are usually used regional and chromosomal anomalies which has given us a great deal of information, these studies are limited by the fact that they require knowledge of the regions of interest and the studies are isolated to specific regions of interest, therefore to overcome this short-coming genome-wide arrays were developed. Pollack et al. (1999) used a cDNA microarray representing 3195 unique cDNA target clones distributed throughout the genome. This study was the first genome-wide profiling array for human cancer genomes pointing out regions of alterations in breast cancer. Genome-wide array CGH using cDNA arrays resulted in significant leaps in the field of cancer genomics.
The scope of CGH is not limited to study genetic basis of adverse phenotypes, for example, Cancer, comparative genomic hybridization has also proved useful in clinical diagnostic services. Using CGH it is possible to identify and characterize chromosomal deletions, duplications, marker chromosomes and unbalanced translocation in prenatal, postnatal and preimplantation sampled. CGH has also been used to revise incorrectly assigned karyotypes. CGH has the ability to precisely define the chromosomal material containing unbalanced translocations and marker chromosomes, this has helped associate critical regions of the chromosome with the respective adverse phenotypic outcomes. This prognostic information is used for genetic counselling and has proved beneficial to couples to make a more informed decision about pregnancy.
Another area that CGH has proved to be a noteworthy tool is in advancing molecular cytogenetics for evaluating mental retardation (Xu et al. , 2002) as mention earlier, CGH analysis is used to further the characterization of unbalanced translocations identified using banding analysis and also screening for “hidden” chromosome anomalies in patients. Figure 2: Array CGH, FISH and G-banging image. The image shows application of molecular cytogenetic tools to detect chromosomal abnormalities in the case of a patient with severe MR, presenting with several dysmorphic facial features prenatal growth deficiency, severe epilepsy, cleft palate, hirsutism, camptodactyly, and syndactyly. ( With the data for the above image a deletion spanning 12 clones with an estimated size of 10 Mb was identified, located at the chromosome band 2q24–31.
Techniques and technologies for chromosomal analysis has greatly improved over the years with the advent of modern cytogenetics, this has led to advancements in clinical diagnostics but also has provided researchers and clinicians with markers to assess prognosis and disease progression. The reason for discussing comparative genomic hybridization at length in this essay is because CGH could possibly be the most significant modern cytogenetic method to have overcome most of the limitations of the traditional cytogenetic methods faced and even then full potential of CGH has not been completely explored.
CGH has proved to a powerful tool in detecting deletions, duplications and amplifications contributing to neoplastic transformations as well as defining chromosomal locations of oncogenes and tumor suppressor genes that are pivotal to development and/or progression of tumors. At this point it is also important to note that there limitations to even CGH, just like any other cytogenetic methodology or technology. CGH is unable to detect balanced chromosomal anomalies such as point mutations, translocations or inversions, in additions to this telomeric, pericentromeric, heterochromatic regions cannot be assessed (Knuutila et al. , 2000). In order for chromosomal imbalances to be detected they most be present in 50% of the cells, so in order to study tumor samples they have to be relatively free of normal cells(Zitzelsberger et al. , 1997). CGH is able to recognize just proportional changes in copy number, the ratio profiles does not recognize the absolute copy-number change. Information on recurring chromosomal anomalies in solid tumors in some hematological cancer is still limited. However with the development of microarray CGH (M-CGH) the limitations faced in classical CGH can be over. Genomic imbalances can be detected with higher resolution also allowing copy-number changes to be associated with individual loci and genomic markers.
The arrival of new FISH-based technologies such as multiplex FISH (M-FISH), comparative genomic hybridization analysis, microarray- CGH (M-CGH), spectral karyotyping (SKY), reverse FISH have managed to fill in the void between conventional cytogenetics and molecular genetics. By combining molecule genetics with traditional cytogenetics, modern cytogenetics has helped unlock a new dimension into understanding disease prognosis, progression and diagnostics, therefore providing powerful tools for research and clinical diagnostic service.
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