Development and Application of Comparative Genomic Hybridisation (CGH)

Genetics, Bioinformatics

Ghaffari, Saeed R

University:University of Glasgow

关键词: Genetics, Bioinformatics;   

Others  :  http://theses.gla.ac.uk/74793/1/11007712.pdf

来源: University of Glasgow

【 摘 要 】

The aims of this study were first, to optimise the comparative genomic hybridisation (CGH) technique and test its reliability as a diagnostic tool in a clinical genetics laboratory, second, to investigate whether any of the current limitations could be overcome, and third, to improve the resolution of the technique beyond the current limits by targeting DNA molecules instead of metaphase chromosomes. During the course of this study, a modified approach to CGH was developed and tested on samples with known and unknown abnormalities in various fields of clinical cytogenetics. The technique was used to detect unbalanced abnormalities in patients with common constitutional abnormalities, marker chromosomes, mosaicism, haematological malignancies and solid tumours. In all fields CGH detected some abnormalities which had remained undetected using conventional cytogenetics or molecular cytogenetic techniques. During the optimisation 94 samples were used. Samples with known karyotypes were the subject of CGH using different conditions of slide preparation, slide treatment, DNA isolation (from different samples including fixed cells) DNA labelling, hybridisation, detection and washing approaches. Some modifications in the standard procedures were adopted and then the modified approach was applied to 56 cases with different abnormalities. Overnight CGH was successfully developed and applied during this study. Nineteen experiments using 10 test DNAs all correctly detected the abnormalities in the cases. 10 samples were the subject of CGH using samples amplified by degenerate oligonucletides primers PCR (DOP-PCR). CGH reliably detected unbalanced abnormalities in the affected cases using as little as 1 ng of DNA. CGH was applied on 14 samples from patients with haematological malignancies. In 2 cases CGH did not detect the abnormality found by conventional cytogenetics because of the balanced nature of the rearrangements. Both CGH and conventional cytogenetics found no abnormality in 5 samples. In 3 abnormal cases CGH and G-banding results were compatible. In 3 patients, CGH detected abnormalities missed by routine cytogenetic analysis. These abnormalities were detected in mosaicism of as low as 30% of abnormal cells. CGH detected new abnormalities that were not detected by conventional cytogenetics in a case of follicular lymphoma. A new site of DNA amplification at chromosome 3p26 was also detected. A new method for defining the copy number differences in cases with ploidy changes was suggested and was successfully applied in a case with hypotriploidy. 13 mentally handicapped patients with normal karyotypes without any clinical clue to a specific syndrome were the subjects of CGH at the next stage of this study. Using a new approach CGH detected telomeric gains and losses in 5 patients from 3 families. Those abnormalities were then confirmed using targeted FISH. Balanced carriers of the translocations in three generations were then detected using targeted FISH. Comparative genomic hybridisation revealed the origin of a mosaic supernumerary ring chromosome 19 in a 72 year-old woman with mental retardation. That abnormality could not be identified by conventional banding methods over many years and, more recently, fluorescence in situ hybridisation (FISH) studies which systematically employed different chromosome probes. In a female with mental retardation and a putative iXp, CGH ruled out that diagnosis by detecting the presence of Xq in the DNA material from the patient. CGH then reliably defined the abnormality as an Xp;Xq translocation. To improve the resolution of the CGH technique combed DNA fibres instead of metaphase chromosomes were used as the target DNA for the hybridisation of specific probes. CGH was then successfully performed on high-density DNA fibres of 35 kb nucleotides. At the final stage of this study CGH was performed on spots of single stranded DNA arrays of ~300-500 bp corresponding to chromosomes X, Y, and chromosome 2 on glass slides. Preliminary results showed that this approach could differentiate the copy number differences using a statistical analysis. This study shows that CGH is a powerful adjunct to the current techniques in clinical cytogenetics for detection of unbalanced abnormalities in the various fields of clinical genetics. This study also presents several modifications in CGH technique and image analysis which improve the resolution and the reliability of the technique and make it faster and less complex. It also provides an opportunity for future studies for CGH on DNA microarray targets that would be a very important step towards the complete automation of the cytogenetics.