| MICROARRAY
GENE EXPRESSION IN CELLS WITH A MUTATION IN THE HIGH RISK PROSTATE
CANCER-PREPOSITION GENE, BRAC2; STUDIES TO ASSESS RESPONSES TO
IRRADIATION.
Zsofia Kote-Jarai, Sarah Jugurnauth and Rosalind Eeles Institute of Cancer Research |
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Summary final report – Project 2004/03 – Zsofia Kote–Jarai
Aims of this study:
1. To determine if microarray expression profiling after irradiation can distinguish between normal cells from male BRCA1 and BRCA2 mutation carriers and non‑carriers.
2. To determine if the assays above can be used to distinguish between pathogenic mutations from non‑pathogenic variants in these genes.
3. To determine if there are any differences in (1) and (2) between BRCA1 and BRCA2 which may account for the differences in prostate cancer risk profiles between these genes when mutated.
At the Institute of Cancer Research and Royal Marsden NHS Foundation Trust, we have established a unique multidisciplinary clinic for the management of cancer predisposition gene carriers, which follows up 350 BRCA1/2 gene carriers. Via this clinic, and throughout the UK, we have identified 59 Male carriers of BRCA1 and BRCA2 mutations who have an increased risk of developing prostate cancer and who would be available for this study.
Sample collection:
We have collected fresh blood samples from 10 BRCA1, 10 BRAC2 mutation carriers and 10 control individuals. The controls were tested negative for known BRCA1 or BRCA2 mutations in their families. This is the sample set which is needed for this study. In addition we have also collected 1 individual with a VUS (Variant of Uncertain Significance) mutation; the collection of these is still ongoing (see details in Table 1). Lymphocytes have been separated from all the blood samples within 24 hours using Lymphoprep and short term cultures have been established. Lymphocytes have been cultured in complete RPM1 medium for 5‑6 days and than treated with 8 Gy high dose X‑ray irradiation (1.5 Gy/min) to induce DNA damage. Total RNA was isolated from the cells 1 hour after this treatment using a Qiagen RNeasy kit. The concentration of the RNA samples was determined and the RNAs are being stored at –800C until the labelling procedure (see below). In addition lymphocytes from all individuals have been cryo‑preserved and are stored in liquid nitrogen (‑1960C) for further studies.
Gene expression profiling:
For this study we have started to use the 30 K Genome‑wide spotted cDNA arrays provided by the Cancer Research UK microarray facility at our Institute. RNA (5ug) from the samples above and Universal Human Reference RNA (Stratagene) has been labelled using Cy3 and Cy5 fluorescent dyes in a reverse transcription reaction. Each Cy3 labelled sample RNA is then mixed with an equal amount of Cy5 labelled reference RNA and co‑hybridized onto the microarray slides. After washing, the slides are scanned using the GenePix 4000B (Axon Instruments) scanner and image analysis is done by the GenePix Pro 5 software.
Results
We have analysed the expression profiles of lymphocyte cultures from 10 BRCA1 and 10 BRCA2 male mutation carriers and compared these with the profiles of 10 non-carrier control samples. All these samples were short-term primary cultures established from fresh blood samples. Cell cultures were irradiated (8 Gy) to induce DNA damage and the expression profiles of all 30 samples were analysed.
In the comparison of the BRCA1 mutation carriers and non-carriers, using statistical analysis we identified 737 genes, of which we were able to identify 56 statistically significant genes which discriminate between these two classes.
Similarly in a significance analysis of the BRCA2 carriers and non-carriers we have identified 575 genes, with 48 statistically significant genes discriminating the samples by their genotype.
If we compared the expression profile of the BRCA1 carriers with the BRCA2 carriers we could also identify significantly differently expressed genes. 534 genes were selected, and 59 genes were identified. The Hierarchical Clustering diagrams relating to the above are available in the full report.
The significant genes involved included transcription factors, cell cycle regulators, cytokines and putative tumour suppressor genes. Some of these might provide targets for treatments for these individuals who are at an increased prostate cancer risk due to a genetic predisposition.
We can conclude that gene expression profiling after induced DNA damage can be used to distinguish the lymphocytes from BRCA1 and BRCA2 mutation carriers from controls and from each other, in men. We have been able to produced HC analyses which show a difference in the profiling but we will also carry out further in-depth analysis of these data with our collaborator, Colin Campbell, at the Bristol University Computational Intelligence Unit.
Supervised and unsupervised class predictions and statistical validations are being planned to identify the most significant targets from these results for potential candidates for the cause of susceptibility to the disease and differential effects from radiation.
A paper will be submitted in the summer of 2007.We should like to thank Prostate Research Campaign UK for their support.
Summary of the final report dated 31 January 2007
Project 2004/03