The Breakthrough Generations Study and Laboratory was set up in 2007 to study the epigenetics of breast cancer in women and men in the United Kingdom over a potential forty year prospective period from 2006 to 2046. The remit was to collect blood samples, lifestyle and medical information from volunteers and then to compare the genetic analysis of the samples to the data collected to look for trends and patterns. The blood samples were stored in a relatively unique format, being in 500 μl buffy coat samples in micro-capillary tubes and at the time of writing 4 million aliquots were in storage. This project had two main aims. Firstly to develop a modified micro-column genomic DNA extraction method that could process the frozen buffy coat samples, and process a high throughput of sample numbers in any given extraction batch. Secondly to use this validated method to extract DNA from male breast cancer patient samples and thereafter genotype these for the FGFR2 single nucleotide polymorphism which has been shown to have a high association with breast cancer in women. Commercially available genomic deoxyribonucleic acid (gDNA) extraction micro-column kits were modified, by altering incubation times and reagent volumes in stages and tested to validate yield and reproducibility, to allow for a greater sample through put than normally processed at one time. In addition, a purpose-designed centrifuge rack dual-level multi tube holder was developed and tested to allow for 96 samples to be processed in one batch. This allowed for the uniquely stored Generations Study samples to be processed, and for the high sample throughput required of such a large study, where batches of 20,000 were often processed at any given time. Male breast cancer blood samples and controls, to a total of 900 samples, were processed using the validated DNA extraction method and the resultant gDNA was genotyped by Taqman™ analysis for the FGFR2 point mutation or SNP at position 116,968,271 in chromosome 10q26. Relative Odds Ratios (OR) for the heterozygote mutant allele were 1.012 and for the homozygote allele were 1.289 – the p-values for these results, however, showed a lack of statistical significance. To ensure this was not due to lack of sample size or power, an additional 1,148 samples were acquired from UK Genetic Lung Cancer Predisposition Study and genotyped with statistically significant OR of 1.06 and 1.43 for homozygote and heterozygote mutations respectively, where Fisher exact test gave p-value of 0.03. This indicated that the FGFR2 SNP has a positive risk association in men, as it has been shown to have in women. The developed extraction method will allow the processing of further samples in high numbers, to genotype further SNPs in male cases. It may provide a direct comparison to the genetic risk association of breast cancer in men to women. This initial set of results for the FGFR2 SNP is the first step in the epigenetic analysis of breast cancer in men in the UK, and the start of the comparison of the epigenetics of breast cancer across the sexes.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:658653 |
| Date | January 2014 |
| Creators | Chilcott-Burns, Sarah |
| Contributors | Mills, Graham ; Dewey, Ann |
| Publisher | University of Portsmouth |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | https://researchportal.port.ac.uk/portal/en/theses/association-of-single-nucleotide-polymorphisms-in-fgfr2-with-male-breast-cancer-following-modified-dna-extraction-methodology(25ace0dd-234b-4e30-94e6-834f81ca5198).html |
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