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Allele-specific up-regulation of FGFR2 increases susceptibility to breast cancer.

Meyer KB, Maia AT, O'Reilly M, Teschendorff AE, Chin SF, Caldas C, Ponder BA - PLoS Biol. (2008)

Bottom Line: This trend was confirmed using real-time (RT) PCR, with the difference between the rare and the common homozygotes yielding a Wilcox p-value of 0.028.In transient transfection experiments, the two SNPs can synergize giving rise to increased FGFR2 expression.We propose a model in which the Oct-1/Runx2 and C/EBPbeta binding sites in the disease-associated allele are able to lead to an increase in FGFR2 gene expression, thereby increasing the propensity for tumour formation.

View Article: PubMed Central - PubMed

Affiliation: Cancer Research UK, Cambridge Research Institute, Li Ka Shing Centre, Cambridge, United Kingdom. Kerstin.Meyer@cancer.org.uk

ABSTRACT
The recent whole-genome scan for breast cancer has revealed the FGFR2 (fibroblast growth factor receptor 2) gene as a locus associated with a small, but highly significant, increase in the risk of developing breast cancer. Using fine-scale genetic mapping of the region, it has been possible to narrow the causative locus to a haplotype of eight strongly linked single nucleotide polymorphisms (SNPs) spanning a region of 7.5 kilobases (kb) in the second intron of the FGFR2 gene. Here we describe a functional analysis to define the causative SNP, and we propose a model for a disease mechanism. Using gene expression microarray data, we observed a trend of increased FGFR2 expression in the rare homozygotes. This trend was confirmed using real-time (RT) PCR, with the difference between the rare and the common homozygotes yielding a Wilcox p-value of 0.028. To elucidate which SNPs might be responsible for this difference, we examined protein-DNA interactions for the eight most strongly disease-associated SNPs in different breast cell lines. We identify two cis-regulatory SNPs that alter binding affinity for transcription factors Oct-1/Runx2 and C/EBPbeta, and we demonstrate that both sites are occupied in vivo. In transient transfection experiments, the two SNPs can synergize giving rise to increased FGFR2 expression. We propose a model in which the Oct-1/Runx2 and C/EBPbeta binding sites in the disease-associated allele are able to lead to an increase in FGFR2 gene expression, thereby increasing the propensity for tumour formation.

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Diagram of the FGFR2 GeneGenetic linkage is taken from HapMap, and the positions of the eight candidate SNPs (red lines) within intron 2 are indicated. The two SNPs for which data are presented in this study are shown in green. Red circle: original tagging SNP rs2981582.
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pbio-0060108-g001: Diagram of the FGFR2 GeneGenetic linkage is taken from HapMap, and the positions of the eight candidate SNPs (red lines) within intron 2 are indicated. The two SNPs for which data are presented in this study are shown in green. Red circle: original tagging SNP rs2981582.

Mentions: Two independent studies have identified FGFR2 as risk factor in breast cancer [10,11]. We have shown that in Europeans, the minor disease-predisposing allele of FGFR2 is inherited as a haplotype of eight single nucleotide polymorphisms (SNPs) covering a region of 7.5 kb within intron 2 of the gene [10] (Figure 1), in a haplotype block with no linkage disequilibrium with the coding region of the gene. Microarray gene expression analysis on the Nottingham City Hospital cohort, using both the Agilent [12] and the Illumina [13] platforms, indicated that FGFR2 is expressed at higher levels by tumours that are homozygous for the minor alleles than by those with the common alleles (Wilcox p < 0.05). Analysed tumours were all diploid for this region based on array-comparative genome hybridization data [14]. This correlation was independent of either ER expression or p53 mutation status of the cells. Quantitative TaqMan PCR analysis confirmed a significant increase in FGFR2 expression in rare homozygotes, as compared to common homozygotes (Wilcox p = 0.028) (Figure 2). We also examined expression of the FGFR2 ligands FGF-7, FGF-10, and FGF-22, which are usually produced by the surrounding stroma, in 45 normal breast samples as well as the microarray data on tumours, but we found no correlation with genotype. Furthermore, FGFR2 displays a very complex splicing pattern with the most commonly expressed variants of the N terminus of the gene either including exons 1, 2, and 3 or including exons 1 and 2, but lacking exon 3. Again, no correlation was observed between genotype and the presence or absence of exon 3. Thus, the risk genotype correlates with FGFR2 expression itself, rather than affecting its function through receptor-ligand interactions.


Allele-specific up-regulation of FGFR2 increases susceptibility to breast cancer.

Meyer KB, Maia AT, O'Reilly M, Teschendorff AE, Chin SF, Caldas C, Ponder BA - PLoS Biol. (2008)

Diagram of the FGFR2 GeneGenetic linkage is taken from HapMap, and the positions of the eight candidate SNPs (red lines) within intron 2 are indicated. The two SNPs for which data are presented in this study are shown in green. Red circle: original tagging SNP rs2981582.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2365982&req=5

pbio-0060108-g001: Diagram of the FGFR2 GeneGenetic linkage is taken from HapMap, and the positions of the eight candidate SNPs (red lines) within intron 2 are indicated. The two SNPs for which data are presented in this study are shown in green. Red circle: original tagging SNP rs2981582.
Mentions: Two independent studies have identified FGFR2 as risk factor in breast cancer [10,11]. We have shown that in Europeans, the minor disease-predisposing allele of FGFR2 is inherited as a haplotype of eight single nucleotide polymorphisms (SNPs) covering a region of 7.5 kb within intron 2 of the gene [10] (Figure 1), in a haplotype block with no linkage disequilibrium with the coding region of the gene. Microarray gene expression analysis on the Nottingham City Hospital cohort, using both the Agilent [12] and the Illumina [13] platforms, indicated that FGFR2 is expressed at higher levels by tumours that are homozygous for the minor alleles than by those with the common alleles (Wilcox p < 0.05). Analysed tumours were all diploid for this region based on array-comparative genome hybridization data [14]. This correlation was independent of either ER expression or p53 mutation status of the cells. Quantitative TaqMan PCR analysis confirmed a significant increase in FGFR2 expression in rare homozygotes, as compared to common homozygotes (Wilcox p = 0.028) (Figure 2). We also examined expression of the FGFR2 ligands FGF-7, FGF-10, and FGF-22, which are usually produced by the surrounding stroma, in 45 normal breast samples as well as the microarray data on tumours, but we found no correlation with genotype. Furthermore, FGFR2 displays a very complex splicing pattern with the most commonly expressed variants of the N terminus of the gene either including exons 1, 2, and 3 or including exons 1 and 2, but lacking exon 3. Again, no correlation was observed between genotype and the presence or absence of exon 3. Thus, the risk genotype correlates with FGFR2 expression itself, rather than affecting its function through receptor-ligand interactions.

Bottom Line: This trend was confirmed using real-time (RT) PCR, with the difference between the rare and the common homozygotes yielding a Wilcox p-value of 0.028.In transient transfection experiments, the two SNPs can synergize giving rise to increased FGFR2 expression.We propose a model in which the Oct-1/Runx2 and C/EBPbeta binding sites in the disease-associated allele are able to lead to an increase in FGFR2 gene expression, thereby increasing the propensity for tumour formation.

View Article: PubMed Central - PubMed

Affiliation: Cancer Research UK, Cambridge Research Institute, Li Ka Shing Centre, Cambridge, United Kingdom. Kerstin.Meyer@cancer.org.uk

ABSTRACT
The recent whole-genome scan for breast cancer has revealed the FGFR2 (fibroblast growth factor receptor 2) gene as a locus associated with a small, but highly significant, increase in the risk of developing breast cancer. Using fine-scale genetic mapping of the region, it has been possible to narrow the causative locus to a haplotype of eight strongly linked single nucleotide polymorphisms (SNPs) spanning a region of 7.5 kilobases (kb) in the second intron of the FGFR2 gene. Here we describe a functional analysis to define the causative SNP, and we propose a model for a disease mechanism. Using gene expression microarray data, we observed a trend of increased FGFR2 expression in the rare homozygotes. This trend was confirmed using real-time (RT) PCR, with the difference between the rare and the common homozygotes yielding a Wilcox p-value of 0.028. To elucidate which SNPs might be responsible for this difference, we examined protein-DNA interactions for the eight most strongly disease-associated SNPs in different breast cell lines. We identify two cis-regulatory SNPs that alter binding affinity for transcription factors Oct-1/Runx2 and C/EBPbeta, and we demonstrate that both sites are occupied in vivo. In transient transfection experiments, the two SNPs can synergize giving rise to increased FGFR2 expression. We propose a model in which the Oct-1/Runx2 and C/EBPbeta binding sites in the disease-associated allele are able to lead to an increase in FGFR2 gene expression, thereby increasing the propensity for tumour formation.

Show MeSH
Related in: MedlinePlus