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Loss of expression and promoter methylation of SLIT2 are associated with sessile serrated adenoma formation.

Beggs AD, Jones A, Shepherd N, Arnaout A, Finlayson C, Abulafi AM, Morton DG, Matthews GM, Hodgson SV, Tomlinson IP - PLoS Genet. (2013)

Bottom Line: An initial panel of 9 sessile serrated adenomas (SSA) and one TSA were analysed using Illumina Goldengate HumanLinkage panel arrays to ascertain regions of loss of heterozygosity.Genes of interest in this region were PDCH7 and SLIT2, and combined MSP/IHC analysis of these genes revealed significant loss of SLIT2 expression associated with promoter methylation of SLIT2.Loss of expression of SLIT2 by promoter hypermethylation and loss of heterozygosity events is significantly associated with serrated adenoma development, and SLIT2 may represent a epimutated tumour suppressor gene according to the Knudson "two hit" hypothesis.

View Article: PubMed Central - PubMed

Affiliation: Molecular and Population Genetics Laboratory, Wellcome Trust Centre for Human Genetics, University of Oxford, Oxford, United Kingdom. abeggs@well.ox.ac.uk

ABSTRACT
Serrated adenomas form a distinct subtype of colorectal pre-malignant lesions that may progress to malignancy along a different molecular pathway than the conventional adenoma-carcinoma pathway. Previous studies have hypothesised that BRAF mutation and promoter hypermethylation plays a role, but the evidence for this is not robust. We aimed to carry out a whole-genome loss of heterozygosity analysis, followed by targeted promoter methylation and expression analysis to identify potential pathways in serrated adenomas. An initial panel of 9 sessile serrated adenomas (SSA) and one TSA were analysed using Illumina Goldengate HumanLinkage panel arrays to ascertain regions of loss of heterozygosity. This was verified via molecular inversion probe analysis and microsatellite analysis of a further 32 samples. Methylation analysis of genes of interest was carried out using methylation specific PCR (verified by pyrosequencing) and immunohistochemistry used to correlate loss of expression of genes of interest. All experiments used adenoma samples and normal tissue samples as control. SSA samples were found on whole-genome analysis to have consistent loss of heterozygosity at 4p15.1-4p15.31, which was not found in the sole TSA, adenomas, or normal tissues. Genes of interest in this region were PDCH7 and SLIT2, and combined MSP/IHC analysis of these genes revealed significant loss of SLIT2 expression associated with promoter methylation of SLIT2. Loss of expression of SLIT2 by promoter hypermethylation and loss of heterozygosity events is significantly associated with serrated adenoma development, and SLIT2 may represent a epimutated tumour suppressor gene according to the Knudson "two hit" hypothesis.

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Photomicrographs.(a) Photomicrographs of IHC staining for PCDH7 and SLIT2 in normal colonic mucosa, colonic adenoma and serrated adenoma tissue. Expression of both PCDH7 and SLIT2 are shown by brown staining in the image. (b) 5×, 10× and 20× photomicrographs of SLIT2 & PCDH7 expression in serrated adenomas. Expression of both PCDH7 and SLIT2 are shown by brown staining in the image.
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pgen-1003488-g004: Photomicrographs.(a) Photomicrographs of IHC staining for PCDH7 and SLIT2 in normal colonic mucosa, colonic adenoma and serrated adenoma tissue. Expression of both PCDH7 and SLIT2 are shown by brown staining in the image. (b) 5×, 10× and 20× photomicrographs of SLIT2 & PCDH7 expression in serrated adenomas. Expression of both PCDH7 and SLIT2 are shown by brown staining in the image.

Mentions: In Figure 4a (5× magnification) & 4b (5×, 10×, 20× magnification), normal expression of SLIT2 can be seen in both the conventional adenoma and normal tissue, whereas there is complete loss of expression in the serrated adenoma. A particularly interesting result was found in a polyp with mixed adenomatous/serrated architecture, as demonstrated in Figure 5. In this image, there was very reduced expression of SLIT2 within the crypts with serrated morphology but normal expression of SLIT2 within the crypts with “classical” adenomatous morphology.


Loss of expression and promoter methylation of SLIT2 are associated with sessile serrated adenoma formation.

Beggs AD, Jones A, Shepherd N, Arnaout A, Finlayson C, Abulafi AM, Morton DG, Matthews GM, Hodgson SV, Tomlinson IP - PLoS Genet. (2013)

Photomicrographs.(a) Photomicrographs of IHC staining for PCDH7 and SLIT2 in normal colonic mucosa, colonic adenoma and serrated adenoma tissue. Expression of both PCDH7 and SLIT2 are shown by brown staining in the image. (b) 5×, 10× and 20× photomicrographs of SLIT2 & PCDH7 expression in serrated adenomas. Expression of both PCDH7 and SLIT2 are shown by brown staining in the image.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3649993&req=5

pgen-1003488-g004: Photomicrographs.(a) Photomicrographs of IHC staining for PCDH7 and SLIT2 in normal colonic mucosa, colonic adenoma and serrated adenoma tissue. Expression of both PCDH7 and SLIT2 are shown by brown staining in the image. (b) 5×, 10× and 20× photomicrographs of SLIT2 & PCDH7 expression in serrated adenomas. Expression of both PCDH7 and SLIT2 are shown by brown staining in the image.
Mentions: In Figure 4a (5× magnification) & 4b (5×, 10×, 20× magnification), normal expression of SLIT2 can be seen in both the conventional adenoma and normal tissue, whereas there is complete loss of expression in the serrated adenoma. A particularly interesting result was found in a polyp with mixed adenomatous/serrated architecture, as demonstrated in Figure 5. In this image, there was very reduced expression of SLIT2 within the crypts with serrated morphology but normal expression of SLIT2 within the crypts with “classical” adenomatous morphology.

Bottom Line: An initial panel of 9 sessile serrated adenomas (SSA) and one TSA were analysed using Illumina Goldengate HumanLinkage panel arrays to ascertain regions of loss of heterozygosity.Genes of interest in this region were PDCH7 and SLIT2, and combined MSP/IHC analysis of these genes revealed significant loss of SLIT2 expression associated with promoter methylation of SLIT2.Loss of expression of SLIT2 by promoter hypermethylation and loss of heterozygosity events is significantly associated with serrated adenoma development, and SLIT2 may represent a epimutated tumour suppressor gene according to the Knudson "two hit" hypothesis.

View Article: PubMed Central - PubMed

Affiliation: Molecular and Population Genetics Laboratory, Wellcome Trust Centre for Human Genetics, University of Oxford, Oxford, United Kingdom. abeggs@well.ox.ac.uk

ABSTRACT
Serrated adenomas form a distinct subtype of colorectal pre-malignant lesions that may progress to malignancy along a different molecular pathway than the conventional adenoma-carcinoma pathway. Previous studies have hypothesised that BRAF mutation and promoter hypermethylation plays a role, but the evidence for this is not robust. We aimed to carry out a whole-genome loss of heterozygosity analysis, followed by targeted promoter methylation and expression analysis to identify potential pathways in serrated adenomas. An initial panel of 9 sessile serrated adenomas (SSA) and one TSA were analysed using Illumina Goldengate HumanLinkage panel arrays to ascertain regions of loss of heterozygosity. This was verified via molecular inversion probe analysis and microsatellite analysis of a further 32 samples. Methylation analysis of genes of interest was carried out using methylation specific PCR (verified by pyrosequencing) and immunohistochemistry used to correlate loss of expression of genes of interest. All experiments used adenoma samples and normal tissue samples as control. SSA samples were found on whole-genome analysis to have consistent loss of heterozygosity at 4p15.1-4p15.31, which was not found in the sole TSA, adenomas, or normal tissues. Genes of interest in this region were PDCH7 and SLIT2, and combined MSP/IHC analysis of these genes revealed significant loss of SLIT2 expression associated with promoter methylation of SLIT2. Loss of expression of SLIT2 by promoter hypermethylation and loss of heterozygosity events is significantly associated with serrated adenoma development, and SLIT2 may represent a epimutated tumour suppressor gene according to the Knudson "two hit" hypothesis.

Show MeSH
Related in: MedlinePlus