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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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Example gel image of PCDH7 promoter methylation-specific PCR (top gel) and SLIT2 methylation-specific PCR (bottom and middle gels).MSP reactions row refers to wells for the methylated and unmethylated reactions for the methylation specific PCR. Methylation status refers to the final determination of methylation within each serrated adenoma sample. Row key: MSP reactions – M = methylation specific reaction, U = unmethylated specific reaction; Methylation status – M = methylated, U = unmethylated.
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pgen-1003488-g003: Example gel image of PCDH7 promoter methylation-specific PCR (top gel) and SLIT2 methylation-specific PCR (bottom and middle gels).MSP reactions row refers to wells for the methylated and unmethylated reactions for the methylation specific PCR. Methylation status refers to the final determination of methylation within each serrated adenoma sample. Row key: MSP reactions – M = methylation specific reaction, U = unmethylated specific reaction; Methylation status – M = methylated, U = unmethylated.

Mentions: Methylation specific PCR of the PCDH7 and SLIT2 promoter regions was carried out in the verification group only. No further examination of other gene specific promoter regions in this area (i.e. GPR125,PACRGL,DHX15,SOD3 or RBPJ) was carried out as a visual examination of the ENCODE DNA methylation tracks in the UCSC Genome Browser did not suggest any long range methylation in this region. For PCDH7, 12/32 (37.5%) SAs had methylation (12/29 SSAs, 0/1 mixed SSA/TSA, 0/2 TSA), whereas none of the classical adenoma (0/10) samples and 1/5 (20%) of the normal mucosa samples were methylated (Figure 2). For SLIT2, 31/32 (96.9%) SAs had methylation (29/29 SSA's, 1/1 mixed TSA/SSA, and 0/2 TSAs). There was no methylation in the adenoma (0/10) or normal mucosa samples (0/5) (Figure 3). No methylation analysis of additional normal mucosa samples was carried out at this point as the frequency of methylation observed in SLIT2/PCDH7 was so low it was thought unlikely to add any additional information. The MSP result was verified in a limited subset of samples using pyrosequencing. Five CpG dinucleotides were interrogated in the pyrosequencing PCR, based on the coverage of the MSP, and abnormal methylation was defined as a methylation percentage greater than two times the standard deviation of the average methylation in normal mucosa. It was found that in SSAs, there was consistent and informative promoter methylation at the 2nd, 3rd and 4th CpG's within the MSP region (Table 2). There was no hypermethylation in a caecal polyp, nor in normal mucosa. As part of a separate experiment conducted in the laboratory, it was found that there was hypermethylation in SLIT2 within a panel of cell lines (Table 2). Using a separate pyrosequencing primer for SLIT2 (PM00018634) , the SSA were checked for long range methylation in the serrated adenomas further down the CpG island. There was no methylation seen (Table 2).


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)

Example gel image of PCDH7 promoter methylation-specific PCR (top gel) and SLIT2 methylation-specific PCR (bottom and middle gels).MSP reactions row refers to wells for the methylated and unmethylated reactions for the methylation specific PCR. Methylation status refers to the final determination of methylation within each serrated adenoma sample. Row key: MSP reactions – M = methylation specific reaction, U = unmethylated specific reaction; Methylation status – M = methylated, U = unmethylated.
© Copyright Policy
Related In: Results  -  Collection

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

pgen-1003488-g003: Example gel image of PCDH7 promoter methylation-specific PCR (top gel) and SLIT2 methylation-specific PCR (bottom and middle gels).MSP reactions row refers to wells for the methylated and unmethylated reactions for the methylation specific PCR. Methylation status refers to the final determination of methylation within each serrated adenoma sample. Row key: MSP reactions – M = methylation specific reaction, U = unmethylated specific reaction; Methylation status – M = methylated, U = unmethylated.
Mentions: Methylation specific PCR of the PCDH7 and SLIT2 promoter regions was carried out in the verification group only. No further examination of other gene specific promoter regions in this area (i.e. GPR125,PACRGL,DHX15,SOD3 or RBPJ) was carried out as a visual examination of the ENCODE DNA methylation tracks in the UCSC Genome Browser did not suggest any long range methylation in this region. For PCDH7, 12/32 (37.5%) SAs had methylation (12/29 SSAs, 0/1 mixed SSA/TSA, 0/2 TSA), whereas none of the classical adenoma (0/10) samples and 1/5 (20%) of the normal mucosa samples were methylated (Figure 2). For SLIT2, 31/32 (96.9%) SAs had methylation (29/29 SSA's, 1/1 mixed TSA/SSA, and 0/2 TSAs). There was no methylation in the adenoma (0/10) or normal mucosa samples (0/5) (Figure 3). No methylation analysis of additional normal mucosa samples was carried out at this point as the frequency of methylation observed in SLIT2/PCDH7 was so low it was thought unlikely to add any additional information. The MSP result was verified in a limited subset of samples using pyrosequencing. Five CpG dinucleotides were interrogated in the pyrosequencing PCR, based on the coverage of the MSP, and abnormal methylation was defined as a methylation percentage greater than two times the standard deviation of the average methylation in normal mucosa. It was found that in SSAs, there was consistent and informative promoter methylation at the 2nd, 3rd and 4th CpG's within the MSP region (Table 2). There was no hypermethylation in a caecal polyp, nor in normal mucosa. As part of a separate experiment conducted in the laboratory, it was found that there was hypermethylation in SLIT2 within a panel of cell lines (Table 2). Using a separate pyrosequencing primer for SLIT2 (PM00018634) , the SSA were checked for long range methylation in the serrated adenomas further down the CpG island. There was no methylation seen (Table 2).

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