Table 1.
Mutations known for each sample are listed. SSP-1 to SSP-8 are exome sequenced. Mutations in other samples are tested by OncoCarta3. BRAF-V600E mutation and KRAS codon 12 and 13 mutations are tested by PCR-sequencing in all samples.
Fig 1.
Hierarchical clustering of all CpGs in 450K methyl-array on colon samples.
The SSP samples are clustered together based on DNA methylation. The TSA-2 sample that is mutant for BRAF gene also clustered with SSPs.
Fig 2.
Global methylation changes in SSP and FAP.
(a) Comparison of FAP with normal samples shows only few DNA methylation changes. From 395,899 CpGs tested on the Infinium array, only 52 CpGs show more than 2 fold increase in methylation in FAP compared to normal colon tissue and 6 CpGs show more than 2 fold reduction in methylation (p-value <0.05). (b) In contrast comparison of SSP with normal samples shows significant DNA methylation changes. From 395,899 CpGs tested on the Infinium array, 42,965 (10.85%) show more than 2 fold increase in methylation in SSP compared to normal colon tissue and 19,019 (4.80%) show more than 2 fold reduction in methylation (p-value <0.05).
Fig 3.
The workflow to determine the SSP-specific DNA methylation markers.
The workflow shows the steps from sample collection to determination of the SSP-specific DNA methylation markers.
Table 2.
DNA methylation markers for SSP characterization and early diagnosis.
Fig 4.
Global loss of methylation and partial methylation in SSP.
(A) Global level of cytosine methylation in SSP, GU and blood. (B) Global level of CpG methylation in SSP, GU and blood. Histogram of fraction methylation in colon SSP, GU and blood (C-E). (C) In normal tissues most of the CpGs are either unmethylated or highly methylated (75–95% methylated). In SSP the very high level of methylation is lost and methylation level shows a more disperse distribution (35–80% methylated). (D) The EMRs show mostly 5–15% methylation in uninvolved colon tissue, while they are 15–45% methylated in SSP. (E) The RMRs in SSP were originally 65–90% methylated in uninvolved colon but this level is reduced to 15–40% in SSP.
Fig 5.
SSP-specific gain of methylation targets CGIs.
(A) The global loss of methylation in SSP genome is detected all over the genome except at CGIs. The CGIs show the lowest level of DNA methylation in GU tissue, and gain methylation in the SSP tissue. The TSS 4Kb flanking regions and 5’UTRs show lower level of methylation loss compared to other regions of the genome. (B) TSS flanking regions that are located at CGIs have low level of CpG methylation. Although TSS flanking regions that are not part of CGIs show methylation level close to the genomic level. CGIs that are located at TSS flanking regions show lower level of methylation than CGIs that are not located around TSS. (C) Average of methylation on 4Kb TSS flanking regions of all Ensemble transcripts. (D) Average of methylation on 4Kb TSS flanking regions of Ensemble transcripts that include CGIs in that 4 Kb. (E) Average of methylation on 4Kb TSS flanking regions of Ensemble transcripts that do not include CGIs in that 4 Kb.
Fig 6.
Gene expression profiling of colon samples.
Hierarchical clustering and sample-to-sample distance heatmap of the expression of protein coding genes. SSP samples are clustered together, FAP samples also clustered together. Gene expression in TSA-2 that has both APC and BRAF mutation is more similar to the pattern of SSP expression cluster. The gene expression pattern of FAP show about 50% similarity to the gene expression of SSP, although the DNA methylation patterns of these two polyp types differ significantly. Therefore studying the DNA methylation marker can lead to detect SSP more specifically than studying the gene expression.