Tumour-specific methylation of PTPRG intron 1 locus in sporadic and Lynch syndrome colorectal cancer

Abstract

DNA methylation is a hallmark in a subset of right-sided colorectal cancers. Methylation-based screening may improve prevention and survival rate for this type of cancer, which is often clinically asymptomatic in the early stages. We aimed to discover prognostic or diagnostic biomarkers for colon cancer by comparing DNA methylation profiles of right-sided colon tumours and paired normal colon mucosa using an 8.5 k CpG island microarray. We identified a diagnostic CpG-rich region, located in the first intron of the protein-tyrosine phosphatase gamma gene (PTPRG) gene, with altered methylation already in the adenoma stage, that is, before the carcinoma transition. Validation of this region in an additional cohort of 103 sporadic colorectal tumours and 58 paired normal mucosa tissue samples showed 94% sensitivity and 96% specificity. Interestingly, comparable results were obtained when screening a cohort of Lynch syndrome-associated cancers. Functional studies showed that PTPRG intron 1 methylation did not directly affect PTPRG expression, however, the methylated region overlapped with a binding site of the insulator protein CTCF. Chromatin immunoprecipitation (ChIP) showed that methylation of the locus was associated with absence of CTCF binding. Methylation-associated changes in CTCF binding to PTPRG intron 1 could have implications on tumour gene expression by enhancer blocking, chromosome loop formation or abrogation of its insulator function. The high sensitivity and specificity for the PTPRG intron 1 methylation in both sporadic and hereditary colon cancers support biomarker potential for early detection of colon cancer.

Figure 1

Identification and validation of the differentially methylated locus PTPRGint1. (a) Trend plot of the top-20 differentially methylated microarray clones (FDR ≤0.01%) showing the average log10 ratios in the normal, adenoma and carcinoma samples compared with the colorectal cancer cell line reference panel. Clone 47B02, corresponding to PTPRGint1, had a similarly increased log ratio in adenomas and carcinomas compared with normals (red solid line). (b) BiQ summary of direct BSA of PTPRGint1 (CpG dinucleotides 1–10) in paired normal colon mucosa (n=19, upper panel) and colon tumours (n=18, 12 right- and left-sided carcinomas and six adenomas, lower panel) showed highest specificity and sensitivity in the four most 3′ CpGs measured. Each box corresponds to one CpG position in the genomic sequence, while colours summarise the methylation states of all sequenced samples at that position.

Figure 2

PTPRGint1 methylation detected by MS-MLPA. (a) GeneMapper output of the custom MS-MLPA to analyse PTPRGint1 CpG9 methylation in a normal mucosa sample. Upper panel: The PTPRGint1 peak was located at ∼84 bp, the control peak was located at ∼110 bp. Lower panel: Loss of PTPRGint1 signal after HhaI digestion indicated an unmethylated CpG9. (b) GeneMapper output of the custom PTPRGint1 MS-MLPA in the corresponding colon cancer sample. Upper panel: Undigested. Lower panel: HhaI digestion. Retention of the PTPRGint1 marker signal indicated protection against HhaI digestion by CpG9 methylation. (c) Frequency of PTPRGint1 CpG9 methylation in precursor lesions (hyperplastic polyps and serrated adenoma), early- and advanced adenomas, carcinomas and corresponding normal mucosal tissue for the latter three groups. The number of samples typed as methylated (dark), partially methylated (striped) and unmethylated (white) in the MS-MLPA assay is indicated on the y axis.

Figure 3

Scatter plot of the relative PTPRG expression against the PTPRGint1 methylation ratio according to the MS-MLPA. The vertical line at 0.22 indicates the cut-off for unmethylated samples. Plotted data is representative of two independent experiments.

Figure 4

(a) CTCF binding to PTPRGint1, positive- and negative control regions in normal colon mucosa (light grey), KP7038f (black) and KP7038t (dotted). The histone H3 normalised values of the CTCF antibody and IgG negative control antibody pull downs are given per primer pair. Standard errors represent the variability of duplicate PCR reactions. This is a representative experiment of three ChIPs. (b) The PTPRGint1/positive control ratio for normal colon mucosa, KP7038f, KP7038t (also shown in a), SW480 (vertically striped) and RKO (diagonally striped).