Investigating the potential role of genetic and epigenetic variation of DNA methyltransferase genes in hyperplastic polyposis syndrome

Abstract

Background: Hyperplastic Polyposis Syndrome (HPS) is a condition associated with multiple serrated polyps, and an increased risk of colorectal cancer (CRC). At least half of CRCs arising in HPS show a CpG island methylator phenotype (CIMP), potentially linked to aberrant DNA methyltransferase (DNMT) activity. CIMP is associated with methylation of tumor suppressor genes including regulators of DNA mismatch repair (such as MLH1, MGMT), and negative regulators of Wnt signaling (such as WIF1). In this study, we investigated the potential for interaction of genetic and epigenetic variation in DNMT genes, in the aetiology of HPS.

Methods: We utilized high resolution melting (HRM) analysis to screen 45 cases with HPS for novel sequence variants in DNMT1, DNMT3A, DNMT3B, and DNMT3L. 21 polyps from 13 patients were screened for BRAF and KRAS mutations, with assessment of promoter methylation in the DNMT1, DNMT3A, DNMT3B, DNMT3L MLH1, MGMT, and WIF1 gene promoters.

Results: No pathologic germline mutations were observed in any DNA-methyltransferase gene. However, the T allele of rs62106244 (intron 10 of DNMT1 gene) was over-represented in cases with HPS (p<0.01) compared with population controls. The DNMT1, DNMT3A and DNMT3B promoters were unmethylated in all instances. Interestingly, the DNMT3L promoter showed low levels of methylation in polyps and normal colonic mucosa relative to matched disease free cells with methylation level negatively correlated to expression level in normal colonic tissue. DNMT3L promoter hypomethylation was more often found in polyps harbouring KRAS mutations (p = 0.0053). BRAF mutations were common (11 out of 21 polyps), whilst KRAS mutations were identified in 4 of 21 polyps.

Conclusions: Genetic or epigenetic alterations in DNMT genes do not appear to be associated with HPS, but further investigation of genetic variation at rs62106244 is justified given the high frequency of the minor allele in this case series.

Figure 1. Normalized and temp-shifted difference plot for DNMT1 gene SNP rs62106244.

*Seven cases were heterozygous for rs62106244. The top arrow shows the sequence of the DNMT1 gene.

Figure 2. Unsupervised hierarchical clustering of the level of methylation of DNA from LCLs and somatic DNA in HPS cases for CpG_1 (analytic unit 1), CpG_6.7 (unit 2), CpG_8 (unit 3) and CpG_10 (unit 6) of the DNMT3L promoter region (A); Box and whisker plot comparing quantitative methylation between polyp tissue (white boxes) and peripheral blood (grey boxes) for each CpG site (B).

Figure 3. DNMT3L bisulphite sequencing: HP40RH_LCL lymphoblastic cell line DNA, HP40RH_P polyp DNA and normal mucosa DNA HP40RH_N.

Each line represents a sequenced clone and DNA methylation information is presented as a circle denoting the CpG site where methylation could occur. Black circles represent methylated CpG's while white circles represent unmethylated CpG's. The promoter of DNMT3L was observed to be less methylated in normal mucosa and polyp tissue but methylated in lymphoblastic cell line DNA.

Figure 4. Correlation between mean methylation of DNMT3L promoter and gene expression level in control mucosa tissue.

Mean was calculated from the combined methylation values for CpG units, 1, 2, 3 and 6.