Comprehensive methylation analysis of imprinting-associated differentially methylated regions in colorectal cancer

Abstract

Background: Imprinted genes are regulated by DNA methylation at imprinting-associated differentially methylated regions (iDMRs). Abnormal expression of imprinted genes is implicated in imprinting disorders and tumors. In colorectal cancer (CRC), methylation and imprinting status of the IGF2/H19 domain have been studied. However, no comprehensive methylation analysis of iDMRs in CRC has been reported. Furthermore, the relationship between iDMR methylation status and other methylation-related issues, such as CpG island methylator phenotype (CIMP) and long interspersed element-1 (LINE-1) methylation, remains unclear.

Results: We analyzed the methylation status of 38 iDMRs in 106 CRC patients. We also investigated CIMP, LINE-1 methylation, KRAS and BRAF gene mutations, and loss of imprinting (LOI) of IGF2. We further examined the relationship between these factors and clinicopathological factors. The overall trend in iDMR methylation was towards hypermethylation, and iDMRs could be grouped into three categories: susceptible, resistant, and intermediate-to-aberrant methylation. The susceptible and resistant iDMRs consisted of all types of iDMR (gametic and somatic, maternally and paternally methylated). Hypermethylation of multiple iDMRs (HyMiD)-positive status was statistically associated with CIMP-positive status, but not associated with mutations in the BRAF and KRAS genes. HyMiD-positive status was inversely associated with LINE-1 methylation. Among four iDMRs within the IGF2/H19 domain, IGF2-DMR0 hypomethylation occurred most frequently, but was not associated with IGF2 LOI. Finally, we statistically calculated predictive prognostic scores based on aberrant methylation status of three iDMRs.

Conclusion: In CRC tissues, some iDMRs were susceptible to hypermethylation independent of the type of iDMR and genomic sequence. Although HyMiD-positive status was associated with CIMP-positive status, this was independent of the BRAF and KRAS pathways, which are responsible for CIMP. Since IGF2-DMR0 hypomethylation and aberrant methylation of other iDMRs within the IGF2/H19 domain were not associated with IGF2 LOI, dysfunction of any of the molecular components related to imprinting regulation may be involved in IGF2 LOI. The prognostic score calculated based on aberrant methylation of three iDMRs has potential clinical applications as a prognostic predictor in patients. Further study is required to understand the biological significance of, and mechanisms behind, aberrant methylation of iDMRs and IGF2 LOI in CRCs.

Keywords: BRAF mutation; CIMP; DNA methylation; Genomic imprinting; IGF2 LOI; IGF2-DMR0; KRAS mutation; LINE-1; iDMR.

Fig. 1

Methylation statuses of 38 iDMRs, CIMP status, and BRAF and KRAS mutations in 106 CRC patients. The three-digit number in the leftmost column indicates each patient’s ID. Hypermethylation and hypomethylation of iDMR are indicated by red and blue, respectively. CIMP-positive samples are indicated in green in the column on the right. The BRAF mutation (V600E) is indicated in orange. The KRAS (G13D) mutation and other KRAS mutations are indicated in black and gray, respectively. G, gametic iDMR; S, somatic iDMR; M, maternally methylated iDMR; P, paternally methylated iDMR; U, unidentified; ZNF597 (TSS), transcription start site region of ZNF597; ZNF597 (3′), 3′ region of ZNF597; ZNF331 (pro), promoter region of ZNF331; ZNF331 (int), intron of ZNF331. Definitions of aberrant methylation of iDMR and CIMP-positive are provided in the “Methods” section

Fig. 2

Frequency of iDMR aberrant methylation. a The distribution of hypermethylated iDMRs in tumors by number. b The distribution of hypomethylated iDMRs in tumors by number. The data indicate that iDMRs are more susceptible to hypermethylation than hypomethylation in CRC tissues

Fig. 3

Inverse relationship between HyMiD status and LINE-1 methylation. LINE-1 methylation in HyMiD-positive tumors (median 55.1%, 22.1–64.8%) was significantly lower than in HyMiD-negative tumors (median 60.2%, 43.7–72.1%), indicating that an HyMiD-positive status was inversely correlated with LINE-1 methylation in CRC tissues. (p = 0.0001, Mann-Whitney U test)

Fig. 4

Aberrant methylation of four iDMRs within the IGF2/H19 domain in tumors. Hypermethylation of the H19-promoter and H19-DMR were found in only a few samples. However, hypomethylation of IGF2-DMRs, especially DMR0, was comparatively overrepresented among iDMRs (*p = 0.0061, **p < 0.0001, χ² test)

Fig. 5

Cox proportional hazards regression analysis. a Model fitting summary. The prognosis score was computed as Z = − 2.011 × LOC728024 + 1.072 × RB1 – 1.187 × ZNF331-promoter. When aberrant methylation (hyper- or hypo-) at LOC728024, RB1, or ZNF331-promoter is found, the corresponding term is assigned a value of 1. coef: coefficient; se: standard error. z value = coef/se(coef). b Kaplan-Meier survival estimates. High prognostic scores (Z ≥ 0.332) were significantly associated with poor clinical prognosis compared low scores (Z < 0.332) (p = 0.0063, log-rank test, HR = 0.102 (95% CI 0.014–0.765))