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. 2012 Jul;90(7):827-35.
doi: 10.1007/s00109-011-0854-z. Epub 2012 Jan 8.

LINE-1 hypomethylation in familial and sporadic cancer

Walter Pavicic  1 , Emmi I Joensuu, Taina Nieminen, Päivi Peltomäki
Affiliations

LINE-1 hypomethylation in familial and sporadic cancer

Walter Pavicic et al. J Mol Med (Berl). 2012 Jul.

Abstract

Increased and decreased methylation at specific sequences (hypermethylation and hypomethylation, respectively) is characteristic of tumor DNA compared to normal DNA and promotes carcinogenesis in multiple ways including genomic instability. Long interspersed element (LINE), an abundant class of retrotransposons, provides a surrogate marker for global hypomethylation. We developed methylation-specific multiplex ligation-dependent probe amplification assays to study LINE-1 methylation in cases of colorectal, gastric, and endometrial cancer (N = 276), stratified by patient category [sporadic; Lynch syndrome (LS); familial colorectal cancer type X (FCCX)] and microsatellite instability status. Within each patient group, LINE-1 showed lower methylation in tumor DNA relative to paired normal DNA and hypomethylation was statistically significant in most cases. Interestingly, normal colorectal mucosa samples from different patient groups displayed differences in LINE-1 methylation that mirrored differences between the respective tumor tissues, with a decreasing trend for LINE-1 methylation from patients with sporadic colorectal cancer to LS to FCCX. Despite the fact that the degree of LINE-1 methylation is generally tissue specific, normal colorectal mucosa, gastric mucosa, and endometrium from LS patients showed similar levels of LINE-1 methylation. Our results suggest that the degree of LINE-1 methylation may constitute a "field defect" that may predispose normal tissues for cancer development.

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Figures

Fig. 1
Fig. 1
Diagram of the CpG island promoter (GenBank accession no. M80343, nucleotide position 108-520 bp) associated with the full length LINE-1. The 5´UTR, 3´UTR and two open reading frames (ORF) of LINE-1 are shown. Location of all ten probe pairs included in the present MS-MLPA assay are indicated below the LINE-1 bar by black/grey and black squares (the so-called L1-m and control probes, respectively). Single CpG sites in the region containing the L1-m probes are shown by vertical lines in the lower part of the diagram. Numbered arrows indicate the location of primers: 1- used for bisulfite PCR in COBRA assay [in the present work and by Chalitchagorn et al. [24], see “Materials and methods”]; 2- used for pyrosequencing by Goel et al. [14]. The exact CpG sites that are part of HhaI recognition sequences in the three L1-m probes as well as the two CpG sites recognized by restriction enzymes used in COBRA are shown by thick vertical lines. LPO and RPO denote sequences homologous to LINE-1 and correspond to the underlined sequences in Table 2
Fig. 2
Fig. 2
a, LINE-1 CpG island promoter methylation analysis by custom-made MS-MLPA kit. Results from an unmethylated sample (WGA) and a methylated sample (FFPE tumor DNA) are shown. Peaks specific to L1-m probes are indicated by arrowheads and the respective Dm values are boxed. Peaks without a label correspond to control probes. b, Graphical comparison of results from LINE-1 promoter methylation analysis by MS-MLPA (Dm, Y- axis) and COBRA (fraction of methylated LINE-1 sequences, X - axis) revealed a good correlation as indicated by the Pearson test
Fig. 3
Fig. 3
Average methylation dosage ratio ± one standard deviation for LINE-1 in tumor (T) vs. normal (N) tissues. The results are given separately for each patient category out of 8 (listed in the key). Please see Supplementary Table 1 for the exact numerical values and Supplementary Fig. 2 for the relationship between T and N case by case. Asterisks indicate statistical significance for the differences between T and N within each patient category (intra-group comparison) on the level of P < 0.05 (*), P < 0.01 (**), and P < 0.001 (***)
Fig. 4
Fig. 4
Average methylation dosage ratio ± one standard deviation for LINE-1 in tumor (T) vs. normal colorectal mucosa or endometrium (N) vs. blood (B) from patients with T, N, and B available. Please see Supplementary Table 2 for the exact numerical values and Supplementary Fig. 3 for the relationship between T and N case by case. Asterisks indicate statistical significance for the differences in pairwise comparisons of T, N, and B within each patient category (intra-group comparison) on the level of P < 0.01 (**) and P < 0.001 (***) (-, nonsignificant)
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