LINE-1 Hypermethylation in Serum Cell-Free DNA of Relapsing Remitting Multiple Sclerosis Patients

Abstract

Concentrations of cell-free DNA (cfDNA) circulating in blood and its epigenetic variation, such as DNA methylation, may provide useful diagnostic or prognostic information. Long interspersed nuclear element-1 (LINE-1) constitutes approximately 20% of the human genome and its 5'UTR region is CpG rich. Due to its wide distribution, the methylation level of the 5'UTR of LINE-1 can serve as a surrogate marker of global genomic DNA methylation. The aim of the current study was to investigate whether the methylation status of LINE-1 elements in serum cell-free DNA differs between relapsing remitting multiple sclerosis (RRMS) patients and healthy control subjects (CTR). Serum DNA samples of 6 patients and 6 controls were subjected to bisulfite sequencing. The results showed that the methylation level varies among distinct CpG sites in the 5'UTR of LINE-1 repeats and revealed differences in the methylation state of specific sites in this element between patients and controls. The latter differences were largely due to CpG sites in the L1PA2 subfamily, which were more frequently methylated in the RRMS patients than in the CTR group, whereas such differences were not observed in the L1HS subfamily. These data were verified by quantitative PCR using material from 18 patients and 18 control subjects. The results confirmed that the methylation level of a subset of the CpG sites within the LINE-1 promoter is elevated in DNA from RRMS patients in comparison with CTR. The present data suggest that the methylation status of CpG sites of LINE repeats could be a basis for development of diagnostic or prognostic tests.

Keywords: Cell-free DNA; CpG; DNA methylation; LINE-1; Multiple sclerosis.

Fig. 1

Sequence alignment of CpG islands of the L1PA2 and L1HS subfamilies. The positions of each CpG site are indicated in red. The L1PA2 and L1HS subfamily contain 27 and 25 CpG sites, respectively

Fig. 2

L1PA2 and L1HS methylation status in cfDNA determined by bisulfite sequencing. cfDNA was isolated from the serum of 6 healthy controls and 6 RRMS patients. After bisulfite treatment and cloning of the resulting DNA, 6 clones for each individual were selected and subjected to DNA sequence analysis. The methylation analysis was performed by Quantification Tool for Methylation Analysis (QUMA). The schemes show the methylation levels as pie charts for each individual CpG site in the L1PA2 subfamily and the L1HS subfamily. Open circles represent completely unmethylated sites and filled circles represent full methylation. The methylation level of each individual CpG site in RRMS and CTR was compared using Fischer’s exact test. *p < 0.05., CpG sites show a trend for higher methylation levels but the differences do not reach significance ^# p = 0.08, p = 0.09, and p = 0.08, respectively. The numbers below each CpG site indicate the position of the respective CpG site in the L1PA2/L1HS element

Fig. 3

LINE-1 promoter CpG site analysis by methylation-specific quantitative PCR assay. a Analysis of PCR products by agarose gel electrophoresis to check primer specificity. U completely unmethylated DNA; M fully methylated DNA, NTC no template control, DNA size markers (base pairs) are shown on the left. The size (base pairs) of the amplicons is indicated on the right. b Methylation level for L1PA2 CpG sites 10, 18, 24, and 27 determined by qPCR using samples from RRMS patients and CTR. The horizontal bars indicate the median with interquartile range. The two groups were compared using unpaired Mann-Whitney tests