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Updated: Jan 11, 2026
Comprehensive DNA Methylation Analysis Using a Methyl-CpG-binding Domain Capture-based Method in Chronic Lymphocytic Leukemia Patients
Published on: June 16, 2017
A comprehensive long-read sequencing system to assess DNA methylation at differentially methylated regions and
Tatsuki Urakawa1,2, Atsushi Hattori1,3, Yasuko Ogiwara1
1Department of Molecular Endocrinology, National Research Institute for Child Health and Development, 2-10-1 Okura, Setagaya-Ku, Tokyo, 157-8535, Japan.
Background:
Imprinted genes are expressed in a parental-origin-specific manner. The imprinted regions including imprinted genes have differentially methylated regions (DMRs) with different 5-methylcytosine (5mC) patterns for CpGs on each parental allele, and DMRs function as imprinting control centers. Aberrant expression of the imprinted genes caused by structural variants involving DMRs, single-nucleotide variants in imprinted genes, uniparental disomy, and epimutation lead to imprinting disorders (IDs). Nanopore-based targeted long-read sequencing (T-LRS) can obtain sequence reads 10-100 kb long together with information on DNA methylation in each CpG and is cost-effective compared to whole-genome LRS. T-LRS is a valuable tool for efficient genetic testing for IDs and has great potential to elucidate the regulatory mechanisms in the imprinted regions. However, there is no T-LRS system targeting all ID-related regions.
Methods:
We conducted T-LRS targeting 78 DMRs and 22 genes in peripheral blood leukocytes from six healthy controls and set the normal range of methylation index (MI) for each CpG within the DMRs. To clarify the properties of DMRs, we compared MIs in CpGs within DMRs between haplotypes in 78 DMRs. To evaluate the usefulness of T-LRS, we conducted T-LRS on two previously reported patients with multi-locus imprinting disturbance (MLID) having pathogenic variants in MLID-causative genes and compared the MIs in CpGs within DMRs with those measured by array-based methylation analysis.
Results:
The median number of reads with 5mC and unmethylated cytosine in all DMRs in the six controls was over 40. We defined the normal range of MI for all CpGs in each allele and the total, and classified 78 DMRs into three categories, namely, 33 Complete-DMRs, 25 Partial-DMRs, and 20 Non-DMRs, based on the average of six controls for the median of differences of MIs in CpGs between haplotypes. We confirmed by T-LRS pathogenic variants in MLID-causative genes in patients with MLID. The patients' methylation defect patterns in T-LRS were similar to those in array-based methylation analysis, although T-LRS showed additional aberrantly methylated DMRs.
Conclusions:
We established a T-LRS system targeting all ID-related regions, defined standard MI ranges in CpGs on each parental allele, and demonstrated the usefulness of T-LRS.
