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Background Breast cancer, the most common malignancy in women, has been proved to have both altered plasma cell-free DNA (cfDNA) methylation and fragmentation profile, nevertheless, simultaneously detecting both of them for breast cancer diagnosis has never been reported. Moreover, although it is known that fragmentation pattern of cfDNA is determined by nuclease digestion of chromatin, structure of which may be affected by DNA methylation, whether cfDNA methylation and fragmentation are biologically related or not still remains unclear.Methods A total of 25 plasm samples from both healthy individuals and patients with breast cancer were divided into discovery cohort and validation cohort. Improved cfMeDIP-seq were utilized to simultaneously profile both cfDNA methylation and fragments size, short fragments ratio were investigated in differentially methylated regions (DMRs). The feasibility of using cfDNA fragmentation profile in hypo- and hyper- methylated regions as a diagnostic marker for breast cancer was evaluated.Results Mean cfDNA fragments size ranging from 100 to 220 base pairs (bp) was found to increase from 170.06 (Input libraries) to 173.04 (IP libraries) bp in healthy individuals, which was not observed in patients with breast cancer (170.51 to 170.71 bp). Furthermore, mean size of cfDNA fragments mapped to hypomethylated regions decreased more in patients with breast cancer (4.60 bp, 172.33 bp in hypermethylated regions to 167.73 bp in hypomethylated regions) than healthy individuals (2.87 bp, 174.54 bp in hypermethylated regions to 171.67 bp in hypomethylated regions). An approach called ‘correlation assessment of DMRs-dependent cfDNA fragmentation profile’ was developed to evaluate the feasibility of using abnormality of short cfDNA fragments ratio in hypomethylated genomic windows for diagnosis of breast cancer in validation cohort. 7 out of 11 patients were detected as having breast cancer (63.6% sensitivity), whereas no healthy individuals were mis-detected (100% specificity).Conclusion We identified enriched short cfDNA fragments after 5mC-immunoprecipitation (IP) in patients with breast cancer, and demonstrated the enriched short cfDNA fragments might originated from hypomethylated genomic regions. Furthermore, we proved the feasibility of using differentially methylated regions (DMRs)-dependent cfDNA fragmentation profile for breast cancer diagnosis. |
