乳腺癌是全球妇女最常见的恶性肿瘤，在其诊断中，已经证实乳腺癌患者的血浆细胞游离DNA（cfDNA）的甲基化和断裂特征发生改变。然而，迄今为止尚未报道有关同时检测两者以用于乳腺癌诊断的研究。此外，尽管cfDNA的断裂模式是由核酸酶对染色质的消化所决定，而染色质的结构可能会受到DNA甲基化的影响，但是cfDNA的甲基化和断裂是否具有生物学相关性仍然不清楚。 我们采用改进的cfMeDIP-seq技术对49个来自健康人和乳腺癌患者的血浆样本进行了全面的cfDNA甲基化和断裂特征分析，并评估了使用cfDNA断裂特征作为乳腺癌诊断标志物的可行性。 结果显示，在与健康人相比，乳腺癌患者低甲基化区域的cfDNA片段平均长度（100-220 bp）缩短更多（乳腺癌患者：4.60 bp，172.33 bp至167.73 bp；健康人：2.87 bp，174.54 bp至171.67 bp）。此外，我们发现，与高甲基化区域相比，乳腺癌患者低甲基化区域中短cfDNA片段（100-150 bp）的比例也增加更多，这个结果在两个独立发现队列中得到了证实。我们进一步评估了在验证队列中使用低甲基化基因组区域中短cfDNA片段比例异常作为乳腺癌诊断的可行性。11名患者中有7名被检测出患有乳腺癌（敏感性为63.6%），而健康人则未被错误检测（特异性为100%）。 我们鉴别出乳腺癌患者中富集的5mC-免疫沉淀（IP）后的短cfDNA片段，并证明这些富集的短cfDNA片段可能来自低甲基化的基因组区域。此外，我们还证实了使用差异甲基化区域（DMRs）相关的cfDNA断裂特征进行乳腺癌诊断的可行性。 ©2023年。BioMed Central Ltd., Springer Nature部分。

Breast cancer, the most common malignancy in women worldwide, has been proven to have both altered plasma cell-free DNA (cfDNA) methylation and fragmentation profiles. Nevertheless, simultaneously detecting both of them for breast cancer diagnosis has never been reported. Moreover, although 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.Improved cfMeDIP-seq were utilized to characterize both cfDNA methylation and fragmentation profiles in 49 plasma samples from both healthy individuals and patients with breast cancer. The feasibility of using cfDNA fragmentation profile in hypo- and hypermethylated regions as diagnostic markers for breast cancer was evaluated.Mean size of cfDNA fragments (100-220 bp) mapped to hypomethylated regions decreased more in patients with breast cancer (4.60 bp, 172.33 to 167.73 bp) than in healthy individuals (2.87 bp, 174.54 to 171.67 bp). Furthermore, proportion of short cfDNA fragments (100-150 bp) in hypomethylated regions when compared with it in hypermethylated regions was found to increase more in patients with breast cancer in two independent discovery cohort. The feasibility of using abnormality of short cfDNA fragments ratio in hypomethylated genomic regions for breast cancer diagnosis in validation cohort was evaluated. 7 out of 11 patients were detected as having breast cancer (63.6% sensitivity), whereas no healthy individuals were mis-detected (100% specificity).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.© 2023. BioMed Central Ltd., part of Springer Nature.