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Previous work has shown that DNA methylation in peripheral blood may be associated with malignancy; however, these studies have mainly been conducted within Caucasian populations. Here, we investigated the association between blood-based methylation of S100 calcium-binding protein P gene (S100P) and hyaluronoglucosaminidase 2 gene (HYAL2) and breast cancer (BC) via mass spectrometry in two independent case-control studies of the Chinese population with a total of 351 BC cases and 427 cancer-free female controls. In Study I, in which subjec