Infinium 450k human methylation beadchip

In the present study, we included a total of seven BC methylation studies. The DNA methylation profile of The Cancer Genome Atlas Breast Invasive Carcinoma (TCGA-BRCA) (Cancer Genome Atlas, 2012 (link); Ciriello et al., 2015 (link)), measured with Illumina Human Methylation 450 BeadChip, included a total of 890 BC samples and associated clinical information of BC patients and was downloaded from UCSC Xena¹. A total of 62, 118, 188, 70, and 58 BC samples were included in GSE37754 (Putluri et al., 2014 (link); Terunuma et al., 2014 (link); Tang et al., 2018 ), GSE72245 (Jeschke et al., 2017 (link)), GSE75067 (Holm et al., 2016 ), GSE78754 (Mathe et al., 2016 ), and GSE72251 (Jeschke et al., 2017 (link)), respectively, and there were 40 normal breast samples and 80 BC samples in GSE66695². Bisulfite converted DNA from the breast samples in the above datasets were hybridized to the Illumina Infinium 450k Human Methylation Beadchip. mRNA expression profile of the TCGA-BRCA including 1,217 samples and somatic variant data in “maf” format, were downloaded from GDC Data Portal³. The inclusion criteria of this study were as follows: the patients were newly diagnosed with BC, the patient’s survival information was well documented or the study contained both normal breast sample and BC samples, and the relevant BC patients received Illumina Human Methylation 450 BeadChip profiling.

Placentae were obtained and dissected within 1 hour of delivery. A 3 cm diameter core was taken from the fetal surface through to the maternal surface halfway between the umbilical cord and the placental margin. Only the fetal placental portion was studied after it was washed in saline and stored at -80C. Please see S1 Text for additional details.
Genomic DNA was isolated (DNeasy, Qiagen) and processed by the University of Cincinnati Genomics, Epigenomics and Sequencing Core including bisulfite conversion for Illumina’s Infinium 450K Human Methylation Bead Chip. The methylation status, β, was calculated at each probe site as β = M/(M+U), where M = methylated and U = unmethylated. Differential methylation was calculated for each sample pair and averaged across all pairs for each probe site to determine the absolute value of the difference in methylation between DDP and control (AVDM). CpGs on the X chromosome were excluded based on complications from X inactivation and the high proportion of tandem repeat DNA [14 (link)]. Genes with differential methylation at multiple probe sites were validated with bisulfite pyrosequencing or Mass Array Epityper. (S1 Text and S1 Table).