Fig. 1

a Methylation frequency of candidate metastatic suppressor genes in breast-to-brain metastases (BBM) (n = 30) versus primary breast tumours (n = 40). Out of the 25 genes that were frequently methylated in brain metastases, three genes (GALNT9, CCDC8 and BNC1) were infrequently methylated in a cohort of independent primary tumours with statistical significance (p = 0.0001, 0.01 and 0.0001, respectively). b, c Expression of GALNT9, CCDC8 and BNC1 correlates with promoter methylation in BBM. Reverse transcription PCR (RT-PCR) for GALNT9, CCDC8 and BNC1 in BBM shows that these genes were expressed in tumours where their promoters are unmethylated (U) and silenced in methylated (M) tumours (see Additional file 7: Figs S3 and Additional file 9: Figure S5 for representative methylation analysis). Expression of β-actin was determined to ensure equal loading for all samples. c Expression levels of each gene were quantified in relation to the expression of β-actin. The methylation status was determined by either CoBRA or sequencing of individual alleles to determine the methylation index (MI) for individual tumours. High levels of expression were not associated with high levels of methylation in the region analysed. A full set of methylation/expression analysis can be seen in Additional file 10: Figure S6 (BM brain metastasis, M methylated, U unmethylated, − analysis was not done)