Promoter methylation assay
The promoter methylation assay consisted of an initial bisulfite conversion of 1μg of DNA using the EpiTect Bisulfite Kit (Qiagen), followed by a methylation specific PCR (MSP) using the EpiTect MSP Kit (Qiagen) and two different sets of specific primers targeting the promoter region of PTPRT . Each set was composed of 2 pairs of primers, which specifically detected methylated or unmethylated DNA and had been previously described by Laczmanska et al. (Laczmanska et al., 2013) (in CRC) or by Peyser et al. (Peyser et al., 2016) (in head and neck carcinoma) (Supplementary Table S2). Tumor DNA from twoPTPRT mutation carriers was used for this assay, while DNA obtained from healthy colon or breast tissue was used as a control. The bisulfite conversion and MSP were carried out following the manufacturers’ instructions, and the PCR products were then analyzed by agarose gel electrophoresis.