Pyrosequencing
A pyrosequencing assay was also used for the measurement of PTPRTpromoter methylation. For that purpose, the bisulfite conversion of 1μg of DNA using the EpiTect Bisulfite Kit (Qiagen) was followed by a PCR targeting a fraction of PTPRT promoter. Finally, the pyrosequencing took place in a PyroMark Q96 MD sequencer (Biotage, Qiagen), taking advantage of the PyroMark Q96 reagents and buffers and following the manufacturer’s instructions. Tumor or healthy tissue DNA from PTPRT mutation carriers was used for this assay, while commercial high-methylated and non-methylated DNA was used as a control (CpGenome Human Methylated/Non-Methylated DNA Standard, Millipore). The PCRs and subsequent pyrosequencing were performed in triplicates, and the relative methylation (%) and standard deviation were represented. The sequences of the PCR and biosynthesis primers are shown in Supplementary Table S2.