Supplementary Materials and Methods

Tumor samples

The 88 samples analyzed in this study consisted of 11 diffuse astrocytomas (DA), 3 oligoastrocytomas (OA), 1 oligodendrogliomas (OL), 12 anaplastic astrocytomas (AA), 6 anaplastic oligoastrocytomas (AOA), 5 anaplastic oligodendrogliomas (AO), 48 primary glioblastomas and 2 secondary glioblastomas. The patient age ranged from 21 to 82 years old. The mutational status of the TERT promoter has previously been reported for these cases[1] ; 5/23 astrocytic tumors (DA and AA), 9/15 oligodendroglial tumors (OL, AO, OA and AOA) and 29/50 glioblastomas harbored TERT promoter mutations.

Targeted regions for pyrosequencing

The CpG island that encompasses the entire exon 1 and 2 of TERT spans over a 6,695 bp area. Three regions (Regions 1-3) within the CpG island were selected for methylation analysis. Region 1 is identical to the region pyrosequenced by Castelo-Branco et al for methylation screening [2]. They showed that hypermethylation in this region was associated with increased TERT expression in pediatric brain tumors. Region 2 includes the proximal 7 CpGs of the region that was hypermethylated in various cancer cell lines and tumor tissues in the study by Guilleret et al [5]. It overlaps with the distal end of the core promoter region [4,13]. Region 3 encompasses the two mutation hotspots in the TERT promoter [1,9]. This region is also part of the core promoter region and methylated in cervical cancer cell lines [4,7].

Bisulfite modification

For each sample, genomic DNA was bisulfite modified using an EZ DNA methylation kit (Zymo Research, Orange, CA) as per manufacturer's recommendations. Commercially available methylated or de-methylated human genomic DNA (EpiTect PCR Control DNA Set, Qiagen, Crawley, UK) were used as positive and negative control.

Amplification

The bisulfite-modified DNA was amplified using PyroMark PCR Kit (Qiagen, Tokyo, Japan). The primer sequences are described below. For Region 1, the targeted sequence was amplified using the forward and reverse primers described in Castelo-Branco et al. [2] .

Pyrosequencing

Single-stranded templates for pyrosequencing were prepared using PSQ 96 Sample Prep Workstation Low (Qiagen, Tokyo, Japan) according to the manufacturer's recommendations.

Pyrosequencing was performed using PyroGold Q96 SQA Reagents and the Pyro Mark Q96 software on a PSQ96 pyrosequencer (Qiagen, Tokyo, Japan) as per manufacturer's recommendations. The data were analyzed using the Pyro Mark Q96 software. The detailed primer design and dispensation order are described in Supplementary Table 1.

Statistical analysis

The Mann-Whitney U test was used to compare mRNA expression levels evaluated by binary logarithm or methylation levels between two groups of samples. The Pearson’s Chi-square test was used for comparing two subsets by dichotomous variable. The relationship between methylation level and expression level was analyzed with a linear regression model. A Multiple regression analysis using standard least squares was used to identify factors associated with TERT expression among multiple variables. All analyses were performed using a JMP version10 software (SAS Institute, Cary, NC, USA). A p-value below 0.05 was considered statistically significant.