TERT promoter mutations and monoallelic activation of TERT in cancer

Abstract

Here we report that promoter mutations in telomerase (TERT), the most common noncoding mutations in cancer, give rise to monoallelic expression of TERT. Through deep RNA sequencing, we find that TERT activation in human cancer cell lines can occur in either mono- or biallelic manner. Without exception, hotspot TERT promoter mutations lead to the re-expression of only one allele, accounting for approximately half of the observed cases of monoallelic TERT expression. Furthermore, we show that monoallelic TERT expression is highly prevalent in certain tumor types and widespread across a broad spectrum of cancers. Taken together, these observations provide insights into the mechanisms of TERT activation and the ramifications of noncoding mutations in cancer.

Figure 1

Identification of cancer cell lines with monoallelic TERT expression. (a) Fraction of sequencing reads harboring nucleotide that differs from reference human genome for heterozygous anchor SNPs. DNA and RNA alternate allelic fractions are plotted along the x axis and y axis, respectively. Analysis was performed on 88 cell lines with a DNA alternate allelic fraction between 0.25 and 0.75 in the DNA sequences of expressed exonic and untranslated regions of the TERT gene. Strong deviation from the diagonal indicates allelic bias in expression. Individual points represent samples, and those harboring hotspot promoter mutations are highlighted. Nineteen cell lines harbored one of the hotspot promoter mutations C228T, C228A or C250T and were found within the 88 cell lines that met the criteria for assessment of allelic bias. (b) Bar plot of promoter mutation status in cell lines with monoallelic and biallelic expression of the TERT gene. TERT expression was classified as monoallelic in samples with heterozygous anchor SNPs in the TERT gene for which expression of a major allele was >10-fold higher than expression of the minor allele in RNA versus DNA.

Figure 2

Distribution of cell lines with monoallelic TERT expression across tissue lineages. We found a highly significant difference in the distribution of samples with monoallelic TERT expression across lineages when compared with cell lines with biallelic TERT expression for both promoter-mutant and promoter-wild-type cell lines (P=1.59 × 10⁻⁶ and P=0.006, respectively; Fisher–Freeman–Halton test).

Figure 3

Relationship between TERT promoter mutations and allele-specific expression. DNA was isolated from cell lines with a TERT promoter mutation and nearby heterozygous exon 2 anchor SNP (first column) using the DNeasy Blood and Tissue Kit (Qiagen, Hilden, Germany). The genomic region containing the TERT promoter and anchor SNP was PCR amplified, gel purified and cloned using the Zero Blunt PCR Cloning Kit (Life Technologies, Carlsbad, CA, USA). Sanger sequencing chromatograms of individual clones demonstrate the TERT promoter mutation (second column) in cis with one of the alleles at the anchor SNP (third column). RNA sequencing confirmed allele-specific expression by identifying exclusive expression of the SNP that was in cis with the TERT promoter mutation (fourth column).