TERT Promoter Mutations in Familial and Sporadic Melanoma

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Science  22 Feb 2013:
Vol. 339, Issue 6122, pp. 959-961
DOI: 10.1126/science.1230062


Cutaneous melanoma occurs in both familial and sporadic forms. We investigated a melanoma-prone family through linkage analysis and high-throughput sequencing and identified a disease-segregating germline mutation in the promoter of the telomerase reverse transcriptase (TERT) gene, which encodes the catalytic subunit of telomerase. The mutation creates a new binding motif for Ets transcription factors and ternary complex factors (TCFs) near the transcription start and, in reporter gene assays, caused up to twofold increase in transcription. We then screened the TERT promoter in sporadic melanoma and observed recurrent ultraviolet signature somatic mutations in 125 of 168 (74%) of human cell lines derived from metastatic melanomas, 45 of 53 corresponding metastatic tumor tissues (85%), and 25 of 77 (33%) primary melanomas. The majority of those mutations occurred at two positions in the TERT promoter and also generated binding motifs for Ets/TCF transcription factors.

The identification of germline mutations that cosegregate with disease in cancer-prone families often provides genetic and mechanistic insights into the more common, sporadically arising cancers. In a study of cutaneous melanoma, the most malignant skin cancer, we investigated a large pedigree with 14 related melanoma patients who were not carriers of germline mutations in CDKN2A or CDK4, two known melanoma genes (Fig. 1). Multipoint linkage analysis showed a possible 2.2-Mb linkage region on chromosome 5p with maximal logarithm of the odds ratio for linkage scores of 2.35 at rs1379917 and 2.45 at rs1968011. Target-enriched high-throughput sequencing (HTS) of the region was carried out on constitutional DNA from the four affected and four unaffected members of the family with an average coverage between 55- and 108-fold (table S1) (1). The HTS data revealed a single promoter variant, three intronic variants, and three nongene variants previously unknown and unique to the DNA sequences of the affected individuals (table S2). The disease segregating variants, seven in total, were validated by Sanger sequencing of DNA from the individuals sequenced by HTS and of DNA from additional unaffected members of the family. The new variants were also detected in an unaffected member (754, table S3), who was 36 years old and carried multiple nevi. DNA from affected individuals other than those sequenced by HTS was not available for testing.

Fig. 1

Pedigree of melanoma-prone family. Four generations were affected by melanoma (solid symbols; circles represent females, and squares represent males). After linkage analysis carried out on 15 family members (L), HTS was performed on four affected and four unaffected individuals (boxed samples). A mutation in the TERT promoter was identified in all affected members and one unaffected individual (stars). Strikethrough symbols indicate deceased individuals. Two-digit numbers are age at onset of melanoma and age at death; Unk, unknown; Rs, rs2853669 observed in heterozygous form; three-digit numbers, DNA available; #, affected by other cancers; and index, index patient.

Of the seven unique variants identified, one variant (T>G), was located in the promoter at –57 base pairs (bp) from ATG translation start site of the telomerase reverse transcriptase (TERT) gene. The TERT gene encodes the catalytic reverse transcriptase subunit of telomerase, the ribonucleoprotein complex that maintains telomere length. The nucleotide change in the sequence CCTGAA>CCGGAA creates a new binding motif for Ets transcription factors, with a general recognition motif GGA(A/T). Beyond the general motif for Ets transcription factors, the familial mutation also generates a binding motif, CCGGAA, for the ternary complex factors (TCFs) Elk1 and Elk4 (2, 3). To exclude the possibility that the detected promoter mutation in TERT is a common germline variant, we screened germline DNA from 140 sporadic melanoma cases and 165 healthy controls, and none carried the variant. Screening of DNA from index cases from 34 Spanish melanoma families also did not show any mutations. No carriers were found in dbSNP and the 1000 Genomes databases (data available for 18 individuals were obtained from Ensembl).

The familial mutation in the TERT promoter was in complete allelic linkage with a common polymorphism rs2853669 (G>A) at –246 bp upstream from the ATG start site (table S3). In previous work, this polymorphism was reported to disrupt an Ets binding site, and it was associated with low telomerase activity in patients with non–small cell lung cancer (4). In luciferase reporter gene assays, we found that the activity of constructs containing the mutation at –57 bp of the TERT promoter was increased 1.5-fold and 1.2-fold over the wild-type construct in Ma-Mel-86a and human embryonic kidney (HEK) 293T cells, respectively. A construct with both the TERT mutation and the variant allele of the rs2853669 polymorphism showed a 2.2-fold increase in promoter activity in Ma-Mel-86a and and 1.3-fold increase in HEK293 cells (mean from three measurements; details in supplementary text and fig. S1).

The germline occurrence of the promoter mutation, creating an Ets/TCF motif, can result in modification of TERT expression in all tissues expressing Ets/TCF. Highest staining for the TCF Elk1 protein has been reported in female-specific tissues, such as ovary and placenta. The increased expression of TCF Elk1 protein in female-specific tissues may cause gender-related differences in cancer susceptibility among carriers of the TERT mutation (5) (supplementary text). Two affected members of the family developed several different types of cancer (marked with # in Fig. 1). One affected individual presented with ovarian cancer at age 27 and melanoma at age 30. Another individual was diagnosed with melanoma at age 20; later she developed ovarian cancer, renal cell carcinoma, bladder cancer, mammary carcinoma, and finally bronchial carcinoma, leading to her death at age 50.

The mutation in the melanoma-prone family prompted us to screen melanoma cell lines derived from sporadic cases of metastatic melanoma. None of the cell lines carried the mutation detected in the family. However, we identified recurrent ultraviolet (UV)–signature mutations in the TERT core promoter in 74% (125 of 168) of the cell lines. The mutations were located within a 49-bp region starting from –100 bp upstream of the ATG start site (Table 1, Fig. 2, fig. S2, and table S4). There were two frequent mutations at –124 bp (G>A; C>T on opposite strand) and –146 bp (G>A); these mutations were mutually exclusive and occurred in 27 and 38% of cell lines, respectively. Two tandem GG>AA (CC>TT) mutations at positions –124/–125 bp and –138/–139 bp were observed at a frequency of 9%. The tandem mutation at positions –138/–139 bp could also be generated by a single-base mutation at –138 bp, because the base change at –139 bp has been reported as a rare polymorphism (rs35550267). The two most frequent single-base mutations as well as the two tandem mutations also result in the creation of Ets/TCF binding motifs.

Table 1

Most-frequent TERT core promoter mutations in screened metastatic melanoma cell lines and paraffin embedded primary tumors. A total of 169 cell lines were screened. Amplification for the TERT promoter failed for one cell line. Of 168 cell lines examined, 125 carried recurrent mutations. Of 77 primary melanomas examined, 24 carried recurrent mutations, and one carried a rare mutation (table S5). Seven rare mutations occurred at other sites in less than 2% of samples. Details of all mutations and polymorphisms are given in tables S4 and S5. Matched normal control DNA corresponding to 23 cell lines did not show mutations. For primary tumors, matched normal control DNA was not available.

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Fig. 2

The TERT core promoter in melanoma. Mutations creating Ets/TCF binding motifs were found in affected family members (–57 bp) immediately next to the transcription start site and in sporadic metastatic melanoma (–124 to –149 bp; sequence details in fig. S2). Binding sites for c-Myc (E-Box), SP1, and Ets transcription factors are known to exist in the wild-type TERT promoter. Ets2 binding was reported for Ets2 sites at –99 and –243 bp (stars) (4). The plus strand of DNA is shown.

Mutations were confirmed in 45 of 53 (85%) available metastasized tumors corresponding to the cell lines. The somatic nature of the mutations was shown by the absence of mutations in corresponding DNA from peripheral blood mononuclear cells available from 23 patients. Somatic mutations in the TERT promoter were more frequent than the BRAF mutations (53%, 90 of 169), CDKN2A alterations (50%, 84 of 169), and NRAS mutations (23%, 38 of 169; fig. S3). The occurrence of concomitant mutations in the TERT promoter and BRAF was more frequent (47%) than by random chance (40%) with an odds ratio (OR) of 3.2 [95% confidence interval (CI) 1.3 to 8.2]. Concomitant mutations in TERT, BRAF, and CDKN2A were observed in 30% of cell lines compared with the expected frequency of such occurrence of 9% (OR 5.6, 95% CI 2.4 to 13.8). The high recurrence and specificity of the TERT promoter mutations, together with the preliminary evidence from reporter assays that they have a functional effect on transcription, suggest that these mutations are driver rather than passenger events. Extensive functional studies will be required to validate this hypothesis.

The TERT promoter mutations were also detected in 25 out of 77 (33%) paraffin embedded primary melanoma tumors (Table 1 and table S5) at –124 bp (7/77; 9%) and –146 bp (5/77; 7%). Four primary tumors carried the GG>AA tandem mutations at –124/–125 bp, and eight primary tumors carried the GG>AA tandem mutations at –138/–139 bp. Reduced sensitivity to detect mutations in paraffin-embedded primary tumors because of contaminating normal cells cannot be ruled out. Primary tumors harbored five additional mutations in the TERT promoter, which were not present in metastases, and those did not generate Ets/TCF binding motifs. We also screened DNA extracted from 25 melanocytic nevi and only one carried a mutation at –101 bp, which did not create an Ets/TCF motif. For both primary tumors and melanocytic nevi, matched normal control DNA was not available for testing.

The TERT coding region has been reported to be somatically mutated in 1% of cancers (14 cancer types, 1271 unique samples) (6). Mutations creating Ets/TCF binding motifs in the TERT promoter in melanoma have not been described in earlier sequencing projects.

TCFs are a subfamily of Ets transcription factors; two members of this subfamily, Elk1 and Elk4, are downstream targets of BRAF and regulate the expression of many genes (711). Conceivably, TCF may represent a link between telomerase activity and the frequent BRAF activating mutations in melanoma (fig. S4) (12, 13). Lastly, whether TERT promoter mutations occur in other cancer types remains to be determined. We did not detect these mutations in a screen of 22 esophageal squamous cell carcinomas, but further analyses are warranted.

Supplementary Materials

Materials and Methods

Supplementary Text

Figs. S1 to S7

Tables S1 to S7

References (1433)

References and Notes

  1. Materials and methods are available as supplementary materials on Science Online.
  2. Acknowledgments: We thank Eurofins MWG Operon (Ebersberg, Germany) for sequencing service and M. Kircher and U. Stenzel for comments on sequence analysis. The study was partially supported by a grant from Deutsche Forschungsgemeinschaft (DFG). The mutation data reported in this manuscript are deposited in

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