Common telomerase reverse transcriptase promoter mutations in hepatocellular carcinomas from different geographical locations

Abstract

Aim: To determine the mutation status of human telomerase reverse transcriptase gene (TERT) promoter region in hepatocellular carcinoma (HCC) from different geographical regions.

Methods: We analyzed the genomic DNA sequences of 59 HCC samples comprising 15 cell lines and 44 primary tumors, collected from patients living in Asia, Europe and Africa. We amplified a 474 bp DNA fragment of the promoter region of TERT gene including the 1295228 and 1295250 sequence of chromosome 5 by using PCR. Amplicons were then sequenced by Sanger technique and the sequence data were analyzed with by using DNADynamo software in comparison with wild type TERT gene sequence as a reference.

Results: The TERT mutations were found highly frequent in HCC. Eight of the fifteen tested cell lines displayed C228T mutation, and one had C250T mutation with a mutation frequency up to 60%. All of the mutations were heterozygous and mutually exclusive. Ten out of forty-four tumors displayed C228T mutation, and additional five tumors had C250T mutation providing evidence for mutation frequency of 34% in primary tumors. Considering the geographic origins of HCC tumors tested, TERT promoter mutation frequencies were higher in African (53%), when compared to non-African (24%) tumors (P = 0.056). There was also a weak inverse correlation between TERT promoter mutations and murine double minute 2 single nucleotide polymorphism 309 TG polymorphism (P = 0.058). Mutation frequency was nearly two times higher in established HCC cell lines (60%) compared to the primary tumors (34%).

Conclusion: TERT promoter is one of most frequent mutational targets in liver cancer, and hepatocellular carcinogenesis is highly associated with the loss of telomere-dependent cellular senescence control.

Keywords: Cellular immortality; Hepatocellular carcinoma; Liver cancer; Promoter mutation; Telomerase reverse transcriptase; Telomerase reverse transcriptase gene.

Figure 1

Sequence chromatograms are representing telomerase reverse transcriptase promoter mutations. Locations of (C228T) and (C250T) mutations are marked with the arrow.

Figure 2

Geographic distribution of telomerase reverse transcriptase promoter mutations. Tumor samples from Africa have the highest mutation rate (53%), followed by European samples with 30%, and Asian samples with a rate of only 21%. WT: Wild type.