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. 2017 Aug 16;12(8):e0183287.
doi: 10.1371/journal.pone.0183287. eCollection 2017.

Telomere biology and telomerase mutations in cirrhotic patients with hepatocellular carcinoma

Flávia S Donaires  1   2 , Natália F Scatena  1   2 , Raquel M Alves-Paiva  2 , Joshua D Podlevsky  3 , Dhenugen Logeswaran  3 , Barbara A Santana  2 , Andreza C Teixeira  2 , Julian J-L Chen  3 , Rodrigo T Calado  2 , Ana L C Martinelli  2
Affiliations

Telomere biology and telomerase mutations in cirrhotic patients with hepatocellular carcinoma

Flávia S Donaires et al. PLoS One. .

Abstract

Telomeres are repetitive DNA sequences at linear chromosome termini, protecting chromosomes against end-to-end fusion and damage, providing chromosomal stability. Telomeres shorten with mitotic cellular division, but are maintained in cells with high proliferative capacity by telomerase. Loss-of-function mutations in telomere-maintenance genes are genetic risk factors for cirrhosis development in humans and murine models. Telomerase deficiency provokes accelerated telomere shortening and dysfunction, facilitating genomic instability and oncogenesis. Here we examined whether telomerase mutations and telomere shortening were associated with hepatocellular carcinoma (HCC) secondary to cirrhosis. Telomere length of peripheral blood leukocytes was measured by Southern blot and qPCR in 120 patients with HCC associated with cirrhosis and 261 healthy subjects. HCC patients were screened for telomerase gene variants (in TERT and TERC) by Sanger sequencing. Age-adjusted telomere length was comparable between HCC patients and healthy subjects by both Southern blot and qPCR. Four non-synonymous TERT heterozygous variants were identified in four unrelated patients, resulting in a significantly higher mutation carrier frequency (3.3%) in patients as compared to controls (p = 0.02). Three of the four variants (T726M, A1062T, and V1090M) were previously observed in patients with other telomere diseases (severe aplastic anemia, acute myeloid leukemia, and cirrhosis). A novel TERT variant, A243V, was identified in a 65-year-old male with advanced HCC and cirrhosis secondary to chronic hepatitis C virus (HCV) and alcohol ingestion, but direct assay measurements in vitro did not detect modulation of telomerase enzymatic activity or processivity. In summary, constitutional variants resulting in amino acid changes in the telomerase reverse transcriptase were found in a small proportion of patients with cirrhosis-associated HCC.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Fig 1
Fig 1. Distribution of age-matched telomere length in healthy subjects and HCC patients by qPCR.
(A) Telomere length analysis revealed no statistical differences between HCC patients and healthy subjects analyzed by qPCR. Patients and controls were age-matched: Slope: −0.007882±0.0007139; y-intercept when x = 0: 1.080±0.02516). (B) Telomere length was longer in females than in males (**p = 0.01).
Fig 2
Fig 2. The functional consequences of variants on enzyme activity.
(A) Four non-synonymous TERT heterozygous mutations (A243V, T726M, A1062T, and V1090M) were detected in HCC patients in both forward and reverse sequences; in silico analysis was performed by CADD, PolyPhen, and SIFT to predict the impact of each variant on the structure and function of the enzyme. The telomerase enzyme is represented in its three domains: N-terminal (green), Reverse Transcriptase (blue), and C-terminal (orange) with all described mutations in red (adapted from http://telomerase.asu.edu/diseases.html#tert). (B) Analysis of TERT variants’ impact on telomerase activity using PCR-based TRAP assay: representative gel image of telomeric DNA repeats generated from wild-type (WT) and variant telomerases reconstituted in vivo. The cell lysates for TRAP assay were obtained from reconstitution of the WT, empty, or mutated TERT expression vectors in the telomerase-negative VA13 cell line cotransfected with TERC-containing vector. No telomeric DNA repeats were obtained from lysates of VA13 cells and cells transfected with the empty vector (negative controls). (C) Mean intensity (and standard error) of telomeric DNA repeats quantitated from the TRAP gels. Intensities are shown relative to the WT (set as 100%). Cell lysates were obtained from two independent transfections. The TRAP assay was performed for each transfection. (D) Analysis of TERT variants’ impact on telomerase activity and processivity using direct assay: gel image of telomeric DNA repeats generated from WT and variant telomerases reconstituted in vivo and immuno-purified. The decreased total intensity of the DNA repeat products generated by variant telomerases relative to wild-type enzyme reflects slightly impaired enzymatic activity of TERT T726M. Processivity remained similar to WT for the two variants tested (A243V and T726M). The TERT mutation D868N is a negative control, catalytically defective in one of the three essential aspartic acid residues for reverse transcription. (E) Northern blot for TERC levels from immuno-purified telomerases and Western blot for TERT expression levels in cells. Western blot performed with anti-Flag and anti-GAPDH antibodies for ectopically expressed Flag-tagged TERT and endogenous GAPDH, respectively. The greater intensity of the catalytically inactive D868N mutant was due to the presence of a 3×Flag tag in place of a single Flag present for the WT and variant TERT proteins. (F) Mean telomerase activity and processivity derived from four independent activity assays. Enzymes were purified from cell lysates from two separate transfections.
Fig 3
Fig 3. Distribution of age-matched telomere length in healthy subjects and HCC mutated patients.
Healthy subjects (n = 261) are represented as grey circles; patients carrying TERT variants (n = 4) are represented as colored circles. Telomere length given as (A) T/S ratio by qPCR (B) and kilobases (kb) by Southern blot.
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