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Abstract

  1. Top of page
  2. Abstract
  3. Patients and Methods
  4. Results
  5. Discussion
  6. References
  7. Supporting Information

Telomere shortening impairs liver regeneration in mice and is associated with cirrhosis formation in humans with chronic liver disease. In humans, telomerase mutations have been associated with familial diseases leading to bone marrow failure or lung fibrosis. It is currently unknown whether telomerase mutations associate with cirrhosis induced by chronic liver disease. The telomerase RNA component (TERC) and the telomerase reverse transcriptase (TERT) were sequenced in 1,121 individuals (521 patients with cirrhosis induced by chronic liver disease and 600 noncirrhosis controls). Telomere length was analyzed in patients carrying telomerase gene mutations. Functional defects of telomerase gene mutations were investigated in primary human fibroblasts and patient-derived lymphocytes. An increased incidence of telomerase mutations was detected in cirrhosis patients (allele frequency 0.017) compared to noncirrhosis controls (0.003, P value 0.0007; relative risk [RR] 1.859; 95% confidence interval [CI] 1.552-2.227). Cirrhosis patients with TERT mutations showed shortened telomeres in white blood cells compared to control patients. Cirrhosis-associated telomerase mutations led to reduced telomerase activity and defects in maintaining telomere length and the replicative potential of primary cells in culture. Conclusion: This study provides the first experimental evidence that telomerase gene mutations are present in patients developing cirrhosis as a consequence of chronic liver disease. These data support the concept that telomere shortening can represent a causal factor impairing liver regeneration and accelerating cirrhosis formation in response to chronic liver disease. (HEPATOLOGY 2011;)

See Editorial on Page 1430

Cirrhosis formation is one of the main complications at the endstage of chronic liver disease, such as chronic hepatitis B and C virus (HBV, HCV) infection, chronic alcoholism, and chronic cholestatic disease.1-3 Cirrhosis leads to progressive liver failure and portal hypertension. In addition, cirrhosis is the main risk factor for the development of liver cancer.4 Decompensation of liver function at endstage cirrhosis is associated with a sharp increase in mortality5 and liver transplantation represents the only possible treatment at this stage. An understanding of the molecular causes of cirrhosis formation is essential to develop new therapeutic strategies for the treatment or prevention of cirrhosis. In addition, this would help to predict individual prognosis and to optimize the timing of final intervention strategies, such as liver transplantation. The identification of genetic risk factors associated with cirrhosis formation represents one possible way to achieve these goals.6, 7

Two models of cirrhosis formation have developed. One hypothesis indicates that cirrhosis develops as a consequence of a progressive deposition of collagen and scar tissue induced by chronic inflammation and necrosis. Another, not mutually exclusive, hypothesis indicates that telomere shortening represents an underlying cause of cirrhosis.8 Telomeres form the ends of human chromosomes. The main function of telomeres is the maintenance of chromosomal stability. However, telomeres shorten as a consequence of cell division due to the “end replication problem” of DNA polymerase, processing of telomeres during S-phase of cell cycle, and the absence of telomerase expression in most somatic tissues.9 Telomere shortening limits the proliferative life span of human cells to 50-70 cell doublings by induction of a permanent cell cycle arrest (replicative senescence) in response to telomere dysfunction.10, 11 Previous studies have shown that telomere shortening also limits the life span of primary human hepatocytes.12

Studies of human cirrhosis have demonstrated that telomere shortening is a general marker of cirrhosis formation correlating with an accumulation of senescent hepatocytes.13, 14 In addition, studies on telomerase-deficient mice have provided the first experimental evidence that telomere shortening limits the regenerative response to acute and chronic liver injury, accelerating the formation of liver fibrosis and steatosis.15, 16 Together, these studies have led to the telomere model of cirrhosis formation, indicating that chronic liver diseases increase the rate of cell turnover, thus leading to accelerated telomere shortening and regenerative exhaustion.8, 17 In agreement with this hypothesis, it has been recognized that proliferative activity declines after long latencies of chronic liver disease and this decline was associated with the progressive formation of disease.18

Genetic studies have proven that mutations in telomerase are the underlying cause of accelerated telomere shortening and organ failure in some rare human diseases, including some forms of dyskeratosis congenita (DKC)19 and aplastic anemia.20 In addition, 10% of the cases of familial idiopathic lung fibrosis are associated with telomerase mutations.21, 22 In most of these cases heterozygous mutations were found in either the RNA (TERC) or protein component (TERT) of telomerase. Interestingly, familial cases of idiopathic lung fibrosis and bone marrow failure also showed an increased frequency of unexplained liver pathologies, including fibrosis, inflammation, macrovesicular steatosis, and hepatic nodular regeneration.23-25 Some of these patients carried mutations in telomerase genes.25 However, these cases are extremely rare and it remains an open question whether telomerase mutations occur at increased frequency in patients who develop cirrhosis as a consequence of chronic liver disease. It is possible that telomerase mutations would impair the regenerative reserves of hepatocytes in the context of chronic liver damage. Accordingly, an increased frequency of telomerase mutations could be associated with cirrhosis induced by chronic liver diseases.

Here, we sequenced the TERT and TERC genes in a cohort of 1,121 individuals, 521 patients with liver cirrhosis and 600 controls. The analysis revealed a significantly increased frequency of telomerase mutations in cirrhosis patients (14 heterozygous, two homozygous allelic variants in 521 individuals; allele frequency 0.017) compared to controls (three heterozygous sequencing variants in 600 individuals; 0.003, P value 0.0007; Relative risk [RR] 1.859; 95% confidence interval [CI] 1.552-2.227). Cirrhosis-associated telomerase mutations showed functional defects and were associated with the evolution of disease complications. Together, these data provide the first demonstration of a broad involvement of telomerase mutations in the evolution and progression of cirrhosis in response to chronic liver injury. The finding could impact on the future development of molecular therapies and surveillance programs in patient with chronic liver disease.

Patients and Methods

  1. Top of page
  2. Abstract
  3. Patients and Methods
  4. Results
  5. Discussion
  6. References
  7. Supporting Information

Patients.

A total of 1,121 individuals were recruited for the current study. Among them, 521 patients were diagnosed with liver cirrhosis; 600 controls were either healthy individuals (n = 473) or patients with chronic HCV infection who did not develop cirrhosis during follow-up (average time of follow-up: 21 years, n = 127). We included the group of hepatitis C carriers who did not progress towards liver cirrhosis because this cohort provides an important control indicating that telomerase mutations do associate with the development of cirrhosis and not with the occurrence of chronic liver disease per se.

Subjects were recruited from (1) the Liver Unit, Hôpital Jean Verdier in Bondy Cedex, France, (2) the Department of Gastroenterology, Hepatology and Endocrinology of Hannover Medical School in Hannover, Germany, (3) the Henriettenstiftung Hannover, Germany, (4) the Institute for Clinical Transfusion Medicine and Immunogenetics, DRK Blood Donor Service Baden-Württemberg-Hesse, University of Ulm, and (5) the Peking Union Medical College Hospital, Chinese Academy of Medical Sciences. The study was approved by the local Institutional Review Boards and written informed consent was obtained from all subjects. The study was designed in accordance with the principles of the Declaration of Helsinki and patient data were evaluated anonymously.

Polymerase Chain Reaction (PCR) and Sequencing.

We manually amplified and sequenced hTERT and hTERC from genomic DNA prepared from peripheral blood cells and liver samples using PCR amplification.

Telomere Restriction Fragment (TRF) Length Analysis.

Telomere length was determined by southern blotting and quantitative PCR. Southern blot was carried out as described.13

Cell Cultures.

TERT point mutations (hTERT p.P65A, p.P380S, p.G1109R, Supporting Table 2) were generated in the pMSCV retroviral vector containing the wildtype hTERT complementary DNA (cDNA) using the QuickChange site-directed mutagenesis Kit (Stratagene). The wildtype and dominant-negative hTERT cDNAs were kindly provided by Robert Weinberg (Whitehead Institute, MIT). Human BJ fibroblasts (American Tissue Culture Collection, ATCC) were infected with the indicated retroviral pMSCV neo constructs and cultured in Dulbecco's minimal essential medium (Gibco) supplemented with 10% fetal bovine serum (Sigma) and 1% penicillin-streptomycin in 5% CO2 / 20% O2 at 37°C. Lymphocytes were isolated and immortalized from fresh EDTA blood as described.26

Analysis of Telomerase Activity.

Telomerase extraction and telomere repeat amplification protocol (TRAP) assays were performed using the TRAPeze Telomerase Detection System (Millipore) according to the manufacturer's instructions.

Statistical Analysis.

Statistical analysis was performed using Microsoft Excel and GraphPad Prism software. A chi-square test was used to calculate P values in Table 1. Linear regression analysis was used in Fig. 3A and unpaired Student's t test was used in Fig. 3B,D,E,G. In all assays, P values of less than 0.05 or 0.001 were considered statistically significant or highly significant, respectively.

Table 1. Distribution of Telomerase Gene Mutations in the Cirrhosis and Control Groups
GeneNucleotide Variants (mRNA)Amino Acid Variants (Codon)No. of Heterozygotes; No. of Homozygotes (Allele Frequency)χ-Square Test (P Value)
Study Group (n=521)Control Group (n=600)
  • The table depicts mutations that lead to amino acid changes in the TERT gene or are below 1% in the control cohort of the current study and in previous studies. Amino acids are indicated by single-letter abbreviations. Significance (P value) was determined by chi-square test. The cumulative frequency of gene variants was significantly higher in patients with liver cirrhosis (cumulative allele frequency 0.017) than in noncirrhosis controls (0.003, P = 0.0007). Furthermore, the allele frequency of the p.G1109R mutation in the TERT gene is significantly higher in patients (allele frequency 0.008) than in controls (0; P = 0.0072).

  • *

    Highlights novel variants that have not been previously reported.

TERCr.156C>A*1;0 (0.001)0;0 
 r.244C>U*1;0 (0.001)0;0 
 r.264G>A*0;01;0 (0.001) 
TERT 1c.37C>A*p.L13M1;0 (0.001)0;0 
 c.40C>A*p.L14M1;0 (0.001)0;0 
 c.193C>Gp.P65A1;0 (0.001)0;0 
TERT2c.340A>T*p.E113V1;0 (0.001)0;0 
 c.1138C>T*p.P380S1;0 (0.001)0;0 
 c.1336C>A*p.R446S0;02;0 (0.002) 
TERT 10c.2638G>T*p.A880S1;0 (0.001)0;0 
 c.2645C>T*p.T882I1;0 (0.001)0;0 
TERT 16c.3325G>A*p.G1109R4;2 (0.008)0;00.0072
 c.3346G>C*p.E1116Q1;0 (0.001)0;0 
Total  14;2 (0.017)3;0 (0.003)0.0007

Results

  1. Top of page
  2. Abstract
  3. Patients and Methods
  4. Results
  5. Discussion
  6. References
  7. Supporting Information

Telomerase Gene Mutations in Cirrhosis Patients.

Sequence analysis was carried out on DNA samples from a total of 1,121 individuals, 521 patients with cirrhosis and a control cohort of 600 individuals (Table 1). In the cirrhosis group, chronic HCV infection was the main cause of cirrhosis followed by HBV infection and chronic alcoholism (Fig. 1). The control samples were derived from healthy individuals (n = 473) or patients with chronic HCV infection who did not develop cirrhosis during follow-up (n = 127, average time of follow-up: 21 years).

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Figure 1. Distribution of etiologies of liver cirrhosis. The pie charts show the distribution of etiologies of chronic liver disease in the complete study cohort (A) and in mutation carriers (B).

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Sequence analysis was carried out on DNA from peripheral blood cells in most of the cases. The first set of DNA samples (n = 176 cirrhosis patients and n = 54 controls) was completely sequenced (TERT exons 1-16 plus TERC; see Supporting Table 1 for primer design). Subsequent sequencing analysis (TERC and TERT exons 1 and 16 completely, TERT exons 2, 10, and 15 partially) was focused on mutated regions that were detected in the initially sequenced cohort as well as on telomerase mutation regions that were published in previous studies on other human diseases, such as DKC and idiopathic pulmonary fibrosis.21, 22, 24, 27 The sequencing analysis identified a set of TERT gene mutations leading to amino acid changes in the TERT protein as well as two mutations in the TERC (Table 1, Supporting Figs. 1, 2). These mutations have not been listed in the single nucleotide polymorphism database (http://www.ncbi.nlm.gov/projexts/SNP). The overall frequency of these gene mutations was significantly increased in the cirrhosis group (14 heterozygous, two homozygous variants in 521 individuals; allele frequency 0.017, Table 1) compared to the control group (three heterozygous sequencing variants in 600 individuals, allele frequency 0.003, P = 0.0007). Reanalysis of the cirrhosis-associated gene mutations in frozen liver biopsies of two patients verified that these telomerase germline mutations were also detectable in liver (data not shown). Subdividing the control cohort into (1) healthy controls without chronic liver disease (n = 473) and (2) chronic liver disease patients without progression toward cirrhosis (n = 127) revealed that both subgroups exhibited significantly lower allele frequencies of telomerase mutations compared to the cirrhosis group (P = 0.0021 and P = 0.0349, respectively). There was no significant difference in allele frequency of telomerase mutations between the two subgroup control cohorts.

One of the TERT gene mutations (c.3325G>A leading to an amino acid change at position p.G1109R) was found in six out of the 521 cirrhosis patients (four heterozygous mutations, two homozygous, allele frequency 0.008, Table 1) but in none of the control samples (0; P = 0.0072).

The prevalence of telomerase gene mutations was not associated with a specific ethnicity of the patients (Supporting Fig. 3) or a specific etiology of cirrhosis (Table 2, Fig. 1). Aside from these gene mutations, a number of single nucleotide polymorphisms and silent nucleotide mutations (not resulting in amino acid changes) were identified (Supporting Table 3). These gene variants were not present at different frequencies in the cirrhosis group compared to the control group. One example was the previously described c.58G>A variation in the TERC gene, which has previously been described to be associated with African ethnic origin28 and was also associated with African ethnic origin in our study.

Table 2. Clinical Characteristics of Patients Carrying TERC and TERT Mutations
No.VariantZygosityAgeSexRaceDiseaseInitial Dx of Liver CirrhosisInitial Dx of HCCComplicationsTime to DecompensationTime to LTXChild- Pugh
  1. The table shows the indicated clinical characteristics of all cirrhosis patients carrying telomerase gene mutations. Most of the patients showed a rapid progression towards endstage cirrhosis and 5/14 patients developed liver cancer. Note that the overall frequency of endstage disease characterized by advanced cirrhosis stage (CHILD B or C), HCC occurrence, conduction or evaluation of liver transplantation was 93% (13/14) in cirrhosis patients harboring telomerase mutations compared to 62% (327/507) in cirrhosis patients without telomerase mutations (P = 0.042).

1TERC C156Aheterozygous38FAsianHBVn.a.n.a.Ongoing antiviral treatmentn.a.n.a.C
2TERC C244Uheterozygous65MWhiteEtOH2005n.a.Progression of liver impairment and major portal hypertension with iterative bleeding despite complete abstinence (no LTX d/t cardiac contraindication)4 yearsn.a.B
3L13M/L14Mheterozygous54FBlackHCV2007n.a.Ongoing antiviral treatmentn.a.n.a.A
4P65Aheterozygous58FBlackHCV20032004Rapid HCC development, LTX for HCC recurrence in 2006n.a.3 yearsA
5E113Vheterozygous55MAsianHBV1995n.a.Initial Dx of HBV infection in 1985, rapid progression of liver impairment, hepatic decompensation in 199510 yearsn.a.C
6P380Sheterozygous59MAsianHCV20062006HCC occurrence and recurrence after radiofrequency ablation in 2006, on waiting list for LTXn.a.3 yearsA
7A880S/T882Iheterozygous39FAsianHBVn.a.n.a.Ongoing antiviral treatmentn.a.n.a.C
8G1109Rhomozygous52MWhiteHCV, EtOH2004n.a.Rapid progression of liver cirrhosis despite alcohol withdrawal with major atrophy of left lobe liver5 yearsn.a.C
9G1109Rhomozygous47MWhiteHCV, EtOH20022009Rapid progression of liver cirrhosis, bifocal HCC development and progression of liver impairment in 2009, on waiting list for LTX7 years7 yearsB
10G1109Rheterozygous61FWhiteHBV, HCV1994n.a.Rapid progression of liver cirrhosis, initial Dx of HBV and HCV infection in 1986, ascitic decompensation in 1994, kidney TX 1997 for mesangioproliferative glomerulonephritis, LTX in 19988 years12 yearsC
11G1109Rheterozygous85FWhiteHCVn.d.2004Progression of liver impairment and HCC development, died in 2007 of HCC recurrencen.a.n.a.B
12G1109Rheterozygous75MWhiteHCVn.d.1995Nonresponsive to interferon monotherapy in 1989, ascites, encephalopathy and bleeding of esophageal varices in 1993, chemoembolization for multilocular HCC (3 foci), LTX & revision d/t graft failure in 199512 years14 yearsC
13G1109Rheterozygous50FWhiteHCV2004n.a.Died in 2004 d/t variceal bleedingn.a.n.a.B
14E1116Qheterozygous43MAsianHBV1997n.a.Rapid progression of liver impairment, hepatic decompensation in 2004 (portal hypertension with iterative bleeding)7 yearsn.a.C

Localization of Cirrhosis-Associated Telomerase Mutations.

Together, these results indicated that telomerase gene mutation, but not polymorphic gene variants, were associated with the evolution of cirrhosis. The cirrhosis-associated TERC gene mutation (r.156C>A) was located in the pseudoknot domain and the second cirrhosis-associated TERC gene variant (c.244C>T) was located in the paired P5 region of the CR4/CR5 domain of the TERC gene, in close proximity to the recently identified r.323C>T mutation that was associated with bone marrow failure (Fig. 2A).29 Three cirrhosis-associated TERT gene mutations were located in Exon 1 (c.37C>A, c.40C>A, and c.193C>G) (Fig. 2B, Table 1, Supporting Fig. 1). Previous studies have shown that alterations at the N-terminus of TERT can affect the ability of TERT to maintain telomere length in cell culture models.30 The cirrhosis-associated c.37C>A and c.40C>A mutations have not previously been identified; the c.193C>G has been identified in a patient with acute myeloid leukemia.31 In addition, two cirrhosis-associated mutations in Exon 2 and two mutations in Exon 16 of the TERT gene were new mutations that have not been identified in previous studies. The most common mutation in cirrhosis patients was located in the c-terminus of the TERT gene (the c.3325G>A mutation resulting in the amino acid change p.G1109R) (Fig. 2B, Table 1, Supporting Fig. 1G). This mutation is located next to an amino acid change (p.T1110M), which has previously been associated with pulmonary fibrosis.21

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Figure 2. Localization of cirrhosis-associated telomerase gene mutations. Localization of cirrhosis-associated telomerase gene mutations (A) in the TERC gene locus (figure adapted from Podlevsky et al.36) (B) in the TERT gene. Amino acid positions of the N-terminal, reverse transcriptase, and C-terminal domains (amino acids are denoted by single-letter abbreviations). The N-terminal region domains (GQ, VSR, CP, QFP), the central reverse transcriptase motifs (T, 1, 2, A, IFD, B, C, D, E), and the C-terminal region domains (E-I to -IV) are indicated as well. The hypomorphic telomerase reverse transcriptase variants characterized in this study (coding variants only) are displayed in boxes above the corresponding amino acid sequence. The untranslated region (3-UTR) of the gene with respective variants found in the study cohort is also shown.

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Clinical Characteristics of Cirrhosis Patients with Telomerase Mutations.

The mean age of the patients with telomerase mutation was 55.8 years (Table 2) compared to a mean age of 58.7 years in the group of cirrhosis patients without telomerase mutations. There was no obvious overrepresentation of a specific disease etiology or ethnicity in the mutation carriers compared to the controls (see above). However, many of the mutation carriers showed a rapid progression of chronic liver disease toward cirrhosis of liver cancer development (Table 2). Specifically, the frequency of endstage liver disease (defined as Child B or C cirrhosis, hepatocellular carcinoma [HCC] occurrence, and conduction or evaluation for liver transplantation) was significantly higher in the group of cirrhosis patients harboring telomerase mutations (93%, 13 out of 14) compared to cirrhosis patients without telomerase mutations (62%, 327 out of 507, P = 0.042), indicating that telomerase mutations may induce a more aggressive progression of chronic liver disease—a hypothesis that should be addressed in future prospective trials.

Functional Analysis of Cirrhosis-Associated Telomerase Mutations.

Cirrhosis patients carrying telomerase gene mutations had significantly shorter telomeres in peripheral blood cells compared to control patients without telomerase gene mutations (Fig. 3A, P = 0.0004). A significant reduction of telomerase activity was detectable by TRAP assay for three of the investigated, cirrhosis-associated telomerase mutations (p.P65A: P < 0.0001, p.G1109R: P = 0.0035, and p.P380S: P < 0.0001), including the most frequent mutation p.G1109R (Fig. 3B,C). Studies on telomerase-negative, human fibroblasts revealed that the cirrhosis-associated TERT mutations (p.P65A and p.G1109R) did not lose immortalization capacity when overexpressed (Fig. 3D). However, proliferation rates of fibroblast lines expressing these TERT mutants were significantly reduced compared to cells expressing wildtype TERT (Fig. 3D). Similarly, Epstein-Barr virus-immortalized, primary lymphocytes from two patients with a homozygous p.G1109R TERT mutation showed an impaired proliferation capacity (Fig. 3E) and shortened telomeres (Fig. 3F,G) compared to immortalized lymphocytes from cirrhosis patients with wildtype TERT. γH2Ax staining of liver sections of six cirrhosis mutations carriers and eight liver cirrhosis patients without telomerase mutation did not reveal any significant difference in the percentage of γH2Ax-positive hepatocytes, a marker for the induction of DNA double-strand breaks (data not shown).

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Figure 3. Functional analysis of cirrhosis-associated telomerase gene mutations. (A) The scatterplot shows the telomere length in total white blood cells from peripheral blood in correlation with the age of liver cirrhosis patients with and without telomerase gene mutations. Linear regression analysis revealed a significant difference between the two groups (P = 0.0004). Telomere length of p.G1109R TERT mutation carriers is highlighted (green triangles). (B-D) Telomerase-negative human fibroblasts (BJ) that were infected with retroviruses carrying (1) wildtype TERT (wt hTERT); (2) a previously described, dominant-negative TERT mutation (DN hTERT)37; and (3) cirrhosis-associated TERT mutations (p.P65A, p.P380S, or p.G1109R). (B,C) Telomerase activity was measured after infection of telomerase-negative BJ-fibroblasts with wildtype TERT or the indicated TERT-mutant. The histogram shows mean values with error bars indicating standard error of the mean. Note that a significant reduction of telomerase activity was seen for three cirrhosis-associated TERT mutations relative to wildtype TERT (unpaired Student's t test, P < 0.0001 for p.P65A, P = 0.0035 for G1109R, and P < 0.0001 for p.P380S). (C) Gel photograph showing a TRAP assay to determine telomerase activity in serially diluted protein samples of fibroblasts transfected with either wildtype or mutated hTERT in protein concentrations of 50, 100, 150, and 200 ng/μL plus one heat-treated test extract for each cell line. The arrow points to the internal PCR control, the intensity of the ladder (extension products) corresponds to telomerase activity. (D) The line graph shows growth curves of telomerase-negative, human fibroblasts (BJ) that were stably infected with the indicated TERT expression constructs. Note that the two tested, cirrhosis-associated TERT mutations (p.P65A, p.G1109R) did not lose immortalization potential but showed significantly reduced growth rates compared to wildtype TERT infected cells (unpaired Student's t test, P < 0.005, day 59 and day 157). (E-G) B lymphocytes were isolated from EDTA blood of homozygote p.G1109R mutation carriers (age 49.5 ± 2.5, n = 2) and healthy controls (age 56.5 ± 5.5, n = 2) and immortalized by Epstein-Barr virus infection. (E) The line graph shows growth curves of immortalized polyclonal B cell lines in suspension culture. Note that immortalized lymphocytes of homozygous p.G1109R mutation carriers showed significantly reduced growth rates compared to immortalized lymphocytes of controls (unpaired Student's t test, P = 0.004, day 18). (F) Representative southern blot to determine telomere length of immortalized B lymphocytes derived from EDTA blood of homozygous p.G1109R mutation carriers and healthy controls. (G) The histogram shows a reduction in telomere length of immortalized B lymphocytes derived from homozygous p.G1109R mutation carriers (3.47 kb ± 0.67 kb) compared to healthy controls (unpaired Student's t test, 8.15 ± 0.08, P = 0.0198).

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Discussion

  1. Top of page
  2. Abstract
  3. Patients and Methods
  4. Results
  5. Discussion
  6. References
  7. Supporting Information

The current study provides the first evidence that telomerase mutations are associated with the evolution of cirrhosis as a consequence of chronic liver disease. The total allele frequency of telomerase mutations in cirrhosis patients was 0.017, compared to 0.003 in healthy controls or hepatitis C patients without fibrosis progression (P = 0.0007). Furthermore, subgroup analysis (number of identified mutations in healthy controls compared to the cirrhosis group: P = 0.0021, number of mutations in chronic liver disease patients without cirrhosis compared to the cirrhosis group: P = 0.0349) reconfirmed that the identified telomerase mutations are associated with cirrhosis but do not occur in healthy controls or patients with indolent HCV infection. To our knowledge, these data represent the first association of telomerase mutations with the evolution and progression of cirrhosis in response to chronic liver injury. The ethnic group in our study consisted mainly of whites (70.1%). It remains to be analyzed whether telomerase mutations occur with similar frequency in other ethnic groups.

The study shows that cirrhosis-associated TERT mutations exhibit an impaired function compared to wildtype TERT. Cirrhosis-associated telomerase mutations result in reduced telomerase activity, impaired telomere maintenance, and reduced growth rates of fibroblasts and lymphocytes. Moreover, a reduction in telomere length was seen in peripheral blood and immortalized lymphocytes of mutation carriers compared to controls. We did not see any significant difference in the percentage of γH2Ax-positive hepatocytes between liver cirrhosis patients with and without telomerase mutations. This, however, does not argue against an involvement of telomerase mutations in cirrhosis. Previous studies have demonstrated that telomere shortening and senescence are general signs of cirrhosis induced by different etiologies.13, 14 We propose that telomerase mutations can lead to accelerated telomere shortening, thus shortening the time to progression of chronic liver disease toward cirrhosis. In addition, telomerase mutations may have extratelomeric effects influencing disease progression. Recent studies have revealed telomere length-independent effects of TERT in regulating the transcriptional function of the Wnt-signaling pathway and stem cell activity in mice.32 It remains to be seen whether cirrhosis-associated TERT mutations show defects in these noncanonical TERT-pathways and whether TERT mutations affect the latency of cirrhosis development in patients with chronic liver disease.

Several lines of argument indicate that the current study likely underestimated the true rate of telomere-related mutations in cirrhosis: (1) other components of the telomerase enzyme complex have been shown to be essential for telomerase activity33 and mutations in one of these components (dyskerin) have been associated with telomere shortening and human disease34; (2) mutations in telomere-binding proteins can impair the function of telomeres, and a first report has recently linked mutation in the telomere-binding protein, TIN2, to the evolution of aplastic anemia35; (3) mutations in noncoding sequences could impair the expression of both TERT and TERC. Together, we anticipate that full coverage sequencing (coding and noncoding sequences) of all known components of the telomerase enzyme complex and the shelterin complex of telomere-binding proteins will reveal an increasing percentage of telomere-related mutations in human cirrhosis.

It remains to be analyzed whether TERT mutations influence the development of HCC. Cirrhosis represents one of the main risk factors for HCC formation. There is ample evidence that telomere shortening increases the risk of cancer formation in humans by inducing chromosomal instability. Therefore, TERT mutations that increase the risk of cirrhosis formation may increase the risk of HCC. In contrast, tumor cells need to activate telomere maintenance mechanisms in order to gain immortal growth capacity—a prerequisite for tumor formation. Thus, it is possible that TERT mutations could also protect individuals from cancer formation. In the current cohort, 5/14 (36%) cirrhosis patients with telomerase mutations (including one patient with a homozygous p.G1109R TERT mutation) developed HCC, indicating that telomerase mutations did not prevent cancer formation.

Together, the current findings represent the first evidence for telomerase mutations in cirrhosis induced by chronic liver disease. The results indicate that telomerase mutations represent confounding factors increasing the risk of cirrhosis formation in the context of chronic liver disease. The study results improve our understanding on the molecular causes of cirrhosis. These findings will influence the future development of molecular therapies for cirrhosis patients and may also have an impact on future surveillance programs and decision making in treatment of patients with chronic liver disease and cirrhosis.

References

  1. Top of page
  2. Abstract
  3. Patients and Methods
  4. Results
  5. Discussion
  6. References
  7. Supporting Information

Supporting Information

  1. Top of page
  2. Abstract
  3. Patients and Methods
  4. Results
  5. Discussion
  6. References
  7. Supporting Information

Additional Supporting Information may be found in the online version of this article.

FilenameFormatSizeDescription
HEP_24217_sm_suppinfofig1-1.tif1519KSupporting Fig. 1. (A-F). Representative forward and reverse sequencing chromatograms from the indicated gene regions. The respective amino acid substitutions are indicated in the lines above the chromatograms: (A) wild-type TERT exon 1 (left side) and a patient with a heterozygous c.37C>A and c.40C>A variant (right side); (B) wild-type TERT exon 1 (left side) and an individual with a heterozygous c.193C>G variant (right side); (C) wild-type TERT exon 2 (left side) and a patient with a heterozygous c.1138C>T variant (right side); (D) wild-type TERT exon 2 (left side) and an individual with a heterozygous c.1336C>A variant (right side); (E) wild-type TERT exon 10 (left side) and a patient with a heterozygous c.2638G>T and c.2345 C>T variant (right side); (F) wild-type TERT exon 16 (left side) and an individual with a heterozygous c.3346G>C variant (right side); (G) wild-type TERT exon 16 (left side), a patient with a heterozygous c.3325G>A variant (middle), and a patient with a homozygous c.3325G>A variant (right side). The homozygous nature of the mutation is evident by the presence of a mutant allele sequence only while the heterozygous form shows both wild-type and mutant allele sequences.
HEP_24217_sm_suppinfofig1-2.tif2237KSupporting Information Figure 1-2
HEP_24217_sm_suppinfofig2.tif1496KSupporting Fig. 2. The pie charts show the overall allele frequency (left sides) and the distribution (right side) of telomerase gene mutation in (A) cirrhosis patients and (B) non-cirrhotic controls.
HEP_24217_sm_suppinfofig3.tif1093KSupporting Fig. 3. The pie charts show the distribution of ethnicities of the complete study cohort (A) and mutation carriers (B).
HEP_24217_sm_suppinfotab1.doc59KSupporting Table 1. Primers used for sequencing of indicated exons of TERT and of the TERC gene
HEP_24217_sm_suppinfotab2.doc34KSupporting Table 2. Primers used to generate expression constructs for cirrhosis-associated TERT-mutations using site-directed mutagenesis
HEP_24217_sm_suppinfotab3.doc139KSupporting Table 3. Distribution of hypomorphic telomerase reverse transcriptase variants in the study and control groups

Please note: Wiley Blackwell is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.