A decrease in the age at cancer onset and increase in cancer incidence in successive generations in Li-Fraumeni syndrome (LFS) families with germline TP53 mutations have been previously described. In the current study a possible relation was analyzed between telomere length and cancer onset in TP53 mutation carriers.
Telomere length was measured using real-time quantitative polymerase chain reaction (PCR) in 20 carriers of germline TP53 mutations and in 83 unrelated healthy individuals. According to the age at blood sampling, patients and controls were divided into 2 age groups, children and adults. Telomere length was correlated to TP53 mutation status and telomere shortening in patients to the age at cancer onset. A t-test and linear regression were used to analyze the data.
Compared with healthy controls, telomere length was significantly shorter both in the child (P = .001) and adult (P = .034) germline T53 mutation carriers. Although a statistically significant correlation between telomere shortening and the age at cancer onset was not observed, there was a trend of shorter telomeres in mutation carriers affected in childhood compared with those affected later in life. Neither cancer therapy nor sex differences were likely to affect the results.
The TP53 tumor suppressor gene encodes the p53 protein, a transcriptional regulator controlling cell cycle progression and apoptosis. In response to DNA damage or cellular stress, TP53 activity increases, initiating a cascade of events that block cell division and may lead to apoptosis. The p53 pathway is often derailed in cancer. Somatic mutations of the TP53 gene occur in over 50% of human tumors.1 Germline TP53 mutations have been reported in families with Li-Fraumeni syndrome (LFS), a rare autosomal dominant disorder characterized by predisposition to a broad spectrum of cancers (mainly soft-tissue sarcomas, osteosarcomas, brain tumors, breast, and adrenocortical carcinomas).2, 3 The penetrance and phenotypic expression of this disorder is very variable, with high heterogeneity in the age at cancer onset and tumor spectrum.2, 4 Although collection of large enough patient samples and correction for possible sampling biases is difficult in LFS, a decrease in the age at cancer onset and an increase in cancer incidence in successive generations of germline TP53 mutation carriers have been suggested.5, 6 The biological basis of anticipation in LFS remains unknown. In multiple neurodegenerative disorders and several other hereditary diseases anticipation was associated with unstable trinucleotide repeats and their progressive expansion between generations. A second mechanism of anticipation was recently identified in families with dyskeratosis congenita (DC), a multisystem disorder characterized by cutaneous abnormalities, bone marrow failure, and increased predisposition to cancer. Here anticipation was associated with progressive telomere shortening.7
Human telomeres are nucleoprotein complexes at chromosome ends, consisting of TTAGGG repeats and associated telomere binding proteins. In germ cells, telomeres range from 10 to 15 kb in length, whereas in somatic cells they are several kb shorter.8 The telomeric DNA has a 3′ single-strand overhang that loops back on the double-stranded DNA, invades the duplex, and forms a t-loop.9 Telomeres protect chromosomes from nuclease degradation and chromosome rearrangements and serve as mitotic clocks that monitor the number of cell divisions.10 During DNA replication, DNA polymerase is unable to copy the 3′ terminal segment of each DNA strand, and in most human somatic tissues chromosomes shorten with each round of cell division. This shortening ultimately leads to senescence in a p53-dependent manner.11, 12 To compensate for telomere loss, germinal cells and some stem cells employ telomerase, an enzyme consisting of an RNA component (TERC), serving as a template for additional DNA synthesis, and a protein component, the telomerase reverse transcriptase (TERT).13 Haploinsufficiency of TERC causes progressive telomere shortening in DC.7 Conversely, reactivation of telomerase is 1 of the most commonly observed features of cancer seen in more than 80% of human tumors.14 Recent cytogenetic and molecular studies have also provided strong evidence that telomere dynamics can contribute to genomic instability, particularly early in tumorigenesis.15
Interestingly, short telomeres have been described in 2 cell lines derived from LFS patients16 and in p53+/− and p53−/− knockout mice.17 In addition, the p53 protein has been shown to bind the t-loop junctions.18 The possible link between p53, telomeres, cancer predisposition, and disease anticipation prompted us to analyze telomeres in a sample of individuals with germline TP53 mutations. Our data show that the telomere length in peripheral blood cells of germline TP53 mutation carriers is shorter than that of normal individuals of the same age.
MATERIALS AND METHODS
Patients and the Control Group
Twenty germline TP53 mutation carriers from 13 cancer-prone families tested at our department were enrolled in the study. Several of these families were described previously (Table 1). Five families met the clinical criteria for LFS,2 4 families met the Birch criteria for Li-Fraumeni-like syndrome (LFL),19 3 families had a family history of cancer but did not meet any of the above criteria, and 1 patient had no family history of cancer. Eighteen patients suffered from cancer (mean age at diagnosis was 15.5 years, range, 0.5–34 years), 4 of them developed a second primary cancer, and 2 had a third primary cancer. Four mutation carriers were asymptomatic at the time of testing but 2 of them have developed tumors since then. The mean age of the patients at the time of blood sampling was 22.3 years (Table 1). The patients clearly separated into 2 discrete subgroups according to the age at sampling: children (2–16 years) and adults (26–41 years). These subgroups corresponded to the younger and older generations in the families (Table 1, Fig. 1). The sample included 6 parent-child pairs (3 mother-daughter, 1 mother-son, 2 father-son) with parent-to-child transmission of the mutation. Detailed information on cancer therapy was available for all children but only for a fraction of adults. Among the children, 4 patients were sampled for DNA testing before chemo/radiotherapy, 1 patient underwent only surgical treatment, and the remaining 4 patients were sampled after therapy (2.5 to 6.5 years after its completion) (Table 1). Four adults were sampled before development of cancer and 2 remained unaffected during the whole study. The control group consisted of 83 healthy individuals from 2 age subgroups: 33 healthy children (9 females and 24 males, mean age 8.1 years, range, 2–17 years) referred to minor surgery at our hospital, and 50 unrelated healthy adults (42 females and 8 males, mean age 36.0 years, range, 26–43 years), women attending checks at the Department of Obstetrics and Gynecology, and parents accompanying children during hospital visits. All samples were obtained with informed consent of the subjects or their parents.
Table 1. Patient Characteristics
Age at onset of cancer
Age at sampling
Family classification: LFS indicates Li-Fraumeni syndrome, LFL, Li-Fraumeni-like syndrome, FH, family history of cancer, N, no history of cancer. Sex: F indicates female, M, male. Tumor: ACT indicates adrenocortical tumor, RMS, rhabdomyosarcoma, CHS, chondrosarcoma, BR, breast cancer, OV, ovarian cancer, LS, liposarcoma, CC, colon cancer, CPC, choroid plexus carcinoma, HE, hemangioendothelioma, NB, neuroblastoma, BL, Burkitt lymphoma, TC, thyroid cancer, MB, medulloblastoma, OS, osteosarcoma. Treatment: N indicates no chemo/radiotherapy before blood sampling, Y, treatment finished before blood sampling (years).
Telomere Length Measurement by Quantitative Real-Time Polymerase Chain Reaction (PCR)
Genomic DNA was extracted from peripheral blood samples using a commercial kit (Puregene, Gentra Systems, Minneapolis, Minn). Relative telomere length was measured using a real-time quantitative PCR method described by Cawthon,20 with a revised primer set (Tel1 and Tel2) for telomere amplification21 and acidic ribosomal phosphoprotein P0 (RPLP0) gene primers (36B4) used as a single-copy gene reference.20 Each 25-μL PCR reaction included 12.5 μL Syber Green PCR Master Mix (Applied Biosystems, Foster City, Calif), primers at final concentrations: 270 nM Tel1 and 900 nM Tel2 or 500 nM each of the forward and reverse 36B4 primers, and 20 ng genomic DNA. The amplification was performed in an ABIPrism 7000 sequence detection system (Applied Biosystems) using the following conditions: 95°C for 10 minutes followed by 30 or 36 cycles for telomeres or the RPLP0 gene, respectively, of 15 seconds denaturation at 95°C and 1 minute annealing/extension at 56°C. To determine the cycle threshold (Ct) value, 2 separate PCR runs were performed for each sample and primer pair. For each run a standard curve was generated using a reference human genomic DNA (Roche Diagnostics, Nutley, NJ) diluted to 2.5 to 50 ng per well.
Calculation of the Relative T/S Ratio and Statistical Analyses
SDS v. 1.1 software (Applied Biosystems) was used for the construction of the standard curve and for the calculation of the Ct values. A point on the standard curve at a concentration corresponding to the average DNA concentration of the samples was used as a calibrator. The relative telomere/single copy gene ratio (T/S value) was calculated using the formula T/S = 2ΔΔCt, where ΔΔCt = ΔCtsample− ΔCtcalibrator, and ΔCt = Ct36B4 − CtTel. The mean values of T/S of various groups of individuals were compared using an independent sample t-test. The T/S values were plotted against the age at blood sampling for individuals from the control group and the relation between these parameters (linear regression) was generated using StatView v. 5.0 (SAS Institute, Cary, NC). The differences ΔTEL between the actual T/S value of each patient and the T/S value predicted for his/her age at sampling from the analysis of controls were calculated and used as a measure of telomere shortening in each patient. In the analysis of parent-child pairs, negative and positive values of ΔTELchild − ΔTELparent indicated shortening and lengthening, respectively, of telomeres between generations.7
The relative T/S values of patients and controls were plotted against their age at blood sampling (Fig. 1). The negative slope of the best-fitted line for the control samples clearly indicated a decrease of the telomere length with the age in the controls (R = −0.388, P < .001). The T/S values of the majority of the TP53 mutation carriers were clearly lower than those of the controls: the ΔTEL was negative for all child patients and all but 3 adult patients. The difference in the mean relative T/S values between the control group and the patient group was statistically significant both for the children (P = .001) and for the adults (P = .034) (Table 2, Fig. 2). There was no significant difference in the mean age at sampling between controls and patients in any of the 2 age categories, and no significant difference was observed between female and male relative T/S ratio among the controls or patients of any age (Table 2).
Table 2. Statistical Analysis of the Relative T/S Ratio
Four out of 9 child patients were treated by chemotherapy or both radiotherapy and chemotherapy before blood sampling. Their relative T/S values were not statistically different from 5 child patients without treatment (P = .246), whereas the mean T/S ratios of the treated and untreated children were significantly different from values obtained from control children (P = .006 and P = .026, respectively) (Table 2). Because of missing clinical data, similar analysis in the adult patients was more limited. We could only separate 4 patients who were unaffected (and thus untreated) at the time of blood sampling from the remaining 7 adult patients in whom information on treatment was missing. Between these 2 groups the relative T/S ratio was significantly lower in the unaffected patients (P = .010). Compared with controls, the relative T/S ratio was significantly lower in the unaffected patients (P = .002) but not significantly different in the remaining patients (P = .629) (Table 2).
The mean ΔTEL was −0.335 for the child patients and −0.186 for the adult patients. Although we observed a clear trend of lower ΔTEL in patients with early onset of cancer, this correlation did not reach statistical significance (R = 0.248, P = .326) (Fig. 3). The values of ΔTELchild − ΔTELparent for the 6 parent-child pairs studied (Fig. 1) were −0.308, −0.097, 0.003, 0.039, 0.212, and 0.308, with a mean of 0.026.
We describe an analysis of telomere length in germline TP53 mutation carriers. We were prompted to perform this study by several observations. Anticipation and/or cohort effect in the age at tumor onset or in disease severity were proposed in LFS.5, 6 We suggested previously that increased susceptibility to cancer in successive generations of mutation carriers may be based on some kind of cumulative damage to the genome in the germline due to gain-of-function mutations and/or reduced dosage of normal TP53 gene expression, possibly linked to the role of p53 in genome stability.5 Interestingly, short telomeres were described in 2 LFS cell lines16 and in p53+/− and p53−/− knockout mice,17 and progressive telomere shortening was identified as a second mechanism of anticipation (after triplet repeat expansions) in human hereditary disorders.7 At the same time, p53 was shown to directly associate with the t-loop junctions.18 Together, these observations suggest a possible link between p53, telomeres, tumor initiation, and anticipation in LFS.
Our data clearly show that telomere length in peripheral blood cells of germline TP53 mutation carriers is shorter than that of normal individuals of corresponding age. This difference is more pronounced in children (about a 34% decrease) than in adults (about a 19% decrease). This discrepancy between the 2 age groups does not imply that age-related telomere shortening must be slower in mutation carriers than in controls—rather, the subset of patients affected by cancer in childhood may have shorter telomeres during their whole life compared with individuals affected in adulthood. With respect to disease anticipation, in our small sample we could not show progressive telomere shortening between successive generations of mutation carriers. Of the 6 parent-child pairs studied, 2 transmissions were associated with no change, 2 with a decrease, and 2 with an increase of ΔTEL. However, we could observe a trend, although not statistically significant, of earlier onset of cancer in individuals with shorter telomeres and vice versa.
For the telomere length assessment we used a real-time quantitative PCR assay.20 Compared with the more widely used terminal restriction fragment (TRF) analysis by Southern blotting,22 this simple, inexpensive, and rapid assay from small amounts of DNA addresses only the telomeric repeat, and is thus more sensitive to variations in telomere lengths and less sensitive to any polymorphisms in subtelomeric regions.
The results of our study could be influenced by several factors. The size of the patient sample was small. LFS is a very rare disorder and the families are decimated by high penetrance and often very unfavorable course of the disease. This also meant that most germline TP53 mutation carriers available for our analysis were sampled after the occurrence of cancer and after therapy. The total absence of unaffected child carriers was due to common policy to postpone TP53 testing of children until adulthood. Literature reports show that if treatment is toxic to the hematopoietic system, the reconstitution requires an extra number of cell divisions, and may thus lead to telomere shortening.23 Such a trend was found in individuals exposed to chemotherapy of childhood solid tumors and breast cancer, but the changes were highly variable between individuals.24, 25 As different blood cell types differ in telomere dynamics and length,26 the change of their mutual proportions after therapy represents another mechanism that may influence the analysis. Of the 9 children examined in our study, all suffered from cancer, and 4 were sampled after therapy. Although telomeres in the latter subgroup were shorter, the difference was not significant, and both these subgroups had significantly shorter telomeres compared with controls. In the adult group not enough information on therapy was available, but 4 carriers were sampled before development of cancer. Interestingly, these 4 untreated adult carriers were among those with the shortest telomeres in the whole group and, paradoxically, their telomeres were significantly shorter than those of controls, whereas the difference between affected patients and controls did not reach statistical significance. Therefore, our data do not support the observation of significantly greater telomere loss in response to treatment in older patients,27 and we do not think that the telomere shortening observed in our study can be an artifact caused by cancer therapy.
Telomere length and dynamics may also differ between sexes. Due to a gender difference in telomere attrition rate in blood, women lose fewer repeats per year than men (19 bp vs 24 bp),28 and their average telomere length is larger. Other studies report larger yearly losses of 84 bp.29 The telomere length at birth is not different between sexes, and the differences have to arise later in life.30 According to another study, telomeres in women were 3.5% longer than in men, but no significant difference in the shortening rate between sexes could be identified.31 In our study, no significant difference was observed between females and males, and we think our results cannot be influenced by different sex ratios in different groups. Finally, the total of 20 TP53 mutation carriers analyzed here included 6 parent-child pairs. Two of these were father-son transmissions, the rest were maternal pairs. A significant hereditary correlation between telomere lengths in paternal but not in maternal transmissions was demonstrated.21 Although this influence is unlikely to be of significance, the data points we obtained cannot be considered strictly independent.
To establish firmly the causal links between germline TP53 mutations, short telomeres, and anticipation in the age at cancer onset in LFS families, 2 main requirements have to be met. First, haploinsufficiency for the functional TP53 allele and/or the presence of dominant-negative or gain-of-function TP53 mutations should lead to gradual and heritable telomere shortening in the germline of the lineage of heterozygous individuals. Second, shorter telomeres in the germline (and somatic cells) in successive generations should lead (in conjunction with the constitutional TP53 mutation) to a decrease in the age at cancer onset. Our data indicate that telomeres are significantly shorter in mutation carriers, and although we could not directly observe any intergenerational shortening, such a shift is very likely to happen between the normal population with a normal telomere length and the mutation carriers. Also, although not statistically significant, our data indicate a trend of earlier cancer onset in individuals with shorter telomeres. These findings, observations on human and mouse LFS models,16, 17 and multiple lines of published evidence may thus support the above scenario.
Due to the broad pleiotropy of p53 effects, multiple mechanisms can be envisaged that could individually or simultaneously influence telomere length in heterozygous cells and individuals. These may be related to cell proliferation or turnover, apoptosis, or any interaction of p53 with telomere metabolism. Deregulation of apoptosis was associated with telomere shortening in Fanconi anemia.32, 33 A role of p53 in telomere structure is supported by its localization at the t-loop both in the presence and absence of the telomere repeat binding factor TRF2. This interaction was suggested to prevent telomere loss.18 TRF2 has a crucial role in the formation of t-loop, and removal of TRF2 ‘uncaps’ telomeres, resulting in end-to-end chromosome fusions.34 A role of p53 in telomere maintenance was proposed long ago based on the observation of telomeric associations as early events in genomic instability of LFS fibroblasts.35 In some settings, inhibition of TRF2 also activates the p53/ATM pathway and induces apoptosis.34 Gene transfer of p53 inhibits telomerase activity via overexpression of p53,36 and interactions observed in vitro suggested that telomerase may be regulated by p53, down-regulation of which would in turn favor up-regulation of telomerase in tumors.37 These effects were cell type-specific and most likely indirect.38 The biological significance of telomerase regulation by p53 under physiological conditions remains to be determined. Loss of tumor suppressor responses of apoptosis or senescence due to a TP53 mutation and impaired telomere function thus confer a risk for neoplastic transformation. Although shorter telomeres are not necessarily dysfunctional, they were associated with higher radiation sensitivity, genome instability, and increased apoptosis in irradiated animals.39 Shorter telomeres also accelerated oncogenesis, especially in the early stages of cancer and on a background of tumor suppressor deficiency.40–42 On the contrary, cells without p53 function try to stabilize their telomeres, most frequently by reactivation of telomerase. Thus, telomeres can both suppress and facilitate cancer.43, 44 Investigations into DC with haploinsufficiency of telomerase provided another functional connection between defective telomeres, bone marrow failure, aging, and cancer,45 and on the genomic level telomere dysfunction was recently shown to induce numerous amplifications and deletions in regions of cancer mutation hot-spots.46
During the finalization of this article a report was published describing a study of telomere length in 20 TP53 mutation carriers based on TRF analysis.50 The authors observed shorter telomeres in affected children and adults compared with normal individuals from the same age group (approximately 14% and 21% shortening, respectively) but much smaller shortening in unaffected mutation carriers. A hypothesis was proposed that the main mechanism of increased cancer predisposition is the faster telomere attrition during the life of the mutation carriers.50 The overall lower telomere shortening compared with our study can be explained by different sensitivity of the 2 methods to changes in the telomeric repeat length. However, the ratio of shortening between children and adults is not in accord between the 2 studies, and it is not clear if the nature of the data allows clear-cut inferences about telomere dynamics during life. In conclusion, although influenced by small sample sizes and inherent high variability in telomere length, both studies clearly show that telomere length is shorter in carriers of germline TP53 mutations, and that this shortening may underlie anticipation in the age at cancer onset in LFS. The results have to be confirmed by longitudinal studies on large groups of patients and large numbers of parent-child transmissions to address the dynamics of telomere shortening during life and especially between successive generations, which is the key point to the phenomenon of anticipation. In parallel, functional studies are needed to elucidate the biological mechanisms of interaction between p53 and telomeres. It is tempting to propose telomere shortening as a potential predictor of the age at cancer onset in LFS, but high variability of telomere length may complicate this concept.
We thank Dr. Ondrej Cinek for providing control samples, Dr. Roman Krejci for statistical analyses, and Dr. Eva Fronkova for assistance with the real-time PCR technique.