Telomere Biology and the Risk of Cancer

Telomeres consist of long TTAGGG nucleotide repeats and associated proteins at the ends of chromosomes that are essential for the maintenance of chromosomal integrity. In order to preserve the chromosome end, the telomerase reverse transcriptase (TERT), its RNA component (TERC) and an ordered protein complex, termed shelterin, consisting of six proteins (gene names: TERF1, TERF2, TINF2, TERF2IP, ACD and POT1) protect the telomere from aberrant end-to-end fusion and recombination. Telomeric repeats are lost with each cell division, in part due to incomplete replication of the 3' end of the chromosome. Telomeric attrition eventually results in critically short telomeres, prompting cellular senescence or cellular crisis, which result in apoptosis, genomic instability or a reduction in cellular lifespan.

Dyskeratosis congenita (DC) study:

DC is an inherited bone marrow failure syndrome (IBMFS) and cancer predisposition disorder characterized by abnormalities in telomere biology and caused by germ-line mutations in one of several genes in the telomere maintenance pathway. We recently showed that telomere length, as measured by flow cytometry-FISH was both sensitive and specific for distinguishing DC from other IBMFS.

Identifying novel genes which might account for the 60% of DC patients who currently did not have detectable mutations in DKC1, TERC or TERT was (and remains) one of the major goals of this study. We performed a linkage study with a large, extended mutation-unknown DC family, which identified TINF2 - a component of the telomere-related shelterin complex - as mutated in that and several additional families with DC. Subsequently, two additional telomere-related genes - NOP2 and NOP3 - have been identified as rare causes of an autosomal recessive variant of DC. In the CGB series, TINF2 accounts for the largest fraction of mutation-positive DC families, but 40% of DC families still have no detectable mutation. Consequently, discovery of new high-penetrance susceptibility genes as well as low-penetrance genetic modifiers of the major genes, remains a major focus of this project.

This study also focuses on comprehensive, systematic clinical phenotyping of DC patients, aimed at refining our understanding of the features which characterize this rare disorder. We have identified novel oral cavity and dental abnormalities as part of the DC spectrum of abnormalities. A comprehensive study of dysmorphology in DC is also underway. These studies will provide more specific data on genotype-phenotype interactions and aid in diagnosis of DC.

We recently performed a quantitative analysis of the reported literature on DC-related malignancy, and published the first quantitative estimates of the risks of acute leukemia, squamous cell cancers of the skin, head/neck and anogenital region in this disorder. The cumulative cancer risk in DC patients are nearly as high as those seen in patients with Fanconi anemia.

Study-Related Publications

Telomere length in target tissues:

We are conducting small, targeted methodological studies that seek to clarify intra-individual variability in telomere length, with the ultimate goal being improved understanding of comparability and reproducibility of telomere length measurement when different cell types and analytic methods are employed. Epidemiologic studies typically use DNA isolated from either blood or buccal cells, yet direct comparisons of telomere length in blood and buccal cell DNA have not been published.

In addition, there are numerous methods for measuring telomere length, including terminal restriction fragment (TRF) measurement, fluorescence in situ hybridization with flow-cytometry, (flow-FISH), which measures telomere lengths in white blood cell subsets, and quantitative PCR (Q-PCR) which measures telomere length as a ratio of telomere repeat copy number (T) to single copy gene copy number (S). Until recently, many studies of telomere length in cancer and other diseases have only evaluated the abnormal tissue, and used the TRF measurement method. Telomere length measurements by Q-PCR are now readily available for epidemiologic studies and flow-FISH is becoming more widely used. However, these studies typically measure telomere length in DNA isolated from whole blood or buffy coats, not directly in the tissue of interest (e.g., the ovary, in ovarian cancer cases). It is important to understand how telomere lengths measured in commonly used specimens (blood and buccal cells) compare with each other and with telomere lengths in a tissue at risk of cancer development (i.e., target tissue). Since Q-PCR and flow-FISH are becoming more widely used, it is also important to understand the intra-individual comparability of these techniques.

This study will address the intra-individual variability in telomere length of blood and buccal cell DNA determined by Q-PCR healthy subjects and evaluate assay reproducibility. Intra-individual differences in telomere length between specimen trios (i.e., blood, buccal cell, and fibroblast DNA) measured by Q-PCR are being evaluated in subjects from the NCI's IBMFS study. Correlations with telomere length determined by flow-FISH are also underway. We are also studying the telomere length differences between buccal cell and blood DNA in healthy controls from an ovarian cancer case-control study. This methodological study will form the basis for larger epidemiologic studies of telomere length as a risk factor for cancer and other illnesses.

Telomere length and gene variants as risk factors for specific cancers:

Telomeres, consisting of TTAGGG nucleotide repeats and a protein complex at chromosome ends, are critical for maintaining chromosomal stability. Since telomeres are essential for genomic integrity, abnormally short telomeres may contribute to genomic instability and carcinogenesis. This hypothesis is supported by observed preliminary associations between short leukocyte or buccal cell telomeres and proposed increases in the risk of several cancers, including bladder, breast, lung, and renal cancer.

Prostate Cancer

Telomeres, telomere shortening and telomerase activity have emerged as important factors in prostate carcinogenesis. The earliest phase of human prostate carcinogenesis may proceed as a consequence of chromosomal instability mediated by shortened, dysfunctional telomeres. We are analyzing data from a case-control study of prostate cancer conducted within the PLCO cohort. Telomere length was determined on ~1,200 controls and ~700 cases of advanced prostate cancer. Strong associations between telomere length and prostate cancer risk were not identified. However, longer telomeres were associated with a healthier lifestyle in study controls, suggesting that telomere length may be influenced by exogenous as well as endogenous factors. Analysis of the relationship between variants in telomere pathway genes and prostate cancer risk is underway.

Ovarian Cancer

Increased telomerase activity and shorter telomeres have been reported in ovarian cancer tissues, findings which provide a basis for hypothesizing that these abnormalities may be related to ovarian cancer risk. In a pilot study, we have investigated leukocyte telomere length in 99 women with serous ovarian adenocarcinoma and 100 age-matched cancer-free controls from the Poland population-based ovarian cancer case-control study. Cases tended to have shorter telomeres than controls. In comparison with subjects in the longest telomere length tertile, those in the middle and shortest tertiles showed stepwise increases in odds ratios. The strongest association was observed in poorly-differentiated carcinomas. This study suggests that short telomeres are associated with increased risk of serous ovarian adenocarcinoma. Plans are currently underway to pursue this observation in a larger cohort of cases.

Breast Cancer

Genomic instability, following telomere crisis, may contribute to breast cancer pathogenesis. Many genes critical in telomere biology have limited nucleotide diversity, thus, single nucleotide polymorphisms (SNPs) in this pathway could contribute to breast cancer risk. In a population-based study of 1,995 unselected breast cancer cases and 2,296 controls from Poland, 24 SNPs representing common variation in five telomere maintenance genes - POT1, TEP1, TERF1, TERF2 and TERT - were genotyped. We did not identify any significant associations between individual SNPs or haplotypes and overall breast cancer risk; however, data suggested that three correlated SNPs in TERT (-1381C>T, -244C>T, and Ex2-659G>A) may be associated with a reduced risk of breast cancer among individuals with a family history of breast cancer (odds ratios 0.73, 0.66, and 0.57, 95% confidence intervals 0.53-1.00, 0.46-0.95 and 0.39-0.84, respectively). Although our data do not support substantial overall associations between SNPs in telomere pathway genes and breast cancer risk, intriguing associations with TERT variants among women with a family history of breast cancer warrant follow-up in independent studies.

Osteogenic Sarcoma

The Bone Disease and Injury Study of Osteosarcoma (BDISO) is a relatively small, hospital-based case-control study of osteogenic sarcoma (OS) -- undertaken in collaboration with investigators from the Harvard Dental School -- that has provided both data and biological samples for a series of pilot studies exploring genetic variants in biologically-plausible candidate genes as possible OS risk modifiers. Telomere length and telomere maintenance gene SNPs are the focus of ongoing analysis. Preliminary data suggest a possible protective effect associated with specific variants in several different telomere pathway genes.

Novel genetic determinants of telomere length:

The same subjects included in the analysis of telomere length in prostate cancer described above were also part of a genome-wide association study (CGEMS, Cancer Genetic Markers of Susceptibility). In collaboration with Drs. Immaculata DeVivo and David Hunter (Harvard School of Public Health), we will add to these normal men 1,200 healthy controls from the Nurses' Health Study, who were part of the CGEMS breast cancer whole-genome scan. We are evaluating the relationship between genetic variants measured on the same genomics platform in the CGEMS GWAS and telomere length (measured in the same laboratory) among healthy controls: 1200 men and 1200 women. Lastly, interactions between genotypes affecting telomere length will be assessed in the prostate cancer cases and controls.

Population genetics of telomere genes:

We have previously shown that nucleotide diversity in genes coding for critical proteins in the telomere maintenance pathway is limited when compared with other types of genes. They are also highly conserved between species. Because these genes appear to be under evolutionary constraint, it is possible that germ-line genetic variation (i.e., single nucleotide polymorphisms, SNPs) in these genes could be a significant risk factor for cancer or other diseases. Therefore, we are expanding our studies to include evaluation of genetic variation in a larger number of genes in the pathway, and plan to evaluate the world-wide genetic diversity present in these genes. More than 30 telomere pathway genes are being analyzed in DNA samples from the Human Genome Diversity Panel (1,000 subjects from individuals around the world). We hope to gain insight into the evolutionary history of these genes regarding both potential functional implications and to facilitate more informed SNP selection in future genetic association studies.

Study-Related Publications