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Int J Mol Epidemiol Genet. Jan 1, 2011; 2(1): 19–29.
Published online Nov 23, 2010.
PMCID: PMC3077235
Telomere length and variation in telomere biology genes in individuals with osteosarcoma
Lisa Mirabello,1 Elliott G Richards,1 Linh M Duong,1,2 Kai Yu,1 Zhaoming Wang,3 Richard Cawthon,4 Sonja I Berndt,1 Laurie Burdett,3 Salma Chowdhury,3 Kedest Teshome,3 Chester Douglass,5 Sharon A Savage,1 and the National Osteosarcoma Etiology Study Group6
1Division of Cancer Epidemiology and Genetics, National Cancer Institute, National Institutes of Health, Rockville, MD, USA
2Currently with the Division of Cancer Prevention and Control, National Center for Chronic Disease Prevention and Health Promotion, Centers for Disease Control and Prevention, Atlanta, GA, This work was completed while Ms. Duong was a fellow at NIH, USA
3Core Genotyping Facility, National Cancer Institute, SAIC-Frederick, Gaithersburg, MD, USA
4Department of Human Genetics, University of Utah, 15 N 2030 E, Room 2100, Salt Lake City, UT, USA
5Department of Epidemiology, Harvard School of Public Health, Boston, MA, USA
6The National Osteosarcoma Etiology Study Group was represented by Michael A. Simon (University of Chicago), Marc C. Gebhardt (Massachusetts General Hospital), Mark T. Scarborough (Shands Medical Center, University of Florida), Steven Gitelis (Rush Presbyterian and St. Lukes Medical Center), Jeffrey J. Eckardt (University of California at Los Angeles School of Medicine), James R. Neff (Nebraska Health System), Michael J. Joyce (Cleveland Clinic Foundation), Martin Malawer (Washington Cancer Institute), Michael McGuire, (Creighton University), and H. Clarke Anderson (University of Kansas Medical Center)
Please address correspondence to: Yong Zhu, Department of Epidemiology and Public Health, Yale University School of Medicine, New Haven, CT 06520. Phone: (203) 785-4844; Fax: (203) 737-6023; Email: yong.zhu/at/yale.edu
Received October 15, 2010; Accepted November 18, 2010.
Osteosarcoma, the most common primary bone tumor, occurs most frequently in adolescents. Chromosomal aneuploidy is common in osteosarcoma cells, suggesting underlying chromosomal instability. Telomeres, located at chromosome ends, are essential for genomic stability; several studies have suggested that germline telomere length (TL) is associated with cancer risk. We hypothesized that TL and/or common genetic variation in telomere biology genes may be associated with risk of osteosarcoma. We investigated TL in peripheral blood DNA and 713 single nucleotide polymorphisms (SNPs) from 39 telomere biology genes in 98 osteosarcoma cases and 69 orthopedic controls. For the genotyping component, we added 1363 controls from the Prostate, Lung, Colorectal, and Ovarian Cancer ScreeningTrial. Short TL was not associated with osteosarcoma risk overall (OR 1.39, P=0.67), although there was a statistically significant association in females (OR 4.35, 95% Cl 1.20-15.74, P=0.03). Genotype analyses identified seven SNPs in TERF1 significantly associated with osteosarcoma risk after Bonferroni correction by gene. These SNPs were highly linked and associated with a reduced risk of osteosarcoma (OR 0.48-0.53, P=0.0001-0.0006). We also investigated associations between TL and telomere gene SNPs in osteosarcoma cases and orthopedic controls. Several SNPs were associated with TL prior to Bonferroni correction; one SNP in NOLA2 and one in MEN1 were marginally non-significant after correction (Padj=0.057 and 0.066, respectively). This pilot-study suggests that females with short telomeres may be at increased risk of osteosarcoma, and that SNPs in TERF1 are inversely associated with osteosarcoma risk.
Keywords: Osteosarcoma, telomere, single nucleotide polymorphism, epidemiology, telomere length
Osteosarcoma is the most common primary bone tumor; it occurs mainly in adolescents and young adults [1]. The etiology of osteosarcoma is not well understood. Epidemiologic studies suggest that height [2] and/or birth weight [3] may be associated with risk, but the data are inconsistent [4,5]. Osteosarcoma occurs at increased frequency in certain hereditary cancer predisposition syndromes [6], such as Li-Fraumeni syndrome, Werner syndrome, and Rothmund Thomson syndrome, but the genetic contribution to apparently sporadic osteosarcoma is not well understood.
Studies of common genetic variants in osteosarcoma have identified several potential candidate genetic variants. Positive associations between osteosarcoma and single nucleotide polymorphisms (SNPs) have been noted with the Fokl genotype of the vitamin D receptor gene [5], and with SNPs in IGFR2 [7], FAS [8], MDM2 [9], and TGFBR1 [10]. An inverse association between osteosarcoma and a TNF promoter variant (-238 SNP) was noted [11]. Null, or equivocal studies of the estrogen receptor and collagen lα1 genes and TP53 have also been reported [5,12].
Telomere epidemiology is a growing field which seeks to study associations between telomere length (TL) and disease or environmental exposures. Telomeres are comprised of (TTAGGG)n nucleotide repeats and a protein complex at chromosome ends, and are key components in the maintenance of chromosomal stability [13]. Several studies suggest that blood or buccal cell -derived DNA TL is associated with certain cancers, for example, bladder cancer [14-16], eso-phageal cancer [17,18], and gastric cancer [19,20]. However, TL was not associated with prostate [21] orcolorectal [22] cancer risk.
Telomere dysfunction has been shown to result in numerous chromosomal abnormalities, including aneuploidy and translocations [23]. Somatic osteosarcoma cells often have significant chromosomal aneuploidy suggestive of underlying DNA instability [24]. While most cancer cells overcome cellular crisis through the upregulation of telomerase, the enzyme that extends nucleotide repeats, osteosarcoma cells use the alternative lengthening of telomeres mechanism (ALT) [25,26]. Although a small study did not identify mutations in telomere biology genes in osteosarcoma cell lines [27], no one has examined whether common germline variants influence the risk of developing osteosarcoma.
In this study, we hypothesized that TL and/or common germline genetic variation in telomere biology genes may be associated with risk of osteosarcoma because of the chromosomal instability inherent in osteosarcoma tumors. We conducted a case-control association study of both TL in peripheral blood DNA and common SNPs from telomere biology genes as potential osteosarcoma risk factors.
Study design
The Bone Disease and Injury Study of Osteosarcoma (BDISO) is a hospital-based prospective case-control study which was conducted in the orthopedic surgery departments in 10 United States medical centers between 1994 and 2000 [3]. The study collected blood samples and questionnaire data on patients with osteosarcoma at the time of limb salvage surgery. There were no identified cases of Paget disease of the bone in this study. Orthopedic controls were derived from individuals treated for non-neoplastic conditions including benign tumors (26%) and other non-neoplastic conditions, such as inflammatory diseases, cysts, and trauma, excluding those with hip fracture or osteoporosis. Institutional review boards at each of the medical centers approved the study protocol and informed consent was obtained from all study subjects. The current analysis was limited to individuals who were self-identified whites (98 osteosarcoma cases and 69 orthopedic controls) in order to reduce potential effects of population stratification. The cases included in our study represent 79% of all cases in the BDISO with DNA available to analyze.
For the genotyping component of this study, an additional 1365 cancer-free white control subjects were selected from the Prostate, Lung, Colorectal, Ovarian (PLCO) Cancer Screening Trial [28]. Men and women, ages 55-74 years, were enrolled in the screening trial from 10 different centers in the U.S. between 1993 and 2001. All subjects included for this study were required to have completed a baseline questionnaire, provided a blood specimen, and consented to participate in etiologic studies of cancer and related diseases. Controls were limited to whites living in the continental U.S. without a diagnosis of colon adenoma or cancer at baseline. DNA was extracted from blood specimens using standard procedures. The institutional review boards at the National Cancer Institute and 10 screening centers approved the study.
Telomere length measurement
Genomic DNA was extracted from buffy coat fractions by standard procedures (Gentra Auto-pure). Relative TL was measured using a multiplexed quantitative polymerase chain reaction (Q-PCR) method previously described [29,30]. Briefly, the average, relative TL was estimated from the ratio of the telomere (T) repeat copy number to a single gene copy number (human β -globin gene; S), expressed as the T/S ratio for each sample using standard curves. All PCR reactions were performed on the Bio-Rad MyiQ Single Color Real-Time PCR detection system. TL in base-pairs (bp) for a T/S ratio of 1.0 is approximately 3.3 kb [29]. Ten blinded quality control samples were included to assess variability, and each sample was run in triplicate. The coefficients of variation (CV) within triplicates of the telomere and single-gene assay were 4.1% and 6.3%, respectively, and the CVs for repeats were 5.1% and 7.9%, respectively.
Gen otyping
743 SNPs were derived from genes which code for proteins previously shown to either directly interact with telomeric DNA or to regulate these proteins ﹛ACD, ATM, BLM, DDX1, DDX11, MCM4, MEN1, MRE11A, MYC, NBN, NOLA1, N0LA2, N0LA3, PARP1, PARP2, PIK3C3, PINX1, POT1, PRKDC, RAD50, RAD51AP1, RAD51C, RAD51L3, RAD54L, RECQL, RECQL4, RECQL5, RTEL1, TEP1, TERC, TERF1, TERF2, TERF2IP, TERT, TINF2, TNKS, TNKS2, WRN, XRCC6). Genotyping was conducted on a Custom Infin-ium® BeadChip (iSelect)™ from Illumina, Inc. The iSelect panel was created by investigators in the Division of Cancer Epidemiology and Genetics, National Cancer Institute (NCI) to target genetic variation in genes potentially important in carcinogenesis and cancer risk. Tag SNPs were identified from the HapMap CEU population assuming an r2 threshold of 0.80, using the Tagzilla module of the GLU software package (http://code.google.eom/p/glu-genetics/), across the regions of interest.
The concordance rates between 10 duplicate BDISO and 195 duplicate PLCO samples on the iSelect panel were 99.5% and 99.9%, respectively. SNPs were excluded if they had less than a 90% genotyping rate, or if they failed the Hardy-Weinberg equilibrium test or genotyping validation. Individuals with more than 10% missing genotypes were excluded.
A principal component analysis was performed using a set of 3,843 independent SNPs selected from the iSelect BeadChip (27,905 SNPs) to evaluate population substructure among the BDISO individuals and the PLCO controls. There was no evidence of significant population stratification. However, 6 BDISO individuals and 2 PLCO controls were considered genetic outliers and excluded from the genotyping analyses. Two BDISO individuals were also excluded due to missing genotype data, fora final sample size for the genotyping analyses of: 96 cases, 63 orthopedic controls, and 1363 PLCO controls.
Statistical analyses
Spearman rank correlations and general linear models were used to investigate the association between TL and age and gender in control subjects, adjusting for age or gender. TL was analyzed as a continuous and as a categorical variable. The Wilcoxon rank-sum test was used to compare TL among case and controls as a continuous variable. Logistic regression models were used to obtain the odds ratio (OR) and 95% confidence intervals (Cl) for the strength of the association between osteosarcoma and TL, adjusting for age and/or gender. TL was considered as a categorical variable by dichotomizing it at the median according to the distribution in control subjects, with longer length as the referent.
Logistic regression models were used to estimate the OR and 95% Cl for the association between osteosarcoma risk independently for each SNP, adjusting for gender. The common allele or the homozygote of the common allele was used as the referent category for the log-additive or dominant model, respectively. We evaluated the log-additive genetic model (log-additive effect of each minor allele) and a dominant inheritance model for each SNP in relationship to osteosarcoma case status. For rare SNPs, we also used the Fisher's Exact Test to evaluate the significance of the allelic associations. We conducted gene-level and pathway-level analyses based on Yu etal [31]. The gene-level analysis is a global test for the association between the outcome and a subset of SNPs within a given gene or region. The pathway-level analysis is a global test for the association between the outcome and any subset of genes within a given pathway. P-values for these analyses were estimated with 20,000 permutation steps according to the algorithm given in Yu et al [31].
Linear regression models were used to estimate the association between TL as a continuous variable and each SNP independently, adjusting for age and gender. The common allele was used as the referent category using an additive model to evaluate the additive effect of each minor allele. Bonferroni adjustments (Padj) were conducted by gene (for all SNPs in a gene) for correction of multiple tests.
Statistical power was calculated with Quanta [32] using the log-additive and dominant models, 96 cases and 1426 controls, baseline population risk of 0.0000001, and type 1 error of 0.05. For the log-additive model, power was greater than 80% for the following minor allele frequencies (MAF): MAF of 0.1 could detect an OR of 1.82 and MAF of 0.3 could detect an OR of 1.53 or higher.
We evaluated the correlation between SNPs [linkage disequilibrium (LD)] with Haploview version 4.1 [33]. Statistical analyses were performed using SAS software, version 9.1 (SAS Institute, Cary, NC), R language, and PLINK software, version 1.06 (http://pngu.mgh.harvard.edu/purcell/plink/).
Characteristics of study subjects
The characteristics of all study participants are shown in Table 1. Subjects evaluated in the TL only component of this study consisted of 98 osteosarcoma cases, median age was 19.5 years (range 7-76), and 69 orthopedic controls, median age was 18.5 (range 7-68). Osteosarcoma cases and orthopedic controls had nearly equal numbers of males and females. For the genotyping component of the study we augmented the sample size through the addition of 1,363 controls from PLCO. These individuals were older than the BDISO participants with a median age of 62.6 (range 55-75). There were more males (63.8%) than females (36.2%) in the PLCO controls. All participants were self-identified whites from the continental United States.
Table 1
Table 1
Characteristics of study subjects
Telomere length in osteosarcoma cases and controls
We measured relative TL in buffy coat DNA derived from osteosarcoma cases and orthopedic controls in BDISO to test the association between TL and osteosarcoma risk. The mean TL for osteosarcoma cases (1.997, standard deviation [SD] 0.32) and controls (2.012, SD 0.33) were not different (Pwilcoxon = 0.42). As expected, TL declined with increasing age (correlation coefficient = -0.489, P < 0.0001). TL was significantly different between male and female controls (1.93 vs. 2.11, P = 0.012), after adjusting for age, with females having longer telomeres. Due to the small sample size, we evaluated TL dichotomized at the median and compared long (above the median) to short (below the median) TL. TL was not associated with risk of osteosarcoma overall or when subjects were grouped by age (Table 2). When males and females were evaluated separately, a statistically significant association between short telomeres and osteosarcoma was noted only in females, with an OR of 4.35 (95% Cl 1.20-15.74, P=0.03).
Table 2
Table 2
Association of osteosarcoma risk with relative leukocyte telomere length dichotomized at the median
Association of genetic variation in telomere biology genes in osteosarcoma
We evaluated associations between individual SNPs in telomere biology genes and risk of osteosarcoma in the BDISO subjects, and included 1,363 controls from PLCO in order to improve statistical power. In total, 713 SNPs from 39 telomere biology genes were analyzed. These genes are described in Supplemental Table 1. Forty-one SNPs were significantly (P < 0.05) associated with osteosarcoma risk before correction for multiple tests by gene (Supplemental Table 2). There were 6 significant SNPs in PARP2, and 9 in TERF1 and TNKS. Only 7 SNPs in TERF1 remained significant after Bonferonni correction (by gene) (Table 3). They had an inverse association with osteosarcoma (OR 0.48-0.53, P = 0.0001-0.0006). These SNPs were highly correlated in our controls (r2 = 0.9-0.99).
Table 3
Table 3
Significant SNPs associated with risk of osteosarcoma after Bonferroni correction by gene (Padj) using a log-additive genetic model
We also conducted global tests by gene and functional pathway (including all telomere biology genes). Three genes were significantly associated with risk of osteosarcoma (Table 3 and Supplemental Table 2): TERF1 (Gene P = 0.0009), PARP2 (Gene P = 0.034), and TNKS (Gene P = 0.043). However, if we corrected for multiple tests (39 genes), only TERF1 remained significant (Padj = 0.035). As a whole, the telomere biology pathway was not significantly associated with osteosarcoma (P = 0.152).
Relative telomere length and genetic variation in telomere biology genes
Potential associations between TL in the BDISO subjects (n = 159) and genetic variation in the 39 telomere biology genes were also evaluated in this study. For this analysis, we combined osteosarcoma cases and BDISO orthopedic controls, because there was no primary association between TL and osteosarcoma. Linear regression models were used to estimate the effect of each SNP, and the direction of the regression coefficient corresponded to each minor allele increasing or decreasing TL. There were 20 SNPs significantly associated with TL before correction for multiple tests (P < 0.05; Table 4), including multiple SNPs in BLM, NOLA2, POT1, TEP1, and TERC. No associations remained significant after Bonferroni correction by gene; one SNP in N0LA2 and MEN1 were marginally non-significant (Padj=0.057 and 0.066, respectively).
Table 4
Table 4
Significant SNPs (P < 0.05) in telomere biology genes associated with telomere length before correction for multiple tests
Our study had three primary goals, to: 1) determine if germline TL was associated with risk of osteosarcoma, 2) identify associations between SNPs in telomere biology genes and osteosarcoma risk, and 3) determine if those SNPs were associated with TL. We hypothesized that since osteosarcoma somatic cells typically have significant chromosomal abnormalities and often use the alternative lengthening of telomeres pathway for telomere maintenance aberrations in telomere biology could contribute to osteosarcoma risk. Overall, we found that short TL was associated with osteosarcoma risk in females, SNPs in TERF1 were associated with decreased osteosarcoma risk, and that telomere biology gene SNPs were not strongly associated with TL.
TL in surrogate tissues (e.g., blood or buccal cells) has been postulated to be a biomarker of cancer risk. Several case-control studies have found statistically significant associations between shorter telomeres and risk of cancers such as bladder [14-16], esophageal [17,18], gastric [19,20], head and neck [16], lung [16,34], ovarian [35], and renal [16,36]. A few studies have also suggested that longer telomeres are associated with risk of melanoma [37], non-Hodgkin lymphoma [38], and breast cancer [39,40], although the breast cancer TL association studies have been inconsistent [39,41-43]. Null associations with TL were reported in prospective studies of prostate [21] and colorectal cancers [22]. Overall, significant differences in TL between osteosarcoma cases and controls were not identified.
Our study and others suggest that healthy females have longer telomeres than males [44-47]. We also found a statistically significant association between shorter TL and risk of osteosarcoma in females. This association was not noted in males or in the combined male-female dataset. This gender difference might reflect the effects of estrogen on telomere dynamics, possibly through the activation of the hTERT gene promoter [48], posttranslational regulation of hTERT [49], or through its antioxidative capacity [50]. It is also possible that this finding is a false positive due to small sample size. Alternatively, one could theorize that females with telomeres that are shorter than expected for their gender might be at even higher risk of cancer related to telomere shortening than males, as others have observed for other cancers [16,44]. It is also possible that females could have different osteosarcoma risk factors than males. A recent study of the Pro72Arg TP53 polymorphism in osteosarcoma found that the variant allele was associated with osteosarcoma only in females [9].
This pilot study was the first to explore the association between SNPs in telomere biology genes and osteosarcoma risk. We were able to augment our statistical power through the addition of controls from the PLCO study. With the addition of these controls, there was 80% power to detect an OR of 1.82 for SNPs with MAFs of at least 0.1. We chose to interpret the SNP data conservatively, by using the Bonferroni correction based on the number of SNPs per gene, because of the study's small sample size, and we used global gene- and pathway-level analyses to comprehensively evaluate our data.
This approach identified seven statistically significant SNPs in TERF1 after Bonferroni correction for the number of SNPs per gene. However, no associations remained significant if corrected for all 713 SNPs in the study. The SNPs in TERF1 were all inversely associated with osteosarcoma risk and were strongly correlated with each other. At the gene-level, TERF1 was also significantly associated with osteosarcoma after correction for multiple tests. TERF1 encodes TRF1, a member of the shelterin telomere protection complex which protects telomeres from degradation and inappropriate DNA repair [51]. The role of TERF1 in osteosarcoma pathogenesis is not known. One small study did not find TERF1 mutations in osteosarcoma cell lines [52].
We also evaluated the association between TL and SNPs in telomere biology genes in the BDISO participants, to better understand the role of common SNPs in TL regulation. A total of 20 SNPs in 13 genes were statistically significantly associated with TL before Bonferroni correction, but none remained significant (P < 0.05) after this conservative statistical correction (Table 4). A recent genome-wide association study (GWAS) identified a SNP in the TERC locus, rs 12696304, that was inversely associated with TL [53]. This SNP was also associated with a reduction in TL in our dataset which was significant before correction (Beta -0.105, SE 0.05, P = 0.034). Two other SNPs in our data-set in this region were also significant before Bonferroni correction. These three TERC SNPs were all highly correlated in our dataset (r2 = 0.8-0.98). Recent genome-wide association studies have found variants in the TERT-CLPTM1L locus associated with cancer risk [54,55]. We evaluated 16 SNPs in the TERT locus and did not find associations with osteosarcoma orTL.
Other studies have mapped loci influencing TL to chromosome 14q23.2 [56], and to variants in the BICD1 [57], DDX11 [56], and VPS34/PIKC3C [58] genes. Of these genes, only DDX11 was in our data set and its SNPs were not associated with TL. Another candidate gene study of TL and SNPs in 43 telomere biology genes found that SNPs in MEN1 were associated with TL [59]. A SNP in MEN1 that was in both studies, rs670358, was significantly associated with TL before Bonferroni correction (P = 0.008) in our study. In the current study this SNP was associated with an increase in TL, but the converse was true in the other study. This discrepancy may be due in part to differences in the age of the study populations (median age of 19 years in this study compared with 62 years in the other).
In summary, this pilot-study explored the potential role of telomere biology in osteosarcoma etiology. The results were very conservatively interpreted using Bonferroni correction which reduces the potential for false positive findings, but may be too stringent. The role of SNPs in TL regulation is an area of active investigation. This study confirms some of those associations, including an association between TL and SNPs in MEN1 and TERC. Common variants in TERF1 were inversely associated with risk of osteosarcoma. Additional studies of the role of TERF1 and other components of shelterin in osteosarcoma are warranted. Lastly, we found that females with shorter teiomeres had higher risks of osteosarcoma than males. The sample size was small and larger studies are required to better understand this gender difference.
Acknowledgments
We are grateful to the BDISO and PLCO partici pants for their valuable contributions. We thank Bill Wheeler at IMS for his assistance with data management and global analyses. The content of this publication does not necessarily reflect the views or policies of the Department of Health and Human Services, nor does mention of trade names, commercial products, or organi zations imply endorsement by the U.S. Govern ment. These findings and conclusions in this report are those of the authors and do not nec essarily represent the official position of the Centers for Disease Control and Prevention. Grant Support: This work was supported in part by the intramural research program of the Na tional Institutes of Health and the National Can cer Institute. This project has been funded in whole or in part with federal funds from the Na tional Cancer Institute, National Institutes of Health, under Contract No. HHSN261200800001E.
supplemental data
Supplemental Table 1
Supplemental Table 1
Description of the 39 telomere genes included in our study
Supplemental Table 2
Supplemental Table 2
Significant SNPs (P < 0.05) in telomere biology genes associated with osteosarcoma before correction for multiple tests
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