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. 2020 Feb 10;15(2):e0228887. doi: 10.1371/journal.pone.0228887

Genetic variation in POT1 and risk of thyroid subsequent malignant neoplasm: A report from the Childhood Cancer Survivor Study

Melissa A Richard 1, Philip J Lupo 1, Lindsay M Morton 2, Yutaka A Yasui 3, Yadav A Sapkota 3, Michael A Arnold 4, Geraldine Aubert 5, Joseph P Neglia 6, Lucie M Turcotte 6, Wendy M Leisenring 7, Joshua N Sampson 2, Stephen J Chanock 2,8, Melissa M Hudson 9, Gregory T Armstrong 3, Leslie L Robison 3, Smita Bhatia 10, Maria Monica Gramatges 1,*
Editor: Arthur J Lustig11
PMCID: PMC7010302  PMID: 32040538

Abstract

Background

Telomere length is associated with risk for thyroid subsequent malignant neoplasm in survivors of childhood cancer. Here, we investigated associations between thyroid subsequent malignant neoplasm and inherited variation in telomere maintenance genes.

Methods

We used RegulomeDB to annotate the functional impact of variants mapping to 14 telomere maintenance genes among 5,066 five-or-more year survivors who participate in the Childhood Cancer Survivor Study (CCSS) and who are longitudinally followed for incidence of subsequent cancers. Hazard ratios for thyroid subsequent malignant neoplasm were calculated for 60 putatively functional variants with minor allele frequency ≥1% in or near telomere maintenance genes. Functional impact was further assessed by measuring telomere length in leukocyte subsets.

Results

The minor allele at Protection of Telomeres-1 (POT1) rs58722976 was associated with increased risk for thyroid subsequent malignant neoplasm (adjusted HR = 6.1, 95% CI: 2.4, 15.5, P = 0.0001; Fisher’s exact P = 0.001). This imputed SNP was present in three out of 110 survivors who developed thyroid cancer vs. 14 out of 4,956 survivors who did not develop thyroid cancer. In a subset of 83 survivors with leukocyte telomere length data available, this variant was associated with longer telomeres in B lymphocytes (P = 0.004).

Conclusions

Using a functional variant approach, we identified and confirmed an association between a low frequency intronic regulatory POT1 variant and thyroid subsequent malignant neoplasm in survivors of childhood cancer. These results suggest that intronic variation in POT1 may affect key protein binding interactions that impact telomere maintenance and genomic integrity.

Introduction

Over 80% of individuals diagnosed and treated for cancer as children will survive five or more years after completing cancer treatment. An increased risk for subsequent malignant neoplasms (SMN), including SMNs of the thyroid (thyroid SMN), is a known late effect of childhood cancer treatment.[1] Higher risk for thyroid SMN is observed among female survivors, those diagnosed with primary cancer at a younger age, and those exposed to radiation and certain chemotherapeutic agents.[2] However, these clinical risk factors do not fully explain variability in thyroid SMN risk, suggesting a likely role for inherited genetic factors.

Telomeres are repetitive DNA-protein structures localized to chromosome ends, protecting chromosome integrity and loss of proximal terminal coding regions during DNA replication. Telomere length is determined by environmental and hereditary factors, shortens with age, and is maintained by telomerase and associated proteins. The majority of population-based studies have noted an inverse association between cancer risk over time and leukocyte telomere length when measured directly through standard methodologies.[3, 4] However, data from genome wide association studies suggest that longer telomere length may also confer risk for a number of cancers.[5] This apparent paradox is hypothesized to result from multi-stage mechanisms underlying malignant transformation: specifically, accumulation of random mutational events during stem cell replication may lead to an increase in sporadic cancer risk that occurs with physiologic aging, for which telomere shortening is a proxy. Longer telomeres confer a greater capacity for cellular clonal expansion and proliferation, so that individuals with very long telomeres may also be at especially high risk for carcinogenesis.[6]

Exposure to ionizing radiation induces DNA damage and may lead to telomere dysfunction,[7–10] providing rationale to suggest a relationship between telomere length and risk for SMN among radiation-exposed cancer survivors. We previously reported an increased risk for thyroid SMN in radiation-exposed survivors of childhood cancer with reduced leukocyte telomere content.[11] We subsequently observed no association between genotypically-estimated telomere length, determined from variation in nine common telomere length-associated SNPs, and thyroid SMN.[12] These prior works prompted a functional variant approach to further interrogate the relationship between variation in genes related to telomere maintenance and thyroid SMN in the Childhood Cancer Survivor Study (CCSS).

Materials and methods

Subjects

The CCSS is a multi-center cohort of individuals diagnosed <21 years of age with childhood cancer between 1970 and 1986, and who survived five or more years after completion of cancer treatment.[13] After enrollment to the CCSS, survivors are prospectively followed through self-report questionnaires to ascertain late effects of cancer treatment. Thyroid SMN was defined as any SMN of the thyroid gland occurring as the first subsequent neoplasm in a CCSS participant, with a diagnosis and date of diagnosis that had been verified from the original pathology report by a CCSS-designated pathologist. All subjects provided written consent to participate in the CCSS, and each of the 26 participating institutions obtained approval to conduct this research through their institutional IRB. Diagnosis and treatment data were initially abstracted from the medical record by the participating treating institution and submitted, fully anonymized, to CCSS. The work described in this study utilized fully anonymized data. The study was conducted in accordance with the Declarations of Helsinki.

Genetic data

This study leveraged genetic data from 5,066 CCSS participants with complete follow-up for SMN. DNA was extracted using standard methods from blood, saliva (Oragene), or buccal cells (mouthwash), collected at least five years from diagnosis and genotyped using the Illumina HumanOmni5Exome array at the Cancer Genomics Research Laboratory of the National Cancer Institute. All survivors were imputed to the 1000 Genomes reference haplotypes.[14] We mapped 3,499 variants to a 100 bp region flanking genes implicated in a telomere biology disorder: ACD, CTC1, DKC1, NAF1, NHP2, NOP10, PARN, POT1, RTEL1, STN1, TERC, TERT, TINF2, WRAP53 (Table 1).[15–31] We restricted our analyses to include only functional SNPs with minor allele frequency (MAF) ≥ 1% in or near these 14 genes that were considered most likely to affect transcriptional factor binding, defined by a RegulomeDB score ≤ 2, which signifies localization to transcriptional factor binding and motifs, DNase footprints and peaks, or identification as a quantitative trait locus for gene expression (eQTL) across multiple tissues.[32] Genetic variants were coded as imputed genotype dosages and filtered for imputation quality >0.7.

Table 1. Genes currently implicated in telomere biology disorders.

Gene name (HGNCa Symbol) Related telomere biology disorder(s)b Role in telomere maintenance
ACD HHS, AA, familial cancers Part of the shelterin complex
CTC1 DC, Coats Plus, cerebroretinal microangiopathy Part of the CST complex
DKC1 DC, HHS Part of the telomerase holoenzyme
NAF1 PF Part of the telomerase holoenzyme
NHP2 DC Part of the telomerase holoenzyme
NOP10 DC Part of the telomerase holoenzyme
PARN DC, PF, HHS Ribonuclease interacting with TERC
POT1 Coats Plus, familial cancers Part of the shelterin complex
RTEL1 DC, PF, HHS DNA helicase interacting with shelterin
STN1 (OBFC1) Coats Plus Part of the CST complex
TERC DC, PF, MDS, HHS, AA Part of the telomerase holoenzyme
TERT DC, PF, AML, MDS, HHS, AA Part of the telomerase holoenzyme
TINF2 DC, HHS, RS, AA Part of the shelterin complex
WRAP53 DC Protein that binds to TERC
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aHUGO Gene Nomenclature Committee

bHHS = Hoyeraal Hreidersson Syndrome, DC = dyskeratosis congenita, PF = pulmonary fibrosis, MDS = myelodysplastic syndrome, AA = aplastic anemia, AML = acute myeloid leukemia, RS = Revesz Syndrome

Statistical analysis

We conducted time-to-event Cox regression for thyroid SMN as a first SMN using the survival package in R v3.5.2. The at-risk period began with the date of initial cancer diagnosis and ended at the date of thyroid SMN diagnosis, or the earliest first report of other SMN, death, and/or date of last follow up. Relative risk of thyroid SMN was estimated using hazard ratios (HR), adjusted for demographic and clinical factors including sex, birth year before or after 1970, age at primary cancer diagnosis, primary cancer diagnosis, radiation exposure (yes/no), neck radiation exposure (yes/no), alkylating agent exposure (yes/no), and thyroid nodules (yes/no). Genetic ancestry proportions were estimated using three continental ancestries (CEU, AFR, and ASN) in STRUCTURE.[33] We used an 80% threshold to define individuals of a predominant ancestry and performed secondary analyses 1) in the total sample additionally adjusted for estimated European and African proportions and 2) restricted to individuals of European ancestry. Statistical significance for association with risk for thyroid SMN was defined by the Bonferroni correction for the number of variants tested (α = 0.00083). For lower frequency variants (MAF 1–5%) that were statistically significant in the Cox regression model, we validated the regression model using a Fisher’s exact test to evaluate differences in allele frequency between those with and without thyroid SMN.[34]

Measurement of telomere length in hematopoietic cells

Viably frozen leukocyte samples were available for 83 CCSS subjects included in this study. Leukocyte telomere length was measured by flow cytometry fluorescence in situ hybridization (flow FISH) following established procedures.[35] Briefly, leukocyte telomere length was assessed against that of control bovine thymocytes after denaturation in formamide at 87°C. Quantitative hybridization with a fluorescein-conjugated (CCCTAA)3 peptide nucleic acid (PNA) probe specific for telomere repeats (in-house synthesis) was then performed and counterstained with LDS751 DNA dye (Exciton), followed by analysis with flow cytometry. Results were transformed to age-adjusted percentiles based on the date of sample collection. For significant functional variants, we compared the proportions of age-based relative telomere length categories (very low, low, normal, high, or very high) by cell type between carriers and non-carriers of the risk allele using Fisher’s exact test.

Results

We identified 110 CCSS participants who developed thyroid SMN five or more years after completion of cancer treatment and 4,956 survivors without thyroid SMN. Survivors who developed thyroid SMN were more likely to be female (thyroid SMN: 62.7%, non-cases: 51.6%) and to have thyroid nodules (thyroid SMN: 84.5%, non-cases: 10.0%). Thyroid SMN also occurred more frequently for those with older age at childhood cancer diagnosis (thyroid SMN cases: mean 9.0 years, non-cases: mean 7.9 years) and a primary diagnosis of Hodgkin lymphoma (thyroid SMN cases: 32.7%, non-cases: 12.6%). Among primary cancer treatment characteristics, radiation treatment to the neck and exposure to alkylating chemotherapy was also more likely to have occurred among thyroid SMN cases than survivors without thyroid SMN (Table 2).

Table 2. Characteristics of the Childhood Cancer Survivor Study participants by development of subsequent malignant neoplasm of the thyroid.

Thyroid SMN cases n = 110 Non-cases n = 4,956
n (%) n (%)
Age at first malignancy, years (mean ± SD) 9.0 ± 5.5 7.9 ± 5.9
Year of birth
Before 1970 68 61.8% 2,227 44.9%
After 1970 42 38.2% 2,729 55.1%
Sex
Male 41 37.3% 2,397 48.4%
Female 69 62.7% 2,559 51.6%
Type of first malignancy
Leukemia 30 27.3% 1,589 32.1%
Central nervous system 12 10.9% 591 11.9%
Hodgkin lymphoma 36 32.7% 623 12.6%
Non-Hodgkin lymphoma 5 4.5% 397 8.0%
Kidney/Wilms tumor 5 4.5% 486 9.8%
Neuroblastoma 5 4.5% 366 7.4%
Soft tissue sarcoma 6 5.5% 463 9.3%
Bone 11 10.0% 441 8.9%
Alkylating chemotherapy 69 62.7% 2,542 51.3%
Any radiation treatment 94 85.5% 3,144 63.4%
Radiation treatment to the neck 66 60.0% 1,041 21.0%
Thyroid nodules 93 84.5% 498 10.0%
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There were 60 SNPs included in our analyses located in or near telomere candidate genes that had both 1) a RegulomeDB score ≤2 signifying high likelihood for affecting transcriptional regulation and 2) a general population MAF <1%. Only one imputed variant (imputation quality r2 = 0.95) in an intronic region of POT1 (Protection of Telomeres 1), rs58722976, met Bonferroni criteria for statistical significance (adjusted HR = 6.1, 95% CI: 2.4, 15.5, P = 0.0001). The risk-associated G allele was present in three individuals with thyroid SMN and 14 individuals without thyroid SMN (Table 3). Comparing the risk allele frequencies between cases and non-cases also supported association of rs58722976 with thyroid SMN (Fisher’s exact P = 0.001). Estimated allele frequency at rs58722976 in the Genome Aggregation Database (gnomAD) suggests variation at rs58722976 occurs at highest frequency in individuals of African ancestry (AFR f(G) = 4.8%). In secondary analyses, we identified a consistent association of rs58722976 with thyroid SMN both when additionally adjusted for ancestry proportions (ancestry-adjusted HR = 8.0, 95% CI: 2.3, 27.2, p = 0.0009) and when restricted to European ancestry CCSS participants (CEU only HR = 18.9, 95% CI: 3.5, 101.7, p = 0.0006; median CEU proportion = 95.7%).

Table 3. Genotype frequencies and Cox regression estimates for POT1 rs58722976 and risk of subsequent malignant neoplasm of the thyroid in the Childhood Cancer Survivor Study.

Genotypes for thyroid SMN cases Genotypes for non-cases Cox regression estimates
SNP Population GG AG AA GG AG AA HR 95% CI P-value
rs58722976 total sample 1 2 107 0 14 4,942 6.1 (2.4, 15.5) 0.0001
AFR and CEU adjusted 1 2 107 0 14 4,942 8.0 (2.3, 27.2) 0.0009
CEU only 0 2 102 0 2 4,621 18.9 (3.5, 101.7) 0.0006
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All models are adjusted for sex, age at primary cancer diagnosis, primary cancer diagnosis, decade of birth, and treatment exposures.

Lastly, telomere length was assessed in leukocyte populations in a subset of 83 survivors of childhood cancer enrolled to the CCSS. Only two were heterozygous for the risk allele in our top SNP. Telomere length was increased for the two heterozygous subjects across all leukocyte subsets compared with the median telomere length for subjects without the risk allele, a difference that was statistically significant for B lymphocytes (Fig 1, P = 0.004).

Fig 1. Deviation from the age-based mean leukocyte telomere length measured by flow-FISH in survivors of childhood cancer.

Fig 1

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Two of these survivors (one CEU and one YRI) were heterozygous for the high risk POT1 rs58722976-G allele (denoted by circles) and demonstrate telomere length that is above the median for all leukocyte subsets and significantly longer among B lymphocytes than telomere length in survivors who lack this variant. Boxes include values falling between the 25th and 75th percentile deviation in telomere length from the age-based mean.

Discussion

POT1 is a highly conserved gene encoding a key component of the shelterin complex, which protects telomere ends against DNA damage recognition and facilitates telomerase-mediated telomere maintenance. POT1 rs58722976 is an intronic variant identified by the ENCODE Consortium as a strong enhancer and DNase I hypersensitive site in multiple tissues, including the hematopoietic compartment, and may affect protein binding in components of the cohesion complex that play key roles in cancer etiology and maintaining genomic integrity.[36] Germline variants in POT1 have been described in association with various cancer types[37] including familial glioma,[38] familial melanoma,[39–41] colorectal, ovarian, and lung cancer,[42] chronic lymphocytic leukemia,[43] multiple myeloma,[44] and non-TP53 familial cancer syndromes.[45] Similar to our analyses, many of these genetic association studies note longer leukocyte telomere length among affected individuals compared with those who are unaffected.[38, 40, 41, 45] Recent data suggest that mutation-induced disruptions in the POT1-TPP1 complex, both components of shelterin, affect the ability of this complex to bind to telomeric DNA, leading to longer and more fragile telomeres that may promote genomic instability and cancer risk.[46]

This study was conducted within the CCSS, the largest genotyped population of survivors of childhood cancer. However, the low frequency of this variant precludes assessment of gene-environment interactions and adequately-sized genotyped survivor populations for replication or stratification among non-white ancestries. For example, all risk allele carriers excluded from CEU-only analysis were of primarily African ancestry (one homozygous individual with thyroid SMN and 12 carriers without thyroid SMN). Although thyroid cancer incidence is highest among individuals of European ancestry,[47] African ancestry confers a higher risk for the follicular variant of papillary thyroid cancer,[48] which was the SMN subtype observed in the survivor with thyroid SMN that was homozygous for the rs58722976 risk allele.

Using an approach that mapped functional variants to candidate genes, we identified an association between a low frequency intronic regulatory variant in POT1 and risk for thyroid SMN in survivors of childhood cancer. We provide evidence that genetic variation at this locus may related to longer telomere length, in line with prior observations of longer leukocyte telomere length in association with cancers characterized by germline mutations in POT1. Our findings support a potential role for genetic variation in POT1 affecting telomere maintenance and risk for thyroid SMN in survivors, suggesting the need for further study as larger genotyped survivor datasets emerge.

Acknowledgments

The authors would like to thank the survivors and their families who participate in the Childhood Cancer Survivor Study.

Data Availability

The CCSS genome-wide association study (GWAS) dataset is available to investigate the role of genetic susceptibility in the development of malignant and non-malignant treatment-related outcomes in cancer survivors. This process is open to investigators through collaboration with CCSS and National Cancer Institute investigators in the use of existing GWAS data and corresponding outcomes-related data to address innovative research questions relating to potential genetic contributions to risk for treatment-related outcomes through submission of an Application of Intent (https://ccss.stjude.org/develop-a-study/gwas-data-resource.html). Genotype data for the CCSS are also available through dbGaP, accession phs001327.v1.p1.

Funding Statement

This work was supported by the National Cancer Institute (NCI) (CA194473: M.M.G., Principal Investigator) and a CCSS Career Development Award to P.J.L. CCSS is supported by the NCI (CA55727: G.T.A., Principal Investigator). Genotyping for CCSS was supported by the Intramural Research Program of the NCI, National Institutes of Health. The sponsors played no role in study design, data collection, analysis, decision to publish, or manuscript preparation.

References

  • 1.Turcotte LM, Liu Q, Yasui Y, Arnold MA, Hammond S, Howell RM, et al. Temporal Trends in Treatment and Subsequent Neoplasm Risk Among 5-Year Survivors of Childhood Cancer, 1970–2015. Jama. 2017;317(8):814–24. 10.1001/jama.2017.0693 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 2.Veiga LH, Lubin JH, Anderson H, de Vathaire F, Tucker M, Bhatti P, et al. A pooled analysis of thyroid cancer incidence following radiotherapy for childhood cancer. Radiation research. 2012;178(4):365–76. 10.1667/rr2889.1 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 3.Ma H, Zhou Z, Wei S, Liu Z, Pooley KA, Dunning AM, et al. Shortened telomere length is associated with increased risk of cancer: a meta-analysis. PLoS One. 2011;6(6):e20466 10.1371/journal.pone.0020466 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 4.Wentzensen IM, Mirabello L, Pfeiffer RM, Savage SA. The association of telomere length and cancer: a meta-analysis. Cancer Epidemiol Biomarkers Prev. 2011;20(6):1238–50. 10.1158/1055-9965.EPI-11-0005 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 5.Telomeres Mendelian Randomization C, Haycock PC, Burgess S, Nounu A, Zheng J, Okoli GN, et al. Association Between Telomere Length and Risk of Cancer and Non-Neoplastic Diseases: A Mendelian Randomization Study. JAMA Oncol. 2017;3(5):636–51. 10.1001/jamaoncol.2016.5945 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 6.Aviv A, Anderson JJ, Shay JW. Mutations, Cancer and the Telomere Length Paradox. Trends Cancer. 2017;3(4):253–8. 10.1016/j.trecan.2017.02.005 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 7.Maeda T, Nakamura K, Atsumi K, Hirakawa M, Ueda Y, Makino N. Radiation-associated changes in the length of telomeres in peripheral leukocytes from inpatients with cancer. International journal of radiation biology. 2013;89(2):106–9. 10.3109/09553002.2013.734945 [DOI] [PubMed] [Google Scholar]
  • 8.Fumagalli M, Rossiello F, Clerici M, Barozzi S, Cittaro D, Kaplunov JM, et al. Telomeric DNA damage is irreparable and causes persistent DNA-damage-response activation. Nat Cell Biol. 2012;14(4):355–65. 10.1038/ncb2466 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 9.Li P, Hou M, Lou F, Bjorkholm M, Xu D. Telomere dysfunction induced by chemotherapeutic agents and radiation in normal human cells. Int J Biochem Cell Biol. 2012;44(9):1531–40. 10.1016/j.biocel.2012.06.020 [DOI] [PubMed] [Google Scholar]
  • 10.Lustig A, Shterev I, Geyer S, Shi A, Hu Y, Morishita Y, et al. Long term effects of radiation exposure on telomere lengths of leukocytes and its associated biomarkers among atomic-bomb survivors. Oncotarget. 2016;7(26):38988–98. 10.18632/oncotarget.8801 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 11.Gramatges MM, Liu Q, Yasui Y, Okcu MF, Neglia JP, Strong LC, et al. Telomere content and risk of second malignant neoplasm in survivors of childhood cancer: a report from the Childhood Cancer Survivor Study. Clinical cancer research: an official journal of the American Association for Cancer Research. 2014;20(4):904–11. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 12.Gramatges MM, Morton LM, Yasui Y, Arnold MA, Neglia JP, Leisenring WM, et al. Telomere Length-Associated Genetic Variants and the Risk of Thyroid Cancer in Survivors of Childhood Cancer: A Report from the Childhood Cancer Survivor Study (CCSS). Cancer Epidemiol Biomarkers Prev. 2019;28(2):417–9. 10.1158/1055-9965.EPI-18-0972 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 13.Robison LL, Armstrong GT, Boice JD, Chow EJ, Davies SM, Donaldson SS, et al. The Childhood Cancer Survivor Study: a National Cancer Institute-supported resource for outcome and intervention research. Journal of clinical oncology: official journal of the American Society of Clinical Oncology. 2009;27(14):2308–18. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 14.Morton LM, Sampson JN, Armstrong GT, Chen T, Hudson MM, Dagnall CL, et al. Genome-wide association study identifies susceptibility loci that modify radiation-related risk for breast cancer after childhood cancer. J Natl Cancer Inst. 2017;in press(in press). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 15.Niewisch MR, Savage SA. An update on the biology and management of dyskeratosis congenita and related telomere biology disorders. Expert Rev Hematol. 2019:1–16. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 16.Heiss NS, Knight SW, Vulliamy TJ, Klauck SM, Wiemann S, Mason PJ, et al. X-linked dyskeratosis congenita is caused by mutations in a highly conserved gene with putative nucleolar functions. Nat Genet. 1998;19(1):32–8. 10.1038/ng0598-32 [DOI] [PubMed] [Google Scholar]
  • 17.Mitchell JR, Wood E, Collins K. A telomerase component is defective in the human disease dyskeratosis congenita. Nature. 1999;402(6761):551–5. 10.1038/990141 [DOI] [PubMed] [Google Scholar]
  • 18.Vulliamy T, Marrone A, Szydlo R, Walne A, Mason PJ, Dokal I. Disease anticipation is associated with progressive telomere shortening in families with dyskeratosis congenita due to mutations in TERC. Nat Genet. 2004;36(5):447–9. 10.1038/ng1346 [DOI] [PubMed] [Google Scholar]
  • 19.Ballew BJ, Yeager M, Jacobs K, Giri N, Boland J, Burdett L, et al. Germline mutations of regulator of telomere elongation helicase 1, RTEL1, in Dyskeratosis congenita. Hum Genet. 2013;132(4):473–80. 10.1007/s00439-013-1265-8 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 20.Walne AJ, Vulliamy T, Kirwan M, Plagnol V, Dokal I. Constitutional mutations in RTEL1 cause severe dyskeratosis congenita. Am J Hum Genet. 2013;92(3):448–53. 10.1016/j.ajhg.2013.02.001 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 21.Zhong F, Savage SA, Shkreli M, Giri N, Jessop L, Myers T, et al. Disruption of telomerase trafficking by TCAB1 mutation causes dyskeratosis congenita. Genes Dev. 2011;25(1):11–6. 10.1101/gad.2006411 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 22.Anderson BH, Kasher PR, Mayer J, Szynkiewicz M, Jenkinson EM, Bhaskar SS, et al. Mutations in CTC1, encoding conserved telomere maintenance component 1, cause Coats plus. Nat Genet. 2012;44(3):338–42. 10.1038/ng.1084 [DOI] [PubMed] [Google Scholar]
  • 23.Tummala H, Walne A, Collopy L, Cardoso S, de la Fuente J, Lawson S, et al. Poly(A)-specific ribonuclease deficiency impacts telomere biology and causes dyskeratosis congenita. J Clin Invest. 2015;125(5):2151–60. 10.1172/JCI78963 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 24.Vulliamy T, Beswick R, Kirwan M, Marrone A, Digweed M, Walne A, et al. Mutations in the telomerase component NHP2 cause the premature ageing syndrome dyskeratosis congenita. Proc Natl Acad Sci U S A. 2008;105(23):8073–8. 10.1073/pnas.0800042105 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 25.Walne AJ, Vulliamy T, Marrone A, Beswick R, Kirwan M, Masunari Y, et al. Genetic heterogeneity in autosomal recessive dyskeratosis congenita with one subtype due to mutations in the telomerase-associated protein NOP10. Hum Mol Genet. 2007;16(13):1619–29. 10.1093/hmg/ddm111 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 26.Savage SA, Giri N, Baerlocher GM, Orr N, Lansdorp PM, Alter BP. TINF2, a component of the shelterin telomere protection complex, is mutated in dyskeratosis congenita. Am J Hum Genet. 2008;82(2):501–9. 10.1016/j.ajhg.2007.10.004 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 27.Guo Y, Kartawinata M, Li J, Pickett HA, Teo J, Kilo T, et al. Inherited bone marrow failure associated with germline mutation of ACD, the gene encoding telomere protein TPP1. Blood. 2014;124(18):2767–74. 10.1182/blood-2014-08-596445 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 28.Kocak H, Ballew BJ, Bisht K, Eggebeen R, Hicks BD, Suman S, et al. Hoyeraal-Hreidarsson syndrome caused by a germline mutation in the TEL patch of the telomere protein TPP1. Genes Dev. 2014;28(19):2090–102. 10.1101/gad.248567.114 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 29.Simon AJ, Lev A, Zhang Y, Weiss B, Rylova A, Eyal E, et al. Mutations in STN1 cause Coats plus syndrome and are associated with genomic and telomere defects. J Exp Med. 2016;213(8):1429–40. 10.1084/jem.20151618 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 30.Takai H, Jenkinson E, Kabir S, Babul-Hirji R, Najm-Tehrani N, Chitayat DA, et al. A POT1 mutation implicates defective telomere end fill-in and telomere truncations in Coats plus. Genes Dev. 2016;30(7):812–26. 10.1101/gad.276873.115 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 31.Stanley SE, Gable DL, Wagner CL, Carlile TM, Hanumanthu VS, Podlevsky JD, et al. Loss-of-function mutations in the RNA biogenesis factor NAF1 predispose to pulmonary fibrosis-emphysema. Sci Transl Med. 2016;8(351):351ra107 10.1126/scitranslmed.aaf7837 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 32.Boyle AP, Hong EL, Hariharan M, Cheng Y, Schaub MA, Kasowski M, et al. Annotation of functional variation in personal genomes using RegulomeDB. Genome Res. 2012;22(9):1790–7. 10.1101/gr.137323.112 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 33.Pritchard JK, Stephens M, Donnelly P. Inference of population structure using multilocus genotype data. Genetics. 2000;155(2):945–59. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 34.Derkach A, Lawless JF, Sun L. Robust and powerful tests for rare variants using Fisher's method to combine evidence of association from two or more complementary tests. Genet Epidemiol. 2013;37(1):110–21. 10.1002/gepi.21689 [DOI] [PubMed] [Google Scholar]
  • 35.Baerlocher GM, Vulto I, de Jong G, Lansdorp PM. Flow cytometry and FISH to measure the average length of telomeres (flow FISH). Nat Protoc. 2006;1(5):2365–76. 10.1038/nprot.2006.263 [DOI] [PubMed] [Google Scholar]
  • 36.Hill VK, Kim JS, Waldman T. Cohesin mutations in human cancer. Biochimica et biophysica acta. 2016;1866(1):1–11. 10.1016/j.bbcan.2016.05.002 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 37.Calvete O, Garcia-Pavia P, Dominguez F, Bougeard G, Kunze K, Braeuninger A, et al. The wide spectrum of POT1 gene variants correlates with multiple cancer types. Eur J Hum Genet. 2017;25(11):1278–81. 10.1038/ejhg.2017.134 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 38.Bainbridge MN, Armstrong GN, Gramatges MM, Bertuch AA, Jhangiani SN, Doddapaneni H, et al. Germline mutations in shelterin complex genes are associated with familial glioma. J Natl Cancer Inst. 2015;107(1):384 10.1093/jnci/dju384 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 39.Wong K, Robles-Espinoza CD, Rodriguez D, Rudat SS, Puig S, Potrony M, et al. Association of the POT1 Germline Missense Variant p.I78T With Familial Melanoma. JAMA dermatology. 2018. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 40.Robles-Espinoza CD, Harland M, Ramsay AJ, Aoude LG, Quesada V, Ding Z, et al. POT1 loss-of-function variants predispose to familial melanoma. Nat Genet. 2014;46(5):478–81. 10.1038/ng.2947 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 41.Shi J, Yang XR, Ballew B, Rotunno M, Calista D, Fargnoli MC, et al. Rare missense variants in POT1 predispose to familial cutaneous malignant melanoma. Nat Genet. 2014;46(5):482–6. 10.1038/ng.2941 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 42.Karami S, Han Y, Pande M, Cheng I, Rudd J, Pierce BL, et al. Telomere structure and maintenance gene variants and risk of five cancer types. Int J Cancer. 2016;139(12):2655–70. 10.1002/ijc.30288 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 43.Speedy HE, Di Bernardo MC, Sava GP, Dyer MJ, Holroyd A, Wang Y, et al. A genome-wide association study identifies multiple susceptibility loci for chronic lymphocytic leukemia. Nature genetics. 2014;46(1):56–60. 10.1038/ng.2843 [DOI] [PubMed] [Google Scholar]
  • 44.Went M, Sud A, Forsti A, Halvarsson BM, Weinhold N, Kimber S, et al. Identification of multiple risk loci and regulatory mechanisms influencing susceptibility to multiple myeloma. Nat Commun. 2018;9(1):3707 10.1038/s41467-018-04989-w [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 45.Calvete O, Martinez P, Garcia-Pavia P, Benitez-Buelga C, Paumard-Hernandez B, Fernandez V, et al. A mutation in the POT1 gene is responsible for cardiac angiosarcoma in TP53-negative Li-Fraumeni-like families. Nature communications. 2015;6:8383 10.1038/ncomms9383 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 46.Rice C, Shastrula PK, Kossenkov AV, Hills R, Baird DM, Showe LC, et al. Structural and functional analysis of the human POT1-TPP1 telomeric complex. Nat Commun. 2017;8:14928 10.1038/ncomms14928 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 47.Magreni A, Bann DV, Schubart JR, Goldenberg D. The effects of race and ethnicity on thyroid cancer incidence. JAMA Otolaryngol Head Neck Surg. 2015;141(4):319–23. 10.1001/jamaoto.2014.3740 [DOI] [PubMed] [Google Scholar]
  • 48.Mehta V, Ow TJ, Kim S, Tharakan T, Schiff B, Smith RV, et al. Significant racial differences in the incidence and behavior of the follicular variant of papillary thyroid carcinoma. Head Neck. 2019;41(5):1403–11. 10.1002/hed.25596 [DOI] [PubMed] [Google Scholar]

Decision Letter 0

Arthur J Lustig

10 Jan 2020

PONE-D-19-33962

Genetic variation in POT1 and risk of thyroid subsequent malignant neoplasm: A report from the Childhood Cancer Survivor Study

PLOS ONE

Dear Dr. Gramatges,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

This manuscript is both a well-designed study and a well-written manuscript.  However, we need the authors to include the source of the data used in this study within a Supplemental Data section that can be accessed and interpreted by readers.. This may include sites of source data or, if not present at a site, raw data. 

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Kind regards,

Arthur J. Lustig, PhD

Academic Editor

PLOS ONE

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'All subjects provided written consent to participate in the CCSS, and each participating institution obtained approval to conduct this research through their institutional IRB. The study was conducted in accordance with the Declarations of Helsinki.'

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[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

Reviewer #2: Yes

**********

2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: Yes

**********

3. Have the authors made all data underlying the findings in their manuscript fully available?

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Reviewer #1: Yes

Reviewer #2: No

**********

4. Is the manuscript presented in an intelligible fashion and written in standard English?

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Reviewer #1: Yes

Reviewer #2: Yes

**********

5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: I was specifically asked to review the statistical procedures in the paper “Genetic variation in POT1 and risk of thyroid subsequent malignant neoplasm: A report from the Childhood Cancer Survivor Study”

I find the statistical approaches to be sophisticated and appropriate for the types of analyses discussed. Appropriate methods and corrections have been used and p-values address clearly delineated null hypotheses.

Reviewer #2: Overall the manuscript is well written. The study design is appropriate and the authors address the short comings (the low frequency of the variant limiting gene-environment interactions). This manuscript is a small report but presents a new perspective a on the potential involvement of the POT1 gene that has been previously been show to be linked to a variety of cancers.

**********

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Reviewer #1: No

Reviewer #2: No

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files to be viewed.]

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email us at figures@plos.org. Please note that Supporting Information files do not need this step.

PLoS One. 2020 Feb 10;15(2):e0228887. doi: 10.1371/journal.pone.0228887.r002

Author response to Decision Letter 0

16 Jan 2020

January 14, 2020

Editorial Board

PLOS One

Re: Research Article submission

Dear Editorial Board,

Thank you for reviewing our manuscript and for your comments. We have addressed the request for revisions as follows:

• Query: We need the authors to include the source of the data used in this study

• Response: Genotype data for the Childhood Cancer Survivor Study are available through dbGaP accession phs001327.v1.p1. I have amended the Data Availability Statement to reflect the accession number, as these data have already been deposited.

In addition, we have addressed recommendations with respect to the PLOS ONE style requirements noted in the decision letter. Specifically:

• With respect to the request for modification of the Ethics Statement to include the full names of all participating institutional review boards (IRBs), there are 26 institutions who participate in CCSS, and hence 26 separate IRBs who approved the CCSS study. We respectfully request to leave this statement unchanged, in line with a prior CCSS cohort publication by Yang et al. that was accepted to PLOS One, PMID 25764003. If the journal preference is to include the names of all 26 IRBs, we will gladly provide this information.

• We have re-named the figure file to adhere to journal policy

• We have modified the Ethics Statement to provide additional information about the patient records/samples that were used as follows: “All subjects provided written consent to participate in the CCSS, and each of the 26 participating institutions obtained approval to conduct this research through their institutional IRB. Diagnosis and treatment data were initially abstracted from the medical record by the participating treating institution and submitted, fully anonymized, to CCSS. The work described in this study utilized fully anonymized data. The study was conducted in accordance with the Declarations of Helsinki.”

• The Methods section has been modified to include details on reagents used for telomere length assessment.

• By providing the accession number for these data, we have clarified our statement that anonymized genotype data are publicly available.

• For the request regarding the Competing Interests section, we can affirm that the employment of Dr. Aubert does not alter our adherence to PLOS ONE policies on sharing data and materials. Thank you for your willingness to change the online submission on our behalf.

Thank you again for your consideration of this work for publication as a Research Article in PLOS One.

Sincerely,

M. Monica Gramatges, MD, PhD

Associate Professor of Pediatrics

Decision Letter 1

Arthur J Lustig

27 Jan 2020

Genetic variation in POT1 and risk of thyroid subsequent malignant neoplasm: A report from the Childhood Cancer Survivor Study

PONE-D-19-33962R1

Dear Dr. Gramatges,

We are pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it complies with all outstanding technical requirements.

Within one week, you will receive an e-mail containing information on the amendments required prior to publication. When all required modifications have been addressed, you will receive a formal acceptance letter and your manuscript will proceed to our production department and be scheduled for publication.

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With kind regards,

Arthur J. Lustig, PhD

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments:

Acceptance letter

Arthur J Lustig

28 Jan 2020

PONE-D-19-33962R1

Genetic variation in POT1 and risk of thyroid subsequent malignant neoplasm: A report from the Childhood Cancer Survivor Study

Dear Dr. Gramatges:

I am pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

If your institution or institutions have a press office, please notify them about your upcoming paper at this point, to enable them to help maximize its impact. If they will be preparing press materials for this manuscript, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org.

For any other questions or concerns, please email plosone@plos.org.

Thank you for submitting your work to PLOS ONE.

With kind regards,

PLOS ONE Editorial Office Staff

on behalf of

Dr. Arthur J. Lustig

Academic Editor

PLOS ONE

Associated Data

    This section collects any data citations, data availability statements, or supplementary materials included in this article.

    Data Availability Statement

    The CCSS genome-wide association study (GWAS) dataset is available to investigate the role of genetic susceptibility in the development of malignant and non-malignant treatment-related outcomes in cancer survivors. This process is open to investigators through collaboration with CCSS and National Cancer Institute investigators in the use of existing GWAS data and corresponding outcomes-related data to address innovative research questions relating to potential genetic contributions to risk for treatment-related outcomes through submission of an Application of Intent (https://ccss.stjude.org/develop-a-study/gwas-data-resource.html). Genotype data for the CCSS are also available through dbGaP, accession phs001327.v1.p1.

    Articles from PLoS ONE are provided here courtesy of PLOS

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