Non-medullary thyroid cancer (NMTC) is a common endocrine malignancy with a genetic basis that has yet to be unequivocally established. In a recent whole-genome sequencing study of five families with occurrence of NMTCs, we shortlisted promising variants with the help of bioinformatics tools. Here, we report in silico analyses and in vitro experiments on a novel germline variant (p.V29L) in the highly conserved oligonucleotide/oligosaccharide binding domain of the Protection of Telomeres 1 (POT1) gene in one of the families. The results showed a reduction in telomere-bound POT1 levels in the mutant protein as compared to its wild-type counterpart. HEK293T cells carrying POT1 p.V29L showed increased telomere length in comparison to wild-type cells, suggesting that the mutation causes telomere dysfunction and may play a role in predisposition to NMTC in this family. While one germline mutation in POT1 has already been reported in a melanoma-prone family with prevalence of thyroid cancers, we report the first of such mutations in a family affected solely by NMTCs, thus expanding current knowledge on shelterin complex-associated cancers.

Computational Oncology Molecular Diagnostics Program National Center for Tumor Diseases 69120 Heidelberg Germany

Department of Medicine 1 and Clinical Chemistry University Hospital of Heidelberg 69120 Heidelberg Germany

Division of Molecular Genetic Epidemiology German Cancer Research Center 69120 Heidelberg Germany

Division of Pediatric Neurooncology German Cancer Research Center 69120 Heidelberg Germany

Faculty of Medicine and Biomedical Center in Pilsen Charles University Prague 30605 Pilsen Czech Republic

Hopp Children's Cancer Center 69120 Heidelberg Germany

Institute of Bioinformatics International Technology Park Bangalore 560066 India

Manipal Academy of Higher Education Manipal 576104 Karnataka India

Medical Faculty Heidelberg University 69120 Heidelberg Germany

Unit of Medical Genetics Department of Medical and Surgical Sciences S Orsola Malpighi Hospital University of Bologna 40138 Bologna Italy

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Bray F., Ferlay J., Soerjomataram I., Siegel R.L., Torre L.A., Jemal A. Global cancer statistics 2018: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries. CA Cancer J. Clin. 2018;68:394–424. doi: 10.3322/caac.21492. PubMed DOI

Hincza K., Kowalik A., Kowalska A. Current Knowledge of Germline Genetic Risk Factors for the Development of Non-Medullary Thyroid Cancer. Genes. 2019;10:482. doi: 10.3390/genes10070482. PubMed DOI PMC

Peiling Yang S., Ngeow J. Familial non-medullary thyroid cancer: Unraveling the genetic maze. Endocr. Relat. Cancer. 2016;23:R577–R595. doi: 10.1530/ERC-16-0067. PubMed DOI

Fallah M., Pukkala E., Tryggvadottir L., Olsen J.H., Tretli S., Sundquist K., Hemminki K. Risk of thyroid cancer in first-degree relatives of patients with non-medullary thyroid cancer by histology type and age at diagnosis: A joint study from five Nordic countries. J. Med. Genet. 2013;50:373–382. doi: 10.1136/jmedgenet-2012-101412. PubMed DOI

El Lakis M., Giannakou A., Nockel P.J., Wiseman D., Gara S.K., Patel D., Sater Z.A., Kushchayeva Y.Y., Klubo-Gwiezdzinska J., Nilubol N., et al. Do patients with familial nonmedullary thyroid cancer present with more aggressive disease? Implications for initial surgical treatment. Surgery. 2019;165:50–57. doi: 10.1016/j.surg.2018.05.075. PubMed DOI PMC

Capezzone M., Cantara S., Marchisotta S., Filetti S., De Santi M.M., Rossi B., Ronga G., Durante C., Pacini F. Short telomeres, telomerase reverse transcriptase gene amplification, and increased telomerase activity in the blood of familial papillary thyroid cancer patients. J. Clin. Endocrinol. Metab. 2008;93:3950–3957. doi: 10.1210/jc.2008-0372. PubMed DOI

Srivastava A., Kumar A., Giangiobbe S., Bonora E., Hemminki K., Forsti A., Bandapalli O.R. Whole Genome Sequencing of Familial Non-Medullary Thyroid Cancer Identifies Germline Alterations in MAPK/ERK and PI3K/AKT Signaling Pathways. Biomolecules. 2019;9:605. doi: 10.3390/biom9100605. PubMed DOI PMC

de Lange T. Shelterin-Mediated Telomere Protection. Annu. Rev. Genet. 2018;52:223–247. doi: 10.1146/annurev-genet-032918-021921. PubMed DOI

Robles-Espinoza C.D., Harland M., Ramsay A.J., Aoude L.G., Quesada V., Ding Z., Pooley K.A., Pritchard A.L., Tiffen J.C., Petljak M., et al. POT1 loss-of-function variants predispose to familial melanoma. Nat. Genet. 2014;46:478–481. doi: 10.1038/ng.2947. PubMed DOI PMC

Shi J., Yang X.R., Ballew B., Rotunno M., Calista D., Fargnoli M.C., Ghiorzo P., Bressac-de Paillerets B., Nagore E., Avril M.F., et al. Rare missense variants in POT1 predispose to familial cutaneous malignant melanoma. Nat. Genet. 2014;46:482–486. doi: 10.1038/ng.2941. PubMed DOI PMC

Potrony M., Puig-Butille J.A., Ribera-Sola M., Iyer V., Robles-Espinoza C.D., Aguilera P., Carrera C., Malvehy J., Badenas C., Landi M.T., et al. POT1 germline mutations but not TERT promoter mutations are implicated in melanoma susceptibility in a large cohort of Spanish melanoma families. Br. J. Dermatol. 2019;181:105–113. doi: 10.1111/bjd.17443. PubMed DOI PMC

Wong K., Robles-Espinoza C.D., Rodriguez D., Rudat S.S., Puig S., Potrony M., Wong C.C., Hewinson J., Aguilera P., Puig-Butille J.A., et al. Association of the POT1 Germline Missense Variant p.I78T with Familial Melanoma. JAMA Dermatol. 2019;155:604–609. doi: 10.1001/jamadermatol.2018.3662. PubMed DOI PMC

Bainbridge M.N., Armstrong G.N., Gramatges M.M., Bertuch A.A., Jhangiani S.N., Doddapaneni H., Lewis L., Tombrello J., Tsavachidis S., Liu Y., et al. Germline mutations in shelterin complex genes are associated with familial glioma. J. Natl.Cancer Inst. 2015;107:384. doi: 10.1093/jnci/dju384. PubMed DOI PMC

Calvete O., Martinez P., Garcia-Pavia P., Benitez-Buelga C., Paumard-Hernández B., Fernandez V., Dominguez F., Salas C., Romero-Laorden N., Garcia-Donas J., et al. A mutation in the POT1 gene is responsible for cardiac angiosarcoma in TP53-negative Li–Fraumeni-like families. Nat. Commun. 2015;6:8383. doi: 10.1038/ncomms9383. PubMed DOI PMC

Chubb D., Broderick P., Dobbins S.E., Frampton M., Kinnersley B., Penegar S., Price A., Ma Y.P., Sherborne A.L., Palles C., et al. Rare disruptive mutations and their contribution to the heritable risk of colorectal cancer. Nat. Commun. 2016;7:11883. doi: 10.1038/ncomms11883. PubMed DOI PMC

Speedy H.E., Kinnersley B., Chubb D., Broderick P., Law P.J., Litchfield K., Jayne S., Dyer M.J.S., Dearden C., Follows G.A., et al. Germ line mutations in shelterin complex genes are associated with familial chronic lymphocytic leukemia. Blood. 2016;128:2319–2326. doi: 10.1182/blood-2016-01-695692. PubMed DOI PMC

McMaster M.L., Sun C., Landi M.T., Savage S.A., Rotunno M., Yang X.R., Jones K., Vogt A., Hutchinson A., Zhu B., et al. Germline mutations in Protection of Telomeres 1 in two families with Hodgkin lymphoma. Br. J. Haematol. 2018;181:372–377. doi: 10.1111/bjh.15203. PubMed DOI PMC

Lei M., Podell E.R., Baumann P., Cech T.R. DNA self-recognition in the structure of Pot1 bound to telomeric single-stranded DNA. Nature. 2003;426:198–203. doi: 10.1038/nature02092. PubMed DOI

Gu P., Wang Y., Bisht K.K., Wu L., Kukova L., Smith E.M., Xiao Y., Bailey S.M., Lei M., Nandakumar J., et al. Pot1 OB-fold mutations unleash telomere instability to initiate tumorigenesis. Oncogene. 2017;36:1939–1951. doi: 10.1038/onc.2016.405. PubMed DOI PMC

Ramsay A.J., Quesada V., Foronda M., Conde L., Martinez-Trillos A., Villamor N., Rodriguez D., Kwarciak A., Garabaya C., Gallardo M., et al. POT1 mutations cause telomere dysfunction in chronic lymphocytic leukemia. Nat. Genet. 2013;45:526–530. doi: 10.1038/ng.2584. PubMed DOI

Cantara S., Pisu M., Frau D.V., Caria P., Dettori T., Capezzone M., Capuano S., Vanni R., Pacini F. Telomere Abnormalities and Chromosome Fragility in Patients Affected by Familial Papillary Thyroid Cancer. J. Clin. Endocrinol. Metab. 2012;97:E1327–E1331. doi: 10.1210/jc.2011-2096. PubMed DOI

Calvete O., Garcia-Pavia P., Dominguez F., Bougeard G., Kunze K., Braeuninger A., Teule A., Lasa A., Ramon Y.C.T., Llort G., et al. The wide spectrum of POT1 gene variants correlates with multiple cancer types. Eur. J. Hum. Genet. 2017;25:1278–1281. doi: 10.1038/ejhg.2017.134. PubMed DOI PMC

He M., Bian B., Gesuwan K., Gulati N., Zhang L., Nilubol N., Kebebew E. Telomere length is shorter in affected members of families with familial nonmedullary thyroid cancer. Thyroid. 2013;23:301–307. doi: 10.1089/thy.2012.0270. PubMed DOI PMC

Li J., An C., Zheng H., Lei T., Zhang N., Zheng Y., Yang M. Leukocyte Telomere Length and Risk of Papillary Thyroid Carcinoma. J. Clin. Endocrinol. Metab. 2019;104:2712–2718. doi: 10.1210/jc.2018-02471. PubMed DOI

McNally E.J., Luncsford P.J., Armanios M. Long telomeres and cancer risk: The price of cellular immortality. J. Clin. Investig. 2019;130:3474–3481. doi: 10.1172/JCI120851. PubMed DOI PMC

Richard M.A., Lupo P.J., Morton L.M., Yasui Y.A., Sapkota Y.A., Arnold M.A., Aubert G., Neglia J.P., Turcotte L.M., Leisenring W.M., et al. Genetic variation in POT1 and risk of thyroid subsequent malignant neoplasm: A report from the Childhood Cancer Survivor Study. PLoS ONE. 2020;15:e0228887. doi: 10.1371/journal.pone.0228887. PubMed DOI PMC

Orois A., Badenas C., Reverter J.L., Lopez V., Potrony M., Mora M., Halperin I., Oriola J. Lack of Mutations in POT1 Gene in Selected Families with Familial Non-Medullary Thyroid Cancer. Horm. Cancer. 2020 doi: 10.1007/s12672-020-00383-5. PubMed DOI PMC

Jegerlehner S., Bulliard J.L., Aujesky D., Rodondi N., Germann S., Konzelmann I., Chiolero A., Group N.W. Overdiagnosis and overtreatment of thyroid cancer: A population-based temporal trend study. PLoS ONE. 2017;12:e0179387. doi: 10.1371/journal.pone.0179387. PubMed DOI PMC

Li H., Durbin R. Fast and accurate short read alignment with Burrows-Wheeler transform. Bioinformatics. 2009;25:1754–1760. doi: 10.1093/bioinformatics/btp324. PubMed DOI PMC

Rimmer A., Phan H., Mathieson I., Iqbal Z., Twigg S.R.F., Consortium W.G.S., Wilkie A.O.M., McVean G., Lunter G. Integrating mapping-, assembly- and haplotype-based approaches for calling variants in clinical sequencing applications. Nat. Genet. 2014;46:912–918. doi: 10.1038/ng.3036. PubMed DOI PMC

Lek M., Exome Aggregation Consortium. Karczewski K.J., Minikel E.V., Samocha K.E., Banks E., Fennell T., O’Donnell-Luria A.H., Ware J.S., Hill A.J., et al. Analysis of protein-coding genetic variation in 60,706 humans. Nature. 2016;536:285–291. doi: 10.1038/nature19057. PubMed DOI PMC

Smigielski E.M. dbSNP: a database of single nucleotide polymorphisms. Nucleic Acids Res. 2000;28:352–355. doi: 10.1093/nar/28.1.352. PubMed DOI PMC

The 1000 Genomes Project Consortium A global reference for human genetic variation. Nature. 2015;526:68–74. doi: 10.1038/nature15393. PubMed DOI PMC

Wang K., Li M., Hakonarson H. ANNOVAR: functional annotation of genetic variants from high-throughput sequencing data. Nucleic Acids Res. 2010;38:e164. doi: 10.1093/nar/gkq603. PubMed DOI PMC

Kumar A., Bandapalli O.R., Paramasivam N., Giangiobbe S., Diquigiovanni C., Bonora E., Eils R., Schlesner M., Hemminki K., Forsti A. Familial Cancer Variant Prioritization Pipeline version 2 (FCVPPv2) applied to a papillary thyroid cancer family. Sci. Rep. 2018;8:11635. doi: 10.1038/s41598-018-29952-z. PubMed DOI PMC

Kircher M., Witten D.M., Jain P., O’Roak B.J., Cooper G.M., Shendure J. A general framework for estimating the relative pathogenicity of human genetic variants. Nat. Genet. 2014;46:310–315. doi: 10.1038/ng.2892. PubMed DOI PMC

Cooper G.M., Stone E.A., Asimenos G., Program N.C.S., Green E.D., Batzoglou S., Sidow A. Distribution and intensity of constraint in mammalian genomic sequence. Genome Res. 2005;15:901–913. doi: 10.1101/gr.3577405. PubMed DOI PMC

Siepel A., Bejerano G., Pedersen J.S., Hinrichs A.S., Hou M., Rosenbloom K., Clawson H., Spieth J., Hillier L.W., Richards S., et al. Evolutionarily conserved elements in vertebrate, insect, worm, and yeast genomes. Genome Res. 2005;15:1034–1050. doi: 10.1101/gr.3715005. PubMed DOI PMC

Pollard K.S., Hubisz M.J., Rosenbloom K.R., Siepel A. Detection of nonneutral substitution rates on mammalian phylogenies. Genome Res. 2010;20:110–121. doi: 10.1101/gr.097857.109. PubMed DOI PMC

Liu X., Wu C., Li C., Boerwinkle E. dbNSFP v3.0: A One-Stop Database of Functional Predictions and Annotations for Human Nonsynonymous and Splice-Site SNVs. Hum. Mutat. 2016;37:235–241. doi: 10.1002/humu.22932. PubMed DOI PMC

Petrovski S., Wang Q., Heinzen E.L., Allen A.S., Goldstein D.B. Genic intolerance to functional variation and the interpretation of personal genomes. PLoS Genet. 2013;9:e1003709. doi: 10.1371/annotation/32c8d343-9e1d-46c6-bfd4-b0cd3fb7a97e. PubMed DOI PMC

Robinson J.T., Thorvaldsdottir H., Wenger A.M., Zehir A., Mesirov J.P. Variant Review with the Integrative Genomics Viewer. Cancer Res. 2017;77:e31–e34. doi: 10.1158/0008-5472.CAN-17-0337. PubMed DOI PMC

Hecht M., Bromberg Y., Rost B. Better prediction of functional effects for sequence variants. BMC Genom. 2015;16:S1. doi: 10.1186/1471-2164-16-S8-S1. PubMed DOI PMC

Yachdav G., Kloppmann E., Kajan L., Hecht M., Goldberg T., Hamp T., Honigschmid P., Schafferhans A., Roos M., Bernhofer M., et al. PredictProtein--an open resource for online prediction of protein structural and functional features. Nucleic Acids Res. 2014;42:W337–W343. doi: 10.1093/nar/gku366. PubMed DOI PMC

Pires D.E.V., Ascher D.B., Blundell T.L. mCSM: Predicting the effects of mutations in proteins using graph-based signatures. Bioinformatics (Oxford, England) 2014;30:335–342. doi: 10.1093/bioinformatics/btt691. PubMed DOI PMC

Notredame C., Higgins D.G., Heringa J. T-Coffee: A novel method for fast and accurate multiple sequence alignment. J. Mol. Biol. 2000;302:205–217. doi: 10.1006/jmbi.2000.4042. PubMed DOI

Loayza D., De Lange T. POT1 as a terminal transducer of TRF1 telomere length control. Nature. 2003;423:1013–1018. doi: 10.1038/nature01688. PubMed DOI

Hosen I., Rachakonda P.S., Heidenreich B., Sitaram R.T., Ljungberg B., Roos G., Hemminki K., Kumar R. TERT promoter mutations in clear cell renal cell carcinoma. Int. J. Cancer. 2015;136:2448–2452. doi: 10.1002/ijc.29279. PubMed DOI

Liu F., Feng X., Ma W. Analysis of Telomere Proteins by Chromatin Immunoprecipitation (ChIP) Methods Mol. Biol. 2017;1587:205–214. doi: 10.1007/978-1-4939-6892-3_19. PubMed DOI

Wilson T.L., Hattangady N., Lerario A.M., Williams C., Koeppe E., Quinonez S., Osborne J., Cha K.B., Else T. A new POT1 germline mutation-expanding the spectrum of POT1-associated cancers. Fam. Cancer. 2017;16:561–566. doi: 10.1007/s10689-017-9984-y. PubMed DOI

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