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Rare missense variants in POT1 predispose to familial cutaneous malignant melanoma

Abstract

Although CDKN2A is the most frequent high-risk melanoma susceptibility gene, the underlying genetic factors for most melanoma-prone families remain unknown. Using whole-exome sequencing, we identified a rare variant that arose as a founder mutation in the telomere shelterin gene POT1 (chromosome 7, g.124493086C>T; p.Ser270Asn) in five unrelated melanoma-prone families from Romagna, Italy. Carriers of this variant had increased telomere lengths and numbers of fragile telomeres, suggesting that this variant perturbs telomere maintenance. Two additional rare POT1 variants were identified in all cases sequenced in two separate Italian families, one variant per family, yielding a frequency for POT1 variants comparable to that for CDKN2A mutations in this population. These variants were not found in public databases or in 2,038 genotyped Italian controls. We also identified two rare recurrent POT1 variants in US and French familial melanoma cases. Our findings suggest that POT1 is a major susceptibility gene for familial melanoma in several populations.

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Figure 1: Pedigrees of CMM-prone families with the POT1 variant g.124493086C>T encoding p.Ser270Asn.
Figure 2: Structural impact of rare variants in POT1.
Figure 3: Telomere length in PBMCs from individuals with the POT1 variant encoding p.Ser270Asn.

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Acknowledgements

This work was supported by the Intramural Research Program of the US National Institutes of Health (NIH), National Cancer Institute (NCI), Division of Cancer Epidemiology and National Institute on Aging (NIA), Division of Molecular Gerontology. The samples from the Instituto Valenciano de Oncología were retrieved from the Biobanco del Instituto Valenciano de Oncología. The Genoa collection was partly supported by Universitá degli Studi di Genova Progetti di Ricerca di Ateneo PRA 2012-2013 and IRCCS Azienda Ospedaliera Universitaria San Martino–IST Istituto Nazionale per la Ricerca sul Cancro, 5 per 1000 per la Ricerca Corrente. The French MELARISK collection was partly supported by Programme Hospitalier de Recherche Clinique (AOM-07-195) and Ligue Nationale Contre le Cancer (PRE 09/FD). We acknowledge the contribution of Institut Gustave-Roussy (IGR) Biobank and Fondation Jean Dausset-CEPH Biobank in providing samples for melanoma-prone families or individual's. The exome sequencing of French samples was supported by a grant from Génome Québec, le Ministére de l'Enseignement Supérieur, de la Recherche, de la Science et de la Technologie (MESRST) du Québec and McGill University. This project has also been funded in part with federal funds from the US NIH, NCI, under contract HHSN261200800001E. The content of this publication does not necessarily reflect the views or policies of the US Department of Health and Human Services, nor does mention of trade names, commercial products or organizations imply endorsement by the US government.

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J.S., X.R.Y., M.R., X.H., A.M.G., L.R.G., J.N.S., F.D., A.V., H.M. and M.T.L. performed and interpreted genetics analyses. B.B., S.A.S. and S.R. performed functional prediction analyses. D.C., M.C.F., P.G., B.B.-d.P., E.N., M.F.A., W.B., L.P., P.Q., J.B.-R., Z.G.-C., F.J., K.P., G.B.S., G.L., M.A.T. and M.T.L. directed the clinical work for the melanoma cases from Italy, Spain, the United States and France. N.E.C. and M.L.M. directed the clinical work for the LPD cases. M.Y., M.A.T. and S.J.C. directed all sequencing analyses at NCI DCEG CGR. J.H., M.C., Z.W., X.Z. and the NCI DCEG Cancer Genomics Research Laboratory conducted the whole-exome and targeted sequencing and the genotyping analyses for the Italian, US, Spanish and part of the French melanoma cases and controls. The NCI DCEG Cancer Sequencing Working Group examined and enrolled families without melanoma and LPD for whole-exome sequencing analyses. The French Familial Melanoma Study Group examined and enrolled melanoma cases and controls from France. M.L., Y.R. and M.F. conducted whole-exome sequencing and genotyping for the French melanoma cases and individuals with other non-cancer diseases. P.L.H., P.M., C.P. and A.E. oversaw sample preparation for the laboratory studies. J.Y., H.V., W.C. and Y.L. performed functional analyses of POT1. M.T.L. designed the overall study. X.R.Y., J.S., B.B., A.M.G. and M.T.L. drafted the manuscript. All authors contributed to the final manuscript.

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Correspondence to Maria Teresa Landi.

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A full list of members appears in the Supplementary Note.

A full list of members appears in the Supplementary Note.

Integrated supplementary information

Supplementary Figure 1 Heat map of the estimated kinship coefficients for POT1 Ser270Asn variant carriers.

POT1 Ser270Asn variant carriers (from Italian families (F1–F5) plus a sporadic case (S1)) and Environment And Genetics in Lung cancer Etiology (EAGLE) controls were genotyped using the Illumina HumanOmniExpress BeadChip (>700,000 common SNPs) and the HumanHap550 BeadChip (~550,000 SNPs), respectively. 306,684 autosomal SNPs that were on both platforms were used for the estimation of kinship coefficients using PLINK. Each block represents a family. None of the subject pairs from different families had an estimated kinship coefficient greater than 0.05.

Supplementary Figure 2 Estimation of the age of the most recent common ancestor (MRCA) carrying the Ser270Asn variant.

All analyses were based on the five probands from the Italian CMM families (F1-1, F2-1, F3-1, F4-1, F5-1) and the one sporadic case (S1) who carried the Ser270Asn variant. (a) Estimated kinship coefficients for Ser270Asn variant carriers. F1-1, F4-1 and S1 show weak correlation. (b) Subjects F1-1, F4-1 and S1 shared long haplotypes encompassing the POT1 region (horizontal blue lines). GERMLINE analysis revealed that multiple additional genomic segments were also shared by these three subjects (data not shown), suggesting that they descended from a common ancestor. Megabase locations are provided for the shared haplotype boundaries and for POT1. The red triangle shows the POT1 location at position 124.5 Mb based on hg19. (c) The lineage used to estimate the age of the MRCA carrying the Ser270Asn variant. From panels a and b, F1-1, F4-1 and S1 descended from a common ancestor, designated as A. A, F2-1, F3-1 and F5-1 descended independently from the MRCA. (d) To estimate the age of the MRCA, we used the independent haplotypes from subjects F2-1, F3-1, F5-1 and from F1-1, which we used to represent the subjects who descended from common ancestor A. The genetic distance (numbers with cM in parentheses) and megabase location for the boundaries of the haplotypes and POT1 (in red) are also provided.

Supplementary Figure 3 Structural illustrations of POT1 rare variants in the N-terminal OB domains.

(a) Ser270 is an exposed residue and from the crystal structure (1XJV) it is evident that it interacts with the phosphate (PI) backbone of nucleotide guanine 6 (DG6) of the telomeric ssDNA using hydrogen bonds (dotted green lines). Important residues surrounding Ser270 (ball and stick) are shown in stick form. (b) Asp224 may form coordinated interactions with Gly267, which mimic the role of guanine-cytosine base-pair interactions and appear to be further strengthened by the favorable positioning of Tyr233, which forms energetically favorable PI-PI interactions (orange lines) with DG10 from the ssDNA (PDB 1XJV). (c) Key residues surrounding Arg137 (highlighted in yellow) are shown in ball-and-stick mode. Arg137 is involved in hydrogen bond interactions (dotted green lines) with Pro113 and Glu107 and also has the possibility of forming a salt bridge with the latter. These concerted interactions could play a key role in the structural integrity of the fold and indirectly influence DNA binding when mutated. RCSB PDB 1XJV was used for highlighting the interactions.

Supplementary Figure 4 Pedigrees of CMM-prone families with rare variants in POT1.

Solid squares and circles, CMM cases; gray, other cancers; circles, females; squares, males. “y” indicates a mutation carrier, and “n” indicates a non-carrier. Age is the sage at diagnosis for CMM cases and the age at exam for unaffected family members.

Supplementary Figure 5 Telomere length in ex vivo stimulated PBMCs from individuals with POT1 variants.

Telomere signal intensity reflecting telomere length was quantified by Q-FISH in ex vivo stimulated PBMCs from POT1-variant carriers (n = 4, including 2 with Ser270Asn and 1 each with Arg137His and Gln623His) and controls (age-matched melanoma cases without POT1 variants; n = 3). Bars (in red) denote mean telomere signal intensity depicted in arbitrary units (AU) in each group. Error bars (in yellow) indicate standard deviation of errors. P values were obtained from two-sided Student's t tests.

Supplementary Figure 6 Incidence of fragile telomeres in ex vivo stimulated PBMCs from individuals with POT1 variants.

Number of fragile telomeres per metaphase spread of ex vivo stimulated PBMCs from POT1-variant carriers (n = 4, including 2 with Ser270Asn and 1 each with Arg137His and Gln623His) and controls (age-matched melanoma cases without POT1 variants; n = 3). At least 30 metaphases per sample were counted. Error bars indicate standard deviation. P values were obtained from two-sided Student's t tests.

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Shi, J., Yang, X., Ballew, B. et al. Rare missense variants in POT1 predispose to familial cutaneous malignant melanoma. Nat Genet 46, 482–486 (2014). https://doi.org/10.1038/ng.2941

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