Identification and functional validation of a novel pathogenic POT1 germline variant p.G95V in familial melanoma

Abstract POT1 variants have been identified in familial melanoma (FM) as well as a number of other germline and somatic malignancies. The functional validation of variants identified from the screening of patients with melanoma gene susceptibility panels is key to understanding the clinical significance of identified variants. Here we report a novel, likely pathogenic POT1 missense variant (p.G95V) in FM and investigate its functional impact. We demonstrate loss of function owing to the inability of the mutant POT1 protein to bind telomeric DNA compared to its wild‐type counterpart. This study provides important functional validation of a novel POT1 variant in FM.


INTRODUCTION
Familial melanoma (FM) is an inherited form of the diease in which at least two first-degree relatives are diagnosed with melanoma.Occurring in 1−10% of all melanoma patients, it is associated with several highpenetrance susceptibility genes, most commonly cyclindependent kinase inhibitor 2 A (CDKN2A) in 20−40% of FM cases. 1 Less commonly CDK4 and BAP1 have also been identified 2 and recently, genes involved in telomere maintenance: POT1, ACD and TERF2IP, 3 but over 50% of suspected FM cases have no identifiable germline mutation.Telomere instabilility is a key feature of melanoma given the high frequency of somatic TERT promoter variants identified in sporadic melanoma, however, TERT germline variants are only detected rarely in FM. 4 Protection of Telomeres 1 (POT1) is a component of the Shelterin complex (POT1, TRF1, TRF2, TIN2, TPP1 and RAP1) which binds to the ends of telomeres, critical for telomere end protection.POT1 specifically binds to the repetitive sequence of single stranded DNA at the very end of the telomere (5′TTAGGG3′) via its N-terminal oligonucleotide binding (OB) domains.As such, POT1 plays a vital role in maintaining chromosomal stability by preventing aberrant DNA damage pathway activation at telomeres and regulating telomerase activity.
There is mounting evidence that loss of function POT1 variants are implicated in malignancy.6][7] POT1 germline variants have been found in 2−4% of CDKN2A/CDK4-negative FM pedigrees, indicating that POT1 is the second major melanoma susceptibility gene following CDKN2A. 8DK4 mutations have been reported with a prevalence of 0.68%. 2 POT1 mutations are rare and contribute to a 0.5% melanoma risk burden in the general population.However, functional validation of variants is critical in light of increased melanoma genetic screening in the clinic.POT1 germline variants have also been identified in familial glioma, including p.G65C, which occurs at the same amino acid residue as the one reported in this study but has not been functionally validated to date.Germline mutations have also been reported in papillary thyroid cancer, one of which (p.V29L) has been shown to have disrupted telomere binding. 6,9

CASE REPORT
A 79 year-old lady presented in 2008 with a history of 12 melanomas from age 32 and during subsequent surveillance she was diagnosed with five early stage melanomas (stage pT1a), nine in-situ melanomas and seven basal cell carcinomas.She developed a papillary thyroid cancer (pT1N0M0), surgically excised in 2010.
The patient had a strong family history of malignancy.Her daughter had four melanomas from the age of 30 and died of gastric cancer age 45.Her son died aged 37 from a glioblastoma.In view of her personal and family history of multiple melanomas, she underwent germline genetic testing following counselling by the clinical genetics team with a melanoma susceptibility gene panel and a variant of uncertain significance in the POT1 gene (c.284G > T p.(Gly95Val)) was identified.

DISCUSSION
There is strong evidence that this novel germline variant is likely to be a variant of significance.Firstly, the affected amino acid resides in the functionally critical OB1 domain (Figure 1a).Furthermore, it is predicted to be damaging by a consensus of five independent bioinformatic tools (Figure 1b) and is classified as pathogenic according to ACMG guidelines. 10The mutated amino acid residue is phylogenetically conserved which suggests it is critical to POT1 function (Figure 1c).In addition, the ability of in-vitro translated POT1 p.G95V to bind to telomeric DNA was investigated using electrophoretic mobility shift assay (EMSA) (Figure 2), where in contrast to wild type protein, a complete lack of POT1-DNA complex formation was observed.
For the first time, this study has functionally validated the p.G95V POT1 variant.We provide evidence that this is a loss of function variant owing to its inability to bind to telomeres, a finding consistent with other reported OB domain POT1 variants. 3,7,9Interestingly, an identical somatic POT1 variant has been identified in chronic lymphocytic leukemia, which further highlights its likely driver mutation status. 11Furthermore, a POT1 variant (p.G65C) which occurs at the same amino acid residue as the one reported in this study, has been identified in familial glioma and is also predicted to disrupt telomere binding. 6OT1 loss has been associated with activation of the Ataxia Telangiectasia and Rad3-related protein (ATR) pathway. 12Given melanoma is a UV driven malignancy it is unsurprising that mutations in genes encoding DNA damage repair proteins have been reported in a subset of melanomas.In one study, mutations affecting the ATR pathway (ATR, Chk1, 53BP1, RPA) occur in 16−25% of cases and the ATM pathway (ATM, MDC1, Chk2) in 8−22% of cases. 13As ATR inhibitors are currently under evaluation in clinical trials for the management of a variety of solid tumours, targeting this pathway could be relevant in patients harbouring POT1 variants.
In summary, the identification of a pathogenic POT1 gene variant with demonstrated functional relevance, along with the advent of new potential therapeutic agents that target DNA damage repair pathways, could have wider implications for genetic screening and the management of other FM patients with POT1 variants.In addition, this patient's family history suggests that POT1 loss of function germline variants can also predispose to a variety of other malignancies.

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I G U R E 1 The POT1 p.G95V missense variant is predicted to be pathogenic and disrupts telomere binding.(a) Mutant localisation to the oligonucleotide binding domain (OB1) on a schematic of the protein.(b) p.G95V is predicted to have a deleterious effect on protein structure and/or function by four independent pathogenicity prediction tools (MutPred2, Provean, Polyphen2 and Mutation taster).(c) Multiple sequence alignment of the POT1 protein in humans and 12 other evolutionary diverse species.Amino acid positions 89−148, relative to human POT1, are shown with the arrow indicating the highly conserved p.G95V residue.POT1 GERMLINE VARIANT P.G95V IN FAMILIAL MELANOMA | 1237