POT1 loss-of-function variants predispose to familial melanoma

Abstract

Deleterious germline variants in CDKN2A account for around 40% of familial melanoma cases, and rare variants in CDK4, BRCA2, BAP1 and the promoter of TERT have also been linked to the disease. Here we set out to identify new high-penetrance susceptibility genes by sequencing 184 melanoma cases from 105 pedigrees recruited in the UK, The Netherlands and Australia that were negative for variants in known predisposition genes. We identified families where melanoma cosegregates with loss-of-function variants in the protection of telomeres 1 gene (POT1), with a proportion of family members presenting with an early age of onset and multiple primary tumors. We show that these variants either affect POT1 mRNA splicing or alter key residues in the highly conserved oligonucleotide/oligosaccharide-binding (OB) domains of POT1, disrupting protein-telomere binding and leading to increased telomere length. These findings suggest that POT1 variants predispose to melanoma formation via a direct effect on telomeres.

Fig. 1. Rare variants in POT1 found in familial melanoma pedigrees

a) We identified four pedigrees carrying deleterious variants in the protection of telomeres 1 (POT1) gene. Shown are a 5-(UF20) and a 6-(AF1) member pedigree carrying the disruptive Tyr89Cys OB domain variant and a splice acceptor variant, respectively. Please note that pedigrees have been adjusted to protect the identity of the families without a loss of scientific integrity. Genotypes for all samples available for testing are shown in blue. CMM; cutaneous malignant melanoma. CLL; chronic lymphocytic leukemia. Sp.A; splice acceptor variant. Circles represent females; squares represent males; diamonds represent undisclosed gender. The patients that were sequenced have a red outline. All melanomas were confirmed by histological analysis with the exception of two cases (*). The number of primary melanomas in each patient is indicated; age of onset is shown in parentheses. b) Highly conserved residues of POT1 are mutated in familial melanoma. Shown are the positions of the variants identified on the POT1 protein (top panel), and on an amino acid alignment (missense variants, bottom panel).

Fig. 2. Missense variants in POT1 disrupt the interaction between POT1 and single-stranded DNA and lead to elongated telomeres

a) Shown is the location of the POT1 residues Tyr89, Gln94 and Arg273 in the N-terminal two oligonucleotide-/oligosaccharide-binding (OB) domains, in green. A telomere-like polynucleotide sequence is shown in orange. Interacting nucleotides in the telomeric sequence are labeled in gray. All three substitutions are predicted to disrupt the association of POT1 with telomeres. b) Mutant Tyr89Cys, Gln94Glu and Arg273Leu POT1 proteins are unable to bind telomeric (TTAGGG)₃ sequences as revealed by an electromobility shift assay. The Tyr223Cys POT1 mutant was used as a positive control representing a known disruptive mutation. c) Calculation of telomere length from exome sequence data. The method used is analogous to the one described in Ref. . Adjusted relative telomere lengths for the three sequenced members of pedigree UF20 are shown alongside the mean telomere length of 38 (all) other melanoma cases who were sequenced alongside, but were wildtype for POT1. The error bar indicates one standard deviation. A Wilcoxon rank sum test was performed comparing the telomere length of the 3 Tyr89Cys cases to the 38 non-carrier controls. d) PCR-based estimate of telomere length. Adjusted mean negative ΔCt values, which correlate positively with telomere length, for POT1 missense variant carriers and non-carrier family controls are shown against a distribution of values from 252 melanoma cases recruited from the Leeds Melanoma cohort that are wildtype at the abovementioned positions (Online Methods). All measurements have been adjusted for age at blood draw and gender. The black line represents a Gaussian kernel density estimate for this set using Silverman’s “rule-of-thumb” for bandwidth smoothing. Orange dots represent members of pedigree UF20; pink, UF31; blue, UF23; red, individual CT1663 from the Leeds Melanoma Case-control study carrying the Arg273Leu variant (Supplementary Table 5). The number of biological replicates for each case ranged from 1 to 4, each with two technical replicates for the POT1 missense variant carriers and non-carrier family controls. Two technical replicates were performed for the 252 POT1 non-carrier cases. Error bars indicate the standard error of the mean.