Judith Symmons

A novel recurrent mutation in MITF predisposes to familial and sporadic melanoma

So far, two genes associated with familial melanoma have been identified, accounting for a minority of genetic risk in families. Mutations in CDKN2A account for approximately 40% of familial cases, and predisposing mutations in CDK4 have been reported in a very small number of melanoma kindreds. Here we report the whole-genome sequencing of probands from several melanoma families, which we performed in order to identify other genes associated with familial melanoma. We identify one individual carrying a novel germline variant (coding DNA sequence c.G1075A; protein sequence p.E318K; rs149617956) in the melanoma-lineage-specific oncogene microphthalmia-associated transcription factor (MITF). Although the variant co-segregated with melanoma in some but not all cases in the family, linkage analysis of 31 families subsequently identified to carry the variant generated a log of odds (lod) score of 2.7 under a dominant model, indicating E318K as a possible intermediate risk variant. Consistent with this, the E318K variant was significantly associated with melanoma in a large Australian case–control sample. Likewise, it was similarly associated in an independent case–control sample from the United Kingdom. In the Australian sample, the variant allele was significantly over-represented in cases with a family history of melanoma, multiple primary melanomas, or both. The variant allele was also associated with increased naevus count and non-blue eye colour. Functional analysis of E318K showed that MITF encoded by the variant allele had impaired sumoylation and differentially regulated several MITF targets. These data indicate that MITF is a melanoma-predisposition gene and highlight the utility of whole-genome sequencing to identify novel rare variants associated with disease susceptibility.

POT1 loss-of-function variants predispose to familial melanoma

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.

Deep sequencing of uveal melanoma identifies a recurrent mutation in PLCB4.

Next generation sequencing of uveal melanoma (UM) samples has identified a number of recurrent oncogenic or loss-of-function mutations in key driver genes including: GNAQ, GNA11, EIF1AX, SF3B1 and BAP1. To search for additional driver mutations in this tumor type we carried out whole-genome or whole-exome sequencing of 28 tumors or primary cell lines. These samples have a low mutation burden, with a mean of 10.6 protein changing mutations per sample (range 0 to 53). As expected for these sun-shielded melanomas the mutation spectrum was not consistent with an ultraviolet radiation signature, instead, a BRCA mutation signature predominated. In addition to mutations in the known UM driver genes, we found a recurrent mutation in PLCB4 (c.G1888T, p.D630Y, NM_000933), which was validated using Sanger sequencing. The identical mutation was also found in published UM sequence data (1 of 56 tumors), supporting its role as a novel driver mutation in UM. PLCB4 p.D630Y mutations are mutually exclusive with mutations in GNA11 and GNAQ, consistent with PLCB4 being the canonical downstream target of the former gene products. Taken together these data suggest that the PLCB4 hotspot mutation is similarly a gain-of-function mutation leading to activation of the same signaling pathway, promoting UM tumorigenesis.

Nevi, Family History, and Fair Skin Increase the Risk of Second Primary Melanoma

Although risk factors for primary cutaneous melanoma are well defined, relatively little is known about predictors for second primary melanoma. Given the rising incidence of this cancer, coupled with improvements in survival, there is a prevalent and growing pool of patients at risk of second primary melanomas. To identify the predictors of second primary melanoma, we followed a cohort of 1,083 Queensland patients diagnosed with incident melanoma between 1982 and 1990 and who completed a baseline questionnaire. During a median follow-up of 16.5 years, 221 patients were diagnosed with at least one additional primary melanoma. In multivariate analyses, second primary melanomas were associated with high nevus count (hazard ratio (HR), 2.91; 95% confidence interval (CI) 1.94-4.35), high familial melanoma risk (HR, 2.12; 95% CI 1.34-3.36), fair skin (HR, 1.51; 95% CI 1.06-2.16), inability to tan (HR, 1.66; 95% CI 1.13-2.43), an in situ first primary melanoma (HR, 1.36; 95% CI 0.99-1.87), and male sex (HR, 1.49; 95% CI 1.12-2.00). Patients whose first primary was lentigo maligna melanoma (HR, 1.80; 95% CI 1.05-3.07) or nodular melanoma (HR, 2.13; 95% CI 1.21-3.74) had higher risks of subsequent primaries than patients whose first primary tumor was superficial spreading melanoma. These characteristics could be assessed in patients presenting with first primary melanoma to evaluate risk of developing a second primary.

POLE mutations in families predisposed to cutaneous melanoma

Germline mutations in the exonuclease domain of POLE have been shown to predispose to colorectal cancers and adenomas. POLE is an enzyme involved in DNA repair and chromosomal DNA replication. In order to assess whether such mutations might also predispose to cutaneous melanoma, we interrogated whole-genome and exome data from probands of 34 melanoma families lacking pathogenic mutations in known high penetrance melanoma susceptibility genes: CDKN2A, CDK4, BAP1, TERT, POT1, ACD and TERF2IP. We found a novel germline mutation, POLE p.(Trp347Cys), in a 7-case cutaneous melanoma family. Functional assays in S. pombe showed that this mutation led to an increased DNA mutation rate comparable to that seen with a Pol ε mutant with no exonuclease activity. We then performed targeted sequencing of POLE in 1243 cutaneous melanoma cases and found that a further ten probands had novel or rare variants in the exonuclease domain of POLE. Although this frequency is not significantly higher than that in unselected Caucasian controls, we observed multiple cancer types in the melanoma families, suggesting that some germline POLE mutations may predispose to a broad spectrum of cancers, including melanoma. In addition, we found the first mutation outside the exonuclease domain, p.(Gln520Arg), in a family with an extensive history of colorectal cancer.

Prevalence of germline BAP1, CDKN2A, and CDK4 mutations in an australian population-based sample of cutaneous melanoma cases

Mutations in Cyclin-Dependent Kinase Inhibitor 2A (CDKN2A) and Cyclin-Dependent Kinase 4 (CDK4) contribute to susceptibility in approximately 40% of high-density cutaneous melanoma (CMM) families and about 2% of unselected CMM cases. BRCA-1 associated protein-1 (BAP1) has been more recently shown to predispose to CMM and uveal melanoma (UMM) in some families; however, its contribution to CMM development in the general population is unreported. We sought to determine the contribution of these genes to CMM susceptibility in a population-based sample of cases from Australia. We genotyped 1,109 probands from Queensland families and found that approximately 1.31% harbored mutations in CDKN2A, including some with novel missense mutations (p.R22W, p.G35R and p.I49F). BAP1 missense variants occurred in 0.63% of cases but no CDK4 variants were observed in the sample. This is the first estimate of the contribution of BAP1 and CDK4 to a population-based sample of CMM and supports the previously reported estimate of CDKN2A germline mutation prevalence.

Assessment of PALB2 as a Candidate Melanoma Susceptibility Gene

Partner and localizer of BRCA2 (PALB2) interacts with BRCA2 to enable double strand break repair through homologous recombination. Similar to BRCA2, germline mutations in PALB2 have been shown to predispose to Fanconi anaemia as well as pancreatic and breast cancer. The PALB2/BRCA2 protein interaction, as well as the increased melanoma risk observed in families harbouring BRCA2 mutations, makes PALB2 a candidate for melanoma susceptibility. In order to assess PALB2 as a melanoma predisposition gene, we sequenced the entire protein-coding sequence of PALB2 in probands from 182 melanoma families lacking pathogenic mutations in known high penetrance melanoma susceptibility genes: CDKN2A, CDK4, and BAP1. In addition, we interrogated whole-genome and exome data from another 19 kindreds with a strong family history of melanoma for deleterious mutations in PALB2. Here we report a rare known deleterious PALB2 mutation (rs118203998) causing a premature truncation of the protein (p.Y1183X) in an individual who had developed four different cancer types, including melanoma. Three other family members affected with melanoma did not carry the variant. Overall our data do not support a case for PALB2 being associated with melanoma predisposition.

Germline mutations in candidate predisposition genes in individuals with cutaneous melanoma and at least two independent additional primary cancers

Background While a number of autosomal dominant and autosomal recessive cancer syndromes have an associated spectrum of cancers, the prevalence and variety of cancer predisposition mutations in patients with multiple primary cancers have not been extensively investigated. An understanding of the variants predisposing to more than one cancer type could improve patient care, including screening and genetic counselling, as well as advancing the understanding of tumour development. Methods A cohort of 57 patients ascertained due to their cutaneous melanoma (CM) diagnosis and with a history of two or more additional non-cutaneous independent primary cancer types were recruited for this study. Patient blood samples were assessed by whole exome or whole genome sequencing. We focussed on variants in 525 pre-selected genes, including 65 autosomal dominant and 31 autosomal recessive cancer predisposition genes, 116 genes involved in the DNA repair pathway, and 313 commonly somatically mutated in cancer. The same genes were analysed in exome sequence data from 1358 control individuals collected as part of non-cancer studies (UK10K). The identified variants were classified for pathogenicity using online databases, literature and in silico prediction tools. Results No known pathogenic autosomal dominant or previously described compound heterozygous mutations in autosomal recessive genes were observed in the multiple cancer cohort. Variants typically found somatically in haematological malignancies (in JAK1, JAK2, SF3B1, SRSF2, TET2 and TYK2) were present in lymphocyte DNA of patients with multiple primary cancers, all of whom had a history of haematological malignancy and cutaneous melanoma, as well as colorectal cancer and/or prostate cancer. Other potentially pathogenic variants were discovered in BUB1B, POLE2, ROS1 and DNMT3A. Compared to controls, multiple cancer cases had significantly more likely damaging mutations (nonsense, frameshift ins/del) in tumour suppressor and tyrosine kinase genes and higher overall burden of mutations in all cancer genes. Conclusions We identified several pathogenic variants that likely predispose to at least one of the tumours in patients with multiple cancers. We additionally present evidence that there may be a higher burden of variants of unknown significance in ‘cancer genes’ in patients with multiple cancer types. Further screens of this nature need to be carried out to build evidence to show if the cancers observed in these patients form part of a cancer spectrum associated with single germline variants in these genes, whether multiple layers of susceptibility exist (oligogenic or polygenic), or if the occurrence of multiple different cancers is due to random chance.

Abstract 20: POT1 mutations predispose to familial melanoma

Mutations in CDKN2A account for approximately 40% of familial melanoma cases, and rare mutations in CDK4, BRCA2, BAP1 and in the promoter of TERT also contribute to the disease. However, about half of familial melanoma cases remain unaccounted for. Here we set out to identify high-penetrance susceptibility genes in these unexplained cases. To achieve this, we sequenced 184 melanoma cases from 105 pedigrees (168 exomes and 16 whole genomes) recruited in the United Kingdom, the Netherlands, and Australia that had been screened and found negative for pathogenetic variants in CDKN2A and CDK4. These patients came from pedigrees with between two and eleven cases of melanoma or were single cases that presented with either multiple primary melanomas, multiple primary cancers, one of which was melanoma, and/or an early age of onset ( We found three missense and one splice acceptor mutation, each co-segregating in a different pedigree, in the protection of telomeres 1 (POT1) gene. Importantly, the missense mutations were all located in the highly conserved N-terminal oligonucleotide-/oligosaccharide-binding (OB) domains of POT1, which function to mediate protein – DNA binding. We show that these mutations completely abolish the POT1-DNA complex. Furthermore, we use two methods (one bioinformatic, the other experimental) to assess telomere length in POT1 missense mutation carriers and non-carriers, conclusively showing that individuals with POT1 mutations have substantially longer telomeres than controls. We also amplified and sequenced the POT1 gene product in two of the splice acceptor mutation carriers, showing that the mutation does lead to aberrant splicing. The families that carry POT1 mutations in this study present not only with melanoma but also with other cancers, namely breast, small cell lung, endometrial and brain tumours, suggesting a possible role for germline POT1 mutations in susceptibility to a range of cancers in addition to melanoma. Furthermore, genotyping across the four identified positions in a melanoma case-control series (1,739 cases and 2,402 controls) revealed that each of two cases carried one of these mutations, whereas no mutations were found in controls, suggesting that POT1 mutations could also account for sporadic melanoma cases. In this study we describe germline mutations in the gene encoding the telomere-associated protein POT1 in almost 4% of CDKN2A/CDK4-negative familial melanoma pedigrees and in almost 6% of pedigrees with five or more melanoma cases, making POT1 the second most frequently mutated high-penetrance familial melanoma gene reported to date. In combination with the recently described TERT promoter mutation, these findings significantly extend our understanding of a novel mechanism predisposing to the development of familial melanoma. Since the dysregulation of telomere protection by POT1 has recently been identified as a target for potential therapeutic intervention, in the future, it may be possible that early identification of families with POT1 mutations may facilitate better management of their disease. Citation Format: Carla Daniela Robles-Espinoza, Mark Harland, Andrew J. Ramsay, Lauren G. Aoude, Victor Quesada, Zhihao Ding, Karen A. Pooley, Antonia L. Pritchard, Jessamy C. Tiffen, Mia Petljak, Jane M. Palmer, Judith Symmons, Peter Johansson, Mitchell S. Stark, Michael G. Gartside, Helen Snowden, Grant W. Montgomery, Nicholas G. Martin, Jimmy Z. Liu, Jiyeon Choi, Matthew Makowski, Kevin M. Brown, Alison M. Dunning, Thomas M. Keane, Carlos Lopez-Otin, Nelleke A. Gruis, Nicholas K. Hayward, D. Timothy Bishop, Julia A. Newton-Bishop, David J. Adams. POT1 mutations predispose to familial melanoma. [abstract]. In: Proceedings of the AACR Special Conference: Cancer Susceptibility and Cancer Susceptibility Syndromes; Jan 29-Feb 1, 2014; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(23 Suppl):Abstract nr 20. doi:10.1158/1538-7445.CANSUSC14-20

From GWAS to genome sequencing: complementary approaches to identify melanoma predisposition genes

Family and twin studies indicate that melanoma susceptibility has a strong genetic component. Very rarely, melanoma runs in families in which there is an inherited mutation in a single ‘high penetrance’ gene, but in the general population melanoma susceptibility is thought to be governed by variation in a series of ‘low penetrance’ genes. We sought to identify new melanoma risk genes of both classes by conducting an Australian genome-wide association study (GWAS) of ~2200 melanoma cases and ~4300 matched controls (from the AMFS and Q-MEGA studies), in parallel with whole-genome sequencing of cases from densely affected melanoma families with follow up genotyping of interesting variants in the GWAS sample and other highly case-loaded melanoma families. Genotyping of the GWAS sample was carried out using Illumina Hap610K or OMNI 1M arrays. All 25 SNPs that reached genome-wide statistical significance (i.e. p<5 x 10-8) map to chromosomal regions/genes previously associated with melanoma (e.g. MC1R, ASIP, OCA2, MTAP/CDKN2A and SLC45A2). However, two other genomic regions had multiple adjacent SNPs with low p values. These were the focus of a replication study using melanoma GWAS data generated by groups from the MD Anderson Cancer Center and the International Melanoma Genetics Consortium (GenoMEL). Both independent GWAS data sets support the original Australian findings and thus indicate that the PARP1 gene and another broad region on 1q (which includes SETDB1, a recently identified melanoma oncogene) are novel low penetrance melanoma risk loci. The three known high penetrance melanoma susceptibility genes (CDKN2A, CDK4 and ARF) account for less than half of all ‘familial’ melanoma. We sought to identify other genes responsible for susceptibility in multi-case melanoma families using a next-generation sequencing approach. Families were chosen on the basis that they did not have a mutation in CDKN2A, CDK4 or ARF; and had at least 5 melanoma cases. Whole-genome or exome sequencing was carried out on X cases from Y families. No convincing evidence has yet been obtained to support the identification of a new familial melanoma gene, however, follow up genotyping of several novel SNPs in the GWAS sample showed that a non-synonymous variant in the MITF gene is associated with melanoma in the general population. This type of integrated approach should help accelerate the discovery of new loci that play a role in the aetiology of melanoma.