Genetics of familial melanoma: 20 years after CDKN2A

Acc

epte

d A

rtic

le

This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process, which may lead to differences between this version and the Version of Record. Please cite this article as doi: 10.1111/pcmr.12333 This article is protected by copyright. All rights reserved.

Received Date : 30-Sep-2014 Revised Date : 19-Nov-2014 Accepted Date : 24-Nov-2014 Article Type : Review

Manuscript Category: Prevention & Epidemiology (PRE)

Genetics of Familial Melanoma: 20 years after CDKN2A

Lauren G. Aoude1,2, Karin A.W. Wadt3, Antonia L. Pritchard1, Nicholas K. Hayward1

1QIMR Berghofer Medical Research Institute, Brisbane, Australia

2University of Queensland, Brisbane, Australia

3Department of Clinical Genetics, Rigshospitalet, Copenhagen, Denmark

Corresponding author : [email protected]

Corresponding author: Nicholas K. Hayward, QIMR Berghofer Medical Research Institute, 300 Herston Rd, Herston, QLD 4029, Australia.

Short title: Familial Melanoma Genetics

SUMMARY

Twenty years ago the first familial melanoma susceptibility gene, CDKN2A, was identified. Two years later, another high penetrance gene, CDK4, was found to be responsible for melanoma development in some families. Progress in identifying new familial melanoma genes was subsequently slow; however, with the advent of next generation sequencing, a small number of new high penetrance genes have recently been uncovered. This approach has identified the lineage specific oncogene MITF as a susceptibility gene both in melanoma families and the general population, as

Acc

epte

d A

rtic

le

This article is protected by copyright. All rights reserved.

well as the discovery of telomere maintenance as a key pathway underlying melanoma predisposition. Given these rapid recent advances, this approach seems likely to continue to pay dividends. Here we review the currently known familial melanoma genes, providing evidence that most additionally confer risk to other cancers, indicating that they are likely general tumour suppressor genes or oncogenes, which has significant implications for surveillance and screening.

INTRODUCTION

There are many environmental and genetic factors that play a part in melanomagenesis. Although exposure to ultraviolet radiation (UVR) plays a significant role in development of cutaneous malignant melanoma (CMM), twin studies estimate 55% of the variation in CMM liability is due to genetic effects (Shekar et al., 2009). While approximately 1% of CMM cases occur in individuals with a strong family history of melanoma, high-risk genes account for melanoma susceptibility in only a small proportion of these families (de Snoo and Hayward, 2005; Hayward, 2000). Genome-wide association studies have identified a growing number of genes contributing to melanoma risk in the general population, however, these risk alleles have only very modest effects (hazard ratios of 1.1 – 2.5). Collectively, identification of these susceptibility genes has been important, because they inform on pathways of melanoma susceptibility other than those mediated by phenotypic risk factors, such as pigmentation, freckling and naevi. Additionally, in principle, they will allow assessment of whether familial clustering of melanoma cases is due to carriage of multiple low penetrance risk variants rather than a single dominant Mendelian mutation, a statistical exercise that has yet to be formally conducted.

Technologies have shifted greatly over the past 20 years. Melanoma predisposition genes were initially found through linkage analysis and positional cloning. This progressed to a candidate gene approach that targeted genes because of their role in the same pathway as a previously identified familial melanoma gene. More recently whole-genome and exome sequencing has revolutionized and expedited the identification of cancer predisposition genes. It has been particularly valuable in instances where mutations are rare, occurring in only a very small number of families. By discovering mutations that segregate within highly melanoma-dense families and then extending analysis to a large collection of lower density families, significant advances in identification of genes conferring CMM predisposition have recently been achieved.

Acc

epte

d A

rtic

le


A detailed review of the identified high penetrance melanoma susceptibility genes, the contribution of each to melanoma development, and where relevant, other cancers, is summarized below.

1994 to 1996 - a promising start

CDKN2A

Role of CDKN2A in melanoma

Cyclin-dependent kinase inhibitor 2A (CDKN2A, MIM: 600160, 9p21.3) was the first familial melanoma gene identified (Hussussian et al., 1994; Kamb et al., 1994) and accounts for the majority of high density melanoma-prone families. CDKN2A is responsible for susceptibility in ~10% of 2-case melanoma families and 30-40% of families with 3 or more cases of melanoma (Goldstein et al., 2007). The connection between CDKN2A mutation and melanoma predisposition was established in 1994 through a combination of linkage studies and a positional cloning approach (Hussussian et al., 1994; Kamb et al., 1994). CDKN2A encodes two distinct proteins, p16INK4A (p16) and p14ARF (p14) (Figure 1), which both function in cell cycle regulation (de Snoo and Hayward, 2005). The p16 and p14 mRNAs are transcribed from alternative first exons (1α and 1β) but utilise the same second and third exons. The two products share no amino acid sequence since they are translated in different reading frames (ARF = alternative reading frame). CDKN2A germline mutations in exon 1α affect only the p16 transcript, whereas some of those occurring in exon 2 can affect both p16 and p14. p16 is a tumour suppressor that modulates pRb regulated G1 to S phase transition by inhibiting the kinase activity of CDK4 and CDK6. Engaging this pathway leads to senescence and regulation of damaged cells (Palmieri et al., 2009). p14 is also a tumour suppressor, acting through the stabilisation of p53, the “guardian of the genome”, via inhibition of MDM2-induced p53 degradation (Zhang et al., 1998).

Mutations occurring in p16 are predominantly loss of function missense mutations distributed along the entire length of the protein (Goldstein et al., 2006; Hussussian et al., 1994; Kamb et al., 1994). In rare instances, a germline CDKN2A promoter mutation (c.-34G>T) that causes an aberrant initiation codon, or a ‘deep’ intronic mutation (IVS2-105A>G), which affects splicing, also predisposes to melanoma (Harland et al., 2000; Liu et al., 1999). In contrast, inactivating mutations of p14 are whole gene deletions, insertions or splice mutations (Harland et al., 2005; Mistry et al., 2005; Randerson-Moor et al., 2001; Rizos et al., 2001), with no missense variants having yet been reported (Table 1). There are several p16 founder mutations (Figure 1) that occur in distinct geographical regions (Goldstein et al., 2006). The p.R112_L113insR mutation occurs in 92% of Swedish CMM families and

Acc

epte

d A

rtic

le


the p.A76fsX70 deletion mutation is present in 90% of Dutch CMM families. There is also a founder mutation in families from France, Spain and Italy (p.G101W); and families from Australia and the UK have several common founders (p.R24P, p.L32P, p.M53I and IVS2-105A>G).

The incidence of germline CDKN2A mutations in the general population is very low. In the first population-based study to address this topic, which analyzed melanoma cases from Queensland, Australia, it was estimated that just 0.2% of all melanoma cases were due to CDKN2A mutations (Aitken et al., 1999). In a subsequent population-based study of CMM cases from North America, Europe and Australia, CDKN2A mutation prevalence was found to be 2% (Begg et al., 2005), and in a recent Greek hospital-based population study, 5% of CMM cases harboured a CDKN2A mutation (Nikolaou et al., 2011). Recent genotyping of 1109 probands from a population-based sample from Queensland found 1.4% of CMM cases to harbour mutations in CDKN2A (Aoude et al., unpublished observations).

Role of CDKN2A in non-melanoma cancers

Somatic mutations of CDKN2A have been described in a wide variety of different tumour types, including oral squamous cell carcinoma, leukaemia and bladder cancer (Foulkes et al., 1997; Ruas and Peters, 1998; Smith-Sorensen and Hovig, 1996), suggesting that CDKN2A might play a key tumorigenic ‘gatekeeper’ role. This led to the investigation of whether germline CDKN2A mutations also increased risk of non-melanoma cancers. The first evidence of a broader tumour spectrum associated with germline CDKN2A mutations was that of an increased risk of pancreatic cancer (Table 1) (Goldstein et al., 1995). There was an estimated 21.8-fold relative risk of pancreatic cancer in four families carrying CDKN2A mutations, with 7 cases observed versus 0.32 cases expected. Many other studies have since identified an increased occurrence of pancreatic cancer in CDKN2A mutation-positive melanoma families (Borg et al., 1996; Borg et al., 2000; Ciotti et al., 1996; Ghiorzo et al., 1999; Goldstein et al., 2000; Gruis et al., 1995; Lal et al., 2000a; Lal et al., 2000b; Liu et al., 1999; Lynch et al., 2002; Moskaluk et al., 1998; Soufir et al., 1998; Whelan et al., 1995). Pancreatic cancer is more strongly associated with CDKN2A mutations in CMM families from North America and Europe than from Australia (Goldstein et al., 2007), suggesting an interplay between modifier genes and/or environmental risk factors. An additional underlying genotype-phenotype correlation has been identified between the position of the mutation within p16 and increased risk of pancreatic cancer, with mutations affecting ankyrin repeats 3-4 (Figure 1), increasing risk of pancreatic cancer, compared to mutations affecting repeats 1-2 (Goldstein, 2004).

Acc

epte

d A

rtic

le


There is also some evidence of an increased risk of breast, prostate, colon and lung cancers in CDKN2A mutation carriers, but this has not been formally demonstrated. A systematic statistical analysis of the risks of non-melanoma cancers in families segregating CDKN2A mutations is clearly needed. Interestingly, CDKN2A has been documented in only one case of uveal melanoma (UMM), who happens to be a member of a family with CMM (Kannengiesser et al., 2003). This indicates that the major predisposition locus for CMM does not play a significant role in UMM.

Interestingly, the Swedish CDKN2A founder mutation, p.R112_L113insR, within the ankyrin repeats of p16, has been linked to increased risk of developing cancer in respiratory and upper digestive tissue, in addition to melanoma and pancreatic cancer. Risk of the former cancers was increased 9-fold in ever-smokers compared to mutation carriers who never smoked. This is in agreement with the frequent finding of somatic mutations in CDKN2A in pancreatic, respiratory and upper digestive tumours (Carter et al., 2010; Dulak et al., 2013; Helgadottir et al., 2014; Lee et al., 2012). Further studies are needed to examine if this is a mutation specific effect, and if regular surveillance for these cancers should be offered to CDKN2A mutation carriers who smoke.

Evidence suggests that p14 mutations predispose to neural system tumours (NSTs) as well as melanoma (Table 1). This combination of tumours has been proposed as a discrete syndrome (OMIM: 155755) by several investigators (Azizi et al., 1995; Bahuau et al., 1998; Bahuau et al., 1997; Kaufman et al., 1993). Constitutional deletions of exon 1β (encoding a large part of p14ARF) have been found in patients with a wide range of NSTs, including: plexiform neurofibroma (Petty et al., 1993), astrocytoma, meningioma, and schwanomma (Bahuau et al., 1998); neurofibroma (Bahuau et al., 1998; Petronzelli et al., 2001) and neurilemmoma (Randerson-Moor et al., 2001).

CDK4

Role of CDK4 in melanoma

Following the identification of CDKN2A, a candidate gene screening approach was taken to identify other possible familial melanoma genes by screening p16 interacting partners. This fairly rapidly led to the identification of mutations in CDK4 (Puntervoll et al., 2013; Soufir et al., 1998; Zuo et al., 1996), which so far have been documented in only a small number (n = 17) of melanoma families (Table 1) (Puntervoll et al., 2013; Soufir et al., 1998; Zuo et al., 1996). Phenotypically, CDK4 and CDKN2A families are similar, with cases of early onset CMM, multiple primary melanomas and atypical naevi (Puntervoll et al., 2013). All mutations occur in codon

Acc

epte

d A

rtic

le


24, with families either carrying a p.R24C substitution (Zuo et al., 1996) or a p.R24H substitution (Soufir et al., 1998). The clustering of pathogenic mutations within codon 24 indicates the importance of this residue in the function of CDK4. The arginine normally at this position is critical for allowing p16 to bind to CDK4; substitutions of this amino acid therefore abrogate the capacity for p16 to inactivate the kinase. This removal of CDK4 kinase control, results in increased phosphorylation of pRb bound to E2F transcription factors, causing increased E2F release. E2F subsequently activates the transcription of pro-S phase cell cycle genes, promoting G1-S phase transition; CDK4 mutations are thus oncogenic. The p.R24C or p.R24H mutations in CDK4 have been described in families from multiple countries, but on different haplotypes (Puntervoll et al., 2013; Soufir et al., 1998; Zuo et al., 1996). This observation indicates the mutation has arisen independently several times in human evolutionary history and implies a mutational hotspot at this codon (Molven et al., 2005). A CDK4 founder mutation has, however, been documented in Latvia, where CDKN2A mutations are very rare (Veinalde et al., 2013).

Role of CDK4 in non-melanoma tumours

Puntervoll and colleagues documented a host of different cancer types (including pancreatic cancer) in families with CDK4 mutations, but were unable to perform statistical analysis due to non-standardized data collection between study centres (Puntervoll et al., 2013). However, formal assessment of the risk of non-melanoma cancers among CDK4 mutation carriers cannot be performed meaningfully at present due to the rarity of the mutations providing insufficient power.

1996 to 2010 – the dark years

For 15 years, between the mid-90s and 2010, progress in identifying further familial melanoma genes was limited, with only linkage analysis providing a glimmer of hope. A region on 1p22 was found to be linked to early-onset CMM (Gillanders et al., 2003), and chromosome band 9q21.32 showed evidence for linkage in 3 Danish families with a combination of CMM and UMM (Jonsson et al., 2005). However, despite extensive sequencing of genes in these regions, neither has provided a mutated candidate gene showing segregation in linked families.

2011 – return of the light

The advent of new genomic sequencing technologies broke the drought in the identification of familial melanoma genes. In a rapid turnaround of fortunes, the past four years has seen significant advancements in the field, with the identification of gene mutations that have implicated new pathways in melanoma susceptibility.

Acc

epte

d A

rtic

le


BAP1

BRCA1 associated protein-1 (BAP1 MIM: 603089, 3p21.1) was initially identified as a tumour suppressor gene playing a role in melanoma by exome capture coupled with massively parallel sequencing of UMM (Harbour et al., 2010). Subsequently, this observation led to Wiesner and colleagues testing BAP1 as a candidate familial UMM susceptibility locus. They identified high penetrance BAP1 mutations in two families with a syndrome characterized by multiple skin-coloured elevated melanocytic tumours, with some individuals also developing UMM or CMM (Wiesner et al., 2012; Wiesner et al., 2011). At the same time, Testa and colleagues reported germline BAP1 mutations in two families with multiple mesothelioma cases, as well as individuals diagnosed with other cancers including UMM (Testa et al., 2011). Subsequently, Abdel-Rahman and colleagues described a UMM family with cases of CMM carrying a BAP1 mutation (Abdel-Rahman et al., 2011). While all five families described by these three studies carry BAP1 mutations resulting in protein truncation (Figure 2), we have subsequently reported a Danish family with multiple UMM and mesothelioma cases, as well as several other cancers including CMM and paraganglioma, carrying a missense mutation of BAP1 resulting in the creation of a strong cryptic splice donor site, aberrant splicing, and a truncating frameshift of the BAP1 transcript (Wadt et al., 2012). Additionally, Popova and colleagues (Popova et al., 2013) reported another family with a missense BAP1 mutation causing aberrant splicing, where the phenotype was renal cell carcinoma (RCC). They identified 10 other families carrying BAP1 mutations with accumulation of RCC, UMM, and CMM (Table 1). In summary, germline mutations in BAP1 account for susceptibility in a small percentage of CMM families and UMM families. Additionally BAP1 increases predisposition to a spectrum of other cancer types, including: mesothelioma, RCC, a distinct type of benign melanocytic tumour, and basal cell carcinoma (BCC) (Abdel-Rahman et al., 2011; Aoude et al., 2013; Carbone et al., 2012; Cheung et al., 2013; de la Fouchardiere et al., 2014; Farley et al., 2013; Harbour et al., 2010; Hoiom et al., 2013; Maerker et al., 2014; Njauw et al., 2012; Pilarski et al., 2014; Popova et al., 2013; Ribeiro et al., 2013; Testa et al., 2011; Wadt et al., 2014; Wiesner et al., 2011).

BAP1 has been reported to regulate differentiation of melanocytes (Matatall et al., 2013) and has been shown to function as part of the DNA damage response by promoting double-strand break repair (Ismail et al., 2014). Further, by generation of a Bap1 knockout mouse to assess mesothelioma development following exposure to asbestos, Xu and colleagues demonstrated that tumour and normal mesothelial cells from these mice had decreased expression of the Rb1 tumour suppressor mRNA, phosphorylated protein and total protein, due to hypermethylation of the Rb1 promoter compared to wild type mice (Xu et al., 2014).

Acc

epte

d A

rtic

le


While studies have confirmed that CMM is part of the phenotype associated with the BAP1 tumour spectrum, its contribution to a population based-sample of CMM cases has not been reported. To date, only 15% of BAP1 mutation carriers are reported with CMM (Carbone et al, 2013), indicating that BAP1 is a medium penetrance risk gene for CMM. Njauw and colleagues reported a very low frequency of BAP1 mutations (0.52%) in dense CMM families. In CMM-UMM families however, they found a much higher mutation frequency (28.5%). In UMM, the population-based frequency of BAP1 mutation is approximately 3% (Aoude et al., 2013; Njauw et al., 2012). Since penetrance of germline BAP1 mutations have a high level of inter- and intra-familial variability there is a high probability of environmental components or modifier genes affecting the phenotype. To date, no genotype-phenotype correlations have been identified (Cheung et al., 2013).

Other high penetrance genes for susceptibility to UMM (with or without CMM) have yet to be identified, although it should be noted that occasionally UMM cases have been found in breast cancer families segregating BRCA2 mutations, and a very small proportion of sporadic UMM cases have also been shown to have mutations of this gene (Easton et al., 1997; Iscovich et al., 2002; Sinilnikova et al., 1999). Given that overall only a small number of UMM cases have been observed in BRCA2 mutation-positive families, the evidence for BRCA2 being a high-penetrance UMM susceptibility gene remains inconclusive.

MITF

Detection of cutaneous DNA damage by keratinocytes leads them to increase production of melanocyte stimulating hormone (MSH) from the proopiomelancortin gene (POMC, MIM: 176830) (Garibyan et al. 2010). MSH secreted by keratinocytes binds to the melanocortin-1-receptor (MC1R, MIM: 155555, 16q24.3) on melanocytes and leads to the induction of the microphthalmia-associated transcription factor gene (MITF, MIM: 156845, 3p14.2-p14.1), which results in blocking of cell cycle progression and increased melanin production, protecting the cells against further UVR DNA damage (Giles et al., 2011). MITF regulates transcription of a suite of genes involved in cell cycle control and melanogenesis (Cheli et al., 2010). These functions allow MITF to mediate differentiation and survival of melanocytes while limiting their uncontrolled progression. Germline loss of MITF abolishes melanocyte formation in mice, while its loss in established melanocyte lines leads to their expansion (Cheli et al., 2010). MITF induces senescence through expression of p16, p21 and anti-apoptosis genes such as BCL2 and APEX1 (Cheli et al., 2010). In humans, germline splice-site/loss-of-function mutations in MITF are responsible for the development of the autosomal dominant Waardenburg Syndrome type 2, which is characterised by pigment abnormalities

Acc

epte

d A

rtic

le


that involve the skin, hair and eyes, hearing loss, as well as minor defects in the structures that develop from the neural crest (Tassabehji et al., 1994).

MITF has been shown to be somatically altered in melanoma. Cronin and coworkers found MITF to be mutated in 2/26 primary melanomas, 8% of melanoma cell lines derived from metastatic tumours and amplified in a further 8% of cell lines (Cronin et al., 2009). In a separate study, MITF was shown to be amplified in 10% of primary CMM, 21% of metastatic tumours and not amplified in benign naevi, suggestive of MITF being a lineage-specific oncogene (Garraway et al., 2005).

MITF became the first gene to be identified in CMM predisposition using next generation sequencing methods. Two independent groups identified the same rare functional non-synonymous variant in MITF, rs149617956 (p.E318K), which alters MITF transcriptional activity through abrogating a sumoylation motif. The variant has been shown to confer a population-wide melanoma risk in three out of four published studies, with estimated odds ratios of 2.19 (95% CI 1.41, 3.45) (Yokoyama et al., 2011), 4.78 (95% CI 2.05, 11.75) (Bertolotto et al., 2011) and 1.7 (95% CI 1.1, 2.7) (Berwick et al., 2014). In a Polish cohort of 4226 patients with one of six cancer types it was not significantly associated with cancer or melanoma development compared to 2114 population controls (Gromowski et al., 2014). In the other populations however, MITF rs149617956 has about the same effect size on melanoma risk as MC1R R alleles, the gene variants predisposing to red hair, thus making it an intermediate penetrance risk variant for CMM. Bertolotto and colleagues (Bertolotto et al., 2011) additionally observed that rs149617956 associates with RCC, likely due to an isoform of MITF regulating kidney development (Table 1). This variant is also associated with increased naevus count and non-blue eye colour, in line with its altered transcriptional activity (Yokoyama et al., 2011). Adjusting for these traits reduced (OR 1.82, 95% CI 0.85, 3.92), but did not abolish, the rs149617956 association with CMM (Yokoyama et al., 2011), suggesting that while p.E318K risk may be mediated in part via one or both of these phenotypes, there is still significant risk conferred by this variant through an independent mechanism. The frequency of the variant allele in Caucasian populations (approximately 0.0079 in the general population and 0.0173 in unselected melanoma cases) means that it contributes quite substantially to the overall burden of melanoma. The population attributable fraction is estimated to be 0.009, i.e. ~1% of all melanomas.

TERT

A novel mutation occurring in the promoter region of the telomerase reverse transcriptase gene (TERT, MIM: 187270, 5p15.33) was recently found to be

Acc

epte

d A

rtic

le


associated with familial CMM. The mutation, located -57 bp from the ATG translation start site, segregated with disease in a 14-case family from Germany (Horn et al., 2013). Functionally, it created a new binding motif for ETS transcription factors and ternary complex factors (TCF), which resulted in a two-fold increase in transcription of TERT. This is the first documentation of a new high-risk familial melanoma oncogene since CDK4 in 1996 (Zuo et al., 1996).

As observed in families with mutations in other high-penetrance melanoma genes, some carriers of the TERT promoter mutation developed multiple cancer types including those of the ovary, kidney, bladder, breast and lung. Horn and colleagues (Horn et al., 2013) analysed tumours derived from sporadic CMM cases and found that 85% of metastatic tumours and 33% of primary tumours carried somatic mutations in the promoter of TERT, with the majority of these mutations occurring at two hotspots (at positions -124 bp and -146 bp relative to the initiation codon).

Huang and colleagues (Huang et al., 2013) showed further support for the role of TERT in melanoma. They used whole-genome sequencing to discover recurrent somatic TERT promoter mutations (-124 bp and -146 bp) in 17 of 19 melanoma cell lines established from metastasised tumours. Since these initial reports, somatic TERT promoter mutations have been reported in a wide variety of cancer types, including: central nervous system, bladder, thyroid, glioma, hepatocellular, squamous cell carcinoma and CMM (Killela et al., 2013; Vinagre et al., 2013). Findings suggest that mutations in this region cause increased expression of telomerase, eventually resulting in tumorigenesis, presumably by stabilisation of cell aging, turnover and senescence.

TERT is not only involved in familial melanoma, but also has complex associations in the general population with melanoma and with naevus count. A meta-analysis of GWAS datasets for the low penetrance melanoma-associated variant rs401681 upstream of TERT showed the C allele, under a random effects model, to have a protective effect on melanoma development (odds ratio (OR) = 0.873; 95% CI 0.812, 0.939) and an association with low self-reported naevus count (p=0.00042) (Law et al., 2011). The melanoma association does not alter when controlling for naevus count (Law et al., 2011), an observation confirmed by Barrett and colleagues (Barrett et al., 2011), which suggests a complex mechanism of functional association between TERT, melanoma risk and naevus density. Part of this complexity might be due to different mechanisms of cancer risk associated with either long, or short, telomeres. Long telomeres may effectively confer immortality to cancer cells, whereas short telomeres may be associated with chromosomal instability. Both

Acc

epte

d A

rtic

le


outcomes favouring cancer growth, maintenance and evolution but the preferred mechanism differing by cancer type.

Germline protein altering mutations in TERT, causing decreased telomerase activity, have been linked to dyskeratosis congenita (DKC), a rare congenital disorder with phenotypic characteristics resembling premature aging. Its clinical features include abnormal skin pigmentation, with cases presenting with hyper- or hypo-pigmentation, nail dystrophy, oral leukoplakia and bone marrow failure (Yang et al., 2011). Cases also have an elevated risk of malignancy, with an over-representation of BCC, Hodgkin lymphoma, gastrointestinal cancers and bronchial cancers. Thus, as with other familial melanoma genes, mutations affecting TERT may also predispose to a range of cancer types.

POT1

Protection of telomeres 1 (POT1, MIM: 606478, 7q31.33) encodes a member of the shelterin complex (Figure 3). Shelterin protects chromosomal ends by regulating how the telomerase complex interacts with telomeres. It is made up of 6 components, encoded by the genes ACD, POT1, TERF1, TERF2, TERF2IP and TINF2. Collectively, they are necessary for all telomere functions, which include the protection of telomeres from degradation or aberrant recombination, as well as from being inappropriately processed by the DNA-repair pathway (de Lange, 2005). POT1 is the most conserved of the shelterin complex genes and in conjunction with ACD, enables the formation of t-loops, which protect telomere ends by looping the single-stranded 3' strand back to anneal to double-stranded telomere hexamer repeats (Xin et al., 2007). POT1 specifically binds to telomeric single-stranded DNA (ssDNA) through two OB (oligonucleotide/oligosaccharide-binding) domains, through which it mediates access of the telomerase complex (Zhong et al., 2012), and the ACD-POT1 sub-complex also has a higher binding affinity to ssDNA than POT1 does on its own (Wang et al., 2007).

Exome sequencing was recently used to identify loss-of-function mutations in POT1 in high-density CMM families from Australia, The UK and The Netherlands (Robles-Espinoza et al., 2014). A total of 184 individuals from 105 CMM families were interrogated and three were found to harbour missense mutations (p.Y89C, p.Q94E and p.R273L) in the highly conserved oligonucleotide binding (OB) domains of POT1 (Figure 4). Structural modelling predicted that the missense mutations would disrupt the binding of the OB domains with telomeric DNA, which was confirmed by an electrophoretic mobility shift assay. Given the disruption of POT1 binding to telomeric DNA, it is not surprising that POT1 mutation carriers have longer telomeres

Acc

epte

d A

rtic

le


(assessed by analysis of exome sequence reads and quantitative PCR). This is hypothesised to be via the abolition of the ability of shelterin to protect the telomeric ssDNA repeats, allowing access by components of the DNA damage response pathway and/or the telomerase complex (Figure 3). Furthermore, experimental data from exome analysis and quantitative PCR has shown that carriers of POT1 missense mutations have longer telomeres, which may be a major contribution to CMM development. A splice acceptor mutation (c.1687-1G>A) that results in a truncated protein 12 amino acids after the mutation site was also reported in a fourth family. Families with POT1 mutations often present with early onset and multiple primary melanoma. Sequencing of 1739 population-based sporadic CMM cases found a single case with a p.R273L variant that presented with an early age of onset and multiple primary melanomas, i.e. a phenotype similar to that seen in the familial setting.

A concurrent study described a founder mutation (p.S270N) in POT1 that occurred in 5 Italian melanoma families, with mutation carriers having longer telomeres than non-carriers (Shi et al., 2014). Additionally, without shelterin present, telomeres resemble unstable genomic DNA, known as fragile sites, and result in the formation of aberrant DNA structures (Sfeir et al., 2009); these were present in mutation carriers, adding further proof this mutations confers changes to the function of the shelterin complex (Shi et al., 2014). Furthermore, they described two rare mutations recurring in families from France and the USA (see Figure 4). Additionally, case-control analysis showed a significant increase in frequency of rare exonic variants in POT1 in cases (n = 768) compared to controls (n = 768), giving an odds ratio of 5.4 (p = 0.0021) for POT1 variants being associated with melanoma predisposition.

While POT1 has not been found to be somatically mutated in a high proportion of melanomas, 3.5% of all chronic lymphocytic leukaemias (CLL) and 9% with an aggressive CLL subtype, harbour mutations in this gene (Ramsay et al., 2013). Functional experiments showed that the CLL cells with mutated POT1 acquired numerous chromosomal abnormalities in comparison to POT1 wild type controls. Interestingly, the vast majority of mutations detected in these cases lie within the OB domains, as seen in CMM families.

ACD AND TERF2IP

Following the discovery that inactivating germline mutations in POT1 predisposed to CMM, a search for germline mutations in the other five components of the shelterin complex (Figure 3) was conducted (Aoude et al., 2014, in press). This resulted in the discovery of the most recent high-risk loci to be associated with familial CMM:

Acc

epte

d A

rtic

le


adrenocortical dysplasia homolog (ACD, MIM: 609377, 16q22.1) and telomeric repeat binding factor 2, interacting protein (TERF2IP, MIM: 605061, 16q23.1). Novel mutations were also observed in TERF1, TERF2 and TINF2, but were not convincingly associated with melanoma.

Whole-genome, exome and targeted sequencing methods were used to screen 510 melanoma families for mutations in these five genes. Six families were found to carry novel ACD mutations, including a segregating nonsense mutation, p.Q320X (Table 1; Figure 4). This sample of families included a founder mutation (p.N249S) segregating in a 12-case family from Australia and present in 5 out of 6 cases in a Danish family. Of five distinct mutations in ACD, four clustered in the POT1 binding domain, including p.Q320X. This clustering of novel mutations in the POT1 binding domain of ACD was statistically higher (p = 0.005) in melanoma probands (5/510) compared to population controls (16/6785), as were all novel and rare variants in ACD (p = 0.040).

Four families carried TERF2IP variants (Table 1; Figure 4), which included a nonsense mutation (p.R364X) and point mutations that co-segregated with melanoma. Novel and rare variants in TERF2IP were statistically enriched (p = 0.022) in melanoma probands compared to population controls.

As seen with other CMM loci, families carrying ACD and TERF2IP mutations were associated with early onset and multiple primary melanomas. Furthermore they were enriched with other cancer types, including those of the breast, lung, cervix, colon, bowel, and ovary, as well as B-cell lymphoma. This suggests that these mutations predispose to a broader spectrum of cancers than just CMM. Intriguingly, a recent report identified a pedigree in which a germline ACD mutation was the cause of a range of blood disorders including bone marrow failure (Guo et al., 2014). The mutation (p.K170del) was an in-frame deletion of an amino acid in the TEL-patch, a region of the protein essential for binding telomerase. Loss of this residue abrogated recruitment of telomerase to telomeres when the mutant protein was ectopically expressed in 293T cells. Carriers of the mutation had considerably shorter telomeres than age-matched population controls. Another very recent report describes the same mutation, this time associated with DKC, in an individual who also inherited a missense variant (p.P491T) from their mother (Kocak et al., 2014). However, the proband’s father, who carried the p.K170del mutation, did not present with any type of blood disorder and only showed premature hair greying (from the age of 16 years) and oral lichen planus. Collectively, these data point to a complex relationship between ACD mutations and disease, and suggest that the type of mutation, its position, and presumably modifier genes, influence the phenotypic outcome.

Acc

epte

d A

rtic

le


Mechanistically, ACD, in a subunit with POT1, mediates the interaction between the shelterin protein complex and TERT via its OB domains (Xin et al., 2007). When this subunit is inhibited, the telomerase complex increases telomere length, indicating that shelterin is required to inhibit the elongation of chromosome ends (Liu et al., 2004; Loayza and De Lange, 2003; Ye and de Lange, 2004; Ye et al., 2004; Zhong et al., 2012). Additionally, ACD links POT1 to other members of the shelterin complex, increasing the affinity of POT1 for telomeric ssDNA (Wang et al., 2007). TERF2IP is additionally necessary for the repression of homology-directed repair of double strand chromosomal breaks at telomeres, a role distinct from the shelterin complex (Sfeir et al., 2010).

Overall, mutations in three of the shelterin genes: ACD, TERF2IP and POT1, account for ~9% of high-density CMM families lacking mutations in previously identified high-penetrance predisposition genes (Robles-Espinoza et al., 2014; Shi et al., 2014) (Aoude, in press). Taken together with the finding of germline mutations in the TERT promoter in a CMM family (Horn et al., 2013; Huang et al., 2013), these data collectively point to telomere dysregulation as an important new pathway contributing to predisposition in a substantial proportion of high-risk CMM families.

CONCLUSIONS

Twenty years on from the identification of CDKN2A mutations in melanoma pedigrees it is pertinent to ask what is the current landscape of familial melanoma genetics? CDKN2A is still the only common cause of melanoma susceptibility, with mutations in approximately 2% of all melanoma cases and ~40% of high-density families. Mutations in CDK4, MITF, BAP1, TERT, POT1, ACD, TERF2IP, are rare, with each contributing to <1% of all familial clustering of melanoma. Thus, the causes of predisposition in around half of all melanoma-prone families have yet to be identified. It is possible that some of the co-occurrence of melanoma in families is due to a shared environment, or simply by chance; it is also likely that for some families with only a few cases of melanoma that predisposition is due to carriage of multiple low penetrance susceptibility alleles. However, other rare high-penetrance genes are likely to exist, and exome or genome sequencing is arguably the most unbiased and cost-effective way to uncover them. Importantly, the finding of mutations within the TERT promoter points to regulatory regions of the genome being pathogenic and playing an important role in melanoma predisposition. Hence, in the future, investigators will not only need to assess mutational mechanisms that alter protein sequences, including translocations causing gene fusions, but also consider regulatory mutations. Next generation sequencing methods will also allow identification of rare, high penetrance risk alleles outside of the familial setting, e.g. via case-control studies.

Acc

epte

d A

rtic

le


Evidence exists for several familial melanoma genes being responsible for predisposition to cancers other than melanoma, thus raising the concept that some ‘melanoma’ susceptibility genes are more general tumour suppressor genes. This is particularly evident for BAP1, which is associated with mesothelioma and a very broad range of other cancer types; however associations between CDKN2A (p16) and pancreatic cancer, CDKN2A (p14) and NSTs, MITF and RCC, and ACD and glioma, point to this being a common phenomenon. This has implications for clinical testing and surveillance; clinicians should lookout for these combinations of cancers in order to conduct focused candidate gene sequencing, otherwise the most cost-effective clinical testing will be exome sequencing. Moreover, regular screening examinations such as MRI or ultrasound scans may be warranted in mutation carriers with elevated risk of particular internal cancers. However, it is important to emphasize that the potential to implement such screening recommendations will require additional multicenter studies to determine the penetrance of these rare mutations and the potential benefits of screening protocols. Genetic testing and counselling for members of families with known high penetrance melanoma predisposing mutations should still be primarily in the context of informing carriers of appropriate lifestyle changes to minimize UVR exposure and to encourage routine skin self-examinations, as well as regular formal dermatological screening. Heightened surveillance in such individuals should result in detection and removal of cutaneous lesions at premalignant (dysplastic naevus or melanoma in situ) stages, or at worse, when melanomas are thin and hence associated with favourable prognosis.

In conclusion, after the initial identification of two high penetrance familial melanoma susceptibility genes, there was a long period of time without further advances. The advent of next generation sequencing technology has brought rapid developments in the field, with a further 10% of familial risk being explained by rare mutations, and new melanoma development pathways being identified. This is an exciting time to be investigating familial melanoma genetics and it is likely that the coming years will deliver further important melanoma-associated mutations.

CONFLICTS OF INTEREST

The authors do not have any conflicts of interest to disclose.

ACKNOWLEDGEMENTS

N.K.H. and A.L.P are supported by fellowships from the National Health and Medical Research Council of Australia and Cure Cancer Australia respectively. L.G.A. and K.W. were supported by PhD scholarships from the Australia and New Zealand

Acc

epte

d A

rtic

le


Banking Group Limited Trustees and Rigshospitalet, University Hospital of Copenhagen, respectively.

FIGURE LEGENDS

Figure 1: CDKN2A

Depicts how alternative splicing of CDKN2A leads to distinct proteins p16INK4A (red exons) and p14ARF (blue exons). The position of the four ankyrin repeats in p16INK4A are marked by red bars. Founder mutations are represented in relation to amino acid and exon. The black bars indicate a deletion of exon 1β or exon 2 in p14ARF.

Figure 2: BAP1

Location of germline BAP1 truncating mutations in relation to protein domains. UCH is the ubiquitin carboxy-terminal hydrolase domain; HBM is the HCFC1 binding motif; ULD is the UCH37-like domain. Binding sites for BARD1, BRCA1 and YY1 are depicted by red bars. Mutation coloured red indicates family without either CMM or UMM.

Figure 3: Shelterin complex

A) The shelterin complex (TERF2IP, TERF1, TERF2, TINF2, ACD and POT1) mediates the interaction between telomeres and the telomerase complex (TERT, NOP10, NHP2, DKC1, GAR1) and recruits the TERC RNA template in order to increase telomere length. Shelterin binds to the telomere hexamer repeat (TTAGGG)n through TERF1 and TERF2. POT1 binding to the 3’ single stranded DNA overhang prevents access of telomerase to telomeres. When POT1 is unbound, the telomerase complex is able to extend telomeres using the TERC RNA template. B) The shelterin complex forms a protective ‘t-loop’ when POT1 binds to the 3’ single-stranded DNA causing it to loop back and anneal to the double-stranded hexamer repeats. The t-loop prevents the telomere ends from being recognized as break points by the DNA repair machinery and inhibits the telomerase complex from being able to bind.

Figure 4: POT1, ACD and TERF2IP

Location of germline mutations in POT1, ACD and TERF2IP in relation to protein domains. Mutations coloured red indicate families without either CMM or UMM.

Acc

epte

d A

rtic

le


POT1 p.S270N is an Italian founder mutation. POT1 p.A532P is a French founder mutation. POT1 p.D224N is a recurrent mutation occurring in a US family and an Italian sporadic melanoma case. ACD p.N249S is a recurrent mutation occurring in an Australian and a Danish family.

REFERENCES

Abdel-Rahman, M. H., Pilarski, R., Cebulla, C. M., Massengill, J. B., Christopher, B. N., Boru, G.,

Hovland, P., and Davidorf, F. H. (2011). Germline BAP1 mutation predisposes to uveal melanoma, lung adenocarcinoma, meningioma, and other cancers. J Med Genet 48, 856-9.

Aitken, J., Welch, J., Duffy, D., Milligan, A., Green, A., Martin, N., and Hayward, N. (1999). CDKN2A variants in a population-based sample of Queensland families with melanoma. J Natl Cancer Inst 91, 446-52.

Aoude, L. G., Vajdic, C. M., Kricker, A., Armstrong, B., and Hayward, N. K. (2013). Prevalence of germline BAP1 mutation in a population-based sample of uveal melanoma cases. Pigment Cell Melanoma Res 26, 278-9.

Azizi, E., Friedman, J., Pavlotsky, F., Iscovich, J., Bornstein, A., Shafir, R., Trau, H., Brenner, H., and Nass, D. (1995). Familial cutaneous malignant melanoma and tumors of the nervous system. A hereditary cancer syndrome. Cancer 76, 1571-8.

Bahuau, M., Vidaud, D., Jenkins, R. B., Bieche, I., Kimmel, D. W., Assouline, B., Smith, J. S., Alderete, B., Cayuela, J. M., Harpey, J. P., et al. (1998). Germ-line deletion involving the INK4 locus in familial proneness to melanoma and nervous system tumors. Cancer Res 58, 2298-303.

Bahuau, M., Vidaud, D., Kujas, M., Palangie, A., Assouline, B., Chaignaud-Lebreton, M., Prieur, M., Vidaud, M., Harpey, J. P., Lafourcade, J., et al. (1997). Familial aggregation of malignant melanoma/dysplastic naevi and tumours of the nervous system: an original syndrome of tumour proneness. Annales de genetique 40, 78-91.

Barrett, J. H., Iles, M. M., Harland, M., Taylor, J. C., Aitken, J. F., Andresen, P. A., Akslen, L. A., Armstrong, B. K., Avril, M. F., Azizi, E., et al. (2011). Genome-wide association study identifies three new melanoma susceptibility loci. Nat Genet 43, 1108-13.

Begg, C. B., Orlow, I., Hummer, A. J., Armstrong, B. K., Kricker, A., Marrett, L. D., Millikan, R. C., Gruber, S. B., Anton-Culver, H., Zanetti, R., et al. (2005). Lifetime risk of melanoma in CDKN2A mutation carriers in a population-based sample. J Natl Cancer Inst 97, 1507-15.

Bertolotto, C., Lesueur, F., Giuliano, S., Strub, T., De Lichy, M., Bille, K., Dessen, P., D'hayer, B., Mohamdi, H., Remenieras, A., et al. (2011). A SUMOylation-defective MITF germline mutation predisposes to melanoma and renal carcinoma. Nature.

Berwick, M., Macarthur, J., Orlow, I., Kanetsky, P., Begg, C. B., Luo, L., Reiner, A., Sharma, A., Armstrong, B. K., Kricker, A., et al. (2014). MITF E318K's effect on melanoma risk independent of, but modified by, other risk factors. Pigment cell & melanoma research 27, 485-8.

Borg, A., Johannsson, U., Johannsson, O., Hakansson, S., Westerdahl, J., Masback, A., Olsson, H., and Ingvar, C. (1996). Novel germline p16 mutation in familial malignant melanoma in southern Sweden. Cancer Res 56, 2497-500.

Borg, A., Sandberg, T., Nilsson, K., Johannsson, O., Klinker, M., Masback, A., Westerdahl, J., Olsson, H., and Ingvar, C. (2000). High frequency of multiple melanomas and breast and pancreas carcinomas in CDKN2A mutation-positive melanoma families. J Natl Cancer Inst 92, 1260-6.

Acc

epte

d A

rtic

le


Carbone, M., Korb Ferris, L., Baumann, F., Napolitano, A., Lum, C. A., Flores, E. G., Gaudino, G., Powers, A., Bryant-Greenwood, P., Krausz, T., et al. (2012). BAP1 cancer syndrome: malignant mesothelioma, uveal and cutaneous melanoma, and MBAITs. J Transl Med 10, 179.

Carter, H., Samayoa, J., Hruban, R. H., and Karchin, R. (2010). Prioritization of driver mutations in pancreatic cancer using cancer-specific high-throughput annotation of somatic mutations (CHASM). Cancer biology & therapy 10, 582-7.

Cheli, Y., Ohanna, M., Ballotti, R., and Bertolotto, C. (2010). Fifteen-year quest for microphthalmia-associated transcription factor target genes. Pigment Cell Melanoma Res 23, 27-40.

Cheung, M., Talarchek, J., Schindeler, K., Saraiva, E., Penney, L. S., Ludman, M., and Testa, J. R. (2013). Further evidence for germline BAP1 mutations predisposing to melanoma and malignant mesothelioma. Cancer genetics 206, 206-10.

Ciotti, P., Strigini, P., and Bianchi-Scarra, G. (1996). Familial melanoma and pancreatic cancer. Ligurian Skin Tumor Study Group. N Engl J Med 334, 469-70; author reply 471-2.

Cronin, J. C., Wunderlich, J., Loftus, S. K., Prickett, T. D., Wei, X., Ridd, K., Vemula, S., Burrell, A. S., Agrawal, N. S., Lin, J. C., et al. (2009). Frequent mutations in the MITF pathway in melanoma. Pigment Cell Melanoma Res 22, 435-44.

De La Fouchardiere, A., Cabaret, O., Savin, L., Combemale, P., Schvartz, H., Penet, C., Bonadona, V., Soufir, N., and Bressac-De Paillerets, B. (2014). Germline BAP1 mutations predispose also to multiple basal cell carcinomas. Clin Genet.

De Lange, T. (2005). Shelterin: the protein complex that shapes and safeguards human telomeres. Genes Dev 19, 2100-10.

De Snoo, F. A., and Hayward, N. K. (2005). Cutaneous melanoma susceptibility and progression genes. Cancer Lett 230, 153-86.

Dulak, A. M., Stojanov, P., Peng, S., Lawrence, M. S., Fox, C., Stewart, C., Bandla, S., Imamura, Y., Schumacher, S. E., Shefler, E., et al. (2013). Exome and whole-genome sequencing of esophageal adenocarcinoma identifies recurrent driver events and mutational complexity. Nat Genet 45, 478-86.

Easton, D. F., Steele, L., Fields, P., Ormiston, W., Averill, D., Daly, P. A., Mcmanus, R., Neuhausen, S. L., Ford, D., Wooster, R., et al. (1997). Cancer risks in two large breast cancer families linked to BRCA2 on chromosome 13q12-13. Am J Hum Genet 61, 120-8.

Farley, M. N., Schmidt, L. S., Mester, J. L., Pena-Llopis, S., Pavia-Jimenez, A., Christie, A., Vocke, C. D., Ricketts, C. J., Peterson, J., Middelton, L., et al. (2013). A novel germline mutation in BAP1 predisposes to familial clear-cell renal cell carcinoma. Molecular cancer research : MCR 11, 1061-71.

Foulkes, W. D., Flanders, T. Y., Pollock, P. M., and Hayward, N. K. (1997). The CDKN2A (p16) gene and human cancer. Molecular medicine 3, 5-20.

Garraway, L. A., Widlund, H. R., Rubin, M. A., Getz, G., Berger, A. J., Ramaswamy, S., Beroukhim, R., Milner, D. A., Granter, S. R., Du, J., et al. (2005). Integrative genomic analyses identify MITF as a lineage survival oncogene amplified in malignant melanoma. Nature 436, 117-22.

Ghiorzo, P., Ciotti, P., Mantelli, M., Heouaine, A., Queirolo, P., Rainero, M. L., Ferrari, C., Santi, P. L., De Marchi, R., Farris, A., et al. (1999). Characterization of ligurian melanoma families and risk of occurrence of other neoplasia. Int J Cancer 83, 441-8.

Giles, N., Pavey, S., Pinder, A., and Gabrielli, B. (2011). Multiple melanoma susceptibility factors function in a UVR response pathway in skin. Br J Dermatol.

Gillanders, E., Juo, S. H., Holland, E. A., Jones, M., Nancarrow, D., Freas-Lutz, D., Sood, R., Park, N., Faruque, M., Markey, C., et al. (2003). Localization of a novel melanoma susceptibility locus to 1p22. Am J Hum Genet 73, 301-13.

Goldstein, A. M. (2004). Familial melanoma, pancreatic cancer and germline CDKN2A mutations. Hum Mutat 23, 630.

Acc

epte

d A

rtic

le


Goldstein, A. M., Chan, M., Harland, M., Gillanders, E. M., Hayward, N. K., Avril, M. F., Azizi, E., Bianchi-Scarra, G., Bishop, D. T., Bressac-De Paillerets, B., et al. (2006). High-risk melanoma susceptibility genes and pancreatic cancer, neural system tumors, and uveal melanoma across GenoMEL. Cancer Res 66, 9818-28.

Goldstein, A. M., Chan, M., Harland, M., Hayward, N. K., Demenais, F., Bishop, D. T., Azizi, E., Bergman, W., Bianchi-Scarra, G., Bruno, W., et al. (2007). Features associated with germline CDKN2A mutations: a GenoMEL study of melanoma-prone families from three continents. J Med Genet 44, 99-106.

Goldstein, A. M., Fraser, M. C., Struewing, J. P., Hussussian, C. J., Ranade, K., Zametkin, D. P., Fontaine, L. S., Organic, S. M., Dracopoli, N. C., Clark, W. H., Jr., et al. (1995). Increased risk of pancreatic cancer in melanoma-prone kindreds with p16INK4 mutations. N Engl J Med 333, 970-4.

Goldstein, A. M., Struewing, J. P., Chidambaram, A., Fraser, M. C., and Tucker, M. A. (2000). Genotype-phenotype relationships in U.S. melanoma-prone families with CDKN2A and CDK4 mutations. J Natl Cancer Inst 92, 1006-10.

Gromowski, T., Masojc, B., Scott, R. J., Cybulski, C., Gorski, B., Kluzniak, W., Paszkowska-Szczur, K., Rozmiarek, A., Debniak, B., Maleszka, R., et al. (2014). Prevalence of the E318K and V320I MITF germline mutations in Polish cancer patients and multiorgan cancer risk-a population-based study. Cancer genetics 207, 128-32.

Gruis, N. A., Van Der Velden, P. A., Sandkuijl, L. A., Prins, D. E., Weaver-Feldhaus, J., Kamb, A., Bergman, W., and Frants, R. R. (1995). Homozygotes for CDKN2 (p16) germline mutation in Dutch familial melanoma kindreds. Nat Genet 10, 351-3.

Guo, Y., Kartawinata, M., Li, J., Pickett, H. A., Teo, J., Kilo, T., Barbaro, P. M., Keating, B., Chen, Y., Tian, L., et al. (2014). Inherited bone marrow failure associated with germline mutation of ACD, the gene encoding telomere protein TPP1. Blood.

Harbour, J. W., Onken, M. D., Roberson, E. D., Duan, S., Cao, L., Worley, L. A., Council, M. L., Matatall, K. A., Helms, C., and Bowcock, A. M. (2010). Frequent mutation of BAP1 in metastasizing uveal melanomas. Science 330, 1410-3.

Harland, M., Holland, E. A., Ghiorzo, P., Mantelli, M., Bianchi-Scarra, G., Goldstein, A. M., Tucker, M. A., Ponder, B. A., Mann, G. J., Bishop, D. T., et al. (2000). Mutation screening of the CDKN2A promoter in melanoma families. Genes Chromosomes Cancer 28, 45-57.

Harland, M., Taylor, C. F., Chambers, P. A., Kukalizch, K., Randerson-Moor, J. A., Gruis, N. A., De Snoo, F. A., Ter Huurne, J. A., Goldstein, A. M., Tucker, M. A., et al. (2005). A mutation hotspot at the p14ARF splice site. Oncogene 24, 4604-8.

Hayward, N. (2000). New developments in melanoma genetics. Current oncology reports 2, 300-6. Helgadottir, H., Hoiom, V., Jonsson, G., Tuominen, R., Ingvar, C., Borg, A., Olsson, H., and Hansson, J.

(2014). High risk of tobacco-related cancers in CDKN2A mutation-positive melanoma families. J Med Genet 51, 545-52.

Hoiom, V., Edsgard, D., Helgadottir, H., Eriksson, H., All-Ericsson, C., Tuominen, R., Ivanova, I., Lundeberg, J., Emanuelsson, O., and Hansson, J. (2013). Hereditary uveal melanoma: a report of a germline mutation in BAP1. Genes Chromosomes Cancer 52, 378-84.

Horn, S., Figl, A., Rachakonda, P. S., Fischer, C., Sucker, A., Gast, A., Kadel, S., Moll, I., Nagore, E., Hemminki, K., et al. (2013). TERT promoter mutations in familial and sporadic melanoma. Science 339, 959-61.

Huang, F. W., Hodis, E., Xu, M. J., Kryukov, G. V., Chin, L., and Garraway, L. A. (2013). Highly recurrent TERT promoter mutations in human melanoma. Science 339, 957-9.

Hussussian, C. J., Struewing, J. P., Goldstein, A. M., Higgins, P. A., Ally, D. S., Sheahan, M. D., Clark, W. H., Jr., Tucker, M. A., and Dracopoli, N. C. (1994). Germline p16 mutations in familial melanoma. Nat Genet 8, 15-21.

Iscovich, J., Abdulrazik, M., Cour, C., Fischbein, A., Pe'er, J., and Goldgar, D. E. (2002). Prevalence of the BRCA2 6174 del T mutation in Israeli uveal melanoma patients. Int J Cancer 98, 42-4.

Acc

epte

d A

rtic

le


Ismail, I. H., Davidson, R., Gagne, J. P., Xu, Z. Z., Poirier, G. G., and Hendzel, M. J. (2014). Germline Mutations in BAP1 Impair Its Function in DNA Double-Strand Break Repair. Cancer Res.

Jonsson, G., Bendahl, P. O., Sandberg, T., Kurbasic, A., Staaf, J., Sunde, L., Cruger, D. G., Ingvar, C., Olsson, H., and Borg, A. (2005). Mapping of a novel ocular and cutaneous malignant melanoma susceptibility locus to chromosome 9q21.32. J Natl Cancer Inst 97, 1377-82.

Kamb, A., Shattuck-Eidens, D., Eeles, R., Liu, Q., Gruis, N. A., Ding, W., Hussey, C., Tran, T., Miki, Y., Weaver-Feldhaus, J., et al. (1994). Analysis of the p16 gene (CDKN2) as a candidate for the chromosome 9p melanoma susceptibility locus. Nat Genet 8, 23-6.

Kannengiesser, C., Avril, M. F., Spatz, A., Laud, K., Lenoir, G. M., and Bressac-De-Paillerets, B. (2003). CDKN2A as a uveal and cutaneous melanoma susceptibility gene. Genes Chromosomes Cancer 38, 265-8.

Kaufman, D. K., Kimmel, D. W., Parisi, J. E., and Michels, V. V. (1993). A familial syndrome with cutaneous malignant melanoma and cerebral astrocytoma. Neurology 43, 1728-31.

Killela, P. J., Reitman, Z. J., Jiao, Y., Bettegowda, C., Agrawal, N., Diaz, L. A., Jr., Friedman, A. H., Friedman, H., Gallia, G. L., Giovanella, B. C., et al. (2013). TERT promoter mutations occur frequently in gliomas and a subset of tumors derived from cells with low rates of self-renewal. Proc Natl Acad Sci U S A 110, 6021-6.

Kocak, H., Ballew, B. J., Bisht, K., Eggebeen, R., Hicks, B. D., Suman, S., O'neil, A., Giri, N., Laboratory, N. D. C. G. R., Group, N. D. C. S. W., et al. (2014). Hoyeraal-Hreidarsson syndrome caused by a germline mutation in the TEL patch of the telomere protein TPP1. Genes Dev.

Lal, G., Liu, G., Schmocker, B., Kaurah, P., Ozcelik, H., Narod, S. A., Redston, M., and Gallinger, S. (2000a). Inherited predisposition to pancreatic adenocarcinoma: role of family history and germ-line p16, BRCA1, and BRCA2 mutations. Cancer Res 60, 409-16.

Lal, G., Liu, L., Hogg, D., Lassam, N. J., Redston, M. S., and Gallinger, S. (2000b). Patients with both pancreatic adenocarcinoma and melanoma may harbor germline CDKN2A mutations. Genes Chromosomes Cancer 27, 358-61.

Law, M. H., Montgomery, G. W., Brown, K. M., Martin, N. G., Mann, G. J., Hayward, N. K., and Macgregor, S. (2011). Meta-Analysis Combining New and Existing Data Sets Confirms that the TERT-CLPTM1L Locus Influences Melanoma Risk. J Invest Dermatol.

Lee, J., Van Hummelen, P., Go, C., Palescandolo, E., Jang, J., Park, H. Y., Kang, S. Y., Park, J. O., Kang, W. K., Macconaill, L., et al. (2012). High-throughput mutation profiling identifies frequent somatic mutations in advanced gastric adenocarcinoma. PLoS One 7, e38892.

Liu, D., Safari, A., O'connor, M. S., Chan, D. W., Laegeler, A., Qin, J., and Songyang, Z. (2004). PTOP interacts with POT1 and regulates its localization to telomeres. Nat Cell Biol 6, 673-80.

Liu, L., Dilworth, D., Gao, L., Monzon, J., Summers, A., Lassam, N., and Hogg, D. (1999). Mutation of the CDKN2A 5' UTR creates an aberrant initiation codon and predisposes to melanoma. Nat Genet 21, 128-32.

Loayza, D., and De Lange, T. (2003). POT1 as a terminal transducer of TRF1 telomere length control. Nature 423, 1013-8.

Lynch, H. T., Brand, R. E., Hogg, D., Deters, C. A., Fusaro, R. M., Lynch, J. F., Liu, L., Knezetic, J., Lassam, N. J., Goggins, M., et al. (2002). Phenotypic variation in eight extended CDKN2A germline mutation familial atypical multiple mole melanoma-pancreatic carcinoma-prone families: the familial atypical mole melanoma-pancreatic carcinoma syndrome. Cancer 94, 84-96.

Maerker, D. A., Zeschnigk, M., Nelles, J., Lohmann, D. R., Worm, K., Bosserhoff, A. K., Krupar, R., and Jagle, H. (2014). BAP1 germline mutation in two first grade family members with uveal melanoma. Br J Ophthalmol 98, 224-7.

Matatall, K. A., Agapova, O. A., Onken, M. D., Worley, L. A., Bowcock, A. M., and Harbour, J. W. (2013). BAP1 deficiency causes loss of melanocytic cell identity in uveal melanoma. BMC Cancer 13, 371.

Acc

epte

d A

rtic

le


Mistry, S. H., Taylor, C., Randerson-Moor, J. A., Harland, M., Turner, F., Barrett, J. H., Whitaker, L., Jenkins, R. B., Knowles, M. A., Bishop, J. A., et al. (2005). Prevalence of 9p21 deletions in UK melanoma families. Genes Chromosomes Cancer 44, 292-300.

Molven, A., Grimstvedt, M. B., Steine, S. J., Harland, M., Avril, M. F., Hayward, N. K., and Akslen, L. A. (2005). A large Norwegian family with inherited malignant melanoma, multiple atypical nevi, and CDK4 mutation. Genes Chromosomes Cancer 44, 10-8.

Moskaluk, C. A., Hruban, H., Lietman, A., Smyrk, T., Fusaro, L., Fusaro, R., Lynch, J., Yeo, C. J., Jackson, C. E., Lynch, H. T., et al. (1998). Novel germline p16(INK4) allele (Asp145Cys) in a family with multiple pancreatic carcinomas. Mutations in brief no. 148. Online. Hum Mutat 12, 70.

Nikolaou, V., Kang, X., Stratigos, A., Gogas, H., Latorre, M. C., Gabree, M., Plaka, M., Njauw, C. N., Kypreou, K., Mirmigi, I., et al. (2011). Comprehensive mutational analysis of CDKN2A and CDK4 in Greek patients with cutaneous melanoma. The British journal of dermatology 165, 1219-22.

Njauw, C. N., Kim, I., Piris, A., Gabree, M., Taylor, M., Lane, A. M., Deangelis, M. M., Gragoudas, E., Duncan, L. M., and Tsao, H. (2012). Germline BAP1 Inactivation Is Preferentially Associated with Metastatic Ocular Melanoma and Cutaneous-Ocular Melanoma Families. PLoS One 7, e35295.

Palmieri, G., Capone, M., Ascierto, M. L., Gentilcore, G., Stroncek, D. F., Casula, M., Sini, M. C., Palla, M., Mozzillo, N., and Ascierto, P. A. (2009). Main roads to melanoma. J Transl Med 7, 86.

Petronzelli, F., Sollima, D., Coppola, G., Martini-Neri, M. E., Neri, G., and Genuardi, M. (2001). CDKN2A germline splicing mutation affecting both p16(ink4) and p14(arf) RNA processing in a melanoma/neurofibroma kindred. Genes Chromosomes Cancer 31, 398-401.

Petty, E. M., Bolognia, J. L., Bale, A. E., and Yang-Feng, T. (1993). Cutaneous malignant melanoma and atypical moles associated with a constitutional rearrangement of chromosomes 5 and 9. American journal of medical genetics 45, 77-80.

Pilarski, R., Cebulla, C. M., Massengill, J. B., Rai, K., Rich, T., Strong, L., Mcgillivray, B., Asrat, M. J., Davidorf, F. H., and Abdel-Rahman, M. H. (2014). Expanding the clinical phenotype of hereditary BAP1 cancer predisposition syndrome, reporting three new cases. Genes Chromosomes Cancer 53, 177-82.

Popova, T., Hebert, L., Jacquemin, V., Gad, S., Caux-Moncoutier, V., Dubois-D'enghien, C., Richaudeau, B., Renaudin, X., Sellers, J., Nicolas, A., et al. (2013). Germline BAP1 Mutations Predispose to Renal Cell Carcinomas. Am J Hum Genet.

Puntervoll, H. E., Yang, X. R., Vetti, H. H., Bachmann, I. M., Avril, M. F., Benfodda, M., Catricala, C., Dalle, S., Duval-Modeste, A. B., Ghiorzo, P., et al. (2013). Melanoma prone families with CDK4 germline mutation: phenotypic profile and associations with MC1R variants. J Med Genet 50, 264-70.

Ramsay, A. J., Quesada, V., Foronda, M., Conde, L., Martinez-Trillos, A., Villamor, N., Rodriguez, D., Kwarciak, A., Garabaya, C., Gallardo, M., et al. (2013). POT1 mutations cause telomere dysfunction in chronic lymphocytic leukemia. Nat Genet 45, 526-30.

Randerson-Moor, J. A., Harland, M., Williams, S., Cuthbert-Heavens, D., Sheridan, E., Aveyard, J., Sibley, K., Whitaker, L., Knowles, M., Bishop, J. N., et al. (2001). A germline deletion of p14(ARF) but not CDKN2A in a melanoma-neural system tumour syndrome family. Hum Mol Genet 10, 55-62.

Ribeiro, C., Campelos, S., Moura, C. S., Machado, J. C., Justino, A., and Parente, B. (2013). Well-differentiated papillary mesothelioma: clustering in a Portuguese family with a germline BAP1 mutation. Ann Oncol.

Rizos, H., Puig, S., Badenas, C., Malvehy, J., Darmanian, A. P., Jimenez, L., Mila, M., and Kefford, R. F. (2001). A melanoma-associated germline mutation in exon 1beta inactivates p14ARF. Oncogene 20, 5543-7.

Acc

epte

d A

rtic

le


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. (2014). POT1 loss-of-function variants predispose to familial melanoma. Nat Genet.

Ruas, M., and Peters, G. (1998). The p16INK4a/CDKN2A tumor suppressor and its relatives. Biochim Biophys Acta 1378, F115-77.

Sfeir, A., Kabir, S., Van Overbeek, M., Celli, G. B., and De Lange, T. (2010). Loss of Rap1 induces telomere recombination in the absence of NHEJ or a DNA damage signal. Science 327, 1657-61.

Sfeir, A., Kosiyatrakul, S. T., Hockemeyer, D., Macrae, S. L., Karlseder, J., Schildkraut, C. L., and De Lange, T. (2009). Mammalian telomeres resemble fragile sites and require TRF1 for efficient replication. Cell 138, 90-103.

Shekar, S. N., Duffy, D. L., Youl, P., Baxter, A. J., Kvaskoff, M., Whiteman, D. C., Green, A. C., Hughes, M. C., Hayward, N. K., Coates, M., et al. (2009). A population-based study of Australian twins with melanoma suggests a strong genetic contribution to liability. J Invest Dermatol 129, 2211-9.

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. (2014). Rare missense variants in POT1 predispose to familial cutaneous malignant melanoma. Nat Genet.

Sinilnikova, O. M., Egan, K. M., Quinn, J. L., Boutrand, L., Lenoir, G. M., Stoppa-Lyonnet, D., Desjardins, L., Levy, C., Goldgar, D., and Gragoudas, E. S. (1999). Germline brca2 sequence variants in patients with ocular melanoma. Int J Cancer 82, 325-8.

Smith-Sorensen, B., and Hovig, E. (1996). CDKN2A (p16INK4A) somatic and germline mutations. Hum Mutat 7, 294-303.

Soufir, N., Avril, M. F., Chompret, A., Demenais, F., Bombled, J., Spatz, A., Stoppa-Lyonnet, D., Benard, J., and Bressac-De Paillerets, B. (1998). Prevalence of p16 and CDK4 germline mutations in 48 melanoma-prone families in France. The French Familial Melanoma Study Group. Hum Mol Genet 7, 209-16.

Tassabehji, M., Newton, V. E., and Read, A. P. (1994). Waardenburg syndrome type 2 caused by mutations in the human microphthalmia (MITF) gene. Nat Genet 8, 251-5.

Testa, J. R., Cheung, M., Pei, J., Below, J. E., Tan, Y., Sementino, E., Cox, N. J., Dogan, A. U., Pass, H. I., Trusa, S., et al. (2011). Germline BAP1 mutations predispose to malignant mesothelioma. Nat Genet 43, 1022-5.

Veinalde, R., Ozola, A., Azarjana, K., Molven, A., Akslen, L. A., Donina, S., Proboka, G., Cema, I., Baginskis, A., and Pjanova, D. (2013). Analysis of Latvian familial melanoma patients shows novel variants in the noncoding regions of CDKN2A and that the CDK4 mutation R24H is a founder mutation. Melanoma Res 23, 221-6.

Vinagre, J., Almeida, A., Populo, H., Batista, R., Lyra, J., Pinto, V., Coelho, R., Celestino, R., Prazeres, H., Lima, L., et al. (2013). Frequency of TERT promoter mutations in human cancers. Nat Commun 4, 2185.

Wadt, K., Choi, J., Chung, J. Y., Kiilgaard, J., Heegaard, S., Drzewiecki, K. T., Trent, J. M., Hewitt, S. M., Hayward, N. K., Gerdes, A. M., et al. (2012). A cryptic BAP1 splice mutation in a family with uveal and cutaneous melanoma, and paraganglioma. Pigment Cell Melanoma Res.

Wadt, K. A., Aoude, L. G., Johansson, P., Solinas, A., Pritchard, A., Crainic, O., Andersen, M. T., Kiilgaard, J. F., Heegaard, S., Sunde, L., et al. (2014). A recurrent germline BAP1 mutation and extension of the BAP1 tumor predisposition spectrum to include basal cell carcinoma. Clin Genet.

Wang, F., Podell, E. R., Zaug, A. J., Yang, Y., Baciu, P., Cech, T. R., and Lei, M. (2007). The POT1-TPP1 telomere complex is a telomerase processivity factor. Nature 445, 506-10.

Whelan, A. J., Bartsch, D., and Goodfellow, P. J. (1995). Brief report: a familial syndrome of pancreatic cancer and melanoma with a mutation in the CDKN2 tumor-suppressor gene. N Engl J Med 333, 975-7.

Acc

epte

d A

rtic

le


Wiesner, T., Fried, I., Ulz, P., Stacher, E., Popper, H., Murali, R., Kutzner, H., Lax, S., Smolle-Juttner, F., Geigl, J. B., et al. (2012). Toward an improved definition of the tumor spectrum associated with BAP1 germline mutations. J Clin Oncol 30, e337-40.

Wiesner, T., Obenauf, A. C., Murali, R., Fried, I., Griewank, K. G., Ulz, P., Windpassinger, C., Wackernagel, W., Loy, S., Wolf, I., et al. (2011). Germline mutations in BAP1 predispose to melanocytic tumors. Nat Genet 43, 1018-21.

Xin, H., Liu, D., Wan, M., Safari, A., Kim, H., Sun, W., O'connor, M. S., and Songyang, Z. (2007). TPP1 is a homologue of ciliate TEBP-beta and interacts with POT1 to recruit telomerase. Nature 445, 559-62.

Xu, J., Kadariya, Y., Cheung, M., Pei, J., Talarchek, J., Sementino, E., Tan, Y., Menges, C. W., Cai, K. Q., Litwin, S., et al. (2014). Germline Mutation of Bap1 Accelerates Development of Asbestos-Induced Malignant Mesothelioma. Cancer Res.

Yang, D., He, Q., Kim, H., Ma, W., and Songyang, Z. (2011). TIN2 protein dyskeratosis congenita missense mutants are defective in association with telomerase. J Biol Chem 286, 23022-30.

Ye, J. Z., and De Lange, T. (2004). TIN2 is a tankyrase 1 PARP modulator in the TRF1 telomere length control complex. Nat Genet 36, 618-23.

Ye, J. Z., Hockemeyer, D., Krutchinsky, A. N., Loayza, D., Hooper, S. M., Chait, B. T., and De Lange, T. (2004). POT1-interacting protein PIP1: a telomere length regulator that recruits POT1 to the TIN2/TRF1 complex. Genes Dev 18, 1649-54.

Yokoyama, S., Woods, S. L., Boyle, G. M., Aoude, L. G., Macgregor, S., Zismann, V., Gartside, M., Cust, A. E., Haq, R., Harland, M., et al. (2011). A novel recurrent mutation in MITF predisposes to familial and sporadic melanoma. Nature, in press.

Zhang, Y., Xiong, Y., and Yarbrough, W. G. (1998). ARF promotes MDM2 degradation and stabilizes p53: ARF-INK4a locus deletion impairs both the Rb and p53 tumor suppression pathways. Cell 92, 725-34.

Zhong, F. L., Batista, L. F., Freund, A., Pech, M. F., Venteicher, A. S., and Artandi, S. E. (2012). TPP1 OB-fold domain controls telomere maintenance by recruiting telomerase to chromosome ends. Cell 150, 481-94.

Zuo, L., Weger, J., Yang, Q., Goldstein, A. M., Tucker, M. A., Walker, G. J., Hayward, N., and Dracopoli, N. C. (1996). Germline mutations in the p16INK4a binding domain of CDK4 in familial melanoma. Nat Genet 12, 97-9.

Table 1. Familial melanoma genes: mutation type, mutation prevalence and association with non-melanoma cancers.

Gene Mutation type Reported families Other cancers

ACD inactivating missense and nonsense 6 families glioma

BAP1 inactivating frameshift and splicing 24 families

uveal melanoma, mesothelioma, renal cell carcinoma, cholangiocarcinoma, basal cell carcinoma

Acc

epte

d A

rtic

le


CDK4 activating missense 17 families -

CDKN2A (p16)

inactivating missense, nonsense, promoter, frameshift, in-frame insertion, splicing and gene deletion

~40% of families pancreas

CDKN2A (p14)

inactivating frameshift, splicing and gene deletion

~1% of families

neural system tumours

MITF inactivating missense PAF† 0.9% of CMM

renal cell carcinoma

POT1 inactivating missense and splicing 12 families -

TERF2IP inactivating missense and nonsense

5 families -

TERT activating promoter 1 family -

† PAF – population attributable fraction

Acc

epte

d A

rtic

le


Acc

epte

d A

rtic

le


Acc

epte

d A

rtic

le


Documents

Genetics of familial melanoma: 20 years after CDKN2A