High prevalence of the G101W germline mutation in the CDKN2A (P16(ink4a)) gene in 62 Italian malignant melanoma families.

CDKN2A germline mutation frequency estimates are commonly based on families with several melanoma cases. When we started counseling in a research setting on gene susceptibility analysis in northern and central Italy, however, we mostly found small families with few cases. Here we briefly characterize those kindred, estimate CDKN2A/CDK4 mutation test yields, and provide indications on the possibility of implementing formal DNA testing for melanoma-prone families in Italy. In September 1995 we started genetic counseling in a research setting at our Medical Genetics Center. Screening for CDKN2A/CDK4 mutations was performed on families with two melanoma patients, one of whom was younger than 50 years at onset, the other complying with one of the following: 1) being a first-degree relative, 2) having an additional relative with pancreatic cancer, or 3) having multiple primary melanomas. Sixty-two of 67 (80%) melanoma cases met our criteria. Four previously described CDKN2A mutations (G101W, R24P, V126D, and N71S) were found in 21 of the 62 families (34%) with a high prevalence of G101W (18/21). The percentage of families with two melanoma cases/family harboring a mutation was low (7%, 2/27), but rose to 45% (9/20) if one of the melanoma patients carried multiple melanomas or if pancreatic cancer was present in that family. In the 15 families with three melanoma cases the presence of a mutation was higher (67%, 10/15) and reached 100% in the 4 families with four or more melanoma cases. Our results suggest that CDKN2A/CDK4 counseling-based mutational analysis may be reasonably efficient also for families with two melanoma cases, if one patient carries multiple melanomas or if pancreatic cancer is present in the family.     

High prevalence of the G101W germline mutation in the CDKN2A (P16ink4a) gene in 62 Italian malignant melanoma families

CDKN2A germline mutation frequency estimates are commonly based on families with several melanoma cases. When we started counseling in a research setting on gene susceptibility analysis in northern and central Italy, however, we mostly found small families with few cases. Here we briefly characterize those kindred, estimate CDKN2A/CDK4 mutation test yields, and provide indications on the possibility of implementing formal DNA testing for melanoma‐prone families in Italy. In September 1995 we started genetic counseling in a research setting at our Medical Genetics Center. Screening for CDKN2A/CDK4 mutations was performed on families with two melanoma patients, one of whom was younger than 50 years at onset, the other complying with one of the following: 1) being a first‐degree relative, 2) having an additional relative with pancreatic cancer, or 3) having multiple primary melanomas. Sixty‐two of 67 (80%) melanoma cases met our criteria. Four previously described CDKN2A mutations (G101W, R24P, V126D, and N71S) were found in 21 of the 62 families (34%) with a high prevalence of G101W (18/21). The percentage of families with two melanoma cases/family harboring a mutation was low (7%, 2/27), but rose to 45% (9/20) if one of the melanoma patients carried multiple melanomas or if pancreatic cancer was present in that family. In the 15 families with three melanoma cases the presence of a mutation was higher (67%, 10/15) and reached 100% in the 4 families with four or more melanoma cases. Our results suggest that CDKN2A/CDK4 counseling‐based mutational analysis may be reasonably efficient also for families with two melanoma cases, if one patient carries multiple melanomas or if pancreatic cancer is present in the family. © 2002 Wiley‐Liss, Inc.     

Population-based prevalence of CDKN2A and CDK4 mutations in patients with multiple primary melanomas

The presence of multiple primary cutaneous melanomas (MPM) has been advocated as guidance to identifying melanoma families. Frequencies of CDKN2A mutations in materials of sporadic MPM cases from pigmented lesion clinics vary between 8 and 15%. Patients with MPM have therefore been regarded as good candidates for CDKN2A mutational screening. We describe a population-based study where all persons in Norway diagnosed with MPM between 1953 and 2004 (n = 738 alive per April 2004) were invited to participate. Three-hundred-and-ninety patients (52.8%) responded confidentially. Mutations in CDKN2A were found in 6.9% of the respondents. Eighty-one MPM patients (20.8%) reported that they belonged to melanoma families, and 17 (21.0%) of these harboured a CDKN2A mutation, compared to 3.2% of the nonfamilial cases. The probability of finding a CDKN2A mutation increased when the patients had three or more melanomas, or a young age of onset of first melanoma. We identified five novel CDKN2A variants (Ala57Gly, Pro81Arg, Ala118Val, Leu130Val, and Arg131Pro) and four that previously have been reported in melanoma families (Glu27X, Met53Ile, Arg87Trp, and Ala127Pro). A large deletion (g.13623_23772del10150) encompassing exon 1alpha and the 5' part of exon 2 was detected in six patients with a family history of melanoma. Three patients, belonging to the same family, had the CDK4 Arg24His mutation. The frequency of CDKN2A mutations was lower than previously reported in other studies, an observation which probably is due to the population-based design of our study.     

A large Norwegian family with inherited malignant melanoma, multiple atypical nevi, and CDK4 mutation

Mutations in two loci encoding cell-cycle-regulatory proteins have been shown to cause familial malignant melanoma. About 20% of melanoma-prone families bear a mutation in the CDKN2A locus, which encodes two unrelated proteins, p16INK4A and p14ARF. Mutations in the other locus, CDK4, are much rarer and have been linked to the disease in only three families worldwide. In the 1960s, a large Norwegian pedigree with multiple atypical nevi and malignant melanomas was identified. Subsequently, six generations and more than 100 family members were traced and 20 cases of melanoma verified. In this article, we report that CDK4 codon 24 is mutated from CGT to CAT (Arg24His) in this unusually large melanoma kindred. Intriguingly, one of the family members had ocular melanoma, but the CDK4 mutation could not be detected in archival tissue samples from this subject. Thus, the case of ocular melanoma in this family was sporadic, suggesting an etiology different from that of the skin tumors. The CDK4 mutation in the Norwegian family was identical to that in melanoma families in France, Australia, and England. Haplotype analysis using microsatellite markers flanking the CDK4 gene and single-nucleotide polymorphisms within the gene did not support the possibility that there was a common founder, but rather indicated at least two independent mutational events. All CDK4 melanoma families known to date have a substitution of amino acid 24. In addition to resulting from selection pressure, this observation may be explained by the CG dinucleotide of codon 24 representing a mutational hot spot in the CDK4 gene.     

Amplification of CDK4 and MDM2 in malignant melanoma

Amplification of the 12q13-15 region is a common event in several human tumors including liposarcomas, gliomas, and osteosarcomas. We have demonstrated high-level amplification of 12q14 in a subset of uncultured malignant melanomas (3 of 53). High-resolution mapping of the amplicon using quantitative PCR revealed a bipartite amplicon consisting of a primary 50-kb amplicon centered on CDK4 and a secondary amplicon centered on MDM2, without amplification of the intervening 11 Mb of genomic DNA. Analysis of mRNA and protein levels in melanomas with 12q14 amplification demonstrated overexpression of target genes CDK4 and MDM2 without loss of CDKN2A-P16 (P16INK4A) or CDKN2A-P14ARF (P14ARF) expression, important regulators of the RB1 and TP53 pathways, which are commonly lost or mutated in melanoma. These results suggest that coamplification of CDK4 and MDM2 may substitute for loss of P16INK4A and P14ARF function in a subset of melanomas.     

Sporadic multiple primary melanoma cases: CDKN2A germline mutations with a founder effect

Multiple primary cancers are one of the hallmarks of inherited predisposition. Outside the familial context, multiple primary tumors could be related either to germline de novo mutations or to low-penetrance mutations, in predisposing genes. We selected 100 patients who displayed multiple primary melanoma (MPM) without any known melanoma cases recorded within their families and looked for germline mutations in the two melanoma-predisposing genes identified to date, CDKN2A and CDK4 exon 2. Nine patients (9%) had germline mutations in CDKN2A, whereas none carried germline mutations in exon 2 of CDK4. Seven cases displayed a recurrent missense mutation, G101W, already described in more than 20 melanoma-prone families; one case carried a missense mutation never reported to date (P114S), and the last case was a carrier of a 6 bp insertion at nucleotide 57 resulting in a duplication of codons 18 and 19. To ascertain whether the G101W was a mutational hot spot for de novo mutations or a common founder mutation, we genotyped eight microsatellite markers flanking the CDKN2A gene. After allowing for recombination over time, haplotype sharing provided evidence for an original G101W mutation common to 6 out of 7 sporadic MPM cases. Therefore, it can be concluded that de novo germline CDKN2A mutations associated with MPM are rare.     

Predicting functional significance of cancer‐associated p16INK4a mutations in CDKN2A

Inherited mutations affecting the INK4a/ARF locus (CDKN2A) are associated with melanoma susceptibility in 40% of multiple case melanoma families. Over 60 different germline INK4a/ARF mutations have been detected in more than 190 families worldwide. The majority of these alterations are missense mutations affecting p16^INK4a, and only 25% of these have been functionally assessed. There is therefore a need for an accurate and rapid assay to determine the functional significance of p16^INK4a mutations. We reviewed the performance of several in vivo functional assays that measure critical aspects of p16^INK4a function, including subcellular location, CDK binding and cell cycle inhibition. In this report the function of 28 p16^INK4a variants, many associated with melanoma susceptibility were compared. We show that assessment of CDK4 binding and subcellular localization can accurately and rapidly determine the functional significance of melanoma‐associated p16^INK4a mutations. p16^INK4a‐CDK6 binding affinity was unhelpful, as no disease‐associated mutation showed reduced CDK6 affinity while maintaining the ability to bind CDK4. Likewise, in silico analyses did not contribute substantially, with only 12 of 25 melanoma‐associated missense variants consistently predicted as deleterious. The ability to determine variant functional activity accurately would identify disease‐associated mutations and facilitate effective genetic counselling of individuals at high risk of melanoma. Hum Mutat 31:1–10, 2010. © 2010 Wiley‐Liss, Inc.     

Germline mutations of the CDKN2 gene in UK melanoma families

Germline mutations in CDKN2 on chromosome 9p21, which codes for the cyclin D kinase inhibitor p16, and more rarely, mutations in the gene coding for CDK4, the protein to which p16 binds, underlie susceptibility in some melanoma families. We have sequenced all exons of CDKN2 and analysed the CDK4 gene for mutations in 27 UK families showing evidence of predisposition to melanoma. Five different germline mutations in CDKN2 were found in six families. Three of the mutations (Met53Ile, Arg24Pro and 23ins24) have been reported previously. We have identified two novel CDKN2 mutations (88delG and Ala118Thr) which are likely to be associated with the development of melanoma, because of their co-segregation with the disease and their likely functional effect on the CDKN2 protein. In binding assays the protein expressed from the previously described mutation, Met53Ile, did not bind to CDK4/CDK6, confirming its role as a causal mutation in the development of melanoma. Ala118Thr appeared to be functional in this assay. Arg24Pro appeared to bind to CDK6, but not to CDK4. No mutations were detected in exon 2 of CDK4, suggesting that causal mutations in this gene are uncommon. The penetrance of these mutant CDKN2 genes is not yet established, nor is the risk of non-melanoma cancer to gene carriers.      

A single Mediterranean,possibly Jewish,origin for the Val59Gly CDKN2A mutation in four melanoma-prone families

We have screened for CDKN2A germline mutations in 49 Jewish families with two or more cases of melanoma. The Val59Gly mutation, one of the three different alterations identified among these families, was also detected independently in two kindreds from France and one from Spain. The impact of the Val59Gly substitution on the function of the cyclin-dependent kinase inhibitor p16(INK4a), a product of the CDKN2A gene, was assessed by protein-protein interaction and cell proliferation assays and related to potential structural alterations predicted by molecular modeling. Seven microsatellite markers in the vicinity of the CDKN2A gene were used to determine whether the mutation in these families is identical by descent, or represents a mutational hotspot in the CDKN2A gene. Our results show that the Val59Gly substitution impairs p16(INK4a) function, and this dysfunction is consistent with structural predictions. All melanoma-affected individuals tested in the families under study harbor this mutation. Interestingly, the Israeli pedigree includes an affected individual who is homozygous for the Val59Gly mutation. A common haplotype of microsatellite markers has been demonstrated for mutation carriers in all four pedigrees. The Israeli pedigree and one of the French melanoma families are of Moroccan and Tunisian Jewish descent, respectively, and the other families originate from regions of France and Spain close to the Pyrenees. We conclude that the Val59Gly mutation is a major contributor to melanoma risk in the families under study and that it may derive from a single ancestral founder of Mediterranean (possibly Jewish) origin.     

Functional,structural, and genetic evaluation of 20 CDKN2A germ line mutations identified in melanoma‐prone families or patients

Germline mutations of the CDKN2A gene are found in melanoma‐prone families and individuals with multiple sporadic melanomas. The encoded protein, p16^INK4A, comprises four ankyrin‐type repeats, and the mutations, most of which are missense and occur throughout the entire coding region, can disrupt the conformation of these structural motifs as well as the association of p16^INK4a with its physiological targets, the cyclin‐dependent kinases (CDKs) CDK4 and CDK6. Assessing pathogenicity of nonsynonymous mutations is critical to evaluate melanoma risk in carriers. In the current study, we investigate 20 CDKN2A germline mutations whose effects on p16^INK4A structure and function have not been previously documented (Thr18_Ala19dup, Gly23Asp, Arg24Gln, Gly35Ala, Gly35Val, Ala57Val, Ala60Val, Ala60Arg, Leu65dup, Gly67Arg, Gly67_Asn71del, Glu69Gly, Asp74Tyr, Thr77Pro, Arg80Pro, Pro81Thr, Arg87Trp, Leu97Arg, Arg99Pro, and [Leu113Leu;Pro114Ser]). By considering genetic information, the predicted impact of each variant on the protein structure, its ability to interact with CDK4 and impede cell proliferation in experimental settings, we conclude that 18 of the 20 CDKN2A variants can be classed as loss of function mutations, whereas the results for two remain ambiguous. Discriminating between mutant and neutral variants of p16^INK4A not only adds to our understanding of the functionally critical residues in the protein but provides information that can be used for melanoma risk prediction. Hum Mutat 0, 1–11, 2009. © 2009 Wiley‐Liss, Inc.     

Prevalence of p16 and CDK4 germline mutations in 48 melanoma-prone families in France. The French Familial Melanoma Study Group [published erratum appears in Hum Mol Genet 1998 May;7(5):941]

Germline mutations in the p16 and CDK4 genes have been reported in a subset of melanoma pedigrees, but their prevalence is not well known. We searched for such germline mutations in 48 French melanoma-prone families selected according to two major criteria: families with at least three affected members (n = 20) or families with two affected members, one of them affected before the age of 50 (n = 28), and one additional minor criterion. Sixteen different p16 germline mutations were found in 21 families, while one germline mutation, Arg24His, was detected in the CDK4 gene. The frequency of p16 gene mutation in our sample (44%) is among the highest rates yet reported and the CDK4 mutation is the second mutation detected in this gene worldwide. In summary, our results show frequent involvement of the p16 gene in familial melanoma and confirm the role of the CDK4 gene as a melanoma- predisposing gene.      

CDKN2A mutations in Spanish cutaneous malignant melanoma families and patients with multiple melanomas and other neoplasia

The CDKN2A gene has been implicated in cutaneous malignant melanoma (CMM) in about 40% of families with linkage to chromosome 9p21, while a small proportion of families have mutations in the CDK4 gene. In order to estimate the importance of these genes in the predisposition to CMM in Spanish families and patients we have analysed, by SSCA, a total of 56 subjects belonging to 34 CMM families, and nine patients with multiple CMM and other neoplasia. We have detected germline CDKN2A mutations in six out of the 34 families (17%). A frameshift mutation (358delG) and four missense mutations (G59V, G101W (two cases), D84Y, and R87W) were identified. Five CMM patients from different families (14%) carried the A148T variant, which is known not to affect p16 activity. No mutations were detected in the patients with multiple CMM or other neoplasms. We have not found mutations either in exon 1 beta of the CDKN2A gene or in exon 2A of CDK4. Linkage analysis of the 9p21 region showed exclusion for one of the families for CMM and for four families for CMM/dysplastic naevi. This study indicates a small role for CDKN2A in Spanish CMM families and suggests that other genes are also responsible for CMM predisposition.     

New founder germline mutations of CDKN2A in melanoma-prone families and multiple primary melanoma development in a patient receiving levodopa treatment

Germline mutations in the CDKN2A gene have been shown to predispose individuals to cutaneous malignant melanoma. Here, we describe three melanoma-prone families and one isolated patient affected by multiple melanoma who carried a tandem germline mutation of CDKN2A at the nucleotide level, [c.339G>C;c.340C>T], [p.Leu113Leu;p.Pro114Ser]. We also describe three other melanoma-prone families that carried a missense germline CDKN2A mutation, c.167G>T, p.Ser56Ile. All these families and patients resided in southeast France. We analyzed six 9p21 markers where the CDKN2A gene is located and found that carrier haplotypes for both mutations were consistent with two respective common founder ancestors. In one family, we identified two fourth-degree relatives homozygous for the Ser56Ile mutation, indicating a possible consanguinity. Furthermore, we observed that a carrier of the founder CDKN2A [p.Leu113Leu;p.Pro114Ser] mutation as well as two MC1R moderate-risk variants, [p.Arg151Cys(+)p.Arg163Gln] developed 22 primary melanomas in the three years that followed initiation of levodopa therapy for Parkinson's disease. This observation suggests that there is a need for reconsideration of the hypothesis that levodopa may play a role in melanoma development, at least when in the context of a high-risk genetic background.     

Decreased expression of the INK4 family of cyclin-dependent kinase inhibitors in Wilms tumor

Cyclin-dependent kinase (CDK) inhibitors represented by the INK4 family (including p16(INK4a, CDKN2A), p15(INK4b, CDKN2B), p18(INK4c, CDKN2C), and p19(INK4d, CDKN2D)) are regulators of the cell cycle shown to be aberrant in many types of human cancer. We tested the hypothesis that these CDK inhibitors are a target for altered gene expression in Wilms tumor. Using RT-PCR, gene expression of the INK4 family was found to be decreased in 9 of 38 Wilms tumor samples obtained from the National Wilms Tumor Study Group (NWTSG) tissue bank. All the affected tumor samples were of favorable histology. Methylation-specific PCR revealed that methylation in the p16 promoter region may be responsible for altered expression. The incidence of loss of p16 expression may increase with increasing tumor stage, i.e., 1/10 (10%) with stage I/II FH Wilms tumor, 2/10 (20%) with stage III FH Wilms tumor, and 4/10 (40%) with stage IV FH Wilms tumor. Thus, determining the expression status of the INK4 family may have potential prognostic value in the management of Wilms tumor.     

Prevalence of CDKN2A mutations in pancreatic cancer patients: implications for genetic counseling

Germline mutations in CDKN2A have been reported in pancreatic cancer families, but genetic counseling for pancreatic cancer risk has been limited by lack of information on CDKN2A mutation carriers outside of selected pancreatic or melanoma kindreds. Lymphocyte DNA from consecutive, unselected white non-Hispanic patients with pancreatic adenocarcinoma was used to sequence CDKN2A. Frequencies of mutations that alter the coding of p16INK4 or p14ARF were quantified overall and in subgroups. Penetrance and likelihood of carrying mutations by family history were estimated. Among 1537 cases, 9 (0.6%) carried germline mutations in CDKN2A, including three previously unreported mutations. CDKN2A mutation carriers were more likely to have a family history of pancreatic cancer (P=0.003) or melanoma (P=0.03), and a personal history of melanoma (P=0.01). Among cases who reported having a first-degree relative with pancreatic cancer or melanoma, the carrier proportions were 3.3 and 5.3%, respectively. Penetrance for mutation carriers by age 80 was calculated to be 58% for pancreatic cancer (95% confidence interval (CI) 8–86%), and 39% for melanoma (95% CI 0–80). Among cases who ever smoked cigarettes, the risk for pancreatic cancer was higher for carriers compared with non-carriers (HR 25.8, P=2.1 × 10⁻¹³), but among nonsmokers, this comparison did not reach statistical significance. Germline mutations in CDKN2A among unselected pancreatic cancer patients are uncommon, although notably penetrant, especially among smokers. Carriers of germline mutations of CDKN2A should be counseled to avoid tobacco use to decrease risk of pancreatic cancer in addition to taking measures to decrease melanoma risk.     

Mutational analysis of the N-ras, p53, p16INK4a, CDK4, and MC1R genes in human congenital melanocytic naevi

Eighteen human congenital melanocytic naevi (CMN) from 17 patients were screened for activating point mutations in the oncogenes N-ras and CDK4 and for sequence variants in the MC1R gene by combined RFLP-PCR/SSCP analysis. In addition, all lesions were screened for deletions and point mutations in the tumour suppressor genes p53 and p16INK4a (CDKN2A) by combined multiplex PCR/SSCP analysis. Positive screening data were specified by sequencing of the corresponding PCR product. Activating point mutations in the N-ras gene (nine CAA (Gln) to AAA (Lys) transversions and one CAA (Gln) to CGA (Arg) transition at codon 61) were detected at high frequency (56%). Furthermore, three missense mutations (V92M) and two silent mutations (CGA (Arg) to CGG (Arg), codon 213, exon 6) were found in the MC1R and p53 genes, respectively. No mutations were found in p16 or CDK4. The activated N-ras oncogene, which is also found in human cutaneous melanomas, may constitute a potential risk factor for melanoma formation within CMN.