High prevalence of germline CDKN2A mutations in Slovenian cutaneous malignant melanoma families

Aim

To prospectively determine the prevalence of germline CDKN2A mutations in the Slovenian cutaneous malignant melanoma (CMM) families.

Methods

From January 2001 till the end of 2003 we prospectively screened 19 individuals from 11 CMM families, as well as 3 children with CMM aged from 6 to 13 years, with a negative family history.

Results

Five distinct mutations were detected in 5 out of 11 screened families (10/19 individuals) and a previously recognized polymorphism was detected in a single family. Detected mutations were functionally deleterious (T281A, G68A, G301T, G71C and IVS – 1g > a). No mutations could be detected in 3 children.

Conclusions

The prevalence of CDKN2A mutations among Slovenian CMM families was high, indicating the need for genetic counseling.The CDKN2A gene is the most common cause of inherited cutaneous malignant melanoma (CMM) (1). The presence and frequency of CDKN2A gene mutations in familial CMM vary considerably in different populations. Yakobson (2) did not find even a single case of germline CDKN2A mutation among 31 Israeli CMM families. However, Mantelli (3) reported a high prevalence of 34% of the germline CDKN2A mutations in his study of 62 Italian CMM families. Overall, the CDKN2A mutations were detected in approximately 20% of the tested CMM families (1).The aim of our study was to prospectively determine the prevalence of germline CDKN2A mutations in the Slovenian CMM families.     

Intronic sequence variants of the CDKN2A gene in melanoma pedigrees

Germ-line mutations of the tumor-suppressor gene CDKN2A predispose individuals to melanoma in families worldwide. However, coding mutations of CDKN2A have not been detected in a significant proportion of those affected. The identification of a disease-associated intronic mutation of CDKN2A in UK families, which has proved to be the most common CDKN2A mutation as yet identified in this population, has highlighted the possibility that additional causal mutations may lie within the intronic sequence of the gene. In this article, we describe the comprehensive screening of 109 English and 26 Australian melanoma pedigrees for intronic mutations of CDKN2A. In total, 24 sequence variants were identified across the two introns of the gene. We show evidence that two of the CDKN2A intronic variants (IVS1 + 1104 C > A and IVS1 - 1104 C > G) predispose to melanoma. IVS1 + 1104 was shown to result in the aberrant splicing of both p16(INK4a) and p14(ARF) mRNA. Overall, however, the proportion of English melanoma families with these variants is small.     

CDKN2A is the main susceptibility gene in Italian pancreatic cancer families

Background Most familial pancreatic cancer (FPC) remains unexplained. The identification of individuals with a high genetic risk of developing pancreatic adenocarcinoma (PC) is important to elucidate its biological basis and is critical to better define emerging strategies for the detection of early pancreatic neoplasms. Patients and methods A series of 225 consecutively enrolled patients with PC were tested for CDKN2A mutations. After personal and family cancer histories of all the patients had been reviewed, a subset of the patients were classified as FPC and were also tested for mutations in PALLD, PALB2, BRCA1 and BRCA2 as FPC candidate genes. Results The CDKN2A mutation rate in the 225 PC cases was 5.7%. The CDKN2A founder mutations, p.E27X and p.G101W, were predominant, but the mutation spectrum also included p.L65P, p.G67R and two novel, potentially pathogenic variants, promoter variant c.-201ACTC>CTTT and p.R144C. None of the patients with FPC harboured germline mutations in PALLD, PALB2 or BRCA2. One family was positive for the BRCA1 UV variant p.P727L. Strikingly, five of 16 patients with FPC (31%) carried CDKN2A mutations. Conclusion These findings suggest that a sizeable subset of Italian FPC families may carry CDKN2A mutations. This result may be of value for identifying the best candidates for future PC screening trials in Italy.     

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.     

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.      

Cytokine gene single nucleotide polymorphisms and susceptibility to and prognosis in cutaneous malignant melanoma

Cutaneous malignant melanoma (CMM) is a potentially fatal malignancy in which exposure to UV light is the most important risk factor. Several lines of evidence suggest that CMM patients develop an immune response to their tumours, although, in most cases, anti‐tumour immune responses are insufficient to abrogate tumour development. Polymorphism in genes regulating the immune response and cell growth may result in increased susceptibility to and/or poorer prognosis in certain individuals. In this study, we addressed whether single nucleotide polymorphisms (SNPs) associated with differential expression of selected pro‐ and anti‐inflammatory cytokines and growth factors [interleukin (IL)‐1β?35 and ?511, IL‐2 ?330, IL‐4 ?590, IL‐6 ?174, IL‐8 ?251, interferon (IFN)‐γ+874 and transforming growth factor (TGF)β1 +915] or as markers of candidate cytokine genes (IL‐12 +1188) are associated with susceptibility to or known prognostic indicators (e.g. initial tumour growth phase, Breslow thickness, mitotic count in vertical growth phase tumours, tumour regression) in CMM. One hundred and sixty‐nine British caucasian CMM patients and 261 controls were included in the study and all SNPs were genotyped by ARMS–PCR. No SNP genotypes or alleles showed significant associations with CMM susceptibility and only the IL‐1β?511 TT genotype was associated with thinner invasive tumours at presentation, as assessed by Breslow thickness at the clinically significant cut‐off point of 1.5 mm [occurring in 2/51 (3.9%) thicker vs. 14/78 (17.9%) thinner tumours (P = 0.03; relative risk = 0.29 (95% confidence interval 0.05–0.95)]. These findings suggest that — with the possible exception of IL‐1β— genetic variation associated with differential expression of the selected pro‐ and anti‐inflammatory cytokines is unlikely to play a major role in susceptibility to and prognosis in CMM.     

Familial melanoma, pancreatic cancer and germline CDKN2A mutations

Germline CDKN2A mutations have been observed in approximately 20 percent of familial melanoma kindreds from North America, Europe and Australasia. There is also an increased risk of pancreatic cancer in a subset of families with mutations, however, the precise relationship between the CDKN2A gene and pancreatic cancer remains unknown. The relationships between familial melanoma, pancreatic cancer and germline CDKN2A mutations were examined using published data. There were 67 different CDKN2A mutations in 189 melanoma-prone families. Forty-two families (18 mutations) had pancreatic cancer reported. For families without reported pancreatic cancer, the most common types of mutations were missense (56%), frameshift (12%), and deletions (12%). For families with pancreatic cancer, missense (56%), splicing (17%), and frameshift (11%) mutations were most common. Seventy percent of the mutations were observed only once, while the remainder recurred in different families. Comparison of 147 melanoma-prone families without pancreatic cancer to the 42 families that had pancreatic cancer reported showed no significant differences in the types or locations of mutations. However, there was a significant difference (p=0.002) in the distribution of families across the four ankyrin repeats. This finding primarily resulted from the six most frequent mutations where the distribution of pancreatic cancer varied significantly (p=0.02) from at least 30% in c.301G>T (p.G101W), c.225_243del19 (p.P75fs), c.337_338insGTC (p.R112_L113insR), and c.377T>A (p.V126D) families to less than 10% in c.71G>C (p.R24P) and c.159G>C (p.M53I) families. Further research utilizing individual-specific data will be required to determine whether these patterns represent etiologic differences or incomplete reporting of cancer and mutation data.     

Cytokine gene single nucleotide polymorphisms and susceptibility to and prognosis in cutaneous malignant melanoma.

Cutaneous malignant melanoma (CMM) is a potentially fatal malignancy in which exposure to UV light is the most important risk factor. Several lines of evidence suggest that CMM patients develop an immune response to their tumours, although, in most cases, anti-tumour immune responses are insufficient to abrogate tumour development. Polymorphism in genes regulating the immune response and cell growth may result in increased susceptibility to and/or poorer prognosis in certain individuals. In this study, we addressed whether single nucleotide polymorphisms (SNPs) associated with differential expression of selected pro- and anti-inflammatory cytokines and growth factors [interleukin (IL)-1beta-35 and -511, IL-2 -330, IL-4 -590, IL-6 -174, IL-8 -251, interferon (IFN)-gamma+874 and transforming growth factor (TGF)beta1 +915] or as markers of candidate cytokine genes (IL-12 +1188) are associated with susceptibility to or known prognostic indicators (e.g. initial tumour growth phase, Breslow thickness, mitotic count in vertical growth phase tumours, tumour regression) in CMM. One hundred and sixty-nine British caucasian CMM patients and 261 controls were included in the study and all SNPs were genotyped by ARMS-PCR. No SNP genotypes or alleles showed significant associations with CMM susceptibility and only the IL-1beta-511 TT genotype was associated with thinner invasive tumours at presentation, as assessed by Breslow thickness at the clinically significant cut-off point of 1.5 mm [occurring in 2/51 (3.9%) thicker vs. 14/78 (17.9%) thinner tumours (P = 0.03; relative risk = 0.29 (95% confidence interval 0.05-0.95)]. These findings suggest that - with the possible exception of IL-1beta- genetic variation associated with differential expression of the selected pro- and anti-inflammatory cytokines is unlikely to play a major role in susceptibility to and prognosis in CMM.     

CDKN2A基因变异与肿瘤

细胞周期依赖性激酶抑制基因（CDKN2A）为抑癌基因，编码两种周期抑制蛋白p16INK4a和p14ARF；进而通过p16INK4a—CDK4（和CDK6）-pRb途径和p14ARF—mdm2-p53途径发挥细胞周期调控作用。研究发现在多种肿瘤中均存在CDKN2A的基因变异，现就近年来的研究状况做一简要概述。     

Mutation screening of the CDKN2A promoter in melanoma families

Germline mutations of CDKN2A, at 9p21, are responsible for predisposition to melanoma in some families. However, evidence of linkage to 9p21 has been demonstrated in a significant proportion of kindreds with no detectable mutations in CDKN2A. It is possible that mutations in noncoding regions may be responsible for predisposition to melanoma in these families. We have analyzed approximately 1 kb of the CDKN2A promoter upstream of the start codon in an attempt to identify causal mutations in 107 melanoma families. Four sequence variants were detected. Two of these (A-191G and A-493T) did not segregate with disease and were present in a control population at a comparable frequency, indicating that they are unlikely to predispose to melanoma. The A-493T variant appeared to be in linkage disequilibrium with the previously described CDKN2A polymorphism Ala148Thr. The variant G-735A was detected in the control population, but segregation of this variant with melanoma within families could not be discounted. The fourth variant (G-34T), located in the 5' UTR, creates an aberrant initiation codon. This variant appeared to segregate with melanoma and was not detected in a control population. G-34T has recently been identified in a subset of Canadian melanoma families and was concluded to be associated with predisposition to melanoma. The creation of an aberrant initiation site in the 5' UTR may have an important role in carcinogenesis in a small percentage of families; however, mutations in the CDKN2A promoter appear to have a limited role in predisposition to melanoma.     

Use of fluorescence in situ hybridization to distinguish metastatic uveal from cutaneous melanoma

Metastatic lesions of malignant melanoma can on occasion be difficult to classify with regard to the primary site of origin. Given the lack of specificity of light microscopic features, ancillary studies are needed. In this study, the authors explored the possibility of distinguishing metastatic tumors derived from uveal primaries from those known to have originated from a cutaneous melanoma by fluorescence in situ hybridization (FISH) using probes for chromosome 3, 8q24, and 1p36. A total of 32 metastatic tumors were analyzed by FISH. Monosomy 3 was detected in 9 out of 16 (56.3%) cases of metastatic uveal melanoma but was not found in any of the 16 metastatic cutaneous melanomas (P < .001). With regard to 1p36, amplifications were found in 8 out of 16 (50%) cases of metastatic cutaneous melanoma but not in any case of uveal melanoma (P < .05). 1p36 was deleted in 3 cases of uveal and 1 case of cutaneous melanoma. Amplifications of 8q were found in 15 out of 16 (94%) cases of uveal melanoma metastases and in 12 out of 16 (75%) cases of cutaneous metastases. The findings suggest that FISH for monosomy 3 is a useful adjunct tool in the differential diagnosis of metastatic uveal versus cutaneous melanoma.     

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.     

Mutations of the CDKN2/p16INK4 gene in Australian melanoma kindreds

The cyclin dependent kinase inhibitor 2 (CDKN2) gene on chromosome9p21 is potentially involved in the genesis of many cancersand is currently under intense investigation as a possible melanomasusceptibility locus. We have analyzed 18 Australian melanomakindreds for mutations within the coding and neighboring splicejunction portions of the CDKN2 gene. In seven kindreds (includingour six largest), CDKN2 mutations were found to segregate withthe putative melanoma chromosome previously assigned by 9p haplotypeanalysis. These changes included the duplication of a 24 bprepeat, a deleted C residue resulting in the introduction ofa premature stop codon, and four single basepair changes causingamino acid substitutions. Mutations segregated to 46 of 51 affectedindividuals in these seven klndreds, with three apparent sporadiccases In one famiiy and one in each of another two families.Penetrance was variable (55–100%) among the differentmutations. These data provide additional strong support thatthe CDKN2 gene is the chromosome 9p21 familial melanoma locus.     

BAP1 cancer syndrome: malignant mesothelioma, uveal and cutaneous melanoma, and MBAITs

ABSTRACT: BACKGROUND: BRCA1--associated protein 1 (BAP1) is a tumor suppressor gene located on chromosome 3p21. Germline BAP1 mutations have been recently associated with an increased risk of malignant mesothelioma, atypical melanocytic tumors and other neoplasms. To answer the question if different germline BAP1 mutations may predispose to a single syndrome with a wide phenotypic range or to distinct syndromes, we investigated the presence of melanocytic tumors in two unrelated families (L and W) with germline BAP1 mutations and increased risk of malignant mesothelioma. METHODS: Suspicious cutaneous lesions were clinically and pathologically characterized and compared to those present in other families carrying BAP1 mutations. We then conducted a meta-analysis of all the studies reporting BAP1-mutated families to survey cancer risk related to the germline BAP1 mutation (means were compared using t-test and proportions were compared with Pearson Chi2 test or two-tailed Fisher's exact test). RESULTS: Melanocytic tumors: of the five members of the L family studied, four (80%) carried a germline BAP1 mutation (p.Gln684*) and also presented one or more atypical melanocytic tumors; of the seven members of W family studied, all carried a germline BAP1 mutation (p.Pro147fs*48) and four of them (57%) presented one or more atypical melanocytic tumors, that we propose to call "melanocytic BAP1-mutated atypical intradermal tumors" (MBAITs). Meta-analysis: 118 individuals from seven unrelated families were selected and divided into a BAP1-mutated cohort and a BAP1-non-mutated cohort. Malignant mesothelioma, uveal melanoma, cutaneous melanoma, and MBAITs prevalence was significantly higher in the BAP1-mutated cohort (p [LESS-THAN OR EQUAL TO] 0.001). CONCLUSIONS: Germline BAP1 mutations are associated with a novel cancer syndrome characterized by malignant mesothelioma, uveal melanoma, cutaneous melanoma and MBAITs, and possibly by other cancers. MBAITs provide physicians with a marker to identify individuals who may carry germline BAP1 mutations and thus are at high risk of developing associated cancers.     

Patients with both pancreatic adenocarcinoma and melanoma may harbor germline CDKN2A mutations

Germline mutations of the CDKN2A tumor suppressor gene have been identified in melanoma kindreds linked to 9p21, and pancreatic adenocarcinoma is the second most common malignancy in some of these families. We hypothesized that unselected patients with both primary cancers, i.e., pancreatic cancer and malignant melanoma, have a genetic predisposition to tumor development, and that this susceptibility may be due to germline CDKN2A mutations. Fourteen patients, with both pathologically verified pancreatic adenocarcinoma and melanoma, were assessed for germline CDKN2A mutations by polymerase chain reaction amplification and sequencing of six overlapping fragments encompassing exons 1alpha and 2. A yeast two-hybrid assay was used to assess the functional consequences of CDKN2A variants. Germline CDKN2A mutations were identified in 2/14 patients: I49S, a novel substitution in exon 1alpha, and M53I, a previously reported missense mutation in exon 2. Both variants lead to compromised CDKN2A function. We conclude that the occurrence of both pancreatic cancer and melanoma, in the same patient, signals an inherited susceptibility to cancer, and that this predisposition is, in some cases, due to germline CDKN2A mutations. This finding has important implications not only for the proband, but also for other family members.     

Significant impact of promoter hypermethylation and the 540 C>T polymorphism of CDKN2A in cutaneous melanoma of the vertical growth phase

Promoter hypermethylation, mutations, and loss of heterozygosity in the CDKN2A gene as well as polymorphisms at the 3'-untranslated region were determined in vertical growth phase melanomas. Methylation-specific polymerase chain reaction in soluti and in situ showed that 19% of the cases were hypermethylated at the CDKN2A promoter region, and some of these cases were heterogeneous with both methylated and unmethylated tumor cells. Methylation was associated with increased tumor cell proliferation by Ki-67 expression (P = 0.01) and decreased patient survival (P = 0.025). Point mutations in CDKN2A were found in 4% of the cases, whereas 90% had loss of heterozygosity at one or more of 4 markers studied. Furthermore, presence of the 540 C>T polymorphism at the 3'-untranslated region of CDKN2A (23%) was associated with improved survival in multivariate analysis (hazard ratio, 2.6; P = 0.02). Our results suggest that promoter methylation of the CDKN2A gene is present in a subgroup of the tumors and associated with increased tumor cell proliferation and reduced survival. Further, the 540 C>T polymorphism might define a distinct subgroup of low-grade vertical growth phase melanomas. These findings support a significant role of the CDKN2A gene in melanoma progression.     

Genome-wide association study identifies nidogen 1 (NID1) as a susceptibility locus to cutaneous nevi and melanoma risk

We conducted a genome-wide association study on the number of melanocytic nevi reported by 9136 individuals of European ancestry, with follow-up replication in 3581 individuals. We identified the nidogen 1 (NID1) gene on 1q42 associated with nevus count (two linked single nucleotide polymorphisms with r(2) > 0.9: rs3768080 A allele associated with reduced count, P = 6.5 × 10(-8); and rs10754833 T allele associated with reduced count, P = 1.5 × 10(-7)). We further determined that the rs10754833 [T] was associated with a decreased melanoma risk in 2368 melanoma cases and 7432 controls [for CT genotype: odds ratio (OR) = 0.86, 95% confidence interval (CI) = 0.75-0.99, P = 0.04; for TT genotype: OR = 0.84, 95% CI = 0.71-0.98, P = 0.03]. Expression level of the NID1 locus was 2-fold higher for the rs10754833 T allele carriers than that with the CC genotype (P = 0.017) in the 87 HapMap CEU cell lines. The NID1 gene is a biologically plausible locus for nevogenesis and melanoma development, with decreased expression levels of NID1 in benign nevi (P = 3.5 × 10(-6)) and in primary melanoma (P = 4.6 × 10(-4)) compared with the normal skin.