P110 Mutational analysis of CDKN2A and CDK4 genes in a hospital-based series of Greek patients with melanoma
Vasiliki NikolaouXinyi KangHelen GogasM. PlakaJ. NjauwK. KypraiouI. MirmigiIrene StefanakiAlexander StratigosHui-Chen Tsao
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Background CDKN2A has been identified as a high penetrance melanoma susceptibility gene based on the presence of germline mutations in up to 25% of melanoma – prone families (FM) and in 15% of patients with multiple primary melanoma (MPM). The CDK4 gene represents an additional melanoma susceptibility locus, with activating mutations reported in a few families worldwide. Aim To investigate the CDKN2A and CDK4 genes for germline mutations in Greek patients with cutaneous melanoma. Methods We studied all melanoma cases diagnosed in a 5-year period at a melanoma referral center in Athens, Greece. Blood samples were collected and direct sequencing of the CDKN2A exons 1α, 1β, and 2 and of exon 2 of the CDK4 gene was performed. Results Three hundred fifty eight patients were diagnosed with invasive melanoma, including 16 patients belonging to 14 families with familial melanoma (3.9% of all cases) and 10 patients with MPM (2.8% of all cases). Two of the familial cases had multiple primaries as well. Genetic screening was done in 298 patients including 9 patients with FM and 7 patients with MPM. Overall, we detected germline CDKN2A mutations in 13 out of the 298 patetients (4.4%). Two of the 14 mutation carriers, were FM cases (2/9, 22.2%), and 4 had MPM (4/7, 57%). One familial case positive for CDKN2A mutation had MPM as well. The mutation rate of sporadic melanoma cases was 2.5% (7/282). The mutations detected included 4 missence mutations (R24P-8 cases, G101R-1 patient, G101E – 1 case, R87W- 1 case), a single Trp110Stop alteration and a novel C.41_43 deletion and insertion 20bp mutation in exon 1a. We also detected the G>T alteration at position -34 in the 5′UTR in one case. The A148 T polymorphism was detected in 24 patients (8%). Finally, an R24H substitution of the CDK4 gene was detected in 1 patient with FM and MPM. Conclusions Our results show that in the greek population, CDKN2A mutations occur more frequently in genetically predisposed groups as well as in sporadic melanoma cases compared to previously reported data. We showed a low rate of familial/multiple primary melanoma cases, but a higher than expected prevalence of CDKN2A/CDK4 mutations in these cases, suggesting a stronger influence of genetic mutations on melanoma-prone individuals in countries with a low incidence rate of the disease.Keywords:
Penetrance
Germline mutation analysis was performed in 469 VHL families from North America, Europe, and Japan. Germline mutations were identified in 300/469 (63%) of the families tested; 137 distinct intragenic germline mutations were detected. Most of the germline VHL mutations (124/137) occurred in 1-2 families; a few occured in four or more families. The common germline VHL mutations were: delPhe76, Asn78Ser, Arg161Stop, Arg167Gln, Arg167Trp, and Leu178Pro. In this large series, it was possible to compare the effects of identical germline mutations in different populations. Germline VHL mutations produced similar cancer phenotypes in Caucasian and Japanese VHL families. Germline VHL mutations were identified that produced three distinct cancer phenotypes: (1) renal carcinoma without pheochromocytoma, (2) renal carcinoma with pheochromocytoma, and (3) pheochromocytoma alone. The catalog of VHL germline mutations with phenotype information should be useful for diagnostic and prognostic studies of VHL and for studies of genotype-phenotype correlations in VHL.
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Study of BRAF mutations in melanoma and nevi from patients with germline mutation in the CDKN2A gene
10075 Background: Germline mutations in the CDKN2A tumor suppressor gene is a rare condition associated with a high risk of melanoma. Somatic activating mutations of the BRAF gene are frequently observed in cutaneous melanoma (40–60%) but their occurrence vary according to melanoma subtypes (cutaneous, vs mucosal or uveal) and are less frequent in melanoma located on chronically sun-exposed skin. It is thought that melanomas occurring in patients carrier of a CDKN2A germline mutation are associated a second inactivating genetic event in the tumor, facilitating cellular transformation by loss of function this crucial gatekeeper of the G1-S checkpoint. However, the potential role of the MAP-kinase pathway, and more precisely, the involvement of BRAF activation, in this particular population of patients, are still unknown. Thus, our objective was to evaluate the frequency of BRAF somatic mutations in melanoma and nevi developed by patients carrier of a CDKN2A germline mutation. Methods: DNA was extracted from paraffin-embedded tissues of 36 primary melanomas, 9 metastases and 20 nevi from 31 patients with CDKN2A germline mutation. Ten sporadic melanoma from patients harbouring no CDKN2A mutation were also studied as a control population. BRAF mutations were screened by direct sequencing of exon 11 and 15. Results: BRAF mutations (V599E) were found with a significantly lower frequency of 14% (5/36) in melanomas from patients with proven CDKN2A germline mutation, as compared to a frequency of 55% (5/9) in patients with no genetic predisposition (two-sided Fishers exact test, p=0.02). Frequencies of BRAF mutations in metastases and nevi from patients with CDKN2A germline mutation were 33%(3/9) and 10% (2/20) respectively. Conclusions: BRAF mutations seem to be less frequent in melanoma from patients with familial predisposition harbouring CDKN2A germ line mutation than in sporadic melanoma. These results suggest that CDKN2A loss of function gives rise to tumorigenic pathways where genetic events distinct from BRAF activating mutation can induce cell transformation. Additional biological studies are performed on this rare population of melanoma samples in order to better characterize the specificity of cellular transformation in the context of CDKN2A germ line mutation. No significant financial relationships to disclose.
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To date, genes receiving greatest attention from melanoma epidemiologists have been the cell cycle gene CDKN2A and the melanocortin-1 receptor gene MC1R. Complex interactions between these genes and as yet unknown factors likely play roles in this malignancy. Currently, epidemiologists rely on either large family studies, such as GenoMEL, or population-based studies, such as GEM and the Queensland Melanoma Study, to evaluate genetic factors associated with disease. Bishop et al. (2002) conducted an international evaluation of CDKN2A penetrance within families in which multiple members had melanoma and harboured CDKN2A mutations and determined penetrance to be high – 67 per cent overall by age 80 – and potentially modified by solar exposure. Begg et al. (2005) conducted an international study of CDKN2A penetrance within families identified from melanoma cases based on population-based registries and found penetrance to be significantly lower – 28 per cent by age 80. MC1R was not analysed in either study. These differing results suggest that studies based on carrier families from a population-based sample generally lead to considerably lower risk estimates than do genetic studies based on families. Begg suggests that other important risk factors may be more prevalent in carriers identified through familial as opposed to population-based ascertainment. A recent GenoMEL analysis published in the Journal of the National Cancer Institute (Demenais et al., 2010) supports this idea: the authors found higher risks for additional genetic variants among individuals identified from families and concluded that the validity of inferences from the two study designs may greatly depend on the method of ascertainment. A subset of MC1R variants are considered a ‘moderate’ risk for melanoma and are strongly associated with red hair colour. It is not yet clear whether MC1R gene variants important in melanoma are associated with or independent of hair colour. In the new study, Demenais and colleagues identified 815 CDKN2A mutation carriers (473 affected and 342 unaffected by melanoma) from 187 families throughout Europe, North America and Australia. They also assessed the most frequent MC1R variants (V60L, V92M, R151C and R160W), also known as ‘red hair variants’ (RHC), with pigmentation phenotypes (such as hair colour, propensity to sunburn and the number of nevi). They observed a statistically significant ‘joint association’ between MC1R RHC variants, hair colour, number of nevi and melanoma risk, the latter reaching an odds ratio (OR) of 4.23 (95% Confidence Interval (CI) 2.27, 7.87) for individuals with more than two variants, blond or red hair, a propensity to sunburn and a high number of nevi. Although this finding is based on a relatively small number of individuals with more than one ‘red hair’ variant, it is important for risk estimation among CDKN2A carriers. Interestingly, the authors report that melanoma risk associated with at least two MC1R RHC variants was 2.6 times higher than with only one variant, that is, 5.8 (95% CI 3.6, 9.5). In a study of melanoma patients in Queensland, Palmer et al. (2000) had reported a similar association in which each additional ‘red hair’ variant doubled melanoma risk, unselected by family history or CDKN2A status. This unanticipated correlation, in which MC1R variants are similar in population-based series unselected for family history, is further illustrated in another large international study in which the proportional distribution of variants in CDKN2A carriers described by Demenais et al. was highly similar to that seen in population-based single primary melanoma cases (Kanetsky et al., 2006). The fact that MC1R is one of the most highly variable genes, both between and within populations and across continents, likely underlies contradictory reports as to whether knowledge of MC1R status aids or is irrelevant to risk prediction. Other investigators have noted that MC1R variants among individuals traditionally perceived to be at lower risk, such as those with darker phenotypes, are critical for risk evaluation (Kanetsky et al., 2010). Actually, these data, when showing analyses stratified by hair colour only, the individual MC1R variants associated with brown or black hair confer significantly higher risk than do those associated with red or blond hair. Clearly, MC1R likely plays a role in the aetiology of melanoma through non-pigmentary as well as pigmentary pathways governing cell survival and apoptosis, DNA repair and interaction of melanocytes with the extracellular matrix (Robinson et al., 2010). An important contribution of Demenais’ work is evaluation of the relationship between MC1R status and specific CDKN2A mutations. Comparing p16INK4a and p14ARF, the authors report that joint risk is highest among p16INK4a variants: in that case 1 MC1R RHC variant increases risk 3.6-fold and 2 or more variants increases risk almost 8.4-fold, compared to a 1.9-fold increase and a 5.9-fold increase in the case of p14ARFvariants. Understanding this relationship requires further investigation. The authors are careful to note that their results apply to CDKN2A carriers and may not be applicable to other populations. Nonetheless, their findings are relevant to risk estimation among families that constitute approximately 10% of all melanoma cases. Furthermore, genome-wide association studies and population-based studies of melanoma are developing risk prediction models that should become more precise as more data becomes available, greatly improving our understanding of melanoma biology and suggesting more effective ways to prevent this aggressive malignancy.
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Germline mutations in the CDKN2A gene, which encodes two proteins (p16INK4A and p14ARF), are the most common cause of inherited susceptibility to melanoma. We examined the penetrance of such mutations using data from eight groups from Europe, Australia and the United States that are part of The Melanoma Genetics Consortium.We analyzed 80 families with documented CDKN2A mutations and multiple cases of cutaneous melanoma. We modeled penetrance for melanoma using a logistic regression model incorporating survival analysis. Hypothesis testing was based on likelihood ratio tests. Covariates included gender, alterations in p14ARF protein, and population melanoma incidence rates. All statistical tests were two-sided.The 80 analyzed families contained 402 melanoma patients, 320 of whom were tested for mutations and 291 were mutation carriers. We also tested 713 unaffected family members for mutations and 194 were carriers. Overall, CDKN2A mutation penetrance was estimated to be 0.30 (95% confidence interval (CI) = 0.12 to 0.62) by age 50 years and 0.67 (95% CI = 0.31 to 0.96) by age 80 years. Penetrance was not statistically significantly modified by gender or by whether the CDKN2A mutation altered p14ARF protein. However, there was a statistically significant effect of residing in a location with a high population incidence rate of melanoma (P =.003). By age 50 years CDKN2A mutation penetrance reached 0.13 in Europe, 0.50 in the United States, and 0.32 in Australia; by age 80 years it was 0.58 in Europe, 0.76 in the United States, and 0.91 in Australia.This study, which gives the most informed estimates of CDKN2A mutation penetrance available, indicates that the penetrance varies with melanoma population incidence rates. Thus, the same factors that affect population incidence of melanoma may also mediate CDKN2A penetrance.
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Germline mutation analysis was performed in 469 VHL families from North America, Europe, and Japan. Germline mutations were identified in 300/469 (63%) of the families tested; 137 distinct intragenic germline mutations were detected. Most of the germline VHL mutations (124/137) occurred in 1–2 families; a few occured in four or more families. The common germline VHL mutations were: delPhe76, Asn78Ser, Arg161Stop, Arg167Gln, Arg167Trp, and Leu178Pro. In this large series, it was possible to compare the effects of identical germline mutations in different populations. Germline VHL mutations produced similar cancer phenotypes in Caucasian and Japanese VHL families. Germline VHL mutations were identified that produced three distinct cancer phenotypes: (1) renal carcinoma without pheochromocytoma, (2) renal carcinoma with pheochromocytoma, and (3) pheochromocytoma alone. The catalog of VHL germline mutations with phenotype information should be useful for diagnostic and prognostic studies of VHL and for studies of genotype-phenotype correlations in VHL. © 1996 Wiley-Liss, Inc.
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Abstract Germline mutations in CDKN2A gene predispose to melanoma with high but incomplete penetrance. Penetrance of CDKN2A gene was found to be significantly influenced by host factors (nevus phenotypes and sunburn) on one hand and by variants of MC1R gene (RHC variants consistently associated with red hair and fair skin) on the other hand. Our goal was to examine the joint effects of MC1R variants and other potential risk factors [total nevi, dysplastic nevi, pigmentary traits (skin, hair and eye color), skin reactions to sunlight, and degree of sun exposure] on CDKN2A penetrance. Clinical, genetic, and covariate data were recorded in 20 French melanoma-prone families with cosegregating CDKN2A mutations. Analysis of the cotransmission of melanoma and CDKN2A mutations was conducted by likelihood-based methods using the regressive logistic models, which can account for a variation of disease risk with age and can include the aforementioned risk factors as covariates. RHC variants, considered either alone or in the presence of pigmentation and nevus phenotypes, were found to increase significantly CDKN2A penetrance. Multivariate analysis, using a stepwise selection procedure, showed significant effects of two factors on melanoma risk in CDKN2A mutations carriers: RHC variants [odds ratio of hazard function (OR), 2.21; P = 0.03] and dysplastic nevi (OR, 2.93; P < 0.01). Such results may have important consequences to improve the prediction of melanoma risk in families.
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Melanocortin 1 receptor
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Abstract The germline mutation rate has been extensively studied and has been found to vary greatly between species, but much less is known about the somatic mutation rate in multicellular organisms, which remains very difficult to determine. Here, we present data on somatic mutation rates in mice and humans, obtained by sequencing single cells and clones derived from primary fibroblasts, which allows us to make the first direct comparison with germline mutation rates in these two species. The results indicate that the somatic mutation rate is almost two orders of magnitude higher than the germline mutation rate and that both mutation rates are significantly higher in mice than in humans. Our findings demonstrate both the privileged status of germline genome integrity and species-specific differences in genome maintenance.
Multicellular organism
Mutation Accumulation
Germline mosaicism
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Brief Summary: This study investigated the mutational background of somatic cells and rates of mutation in 29 distinct anatomical structures and compared these with the male germline from the same donor. The rate of mutation was lowest in spermatogonia.
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