Germline CDKN2A Mutations Research Articles

Prevalence and predictors of germline CDKN2A mutations for melanoma cases from Australia, Spain and the United Kingdom.

BackgroundMutations in the CDKN2A and CDK4 genes predispose to melanoma. From three case-control studies of cutaneous melanoma, we estimated the prevalence and predictors of these mutations for people from regions with widely differing latitudes and melanoma incidence.MethodsPopulation-based cases and controls from the United Kingdom (1586 cases, 499 controls) and Australia (596 early-onset cases, 476 controls), and a hospital-based series from Spain (747 cases, 109 controls), were screened for variants in all exons of CDKN2A and the p16INK4A binding domain of CDK4.ResultsThe prevalence of mutations for people with melanoma was similar across regions: 2.3%, 2.5% and 2.0% for Australia, Spain and the United Kingdom respectively. The strongest predictors of carrying a mutation were having multiple primaries (odds ratio (OR) = 5.4, 95% confidence interval (CI: 2.5, 11.6) for 2 primaries and OR = 32.4 (95% CI: 14.7, 71.2) for 3 or more compared with 1 primary only); and family history (OR = 3.8; 95% CI:1.89, 7.5) for 1 affected first- or second-degree relative and OR = 23.2 (95% CI: 11.3, 47.6) for 2 or more compared with no affected relatives). Only 1.1% of melanoma cases with neither a family history nor multiple primaries had mutations.ConclusionsThere is a low probability (<2%) of detecting a germline CDKN2A mutation in people with melanoma except for those with a strong family history of melanoma (≥2 affected relatives, 25%), three or more primary melanomas (29%), or more than one primary melanoma who also have other affected relatives (27%).Electronic supplementary materialThe online version of this article (doi:10.1186/1897-4287-12-20) contains supplementary material, which is available to authorized users.

A short acidic motif in ARF guards against mitochondrial dysfunction and melanoma susceptibility.

ARF is a small, highly basic protein that can be induced by oncogenic stimuli and exerts growth-inhibitory and tumour-suppressive activities through the activation of p53. Here we show that, in human melanocytes, ARF is cytoplasmic, constitutively expressed, and required for maintaining low steady-state levels of superoxide under conditions of mitochondrial dysfunction. This mitochondrial activity of ARF is independent of its known autophagic and p53-dependent functions, and involves the evolutionarily conserved acidic motif GHDDGQ, which exhibits weak homology to BCL-2 homology 3 (BH3) domains and mediates interaction with BCL-xL--an important regulator of mitochondrial redox homeostasis. Melanoma-predisposing CDKN2A germline mutations, which affect conserved glycine and aspartate residues within the GHDDGQ motif, impair the ability of ARF to control superoxide production and suppress growth of melanoma cells in vivo. These results reveal an important cell-protective function of ARF that links mitochondrial dysfunction and susceptibility to melanoma.

Genome-wide DNA methylation profile of leukocytes from melanoma patients with and without CDKN2A mutations

Melanoma is a highly aggressive cancer, accounting for up to 75% of skin cancer deaths. A small proportion of melanoma cases can be ascribed to the presence of highly penetrant germline mutations, and approximately 40% of hereditary melanoma cases are caused by CDKN2A mutations. The current study sought to investigate whether the presence of germline CDKN2A mutations or the occurrence of cutaneous melanoma would result in constitutive genome-wide DNA methylation changes. The leukocyte methylomes of two groups of melanoma patients (those with germline CDKN2A mutations and those without CDKN2A mutations) were analyzed together with the profile of a control group of individuals. A pattern of DNA hypomethylation was detected in the CDKN2A-negative patients relative to both CDKN2A-mutated patients and controls. Additionally, we delineated a panel of 90 CpG sites that were differentially methylated in CDKN2A-mutated patients relative to controls. Although we identified a possible constitutive epigenetic signature in CDKN2A-mutated patients, the occurrence of reported SNPs at the detected CpG sites complicated the data interpretation. Thus, further studies are required to elucidate the impact of these findings on melanoma predisposition and their possible effect on the penetrance of CDKN2A mutations.

CDKN2A mutations could influence the dermoscopic pattern of presentation of multiple primary melanoma: a clinical dermoscopic genetic study.

Patients who develop cutaneous melanoma are at increased risk of developing a second primary melanoma. There are many aetiological reasons by which the risk of a second melanoma increases. Among others, genetic factors may contribute to modulating this risk. The risk of identifying a CDKN2A germline mutation increases with the number of primary melanomas and with the presence of familial history of melanoma. Patients with melanoma are especially encouraged to have regular follow-up visits with their dermatologist to perform clinical and dermatoscopic examination. In particular, dermoscopy could be very useful in multiple primary melanoma (MPM) patients. To analyse the clinical and dermatoscopic features of multiple melanomas, focusing on those features that are more frequently found in the same patient to recognize them earlier and understand whether they appear with the similar peculiar dermatoscopic features, especially in CDKN2A carriers. Medical records of MPM patients were selected from a database including 1065 patients with histopathologically proven melanoma diagnosis, all treated at the dermatology clinic of the University of Florence from 2000 to 2013. Pictures of melanoma were independently and blindly administered to three dermatologist experts in dermoscopy to evaluate the presence or absence of ABCD criteria for each clinical image, and the main pattern for the dermoscopic images. The results were then analyzed and crossed to rate the clinical and dermoscopic features of MPM. Seventy five (7.0%) of 1065 patients included in our database were found to carry an MPM disease. Among them, we selected 12 (16%) patients with three or more MPMs. The presence of the CDKN2A melanoma susceptibility gene was observed in 4/12 (33.33%) patients; two patients presented the C500G and c.5+1delG polymorphisms in the CDKN2A gene. In CDKN2A carriers, each patient showed a similar and specific dermatoscopic pattern in their lesions. Even being aware of the limitations of this study, according to hereditary characters and their modes of transmissions, we could speculate that for each patient with a CDKN2A germline mutation, it is possible to find the same kind of dermoscopical pattern among their melanocytic tumours.

Germline CDKN2A mutations in Brazilian patients of hereditary cutaneous melanoma.

Approximately 10 % of all cutaneous melanoma cases occur in a familial context. The major susceptibility gene for familial melanoma is CDKN2A. In Latin America, genetic studies investigating melanoma predisposition are scarce. The aim of this work was to investigate germline CDKN2A point mutations and genomic rearrangements in a cohort of 59 Brazilian melanoma-prone patients. Screening of CDKN2A alterations was performed by sequencing and multiplex ligation probe amplification. Germline CDKN2A mutations affecting p16(INK4a) were detected in 8 unrelated probands (13.6 %), including 7 familial cases and one patient with multiple melanomas; 4 out of 8 mutation carriers met the criteria for familial melanoma and had multiple primary lesions. Although this study adds to the literature on melanoma susceptibility in Latin America, it is limited by the small size of the cohort. Our findings suggest that stringent inclusion criteria led to a substantially increased rate of CDKN2A mutation detection. This consideration should be taken into account when referring patients for genetic screening in a setting of limited budget, such as in developing countries.

High risk of tobacco-related cancers in CDKN2A mutation-positive melanoma families

BackgroundGermline mutations in the tumour suppressor gene CDKN2A occur in 5–20% of familial melanoma cases. A single founder mutation, p.Arg112dup, accounts for the majority of CDKN2A mutations in Swedish carriers....

Risk of tobacco-related cancers in CDKN2A mutation-positive melanoma families.

1513 Background: Germline mutations in the tumor suppressor gene CDKN2A occur in 5-20% of familial melanoma cases. A single founder mutation, p.Arg112dup, accounts for the majority of CDKN2A mutati...

Assessment of germline and somatic alterations in main candidate genes among patients with multiple primary melanoma

Background A series of patients with multiple primary melanoma (MPM) has been studied for assessing frequency and distribution of alterations in candidate genes involved in susceptibility (CDKN2A) and pathogenesis (BRAF, cKIT, CyclinD1) of such a disease. Methods Two hundred and twenty-seven genomic DNA samples from paired synchronous and/or asynchronous tumour tissues of 106 MPM patients (92 cases with two, 13 with three, and 1 with four primary melanomas) were screened for somatic mutations in BRAF gene and FISHbased amplifications in cKIT and CyclynD1 genes. For 87 (84%) MPM patients from our series, peripheral blood was available and germline DNAs was analyzed for mutations in p16CDKN2A and p14CDKN2A genes. All mutation analyses we performed by direct automated DNA sequencing. Family history for melanoma was defined according to standardized criteria. Results At somatic level, BRAF mutations were identified in 108/ 227 (48%) primary melanoma tissues, whereas amplification of cKIT and CyclinD1 genes was observed in 8/208 (4%) and 28/204 (14%) analyzed tissue samples, respectively. Considering all types of genetic events, paired samples presented a poorly consistent distribution of somatic alterations in same patients [55/106 (52%) discrepant cases). Among them, 35/106 (33%) patients presented discrepant MPM lesions according to the BRAF mutation status. Among the 87 MPM patients whose germline DNA was available, 8 (9%) of them showed different CDKN2A germline mutations: 7 in p16CDKN2A and 1 in p14CDKN2A. Assessment of family history for melanoma revealed that 13/87 (15%) patients presented at least one additional family member affected; a total of ≥ 3 melanomas in family was observed in 20/87 (23%) cases of our series. The CDKN2A germline mutations were found significantly more frequent in patients with familial history of melanoma (7/13; 54%) compared with patients without (1/74; 1.4%) (P<0.001); analogously, CDKN2A mutations were observed in 1/67 (1.5%) and 7/20 (35%) patients with 2 and 3 or more melanomas in family, respectively (P<0.001). Conclusions The low consistency in mutation patterns at somatic level among MPM lesions from the same patients provide further evidence that melanomagenesis is heterogeneous and molecularly different cell types may participate to the development of multiple melanomas. Our findings on germline DNA indicate that occurrence of at least 3 melanomas (in patients or families) or familial recurrence of melanoma may represent strong indicators to address patients to CDKN2A mutation screening.

Clinical and Histopathological Characteristics between Familial and Sporadic Melanoma in Barcelona, Spain.

BackgroundAbout 6 to 14% of melanoma cases occur in a familial setting. Germline mutations in CDKN2A are detected in 20 to 40% of melanoma families.ObjectiveTo characterise the clinical and histopathological characteristics of familial melanoma thus providing more information to clinicians and contribute to the understanding of the genetic-environment interplay in the pathogenesis of melanoma.MethodsClinical, histological and immunohistochemical characteristics of 62 familial melanomas were compared with 127 sporadic melanomas.Resultsvariables associated with familial melanoma were earlier age at diagnosis (OR 1.036; 95% CI 1.017–1.055), lower Breslow thickness (OR 1.288; 95% CI 1.013–1.683) and in situ melanomas (OR 2.645; 95% CI 1.211–5.778). Variables associated with CDKN2A mutation carriers were earlier age at diagnosis (OR 1.060; 95% CI 1.016–1.105), in situ melanomas (OR 6.961; 95% CI 1.895–25.567), the presence of multiple melanomas (OR 8.920; 95% CI 2.399–33.166) and the immunopositivity of the tumours for cytoplasmic survivin (OR 9.072; 95% CI 1.025–85.010).ConclusionsFamilial melanoma was significantly associated with the earlier age of onset, lower Breslow thickness and with a higher number of in situ melanomas; and also carriers of CDKN2A mutations were associated with a higher risk of multiple melanomas and cytoplasmic survivin immunostaining.

Surveillance of Second-Degree Relatives from Melanoma Families with a CDKN2A Germline Mutation

Lifetime melanoma risk of mutation carriers from families with a germline mutation in the CDKN2A gene is estimated to be 67%. The necessity to include family members in a melanoma surveillance program is widely endorsed, but there is no consensus on which family members should be invited. In a retrospective follow-up study, we investigated the yield of surveillance of first- and second-degree relatives of melanoma and pancreatic cancer patients from 21 families with the "p16-Leiden" CDKN2A mutation. Melanoma incidence rates were compared with the general population. Three-hundred and fifty-four first-degree relatives and 391 second-degree relatives were included. Forty-five first-degree relatives and 11 second-degree relatives were diagnosed with melanoma. Most (72%) of second-degree relatives diagnosed with melanoma had become a first-degree relative before diagnosis, due to the occurrence of a melanoma in a parent or sibling. Overall, melanoma incidence rate was 2.1 per 1,000 person years [95% confidence interval (CI), 1.2-3.8] in family members still being second-degree relatives at diagnosis, compared with 9.9 per 1,000 person years (95% CI, 7.4-13.3) in first-degree relatives. The standardized morbidity ratio for melanoma of second-degree relatives compared with the general population was 12.9 (95% CI, 7.2-23.4). Second-degree relatives from families with the p16-Leiden mutation in CDKN2A have a considerably increased melanoma risk compared with the general population. This study provides justification for the surveillance of second-degree relatives from families with a CDKN2A germline mutation.

Genital and anorectal mucosal melanoma is associated with cutaneous melanoma in patients and in families

Genital and anorectal mucosal melanomas (GAMMs) are rare compared with cutaneous melanoma (CM). Many epidemiological and genetic studies have been carried out on CM. In contrast, the genetic and environmental risk factors for GAMM have been poorly documented up to now. To compare the distribution of pigmentation and naevus phenotypes, sun exposure and family history of melanoma between patients with GAMM and CM. We compared two series of patients, 81 with GAMM and 293 with CM. Patients with GAMM and CM did not show significant differences for phenotypic risk factors. However, patients with GAMM tended to display red hair (11% vs. 5·5%, P = 0·08) and a poor tanning ability (22% vs. 13·3%, P = 0·06) at a higher frequency than patients with CM. A family history of melanoma was significantly more frequent with GAMM than with CM (18% vs. 7·5%, P = 0·005). Apart from the GAMM index case, affected relatives had CM except in one family. The frequency of multiple primary melanomas (MPMs) was similar in the GAMM and CM series (6% vs. 5·3%, P = 0·43). All patients with GAMM and MPM had only one GAMM primary, while the other primary was cutaneous. No CDKN2A germline mutation was detected in patients with GAMM. This study shows that GAMM and CM may occur in the same patient, and GAMM may develop in a familial setting. The association of both GAMM and CM in patients and families suggests shared genetic factors by these two types of melanoma.

Rapidly growing pancreatic ductal adenocarcinoma in a patient with metastatic melanoma and harbouring CDKN2A germline mutation

Departments of aGeneral Dermatology and Skin Cancer bRadiology, Ambroise Paré Hospital, APHP and University of Versailles SQY, Boulogne-Billancourt, France Correspondence to Sophie Lipowicz, MD, Department of General Dermatology and Skin Cancer, Ambroise Paré Hospital, APHP and University of Versailles SQY, 9 Avenue du Général-de-Gaulle, 92104 Boulogne-Billancourt, Cedex, France Tel: +33 1 49 09 56 73; fax: +33 1 49 09 56 85; e-mail: [email protected] Received September 4, 2012 Accepted March 10, 2013

Melanoma prone families with CDK4 germline mutation: phenotypic profile and associations with MC1R variants

BackgroundCDKN2A and CDK4 are high risk susceptibility genes for cutaneous malignant melanoma. Melanoma families with CDKN2A germline mutations have been extensively characterised, whereas CDK4 families are rare and lack a...

LINE-1 methylation in peripheral blood and the risk of melanoma in melanoma-prone families with and without CDKN2A mutations

Cutaneous malignant melanoma (CMM) is an etiologically heterogenous disease with genetic, environmental (sun exposure), and host (pigmentation/nevi) factors and their interactions contributing to risk. Recently, epigenetic changes involving reduced levels of global DNA methylation in blood have been associated with genomic instability and cancer risk. We thus examined whether global methylation was associated with CMM risk in individuals from melanoma-prone families with and without CDKN2A germline mutations. We measured global DNA methylation using bisulfite pyrosequencing at four CpG sites of the long interspersed nucleotide element-1 (LINE-1) sequences in peripheral blood mononuclear cells (PBMCs) from individuals in 64 melanoma-prone families including 114 CMM cases (45 CDKN2A-positive and 69 CDKN2A-negative) and 121 unaffected individuals (31 CDKN2A-positive and 90 CDKN2A-negative). We used unconditional logistic regression to evaluate the association between CMM status and LINE-1 methylation levels, adjusting for age at blood draw and accounting for familial correlation in the variance. We found that male sex was significantly associated with higher overall LINE-1 methylation (P=0.0001). However, the overall and site-specific levels of LINE-1 methylation did not vary significantly by CMM status (overall odds ratio: 1.57, 95% confidence interval: 0.84-2.95, P=0.16; comparing lowest to highest or reference methylation group). Similar results were obtained when CDKN2A-positive and CDKN2A-negative families were analyzed separately. Our findings did not support a significant association between constitutional LINE-1 methylation in PBMCs and risk of CMM in melanoma-prone families with or without CDKN2A mutations.

1118PD - Multiple Primary Melanomas from Same Patients Present Discrepant Somatic Alterations in Main Candidate Genes

Background A series of patients with multiple primary melanoma (MPM) were screened for the involvement of the key-regulator genes in susceptibility (CDKN2A) and pathogenesis (BRAF, cKIT, CyclinD1) of such a disease. Methods Genomic DNA from peripheral blood of 63 MPM patients (54 cases with two primary melanomas, 8 with three, and 1 with four) were screened for germline mutations in p16 CDKN2A and p14 CDKN2A genes by automated DNA sequencing. Melanoma families were identified according to standardized criteria: 9 (14%) patients were classified as familial cases. Paired synchronous and/or asynchronous MPM tissues (N = 100) from same patients (N = 46) were analyzed for somatic mutations in BRAF gene and FISH-based amplifications in cKIT and CyclynD1 genes. Results Overall, 6 (10%) different CDKN2A germline mutations were identified: 5 inp1 6 CDKN2A and 1 inp 14 CDKN2A . The age of onset was significantly lower and the number of primary melanomas higher in patients with mutations. CDKN2A mutations were significantly more frequent in patients with familial history of melanoma (5/9; 56%) compared with patients without (1/54; 2%) (P < 0.001), and in patients with more than two melanomas (3/9; 33%) compared with patients with only two melanomas (3/54; 6%) (P = 0.012). The debated A148T polymorphism was found at low level (2/54; 4%) in our series. Regarding genetic alterations at somatic level, BRAF mutations were identified in 36/100 (36%) primary melanoma tissues, whereas amplification of cKIT and CyclinD1 genes was observed in 2/88 (2%) and 10/88 (11%) analyzed tissue samples, respectively. Considering all types of genetic events, paired samples presented a poorly consistent distribution of somatic alterations in same patients (52% consistency). Conclusions Coexistence of MPM and familial recurrence of melanoma as well as the presence of more than two melanomas seem to be strong indications to address patients to CDKN2A mutational screening. The low consistency in genetic patterns of primary tumors from the same patients provide additional evidence that pathogenetic mechanisms of melanomagenesis are heterogeneous and molecularly different cell types may be generated in multiple primary melanoma.

Assessment of germ-line and somatic alterations in main candidate genes among patients with multiple primary melanoma.

8581 Background: We have studied a series of patients with multiple primary melanoma (MPM) for the involvement of the key-regulator genes in susceptibility (CDKN2A) and pathogenesis (BRAF, cKIT, CyclinD1) of such a disease. Methods: Genomic DNA from peripheral blood of 63 MPM patients (54 cases with two primary melanomas, 8 with three, and 1 with four) were screened for germline mutations in p16CDKN2A and p14CDKN2A genes by automated DNA sequencing. Melanoma families were identified according to standardized criteria: 9 (14%) patients were classified as familial cases. Paired synchronous and/or asynchronous MPM tissues (N=100) from same patients (N=46) were analyzed for somatic mutations in BRAF gene and FISH-based amplifications in cKIT and CyclynD1 genes. Results: Overall, 6 (10%) different CDKN2A germline mutations were identified: 5 in p16CDKN2A and 1 in p14CDKN2A. The age of onset was significantly lower and the number of primary melanomas higher in patients with mutations. CDKN2A mutations were significantly more frequent in patients with familial history of melanoma (5/9; 56%) compared with patients without (1/54; 2%) (P&lt;0.001), and in patients with more than two melanomas (3/9; 33%) compared with patients with only two melanomas (3/54; 6%) (P=0.012). The debated A148T polymorphism was found at low level (2/54; 4%) in our series. Regarding genetic alterations at somatic level, BRAF mutations were identified in 36/100 (36%) primary melanoma tissues, whereas amplification of cKIT and CyclinD1 genes was observed in 2/88 (2%) and 10/88 (11%) analyzed tissue samples, respectively. Considering all types of genetic events, paired samples presented a poorly consistent distribution of somatic alterations in same patients (52% consistency). Conclusions: Coexistence of MPM and familial recurrence of melanoma as well as the presence of more than two melanomas seem to be strong indications to address patients to CDKN2A mutational screening. The low consistency in genetic patterns of primary tumors from the same patients provide additional evidence that pathogenetic mechanisms of melanomagenesis are heterogeneous and molecularly different cell types may be generated in multiple primary melanoma.

Constitutive mitochondrial DNA copy number in peripheral blood of melanoma families with and without CDKN2A mutations.

Quantitative changes in mitochondrial DNA (mtDNA) have been associated with the risk of a number of human cancers; however, the relationship between constitutive mtDNA copy number in blood and the risk of familial cutaneous malignant melanoma (CMM) has not been reported. We measured mtDNA copy number using quantitative PCR in blood-derived DNA from 136 CMM cases and 302 controls in 53 melanoma-prone families (23 segregating CDKN2A germline mutations). MtDNA copy number did not vary by age, sex, pigmentation characteristics, or CMM status. However, germline CDKN2A mutation carriers had significantly higher mean mtDNA copy number compared to non-carriers, particularly among CMM cases (geometric mean mtDNA copy number of 144 and 111 for carrier versus non-carrier, respectively; P= 0.02). When adjusting for age, sex, and familial correlation, having increasing mtDNA copy number was significantly associated with CDKN2A mutation status among CMM cases (OR=1.47, Ptrend=0.024). In particular, individuals with specific CDKN2A mutations with the potential to inactivate or reduce the level of the p16-INK4 reactive oxygen species (ROS) protective function had significantly increased mtDNA copy number levels (P=0.035). Future research in prospective studies is required to validate these findings and to further investigate mtDNA copy number in both blood and melanoma tissues in relation to CMM risk and CDKN2A mutation status.

Novel germline CDKN2A mutation associated with head and neck squamous cell carcinomas and melanomas

The ability to identify individuals at increased risk of cancer is of immediate clinical relevance. Germline mutations in the CDKN2A locus, encoding the key tumor suppressor proteins p16/INK4A and p14/ARF, are frequently present in kindreds with hereditary cutaneous melanoma but have seldom been reported in families with genetic susceptibility to head and neck squamous cell carcinomas (HNSCC). We report the pedigree of a patient with an unusually high incidence of HNSCC and melanomas. CDKN2A mutation analysis was performed with standard capillary sequencing and multiplex ligation-dependent probe amplification. A previously unreported germline CDKN2A mutation affecting only the p16/INK4A open reading frame, c.106delG (p.Ala36ArgfsX17), was detected in the proband. This mutation causes a premature termination codon. Our report emphasizes the need to consider germinal CDKN2A mutations in the differential diagnosis of familial HNSCC and the importance of awareness of these tumors in carriers of CDKN2A mutations.

Comprehensive mutational analysis of CDKN2A and CDK4 in Greek patients with cutaneous melanoma.

The penetrance of CDKN2A mutations is subject to geographical and latitudinal variation and is presumably dictated by ultraviolet radiation exposure and possibly other co-inherited genetic factors. The frequency of mutations increases with the number of family members affected and the number of primary tumours, and also fluctuates with geography. To date, little is known about the prevalence of CDKN2A mutations in patients with melanoma from Greece. To characterize the frequency of CDKN2A and CDK4 mutations in a hospital-based population of Greek patients with melanoma. Three hundred and four consecutive single primary melanoma (SPM), nine familial melanoma (FM) and seven multiple primary melanoma cases (MPM) were assessed for sequence variants in exons 1α, 1β and 2 of CDKN2A and exon 2 of CDK4. Germline CDKN2A mutations were detected in 10 of 304 SPM (3·3%), in four of seven MPM (57%) and in two of nine FM (22%) cases. The most common mutation was a Northern European allele (p16 p.R24P) detected in eight individuals. Five previously unreported CDKN2A variants were also identified: -34G>C, c.41_43delins20bp, c.301G>C (p.G101R), c.301G>A (p.G101E) and c.296_297insGACC. We also describe the first report of a CDK4 p.R24H substitution in a Greek family. The Greek population appears to harbour a higher prevalence of the CDKN2A mutation than other reported cohorts. This supports the notion that genetic susceptibility may play a stronger influence in a country with a relatively low incidence of melanoma. Furthermore, the identification of Northern European alleles suggests that gene migration may be responsible, in part, for the observed cases in Greece.

Abstract A84: Reduced LINE-1 methylation in peripheral blood mononuclear cells is associated with risk of melanoma in families segregating CDKN2A germline mutations

Abstract Background: CDKN2A is a major susceptibility gene for familial melanoma, but its incomplete penetrance suggests that other factors may modify the effect of this gene in melanoma-prone families. Epigenetic changes involving reduced levels of global DNA methylation in blood have been associated with genomic instability and cancer risk. In addition, genetic background has the potential to influence epigenetic processes through cis- or trans-regulatory variation. Therefore, we hypothesize that the CDKN2A carrier mutation state may be an important factor influencing global DNA methylation in predisposed individuals and that aberrant methylation may be an indicator of modified risk in individuals from melanoma-prone families segregating CDKN2A germline mutations. Methods: We measured the overall level of genomic DNA methylation using bisulfite pyrosequencing at four CpG sites (CpG1, CpG2, CpG3 and CpG4) of the Long Interspersed Nucleotide Element-1 (LINE-1) sequences in peripheral blood mononuclear cells (PBMCs) from 42 CDKN2A mutation positive cases (20 males and 22 females) and 73 controls (29 CDKN2A carrier controls [9 males and 20 females] and 44 non-carrier controls [19 males and 25 females]) in 26 families with CDKN2A mutations. LINE-1 methylation was reported as the average (of all 4 CpGs) and as site-specific percentage methylation. Odds' ratios (OR) and P-values were obtained by comparing melanoma cases to controls using unconditional logistic regression with adjustment for family correlation (using the generalized estimating equations [GEE] approach), age, sex, number of moles and with melanoma status as the outcome variable. Percentage LINE-1 methylation was categorized using tertiles with the highest tertile as the reference. Results: Male gender was significantly associated with higher average LINE-1 methylation (P=&amp;lt;0.001) and methylation at all CpG sites (P=&amp;lt;0.03). In contrast, age, solar injury, dysplastic nevi (DN) and number of moles were not associated with average LINE-1 methylation. Although mean levels of LINE-1 methylation were similar in melanoma cases, carrier controls, and non-carrier controls, after adjusting for family correlation, age, sex and number of moles, low average LINE-1 methylation (tertile 1) was significantly associated with increased risk of melanoma (OR=3.87, 95% confidence interval [CI]: 1.11–13.5, P=0.033, compared to all controls; OR=1.83, 95% CI: 1.09–11.7, P=0.035, compared to carrier controls; and OR=3.43, 95% CI: 1.17–10.0, P= 0.024, compared to non-carrier controls). This association was observed for most CpG sites with the most significant association seen for site 3 and risk of melanoma compared to all controls (OR=5.18, 95% CI: 1.54–17.4, P=0.008). We observed no significant association between average or CpG site-specific LINE-1 methylation and CDKN2A mutation status among controls although the comparison was limited by small sample size. Conclusions: Melanoma cases from families segregating CDKN2A germline mutations have significantly lower LINE-1 global methylation in their PBMCs compared to controls. Changes in LINE-1 methylation in PBMCs from CDKN2A mutation positive cases may reflect a potential genetic background influence on global DNA methylation in these individuals. Further studies are needed to clarify these preliminary observations. Citation Information: Cancer Prev Res 2011;4(10 Suppl):A84.