Germline ATM variants predispose to melanoma: a joint analysis across the GenoMEL and MelaNostrum consortia.

PURPOSE: Ataxia-Telangiectasia Mutated (ATM) has been implicated in the risk of several cancers, but establishing a causal relationship is often challenging. Although ATM single-nucleotide polymorphisms have been linked to melanoma, few functional alleles have been identified. Therefore, ATM impact on melanoma predisposition is unclear. METHODS: From 22 American, Australian, and European sites, we collected 2,104 familial, multiple primary (MPM), and sporadic melanoma cases who underwent ATM genotyping via panel, exome, or genome sequencing, and compared the allele frequency (AF) of selected ATM variants classified as loss-of-function (LOF) and variants of uncertain significance (VUS) between this cohort and the gnomAD non-Finnish European (NFE) data set. RESULTS: LOF variants were more represented in our study cohort than in gnomAD NFE, both in all (AF = 0.005 and 0.002, OR = 2.6, 95% CI = 1.56-4.11, p < 0.01), and familial + MPM cases (AF = 0.0054 and 0.002, OR = 2.97, p < 0.01). Similarly, VUS were enriched in all (AF = 0.046 and 0.033, OR = 1.41, 95% CI = 1.6-5.09, p < 0.01) and familial + MPM cases (AF = 0.053 and 0.033, OR = 1.63, p < 0.01). In a case-control comparison of two centers that provided 1,446 controls, LOF and VUS were enriched in familial + MPM cases (p = 0.027, p = 0.018). CONCLUSION: This study, describing the largest multicenter melanoma cohort investigated for ATM germline variants, supports the role of ATM as a melanoma predisposition gene, with LOF variants suggesting a moderate-risk.

TET2 binds the androgen receptor and loss is associated with prostate cancer.

Genetic alterations associated with prostate cancer (PCa) may be identified by sequencing metastatic tumour genomes to identify molecular markers at this lethal stage of disease. Previously, we characterized somatic alterations in metastatic tumours in the methylcytosine dioxygenase ten-eleven translocation 2 (TET2), which is altered in 5-15% of myeloid, kidney, colon and PCas. Genome-wide association studies previously identified non-coding risk variants associated with PCa and melanoma. We perform fine-mapping of PCa risk across TET2 using genotypes from the PEGASUS case-control cohort and identify six new risk variants in introns 1 and 2. Oligonucleotides containing two risk variants are bound by the transcription factor octamer-binding protein 1 (Oct1/POU2F1) and TET2 and Oct1 expression are positively correlated in prostate tumours. TET2 is expressed in normal prostate tissue and reduced in a subset of tumours from the Cancer Genome Atlas (TCGA). Small interfering RNA-mediated TET2 knockdown (KD) increases LNCaP cell proliferation, migration and wound healing, verifying loss drives a cancer phenotype. Endogenous TET2 bound the androgen receptor (AR) and AR-coactivator proteins in LNCaP cell extracts, and TET2 KD increases prostate-specific antigen (KLK3/PSA) expression. Published data reveal TET2 binding sites and hydroxymethylcytosine proximal to KLK3. A gene co-expression network identified using TCGA prostate tumour RNA-sequencing identifies co-regulated cancer genes associated with 2-oxoglutarate (2-OG) and succinate metabolism, including TET2, lysine demethylase (KDM) KDM6A, BRCA1-associated BAP1, and citric acid cycle enzymes IDH1/2, SDHA/B, and FH. The co-expression signature is conserved across 31 TCGA cancers suggesting a putative role for TET2 as an energy sensor (of 2-OG) that modifies aspects of androgen-AR signalling. Decreased TET2 mRNA expression in TCGA PCa tumours is strongly associated with reduced patient survival, indicating reduced expression in tumours may be an informative biomarker of disease progression and perhaps metastatic disease.

Revisiting the cost of carnivory in mammals.

Predator-prey relationships play a key role in the evolution and ecology of carnivores. An understanding of predator-prey relationships and how this differs across species and environments provides information on how carnivorous strategies have evolved and how they may change in response to environmental change. We aim to determine how mammals overcame the challenges of living within the marine environment; specifically, how this altered predator-prey body mass relationships relative to terrestrial mammals. Using predator and prey mass data collected from the literature, we applied phylogenetic piecewise regressions to investigate the relationship between predator and prey size across carnivorous mammals (51 terrestrial and 56 marine mammals). We demonstrate that carnivorous mammals have four broad dietary groups: small marine carnivores (< 11 000 kg) and small terrestrial carnivores (< 11 kg) feed on prey less than 5 kg and 2 kg, respectively. On average, large marine carnivores (> 11 000 kg) feed on prey equal to 0.01% of the carnivore's body size, compared to 45% or greater in large terrestrial carnivores (> 11 kg). We propose that differences in prey availability, and the relative ease of processing large prey in the terrestrial environment and small prey in marine environment, have led to the evolution of these novel foraging behaviours. Our results provide important insights into the selection pressures that may have been faced by early marine mammals and ultimately led to the evolution of a range of feeding strategies and predatory behaviours.

Reliability of 24-h void frequency as an index of hydration status when euhydrated and hypohydrated.

BACKGROUND/OBJECTIVES: Void frequency (VF) is significantly correlated to hydration status, but it is unknown whether VF is reliable when an individual is repeatedly euhydrated (EU) or hypohydrated (HY). The purpose of this study was to test the reliability of VF when individuals were EU or HY on multiple occasions. SUBJECTS/METHODS: Fourteen males (age 22±2 years, mass 79.1±12.8 kg) completed three EU trials achieved with 24-h ad libitum fluid intake, and 14 males (age 22±2 years, mass 78.6±10.4 kg) completed three HY trials achieved with 24-h fluid restriction. Twenty-four hour urine was collected and analyzed for specific gravity (USG) and VF. Subjects voided at a 'normal urgency' (rated a '2' on a 0-4 perceptual scale) throughout each 24-h period. RESULTS: Twenty-four hour USG was greater and VF was lower when HY (1.026±0.003 and 5±2, respectively) versus EU (1.014±0.003 and 7±2; both P<0.05). Intra-class correlations for VF between the three trials at each hydration status were deemed acceptable (0.863 and 0.849 for EU and HY, respectively). Within-subject coefficients of variation for VF were 15±9 and 21±14% for the EU and HY trials. CONCLUSIONS: VF is a reliable index of 24-h hydration status when healthy young males are EU or HY and voiding at a consistent 'urgency'.

Effect of passive heat stress on arterial stiffness in smokers versus non-smokers.

In non-smokers, passive heat stress increases shear stress and vasodilation, decreasing arterial stiffness. Smokers, who reportedly have arterial dysfunction, may have similar improvements in arterial stiffness with passive heat stress. Therefore, we examined the effects of an acute bout of whole-body passive heat stress on arterial stiffness in smokers vs. non-smokers. Thirteen smokers (8.8 ± 5.5 [median = 6] cigarettes per day for > 4 years) and 13 non-smokers matched for age, mass, height, and exercise habits (27 ± 8 years; 78.8 ± 15.4 kg; 177.6 ± 6.7 cm) were passively heated to 1.5 °C core temperature (T C) increase. At baseline and each 0.5 °C T C increase, peripheral (pPWV) and central pulse wave velocity (cPWV) were measured via Doppler ultrasound. No differences existed between smokers and non-smokers for any variables (all p > .05), except cPWV slightly increased from baseline (526.7 ± 81.7 cm · s(-1)) to 1.5 °C ΔT C (579.7 ± 69.8 cm · s(-1); p < 0.005), suggesting heat stress acutely increased central arterial stiffness. pPWV did not change with heating (grand mean: baseline = 691.9 ± 92.9 cm · s(-1); 1.5 °C ΔT C = 691.9 ± 79.5 cm · s(-1); p > 0.05). Changes in cPWV and pPWV during heating correlated (p < 0.05) with baseline PWV in smokers (cPWV: r = -0.59; pPWV: r = -0.62) and non-smokers (cPWV: r = -0.45; pPWV: r = -0.77). Independent of smoking status, baseline stiffness appears to mediate the magnitude of heating-induced changes in arterial stiffness.

24-h Void number as an indicator of hydration status.

BACKGROUND/OBJECTIVES: Few user-friendly hydration assessment techniques exist for the general population to use on a daily basis. The present study evaluated void number over 24 h as a potential hydration assessment tool. SUBJECTS/METHODS: Male and female subjects collected urine for 24 h while adequately hydrated (n=44; 22 ± 4 years, 168 ± 16 cm, 73 ± 15 kg) or fluid restricted (n=43; 22 ± 3 years, 175 ± 10 cm, 81 ± 24 kg). As a control, participants were asked to void when feeling the 'first urge to void' on a commonly used urge scale and noted the volume of each void. For each sample, 24-h urine volume, osmolality (U(OSM)), specific gravity (U(SG)) and color were measured in the laboratory. RESULTS: As designed, the level of urge upon voiding was consistent throughout the study (2 ± 0; 'first urge to void'). Samples were classified by U(SG) as either euhydrated (U(SG)<1.020) or hypohydrated (U(SG) ⩾ 1.020). Grouping by U(OSM) did not change results. Euhydrated versus hypohydrated individuals had greater 24-h urine volume (1933 ± 864 versus 967 ± 306 ml, respectively) and lower urine color (2 ± 1 versus 5 ± 1), U(SG) (1.012 ± 0.004 versus 1.025 ± 0.004) and UOSM (457 ± 180 versus 874 ± 175 mOsm/kg H2O; all P<0.001). Euhydrated individuals voided more than hypohydrated individuals over the 24-h period (5 ± 2 versus 3 ± 1 voids; P<0.001). Additionally, void number inversely correlated with hydration status as identified by U(SG) (r=-0.50; P<0.05) and U(OSM) (r=-0.56; P<0.05). CONCLUSIONS: In conclusion, over 24 h, individuals with a higher void number were euhydrated (that is, had less concentrated hydration biomarkers) than those with a lower void number. Based on these data, void number might be utilized as a simple and feasible hydration assessment for the general public, as it utilizes no equipment or technical expertise.

Breast cancer risk after radiotherapy for heritable and non-heritable retinoblastoma: a US-UK study.

BACKGROUND: Retinoblastoma is a rare childhood eye cancer caused by germline or somatic mutations in the RB1 gene. Previous studies observed elevated breast cancer risk among retinoblastoma survivors. However, there has been no research on breast cancer risk in relation to radiation (primarily scatter radiation from the primary treatment) and genetic susceptibility of retinoblastoma survivors. METHODS: Two groups of retinoblastoma survivors from the US and UK were selected, and breast cancer risk analysed using a case-control methodology, nesting within the respective cohorts, matching on heritability (that is to say, having bilateral retinoblastoma or being unilateral cases with at least one relative with retinoblastoma), and using exact statistical methods. There were a total of 31 cases and 77 controls. RESULTS: Overall there was no significant variation of breast cancer risk with dose (P>0.5). However, there was a pronounced and significant (P=0.047) increase in the risk of breast cancer with increasing radiation dose for non-heritable retinoblastoma patients and a slight and borderline significant (P=0.072) decrease in risk of breast cancer with increasing radiation dose for heritable retinoblastoma patients, implying significant (P=0.024) heterogeneity in radiation risk between the heritable and non-heritable retinoblastoma groups; this was unaffected by the blindness status. There was no significant effect of any type of alkylating-agent chemotherapy on breast cancer risk (P>0.5). CONCLUSIONS: There is significant radiation-related risk of breast cancer for non-heritable retinoblastoma survivors but no excess risk for heritable retinoblastoma survivors, and no significant risk overall. However, these results are based on very small numbers of cases; therefore, they must be interpreted with caution.

Common genetic variants in the 9p21 region and their associations with multiple tumours.

BACKGROUND: The chromosome 9p21.3 region has been implicated in the pathogenesis of multiple cancers. METHODS: We systematically examined up to 203 tagging SNPs of 22 genes on 9p21.3 (19.9-32.8 Mb) in eight case-control studies: thyroid cancer, endometrial cancer (EC), renal cell carcinoma, colorectal cancer (CRC), colorectal adenoma (CA), oesophageal squamous cell carcinoma (ESCC), gastric cardia adenocarcinoma and osteosarcoma (OS). We used logistic regression to perform single SNP analyses for each study separately, adjusting for study-specific covariates. We combined SNP results across studies by fixed-effect meta-analyses and a newly developed subset-based statistical approach (ASSET). Gene-based P-values were obtained by the minP method using the Adaptive Rank Truncated Product program. We adjusted for multiple comparisons by Bonferroni correction. RESULTS: Rs3731239 in cyclin-dependent kinase inhibitors 2A (CDKN2A) was significantly associated with ESCC (P=7 × 10(-6)). The CDKN2A-ESCC association was further supported by gene-based analyses (Pgene=0.0001). In the meta-analyses by ASSET, four SNPs (rs3731239 in CDKN2A, rs615552 and rs573687 in CDKN2B and rs564398 in CDKN2BAS) showed significant associations with ESCC and EC (P<2.46 × 10(-4)). One SNP in MTAP (methylthioadenosine phosphorylase) (rs7023329) that was previously associated with melanoma and nevi in multiple genome-wide association studies was associated with CRC, CA and OS by ASSET (P=0.007). CONCLUSION: Our data indicate that genetic variants in CDKN2A, and possibly nearby genes, may be associated with ESCC and several other tumours, further highlighting the importance of 9p21.3 genetic variants in carcinogenesis.

Association of MC1R variants and host phenotypes with melanoma risk in CDKN2A mutation carriers: a GenoMEL study.

BACKGROUND: Carrying the cyclin-dependent kinase inhibitor 2A (CDKN2A) germline mutations is associated with a high risk for melanoma. Penetrance of CDKN2A mutations is modified by pigmentation characteristics, nevus phenotypes, and some variants of the melanocortin-1 receptor gene (MC1R), which is known to have a role in the pigmentation process. However, investigation of the associations of both MC1R variants and host phenotypes with melanoma risk has been limited. METHODS: We included 815 CDKN2A mutation carriers (473 affected, and 342 unaffected, with melanoma) from 186 families from 15 centers in Europe, North America, and Australia who participated in the Melanoma Genetics Consortium. In this family-based study, we assessed the associations of the four most frequent MC1R variants (V60L, V92M, R151C, and R160W) and the number of variants (1, ≥2 variants), alone or jointly with the host phenotypes (hair color, propensity to sunburn, and number of nevi), with melanoma risk in CDKN2A mutation carriers. These associations were estimated and tested using generalized estimating equations. All statistical tests were two-sided. RESULTS: Carrying any one of the four most frequent MC1R variants (V60L, V92M, R151C, R160W) in CDKN2A mutation carriers was associated with a statistically significantly increased risk for melanoma across all continents (1.24 × 10(-6) ≤ P ≤ .0007). A consistent pattern of increase in melanoma risk was also associated with increase in number of MC1R variants. The risk of melanoma associated with at least two MC1R variants was 2.6-fold higher than the risk associated with only one variant (odds ratio = 5.83 [95% confidence interval = 3.60 to 9.46] vs 2.25 [95% confidence interval = 1.44 to 3.52]; P(trend) = 1.86 × 10(-8)). The joint analysis of MC1R variants and host phenotypes showed statistically significant associations of melanoma risk, together with MC1R variants (.0001 ≤ P ≤ .04), hair color (.006 ≤ P ≤ .06), and number of nevi (6.9 × 10(-6) ≤ P ≤ .02). CONCLUSION: Results show that MC1R variants, hair color, and number of nevi were jointly associated with melanoma risk in CDKN2A mutation carriers. This joint association may have important consequences for risk assessments in familial settings.

Daytime napping: Effects on human direct associative and relational memory.

Sleep facilitates declarative memory processing. However, we know little about whether sleep plays a role in the processing of a fundamental feature of declarative memory, relational memory - the flexible representation of items not directly learned prior to sleep. Thirty-one healthy participants first learned at 12 pm two sets of face-object photograph pairs (direct associative memory), in which the objects in each pair were common to both lists, but paired with two different faces. Participants either were given approximately 90 min to have a NREM-only daytime nap (n=14) or an equivalent waking period (n=17). At 4:30 pm, participants who napped demonstrated significantly better retention of direct associative memory, as well as better performance on a surprise task assessing their relational memory, in which participants had to associate the two faces previously paired with the same object during learning. Particularly noteworthy, relational memory performance was correlated with the amount of NREM sleep during the nap, with only slow-wave sleep predicting relational memory performance. Sleep stage data did not correlate with direct associative memory retention. These results suggest an active role for sleep in facilitating multiple processes that are not limited to the mere strengthening of rote memories, but also the binding of items that were not directly learned together, reorganizing them for flexible use at a later time.

Adverse effects of trichothiodystrophy DNA repair and transcription gene disorder on human fetal development.

The effects of DNA repair and transcription gene abnormalities in human pre-natal life have never been studied. Trichothiodystrophy (TTD) is a rare (affected frequency of 10(-6)) recessive disorder caused by mutations in genes involved in nucleotide excision repair (NER) pathway and in transcription. Based on our novel clinical observations, we conducted a genetic epidemiologic study to investigate gestational outcomes associated with TTD. We compared pregnancies resulting in TTD-affected offspring (n = 24) with respect to abnormalities during their antenatal and neonatal periods to pregnancies resulting in their unaffected siblings (n = 18), accounting for correlation, and to population reference values. Significantly higher incidence of several severe gestational complications was noted in TTD-affected pregnancies. Small for gestational age (SGA) <10th percentile [Relative risk (RR ) = 9.3, 95% CI = 1.4-60.5, p = 0.02], SGA <3rd percentile (RR = 7.2, 95% CI = 1.1-48.1, p = 0.04), and neonatal intensive care unit (NICU) hospitalization (RR = 6.4, 95% CI = 1.4-29.5, p = 0.02) occurred more frequently among TTD-affected neonates compared with their unaffected siblings. Compared with reference values from general obstetrical population, pregnancies that resulted in TTD-affected infants were significantly more likely to be complicated by hemolysis, elevated liver enzymes and low platelets (HELLP) syndrome (RR = 35.7, 95% CI = 7.6-92.5, p = 0.0002), elevated mid-trimester maternal serum human chorionic gonadotropin (hCG) levels (RR = 14.3, 95% CI = 7.0-16.6, p < 0.0001), SGA <3rd percentile (RR = 13.9, 95% CI = 7.4-21.1, p < 0.0001), pre-term delivery (<32 weeks) (RR = 12.0, 95% CI = 4.9-21.6, p < 0.0001), pre-eclampsia (RR = 4.0, 95% CI = 1.6-7.4, p = 0.006), and decreased fetal movement (RR = 3.3, 95% CI = 1.6-5.2, p = 0.0018). Abnormal placental development is an underlying mechanism that may explain the constellation of observed complications in our study. Thus, we hypothesize that TTD DNA repair and transcription genes play an important role in normal human placental development.

CDKN2A mutations and melanoma risk in the Icelandic population.

BACKGROUND: Germline CDKN2A mutations have been observed in 20-40% of high risk, melanoma prone families; however, little is known about their prevalence in population based series of melanoma cases and controls. METHODS: We resequenced the CDKN2A gene, including the p14ARF variant and promoter regions, in approximately 703 registry ascertained melanoma cases and 691 population based controls from Iceland, a country in which the incidence of melanoma has increased rapidly. RESULTS: We identified a novel germline variant, G89D, that was strongly associated with increased melanoma risk and appeared to be an Icelandic founder mutation. The G89D variant was present in about 2% of Icelandic invasive cutaneous malignant melanoma cases. Relatives of affected G89D carriers were at significantly increased risk of melanoma, head and neck cancers, and pancreatic carcinoma compared to relatives of other melanoma patients. Nineteen other germline variants were identified, but none conferred an unequivocal risk of melanoma. CONCLUSIONS: This population based study of Icelandic melanoma cases and controls showed a frequency of disease related CDKN2A mutant alleles ranging from 0.7% to 1.0%, thus expanding our knowledge about the frequency of CDKN2A mutations in different populations. In contrast to North America and Australia where a broad spectrum of mutations was observed at a similar frequency, in Iceland, functional CDKN2A mutations consist of only one or two different variants. Additional genetic and/or environmental factors are likely critical for explaining the high incidence rates for melanoma in Iceland. This study adds to the geographic regions for which population based estimates of CDKN2A mutation frequencies are available.

Comprehensive analysis of CDKN2A (p16INK4A/p14ARF) and CDKN2B genes in 53 melanoma index cases considered to be at heightened risk of melanoma.

OBJECTIVE: Comprehensive analysis of the 9p21 locus including the CDKN2A, ARF, and CDKN2B genes in 53 individuals from melanoma index cases considered to be at heightened risk of melanoma. METHODS AND RESULTS: Using a combination of DNA sequencing, gene copy number by real time quantitative PCR, linkage analysis, and transcript analysis in haploid somatic cell hybrids, we found no evidence for germline alteration in either coding or non-coding domains of CDKN2A and CDKN2B. However, we identified a p14ARF exon 1beta missense germline mutation (G16D) in a melanoma-neural system tumour syndrome (CMM+NST) family and a 8474 bp germline deletion from 196 bp upstream of p14ARF exon 1beta initiation codon to 11233 bp upstream of exon 1alpha of p16(INK4A) in a family with five melanoma cases. For three out of 10 families with at least three melanoma cases, the disease gene was unlinked to the 9p21 region, while linkage analysis was not fully conclusive for seven families. CONCLUSIONS: These data reinforce the hypothesis that ARF is a melanoma susceptibility gene and suggest that germline deletions specifically affecting p14ARF may not be solely responsible for NST susceptibility. Predisposition to CMM+NST could either be due to complete disruption of the CDKN2A locus or be the result of more complex genetic inheritance. In addition, the absence of any genetic alteration in 50 melanoma prone families or patients suggests the presence of additional tumour suppressor genes possibly in the 9p21 region, and on other chromosomes.

Prospective risk of cancer in CDKN2A germline mutation carriers.

BACKGROUND: The CDKN2A gene is the major known high-risk melanoma susceptibility gene. Susceptibility to other cancers has also been suggested. However, most studies examining the risks of other cancers classified individuals according to the family's CDKN2A mutation rather than determining individual mutation status. For non-population-based studies, risks could also be biased because of cancer occurrence prior to family ascertainment. METHODS: We examined the risk of non-melanoma cancer in 117 mutation-positive and 136 mutation-negative members from 15 families that had at least two first degree relatives with melanoma and CDKN2A mutations restricting the analysis to the period after the families were ascertained (that is, the prospective period) and using individual mutation data. The families have been followed prospectively for 4-26 years starting in the 1970s. RESULTS: Overall, there was no significant association for mutation-negative subjects (Obs/Exp = 0.3, 95% confidence interval (CI) 0.0 to 1.2) although this group had only two observed cancers. In contrast, mutation-positive subjects had a significantly increased risk for all cancers combined (Obs/Exp = 12/5.5 = 2.2, 95% CI 1.1 to 3.8) primarily because of digestive system tumours, particularly pancreatic cancer. No other organ systems or individual tumour sites showed significantly increased risks. CONCLUSIONS: Differences in CDKN2A-non-melanoma cancer associations across studies may result from variation in genetic backgrounds, insufficient follow up, misclassification of mutation carriers, or the presence of other genetic and/or environmental risk factors in both CDKN2A mutation carriers and non-carriers. Larger sample sizes, prospective follow up, and individual mutation data will be required to understand these differences.

Rarity of CDK4 germline mutations in familial melanoma.

To date, two genes have been implicated in melanoma pathogenesis. The first, CDKN2A, is a tumour suppressor gene with germline mutations detected in 20% of melanoma-prone families. The second, CDK4, is an oncogene with co-segregating germline mutations detected in only three kindreds worldwide. We examined 16 American melanoma-prone families for mutations in all coding exons of CDK4 and screened additional members of two previously reported families with the Arg24Cys germline CDK4 mutation to evaluate the penetrance of the mutation. No new CDK4 mutations were identified. In the two Arg24Cys families, the penetrance was estimated to be 63%. Overall, 12 out of 12 invasive melanoma patients, none out of one in situ melanoma patient, five out of 13 dysplastic naevi patients, two out of 15 unaffected family members, and none out of 10 spouses carried the Arg24Cys mutation. Dysplastic naevi did not strongly co-segregate with the Arg24Cys mutation. Thus the phenotype observed in melanoma-prone CDK4 families appears to be more complex than just the CDK4 mutation. Both genetic and environmental factors are likely to contribute to the occurrence of melanoma and dysplastic naevi in these families. In summary, although CDK4 is a melanoma susceptibility gene, it plays a minor role in hereditary melanoma.

Combined risk factors for melanoma in a Mediterranean population.

A case-control study of non-familial melanoma including 183 incident cases and 179 controls was conducted in North-Eastern Italy to identify important risk factors and determine how combination of these affects risk in a Mediterranean population. Presence of dysplastic nevi (OR = 4.2, 95% CI = 2.4-7.4), low propensity to tan (OR = 2.4, 95% CI = 1.1-5.0), light eye (OR = 2.4, 95% CI = 1.1-5.2), and light skin colour (OR = 4.1, 95% CI = 1.4-12.1) were significantly associated with melanoma risk after adjustment for age, gender and pigmentation characteristics. A chart which identifies melanoma risk associated with combinations of these factors is presented; it can be used to identify subjects who would most benefit from preventive measures in Mediterranean populations. According to the combination of these factors, a relative risk range from 1 to 98.5 was found. Light skin colour, high number of sunburns with blistering, and low propensity to tan were significantly associated with melanoma thickness, possibly indicating that individuals with these characteristics underestimate their risk and seek attention when their lesion is already advanced.