Extracellular Vesicles and Their miRNA Content in Amniotic and Tracheal Fluids of Fetuses with Severe Congenital Diaphragmatic Hernia Undergoing Fetal Intervention.

Infants with congenital diaphragmatic hernia (CDH) are at high risk of postnatal mortality due to lung hypoplasia and arterial pulmonary hypertension. In severe cases, prenatal intervention by fetal endoscopic tracheal occlusion (FETO) can improve survival by accelerating lung growth. However, postnatal mortality remains in the range of about 50% despite fetal treatment, and there is currently no clear explanation for this different clinical response to FETO. We evaluated the concentration of extracellular vesicles (EVs) and associated microRNA expression in amniotic and tracheal fluids of fetuses with CDH undergoing FETO, and we examined the association between molecular findings and postnatal survival. We observed a higher count of EVs in the amniotic fluid of non-survivors and in the tracheal fluid sampled in utero at the time of reversal of tracheal occlusion, suggesting a pro-inflammatory lung reactivity that is already established in utero and that could be associated with a worse postnatal clinical course. In addition, we observed differential regulation of four EV-enclosed miRNAs (miR-379-5p, miR-889-3p; miR-223-3p; miR-503-5p) in relation to postnatal survival, with target genes possibly involved in altered lung development. Future research should investigate molecular therapeutic agents targeting differentially regulated miRNAs to normalize their expression and potentially improve clinical outcomes.

The Independent Role of Body Mass Index (BMI) and Severity of Depressive Symptoms on Biological Changes of Women Affected by Overweight/Obesity.

Background: Both obesity and depression are medical conditions associated with severe disability and biological abnormalities. Our aim was to study associations between Body Mass Index (BMI), depression and biological changes in women affected by overweight or obesity. Methods: Depressive symptoms were evaluated by the Beck Depression Inventory II (BDI-II) questionnaire in 200 women affected by overweight/obesity (mean age of the sample 52.7 ± 12.9 years, BMI 33.8 ± 5.5 kg/m2). A blood sample was obtained for evaluation of biochemical (oxytocin and vitamin D), inflammatory and epigenetic (methylation of clock genes) parameters. Multivariable linear regression models were used to study the association between BMI or severity of depressive symptoms (BDI-II scores) with different biomarkers. Results: BMI was found to be associated with severity of depressive symptoms (p = 0.050). Severity of obesity resulted to be associated with lower plasma levels of oxytocin (p = 0.053), vitamin D deficiency (p = 0.006) and higher plasma levels of IFN-γ (p = 0.004), IL-6 (p = 0.013), IL-7 (p = 0.013), TNF-alpha (p = 0.036) and chemokine ligand 3 (CCL3) (p = 0.013, R2 = 0.03). Severity of depression was significantly associated with more methylation of clock genes CRY1 (p = 0.034, R2 = 0.16) and CRY2 (p = 0.019, R2 = 0.47). More severe depression together with higher levels of IL-8 strongly predicted lower methylation of CLOCK gene (p = 0.009); Conclusions: Different biological abnormalities have been found to be independently associated with BMI and severity of depressive symptoms in women affected by overweight/obesity. The complex interplay between overweight, depression and biological changes will have to be better clarified by future studies.

Is There an Association Between Oxytocin Levels in Plasma and Pregnant Women's Mental Health?

BACKGROUND: Mood and anxiety disorders are prevalent in women during peripartum. AIMS: Purpose of the present article was to study the relationship between oxytocin (OT) plasma levels and affective symptoms in women during the third trimester of pregnancy. METHODS: Thirty-four pregnant women (13 with an affective disorder, 9 with preeclampsia, and 12 controls) were evaluated through the Edinburgh Postnatal Depression Scale (EPDS), the State/Trait Anxiety Inventory Form Y (STAI-Y), and the Prenatal Attachment Inventory (PAI). A blood sample was collected from all participants, and OT plasma levels have been compared between diagnostic groups. The total sample has been divided into two groups, according to OT median plasma levels, and compared using (a) χ2 tests for qualitative variables and (b) a multivariate analysis of covariance for quantitative ones. RESULTS: No statistically significant difference was found among the diagnostic groups in terms of OT plasma levels (F = 0.49, p = .62). Women with lower OT plasma levels, independent from the presence of preeclampsia or an affective disorder, showed worse EPDS and STAI-S total scores than individuals with higher hormone levels (F = 5.93, p = .02 and F = 7.57, p = .01, respectively). CONCLUSIONS: OT may play a role in the etiology of anxious/depressive symptoms during perinatal period independent from a medical or psychiatric diagnosis. This result has a clear effect on the quality of the relationship of patients with mental health professionals, including nurses, and higher levels of this hormone, in the light of its anxiolytic and antidepressive effect, may make easier medical and nursing procedures.

Blood-derived extracellular vesicles isolated from healthy donors exposed to air pollution modulate in vitro endothelial cells behavior.

The release of Extracellular Vesicles (EVs) into the bloodstream is positively associated with Particulate Matter (PM) exposure, which is involved in endothelial dysfunction and related to increased risk of cardiovascular disease. Obesity modifies the effects of PM exposure on heart rate variability and markers of inflammation, oxidative stress, and acute phase response. We isolated and characterized plasmatic EVs from six healthy donors and confirmed a positive association with PM exposure. We stratified for Body Mass Index (BMI) and observed an increased release of CD61+ (platelets) and CD105+ (endothelium) derived-EVs after high PM level exposure in Normal Weight subjects (NW) and no significant variations in Overweight subjects (OW). We then investigated the ability to activate endothelial primary cells by plasmatic EVs after both high and low PM exposure. NW-high-PM EVs showed an increased endothelial activation, measured as CD105+/CD62e+ (activated endothelium) EVs ratio. On the contrary, cells treated with OW-high-PM EVs showed reduced endothelial activation. These results suggest the ability of NW plasmatic EVs to communicate to endothelial cells and promote the crosstalk between activated endothelium and peripheral cells. However, this capacity was lost in OW subjects. Our findings contribute to elucidate the role of EVs in endothelial activation after PM exposure.

Nasal Microbiota Modifies the Effects of Particulate Air Pollution on Plasma Extracellular Vesicles.

Air pollution exposure has been linked to modifications of both extracellular vesicle (EV) concentration and nasal microbiota structure (NMB), which might act as the respiratory health gatekeeper. This study aimed to assess whether an unbalanced NMB could modify the effect of particulate matter (PM) exposure on plasmatic EV levels. Due to two different NMB taxonomical profiles characterized by a widely different relative abundance of the Moraxella genus, the enrolled population was stratified into Mor- (balanced NMB) and Mor+ (unbalanced NMB) groups (Moraxella genus's cut-off ≤25% and >25%, respectively). EV features were assessed by nanoparticle tracking analysis (NTA) and flow-cytometry (FC). Multivariable analyses were applied on EV outcomes to evaluate a possible association between PM10 and PM2.5 and plasmatic EV levels. The Mor- group revealed positive associations between PM levels and plasmatic CD105+ EVs (GMR = 4.39 p = 0.02) as for total EV count (GMR = 1.92 p = 0.02). Conversely, the Mor+ group showed a negative association between exposure and EV outcomes (CD66+ GMR = 0.004 p = 0.01; EpCAM+ GMR = 0.005 p = 0.01). Our findings provide an insight regarding how a balanced NMB may help to counteract PM exposure effects in terms of plasmatic EV concentration. Further research is necessary to understand the relationship between the host and the NMB to disentangle the mechanism exerted by inhaled pollutants in modulating EVs and NMB.

Effects of an acute bout of exercise on circulating extracellular vesicles: tissue-, sex-, and BMI-related differences.

BACKGROUND: Exercise is recognized to evoke multisystemic adaptations that, particularly in obese subjects, reduce body weight, improve glucometabolic control, counteract sarcopenia, and lower the risk of cardiometabolic diseases. Understanding the molecular and cellular mechanisms of exercise-induced benefits is of great interest due to the therapeutic implications against obesity. OBJECTIVES AND METHODS: The aim of the present study was to evaluate time-related changes in size distribution and cell origin of extracellular vesicles (EVs) in obese and normal-weight subjects who underwent a moderate-intensity exercise on a treadmill (at 60% of their VO2max). Blood samples were drawn before, immediately at the end of the exercise and during the postexercise recovery period (3 and 24 h). Circulating EVs were analyzed by a nanoparticle tracking analysis and flow cytometry after labeling with the following cell-specific markers: CD14 (monocyte/macrophage), CD61 (platelet), CD62E (activated endothelium), CD105 (total endothelium), SCGA (skeletal muscle), and FABP (adipose tissue). RESULTS: In all subjects, acute exercise reduced the release of total (i.e., 30-700 nm) EVs in circulation, predominantly EVs in the microvesicle size range (i.e., 130-700 nm EVs). The postexercise release of microvesicles was higher in normal-weight than obese subjects; after exercise, circulating levels of exosomes (i.e., 30-130 nm EVs) and microvesicles were, respectively, lower and higher in females than males. In all experimental subgroups (males vs. females and obese vs. normal-weight subjects), acute exercise reduced and increased, respectively, CD61 + and SCGA + EVs, being the effect on CD61 + EVs prolonged up to 24 h after the end of the test with subjects in resting conditions. Total EVs, exosomes, and CD61 + EVs were associated with HOMA-IR. CONCLUSIONS: Though preliminary, the results of the present study show that a single bout of acute exercise modulates the release of EVs in circulation, which are tissue-, sex-, and BMI specific, suggesting that the exercise-related benefits might depend upon a complex interaction of tissue, endocrine, and metabolic factors.

Tiotropium inhibits proinflammatory microparticle generation by human bronchial and endothelial cells.

Tiotropium is a muscarinic antagonist that reduces the risk of acute exacerbations of chronic obstructive pulmonary disease, possibly through an as yet incompletely characterized anti-inflammatory activity. We hypothesized that muscarinic activation of bronchial epithelial cells and endothelial cells causes the release of proinflammatory microparticles and that tiotropium inhibits the phenomenon. Microparticle generation was assessed by a functional assay, by flow cytometry and by NanoSight technology. Immortalized bronchial epithelial cells (16HBE) and umbilical vein endothelial cells were treated with acetylcholine in the presence of varying concentrations of tiotropium. Intracellular calcium concentration, extracellular regulated kinase phosphorylation and chemokine content in the conditioned media were assessed by commercial kits. Acetylcholine causes microparticle generation that is completely inhibited by tiotropium (50 pM). Microparticles generated by acetylcholine-stimulated cells increase the synthesis of proinflammatory mediators in an autocrine fashion. Acetylcholine-induced upregulation of microparticle generation is inhibited by an inhibitor of extracellular regulated kinase phosphorylation and by a phospholipase C inhibitor. Tiotropium blocks both extracellular regulated kinase phosphorylation and calcium mobilization, consistent with the hypothesis that the drug prevents microparticle generation through inhibition of these critical pathways. These results might contribute to explain the effect of tiotropium in reducing acute exacerbations of chronic obstructive pulmonary disease.

PICALM Gene Methylation in Blood of Alzheimer's Disease Patients Is Associated with Cognitive Decline.

Epigenetic mechanisms might be involved in Alzheimer's disease (AD). Genetic polymorphisms in several genes, including APOE (Apolipoprotein E), PSEN1 (Presenilin 1), CR1 (Complement receptor 1), and PICALM (Phosphatidylinositol binding clathrin assembly protein), have been associated to an increased AD risk. However, data regarding methylation of these specific genes are lacking. We evaluated DNA methylation measured by quantitative bisulfite-PCR pyrosequencing in 43 AD patients and 38 healthy subjects (HS). In a multivariate age- and gender-adjusted model, PICALM methylation was decreased in AD compared to HS (mean = 3.54 and 4.63, respectively, p = 0.007). In AD, PICALM methylation level was also positively associated to Mini-Mental Scale Examination (MMSE) score (percent change 3.48%, p = 0.008). Moreover, a negative association between PICALM methylation and age was observed only in HS (percent change - 2.29%, p = 0.002). In conclusion, our data suggest a possible role of PICALM methylation in AD, particularly related to cognitive function. Given the small study sample and the associative nature of our study, further prospective investigations are required to assess the dynamics of DNA methylation in the early stages of AD development.

Is there a link between air pollution and mental disorders?

Several studies have demonstrated the association between air pollution and different medical conditions including respiratory and cardiovascular diseases. Air pollutants might have a role also in the etiology of mental disorders in the light of their toxicity on central nervous system. Purpose of the present manuscript was to review and summarize available data about an association between psychiatric disorders and air pollution. A research in the main database sources has been conducted to identify relevant papers about the topic. Different air pollutants and in particular PM and nitric oxides have been associated with poor mental health; long exposition to PM2.5 has been associated with an increased risk of new onset of depressive symptoms (Cohen's effect size d: 0.05-0.81), while increased concentration of nitric dioxide in summer with worsening of existing depressive conditions (Cohen's effect size d: 0.05-1.77). However, the interpretation of these finding should take into account the retrospective design of most of studies, different periods of observations, confounding factors such as advanced age or medical comorbidity. Further studies with rigorous methodology are needed to confirm the results of available literature about this topic.

The role of clock genes in the etiology of Major Depressive Disorder: Special Section on "Translational and Neuroscience Studies in Affective Disorders". Section Editor, Maria Nobile MD, PhD. This Section of JAD focuses on the relevance of translational and neuroscience studies in providing a better understanding of the neural basis of affective disorders. The main aim is to briefly summaries relevant research findings in clinical neuroscience with particular regards to specific innovative topics in mood and anxiety disorders.

BACKGROUND: Circadian rhythms are largely dysregulated in Major Depressive Disorder (MDD). The present review provides a summary of the findings about the role of clock genes in the etiology of MDD. METHODS: A careful search of articles on Pubmed, PsycINFO, Isi Web of Knowledge was performed in order to obtain a comprehensive review about the topic. RESULTS: The studies reported contrasting results about the association of different single nucleotide polymorphisms (SNPs) in clock genes and MDD. The most consistent result reported the association between SNP rs2287161 of CRY1 and MDD development. LIMITATIONS: Most of the published papers on the topic show bias as a prevalence of Asian ethnicity or not blinded conditions of laboratory experiments with respect to subjects' conditions (healthy controls or MDD). CONCLUSION: Further epigenetic and genome-wide studies are necessary to have a more clear idea about the role of clock genes in the etiology of MDD.

Extracellular vesicle-packaged miRNA release after short-term exposure to particulate matter is associated with increased coagulation.

BACKGROUND: Exposure to particulate matter (PM) is associated with increased incidence of cardiovascular disease and increased coagulation, but the molecular mechanisms underlying these associations remain unknown. Obesity may increase susceptibility to the adverse effects of PM exposure, exacerbating the effects on cardiovascular diseases. Extracellular vesicles (EVs), which travel in body fluids and transfer microRNAs (miRNAs) between tissues, might play an important role in PM-induced cardiovascular risk. We sought to determine whether the levels of PM with an aerodynamic diameter ≤ 10 µm (PM10) are associated with changes in fibrinogen levels, EV release, and the miRNA content of EVs (EV-miRNAs), investigating 1630 overweight/obese subjects from the SPHERE Study. RESULTS: Short-term exposure to PM10 (Day before blood drawing) was associated with an increased release of EVs quantified by nanoparticle tracking analysis, especially EVs derived from monocyte/macrophage components (CD14+) and platelets (CD61+) which were characterized by flow cytometry. We first profiled miRNAs of 883 subjects by the QuantStudio™ 12 K Flex Real Time PCR System and the top 40 EV-miRNAs were validated through custom miRNA plates. Nine EV-miRNAs (let-7c-5p; miR-106a-5p; miR-143-3p; miR-185-5p; miR-218-5p; miR-331-3p; miR-642-5p; miR-652-3p; miR-99b-5p) were downregulated in response to PM10 exposure and exhibited putative roles in cardiovascular disease, as highlighted by integrated network analysis. PM10 exposure was significantly associated with elevated fibrinogen levels, and five of the nine downregulated EV-miRNAs were mediators between PM10 exposure and fibrinogen levels. CONCLUSIONS: Research on EVs opens a new path to the investigation of the adverse health effects of air pollution exposure. EVs have the potential to act both as markers of PM susceptibility and as potential molecular mechanism in the chain of events connecting PM exposure to increased coagulation, which is frequently linked to exposure and CVD development.

Short-term particulate matter exposure induces extracellular vesicle release in overweight subjects.

BACKGROUND: Extracellular vesicles (EVs) represent a plausible molecular mechanism linking particulate matter (PM) inhalation to its systemic effects. Microvesicles (MVs) are released from many cell types in response to various stimuli. Increased body mass index (BMI) could modify the response to PM exposure due to enhanced PM uptake and/or an underlying pro-oxidative state. We investigated the relationship between EV release and PM10/PM2.5 exposure in a cohort of 51 volunteers. Subjects were stratified based on their BMI to evaluate whether overweight BMI is a determinant of hypersusceptibility to PM effects. RESULTS: Exposure to PM10/PM2.5 was assessed with a personal sampler worn for 24hours before plasma collection and confirmed with monitoring station data. Size and cellular origin of plasma EVs were characterized by Nanosight analysis and flow cytometry, respectively. Multivariate regression models were run after log-transformation, stratifying subjects based on BMI (≥ or <25kg/m2). PM exposure resulted in increased release of EVs, with the maximum observed effect for endothelial MVs. For PM10 and PM2.5, the adjusted geometric mean ratio and 95% confidence interval were 3.47 (1.30, 9.27) and 3.14 (1.23, 8.02), respectively. Compared to those in normal subjects, PM-induced EV alterations in overweight subjects were more pronounced, with visibly effect in all MV subtypes, particularly endothelial MVs. CONCLUSIONS: Our findings emphasize the role of EV release after PM exposure and the susceptibility of overweight subjects. Larger studies with accurate exposure assessment and complete EVs characterization/content analysis, could further clarify the molecular mechanism responsible for PM effects and of hypersusceptibility of overweight subjects.

Extracellular vesicle-driven information mediates the long-term effects of particulate matter exposure on coagulation and inflammation pathways.

BACKGROUND: Continuous exposure to particulate air pollution (PM) is a serious worldwide threat to public health as it coherently links with increased morbidity and mortality of cardiorespiratory diseases (CRD), and of type 2 diabetes (T2D). Extracellular vesicles (EVs) are circular plasma membrane fragments released from human cells that transfer microRNAs between tissues. In the present work it was explored the hypothesis that EVs with their encapsulated microRNAs (EVmiRNAs) contents might mediate PM effects by triggering key pathways in CRD and T2D. METHODS: Expression of EVmiRNAs analyzed by real-time PCR was correlated with oxidative stress, coagulation and inflammation markers, from healthy steel plant workers (n=55) with a well-characterized exposure to PM and PM-associated metals. All p-values were adjusted for multiple comparisons. In-silico Ingenuity Pathway Analysis (IPA) was performed to identify biological pathways regulated by PM-associated EVmiRNAs. RESULTS: Increased expression in 17 EVmiRNAs is associated with PM and metal exposure (p<0.01). Mir-196b that tops the list, being related to 9 different metals, is fundamental in insulin biosynthesis, however three (miR-302b, miR-200c, miR-30d) out of these 17 EVmiRNAs are in turn also related to disruptions (p<0.01) in inflammatory and coagulation markers. CONCLUSIONS: The study's findings support the hypothesis that adverse cardiovascular and metabolic effects stemming from inhalation exposures in particular to PM metallic component may be mediated by EVmiRNAs that target key factors in the inflammation, coagulation and glucose homeostasis pathways.

Particulate matter induces prothrombotic microparticle shedding by human mononuclear and endothelial cells.

Particulate airborne pollution is associated with increased cardiopulmonary morbidity. Microparticles are extracellular vesicles shed by cells upon activation or apoptosis involved in physiological processes such as coagulation and inflammation, including airway inflammation. We investigated the hypothesis that particulate matter causes the shedding of microparticles by human mononuclear and endothelial cells. Cells, isolated from the blood and the umbilical cords of normal donors, were cultured in the presence of particulate from a standard reference. Microparticles were assessed in the supernatant as phosphatidylserine concentration. Microparticle-associated tissue factor was assessed by an one-stage clotting assay. Nanosight technology was used to evaluate microparticle size distribution. Particulate matter induces a dose- and time- dependent, rapid (1h) increase in microparticle generation in both cells. These microparticles express functional tissue factor. Particulate matter increases intracellular calcium concentration and phospholipase C inhibition reduces microparticle generation. Nanosight analysis confirmed that upon exposure to particulate matter both cells express particles with a size range consistent with the definition of microparticles (50-1000 nm). Exposure of mononuclear and endothelial cells to particulate matter upregulates the generation of microparticles at least partially mediated by calcium mobilization. This observation might provide a further link between airborne pollution and cardiopulmonary morbidity.

Microvesicle-associated microRNA expression is altered upon particulate matter exposure in healthy workers and in A549 cells.

Cardiovascular disease risk has been consistently linked with particulate matter (PM) exposure. Cell-derived microvesicles (MVs) are released into plasma and transfer microRNAs (miRNAs) between tissues. MVs can be produced by the respiratory system in response to proinflammatory triggers, enter the circulatory system and remotely modify gene expression in cardiovascular tissues. However, whether PM affects MV signaling has never been investigated. In this study, we evaluated expression of microRNAs contained within plasma MVs upon PM exposure both in vivo and in vitro. In the in vivo study, we isolated plasma MVs from healthy steel plant workers before and after workplace PM exposure. We measured the expression of 88 MV-associated miRNAs by real-time polymerase chain reaction. To assess a possible source of the MV miRNAs identified in vivo, we measured their miRNA expression in PM-treated A549 pulmonary cell lines in vitro. MiRNA profiling of plasma MVs showed 5.62- and 13.95-fold increased expression of miR-128 and miR-302c, respectively, after 3 days of workplace PM exposure (P < 0.001). According to Ingenuity Pathway Analysis, miR-128 is part of coronary artery disease pathways, and miR-302c is part of coronary artery disease, cardiac hypertrophy and heart failure pathways. In vitro experiments confirmed a dose-dependent expression of miR-128 in MVs released from A549 cells after 6 h of PM treatment (P = 0.030). MiR-302c was expressed neither from A549 cells nor in reference lung RNA. These results suggest novel PM-activated molecular mechanisms that may mediate the effects of air pollution and could lead to the identification of new diagnostic and therapeutic interventions.

Susceptibility to particle health effects, miRNA and exosomes: rationale and study protocol of the SPHERE study.

BACKGROUND: Despite epidemiological findings showing increased air pollution related cardiovascular diseases (CVD), the knowledge of the involved molecular mechanisms remains moderate or weak. Particulate matter (PM) produces a local strong inflammatory reaction in the pulmonary environment but there is no final evidence that PM physically enters and deposits in blood vessels. Extracellular vesicles (EVs) and their miRNA cargo might be the ideal candidate to mediate the effects of PM, since they could be potentially produced by the respiratory system, reach the systemic circulation and lead to the development of cardiovascular effects.The SPHERE ("Susceptibility to Particle Health Effects, miRNAs and Exosomes") project was granted by ERC-2011-StG 282413, to examine possible molecular mechanisms underlying the effects of PM exposure in relation to health outcomes. METHODS/DESIGN: The study population will include 2000 overweight (25 < BMI < 30 kg/cm2) or obese (BMI ≥ 30 kg/cm2) subjects presenting at the Center for Obesity and Work (Fondazione IRCCS Ca' Granda Ospedale Maggiore Policlinico, Milan, Italy).Each subject donates blood, urine and hair samples. Extensive epidemiological and clinical data are collected. Exposure to PM is assigned to each subject using both daily PM10 concentration series from air quality monitors and pollutant levels estimated by the FARM (Flexible air Quality Regional Model) modelling system and elaborated by the Regional Environmental Protection Agency.The recruitment period started in September 2010 and will continue until 2015. At December 31, 2013 we recruited 1250 subjects, of whom 87% lived in the province of Milan.Primary study outcomes include cardiometabolic and respiratory health effects. The main molecular mechanism we are investigating focuses on EV-associated microRNAs. DISCUSSION: SPHERE is the first large study aimed to explore EVs as a novel potential mechanism of how air pollution exposure acts in a highly susceptible population. The rigorous study design, the availability of banked biological samples and the potential to integrate epidemiological, clinical and molecular data will also furnish a powerful base for investigating different complementary molecular mechanisms. Our findings, if confirmed, could lead to the identification of potentially reversible alterations that might be considered as possible targets for new diagnostic and therapeutic interventions.

Blood DNA methylation, nevi number, and the risk of melanoma.

Germline mutations determining increased cutaneous malignant melanoma (CMM) risk have been identified in familial and sporadic CMM cases, but they account only for a small proportion of CMM cases. Recent evidence suggests that germline epimutations (e.g. DNA methylation alterations), which can be inherited similarly to genomic mutations and can be detected in normal body cells (including blood), might increase susceptibility to cancer. The aim of the study was to identify germline epimutations of genes that were found to be mutated in familial CMM (p16, p14, CDK4, MC1R, hTERT), immune and inflammatory genes (ICAM-1, TNFα), DNA mismatch repair gene (MLH1), and repetitive elements (ALU, LINE-1, HERV-w). We measured DNA methylation using bisulfite pyrosequencing in peripheral blood mononuclear cells from 167 CMM cases and 164 sex-matched and age-matched controls. We used multivariable logistic regression models to evaluate the association between methylation levels and CMM status or presence of dysplastic nevi. We found an association between the risk of CMM and peripheral blood mononuclear cell methylation levels of TNFα [odds ratio (OR)=1.11, 95% confidence interval (CI)=1.03-1.18], CDK4 (OR=0.76, 95% CI=0.64-0.91), and MLH1 (OR=1.12, 95% CI=1.02-1.22). In control participants, the risk of developing dysplastic nevi was associated with methylation levels of TNFα (OR=0.81, 95% CI=0.69-0.95), hTERT (OR=0.90, 95% CI=0.82-0.99), and ALU (OR=1.56, 95% CI=1.02-2.39). Epimutations in CMM susceptibility genes and in genes involved in response to oxidative damage are associated with the risk of developing CMM or dysplastic nevi. Further studies measuring methylation levels of these genes in prospectively collected samples are warranted to further elucidate their role in the development and progression of CMM.