DNA Methylation Biomarkers for Young Children with Idiopathic Autism Spectrum Disorder: A Systematic Review.

Autism spectrum disorder (ASD) is a complex neurodevelopmental condition, the underlying pathological mechanisms of which are not yet completely understood. Although several genetic and genomic alterations have been linked to ASD, for the majority of ASD patients, the cause remains unknown, and the condition likely arises due to complex interactions between low-risk genes and environmental factors. There is increasing evidence that epigenetic mechanisms that are highly sensitive to environmental factors and influence gene function without altering the DNA sequence, particularly aberrant DNA methylation, are involved in ASD pathogenesis. This systematic review aimed to update the clinical application of DNA methylation investigations in children with idiopathic ASD, investigating its potential application in clinical settings. To this end, a literature search was performed on different scientific databases using a combination of terms related to the association between peripheral DNA methylation and young children with idiopathic ASD; this search led to the identification of 18 articles. In the selected studies, DNA methylation is investigated in peripheral blood or saliva samples, at both gene-specific and genome-wide levels. The results obtained suggest that peripheral DNA methylation could represent a promising methodology in ASD biomarker research, although further studies are needed to develop DNA-methylation-based clinical applications.

Increase in Mitochondrial D-Loop Region Methylation Levels in Mild Cognitive Impairment Individuals.

Methylation levels of the mitochondrial displacement loop (D-loop) region have been reported to be altered in the brain and blood of Alzheimer's disease (AD) patients. Moreover, a dynamic D-loop methylation pattern was observed in the brain of transgenic AD mice along with disease progression. However, investigations on the blood cells of AD patients in the prodromal phases of the disease have not been performed so far. The aim of this study was to analyze D-loop methylation levels by means of the MS-HRM technique in the peripheral blood cells of 14 mild cognitive impairment (MCI) patients, 18 early stage AD patients, 70 advanced stage AD patients, and 105 healthy control subjects. We found higher D-loop methylation levels in MCI patients than in control subjects and AD patients. Moreover, higher D-loop methylation levels were observed in control subjects than in AD patients in advanced stages of the disease, but not in those at early stages. The present pilot study shows that peripheral D-loop methylation levels differ in patients at different stages of AD pathology, suggesting that further studies deserve to be performed in order to validate the usefulness of D-loop methylation analysis as a peripheral biomarker for the early detection of AD.

Association of Polymorphisms in Genes Involved in One-Carbon Metabolism with MTHFR Methylation Levels.

Methylenetetrahydrofolate reductase (MTHFR) is a pivotal enzyme in the one-carbon metabolism, a metabolic pathway required for DNA synthesis and methylation reactions. MTHFR hypermethylation, resulting in reduced gene expression, can contribute to several human disorders, but little is still known about the factors that regulate MTHFR methylation levels. We performed the present study to investigate if common polymorphisms in one-carbon metabolism genes contribute to MTHFR methylation levels. MTHFR methylation was assessed in peripheral blood DNA samples from 206 healthy subjects with methylation-sensitive high-resolution melting (MS-HRM); genotyping was performed for MTHFR 677C>T (rs1801133) and 1298A>C (rs1801131), MTRR 66A>G (rs1801394), MTR 2756A>G (rs1805087), SLC19A1 (RFC1) 80G>A (rs1051266), TYMS 28-bp tandem repeats (rs34743033) and 1494 6-bp ins/del (rs34489327), DNMT3A -448A>G (rs1550117), and DNMT3B -149C>T (rs2424913) polymorphisms. We observed a statistically significant effect of the DNMT3B -149C>T polymorphism on mean MTHFR methylation levels, and particularly CT and TT carriers showed increased methylation levels than CC carriers. The present study revealed an association between a functional polymorphism of DNMT3B and MTHFR methylation levels that could be of relevance in those disorders, such as inborn defects, metabolic disorders and cancer, that have been linked to impaired DNA methylation.

Current Knowledge on Endocrine Disrupting Chemicals (EDCs) from Animal Biology to Humans, from Pregnancy to Adulthood: Highlights from a National Italian Meeting.

Wildlife has often presented and suggested the effects of endocrine disrupting chemicals (EDCs). Animal studies have given us an important opportunity to understand the mechanisms of action of many chemicals on the endocrine system and on neurodevelopment and behaviour, and to evaluate the effects of doses, time and duration of exposure. Although results are sometimes conflicting because of confounding factors, epidemiological studies in humans suggest effects of EDCs on prenatal growth, thyroid function, glucose metabolism and obesity, puberty, fertility, and on carcinogenesis mainly through epigenetic mechanisms. This manuscript reviews the reports of a multidisciplinary national meeting on this topic.

A panel of in vitro tests to evaluate genotoxic and morphological neoplastic transformation potential on Balb/3T3 cells by pristine and remediated titania and zirconia nanoparticles.

The FP7 Sanowork project was aimed to minimise occupational hazard and exposure to engineered nanomaterials (ENM) through the surface modification in order to prevent possible health effects. In this frame, a number of nanoparticles (NP) have been selected, among which zirconium (ZrO2) and titanium (TiO2) dioxide. In this study, we tested ZrO2 NP and TiO2 NP either in their pristine (uncoated) form, or modified with citrate and/or silica on their surface. As benchmark material, Aeroxide® P25 was used. We assessed cytotoxicity, genotoxicity and induction of morphological neoplastic transformation of NP by using a panel of in vitro assays in an established mammalian cell line of murine origin (Balb/3T3). Cell viability was evaluated by means of colony-forming efficiency assay (CFE). Genotoxicity was investigated by cytokinesis-block micronucleus cytome assay (CBMN cyt) and comet assay, and by the use of the restriction enzymes EndoIII and Fpg, oxidatively damaged DNA was detected; finally, the morphological neoplastic transformation of NP was assayed in vitro by cell transformation assay (CTA). Our results show that the surface remediation has not been effective in modifying cyto- and genotoxic properties of the nanomaterials tested; indeed, in the case of remediation of zirconia and titania with citrate, there is a tendency to emphasise the toxic effects. The use of a panel of assays, such as those we have employed, allowing the evaluation of multiple endpoints, including cell transformation, seems particularly advisable especially in the case of long-term exposure effects in the same cell type.

Gene-Specific Methylation Analysis in Thymomas of Patients with Myasthenia Gravis.

Thymomas are uncommon neoplasms that arise from epithelial cells of the thymus and are often associated with myasthenia gravis (MG), an autoimmune disease characterized by autoantibodies directed to different targets at the neuromuscular junction. Little is known, however, concerning epigenetic changes occurring in thymomas from MG individuals. To further address this issue, we analyzed DNA methylation levels of genes involved in one-carbon metabolism (MTHFR) and DNA methylation (DNMT1, DNMT3A, and DNMT3B) in blood, tumor tissue, and healthy thymic epithelial cells from MG patients that underwent a surgical resection of a thymic neoplasm. For the analyses we applied the methylation-sensitive high-resolution melting technique. Both MTHFR and DNMT3A promoters showed significantly higher methylation in tumor tissue with respect to blood, and MTHFR also showed significantly higher methylation levels in tumor tissue respect to healthy adjacent thymic epithelial cells. Both DNMT1 and DNMT3B promoter regions were mostly hypomethylated in all the investigated tissues. The present study suggests that MTHFR methylation is increased in thymomas obtained from MG patients; furthermore, some degrees of methylation of the DNMT3A gene were observed in thymic tissue with respect to blood.

Towards a systemic paradigm in carcinogenesis: linking epigenetics and genetics.

For at least 30 years cancer has been defined as a genetic disease and explained by the so-called somatic mutation theory (SMT), which has dominated the carcinogenesis field. Criticism of the SMT has recently greatly increased, although still not enough to force all SMT supporters to recognize its limits. Various researchers point out that cancer appears to be a complex process concerning a whole tissue; and that genomic mutations, although variably deleterious and unpredictably important in determining the establishment of the neoplastic phenotype, are not the primary origin for a malignant neoplasia. We attempt to describe the inadequacies of the SMT and demonstrate that epigenetics is a more logical cause of carcinogenesis. Many previous models of carcinogenesis fall into two classes: (i) in which some biological changes inside cells alone lead to malignancy; and (ii) requiring changes in stroma/extracellular matrix. We try to make clear that in the (ii) model genomic instability is induced by persistent signals coming from the microenvironment, provoking epigenetic and genetic modifications in tissue stem cells that can lead to cancer. In this perspective, stochastic mutations of DNA are a critical by-product rather then the primary cause of cancer. Indirect support for such model of carcinogenesis comes from the in vitro and vivo experiments showing apparent 'reversion' of cancer phenotypes obtained via physiological factors of cellular differentiation (cytokines and other signaling molecules) or drugs, even if the key mutations are not 'reversed'.

Obesity and diabetes: from genetics to epigenetics.

Obesity is becoming an epidemic health problem. During the last years not only genetic but also, and primarily, environmental factors have been supposed to contribute to the susceptibility to weight gain or to develop complications such as type 2 diabetes. In spite of the intense efforts to identify genetic predisposing variants, progress has been slow and success limited, and the common obesity susceptibility variants identified only explains a small part of the individual variation in risk. Moreover, there is evidence that the current epidemic of obesity and diabetes is environment-driven. Recent studies indicate that normal metabolic regulation during adulthood besides requiring a good balance between energy intake and energy expenditure, can be also affected by pre- and post-natal environments. In fact, maternal nutritional constraint during pregnancy can alter the metabolic phenotype of the offspring by means of epigenetic regulation of specific genes, and this can be passed to the next generations. Studies focused on epigenetic marks in obesity found altered methylation and/or histone acetylation levels in genes involved in specific but also in more general metabolic processes. Recent researches point out the continuous increase of "obesogens", in the environment and food chains, above all endocrine disruptors, chemicals that interfere with many homeostatic mechanisms. Taken into account the already existing data on the effects of obesogens, and the multiple potential targets with which they might interfere daily, it seems likely that the exposure to obesogens can have an important role in the obesity and diabesity pandemic.

The MTRR 66A>G polymorphism and maternal risk of birth of a child with Down syndrome in Caucasian women: a case-control study and a meta-analysis.

We performed a large case-control study and a meta-analysis of the literature to address the role of the methionine synthase reductase (MTRR) c.66A>G polymorphism as a maternal risk factor for the birth of a child with Down Syndrome (DS) among Caucasian women. A total of 253 mothers of a DS child (MDS) and 298 control mothers of Italian origin were included in the case-control study. The meta-analysis of previous and present data involved a total of seven studies performed in Caucasian populations (971 MDS and 1,387 control mothers). Results from the meta-analysis indicated overall a positive significant association between MTRR c.66A>G genotype [OR 1.36 (95 % CI 1.10-1.68), dominant model] and allele frequencies [OR 1.26 (95 % CI 1.04-1.51), allele contrast model] and maternal risk of birth of a child with DS. A sensitivity analysis revealed some interesting differences between Europeans, Caucasians of European descent, and inhabitants of Mediterranean regions, suggesting the possibility of population-specific modifying factors. The case-control study revealed association of the polymorphism with increased folate levels, and a possible interaction with the methionine synthase (MTR) c.2756A>G one, that resulted in a borderline significant maternal risk of birth of a child with DS for the double heterozygous MTR 2756AG/MTRR 66AG genotype [OR 1.79 (95 % CI 1.00-3.18)]. Overall, present data suggest that the MTRR c.66A>G polymorphism represents a risk factor for the birth of a child with DS among white Caucasian women. However, the combined presence of other genetic factors and interactions with geographic and environmental ones, can modify the effect of the single polymorphism alone, leading to population specific effect sizes.

Genotoxic and epigenetic mechanisms in arsenic carcinogenicity.

Arsenic is a human carcinogen with weak mutagenic properties that induces tumors through mechanisms not yet completely understood. People worldwide are exposed to arsenic-contaminated drinking water, and epidemiological studies showed a high percentage of lung, bladder, liver, and kidney cancer in these populations. Several mechanisms by which arsenical compounds induce tumorigenesis were proposed including genotoxic damage and chromosomal abnormalities. Over the past decade, a growing body of evidence indicated that epigenetic modifications have a role in arsenic-inducing adverse effects on human health. The main epigenetic mechanisms are DNA methylation in gene promoter regions that regulate gene expression, histone tail modifications that regulate the accessibility of transcriptional machinery to genes, and microRNA activity (noncoding RNA able to modulate mRNA translation). The "double capacity" of arsenic to induce mutations and epimutations could be the main cause of arsenic-induced carcinogenesis. The aim of this review is to better clarify the mechanisms of the initiation and/or the promotion of arsenic-induced carcinogenesis in order to understand the best way to perform an early diagnosis and a prompt prevention that is the key point for protecting arsenic-exposed population. Studies on arsenic-exposed population should be designed in order to examine more comprehensively the presence and consequences of these genetic/epigenetic alterations.

Mitochondrial D-loop methylation levels inversely correlate with disease duration in amyotrophic lateral sclerosis.

Aim: To correlate mitochondrial D-loop region methylation levels and mtDNA copy number with disease duration in familial amyotrophic lateral sclerosis (ALS) patients. Patients & methods: The study population included 12 ALS patients with a mutation in SOD1 and 13 ALS patients with the C9orf72 hexanucleotide repeat expansion. Methylation levels of the D-loop region and mtDNA copy number were quantified using pyrosequencing and quantitative PCR, respectively. Results: We observed that D-loop methylation levels inversely correlated while mtDNA copy number positively correlated with disease duration. Conclusion: Considering the central role played by mitochondria in ALS, this preliminary study provides new knowledge for future studies aimed at identifying biomarkers of disease progression and new targets for therapeutic interventions.

Amyotrophic lateral sclerosis is a devastating neurodegenerative disease which leads to the patient's death a few years after the onset of the first symptoms. There are currently no treatments to cure the disease, and the only drugs available are able to prolong patients' lives by only a few months. Patients may have much variability in the presentation of symptoms, including different duration of disease. This study aims to research whether mitochondrial DNA methylation, a mechanism involved in the biology of the mitochondrion, is associated with the duration of the disease. We observed that methylation of mitochondrial DNA inversely correlates with the disease duration, providing new knowledge for future studies aimed at identifying biomarkers of disease progression.

DNMT3B promoter polymorphisms and maternal risk of birth of a child with Down syndrome.

STUDY QUESTION: Are DNMT3B promoter polymorphisms among maternal risk factors for the birth of a child with Down syndrome (DS)? SUMMARY ANSWER: Present results suggest that combinations of functional DNMT3B promoter polymorphisms might modulate maternal risk of birth of a child with DS. WHAT IS KNOWN ALREADY: The DNMT3B gene codes for DNA methyltransferase 3b (DNMT3b), a protein required for genome-wide de novo methylation, for the establishment of DNA methylation patterns during development and for regulating the histone code and DNA methylation at centromeric regions. Two common functional DNMT3B promoter polymorphisms, namely -149 C > T (rs2424913) and -579 G > T (rs1569686), have been extensively investigated in cancer genetic association studies but less is known about their role in non-cancer diseases. Early in 1999, it was supposed that impaired DNA methylation of pericentromeric regions might represent a maternal risk factor for having a baby with DS. STUDY DESIGN, SIZE AND DURATION: We aimed to investigate DNMT3B -149 C > T and -579 G > T polymorphisms as maternal risk factors for the birth of a child with DS. The study was performed on DNA samples from 172 mothers of DS individuals (135 aged <35 years when they conceived) and 157 age-matched mothers of unaffected individuals. PARTICIPANTS/MATERIALS, SETTING AND METHODS: Genotyping was performed by means of the PCR-RFLP technique. MAIN RESULTS AND THE ROLE OF CHANCE: The DNMT3B -579T allele [odds ratio (OR) = 0.68; 95% confidence interval (CI) = 0.48-0.94, P = 0.02], the DNMT3B -579 GT genotype (OR = 0.55; 95% CI = 0.35-0.87 , P = 0.01) and the combined DNMT3B -579 GT + TT genotype (OR = 0.55; 95% CI = 0.36-0.86 , P = 0.008) were associated with reduced risk of birth of a child with DS. A joint effect of the two polymorphisms was observed and the combined -579 GT/-149 CC genotype resulted in decreased DS risk (OR = 0.22; 95% CI = 0.08-0.64, P = 0.003). The effect remained statistically significant after Bonferroni's correction for multiple comparisons. Similar results were obtained when the analysis was restricted to women who conceived a DS child before 35 years of age. LIMITATIONS AND REASONS FOR CAUTION: To the best of our knowledge, this is the first genetic association study aimed at evaluating DNMT3B polymorphisms as maternal risk factors for DS. Replication of the findings in other populations is required. WIDER IMPLICATIONS OF THE FINDINGS: If confirmed in subsequent studies, DNMT3B promoter polymorphisms might be additional markers to be taken into account when evaluating the contribution of one-carbon (folate) metabolism to the maternal risk of birth of a child with DS.

Multiple cytotoxic and genotoxic effects induced in vitro by differently shaped copper oxide nanomaterials.

In nanotoxicology, the capacity of nanoparticles of the same composition but different shape to induce cytotoxicity and genotoxicity is largely unknown. A series of cytotoxic and genotoxic responses following in vitro exposure to differently shaped CuO nanoparticles (CuO NPs, mass concentrations from 0.1 to 100 µg/ml) were assessed in murine macrophages RAW 264.7 and in peripheral whole blood from healthy volunteers. Cytotoxicity, cytostasis and genotoxicity were evaluated by the colorimetric assay of formazan reduction [3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT)] and by the cytokinesis-block micronucleus cytome (CBMN Cyt) assay. The comet assay was applied for detecting DNA strand breaks and information on oxidative damage to DNA (oxidised purines and pyrimidines). The MTT assay revealed a decrease in cell viability in RAW 264.7 cells and peripheral blood lymphocytes (PBL) with significant dose-effect relationships for the different CuO NP shapes. The comet assay revealed a dose-dependent increase in primary DNA damage, and a significant increase in oxidative damage to DNA was also detectable, as well as increased frequency of micronuclei in binucleated cells, often in a dose-related manner. Proliferative activity, cytotoxicity and apoptotic markers showed a significant trend in the two cell types. Finally, we have differentiated clastogenic events from aneugenic events by fluorescence in situ hybridisation with human and murine pancentromeric probes, revealing for the first time characteristic aneugenic responses related to the shape of CuO NPs and cell type. Independently of size and shape, all CuO NPs revealed a clear-cut cytotoxic and genotoxic potential; this suggests that CuO NPs are good candidates for positive controls in nanotoxicology.

The MTR 2756A>G polymorphism and maternal risk of birth of a child with Down syndrome: a case-control study and a meta-analysis.

Methionine synthase (MTR) is required for the conversion of homocysteine (hcy) to methionine in the one-carbon metabolic pathway. Previous studies investigating a common MTR 2756A>G polymorphism as a maternal risk factor for the birth of a child with Down syndrome (DS) are conflicting and limited by small case-control cohorts, and its contribution to circulating hcy levels is still debated. We performed a large case-control study and a meta-analysis of the literature to further address the role of MTR 2756A>G as a maternal risk factor for the birth of a child with DS. 286 mothers of a DS child (MDS) and 305 control mothers of Italian origin were included in the case-control study. Genotyping was performed by means of PCR/RFLP technique. Data on circulating levels of hcy, folates, and vitamin B12 were available for 189 MDS and 194 control mothers. The meta analysis of previous and present data involved a total of 8 studies (1,171 MDS and 1,402 control mothers). Both the case-control study and the meta-analysis showed no association of MTR 2756A>G with the maternal risk of birth of a child with DS (OR = 1.15; 95 % CI 0.85-1.55, and OR = 1.08; 95 % CI 0.93-1.25, respectively), even after stratification of the overall data available for the meta-analysis into ethnic groups. No association of the studied polymorphism with circulating levels of hcy, folates, and vitamin B12 was observed. Present data do not support a role for MTR 2756A>G as independent maternal risk factor for a DS birth.

Efficacy and feasibility of the epithelial cell adhesion molecule (EpCAM) immunomagnetic cell sorter for studies of DNA methylation in colorectal cancer.

The aim of this work was to assess the impact on measurements of methylation of a panel of four cancer gene promoters of purifying tumor cells from colorectal tissue samples using the epithelial cell adhesion molecule (EpCAM)-immunomagnetic cell enrichment approach. We observed that, on average, methylation levels were higher in enriched cell fractions than in the whole tissue, but the difference was significant only for one out of four studied genes. In addition, there were strong correlations between methylation values for individual samples of whole tissue and the corresponding enriched cell fractions. Therefore, assays on whole tissue are likely to provide reliable estimates of tumor-specific methylation of cancer genes. However, tumor cell tissue separation using immunomagnetic beads could, in some cases, give a more accurate value of gene promoter methylation than the analysis of the whole cancer tissue, although relatively expensive and time-consuming. The efficacy and feasibility of the immunomagnetic cell sorting for methylation studies are discussed.

Prenatal Environmental Stressors and DNA Methylation Levels in Placenta and Peripheral Tissues of Mothers and Neonates Evaluated by Applying Artificial Neural Networks.

Exposure to environmental stressors during pregnancy plays an important role in influencing subsequent susceptibility to certain chronic diseases through the modulation of epigenetic mechanisms, including DNA methylation. Our aim was to explore the connections between environmental exposures during gestation with DNA methylation of placental cells, maternal and neonatal buccal cells by applying artificial neural networks (ANNs). A total of 28 mother-infant pairs were enrolled. Data on gestational exposure to adverse environmental factors and on mother health status were collected through the administration of a questionnaire. DNA methylation analyses at both gene-specific and global level were analyzed in placentas, maternal and neonatal buccal cells. In the placenta, the concentrations of various metals and dioxins were also analyzed. Analysis of ANNs revealed that suboptimal birth weight is associated with placental H19 methylation, maternal stress during pregnancy with methylation levels of NR3C1 and BDNF in placentas and mother's buccal DNA, respectively, and exposure to air pollutants with maternal MGMT methylation. Associations were also observed between placental concentrations of lead, chromium, cadmium and mercury with methylation levels of OXTR in placentas, HSD11B2 in maternal buccal cells and placentas, MECP2 in neonatal buccal cells, and MTHFR in maternal buccal cells. Furthermore, dioxin concentrations were associated with placental RELN, neonatal HSD11B2 and maternal H19 gene methylation levels. Current results suggest that exposure of pregnant women to environmental stressors during pregnancy could induce aberrant methylation levels in genes linked to several pathways important for embryogenesis in both the placenta, potentially affecting foetal development, and in the peripheral tissues of mothers and infants, potentially providing peripheral biomarkers of environmental exposure.

Gene-environment interactions in Alzheimer disease: the emerging role of epigenetics.

With the exception of a few monogenic forms, Alzheimer disease (AD) has a complex aetiology that is likely to involve multiple susceptibility genes and environmental factors. The role of environmental factors is difficult to determine and, until a few years ago, the molecular mechanisms underlying gene-environment (G × E) interactions in AD were largely unknown. Here, we review evidence that has emerged over the past two decades to explain how environmental factors, such as diet, lifestyle, alcohol, smoking and pollutants, might interact with the human genome. In particular, we discuss how various environmental AD risk factors can induce epigenetic modifications of key AD-related genes and pathways and consider how epigenetic mechanisms could contribute to the effects of oxidative stress on AD onset. Studies on early-life exposures are helping to uncover critical time windows of sensitivity to epigenetic influences from environmental factors, thereby laying the foundations for future primary preventative approaches. We conclude that epigenetic modifications need to be considered when assessing G × E interactions in AD.

Relationship between pregnancy and coronavirus: what we know.

The identification in China in December 2019 of a new coronavirus (SARS-CoV-2) immediately rekindled the spotlight on a problem also addressed in the past during the epidemics of SARS in 2002-2003 and MERS in 2012: the implications of a possible infection during pregnancy, both for pregnant women and for fetuses and infants. Pregnancy is characterized by some changes involving both the immune system and the pulmonary physiology, exposing the pregnant woman to a greater susceptibility to viral infections and more serious complications. The objective of this review is therefore to analyze the relationship between pregnancy and known coronaviruses, with particular reference to SARS-CoV-2.

Artificial neural networks reveal sex differences in gene methylation, and connections between maternal risk factors and symptom severity in autism spectrum disorder.

Aim and methods: Artificial neural networks were used to unravel connections among blood gene methylation levels, sex, maternal risk factors and symptom severity evaluated using the Autism Diagnostic Observation Schedule 2 (ADOS-2) score in 58 children with autism spectrum disorder (ASD). Results: Methylation levels of MECP2, HTR1A and OXTR genes were connected to females, and those of EN2, BCL2 and RELN genes to males. High gestational weight gain, lack of folic acid supplements, advanced maternal age, preterm birth, low birthweight and living in rural context were the best predictors of a high ADOS-2 score. Conclusion: Artificial neural networks revealed links among ASD maternal risk factors, symptom severity, gene methylation levels and sex differences in methylation that warrant further investigation in ASD.

Preferential X Chromosome Inactivation as a Mechanism to Explain Female Preponderance in Myasthenia Gravis.

Myasthenia gravis (MG) is a neuromuscular autoimmune disease characterized by prevalence in young women (3:1). Several mechanisms proposed as explanations for gender bias, including skewed X chromosome inactivation (XCI) and dosage or sex hormones, are often involved in the development of autoimmunity. The skewed XCI pattern can lead to an unbalanced expression of some X-linked genes, as observed in several autoimmune disorders characterized by female predominance. No data are yet available regarding XCI and MG. We hypothesize that the preferential XCI pattern may contribute to the female bias observed in the onset of MG, especially among younger women. XCI analysis was performed on blood samples of 284 women between the ages of 20 and 82. XCI was tested using the Human Androgen Receptor Assay (HUMARA). XCI patterns were classified as random (XCI < 75%) and preferential (XCI ≥ 75%). In 121 informative patients, the frequency of skewed XCI patterns was 47%, significantly higher than in healthy controls (17%; p ≤ 0.00001). Interestingly, the phenomenon was observed mainly in younger patients (<45 years; p ≤ 0.00001). Furthermore, considering the XCI pattern and the other clinical characteristics of patients, no significant differences were found. In conclusion, we observed preferential XCI in MG female patients, suggesting its potential role in the aetiology of MG, as observed in other autoimmune diseases in women.