Genetic and epigenetic methylation defects and implication of the ERMN gene in autism spectrum disorders.
ABSTRACT: Autism spectrum disorders (ASD) are highly heritable and genetically complex conditions. Although highly penetrant mutations in multiple genes have been identified, they account for the etiology of <1/3 of cases. There is also strong evidence for environmental contribution to ASD, which can be mediated by still poorly explored epigenetic modifications. We searched for methylation changes on blood DNA of 53 male ASD patients and 757 healthy controls using a methylomic array (450K Illumina), correlated the variants with transcriptional alterations in blood RNAseq data, and performed a case-control association study of the relevant findings in a larger cohort (394 cases and 500 controls). We found 700 differentially methylated CpGs, most of them hypomethylated in the ASD group (83.9%), with cis-acting expression changes at 7.6% of locations. Relevant findings included: (1) hypomethylation caused by rare genetic variants (meSNVs) at six loci (ERMN, USP24, METTL21C, PDE10A, STX16 and DBT) significantly associated with ASD (q-value <0.05); and (2) clustered epimutations associated to transcriptional changes in single-ASD patients (n=4). All meSNVs and clustered epimutations were inherited from unaffected parents. Resequencing of the top candidate genes also revealed a significant load of deleterious mutations affecting ERMN in ASD compared with controls. Our data indicate that inherited methylation alterations detectable in blood DNA, due to either genetic or epigenetic defects, can affect gene expression and contribute to ASD susceptibility most likely in an additive manner, and implicate ERMN as a novel ASD gene.
Project description:Autism spectrum disorder (ASD) is a severe neurodevelopmental disorder that involves social interaction defects, impairment of non-verbal and verbal interactions, and limited interests along with stereotypic activities. Its incidence has been increasing rapidly in recent decades. Despite numerous attempts to understand the pathophysiology of ASD, its exact etiology is still unclear. Recent data shows the role of accurate myelination and translational regulation in ASD's pathogenesis. In this study, we assessed Ermin (<i>ERMN</i>) and Listerin E3 Ubiquitin Protein Ligase 1 (<i>LTN1</i>) genes expression in Iranian ASD patients and age- and gender-matched healthy subjects' peripheral blood using quantitative real-time PCR to recognize any probable dysregulation in the expression of these genes and propose this disorder's mechanisms. Analysis of the expression demonstrated a significant <i>ERMN</i> downregulation in total ASD patients compared to the healthy individuals (posterior beta = -0.794, adjusted <i>P</i>-value = 0.025). <i>LTN1</i> expression was suggestively higher in ASD patients in comparison with the corresponding control individuals. Considering the gender of study participants, the analysis showed that the mentioned genes' different expression levels were significant only in male subjects. Besides, a significant correlation was found between expression of the mentioned genes (<i>r</i> = -0.49, <i>P</i> < 0.0001). The present study provides further supports for the contribution of <i>ERMN</i> and <i>LTN1</i> in ASD's pathogenesis.
Project description:DNA methylation is a heritable epigenetic mark that controls gene expression, is responsive to environmental stresses, and, in plants, may also play a role in heterosis. To determine the degree to which DNA methylation is inherited in rice, and how it both influences and is affected by transcription, we performed genome-wide measurements of these patterns through an integrative analysis of bisulfite-sequencing, RNA-sequencing, and siRNA-sequencing data in two inbred parents of the Nipponbare (NPB) and indica (93-11) varieties of rice and their hybrid offspring. We show that SNPs occur at a rate of about 1/253 bp between the two parents and that these are faithfully transmitted into the hybrids. We use the presence of these SNPs to reconstruct the two chromosomes in the hybrids according to their parental origin. We found that, unlike genetic inheritance, epigenetic heritability is quite variable. Cytosines were found to be differentially methylated (epimutated) at a rate of 7.48% (1/15 cytosines) between the NPB and 93-11 parental strains. We also observed that 0.79% of cytosines were epimutated between the parent and corresponding hybrid chromosome. We found that these epimutations are often clustered on the chromosomes, with clusters representing 20% of all epimutations between parental ecotypes, and 2-5% in F1 plants. Epimutation clusters are also strongly associated with regions where the production of siRNA differs between parents. Finally, we identified genes with both allele-specific expression patterns that were strongly inherited as well as those differentially expressed between hybrids and the corresponding parental chromosome. We conclude that much of the misinheritance of expression levels is likely caused by epimutations and trans effects.
Project description:BACKGROUND:Exposure to anesthetics during early life may impair cognitive functions. However, the underlying mechanisms remain largely unknown. We set out to determine effects of sevoflurane anesthesia on folate metabolism and myelination in young non-human primates, mice and children. METHODS:Young rhesus macaque and mice received 2.5 to 3% sevoflurane daily for three days. DNA and RNA sequencing and immunohistochemistry among others were used in the studies. We performed unbiased transcriptome profiling in prefrontal cortex of rhesus macaques and mice after the sevoflurane anesthesia. We constructed a brain blood barrier-crossing AAV-PHP.EB vector to harbor ERMN expression in rescue studies. We measured blood folate levels in children after anesthesia and surgery. FINDINGS:We found that thymidylate synthase (TYMS) gene was downregulated after the sevoflurane anesthesia in both rhesus macaque and mice. There was a reduction in blood folate levels in children after the anesthesia and surgery. Combined with transcriptome and genome-wide DNA methylation analysis, we identified that ERMN was the primary target of the disrupted folate metabolism. Myelination was compromised by the anesthesia in the young mice, which was rescued by systematic administration of folic acid or expression of ERMN in the brain through brain-specific delivery of the adeno-associated virus. Moreover, folic acid and expression of ERMN alleviated the cognitive impairment caused by the sevoflurane anesthesia in the mice. INTERPRETATION:General anesthesia leads to disrupted folate metabolism and subsequently defects in myelination in the developmental brain, and ERMN is the important target affected by the anesthesia via epigenetic mechanisms.
Project description: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, TNF?), 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 TNF? [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 TNF? (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.
Project description:Protein-lysine methylation is a common posttranslational modification (PTM) throughout the human proteome that plays important roles in diverse biological processes. In humans, there are >100 known and candidate protein lysine methyltransferases (PKMTs), many of which are linked to human diseases. Methyltransferase-like protein 21C (METTL21C) is a PKMT implicated in muscle biology that has been reported to methylate valosin-containing protein/p97 (VCP) and heat shock 70-kDa protein 8 (HSPA8). However, a clear <i>in vitro</i> methyltransferase activity for METTL21C remains yet to be demonstrated, and whether it is an active enzyme that directly methylates substrate(s) <i>in vivo</i> is unclear. Here, we used an unbiased biochemistry-based screening assay coupled to MS, which identified alanine tRNA synthetase 1 (AARS1) as a direct substrate of METTL21C. We found that METTL21C catalyzes methylation of Lys-943 of AARS1 (AARS1-K943me) both <i>in vitro</i> and <i>in vivo</i> <i>In vitro</i> METTL21C-mediated AARS1 methylation was independent of ATP or tRNA molecules. Unlike for AARS1, and in conflict with previous reports, we did not detect METTL21C methylation of VCP and HSPA8. AARS1-K943 methylation in HEK293T cells depends upon METTL21C levels. Finally, METTL2C was almost exclusively expressed in muscle tissue, and, accordingly, we detected METTL21C-catalyzed methylation of AARS1 in mouse skeletal muscle tissue. These results reveal that AARS1 is a <i>bona fide in vitro</i> substrate of METTL21C and suggest a role for the METTL21C-AARS1 axis in the regulation of protein synthesis in muscle tissue. Moreover, our study describes a straightforward protocol for elucidating the physiological substrates of poorly characterized or uncharacterized PKMTs.
Project description:The chicken is a common type of poultry that is economically important both for its medicinal and nutritional values. Previous studies have found that free-range chickens have more skeletal muscle mass. The methyltransferase-like 21C gene (<i>METTL21C)</i> plays an important role in muscle development; however, there have been few reports on the role of <i>METTL21C</i> in chickens. In this study, we performed a genome-wide identification of chicken <i>METTL21C</i> genes and analyzed their phylogeny, transcriptional expression profile, and real-time quantitative polymerase chain reaction (qPCR). We identified 10 <i>GgMETTL21C</i> genes from chickens, 11 from mice, and 32 from humans, and these genes were divided into six groups, which showed a large amount of variation among these three species. A total of 15 motifs were detected in <i>METTL21C</i> genes, and the intron phase of the gene structure showed that the <i>METTL21C</i> gene family was conservative in evolution. Further, both the transcript data and qPCR showed that a single gene's (<i>GgMETTL21C3)</i> expression level increased with the muscle development of chickens, indicating that the <i>METTL21C</i> genes are involved in the development of chicken muscles. Our results provide some reference value for the subsequent study of the function of <i>METTL21C.</i>
Project description:Regulation of p53 by ubiquitination and deubiquitination is important for its function. In this study, we demonstrate that USP24 deubiquitinates p53 in human cells. Functional USP24 is required for p53 stabilization, and p53 destabilization in USP24-depleted cells can be corrected by the forced expression of USP24. We show that USP24 depletion renders cells resistant to apoptosis after UV irradiation, consistent with the requirement of USP24 for p53 stabilization and PUMA activation in vivo. Additionally, purified USP24 protein is able to cleave ubiquitinated p53 in vitro. Importantly, cells with USP24 depletion exhibited significantly elevated mutation rates at the endogenous HPRT locus, implying an important role for USP24 in maintaining genome stability. Our data reveal that the USP24 deubiquitinase regulates the DNA damage response by directly targeting the p53 tumor suppressor.
Project description:Rare inherited variations in multiplex families with autism spectrum disorder (ASD) are suggested to play a major role in the genetic etiology of ASD. To further investigate the role of rare inherited variations, we performed whole-exome sequencing (WES) in two families, each with three affected siblings. We also performed a two-stage follow-up case-control study in a Japanese population. WES of the six affected siblings identified six novel rare missense variations. Among these variations, CLN8 R24H was inherited in one family by three affected siblings from an affected father and thus co-segregated with ASD. In the first stage of the follow-up study, we genotyped the six novel rare missense variations identified by WES in 241 patients and 667 controls (the Niigata sample). Only CLN8 R24H had higher mutant allele frequencies in patients (1/482) compared with controls (1/1334). In the second stage, this variation was further genotyped, yet was not detected in a sample of 309 patients and 350 controls (the Nagoya sample). In the combined Niigata and Nagoya samples, there was no significant association (odds ratio = 1.8, 95% confidence interval = 0.1-29.6). These results suggest that CLN8 R24H plays a role in the genetic etiology of ASD, at least in a subset of ASD patients.
Project description:<h4>Simple Summary</h4> Epigenetic mechanisms contribute to the regulation of gene expression. However, when they fail, they result in diseases such as cancer. Among these effects is aberrant DNA methylation caused by inherited mutations in cis of the affected gene, referred to as constitutional secondary epimutations. Little is known about this phenomenon, in which hypermethylation promotes transcriptional silencing of tumor suppressor genes in patients with inherited cancers that do not have pathogenic variants in the coding region of cancer susceptibility genes. Here we discuss these hereditary alterations and their effect during the early stages of tumorigenesis, as well as their contribution to disease historically and from a molecular perspective. <h4>Abstract</h4> Epigenetics affects gene expression and contributes to disease development by alterations known as epimutations. Hypermethylation that results in transcriptional silencing of tumor suppressor genes has been described in patients with hereditary cancers and without pathogenic variants in the coding region of cancer susceptibility genes. Although somatic promoter hypermethylation of these genes can occur in later stages of the carcinogenic process, constitutional methylation can be a crucial event during the first steps of tumorigenesis, accelerating tumor development. Primary epimutations originate independently of changes in the DNA sequence, while secondary epimutations are a consequence of a mutation in a cis or trans-acting factor. Secondary epimutations have a genetic basis in cis of the promoter regions of genes involved in familial cancers. This highlights epimutations as a novel carcinogenic mechanism whose contribution to human diseases is underestimated by the scarcity of the variants described. In this review, we provide an overview of secondary epimutations and present evidence of their impact on cancer. We propose the necessity for genetic screening of loci associated with secondary epimutations in familial cancer as part of prevention programs to improve molecular diagnosis, secondary prevention, and reduce the mortality of these diseases.
Project description:Pseudohypoparathyroidism (PHP) is caused by (epi)genetic defects in the imprinted GNAS cluster. Current classification of PHP patients is hampered by clinical and molecular diagnostic overlaps. The European Consortium for the study of PHP designed a genome-wide methylation study to improve molecular diagnosis.The HumanMethylation 450K BeadChip was used to analyze genome-wide methylation in 24 PHP patients with parathyroid hormone resistance and 20 age- and gender-matched controls. Patients were previously diagnosed with GNAS-specific differentially methylated regions (DMRs) and include 6 patients with known STX16 deletion (PHP(?stx16)) and 18 without deletion (PHP(neg)).The array demonstrated that PHP patients do not show DNA methylation differences at the whole-genome level. Unsupervised clustering of GNAS-specific DMRs divides PHP(?stx16) versus PHP(neg) patients. Interestingly, in contrast to the notion that all PHP patients share methylation defects in the A/B DMR while only PHP(?stx16) patients have normal NESP, GNAS-AS1 and XL methylation, we found a novel DMR (named GNAS-AS2) in the GNAS-AS1 region that is significantly different in both PHP(?stx16) and PHP(neg), as validated by Sequenom EpiTYPER in a larger PHP cohort. The analysis of 58 DMRs revealed that 8/18 PHP(neg) and 1/6 PHP(?stx16) patients have multi-locus methylation defects. Validation was performed for FANCC and SVOPL DMRs.This is the first genome-wide methylation study for PHP patients that confirmed that GNAS is the most significant DMR, and the presence of STX16 deletion divides PHP patients in two groups. Moreover, a novel GNAS-AS2 DMR affects all PHP patients, and PHP patients seem sensitive to multi-locus methylation defects.