A novel Werner Syndrome mutation: pharmacological treatment by read-through of nonsense mutations and epigenetic therapies

Werner Syndrome (WS) is a rare inherited disease characterized by premature aging and increased propensity for cancer. Mutations in the WRN gene can be of several types, including nonsense mutations, leading to a truncated protein form. WRN is a RecQ family member with both helicase and exonuclease activities, and it participates in several cell metabolic pathways, including DNA replication, DNA repair, and telomere maintenance. Here, we reported a novel homozygous WS mutation (c.3767 C > G) in 2 Argentinian brothers, which resulted in a stop codon and a truncated protein (p.S1256X). We also observed increased WRN promoter methylation in the cells of patients and decreased messenger WRN RNA (WRN mRNA) expression. Finally, we showed that the read-through of nonsense mutation pharmacologic treatment with both aminoglycosides (AGs) and ataluren (PTC-124) in these cells restores full-length protein expression and WRN functionality.     

DNA repair-deficient premature aging models display accelerated epigenetic age

Several premature aging mouse models have been developed to study aging and identify interventions that can delay age-related diseases. Yet, it is still unclear whether these models truly recapitulate natural aging. Here, we analyzed DNA methylation in multiple tissues of four previously reported mouse models of premature aging (Ercc1, LAKI, Polg, and Xpg). We estimated DNA methylation (DNAm) age of these samples using the Horvath clock. The most pronounced increase in DNAm age could be observed in Ercc1 mice, a strain which exhibits a deficit in DNA nucleotide excision repair. Similarly, we detected an increase in epigenetic age in fibroblasts isolated from patients with progeroid syndromes associated with mutations in DNA excision repair genes. These findings highlight that mouse models with deficiencies in DNA repair, unlike other premature aging models, display accelerated epigenetic age, suggesting a strong connection between DNA damage and epigenetic dysregulation during aging.     

The clinical characteristics of Werner syndrome: molecular and biochemical diagnosis

Werner syndrome (WS) is an adult onset segmental progeroid syndrome caused by mutations in the WRN gene. The WRN gene encodes a 180 kDa nuclear protein that possesses helicase and exonuclease activities. The absence of WRN protein leads to abnormalities in various DNA metabolic pathways such as DNA repair, replication and telomere maintenance. Individuals with WS generally develop normally until the third decade of life, when premature aging phenotypes and a series of age-related disorders begin to manifest. In Japan, where a founder effect has been described, the frequency of Werner heterozygotes appears to be as high as 1/180 in the general population. Due to the relatively non-specific nature of the symptoms and the lack of awareness of the condition, this disease may be under-diagnosed in other parts of the world. Genetic counseling of WS patients follows the path of other autosomal recessive disorders, with special attention needed for cancer surveillance in relatives. Molecular diagnosis of WS is made by nucleotide sequencing and, in some cases, protein analysis. It is also of potential interest to measure WRN activities in WS patients. More than 50 different disease-causing mutations in the WRN gene have been identified in WS patients from all over the world. All but one of these cases has mutations that result in the premature termination of the protein. Here we describe the clinical, molecular and biochemical characteristics of WS for use by medical professionals in a health care setting. Additional information is available through the International Registry of WS ().     

MPTP诱导的帕金森病小鼠模型黑质脑组织DNA甲基化研究

为研究DNA甲基化在帕金森病发病机制中的作用,本研究用环境毒素1-甲基-4-苯基-1,2,3,6-四氢吡啶(MPTP)连续腹腔给药诱导小鼠帕金森病(Parkison's disease,PD)模型,应用ELISA检测小鼠黑质脑组织总体甲基化水平,应用实时荧光定量PCR方法检测DNA甲基转移酶表达水平,探讨MPTP诱导的小鼠PD模型黑质部位是否存在DNA甲基化异常.进一步应用甲基化DNA免疫共沉淀结合DNA甲基化芯片方法,构建MPTP诱导的小鼠PD模型黑质脑组织DNA甲基化谱,并寻找DNA甲基化修饰异常的PD相关基因对其进行验证.结果表明,模型组小鼠黑质脑组织DNA总体甲基化水平较对照组显著降低,Dnmt1的表达水平显著增高.利用DNA甲基化芯片在全基因组内筛选出甲基化差异修饰位点共48个,涉及44个基因,这些甲基化差异基因参与信号转导、分子转运、转录调控、发育、细胞分化、凋亡调控、氧化应激、蛋白质降解等生物学过程.在甲基化差异修饰基因中,对Uchl1基因及Arih2基因进行了甲基化水平以及表达水平的验证.结果表明,模型组小鼠黑质脑组织Uchl1启动子区域甲基化水平较对照组增高,m RNA及蛋白质表达水平降低,Arih2启动子区域甲基化水平较对照组降低,m RNA及蛋白质表达水平增高.实验结果进一步证实,DNA甲基化修饰异常在帕金森病发病机制中有重要作用,环境因素(如MPTP)可以通过改变DNA甲基化修饰参与帕金森病的发生发展.     

Genetic correction of Werner syndrome gene reveals impaired pro‐angiogenic function and HGF insufficiency in mesenchymal stem cells

WRN mutation causes a premature aging disease called Werner syndrome (WS). However, the mechanism by which WRN loss leads to progeroid features evident with impaired tissue repair and regeneration remains unclear. To determine this mechanism, we performed gene editing in reprogrammed induced pluripotent stem cells (iPSCs) derived from WS fibroblasts. Gene correction restored the expression of WRN. WRN^+/+ mesenchymal stem cells (MSCs) exhibited improved pro‐angiogenesis. An analysis of paracrine factors revealed that hepatocyte growth factor (HGF) was downregulated in WRN^?/? MSCs. HGF insufficiency resulted in poor angiogenesis and cutaneous wound healing. Furthermore, HGF was partially regulated by PI3K/AKT signaling, which was desensitized in WRN^?/? MSCs. Consistently, the inhibition of the PI3K/AKT pathway in WRN^+/+ MSC resulted in reduced angiogenesis and poor wound healing. Our findings indicate that the impairment in the pro‐angiogenic function of WS‐MSCs is due to HGF insufficiency and PI3K/AKT dysregulation, suggesting trophic disruption between stromal and epithelial cells as a mechanism for WS pathogenesis.     

Rapamycin decreases DNA damage accumulation and enhances cell growth of WRN‐deficient human fibroblasts

Werner syndrome (WS), caused by mutations at the WRN helicase gene, is a progeroid syndrome characterized by multiple features consistent with accelerated aging. Aberrant double‐strand DNA damage repair leads to genomic instability and reduced replicative lifespan of somatic cells. We observed increased autophagy in WRN knockdown cells; this was further increased by short‐term rapamycin treatment. Long‐term rapamycin treatment resulted in improved growth rate, reduced accumulation of DNA damage foci and improved nuclear morphology; autophagy markers were reduced to near‐normal levels, possibly due to clearance of damaged proteins. These data suggest that protein aggregation plays a role in the development of WS phenotypes and that the mammalian target of rapamycin complex 1 pathway is a potential therapeutic target of WS.     

Genome-wide DNA methylation changes in skeletal muscle between young and middle-aged pigs

Background

Age-related physiological, biochemical and functional changes in mammalian skeletal muscle have been shown to begin at the mid-point of the lifespan. However, the underlying changes in DNA methylation that occur during this turning point of the muscle aging process have not been clarified. To explore age-related genomic methylation changes in skeletal muscle, we employed young (0.5 years old) and middle-aged (7 years old) pigs as models to survey genome-wide DNA methylation in the longissimus dorsi muscle using a methylated DNA immunoprecipitation sequencing approach.

Results

We observed a tendency toward a global loss of DNA methylation in the gene-body region of the skeletal muscle of the middle-aged pigs compared with the young group. We determined the genome-wide gene expression pattern in the longissimus dorsi muscle using microarray analysis and performed a correlation analysis using DMR (differentially methylated region)-mRNA pairs, and we found a significant negative correlation between the changes in methylation levels within gene bodies and gene expression. Furthermore, we identified numerous genes that show age-related methylation changes that are potentially involved in the aging process. The methylation status of these genes was confirmed using bisulfite sequencing PCR. The genes that exhibited a hypomethylated gene body in middle-aged pigs were over-represented in various proteolysis and protein catabolic processes, suggesting an important role for these genes in age-related muscle atrophy. In addition, genes associated with tumorigenesis exhibited aged-related differences in methylation and expression levels, suggesting an increased risk of disease associated with increased age.

Conclusions

This study provides a comprehensive analysis of genome-wide DNA methylation patterns in aging pig skeletal muscle. Our findings will serve as a valuable resource in aging studies, promoting the pig as a model organism for human aging research and accelerating the development of comparative animal models in aging research.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-653) contains supplementary material, which is available to authorized users.     

Age-related epigenome-wide DNA methylation and hydroxymethylation in longitudinal mouse blood

DNA methylation at cytosine-phosphate-guanine (CpG) dinucleotides changes as a function of age in humans and animal models, a process that may contribute to chronic disease development. Recent studies have investigated the role of an oxidized form of DNA methylation – 5-hydroxymethylcytosine (5hmC) – in the epigenome, but its contribution to age-related DNA methylation remains unclear. We tested the hypothesis that 5hmC changes with age, but in a direction opposite to 5-methylcytosine (5mC), potentially playing a distinct role in aging. To characterize epigenetic aging, genome-wide 5mC and 5hmC were measured in longitudinal blood samples (2, 4, and 10 months of age) from isogenic mice using two sequencing methods – enhanced reduced representation bisulfite sequencing and hydroxymethylated DNA immunoprecipitation sequencing. Examining the epigenome by age, we identified 28,196 unique differentially methylated CpGs (DMCs) and 8,613 differentially hydroxymethylated regions (DHMRs). Mouse blood showed a general pattern of epigenome-wide hypermethylation and hypo-hydroxymethylation with age. Comparing age-related DMCs and DHMRs, 1,854 annotated genes showed both differential 5mC and 5hmC, including one gene – Nfic – at five CpGs in the same 250 bp chromosomal region. At this region, 5mC and 5hmC levels both decreased with age. Reflecting these age-related epigenetic changes, Nfic RNA expression in blood decreased with age, suggesting that age-related regulation of this gene may be driven by 5hmC, not canonical DNA methylation. Combined, our genome-wide results show age-related differential 5mC and 5hmC, as well as some evidence that changes in 5hmC may drive age-related DNA methylation and gene expression.     

DNA methylation-associated colonic mucosal immune and defense responses in treatment-naïve pediatric ulcerative colitis

Inflammatory bowel diseases (IBD) are emerging globally, indicating that environmental factors may be important in their pathogenesis. Colonic mucosal epigenetic changes, such as DNA methylation, can occur in response to the environment and have been implicated in IBD pathology. However, mucosal DNA methylation has not been examined in treatment-naïve patients. We studied DNA methylation in untreated, left sided colonic biopsy specimens using the Infinium HumanMethylation450 BeadChip array. We analyzed 22 control (C) patients, 15 untreated Crohn’s disease (CD) patients, and 9 untreated ulcerative colitis (UC) patients from two cohorts. Samples obtained at the time of clinical remission from two of the treatment-naïve UC patients were also included into the analysis. UC-specific gene expression was interrogated in a subset of adjacent samples (5 C and 5 UC) using the Affymetrix GeneChip PrimeView Human Gene Expression Arrays. Only treatment-naïve UC separated from control. One-hundred-and-twenty genes with significant expression change in UC (> 2-fold, P < 0.05) were associated with differentially methylated regions (DMRs). Epigenetically associated gene expression changes (including gene expression changes in the IFITM1, ITGB2, S100A9, SLPI, SAA1, and STAT3 genes) were linked to colonic mucosal immune and defense responses. These findings underscore the relationship between epigenetic changes and inflammation in pediatric treatment-naïve UC and may have potential etiologic, diagnostic, and therapeutic relevance for IBD.     

Identification of DNA methylation signature to predict prognosis in gastric adenocarcinoma

Gastric adenocarcinoma is an important death-related cancer. To find factors related to survival and prognosis, and thus improve recovery prospects, a powerful signature is needed. DNA methylation plays an important role in gastric adenocarcinoma processes and development, and here we report on the search for a significant DNA methylation gene to aid with the earlier diagnosis of gastric adenocarcinoma patients. A Cox proportional risk regression analysis and random survival forest algorithm were used to analyze gastric adenocarcinoma patients’ DNA methylation data from The Cancer Genome Atlas, a public database. DNA methylation gene signature consisting of five genes (SERPINA3, AP000357.4, GZMA, AC004702.2, and GREB1L) were selected. As the most accurate predictor, the area under the curve in the training and test group were 0.72 and 0.61, respectively. The signature was able to sort patients into high- and low-risk groups with meaningful overall survival rates (median: 18.36 vs 72.23 months, log-rank test, P < 0.001) in the training group, which predictive ability was validated in a test data set (median: 25.56 vs 58.80 months, log-rank test, P < 0.016). A multivariate Cox regression analysis showed the significant DNA methylation was an independent prediction prognostic factor for gastric adenocarcinoma patients. Functional analysis suggests that these signature genes may be related to pathways and biological processes associated with tumorigenesis. The significant DNA methylation gene could be a novel prediction and prognostic biomarker that both aids in the treatment and predicts the overall survival likelihoods of gastric adenocarcinoma patients.     

Signatures of altered DNA methylation gene expression after central and peripheral nerve injury

Nerve damage can lead to movement and sensory dysfunction, with high morbidity and disability rates causing severe burdens on patients, families, and society. DNA methylation is a kind of epigenetics, and a great number of previous studies have demonstrated that DNA methylation plays an important role in the process of nerve regeneration and remodeling. However, compared with the central nervous system, the peripheral nervous system shows stronger recovery after injury, which is related to the complex microenvironment and epigenetic changes occurring at the site of injury. Therefore, what common epigenetic changes between the central and peripheral nervous systems remain to be elucidated. We first screened differential methylation genes after spinal cord injury and sciatic nerve injury using whole-genome bisulfite sequencing and methylated DNA immunoprecipitation sequencing, respectively. Subsequently, a total of 16 genes had the same epigenetic changes after spinal cord injury and sciatic nerve injury. The Gene Ontology analysis and Kyoto Encyclopedia of Genes and Genomes enrichment analysis were performed to identify the critical biological processes and pathways. Furthermore, a protein−protein interaction network analysis indicated that Dnm3, Ntrk3, Smurf1, Dpysl2, Kalrn, Shank1, Dlg2, Arsb, Reln, Bmp5, Numbl, Prickle2, Map6, and Htr7 were the core genes. These outcomes may provide novel insights into the molecular mechanism of the subacute phase of nerve injury. These verified genes can offer potential diagnostic and therapeutic targets for nerve injury.     

Human metastable epiallele candidates link to common disorders

Metastable epialleles (MEs) are mammalian genomic loci where epigenetic patterning occurs before gastrulation in a stochastic fashion leading to systematic interindividual variation within one species. Importantly, periconceptual nutritional influences may modulate the establishment of epigenetic changes, such as DNA methylation at MEs. Based on these characteristics, we exploited Infinium HumanMethylation450 BeadChip kits in a 2-tissue parallel screen on peripheral blood leukocyte and colonic mucosal DNA from 10 children without identifiable large intestinal disease. This approach led to the delineation of 1776 CpG sites meeting our criteria for MEs, which associated with 1013 genes. The list of ME candidates exhibited overlaps with recently identified human genes (including CYP2E1 and MGMT, where methylation has been associated with Parkinson disease and glioblastoma, respectively) in which perinatal DNA methylation levels where linked to maternal periconceptual nutrition. One hundred 18 (11.6%) of the ME candidates overlapped with genes where DNA methylation correlated (r > 0.871; p < 0.055) with expression in the colon mucosa of 5 independent control children. Genes involved in homophilic cell adhesion (including cadherin-associated genes) and developmental processes were significantly overrepresented in association with MEs. Additional filtering of gene expression-correlated MEs defined 35 genes, associated with 2 or more CpG sites within a 10 kb genomic region, fulfilling the ME criteria. DNA methylation changes at a number of these genes have been linked to various forms of human disease, including cancers, such as asthma and acute myeloid leukemia (ALOX12), gastric cancer (EBF3), breast cancer (NAV1), colon cancer and acute lymphoid leukemia (KCNK15), Wilms tumor (protocadherin gene cluster; PCDHAs) and colorectal cancer (TCERG1L), suggesting a potential etiologic role for MEs in tumorigenesis and underscoring the possible developmental origins of these malignancies. The presented compendium of ME candidates may accelerate our understanding of the epigenetic origins of common human disorders.     

DNA methylation biomarkers for head and neck squamous cell carcinoma

DNA methylation plays an important role in the etiology and pathogenesis of head and neck squamous cell carcinoma (HNSCC). The current study aimed to identify aberrantly methylated-differentially expressed genes (DEGs) by a comprehensive bioinformatics analysis. In addition, we screened for DEGs affected by DNA methylation modification and further investigated their prognostic values for HNSCC. We included microarray data of DNA methylation (GSE25093 and GSE33202) and gene expression (GSE23036 and GSE58911) from Gene Expression Omnibus. Aberrantly methylated-DEGs were analyzed with R software. The Cancer Genome Atlas (TCGA) RNA sequencing and DNA methylation (Illumina HumanMethylation450) databases were utilized for validation. In total, 27 aberrantly methylated genes accompanied by altered expression were identified. After confirmation by The Cancer Genome Atlas (TCGA) database, 2 hypermethylated-low-expression genes (FAM135B and ZNF610) and 2 hypomethylated-high-expression genes (HOXA9 and DCC) were identified. A receiver operating characteristic (ROC) curve confirmed the diagnostic value of these four methylated genes for HNSCC. Multivariate Cox proportional hazards analysis showed that FAM135B methylation was a favorable independent prognostic biomarker for overall survival of HNSCC patients.