DNA Methylation Levels Of Genes Research Articles

Prospective study of the association between chronotypes and depressive symptoms in Chinese university students: Moderating effects of PER1 gene DNA methylation

ABSTRACT Most studies have shown a link between chronotypes and mental health and have identified evening chronotypes (E-types) as a potential risk for depressive symptoms. However, the mechanisms behind this association remain unknown. Abnormal expression of the PER1 gene was not only associated with circadian rhythm disturbance, but also closely related to mental illness. Therefore, this study aimed to examine the association of chronotype with depressive symptoms, and further explore the moderating effects of the PER1 gene DNA methylation on chronotypes and depressive symptoms in Chinese university students. In a stratified cluster sampling design, chronotype and depressive symptoms were assessed in 1 042 university students from 2 universities in a two-year prospective survey from April 2019 to October 2020. The survey was conducted once every 6 months, corresponding to the time points in April 2019 (T0), October 2019 (T1), April 2020 (T2), and October 2020 (T3). At T0, the Morning and Evening Questionnaire 5 (MEQ-5) was adopted to assess chronotype. At T0-T3, the Patient Health Questionnaire 9 (PHQ-9) was adopted to investigate depressive symptoms. Meanwhile, at T0, participants were subjected to a health check-up trip in the hospital, and blood samples were taken from the students to measure the PER1 gene DNA methylation levels. Binary logistic regression was used to analyze the association of chronotypes with depressive symptoms. The depression/total depression group was coded as 1, while the remaining participants was defined as one group, and was coded as 0. The PROCESS plug-in of SPSS software was used to analyze the moderating effects of PER1 gene DNA methylation on the association of chronotype with depressive symptoms. After adjusting for covariates, the results indicated that T0 E-types were positively correlated with T0-T3 depression/total depression in female university students. Furthermore, the PER1 gene DNA methylation has negative moderating effects between T0 chronotype and T3 depressive symptoms and has a sex difference. This study can provide more favorable scientific value for the prevention and control of depression in university students.

Effect of Aerobic/Strength Training on RANKL Gene DNA Methylation Levels.

The osteoclastogenesis RANKL gene plays a key role in bone remodeling. The hypomethylation of its promoter region may cause osteoporosis. The present study aimed to elucidate the influence of physical activity on DNA methylation changes of RANKL promoter cytosine-phosphate-guanine (CpG)-rich region in active and sedentary adults and to assess the effect of aerobic and strength training on RANKL DNA methylation changes among Tunisian-North African adults. A total of 104 participants including 52 adults (58% males and 42% females) and 52 adults (31% males and 69% females) were recruited for the observational and interventional part of the study, respectively. The intervention consisted of 12weeks of aerobic training (30min/session) followed by 10minutes of strengthening exercises. All participants completed the International Physical Activity Questionnaire and provided blood samples for quantitative methylation-specific polymerase chain reaction (PCR) analysis. The study revealed a significant difference (P = 6 × 10-10) in the methylation level of the RANKL promoter region between active and sedentary adults, with a 6.68-fold increase observed in the active group. After the intervention, both the trained (P = 41 × 10-5) and untrained (P = .002) groups displayed high methylation levels in the RANKL promoter region. In addition, the trained group exhibited significant improvements in heart rate (P = 2.2 × 10-16), blood pressure (P = 39 × 10-3), maximal oxygen uptake (P = 1.5 × 10-7), and fat mass (P = 7 × 10-4). Exploring epigenetic modifications in the RANKL promoter region may contribute to a more comprehensive understanding of the complexity of osteoporosis. This suggests that aerobic/strength training could potentially improve the bone system, reducing its vulnerability to osteoporosis by increasing RANKL DNA methylation.

0608 PAX8/PAX8-AS1 DNA methylation levels are not associated with sleep duration in medicated patients with unexplained hypersomnolence

Abstract Introduction The biological basis of daytime sleepiness and excessive sleep duration in patients with unexplained hypersomnolence is unknown. However, a growing body of evidence suggests modulations in the epigenome coincide with objective measures of sleep, suggesting epigenetic mechanisms may be related to hypersomnolence. Previously, we identified significant correlations between saliva DNA methylation abundance at nine CpG sites located in the PAX8/PAX8-AS1 genes and total sleep time measured using polysomnography in unmedicated, clinical patients with unexplained hypersomnolence. Since psychotropic medications frequently affect clinical sleep testing and may also affect DNA methylation, here we attempted to replicate prior findings in a separate, medicated sample. Methods Forty-three medicated clinical patients with unexplained hypersomnolence were drawn from a larger study of a multimodal sleep assessment that included ad libitum overnight polysomnography. Pyrosequencing quantified DNA methylation at the previously identified CpG sites in PAX8/PAX8-AS1. Multivariate linear models, adjusting for age, sex, and body mass index (BMI), assessed relationships between total sleep time and DNA methylation at these prespecified CpG sites. Results Participants were young -to middle-aged (Mean Age = 32.2±10 years) and predominantly female (Percent Female = 95.3%), with a mean BMI of 27.8±5.4kg/m2. Epworth Sleepiness Scale (ESS) score was consistent with complaints of daytime sleepiness (Mean ESS =15.1±-4.3). Total sleep time on ad libitum polysomnography was 9.2±1.7 hours (range 5.6-12.8 hours). No significant associations between DNA methylation at the PAX8/PAX8-AS1 CpG sites and sleep duration were identified in adjusted models. Conclusion We were unable to replicate findings of an association between sleep duration and DNA methylation levels at specific PAX8/PAX8-AS1 CpG sites in medicated patients with unexplained hypersomnolence. Further research to clarify whether this is a result of medications affecting the DNA methylation levels at these CpG sites or previously identified associations between DNA methylation of PAX8/PAX8-AS1 and sleep length are spurious will require replication of prior findings in a novel unmedicated cohort and quantification of within-subjects effects of psychotropic medications on DNA methylation levels in genes of interest. Support (if any) AASM Foundation (229 SR-20 - Diversity Supplement Grant)

Moderating effects of PER3 gene DNA methylation on the association of sleep quality with mental health in Chinese young adults

BackgroundPoor sleepers have a higher prevalence of mental health problems, and vice versa. However, the mechanisms underlying this association remain unknown. We aimed to examine the bidirectional association between sleep quality and mental health, and further explore the moderating effects of PER3 gene DNA methylation on the association. MethodsThis prospective longitudinal study was conducted from April 2019 to May 2021, integrated questionnaire and blood sample data from 2 universities in Anhui and Jiangxi Provinces. The current study recruited 1179 young adults at baseline and conducted the follow-up survey among 1135 half a year later. The sleep quality and depressive symptoms, anxiety symptoms, and stress symptoms were assessed using a questionnaire at baseline and follow-up. Blood samples were collected at baseline, and MethyTarget™ was used to detect the PER3 gene DNA methylation level. A cross-lag model was used to examine the bidirectional association between sleep quality and mental health. The PROCESS plug-in of SPSS software was used to analyse the moderating effects of PER3 gene DNA methylation. ResultsCross-lagged analyses suggested a significant bidirectional relationship between poor sleep quality and depressive symptoms, anxiety symptoms, and stress symptoms. Sleep quality at baseline was a significant predictor of depressive symptoms (β = 0.344, P < 0.001), anxiety symptoms (β = 0.348, P < 0.001), and stress symptoms (β = 0.324, P < 0.001) half a year later. Depressive symptoms (β = 0.049, P < 0.001), anxiety symptoms (β = 0.055, P < 0.001), and stress symptoms (β = 0.063, P < 0.001) at baseline were also significant predictors of poor sleep quality half a year later. Furthermore, PER3 gene DNA methylation has negative moderating effects between sleep quality at baseline and depressive symptoms (β = −11.706, P = 0.012), anxiety symptoms (β = −10.289, P = 0.019), and stress symptoms (β = −10.799, P = 0.024) half a year later and a sex difference. Among boys, PER3 gene DNA methylation has positive moderating effects between anxiety symptoms at baseline and sleep quality (β = 3.337, P = 0.018) half a year later. However, there was no association between mental health at baseline and sleep quality half a year later among girls. ConclusionBidirectional relationships were identified between sleep quality and mental health among Chinese young adults during the study period. DNA methylation evidence supports a negative moderating effect of PER3 gene DNA methylation on the relationship between sleep quality at baseline and mental health half a year later and had sex differences. Among boys, PER3 gene DNA methylation had positive moderating effects between anxiety symptoms at baseline and sleep quality half a year later. These findings point to the importance of circadian clock gene DNA methylation in the relationship between sleep quality and mental health.

The Cannabis-Induced Epigenetic Regulation of Genes Associated with Major Depressive Disorder.

The prevalence of depression is increasing worldwide, as is the number of people suffering from treatment-resistant depression; these patients constitute 30% of those treated. Unfortunately, there have not been significant advances in the treatment of this disorder in the past few decades. Exposure to cannabis and cannabis-derived compounds impacts depression symptomatology in different ways, with evidence indicating that cannabidiol has antidepressant effects; there have been mixed results with medical cannabis. Even though the exact molecular mechanisms of the action underlying changes in depression symptomatology upon exposure to cannabis and cannabis-derived compounds are still unknown, there is strong evidence that these agents have a widespread impact on epigenetic regulation. We hypothesized that exposure to cannabis or cannabis-derived compounds changes the DNA methylation levels of genes associated with depression. To test this hypothesis, we first performed a literature search to identify genes that are differentially methylated upon exposure to cannabis and cannabis-derived compounds, as reported in methylome-wide association studies. We next checked whether genes residing in loci associated with depression, as identified in the largest currently available genome-wide association study of depression, were reported to be epigenetically regulated by cannabis or cannabis-related compounds. Multiple genes residing in loci associated with depression were found to be epigenetically regulated by exposure to cannabis or cannabis-derived compounds. This epigenomic regulation of depression-associated genes by cannabis or cannabis-derived compounds was reported across diverse organisms, tissues, and developmental stages and occurred in genes crucial for neuronal development, functioning, survival, and synapse functioning, as well as in genes previously implicated in other mental disorders.

Cryopreservation did not affect sperm DNA methylation levels of genes related to fertilization and embryonic development of cynomolgus macaque (Macaca fascicularis)

DNA methylation alters gene expression in numerous biological processes, including embryonic development. It is little known about the effect of cryopreservation on sperm DNA methylation. The present study has investigated whether cryopreservation causes abnormal DNA methylation in cynomolgus macaque sperm for five critical genes that includes the maternally imprinted gene (SNRPN), genes associated with male infertility (HSPA1L, MTHFR) and genes involved in embryonic development (TET3, LZTR1). Our results showed that sperm motility, the percentage of acrosomal integrity, DNA integrity and mitochondrial membrane potential were decreased after cryopreservation either being frozen with penetrating cryoprotectant, glycerol (Gly) or ethylene glycol (EG), compared to fresh sperm (p = 0.000), but the methylation patterns of the five target genes from cynomolgus macaque sperm samples were not affected after cryopreservation as evaluated by the Bisulfite Sequencing PCR (BSP) method. The data indicates that the current protocol for sperm cryopreservation of cynomolgus macaque is safe in terms of DNA methylation levels in these genes related to critical sperm functions.

ABCB9 polymorphism rs61955196 is associated with schizophrenia in a Chinese Han population.

BACKGROUNDSchizophrenia (SCZ) is a complex disease which can be affected by both genetic and environmental factors. Prenatal famine exposure may cause changes in DNA methylation levels of genes. Meanwhile, maternal nutrition during pregnancy is a pivotal environmental factor in the development of SCZ. DNA methylation may be an intermediate factor mediating exposure to famine during pregnancy and SCZ, and DNA methylation quantitative trait loci might serve as a promising tool for linking SCZ and prenatal famine. AIMTo analyze the association between prenatal famine exposure and SCZ risk in Northeast Han Chinese through analysis of DNA methylation related loci.METHODSA total of 954 Han Chinese from Northeast China were recruited, including 443 patients with SCZ and 511 healthy controls. The participants were further divided into famine (born in 1960-1962) and non-famine (born in 1963-1965) groups to investigate the effect of prenatal famine exposure. Four single-nucleotide polymorphisms (SNPs) selected according to the relevant literature were genotyped, namely, rs11917047 in PTPRG, rs2239681 in IGF2, rs3842756 in INSIGF, and rs61955196 in ABCB9. DNA were extracted from peripheral blood samples, and the genotypes of these SNP loci were detected using the improved Multiple Ligase Detection Reaction multiple SNP typing technique. The associations of the DNA methylation related SNPs with SCZ risk and prenatal famine, and their interactions were analyzed using logistic regression analysis and generalized multifactor dimensionality reduction (GMDR) software.RESULTSBased on the sequencing data, genotype distributions and allele frequencies of the four selected SNPs were determined. All genotype frequencies of the four SNPs in the healthy control group were tested for deviation from Hardy-Weinberg equilibrium (P > 0.05). Logistic regression analysis showed that rs61955196 was significantly associated with SCZ risk in the log-additive model [odds ratio (OR): 1.22; 95% confidence interval (CI): 1.01-1.48; P = 0.040]. We also found that the rs61955196 allele was related with an enhanced risk of SCZ (G>C, OR: 1.22; 95%CI: 1.01-1.47; P = 0.042). However, no associations were observed between rs11917047, rs2239681, or rs3842756 and SCZ risk. Under the optimal genetic model, no significant association of famine with the four SNPs was seen. Though the gene–gene interactions between rs2239681 and rs61955196 were found in GMDR analysis, none of the gene-gene interactions and gene-famine interactions were associated with the risk of SCZ.CONCLUSIONOur study suggested that rs61955196 in ABCB9 is associated with SCZ susceptibility in Northeast Han Chinese, providing insight into genetic effects on SCZ.

A prognostic risk model based on DNA methylation levels of genes and lncRNAs in lung squamous cell carcinoma.

BackgroundRecurrence is a risk factor for the prognosis of lung squamous carcinoma (LUSC). DNA methylation levels of RNAs are also associated with LUSC prognosis. This study aimed to construct a prognostic model with high performance in predicting LUSC prognosis using the methylation levels of lncRNAs and genes.MethodsThe differentially expressed RNAs (DERs) and differentially methylated RNAs (DMRs) between the recurrent and non-recurrent LUSC tissues in The Cancer Genome Atlas (TCGA; training dataset) were identified. Weighted correlation network analysis was performed to identify co-methylation networks. Differentially methylated genes and lncRNAs with opposite expression-methylation levels were used for the screening of prognosis-associated RNAs. The prognostic model was constructed and its performance was validated in the GSE39279 dataset.ResultsA total of 664 DERs and 981 DMRs (including 972 genes) in recurrent LUSC tissues were identified. Three co-methylation modules, including 226 differentially methylated genes, were significantly associated with LUSC. Among prognosis-associated RNAs, 18 DERs/DMRs with opposite methylation-expression levels were included in the methylation prognostic risk model. LUSC patients with high risk scores had a poor prognosis compared with patients who had low risk scores (TCGA: HR = 3.856, 95% CI [2.297–6.471]; GSE39279: HR = 3.040, 95% CI [1.435–6.437]). This model had a high accuracy in predicting the prognosis (AUC = 0.903 and 0.800, respectively), equivalent to the nomogram model inclusive of clinical variables.ConclusionsReferring to the methylation levels of the 16-RNAs might help to predict the survival outcomes in LUSC.

Effects of different energy diets on DNA methylation and mRNA expression in follicle stimulating hormone receptor gene promoter region of Duolang sheep during estrus.

The aim of this study was to study the relationship between the methylation level of the promoter region of follicle-stimulating hormone receptor (FSHR) gene and the mRNA expression of Duolang sheep fed different energy diets. In this study, polyembryo estrus Duolang sheep under different energy levels were selected as the experimental subjects. Dietary nutrient level reference (NY/T 816-2004), medium energy level was 10.88MJ/day, high and low energy groups were increased and decreased by 15% on the basis of medium energy level, respectively 12.51MJ/day, 9.25MJ/day. Through RNA and DNA extraction, qPCR, bisulfitegenomicse-quencing PCR (BSP), sequence matching and other analysis of ovarian tissue of Duolang sheep. The difference of DNA methylation level and mRNA expression of FSHR gene during estrus in Duolang sheep fed with different energy diets was detected. The results showed the expression level of FSHR in high energy group was significantly higher than that in low energy group (P < 0.01), the expression level of FSHR in high energy group was significantly higher than that in medium energy group (P < 0.05), and the expression level of FSHR in medium energy group was significantly higher than that in low energy group (P < 0.05). In the target fragment of the promoter region of the FSHR gene, the methylation rate was 25% in the high energy group, 50% in the normal group, and 75% in the low energy group. This study revealed that different dietary energy levels had certain effects on the FSHR gene DNA methylation level and mRNA expression, and the expression level was negatively correlated with methylation level.

Epigenome-wide association study of mitochondrial genome copy number.

We conducted cohort- and race-specific epigenome-wide association analyses of mitochondrial deoxyribonucleic acid (mtDNA) copy number (mtDNA CN) measured in whole blood from participants of African and European origins in five cohorts (n = 6182, mean age = 57-67years, 65% women). In the meta-analysis of all the participants, we discovered 21 mtDNA CN-associated DNA methylation sites (CpG) (P < 1 × 10-7), with a 0.7-3.0 standard deviation increase (3 CpGs) or decrease (18 CpGs) in mtDNA CN corresponding to a 1% increase in DNA methylation. Several significant CpGs have been reported to be associated with at least two risk factors (e.g. chronological age or smoking) for cardiovascular disease (CVD). Five genes [PR/SET domain 16, nuclear receptor subfamily 1 group H member 3 (NR1H3), DNA repair protein, DNA polymerase kappa and decaprenyl-diphosphate synthase subunit 2], which harbor nine significant CpGs, are known to be involved in mitochondrial biosynthesis and functions. For example, NR1H3 encodes a transcription factor that is differentially expressed during an adipose tissue transition. The methylation level of cg09548275 in NR1H3 was negatively associated with mtDNA CN (effect size = -1.71, P = 4 × 10-8) and was positively associated with the NR1H3 expression level (effect size = 0.43, P = 0.0003), which indicates that the methylation level in NR1H3 may underlie the relationship between mtDNA CN, the NR1H3 transcription factor and energy expenditure. In summary, the study results suggest that mtDNA CN variation in whole blood is associated with DNA methylation levels in genes that are involved in a wide range of mitochondrial activities. These findings will help reveal molecular mechanisms between mtDNA CN and CVD.

Peripheral Blood-Based DNA Methylation of Long Non-Coding RNA H19 and Metastasis-Associated Lung Adenocarcinoma Transcript 1 Promoters are Potential Non-Invasive Biomarkers for Gastric Cancer Detection.

IntroductionThe early diagnosis and detection could greatly improve the clinical outcome of gastric cancer (GC) patients. However, the non-invasive biomarkers for GC detection remain to be identified.MethodWe used online databases (GEPIA, UALCAN, Kaplan-Meier plotter, TIMER, and MEXPRESS) to explore the association between H19 or metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) expression in tissues and the occurrence, development, prognosis, the levels of immune cell infiltration, and methylation of GC; the correlation between mRNA expression and DNA methylation levels of genes were also examined. Methylation levels of H19 or MALAT1 in peripheral blood were compared between 150 GC patients and 100 healthy controls (HCs). Predictive nomograms were constructed among female and male groups for GC diagnosis. The calibration curves, Hosmer–Lemeshow test, and decision curve analysis were also used to examine the nomograms’ predictive ability and clinical values.ResultsUsing multiple online databases, we found that the mRNA expressions of H19 and MALAT1 in tissues were related to the occurrence of GC, and such expressions were associated with immune cell infiltration of GC and negatively correlated with DNA methylation levels of H19 and MALAT1. H19 gene, H19C island, and MALAT1B island, as well as 20 CpG sites were hypermethylated in peripheral blood of GC patients compared with HCs; similar results were also found in female and male groups (P < .05 for all). The combination of H19c3, H19c4, MALAT1b12, and age, as well as the combination of H19b7, H19c1, H19c5, and age in the nomograms could distinguish GC patients from HCs in the female group and male group, respectively.ConclusionWe found statistically significant hypermethylation of H19 and MALAT1 promoters in GC patients, and meaningful sensitivity and specificity of MALAT1 and H19 methylation in discriminating GC and HCs were observed in both female and male groups, which indicates that the peripheral blood-based DNA methylation of H19 and MALAT1 could act as potential non-invasive biomarkers for the diagnosis of GC.

Thioguanine Induces Apoptosis in Triple-Negative Breast Cancer by Regulating PI3K-AKT Pathway.

Triple-negative breast cancer (TNBC) is notoriously difficult to treat due to the lack of biological targets and poor sensitivity to conventional therapies. Chemotherapy is the main clinical therapy, but the effective screening strategy for chemotherapy drugs is poorly investigated. Drug repositioning has been the center of attention in recent years attracting numerous studies. Here, we firstly found multiple common features between leukemia and TNBC by analyzing the global transcriptome profiles based on the transformed comparison data from NCI60. Therefore, we investigated the role of the classic leukemia drug thioguanine (6-TG) in TNBC cancer cells. Our results indicated that 6-TG inhibited cell proliferation and tumor cell progression by suppressing PI3K–AKT pathway via downregulating the DNA methylation level of PTEN. Moreover, apoptosis was induced via the activation of PI3K-AKT downstream TSC1 and the downregulation of methylation levels of DAXX, TNF, FADD and CASP8 etc. These findings indicated 6-TG exerts its anti-tumor effects in vitro and in vivo through regulating the DNA methylation levels of genes involved in PI3K–AKT and apoptosis pathway. Meanwhile, our study suggested that transcriptome-based drug screening has potential implications for breast cancer therapy and drug selection.

Epigenome-wide association study of depression symptomatology in elderly monozygotic twins

Depression is a severe and debilitating mental disorder diagnosed by evaluation of affective, cognitive and physical depression symptoms. Severity of these symptoms strongly impacts individual’s quality of life and is influenced by a combination of genetic and environmental factors. One of the molecular mechanisms allowing for an interplay between these factors is DNA methylation, an epigenetic modification playing a pivotal role in regulation of brain functioning across lifespan. The aim of this study was to investigate if there are DNA methylation signatures associated with depression symptomatology in order to identify molecular mechanisms contributing to pathophysiology of depression. We performed an epigenome-wide association study (EWAS) of continuous depression symptomatology score measured in a cohort of 724 monozygotic Danish twins (346 males, 378 females). Through EWAS analyses adjusted for sex, age, flow-cytometry based blood cell composition, and twin relatedness structure in the data we identified depression symptomatology score to be associated with blood DNA methylation levels in promoter regions of neuropsin (KLK8, p-value = 4.7 × 10−7) and DAZ associated protein 2 (DAZAP2, p-value = 3.13 × 10−8) genes. Other top associated probes were located in gene bodies of MAD1L1 (p-value = 5.16 × 10−6), SLC29A2 (p-value = 6.15 × 10−6) and AKT1 (p-value = 4.47 × 10−6), all genes associated before with development of depression. Additionally, the following three measures (a) DNAmAge (calculated with Horvath and Hannum epigenetic clock estimators) adjusted for chronological age, (b) difference between DNAmAge and chronological age, and (c) DNAmAge acceleration were not associated with depression symptomatology score in our cohort. In conclusion, our data suggests that depression symptomatology score is associated with DNA methylation levels of genes implicated in response to stress, depressive-like behaviors, and recurrent depression in patients, but not with global DNA methylation changes across the genome.

TSPYL5 Depletion Induces Specific Death of ALT Cells through USP7-Dependent Proteasomal Degradation of POT1.

A significant fraction (∼10%) of cancer cells maintain their telomere length via a telomerase-independent mechanism known as alternative lengthening of telomeres (ALT). There are no known molecular, ALT-specific, therapeutic targets. We have identified TSPYL5 (testis-specific Y-encoded-like protein 5) as a PML body component, co-localizing with ALT telomeres and critical for ALT+ cell viability. TSPYL5 was described as an inhibitor of the USP7 deubiquitinase. We report that TSPYL5 prevents the poly-ubiquitination of POT1-a shelterin component-and protects POT1 from proteasomal degradation exclusively in ALT+ cells. USP7 depletion rescued POT1 poly-ubiquitination and loss, suggesting thatthe deubiquitinase activates POT1 E3 ubiquitin ligase(s). Similarly, PML depletion suppressed POT1 poly-ubiquitination, suggesting an interplay between USP7 and PML to trigger POT1 degradation in TSPYL5-depleted ALT+ cells. We demonstrate that ALT telomeres need to be protected from POT1 degradation in ALT-associated PML bodies and identify TSPYL5 as an ALT+ cancer-specific therapeutic target.

DMPK gene DNA methylation levels are associated with muscular and respiratory profiles in DM1

ObjectiveTo assess the effects of dystrophia myotonica protein kinase (DMPK) DNA methylation (DNAme) epivariation on muscular and respiratory profiles in patients with myotonic dystrophy type 1 (DM1).MethodsPhenotypes were assessed with standardized measures. Pyrosequencing of bisulfite-treated DNA was used to quantify DNAme levels in blood from 90 patients with DM1 (adult form). Modal CTG repeat length was assessed using small-pool PCR. The presence of Acil-sensitive variant repeats was also tested.ResultsDNAme levels upstream of the CTG expansion (exon and intron 11) were correlated with modal CTG repeat length (rs = −0.224, p = 0.040; rs = −0.317, p = 0.003; and rs = −0.241, p = 0.027), whereas correlations were observed with epivariations downstream of the CTG repeats (rs = 0.227; p = 0.037). The presence of a variant repeat was associated with higher DNAme levels at multiple CpG sites (up to 10% higher; p = 0.001). Stepwise multiple linear regression modeling showed that DNAme contributed significantly and independently to explain phenotypic variability in ankle dorsiflexor (3 CpGs: p = 0.001, 0.013, and 0.001), grip (p = 0.089), and pinch (p = 0.028) strengths and in forced vital capacity (2 CpGs: p = 0.002 and 0.021) and maximal inspiratory pressure (p = 0.012).ConclusionsIn addition to the CTG repeat length, DMPK epivariations independently explain phenotypic variability in DM1 and could thus improve prognostic accuracy for patients.

11 C]Choline as a Novel PET/CT Biomarker of Liver Cirrhosis: A Prospective Pilot Study

Aim: Choline plays a role in hepatic mitochondrial impairment, oxidative stress and DNA methylation levels of genes involved in lipid metabolism. The aim of this study was to compare findings between patients with liver cirrhosis and subjects with a normal liver on a [11C]choline PET/CT. Methods: This prospective pilot study was conducted between the years 2012-2016. The cohort included 14 patients with prostate cancer (reference group) and 11 patients with cirrhosis attending a tertiary medical center. Demographic, clinical and laboratory data were obtained from the medical files. All participants underwent a dynamic [11C]choline PET/CT (Discovery ST, GE Medical Systems, Milwaukee WI). We compared the maximal standard uptake values (SUVmax) and the area under the curve (AUC) at 1110 seconds in both groups. Results: The mean age of the cirrhosis group (63.4% men) was 68.4 ± 10.7 and the control group, 69.7 ± 7.3 years. The mean SUVmax was significantly higher in the cirrhosis group than in the controls (right lobe, 10.06 ± 12 vs. 6.3 ± 1.6, P=0.011; left lobe, 8.6 ± 11.6 vs. 5.4 ± 0.9, P=0.024; spleen 17.99 ± 27.8 vs. 13.4 ± 2.6, P=0.027; kidney, 35.9 ± 59.5 vs. 19.3 ± 4.8, P=0.025). The corresponding AUC values at 1110 seconds was significantly distinguished between the groups (right lobe, 13538 ± 20020 vs. 8427.3 ± 1557.9, P=0.026; left lobe 12304 ± 18871 vs. 6878.9 ± 1294.3, P=0.024; spleen, 12875 ± 17930 vs. 8263.9 ± 1279.2, P=0.023; kidney, 24623 ± 36025 vs. 13667 ± 3873.9, P=0.032). No correlations were found between the clinical characteristics and the imaging-derived parameters in the patients with cirrhosis. Conclusions: Our findings suggest a role for [11C]choline PET/CT as a noninvasive biomarker of cirrhosis. Further larger-scale studies are needed to confirm these observations.

Occupational exposure to gases/fumes and mineral dust affect DNA methylation levels of genes regulating expression.

Many workers are daily exposed to occupational agents like gases/fumes, mineral dust or biological dust, which could induce adverse health effects. Epigenetic mechanisms, such as DNA methylation, have been suggested to play a role. We therefore aimed to identify differentially methylated regions (DMRs) upon occupational exposures in never-smokers and investigated if these DMRs associated with gene expression levels. To determine the effects of occupational exposures independent of smoking, 903 never-smokers of the LifeLines cohort study were included. We performed three genome-wide methylation analyses (Illumina 450 K), one per occupational exposure being gases/fumes, mineral dust and biological dust, using robust linear regression adjusted for appropriate confounders. DMRs were identified using comb-p in Python. Results were validated in the Rotterdam Study (233 never-smokers) and methylation-expression associations were assessed using Biobank-based Integrative Omics Study data (n = 2802). Of the total 21 significant DMRs, 14 DMRs were associated with gases/fumes and 7 with mineral dust. Three of these DMRs were associated with both exposures (RPLP1 and LINC02169 (2×)) and 11 DMRs were located within transcript start sites of gene expression regulating genes. We replicated two DMRs with gases/fumes (VTRNA2-1 and GNAS) and one with mineral dust (CCDC144NL). In addition, nine gases/fumes DMRs and six mineral dust DMRs significantly associated with gene expression levels. Our data suggest that occupational exposures may induce differential methylation of gene expression regulating genes and thereby may induce adverse health effects. Given the millions of workers that are exposed daily to occupational exposures, further studies on this epigenetic mechanism and health outcomes are warranted.

Epigenetic modulation of intestinal Na+/H+ exchanger-3 expression.

Na+/H+ exchanger-3 (NHE3) is crucial for intestinal Na+ absorption, and its reduction has been implicated in infectious and inflammatory bowel diseases (IBD)-associated diarrhea. Epigenetic mechanisms such as DNA methylation are involved in the pathophysiology of IBD. Whether changes in DNA methylation are involved in modulating intestinal NHE3 gene expression is not known. Caco-2 and HuTu 80 cells were used as models of human intestinal epithelial cells. Normal C57/BL6, wild-type, or growth arrest and DNA damage-inducible 45b (GADD45b) knockout (KO) mice were used as in vivo models. NHE3 gene DNA methylation levels were assessed by MBDCap (MethyMiner) assays. Results demonstrated that in vitro methylation of NHE3 promoter construct (p-1509/+127) cloned into a cytosine guanine dinucleotide-free lucia vector decreased the promoter activity in Caco-2 cells. DNA methyltransferase inhibitor 5-azacytidine (10 μM, 24 h) caused a significant decrease in DNA methylation of the NHE3 gene and concomitantly increased NHE3 expression in Caco-2 cells. Similarly, 5-azacytidine treatment increased NHE3 mRNA levels in HuTu 80 cells. 5-Azacytidine treatment for 3 wk (10 mg/kg body wt ip, 3 times/wk) also resulted in an increase in NHE3 expression in the mouse ileum and colon. Small-interfering RNA knockdown of GADD45b (protein involved in DNA demethylation) in Caco-2 cells decreased NHE3 mRNA expression. Furthermore, there was a significant decrease in NHE3 mRNA and protein expression in the ileum and colon of GADD45b KO mice. Our findings demonstrate that NHE3 gene expression is regulated by changes in its DNA methylation. NEW & NOTEWORTHY Our studies for the first time demonstrate that Na+/H+ exchanger-3 gene expression is regulated by an epigenetic mechanism involving DNA methylation.

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.

Abstract 4439: Relationship between DNA methylation of TET genes and levels of 5-methyl-cytosine and 5-hydroxymethyl-cytosine in hepatocellular carcinoma

Abstract Long-term survival after hepatocellular carcinoma (HCC) diagnosis has clearly improved as a result of treatment by means of a liver transplant. However, a large number of cases do not meet the transplant criteria and undergo resection. In these patients, loss of global DNA hydroxymethylation has been linked to worse prognosis, but no association has been found in transplant patients. Differential epigenetic processing is a possible reason for this, and has not been previously explored. The process of DNA demethylation is mediated by the family of ten-eleven translocation (TET) proteins, that catalyze the conversion of 5-methylcytosine (5mC) into 5-hydroxymethylcytosine (5hmC). There are three different TET protein variants: TET1, TET2 and TET3. Studies have shown that expression of at least TET1 is epigenetically regulated. In this study we investigated the association between DNA methylation of TET genes and global DNA methylation and hydroxymethylation in 66 tumor (T) and non-tumor (NT) paired samples of individuals treated at Columbia Presbyterian Medical Center in New York. Levels of 5mC and 5hmC were determined by UPLC/MS/MS, and DNA methylation of the TET promoters was obtained from Illumina Infinium 450k CpG array data. Mean levels of 5mC (T = 3.15±0.49% vs. NT = 3.80±0.12% (p&amp;lt;0.0001) and 5hmC (T = 0.11±0.04% vs. NT = 0.23±0.07%, p&amp;lt;0.0001) in tumor tissues were statistically significantly lower than in non-tumor tissues. In contrast, average DNA methylation levels of TET1 (T = 0.40±0.06% vs. NT = 0.35±0.02%, p&amp;lt;0.0001) were statistically significantly lower in non-tumor tissues than in tumor tissues. However, this was not the case for TET2 or TET3. TET1 DNA methylation levels negatively correlate with global DNA 5mC levels in tumor and non-tumor tissue (Spearman correlation coefficient: T = -0.47 (p = 0.002) and NT = -0.60 (p&amp;lt;0.0001)). In contrast, associations between gene DNA methylation and 5hmC DNA levels were different for the tumor and non-tumor tissue samples. 5hmC levels positively and statistically significantly correlated with TET1 and TET2 DNA methylation (Spearman corr. coef TET1 NT = 0.57 (p&amp;lt;0.0001), and TET2 NT = 0.56 (p&amp;lt;0.0001)) in non-tumor tissue while there was a statistically significant negative correlation with TET3 (Spearman corr. coef. TET3 T = -0.37 (p = 0.005)). No other associations were found between 5mC and 5hmC levels and TET gene DNA methylation levels. These findings suggest higher expression of TET1 correlates with a hypomethylated state and that the accumulation of 5hmC possibly results from a downregulation of TET1 and TET2 in non-tumor tissue. HCC carcinogenesis alters this process, and while further studies need to be conducted, TET3 seems to play a larger role in tumor cells. Our sample size was limited, but these results suggest that future research of 5hmC might help elucidate HCC underlying molecular events and aid in the design of prognostic markers. Citation Format: Lissette Delgado-Cruzata, Hui-Chen Wu, Jin Shen, Tiffany Thomas, Abby B. Siegel, Yu-Jing Zhang, Abhishek Goyal, Christine C. Hsu, Helen E. Remotti, Regina M. Santella. Relationship between DNA methylation of TET genes and levels of 5-methyl-cytosine and 5-hydroxymethyl-cytosine in hepatocellular carcinoma. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4439.