Alu Repetitive Elements Research Articles

Toward Deep Learning Approaches for Learning Structure Motifs and Classifying Biological Sequences From RNA A-to-I Editing Events

RNA editing is a post-transcriptional RNA sequence modification that alters the mature RNA sequence from its template DNA sequences. RNA editing events are critical in various biological and biochemical mechanisms, and can expand the transcriptomic and proteomic diversity from altered gene regulation to mutations. A-to-I RNA editing is now being vastly detected and quantified on a global scale and gained much attention. A deeper understanding of this process with insufficient genomic annotations and prior knowledge-based filtering steps, such as high-throughput next-generation sequencing techniques, are needed, in addition to data regarding whether an editing location is located in one of the following three main classes of RNA editing sites: ALU in Alu repetitive elements, REP in non-Alu repetitive elements, and NONREP in non-repetitive regions. This study proposes deep learning approaches to ameliorate these issues by learning motif patterns and identifying regions of A-to-I editing events in biological sequences. Using datasets derived from the public REDI portal, 300,000 editing sites were equally divided and used for learning sequence and structure motifs visualized by convolutional kernels. We explored the RNA editing pattern changes using information of positional and class enrichment of learned motifs in the three aforementioned classes. We demonstrated that these newly investigated approaches using large-scale RNA sequencing data offer excellent classification accuracy with a well-optimized convolutional neural network and recurrent neural network classifiers that obtained average area under curves of 0.960 and 0.962, respectively. The findings further decipher the principles underlying RNA editing events and will facilitate more effective RNA sequencing research.

Dietary intake of one-carbon metabolism nutrients and DNA methylation in peripheral blood

Folate and other one-carbon metabolism nutrients are essential to enable DNA methylation to occur, but the extent to which their dietary intake influences methylation in adulthood is unclear. We assessed associations between dietary intake of these nutrients and DNA methylation in peripheral blood, overall and at specific genomic locations. We conducted a cross-sectional study using baseline data and samples from 5186 adult participants in the Melbourne Collaborative Cohort Study (MCCS). Nutrient intake was estimated from a food-frequency questionnaire. DNA methylation was measured by using the Illumina Infinium HumanMethylation450 BeadChip array (HM450K). We assessed associations of intakes of folate, riboflavin, vitamins B-6 and B-12, methionine, choline, and betaine with methylation at individual cytosine-guanine dinucleotides (CpGs), and with median (genome-wide) methylation across all CpGs, CpGs in gene bodies, and CpGs in gene promoters. We also assessed associations with methylation at long interspersed nuclear element 1 (LINE-1), satellite 2 (Sat2), and Arthrobacter luteus restriction endonuclease (Alu) repetitive elements for a subset of participants. We used linear mixed regression, adjusting for age, sex, country of birth, smoking, energy intake from food, alcohol intake, Mediterranean diet score, and batch effects to assess log-linear associations with dietary intake of each nutrient. In secondary analyses, we assessed associations with low or high intakes defined by extreme quintiles. No evidence of log-linear association was observed at P<10-7 between the intake of one-carbon metabolism nutrients and methylation at individual CpGs. Low intake of riboflavin was associated with higher methylation at CpG cg21230392 in the first exon of PROM1 (P=5.0×10-8). No consistent evidence of association was observed with genome-wide or repetitive element measures of methylation. Our findings suggest that dietary intake of one-carbon metabolism nutrients in adulthood, as measured by a food-frequency questionnaire, has little association with blood DNA methylation. An association with low intake of riboflavin requires replication in independent cohorts. This study was registered at http://www.clinicaltrials.gov as NCT03227003.

Prenatal phthalate exposure, birth outcomes and DNA methylation of Alu and LINE-1 repetitive elements: A pilot study in China

BackgroundEpigenetic mechanisms, such as altered DNA methylation, may participate in the relationship between prenatal phthalate exposure and adverse birth outcomes. ObjectiveTo explore the mediation effect of DNA methylation in the associations of phthalate exposure before delivery with birth outcomes in a Chinese cohort. MethodsEight phthalate metabolites in maternal urine before delivery and DNA methylation of Alu and long interspersed nucleotide elements (LINE-1) in cord blood were determined among 106 mother-infant pairs. General additive models were used to assess the associations of maternal urinary phthalate metabolites with birth outcomes and DNA methylation; the mediating role of DNA methylation in cord blood was evaluated by mediation analysis. ResultsWe found sex-specific associations between prenatal phthalate exposure and birth outcomes and DNA methylation of cord blood. For example, the molar sum of di-2-(ethylhexyl) phthalate (∑DEHPm) metabolites in maternal urine was positively associated with gestational age among male newborns only (P < 0.05); maternal urinary monobenzyl phthalate (MBzP) was negatively associated with Alu methylation among female newborns only (P < 0.05). Mediation analysis did not find that methylation of Alu and LINE-1 to be a direct mediator in the relationships between maternal urinary phthalate metabolites before delivery and birth outcomes. ConclusionPrenatal exposure to certain phthalates was associated with altered birth outcomes and decreased repetitive element methylation of newborns. However, the altered birth outcomes exerted by prenatal phthalate exposure does not seem to be directly mediated through repetitive element methylation in cord blood.

Methylation status of alu repetitive elements in children with tuberculosis disease

Investigation of DNA methylation in Alu repetitive elements (REs) was shown to be a promising field to explore transcriptional changes in human genome under disease condition. To scrutinize the association between Alu methylation and tuberculosis (TB) disease in children, the difference in Alu DNA methylation level was compared with healthy controls. Whole-blood genomic DNA from 36 TB-infected children and 32 healthy controls was isolated, and the level of Alu repeat DNA methylation was examined by methylation-specific polymerase chain reaction. The median Alu methylation level in TB patients was 30% (Interquartile range [IQR], 25-30%), whereas in healthy controls, it was 75% (IQR, 50-75%) (P < 0.0001). The median level of DNA methylation of Alu RE in TB cases was significantly lower than healthy controls. Receiver operating characteristic curve analysis showed that the area under the curve for diagnosis was 0.969 (95% confidence interval, 0.936-1) (P < 0.0001), with 100% sensitivity and 84% specificity. Our results point out that detection of Alu DNA methylation in whole-blood DNA may be clinically useful tool for the diagnosis and prognosis of TB disease in children.

Prenatal urinary polycyclic aromatic hydrocarbon metabolites, global DNA methylation in cord blood, and birth outcomes: A cohort study in China.

Prenatal exposure to polycyclic aromatic hydrocarbons (PAHs) is a potential risk factor for adverse birth outcomes. Epigenetic mechanisms may play a key role in which PAHs exert its effects. Our study aimed to examine whether prenatal PAH exposure was associated with adverse birth outcomes and altered DNA methylation and to explore potential mediating roles of DNA methylation. Ten urinary PAH metabolites were measured from 106 pregnant women during late pregnancy in a Chinese cohort study. Cord blood DNA methylation in long interspersed nucleotide element-1 (LINE-1) and Alu repetitive elements as surrogates of global DNA methylation was analyzed by bisulfite pyrosequencing. Multivariable linear regression was used to estimate the associations of urinary PAH metabolites with birth outcomes and DNA methylation, and a mediation analysis was also conducted. Prenatal urinary 2-hydroxynaphthalene (2-OHNa), ∑OHNa (sum of 1- and 2-OHNa), and sum of monohydroxy-PAH (∑OH-PAHs) were associated with lower birth length (e.g.,-0.80%, 95% CI:-1.39%,-0.20% for the third vs. first tertile of 2-OHNa; p for trend=0.01). Prenatal urinary 2-OHNa and 1-hydroxyphenanthrene (1-OHPh) were associated with lower Alu and LINE-1 methylation (e.g.,-1.88%, 95% CI:-3.73%,-0.10% for the third vs. first tertile tertile of 2-OHNa in Alu methylation; p for trend=0.04). Mediation analysis failed to show a mediator effect of global DNA methylation in the association between prenatal urinary OH-PAHs and birth outcomes. Prenatal specific PAH exposures are associated with decreased birth length and global DNA methylation. However, global DNA methylation does not mediate the associations of prenatal PAH exposure with birth outcomes. Further studies are needed to confirm the results.

Evaluation of Streck BCT and PAXgene Stabilised Blood Collection Tubes for Cell-Free Circulating DNA Studies in Plasma

Blood samples for studies of circulating DNA in disease are often collected in clinical settings where prompt processing of samples is not possible. In order to avoid problems associated with leukocyte lysis after prolonged blood storage, stabilised blood tubes have been developed containing preservatives that prevent cell lysis. We evaluated Streck BCT tubes and PAXgene ccfDNA tubes, as well as standard EDTA blood collection tubes, in terms of DNA yield and fragment size. Blood was collected in EDTA, Streck BCT or PAXgene ccfDNA tubes and stored for 1h at 4°C, or 4days at room temperature. DNA was extracted using the QIAamp Circulating Nucleic Acids kit, and visualised on an agarose gel or quantitated by qPCR. Ratios of a 247-base and a 115-base amplicon of the Alu repetitive element were used to infer size distribution. While plasma DNA in EDTA tube blood samples increased by ~10- to 20-fold after 4days of storage at room temperature, both Streck BCT tubes and PAXgene ccfDNA tubes maintained stable plasma DNA concentrations. A slight decrease in DNA yield following 1h of blood storage at 4°C was observed in Streck BCT and PAXgene ccfDNA tubes relative to EDTA tubes. This decrease was reversed by increasing the proteinase digest step of the DNA extraction protocol to 60min, as recommended by Streck tube product literature. Visualisation of the extracted DNA on an agarose gel showed that after 4days of room temperature storage, samples collected in EDTA tubes contained abundant high-molecular weight DNA, which was partially fragmented in a ladder pattern. A slight increase in high-molecular weight DNA in samples stored for 4days at room temperature in Streck BCT tubes was also observed, but this was not reflected in a change in large and small Alu fragment ratios as measured by qPCR. Tubes containing preservative to prevent cell lysis can extend the scope for blood collection in clinical settings; however, slight differences between samples collected in different tube types underscore the requirement for standardised protocols, as well as attention to sample handling.

Cell-free circulating DNA integrity is an independent predictor of impending breast cancer recurrence.

Non-invasive blood-based molecule markers are evaluated as promising biomarkers these days. Here we investigated the potential of cell-free circulating DNA Integrity (cfDI) as blood-based marker for the prediction of recurrence during the follow-up of breast cancer patients within a prospective study cohort. cfDI was determined in plasma of 212 individuals, by measuring ALU and LINE1 repetitive DNA elements using quantitative PCR. A significant decrease of cfDI in recurrent breast cancer patients was observed. The group of patients who had impending recurrence during the follow-up had significant lower cfDI compared to the group of non-recurrent patients (P < 0.001 for ALU and LINE1 cfDI). cfDI could differentiate recurrent breast cancer patients from non-recurrent breast cancer subjects (area under the curve, AUC = 0.710 for ALU and 0.704 for LINE1). Univariate and multivariate analysis confirmed a significant association of recurrence and cfDI. Breast cancer patients with a lower cfDI had a much higher risk to develop recurrence than the patients with a higher cfDI (P = 0.020 for ALU cfDI and P = 0.019 for LINE1 cfDI, respectively). Further we show that cfDI is an independent predictor of breast cancer recurrence. In combination with other molecular markers, cfDI might be a useful biomarker for the prediction for breast cancer recurrence in clinic utility. We propose that cfDI might also be useful for the prediction of recurrence during the follow-up of other cancers.

Prenatal exposure to drinking water disinfection by-products and DNA methylation in cord blood.

Maternal exposure to drinking water disinfection by-products (DBPs) during pregnancy has been related to adverse birth outcomes. While experimental studies have shown that exposure to DBPs induce DNA hypomethylation, evidence from humans is limited. This study aimed to examine whether prenatal exposure to drinking water DBPs was associated with DNA methylation in cord blood. Maternal biomarkers of exposure to drinking water DBPs including blood trihalomethanes [THMs, including chloroform (TCM), bromodichloromethane (BDCM), dibromochloromethane (DBCM), and bromoform (TBM)] and urinary trichloroacetic acid (TCAA) were measured during late pregnancy. DNA methylation in Alu and long interspersed nucleotide element-1 (LINE-1) repetitive elements from cord blood samples (n=115) was measured by pyrosequencing. We used multivariable linear regression to estimate the associations of DNA methylation in cord blood with maternal blood THMs and urinary TCAA. We found no statistically significant association between urinary TCAA and DNA methylation. However, we found that blood TBM was associated with decreased Alu methylation (-0.39%; 95% CI: -0.83%, 0.05% for the highest versus lowest exposure group; p for trend=0.08) and decreased LINE-1 methylation (-1.27%; 95% CI: -2.91%, 0.36% for the highest versus lowest exposure group; p for trend=0.06). Our results suggest that prenatal exposure to drinking water TBM is associated with DNA hypomethylation in cord blood. However, further studies are needed to confirm our findings.

Long-term outdoor air pollution and DNA methylation in circulating monocytes: results from the Multi-Ethnic Study of Atherosclerosis (MESA).

BackgroundDNA methylation may mediate effects of air pollution on cardiovascular disease. The association between long-term air pollution exposure and DNA methylation in monocytes, which are central to atherosclerosis, has not been studied. We investigated the association between long-term ambient air pollution exposure and DNA methylation (candidate sites and global) in monocytes of adults (aged ≥55).MethodsOne-year average ambient fine particulate matter (PM2.5) and oxides of nitrogen (NOX) concentrations were predicted at participants’ (n = 1,207) addresses using spatiotemporal models. We assessed DNA methylation in circulating monocytes at 1) 2,713 CpG sites associated with mRNA expression of nearby genes and 2) probes mapping to Alu and LINE-1 repetitive elements (surrogates for global DNA methylation) using Illumina’s Infinium HumanMethylation450 BeadChip. We used linear regression models adjusted for demographics, smoking, physical activity, socioeconomic status, methyl-nutrients, and technical variables. For significant air pollution-associated methylation sites, we also assessed the association between expression of gene transcripts previously associated with these CpG sites and air pollution.ResultsAt a false discovery rate of 0.05, five candidate CpGs (cg20455854, cg07855639, cg07598385, cg17360854, and cg23599683) had methylation significantly associated with PM2.5 and none were associated with NOX. Cg20455854 had the smallest p-value for the association with PM2.5 (p = 2.77 × 10−5). mRNA expression profiles of genes near three of the PM2.5-associated CpGs (ANKHD1, LGALS2, and ANKRD11) were also significantly associated with PM2.5 exposure. Alu and LINE-1 methylation were not associated with long-term air pollution exposure.ConclusionsWe observed novel associations between long-term ambient air pollution exposure and site-specific DNA methylation, but not global DNA methylation, in purified monocytes of a multi-ethnic adult population. Epigenetic markers may provide insights into mechanisms underlying environmental factors in complex diseases like atherosclerosis.Electronic supplementary materialThe online version of this article (doi:10.1186/s12940-016-0202-4) contains supplementary material, which is available to authorized users.

DNA hypomethylation and its mediation in the effects of fine particulate air pollution on cardiovascular biomarkers: A randomized crossover trial

BackgroundShort-term exposure to fine particulate matter (PM2.5) air pollution has been associated with altered DNA methylation in observational studies, but it remains unclear whether this change mediates the effects on cardiovascular biomarkers. ObjectiveTo examine the impact of ambient PM2.5 on gene-specific DNA methylation and its potential mediation in the acute effects of PM2.5 on cardiovascular biomarkers. MethodsWe designed a randomized, double-blind crossover trial using true or sham air purifiers for 48h among 35 healthy college students in Shanghai, China, in 2014. We measured blood global methylation estimated in long interspersed nucleotide element-1 (LINE‑1) and Alu repetitive elements, methylation in ten specific genes, and ten cardiovascular biomarkers. We used linear mixed-effect models to examine the associations between PM2.5 and methylation. We also performed causal mediation analyses to evaluate the potential mediation of methylation in the associations between PM2.5 and biomarkers. ResultsAir purification increased DNA methylation in repetitive elements and all candidate genes. An IQR increase (64μg/m3) in PM2.5 was significantly associated with reduction of methylation in LINE-1 (1.44%), one pro-inflammatory gene (CD40LG, 9.13%), two pro-coagulant genes (F3, 15.20%; SERPINE1, 3.69%), and two pro-vasoconstriction genes (ACE, 4.64%; EDN1, 9.74%). There was a significant mediated effect (17.82%, P=0.03) of PM2.5 on sCD40L protein through CD40LG hypomethylation. Hypomethylation in other candidate genes generally showed positive but non-significant mediation. ConclusionsThis intervention study provided robust human evidence that ambient PM2.5 could induce rapid decreases in DNA methylation and consequently partly mediate its effects on cardiovascular biomarkers.

Maternal phthalate exposure during pregnancy is associated with DNA methylation of LINE-1 and Alu repetitive elements in Mexican-American children

Phthalates are frequently used in personal care products and plasticizers and phthalate exposure is ubiquitous in the US population. Exposure to phthalates during critical periods in utero has been associated with a variety of adverse health outcomes but the biological mechanisms linking these exposures with disease are not well characterized. In this study, we examined the relationship of in utero phthalate exposure with repetitive element DNA methylation, an epigenetic marker of genome instability, in children from the longitudinal birth cohort CHAMACOS. Methylation of Alu and long interspersed nucleotide elements (LINE-1) was determined using pyrosequencing of bisulfite-treated DNA isolated from whole blood samples collected from newborns and 9 year old children (n=355). Concentrations of eleven phthalate metabolites were measured in urine collected from pregnant mothers at 13 and 26 weeks gestation. We found a consistent inverse association between prenatal concentrations of monoethyl phthalate, the most frequently detected urinary metabolite, with cord blood methylation of Alu repeats (β(95%CI): −0.14 (−0.28,0.00) and −0.16 (−0.31, −0.02)) for early and late pregnancy, respectively, and a similar but weaker association with LINE-1 methylation. Additionally, increases in urinary concentrations of di-(2-ethylhexyl) phthalate metabolites during late pregnancy were associated with lower levels of methylation of Alu repeats in 9 year old blood (significant p-values ranged from 0.003 to 0.03). Our findings suggest that prenatal exposure to some phthalates may influence differences in repetitive element methylation, highlighting epigenetics as a plausible biological mechanism through which phthalates may affect health.

DNA methylation of LINE-1 and Alu repetitive elements in relation to sex hormones and pubertal timing in Mexican-American children.

BackgroundThe molecular mechanisms linking environmental exposures to earlier pubertal development are not well characterized. Epigenetics may play an important role, but data on the relationship between epigenetic marks and puberty, particularly in humans, is limited.MethodsWe used pyrosequencing to measure Alu and long interspersed nucleotide elements (LINE-1) methylation in DNA isolated from whole blood samples collected from newborns and 9-year-old children (n=266). Tanner staging was completed six times between ages 9 and 12 years to determine pubertal status, and hormone levels were measured in 12-year-old boys.ResultsAmong girls, we observed a suggestive trend of increased odds of breast and pubic hair development with higher Alu and LINE-1 methylation in 9-year-old blood, respectively. The strongest association identified was an inverse association of LINE-1 methylation in 9-year-old girls with odds of experiencing menarche by age 12 (OR(95%CI): 0.63(0.46,0.87); p=0.005). We observed a consistent inverse relationship for Alu and LINE-1 methylation at 9 years with luteinizing hormone (LH), testosterone and follicle stimulating hormone levels in boys but it was only significant between LINE-1 and LH.ConclusionDNA methylation of Alu and LINE-1 may be involved in puberty initiation and development. This relationship should be confirmed in future studies.

A knowledgebase of the human Alu repetitive elements

Alu elements are the most abundant retrotransposons in the human genome with more than one million copies. Alu repeats have been reported to participate in multiple processes related with genome regulation and compartmentalization. Moreover, they have been involved in the facilitation of pathological mutations in many diseases, including cancer. The contribution of Alus and other repeats in genomic regulation is often overlooked because their study poses technical and analytical challenges hardly attainable with conventional strategies. Here we propose the integration of ontology-based semantic methods to query a knowledgebase for the human Alus.The knowledgebase for the human Alus leverages Sequence (SO) and Gene Ontologies (GO) and is devoted to address functional and genetic information in the genomic context of the Alus. For each Alu element, the closest gene and transcript are stored, as well their functional annotation according to GO, the state of the chromatin and the transcription factors binding sites inside the Alu. The model uses Web Ontology Language (OWL) and Semantic Web Rule Language (SWRL). As a case of use and to illustrate the utility of the tool, we have evaluated the epigenetic states of Alu repeats associated with gene promoters according to their transcriptional activity.The ontology is easily extendable, offering a scaffold for the inclusion of new experimental data. The RDF/XML formalization is freely available at http://aluontology.sourceforge.net/.

Prospective study of DNA methylation at LINE-1 and Alu in peripheral blood and the risk of prostate cancer.

Evidence suggests that global blood DNA methylation levels may be associated with the risk of various cancers, but no studies have evaluated this relationship for prostate cancer. We used pyrosequencing to quantify DNA methylation levels at the long interspersed nuclear element 1 (LINE-1) and Alu repetitive elements in pre-diagnostic blood samples from 694 prostate cancer cases and 703 controls from the Prostate, Lung, Colorectal, and Ovarian Cancer Screening Trial. We evaluated prostate cancer risk associated with the mean methylation level for each element using logistic regression, adjusting for potential confounders. We did not observe a significant association with prostate cancer for LINE-1 [odds ratio (OR) for the highest compared to the lowest quartile = 1.01, 95% confidence interval (CI): 0.73-1.39, Ptrend = 0.99] or Alu (OR = 0.94, 95% CI: 0.68-1.29, Ptrend = 0.69) methylation levels overall. However, for Alu, we observed that higher DNA methylation levels were associated with a significant increased risk for those diagnosed 4 or more years after blood draw (OR = 2.26, 95% CI: 1.27-4.00, Ptrend = 4.4 × 10(-3) ). In contrast, there was no association for those diagnosed 2 (OR = 1.13, 95% CI: 0.67-1.90, Ptrend = 0.64) or 3 years after draw (OR = 1.22, 95% CI: 0.71-2.07, Ptrend = 0.32), and a decreased risk for those diagnosed less than 2 years after draw (OR = 0.40, 95% CI: 0.25-0.65, Ptrend = 3.8 × 10(-5) ; Pheterogeneity = 5.3 × 10(-6) ). Although LINE-1 DNA methylation levels were not associated with prostate cancer, we observed an association for Alu that varied by time from blood draw to diagnosis. Our study suggests that elevated Alu blood DNA methylation levels several years before diagnosis may be associated with an increased prostate cancer risk.

Decreased serum cell-free DNA levels in rheumatoid arthritis.

PurposeRecent studies have demonstrated that serum/plasma DNA and RNA molecules in addition to proteins can serve as biomarkers. Elevated levels of these nucleic acids have been found not only in acute, but also in chronic conditions, including autoimmune diseases. The aim of this study was to assess cell-free DNA (cfDNA) levels in sera of rheumatoid arthritis (RA) patients compared to controls.MethodscfDNA was extracted from sera of patients with early and established RA, relapsing-remitting multiple sclerosis patients (RRMS) and healthy subjects, and its concentration was determined by quantitative PCR using two amplicons, Alu115 and β-actin205, corresponding to Alu repetitive elements and the β-actin single-copy gene, respectively. Serum DNase activity was measured by a single radial enzyme diffusion method.ResultsReduced levels of cfDNA were observed in patients with established RA in comparison with healthy controls, early RA patients and RRMS patients. There were no significant differences in cfDNA concentration between healthy controls, early RA and RRMS patients. Total DNase activity appeared to be similar in the sera of all tested groups.ConclusionsOur results demonstrate that cfDNA levels are strongly reduced in the sera of established RA patients, which is not caused by changes in DNase activity. Measurement of cfDNA can distinguish established RA patients from early RA patients. Thus, cfDNA may serve as a biomarker in RA.Electronic supplementary materialThe online version of this article (doi:10.1007/s13317-015-0066-6) contains supplementary material, which is available to authorized users.

Alu-mediated diverse and complex pathogenic copy-number variants within human chromosome 17 at p13.3.

Alu repetitive elements are known to be major contributors to genome instability by generating Alu-mediated copy-number variants (CNVs). Most of the reported Alu-mediated CNVs are simple deletions and duplications, and the mechanism underlying Alu-Alu-mediated rearrangement has been attributed to non-allelic homologous recombination (NAHR). Chromosome 17 at the p13.3 genomic region lacks extensive low-copy repeat architecture; however, it is highly enriched for Alu repetitive elements, with a fraction of 30% of total sequence annotated in the human reference genome, compared with the 10% genome-wide and 18% on chromosome 17. We conducted mechanistic studies of the 17p13.3 CNVs by performing high-density oligonucleotide array comparative genomic hybridization, specifically interrogating the 17p13.3 region with ∼150 bp per probe density; CNV breakpoint junctions were mapped to nucleotide resolution by polymerase chain reaction and Sanger sequencing. Studied rearrangements include 5 interstitial deletions, 14 tandem duplications, 7 terminal deletions and 13 complex genomic rearrangements (CGRs). Within the 17p13.3 region, Alu-Alu-mediated rearrangements were identified in 80% of the interstitial deletions, 46% of the tandem duplications and 50% of the CGRs, indicating that this mechanism was a major contributor for formation of breakpoint junctions. Our studies suggest that Alu repetitive elements facilitate formation of non-recurrent CNVs, CGRs and other structural aberrations of chromosome 17 at p13.3. The common observation of Alu-mediated rearrangement in CGRs and breakpoint junction sequences analysis further demonstrates that this type of mechanism is unlikely attributed to NAHR, but rather may be due to a recombination-coupled DNA replicative repair process.

Short-term diesel exhaust inhalation in a controlled human crossover study is associated with changes in DNA methylation of circulating mononuclear cells in asthmatics.

BackgroundChanges in DNA methylation have been associated with traffic-related air pollution in observational studies, but the specific mechanisms and temporal dynamics therein have not been explored in a controlled study of asthmatics. In this study, we investigate short-term effects of diesel exhaust inhalation on DNA methylation levels at CpG sites across the genome in circulating blood in asthmatics.MethodsA double-blind crossover study of filtered air and diesel exhaust exposures was performed on sixteen non-smoking asthmatic subjects. Blood samples were collected pre-exposure, and then 6 and 30 hours post-exposure. Peripheral blood mononuclear cell DNA methylation was interrogated using the Illumina Infinium HumanMethylation450 Array. Exposure-related changes in DNA methylation were identified. In addition, CpG sites overlapping with Alu or LINE1 repetitive elements and candidate microRNA loci were also analyzed.ResultsDNA methylation at 2827 CpG sites were affected by exposure to diesel exhaust but not filtered air; these sites enriched for genes involved in protein kinase and NFkB pathways. CpG sites with significant changes in response to diesel exhaust exposure primarily became less methylated, with a site residing within GSTP1 being among the significant hits. Diesel exhaust-associated change was also found for CpG sites overlapping with Alu and LINE1 elements as well as for a site within miR-21.ConclusionShort-term exposure to diesel exhaust resulted in DNA methylation changes at CpG sites residing in genes involved in inflammation and oxidative stress response, repetitive elements, and microRNA. This provides plausibility for the role of DNA methylation in pathways by which airborne particulate matter impacts gene expression and offers support for including DNA methylation analysis in future efforts to understand the interactions between environmental exposures and biological systems.Electronic supplementary materialThe online version of this article (doi:10.1186/s12989-014-0071-3) contains supplementary material, which is available to authorized users.

Development of two highly sensitive forensic sex determination assays based on human DYZ1 and Alu repetitive DNA elements.

Sex determination is a critical component of forensic identification, the standard genetic method for which is detection of the single copy amelogenin gene that has differing homologues on the X and Y chromosomes. However, this assay may not be sensitive enough when DNA samples are minute or highly compromised, thus other strategies for sex determination are needed. In the current research, two ultrasensitive sexing assays, based on real-time PCR and pyrosequencing, were developed targeting the highly repetitive elements DYZ1 on the Y chromosome and Alu on the autosomes. The DYZ1/Alu strategy was compared to amelogenin for overall sensitivity based on high molecular weight and degraded DNA, followed by assaying the sex of 34 touch DNA samples and DNA from 30 hair shafts. The real-time DYZ1/Alu assay proved to be approximately 1500 times more sensitive than its amelogenin counterpart based on high molecular weight DNA, and even more sensitive when sexing degraded DNA. The pyrosequencing DYZ1/Alu assay correctly sexed 26 of the touch DNAs, compared to six using amelogenin. Hair shaft DNAs showed equally improved sexing results using the DYZ1/Alu assays. Overall, both DYZ1/Alu assays were far more sensitive and accurate than was the amelogenin assay, and thus show great utility for sexing poor quality and low quantity DNA evidence.

Association between hypermethylation of DNA repetitive elements in white blood cell DNA and pancreatic cancer

Pancreatic cancer is a leading cause of cancer-related deaths worldwide. Methylation of DNA may influence risk or be a marker of early disease. The aim of this study was to measure the association between methylation of three DNA repetitive elements in white blood cell (WBC) DNA and pancreatic cancer.DNA from WBCs of pancreatic cancer cases (n=559) and healthy unrelated controls (n=603) were tested for methylation of the LINE-1, Alu and Sat2 DNA repetitive elements using MethyLight quantitative PCR assays. Odds ratios (ORs) and 95% confidence intervals (95%CI) between both continuous measures of percent of methylated sample compared to a reference (PMR) or quintiles of PMR and pancreatic cancer, adjusted for age, sex, smoking, BMI, alcohol and higher education, were estimated.The PMR for each of the three markers was higher in cases than in controls, although only LINE-1 was significantly associated with pancreatic cancer (OR per log unit=1.37, 95%CI=1.16–1.63). The marker methylation score for all three markers combined was significantly associated with pancreatic cancer (p-trend=0.0006). There were no associations between measures of PMR and either presence of metastases, or timing of blood collection in relation to diagnosis, surgery, chemotherapy or death (all p>0.1).We observed an association between methylation of LINE-1 in WBC DNA and risk of pancreatic cancer. Further studies are needed to confirm this association.

The prognostic value of global DNA hypomethylation in cancer: a meta-analysis.

BackgroundAberrant methylation of the global genome has been investigated as a prognostic indicator in various cancers, but the results are controversial and ambiguous.Methods and FindingsThis meta-analysis presents pooled estimates of the evidence to elucidate this issue. We searched the electronic databases: PubMed, Embase, ISI Web of Science and the Cochrane library (up to August 2013) to identify all of the relevant studies. The association between the level of surrogates' indexes of genome-wide hypomethylation (LINE-1, Alu and Sat–α) and the overall survival (OS) of cancer patients was examined. In addition, the pooled hazard ratios (HRs) with their 95% confidence interval (95%CI) were calculated to estimate the influences through fixed-effects and random-effects model. Finally, twenty studies with total population of 5447 met the inclusion criteria. The results indicate that the summary HRs for the studies employing LINE-1, Alu, and Sat-α repetitive elements also show that the global DNA hypomethylation have significant desirable effects on the tumour prognostic value. The pooled HRs (and CIs) of LINE-1, Alu and Sat-α were 1.83 (1.38–2.44), 2.00 (1.16–3.45), and 2.92 (1.04–8.25), with a heterogeneity measure index of I2 (and p-value) shows of 66.6% (p = 0.001), 57.1% (p = 0.053) and 68.2% (p = 0.076) respectively. The meta-regression and subgroup analysis indicated that the percentage of hypomethylated sample of cancer patients is one source of heterogeneity.ConclusionOur meta-analysis findings support the hypothesis that the global DNA hypomethylation is associated with a detrimental prognosis in tumour patients.