Metastable DNA methylation sites associated with longitudinal lung function decline and aging in humans: an epigenome-wide study in the NAS and KORA cohorts.

DNA methylation is an epigenetic regulator of gene transcription, which has been found to be both metastable and variable within human cohort studies. Currently, few studies have been done to identify metastable DNA methylation biomarkers associated with longitudinal lung function decline in humans. The identification of such biomarkers is important for screening vulnerable populations. We hypothesized that quantifiable blood-based DNA methylation alterations would serve as metastable biomarkers of lung function decline and aging, which may help to discover new pathways and/or mechanisms related to pulmonary pathogenesis. Using linear mixed models, we performed an Epigenome Wide Association Study (EWAS) between DNA methylation at CpG dinucleotides and longitudinal lung function (FVC, FEV1, FEF25-75%) decline and aging with initial discovery in the Normative Aging Study, and replication in the Cooperative Health Research in the Region of Augsburg cohort. We identified two metastable epigenetic loci associated with either poor lung function and aging, cg05575921 (AHRR gene), or lung function independently of aging, cg06126421 (IER3 gene). These loci may inform basic mechanisms associated with pulmonary function, pathogenesis, and aging. Human epigenomic variation, may help explain features of lung function decline and related pathophysiology not attributable to DNA sequence alone, such as accelerated pulmonary decline in smokers, former smokers, and perhaps non-smokers. Our EWAS across two cohorts, therefore, will likely have implications for the human population, not just the elderly.

A longitudinal study of DNA methylation as a potential mediator of age-related diabetes risk.

DNA methylation (DNAm) has been found to show robust and widespread age-related changes across the genome. DNAm profiles from whole blood can be used to predict human aging rates with great accuracy. We sought to test whether DNAm-based predictions of age are related to phenotypes associated with type 2 diabetes (T2D), with the goal of identifying risk factors potentially mediated by DNAm. Our participants were 43 women enrolled in the Women's Health Initiative. We obtained methylation data via the Illumina 450K Methylation array on whole blood samples from participants at three timepoints, covering on average 16 years per participant. We employed the method and software of Horvath, which uses DNAm at 353 CpGs to form a DNAm-based estimate of chronological age. We then calculated the epigenetic age acceleration, or Δage, at each timepoint. We fit linear mixed models to characterize how Δage contributed to a longitudinal model of aging and diabetes-related phenotypes and risk factors. For most participants, Δage remained constant, indicating that age acceleration is generally stable over time. We found that Δage associated with body mass index (p = 0.0012), waist circumference (p = 0.033), and fasting glucose (p = 0.0073), with the relationship with BMI maintaining significance after correction for multiple testing. Replication in a larger cohort of 157 WHI participants spanning 3 years was unsuccessful, possibly due to the shorter time frame covered. Our results suggest that DNAm has the potential to act as a mediator between aging and diabetes-related phenotypes, or alternatively, may serve as a biomarker of these phenotypes.

Blood Epigenetic Age may Predict Cancer Incidence and Mortality.

Biological measures of aging are important for understanding the health of an aging population, with epigenetics particularly promising. Previous studies found that tumor tissue is epigenetically older than its donors are chronologically. We examined whether blood Δage (the discrepancy between epigenetic and chronological ages) can predict cancer incidence or mortality, thus assessing its potential as a cancer biomarker. In a prospective cohort, Δage and its rate of change over time were calculated in 834 blood leukocyte samples collected from 442 participants free of cancer at blood draw. About 3-5 years before cancer onset or death, Δage was associated with cancer risks in a dose-responsive manner (P = 0.02) and a one-year increase in Δage was associated with cancer incidence (HR: 1.06, 95% CI: 1.02-1.10) and mortality (HR: 1.17, 95% CI: 1.07-1.28). Participants with smaller Δage and decelerated epigenetic aging over time had the lowest risks of cancer incidence (P = 0.003) and mortality (P = 0.02). Δage was associated with cancer incidence in a 'J-shaped' manner for subjects examined pre-2003, and with cancer mortality in a time-varying manner. We conclude that blood epigenetic age may mirror epigenetic abnormalities related to cancer development, potentially serving as a minimally invasive biomarker for cancer early detection.

CpGFilter: model-based CpG probe filtering with replicates for epigenome-wide association studies.

SUMMARY: The development of the Infinium HumanMethylation450 BeadChip enables epigenome-wide association studies at a reduced cost. One observation of the 450K data is that many CpG sites the beadchip interrogates have very large measurement errors. Including these noisy CpGs will decrease the statistical power of detecting relevant associations due to multiple testing correction. We propose to use intra-class correlation coefficient (ICC), which characterizes the relative contribution of the biological variability to the total variability, to filter CpGs when technical replicates are available. We estimate the ICC based on a linear mixed effects model by pooling all the samples instead of using the technical replicates only. An ultra-fast algorithm has been developed to address the computational complexity and CpG filtering can be completed in minutes on a desktop computer for a 450K data set of over 1000 samples. Our method is very flexible and can accommodate any replicate design. Simulations and a real data application demonstrate that our whole-sample ICC method performs better than replicate-sample ICC or variance-based method. AVAILABILITY AND IMPLEMENTATION: CpGFilter is implemented in R and publicly available under CRAN via the R package 'CpGFilter'. CONTACT: chen.jun2@mayo.edu or xlin@hsph.harvard.edu SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Central role of ULK1 in type I interferon signaling.

We provide evidence that the Unc-51-like kinase 1 (ULK1) is activated during engagement of the type I interferon (IFN) receptor (IFNR). Our studies demonstrate that the function of ULK1 is required for gene transcription mediated via IFN-stimulated response elements (ISRE) and IFNγ activation site (GAS) elements and controls expression of key IFN-stimulated genes (ISGs). We identify ULK1 as an upstream regulator of p38α mitogen-activated protein kinase (MAPK) and establish that the regulatory effects of ULK1 on ISG expression are mediated possibly by engagement of the p38 MAPK pathway. Importantly, we demonstrate that ULK1 is essential for antiproliferative responses and type I IFN-induced antineoplastic effects against malignant erythroid precursors from patients with myeloproliferative neoplasms. Together, these data reveal a role for ULK1 as a key mediator of type I IFNR-generated signals that control gene transcription and induction of antineoplastic responses.

Evaluation of commercially available RNA amplification kits for RNA sequencing using very low input amounts of total RNA.

This article includes supplemental data. Please visit http://www.fasebj.org to obtain this information.Multiple recent publications on RNA sequencing (RNA-seq) have demonstrated the power of next-generation sequencing technologies in whole-transcriptome analysis. Vendor-specific protocols used for RNA library construction often require at least 100 ng total RNA. However, under certain conditions, much less RNA is available for library construction. In these cases, effective transcriptome profiling requires amplification of subnanogram amounts of RNA. Several commercial RNA amplification kits are available for amplification prior to library construction for next-generation sequencing, but these kits have not been comprehensively field evaluated for accuracy and performance of RNA-seq for picogram amounts of RNA. To address this, 4 types of amplification kits were tested with 3 different concentrations, from 5 ng to 50 pg, of a commercially available RNA. Kits were tested at multiple sites to assess reproducibility and ease of use. The human total reference RNA used was spiked with a control pool of RNA molecules in order to further evaluate quantitative recovery of input material. Additional control data sets were generated from libraries constructed following polyA selection or ribosomal depletion using established kits and protocols. cDNA was collected from the different sites, and libraries were synthesized at a single site using established protocols. Sequencing runs were carried out on the Illumina platform. Numerous metrics were compared among the kits and dilutions used. Overall, no single kit appeared to meet all the challenges of small input material. However, it is encouraging that excellent data can be recovered with even the 50 pg input total RNA.

Recurrent DGCR8, DROSHA, and SIX homeodomain mutations in favorable histology Wilms tumors.

We report the most common single-nucleotide substitution/deletion mutations in favorable histology Wilms tumors (FHWTs) to occur within SIX1/2 (7% of 534 tumors) and microRNA processing genes (miRNAPGs) DGCR8 and DROSHA (15% of 534 tumors). Comprehensive analysis of 77 FHWTs indicates that tumors with SIX1/2 and/or miRNAPG mutations show a pre-induction metanephric mesenchyme gene expression pattern and are significantly associated with both perilobar nephrogenic rests and 11p15 imprinting aberrations. Significantly decreased expression of mature Let-7a and the miR-200 family (responsible for mesenchymal-to-epithelial transition) in miRNAPG mutant tumors is associated with an undifferentiated blastemal histology. The combination of SIX and miRNAPG mutations in the same tumor is associated with evidence of RAS activation and a higher rate of relapse and death.

Identification of eQTLs for hepatic Xbp1s and Socs3 gene expression in mice fed a high-fat, high-caloric diet.

Nonalcoholic fatty liver disease (NAFLD) is a highly prevalent form of human hepatic disease and feeding mice a high-fat, high-caloric (HFHC) diet is a standard model of NAFLD. To better understand the genetic basis of NAFLD, we conducted an expression quantitative trait locus (eQTL) analysis of mice fed a HFHC diet. Two-hundred sixty-five (A/J × C57BL/6J) F2 male mice were fed a HFHC diet for 8 wk. eQTL analysis was utilized to identify genomic regions that regulate hepatic gene expression of Xbp1s and Socs3. We identified two overlapping loci for Xbp1s and Socs3 on Chr 1 (164.0-185.4 Mb and 174.4-190.5 Mb, respectively) and Chr 11 (41.1-73.1 Mb and 44.0-68.6 Mb, respectively), and an additional locus for Socs3 on Chr 12 (109.9-117.4 Mb). C57BL/6J-Chr 11(A/J)/ NaJ mice fed a HFHC diet manifested the A/J phenotype of increased Xbp1s and Socs3 gene expression (P < 0.05), whereas C57BL/6J-Chr 1(A/J)/ NaJ mice retained the C57BL/6J phenotype. In addition, we replicated the eQTLs on Chr 1 and Chr 12 (LOD scores ≥3.5) using mice from the BXD murine reference panel challenged with CCl4 to induce chronic liver injury and fibrosis. We have identified overlapping eQTLs for Xbp1 and Socs3 on Chr 1 and Chr 11, and consomic mice confirmed that replacing the C57BL/6J Chr 11 with the A/J Chr 11 resulted in an A/J phenotype for Xbp1 and Socs3 gene expression. Identification of the genes for these eQTLs will lead to a better understanding of the genetic factors responsible for NAFLD and potentially other hepatic diseases.

Assessing technical performance in differential gene expression experiments with external spike-in RNA control ratio mixtures.

There is a critical need for standard approaches to assess, report and compare the technical performance of genome-scale differential gene expression experiments. Here we assess technical performance with a proposed standard 'dashboard' of metrics derived from analysis of external spike-in RNA control ratio mixtures. These control ratio mixtures with defined abundance ratios enable assessment of diagnostic performance of differentially expressed transcript lists, limit of detection of ratio (LODR) estimates and expression ratio variability and measurement bias. The performance metrics suite is applicable to analysis of a typical experiment, and here we also apply these metrics to evaluate technical performance among laboratories. An interlaboratory study using identical samples shared among 12 laboratories with three different measurement processes demonstrates generally consistent diagnostic power across 11 laboratories. Ratio measurement variability and bias are also comparable among laboratories for the same measurement process. We observe different biases for measurement processes using different mRNA-enrichment protocols.

Multi-platform assessment of transcriptome profiling using RNA-seq in the ABRF next-generation sequencing study.

High-throughput RNA sequencing (RNA-seq) greatly expands the potential for genomics discoveries, but the wide variety of platforms, protocols and performance capabilitites has created the need for comprehensive reference data. Here we describe the Association of Biomolecular Resource Facilities next-generation sequencing (ABRF-NGS) study on RNA-seq. We carried out replicate experiments across 15 laboratory sites using reference RNA standards to test four protocols (poly-A-selected, ribo-depleted, size-selected and degraded) on five sequencing platforms (Illumina HiSeq, Life Technologies PGM and Proton, Pacific Biosciences RS and Roche 454). The results show high intraplatform (Spearman rank R > 0.86) and inter-platform (R > 0.83) concordance for expression measures across the deep-count platforms, but highly variable efficiency and cost for splice junction and variant detection between all platforms. For intact RNA, gene expression profiles from rRNA-depletion and poly-A enrichment are similar. In addition, rRNA depletion enables effective analysis of degraded RNA samples. This study provides a broad foundation for cross-platform standardization, evaluation and improvement of RNA-seq.

Genome-wide DNA methylation analysis predicts an epigenetic switch for GATA factor expression in endometriosis.

Endometriosis is a gynecological disease defined by the extrauterine growth of endometrial-like cells that cause chronic pain and infertility. The disease is limited to primates that exhibit spontaneous decidualization, and diseased cells are characterized by significant defects in the steroid-dependent genetic pathways that typify this process. Altered DNA methylation may underlie these defects, but few regions with differential methylation have been implicated in the disease. We mapped genome-wide differences in DNA methylation between healthy human endometrial and endometriotic stromal cells and correlated this with gene expression using an interaction analysis strategy. We identified 42,248 differentially methylated CpGs in endometriosis compared to healthy cells. These extensive differences were not unidirectional, but were focused intragenically and at sites distal to classic CpG islands where methylation status was typically negatively correlated with gene expression. Significant differences in methylation were mapped to 403 genes, which included a disproportionally large number of transcription factors. Furthermore, many of these genes are implicated in the pathology of endometriosis and decidualization. Our results tremendously improve the scope and resolution of differential methylation affecting the HOX gene clusters, nuclear receptor genes, and intriguingly the GATA family of transcription factors. Functional analysis of the GATA family revealed that GATA2 regulates key genes necessary for the hormone-driven differentiation of healthy stromal cells, but is hypermethylated and repressed in endometriotic cells. GATA6, which is hypomethylated and abundant in endometriotic cells, potently blocked hormone sensitivity, repressed GATA2, and induced markers of endometriosis when expressed in healthy endometrial cells. The unique epigenetic fingerprint in endometriosis suggests DNA methylation is an integral component of the disease, and identifies a novel role for the GATA family as key regulators of uterine physiology-aberrant DNA methylation in endometriotic cells correlates with a shift in GATA isoform expression that facilitates progesterone resistance and disease progression.

Genome-wide analysis of small RNAs expressed by Yersinia pestis identifies a regulator of the Yop-Ysc type III secretion system.

Small noncoding RNA (sRNA) molecules are integral components of the regulatory machinery for many bacterial species and are known to posttranscriptionally regulate metabolic and stress-response pathways, quorum sensing, virulence factors, and more. The Yop-Ysc type III secretion system (T3SS) is a critical virulence component for the pathogenic Yersinia species, and the regulation of this system is tightly controlled at each step from transcription to translocation of effectors into host cells. The contribution of sRNAs to the regulation of the T3SS in Yersinia has been largely unstudied, however. Previously, our lab identified a role for the sRNA chaperone protein Hfq in the regulation of components of the T3SS in the gastrointestinal pathogen Yersinia pseudotuberculosis. Here we present data demonstrating a similar requirement for Hfq in the closely related species Yersinia pestis. Through deep sequencing analysis of the Y. pestis sRNA-ome, we found 63 previously unidentified putative sRNAs in this species. We identified a Yersinia-specific sRNA, Ysr141, carried by the T3SS plasmid pCD1 that is required for the production of multiple T3SS proteins. In addition, we show that Ysr141 targets an untranslated region upstream of yopJ to posttranscriptionally activate the synthesis of the YopJ protein. Furthermore, Ysr141 may be an unstable and/or processed sRNA, which could contribute to its function in the regulation of the T3SS. The discovery of an sRNA that influences the synthesis of the T3SS adds an additional layer of regulation to this tightly controlled virulence determinant of Y. pestis.

The inflammatory gene pathway is not a major contributor to polycystic ovary snydrome.

CONTEXT: Although inflammation is clearly associated with obesity, diabetes, and insulin resistance, the role of chronic inflammation in the etiology of polycystic ovary syndrome (PCOS) is unclear. OBJECTIVE: To determine whether chronic inflammation plays a causal role in the etiology of PCOS, we tested for an association between PCOS and genetic markers mapping to 80 members of the inflammatory pathway. DESIGN: This was a case-control association study. SETTING: The setting was an academic medical center. PATIENTS OR PARTICIPANTS: A total of 905 index case patients with PCOS and 955 control women (108 intensively phenotyped subjects with normal androgen levels and regular menses and 847 minimally phenotyped subjects with regular menses and no history of PCOS). INTERVENTIONS: Subjects were genotyped at single nucleotide polymorphisms mapping to 80 inflammatory genes. Logistic regression was used to test for an association between 822 single nucleotide polymorphisms and PCOS after adjustment for population stratification, body mass index, and/or age. In the index patients, we also tested for association with 11 quantitative traits (body mass index and testosterone, fasting insulin, fasting glucose, 2-hour postchallenge glucose, LH, FSH, total cholesterol, high-density lipoprotein, low-density lipoprotein, and triglyceride levels). MAIN OUTCOME MEASURES: The evidence for an association with PCOS and with 11 quantitative traits was investigated. RESULTS: Nominally significant evidence for an association was observed with MAP3K7, IKBKG, TNFRS11A, AKT2, IL6R, and IRF1, but no results remained statistically significant after adjustment for multiple testing. CONCLUSIONS: Genetic variation in the inflammatory pathway is not a major contributor to the etiology of PCOS or related quantitative traits in women with PCOS.

Functional specialization of the small interfering RNA pathway in response to virus infection.

In Drosophila, post-transcriptional gene silencing occurs when exogenous or endogenous double stranded RNA (dsRNA) is processed into small interfering RNAs (siRNAs) by Dicer-2 (Dcr-2) in association with a dsRNA-binding protein (dsRBP) cofactor called Loquacious (Loqs-PD). siRNAs are then loaded onto Argonaute-2 (Ago2) by the action of Dcr-2 with another dsRBP cofactor called R2D2. Loaded Ago2 executes the destruction of target RNAs that have sequence complementarity to siRNAs. Although Dcr-2, R2D2, and Ago2 are essential for innate antiviral defense, the mechanism of virus-derived siRNA (vsiRNA) biogenesis and viral target inhibition remains unclear. Here, we characterize the response mechanism mediated by siRNAs against two different RNA viruses that infect Drosophila. In both cases, we show that vsiRNAs are generated by Dcr-2 processing of dsRNA formed during viral genome replication and, to a lesser extent, viral transcription. These vsiRNAs seem to preferentially target viral polyadenylated RNA to inhibit viral replication. Loqs-PD is completely dispensable for silencing of the viruses, in contrast to its role in silencing endogenous targets. Biogenesis of vsiRNAs is independent of both Loqs-PD and R2D2. R2D2, however, is required for sorting and loading of vsiRNAs onto Ago2 and inhibition of viral RNA expression. Direct injection of viral RNA into Drosophila results in replication that is also independent of Loqs-PD. This suggests that triggering of the antiviral pathway is not related to viral mode of entry but recognition of intrinsic features of virus RNA. Our results indicate the existence of a vsiRNA pathway that is separate from the endogenous siRNA pathway and is specifically triggered by virus RNA. We speculate that this unique framework might be necessary for a prompt and efficient antiviral response.

Evidence for chromosome 2p16.3 polycystic ovary syndrome susceptibility locus in affected women of European ancestry.

CONTEXT: A previous genome-wide association study in Chinese women with polycystic ovary syndrome (PCOS) identified a region on chromosome 2p16.3 encoding the LH/choriogonadotropin receptor (LHCGR) and FSH receptor (FSHR) genes as a reproducible PCOS susceptibility locus. OBJECTIVE: The objective of the study was to determine the role of the LHCGR and/or FSHR gene in the etiology of PCOS in women of European ancestry. DESIGN: This was a genetic association study in a European ancestry cohort of women with PCOS. SETTING: The study was conducted at an academic medical center. PARTICIPANTS: Participants in the study included 905 women with PCOS diagnosed by National Institutes of Health criteria and 956 control women. INTERVENTION: We genotyped 94 haplotype-tagging single-nucleotide polymorphisms and two coding single-nucleotide polymorphisms mapping to the coding region of LHCGR and FSHR plus 20 kb upstream and downstream of the genes and test for association in the case control cohort and for association with nine quantitative traits in the women with PCOS. RESULTS: We found strong evidence for an association of PCOS with rs7562215 (P = 0.0037) and rs10495960 (P = 0.0046). Although the marker with the strongest association in the Chinese PCOS genome-wide association study (rs13405728) was not informative in the European populations, we identified and genotyped three markers (rs35960650, rs2956355, and rs7562879) within 5 kb of rs13405728. Of these, rs7562879 was nominally associated with PCOS (P = 0.020). The strongest evidence for association mapping to FSHR was observed with rs1922476 (P = 0.0053). Furthermore, markers with the FSHR gene region were associated with FSH levels in women with PCOS. CONCLUSIONS: Fine mapping of the chromosome 2p16.3 Chinese PCOS susceptibility locus in a European ancestry cohort provides evidence for association with two independent loci and PCOS. The gene products LHCGR and FSHR therefore are likely to be important in the etiology of PCOS, regardless of ethnicity.

Technical reproducibility of genotyping SNP arrays used in genome-wide association studies.

During the last several years, high-density genotyping SNP arrays have facilitated genome-wide association studies (GWAS) that successfully identified common genetic variants associated with a variety of phenotypes. However, each of the identified genetic variants only explains a very small fraction of the underlying genetic contribution to the studied phenotypic trait. Moreover, discordance observed in results between independent GWAS indicates the potential for Type I and II errors. High reliability of genotyping technology is needed to have confidence in using SNP data and interpreting GWAS results. Therefore, reproducibility of two widely genotyping technology platforms from Affymetrix and Illumina was assessed by analyzing four technical replicates from each of the six individuals in five laboratories. Genotype concordance of 99.40% to 99.87% within a laboratory for the sample platform, 98.59% to 99.86% across laboratories for the same platform, and 98.80% across genotyping platforms was observed. Moreover, arrays with low quality data were detected when comparing genotyping data from technical replicates, but they could not be detected according to venders' quality control (QC) suggestions. Our results demonstrated the technical reliability of currently available genotyping platforms but also indicated the importance of incorporating some technical replicates for genotyping QC in order to improve the reliability of GWAS results. The impact of discordant genotypes on association analysis results was simulated and could explain, at least in part, the irreproducibility of some GWAS findings when the effect size (i.e. the odds ratio) and the minor allele frequencies are low.

Genome-wide study of DNA methylation alterations in response to diazinon exposure in vitro.

Pesticide exposure has repeatedly been associated with cancers. However, molecular mechanisms are largely undetermined. In this study, we examined whether exposure to diazinon, a common organophosphate that has been associated with cancers, could induce DNA methylation alterations. We conducted genome-wide DNA methylation analyses on DNA samples obtained from human hematopoietic K562 cell exposed to diazinon and ethanol using the Illumina Infinium HumanMethylation27 BeadChip. Bayesian-adjusted t-tests were used to identify differentially methylated gene promoter CpG sites. We identified 1069 CpG sites in 984 genes with significant methylation changes in diazinon-treated cells. Gene ontology analysis demonstrated that some genes are tumor suppressor genes, such as TP53INP1 (3.0-fold, q-value <0.001) and PTEN (2.6-fold, q-value <0.001), some genes are in cancer-related pathways, such as HDAC3 (2.2-fold, q-value=0.002), and some remain functionally unknown. Our results provided direct experimental evidence that diazinon may modify gene promoter DNA methylation levels, which may play a pathological role in cancer development.

DNA methylation alterations in response to pesticide exposure in vitro.

Although pesticides are subject to extensive carcinogenicity testing before regulatory approval, pesticide exposure has repeatedly been associated with various cancers. This suggests that pesticides may cause cancer via nonmutagenicity mechanisms. The present study provides evidence to support the hypothesis that pesticide-induced cancer may be mediated in part by epigenetic mechanisms. We examined whether exposure to seven commonly used pesticides (i.e., fonofos, parathion, terbufos, chlorpyrifos, diazinon, malathion, and phorate) induces DNA methylation alterations in vitro. We conducted genome-wide DNA methylation analyses on DNA samples obtained from the human hematopoietic K562 cell line exposed to ethanol (control) and several organophosphate pesticides (OPs) using the Illumina Infinium HumanMethylation27 BeadChip. Bayesian-adjusted t-tests were used to identify differentially methylated gene promoter CpG sites. In this report, we present our results on three pesticides (fonofos, parathion, and terbufos) that clustered together based on principle component analysis and hierarchical clustering. These three pesticides induced similar methylation changes in the promoter regions of 712 genes, while also exhibiting their own OP-specific methylation alterations. Functional analysis of methylation changes specific to each OP, or common to all three OPs, revealed that differential methylation was associated with numerous genes that are involved in carcinogenesis-related processes. Our results provide experimental evidence that pesticides may modify gene promoter DNA methylation levels, suggesting that epigenetic mechanisms may contribute to pesticide-induced carcinogenesis. Further studies in other cell types and human samples are required, as well as determining the impact of these methylation changes on gene expression.

The effect of vorinostat on the development of resistance to doxorubicin in neuroblastoma.

Histone deacetylase (HDAC) inhibitors, especially vorinostat, are currently under investigation as potential adjuncts in the treatment of neuroblastoma. The effect of vorinostat co-treatment on the development of resistance to other chemotherapeutic agents is unknown. In the present study, we treated two human neuroblastoma cell lines [SK-N-SH and SK-N-Be(2)C] with progressively increasing doses of doxorubicin under two conditions: with and without vorinsotat co-therapy. The resultant doxorubicin-resistant (DoxR) and vorinostat-treated doxorubicin resistant (DoxR-v) cells were equally resistant to doxorubicin despite significantly lower P-glycoprotein expression in the DoxR-v cells. Whole genome analysis was performed using the Ilumina Human HT-12 v4 Expression Beadchip to identify genes with differential expression unique to the DoxR-v cells. We uncovered a number of genes whose differential expression in the DoxR-v cells might contribute to their resistant phenotype, including hypoxia inducible factor-2. Finally, we used Gene Ontology to categorize the biological functions of the differentially expressed genes unique to the DoxR-v cells and found that genes involved in cellular metabolism were especially affected.

The role of inflammatory pathway genetic variation on maternal metabolic phenotypes during pregnancy.

BACKGROUND: Since mediators of inflammation are associated with insulin resistance, and the risk of developing diabetes mellitus and gestational diabetes, we hypothesized that genetic variation in members of the inflammatory gene pathway impact glucose levels and related phenotypes in pregnancy. We evaluated this hypothesis by testing for association between genetic variants in 31 inflammatory pathway genes in the Hyperglycemia and Adverse Pregnancy Outcome (HAPO) cohort, a large multiethnic multicenter study designed to address the impact of glycemia less than overt diabetes on pregnancy outcome. RESULTS: Fasting, 1-hour, and 2-hour glucose, fasting and 1-hour C-peptide, and HbA1c levels were measured in blood samples obtained from HAPO participants during an oral glucose tolerance test at 24-32 weeks gestation. We tested for association between 458 SNPs mapping to 31 genes in the inflammatory pathway and metabolic phenotypes in 3836 European ancestry and 1713 Thai pregnant women. The strongest evidence for association was observed with TNF alpha and HbA1c (rs1052248; 0.04% increase per allele C; p-value = 4.4×10(-5)), RETN and fasting plasma glucose (rs1423096; 0.7 mg/dl decrease per allele A; p-value = 1.1×10(-4)), IL8 and 1 hr plasma glucose (rs2886920; 2.6 mg/dl decrease per allele T; p-value = 1.3×10(-4)), ADIPOR2 and fasting C-peptide (rs2041139; 0.55 ug/L decrease per allele A; p-value = 1.4×10(-4)), LEPR and 1-hour C-peptide (rs1171278; 0.62 ug/L decrease per allele T; p-value = 2.4×10(-4)), and IL6 and 1-hour plasma glucose (rs6954897; -2.29 mg/dl decrease per allele G, p-value = 4.3×10(-4)). CONCLUSIONS: Based on the genes surveyed in this study the inflammatory pathway is unlikely to have a strong impact on maternal metabolic phenotypes in pregnancy although variation in individual members of the pathway (e.g. RETN, IL8, ADIPOR2, LEPR, IL6, and TNF alpha,) may contribute to metabolic phenotypes in pregnant women.