Genome-wide Epigenetic Studies Research Articles

Testing for the causal mediation effects of multiple mediators using the kernel machine difference method in genome-wide epigenetic studies

Testing for the causal mediation effects of multiple mediators using the kernel machine difference method in genome-wide epigenetic studies

CpG island definition and methylation mapping of the T2T-YAO genome

Abstract Precisely defining and mapping all cytosine positions and their clusters, known as CpG islands (CGIs), as well as their methylation status are pivotal for genome-wide epigenetic studies, especially when population-centric reference genomes are ready for timely application. Here we first align the two high-quality reference genomes, T2T-YAO and T2T-CHM13, from different ethnic backgrounds in a base-by-base fashion and compute their genome-wide density-defined and position-defined CGIs. Second, mapping some representative genome-wide methylation data from selected organs onto the two genomes, we find that there are about 4.7–5.8% sequence divergency of variable categories depending on quality cutoffs. Genes among the divergent sequences are mostly associated with neurological functions. Moreover, CGIs associated with the divergent sequences are significantly different with respect to CpG density and observed CpG/expected CpG (O/E) ratio between the two genomes. Finally, we find that the T2T-YAO genome not only has a greater CpG site coverage than that of the T2T-CHM13 genome when whole-genome bisulfite sequencing (WGBS) data from the European and American populations are mapped to each reference, but also show more hyper-methylated CpG sites as compared to the T2T-CHM13 genome. Our study suggests that future genome-wide epigenetic studies of the Chinese populations rely on both acquisition of high-quality methylation data and subsequent precision CGI mapping based on the Chinese T2T reference.

Dynamics and recognition of homeodomain containing protein-DNA complex of IRX4.

Iroquois Homeobox 4 (IRX4) belongs to a family of homeobox TFs having roles in embryogenesis, cell specification, and organ development. Recently, large scale genome-wide association studies and epigenetic studies have highlighted the role of IRX4 and its associated variants in prostate cancer. No studies have investigated and characterized the structural aspect of the IRX4 homeodomain and its potential to bind to DNA. The current study uses sequence analysis, homology modeling, and molecular dynamics simulations to explore IRX4 homeodomain-DNA recognition mechanisms and the role of somatic mutations affecting these interactions. Using publicly available databases, gene expression of IRX4 was found in different tissues, including prostate, heart, skin, vagina, and the protein expression was found in cancer cell lines (HCT166, HEK293), B cells, ascitic fluid, and brain. Sequence conservation of the homeodomain shed light on the importance of N- and C-terminal residues involved in DNA binding. The specificity of IRX4 homodimer bound to consensus human DNA sequence was confirmed by molecular dynamics simulations, representing the role of conserved amino acids including R145, A194, N195, S190, R198, and R199 in binding to DNA. Additional N-terminal residues like T144 and G143 were also found to have specific interactions highlighting the importance of N-terminus of the homeodomain in DNA recognition. Additionally, the effects of somatic mutations, including the conserved Arginine (R145, R198, and R199) residues on DNA binding elucidated the importance of these residues in stabilizing the protein-DNA complex. Secondary structure and hydrogen bonding analysis showed the roles of specific residues (R145, T191, A194, N195, R198, and R199) in maintaining the homogeneity of the structure and its interaction with DNA. The differences in relative binding free energies of all the mutants shed light on the structural modularity of this protein and the dynamics behind protein-DNA interaction. We also have predicted that the C-terminal sequence of the IRX4 homeodomain could act as a potential cell-penetrating peptide, emphasizing the role these small peptides could play in targeting homeobox TFs.

Large-Scale Hypothesis Testing for Causal Mediation Effects with Applications in Genome-wide Epigenetic Studies

In genome-wide epigenetic studies, it is of great scientific interest to assess whether the effect of an exposure on a clinical outcome is mediated through DNA methylations. However, statistical inference for causal mediation effects is challenged by the fact that one needs to test a large number of composite null hypotheses across the whole epigenome. Two popular tests, the Wald-type Sobel’s test and the joint significant test using the traditional null distribution are underpowered and thus can miss important scientific discoveries. In this article, we show that the null distribution of Sobel’s test is not the standard normal distribution and the null distribution of the joint significant test is not uniform under the composite null of no mediation effect, especially in finite samples and under the singular point null case that the exposure has no effect on the mediator and the mediator has no effect on the outcome. Our results explain why these two tests are underpowered, and more importantly motivate us to develop a more powerful divide-aggregate composite-null test (DACT) for the composite null hypothesis of no mediation effect by leveraging epigenome-wide data. We adopted Efron’s empirical null framework for assessing statistical significance of the DACT test. We showed analytically that the proposed DACT method had improved power, and could well control Type I error rate. Our extensive simulation studies showed that, in finite samples, the DACT method properly controlled the Type I error rate and outperformed Sobel’s test and the joint significance test for detecting mediation effects. We applied the DACT method to the U.S. Department of Veterans Affairs Normative Aging Study, an ongoing prospective cohort study which included men who were aged 21 to 80 years at entry. We identified multiple DNA methylation CpG sites that might mediate the effect of smoking on lung function with effect sizes ranging from –0.18 to –0.79 and false discovery rate controlled at the level 0.05, including the CpG sites in the genes AHRR and F2RL3. Our sensitivity analysis found small residual correlations (less than 0.01) of the error terms between the outcome and mediator regressions, suggesting that our results are robust to unmeasured confounding factors. Supplementary materials for this article, including a standardized description of the materials available for reproducing the work, are available as an online supplement.

Testing cell-type-specific mediation effects in genome-wide epigenetic studies.

Epigenome-wide mediation analysis aims to identify DNA methylation CpG sites that mediate the causal effects of genetic/environmental exposures on health outcomes. However, DNA methylations in the peripheral blood tissues are usually measured at the bulk level based on a heterogeneous population of white blood cells. Using the bulk level DNA methylation data in mediation analysis might cause confounding bias and reduce study power. Therefore, it is crucial to get fine-grained results by detecting mediation CpG sites in a cell-type-specific way. However, there is a lack of methods and software to achieve this goal. We propose a novel method (Mediation In a Cell-type-Specific fashion, MICS) to identify cell-type-specific mediation effects in genome-wide epigenetic studies using only the bulk-level DNA methylation data. MICS follows the standard mediation analysis paradigm and consists of three key steps. In step1, we assess the exposure-mediator association for each cell type; in step 2, we assess the mediator-outcome association for each cell type; in step 3, we combine the cell-type-specific exposure-mediator and mediator-outcome associations using a multiple testing procedure named MultiMed [Sampson JN, Boca SM, Moore SC, etal. FWER and FDR control when testing multiple mediators. Bioinformatics 2018;34:2418-24] to identify significant CpGs with cell-type-specific mediation effects. We conduct simulation studies to demonstrate that our method has correct FDR control. We also apply the MICS procedure to the Normative Aging Study and identify nine DNA methylation CpG sites in the lymphocytes that might mediate the effect of cigarette smoking on the lung function.

A novel understanding of global DNA methylation in bobcat (Lynx rufus).

Epigenetic mechanisms may provide a novel prospective of bobcat (Lynx rufus) adaptation to habitat loss/fragmentation. Previous research has focused on bobcat behavior and genetics, but epigenetics has not been studied in bobcat. The aim of this study was to determine the quantity of global DNA methylation in the liver of 30 bobcats. DNA was extracted from liver samples obtained from the Vermont Fish and Wildlife Department. The percent of global DNA methylation was quantified and calculated using the MethylFlashTM Methylated DNA 5-mC Quantification Kit from Epigentek (Farmingdale, NY, USA). Age, sex, and carcass weight data were collected at sampling and analyzed with percent of global DNA methylation. Global DNA methylation was found to range from 0.46% to 2.76%. Age ranged from <1 to 12 years old and weight ranged from 3.18 to 13.61 kg. Further analysis of differential methylation may provide insight into novel means of bobcat conservation within different regions of Vermont. These results reinforce the need for genome-wide epigenetic studies in conservation biology.

Peripheral Arterial Disease Genetics: Progress to Date and Challenges Ahead.

In this paper, we review the progress made thus far in research related to the genetics of peripheral arterial disease (PAD) by detailing efforts to date in heritability, linkage analyses, and candidate gene studies. We further summarize more contemporary genome-wide association studies (GWAS) and epigenetic studies of PAD. Finally, we review current challenges and future avenues of advanced research in PAD genetics including whole genome sequencing studies. Studies have estimated the heritability of PAD to be moderate, though the contribution to this heritability that is independent of traditional cardiovascular risk factors remains unclear. Recent efforts have identified SNPs associated with PAD in GWAS analyses, but these have yet to be replicated in independent studies. Much remains to be discovered in the field of PAD genetics. An improved understanding of the genetic foundation for PAD will allow for earlier diagnosis of disease and a more complete pathophysiological understanding of the mechanisms of the disease leading to novel therapeutic interventions. Future avenues for success will likely arise from very large-scale GWAS, whole genome sequencing, and epigenetic studies involving very well-characterized cohorts.

MicroRNAs Responsible for Inflammation in Obesity

Background: In recent years, more and more evidence has accumulated to elucidate the modulating roles of microRNA in immune and inflammatory system. We conducted a novel positional omics integration study to identify microRNAs that could shed further light on the possible links between microRNAs, adipose tissue immunity/inflammation and obesity. Methods: In contrast to previous methodologies employed for integration of heterogeneous OMIC data, we based the integration on genomic positions of alterations in human disease and employed an additional weighing step. A data search for various types of studies on obesity (genome-wide association, meta-analysis, transcriptomic, proteomic studies and epigenetic studies) was conducted to establish the initial data set. Results and discussion: The analysis identified 19 high scoring microRNAs (miR-146, miR-378, miR-143, miR-145, miR-194, miR-1273, miR-190, miR-561, miR-151, miR-215, miR-196, miR-328, miR-208, miR-3155A, miR-933, miR-4685, miR-640, miR-4659, and miR-877). Five (miR-146, miR-378, miR-143, miR-145, and miR-194), which may be directly linked to adipose tissue inflammation or obesity-related diseases, eight other microRNAs (miR-1273, miR-190, miR-561, miR-151, miR-215, miR-196, miR-328, and miR-208) have been identified to play a role in cancer and myocardial infarction, where obesity is a defined risk factor. Conclusion: In this study, we applied a new method of positional integrational analysis of different OMIC-layers and utilized an additional validation step through weighing. Our study yields a number of plausible microRNAs that provide an interesting basis for further research to elucidate underlying mechanisms of obesity. Our detection of common microRNAs which are also related with an increased risk for inflammations, cardiovascular problems and type 2 diabetes, irrespectively of gender and age may provide a path for understanding the inherited or acquired impact of microRNAs on human health and wellbeing. J Endocrinol Metab. 2017;7(3):77-85 doi: https://doi.org/10.14740/jem399w

MicroRNAs Responsible for Inflammation in Obesity

Background: In recent years, more and more evidence has accumulated to elucidate the modulating roles of microRNA in immune and inflammatory system. We conducted a novel positional omics integration study to identify microRNAs that could shed further light on the possible links between microRNAs, adipose tissue immunity/inflammation and obesity. Methods: In contrast to previous methodologies employed for integration of heterogeneous OMIC data, we based the integration on genomic positions of alterations in human disease and employed an additional weighing step. A data search for various types of studies on obesity (genome-wide association, meta-analysis, transcriptomic, proteomic studies and epigenetic studies) was conducted to establish the initial data set. Results and discussion: The analysis identified 19 high scoring microRNAs (miR-146, miR-378, miR-143, miR-145, miR-194, miR-1273, miR-190, miR-561, miR-151, miR-215, miR-196, miR-328, miR-208, miR-3155A, miR-933, miR-4685, miR-640, miR-4659, and miR-877). Five (miR-146, miR-378, miR-143, miR-145, and miR-194), which may be directly linked to adipose tissue inflammation or obesity-related diseases, eight other microRNAs (miR-1273, miR-190, miR-561, miR-151, miR-215, miR-196, miR-328, and miR-208) have been identified to play a role in cancer and myocardial infarction, where obesity is a defined risk factor. Conclusion: In this study, we applied a new method of positional integrational analysis of different OMIC-layers and utilized an additional validation step through weighing. Our study yields a number of plausible microRNAs that provide an interesting basis for further research to elucidate underlying mechanisms of obesity. Our detection of common microRNAs which are also related with an increased risk for inflammations, cardiovascular problems and type 2 diabetes, irrespectively of gender and age may provide a path for understanding the inherited or acquired impact of microRNAs on human health and wellbeing. J Endocrinol Metab. 2017;7(3):77-85 doi: https://doi.org/10.14740/jem399w

Genetic and epigenomic mechanisms of mammalian circadian transcription.

The mammalian molecular clock comprises a complex network of transcriptional programs that integrates environmental signals with physiological pathways in a tissue-specific manner. Emerging technologies are extending knowledge of basic clock features by uncovering their underlying molecular mechanisms, thus setting the stage for a 'systems' view of the molecular clock. Here we discuss how recent data from genome-wide genetic and epigenetic studies have informed the understanding of clock function. In addition to its importance in human physiology and disease, the clock mechanism provides an ideal model to assess general principles of dynamic transcription regulation in vivo.

Re-patterning of H3K27me3, H3K4me3 and DNA methylation during fibroblast conversion into induced cardiomyocytes

Direct conversion of fibroblasts into induced cardiomyocytes (iCMs) offers an alternative strategy for cardiac disease modeling and regeneration. During iCM reprogramming, the starting fibroblasts must overcome existing epigenetic barriers to acquire the CM-like chromatin pattern. However, epigenetic dynamics along this reprogramming process have not been studied. Here, we took advantage of our recently generated polycistronic system and determined the dynamics of two critical histone marks, H3K27me3 and H3K4me3, in parallel with gene expression at a set of carefully selected cardiac and fibroblast loci during iCM reprogramming. We observed reduced H3K27me3 and increased H3K4me3 at cardiac promoters as early as day 3, paralleled by a rapid significant increase in their mRNA expression. In contrast, H3K27me3 at loci encoding fibroblast marker genes did not increase until day 10 and H3K4me3 progressively decreased along the reprogramming process; these changes were accompanied by a gradual decrease in the mRNA expression of fibroblast marker genes. Further analyses of fibroblast-enriched transcription factors revealed a similarly late deposition of H3K27me3 and decreased mRNA expression of Sox9, Twist1 and Twist2, three important players in epithelial−mesenchymal transition. Our data suggest early rapid activation of the cardiac program and later progressive suppression of fibroblast fate at both epigenetic and transcriptional levels. Additionally, we determined the DNA methylation states of representative cardiac promoters and found that not every single CpG was equally demethylated during early stages of iCM reprogramming. Rather, there are specific CpGs, whose demethylation states correlated tightly with transcription activation, that we propose are the major contributing CpGs. Our work thus reveals a differential re-patterning of H3K27me3, H3K4me3 at cardiac and fibroblast loci during iCM reprogramming and could provide future genome-wide epigenetic studies with important guidance such as the appropriate time window and loci to be utilized as positive and negative controls.

Integration and Weighing of Omics Data for Obesity

Objective: OMICs research has become of great interest over the last years and enabled the research community to acquisition an increasing amount of data. In a prior study by our group, we have employed a novel positional integrative approach. For this second study we utilized the same integration method but weighed each individual data source trying to verify our already found regions and/or identify new gene regions of interest for obesity. Method: In contrast to previous methodologies employed for integration of heterogeneous OMIC data, we based the integration on genomic positions of alterations in human disease and employed an additional weighing step.A data search for various types of studies on Obesity (genome-wide association, meta-analysis, transcriptomic,proteomic studies and epigenetic studies) was conducted to establish the initial data set. For this study, five different weighing settings were used, to individually double the input of genomic data, transcriptomic data, proteomic data,microRNA data and epigenetic data in comparison to all the other data sources. Results and discussion: The analysis identified ten gene regions (ATP5O, ALK7, GAPDH, IFFO1, NCAPD2, DDX50, MAOB, ATM, STOX1 and PRRC2A). ATP5O, ALK7 and GADPH were also identified in our prior study as the highest scoring gene regions and prove to be consistent in our data set. Even though, the ten high ranked and discussed genes could not be directly linked to obesity nine of them are associated with Type 2 Diabetes or a neurodegenerative disease. Both disorders show a higher prevalence in obese individuals compared to lean. Conclusion: In this study, we applied a new method of positional integrational analysis of different OMIC-layers and an additional validation step through weighing. our study provides a basis for further research to elucidate underlying mechanisms of these associations and identify new targets for preventive and therapeutic interventions.

CardioGenBase: A Literature Based Multi-Omics Database for Major Cardiovascular Diseases.

Cardiovascular diseases (CVDs) account for high morbidity and mortality worldwide. Both, genetic and epigenetic factors are involved in the enumeration of various cardiovascular diseases. In recent years, a vast amount of multi-omics data are accumulated in the field of cardiovascular research, yet the understanding of key mechanistic aspects of CVDs remain uncovered. Hence, a comprehensive online resource tool is required to comprehend previous research findings and to draw novel methodology for understanding disease pathophysiology. Here, we have developed a literature-based database, CardioGenBase, collecting gene-disease association from Pubmed and MEDLINE. The database covers major cardiovascular diseases such as cerebrovascular disease, coronary artery disease (CAD), hypertensive heart disease, inflammatory heart disease, ischemic heart disease and rheumatic heart disease. It contains ~1,500 cardiovascular disease genes from ~2,4000 research articles. For each gene, literature evidence, ontology, pathways, single nucleotide polymorphism, protein-protein interaction network, normal gene expression, protein expressions in various body fluids and tissues are provided. In addition, tools like gene-disease association finder and gene expression finder are made available for the users with figures, tables, maps and venn diagram to fit their needs. To our knowledge, CardioGenBase is the only database to provide gene-disease association for above mentioned major cardiovascular diseases in a single portal. CardioGenBase is a vital online resource to support genome-wide analysis, genetic, epigenetic and pharmacological studies.

Genetic influences on nicotinic α5 receptor (CHRNA5) CpG methylation and mRNA expression in brain and adipose tissue.

IntroductionThe nicotinic α5 receptor subunit, encoded by CHRNA5, harbors multiple functional single nucleotide polymorphisms (SNPs) that affect mRNA expression and alter the encoded protein. These polymorphisms are most notably associated with drug-taking behaviors and cognition. We previously identified common SNPs in a distant regulatory element (DRE) that increase CHRNA5 mRNA expression in the human prefrontal cortex (PFC) and confer risk for nicotine dependence. Genome-wide epigenetic studies in PFC and adipose tissue find strong effects of the DRE SNPs on CpG methylation. However, it is unclear whether DRE SNPs influence CpG methylation en route to modulating CHRNA5 mRNA expression. It is also unclear whether these polymorphisms affect expression in other brain regions, especially those mediating drug-taking behaviors.ResultsBy measuring total and allelic CHRNA5 mRNA expression in human habenula and putamen autopsy tissues, we found that CHRNA5 DRE variants considerably increase mRNA expression by up to 3.5-fold in both brain regions. Our epigenetic analysis finds no association between CpG methylation and CHRNA5 mRNA expression in the PFC or adipose tissues.ConclusionsThese finding suggests the mechanisms responsible for the genetic modulation of CpG methylation and mRNA expression are independent despite the DRE SNPs being highly associated with both measures. Our findings support a strong association between the DRE SNPs and mRNA expression or CpG methylation in the brain and periphery, but the independence of the two measures leads us to conclude that environmental factors affecting CpG methylation do not appear to directly modulate gene expression.

Nucleated red blood cells impact DNA methylation and expression analyses of cord blood hematopoietic cells.

BackgroundGenome-wide DNA methylation (DNAm) studies have proven extremely useful to understand human hematopoiesis. Due to their active DNA content, nucleated red blood cells (nRBCs) contribute to epigenetic and transcriptomic studies derived from whole cord blood. Genomic studies of cord blood hematopoietic cells isolated by fluorescence-activated cell sorting (FACS) may be significantly altered by heterotopic interactions with nRBCs during conventional cell sorting.ResultsWe report that cord blood T cells, and to a lesser extent monocytes and B cells, physically engage with nRBCs during FACS. These heterotopic interactions resulted in significant cross-contamination of genome-wide epigenetic and transcriptomic data. Formal exclusion of erythroid lineage-specific markers yielded DNAm profiles (measured by the Illumina 450K array) of cord blood CD4 and CD8 T lymphocytes, B lymphocytes, natural killer (NK) cells, granulocytes, monocytes, and nRBCs that were more consistent with expected hematopoietic lineage relationships. Additionally, we identified eight highly differentially methylated CpG sites in nRBCs (false detection rate <5 %, |Δβ| >0.50) that can be used to detect nRBC contamination of purified hematopoietic cells or to assess the impact of nRBCs on whole cord blood DNAm profiles. Several of these erythroid markers are located in or near genes involved in erythropoiesis (ZFPM1, HDAC4) or immune function (MAP3K14, IFIT1B), reinforcing a possible immune regulatory role for nRBCs in early life.ConclusionsHeterotopic interactions between erythroid cells and white blood cells can result in contaminated cell populations if not properly excluded during cell sorting. Cord blood nRBCs have a distinct DNAm profile that can significantly skew epigenetic studies. Our findings have major implications for the design and interpretation of genome-wide epigenetic and transcriptomic studies using human cord blood.Electronic supplementary materialThe online version of this article (doi:10.1186/s13148-015-0129-6) contains supplementary material, which is available to authorized users.

Integration of OMICS Data for Obesity

Introduction: Obesity is a multifactorial condition that results from the interactions among genetic, dietary, environmental, and lifestyle factors. In our study, we have employed a novel integrative approach to identify mechanisms involved in human disease.Method: In contrast to previous methodologies employed for integration of heterogeneous OMIC data, we based the integration on genomic positions of alterations in human disease. A data search for various types of studies on obesity (genome-wide association, meta-analysis, transcriptomic, proteomic studies and epigenetic studies) was conducted in literature sources and OMIC data repositories, using GWAS Central and Medline database with search string (obesity) AND (transcriptome OR proteome OR genome-wide OR microarray OR profiling OR epigenetics). Additionally, Gene Expression Omnibus (GEO) repository, Array Express and Stanford Microarray Database were searched to discover suitable sources of data for inclusion in our initial dataset.Results and Discussion: As a result of the employed high through put technology, 71 high scoring regions were identified. We identified 8 high scoring gene regions (ATP5O, ALK7, CR1, CR2, S100, GAPDH, TLR1 and TLR6) that have not yet been associated to obesity. Interestingly, all of these genes were identified by Gene Ontology and Kyoto Encyclopedia of Genes and Genomes to be implicated in the energy metabolism and the immune response, which are known to be involved in obesity.Conclusion: In our study, we have performed a novel integrative approach to identify candidate regions and genes involved in human disease. The results showed that none of the high scoring genes that were identified were yet associated with obesity per se, but that they were found to be implicated in the immune response or the energy metabolism. Further research will be needed to validate the found gene regions for obesity.

Epigenetic Mechanisms of the Aging Human Retina.

Degenerative retinal diseases, such as glaucoma, age-related macular degeneration, and diabetic retinopathy, have complex etiologies with environmental, genetic, and epigenetic contributions to disease pathology. Much effort has gone into elucidating both the genetic and the environmental risk factors for these retinal diseases. However, little is known about how these genetic and environmental risk factors bring about molecular changes that lead to pathology. Epigenetic mechanisms have received extensive attention of late for their promise of bridging the gap between environmental exposures and disease development via their influence on gene expression. Recent studies have identified epigenetic changes that associate with the incidence and/or progression of each of these retinal diseases. Therefore, these epigenetic modifications may be involved in the underlying pathological mechanisms leading to blindness. Further genome-wide epigenetic studies that incorporate well-characterized tissue samples, consider challenges similar to those relevant to gene expression studies, and combine the genome-wide epigenetic data with genome-wide genetic and expression data to identify additional potentially causative agents of disease are needed. Such studies will allow researchers to create much-needed therapeutics to prevent and/or intervene in disease progression. Improved therapeutics will greatly enhance the quality of life and reduce the burden of disease management for millions of patients living with these potentially blinding conditions.

Neuroblastoma.

Neuroblastoma is the most common extracranial solid tumor in children. Since its first description some 150 years ago, its enigmatic clinical presentation has challenged clinicians and fascinated basic researchers. This article presents an overview of the key clinical features of neuroblastoma and current therapeutic approaches. It also highlights how our understanding of sympathoadrenal developmental biology, coupled with transcriptome analyses, next-generation sequencing, and genome-wide association and epigenetic studies, has illuminated critical signal transduction pathways involved in neuroblastoma tumorigenesis and identified therapeutically tractable targets for clinical development.

A polymorphism in the TH 2 locus control region is associated with changes in DNA methylation and gene expression.

Genomewide association and epigenetic studies found a region within the RAD50 gene on chromosome 5q31 to be associated with total serum IgE levels and asthma. In mice, this region harbors a locus control region for nearby TH 2 cytokines, which is characterized by four Rad50 DNase I hypersensitive sites (RHS4-7). Among these, RHS7 seems to have the strongest impact on TH 2 differentiation. We investigated whether within the human homolog of RHS7, functional polymorphisms exist, which could affect DNA methylation or gene expression in the 5q31 locus and might have an influence on asthma status or IgE regulation. The human RHS7 region was fine mapped using 1000 genomes database information. In silico analysis and electrophoretic mobility shift assays were used to assess SNP function. Allele-specific effects on DNA methylation were evaluated in cord blood (n=73) and at age of 4.5years (n=61) by pyrosequencing. Allele-specific effects on RAD50, IL4, and IL13 expression were analyzed in 100 subjects. Associations with asthma and IgE levels were investigated in the MAGICS/ISAAC II population (n=1145). Polymorphism rs2240032 in the RHS7 region is suggestive of allele-specific transcription factor binding, affects methylation of the IL13 promoter region and influences RAD50 and IL4 expression (lowest P=0.0027). It is also associated with total serum IgE levels (P=0.0227). A functional relevant polymorphism in the TH 2 locus control region, equivalent to RHS7 in mice, affects DNA methylation and gene expression within 5q31 and influences total serum IgE on the population level.

Integrated analysis of genome-wide genetic and epigenetic association data for identification of disease mechanisms

Many human diseases are multifactorial, involving multiple genetic and environmental factors impacting on one or more biological pathways. Much of the environmental effect is believed to be mediated through epigenetic changes. Although many genome-wide genetic and epigenetic association studies have been conducted for different diseases and traits, it is still far from clear to what extent the genomic loci and biological pathways identified in the genetic and epigenetic studies are shared. There is also a lack of statistical tools to assess these important aspects of disease mechanisms. In the present study, we describe a protocol for the integrated analysis of genome-wide genetic and epigenetic data based on permutation of a sum statistic for the combined effects in a locus or pathway. The method was then applied to published type 1 diabetes (T1D) genome-wide- and epigenome-wide-association studies data to identify genomic loci and biological pathways that are associated with T1D genetically and epigenetically. Through combined analysis, novel loci and pathways were also identified, which could add to our understanding of disease mechanisms of T1D as well as complex diseases in general.