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1.  Epigenome-Wide Association Studies for common human diseases 
Nature reviews. Genetics  2011;12(8):529-541.
Despite the success of genome-wide association studies (GWAS) in identifying loci associated with common diseases, a significant proportion of the causality remains unexplained. Recent advances in genomic technologies have placed us in a position to initiate large-scale studies of human disease-associated epigenetic variation, specifically variation in DNA methylation (DNAm). Such Epigenome-Wide Association Studies (EWAS) present novel opportunities but also create new challenges that are not encountered in GWAS. We discuss EWAS study design, cohort and sample selections, statistical significance and power, confounding factors, and follow-up studies. We also discuss how integration of EWAS with GWAS can help to dissect complex GWAS haplotypes for functional analysis.
PMCID: PMC3508712  PMID: 21747404
Epigenomics; Disease Genetics; DNA Methylation; Epigenetics; Quantitative Trait
2.  Functional regulatory T cells produced by inhibiting cyclic nucleotide phosphodiesterase type 3 prevent allograft rejection 
Science translational medicine  2011;3(83):83ra40.
Regulatory T cells (Tregs) manipulated ex vivo have potential as cellular therapeutics in autoimmunity and transplantation. Although it is possible to expand naturally occurring Tregs, an attractive alternative possibility, particularly suited to solid organ and bone marrow transplantation, is the stimulation of total T cell populations with defined allogeneic antigen presenting cells under conditions that lead to the generation or expansion of donor-reactive, adaptive Tregs. Here we demonstrate that stimulation of mouse CD4+ T cells by immature allogeneic dendritic cells (DCs) combined with pharmacological inhibition of phosphodiesterase 3 (PDEi) results in a functional enrichment of Foxp3+ T cells. Without further manipulation or selection, the resultant population delayed skin allograft rejection mediated by polyclonal CD4+ effectors or donor-reactive CD8+ TCR transgenic T cells and inhibited both effector cell proliferation and T cell priming for IFN-γ production. Notably, PDE inhibition also enhanced the enrichment of human Foxp3+ CD4+ T cells driven by allogeneic APC. These cells inhibited T cell proliferation in a standard in vitro mixed lymphocyte assay and importantly, attenuated the development of vasculopathy mediated by autologous PBMC in a functionally relevant humanized mouse transplant model. These data establish a method for the ex vivo generation of graft-reactive, functional mouse and human Tregs that uses a clinically approved agent, making pharmacological PDE inhibition a potential strategy for Treg-based therapies
PMCID: PMC3321352  PMID: 21593400
3.  Genome-Wide Screen for Differential DNA Methylation Associated with Neural Cell Differentiation in Mouse 
PLoS ONE  2011;6(10):e26002.
Cellular differentiation involves widespread epigenetic reprogramming, including modulation of DNA methylation patterns. Using Differential Methylation Hybridization (DMH) in combination with a custom DMH array containing 51,243 features covering more than 16,000 murine genes, we carried out a genome-wide screen for cell- and tissue-specific differentially methylated regions (tDMRs) in undifferentiated embryonic stem cells (ESCs), in in-vitro induced neural stem cells (NSCs) and 8 differentiated embryonic and adult tissues. Unsupervised clustering of the generated data showed distinct cell- and tissue-specific DNA methylation profiles, revealing 202 significant tDMRs (p<0.005) between ESCs and NSCs and a further 380 tDMRs (p<0.05) between NSCs/ESCs and embryonic brain tissue. We validated these tDMRs using direct bisulfite sequencing (DBS) and methylated DNA immunoprecipitation on chip (MeDIP-chip). Gene ontology (GO) analysis of the genes associated with these tDMRs showed significant (absolute Z score>1.96) enrichment for genes involved in neural differentiation, including, for example, Jag1 and Tcf4. Our results provide robust evidence for the relevance of DNA methylation in early neural development and identify novel marker candidates for neural cell differentiation.
PMCID: PMC3196508  PMID: 22028803
4.  Identification of Type 1 Diabetes–Associated DNA Methylation Variable Positions That Precede Disease Diagnosis 
PLoS Genetics  2011;7(9):e1002300.
Monozygotic (MZ) twin pair discordance for childhood-onset Type 1 Diabetes (T1D) is ∼50%, implicating roles for genetic and non-genetic factors in the aetiology of this complex autoimmune disease. Although significant progress has been made in elucidating the genetics of T1D in recent years, the non-genetic component has remained poorly defined. We hypothesized that epigenetic variation could underlie some of the non-genetic component of T1D aetiology and, thus, performed an epigenome-wide association study (EWAS) for this disease. We generated genome-wide DNA methylation profiles of purified CD14+ monocytes (an immune effector cell type relevant to T1D pathogenesis) from 15 T1D–discordant MZ twin pairs. This identified 132 different CpG sites at which the direction of the intra-MZ pair DNA methylation difference significantly correlated with the diabetic state, i.e. T1D–associated methylation variable positions (T1D–MVPs). We confirmed these T1D–MVPs display statistically significant intra-MZ pair DNA methylation differences in the expected direction in an independent set of T1D–discordant MZ pairs (P = 0.035). Then, to establish the temporal origins of the T1D–MVPs, we generated two further genome-wide datasets and established that, when compared with controls, T1D–MVPs are enriched in singletons both before (P = 0.001) and at (P = 0.015) disease diagnosis, and also in singletons positive for diabetes-associated autoantibodies but disease-free even after 12 years follow-up (P = 0.0023). Combined, these results suggest that T1D–MVPs arise very early in the etiological process that leads to overt T1D. Our EWAS of T1D represents an important contribution toward understanding the etiological role of epigenetic variation in type 1 diabetes, and it is also the first systematic analysis of the temporal origins of disease-associated epigenetic variation for any human complex disease.
Author Summary
Type 1 diabetes (T1D) is a complex autoimmune disease affecting >30 million people worldwide. It is caused by a combination of genetic and non-genetic factors, leading to destruction of insulin-secreting cells. Although significant progress has recently been made in elucidating the genetics of T1D, the non-genetic component has remained poorly defined. Epigenetic modifications, such as methylation of DNA, are indispensable for genomic processes such as transcriptional regulation and are frequently perturbed in human disease. We therefore hypothesized that epigenetic variation could underlie some of the non-genetic component of T1D aetiology, and we performed a genome-wide DNA methylation analysis of a specific subset of immune cells (monocytes) from monozygotic twins discordant for T1D. This revealed the presence of T1D–specific methylation variable positions (T1D–MVPs) in the T1D–affected co-twins. Since these T1D–MVPs were found in MZ twins, they cannot be due to genetic differences. Additional experiments revealed that some of these T1D–MVPs are found in individuals before T1D diagnosis, suggesting they arise very early in the process that leads to overt T1D and are not simply due to post-disease associated factors (e.g. medication or long-term metabolic changes). T1D–MVPs may thus potentially represent a previously unappreciated, and important, component of type 1 diabetes risk.
PMCID: PMC3183089  PMID: 21980303
5.  The tammar wallaby major histocompatibility complex shows evidence of past genomic instability 
BMC Genomics  2011;12:421.
The major histocompatibility complex (MHC) is a group of genes with a variety of roles in the innate and adaptive immune responses. MHC genes form a genetically linked cluster in eutherian mammals, an organization that is thought to confer functional and evolutionary advantages to the immune system. The tammar wallaby (Macropus eugenii), an Australian marsupial, provides a unique model for understanding MHC gene evolution, as many of its antigen presenting genes are not linked to the MHC, but are scattered around the genome.
Here we describe the 'core' tammar wallaby MHC region on chromosome 2q by ordering and sequencing 33 BAC clones, covering over 4.5 MB and containing 129 genes. When compared to the MHC region of the South American opossum, eutherian mammals and non-mammals, the wallaby MHC has a novel gene organization. The wallaby has undergone an expansion of MHC class II genes, which are separated into two clusters by the class III genes. The antigen processing genes have undergone duplication, resulting in two copies of TAP1 and three copies of TAP2. Notably, Kangaroo Endogenous Retroviral Elements are present within the region and may have contributed to the genomic instability.
The wallaby MHC has been extensively remodeled since the American and Australian marsupials last shared a common ancestor. The instability is characterized by the movement of antigen presenting genes away from the core MHC, most likely via the presence and activity of retroviral elements. We propose that the movement of class II genes away from the ancestral class II region has allowed this gene family to expand and diversify in the wallaby. The duplication of TAP genes in the wallaby MHC makes this species a unique model organism for studying the relationship between MHC gene organization and function.
PMCID: PMC3179965  PMID: 21854592

Results 1-5 (5)