Ethnicity-specific epigenetic variation in naïve CD4+ T cells and the susceptibility to autoimmunity

Abstract Background Genetic and epigenetic variability contributes to the susceptibility and pathogenesis of autoimmune diseases. T cells play an important role in several autoimmune conditions, including lupus, which is more common and more severe in people of African descent. To investigate inherent epigenetic differences in T cells between ethnicities, we characterized genome-wide DNA methylation patterns in naïve CD4+ T cells in healthy African-Americans and European-Americans, and then confirmed our findings in lupus patients. Results Impressive ethnicity-specific clustering of DNA methylation profiling in naïve CD4+ T cells was revealed. Hypomethylated loci in healthy African-Americans were significantly enriched in pro-apoptotic and pro-inflammatory genes. We also found hypomethylated genes in African-Americans to be disproportionately related to autoimmune diseases including lupus. We then confirmed that these genes, such as IL32, CD226, CDKN1A, and PTPRN2 were similarly hypomethylated in lupus patients of African-American compared to European-American descent. Using patch DNA methylation and luciferase reporter constructs, we showed that methylation of the IL32 promoter region reduces gene expression in vitro. Importantly, bisulfite DNA sequencing demonstrated that cis-acting genetic variants within and directly disrupting CpG sites account for some ethnicity-specific variability in DNA methylation. Conclusion Ethnicity-specific inherited epigenetic susceptibility loci in CD4+ T cells provide clues to explain differences in the susceptibility to autoimmunity and possibly other T cell-related diseases between populations.http://deepblue.lib.umich.edu/bitstream/2027.42/116042/1/13072_2015_Article_37.pd

Whole Exome Sequencing Identifies Rare Protein-Coding Variants in Behçet's Disease

Behçet's disease (BD) is a systemic inflammatory disease with an incompletely understood etiology. Despite the identification of multiple common genetic variants associated with BD, rare genetic variants have been less explored. We undertook this study to investigate the role of rare variants in BD by performing whole exome sequencing in BD patients of European descent. METHODS: Whole exome sequencing was performed in a discovery set comprising 14 German BD patients of European descent. For replication and validation, Sanger sequencing and Sequenom genotyping were performed in the discovery set and in 2 additional independent sets of 49 German BD patients and 129 Italian BD patients of European descent. Genetic association analysis was then performed in BD patients and 503 controls of European descent. Functional effects of associated genetic variants were assessed using bioinformatic approaches. RESULTS: Using whole exome sequencing, we identified 77 rare variants (in 74 genes) with predicted protein-damaging effects in BD. These variants were genotyped in 2 additional patient sets and then analyzed to reveal significant associations with BD at 2 genetic variants detected in all 3 patient sets that remained significant after Bonferroni correction. We detected genetic association between BD and LIMK2 (rs149034313), involved in regulating cytoskeletal reorganization, and between BD and NEIL1 (rs5745908), involved in base excision DNA repair (P = 3.22 × 10(-4) and P = 5.16 × 10(-4) , respectively). The LIMK2 association is a missense variant with predicted protein damage that may influence functional interactions with proteins involved in cytoskeletal regulation by Rho GTPase, inflammation mediated by chemokine and cytokine signaling pathways, T cell activation, and angiogenesis (Bonferroni-corrected P = 5.63 × 10(-14) , P = 7.29 × 10(-6) , P = 1.15 × 10(-5) , and P = 6.40 × 10(-3) , respectively). The genetic association in NEIL1 is a predicted splice donor variant that may introduce a deleterious intron retention and result in a noncoding transcript variant. CONCLUSION: We used whole exome sequencing in BD for the first time and identified 2 rare putative protein-damaging genetic variants associated with this disease. These genetic variants might influence cytoskeletal regulation and DNA repair mechanisms in BD and might provide further insight into increased leukocyte tissue infiltration and the role of oxidative stress in BD

Molecular Analysis of Recurrent Translocations in Mucoepidermoid Carcinoma

Head and neck cancers include a diverse group of malignancies, and pathogenesis is driven by different recurring somatic mutations. In head and neck squamous cell carcinoma (HNSCC), these mutations include single nucleotide variants of several different genes, as well as HPV viral integration. By contrast, many salivary gland tumors are characterized by genomic translocations, resulting in frequent gene fusions. For example, mucoepidermoid carcinomas (MEC) have prevalent CRTC1-MAML2 fusions, while hyalinizing clear cell carcinomas (HCCC) have prevalent EWSR1-ATF1 fusions. Exploring the molecular phenotypes caused by these driver mutations and others will better explain the mechanisms of tumorigenesis and growth in salivary gland tumors, and is therefore necessary to identify potential targets for future patient treatments. In my thesis, I investigate the hypothesis that driver mutations, such as CRTC1-MAML2 and EWSR1-ATF1, alter transcription regulation in salivary gland tumors, varying based on tumor type, fusion status, and grade. In this thesis, I begin by using molecular techniques to differentiate two salivary gland tumors, MEC and HCCC, which are difficult to differentiate by standard histopathology approaches. Using RNA sequencing (RNAseq), I identify a 354 gene signature that differentiates both malignancies. These genes are significantly enriched for an ATF1 binding motif, consistent with the EWSR1-ATF1 fusion found in HCCC. These differentially expressed genes include IGF1R, SGK1, and SGK3, which are elevated in HCCC tumors. This, and other differentially expressed genes in this signature, describe examples of differing molecular pathology between MEC and HCCC. I then seek to further understand the genetic underpinning of MEC. Within MEC tumors, the most common somatic translocation forms the CRTC1-MAML2 fusion. I map the CRTC1-MAML2 breakpoint in four MEC-derived cell lines, via long-read sequencing. I also identify a series of genomic translocations leading to this fusion and uncover a TERT promoter rearrangement in NCI-H292. Subsequent TERT break apart FISH reveals TERT copy number increase and translocation events in all four cell lines tested. These experiments reveal complex genomic rearrangement leading to CRTC1-MAML2 formation and a novel TERT driver mutation. Thus, I discover and validate TERT as a novel MEC driver. While the CRTC1-MAML2 fusion is the most common MEC driver mutation, patients with CRTC1-MAML2, or less commonly CRTC3-MAML2, positive tumors have a better prognosis. Therefore, using RNAseq on 48 MEC tumors, I identify gene expression patterns associated with tumor CRTC1/3-MAML2 fusion status and grade. Gene expression signatures associated with fusion status are enriched for gene sets involving cellular respiration, including oxidative phosphorylation and the electron transport chain. Moreover, changes to T and B cell infiltration are associated with MAML2 fusion status and grade, respectively. Therefore, I perform spatial RNA sequencing to measure the effect of CRTC1-MAML2 activity throughout the MEC tumor microenvironment. I identify spatial overlap between CRTC1-MAML2 associated gene expression and many other transcripts, including VEGFA and CTNNB1. These data suggest that CRTC1-MAML2-associated gene expression affect a variety of biological processes throughout the tumor microenvironment. Overall, these data describe a pattern of gene regulation dependent on tumor type, fusion status, and grade. These gene expression changes, coupled with novel driver mutations, such as TERT translocation, affect multiple cancer phenotypes throughout the tumor microenvironment. These biological processes play a role in the molecular etiology of HCCC and MEC tumors, uncovering several pathways which are opportunities to advance targeted therapies, which may improve the survival of MEC and HCCC patients.PHDCellular & Molecular BiologyUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/176529/1/gensterb_1.pd

Protecting Tumors by Preventing Human Papilloma Virus Antigen Presentation: Insights from Emerging Bioinformatics Algorithms

Recent developments in bioinformatics technologies have led to advances in our understanding of how oncogenic viruses such as the human papilloma virus drive cancer progression and evade the host immune system. Here, we focus our review on understanding how these emerging bioinformatics technologies influence our understanding of how human papilloma virus (HPV) drives immune escape in cancers of the head and neck, and how these new informatics approaches may be generally applicable to other virally driven cancers. Indeed, these tools enable researchers to put existing data from genome wide association studies, in which high risk alleles have been identified, in the context of our current understanding of cellular processes regulating neoantigen presentation. In the future, these new bioinformatics approaches are highly likely to influence precision medicine-based decision making for the use of immunotherapies in virally driven cancers

Ethnicity-specific epigenetic variation in naïve CD4+ T cells and the susceptibility to autoimmunity

BACKGROUND: Genetic and epigenetic variability contributes to the susceptibility and pathogenesis of autoimmune diseases. T cells play an important role in several autoimmune conditions, including lupus, which is more common and more severe in people of African descent. To investigate inherent epigenetic differences in T cells between ethnicities, we characterized genome-wide DNA methylation patterns in naïve CD4+ T cells in healthy African-Americans and European-Americans, and then confirmed our findings in lupus patients. RESULTS: Impressive ethnicity-specific clustering of DNA methylation profiling in naïve CD4+ T cells was revealed. Hypomethylated loci in healthy African-Americans were significantly enriched in pro-apoptotic and pro-inflammatory genes. We also found hypomethylated genes in African-Americans to be disproportionately related to autoimmune diseases including lupus. We then confirmed that these genes, such as IL32, CD226, CDKN1A, and PTPRN2 were similarly hypomethylated in lupus patients of African-American compared to European-American descent. Using patch DNA methylation and luciferase reporter constructs, we showed that methylation of the IL32 promoter region reduces gene expression in vitro. Importantly, bisulfite DNA sequencing demonstrated that cis-acting genetic variants within and directly disrupting CpG sites account for some ethnicity-specific variability in DNA methylation. CONCLUSION: Ethnicity-specific inherited epigenetic susceptibility loci in CD4+ T cells provide clues to explain differences in the susceptibility to autoimmunity and possibly other T cell-related diseases between populations

MOESM1 of Ethnicity-specific epigenetic variation in naĂŻve CD4+ T cells and the susceptibility to autoimmunity

Additional file 1. Supplementary material (Table S1 and Figure S1)

Microbe-Mediated Activation of Toll-like Receptor 2 Drives PDL1 Expression in HNSCC

As immunotherapies targeting the PDL1 checkpoint have become a mainstay of treatment for a subset of head and neck squamous cell carcinoma (HNSCC) patients, a detailed understanding of the mechanisms underlying PDL1-mediated immune evasion is needed. To elucidate factors regulating expression of PDL1 in HNSCC cells, a genome-wide CRISPR profiling approach was implemented to identify genes and pathways conferring altered PDL1 expression in an HNSCC cell line model. Our screen nominated several candidate PDL1 drivers, including Toll-like Receptor 2 (TLR2). Depletion of TLR2 blocks interferon-γ-induced PDL1 expression, and stimulation of TLR2 with either Staphylococcus aureus or a bacterial lipopeptide mimetic, Pam3CSK4, enhanced PDL1 expression in multiple models. The data herein demonstrate a role for TLR2 in modulating the expression of PDL1 in HNSCC models and suggest that microbiota may directly modulate immunosuppression in cancer cells. Our study represents a step toward disentangling the diverse pathways and stimuli regulating PDL1 expression in HNSCC and underscores a need for future work to characterize the complex microbiome in HNSCC patients treated with immunotherapy