A major project that involves the TIS is the definition of molecular biomarkers for autoimmune and autoinflammatory diseases. In collaboration with Dan Kastner and Ivona Aksentijevich, we have measured serum cytokines levels in patients with a novel autoinflammatory disease caused by haploinsufficiency of A20 (encoded by TNFAIP3). The syndrome resembles Behcets disease (BD) and our data showed that these patients have increased expression of NFκB-mediated proinflammatory cytokines. A manuscript reporting these findings is currently being reviewed. With the Goldbach-Mansky group, we studied patients with Chronic Atypical Neutrophilic Dermatosis with Lipodystrophy and Elevated temperature (CANDLE), a Proteasome-Associated Autoinflammatory Syndrome (PRAAS), who were shown to have in the inducible proteasome-subunit, PSMB8/β5i. The TIS was responsible for measuring patient serum cytokine levels, which revealed an increase in type I IFNs as well as inflammatory cytokines such as IL-6 and TNF. We continue to collaborate with NHGRI investigators assessing the cytokine-secreting capabilities of patients with Erdheim-Chester Disease (ECD), a rare, non-familial multisystem disorder characterized by proliferation and infiltration of non-Langerhans histocytes into multiple organs. We compared the cytokine secreted by ECD patients to normal controls after stimulation of PBMCs. The TIS has also been investigating novel approaches for the treatment of autoimmune diseases. In collaboration with the O'Shea group, the Kaplan group, and Pfizer (via a CRADA), we evaluated the efficacy of tofacitinib on a murine model of systemic lupus erythematous (SLE) the MRL/lpr mouse. Tofacitinib treatment results in a significant amelioration of SLE phenotype, besides reducing autoantibody levels, nephritis, skin inflammation and inflammatory cytokines, tofacitinib mitigates the dysregulation of neutrophil function and induces significant improvements in endothelium-dependent vasorelaxation, which is relevant to vascular health. A manuscript reporting these results is currently under review. We renewed the CRADA and received second-generation JAK-selective inhibitors, which we will evaluate for their activity on T cells. We generated induced pluripotent stem cells (iPSC) from a patient with HLA B-27+ Ankylosing Spondylitis and demonstrated their pluripotency. We then differentiated iPSCs into MSC and osteoblasts, and found that MSC-derived osteoblasts from patients showed greater mineralization capacity comparing with normal controls. We have continued the gene-editing project using Crispr-CAS9 techniques to change B*27:04 into B*27:06 (non-disease-associated allele) and assess role of the gene in the disease process. Editing specific HLA alleles has been difficult due to the high levels of sequence homology and dense variability found within the target region of the genome. In order to understand the mechanism(s) by which HLA-B27 contributes to disease, we have tried to delete the HLA-B27 gene in AS patients iPS cells using CRISPR/CAS9. We have used GFP sorting and single colony manual selection and FACS to enrich knock-out of HLA B*27 iPSc populations. After selection we have identified 18 (out of 120) colonies with potential HLA B27 gene knock-out (by FACS). We are currently utilizing RNAseq to confirm our results and decipher the target region. The TIS is also providing support to several projects carried out at NIAMS to improve the understanding of the genetic determinants of autoimmune, autoinflammatory and musculoskeletal diseases using the Illumina HiSeq 2500 and the MiSeq for ultra high-throughput DNA sequencing. With the Sartorelli group we used NGS technology to explore the role of proteins, such as Polycomb proteins, Spt6, the transcription factor Notch or the histone variant macroH2A1.2, differently involved in chromatin modification and transcriptional control. By using chip-Seq and RNA-Seq we identified the histone chaperone Spt6 as a regulator of PRC2 through direct interaction with Suz12. We are also exploring the role of eRNAs in regulating gene expression during muscle differentiation. With Drs. OShea and Kanno we have performed ATAC-Seq to comprehensively map chromatin accessibility among T helper subsets and innate lymphocytes of rare populations ex vivo. Further, we have used GRO-Seq and nascent RNA-Seq to map on-going transcription and reported an unappreciated role of BRD4 to support transcription elongation. With Drs. Casellas and Kieffer we have worked on the role of CTCF in B cell nuclear architecture, creating CTCF zinc finger mutations in ES and B cells and studying their effect by RNA-Seq, ChIP-Seq, and HiC. We are also exploring how B cell activation changes the compaction of chromatin. To this end we have done MNase-Seq, ChIP-Seq, and RNA-Seq. With Dr. Morasso we study the molecular and cellular mechanisms that are altered in absence of Dlx3 by performing RNA-Seq at different time points after deletion of Dlx3 in epidermis. We also utilized ChIP-sequencing to analyze the binding of DLX3 and histone modifications in Pam212 cells and suprabasal murine skin. We are using ATAC-Seq data to identify the re-positioning of nucleosomes and transcription factors involved in epidermal differentiation. With Dr. Rabens lab we evaluate TFE3 therapeutic potential in Pompe disease and we mapped its binding sites across the genome in the disease and lso integrated the ChIP-Seq data with gene expression data obtained from RNA-Seq. With Dr. Blanco in the Kaplan lab we sequenced small RNA present in neutrophils extracellular traps (NETs) derived from healthy controls or lupus patients, with the goal of comparing those NETs data with the sequence obtained from whole neutrophils by sequencing the total RNA from 25 samples containing whole cells. With Drs. Siegel, Deng and Ivovic we are screening somatic mutations in patients with autoimmune diseases by deep sequencing of targeted genes suspected to be involved in the diseases. We are also testing and implementing ultra-sensitive somatic mutation detection methods such as duplex sequencing, circle sequencing and HaloplexHs. With Dr. Ombrello we performed targeted deep resequencing of sJIA patient samples. We validated library quality and performed sequencing runs of small batches of individuals to optimize the pooling of the target capture baits. With Drs. Goldbach-Mansky and Almeida de Jesus we have performed RNA-Seq in libraries generated from samples from patients with diseases such as: chronic atypical dermatosis with lipodystrophy and elevated temperature syndrome; STING-associated vasculopathy with onset in infancy; Aicardi-Goutires syndrome; severe combined immunodeficiency due to ADA1 deficiency; primary immunodeficiency due to STAT1 gain-of-function mutations; systemic sclerosis; and juvenile systemic lupus erythematosus. We also performed sequencing to obtain phase information for patients who carried two heterozygous variants in the same gene, by first performing cDNA PCR amplification of the targeted region followed by tagmentation and indexing of the amplicons using the Illumina Nextera technology. With Drs. Colbert and Gill we performed RNA-Seq of rat cecal tissue to understand host immune regulation in response to microbial dysbiosis during Spondyloarthritis-like disease in rats. Dr. Canela in Dr. Nussenzweig group (NCI) continued studies in the role of MLL4 in lymphomagenesis. The Hafner lab is dissecting post-transcriptional regulatory mechanisms of RNA-binding proteins using next-generation sequencing-based methods. The Mammensgroup performed ChIP-Seq experiments to identify genes regulated by the Nucleosome and Remodeling and Deacetylase (NuRD) complex during the differentiation of myoblasts into myotubes.
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