Identification of somatic mutations in parathyroid tumors using whole-exome sequencing

Context: The underlying molecular alterations causing sporadic parathyroid adenomas that drive primary hyperparathyroidism have not been thoroughly defined. Objective: The aim of the study was to investigate the occurrence of somatic mutations driving tumor formation and progression in sporadic parathyroid adenoma using whole-exome sequencing. Design: Eight matched tumor-constitutional DNA pairs from patients with sporadic parathyroid adenomas underwent whole-exome capture and high-throughput sequencing. Selected genes were analyzed for mutations in an additional 185 parathyroid adenomas. Results: Four of eight tumors displayed a frame shift deletion or nonsense mutation in MEN1, which was accompanied by loss of heterozygosity of the remaining wild-type allele. No other mutated genes were shared among the eight tumors. One tumor harbored a Y641N mutation of the histone methyltransferase EZH2 gene, previously linked to myeloid and lymphoid malignancy formation. Targeted sequencing in the additional 185 parathyroid adenomas revealed a high rate of MEN1 mutations (35%). Furthermore, this targeted sequencing identified an additional parathyroid adenoma that contained the identical, somatic EZH2 mutation that was found by exome sequencing. Conclusion: This study confirms the frequent role of the loss of heterozygosity of chromosome 11 and MEN1 gene alterations in sporadic parathyroid adenomas and implicates a previously unassociated methyltransferase gene, EZH2, in endocrine tumorigenesis.     

Human macrophage and dendritic cell-specific silencing of high-mobility group protein B1 ameliorates sepsis in a humanized mouse model

Hypersecretion of cytokines by innate immune cells is thought to initiate multiple organ failure in murine models of sepsis. Whether human cytokine storm also plays a similar role is not clear. Here, we show that human hematopoietic cells are required to induce sepsis-induced mortality following cecal ligation and puncture (CLP) in the severely immunodeficient nonobese diabetic (NOD)/SCID/IL2Rγ^−/− mice, and siRNA treatment to inhibit HMGB1 release by human macrophages and dendritic cells dramatically reduces sepsis-induced mortality. Following CLP, compared with immunocompetent WT mice, NOD/SCID/IL2Rγ^−/− mice did not show high levels of serum HMGB1 or murine proinflammatory cytokines and were relatively resistant to sepsis-induced mortality. In contrast, NOD/SCID/IL2Rγ^−/− mice transplanted with human hematopoietic stem cells [humanized bone marrow liver thymic mice (BLT) mice] showed high serum levels of HMGB1, as well as multiple human but not murine proinflammatory cytokines, and died uniformly, suggesting human cytokines are sufficient to induce organ failure in this model. Moreover, targeted delivery of HMGB1 siRNA to human macrophages and dendritic cells using a short acetylcholine receptor (AchR)-binding peptide [rabies virus glycoprotein (RVG)-9R] effectively suppressed secretion of HMGB1, reduced the human cytokine storm, human lymphocyte apoptosis, and rescued humanized mice from CLP-induced mortality. siRNA treatment was also effective when started after the appearance of sepsis symptoms. These results show that CLP in humanized mice provides a model to study human sepsis, HMGB1 siRNA might provide a treatment strategy for human sepsis, and RVG-9R provides a tool to deliver siRNA to human macrophages and dendritic cells that could potentially be used to suppress a variety of human inflammatory diseases.Sepsis is an important cause of mortality in intensive-care units, with more than 750,000 individuals developing severe sepsis in North America annually and a mortality rate varying between 35 and 50% (1, 2). The pathogenesis of sepsis includes countless disturbances of the host immune system starting with a harmful, infection-triggered exaggerated inflammatory cascade that results in tissue injury and rapidly leads to massive apoptosis of immune cells (2, 3). This is followed by a secondary immune paralysis phase accompanied by uncontrolled growth of bacteria and tissue damage. Although therapy to suppress the immediate cytokine response, such as treatment with TNF and IL-1β antibodies, have failed in clinical trials (4–6), it has now come to be recognized that, at least in animal models, high-mobility group protein 1 (HMGB1), which is secreted from macrophages and dendritic cells (DCs) but not lymphocytes late in the disease, acts as a master regulator of late and sustained cytokine storm, up-regulating many cytokines including TNF-α, IL-6, IL-1β, and IL-8 (reviewed in refs. 7–11). In fact, injection of mice with HMGB1 is enough to induce the lethal organ damage seen in sepsis (12), whereas treatment with neutralizing HMGB1 antibody can rescue mice and rats from experimental sepsis (13, 14). However, although HMGB1 is also secreted in human sepsis (12), its role in sepsis pathogenesis or the impact of its neutralization on human cells remain unclear.RNA interference can be used to silence virtually any gene, including multiple genes, as long as a way can be found to introduce small interfering (si)RNAs into relevant cell types in vivo without toxicity. Several advances have been made in developing methods to deliver siRNA in vivo to different cell types, most successfully to the liver cells (reviewed in refs. 15–17). A lipid-like nanoparticle called C12-200, which had been developed for liver-specific delivery of siRNA, was recently also shown to deliver siRNA to murine monocytes, and silencing C-C chemokine receptor type 2 (CCR2) in monocytes using this reagent was effective in reducing atherosclerosis, islet transplantation and tumors (18). Whether this reagent also targets human DCs and monocytes/macrophages is unclear. We have reported previously that a short 29-aa peptide derived from the rabies virus glycoprotein (RVG), fused to 9R residues (RVG-9R), can deliver siRNA to murine macrophages and brain cells by specific binding to its ligand acetylcholine receptor (AchR) (19, 20). Because AchR is also expressed on human macrophages and DCs (21) and also because the acetylcholine-binding site on the α7 subunit is highly conserved, we reasoned that RVG-9R might also be used to target human macrophages and DCs. In this study, we validate this hypothesis in vitro, as well as in vivo, using human hematopoietic stem cell–engrafted nonobese diabetic NOD/SCID/IL2Rγ^−/− mice that lack mouse innate and adaptive immune systems (22). More importantly, we also show that silencing human HMGB1 using this delivery reagent in this mouse model substantially reduces human lymphocyte apoptosis and cytokine storm and protects mice from sepsis-induced mortality.