The methodology is implemented in the limma software package for R, available from the CRAN repository http://www.r-project.org

No abstract

We report the discovery of the GATA2 gene as a new myelodysplastic syndrome (MDS)/acute myeloid leukemia (AML) predisposition gene. We found the same, novel heterozygous c.1061C>T (p.Thr354Met) missense mutation in the GATA2 transcription factor gene segregating with the multigenerational transmission of MDS/AML in three families, and a GATA2 c.1063_1065delACA (p.Thr355del) mutation at an adjacent codon in a fourth MDS/AML family. The mutations reside within the second zinc finger of GATA2 which mediates DNA-binding and protein-protein interactions. We show differential effects of the mutants on transactivation of target genes, cellular differentiation, apoptosis and global gene expression. Identification of such predisposing genes to familial forms of MDS and AML is critical for more effective diagnosis and prognosis, counselling, selection of related bone marrow transplant donors, and development of therapies.

Aire-expressing medullary thymic epithelial cells (mTECs) play a key role in preventing autoimmunity by expressing tissue-restricted antigens to help purge the emerging T cell receptor repertoire of self-reactive specificities. Here we demonstrate a novel role for a CD4+3− inducer cell population, previously linked to development of organized secondary lymphoid structures and maintenance of T cell memory in the functional regulation of Aire-mediated promiscuous gene expression in the thymus. CD4+3− cells are closely associated with mTECs in adult thymus, and in fetal thymus their appearance is temporally linked with the appearance of Aire+ mTECs. We show that RANKL signals from this cell promote the maturation of RANK-expressing CD80−Aire− mTEC progenitors into CD80+Aire+ mTECs, and that transplantation of RANK-deficient thymic stroma into immunodeficient hosts induces autoimmunity. Collectively, our data reveal cellular and molecular mechanisms leading to the generation of Aire+ mTECs and highlight a previously unrecognized role for CD4+3−RANKL+ inducer cells in intrathymic self-tolerance.

MicroRNAs (miRNAs) participate in most aspects of cellular differentiation and homeostasis, and consequently have roles in many pathologies, including cancer. These small non-coding RNAs exert their effects in the context of complex regulatory networks, often made all the more extensive by the inclusion of transcription factors as their direct targets. In recent years, the increased availability of gene expression data and the development of methodologies that profile miRNA targets en masse have fuelled our understanding of miRNA functions, and of the sources and consequences of miRNA dysregulation. Advances in experimental and computational approaches are revealing not just cancer pathways controlled by single miRNAs but also intermeshed regulatory networks controlled by multiple miRNAs, which often engage in reciprocal feedback interactions with the targets that they regulate.

Familial platelet disorder with predisposition to acute myelogenous leukemia (FPD/ AML) is an autosomal dominant familial platelet disorder characterized by thrombocytopenia and a propensity to develop AML. Mutation analyses of RUNX1 in 3 families with FPD/AML showing linkage to chromosome 21q22.1 revealed 3 novel heterozygous point mutations (K83E, R135fsX177 (IVS4 ؉ 3delA), and Y260X). Functional investigations of the 7 FPD/ AML RUNX1 Runt domain point mutations described to date (2 frameshift, 2 nonsense, and 3 missense mutations) were performed. Consistent with the position of the mutations in the Runt domain at the RUNX1-DNA interface, DNA binding of all mutant RUNX1 proteins was absent or significantly decreased. In general, missense and nonsense RUNX1 proteins retained the ability to heterodimerize with PEBP2␤/CBF␤ and inhibited transactivation of a reporter gene by wild-type RUNX1. Colocalization of mutant RUNX1 and PEBP2␤/CBF␤ in the cytoplasm was observed. These results suggest that the sequestration of PEBP2␤/CBF␤ by mutant RUNX1 may cause the inhibitory effects. While haploinsufficiency of RUNX1 causes FPD/AML in some families (deletions and frameshifts), mutant RUNX1 proteins (missense and nonsense) may also inhibit wild-type RUNX1, possibly creating a higher propensity to develop leukemia. This is consistent with the hypothesis that a second mutation has to occur, either in RUNX1 or another gene, to cause leukemia among individuals harboring RUNX1 FPD/AML mutations and that the propensity to acquire these additional mutations is determined, at least partially, by the initial RUNX1 mutation. IntroductionThe most frequent mutations associated with leukemia are recurrent somatic chromosomal translocations or inversions, many of which involve the polyomavirus enhancer-binding protein or core-binding factor transcriptional regulation complex (PEBP2/CBF). Several translocations involve the ␣ subunit of this complex, the RUNX1 gene (also called AML1, CBF␣2, or PEBP2␣B) on chromosome 21q22.1 (t(8;21), t(3;21), and t(12;21)). Additionally, the ␤ subunit of the complex, PEBP2␤ also called CBF␤, is disrupted in inv(16)(p13;q22). 1 An abundance of evidence points to the existence of genes that predispose to hematologic malignancies. However, large multiple-generation families with hematologic malignancies alone are rare. 2 Only 2 loci for familial hematologic malignancies have been identified to date, 1 on chromosome 21q22.1 3 and the other on 16q22. 4,5 These loci contain RUNX1 and PEBP2␤/CBF␤, respectively.Studies of families that demonstrate single-gene inheritance for leukemia predisposition should help to identify the genes and mechanisms involved in the first steps of leukemia development. The autosomal dominant familial platelet disorder (FPD)/ AML (acute myelogenous leukemia; Online Mendelian Inheritance in Man no. 601399) is a good model to validate this hypothesis because, in addition to developing thrombocytopenia, patients show a propensity for progression to myelodysplasia and acute myeloid leuke...

Medullary thymic epithelial cells (mTECs) are specialized for inducing central immunological tolerance to self-antigens. To accomplish this, mTECs must adopt a mature phenotype characterized by expression of the autoimmune regulator Aire, which activates the transcription of numerous genes encoding tissue-restricted self-antigens. The mechanisms that control mature Aire(+) mTEC development in the postnatal thymus remain poorly understood. We demonstrate here that, although either CD4(+) or CD8(+) thymocytes are sufficient to sustain formation of a well-defined medulla, expansion of the mature mTEC population requires autoantigen-specific interactions between positively selected CD4(+) thymocytes bearing autoreactive T cell receptor (TCR) and mTECs displaying cognate self-peptide-MHC class II complexes. These interactions also involve the engagement of CD40 on mTECs by CD40L induced on the positively selected CD4(+) thymocytes. This antigen-specific TCR-MHC class II-mediated crosstalk between CD4(+) thymocytes and mTECs defines a unique checkpoint in thymic stromal development that is pivotal for generating a mature mTEC population competent for ensuring central T cell tolerance.

Inactivation of the autoimmune regulator (Aire) gene causes a rare recessive disorder, autoimmune polyendocrine syndrome 1 (APS1), but it is not known if Aire-dependent tolerance mechanisms are susceptible to the quantitative genetic changes thought to underlie more common autoimmune diseases. In mice with a targeted mutation, complete loss of Aire abolished expression of an insulin promoter transgene in thymic epithelium, but had no effect in pancreatic islets or the testes. Loss of one copy of Aire diminished thymic expression of the endogenous insulin gene and the transgene, resulting in a 300% increase in islet-reactive CD4 T cells escaping thymic deletion in T cell receptor transgenic mice, and dramatically increased progression to diabetes. Thymic deletion induced by antigen under control of the thyroglobulin promoter was abolished in Aire homozygotes and less efficient in heterozygotes, providing an explanation for thyroid autoimmunity in APS1. In contrast, Aire deficiency had no effect on thymic deletion to antigen controlled by a systemic H-2K promoter. The sensitivity of Aire-dependent thymic deletion to small reductions in function makes this pathway a prime candidate for more subtle autoimmune quantitative trait loci, and suggests that methods to increase Aire activity would be a potent strategy to lower the incidence of organ-specific autoimmunity.