Claire Mulligan

Acquired mutations in GATA1 in neonates with Down's syndrome with transient myeloid disorder

Transient myeloid disorder is a unique self-regressing neoplasia specific to Downs syndrome. The transcription factor GATA1 is needed for normal growth and maturation of erythroid cells and megakaryocytes. Mutations in GATA1 have been reported in acute megakaryoblastic leukaemia in Downs syndrome. We aimed to investigate changes in GATA1 in patients with Downs syndrome and either transient myeloid disorder (n=10) or acute megakaryoblastic leukaemia (n=6). We recorded mutations eliminating exon 2 from GATA1 in all patients with transient myeloid disorder (age 0-24 days) and in all with acute megakaryoblastic leukaemia (age 14-38 months). The range of mutations did not differ between patients with each disorder. Patients with transient myeloid disorder with mutations in GATA1 can regress spontaneously to complete remission, and mutations do not necessarily predict later acute megakaryoblastic leukaemia.

ETO/MTG8 Is an Inhibitor of C/EBPβ Activity and a Regulator of Early Adipogenesis

ABSTRACT The putative transcriptional corepressor ETO/MTG8 has been extensively studied due to its involvement in a chromosomal translocation causing the t(8;21) form of acute myeloid leukemia. Despite this, the role of ETO in normal physiology has remained obscure. Here we show that ETO is highly expressed in preadipocytes and acts as an inhibitor of C/EBPβ during early adipogenesis, contributing to its characteristically delayed activation. ETO prevents both the transcriptional activation of the C/EBPα promoter by C/EBPβ and its concurrent accumulation in centromeric sites during early adipogenesis. ETO expression rapidly reduces after the initiation of adipogenesis, and this is essential to the normal induction of adipogenic gene expression. These findings define, for the first time, a molecular role for ETO in normal physiology as an inhibitor of C/EBPβ and a novel regulator of early adipogenesis.

Microarray transcript profiling distinguishes the transient from the acute type of megakaryoblastic leukaemia (M7) in Down's syndrome, revealing PRAME as a specific discriminating marker

Transient myeloproliferative disorder (TMD) is a unique, spontaneously regressing neoplasia specific to Downs syndrome (DS), affecting up to 10% of DS neonates. In 20–30% of cases, it reoccurs as progressive acute megakaryoblastic leukaemia (AMKL) at 2–4 years of age. The TMD and AMKL blasts are morphologically and immuno‐phenotypically identical, and have the same acquired mutations in GATA1. We performed transcript profiling of nine TMD patients comparing them with seven AMKL patients using Affymetrix HG‐U133A microarrays. Similar overall transcript profiles were observed between the two conditions, which were only separable by supervised clustering. Taqman analysis on 10 TMD and 10 AMKL RNA samples verified the expression of selected differing genes, with statistical significance (P < 0·05) by Students t‐test. The Taqman differences were also reproduced on TMD and AMKL blasts sorted by a fluorescence‐activated cell sorter. Among the significant differences, CDKN2C, the effector of GATA1‐mediated cell cycle arrest, was increased in AMKL but not TMD, despite the similar level of GATA1. In contrast, MYCN (neuroblastoma‐derived oncogene) was expressed in TMD at a significantly greater level than in AMKL. MYCN has not previously been described in leukaemogenesis. Finally, the tumour antigen PRAME was identified as a specific marker for AMKL blasts, with no expression in TMD. This study provides markers discriminating TMD from AMKL‐M7 in DS. These markers have the potential as predictive, diagnostic and therapeutic targets. In addition, the study provides further clues into the pathomechanisms discerning self‐regressive from the progressive phenotype.

Loss‐of‐function JAK3 mutations in TMD and AMKL of Down syndrome

Acquired mutations activating Janus kinase 3 (jak3) have been reported in Down syndrome (DS) and non‐DS patients with acute megakaryoblastic leukaemia (AMKL). This highlighted jak3‐activation as an important event in the pathogenesis of AMKL, and predicted inhibitors of jak3 as conceptual therapeutics for AMKL. Of 16 DS‐transient myeloproliferative disorder (TMD)/AMKL patients tested, seven showed JAK3 mutations. Three mutations deleted the kinase (JH1) domain, abolishing the main function of jak3. Another patient displayed a mutation identical to a previously reported inherited loss‐of‐function causing severe combined immunodeficiency. Our data suggest that both gain‐, and loss‐of function mutations of jak3 can be acquired in DS‐TMD/AMKL.

Trisomic dose of several chromosome 21 genes perturbs haematopoietic stem and progenitor cell differentiation in Down's syndrome

Children with Downs syndrome (DS) have 20–50-fold higher incidence of all leukaemias (lymphoid and myeloid), for reasons not understood. As incidence of many solid tumours is much lower in DS, we speculated that disturbed early haematopoietic differentiation could be the cause of increased leukaemia risk. If a common mechanism is behind the risk of both major leukaemia types, it would have to arise before the bifurcation to myeloid and lymphoid lineages. Using the transchromosomic system (mouse embryonic stem cells (ESCs)) bearing an extra human chromosome 21 (HSA21)) we analyzed the early stages of haematopoietic commitment (mesodermal colony formation) in vitro. We observed that trisomy 21 (T21) causes increased production of haemogenic endothelial cells, haematopoietic stem cell precursors and increased colony forming potential, with significantly increased immature progenitors. Transchromosomic colonies showed increased expression of Gata-2, c-Kit and Tie-2. A panel of partial T21 ESCs allowed us to assign these effects to HSA21 sub-regions, mapped by 3.5 kbp-resolution tiling arrays. The Gata-2 increase on one side, and c-Kit and Tie-2 increases on the other, could be attributed to two different, non-overlapping HSA21 regions. Using human-specific small interfering RNA silencing, we could demonstrate that an extra copy of RUNX1, but not ETS-2 or ERG, causes an increase in Tie-2/c-Kit levels. Finally, we detected significantly increased levels of RUNX1, C-KIT and PU.1 in human foetal livers with T21. We conclude that overdose of more than one HSA21 gene contributes to the disturbance of early haematopoiesis in DS, and that one of the contributors is RUNX1. As the observed T21-driven hyperproduction of multipotential immature precursors precedes the bifurcation to lymphoid and myeloid lineages, we speculate that this could create conditions of increased chance for acquisition of pre-leukaemogenic rearrangements/mutations in both lymphoid and myeloid lineages during foetal haematopoiesis, contributing to the increased risk of both leukaemia types in DS.

An additional human chromosome 21 causes suppression of neural fate of pluripotent mouse embryonic stem cells in a teratoma model

BackgroundDown syndrome (DS), caused by trisomy of human chromosome 21 (HSA21), is the most common genetic cause of mental retardation in humans. Among complex phenotypes, it displays a number of neural pathologies including smaller brain size, reduced numbers of neurons, reduced dendritic spine density and plasticity, and early Alzheimer-like neurodegeneration. Mouse models for DS show behavioural and cognitive defects, synaptic plasticity defects, and reduced hippocampal and cerebellar neuron numbers. Early postnatal development of both human and mouse-model DS shows the reduced capability of neuronal precursor cells to generate neurons. The exact molecular cause of this reduction, and the role played by increased dosage of individual HSA21 genes, remain unknown.ResultsWe have subcutaneously injected mouse pluripotent ES cells containing a single freely segregating supernumerary human chromosome 21 (HSA21) into syngeneic mice, to generate transchromosomic teratomas. Transchromosomic cells and parental control cells were injected into opposite flanks of thirty mice in three independent experiments. Tumours were grown for 30 days, a time-span equivalent to combined intra-uterine, and early post-natal mouse development. When paired teratomas from the same animals were compared, transchromosomic tumours showed a three-fold lower percentage of neuroectodermal tissue, as well as significantly reduced mRNA levels for neuron specific (Tubb3) and glia specific (Gfap) genes, relative to euploid controls. Two thirds of transchromosomic tumours also showed a lack of PCR amplification with multiple primers specific for HSA21, which were present in the ES cells at the point of injection, thus restricting a commonly retained trisomy to less than a third of HSA21 genes.ConclusionWe demonstrate that a supernumerary chromosome 21 causes Inhibition of Neuroectodermal DIfferentiation (INDI) of pluripotent ES cells. The data suggest that trisomy of less than a third of HSA21 genes, in two chromosomal regions, might be sufficient to cause this effect.

Quantitative Proteomics Characterization of a Mouse Embryonic Stem Cell Model of Down Syndrome

Down syndrome, caused by the trisomy of chromosome 21, is a complex condition characterized by a number of phenotypic features, including reduced neuron number and synaptic plasticity, early Alzheimer disease-like neurodegeneration, craniofacial dysmorphia, heart development defects, increased incidence of childhood leukemia, and powerful suppression of the incidence of most solid tumors. Mouse models replicate a number of these phenotypes. The Tc1 Down syndrome model was constructed by introducing a single supernumerary human chromosome 21 into a mouse embryonic stem cell, and it reproduces a large number of Down syndrome phenotypes including heart development defects. However, little is still known about the developmental onset of the trisomy 21-induced mechanisms behind these phenotypes or the proteins that are responsible for them. This study determined the proteomic differences that are present in undifferentiated embryonic stem cells and are caused by an additional human chromosome 21. A total of 1661 proteins were identified using two-dimensional liquid chromatography followed by tandem mass spectrometry from whole embryonic stem cell lysates. Using isobaric tags for relative and absolute quantification, we found 52 proteins that differed in expression by greater than two standard deviations from the mean when an extra human chromosome 21 was present. Of these, at least 11 have a possible functional association with a Down syndrome phenotype or a human chromosome 21-encoded gene. This study also showed that quantitative protein expression differences in embryonic stem cells can persist to adult mouse as well as reproduce in human Down syndrome fetal tissue. This indicates that changes that are determined in embryonic stem cells of Down syndrome could potentially identify proteins that are involved in phenotypes of Down syndrome, and it shows that these cell lines can be used for the purpose of studying these pathomechanisms.