DNA microarray analysis in down syndrome

Cyanide exerts its toxic effects primarily by inhibiting mitochondrial Complex IV (Cytochrome c oxidase, CCOx). Recent studies have shown that mammalian cells can endogenously produce cyanide from glycine via a lysosomal pathway. At low concentrations, cyanide may play regulatory roles, but at higher levels, it causes metabolic inhibition. Here we show that Down syndrome (DS) cells and tissues exhibit significant overproduction of cyanide, contributing to cellular metabolic suppression. DS rats show elevated blood cyanide levels, and their tissues generate more cyanide than wild-type controls—both under basal conditions and following glycine supplementation. Similarly, human DS fibroblasts produce higher levels of cyanide than healthy control cells. We attribute this increased cyanide production in DS to the marked downregulation of thiosulfate sulfurtransferase (TST, also known as rhodanese), the key enzyme responsible for cyanide detoxification. Importantly, suppression of lysosomal cyanide production in DS cells (through cyanide scavengers, lysosomal deacidification, or inhibition of serine/glycine conversion) improves cellular bioenergetics and/or enhances cell proliferation rates. Previous work has implicated excessive hydrogen sulfide (H2S) production, another endogenous gaseous signaling molecule that inhibits CCOx, in DS-associated metabolic suppression. Our current findings indicate that cyanide overproduction may also contribute to this dysfunction. Cyanide and H2S may act cooperatively on the same molecular target. These results open the possibility of developing therapeutic strategies that target cyanide or both cyanide and H2S to counteract DS-associated metabolic impairment.

Somatic stem cell activity is critical for tissue homeostasis. Defects in stem cells are thought to be involved in many diseases, including inherited disorders and aging (He et al , 2009). In a recent paper published in Nature , Adorno et al (2013) demonstrate that there is a general somatic stem cell defect in Down syndrome (DS), a congenital disorder with triplication of human chromosome 21 (HSA21; Roper and Reeves, 2006; Megarbane et al , 2009). They report that the deubiquitinase Usp16 gene located on HSA21 is a key epigenetic switch that regulates stem cell self‐renewal and senescence in DS, and suggest that inhibiting or reducing HSA21 may be beneficial in treating the sequelae of DS. DS is the most common human chromosomal abnormality with triplication of all or part of HSA21 in humans. Individuals with DS uniformly exhibit varying degrees of developmental delays, mental retardation and premature ageing (Roper and Reeves, 2006; Megarbane et al , 2009). While the underlying mechanisms of these multifactorial alterations in different systems remain to be defined, one possibility is that the functional decline of adult stem cells may partially …

Down syndrome (DS), caused by an extra copy of chromosome 21, is a leading cause of intellectual disability. One of the key factors contributing to this intellectual disability is impaired neurogenesis observed from fetal stages onwards. To study these neurodevelopmental abnormalities, human-induced pluripotent stem cells (hiPSCs) generated using cells obtained from DS patients provide a valuable and relevant model. Here, a comprehensive protocol is described for recapitulating DS-impaired neurogenesis observed during DS fetal stages. This protocol utilizes a pair of DS-hiPSCs having three copies of chromosome 21 and its isogenic euploid hiPSCs having two copies of chromosome 21. Importantly, the protocol described here recapitulates DS-impaired neurogenesis and found that biphasic cell cycle defect, i.e., reduced proliferation of DS neural progenitor cells (NPC) during the early phase of the neurogenic stage followed by increased proliferation of DS NPC during the late phase of the neurogenic stage is the cause of DS impaired neurogenesis. Increased proliferation of DS NPC during the late phase of the neurogenic stage leads to delayed exit from the cell cycle, causing reduced generation of post-mitotic neurons from DS NPCs. This protocol includes detailed steps for the maintenance of hiPSCs, their differentiation into neural lineages displaying biphasic cell cycle defect during the neurogenic stage, and the subsequent validation of reduced neural differentiation in DS cells. By following this methodology, researchers can create a robust experimental system that mimics the neurodevelopmental conditions of DS, enabling them to explore the specific alterations in brain development caused by trisomy 21.

Defective DNA repair and increased chromatin binding of DNA repair factors in Down syndrome fibroblasts

BackgroundDown syndrome (DS) neurons are more susceptible to oxidative stress and previous studies have shown that vitamin E was able to reduce oxidative stress and improve DS neurons' viability. Therefore, this study was done to investigate the protective role of γ-tocotrienol (γT3) in DS neurons from hydrogen peroxide (H2O2) -induced oxidative stress. The pro-apoptosis tendency of γT3 was compared to α-tocopherol (αT) in non-stress condition as well.MethodsPrimary culture of DS and euploid neurons were divided into six groups of treatment: control, H2O2, γT3 pre-treatment with H2O2, γT3 only, αT pre-treatment with H2O2 and αT only. The treatments were assessed by MTS assay and apoptosis assay by single-stranded DNA (ssDNA) apoptosis ELISA assay, Hoechst and Neu-N immunofluorescence staining. The cellular uptake of γT3 and αT was determined by HPLC while protein expressions were determined by Western blot. Comparison between groups was made by the Student's t test, one-way ANOVA and Bonferroni adjustment as well as two-way ANOVA for multiple comparisons.ResultsOne day incubation of γT3 was able to reduced apoptosis of DS neurons by 10%, however γT3 was cytotoxic at longer incubation period (14 days) and at concentrations ≥ 100 μM. Pre-treatment of αT and γT3 only attenuate apoptosis and increase cell viability in H2O2-treated DS and euploid neurons by 10% in which the effects were minimal to maintain most of the DS cells' morphology. γT3 act as a free radical scavenger by reducing ROS generated by H2O2. In untreated controls, DS neurons showed lower Bcl-2/Bax ratio and p53 expression compared to normal neurons, while cPKC and PKC-δ expressions were higher in DS neurons. On the other hand, pre-treatment of γT3 in H2O2-treated DS neurons have reduced Bcl-2/Bax ratio, which was not shown in euploid neurons. This suggests that pre-treatment of γT3 did not promote DS cell survival. Meanwhile γT3 and αT treatments without H2O2 as well as pre-treatment of γT3 and αT induced changes in cPKC and PKC-δ expression in DS neurons suggesting interaction of γT3 and αT with PKC activity.ConclusionOur study suggests that γT3 pre-treatment are not sufficient to protect DS neurons from H2O2-induced oxidative assault, instead induced the apoptosis process.

Trisomy of chromosome 21 (TS21) is the most common autosomal aneuploidy compatible with postnatal survival with a prevalence of 1 in 700 newborns. Its phenotype is highly complex with constant features, such as mental retardation, dysmorphic traits and hypotonia, and variable features including heart defects, susceptibility to Alzheimer’s disease (AD), type 2 diabetes, obesity and immune disorders. Overexpression of genes on chromosome-21 (Hsa21) is responsible for the pathogenesis of Down syndrome (DS) phenotypic features either in a direct or in an indirect manner since many Hsa21 genes can affect the expression of other genes mapping to different chromosomes. Many of these genes are involved in mitochondrial function and energy conversion, and play a central role in the mitochondrial dysfunction and chronic oxidative stress, consistently observed in DS subjects.Recent studies highlight the deep interconnections between mitochondrial dysfunction and DS phenotype. In this short review we first provide a basic overview of mitochondrial phenotype in DS cells and tissues. We then discuss how specific Hsa21 genes may be involved in determining the disruption of mitochondrial DS phenotype and biogenesis. Finally we briefly focus on drugs that affect mitochondrial function and mitochondrial network suggesting possible therapeutic approaches to improve and/or prevent some aspects of the DS phenotype.

SUMMARY Beneficial insects such as honeybees and silkworms are threatened by numerous pests, parasites and pathogens. However, pest insects as m uch as beneficial species also often acquire infectious diseases, which can potentially be used for pest control. To take advantage of pathogens infecting pest species, detailed knowl edge of the immune system of pest insects is necessary. Here, the immune gene Down syndrome cell adhesion molecule (Dscam) is studied in Australian bulldog and jumper ants, whos e venom can cause a fatal anaphylactic shock in humans. In insects, Dscam shows extraordinary genetic diversity through alternative splicing. More than 12000 alternative splice varian ts are estimated being transcribed in the Western honeybee. To understand better the function and evolution of Dscam, one of the four variable exon cassettes was sequenced from six Myrmecia ant species. Using a comparative codon-based model, the results do not indicate any evidence of adaptive evolution in the analysed data set. Corroborating previous studies, the comparison of Dscam in ants and other insects including the Western honeybee and silkworms demonstrates high similarity of this gene across insect orders and suggests a conserved function of this gene.

The Down syndrome cell adhesion molecule (Dscam) family, is within the cell adhesion molecules, a
\nfamily whose members are characterized by being composed of immunoglobulin (Ig) and fibronectin
\ndomains and which are known to play an essential role in the development of the nervous system in both
\nvertebrates and invertebrates.
\nIn insects, one member of the Dscam family diversified extensively due to internal exon duplications
\nand a sophisticated mechanism of mutually exclusive alternative splicing (AS). This enables a single
\nindividual to generate somatically thousands of Dscam isoforms which differ in half of two Ig domains
\nand in another complete Ig domain. That creates a high diversity of adhesion properties which are used by
\nnervous cells and also by immune cells (hemocytes).
\nHow this situation evolved is best understood my means of comparative studies. I have studied aspects
\nof the evolution and expression of this diversified member of the Dscam family mainly in the brachiopod
\ncrustacean Daphnia magna and to lesser extent, in other representatives of the arthropod phyla. I have
\nshown that like in insects, a highly variable Dscam gene evolved in crustaceans, which also express
\nDscam diversity in nervous and in immune cells. Additionally I could demonstrate that not only Dscam’s
\nectodomains are diversified but that several cytoplasmic tails with different signal transduction capacities
\ncan also be expressed. The comparison between Daphnia and insects revealed furthermore that there is
\nhigh amino acid conservation among distantly related species for most Dscam domains except for the Ig
\nregions that are coded by the multiple exons, suggesting that the latter evolved under different selective
\nconstraints.
\nDscam has been proposed as an exciting candidate molecule for mediating specific immune responses
\nin arthropods. Nevertheless, the involvement of Dscam in immunity remains largely elusive. I tested the
\neffect of parasite infection on the expression of total Dscam and on the diversity of some duplicated exons
\nat the RNA level and found no significant effect. Yet, hemocytes expressed reduced transcript diversity
\nrelative to the brain, but each transcript was likely more abundant. This would be consistent with a
\nfunction in the immune system given that each Dscam isoform would be present in higher concentrations
\nwhich would increase their functional capacity.
\nDscam isoforms engage in dimer formation with other identical isoforms, promoting cell-cell
\nrecognition. It has been demonstrated that the variable parts of Dscam coded by the duplicated exons
\nmediate dimer formation. The genetic diversification caused by exon duplication and AS has thus direct
\nfunctional implications. I estimated signatures of selection on some of the regions involved in dimer
\nformation by comparing sequences from different Daphnia magna populations and from different species
\nof Daphnia and Drosophila. The results indicated that diversity created by duplication followed by
\ndivergence is maintained by purifying selection against new mutations and against new gene conversion
\nevents. That is consistent with the essential role of Dscam diversity in the nervous system. Contrastingly, I
\nfound that some parts of the variable regions which are not involved in dimer formation and are oriented
\ntowards the dimer’s external environment, may evolve under positive selection, which would be consistent
\nwith an immune function.
\nTo understand the evolutionary history of the molecule, I searched for Dscam related genes in
\nrepresentatives of chelicerates (Ixodes scapularis) and myriapodes (Strigamia maritima), two other groups
\nof arthropods. In both myriapodes and chelicerates, Dscam diversified extensively by whole gene
\nduplications and by duplications of some internal exons coding for one Ig domain region, but not several,
\nlike in insects and crustaceans. Similar duplications could have provided the raw material from which the
\nhighly diverse Dscam evolved uniquely in the ancestors of crustaceans and insects. I propose a speculative
\nscenario under which the evolution of this remarkable gene might have occurred.

Astrocytes exert adverse effects on the brains of individuals with Down syndrome (DS). Although a neurogenic-to-gliogenic shift in the fate-specification step has been reported, the mechanisms and key regulators underlying the accelerated proliferation of astrocyte precursor cells (APCs) in DS remain elusive. Here, we established a human isogenic cell line panel based on DS-specific induced pluripotent stem cells, the XIST-mediated transcriptional silencing system in trisomic chromosome 21, and genome/chromosome-editing technologies to eliminate phenotypic fluctuations caused by genetic variation. The transcriptional responses of genes observed upon XIST induction and/or downregulation are not uniform, and only a small subset of genes show a characteristic expression pattern, which is consistent with the proliferative phenotypes of DS APCs. Comparative analysis and experimental verification using gene modification reveal dose-dependent proliferation-promoting activity of DYRK1A and PIGP on DS APCs. Our collection of human isogenic cell lines provides a comprehensive set of cellular models for further DS investigations.

Children with Down syndrome (DS) and acute lymphoblastic leukemia (ALL) have an inferior prognosis compared with non-DS ALL patients. We reviewed methotrexate (MTX)/mercaptopurine (6MP) maintenance therapy data for children with DS treated according to the Nordic Society of Pediatric Hematology and Oncology (NOPHO) ALL92 or the NOPHO ALL2000 protocols between 1992 and 2007. The 5-year event-free survival probability (pEFS(5 yr)) for the 66 DS patients was inferior to the 2602 non-DS patients (0.50 ± 0.07 vs 0.77 ± 0.01 (P<0.001)). The 48 DS patients in first remission at the beginning of maintenance therapy had pEFS(10 yr) below that of the 522 non-DS control patients (pEFS(10 yr): 0.58 (95% confidence interval (CI) 0.43-0.77) vs 0.83 (95% CI 0.80-0.86), respectively (P<0.0001)). The DS patients received lower median doses of MTX (median: 11.8 vs 15.4 (P<0.0001)) and 6MP (median: 43.6 vs 59.4 (P<0.0001)). In Cox regression analysis, male gender, presence of DS and high median maintenance therapy white blood cell levels (mWBC) were associated with increased risk for relapse. DS-ALL patients with mWBC above or below 3.5 × 10(9)/l (protocol target) had pEFS(10 yr) of 0.31 and 0.72 (P=0.02), and the mWBC hazard ratio for DS-ALL patients was 2.0 (P<0.0005). We conclude that insufficient treatment intensity during maintenance therapy of DS-ALL patients may contribute to their poor prognosis.

Pathfinding of growing axons to reach their target during brain development is a subtle process needed to build up contacts between neurons. Abnormalities in brain development in Down Syndrome (DS) are described in a couple of morphological reports but the molecular mechanisms underlying abnormal wiring in fetal DS brain are not yet elucidated. We therefore performed a study using the proteomic approach to show differences in protein levels involved in the guidance of axons between control and DS brain in early prenatal life. Proteins obtained from autopsy of human fetal abortus were applied on 2-dimensional gel, identified and quantified. We quantified 5 members of the semaphorin/collapsin family, the dihydropyrimidinase related proteins 1-4 and the collapsin response mediator protein-5 (CRMP-5) in 8 DS and 7 control cortex samples. DRP-1 and CRMP-5 levels were comparable in the control and DS samples. Evaluation of DRP-2, DRP-3 and DRP-4 revealed significantly decreased levels of 2 of the 15 spots assigned to DRP-2 and increased levels of one spot assigned to DRP-3 and increased DRP-4 in DS brain. We conclude that as early as from the 19th week of gestation pathfinding cues of the outgrowing axons are impaired in DS. These findings may help to elucidate mechanisms leading to abnormalities in neural migration of DS brain.

Background: Down syndrome (DS) is a complex genetic disease that is caused by having three copies of chromosome 21. A possible association between polymorphisms in maternal folate metabolism genes and DS has been evaluated. Objectives: It was aimed to first investigate the influence of C677T and A1298C polymorphisms in the methylenetetrahydrofolate reductase gene (MTHFR) and plasma homocysteine (Hcy) on the maternal risk for DS in the southwest of Iran. Methods: The MTHFR C677T and A1298C polymorphisms were genotyped using restriction fragment length polymorphism and Sanger sequencing, respectively. Allele and genotype frequencies and the dominant model of the MTHFR C677T and A1298C polymorphisms were evaluated in 80 mothers of children with DS and 80 control mothers. Eventually, the ELISA test was used to compare the concentration of plasma Hcy in both groups. Results: A significant association was observed in the 677T and 1298C alleles between the mothers of DS and control groups (P = 0.00077 and P = 0.01248, respectively). Further, the median concentrations of Hcy were significantly higher in mothers with DS babies compared to the control group (P &lt; 0.05). Conclusions: There was an association between MTHFR C677T, A1298C, and plasma Hcy concentrations as the maternal risk of mothers with DS children.

An expansive menu of prenatal tests for Down syndrome (DS) is already available to pregnant women around the globe, but new tests are likely to become the most popular entrees....

Atrioventricular septal defect (AVSD) is a clinically significant subtype of congenital heart disease (CHD) that severely influences the health of babies during birth and is associated with Down syndrome (DS). Thus, exploring the differences in functional genes in DS samples with and without AVSD is a critical way to investigate the complex association between AVSD and DS. In this study, we present a computational method to distinguish DS patients with AVSD from those without AVSD using the newly proposed self-normalizing neural network (SNN). First, each patient was encoded by using the copy number of probes on chromosome 21. The encoded features were ranked by the reliable Monte Carlo feature selection (MCFS) method to obtain a ranked feature list. Based on this feature list, we used a two-stage incremental feature selection to construct two series of feature subsets and applied SNNs to build classifiers to identify optimal features. Results show that 2737 optimal features were obtained, and the corresponding optimal SNN classifier constructed on optimal features yielded a Matthew’s correlation coefficient (MCC) value of 0.748. For comparison, random forest was also used to build classifiers and uncover optimal features. This method received an optimal MCC value of 0.582 when top 132 features were utilized. Finally, we analyzed some key features derived from the optimal features in SNNs found in literature support to further reveal their essential roles.

Down syndrome (DS), trisomy of human chromosome 21 (HSA21), is characterized by lifelong cognitive impairments and the development of the neuropathological hallmarks of Alzheimer's disease (AD). The cellular and molecular modifications responsible for these effects are not understood. Here we performed single-nucleus RNA sequencing (snRNA-seq) employing both short- (Illumina) and long-read (Pacific Biosciences) sequencing technologies on a total of 29 DS and non-DS control prefrontal cortex samples. In DS, the ratio of inhibitory-to-excitatory neurons was significantly increased, which was not observed in previous reports examining sporadic AD. DS microglial transcriptomes displayed AD-related aging and activation signatures in advance of AD neuropathology, with increased microglial expression of C1q complement genes (associated with dendritic pruning) and the HSA21 transcription factor gene RUNX1 Long-read sequencing detected vast RNA isoform diversity within and among specific cell types, including numerous sequences that differed between DS and control brains. Notably, over 8,000 genes produced RNAs containing intra-exonic junctions, including amyloid precursor protein (APP) that had previously been associated with somatic gene recombination. These and related results illuminate large-scale cellular and transcriptomic alterations as features of the aging DS brain.