Altered expression of hypothetical proteins in hippocampus of transgenic mice overexpressing human Cu/Zn-superoxide dismutase 1

Background  

Cu/Zn-superoxide dismutase 1 (SOD1), encoded on chromosome 21, is a key enzyme in the metabolism of reactive oxygen species (ROS) and pathogenetically relevant for several disease states including Down syndrome (DS; trisomy 21). Systematically studying protein expression in human brain and animal models of DS we decided to carry out "protein hunting" for hypothetical proteins, i.e. proteins that have been predicted based upon nucleic sequences only, in a transgenic mouse model overexpressing human SOD1.     

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

Background

Down 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.

Results

We 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.

Conclusion

We 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 proteomic analysis of amniocytes reveals potentially dysregulated molecular networks in Down syndrome

Background

Down syndrome (DS), caused by an extra copy of chromosome 21, affects 1 in 750 live births and is characterized by cognitive impairment and a constellation of congenital defects. Currently, little is known about the molecular pathogenesis and no direct genotype-phenotype relationship has yet been confirmed. Since DS amniocytes are expected to have a distinct biological behaviour compared to normal amniocytes, we hypothesize that relative quantification of proteins produced from trisomy and euploid (chromosomally normal) amniocytes will reveal dysregulated molecular pathways.

Results

Chromosomally normal- and Trisomy 21-amniocytes were quantitatively analyzed by using Stable Isotope Labeling of Amino acids in Cell culture and tandem mass spectrometry. A total of 4919 unique proteins were identified from the supernatant and cell lysate proteome. More specifically, 4548 unique proteins were identified from the lysate, and 91% of these proteins were quantified based on MS/MS spectra ratios of peptides containing isotope-labeled amino acids. A total of 904 proteins showed significant differential expression and were involved in 25 molecular pathways, each containing a minimum of 16 proteins. Sixty of these proteins consistently showed aberrant expression from trisomy 21 affected amniocytes, indicating their potential role in DS pathogenesis. Nine proteins were analyzed with a multiplex selected reaction monitoring assay in an independent set of Trisomy 21-amniocyte samples and two of them (SOD1 and NES) showed a consistent differential expression.

Conclusions

The most extensive proteome of amniocytes and amniotic fluid has been generated and differentially expressed proteins from amniocytes with Trisomy 21 revealed molecular pathways that seem to be most significantly affected by the presence of an extra copy of chromosome 21.     

Gene expression variation in Down's syndrome mice allows prioritization of candidate genes

Background  

Down's syndrome (DS), or trisomy 21, is a complex developmental disorder that exhibits many clinical signs that vary in occurrence and severity among patients. The molecular mechanisms responsible for DS have thus far remained elusive. We argue here that normal variation in gene expression in the population contributes to the heterogeneous clinical picture of DS, and we estimated the amplitude of this variation in 50 mouse orthologs of chromosome 21 genes in brain regions of Ts65Dn (a mouse model of DS). We analyzed the RNAs of eight Ts65Dn and eight euploid mice by real-time polymerase chain reaction.     

The Feasibility Study of Non-Invasive Fetal Trisomy 18 and 21 Detection with Semiconductor Sequencing Platform

Objective

Recent non-invasive prenatal testing (NIPT) technologies are based on next-generation sequencing (NGS). NGS allows rapid and effective clinical diagnoses to be determined with two common sequencing systems: Illumina and Ion Torrent platforms. The majority of NIPT technology is associated with Illumina platform. We investigated whether fetal trisomy 18 and 21 were sensitively and specifically detectable by semiconductor sequencer: Ion Proton.

Methods

From March 2012 to October 2013, we enrolled 155 pregnant women with fetuses who were diagnosed as high risk of fetal defects at Xiamen Maternal & Child Health Care Hospital (Xiamen, Fujian, China). Adapter-ligated DNA libraries were analyzed by the Ion Proton™ System (Life Technologies, Grand Island, NY, USA) with an average 0.3× sequencing coverage per nucleotide. Average total raw reads per sample was 6.5 million and mean rate of uniquely mapped reads was 59.0%. The results of this study were derived from BWA mapping. Z-score was used for fetal trisomy 18 and 21 detection.

Results

Interactive dot diagrams showed the minimal z-score values to discriminate negative versus positive cases of fetal trisomy 18 and 21. For fetal trisomy 18, the minimal z-score value of 2.459 showed 100% positive predictive and negative predictive values. The minimal z-score of 2.566 was used to classify negative versus positive cases of fetal trisomy 21.

Conclusion

These results provide the evidence that fetal trisomy 18 and 21 detection can be performed with semiconductor sequencer. Our data also suggest that a prospective study should be performed with a larger cohort of clinically diverse obstetrics patients.     

A patient with partial trisomy 21 and 7q deletion expresses mild Down syndrome phenotype

Backround

Down syndrome (DS) is the most common aneuploidy in live-born individuals and it is well recognized with various phenotypic expressions. Although an extra chromosome 21 is the genetic cause for DS, specific phenotypic features may result from the duplication of smaller regions of the chromosome and more studies need to define genotypic and phenotypic correlations.

Case report

We report on a 26 year old male with partial trisomy 21 presenting mild clinical symptoms relative to DS including borderline intellectual disability. In particular, the face and the presence of hypotonia and keratoconus were suggestive for the DS although the condition remained unnoticed until his adult age array comparative genomic hybridization (aCGH) revealed a 10.1 Mb duplication in 21q22.13q22.3 and a small deletion of 2.2 Mb on chromosomal band 7q36 arising from a paternal translocation t(7;21). The 21q duplication encompasses the gene DYRK1.

Conclusion

Our data support the evidence of specific regions on distal 21q whose duplication results in phenotypes recalling the typical DS face. Although the duplication region contains DYRK1, which has previously been implicated in the causation of DS, our patient has a borderline IQ confirming that their duplication is not sufficient to cause the full DS phenotype.     

First trimester screening for trisomy 21 in gestational week 8-10 by ADAM12-S as a maternal serum marker

Background  

A disintegrin and metalloprotease 12 (ADAM12-S) has previously been reported to be significantly reduced in maternal serum from women with fetal aneuploidy early in the first trimester and to significantly improve the quality of risk assessment for fetal trisomy 21 in prenatal screening. The aim of this study was to determine whether ADAM12-S is a useful serum marker for fetal trisomy 21 using the mixture model.     

Plasma metabolites related to cellular energy metabolism are altered in adults with Down syndrome and Alzheimer's disease

Down syndrome (DS) is a well‐known neurodevelopmental disorder most commonly caused by trisomy of chromosome 21. Because individuals with DS almost universally develop heavy amyloid burden and Alzheimer's disease (AD), biomarker discovery in this population may be extremely fruitful. Moreover, any AD biomarker in DS that does not directly involve amyloid pathology may be of high value for understanding broader mechanisms of AD generalizable to the neurotypical population. In this retrospective biomarker discovery study, we examined banked peripheral plasma samples from 78 individuals with DS who met clinical criteria for AD at the time of the blood draw (DS‐AD) and 68 individuals with DS who did not (DS‐NAD). We measured the relative abundance of approximately 5,000 putative features in the plasma using untargeted mass spectrometry (MS). We found significantly higher levels of a peak putatively annotated as lactic acid in the DS‐AD group (q = .014), a finding confirmed using targeted MS (q = .011). Because lactate is the terminal product of glycolysis and subsequent lactic acid fermentation, we performed additional targeted MS focusing on central carbon metabolism which revealed significantly increased levels of pyruvic (q = .03) and methyladipic (q = .03) acids in addition to significantly lower levels of uridine (q = .007) in the DS‐AD group. These data suggest that AD in DS is accompanied by a shift from aerobic respiration toward the less efficient fermentative metabolism and that bioenergetically derived metabolites observable in peripheral blood may be useful for detecting this shift.     

MTHFR Gene variants C677T, A1298C and association with Down syndrome: A Case-control study from South India

BACKGROUND:

The 5,10-methylenetetrahydrofolate reductase (MTHFR) polymorphisms and low folate levels are associated with inhibition of DNA methyltransferase and consequently DNA hypomethylation. The expanding spectrum of common conditions linked with MTHFR polymorphisms includes certain adverse birth outcome, pregnancy complications, cancers, adult cardiovascular diseases and psychiatric disorders, with several of these associations remaining still controversial. Trisomy 21 or Down syndrome (DS) is the most common genetic cause of mental retardation. It stems predominantly from the failure of chromosome 21 to segregate normally during meiosis. Despite substantial research, the molecular mechanisms underlying non-disjunction leading to trisomy 21 are poorly understood.

MATERIALS AND METHODS:

Two common variants C677T and A1298C of the MTHFR gene were screened in 36 parents with DS children and 60 healthy couples from Tamil Nadu and Karnataka. The MTHFR genotypes were studied by RFLP analysis of PCR-amplified products and confirmed by sequencing.

RESULTS:

The CT genotype was seen in three each (8.3%) of case mothers and fathers. One case father showed TT genotype. All the control individuals exhibited the wild type CC genotype. A similar frequency for the uncommon allele C of the second polymorphism was recorded in case mothers (0.35) and fathers (0.37) in comparison with the control mothers (0.39) and fathers (0.37).

CONCLUSION:

This first report on MTHFR C677T and A1298C polymorphisms in trisomy 21 parents from south Indian population revealed that MTHFR 677CT polymorphism was associated with a risk for Down syndrome.     

Down syndrome: searching for the genetic culprits

Down syndrome (DS) is caused by trisomy of human chromosome 21 (Hsa21) and results in a large number of phenotypes, including learning difficulties, cardiac defects, distinguishing facial features and leukaemia. These are likely to result from an increased dosage of one or more of the ∼310 genes present on Hsa21. The identification of these dosage-sensitive genes has become a major focus in DS research because it is essential for a full understanding of the molecular mechanisms underlying pathology, and might eventually lead to more effective therapy. The search for these dosage-sensitive genes is being carried out using both human and mouse genetics. Studies of humans with partial trisomy of Hsa21 have identified regions of this chromosome that contribute to different phenotypes. In addition, novel engineered mouse models are being used to map the location of dosage-sensitive genes, which, in a few cases, has led to the identification of individual genes that are causative for certain phenotypes. These studies have revealed a complex genetic interplay, showing that the diverse DS phenotypes are likely to be caused by increased copies of many genes, with individual genes contributing in different proportions to the variance in different aspects of the pathology.     

Gene Network Disruptions and Neurogenesis Defects in the Adult Ts1Cje Mouse Model of Down Syndrome

Background

Down syndrome (DS) individuals suffer mental retardation with further cognitive decline and early onset Alzheimer''s disease.

Methodology/Principal Findings

To understand how trisomy 21 causes these neurological abnormalities we investigated changes in gene expression networks combined with a systematic cell lineage analysis of adult neurogenesis using the Ts1Cje mouse model of DS. We demonstrated down regulation of a number of key genes involved in proliferation and cell cycle progression including Mcm7, Brca2, Prim1, Cenpo and Aurka in trisomic neurospheres. We found that trisomy did not affect the number of adult neural stem cells but resulted in reduced numbers of neural progenitors and neuroblasts. Analysis of differentiating adult Ts1Cje neural progenitors showed a severe reduction in numbers of neurons produced with a tendency for less elaborate neurites, whilst the numbers of astrocytes was increased.

Conclusions/Significance

We have shown that trisomy affects a number of elements of adult neurogenesis likely to result in a progressive pathogenesis and consequently providing the potential for the development of therapies to slow progression of, or even ameliorate the neuronal deficits suffered by DS individuals.     

An Excess of Deleterious Variants in VEGF-A Pathway Genes in Down-Syndrome-Associated Atrioventricular Septal Defects

About half of people with trisomy 21 have a congenital heart defect (CHD), whereas the remainder have a structurally normal heart, demonstrating that trisomy 21 is a significant risk factor but is not causal for abnormal heart development. Atrioventricular septal defects (AVSD) are the most commonly occurring heart defects in Down syndrome (DS), and ∼65% of all AVSD is associated with DS. We used a candidate-gene approach among individuals with DS and complete AVSD (cases = 141) and DS with no CHD (controls = 141) to determine whether rare genetic variants in genes involved in atrioventricular valvuloseptal morphogenesis contribute to AVSD in this sensitized population. We found a significant excess (p < 0.0001) of variants predicted to be deleterious in cases compared to controls. At the most stringent level of filtering, we found potentially damaging variants in nearly 20% of cases but fewer than 3% of controls. The variants with the highest probability of being damaging in cases only were found in six genes: COL6A1, COL6A2, CRELD1, FBLN2, FRZB, and GATA5. Several of the case-specific variants were recurrent in unrelated individuals, occurring in 10% of cases studied. No variants with an equal probability of being damaging were found in controls, demonstrating a highly specific association with AVSD. Of note, all of these genes are in the VEGF-A pathway, even though the candidate genes analyzed in this study represented numerous biochemical and developmental pathways, suggesting that rare variants in the VEGF-A pathway might contribute to the genetic underpinnings of AVSD in humans.     

Human neural stem cells: a new tool for studying cortical development in Down's syndrome

The clinical characteristics of Down's syndrome (DS), or trisomy 21, are caused by errors that occur during development. In addition to mental retardation, DS individuals have craniofacial abnormalities, clinical defects of the heart, gut and immune system, as well as predisposition to certain diseases, such as leukemias and Alzheimer's disease. To explain the developmental mechanisms that cause these traits, it is necessary to look at how developmental processes in DS compare to normal development. The neurological characteristics of DS are established during the prenatal and early postnatal period in humans, when the bulk of brain development occurs. Mouse models of DS have provided a useful way of studying DS neural development. However, there are clearly significant differences between rodent and human biology that may not be reflected in mouse models. Recent advances in stem cell biology now allow the generation of human neural tissue in the culture dish ( Ostenfeld & Svendsen 2003 ). Stem cells offer a novel model system to study alterations in neuron development in developmental disorders such as DS.     

Possible risk factors for Down syndrome and sex chromosomal aneuploidy in Mysore, South India

BACKGROUND:

Down syndrome (DS) and sex chromosomal aneuploidy (SA) are common chromosomal anomalies causing congenital malformations and mental retardation in humans. The well-established risk factor, advanced maternal age, was not found in many of the DS and SA cases in India, while the other possible risk factors have not been well studied. In view of this, the present study has been made.

MATERIALS AND METHODS:

During the last 5 years, 150 clinically suspected DS and 25 SA cases were referred to our laboratory for chromosome investigation from major hospitals of Mysore city. Chromosome preparations were made from these patients after informed consent was obtained. Well-spread G-banded metaphase plates were analyzed by automated LEICA KARYO software. Two hundred and 100 randomly selected families belonging to different religions were used as controls for the DS and SA cases, respectively. Statistical analysis was carried out using logistic regression

RESULTS:

Out of the 150 cases of DS, 122 had free trisomy 21, two were mosaic trisomy 21, and one had translocation. Logistic regression of case-control study of DS children revealed that the odds ratio of uncle-niece marriages, or second cousin marriages, or parents lived in rural region, or exposure of the parents to chemicals, or parents education status, or habits (tobacco/ alcohol used) of father, or mother not undergone prenatal scanning, or mothers with previous abortions were significant when all the variables of that category were used one at a time. Exposure of the parents to chemicals, parents’ educational status, habits (tobacco/alcohol use) of the father, mother not undergone prenatal scanning, and history of previous abortions were significant when all the variables of that category were used one at a time. Similarly, except for consanguinity, history of previous abortions, and mother not undergone prenatal scanning, all other factors showed significant odds ratios in SA cases.

CONCLUSION:

Besides the known risk factors, consanguinity, region (rural/urban) of residence of parents, exposure of parents to chemicals, educational status of parents, habits of father, prenatal scanning, and reproductive performance of mother are possible risk factors for chromosomal aneuploidy.     

Inflammatory and Immunological parameters in adults with Down syndrome

Background  

The increase in life expectancy within the general population has resulted in an increasing number of elderly adults, including patients with Down syndrome (DS), with a current life expectancy of about 50 years. We evaluate the parameters of humoral and cellular immune response, the quantitative expression of the regulator of calcineurin1 gene (RCAN1) and the production of cytokines. The study group consisted of adults DS (n = 24) and a control group with intellectual disability without Down syndrome (ID) (n = 21) and living in a similar environmental background. It was evaluated serology, immunophenotyping, the quantitative gene expression of RCAN1 and the production of cytokines.     

CASP3 protein expression by flow cytometry in Down’s syndrome subjects

Down’s syndrome (DS), the most common chromosomal disorder, is caused by 21 trisomy and is featured by intellectual disability. Subjects with DS can develop some traits of Alzheimer disease (AD) at an earlier age than subjects without trisomy 21. Apoptosis is a programmed cell death process under both normal physiological and pathological conditions. Caspase-3 (CASP3) plays an important role in neuronal death during nervous system development and under certain pathological conditions. Furthermore, in vitro and in vivo studies report elevated expression and activation of CASP3 in models of AD. On this account, the expression of CASP3 gene was evaluated in cultures of fibroblasts of DS and normal subjects by flow cytometry. CASP3 protein was up-regulated in fibroblasts of DS. The data obtained from this study strengthen the hypothesis that the over-expression of CASP3 gene could have a role in the activation of the apoptotic pathways acting in the neurodegenerative processes in DS.     

Mutational analyses of the signals involved in the subcellular location of DSCR1

Background  

Down syndrome is the most frequent genetic disorder in humans. Rare cases involving partial trisomy of chromosome 21 allowed a small chromosomal region common to all carriers, called Down Syndrome Critical Region (DSCR), to be determined. The DSCR1 gene was identified in this region and is expressed preferentially in the brain, heart and skeletal muscle. Recent studies have shown that DSCR1 belongs to a family of proteins that binds and inhibits calcineurin, a serine-threonine phosphatase. The work reported on herein consisted of a study of the subcellular location of DSCR1 and DSCR1-mutated forms by fusion with a green fluorescent protein, using various cell lines, including human.     

Isolation and subregional mapping of a human cDNA clone detecting a common RELP on chromosome 12

Summary Peripheral blood lymphocytes from three patients with Down syndrome (DS; trisomy 21; aged 5–6 years) and three age-matched control children were studied for the induction of chromosomal aberrations and sister chromatid exchanges (SCEs).Cells in G₀ were exposed to bleomycin (20–100 g/ml) for 3 h, and then cultured in medium containing 5-bromodeoxyuridine and phytohemagglutinin for 66 h. By the sister chromatid differential staining method, chromosome analyses were performed on metaphase cells that had divided one, two, or three or more times after treatment. The results indicate that DS cells exposed to bleomycin are hypersensitive to the production of dicentric and ring chromosomes compared to normal cells. Bleomycin also led to a dose-related increase in the frequency of SCEs, but no difference was found between the SCE frequencies in DS or normal lymphocytes exposed to bleomycin.     

Early Lineage Priming by Trisomy of Erg Leads to Myeloproliferation in a Down Syndrome Model

Down syndrome (DS), with trisomy of chromosome 21 (HSA21), is the commonest human aneuploidy. Pre-leukemic myeloproliferative changes in DS foetal livers precede the acquisition of GATA1 mutations, transient myeloproliferative disorder (DS-TMD) and acute megakaryocytic leukemia (DS-AMKL). Trisomy of the Erg gene is required for myeloproliferation in the Ts(17¹⁶)65Dn DS mouse model. We demonstrate here that genetic changes specifically attributable to trisomy of Erg lead to lineage priming of primitive and early multipotential progenitor cells in Ts(17¹⁶)65Dn mice, excess megakaryocyte-erythroid progenitors, and malignant myeloproliferation. Gene expression changes dependent on trisomy of Erg in Ts(17¹⁶)65Dn multilineage progenitor cells were correlated with those associated with trisomy of HSA21 in human DS hematopoietic stem and primitive progenitor cells. These data suggest a role for ERG as a regulator of hematopoietic lineage potential, and that trisomy of ERG in the context of DS foetal liver hemopoiesis drives the pre-leukemic changes that predispose to subsequent DS-TMD and DS-AMKL.