Genetic polymorphisms modulate the folate metabolism of Brazilian individuals with Down syndrome

Individuals with Down syndrome (DS) carry three copies of the Cystathionine beta-synthase (C beta S) gene. The increase in the dosage of this gene results in an altered profile of metabolites involved in the folate pathway, including reduced homocysteine (Hcy), methionine, S-adenosylhomocysteine (SAH) and S-adenosylmethionine (SAM). Furthermore, previous studies in individuals with DS have shown that genetic variants in genes involved in the folate pathway influence the concentrations of this metabolism's products. The purpose of this study is to investigate whether polymorphisms in genes involved in folate metabolism affect the plasma concentrations of Hcy and methylmalonic acid (MMA) along with the concentration of serum folate in individuals with DS. Twelve genetic polymorphisms were investigated in 90 individuals with DS (median age 1.29 years, range 0.07-30.35 years; 49 male and 41 female). Genotyping for the polymorphisms was performed either by polymerase chain reaction (PCR) based techniques or by direct sequencing. Plasma concentrations of Hcy and MMA were measured by liquid chromatography-tandem mass spectrometry as previously described, and serum folate was quantified using a competitive immunoassay. Our results indicate that the MTHFR C677T, MTR A2756G, TC2 C776G and BHMT G742A polymorphisms along with MMA concentration are predictors of Hcy concentration. They also show that age and Hcy concentration are predictors of MMA concentration. These findings could help to understand how genetic variation impacts folate metabolism and what metabolic consequences these variants have in individuals with trisomy 21.Fundacao de Amparo a Pesquisa do Estado de Sao Paulo (FAPESP) [04/15944-5, 03/09931-5]Conselho Nacional de Desenvolvimento Cientifico e Tecnologico (CNPq) [302157/2008-5]Coordenacao de Aperfeicoamento de Pessoal de Nivel Superior (CAPES) [CGPP 046/2006

Trisomy 21 alters DNA methylation in parent-of-origin-dependent and independent manners

The supernumerary chromosome 21 in Down syndrome differentially affects the methylation statuses at CpG dinucleotide sites and creates genome-wide transcriptional dysregulation of parental alleles, ultimately causing diverse pathologies. At present, it is unknown whether those effects are dependent or independent of the parental origin of the nondis-joined chromosome 21. Linkage analysis is a standard method for the determination of the parental origin of this aneuploidy, although it is inadequate in cases with deficiency of samples from the progenitors. Here, we assessed the reliability of the epigenetic 5(m)CpG imprints resulting in the maternally (oocyte)-derived allele methylation at a differentially methylated region (DMR) of the candidate imprinted WRB gene for asserting the parental origin of chromosome 21. We developed a methylation-sensitive restriction enzyme-specific PCR assay, based on the WRB DMR, across single nucleotide polymorphisms (SNPs) to examine the methylation statuses in the parental alleles. In genomic DNA from blood cells of either disomic or trisomic subjects, the maternal alleles were consistently methylated, while the paternal alleles were unmethylated. However, the supernumerary chromosome 21 did alter the methylation patterns at the RUNX1 (chromosome 21) and TMEM131 (chromosome 2) CpG sites in a parent-of-origin-independent manner. To evaluate the 5(m)CpG imprints, we conducted a computational comparative epigenomic analysis of transcriptome RNA sequencing (RNA-Seq) and histone modification expression patterns. We found allele fractions consistent with the transcriptional biallelic expression of WRB and ten neighboring genes, despite the similarities in the confluence of both a 17-histone modification activation backbone module and a 5-histone modification repressive module between the WRB DMR and the DMRs of six imprinted genes. We concluded that the maternally inherited 5(m)CpG imprints at the WRB DMR are uncoupled from the parental allele expression of WRB and ten neighboring genes in several tissues and that trisomy 21 alters DNA methylation in parent-of-origin-dependent and -independent manners

Altered expression of immune-related genes in children with Down syndrome.

Individuals with Down syndrome (DS) have a high incidence of immunological alterations with increased susceptibility to bacterial and viral infections and high frequency of different types of hematologic malignancies and autoimmune disorders. In the current study, we profiled the expression pattern of 92 immune-related genes in peripheral blood mononuclear cells (PBMCs) of two different groups, children with DS and control children, to identify differentially expressed genes that might be of pathogenetic importance for the development and phenotype of the immunological alterations observed in individuals with DS. PBMCs samples were obtained from six DS individuals with karyotypically confirmed full trisomy 21 and six healthy control individuals (ages 2-6 years). Gene expression was profiled in duplicate according to the manufacturer's instructions provided by commercially available TaqMan Human Immune Array representing 92 immune function genes and four reference genes on a 96-plex gene card. A set of 17 differentially expressed genes, not located on chromosome 21 (HSA21), involved in immune and inflammatory pathways was identified including 13 genes (BCL2, CCL3, CCR7, CD19, CD28, CD40, CD40LG, CD80, EDN1, IKBKB, IL6, NOS2 and SKI) significantly down-regulated and four genes (BCL2L1, CCR2, CCR5 and IL10) significantly up-regulated in children with DS. These findings highlight a list of candidate genes for further investigation into the molecular mechanism underlying DS pathology and reinforce the secondary effects of the presence of a third copy of HSA21

Differential Expression of Inflammation-Related Genes in Children with Down Syndrome

Objective. The aim of the study was to investigate the expression patterns of a specific set of genes involved in the inflammation process in children with Down Syndrome (DS) and children without the syndrome (control group) to identify differences that may be related to the immune abnormalities observed in DS individuals. Method. RNA samples were obtained from peripheral blood, and gene expression was quantified using the TaqMan® Array Plate Human Inflammation Kit, which facilitated the investigation into 92 inflammation-related genes and four reference genes using real-time polymerase chain reaction (qPCR). Results. Twenty genes showed differential expression in children with DS; 12 were overexpressed (PLA2G2D, CACNA1D, ALOX12, VCAM1, ICAM1, PLCD1, ADRB1, HTR3A, PDE4C, CASP1, PLA2G5, and PLCB4), and eight were underexpressed (LTA4H, BDKRB1, ADRB2, CD40LG, ITGAM, TNFRSF1B, ITGB1, and TBXAS1). After statistically correcting for the false discovery rate, only the genes BDKRB1 and LTA4H showed differential expression, and both were underexpressed within the DS group. Conclusion. DS children showed differential expression of inflammation-related genes that were not located on chromosome 21 compared with children without DS. The BDKRB1 and LTA4H genes may differentiate the case and control groups based on the inflammatory response, which plays an important role in DS pathogenesis

Absolute count of immune cells in children with Down syndrome (DS) compared with controls.

a<p> Data are expressed as mean ± standard deviation, and two-tailed t test was used for comparisons.</p>b<p> Data is expressed as median (interquartile range), and Mann-Whitney test was used.</p><p>Absolute count of immune cells in children with Down syndrome (DS) compared with controls.</p

Common combinatorial histone modification expression patterns around the <i>WRB</i> CGI-2 DMRs.

<p>Graphical representation of the confluence of activating and repressive epigenetics histone modification marks for the known imprinted <i>MEST</i> gene (<b>A</b>) and the candidate imprinted <i>WRB</i> gene (<b>B</b>). Shown are the 17-histone modification activation backbone module and the 5-histone modification repressive module found in human CD4+ T cells [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0154108#pone.0154108.ref054" target="_blank">54</a>]. Highlighted in light blue is the DMR in each gene. Composite of screenshots of the dataset viewed at the UCSC Genome Browser hg18 (<a href="http://genome.ucsc.edu/" target="_blank">http://genome.ucsc.edu</a>).</p

The maternally derived, imprinted 5^mCpG marks at the <i>WRB</i> CGI-2 DMR do not dictate a paternal monoallelic expression in blood.

<p>Qualitative SNuPE allele-specific profiling of the <i>WRB</i> 3´-UTR rs1060180, rs13230 and rs60490159 SNPs in a disomic, informative nuclear family (DNA). The assay reveals a pattern consistent with biallelic transcriptional expression (cDNA) in the child, who is heterozygous for all three SNP variants.</p

5^mCpG statuses at the <i>WRB</i> CGI-2 DMR in a complete androgenetic mole and a human embryonic stem cell line.

<p>A consistent unmethylated pattern of CpG sites at the <i>WRB</i> CGI-2 DMR revealed in a sample of an androgenetic complete hydatidiform mole (<b>A</b>) contrast with the hypermethylated pattern observed in the representative HUES 3 embryonic cell line (<b>B</b>). Electropherograms of the amplimers (see details of the assay in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0154108#pone.0154108.g002" target="_blank">Fig 2</a>) generated from either undigested DNA or <i>Hha</i>I-digested DNA. The numbers in the upper boxes correspond to the amplimer lengths in base pairs while those in the lower boxes refer to the areas under the peak of the amplimer.</p