Bruna Lancia Zampieri

Maternal risk for Down syndrome is modulated by genes involved in folate metabolism

Studies have shown that the maternal risk for Down syndrome (DS) may be modulated by alterations in folate metabolism. The aim of this study was to evaluate the influence of 12 genetic polymorphisms involved in folate metabolism on maternal risk for DS. In addition, we evaluated the impact of these polymorphisms on serum folate and plasma methylmalonic acid (MMA, an indicator of vitamin B12 status) concentrations. The polymorphisms transcobalamin II (TCN2) c.776C>G, betaine-homocysteine S-methyltransferase (BHMT) c.742A>G, methylenetetrahydrofolate reductase (NAD(P)H) (MTHFR) c.677 C>T and the MTHFR 677C-1298A-1317T haplotype modulate DS risk. The polymorphisms MTHFR c.677C>T and solute carrier family 19 (folate transporter), member 1 (SLC19A1) c.80 A>G modulate folate concentrations, whereas the 5-methyltetrahydrofolate-homocysteine methyltransferase reductase (MTRR) c.66A>G polymorphism affects the MMA concentration. These results are consistent with the modulation of the maternal risk for DS by these polymorphisms.

A80G polymorphism of reduced folate carrier 1 (RFC1) and C776G polymorphism of transcobalamin 2 (TC2) genes in Down's syndrome etiology

CONTEXT AND OBJECTIVE There is evidence that polymorphisms of genes involved in folate metabolism may be associated with higher risk that mothers may bear a Downs syndrome (DS) child. This study therefore had the objective of investigating the A80G polymorphism of the reduced folate carrier 1 (RFC1) gene and the C776G polymorphism of the transcobalamin 2 (TC2) gene as maternal risk factors for DS among Brazilian women. DESIGN AND SETTING Analytical cross-sectional study with control group, at Faculdade de Medicina de São José do Rio Preto (Famerp). METHODS Sixty-seven mothers of DS individuals with free trisomy 21, and 113 control mothers, were studied. Molecular analysis of the polymorphisms was performed by means of the polymerase chain reaction with restriction fragment length polymorphism (PCR-RFLP), followed by electrophoresis on 2% agarose gel. RESULTS The frequencies of the polymorphic alleles were 0.51 and 0.52 for RFC1 80G, and 0.34 and 0.34 for TC2 776G, in the case and control groups, respectively. Thus, there were no differences between the groups in relation to either the allele or the genotype frequency, for both polymorphisms (P = 0.696 for RFC1 A80G; P = 0.166 for TC2 C776G; P = 0.268 for combined genotypes). CONCLUSION There was no evidence of any association between the RFC1 A80G and TC2 C776G polymorphisms and the maternal risk of DS in the sample evaluated.

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 manufacturers 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.

BHMT G742A and MTHFD1 G1958A Polymorphisms and Down Syndrome Risk in the Brazilian Population

BACKGROUND Mechanisms underlying meiotic nondisjunction are poorly understood. Attempts to elucidate the causes of Down syndrome (DS) have analyzed the relationship between polymorphism in folate metabolism and DS. AIM The role of methylenetetrahydrofolate dehydrogenase 1 (MTHFD1) G1958A and betaine-homocysteine methyltransferase (BHMT) G742A polymorphisms in DS risk was investigated. METHODS Blood samples were collected from a total of 86 DS mothers and from 161 control mothers. The investigation of the MTHFD1 G1958A polymorphism was performed by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) and by real-time PCR for the BHMT G742A polymorphism. RESULTS The median maternal age of case mothers (30.40; 12.9-46.3 years) was significantly higher (p<0.0005) than in the control group (26.60; 15.4-57.9 years). The frequency of BHMT variant genotypes was significantly lower in DS mothers compared with controls (p=0.047). A significant decreased risk for BHMT 742 AA genotype (odds ratio [OR]=0.30; 95% confidence interval [CI]: 0.10-0.93; p=0.037) was observed. Moreover, when the dominant model was applied (BHMT 742GA or 7428AA versus 742GG), there was also a significant decrease in DS risk (OR=0.58; 95% CI: 0.37-0.98; p=0.042). MTHFD1 G1958A genotype frequencies were not significantly altered in DS mothers (p=0.206). CONCLUSIONS Our study suggests that the polymorphism BHMT G742A may modulate the DS risk in Brazilian mothers.

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 nondisjoined 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 5mCpG 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 5mCpG 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 5mCpG 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.

19-base pair deletion polymorphism of the dihydrofolate reductase (DHFR) gene: maternal risk of Down syndrome and folate metabolism

CONTEXT AND OBJECTIVE Polymorphisms in genes involved in folate metabolism may modulate the maternal risk of Down syndrome (DS). This study evaluated the influence of a 19-base pair (bp) deletion polymorphism in intron-1 of the dihydrofolate reductase (DHFR) gene on the maternal risk of DS, and investigated the association between this polymorphism and variations in the concentrations of serum folate and plasma homocysteine (Hcy) and plasma methylmalonic acid (MMA). DESIGN AND SETTING Analytical cross-sectional study carried out at Faculdade de Medicina de São José do Rio Preto (Famerp). METHODS 105 mothers of individuals with free trisomy of chromosome 21, and 184 control mothers were evaluated. Molecular analysis on the polymorphism was performed using the polymerase chain reaction (PCR) through differences in the sizes of fragments. Folate was quantified by means of chemiluminescence, and Hcy and MMA by means of liquid chromatography and sequential mass spectrometry. RESULTS There was no difference between the groups in relation to allele and genotype frequencies (P = 0.44; P = 0.69, respectively). The folate, Hcy and MMA concentrations did not differ significantly between the groups, in relation to genotypes (P > 0.05). CONCLUSIONS The 19-bp deletion polymorphism of DHFR gene was not a maternal risk factor for DS and was not related to variations in the concentrations of serum folate and plasma Hcy and MMA in the study population.

DHFR 19-bp Deletion and SHMT C1420T Polymorphisms and Metabolite Concentrations of the Folate Pathway in Individuals with Down Syndrome

BACKGROUND Down syndrome (DS) results from the presence and expression of three copies of the genes located on chromosome 21. Studies have shown that, in addition to overexpression of the Cystathionine β-synthase (CBS) gene, polymorphisms in genes involved in folate/homocysteine (Hcy) metabolism may also influence the concentrations of metabolites of this pathway. AIM Investigate the association between Dihydrofolate reductase (DHFR) 19-base pair (bp) deletion and Serine hydroxymethyltransferase (SHMT) C1420T polymorphisms and serum folate and plasma Hcy and methylmalonic acid (MMA) concentrations in 85 individuals with DS. METHODS Molecular analysis of the DHFR 19-bp deletion and SHMT C1420T polymorphisms was performed by polymerase chain reaction (PCR) by difference in the size of fragments and real-time PCR allelic discrimination, respectively. Serum folate was quantified by chemiluminescence and plasma Hcy and MMA by liquid chromatography-tandem mass spectrometry. RESULTS Individuals with DHFR DD/SHMT TT genotypes presented increased folate concentrations (p=0.004) and the DHFR II/SHMT TT genotypes were associated with increased MMA concentrations (p=0.008). In addition, the MMA concentrations were negatively associated with age (p=0.04). CONCLUSION There is an association between DHFR DD/SHMT TT and DHFR II/SHMT TT combined genotypes and folate and MMA concentrations in individuals with DS.

Abnormal Folate Metabolism and Maternal Risk for Down Syndrome

Down syndrome (DS) or trisomy 21 (MIM 190685) is the most common genetic disorder with a prevalence of 1 in 660 live births (Jones, 2006). DS is the leading cause of geneticallydefined intellectual disability (Contestabile et al., 2010) and its phenotype is complex and variable among individuals, who may present with a combination of dysmorphic features (Ahmed et al., 2005; Pavarino-Bertelli et al., 2009), congenital heart disease (Abbag, 2006), neurological abnormalities such as early manifestations of Alzheimer’s disease (Lott & Head, 2005), immunological impairments (Ram & Chinen, 2011), elevated risk of specific types of leukemia (Hasle et al., 2000), and other clinical complications (Venail et al., 2004). Trisomy 21 can be caused by three types of chromosomal abnormalities: free trisomy, translocation, or mosaicism. Mosaicism accounts for the minority of DS cases (about 1%) and is characterized by some cells containing 46 chromosomes and others, 47 chromosomes. Translocations are attributed to 3-4% of the cases, with Robertsonian translocation involving chromosomes 14 and 21 being the most common type. Finally, free trisomy occurs in about 95% of cases (Ahmed et al., 2005; J.M. Biselli et al., 2008b) and is characterized by the presence of three complete copies of chromosome 21. Free trisomy, the main chromosomal abnormality leading to DS, is caused by the failure of normal chromosome 21 segregation during meiosis (meiotic nondisjunction) (Hassold & Hunt, 2000). The parental origin of the extra chromosome 21 is maternal in about 80% of cases (Jyothy et al., 2001), and most (about 77%) occur during the first maternal meiotic division in the maturing oocyte, before conception (Antonarakis et al., 1992).

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.