Systematic identification of placental epigenetic signatures for the noninvasive prenatal detection of Edwards syndrome

Background

Noninvasive prenatal diagnosis of fetal aneuploidy by maternal plasma analysis is challenging owing to the low fractional and absolute concentrations of fetal DNA in maternal plasma. Previously, we demonstrated for the first time that fetal DNA in maternal plasma could be specifically targeted by epigenetic (DNA methylation) signatures in the placenta. By comparing one such methylated fetal epigenetic marker located on chromosome 21 with another fetal genetic marker located on a reference chromosome in maternal plasma, we could infer the relative dosage of fetal chromosome 21 and noninvasively detect fetal trisomy 21. Here we apply this epigenetic-genetic (EGG) chromosome dosage approach to detect Edwards syndrome (trisomy 18) in the fetus noninvasively.

Principal Findings

We have systematically identified methylated fetal epigenetic markers on chromosome 18 by methylated DNA immunoprecipitation (MeDIP) and tiling array analysis with confirmation using quantitative DNA methylation assays. Methylated DNA sequences from an intergenic region between the VAPA and APCDD1 genes (the VAPA-APCDD1 DNA) were detected in pre-delivery, but not post-delivery, maternal plasma samples. The concentrations correlated positively with those of an established fetal genetic marker, ZFY, in pre-delivery maternal plasma. The ratios of methylated VAPA-APCDD1(chr18) to ZFY(chrY) were higher in maternal plasma samples of 9 male trisomy 18 fetuses than those of 27 male euploid fetuses (Mann-Whitney test, P = 0.029). We defined the cutoff value for detecting trisomy 18 fetuses as mean+1.96 SD of the EGG ratios of the euploid cases. Eight of 9 trisomy 18 and 1 of 27 euploid cases showed EGG ratios higher than the cutoff value, giving a sensitivity of 88.9% and a specificity of 96.3%.

Conclusions

Our data have shown that the methylated VAPA-APCDD1 DNA in maternal plasma is predominantly derived from the fetus. We have demonstrated that this novel fetal epigenetic marker in maternal plasma is useful for the noninvasive detection of fetal trisomy 18.     

Phenotypic and cytogenetic spectrum of 9p trisomy

Trisomy 9p is one of the most frequent autosomal anomalies compatible with long survival rate. The spectrum of clinical severity in trisomy 9 roughly correlates with the extent of trisomic chromosome material. Trisomy 9p is a clinically well delineated syndrome and of all stigmata craniofacial dysmorphism is most specific. In this study we report five cases with de novo trisomy 9p. The study aimed at the identification of the genotype/phenotype correlations in patients with different breakpoints. GTG banding, DAPI stain, whole chromosome paint, centromere, telomere and 9p21 specific locus probes demonstrated that partial trisomy 9p in case 1 was due to isochromosome 9p with translocation of the long arm of re-arranged chromosome 9 onto the short arm of chromosome 13, cases 2 and 3 had intrachromosomal duplication of the short arm of chromosome 9 [dup(9)(p21p24)], case 4 had "classical" 9p trisomy and case 5 had duplication of whole short arm and part of the long arm of chromosome 9 (partial 9 trisomy). Although cases 1 to 4 had trisomy involving 9p, cases 1 and 2 exhibited the classical clinical manifestations of 9p trisomy, while cases 3 and 4 had additional features overlapping with Coffin-Siris syndrome. The present study strengthens the association of Coffin-Siris syndrome and 9p, the significance of such observations may point to possible gene location of Coffin-Siris syndrome on 9p. Case 5 had additional manifestations more than those typical of trisomy 9p which could be due to duplication of 9q21 region. Wide gap between 1st and 2nd toes, observed in the studied cases, can be added to the phenotype of this trisomy. Three of our cases had brain malformations, case 3 had dilated ventricles with hypogenesis of corpus callosum, case 4 had agenesis of corpus callosum, and case 5 had Dandy-Walker malformation. We also suggest that dosage effects of genes located in 9pter-q22 contribute to the etiology of Dandy-Walker syndrome. We recommend MRI studies as a routine in all cases with trisomy 9p.     

Gene dosage effect for GALT in 9p trisomy and in 9p tetrasomy with an improved technique for GALT determination

Summary A new born male and a three-year-old female with various dysmorphic features were both found to have a supernumerary chromosome. Clinical and cytogenetic findings confirmed the existence of a pure de novo 9p tetrasomy in the first case and a pure de novo 9p trisomy in the second case. Gene dosage effects were demonstrated for galactose-1-phosphate uridyltransferase GALT (EC 2.7.7.12) using an improved method for the assay of this enzyme in red blood cells. These findings are in agreement with previous results on similar cases and therefore confirm the assignment of the locus for GALT to 9p.     

Dandy-Walker malformations in a case of partial trisomy 9p (p12.1→pter) due to maternal translocation t(9;12)(p12.1;p13.3)

We describe a five-year-old proband presented with Dandy-Walker malformations, right microopthalmia, hamstring contractures, undescended testis with absence of testis in right scrotum in addition to typical trisomy 9p clinical features. Routine cytogenetic studies with GTG - banding showed 46,XY,der(12)t(9;12) (p12;q13.3),mat karyotype (trisomy 9p). Chromosomal analysis of the father was normal and phenotypically normal mother had 46,XX,t(9;12)(p12;q13) karyotype. Fluorescence in situ hybridization analysis with single copy probes bA5OIA2 (9p11.2), bA562M8 (12p12.1) and centromere probes (9) showed break point at 9p12.1 region. The gene dosage effect of Chromosome 9p along with environmental factors might be associated with Dandy- Walker malformations in the patient.     

Cystathionine beta synthase: gene dosage effect in trisomy 21

The enzymatic activity of cystathionine beta synthase has been studied in fibroblasts of nine patients with regular trisomy 21. An excess of CBS activity was found in trisomy 21 with a trisomy 21/normal ratio equal to 1.66. A 1.04 ratio was found in 21q21----21 p ter monosomy; a 1.04 and 0.99 ratio was found in two 21 qter----21q22.3 monosomies; a 1.14 ratio in 21 qter----21q22 monosomy; a 0.89 ratio in a 21q21----21 pter trisomy; an excess of CBS activity was found in a 21q22.1 ----21q21 trisomy with a 1.57 ratio. These results show a gene dosage effect in human fibroblasts trisomic for chromosome 21 and suggest the assignment of human CBS locus between 21q22.1 and 21q21.     

Regional mapping of the locus for hexokinase-1 (HK1) to 10p11→q23 by gene dosage in human fibroblasts

Summary The hexokinase (HK) activity in human fibroblasts was close to that expected for a gene dosage effect in a mosaic cell line with about 86% trisomy 10 cells (64% greater than four control lines with normal karyotypes). There was no dosage effect for HK in the cell line that was trisomic for 10q24qter, nor in the cell line monosomic for 10pterp12. The data suggest an assignment of the HK1 locus (the only hexokinase in fibroblasts) to 10p11q23 by exclusion.     

The effect of trisomy 21 on the patterns of polypeptide synthesis in human fibroblasts.

In addition to direct gene dosage effects, the deleterious phenotypic consequences of aneuploidy may result from secondary regulatory effects on the production and degradation of gene products coded for by other chromosomes. In an initial test of the hypothesis that extensive secondary effects play an important role in the phenotypic consequences of aneuploidy, we have used two-dimensional gel electrophoresis with radioautography to look for such secondary effects among the polypeptides synthesized by human fibroblasts grown in vitro. The polypeptide patterns of fibroblast strains from four trisomy 21 subjects and one trisomy 21/normal mosaic were compared to those from five matched normal subjects. Of approximately 850 polypeptides visualized, only four show a pattern of variation which may be related to trisomy 21. Additional differences in polypeptide concentrations were found among the strains, attributable to genetic heterogeneity between donor individuals and differences in tissue of origin. These results indicate that, at least in fibroblasts in vitro, trisomy 21 does not cause major regulatory changes in the rates of production and degradation of a large number of polypeptides.     

Gene dosage of CuZnSOD and Down''s syndrome: diminished prostaglandin synthesis in human trisomy 21, transfected cells and transgenic mice.

Patients with Down's syndrome (DS) exhibit elevated activity of copper zinc superoxide dismutase (CuZnSOD) caused by the trisomy 21 state. To investigate the possible involvement of CuZnSOD gene dosage in perturbation of prostaglandin biosynthesis we analyzed transfected cells and transgenic mice that express elevated levels of human CuZnSOD. It was found that the synthesis of prostaglandin E2 (PGE2) was diminished in transfected PC12-CuZnSOD cells as well as in fibroblasts from DS patients. Primary cells derived from transgenic CuZnSOD mice showed similar reduction. Impaired biosynthesis of prostaglandins was not confined to cells grown in culture since secretion of PGE2 and PGD2 by kidney and cerebellum of transgenic CuZnSOD was significantly lower than in non-transgenic littermate mice. These findings strongly suggest that overexpression of the CuZnSOD gene induces a demotion in PGE2 and PGD2 formation and establish a connection between alteration of prostaglandin biosynthesis in trisomy 21 cells and gene dosage of CuZnSOD.     

Gene dosage effect for glycinamide ribonucleotide synthetase in human fibroblasts trisomic for chromosome 21

The mean activity of glycinamide ribonucleotide synthetase of fibroblasts from fetuses with trisomy 21 is 1.64 — times that of normal cells. This gene dosage effect is confirmatory of the assignment of the gene for this enzyme to human chromosome 21.     

Interferon induction of (2′–5′) oligoisoadenylate synthetase in diploid and trisomy 21 human fibroblasts: Relation to dosage of the interferon receptor gene (IRFC)

Trisomy 21 human fibroblasts are more sensitive to human interferon-alpha (IFN-alpha) than are diploid controls, consistent with the location of the gene (IFRC) which codes for the IFN-alpha receptor on chromosome 21. When compared in the antiviral assay, the difference in sensitivity is five- to tenfold, much greater than the 50% difference in IFRC gene dosage. An understanding of the mechanism by which this amplification of gene dosage occurs is relevant to the specific pathology of Down's syndrome and as a model system for studying the pathogenic effects of chromosomal aneuploidy. The enzyme (2'-5') oligoisoadenylate synthetase (2-5A synthetase), which is believed to be central to the interferon-induced antiviral response, is induced 50% more in trisomy 21 fibroblasts than in diploid controls. Thus the amplification in response occurs subsequent to the binding of IFN-alpha to its receptor and the triggering of the first set of intracellular events, the latter exemplified by the induction of 2-5A synthetase. Similar results were obtained with IFN-gamma, consistent with other evidence which indicates that a gene coding for a separate IFN-gamma receptor is also located on chromosome 21.     

Matrix metalloproteinases and the ontogeny of scarless repair: the other side of the wound healing balance

Early gestation mammalian fetuses possess the remarkable ability to heal cutaneous wounds in a scarless fashion. Over the past 20 years, scientists have been working to decipher the mechanisms underlying this phenomenon. Much of the research to date has focused on fetal correlates of adult wound healing that promote fibrosis and granulation tissue formation. It is important to remember, however, that wound repair consists of a balance between tissue synthesis, deposition, and degradation. Relatively little attention has been paid to this latter component of the fetal wound healing process.In this study, we examined the ontogeny of ten matrix metalloproteinases (MMPs) and their tissue inhibitors (TIMPs) in nonwounded fetal rat skin and fibroblasts as a function of gestational age. We used a semiquantitative polymerase chain reaction protocol to analyze these important enzymes at time points that represent both the scarless and scar-forming periods of rat gestation. The enzymes evaluated were collagenase-1 (MMP-1), stromelysin-1 (MMP-3), gelatinase A (MMP-2), gelatinase B (MMP-9), membrane-type matrix metalloproteinases (MT-MMPs) 1, 2, and 3, and TIMPs 1, 2, and 3.Results demonstrated marked increases in gene expression for MMP-1, MMP-3 and MMP-9 that correlated with the onset of scar formation in nonwounded fetal skin. Similar results were noted in terms of MMP-9 gene expression in fetal fibroblasts. These results suggest that differences in the expression of these matrix metalloproteinases may have a role in the scarless wound healing phenotype observed early in fetal rat gestation. Furthermore, our data suggest that the differential expression of gelatinase B (MMP-9) may be mediated by the fetal fibroblasts themselves.     

Epigenetic-genetic chromosome dosage approach for fetal trisomy 21 detection using an autosomal genetic reference marker

Background

The putative promoter of the holocarboxylase synthetase (HLCS) gene on chromosome 21 is hypermethylated in placental tissues and could be detected as a fetal-specific DNA marker in maternal plasma. Detection of fetal trisomy 21 (T21) has been demonstrated by an epigenetic-genetic chromosome dosage approach where the amount of hypermethylated HLCS in maternal plasma is normalized using a fetal genetic marker on the Y chromosome as a chromosome dosage reference marker. We explore if this method can be applied on both male and female fetuses with the use of a paternally-inherited fetal single nucleotide polymorphism (SNP) allele on a reference chromosome for chromosome dosage normalization.

Methodology

We quantified hypermethylated HLCS molecules using methylation-sensitive restriction endonuclease digestion followed by real-time or digital PCR analyses. For chromosome dosage analysis, we compared the amount of digestion-resistant HLCS to that of a SNP allele (rs6636, a C/G SNP) that the fetus has inherited from the father but absent in the pregnant mother.

Principal Findings

Using a fetal-specific SNP allele on a reference chromosome, we analyzed 20 euploid and nine T21 placental tissue samples. All samples with the fetal-specific C allele were correctly classified. One sample from each of the euploid and T21 groups were misclassified when the fetal-specific G allele was used as the reference marker. We then analyzed 33 euploid and 14 T21 maternal plasma samples. All but one sample from each of the euploid and T21 groups were correctly classified using the fetal-specific C allele, while correct classification was achieved for all samples using the fetal-specific G allele as the reference marker.

Conclusions

As a proof-of-concept study, we have demonstrated that the epigenetic-genetic chromosome dosage approach can be applied to the prenatal diagnosis of trisomy 21 for both male and female fetuses.     

Possible localization of the glutathione reductase (EC 1.6.4.2) on the 8p21 band]

Glutathione reductase (EC 1.6.4.2.) (GSR) activity was measured in the red cells of patients with different rearrangements of chromosome 8. Of three patients with mosaic trisomy 8, two had a high GSR activity. The lack of correlation between GSR levels and the degree of mosaicism in lymphocytes is discussed. In six patients with trisomy 8qter the mean value of GSR activities was normal. In one patient with trisomy pter leads to q22.1 and in two with trisomy p11 leads to p22, a significant increase (+60%) of GSR activity was observed. In two patients with monosomy p22 leads to pter and p21 leads to pter, respectively, the GSR levels were normal. It is concluded that the gene locus of GSR can be assigned to the 8p11 leads to p22 segment. A comparison of these results with one other case from the literature suggests a more precise assignment of the GSR locus to band p21.     

A case of de novo trisomy 12p syndrome

We described a case of complete trisomy 12p syndrome (non-mosaic) originating de novo in a first born child to healthy 26 year old nonconsanguineous parents. The 12p syndrome has been confirmed by clinical, cytogenetic and gene dosage studies.     

Gene dosage effect for human triosephosphate isomerase and glyceraldehyde-3-phosphate dehydrogenase in partial trisomy 12p13 and trisomy 18p

Summary An 8-year-old girl with profound mental retardation and a neurologic syndrome associated with morphologic abnormalities was found to have a supernumerary small submetacentric chromosome. Several members of her family carried a balanced translocation t(12;18)(p12;q11), and the child's karyotype could be explained by 3:1 maternal segregation (tertiary trisomy). The proband was trisomic for 12p13 and 18p. A gene dosage effect was demonstrated for triosephosphate isomerase and glyceraldehyde-3-phosphate in erythrocytes and leukocytes allowing us to assign the corresponding loci to the tip of the chromosome 12 short arm.     

Plasma placental RNA allelic ratio permits noninvasive prenatal chromosomal aneuploidy detection

Current methods for prenatal diagnosis of chromosomal aneuploidies involve the invasive sampling of fetal materials using procedures such as amniocentesis or chorionic villus sampling and constitute a finite risk to the fetus. Here, we outline a strategy for fetal chromosome dosage assessment that can be performed noninvasively through analysis of placental expressed mRNA in maternal plasma. We achieved noninvasive prenatal diagnosis of fetal trisomy 21 by determining the ratio between alleles of a single-nucleotide polymorphism (SNP) in PLAC4 mRNA, which is transcribed from chromosome 21 and expressed by the placenta, in maternal plasma. PLAC4 mRNA in maternal plasma was fetal derived and cleared after delivery. The allelic ratios in maternal plasma correlated with those in the placenta. Fetal trisomy 21 was detected noninvasively in 90% of cases and excluded in 96.5% of controls.     

A patient with tetrasomy 9p, Dandy-Walker cyst and Hirschsprung disease.

The authors report on a patient with tetrasomy 9p and 9qh due a karyotype 47,XY,+dic(9)(q12) in lymphocytes and a normal karyotype in fibroblasts. Clinical and complementary investigation revealed a malformation syndrome with many anomalies like those of trisomy 9p as well as Dandy-Walker cyst and Hirschsprung disease not previously described in tetrasomy 9p.     

Duplication of the short arm of chromosome 9. Analysis of five cases

Summary Five females with duplication of the short arm of one chromosome 9 are reported, one tetrasomic and four trisomic for 9p. The tetrasomy is due to an isochromosome 9p while the trisomies are due in one case to an intrachromosomal duplication present in lymphocytes but not in fibroblasts, two are secondary to translocations with chromosomes 22 and 13 respectively, and one is a mosaic with a cell line with an additional deleted chromosome 9 present in lymphocytes and fibroblasts. This analysis indicates that duplications 9p may result in impairment of ovarian function. The phenotypic differences between trisomy and tetrasomy 9p are discussed.     

Human microRNA-155 on chromosome 21 differentially interacts with its polymorphic target in the AGTR1 3' untranslated region: a mechanism for functional single-nucleotide polymorphisms related to phenotypes

Animal microRNAs (miRNAs) regulate gene expression through base pairing to their targets within the 3' untranslated region (UTR) of protein-coding genes. Single-nucleotide polymorphisms (SNPs) located within such target sites can affect miRNA regulation. We mapped annotated SNPs onto a collection of experimentally supported human miRNA targets. Of the 143 experimentally supported human target sites, 9 contain 12 SNPs. We further experimentally investigated one of these target sites for hsa-miR-155, within the 3' UTR of the human AGTR1 gene that contains SNP rs5186. Using reporter silencing assays, we show that hsa-miR-155 down-regulates the expression of only the 1166A, and not the 1166C, allele of rs5186. Remarkably, the 1166C allele has been associated with hypertension in many studies. Thus, the 1166C allele may be functionally associated with hypertension by abrogating regulation by hsa-miR-155, thereby elevating AGTR1 levels. Since hsa-miR-155 is on chromosome 21, we hypothesize that the observed lower blood pressure in trisomy 21 is partially caused by the overexpression of hsa-miR-155 leading to allele-specific underexpression of AGTR1. Indeed, we have shown in fibroblasts from monozygotic twins discordant for trisomy 21 that levels of AGTR1 protein are lower in trisomy 21.