Ruby Jiang

Androgenetic/biparental mosaicism causes placental mesenchymal dysplasia

Background: Placental mesenchymal dysplasia (PMD) is a distinct syndrome of unknown aetiology that is associated with significant fetal morbidity and mortality. Intrauterine growth restriction is common, yet, paradoxically, many of the associated fetuses/newborns have been diagnosed with Beckwith-Wiedemann syndrome (BWS). Methods: We report two cases of PMD with high levels of androgenetic (complete paternal uniparental isodisomy) cells in the placenta and document, in one case, a likely androgenetic contribution to the fetus as well. Results: The same haploid paternal complement found in the androgenetic cells was present in coexisting biparental cells, suggesting origin from a single fertilisation event. Conclusions: Preferential allocation of the normal cells into the trophoblast explains the absence of trophoblast overgrowth, a key feature of this syndrome. Interestingly, the distribution of androgenetic cells appears to differ from that reported for artificially created androgenetic mouse chimeras. Androgenetic mosaicism for the first time provides an aetiology for PMD, and may be a novel mechanism for BWS and unexplained intrauterine growth restriction.

MECP2 promoter methylation and X chromosome inactivation in autism

Epigenetic mechanisms have been proposed to play a role in the etiology of autism. This hypothesis is supported by the discovery of increased MECP2 promoter methylation associated with decreased MeCP2 protein expression in autism male brain. To further understand the influence of female X chromosome inactivation (XCI) and neighboring methylation patterns on aberrant MECP2 promoter methylation in autism, multiple methylation analyses were performed on brain and blood samples from individuals with autism. Bisulfite sequencing analyses of a region 0.6 kb upstream of MECP2 in brain DNA samples revealed an abrupt transition from a highly methylated region in both sexes to a region unmethylated in males and subject to XCI in females. Chromatin immunoprecipitation analysis demonstrated that the CCCTC‐binding factor (CTCF) is bound to this transition region in neuronal cells, consistent with a chromatin boundary at the methylation transition. Male autism brain DNA samples displayed a slight increase in methylation in this transition region, suggesting a possible aberrant spreading of methylation into the MECP2 promoter in autism males across this boundary element. In addition, autistic female brain DNA samples showed evidence for aberrant MECP2 promoter methylation as an increase in the number of bisulfite sequenced clones with undefined XCI status for MECP2 but not androgen receptor (AR). To further investigate the specificity of MECP2 methylation alterations in autism, blood DNA samples from females and mothers of males with autism were also examined for XCI skewing at AR, but no significant increase in XCI skewing was observed compared to controls. These results suggest that the aberrant MECP2 methylation in autism brain DNA samples is due to locus‐specific rather than global X chromosome methylation changes.

Skewed X-Chromosome Inactivation Is Associated with Trisomy in Women Ascertained on the Basis of Recurrent Spontaneous Abortion or Chromosomally Abnormal Pregnancies

An increase in extremely skewed X-chromosome inactivation (XCI) (> or = 90%) among women who experienced recurrent spontaneous abortion (RSA) has been previously reported. To further delineate the etiology of this association, we have evaluated XCI status in 207 women who experience RSA. A significant excess of trisomic losses was observed among the women who had RSA with skewed XCI versus those without skewed XCI (P=.02). There was also a significant excess of boys among live births in this group (P=.04), which is contrary to expectations if the cause of skewed XCI was only that these women carried X-linked lethal mutations. To confirm the association between skewed XCI and the risk of trisomy, an independent group of 53 women, ascertained on the basis of a prenatal diagnosis of trisomy mosaicism, were investigated. Only cases for which the trisomy was shown to be of maternal meiotic origin were included. The results show a significantly higher level of extreme skewing (> or = 90%) in women whose pregnancies involved placental trisomy mosaicism (17%) than in either of two separate control populations (n=102 and 99) (P=.02 compared with total control subjects). An additional 11 cases were ascertained on the basis of one or more trisomic-pregnancy losses. When all women in the present study with a trisomic pregnancy (n=103) were considered together, skewed XCI was identified in 18%, as compared with 7% in all controls (n=201) (P=.005). This difference was more pronounced when a cutoff of extreme skewing of 95% was used (10% vs. 1.5% skewed; P=.002). Maternal age was not associated with skewing in either the patient or control populations and therefore cannot account for the association with trisomy. Previous studies have shown that a reduced ovarian reserve is associated with increased risk of trisomic pregnancies. We hypothesize that the association between skewed XCI and trisomic pregnancies is produced by a common mechanism that underlies both and that involves a reduction of the size of the follicular pool.

Genome-wide mapping of imprinted differentially methylated regions by DNA methylation profiling of human placentas from triploidies

BackgroundGenomic imprinting is an important epigenetic process involved in regulating placental and foetal growth. Imprinted genes are typically associated with differentially methylated regions (DMRs) whereby one of the two alleles is DNA methylated depending on the parent of origin. Identifying imprinted DMRs in humans is complicated by species- and tissue-specific differences in imprinting status and the presence of multiple regulatory regions associated with a particular gene, only some of which may be imprinted. In this study, we have taken advantage of the unbalanced parental genomic constitutions in triploidies to further characterize human DMRs associated with known imprinted genes and identify novel imprinted DMRs.ResultsBy comparing the promoter methylation status of over 14,000 genes in human placentas from ten diandries (extra paternal haploid set) and ten digynies (extra maternal haploid set) and using 6 complete hydatidiform moles (paternal origin) and ten chromosomally normal placentas for comparison, we identified 62 genes with apparently imprinted DMRs (false discovery rate <0.1%). Of these 62 genes, 11 have been reported previously as DMRs that act as imprinting control regions, and the observed parental methylation patterns were concordant with those previously reported. We demonstrated that novel imprinted genes, such as FAM50B, as well as novel imprinted DMRs associated with known imprinted genes (for example, CDKN1C and RASGRF1) can be identified by using this approach. Furthermore, we have demonstrated how comparison of DNA methylation for known imprinted genes (for example, GNAS and CDKN1C) between placentas of different gestations and other somatic tissues (brain, kidney, muscle and blood) provides a detailed analysis of specific CpG sites associated with tissue-specific imprinting and gestational age-specific methylation.ConclusionsDNA methylation profiling of triploidies in different tissues and developmental ages can be a powerful and effective way to map and characterize imprinted regions in the genome.

Assessing the role of placental trisomy in preeclampsia and intrauterine growth restriction

Prenatally diagnosed confined placental trisomy is associated with increased risk for intrauterine growth restriction (IUGR) and preeclampsia. However, it is unclear how often this might underlie pregnancy complications. Our objective was to evaluate the frequency and distribution of trisomic cells in placentae ascertained for IUGR and/or preeclampsia.

Methylation of ZNF261 as an assay for determining X chromosome inactivation patterns.

X chromosome inactivation (XCI) occurs early in female development to silence one of the pair of X chromosomes. The choice of chromosome to inactivate is generally random, but is then stably inherited, resulting in females being mosaics of cells with the alternate X chromosome active [Lyon, 1961]. Nonrandom X inactivation can occur due to a selective advantage of one cell population, raremutations altering the inactivation pattern, or by chance, which can be enhanced if there is a decreased number of precursor cells (reviewed in Brown and Robinson, 2000). In addition to use in demonstrating the monoclonal origin of tumors [Linder and Gartler, 1965], or carriers of X-linked diseases [e.g., Puck et al., 1987], skewed inactivationhas recently been reported to be associated with confined placental mosaicism [Lau et al., 1997], recurrent spontaneous abortion [Pegoraro et al., 1998; Sangha et al., 1999], and young breast cancer patients [Kristiansen et al., 2002], although the basis for the latter two correlations is not yet clear. Assays for X-inactivation patterns rely on a polymorphism to distinguish the X chromosomes and either methylation or expression differences to identify the active (expressed) or inactive (methylated) allele. The most commonlyusedassayutilizes ahighlypolymorphic repeat in the androgen receptor (AR) gene that is adjacent to several CpG methylation-sensitive restriction enzyme sites (HpaII and HhaI) that are differentially methylated on the active and inactive X chromosome [Allen et al., 1992]. The advantage to assays using methylation differences is that they utilize DNA, which is often more readily obtained from clinical specimens than RNA. However, these assays can be influenced by any disruption in the correlation between hypermethylation and X chromosome silencing. The polymorphism in AR is informative in over 80% of females [Edwards et al., 1991], but the assay can be difficult to quantitate when the alleles are only separated by a single repeat. Other methylation-based assays are not used as commonly because they require more steps (e.g., MAOA [Hendriks et al., 1992]), are less informative (e.g.,PGK1 [Gilliland et al., 1991]), or are complicated by differential amplification (e.g., FMR1 [Lee et al., 1994]) or inconsistent methylation differences (e.g., M27B [Boyd and Fraser, 1990]). Thus we have adapted the highly polymorphic ZNF261 gene for methylation-based analysis of X inactivation. The ZNF261 (DXS6673E) gene maps proximal to the X inactivation center in Xq13.1. The gene is highly conserved amongst vertebrates, and is a candidate for X-linked mental retardation, since it was shown to be disrupted in a balanced X;13 translocation in amentally retarded female [vanderMaarel et al., 1996].Consistent with this possibility, the gene shows highest expression in brain, although expression is found in all tissues examined. The mouse gene shows alternative splicing with variable tissue distributions of the alternate isoforms [Scheer et al., 2000]. The gene contains a highly polymorphic dinucleotide repeat in the 50 untranslated region that has previously been used to monitor inactivation patterns by comparing expression levels of the two alleles in heterozygous females [Carrel and Willard, 1999]. Two 50 exons have been described in humans, and the repeat is found in the more 50 of these, exon 1A. There is a CpG island associated with the alternative first exon, 1B, but it is 700–800 bp downstream of the polymorphic repeat and, therefore, impractical for a single step assay using PCR. Near the repeat, however, there are two HhaI restriction enzyme sites, and we have designed primers that flank these sites as well as the repeat, and demonstrate that these sites show differential methylation between the active and inactive X chromosomes. The primers (DXS6673E-A: ATG CTA AGG ACC ATC CAG GA and DXS6673E-B: GGA GTT TTC CTC CCT CAC CA) amplify an 275 bp product of exon 1A in both DNA and cDNA. Amplification is sensitive to salt concentration andwe have optimized them using 1 ml of template DNA (10–100 ng DNA) in digestion buffer 4 (New England Biolabs) in a 25 ml PCR with 0.5 mM MgCl2. Product is routinely obtainedafter 30 cycles of (948Cfor 1min; 548C for 1min; 728C for 2min). In Figure 1A, we show that in DNAfromtwo female cell lines previously demonstrated to have nonrandom X inactivation, there is loss of one Grant sponsor: CIHR; Grant numbers: MT-13690, MT-13694.

Patterns of placental development evaluated by X chromosome inactivation profiling provide a basis to evaluate the origin of epigenetic variation

BACKGROUND Inactivation of the maternally or paternally derived X chromosome (XCI) initially occurs in a random manner in early development; however as tissues form, a ‘patchiness’ will occur in terms of which X is inactivated if cells positioned near each other are clonally descended from a common precursor. Determining the relationship between skewed XCI in different tissues and in different samples from the same tissue provides a molecular assessment of the developmental history of a particular tissue that can then be used to understand how genetic and epigenetic variation arises in development. METHODS XCI skewing was evaluated in and compared between amnion, chorion, trophoblast and mesenchyme using multiple sampling sites from 14 term placentae. XCI was also evaluated in chorionic villus samples obtained at multiple sites and depths from four additional term placentae. The pattern of variation was then compared with methylation variation associated with the H19/IGF2 imprinting control region (ICR); promoter regions of KISS1, PTPN6, CASP8 and APC; and LINE-1 elements. RESULTS Mean placental level of skewing for amnion and chorion are correlated, consistent with a common developmental origin of at least a component of these membranes from inner cell mass derivatives subsequent to XCI, while trophoblast appears to be derived independently, consistent with its origin from the trophectoderm. Villus samples taken from different depths spanning the fetal to maternal side of the placenta were highly clonally related. Comparing patterns of clonal growth identified through XCI to the distribution of epigenetic variation in other genomic regions suggests that some variation arises early in development (e.g. LINE-1 methylation), whereas other variation arises predominantly after villus tree formation (e.g. methylation at H19/IGF2 ICR). CONCLUSIONS The patterns of XCI skewing are consistent with a model whereby each biopsied site of chorionic villi represents one or a few individual villus trees, each of which is clonally derived from only one or a few precursor cells. Sampling of placentae to evaluate changes associated with clinical pathology should be done with consideration of the tree-to-tree differences. A limitation of this study is the small number of placentas used and therefore placental-specific differences in variation could not be assessed.

Recurrent trisomy 21: four cases in three generations.

While gonadal mosaicism can lead to recurrence of trisomy 21 (T21) for a single couple, the recurrence of free T21 in multiple members of a single pedigree has rarely been reported. We present an unusual pedigree with four cases of Down syndrome (DS) with free T21 were born to four separate women related through three generations of one family. The mothers were aged 18, 21, 29, and approximately 30 years at the time of the births. Using microsatellite markers, we excluded most of chromosome 21, excepting two small regions within 21q11.1 and 21q22.3, as being shared among the mothers of the DS children. However, two members of the pedigree, including one DS mother with a normal G‐banded karyotype, carried supernumerary alleles at markers 2503J9TG, D21S369, and D21S215, which span the region from 21pter to 21q11.1. Fluorescence in situ hybridization using a centromeric probe hybridizing to chromosomes 13 and 21 did not reveal a novel location, ruling out a cryptic centromeric translocation between chromosome 21 and any chromosome other than chromosome 13. The level of meiotic recombination on chromosome 21 was unusually high in this family as well. We hypothesize that a cryptic rearrangement within the highly repetitive region of 21q11.1 is present in this family, disrupting pairing and leading to an increased risk of non‐disjunction of chromosome 21 in this family.

Variant ATRX syndrome with dysfunction of ATRX and MAGT1 genes.

A 0.8kb intronic duplication in MAGT1 and a single base pair deletion in the last exon of ATRX were identified using a chromosome X‐specific microarray and exome sequencing in a family with five males demonstrating intellectual disability (ID) and unusual skin findings (e.g., generalized pruritus). MAGT1 is an Mg2+ transporter previously associated with primary immunodeficiency and ID, whereas mutations in ATRX cause ATRX‐ID syndrome. In patient cells, the function of ATRX was demonstrated to be abnormal based on altered RNA/protein expression, hypomethylation of rDNA, and abnormal cytokinesis. Dysfunction of MAGT1 was reflected in reduced RNA/protein expression and Mg2+ influx. The mutation in ATRX most likely explains the ID, whereas MAGT1 disruption could be linked to abnormal skin findings, as normal magnesium homeostasis is necessary for skin health. This work supports observations that multiple mutations collectively contribute to the phenotypic variability of syndromic ID, and emphasizes the importance of correlating clinical phenotype with genomic and cell function analyses.