Description of <i>NLRP7</i> mutations with methylation and expression profiling of imprinted loci.

<p>(A) Confirmation of recessive <i>NLRP7</i> mutations in female patients and heterozygous status in the RHM samples. The asterisk (*) on the electropherogram highlights the position of the mutation. For patient 3 the position of the deletion is shown. (B) Circular heat map of the 616 Infinium array probes mapping to 36 ubiquitously imprinted DMRs. The inner circle represents the methylation values of androgenetic HMs, the middle circles normal placental biopsies and the outer circle the RHMs associated with maternal-effect <i>NLRP7</i> mutations. (C) Confirmation of the methylation profile of the <i>NLRP7</i> mutated RHMs at the <i>NAP1L5</i>, <i>PEG10</i>, <i>RB1</i>, <i>L3MBTL1</i> and <i>H19</i> DMRs by bisulphite PCR and subcloning. Each circle represents a single CpG dinucleotide on a DNA strand, a methylated cytosine (●) or an unmethylated cytosine (○). For clarity, only the first 10 CpG dinucleotides from each amplicon are shown with the letters in the parentheses indicating SNP genotype. (D) Allelic expression analysis of imprinted genes <i>NAP1L5</i>, <i>HYMAI</i>, <i>PEG10</i> and <i>PEG3</i> in control placenta samples (PL) and <i>NLRP7</i>-mutated moles (RHM).</p

Identification of additional placenta-specific imprinted DMRs in RHM samples.

<p>(A) A heatmap for the β_mean of the Infinium probes with a methylation difference (>20%, minimum 3 consecutive probes) in RHMs associated with maternal effect <i>NLRP7</i> mutations compared to control placental biopsies. (B) Schematic representation of the methylation-sensitive <i>Hpa</i>II genotyping assay. (C) Methylation profiles as determined by methylation-sensitive genotyping and (D) bisulfite PCR and subcloning on placenta and somatic tissue DNA samples at the <i>SCIN</i>, <i>ST8AIA1</i> and <i>CABIN1</i> promoters. Note that the samples used for methylation-sensitive genotyping and bisulphite PCR maybe different to highlight that methylation is not associated with genotype but parental origin.</p

Methylation and expression analyses of placenta-specific DMRs in RHM samples.

<p>(A) Circular heatmap of the 153 Infinium array probes mapping to the 18 known placenta-specific imprinted DMRs. The inner circles represent the methylation values of androgenetic HMs, the middle circles normal placental biopsies and the outer circle the RHMs associated with maternal-effect <i>NLRP7</i> mutations. (B) Confirmation of the methylation profile at the maternally methylated <i>GLIS3</i>, <i>DNMT1</i> and <i>MCCC1</i> DMRs by bisulphite PCR and subcloning. Each circle represents a single CpG dinucleotide on a DNA strand, a methylated cytosine (●) or an unmethylated cytosine (○). For clarity, only the first 10 CpG dinucleotides from each amplicon are shown with the letters in the parentheses indicating SNP genotype. (C) Allelic expression analysis of imprinted genes <i>MCCC1</i>, <i>LIN28B</i> and <i>GLIS3</i> in control placenta samples (PL) and <i>NLRP7</i>-mutated moles (RHM). (D) Quantitative RT-PCR for <i>H19</i>, <i>DNMT1</i> and <i>AGBL3</i> in RHM samples. The boxplot show the median expression (whiskers 5–95% percentile) determined for 15 control placenta samples with the values of RHMs highlighted.</p

Methylation profiling of human gametes, embryos and tissues.

<p>(<b>A</b>) Heatmap of all 25 CpG dinucleotide tiles with maternal (left) and paternal (right) germline-derived methylation maintaining an intermediate state in blastocysts and arranged in descending order according to their placenta methylation profile. Tiles were partitioned according to their hypo- (<20%), hyper- (>80%) or intermediate (>20%, <80%) methylation. Genomic features are included as separate heatmaps. (<b>B</b>) Violin plots classified as the % repetitive sequences per 20 CpG dinucleotide tile for the paternally methylated, maternally methylated and known imprinted DMRs. The numbers in red represent the tiles with unique sequences (defined as >25% repeats). (<b>C</b>) Two novel maternally methylated ubiquitous DMRs associated with the <i>SVOPL</i> and <i>FANCC</i> genes exhibit promoters that are unmethylated in sperm, hypermethylated in oocytes and intermediately methylated in blastocysts, placenta and somatic tissue in methyl-seq datasets. The vertical black lines in the methyl-seq tracks represent the mean methylation value for individual CpG dinucleotides. Green boxes highlight the position of the gDMRs. (<b>D</b>) Bisulphite PCR and subcloning was used for confirmation. Each circle represents a single CpG dinucleotide on a DNA strand. (•) Methylated cytosine, (o) unmethylated cytosine. Each row corresponds to an individual cloned sequence. If heterozygous for a SNP, the parental-origin of methylation is indicated. For clarity only the first 10 CpG dinucleotides are shown. (E) Allelic RT-PCR for <i>SVOPL</i> reveals transcription from the maternal allele in placenta and monoallelic expression in brain and leukocytes.</p

Methylation profiling of opposing gDMRs using bisulphite PCR in human gametes and blastocysts.

<p>(<b>A</b>) The confirmation that the <i>H19</i> DMR acquires methylation from the sperm and maintains in preimplantation embryos (separated into ICM and TE) and in somatic tissues. The <i>MSCT2</i> DMR shows the opposite profile with sperm devoid of methylation. (<b>B</b>) The bisulphite PCR profiles for the novel ubiquitous <i>FANCC</i> and <i>SVOPL</i> DMRs in human sperm, blastocysts and placenta. (<b>C</b>) Methyl-seq datasets reveal that the <i>R3HCC1</i> gene has two adjacent gDMRs, an upstream paternal gDMR (region 1) that subsequently gains methylation on both alleles during the blastocyst stage and a placenta-specific maternally methylated promoter region (region 2). The vertical black lines in the methyl-seq tracks represent the mean methylation value for individual CpG dinucleotides. Green boxes highlight the position of the gDMRs. (<b>D</b>) Confirmation of the methylation profile by bisulfite PCR and subcloning. Each circle represents a single CpG dinucleotide on a DNA strand. (•) Methylated cytosine, (o) unmethylated cytosine. Each row corresponds to an individual cloned sequence. If informative, the parental-origin of methylation is indicated. For clarity only the first 10 CpG dinucleotides are shown.</p

Analysis of allelic expression for genes associated with novel placenta-specific maternally methylated gDMRs.

<p>(<b>A</b>) Allelic RT-PCR analysis for nine transcripts originating from placenta-specific DMRs in control placenta samples. Monoallelic paternal expression was observed in heterozygous placenta biopsies. (<b>B</b>) The identification of a ~10 kb ncRNA overlapping a placenta-specific gDMR ~12 kb downstream of the <i>TET3</i> gene. The vertical black lines in the methyl-seq tracks represent the mean methylation value for individual CpG dinucleotides. The green box highlights the position of the gDMRs. (<b>C</b>) The 2.7 kb maternally methylated placenta-specific DMR identified by methyl-seq and confirmed with allelic-specific bisulphite PCR and subcloning. Each circle represents a single CpG dinucleotide on a DNA strand. (•) Methylated cytosine, (o) unmethylated cytosine. Each row corresponds to an individual cloned sequence. For clarity only the first 10 CpG dinucleotides are shown. (<b>D</b>) Paternal expression of the RNA-seq peak was determined by RT-PCR, whilst allele-specific RT-PCR revealed that <i>TET3</i> is biallelically expressed in term placenta samples.</p

Analysis of tissue-specific maintenance of germline methylation in different tissues.

<p>(<b>A</b>) A bar graph showing the fate of gDMRs in tissues. The bars represent the profiles of known ubiquitous (black) and placenta-specific (orange) gDMRs with numbers corresponding to the left y-axis. The superimposed line graph represent the profile of all remaining germline difference that are maintained to the blastocyst stage and correspond to the right y-axis. * placenta-specific DMRs identified by Court (2014) [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006427#pgen.1006427.ref010" target="_blank">10</a>] and Sanchez-Delgado (2015) [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006427#pgen.1006427.ref015" target="_blank">15</a>]. (<b>B</b>) A pie graph showing the distribution of individual tissues maintaining a partially methylated profile. (<b>C</b>) The <i>GRID2</i> gene exhibits high inter- and intragenic methylation and several regions with oocyte-derived methylation. The methyl-seq data reveals that a 1.9 kb region overlapping the promoter remains an imprinted gDMR in placenta while it is demethylated in all other tissues. A second oocyte-derived gDMR, consisting largely of an Alu/SINE repeat, becomes fully methylated in all tissues analysed. The vertical black lines in the methyl-seq tracks represent the mean methylation value for individual CpG dinucleotides. A green box highlights the position of the gDMR. (<b>D</b>) Bisulfite PCR and subcloning on heterozygous placenta DNA samples for the <i>GRID2</i> promoter and intragenic regions. Each circle represents a single CpG dinucleotide on a DNA strand. (•) Methylated cytosine, (o) unmethylated cytosine. Each row corresponds to an individual cloned sequence. If informative for a SNP, the parental-origin of methylation is indicated. For clarity only the first 10 CpG dinucleotides are shown.</p

Identification of novel imprinted genes in human embryos using allele-specific RNA-seq datasets.

<p>(<b>A</b>) Schematic drawing of the sequential transcriptome switching from oocyte-derived transcripts to the embryonic genome in human preimplantation embryos. (<b>B</b>) The expression pattern of the <i>ZHX3</i> gene during human preimplantation development. High expression was observed from the zygote to the 8-cell stage, declining in the morula. Paternal expression as observed from the 4-cell stage onwards. (<b>C</b>) Allele-specific RT-PCR was performed on term placenta samples heterozygous for the exonic SNP rs17265513. (<b>D</b>) Methyl-seq traces reveal the location of the placenta-specific maternal gDMR overlapping the <i>ZHX3</i> promoter. The vertical black lines in the methyl-seq tracks represent the mean methylation value for individual CpG dinucleotides. The green box highlights the position of the gDMRs. (<b>E</b>) The methylation profile confirmed using bisulphite PCR and cloning in sperm, blastocysts and placenta. Each circle represents a single CpG dinucleotide on a DNA strand. (•) Methylated cytosine, (o) unmethylated cytosine. Each row corresponds to an individual cloned sequence. For clarity only the first 10 CpG dinucleotides are shown.</p

Molecular and Clinical Studies in 138 Japanese Patients with Silver-Russell Syndrome

<div><p>Background</p><p>Recent studies have revealed relative frequency and characteristic phenotype of two major causative factors for Silver-Russell syndrome (SRS), i.e. epimutation of the <i>H19</i>-differentially methylated region (DMR) and uniparental maternal disomy 7 (upd(7)mat), as well as multilocus methylation abnormalities and positive correlation between methylation index and body and placental sizes in <i>H19</i>-DMR epimutation. Furthermore, rare genomic alterations have been found in a few of patients with idiopathic SRS. Here, we performed molecular and clinical findings in 138 Japanese SRS patients, and examined these matters.</p> <p>Methodology/Principal Findings</p><p>We identified <i>H19</i>-DMR epimutation in cases 1–43 (group 1), upd(7)mat in cases 44–52 (group 2), and neither <i>H19</i>-DMR epimutation nor upd(7)mat in cases 53–138 (group 3). Multilocus analysis revealed hyper- or hypomethylated DMRs in 2.4% of examined DMRs in group 1; in particular, an extremely hypomethylated <i>ARHI</i>-DMR was identified in case 13. Oligonucleotide array comparative genomic hybridization identified a ∼3.86 Mb deletion at chromosome 17q24 in case 73. Epigenotype-phenotype analysis revealed that group 1 had more reduced birth length and weight, more preserved birth occipitofrontal circumference (OFC), more frequent body asymmetry and brachydactyly, and less frequent speech delay than group 2. The degree of placental hypoplasia was similar between the two groups. In group 1, the methylation index for the <i>H19</i>-DMR was positively correlated with birth length and weight, present height and weight, and placental weight, but with neither birth nor present OFC.</p> <p>Conclusions/Significance</p><p>The results are grossly consistent with the previously reported data, although the frequency of epimutations is lower in the Japanese SRS patients than in the Western European SRS patients. Furthermore, the results provide useful information regarding placental hypoplasia in SRS, clinical phenotypes of the hypomethylated <i>ARHI</i>-DMR, and underlying causative factors for idiopathic SRS.</p> </div

Oligonucleotide array CGH in case 73, showing a ∼3.86 Mb deletion at chromosome 17q24.

<p>The black, the red, and the green dots denote signals indicative of the normal, the increased(>+0.5), and the decreased (< –1.0) copy numbers, respectively. The horizontal bar with arrowheads indicates a ∼2.3 Mb deletion identified in a patient with Carney complex and SRS-like phenotype <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0060105#pone.0060105-Blyth1" target="_blank">[44]</a>, and the black square represent a ∼65 kb segment harboring the breakpoint of a <i>de novo</i> translocation 46,XY,t(1;17)(q24;q23–q24) identified in a patient with SRS phenotype <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0060105#pone.0060105-Midro1" target="_blank">[45]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0060105#pone.0060105-Drr1" target="_blank">[46]</a>.</p