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

Analysis of the <i>ARHI</i>-DMR in case 13.

<p>For bisulfite sequencing, each line indicates a single clone, and each circle denotes a CpG dinucleotide; the cytosine residues at the CpG dinucleotides are usually unmethylated after paternal transmission (open circles) and methylated after maternal transmission (filled circles). Electrochromatograms delineate the sequences of the primer binding sites utilized for the methylation analysis.</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

Phenotypic comparison in three groups of patients with Silver-Russell syndrome.

<p>BL: birth length; BW: birth weight; BOFC: birth occipitofrontal circumference; PH: present height; PW: present weight; POFC: present occipitofrontal circumference, and SDS: standard deviation score.</p><p>For body features, the denominators indicate the number of patients examined for the presence or absence of each feature, and the numerators represent the number of patients assessed to be positive for that feature.</p>*<p>Mandatory criteria and †five clinical features utilized as selection criteria for Silver-Russell syndrome proposed by Netchine et al. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0060105#pone.0060105-Netchine1" target="_blank">[14]</a>.</p><p>Significant <i>P</i>-values(<0.05) are boldfaced.</p>a<p>Placental weight SDS is –1.68, –2.55, –2.24, –1.12, –2.14 and –0.60 in case 46, 47, 49, 50, 51 and 52, respectively; the placental weight SDS is –1.95±0.57 in five cases except for case 52 with mosaic upd(7)mat.</p>b<p>Maternal childbearing age is 32, 32, 33, 42, 32, 34, 33, 25 and 36 years in case 44–52, respectively.</p

Correlation analyses in patients with <i>H19</i>-DMR hypomethylations.

<p>SDS: standard deviation score; and OFC: occipitofrontal circumference.</p>*<p>The mean value of MIs for CG5, CG6, CG7, and CG9 obtained by pyrosequencing analysis.</p><p>Significant <i>P</i>-values(<0.05) are boldfaced.</p

Methylation analysis of the <i>H19</i>-DMR, using bisulfite-treated genomic DNA.

<p>A. Schematic representation of a segment encompassing 21 CpG dinucleotides (CG1→CG21) within the <i>H19</i>-DMR. The cytosine residues at the CpG dinucleotides are usually methylated after paternal transmission (filled circles) and unmethylated after maternal transmission (open circles). The CTCF binding site 6 (CTCF6) is indicated with a blue rectangle; the cytosine residue at CG8 constitutes a C/T SNP (indicated with a gray rectangle). For pyrosequencing analysis, a 279 bp segment was PCR-amplified with PyF & PyR primers, and a sequence primer (SP) was hybridized to a single-stranded PCR products. Subsequently, the MIs were obtained for four CpG dinucleotides (CG5–CG7 and CG9) (indicated with a yellow rectangle). For COBRA, a 435 bp region was PCR-amplified with CoF & CoR primers, and the PCR product was digested with methylated allele-specific restriction enzymes to examine the methylation pattern of CG5 ands CG16 (the PCR products is digested with <i>Hpy</i>188I when the cytosine residue at CG5 is methylated and with <i>Afl</i>III when the cytosine residue at CG16 is methylated) (indicated with orange rectangles). <i>IGF2</i> is a paternally expressed gene, and <i>H19</i> is a maternally expressed gene. The stippled ellipse indicates the enhancer for <i>IGF2</i> and <i>H19</i>. B. Pyrosequencing data. Left part: Representative results indicating the MIs for CG5– CG7 and CG9. CG5– CG7 and CG9 are hypomethylated in case 1, and similarly methylated between case 53 and a control subject. Right part: Histograms showing the distribution of the MIs (the horizontal axis: the methylation index; and the vertical axis: the patient number). Forty-three SRS patients with low MIs are shown in red. C. COBRA data. Left part: Representative findings of PCR products loaded onto a DNA 1000 LabChip (Agilent, Santa Clara, CA, USA) after digestion with <i>Hpy</i>188I or <i>Afl</i>III. U: unmethylated clone specific bands; M: methylated clone specific bands; and BWS: Beckwith-Wiedemann syndrome patient with upd(11p15)pat. Right part: Histograms showing the distribution of the MIs.</p

Methylated and unmethylated allele-specific PCR analysis for the <i>MEST</i>-DMR.

<p>A. Schematic representation of the <i>MEST</i>-DMR. The cytosine residues at the CpG dinucleotides are usually unmethylated after paternal transmission (open circles) and methylated after maternal transmission (filled circles). The PCR primers have been designed to hybridize either methylated or unmethylated clones. B. The results of methylation analysis with methylated and unmethylated allele-specific primers.</p

The methylation indices (%) for the <i>H19</i>-DMR.

<p>The position of examined CpG dinucleotides (CG5–7, CG9, and CG16) is shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0060105#pone-0060105-g001" target="_blank">Figure 1A</a>.</p><p>COBRA: combined bisulfite restriction analysis.</p