Visualization by molecular combing of the 17q21 region around <i>BRCA1</i>.

<p>(A) Schematization of the genomic morse code used. The <i>BRCA1</i> Genomic Morse Code (GMC) depicted (v4.0) is an improvement of the published code (v1.0) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0076054#pone.0076054-Cheeseman1" target="_blank">[23]</a>. It covers a genomic region of 200 kb and consist in 17 signals of a distinct color (green, red or blue), each composed of 1 to 3 small horizontal bars corresponding to a single DNA probe. The signals for the flanking probes FP1-4 are each composed of 2 green or blue horizontal bars, while the signal for the <i>RNU2</i> array repeat unit is composed of 1 red horizontal bar. Of note, the probe for the <i>RNU2</i> array cross-reacts with <i>RNU2-4P</i>. (B) Fourteen fibres displaying different numbers of <i>RNU2</i> signals are shown. The first six fibres display the entire bar code from the <i>BRCA1</i> GMC to <i>RNU2-4P</i>, while the followings miss either the beginning of the <i>BRCA1</i> GMC or <i>RNU2-4P</i>.</p

Description of the <i>RNU2</i> array alleles identified in 46 unrelated chromosomes.

<p>Description of the <i>RNU2</i> array alleles identified in 46 unrelated chromosomes.</p

Direct Visualization of the Highly Polymorphic <i>RNU2</i> Locus in Proximity to the <i>BRCA1</i> Gene

<div><p>Although the breast cancer susceptibility gene <i>BRCA1</i> is one of the most extensively characterized genetic loci, much less is known about its upstream variable number tandem repeat element, the <i>RNU2</i> locus. <i>RNU2</i> encodes the U2 small nuclear RNA, an essential splicing element, but this locus is missing from the human genome assembly due to the inherent difficulty in the assembly of repetitive sequences. To fill the gap between <i>RNU2</i> and <i>BRCA1</i>, we have reconstructed the physical map of this region by re-examining genomic clone sequences of public databases, which allowed us to precisely localize the <i>RNU2</i> array 124 kb telomeric to <i>BRCA1</i>. We measured by performing FISH analyses on combed DNA for the first time the exact number of repeats carried by each of the two alleles in 41 individuals and found a range of 6-82 copies and a level of heterozygosity of 98%. The precise localisation of the <i>RNU2</i> locus in the genome reference assembly and the implementation of a new technical tool to study it will make the detailed exploration of this locus possible. This recently neglected macrosatellite could be valuable for evaluating the potential role of structural variations in disease due to its location next to a major cancer susceptibility gene.</p></div

Schematic representation of the chromosome 17q21 region around the <i>BRCA1</i> gene.

<p>(A) Gene locations and physical map distances as reported in the literature <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0076054#pone.0076054-Liu1" target="_blank">[16]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0076054#pone.0076054-Pavelitz2" target="_blank">[19]</a>. (B) Gene locations within a 300 Kb window as shown in the UCSC Genome Browser. Arrows indicate transcription direction. BRCA1P1: <i>BRCA1</i> pseudogene.</p

Localisation of the <i>RNU2</i> macrosatellite within the chromosome 17 sequence assemblies from NCBI Build 37.p10.

<p>(A) Schema of the region surrounding the <i>RNU2</i> array. The location of the portion of the <i>RNU2</i> repeat unit (not comprising the <i>RNU2</i> gene) and of the right junction found in the assemblies are depicted, as well as the probes used in molecular combing experiments that flank the <i>RNU2</i> array (FP1-4), and the <i>NBR1</i> and <i>TMEM106A</i> genes. (B) Clones covering the region. The reference sequence assemblies is based upon the complete sequence of 3 overlapping BACs, RP11-242D8, CTD-3014M21 and RP11-100E5 (AC060780.18, AC109326.11 and AC087650.12 respectively), represented by brown arrows. The complete sequence of the WI2-3095P13 fosmid (AC160862.2, green arrow) matches the reference sequence. The sequence of the ABC10-44487500M2 fosmid (AC231386.2, green arrow) matches the reference sequence up to its centromeric extremity where it contains several <i>RNU2</i> repeat units (depicted in a dotted curl). The five unassembled contigs of the working draft sequence of the RP11-570A16 BAC clone (AC087365.3) showing homology with the reference sequence are represented by a purple arrow. Contig 15 has been mis-assembled, as it contains several <i>RNU2</i> repeat units (depicted in dotted curls) at both its extremities.</p

Visualization by FISH on mitotic metaphase chromosome of the <i>RNU2</i> locus.

<p>Two probes were used, one consisting of the 6.1</p

<i>SHANK</i> variants in patients with ASD and controls.

<p>Coding-sequence variants identified only in patients with ASD (upper panel), shared by patients and controls (lower panel and underlined), and present only in controls (lower panel). Truncating variants are indicated in red. The variants predicted as deleterious or benign are indicated in orange and green, respectively. Coding-sequence variants with a proven <i>in vitro</i> functional impact are indicated with black stars. Conserved domains are represented in color: SPN (yellow), Ankyrin (red), SH3 (orange), PDZ (blue) and SAM (green).</p

Scatter plots of the intellectual quotient and the Autism Diagnostic Interview-Revised (ADI-R) scores of the patients with ASD screened for <i>SHANK1-3</i> mutations.

<p>Mutations in <i>SHANK1-3</i> are associated with a gradient of severity in cognitive impairment. <i>SHANK1</i> mutations were reported in patients without ID (green dots). <i>SHANK2</i> mutations were reported in patients with mild ID (orange dots). <i>SHANK3</i> mutations were found in patients with moderate to severe deficit (red dots). Black dots correspond to the patients enrolled in the PARIS cohort screened for deleterious <i>SHANK1-3</i> mutations (n = 498). In addition to the PARIS cohort <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004580#pgen.1004580-Durand1" target="_blank">[6]</a>, <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004580#pgen.1004580-Pinto1" target="_blank">[8]</a>, <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004580#pgen.1004580-Leblond1" target="_blank">[18]</a>, three patients with a <i>SHANK1</i> deletion <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004580#pgen.1004580-Sato1" target="_blank">[19]</a> and two patients with a <i>SHANK2</i> deletion <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004580#pgen.1004580-Berkel1" target="_blank">[14]</a> were included in the scatter plot. A high score of the ADI-R is associated with a more severe profile. The threshold of the “Social”, “Verbal”, “Non-Verbal” and “Repetitive Behavior” Scores are 10, 8, 7 and 3, respectively.</p

Prevalence and meta-analysis of coding-sequence variant studies in ASD.

<p>A. The prevalence and the confidence interval from a set of single coding-sequence variant studies, and the pooled prevalence and the confidence interval of the meta-analysis. The prevalence is indicated by circles in red, pink, purple and black for “ASD all” (all ASD patients), “ASD IQ<70” (patients with ID; IQ<70), “ASD IQ>70” (patients with normal IQ), and “CTRL” (controls), respectively. Three categories are used to study the prevalence of coding-sequence variants in ASD and controls: all or “A” (all mutation), Damaging or “D” (damaging missense mutation; score obtained from polyphen-2), and Truncating or “T” (mutation altering SHANK protein). The plotted circles are proportional to the corresponding sample size. B. Meta-analysis of coding-sequence variant studies altering <i>SHANK</i> genes. For each study, the Odds ratio and confidence interval is given. Each meta-analysis is calculated using inverse variance method for fixed (IV-FEM) and random effects (IV-REM). The statistics measuring heterogeneity (Q, I² and Tau²) are indicated. The number under the scatter plot correspond to independent studies: 1 = “This study”, 2 = “ Sato et al. (2012) <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004580#pgen.1004580-Sato1" target="_blank">[19]</a>”, 3 = “Berkel et al. (2010) <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004580#pgen.1004580-Berkel1" target="_blank">[14]</a>”, 4 = “Leblond et al. (2012) <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004580#pgen.1004580-Leblond1" target="_blank">[18]</a>”, 5 = “Boccuto et al. (2012) <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004580#pgen.1004580-Boccuto1" target="_blank">[17]</a>”, and 6 = “[This Study and Durand et al. 2007 <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004580#pgen.1004580-Durand1" target="_blank">[6]</a>]”, 7 = “[Gauthier et al. (2009–2010) <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004580#pgen.1004580-Gauthier1" target="_blank">[16]</a>, <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004580#pgen.1004580-Gauthier2" target="_blank">[47]</a>]”, 8 = “Moessner et al. (2007) <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004580#pgen.1004580-Moessner1" target="_blank">[13]</a>”, 9 = “Schaff et al. (2011) <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004580#pgen.1004580-Schaaf1" target="_blank">[35]</a>”. IV, Inverse Variance; FEM, Fixed Effect Method; REM, Random Effect Method; OR, Odds Ratio; CI, Confidence Interval; IQ, Intellectual Quotient; CNV, Copy Number Variant.</p

Summary of the SHANK protein functions and of the main findings obtained for patients with ASD.

<p>The frequency of mutation in patients and control individuals was calculated from the total cohort (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004580#pgen-1004580-t001" target="_blank">Table 1</a>). The frequency of mutation in patients with normal IQ (IQ>70) and low IQ (IQ<70) were calculated for the patients with available IQ scores (copy-number variants for all SHANK: nASD with IQ>70 = 1 638 & nASD with IQ<70 = 917; SHANK1 coding-sequence variants: nASD with IQ>70 = 354 and nASD with IQ<70 = 278; SHANK2 coding-sequence variants: nASD with IQ>70 = 335 & nASD with IQ<70 = 344; SHANK3 coding-sequence variants: nASD with IQ>70 = 667 & nASD with IQ<70 = 611). The mean IQ and standard deviation was given only for patients carrying truncating or <i>de novo</i> mutations. The black star indicates that Schmeisser et al. (2012) <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004580#pgen.1004580-Bonaglia1" target="_blank">[21]</a> found an increase in NMDA currents, while Won et al. (2012) <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004580#pgen.1004580-Betancur2" target="_blank">[22]</a> found a decrease in NMDA currents in two independent SHANK2 knock-out mice.</p><p>Summary of the SHANK protein functions and of the main findings obtained for patients with ASD.</p