<i>Fmr1/</i>Fmrp expression in WT and <i>Fmr1</i> KO retinas.

<p>(<b>A</b>) <i>Fmr1</i> mRNA expression was quantified by qPCR (n = 8 per group). Data are expressed as 2^−ΔCt values and normalized to 18S RNA internal control. Significant expression of <i>Fmr1</i> was only found in WT retina. (<b>B</b>) Fmrp expression was assessed by Western-blot analysis (n = 8 per group). All protein variations were normalized to β-actin. Fmrp was present in WT retinas whereas no detectable protein was observed in <i>Fmr1</i> KO. Data are presented as Mean ± SEM. Three independent experiments were performed with similar results. Student’s t test, ****p</p

Scotopic a-wave characteristic in WT and <i>Fmr1</i> KO mice.

<p>Retinal function was evaluated using to ElectroRetinoGram (ERG) (n = 20 for WT and n = 17 for <i>Fmr1</i> KO). For each (<b>A</b>) typical ERG obtained at light intensity −2.88 log(cd.s.m⁻²), the decreasing part of the a-wave is fitted to calculate the extrapolated (<b>B</b>) maximal a-wave amplitude (<i>A</i>_max) and (<b>C</b>) S</p><p>_A</p> parameter reflecting the photoreceptor sensitivity. In <i>Fmr1</i> KO mice compared to WT mice, <i>A</i>_max is significantly decreased by 26% (p<0.05), and the S<p>_A</p> parameter is not significantly different (p = 0.08). (<b>D</b>) a-wave latency was not different between groups. Data are presented as Mean ± SEM. Student’s t test, *p<p></p

Detection of Chromosomal Breakpoints in Patients with Developmental Delay and Speech Disorders

<div><p>Delineating candidate genes at the chromosomal breakpoint regions in the apparently balanced chromosome rearrangements (ABCR) has been shown to be more effective with the emergence of next-generation sequencing (NGS) technologies. We employed a large-insert (7–11 kb) paired-end tag sequencing technology (DNA-PET) to systematically analyze genome of four patients harbouring cytogenetically defined ABCR with neurodevelopmental symptoms, including developmental delay (DD) and speech disorders. We characterized structural variants (SVs) specific to each individual, including those matching the chromosomal breakpoints. Refinement of these regions by Sanger sequencing resulted in the identification of five disrupted genes in three individuals: guanine nucleotide binding protein, q polypeptide <i>(GNAQ),</i> RNA-binding protein, fox-1 homolog <i>(RBFOX3),</i> unc-5 homolog D (<i>C.elegans) (UNC5D</i>), transmembrane protein 47 (<i>TMEM47</i>), and X-linked inhibitor of apoptosis (<i>XIAP</i>). Among them, <i>XIAP</i> is the causative gene for the immunodeficiency phenotype seen in the patient. The remaining genes displayed specific expression in the fetal brain and have known biologically relevant functions in brain development, suggesting putative candidate genes for neurodevelopmental phenotypes. This study demonstrates the application of NGS technologies in mapping individual gene disruptions in ABCR as a resource for deciphering candidate genes in human neurodevelopmental disorders (NDDs).</p></div

Patient CD10 with translocation t(6;8).

<p>A) The pedigree of patient CD10 is indicated. The familial translocation is inherited from asymptomatic carrier mother and shared with his affected sister (CD11). B) Sanger sequencing analysis refined the chromosomal breakpoint regions and revealed a loss of 11 bp on chromosome 6 and 8 bp on chromosome 8, with a microhomology of 3 bp between the paired breakpoints. C) <i>UNC5D</i> mRNA expression in human tissue panel showed high expression in the fetal brain, adult brain and cerebellum compared to other tissues. D) The translocation breakpoint is located at intron 1 of <i>UNC5D</i> indicated by the black arrow, encompasses 15 CNVs cases described in the DECIPHER.</p

List of SVs obtained after filtration of four patients.

a<p>Discordantly mapped PETs (dPETs) which connect the same two genomic regions are clustered together.</p>b<p>dPET clusters which has passed quality filters to remove sequencing artefacts provided the list of predicted SVs.</p>c<p>Approximately 95% of SVs overlapped with SVs found in normal population (see Methods).</p

Patient CD5 with translocation t(9;17).

<p>A) The pedigree of patient CD5 is indicated. The translocation is transmitted to his two sons (CD21 and CD22). B) Translocation between chromosome 9 and 17 were validated by Sanger sequencing in three translocation carriers. The reference sequence is indicated, showing the fusion of two genes at the genomic level: the first five exons of <i>GNAQ</i> fused to exon 3–14 of <i>RBFOX3</i> and the first two exons of <i>RBFOX3</i> fused to exon 6–7 of <i>GNAQ.</i> C) mRNA expression of <i>GNAQ</i> and <i>RBFOX3</i> showed high expression in fetal brain, adult brain and cerebellum in human tissue panel.</p

Screening of CNVs in cases/controls from published and public datasets.

<p>The total number of cases in Cooper et al <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0090852#pone.0090852-Cooper1" target="_blank">[28]</a> is 15,767 cases, and for DECIPHER is ∼17,000 cases <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0090852#pone.0090852-Swaminathan1" target="_blank">[29]</a>. The total number of controls in Cooper et al. is 8329 controls and for 1000 Genome SV Release set is 185 controls <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0090852#pone.0090852-Mills1" target="_blank">[22]</a>.</p

Patient CD8 with a complex chromosomal inversion.

<p>A) Karyogram of normal chromosome X compared to der(X) in patient CD8. B) FISH validation of 10 SVs shown in Table S5 (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0090852#pone.0090852.s001" target="_blank">Supporting Information S1</a>) with the respective FISH probes: Hybridization of RP1-296G17-Biot (SV15) and RP1-315-Dig (SV16) were localized on the centromere of the patient’s metaphase. Probes for SV17 and SV21 on Xp21 (RP11-330K13-Biot) and Xq25 (W12-499N23-Dig), respectively resulted in a split signal between Xp21 and the centromeric region in the patient’s chromosome. Further FISH analysis was performed by using probe RP11-762M23-Biot on Xq11.1 (SV22) that was found to localize on the upper chromosomal arm. Probe RP11-655E22 on Xp11.2 was localized on the lower arm of derivative chromosome X. C) Reconstructed derivative chromosome X for patient CD8. Normal human chromosome X according to ISCN 2009 with the arrow orientation from <i>a</i> to <i>d</i> and the proposed mechanism of sequential double inversion in patient CD8. Based on our FISH analysis, an inversion occurred first between Xp21 and Xq25, changing the orientation of p and q arm with a shift of the centromere position towards the lower q-arm shown by inverted red arrow <i>b</i> and <i>c</i>. This was followed by the second inversion that occurred between Xq11.1 and Xq25, altering the orientation of the q-arm (inverted green arrow <i>c</i>). D) Expression of <i>TMEM47</i> in human tissue panel assessed by qRT-PCR. E) Expression analysis of four disrupted genes in patient CD8 assessed by qRT-PCR.</p

Validated breakpoints and disrupted candidate genes from DNA-PET sequencing in four patients.

<p>Validated breakpoints and disrupted candidate genes from DNA-PET sequencing in four patients.</p