Summary of cytogenetic and clinical findings in cases with overlapping events at 19p13.12.

<p>Summary of cytogenetic and clinical findings in cases with overlapping events at 19p13.12.</p

Chr19p13.12 duplication breakpoints.

<p>Results from an Affy 6.0 SNP array of Tg64.001 reveal a 760 kb duplication at 19p13.12. The top panel displays the log2 ratio of normalized intensity. The middle panel shows the difference of A allele signal and B allele signal. The bottom panel displays a Gaussian smoothed copy number estimate. Intensity values for probes included on the 44k Agilent array used in initial clinical evaluation are overlaid in green ovals.</p

Characterization the of Tg64 family.

<p>(a) 3dMD photos of all Tg64 duplication carriers: proband (left), carrier sister (middle), carrier mother (right). (b) Carrier status of Tg64 family members for the gain observed at 19p13.12 (chr19:15051982–15809751; hg19) and sequence variant at <i>TPTE / PTEN2</i> (NM_199261.3:c.1357-3_1357-2delTA). (c) Summary of head circumference measurements and developmental concerns for Tg64 family members.</p

Summary of cases with copy number variants at 19p13.12 in relation to RefSeq genes.

<p>Black bars represent the critical interval breakpoints. Deletions and duplications are presented in red and blue, respectively. Cases are encoded as follows: 1- DECIPHER 257523; 2- Gallant et al., 2011; 3- DECIPHER 285763; 4- DECIPHER 283124; 5- DECIPHER 284366; 6- Engels et al., 2007/Bonaglia et al., 2010; 7- Bonaglia et al., 2010; 8- Van der Aa et al., 2010/DECIPHER 255743; 9- DECIPHER 249355; 10- DECIPHER 249428; 11- Sanders et al., 2011; 12- Tg64.100; 13- Tg64.002; 14- Tg64.001; 15- DECIPHER 265764; 16- Jelsig et al., 2012; 17- DECIPHER 250827; 18- DECIPHER 255839; 19- Kosaki et al., 2011.</p

A subset of genes dysregulated as a result of <i>CYFIP1</i> knockdown show striking similarity to known epilepsy genes, and as such are strong candidate disease genes.

<p>A subset of genes dysregulated as a result of <i>CYFIP1</i> knockdown show striking similarity to known epilepsy genes, and as such are strong candidate disease genes.</p

Epilepsy genes dysregulated in response to <i>CYFIP1</i> knockdown are involved in synaptic transmission.

<p>Epilepsy genes dysregulated in response to <i>CYFIP1</i> knockdown are involved in synaptic transmission.</p

<i>CYFIP1</i> knockdown results in dysregulation of hundreds of genes in human neural progenitor cells (NPCs).

<p> (<b>A</b>) RNA-seq and qPCR show that compared to non-silencing control transduced NPC lines (NS, black), cells transduced with an shRNA targeting <i>CYFIP1</i> (<i>CYFIP1</i>^KD, grey and hatched) show a significant reduction in <i>CYFIP1</i> mRNA in lines from three different BP1-2 copy number neutral individuals (C2, red; C4, green; C5, blue, n = 3 per group throughout). P-values for RNA-seq analysis- DESeq2; qPCR- one-tailed Student’s t-test; *p≤ 0.05; ****p≤0.0001. (<b>B</b>) RNA-seq shows that mRNAs for NPC markers are expressed 10 to 100-fold higher than mRNAs for neuronal and glial markers. TPM values are averaged across NS and <i>CYFIP1</i>^KD samples. (<b>C</b>, <b>D</b>) Only a handful of genes are similarly dysregulated in response to <i>CYFIP1</i> knockdown across all three NPC lines evaluated. (<b>E</b>) Unsupervised hierarchical clustering of genes showing nominal differential expression (p<0.05) points to marked between-line differences. (<b>F</b>) RNA-seq and qPCR on mRNA from control and <i>CYFIP1</i>^KD NPCs (<i>CYFIP1</i>^KD RNA-seq, black; <i>CYFIP1</i>^KD qPCR, grey) show reduced levels of genes downregulated in all three cell lines. Abbreviations: <i>CCND1</i>- cyclin D1, <i>GFRA1</i>- GDNF family receptor alpha-1, <i>GPR133</i>- G-protein coupled receptor 133, <i>L1CAM</i>- L1 cell adhesion molecule, <i>PGPEP1</i>- pyroglutamyl-peptidase I, <i>TIPARP</i>- TCDD-inducible poly(ADP-ribose) polymerase.</p

Genes implicated in schizophrenia and epilepsy, but not conditions unrelated to BP1-2 deletions, are overrepresented among <i>CYFIP1</i> knockdown dysregulated mRNAs.

<p>Genes implicated in schizophrenia and epilepsy, but not conditions unrelated to BP1-2 deletions, are overrepresented among <i>CYFIP1</i> knockdown dysregulated mRNAs.</p

Dysregulation of FMRP targets and post-synaptic density genes in response to <i>CYFIP1</i> knockdown in human neural progenitor cells.

<p>Dysregulation of FMRP targets and post-synaptic density genes in response to <i>CYFIP1</i> knockdown in human neural progenitor cells.</p

Dysregulation of cytoskeletal remodeling in response to <i>CYFIP1</i> knockdown in human neural progenitor cells (NPCs).

<p>(<b>A</b>) Genes involved in cytoskeletal remodeling are overrepresented among transcripts downregulated in response to <i>CYFIP1</i> knockdown. (<b>B, C</b>) Western blot shows that relative to non-silencing shRNA transduced NPCs (NS, black), transduction with an shRNA targeted against <i>CYFIP1</i> (<i>CYFIP1</i>^KD, grey) results in a significant 54% reduction of WAVE1/2 protein levels. n = 10 per group. (<b>D, E, F</b>) Phalloidin staining shows that F-actin levels are significantly reduced (11% reduction) in <i>CYFIP1</i>^KD NPCs relative to control cells. n = 12 wells per group. (<b>G, H, I</b>) Nuclear area, quantified by DAPI, is significantly increased by 9% in <i>CYFIP1</i>^KD NPCs relative to control cells. n = 12 wells per group. (<b>J, K, L</b>) Cell area, captured by phalloidin staining, is nominally greater in <i>CYFIP1</i>^KD NPCs relative to control cells (34% increase). n = 12 wells per group. Two-tailed Student’s t-test, *p<0.05 **p≤0.01 ***p≤0.001.</p