Reprogramming-based models of neurodevelopmental disorders and uses thereof
11369577 · 2022-06-28
Assignee
- IEO—Istituto Europeo di Oncologia S.r.l. (Milan, IT)
- I.R.C.C.S. “Casa Sollievo della Sofferenza” (San Giovanni Rotundo, IT)
- Università degli Studi di Milano (Milan, IT)
Inventors
- Giuseppe Testa (Milan, IT)
- Sina Atashpazgargari (Milan, IT)
- Antonio Adamo (Milan, IT)
- Pierre-Luc Germain (Milan, IT)
- Giuseppe Merla (Milan, IT)
- Giuseppe D'Agostino (Milan, IT)
- Matteo Zanella (Milan, IT)
Cpc classification
C12N2740/15041
CHEMISTRY; METALLURGY
C12N2501/06
CHEMISTRY; METALLURGY
C12Q1/6881
CHEMISTRY; METALLURGY
C12N2501/13
CHEMISTRY; METALLURGY
C12Q1/6897
CHEMISTRY; METALLURGY
C12N5/0696
CHEMISTRY; METALLURGY
C12N2501/115
CHEMISTRY; METALLURGY
A61K31/135
HUMAN NECESSITIES
A61K31/00
HUMAN NECESSITIES
C12Q1/6883
CHEMISTRY; METALLURGY
International classification
A61K31/00
HUMAN NECESSITIES
G01N33/50
PHYSICS
C12Q1/6881
CHEMISTRY; METALLURGY
C12Q1/6897
CHEMISTRY; METALLURGY
C12Q1/6883
CHEMISTRY; METALLURGY
Abstract
The present invention relates to iPSC produced from fibroblast obtained from a subject affected by a neurodevelopmental disorder entailing intellectual disability (ID) and/or a disorder belonging to the Autism Spectrum Disorder (ASD) and/or Schizophrenia (SZ) and uses thereof. The present invention also relates to a cortical neural progenitor cell or a terminally differentiated cortical glutamatergic or gabaergic neuronal cell or a neural crest stem cell line, a mesenchymal stem cell line produced from the iPSC or iPSC line. The invention also relates to method for identifying a compound for the treatment and/or prevention of a neurodevelopmental disorder entailing intellectual disability (ID) and/or a disorder belonging to the Autism Spectrum Disorder (ASD) and/or Schizophrenia (SZ) and to a LSD1 inhibitor or a HDAC2 inhibitor for use in the treatment of such disorders.
Claims
1. A method of preventing and/or treating an at least one behavior-cognitive defect in an individual with a copy number variation (CNV) at 7q11.23 , comprising: administering an LSD1 inhibitor to said individual; wherein the said administration disrupts the transcriptional repressive activity of LSD1 on lysine 4 of histone H3 tails; wherein the degree of LSD1 inhibition sufficiently reduces the function of the GTF2I transcriptional co-repressor complex; wherein the disruption of GTF2I function increases BEND4 upregulation; wherein genes which are imputable to GTF2I also change expression significantly upon LSD1 inhibition, such as but not limited to expression of one or more of IGF1R, SDH2, ARRB1, NRK, EPHA4, IGFBP3, SERINC5, PRICKLE1, NLGN2, PTPRD, COX7B, ADCY1, MAP2, EDHA4, and COL9A1, or any combination therein.
2. The method of claim 1, wherein the subject has an intellectual disability and/or Autism Spectrum Disorder.
3. The method of claim 1, wherein the copy number variations (CNV) at 7q11.23 causes 7dupASD.
4. The method of claim 1, wherein the copy number variations (CNV) at 7q11.23 causes a 7q11.23 microduplication-dependent Schizophrenia.
5. The method of claim 1, wherein the LSD1 inhibitor is N-[4-[trans-2-aminocyclopropyl]phenyl]-3-(2-oxooxazolidin-3-yl)benzamide hydrochloride.
6. The method of claim 1, wherein the LSD1 inhibitor is (trans)-N1-((1R, 2S)-2-phenylcyclopropyl)cyclohexane-1,4-diamine hydrochloride.
7. The method of claim 1, wherein treatment with the LSD1 inhibitor significantly increases expression of one or more of IGFBP3, ANXA2, MAP2, ELF3, EPHA4, ATP2A3, PRICKLE1, NRK, CDH2, IGFBP5, GLDC, DPPA2, ARRB1, SERINC5, COL9A1, or COX7B.
8. The method of claim 1, wherein treatment with the LSD1 inhibitor significantly decreases expression of one or more of NLGN2, MYSM1, PTPRD, ATAD5, IGF1R, or RHBDF2.
9. The method of claim 1, wherein treatment with the LSD1 inhibitor significantly alters expression of one or more of NACAD, JAZF1, ADCY1, ZNF578, NID1, ITPR2, FOXN3, or FAM189A2.
10. The method of claim 1, wherein the at least one behavioral-cognitive defect is selected from the group consisting of autistic-like behaviors, attention deficit hyperactivity disorder (ADHD), language impairment, anxiety, intellectual disability, global developmental delay, learning difficulties, impairment in adaptive behavior, language delay, oral motor and speech sound delay, oral motor and speech sound disorder, social anxiety, separation anxiety, social phobia, autism spectrum disorder (ASD), selective mutism, oppositional defiant disorder (ODD), repetitive behaviors/movements, stereotypies, compromised visuo-motor integration, social communication impairment/deficits or any combination thereof.
Description
(1) The invention will be illustrated by means of non limiting examples in reference to the following figures.
(2)
(3)
(4)
(5)
(6)
(7)
(8)
(9)
(10)
(11)
(12)
(13)
(14)
(15)
(16)
(17)
(18)
(19)
(20)
DETAILED DESCRIPTION OF THE INVENTION
(21) Methods
(22) A summary of which experiments were performed on which cell line is given in Table 1.
(23) TABLE-US-00001 TABLE 1 list of performed experiments GTF2I scramble GTF2I sh2 Single Neural interfered interfered cell RNA-seq RNAseq CGH Nano- Neural crest Mesenchymal iPSC & iPSC & Lines adaptation (Ribozero) (PolyA) array string ChiP-seq progenitors stem cell stem cell RNAseq RNAseq WBS1 X WBS1-C2 X X X X X WBS1-C3 X X X X X X X WBS1-C1 X X X X X X X X X WBS2 X WBS2-C3 X WBS2-C1 X X X X WBS2-C2 X X X X X X X X X X X WBS3 X WBS3-C3 X X X X X WBS3-C1 X X X X WBS3-C2 X X X X X X WBS4 X WBS4-C3 X X X X WBS4-C2 X X X X WBS4-C1 X X X X X X X X X X AtWBS1 X AtWBS1-C2 X X X X X X X X X X X AtWBS1-C3 X X X X X X X AtWBS1-C1 X X X X CTL2-C1 X X X X CTL2-C2 X X X X X X X X CTL3-C1 X X X X X X CTL3-C2 X X CTL1R CTL1R-C1 X X X X X X CTL1R-C2 X X X X X X CTL1R-C3 X X X X X X X 7dupASD1 X 7dupASD1-C1 X X X X X 7dupASD1-C3 X X 7dupASD1-C2 X X X X X X X X X X X 7dupASD2 X X 7dupASD2-C4 X 7dupASD2-C2 X X X X X X 7dupASD2-C3 X 7dupASD2-C5 X X X X X X X X X X 7dupASD2-C1
Human Samples
(24) Participation in this study by patients and relatives along with skin biopsy donations and informed consent procedures were approved by the Ethics Committee of the Genomic and Genetic Disorder Biobank (Casa Sollievo della Sofferenza, San Giovanni Rotondo, Italy) and the University of Perugia (Azienda Ospedaliera-Universitaria “S. Maria della Misericordia”, Perugia, Italy).
(25) Fibroblast Culture and Reprogramming
(26) Primary fibroblast cell lines WBS1-2-3-4, 7Dup-ASD2, AtWBS1, CTLR were obtained from Genetic Disease Biobank. 7Dup-ASD1 primary fibroblast was obtained from Azienda Ospedaliera-Universitaria “S. Maria della Misericordia”, Perugia, Italy. Fibroblasts were cultured in HF medium composed as follows: RPMI 1640, 1% L-Glutamine, 1% Pen-Strep, 15% FBS for few passages before reprogramming.
(27) WBS1-2-3-4, 7Dup-ASD1-2, AtWBS1, CTL1R, and CTL2 fibroblast lines were reprogrammed using mRNA Reprogramming Kit (Stemgent). For reprogramming 7Dup-ASD2 and CTL1R lines microRNA Booster Kit (Stemgent) were used to enhance the reprogramming. CTL3 line was reprogrammed using STEMCCA polycistronic lentiviral vector followed by successful cre-mediated excision of the integrated polycistron. For mRNA-mediated reprogramming epithelial-to-mesenchymal transition was monitored from day 5 by tracing GFP-positive cells. Successfully reprogrammed colonies were assayed for pluripotency at day 20 using a live TRA-1-60 antibody (Stemgent) and selected for further expansion as detailed below.
(28) iPSC Culture
(29) iPSC lines were cultured on mitomycin C-inactivated mouse embrionic fibroblasts (MEFs) as previously described.sup.23 in a medium composed as follows: DMEM-F12 (Gibco) in a 1:1 ratio supplemented with 20% KSR, 1% Non Essential Amino Acids, 1% Pen-Strep, 1% Glutamine, 0.1% beta-mercaptoethanol, 10 ng/ml basic fibroblast growth factor (bFGF, Gibco). Colonies were passed and expanded twice by physical fractionation with a sterile needle and replated onto newly seeded MEFs for line establishment. After few passages iPSCs were adapted to grow in feeder-free condition on plates coated with human-qualified Matrigel (BD Biosciences) diluted 1:40 in DMEM-F12 and in mTeSR-1 (StemCell Technologies) medium and were passed by physical fractionation upon a 2 minutes treatment with Dispase (Sigma) at 37° C. Feeder-free iPSCs were also adapted to grow in single cell culture by dissociating them by a 3 minutes treatment with Accutase (Sigma) at 37° C. and finally resuspended in a suitable volume of mTeSR-1 supplemented with 5 μM Y-27632 (Sigma).
(30) Teratoma Assay and Immunohistochemistry
(31) Teratoma assay was performed by subcutaneously injecting 1-3×10.sup.6 iPSCs in human-qualified Matrigel (BD Biosciences) into the dorsal flanks of NOD-SCID IL2RG male mice. Teratomas were isolated when the diameter reached >1.5 cm and fixed in 4% buffered formalin. Samples were then OCT embedded, sectioned and stained for H&E and germ layer specific antibodies: desmin (Dako), S-100 (Dako) and cytokeratin (Dako).
(32) Immunocytochemistry
(33) Cells were fixed in 4% PFA for 20′ and subsequently blocked in 10% FBS+0.1% Triton for 30′ at room temperature. Cells were incubated with primary antibodies overnight at 4° C. and then with secondary antibodies for one hour at room temperature. Primary and secondary antibodies were resuspended in 10% FBS. Primary antibodies used were OCT3/4 (SantaCruz), NANOG (Everest Biotech), SSEA3 (Invitrogen), Tra1-60 (Stemgent), TBR2 (Abcam), SOX2 (R&D), PAX6 (HBDS), NESTIN (Abcam), FOXG1 (StemCulture), ZO1 (Invitrogen), OTX2 (Millipore), Ki67 (Abcam), PHH3 (Millipore) HNK1 (SIGMA), NGFr (p75, Advanced Targeting Systems). Alkaline phosphatase staining was performed using Alkaline Phosphatase Detection Kit (Sigma).
(34) Images were acquired at an Olympus AX70 microscope.
(35) DNA, RNA and Protein Extraction
(36) Genomic DNA was extracted from fibroblasts and feeder-free iPSC lines using the DNeasy Blood and Tissue Kit (Qiagen) according to manufacturer specifications. RNA was extracted from iPSC lines using the RNeasy Micro Plus Kit (Qiagen) according to manufacturer specifications, substituting the genomic DNA elimination column by needle and Dnase treatment (Qiagen). Quality and concentration of DNA and RNA was assessed using a NanoDropSpectrophotometer (NanoDrop Technologies).
(37) Proteins were extracted as follows: cells were scraped from the plate and centrifuged at 1100 g at 4° C. for 3 minutes, then washed in PBS and lysed in RIPA buffer plus protease inhibitors cocktail (Sigma) on a spinning wheel at 4° C. for 30 minutes. Lysates were sonicated using the Bioruptor Sonication System (UCD200) for 3 cycles of 30 seconds with 60 seconds breaks at high power. Lysates were centrifuged at 13000 g for 15 minutes and supernatants were transferred to a new tube. Protein quantification was performed using the Bradford protein assay (BioRad) and following manufacturer instructions.
(38) Immunoblotting
(39) For immunoblotting 20 to 40 μg of protein extract per sample were run on a precast Nupage 4-12% Bis-tris Gel (Life Technologies), transferred on a nitrocellulose membrane and blocked in TBS-T and 5% milk. Antibodies used for detection were GTF2I (Cell signalling), BAZ1B (Abcam) and GAPDH (Abcam). Blots were scanned using a LI-COR Odyssey Infrared Imaging System and bands were quantified using ImageJ software.
(40) Nanostring
(41) Nanostring quantification was performed according to manufacturer instructions and data normalization was performed with the nSolver Analysis Software 1.1, using GAPDH, TUBB, and POLR1B as normalizers.
(42) RNAseq
(43) Library preparation for RNA sequencing was performed using Poly-A, RiboZero and Single Stranded kits (Illumina) according to manufacturer instructions.
(44) For the purpose of differential expression analysis, the inventors complemented their polyA dataset with the transcriptomic profiles of three control iPSC lines available from the literature (GEO samples GSM1153507, GSM1153512, and GSM1153513, from cell lines ADRC40, WT-9 and WT-126 respectively), which were selected on the basis of the similarity of their culture conditions and sequencing protocols to ours. The inventors excluded from the analysis genes (292, of which 43 would otherwise be DEGs between genotypes) that were found differentially-expressed between controls from theircohort and external controls.
(45) Reads were aligned to the hg19 transcriptome using TopHat 2.0.10. The alignment was first performed on the RefSeq transcriptome and all the reads that had an edit distance≥1 were realigned on the genome, allowing a maximum read edit distance of 3 and 3 (100 bp reads) and 2 (50 bp reads) maximum mismatches. 50 bp stranded reads (MSC transcriptomes, as well as transcriptomes of the knockdown and inhibited lines) were analyzed using the “fr-firststrand” option. Quantification of reads over the RefSeq transcriptome was performed with Cufflinks 2.2.1 using sequence-bias and multi-read corrections. Differential gene expression was estimated using Cufflinks 2.2.1, using per-condition dispersion models (except for the knockdown experiment in which a global model was used due to the low number of replicates). For the iPSC stage, given the presence of both polyA and Ribo-zero samples, the inventors considered the union of DEGs identified through a global analysis of all samples (FDR<0.05) with those identified through independent analysis of the polyA and Ribo-zero samples. In the latter case, the inventors considered as differentially expressed genes that had a FDR<0.2 in both datasets (comparing the same genotypes), and for which the change was in the same direction.
(46) Downsampling Test
(47) The vast majority of differentially-expressed genes identified in this study were still found when removing the external controls, and most of the remaining DEGs were close to significance, arguing against the introduction of a major bias through the use of external controls.
(48) In order to assess the effect, on the transcriptional analysis, of having fewer samples, the inventors repeated the analysis of their polyA dataset (focusing on the comparison for which the inventors had the most samples, i.e. the global analysis of WBS vs CTL iPSC), using only subsets of the samples. Random removal of 1 clone per patient lead to a dramatic reduction in the number of DEGs (48 to 76% lost), and to the identification of DEGs that are falsified by the discarded data. The impact of removing all clones from one patient per condition (amounting to fewer samples than removing one clone per patient) was even greater. In contrast, depth of sequencing appeared to make little difference: reducing coverage by half led to the loss of 11% of DEGs and to very few false positives.
(49) Shuffling Tests
(50) To assess the possibility that the observed differential expression might arise due to random variations, the inventors performed a series of differential expression analysis between randomly-selected samples, discarding comparisons in which the two groups were not balanced for sex and/or genotype. A minimum of 3 such combinations were tested per tissue, and the resulting genes were pooled for the purpose of enrichment analysis.
(51) At the iPSC stage (using the polyA dataset), the inventors first randomly assigned all patients to two groups, but could the inventors obtain statistically significant genes in none of the combinations. The inventors therefore gradually removed patients until significant genes were obtained, which did not happen until a comparison involving 6 vs 6 samples (in each group, 3 samples from 2 patients). In contrast, when clones were selected and assigned to groups in a way that maximized the number of patients represented in each group, the inventors had to go down to 3 vs 3 (3 samples per group, coming from 3 different patients) to get statistically significant genes (18 DEGs, showing no significant GO enrichment). Similarly, random allocation of the control samples (including external controls, balanced across groups) yielded very few DEGs (maximum 17) and no significant GO enrichment.
(52) These results suggest that the primary source of “spurious” differential expression is genetic variation between individuals, which only gets mitigated using lines derived from several patients.
(53) Finally, it is interesting to note that despite yielding very few DEGs (maximum 78), some of the 6 vs 6 comparisons showed statistically significant enrichment for the GO categories of extracellular matrix organization and extracellular structure organization (FDR 2.5E-7 in the shuffling test, versus 2.3E-10 in the comparison between genotypes), pointing to these genes as particularly varying in expression between lines and/or individuals.
(54) In differentiated cell types, shuffling tests using (3 combinations of) 3vs3 samples yielded either no significant differential expression (NCSC), or very few genes (NPC) that displayed no significant enrichments, with the exception of the MSC dataset. The union of genes found significant from random combinations of the MSC samples showed several GO enrichments, including (albeit at a lower level) some categories that were found significant between the genetic conditions. However, removal of these genes did not significantly alter the main categories enriched among the DEGs between genetic conditions (Table 2).
(55) TABLE-US-00002 TABLE 2 Gene Ontology category enrichments for differentially expressed genes Enrichment Enrichment Log10(Fdr) Enrichment Log10(Fdr) Enrichment over without category term DEGs DEGs random random random random GO:0001974 blood vessel remodeling 4.90022987 9.57 1.59670852 6.23 1.59 5.88 GO:0014706 striated muscle tissue development 8.74816715 4.22 2.74824703 3.21 1.56 3.00 GO:0050877 neurological system process 5.02609321 2.27 2.80372131 2.59 1.56 1.81 GO:0070371 ERK1 and ERK2 cascade 5.13988542 5.04 1.39744502 3.19 1.55 4.02 GO:0001655 urogenital system development 9.4144022 4.70 2.43853353 3.13 1.54 3.58 GO:0072001 renal system development 9.41840026 5.03 2.5979742 3.35 1.52 3.93 GO:0030510 regulation of BMP signaling pathway 4.4263336 6.70 1.54487389 4.71 1.42 3.94 GO:0048514 blood vessel morphogenesis 7.50827453 3.46 3.37609005 3.09 1.34 2.32 GO:0051216 cartilage development 5.47172473 4.74 1.94938905 3.44 1.30 3.18
cDNA Preparation and qPCR
(56) Retrotranscribed cDNAs have been obtained from 1 μg of total DNA-depleted RNA using the superscript VILO retrotranscription kit from Life Technologies according to manufacturer instructions.
(57) For real time q-PCR analysis a total amount of cDNA corresponding to 10-50 ng of starting RNA has been used for each reaction. FAST SYBR green master mix from Life Technologies and 10 μM primers pair have been used. The qPCR reactions have been performed on an Applied Biosystems® 7500 Real-Time PCR machine following the standard amplification protocol.
(58) The pair oligos used for qPCR were: GAPDH (F: GCACCGTCAAGGCTGAGAAC (SEQ ID No. 1), R: AGGGATCTCGCTCCTGGAA (SEQ ID No. 2)), BEND4 (F: GGAAAAGGAAAAGGTCAGTGC (SEQ ID No. 3), R: GTITATCTGCTCTTCCGAGGG (SEQ ID No. 4)) and GTF2I (F: GATCTTGCAACCCTGAAATGG (SEQ ID No. 5), R: CACCTGGAGATAGTATITGACCTG (SEQ ID No. 6)).
(59) Chromatin Immunoprecipitation Coupled with Sequencing (ChIPseq)
(60) 10.sup.8 iPSCs were used for each immunoprecipitation. Cells were detached using accutase, resuspended in PBS containing 1% formaldehyde for fixation and quenched with 125 mM glycine. Cells were lysed using ChIP lysis buffer (1% SDS, 10 mM EDTA, 50 mM Tris-HCl, pH 8.1) and sonicated using the Covaris Sonicator to generate 250 bp DNA fragments. Soluble chromatin was diluted 10 times in ChIP dilution buffer (0.01% SDS, 1.1% Triton-X100, 1.2 mM EDTA, 16.7 mM Tris-HCl pH 8.1, 167 mM NaCl). Chromatin was incubated overnight at 4° C. with the antibody and recovered the day after using Dynabeads Protein G (Life Technologies). Beads were washed sequentially with Low Salt Wash Buffer (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris-HCl, pH 8.1, 150 mM NaCl), High Salt Wash Buffer (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris-HCl, pH 8.1, 500 mM NaCl), LiC Wash Buffer (0.25M LiC, 1% NP40, 1% deoxycholate, 1 mM EDTA, 10 mM Tris-HCl, pH 8.1) and TE. Immunocomplexes were eluted in ChIP elution buffer (0.1% SDS, 1% Triton-X100, 2 mM EDTA, 20 mM Tris-HCl pH 8.1, 500 mM NaCl) and the decrosslinking was performed overnight at 65° C. DNA was purified using Qiaquick PCR Columns (Qiagen) according to manufacturer instructions.
(61) DNA libraries were prepared according to Blecher-Gonen et al..sup.24 and DNA was sequenced on an Illumina HiSeq 2000 platform.
(62) Validation of ChIPseq results has been performed through qPCR analysis using the following oligo pairs: EOMES (F: GCTGCCATCTTCCTCTGGTAA (SEQ ID No. 7), R: GCTGCCATCTCCTCTGGTAA (SEQ ID No. 8)), SNAP25 (F: CCTCCTGCATAGCTTCAACAAA (SEQ ID No. 9), R: GGTCAGAGGGCAACACAGA (SEQ ID No. 10), DTX3L (F: CACCAGCCCTTGACATCAG (SEQ ID No. 11), R: TCCCTCCAACTCTACCCTAG (SEQ ID No. 12)), HDAC1 (F: TGATGTGGAGAGTTGAGTGG (SEQ ID No. 13), R: GAAAAGATGAGGAAGGGACGG (SEQ ID No. 14)), SEMA4B (F: ATGITAAGGGAGCAGTGAGC (SEQ ID No. 15), R: ATGGTCAGCTCTCAAGAATGG (SEQ ID No. 16)), CRB2 (F: GAACCCAGATCTCTTACGCTG (SEQ ID No. 17), R: CATCTTTAATCCCCTGCCTCTC (SEQ ID No. 18)), MYO1B (F: GGAAACCAGATTAGAGACGGG (SEQ ID No. 19), R: GTTTGTAGTTACCTCTCCAGCG (SEQ ID No. 20)), MFAP2 (F: GAAATCAAGCCTCCCAAAGTG (SEQ ID No. 21), R: TGGAGAGGCAGAAGGAAAAC (SEQ ID No. 22)), ADAM19 (F: AAGCCTTCTCCGGTCATAATG (SEQ ID No. 23), R: TGATGTCCGTGTTCTCAGG (SEQ ID No. 24)), S1PR5 (F: CCTGTGAACTGAGGTTCCTG (SEQ ID No. 25), R: CCACTGAAGACTCCTGCTAAG (SEQ ID No. 26)), JKAMP (F: ACAAGCCCGAAGTCCAAAG (SEQ ID No. 27), R: TCCTGTTCCTGCACAACG (SEQ ID No. 28)), PTPN5 (F: CCTCAACCAGAAACAAGCAG (SEQ ID No. 29), R: CCACCGACCCTTTAGCTTTAG (SEQ ID No. 30)), TMEM20 (F: GCTCCTGTAATTAGTGTCGGG (SEQ ID No. 31), R: GGGATCACTTTCAGGGTCAG (SEQ ID No. 32)).
(63) ChIPseq Analysis
(64) Reads were trimmed for adapter contamination using Scythe 0.981 with a minimum match of 3 before being aligned to the HG19 genome using BowTie 1.0, allowing 2 mismatches and discarding multiply aligning reads. Peaks were called using Macs 2.0.9 with default settings and ignoring duplicated reads. For GTF2I ChIPseq, given the variability between samples, the inventors used two complementary approaches to identify GTF2I binding sites. The inventors considered peaks (independently of FDR) that overlapped between all control and 7dupASDsamples (2079 peaks; 436 target genes) as “conserved peaks”, from which the inventors inferred the core GTF2I targets genes. In addition, the inventors called peaks on the pooled reads from all samples (against the pooled inputs) to identify a broader set of putative binding sites, considering only regions with a fold enrichment over input of at least 5 and FDR <0.001 (10691 peaks; 1554 target genes). In all cases, target genes were identified as genes with a peak in a −2.5 /+1 kb around their TSS.
(65) Bivalent domains were defined as regions having H3K4me3 and H3K27me3 peaks within 1 kb of each other in ENCODE's data from the H9 human embryonic stem cell (hESC) line. To assess the quality of the LSD1 ChIPseq, the inventors compared it to ENCODE's ChIPseq for H3K4me2 in H1 hESC line. As expected, 95% of the LSD1 peaks defined by the inventors overlap with a H3K4me2 peak.
(66) Finally, associations between TSS and distal enhancers were inferred from strong correlation, across ENCODE datasets, between the enhancer chromatin signature and expression at the putative target TSS; more specifically, the inventors considered only associations with a FDR<0.001 and a minimum Pearson correlation of 0.5 with H3K27ac, H3K4me1, and −0.5 with DNA methylation, for a total of 5996 enhancer-TSS associations.
(67) Immunoprecipitation and MS Analysis of the GTF2I Complex
(68) About 25-35 millions of cells were harvested and lysed in low stringency lysis buffer (50 mMTris-HCl, pH 7.5, 120 mMNaCl, 0.5 mM EDTA, 0.5% Nonidet P-40) and sonicated using a Branson 250 digital sonifier.
(69) 3 mg of protein extract are used for the immunoprecipitation with 10 μg of GTF2I antibody (Bethyl) and with unspecific IgG immunoglobulin O.N rotating at 4° C. The day after, 50 μL of NovexDynabeads Protein G (Life Technologies) are added to each sample and incubated rotating for 2 hours at 4° C. After three washing steps using the washing buffer (50 mMTris-HCl, pH 8, 150 mMNaCl, 0,1% Triton, 5% glycerol) the beads are treated with one volume of 4× Novex LDS sample buffer (50 mM DT) (Life Technologies) and the same volume of sample is loaded on a precast 12%-4% Novex gel (Life Technologies) for SDS-PAGE. Gels were stained using Colloidal Blue Coomassie (Fermentas). 7 discrete bands were cut and analyzed via liquid chromatography-tandem MS.
(70) Liquid Chromatography-Tandem MS (LC-MS/MS) Analysis
(71) Bands of interest were cut from gels and trypsinized as previously described by Shevchencko et al.sup.25. Peptides were desalted and concentrated on a homemade stage Tip.sup.26 dried in a Speed-Vac and resuspended in 10 μL of 0.1% formic acid. LC-ESI-MS/MS of 5 μL of each sample was performed on a Fourier transformed-LTQ mass spectrometer (FT-LTQ, Thermo Electron, San Jose, Calif.). Peptides separation was achieved on a linear gradient from 100% solvent A (5% ACN, 0.1% formic acid) to 20% solvent B (acetonitrile, 0.1% formic acid) over 30 min and from 20% to 80% solvent B in 20 min at a constant flow rate of 0.3 μL/min on Agilent chromatographic separation system 1100 (Agilent Technologies, Waldbronn, Germany) where the LC system was connected to a 15 cm fused-silica emitter of 75 μm inner diameter (New Objective, Inc. Woburn, Mass. USA), packed in-house with ReproSil-Pur C18-AQ 3 μm beads (Dr. MaischGmbh, Ammerbuch, Germany) using a high-pressure bomb loader (Proxeon, Odense, Denmark).
(72) Survey MS scans were acquired in the FT from m/z 350-1650 with 100 000 resolution. The five most intense doubly and triply charged ions were automatically selected for fragmentation.
(73) Target ions already selected for the MS/MS were dynamically excluded for 60 s.
(74) Data Processing and Analysis
(75) DATABASE SEARCHING: Raw MS files were converted into peaklist (.msm files) via Raw2msm ver 1.10_2007.06.14. All MS/MS samples were analyzed using Mascot (Matrix Science, London, UK; version 2.3.02) set up with the following parameters: Database UniProt_CP_Human_20130724 database (unknown version, 88378 entries), Taxonomy Homo sapiens, enzyme Trypsin, Max missing cleavage 2, fixed modification carbamidomethyl (C), variable modification oxidation (M), acetyl (protein N-terminus) peptide tolerance 10 ppm, MS/MS tolerance 0.5 Da.
(76) CRITERIA FOR PROTEIN IDENTIFICATION: Scaffold (version Scaffold_4.3.4, Proteome Software Inc., Portland, Oreg.) was used to validate MS/MS based peptide and protein identifications. Peptide identifications were accepted if they could be established as greater than 95.0% probability by the Peptide Prophet algorithm.sup.27 with Scaffold delta-mass correction. Protein identifications were accepted if they could be established as greater than 99.0% probability and contained at least 2 identified peptides. Protein probabilities were assigned by the Protein Prophet algorithm.sup.28. Proteins that contained similar peptides and could not be differentiated based on MS/MS analysis alone were grouped to satisfy the principles of parsimony. Proteins sharing significant peptide evidence were grouped into clusters.
(77) Lentivirus Production
(78) GTF2I knock-down were performed using validated pLKO.1 TRC vector (TRCN0000019315 referred as sh2, TRCN0000364552 referred as sh52 and TRCN0000369208 referred as sh08). A previously described pLKO.1 TRC containing a scrambled short hairpin was used as a negative control.sup.29. Viral particles were produced using psPAX2 and pMD2.G packaging vectors in 293T cells. Viral particles were harvested at 24 and 36 hours post-transfection and concentrated 250× by ultracentrifugation at 24000 g for 2 hours at 16° C. iPSC lines were infected with different amounts of viral particles, and the amount that gave rise to 50% of survival upon 1 μg/mL puromycin treatment for 72 hours was selected for following experiments.
(79) Infected cells were kept in puromycin-containing mTeSR for the whole duration of the experiments.
(80) Differentiation
(81) Differentiation into the dorsal telencephalic lineage was accomplished by dual Smad inhibition in the presence of SB431542 (Tocris) and Noggin (R&D).sup.30,31. Differentiation of NCSC and MSC was performed as previously described.sup.32, through the activation of Wnt signalling and Smad pathway blockade by administering the small molecules GSK3i (Calbiochem) and SB431542 (Tocris).
(82) Embryoid Bodies (EBs) Production and LSD1 Inhibitors Treatments.
(83) In a differentiation experiment about 1.5×10.sup.4 cells/well (CTR1R-C3) have been plated on a 96 wells conic plate in mTeSR medium containing 10 mM of a LSD1 inhibitor: N-[4-[trans-2-aminocyclopropyl]phenyl]-3-(2-oxooxazolidin-3-yl)benzamide hydrochloride
(84) ##STR00001##
or Example 5 as disclosed in WO2013057322: (trans)-N1-((1R,2S)-2-phenylcyclopropyl)cyclohexane-1,4-diamine hydrochloride 96 well plates were centrifuged at 850 g for 10 minutes at room temperature and the forming EBs have been incubated at 37 degree for 24 hours. 10 uM RA has been added at day 0 or at day 4. At day 1 EBs have been plated on a low attachment dish and media was replaced with KO-DMEM (+P/S+Glut)(EB media) 20% FBS containing. Media, RA, inhibitors and vehicle have been changed every other day for the following 6 days. Next, RNA extraction and cDNA preparation has been performed as described above.
(85) LSD1 inhibitor has been also tested in non-differentiating condition according to the following experimental procedure: about 2.5×10.sup.5 cells were seeded at day 0 in feeder-free condition on a 6 cm (diameter) plate coated with diluted human-qualified Matrigel (BD Biosciences) and in mTeSR-1 (StemCell Technologies) medium supplemented with 5 μM Y-27632 (Sigma). mTeSR-1 was replaced at day 1 and day 2. At day 3 medium was replaced with mTeSR-1 containing 10 mM of a LSD1 inhibitor N-[4-[trans-2-aminocyclopropyl]phenyl]-3-(2-oxooxazolidin-3-yl)benzamide hydrochloride and incubated at 37 degree for 24 hours. Next, cells were first dissociated by a 3 minutes treatment with Accutase (Sigma) at 37° C. and then harvested for RNA extraction, cDNA and library preparation as described above.
(86) Flow Cytometry
(87) 1×10.sup.6 cells were fixed in 4% PFA and subsequently blocked in 10% BSA. Cells were incubated for one hour with primary conjugated antibody resuspended in 1-2% BSA. The primary conjugated antibody used were CD57-FITC (HNK1, BD), CD271-647 (NGFR, BD), CD44-APC (EBIOS) and CD73-PE (BD). Analysis were performed on FACSCalibur (BD Biosciences) and data were analyzed with FCS express software (Tree Star inc.).
(88) CGH Array
(89) DNA was isolated from parental fibroblast and iPSC using Qiagen kit as described above. DNA concentration and purity were determinate with a ND-1000 spectrophotometer (NanoDrop Technologies, Berlin, Germany) while whole-genome copy number variations (CNVs) analysis was carried out using the CytoScan HD array platform (Affymetrix, Santa Clara, Calif.). The CytoScan HD assay was performed according to the manufacturer protocol, starting with 250 ng of DNA. Briefly, total genomic DNA was digested with a restriction enzyme (NspI), ligated to an appropriate adapter for the enzyme and subjected to PCR amplification using a single primer. After digestion with DNase I, the PCR products were labeled with a biotinylated nucleotide analogue, using terminal deoxynucleotidyl transferase (TdT) and hybridized to the microarray. Hybridization was carried out in the Hybridization Oven 645 while subsequent washing and staining were performed using the Fluidics Station 450.
(90) CGH Array Analysis
(91) Each array was then scanned with the Scanner 3000 7G and both quality control step and copy number analysis were performed using the Chromosome Analysis Suite Software version 2.0: i) the raw data file (.CEL) was normalized using the default options; ii) an unpaired analysis was performed using as baseline 270 HapMap samples in order to obtain Copy numbers value from .CEL files while the amplified and/or deleted regions were detected using a standard Hidden Markov Model (HMM) method.
(92) Microarray
(93) Microarrayanalysis was performed with the Affy package using Marc Carlson's Hugene 2.1st RefSeq annotation file, version 18. Background normalization was performed using the RMA method, whereas between-sample normalization was performed using the quantile normalization method. Quantification of expression was obtained using perfectly matching probes only with median polish summarization, averaging probesets to obtain gene-level expression. Probesets not assigned to known genes or having log.sub.2 fold changes<0.5 were discarded and differential expression was assessed using a 2-tailed t-test. For the purpose of enrichment analyses, DEGs with a FDR<0.2 were considered.
(94) Gene Ontology Enrichment Analysis
(95) For RNAseq data, enrichment analysis was performed using the R package GOseq in order to correct for transcript length bias considering only categories with at least 10 annotated genes and discarding categories that had less than 8 significant genes. For genes measured by other methods, the enrichment analysis was performed with the package TopGO using the classic algorithm and Fisher's test with the same cutoffs described above. In order to create enrichment treemaps, parent categories that had enriched children were first removed and then maps were created with the package Treemap, using as colors the combination of non-overlapping parent categories accounting for the largest proportion of plotted categories. All reported FDR values were calculated using the Benjamini-Hochberg method.
(96) NGN2 Clonal Line Establishment and Characterization
(97) 1) Prepare lentiviral particles from UbC-rtTA and FUW-TetO-NGN2-EGFP-Puro or FUW-TetO-NGN2-Puro as in Pang et al..sup.33 and concentrate by ultracentrifugation,
(98) 2) Plate 3×10.sup.6 iPSCs per well in a 24-well plate in mTeSR medium, 1 day before the infection and replace exhausted medium with fresh mTeSR and infect cells by adding from 0.5 to 1.5 ul of UbC-rtTA lentiviral concentrate and the same amount of FUW-TetO-NGN2-EGP-Puro/FUW-TetO-NGN2-Puro lentiviral concentrate to each well,
(99) 3) Split confluent, infected and uninduced iPSCs from a 24-well plate well to a 12-well plate well 1:1,
(100) 4) Split cells from a 12-well plate wells to a 6-well plate well 1:1,
(101) 5) Split cells from a 6-well plate to 3 6-cm dish 1:3,
(102) 6) upon daily medium change, exhausted medium is not discarded but is kept at 4° C. It is then filtered and mixed 1:1 with fresh medium, making up the “recovery medium”. This medium is necessary to sustain single cell survival after sorting,
(103) 7) upon reaching confluence in 6-cm dishes, 1 dish is frozen as “unsorted bulk population”. The other 2 dishes are split using Accutase and resuspending in 800 ul PBS with 1% Pen-Strep per dish,
(104) 8) Cells in PBS are sorted as DAPI-negative single cells, each cell in a 96-well plate well and containing 150 ul of “recovery medium” supplemented with 5 uM Rock Inhibitor, using a BD FACSAria II. DAPI-negative cells are alive; the recovery medium with rock inhibitor favors survival of single iPSCs,
(105) 9) Cells grow in the incubator for an average of 10-12 days. Medium is changed by replacing 50% with fresh medium and only after 5 days (changing it before can damage small clusters of cells). Medium is then changed daily until colonies emerge. Colonies are scored by eye under a brightfield microscope for a pluripotent morphology of cells and a round shape of the colony, so as to rule out the presence of two cells in the same well. 3 to 5 wells are chosen to establish new lines; each of these wells gives rise to a stable cell lines which arise from a single iPSC with its own transgene integration profile, hence the term “clonal” lines,
(106) 10) Colonies occupying approximately half of the surface of the 96 well are split by washing cells with 100 ul PBS and then incubating them with 30 ul of Gentle dissociation reagent for 3 minutes at 37° C. Gentle dissociation reagent is discarded and cells are roughly resuspended in 200 ul mTeSR. Resuspension is very brief so as to avoid breaking colony clumps into single cells. Resuspended clumps are then plated in 48-well plate wells, in a total volume of 300 ul of mTeSR without Rock Inhibitor per well,
(107) 11) Cells are subsequently passaged in order to reach 24-, 12- and 6-well plates. This step takes approximately 15 to 20 days as it is important to split cells without diluting them more than 1:5. Each split is done using Gentle dissociation reagent and without Rock inhibitor. Upon passaging cells from the 24-well to 12-well format, cells are split in 2 wells. Two days after splitting, mTeSR is swapped in one of the two wells with Medium 1 with doxycycline,
(108) 12) after doxycycline induction of the single 12-well plate well (preferably after 24 hours), GFP-positivity is scored. GFP-positive clones are then further expanded to 6-well and 6 cm dishes starting from the uninduced well,
(109) 13) Confluent 6 cm dishes of clonal, GFP-positive cell lines are frozen as stocks and can be readily thawed and expanded at will as iPSC lines.
(110) To induce neuronal differentiation it is sufficient to substitute mTeSR with medium 1+doxycycline as follows:
(111) 14) change medium from mTeSR to “Medium 1” as detailed in Zhang et al..sup.34:
(112) N2/DMEM/F12/NEAA
(113) Human BDNF (10 mg/l)
(114) human NT-3 (10 mg/l)
(115) mouse laminin (0.2 mg/l)
(116) Doxycycline (2 mg/l) is added preferably on the same day to induce NGN2 and retained in the medium until the end of the experiment,
(117) 15) after induction (preferably one day), puromycin is added to the medium (1 mg/l) for 24 hours.
(118) 16) after puromycin administration (preferably one day), 6×10.sup.5 mouse astrocytes are added to each wells, and the medium is changed to “Medium 2” as detailed in Zhang et al. 2013:
(119) Neurobasal supplemented with B27/Glutamax
(120) Human BDNF (10 mg/l)
(121) human NT-3 (10 mg/l)
(122) Ara-C (2 g/1)
(123) 17) 8 days after glia addition, FBS (2.5%) is added to the medium to support astrocyte viability.
(124) 18) Cells are assayed by immunofluorescence at day in a range between 14 and 31 after induction.
(125) All plates are coated with hESC-qualified matrigel (diluted 1:40 in DMEM/F12/Gln/Pen-Strep) unless otherwise specified. Cells are always incubated at 37° C. with 3% O2 and 5% CO2. Cells are always split using accutase and plated in mTeSR supplemented with 5 uM Rock Inhibitor, which is then removed after the first media change.
(126) The method optionally comprises extraction of genomic DNA from each stable cell line to quantify by digital PCR or TaqMan the exact amount of UbC-rtTA and TetO-NGN2-EGFP-Puro transgene copy numbers.
(127) The present method allows to: avoid preparing viral particles to infect iPSC lines (an advantage especially if large amounts of iPSCs need to be infected) avoid infecting iPSCs for every differentiation experiment conduct large-scale differentiation experiments—iPSCs can grow as such virtually indefinitely and can then be differentiated simultaneously obtain a homogeneous population as all stable lines come from single integration events obtain reproducible differentiation experiments as two important sources of variability (virus production and iPSC infection) have been suppressed
EXAMPLES
Example 1: Establishment of a Large Cohort of Transgene-Free Induced Pluripotent Stem Cell Lines from WBS and 7dupASD Patients
(128) The inventors selected a highly informative cohort of WBS and 7dupASD patients, whose fibroblast biopsies were deposited in the Genomic and Genetic Disease Biobank (http://www.telethon.it/en/scientists/biobanks) and who were assessed by a multidisciplinary team of specialists for a detailed clinical record (
(129) TABLE-US-00003 TABLE 3 Clinical features of patients. GDB192/ GDB306/ GDB316/ GDB361/ GDB339/ GDB242/ WBS154 WBS276 WBS301 WBS309 WBS302 GDB CF WBS202 Clinical features AtWBS1 WBS4 WBS2 WBS3 WBS1 7dupASD1 7dupASD2 Intellectual disability Moderate Moderate Moderate Moderate Moderate Moderate Moderate Cardiovascular Supravalvular aortic − + − − + NA NA stenosis Peripheral pulmonary − − − + − NA NA stenosis Valvular pulmonic − + − − − NA NA stenosis Hypertension − − NA − + NA NA Others − − − left − NA NA ventricular hypertrophy: interatrial septal defect Craniofacial Wide mouth + − + − + NA NA Prominent ear lobes − − − + + NA + Dolicocephaly − + + − − NA NA Broad forehead − + + − + − NA High narrow forehead − − − − − + NA Microcephaly − + + + + − NA Macrocephaly − − − − − + NA Bitemporal narrowing + + + + + NA NA Periorbital fullness + + + + + NA NA Epicanthal folds − − + + + NA NA Long eyelashes NA NA NA NA NA + NA Stellate Irides + + + + + NA NA Malar flattening + + + − + NA NA High broad nose − − − − − + NA Short upturned nose − + + + + − NA Bulbous nasal tip + − + + + NA NA Bridge nose flattened − − + + + NA NA Long philtrum + + + + + − − Short philtrum − − − − − + + Full lips + + + − + NA NA Full cheeks + + + − + NA NA High arched palate − − − − − + NA Dental abnormalities/ − + + NA + + NA malocclusion Small or unusually − + NA NA + NA NA shaped primary teeth Hypodontia − NA NA NA + NA NA Small mandible NA NA NA NA NA + NA Retrognathia NA NA NA NA NA + NA Endocrine subclinical − + + NA + NA NA hypothyroidism Precocious puberty − NA NA NA − NA NA Hypercalcemia − + − − + NA NA Glucose Intolerance or − − NA NA − NA NA diabetes mellitus Gastrointestinal Feeding difficulties − + − + + NA NA Abnormal weight gain − + − − + NA NA Celiac disease − − − NA NA NA NA Constipation + + − − − NA NA Gastroesophageal − − − − − NA NA reflux Abdominal pain − NA − NA NA NA NA (unclear cause) Rectal prolapse − NA − NA NA NA NA Diverticular disease − − − NA − NA NA Genitourinary Congenital anomalies − + − + − NA NA Enuresis − − − + + NA NA Nephrocalcinosis − − − − − NA NA Ocular Strabismus + − + + + NA NA Hypermetropia − + − NA − NA NA Narrowing of lacrimal − − − + − NA NA duct Muscoloskeletal Kyphosis + − − − − NA NA Lordosis − − − − − NA NA Scollosis + − − − NA NA NA Joint laxity − + − + + NA NA Radiouinar synostosis − − + − − NA NA Umbilical hernia − − − − − NA NA Neurological and Hypotonia − + + + + + + neuropsychatric Hyperrelexia − − − NA − NA NA features (including Cerebellar findings − − − − − NA NA ASD hallmarks) Type 1 Chlari − − − − − NA NA malformation Hoarse voice − + + + + NA NA Hyperacusis + + + NA + NA − Sensorineural hearing + (mild, − + NA − NA − loss right ear) Epilepsy − − − − − NA + Sleep dysregulation − − − NA NA NA − Speech impairment − − − − − + + Social impairment − − − − − + − Stereotypies NA NA NA NA NA + − MRI anomalies Cortical thickening NA NA NA NA NA + + Ventricular dilatation NA NA NA NA NA NA + Simplified gyral pattern NA NA NA NA NA + NA Increased intracranial NA NA NA NA NA NA + volume Decreased NA NA NA NA NA NA + amygdala/intracranial volume ratio No activation of NA NA NA NA NA NA + emotion-processing areas (fMRI) Other Recurrent otitis media − − − − − NA NA Short stature − + − + − NA NA Inguinal hernias − − − − − NA NA Reference Fusco et Torniero Prontera al. el al. et al. EJHG EJHG JADD 2014 2007 2014 NA, data not available. “+” stands for present; stands for not present; “−” stands for not present. * bilateral cryptorchidism, penile hypospadia
(130) This cohort includes: i) four patients carrying the typical WBS deletion; ii) one patient carrying an atypical WBS deletion that spares several genes including BAZ1B, who exhibits milder craniofacial dysmorphisms.sup.35 and lack cardiovascular abnormalities, supporting a role for BAZ1B in neural crest-derived lineages.sup.36; iii) two patients carrying the typical duplication of the 7q11.23 interval associated to language impairment, autism spectrum disorder and craniofacial dysmorphisms.sup.37; and iv) one unaffected relative of a typical WBS patient, chosen as genetically half-matched control (three iPSC lines from two additional unrelated individuals were included as additional controls,
(131) The inventors succeeded in reprogramming skin fibroblasts from each patient/control into iPSC by daily transfection of synthetic mRNAs encoding the five pluripotency factors OCT4 (also known as POU5F1), SOX2, KLF4, LIN28 and c-MYC.sup.23,38
(132) TABLE-US-00004 TABLE 4 Copy-number variations (CNVs) identified through aCGH iPSC line Total CNVs iPSC-specific CNVs (absent in fibroblasts) 7dupASD1-C1 5 chr6: 254253-381137 (−), chr20: 29989418-30665270 (+) 7dupASD1-C2 4 chr6: 254175-381137 (−) 7dupASD1-C3 5 chr6: 254253-381137 (−), chr6: 102252826-102423940 (−) 7dupASD2- C1 20 chr4: 93058279-94067253 (+), chr5: 147088797-147206977 (+), chr6: 267501-381118 (−), chr6: 94409405-94683997 (+), chr6: 73864358- 74468940 (+), chr4: 5525692-5746086 (−), chr7: 121960918-122229095 (+), chr7: 83674322-83967930 (+), chr7: 83043316-83370032 (+), chr11: 125569543-126231307 (+), chr12: 79761650-79874892 (+), chr14: 57301966-57489712 (+), chr14: 37095616-37311723 (+), chr14: 21770209-22001589 (+), chr20: 29652121-32332903 (+) 7dupASD2-C2 5 none significant 7dupASD2-C3 5 none significant 7dupASD2-C5 6 chr10: 53793333-53986201 (+) AtWBS1-C1 17 chr5: 147160867-147211743 (+), chr7: 122088955-122165269 (+), chr10: 53775828-53856760 (+), chr19: 53893835-53991962 (+), chr5: 180378753-180485857 (+), chr19: 54196897-54306189 (+), chr6: 94441864-94581718 (+), chr7: 82958543-83174511 (+), chr7: 121529836-121803926 (+), chr20: 60684625-60960515 (+), chr1: 144081221-144884970 (+), chr1: 147933972-149758028 (+) AtWBS1-C2 7 chr5: 174172854-174216370 (−), chr1: 64098064-64177299 (+), chr5: 180374897-180485857 (+) AtWBS1-C3 17 chr4: 55103916-55159462 (+), chr7: 122088594-122160742 (+), chr7: 121980929-122064412 (+), chr5: 180374483-180485857 (+), chr6: 94445905-94596361 (+), chr7: 83018833-83184441 (+), chr1: 82209385-82379820 (+), chr7: 83435331-83638075 (+), chr19: 54165173-54454992 (+), chr1: 144086896-144884321 (+), chr1: 147831169-149660970 (+) CTL1R-C1 5 chr7: 121960918-122027625 (+), chr7: 122090358-122160742 (+) CTL1R-C2 7 chr8: 1825200-1941407 (+), chr10: 24028502-24165815 (−), chrX: 119999582-120139697 (−), chr6: 33227014-33409781 (+) CTL1R-C3 9 chr5: 147188740-147291626 (+), chr14: 106667034-106931309 (+), chr5: 65445341-66268829 (+), chr1: 237971511-238837154 (+), chr8: 112519123-113612654 (+), chr6: 109789585-111271950 (+) WBS1-C2 7 chr5: 147185452-147246473 (+), chr8: 68346377-68541850 (+), chr7: 137529539-138040330 (+) WBS1-C3 7 chr7: 122017176-122129600 (+), chr3: 77123372-77418651 (+), chr4: 92974531-93517978 (+) WBS2-C1 8 chr4: 93151709-93505123 (+), chr7: 122017096-122293350 (+) WBS2-C2 10 chr4: 93151709-93573786 (+), chr6: 94441864-94817554 (+), chr7: 83018833-83214294 (+), chr7: 121960918-122205630 (+) WBS2-C3 5 chr4: 93099134-94304185 (+), chr7: 121776521-122291289 (+) WBS3-C1 4 none significant WBS3-C2 12 chr6: 294712-381137 (−), chr7: 121695010-121818973 (+), chr20: 15053066-15192669 (−), chr7: 83435331-83607471 (+), chr7: 121962453-122191669 (+), chr7: 82837315-83213300 (+), chr6: 94378742-94822428 (+), chr4: 92992264-93622621 (+) WBS3-C3 8 chr6: 330691-381137 (−), chr7: 83063087-83168747 (+), chr7: 121960918- 122148266 (+), chr6: 94478593-94824268 (+) WBS4-C1 10 chr10: 47055847-47149411 (−), chr7: 74507793-74629034 (−), chr12: 124814373-124974860 (−), chr4: 93150773-93389036 (+), chr4: 85917848-86271752 (−) WBS4-C2 10 chr7: 74530357-74621643 (−), chr1: 234083694-234196919 (+), chr7: 110817057-111296229 (−) WBS4-C3 11 chr1: 234083694-234200257 (+), chr7: 121981127-122129600 (+), chr6: 94405746-94592955 (+), chr6: 40846660-41825053 (−) Summary of the aCGH data on iPSC lines, indicating the genomic coordinates (in the human reference genome version hg19) of iPSC-specific CNVs. “+” indicates a copy-number gain, while “−” indicates a loss. For example, line 7dupASD1-C1 has lost a copy of the region spanning base pairs 254253 to 381137 of chromosome 6.
Example 2: Expression of 7q11.23 Genes Follows Gene Dosage in the Pluripotent State
(133) In order to ascertain whether the pluripotent state represented a meaningful stage at which to probe the effect of 7q11.23 dosage, the inventors first asked whether the mRNA expression of the 7q11.23 genes follows gene dosage. For this the inventors resorted to the high accuracy of Nanostring-based quantitation as well as to RNAseq and found that the expression of all genes of the interval (including those expressed at very low levels) mirrors gene dosage (
Example 3: 7q11.23 Dosage Imbalance Causes Transcriptional Dysregulation in Disease-Relevant Pathways Already at the Pluripotent State
(134) To assess differential expression between genotypes, the inventors profiled by RNAseq the panel of patient- and control-derived iPSC lines, and complemented this dataset also with additional control lines from the literature (hereafter referred to as external controls, see methods), excluding from further analysis the genes that were differentially-expressed between controls from the inventors' cohort and external controls. A pair-wise comparison of the three genotypes identified 757 differentially-expressed genes (DEGs) (
(135)
(136) i) cellular calcium ion homeostasis (FDR˜0.0012), a category of potential relevance across disease areas but that acquires particular salience in light of the high prevalence of hypercalcemia in WBS.sup.46 (DEGs in category: ATP1A2, ATP2A2, ATP7B, CACNA1A, CALCR, CAV1, CCDC47, CCKBR, CD40, DIAPH1, EDNRB, EPHX2, GRIK2, GSTO1, GTF2I, HRC, ITPR3, KDR, RYR1, RYR2, SLC30A1, SLC8A3, TACR1, TRDN);
(137) ii) inner ear morphogenesis (FDR˜0.0046), consistent with the combination of hyperacusis and sensorineural hearing loss that is virtually always present in WBS.sup.47, as well as with the balance and sensory processing disorders found in ASD.sup.48 (DEGs in category: COL11A1, COL2A1, FZD2, GBX2, MAFB, MYO6, NTN1, SOX9, WNT3A, ZEB1, ZIC1);
(138) iii) a number of categories relevant for the craniofacial phenotype, such as skeletal muscle organ development (FDR˜0.0067), migration (FDR˜4.2E-8) and neural crest cell differentiation (FDR˜0.011) (DEGs in any of the three categories;
(139) iv) categories such as blood vessel development (FDR˜0.0068) and cardiovascular system development (FDR˜0.0043), that reflect the wide range of cardiovascular problems in WBS;
(140) v) kidney epithelium development (FDR˜0.0026), in line with the highly prevalent kidney abnormalities of WBS.sup.49.
(141) Importantly, removal of the external controls did not lead to significant changes in the enrichments the inventors obtained (
(142) The inventors found that the majority of DEGs either show a symmetrically opposite pattern in the two conditions or have a fold-change in the same direction over controls, indicating that the symmetrical dosage imbalances mostly affect the same transcriptional programs, either in the same or in symmetrically opposite ways. The inventors thus proceeded to uncover what were the most highly symmetrical genes, taking the DEGs for which the mean expression in control samples was within a 20-80% range between the means of the WBS and 7DupASD (˜39% of the DEGs) and that had an absolute Pearson correlation of at least 0.5 with WBS gene dosage. This high-confidence set included 166 symmetrical DEGs (
Example 4: GTF2I Dosage Accounts for a Significant Fraction of Transcriptional Dysregulation in the Pluripotent State
(143) Convergent evidence from atypical patients and experimental models.sup.13,41,52-54 pointed to general transcription factor GTF2I as a critical gene for the pathogenesis of the cognitive/behavioural deficits of the two conditions. In light of the above findings, the inventors thus asked what quota of 7q11.23 transcriptional dysregulation could be attributed to GTF2I, using lentiviral RNAi to i) revert the GTF2I dosage of 7DupASD to control levels, ii) to model GTF2I haploinsufficiency by knocking-down GTF2I in controls and selecting clones whose residual GTF2I levels were similar to those of WBS lines, iii) to exacerbate the dysregulation of GTFI transcriptional targets that are insensitive to haploinsufficiency by knocking down GTF2I mRNA also in WBS lines (
Example 5: GTF2I Assembles a Transcriptional Co-Repressor Complex Containing LSD1 and HDAC2
(144) In order to elucidate the mechanism through which GTF2I regulates this significant portion of disease-relevant gene expression, the inventors purified its protein complex and defined its genome-wide occupancy. First, the inventors tested the ability of two GTF2I antibodies to immunoprecipitate GTF2I in its native form (
Example 6: Identification of GTF2I Targets in WBS and 7DupASD Patient-Derived iPSCs
(145) The observation that in iPSCs GTF2I associates with transcriptional repressors was unexpected both in light of published reports on its transcriptional effects.sup.59,60 and of inventors' finding that GTF2I-attributable DEGs are evenly distributed among repressed and activated targets. This raises the hypothesis that GTF2I binds directly to only a subset of its imputable targets. Thus, in order to discriminate direct from indirect transcriptional changes, the inventors determined the GTF2I binding profiles by ChIP-seq (Chromatin Immuno-Precipitation coupled with sequencing). The inventors first tested three available antibodies (Cell Signaling, Bethyl and Santa Cruz) on a control iPSC line (
(146) The 436 genes are as follows:
(147) GTF2I Core Targets (Conserved Promoter Peak in all Control Samples):
(148) ABCC3, ACOT12, ACTA2, ACTN3, ADA, ADAM19, ADAMTS10, ADAMTSL5, ADCY3, ADRBK2, AES, AGPAT3, AGXT2L2, AHCY, ALDH18A1, ANO8, APC2, ARAP1, ARF1, ARHGAP40, ARHGEF1, ARHGEF10L, ARHGEF16, ARID3A, ARMC3, ARPC1B, ATE1, ATG16L1, ATG4D, ATP2C2, ATXN10, AVPI1, B3GNT3, B4GALT4, BCL11B, BCL7B, BCL9, BIK, BRF1, BSND, BTBD11, BTBD6, BTBD8, C11orf86, C16orf95, C19orf60, C19orf66, C19orf71, C2orf48, C7orf55, C7orf55-LUC7L2, CACTIN-AS1, CAMK1, CAMK2G, CAND2, CASKIN1, CBFA2T2, CBX3, CCDC34, CCDC71, CCNI2, CCR10, CD209, CD3EAP, CD83, CDC25B, CDH5, CDK9, CEBPG, CELF5, CHST12, CLK3, CNN2, CNTNAP1, COASY, COL16A1, CPA5, CPPED1, CRB2, CREB3L4, CRX, CRYBB1, CRYL1, CSRNP1, CST6, CTDSP1, CYB5B, CYB5R3, DAK, DAPK3, DDB1, DDX17, DEAF1, DEPDC5, DIAPH2, DIRAS1, DOCK6, DPM2, DPP3, DPP4, DPPA2, DRG2, DYRK1A, EARS2, EFHD1, EFNB3, EFR3A, EHD2, EIF4E1B, EIF4G2, ELAVL3, ELL, EMID1, EML2, EPC2, EXOC2, FAM220A, FAM49B, FAM92B, FAS-AS1, FASTKD5, FBLN1, FBLN2, FBN3, FBXO31, FCHO1, FGF22, FHAD1, FOSL2, FOXA3, FSTL5, FUS, FXN, FXYD7, FZD1, FZD2, GADD45B, GALNT10, GALNT11, GAMT, GAREML, GAS2L1, GATSL3, GDPD3, GNA12, GRIP2, GRK6, GRPEL2, GTF2F1, GTPBP3, H6PD, HDAC1, HLA-DPA1, HLCS, HNRNPA2B1, HPS4, ICAM1, IFI30, IQCA1, IQCE, ISOC2, JUNB, JUND, KANK2, KCNK15, KCNS1, KCTD14, KCTD21, KDM4A, KHDRBS3, KIF12, KIF13A, KIF16B, KPTN, KREMEN2, KRTAP10-4, KXD1, L1TD1, LAMC3, LARP1, LDLR, LDLRAP1, LGALS1, LGR6, LINC00620, LINC00862, LINGO1, LMTK3, LOC100128398, LOC100131655, LOC100506082, LOC100506100, LOC100507600, LOC284751, LOC387723, LOC389033, LOXL4, LPAR2, LPIN1, LRRC25, LRRC32, LRRC33, LSR, LUC7L2, MAGI3, MAP1LC3B, MAP4K2, MAPKAP1, MB, MCOLN2, MDS2, MEGF9, MFAP2, MFF, MGAT3, MIAT, MICALL2, MIDN, MIR2861, MIR3188, MIR3190, MIR3191, MIR3529, MIR3960, MIR4281, MIR4497, MIR4632, MIR4736, MKNK2, MMP28, MRPS5, MTFP1, MYO1B, MYOCD, NACC1, NAE1, NAPA-AS1, NCOR2, NDE1, NDUFA3, NEK6, NIPSNAP1, NKD1, NLRX1, NMUR1, NPAS1, NPY4R, NR2F6, NR6A1, NTAN1, NUDT3, NUDT8, NYAP1, OGDHL, OSCAR, PAK4, PAPPA, PASK, PDE4A, PDE8A, PDLIM1, PDLIM7, PEAR1, PEX5, PGPEP1, PHACTR4, PHYH, PIANP, PION, PKN1, PLAUR, PLCD1, PLIN3, PLIN5, PLK5, PLOD1, PNPLA6, POLR3K, PPIA, PPP1R13L, PPP1R16A, PPP1R1A, PPP1R7, PRKCD, PRRT3, PSIP1, PSMG3, PSMG3-AS1, PTGIS, PTPN11, PTPRU, RAB1A, RAB3IL1, RAC1, RAP1GAP, RASGEF1C, RELB, RHPN2, RILPL1, RILPL2, RIN3, RPL18, RPL4, RPS18, RPSAP58, RRM2, SAFB, SAFB2, SCN8A, SCRN1, SEMA4B, SEMA6B, SEPT9, SEPW1, SETD1A, SGSM1, SH2D5, SH3GLB2, SH3PXD2B, SHISA5, SHISA6, SIRPA, SLC12A4, SLC15A3, SLC1A6, SLC29A3, SLC2A1, SLC2A1-AS1, SLC38A10, SLC39A1, SLC39A10, SLC43A1, SLC44A1, SLC52A3, SLC5A5, SLC6A20, SLC6A4, SNORD16, SNORD18A, SNRNP25, SOGA1, SORBS1, SPATA2, SPHK2, SPOCD1, SREBF1, SRPK1, SRPK2, SRRD, SRXN1, SSBP4, ST6GALNAC6, STRBP, STX1B, SWSAP1, SYMPK, SYN1, SYNGR1, SYNJ2, SYT2, TADA1, TAF6L, TAGLN, TBC1D2, TBX6, TBXA2R, TCEA2, TCF3, TCIRG1, TCTN3, TESC, TEX40, TFE3, THUMPD2, TINAGL1, TJP3, TKT, TMC6, TMC7, TMEM120A, TMEM14B, TMEM161A, TMEM194B, TMEM200B, TMEM229B, TMEM80, TNFAIP8L1, TNNT2, TNRC18, TOPORS-AS1, TP53I11, TPM4, TPST2, TRIM26, TRIM59, TRMT1, TRPV4, TSC1, TSPO, TC38, TUB, TVP23A, UBE2Q1, UBFD1, UBOX5, USHBP1, USP35, VAV1, VPS37C, VPS37D, VPS52, VSTM2L, WBSCR27, WDR38, WNT4, WSCD2, XKR7, YIPF1, ZBTB47, ZCCHC14, ZDHHC12, ZDHHC24, ZFAND4, ZMYM6NB, ZNF234, ZNF236, ZNF335, ZNF500, ZNF579, ZNF606, ZNF646, ZNF668, ZNF839, ZNRF4, ZSWIM4, ZWILCH
(149) All GTF2I Targets (Significant Promoter Peak in the Merged Analysis):
(150) ABCA7, ABCC3, ABCD4, ABHD3, ABHD8, ACAD10, ACBD4, ACMSD, ACOT12, ACP5, ACTA2, ACTL8, ACTN3, ACVR2B, ADA, ADAM19, ADAM32, ADAMTS10, ADAMTS14, ADAMTSL5, ADAR, ADCY3, ADM5, ADORA2A, ADRBK2, ADSL, AES, AFAP1L2, AFG3L2, AGPAT3, AGT, AGXT2L2, AHCY, AIM1, AIM1L, AIRE, AK1, AK8, AKNA, ALDH18A1, ALDH1B1, ALDH3B1, ALDH4A1, ALG8, ALS2CL, ALX4, ALYREF, AMICA1, AMN, ANAPC1, ANAPC11, ANGPT1, ANKLE2, ANKMY1, ANKRD24, ANKRD9, ANO8, ANO9, AP4S1, APBA3, APC2, APCDD1L, APCDD1L-AS1, APOE, APOO, ARAP1, ARF1, ARHGAP18, ARHGAP22, ARHGAP23, ARHGAP27, ARHGAP40, ARHGEF1, ARHGEF10L, ARHGEF16, ARHGEF19, ARHGEF3, ARID1A, ARID3A, ARID5A, ARL5A, ARL6IP5, ARMC3, ARPC1B, ARPC4, ARPC4-TTLL3, ARRDC3-AS1, ARSI, ASAP3, ASS1, ASTN2, ATAD2, ATCAY, ATE1, ATE1-AS1, ATG16L1, ATG16L2, ATG4D, ATP1A2, ATP2C2, ATP8B3, ATXN10, AVPI1, AVPR1A, B3GALT5, B3GNT3, B3GNT5, B4GALNT3, B4GALT4, B4GALT5, BAIAP3, BAX, BCAS1, BCAT1, BCL11B, BCL2L13, BCL7B, BCL9, BCL9L, BCS1L, BEND4, BFSP1, BIK, BLVRB, BMP7, BNIP3, BPIFB3, BRAP, BRF1, BSND, BSPRY, BST2, BTBD11, BTBD6, BTBD8, BZRAP1, BZRAP1-AS1, C10orf55, C11orf68, C11orf86, C12orf49, C12orf65, C14orf1, C16orf54, C16orf62, C16orf95, C16orf96, C17orf58, C17orf99, C19orf25, C19orf35, C19orf38, C19orf47, C19orf60, C19orf66, C19orf71, C19orf81, C19orf83, C1orf137, C1QC, C1QTNF6, C21orf88, C2orf48, C2orf50, C3orf18, C3orf55, C6orf100, C7orf55, C7orf55-LUC7L2, C7orf61, C9orf40, C9orf9, CABP1, CACNA1F, CACNA1H, CACNB3, CACTIN, CACTIN-AS1, CAGE1, CALCA, CALR, CAMK1, CAMK2G, CAMKK1, CAND2, CAP1, CAPN12, CAPN2, CARD10, CARHSP1, CARNS1, CASC8, CASKIN1, CATIP-AS1, CBFA2T2, CBLN3, CBX1, CBX3, CBX7, CCDC110, CCDC130, CCDC148, CCDC22, CCDC23, CCDC34, CCDC63, CCDC69, CCDC71, CCL2, CCM2L, CCNI2, CCR10, CD19, CD209, CD276, CD3EAP, CD40LG, CD79B, CD83, CD99L2, CDA, CDAN1, CDC25B, CDC42BPG, CDC42EP2, CDC42EP4, CDH5, CDK2AP2, CDK9, CEACAM16, CEBPE, CEBPG, CECR5, CELF5, CEMIP, CENPA, CEP68, CERS4, CFD, CGN, CHADL, CHDH, CHST12, CHST9, CHUK, CILP2, CIZ1, CLASP, CLDN14, CLEC16A, CLEC17A, CLEC2L, CLEC4G, CLEC4GP1, CLIP3, CLK3, CLMP, CLPSL2, CLSTN1, CLU, CLVS1, CMTM3, CNN1, CNN2, CNOT8, CNP, CNPY4, CNTD2, CNTFR, CNTNAP1, COASY, COL16A1, COL9A3, COQ7, COX10, COX10-AS1, COX17, CPA5, CPLX2, CPNE5, CPPED1, CPSF6, CPT1A, CPT1C, CRABP1, CRB2, CREB3L3, CREB3L4, CRLF1, CROCC, CRX, CRYBA2, CRYBA4, CRYBB1, CRYGN, CRYL1, CSF1, CSK, CSRNP1, CST11, CST3, CST6, CSTB, CTD-2270F17.1, CTDSP1, CTF1, CXorf58, CXXC5, CYB5B, CYB5R3, CYGB, CYHR1, CYLD, CYP20A1, CYP26A1, CYP2W1, CYP4F22, DACT3, DACT3-AS1, DAGLB, DAK, DAND5, DAPK3, DCK, DCLK1, DCTN6, DCTPP1, DDB1, DDIAS, DDX17, DDX54, DEAF1, DEGS2, DEPDC5, DFFA, DHRS12, DHRS13, DHX35, DIAPH2, DIRAS1, DLGAP4, DNAAF3, DNAJB8-AS1, DNAJC17, DNMT1, DNMT3B, DOCK11, DOCK6, DOLPP1, DPEP1, DPM2, DPP3, DPP4, DPPA2, DRAP1, DRC1, DRG2, DUS3L, DUSP28, DUSP8, DYRK1A, E4F1, EARS2, ECD, EEF2K, EFCAB6, EFCAB7, EFHD1, EFNA4, EFNB3, EFR3A, EGR4, EHBP1L1, EHD2, EIF2B3, EIF4E1B, EIF4E2, EIF4E3, EIF4G2, ELAVL3, ELF5, ELFN1, ELFN2, ELL, EMID1, EML2, EML3, ENG, EPB41L3, EPC2, EPS15, ERICH2, ERMAP, ERP44, EVA1B, EXOC2, EYA2, F11R, F2R, FAM110A, FAM149A, FAM160B2, FAM166B, FAM167B, FAM174B, FAM178A, FAM179A, FAM188B, FAM193B, FAM199X, FAM220A, FAM222A, FAM222A-AS1, FAM49B, FAM64A, FAM65A, FAM69B, FAM83A-AS1, FAM92A1, FAM92B, FANCA, FAS-AS1, FASTKD5, FBLN1, FBLN2, FBN3, FBXL19, FBXO21, FBXO31, FBXO46, FBXW8, FCER2, FCHO1, FGF22, FGF8, FGFR1, FGFR2, FHAD1, FIBCD1, FKBP11, FLII, FLJ21408, FLJ26850, FLJ33534, FMN1, FOSL2, FOXA3, FOXN1, FRRS1L, FSD1, FSIP1, FSTL5, FURIN, FUS, FUT8, FXN, FXYD5, FXYD7, FZD1, FZD2, GABRP, GABRR3, GADD45B, GALNT10, GALNT1, GAMT, GAPDHS, GAREML, GAS2L1, GAS7, GATA2, GATSL3, GBGT1, GCM2, GDPD3, GDPD5, GEMIN7, GGT1, GHDC, GINS2, GIPR, GKN2, GNA12, GNB5, GOLGA3, GOLGA5, GOLTA, GOT1, GP9, GPAM, GPBP1L1, GPN2, GPR27, GPR64, GPR68, GPX1, GREB1, GRHL3, GRIN3B, GRIP2, GRK5, GRK6, GRPEL2, GSAP, GSC2, GTDC1, GTF2E2, GTF2F1, GTF3C1, GTPBP3, GUCY1B2, GUCY2C, H6PD, HAPLN4, HAUS5, HCK, HDAC1, HDAC11, HEATR5A, HEMK1, HHEX, HIC1, HIF3A, HIRIP3, HLA-DPA1, HLCS, HM13-AS1, HMCN2, HMGN1, HMHA1, HNRNPA2B1, HNRNPM, HPD, HPN-AS1, HPS1, HPS4, HRH2, HS1BP3, HS3ST3A1, HSPB1, HTR6, ICAM1, ICAM3, ID1, IER3IP1, IFI30, IFT122, IFT43, IGSF1, IL12RB1, IL17RA, IL17RB, IL19, ILRL1, IL21R, IL4R, INF2, INHBA, INO80, INO80E, INPP5J, INSR, INSRR, INTS8, INVS, IP09, IP09-AS1, IQCA1, IQCD, IQCE, IQGAP2, IRF2BP1, IRX6, ISM2, ISOC2, ISYNA1, ITGAL, ITGB3BP, JPH3, JUNB, JUND, KANK2, KANK3, KANTR, KAT2B, KAZN, KCMF1, KCND1, KCNH3, KCNIP3, KCNJ12, KCNJ4, KCNK15, KCNK6, KCNMB1, KCNQ4, KCNS1, KCTD14, KCTD2, KCTD21, KCTD21-AS1, KDELR3, KDM4A, KEAP1, KHDRBS3, KIAA0556, KIAA1191, KIAA1279, KIAA1644, KIAA1671, KIAA1755, KIAA1919, KIAA2018, KIF12, KIF13A, KIF16B, KIF3B, KIF6, KLHL17, KLHL21, KLHL35, KLHL4, KPNA1, KPNB1, KPTN, KREMEN1, KREMEN2, KRTAP10-2, KRTAP10-4, KRTAP5-AS1, KXD1, L1TD1, LAMC2, LAMC3, LAMTOR1, LAPTM5, LARP, LDB1, LDHD, LDLR, LDLRAP1, LEFTY2, LGALS1, LGR6, LHPP, LINC00112, LINC00200, LINC00207, LINC00263, LINC00311, LINC00479, LINC00489, LINC00523, LINC00535, LINC00620, LINC00690, LINC00862, LINC00877, LINC00959, LINC01100, LINC01270, LINC01272, LINGO1, LIPC, LITAF, LMNTD2, LMTK3, LOC100128398, LOC100128531, LOC100128568, LOC100129973, LOC100130238, LOC100131655, LOC100288866, LOC100335030, LOC100505622, LOC100505666, LOC100505942, LOC100506082, LOC100506100, LOC100506119, LOC100506700, LOC100506860, LOC100506985, LOC100507501, LOC100507534, LOC100507600, LOC100996351, LOC101926888, LOC101927124, LOC101927272, LOC101927323, LOC101927380, LOC101927954, LOC101928069, LOC101928093, LOC101928107, LOC101928244, LOC101928736, LOC101928737, LOC101929116, LOC101929125, LOC101929384, LOC101929526, LOC101929567, LOC101929657, LOC102503427, LOC102546298, LOC102577424, LOC102723385, LOC102724020, LOC102724827, LOC149684, LOC150381, LOC253044, LOC284751, LOC387723, LOC388282, LOC389033, LOC401177, LOC440028, LOC643770, LOC728743, LOC729966, LOXL4, LPAR2, LPIN1, LPP, LPP-AS2, LPPR2, LRP5, LRRC25, LRRC32, LRRC33, LRRTM3, LSR, LTBP4, LUC7L2, LUZP6, LYPD6B, MAGI1-AS1, MAGI3, MAN2C1, MAP1LC3B, MAP1S, MAP2K3, MAP4K2, MAP7D1, MAPKAP1, MAPRE1, MARK4, MAST1, MATK, MB, MBD4, MCOLN1, MCOLN2, MDS2, MECR, MED12L, MED16, MED17, MED7, MEF2B, MEGF10, MEGF8, MEGF9, MEIS3, MELK, MEOX1, METTL14, MEX3A, MFAP2, MFF, MFHAS1, MFNG, MFSD4, MGAT3, MIAT, MICALL2, MIDN, MIEF2, MIER1, MIIP, MIR1229, MIR1249, MIR135B, MIR1470, MIR219A2, MIR2861, MIR3188, MIR3190, MIR3191, MIR3193, MIR330, MIR3529, MIR3613, MIR365A, MIR3960, MIR4254, MIR4281, MIR4285, MIR4291, MIR4308, MIR4309, MIR4311, MIR4322, MIR4323, MIR4492, MIR4497, MIR4519, MIR4632, MIR4660, MIR4672, MIR4686, MIR4710, MIR4714, MIR4736, MIR4756, MIR4767, MIR4776-1, MIR4783, MIR5001, MIR548Z, MIR5689, MIR5690, MIR6515, MIR6763, MIR6789, MIR6790, MIR6813, MIR6843, MKL1, MKL2, MKNK2, MMP28, MOGAT1, MPHOSPH9, MPI, MPRIP, MPV17L2, MRPL11, MRPL20, MRPL34, MRPL41, MRPL48, MRPL54, MRPS12, MRPS15, MRPS17, MRPS25, MRPS5, MTFP1, MTFR2, MTPN, MTUS1, MTUS2, MUC12, MYBPC2, MYEOV2, MYH11, MYH9, MYL2, MYLK, MYO1B, MYOCD, MYRF, N4BP3, NAA35, NACC1, NACC2, NAE1, NANS, NAPA-AS1, NATD1, NCAN, NCMAP, NCOR2, NDE1, NDRG4, NDUFA3, NDUFA6, NDUFA6-AS1, NDUFA8, NDUFB4, NDUFS4, NEBL, NEBL-AS1, NEDD1, NEIL1, NEK6, NEK9, NEMF, NES, NEURL1B, NEUROG3, NFATC2, NFE2L3, NFIC, NGFRAP1, NIFK-AS1, NINJ1, NIPAL3, NIPAL4, NIPSNAP1, NKD1, NKX3-1, NLRP14, NLRX1, NMUR1, NOC2L, NOSIP, NPAS1, NPHP3, NPHP3-ACAD11, NPHP3-AS1, NPY4R, NR2C2AP, NR2E1, NR2E3, NR2F6, NR6A1, NRGN, NRROS, NRTN, NT5DC3, NTAN1, NTN1, NTN5, NTRK1, NUCKS1, NUDT21, NUDT3, NUDT8, NUP210, NUP210L, NWD1, NXPH3, NYAP1, OACYLP, OAS2, OCEL1, OCIAD2, OCSTAMP, OGDHL, OGFOD1, OLIG2, ONECUT3, OPCML, OPRL1, OR10H3, OR2D3, OR3A1, OSCAR, OSGIN1, OTUB2, OVOL3, P2RY6, PACSIN3, PADI4, PADI6, PAH, PAK4, PALM, PAPL, PAPLN, PAPPA, PAPPA-AS1, PAQR7, PARP15, PASK, PAWR, PC, PCDH12, PCGF6, PCSK4, PDE2A, PDE4A, PDE4C, PDE8A, PDGFD, PDGFRB, PDLIM1, PDLIM7, PEAR1, PEF1, PEX13, PEX5, PFDN4, PFKFB3, PFKFB4, PGAM5, PGAP2, PGLYRP2, PGPEP1, PHACTR3, PHACTR4, PHF19, PHLDA2, PHOSPHO1, PHYH, PHYKPL, PIANP, PIGB, PION, PITPNM1, PKD2L2, PKN1, PLA2R1, PLAGL2, PLAUR, PLCB3, PLCD1, PLCD3, PLD3, PLEKHA2, PLEKHJ1, PLIN3, PLIN5, PLK5, PLOD1, PLTP, PLXDC2, PLXNC1, PLXND1, PMVK, PNKP, PNPLA4, PNPLA5, PNPLA6, PNPLA7, POFUT1, POLG, POLR3K, POMGNT1, PON2, POP5, POU2F2, PPIA, PPM1H, PPP1R13L, PPP1R16A, PPP1R16B, PPP1R1A, PPP1R7, PPP2R1A, PPP2R5D, PPP4R1, PPP5C, PQLC3, PRCP, PRDM11, PRDM13, PRELID2, PRELP, PRKCD, PROCA1, PROSER2, PRPF40B, PRR15, PRR29, PRR5, PRRC1, PRRG2, PRRT3, PRX, PSIP1, PSMB4, PSMD9, PSMG3, PSMG3-AS1, PTGIS, PTGS1, PTP4A3, PTPDC1, PTPN1, PTPN11, PTPN3, PTPRA, PTPRS, PTPRU, PUS10, PVR, PVRL2, PXMP4, PYGM, PYGO1, PYROXD2, QPCT, QSOX2, R3HDML, RAB11FIP1, RAB1A, RAB25, RAB36, RAB39A, RAB3D, RAB3IL1, RAB4B, RAB4B-EGLN2, RAB8A, RABEP1, RABEPK, RAC1, RAC2, RAD21L1, RAI1, RALGAPB, RALY, RANBP3, RAP GAP, RARA, RARB, RARS, RASD2, RASGEF1A, RASGEF1C, RASGRP2, RASGRP4, RASSF7, RAX2, RBPMS, RBPMS2, RCAN2, RCAN3, RCAN3AS, RCC1, RCCD1, RCN3, RDH13, REEP6, RELB, REPIN1, REPS1, REXO1, RFX1, RGAG1, RGCC, RGS19, RHBDF2, RHBDL2, RHOD, RHOH, RHPN2, RILPL1, RILPL2, RIMS2, RIN1, RIN3, RIOK1, RIPPLY3, RITA1, RNF180, RNF187, RNF44, RNF6, RNU6-33P, RNU6-34P, ROM1, ROR1, RPA2, RPL18, RPL27A, RPL29, RPL4, RPN2, RPP25, RPS11, RPS18, RPS7, RPSAP58, RRM1, RRM2, RSPO4, RTBDN, RTDR1, RTN2, RXRA, RYR1, S100A3, S100A4, S1PR5, SAFB, SAFB2, SAMD4B, SARS2, SBF2-AS1, SCAP, SCML1, SCN1B, SCN8A, SCNN1B, SCNN1G, SCOC, SCRN1, SCRN2, SDC4, SDHAP3, SDK1, SEC14L5, SEC31B, SEMA4B, SEMA4G, SEMA6B, SEPT5, SEPT9, SEPW1, SERPINA1, SERPINB1, SERPINH1, SETD1A, SF3A2, SGK3, SGSM1, SGTA, SH2D2A, SH2D3A, SH2D3C, SH2D5, SH3BP2, SH3GLB2, SH3PXD2B, SHANK2, SHD, SHISA5, SHISA6, SHISA8, SIGLEC1, SIPA1L3, SIRPA, SIRT1, SIRT6, SLC12A3, SLC12A4, SLC13A3, SLC15A3, SLC16A6, SLC1A5, SLC1A6, SLC22A15, SLC22A23, SLC22A7, SLC25A17, SLC25A39, SLC25A42, SLC25A47, SLC29A3, SLC2A1, SLC2A10, SLC2A13, SLC2A1-AS1, SLC31A2, SLC35G1, SLC37A1, SLC38A10, SLC39A1, SLC39A10, SLC3A2, SLC43A1, SLC44A1, SLC52A3, SLC5A5, SLC6A1, SLC6A12, SLC6A2, SLC6A20, SLC6A4, SLC7A10, SLC7A2, SMIM2-AS1, SNHG5, SNN, SNORA45A, SNORD16, SNORD18A, SNORD50A, SNORD50B, SNPH, SNRNP25, SNUPN, SNX19, SNX22, SNX31, SOCS7, SOD3, SOGA1, SORBS1, SORBS2, SOWAHD, SOX2, SP6, SPAG1, SPAG9, SPATA2, SPHK2, SPIB, SPOCD1, SPTBN4, SPTBN5, SRC, SREBF1, SREBF2, SRPK1, SRPK2, SRRD, SRRT, SRSF12, SRXN1, SSBP4, SSC5D, SSH1, ST3GAL5, ST5, ST6GALNAC6, STARD4, STARD4-AS1, STPG1, STRA6, STRADA, STRBP, STRN3, STX1B, STXBP1, SULTIC2, SULT2B1, SULT4A1, SUOX, SUPT4H1, SUSD3, SWSAP1, SYCE2, SYCP3, SYMPK, SYN1, SYNE3, SYNGR1, SYNJ2, SYNPO2, SYNRG, SYT12, SYT2, SYT3, TADA1, TADA3, TAF1B, TAF6, TAF6L, TAGLN, TBC1D16, TBC1D2, TBX6, TBXA2R, TCEA2, TCERG1L, TCF3, TCIRG1, TCL1A, TCL1B, TCTA, TCTE1, TCTN3, TESC, TEX40, TFB1M, TFE3, TFF3, TGFB3, TGM6, TH, THEG, ThOP1, THPO, THUMPD2, TICAM1, TIGD1, TIMM10, TINAGL1, TJP3, TKT, TLE2, TLE6, TM9SF4, TMC6, TMC7, TMC8, TMED2, TMEM114, TMEM119, TMEM120A, TMEM147, TMEM14B, TMEM151A, TMEM161A, TMEM194B, TMEM200B, TMEM211, TMEM229B, TMEM30C, TMEM37, TMEM40, TMEM54, TMEM56, TMEM56-RWDD3, TMEM61, TMEM69, TMEM80, TMEM9, TMEM92, TMEM9B, TMEM9B-AS1, TMIGD2, TMPRSS13, TMPRSS2, TMPRSS6, TNC, TNFAIP8, TNFAIP8L1, TNFSF14, TNNT2, TNPO2, TNRC18, TNRC6B, TOPORS-AS1, TOR1AIP2, TOR1B, TP53I11, TP53RK, TPCN1, TPD52L1, TPM4, TPST2, TRAF1, TRAF5, TRAPPC12, TRAPPC3, TRIB3, TRIM26, TRIM32, TRIM56, TRIM59, TRIOBP, TRIT1, TRMT1, TRMT10C, TRPM6, TRPV4, TSC1, TSPO, TSSC1, TrC25, TTC28, TTC33, TTC38, TTC5, TTLL13, TTLL5, TTYH3, TUB, TUFM, TVP23A, TWSG1, TXN, TXNRD1, TYROBP, UBA1, UBA3, UBAC2, UBAC2-AS1, UBE2Q1, UBE2S, UBFD1, UBOX5, UBXN8, UPF1, UPK1A-AS1, UPK1B, USHBP1, USP35, UTP11L, VAT1L, VAV1, VEGFC, VPS37B, VPS37C, VPS37D, VPS52, VSTM2L, VSTM4, VWA8, VWA8-AS1, WARS, WASF2, WBSCR27, WDR25, WDR34, WDR38, WDR52, WDR78, WDR87, WIPF2, WIPF3, WISP1, WNT2B, WNT4, WRB, WSCD2, XKR7, XPO7, YBX2, YIF1A, YIF1B, YIPF1, ZBTB40, ZBTB47, ZCCHC14, ZCCHC18, ZCCHC8, ZDHHC12, ZDHHC24, ZFAND4, ZFP64, ZFYVE19, ZMIZ1, ZMIZ1-AS1, ZMYM6, ZMYM6NB, ZMYND8, ZNF135, ZNF142, ZNF214, ZNF234, ZNF236, ZNF24, ZNF296, ZNF335, ZNF337, ZNF423, ZNF500, ZNF536, ZNF579, ZNF592, ZNF606, ZNF613, ZNF646, ZNF668, ZNF687, ZNF689, ZNF710, ZNF792, ZNF839, ZNRF4, ZSWIM4, ZUFSP, ZWILCH
(151) GTF2I Targets Differentially-Expressed in iPSC:
(152) ABCA7, ACTN3, ADAMTS10, ALDH18A1, ARL6IP5, ATP1A2, B3GALT5, B3GNT3, BCL7B, BEND4, BST2, CAPN2, CGN, CHST9, CILP2, CNTFR, CNTNAP1, CPT1C, CRABP1, CRLF1, CYP4F22, DAGLB, DPPA2, FAM65A, FZD2, GUCY2C, IL17RA, IQGAP2, KCNK6, KLHL4, LAMC3, LITAF, LOC100130238, LTBP4, MEGF10, MEGF8, MEIS3, NTN1, NUP210, PAWR, PDLIM1, PLOD1, PLXND1, RASD2, RCN3, RHBDF2, RHOH, RPSAP58, RRM2, RYR1, S1PR5, SCAP, SEMA4B, SH2D3A, SHISA6, SLC5A5, STX1B, STXBP1, SYT2, TBC1D2, TCL1A, TLE2, TMEM151A, TMEM161A, TMEM92, TRIB3, TRIT1, UBXN8, VAT1L, VPS37D, WBSCR27.
(153) Importantly, as shown in
(154) Furthermore, the inventors found that GTF2I binds preferentially to bivalent domains (p-value<1,58e-03 for core targets and 1.13e-10 for all targets). Interestingly however, as previously reported.sup.60,61, the distribution of GTF2I binding vis-à-vis the TSS does not correlate with expression (
(155) To understand the discrepancy between GTF2I transcriptional impact and occupancy, the inventors first explored the possibility that GTF2I dosage-dependent transcriptional dysregulation could be mediated by distal binding, looking at GTF2I-bound enhancers associated to putative target genes. The inventors found however only one of the 89 GTF2I-bound enhancers associated to a GTF2I-attributable, differentially-expressed TSS (PHC2), thus excluding that GTF2I acts mostly at enhancers, and supporting the alternative model of a largely indirect impact mediated by a minority of GTF2I direct targets. The inventors thus searched, among the direct GTF2I targets, for candidate transcription factors that could mediate its overall transcriptional impact, and identified BEND4, a TF that is both highly expressed in the brain and is part of a large CNV that has been reported in an ASD patient.sup.62. BEND4 was confirmed to be differentially expressed both between genotypes (
(156) Next, in order to investigate whether GTF2I exerts its repressive effect on BEND4 through the LSD1 component of its associated repressive complex, the inventors tested the effect of LSD1 inhibition on the expression of BEND4 in differentiating iPSC. As shown in
(157) Thus, the inventors decided to profile LSD1 occupancy in iPSCs in order to test whether it could discriminate dosage-dependent and independent GTF2I targets. Comparison between the GTF2I and LSD1 profiles revealed that while GTF2I co-occupies only a relatively small fraction of the LSD1 targets (
(158) Notably, LSD1-bound GTF2I dosage-sensitive targets include further disease-relevant genes, as in the paradigmatic case of CRB2 (
(159) Finally, the inventors extended their observations beyond the specific case of BEND4 (
Example 7: iPSC-Specific Transcriptional Dysregulation is Amplified During Development in a Lineage-Specific Manner
(160) That 7q11.23 CNVs trigger disease-relevant transcriptional dysregulation already at the pluripotent state suggests that these initial conditions may prime the accumulation of even greater transcriptional alterations during development. Further, it predicts that the aggregate dysregulation in iPSC across categories spanning several developmental pathways and organ systems will be channeled upon differentiation, resulting in the selective amplification of specific domains of iPSC-specific dysregulation in a lineage-dependent fashion.
(161) In order to test this hypothesis, the inventors differentiated the present invention's iPSC lines into three lineages of cardinal relevance for the two conditions (
(162) The inventors confirmed that patient-derived NPCs expressed key forebrain markers such as FOXG1, OTX2 and ZO1, and were typically arranged in neural rosettes with TBR2.sup.+ intermediate progenitors surrounding apical Pax6.sup.+ progenitors (
(163) In parallel, the inventors differentiated the same cohort of iPSC lines into NCSC that displayed distinct morphology (
(164) Finally, to define the impact of iPSC-primed transcriptional dysregulation upon further differentiation, the inventors induced patient-derived NCSC towards the MSC fate, characterized by a typical MSC-like morphology (
(165) Given the physiopathological significance of the transcriptional dysregulation in MSC, the inventors next asked what proportion of MSC-specific DEGs were also impacted at the iPSC state. As shown in
(166) Finally, the inventors noted that the proportion of symmetrically dysregulated genes is significantly higher among the DEGs that are in common between iPSC and differentiated lineages (average odd ratio˜1.75, p˜5e-03 evaluated from lineage-specific Chi-squared tests compounded through Fisher's method), supporting the notion that symmetrical patterns, likely under more direct control by 7q11.23 dosage, are particularly relevant for the quota of disease-relevant transcriptional dysregulation that is seeded already in the pluripotent state.
Example 8: A Web Platform for 7q11.23 CNV Syndromes
(167) Finally, the inventors designed a new web platform called WikiWilliamns-7qGeneBase to make their data accessible to the community of scientists and practitioners working on these two diseases, through a user-friendly, gene-centered interface. Besides integrating, in a multi-layered manner, all their results with data from the literature, WikiWilliamns is open to contributions by other groups through submission to the database's curators, with the aim of assembling in one site all molecular data on 7q11.23 syndromes (
Example 9: An Approach to Isolate iPSC-Derived FOXG1-Expressing Cortical Progenitors
(168) The proof of principle of experiments aimed at isolating cortical progenitors by selection and FACS sorting is described in
Example 10: The Screening Assay Based on qRT-PCR
(169) The inventors have already validated the automated workflow for the scoring of the expression of GTF2I and BAZ1B (by multiplex qRT-PCR) in iPSC grown on a 96-well format (
Example 11: Establishment of NGN2-Transduced Clonal Cell Lines
(170) The inventors modified the protocol for the direct differentiation of iPSCs towards the cortical glutamatergic lineage via induction of a lentivirally transduced factor, NGN2.sup.34. Briefly, the original protocol employs two lentiviral vectors to integrate i) the inducible tetracyclin transactivator (rtTA) constitutively expressed under the UbC promoter and ii) an NGN2-EGFP-Puro polypeptide-encoding cDNA whose transcription is driven by the transactivator in the presence of doxycylin. The advantage in the use of this protocol lies in its simplicity, its rapidity and the homogeneity of the resulting differentiated population. To further enhance these characteristics and enable for scaling up neuronal production (for high throughput screenings that require large amounts of homogeneous neurons) the inventors have devised a strategy to establish clonal cell lines that are readily inducible by the addition of doxycylin in the medium. Clones are established by: i) sorting a co-infected iPSC population into a 96-well plate; ii) selecting and expanding colonies with an ES-like (undifferentiated) morphology; iii) splitting selected colonies in two wells and testing one for GFP expression upon induction; iv) expanding and stocking 3-5 GFP positive iPSCs lines.
(171) Such lines have been tested for differentiation (up until day 21 from induction) and display the same mature neuronal markers, including glutamatergic markers, regardless of the genotype (
(172) Discussion
(173) The inventors chose the paradigmatic pair of genetic syndromes caused by symmetrical 7q11.23 CNV 7q11.23 in order to test the potential of iPSCs for defining how gene dosage impacts disease-relevant pathways in early developmental lineages. Their findings have broad significance for the molecular pathogenesis of WBS and 7dupASD as well as for the reprogramming-based disease modeling field as a whole.
(174) First, variability has been recently recognized as a key concern for iPSC-based disease modeling, inviting caution in the interpretation of results from few lines that do not adequately sample variability either across individuals or across lines reprogrammed from the same individual.sup.1. Thus, by presenting the largest cohort of iPSC lines characterized so far for any single genetic condition, combined to the first large scale use of mRNA-based integration-free reprogramming, the present invention benchmarks the possibility of detecting robust dosage-dependent alterations in transcriptional programs, even when these are caused by subtle dosage imbalances.
(175) Second, the size and quality of the present invention's iPSC cohort permitted two key observations on the molecular impact of 7q11.23 CNV: i) that these caused significant transcriptional dysregulation in disease-relevant pathways already in the pluripotent state; and ii) that this dysregulation was selectively amplified in a lineage-specific manner, with disease-relevant pathways preferentially and progressively more affected in differentiated lineages matching specific disease domains. The significance of this observation for the iPSC modeling field lies in the fact that the pluripotent state is by far the best characterized and most standardized one among the human developmental stages captured in vitro. Importantly, it is also the most amenable to high-throughput upscaling. Hence, the observation that the pluripotent state is not only a viable stage in which to measure disease-relevant transcriptional effects of genetic alterations, but that these effects are also predictive of further dysregulation in differentiated lineages, grounds the feasibility of middle-to-high-throughput iPSCs characterization in order to functionally annotate human genomes, prior to selecting lines and assays for more labor-intensive differentiation courses.
(176) In terms of the molecular pathogenesis of WBS and 7dupASD, besides uncovering the impact of 7q11.23 dosage already in the pluripotent state, these results also provide a first entry point for the molecular dissection of the outstanding feature that characterizes these two conditions, namely the coexistence, in the face of symmetrically opposite CNV, of both shared and symmetrically opposite phenotypes. By analyzing many samples from both conditions, the inventors were in fact able to define a subset of DEGs that follows a symmetrically opposite dosage-dependent trend. Importantly, the inventors found that this quota is significantly retained upon differentiation, indicating that symmetrically opposite patterns of gene expression seeded already in the pluripotent state, likely under direct control of 7q11.23 dosage, become increasingly prominent in disease-relevant differentiated lineages, thus providing a strong rationale for studying these two diseases (and by implication other CNV-based symmetric disease pairs) together. Importantly, inventors' analysis of symmetrically dsyregulated targets also uncovered the following genes as prime candidates for mediating the molecular pathogenesis of defining aspects of the two conditions: i) PDLIM1, which has been associated to attention-deficit disorder, neurite outgrowth, cardiovascular defects, and hyperacusis; ii) MYH14, which was involved in hearing impairment; iii) BEND4, a transcription factor harboring the BEN domain that distinguishes a recently characterized family of neural repressors, and that was sensitive to both GTF2I dosage and its LSD1-mediated repressive activity, a finding that resonates also with the inversely correlated pattern of GTF2I and BEND4 gene expression in the human brain.
(177) Third, both epidemiological data from atypical patients as well as mouse studies had pointed to GTF2I as one of the key genes mediating the phenotypes of the two conditions, especially as far as the cognitive behavioral aspects are concerned. By selectively rescuing GTF2I levels in the background of the three 7q11.23 genotypes and profiling GTF2I genome-wide occupancy across the heterogeneity of human samples, the inventors provide a mechanistic dissection of its contribution, finding that GTF2I accounts, mostly indirectly, for a significant and specific proportion of 7q11.23 dosage-dependent transcriptional impact. Importantly, the inventors found that in the human pluripotent state GTF2I assembles a chromatin repressive complex with LSD1 and HDAC2 and binds preferentially to bivalent chromatin promoters, whose timed resolution has been shown by us and several other groups to be essential for normal development, especially along the neural lineage.sup.77-80. This provides the conceptual and experimental framework to investigate the epigenetic and transcriptional impact on GTF2I dosage-dependent targets through development. This result also confirm that LSD1 inhibitors and/or HDAC2 inhibitors may be used for the prevention and/or treatment of a neurodevelopmental disorder entailing intellectual disability (ID) and/or belonging to the Autism Spectrum Disorder (ASD) and/or Schizophrenia (SZ).
(178) Finally, the inventors provide a user-friendly, open source web platform in which the inventors assembled the multi-layered datasets from this first cohort of WBS and 7dupASD samples, and which was designed to integrate ongoing contributions from the entire scientific community working on these two diseases, thus serving also as a first template for data sharing from iPSC-based functional genome annotation.
REFERENCES
(179) 1. Cahan, P. & Daley, G. Q. Origins and implications of pluripotent stem cell variability and heterogeneity. Nat Rev Mol Cell Biol 14, 357-68 (2013). 2. Prilutsky, D. et al. iPSC-derived neurons as a higher-throughput readout for autism: promises and pitfalls. Trends Mol Med 20, 91-104 (2014). 3. Ghosh, A., Michalon, A., Lindemann, L., Fontoura, P. & Santarelli, L. Drug discovery for autism spectrum disorder challenges and opportunities. Nat Rev Drug Discov 12, 777-90 (2013). 4. Marchetto, M. C. et al. A model for neural development and treatment of Rett syndrome using human induced pluripotent stem cells. Cell 143, 527-39 (2010). 5. Pasca, S. P. et al. Using iPSC-derived neurons to uncover cellular phenotypes associated with Timothy syndrome. Nat Med 17, 1657-62 (2011). 6. Chailangkarn, T., Acab, A. & Muotri, A. R. Modeling neurodevelopmental disorders using human neurons. Curr Opin Neurobiol 22, 785-90 (2012). 7. Kim, K. Y., Jung, Y. W., Sullivan, G. J., Chung, L. & Park, I. H. Cellular reprogramming: a novel tool for investigating autism spectrum disorders. Trends Mol Med 18, 463-71 (2012). 8. Sanders, S. J. et al. Multiple recurrent de novo CNVs, including duplications of the 7q11.23 Williams syndrome region, are strongly associated with autism. Neuron 70, 863-85 (2011). 9. Pober, B. R. MEDICAL PROGRESS Williams-Beuren Syndrome (vol 362, pg 239, 2010). New England Journal of Medicine 362, 2142-2142 (2010). 10. Somerville, M. J. et al. Severe expessive-language delay related to duplication of the Williams-Beuren locus. N Engl J Med 353, 1694-701 (2005). 11. Merla, G., Brunetti-Pierri, N., Micale, L. & Fusco, C. Copy number variants at Williams-Beuren syndrome 7q11.23 region. Human Genetics 128, 3-26 (2010). 12. Van der Aa, N. et al. Fourteen new cases contribute to the characterization of the 7q11.23 microduplication syndrome. Eur J Med Genet 52, 94-100 (2009). 13. Osborne, L. R. Animal Models of Williams Syndrome. American Journal of Medical Genetics Part C-Seminars in Medical Genetics 154C, 209-219 (2010). 14. Mervis, C. B. et al. Duplication of GTF2I results in separation anxiety in mice and humans. American journal of human genetics 90, 1064-70 (2012). 15. O'Leary, J. & Osborne, L. R. Global analysis of gene expression in the developing brain of Gtf2ird1 knockout mice. PLoS One 6, e23868 (2011). 16. Campuzano, V. et al. Reduction of NADPH-oxidase activity ameliorates the cardiovascular phenotype in a mouse model of Williams-Beuren Syndrome. PLoS Genet 8, e1002458 (2012). 17. Pinto, D. et al. Convergence of genes and cellular pathways dysregulated in autism spectrum disorders. Am J Hum Genet 94, 677-94 (2014). 18. Jeste, S. S. & Geschwind, D. H. Disentangling the heterogeneity of autism spectrum disorder through genetic findigs. Nat Rev Neurol 10, 74-81 (2014). 19. Spooren, W., Lindemann, L., Ghosh, A. & Santarelli, L. Synapse dysfunction in autism: a molecular medicine approach to drug discovery in neurodevelopmental disorders. Trends Pharmacol Sci 33, 669-84 (2012). 20. Krumm, N., O'Roak, B. J., Shendure, J. & Eichler, E. E. A de novo convergence of autism genetics and molecular neuroscience. Trends Neurosci 37, 95-105 (2014). 21. Dokmanovic, M., Clarke, C. & Marks, P. A. Histone deacetylase inhibitors: overview and perspectives. Mol Cancer Res 5, 981-9 (2007). 22. Kim, H. J. & Bae, S. C. Histone deacetylase inhibitors: molecular mechanisms of action and clinical trials as anti-cancer drugs. Am J Transl Res 3, 166-79 (2011). 23. Takahashi, K. et al. Induction of pluripotent stem cells from adult human fibroblasts by defined factors. Cell 131, 861-72 (2007). 24. Blecher-Gonen, R. et al. High-throughput chromatin immunoprecipitation for genome-wide mapping of in vivo protein-DNA interactions and epigenomic states. Nat Protoc 8, 539-54 (2013). 25. Shevchenko, A., Wilm, M., Vorm, O. & Mann, M. Mass spectrometric sequencing of proteins silver-stained polyacrylamide gels. Anal Chem 68, 850-8 (1996). 26. Rappsilber, J., Ishihama, Y. & Mann, M. Stop and go extraction tips for matrix-assisted laser desorption/ionization, nanoelectrospray, and LC/MS sample pretreatment in proteomics. Anal Chem 75, 663-70 (2003). 27. Keller, A., Nesvizhskii, A. I., Kolker, E. & Aebersold, R. Empirical statistical model to estimate the accuracy of peptide identifications made by MS/MS and database search. Anal Chem 74, 5383-92 (2002). 28. Nesvizhskii, A. I., Keller, A., Kolker, E. & Aebersold, R. A statistical model for identifying proteins by tandem mass spectrometry. Anal Chem 75, 4646-58 (2003). 29. Adamo, A. et al. LSD1 regulates the balance between self-renewal and differentiation in human embryonic stem cells. Nat Cell Biol 13, 652-9 (2011). 30. Shi, Y., Kirwan, P. & Livesey, F. J. Directed differentiation of human pluripotent stem cells to cerebral cortex neurons and neural networks. Nat Protoc 7, 1836-46 (2012). 31. Shi, Y., Kirwan, P., Smith, J., Robinson, H. P. & Livesey, F. J. Human cerebral cortex development from pluripotent stem cells to functional excitatory synapses. Nat Neurosci 15, 477-86, S1 (2012). 32. Menendez, L. et al. Directed differentiation of human pluripotent cells to neural crest stem cells. Nat Protoc 8, 203-12 (2013). 33. Pang, Z. P., Cao, P., Xu, W. & Sudhof, T. C. Calmodulin controls synaptic strength via presynaptic activation of calmodulin kinase II. J Neurosc 30, 4132-42 (2010). 34. Zhang, Y. et al. Rapid single-step induction of functional neurons from human pluripotent stem cells. Neuron 78, 785-98 (2013). 35. Fusco, C. et al. Smaller and larger deletions of the Williams Beuren syndrome region implicate genes involved in mild facial phenotype, epilepsy and autistic traits. Eur J Hum Genet 22, 64-70 (2014). 36. Barnett, C. et al. Williams Syndrome Transcription Factor is critical for neural crest cell function in Xenopus laevis. Mech Dev 129, 324-38 (2012). 37. Donaudy, F. et al. Nonmuscle myosin heavy-chain gene MYH14 is expressed in cochlea and mutated in patients affected by autosomal dominant hearing impairment (DFNA4). Am J Hum Genet 74, 770-6 (2004). 38. Warren, L. et al. Highly efficient reprogramming to pluripotency and directed differentiation of human cells with synthetic modified mRNA. Cell Stem Cell 7, 618-30 (2010). 39. Pasi, C. E. et al. Genomic instability in induced stem cells. Cell Death Differ 18, 745-53 (2011). 40. Abyzov, A. et al. Somatic copy number mosaicism in human skin revealed by induced pluripotent stem cells. Nature 492, 438-42 (2012). 41. Antonell, A. et al. Partial 7q11.23 deletions further implicate GTF2I and GTF2IRD1 as the main genes responsible for the Williams-Beuren syndrome neurocognitive profile. J Med Genet 47, 312-20 (2010). 42. Edelmann, L. et al. An atypical deletion of the Williams-Beuren syndrome interval implicates genes associated with defective visuospatial processing and autism. J Med Genet 44, 136-43 (2007). 43. Hirota, H. et al. Williams syndrome deficits in visual spatial processing linked to GTF2IRD1 and GTF2I on chromosome 7q11.23. Genet Med 5, 311-21 (2003). 44. Lazebnik, M. B., Tussie-Luna, M. I., Hinds, P. W. & Roy, A. L. Williams-Beuren syndrome-associated transcription factor TFII-I regulates osteogenic marker genes. J Biol Chem 284, 36234-9 (2009). 45. Sakurai, T. et al. Haploinsufficiency of Gtf2i, a gene deleted in Williams Syndrome, leads to increases in social interactions. Autism Res 4, 28-39 (2011). 46. Kruse, K., Pankau, R., Gosch, A. & Wohlfahrt, K. Calcium metabolism in Williams-Beuren syndrome. J Pediatr 121, 902-7 (1992). 47. Gothelf, D., Farber, N., Raveh, E., Apter, A. & Attias, J. Hyperacusis in Williams syndrome: characteristics and associated neuroaudiologic abnormalities. Neurology 66, 390-5 (2006). 48. Kern, J. K. et al. The pattern of sensory processing abnormalities in autism. Autism 10, 480-94 (2006). 49. Pankau, R., Partsch, C. J., Winter, M., Gosch, A. & Wessel, A. Incidence and spectrum of renal abnormalities in Williams-Beuren syndrome. Am J Med Genet 63, 301-4 (1996). 50. Wang, K. S., Liu, X., Zhang, Q., Aragam, N. & Pan, Y. Parent-of-origin effects of FAS and PDLIM1 in attention-deficit/hyperactivity disorder. J Psychiatry Neurosc 37, 46-52 (2012). 51. Ohno, K., Kato, H., Funahashi, S., Hasegawa, T. & Sato, K. Characterization of CLP36/Elfn/PDLIM1 in the nervous system. J Neurochem 111, 790-800 (2009). 52. Antonell, A., Vilardell, M. & Perez Jurado, L. A. Transcriptome profile in Williams-Beuren syndrome lymphoblast cells reveals gene pathways implicated in glucose intolerance and visuospatial construction deficits. Hum Genet 128, 27-37 (2010). 53. Ferrero, G. B. et al. An atypical 7q11.23 deletion in a normal IQ Williams-Beuren syndrome patient. Eur J Hum Genet 18, 33-8 (2010). 54. Morris, C. A. et al. GTF2I hemizygosity implicated in mental retardation in Williams syndrome: genotype-phenotype analysis of five families with deletions in the Williams syndrome region. Am J Med Genet A 123A, 405-59 (2003). 55. Gocke, C. B. & Yu, H. ZNF198 stabilizes the LSD1-CoREST-HDAC1 complex on chromatin through its MYM-type zinc fingers. PLoS One 3, e3255 (2008). 56. Hakimi, M. A., Dong, Y., Lane, W. S., Speicher, D. W. & Shiekhattar, R. A candidate X-linked mental retardation gene is a component of a new family of histone deacetylase-containing complexes. J Biol Chem 278, 7234-9 (2003). 57. Ming, G. L. et al. Cellular Reprogramming: Recent Advances in Modeling Neurological Diseases. J Neurosci 31, 16070-16075 (2011). 58. Yang, P. et al. RCOR2 is a subunit of the LSD1 complex that regulates ESC property and substitutes for SOX2 in reprogramming somatic cells to pluripotency. Stem Cells 29, 791-801 (2011). 59. Chimge, N. O., Makeyev, A. V., Ruddle, F. H. & Bayarsaihan, D. Identification of the TFII-I family target genes in the vertebrate genome. Proc Natl Acad Sci, USA 105, 9006-10 (2008). 60. Makeyev, A. V. et al. Diversity and complexity in chromatin recognition by TFII-I transcription factors in pluripotent embryonic stem cells and embryonic tissues. PLoS One 7, e44443 (2012). 61. Fan, A. X. et al. Genomic and proteomic analysis of transcription factor TFII-I reveals insight into the response to cellular stress. Nucleic Acids Res (2014). 62. Sabaratnam, M., Turk, J. & Vroegop, P. Case report: autistic disorder and chromosomal abnormality 46, XX duplication (4) p12-p13. Eur Chld Adolesc Psychiatry 9, 307-11 (2000). 63. Ramasamy, A. et al. Genetic variability in the regulation of gene expression in ten regions of the human brain. Nat Neurosci 17, 1418-28 (2014). 64. Hawrylycz, M. J. et al. An anatomically comprehensive atlas of the adult human brain transcriptome. Nature 489, 391-9 (2012). 65. Letra, A. et al. Follow-up association studies of chromosome region 9q and nonsyndromic cleft lip/palate. Am J Med Genet A 152A, 1701-10 (2010). 66. Bonilla-Claudio, M. et al. Bmp signaling regulates a dose-dependent transcriptional program to control facial skeletal development. Development 139, 709-19 (2012). 67. Cobourne, M. T. et al. Sonic hedgehog signalling inhibits palatogenesis and arrests tooth development in a mouse model of the nevoid basal cell carcinoma syndrome. Dev Biol 331, 38-49 (2009). 68. Dennis, J. F. et al. Mutations in Hedgehog acyltransferase (Hhat) perturb Hedgehog signaling, resulting in severe acrania-holoprosencephaly-agnathia craniofacial defects. PLoS Genet 8, e1002927 (2012). 69. Dobreva, G. et al. SATB2 is a multifunctional determinant of craniofacial patterning and osteoblast differentiation. Cell 125, 971-86 (2006). 70. Kurosaka, H., Iulianella, A., Williams, T. & Trainor, P. A. Disrupting hedgehog and WNT signaling interactions promotes cleft lip pathogenesis. J Clin Invest 124, 1660-71 (2014). 71. Metzis, V. et al. Patchedi is required in neural crest cells for the prevention of orofacial clefs. Hum Mol Genet 22, 5026-35 (2013). 72. Singh, S., Yin, X., Pisano, M. M. & Greene, R. M. Molecular profiles of mitogen activated protein kinase signaling pathways in orofacial development. Birth Defects ResA Clin Mol Teratol 79, 35-44 (2007). 73. Zhao, X. et al. The role of SATB2 in skeletogenesis and human disease. Cytokune Growth Factor Rev 25, 35-44 (2014). 74. Minoux, M. & Rijli, F. M. Molecular mechanisms of cranial neural crest cell migration and patterning in craniofacial development. Development 137, 2605-21 (2010). 75. Phillips, H. M. et al. Neural crest cell survival is dependent on Rho kinase and is required for development of the mid face in mouse embryos. PLoS One 7, e37685 (2012). 76. Ge, X. et al. Modeling supravalvular aortic stenosis syndrome with human induced pluripotent stem cells. Circulation 126, 1695-704 (2012). 77. Burgold, T. et al. The H3K27 Demethylase JMJD3 is Required for Maintenance of the Embryonic Respiratory Neuronal Network, Neonatal Breathing, and Survival. Cell Rep 2, 1244-58 (2012). 78. Mohn, F. et al. Lineage-specific polycomb targets and de novo DNA methylation define restriction and potential of neuronal progenitors. Mol Cell 30, 755-66 (2008). 79. Burgold, T. et al. The histone H3 lysine 27-specific demethylase Jmjd3 is required for neural commitment. PLoS One 3, e3034 (2008). 80. Bernstein, B. E. et al. A bivalent chromatin structure marks key developmental genes in embryonic stem cells. Cell 125, 315-26 (2006).