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Table 2 iPSCs derived from patients with ASD associated CNVs

From: Copy number variants (CNVs): a powerful tool for iPSC-based modelling of ASD

CNV Type and size of CNV Source Reprogramming Number of patients and healthy controls Differentiation protocol Neuronal cell types Associated cellular phenotype Ref.
1p21.3 Deletion
Size of CNV—not available in the paper
Keratinocytes Patient: CytoTune-iPS Sendai Reprogramming Kit
Controls: Constitutive Polycistronic Lentivirus Reprogramming Kit
Patient: 1
Controls: 3
Neurons (cortical neuron differentiation method based on dual SMAD inhibition) Neural precursors
Neural cells
Delay in expression of neuronal markers
Dynamic imbalance in GABA/glutamate cell populations over time
Enrichment of gene networks identified in autism post-mortem brains
Adhya et al., 2019 [38]
1p33 323 kb deletion/+
(chr1:49894000-50224000del) (primary genetic variant)
Other ASD implicated variants detected in patient:
- 2q21.1 516 kb duplication/+
- HTR3A p.G148X/+
Fibroblasts CytoTune-iPS Sendai Reprogramming Kit Patient: 1
Controls: 11 (total in the study—controls: 11; ASD-affected: 14)
Neurons (NGN2 ectopic expression approach) Glutamatergic neurons Reduced weighted mean firing rate Deneault et al., 2019 [39]
2p16.3 Bi-allelic NRXN1-α deletion
- Paternal deletion: exon 1-5, ~ 0.4 kb
- Maternal deletion: exon 1-5, ~ 0.18 kb
Fibroblasts CytoTune-iPS reprogramming kit Patient: 1
Controls: 4
Neuroepithelial stem cells
Neurons (modified dual SMAD inhibition protocol)
Neuroepithelial stem cells
Neurons
Slower proliferation rate
Expression of radial glia-like genes and preferentially differentiation to astroglia
Depressed calcium signalling capacity, lower levels of neurotransmitter, impairment of maturation
Lam et al., 2019 [40]
2p16.3 Deletion
Size of CNV—not available in the paper
Keratinocytes Patients: CytoTune-iPS Sendai Reprogramming Kit
Controls: Constitutive Polycistronic Lentivirus Reprogramming Kit
Patients: 2
Controls: 3
Neurons (cortical neuron differentiation method based on dual SMAD inhibition) Neural precursors
Neural cells
Delay in expression of neuronal markers
Dynamic imbalance in GABA/glutamate cell populations over time
Enrichment of gene networks identified in autism post-mortem brains
Adhya et al., 2019 [38]
2p16.3 De novo 430 kb deletion/+
(chr2:50567944-51057790del)
Fibroblasts Retroviruses expressing OCT4/POU5F1, SOX2, KLF4 and MYC and lentiviral vector containing pluripotency reporter EOS-GFP/PuroR Patient: 1
Controls: 11 (total in the study—controls: 11; ASD-affected: 14)
Neurons (NGN2 ectopic expression approach) Glutamatergic neurons No differences in weighted mean firing rate between patient and pool of all controls Deneault et al., 2019 [39]
3p Deletion
Size of CNV - not available in the paper
Keratinocytes Patient: CytoTune-iPS Sendai Reprogramming Kit
Controls: Constitutive Polycistronic Lentivirus Reprogramming Kit
Patient: 1
Controls: 3
Neurons (cortical neuron differentiation method based on dual SMAD inhibition) Neural precursors
Neural cells
Delay in expression of neuronal markers
Dynamic imbalance in GABA/glutamate cell populations over time
Enrichment of gene networks identified in autism post-mortem brains
Adhya et al., 2019 [38]
3p26.3 ~ 1 Mb microduplication (begins approximately 600 kb upstream of the CNTN6 gene and ends more than 50 kb downstream of its stop codon) Fibroblasts LeGO lentiviral vectors containing OCT4, SOX2, C-MYC and KLF4 Patient: 1
Controls: 2
Neurons (Ngn2 overexpression protocol)
Neurons (through neural rosette stage)
Neural progenitors
Layer 2/3 excitatory
Cortical neurons
Neurons showed the characteristics of mature neurons based on the presence of neuronal markers and their electrophysiological activities Gridina et al., 2018 [41]
de l[5](p14) Microdeletion
Size of CNV -not available in the paper
Peripheral blood mononuclear cells CytoTune-iPS Sendai Reprogramming Kit Patient: 1
Control: 1
/ / / Piovani et al., 2019 [42]
7q11.23 ~ 1.6–1.8 duplication Fibroblasts Synthetic mRNAs encoding the POU5F1 (OCT4), SOX2, KLF4, LIN28A and MYC Patients: 2
Controls: 3
Dorsal telencephalic lineage
Neural crest stem cells
Telencephalic neural progenitor cells
Neural crest stem cells
Disruption of transcriptional circuits in disease-relevant pathways Adamo et al., 2015 [43]
8p23.3 De novo 791 kb duplication/+
(chr8:704001-1535000dup) (primary genetic variant)
Other ASD implicated variants detected in patient:
- 8p22-p21.3 823 kb duplication/+
- RNF148 p.R225X/+
- CHD7 p.E1897K/+
- RAI1 p.G1864R/+
Fibroblasts CytoTune-iPS Sendai Reprogramming Kit Patient: 1
Controls: 11 (2 family controls—unaffected father and affected brother) (total in the study—controls: 11; ASD-affected: 14)
Neurons (NGN2 ectopic expression approach) Glutamatergic neurons No difference in weighted mean firing rate between patient and family controls Deneault et al., 2019 [39]
8q21.12 -
q21.13
Deletion
Size of CNV - not available in the paper
Additional findings detected in patient: 19:41759516 C>T
Keratinocytes Patient: CytoTune-iPS Sendai Reprogramming Kit
Controls: Constitutive Polycistronic Lentivirus Reprogramming Kit
Patient: 1
Controls: 3
Neurons (cortical neuron differentiation method based on dual SMAD inhibition) Neural precursors
Neural cells
Delay in expression of neuronal markers
Dynamic imbalance in GABA/glutamate cell populations over time
Enrichment of gene networks identified in autism post-mortem brains
Adhya et al., 2019 [38]
9q34.3 Mosaic 233 kb microdeletion (proximal breakpoint between exons 4 and 5 of the EHMT1 gene and distal breakpoint between exons 10 and 11 of the CACNA1B gene) Fibroblasts Retroviral vectors expressing OCT4, SOX2, KLF4 and cMYC
CRISPR line was generated by nonintegrating Sendai virus
Patient: 1 (iPS clone harbouring the microdeletion as well as a control clone not carrying the microdeletion were selected)
Controls: 2
Neurons (Ngn2 overexpression protocol) Excitatory cortical layer 2/3 neurons Reduced H3K9me2 immunoreactivity
Network bursts is occurred at a lower frequency and with longer duration
Longer inter-burst interval
Smaller percentage of spikes occurring outside the network bursts
Network burst activity strongly depends on NMDA receptor mediated transmission
Frega et al., 2019 [44];
Willemsen et al., 2011 [45]
11q22.1 Maternal 676 kb deletion/+
(chr11:99477401-100157000del)
Fibroblasts CytoTune-iPS Sendai Reprogramming Kit Patient: 1
Controls: 11 (1 family control) (total in the study: controls: 11; ASD-affected: 14)
Neurons (NGN2 ectopic expression approach) Glutamatergic neurons Increased neuronal activity Deneault et al., 2019 [39]
Deletion in chromosome 14 4.8 kb deletion (chr14:39987476-39992327) Fibroblasts retroviruses containing OCT4, SOX2, KLF4 and c-MYC Patient: 1
Controls:
(a) 2 unaffected, first-degree family members (mother, father)
(b) PGP1-1 iPSC line (Ball et al., 2009)
(c) K3 iPSC line (Si-Tayeb et al., 2010)
Telencephalic organoids Radial glia
Intermediate progenitors
Neurons
Upregulation of genes involved in cell proliferation, neuronal differentiation and synaptic assembly
Decrease in cell-cycle length in iPSCs and neuronal progenitors
Increased neuronal differentiation and synaptic connections
Increase in the number of inhibitory synapses
Overproduction of GABAergic inhibitory neurons
Mariani et al., 2015 [46];
Abyzov et al., 2012 [47]
15q11.2 382 kb microdeletion between BP1 and BP2 Fibroblasts Sendai virus (five constructs -TS7-OCT3/4, -SOX2, -KLF4, -c-MYC and GFP) Patients 2
Control: 1
Neurons Neural progenitor cells
Neurons
Altered dendritic morphology Das et al., 2015 [48]
15q11.2-q13.1 ~ 5.57 Mb duplication (chr15:21,144,837-26,722,409) Peripheral blood mononuclear cells Episomal vectors (pCE-hOCT3/4, pCE-hSK, pCE-hUL, pCE-mp53DD and pCXB-EBNA1) Patient: 1 / / / Arioka et al., 2018 [49]
15q11- q13.1 Isodicentric and interstitial duplications of 15q11-q13 Fibroblasts
Umbilical cord blood
Retroviral, lentiviral or episomal vectors encoding OCT4, SOX2, KLF4, MYC and LIN28 Patients: 4 (two individuals with isodicentric [15], one with a paternally inherited duplication of chromosome 15q11-q13.1 and one individual mosaic for a maternally inherited interstitial duplication of chromosome 15q11-q13.1)
Control: 1
Neuron (embryoid body-based protocol or monolayer differentiation) Vesicular glutamate transporter 1-positive excitatory neurons
Glutamate decarboxylate 65-positive inhibitory
Neurons
Downregulation of genes involved in neuron
development
Upregulation of genes involved in cell cycle and protein catabolic processes in isodicentric chromosome 15 neurons
Germain et al., 2014 [50]
15q13.3 Heterozygous 15q13.3 deletions and duplications
Patient 1—BP4/BP5 duplication (2.1 Mb) (second hit CNVs detected - 6q21 duplication)
Patient 2—BP4/BP5 deletion
Patient 3—BP3/BP5 deletion (second hit CNVs detected -17q12 loss)
Patient 4—BP3/BP5 duplication
Patient 5—D-CHRNA7-LCR/BP5 duplication
Patient 6—BP4/BP5 deletion
Fibroblasts CytoTune-iPS Sendai Reprogramming Kit Patients: 6
Controls: 3
Neural progenitor cells (NPCs) (dual SMAD inhibition protocol) Cortical-like neural progenitor cells α7 nicotinic acetylcholine receptor (α7 nAChR)-associated calcium flux was decreased in 15q13.3 deletion and duplication probands
Increased gene expression of chaperones involved in folding, assembly and trafficking α7 nAChRs in 15q13.3 duplication NPCs
Increased expression of a subset of ER stress markers in 15q13.3 duplication NPCs
Decreased expression of JAK2 in both CNV groups
Gillentine et al., 2017 [51]
16p11.2 De novo 616 kb deletion/+
(chr16:29584000-30200000del)
Fibroblasts Retroviruses expressing OCT4/POU5F1, SOX2, KLF4 and MYC and lentiviral vector containing pluripotency reporter EOS-GFP/PuroR Patient: 1
Controls: 11 (1 family control—unaffected father) (total in the study: Controls: 11; ASD-affected: 14)
Neurons (NGN2 ectopic expression approach) Glutamatergic neurons / Deneault et al., 2019 [39]
16p11.2 Microduplication (1 patient)
Microdeletion (3 patients)
Size of CNV—not available in the paper
Fibroblasts Episomal plasmids pCXLE-hOct3/4-shp53-F, pCXLE-hSox2-Klf4, pCXLE-hcmyc-Lin28 Patient: 4
Controls: 4
Neurons Forebrain cortical neurons 16pdup neurons—reduced neuronal size and dendrite length, less complex dendritic arbors, reduced soma size, reduced synaptic density, increased synaptic strength and lower density of excitatory synapses
16pdel neurons—increased soma size and dendrite length, more extensive dendritic arbors, reduced synaptic density, increased synaptic strength, lower density of excitatory synapses, higher current needed to fire first action potential (AP)
16pdel neurons fired far fewer APs than the control and 16pdup neurons
Reduced voltage responses of 16pdel neurons
Deshpande et al., 2017 [52]
16p13.11 Heterozygous 1.65 Mb microduplication (chr16: 14,892,975-16,544,033)
(de novo loss-of-function variant in TSC2 at 16:2115634:C/T)
Fibroblasts Episomal—plasmids containing Oct4/shP53, SOX2/Klf4 and L-Myc/Lin28 (pCXLE-hSK and pCXLE-hUL)
Control 5—retroviruses containing Oct4, Sox2, Klf4 and c-MYC
Patient: 1
Controls: 5
Anterior neural precursor cells (NPCs)
Cerebral organoids
Anterior neural precursor cells
Cerebral organoids (neural precursor cells and neurons)
Reduced NPC proliferation
Smaller organoids
Organoids
- Far fewer neuronal progenitor cell regions
- Reduced numbers of total dividing neuronal progenitor cells
- Altered planes of cell division
Deficit in the NFκB p65 pathway in NPCs and cerebral organoids
Johnstone et al., 2019 [53]
17p13.3 Patient 1—4.5 Mb deletion
Patient 2—5.7 Mb deletion
Patient 3—2.7 Mb deletion
Fibroblasts Episomal—plasmids encoding OCT3/4, SOX2, KLF4, L-MYC, LIN28 and shRNA for TP53 Patients: 3
Controls: 3
Cerebral organoids Cerebral organoids (neuroepithelial stem cells, neurons) Smaller organoids
Organoids
- Increased apoptosis in cortical ventricular zone-like regions
- Decreased vertical divisions
- Defective neuronal migration
- Increased abundance of deep-layer neurons
Mitotic defect (delay in cell division) in outer radial glia cells
Bershteyn et al., 2017 [54]
22q13 Patient 1—871 kb microdeletion
Patient 2—825 kb microdeletion
Fibroblasts Retroviruses carrying SOX2, OCT3/4, c-MYC, KLF4 Patients: 2
Controls: H9-ESC line, IM23-9 and NH1-1 cell lines (Yazawa et al., 2011; Paşca et al., 2011)
Neurons FoxG1/Pax6- positive telencephalic neuronal precursors
Neurons
Reduced number of neurons
Defects in excitatory synaptic transmission
Reduced number of excitatory synapses
Reduction in the expression of glutamate receptors
Reduced level of SHANK3 protein expression
Shcheglovitov et al., 2013 [55]
22q13.33 Microdeletion in SHANK3 gene
Size of CNV—not available in the paper
Keratinocytes Polycistronic lentiviral construct coexpressing OCT4, SOX2, KLF4 and c-MYC Patients: 2
Controls:3
Neurons (modified version of dual SMAD inhibition protocol) Cortical and olfactory placodal neurons Fewer puncta labelled with both presynaptic and postsynaptic markers
Placodal neurons—smaller cell diameter, higher neurite length and the mean number of neurites
Cortical neurons—shorter neurites
Higher rate of formation of the primary neurite
Lower rate of primary neurite elimination
Higher rate of extension of primary neurite length
Lower rate of primary neurite length retraction
Reduced soma speed
Slower growth
Kathuria et al., 2018 [56];
Cocks et al., 2014 [57]
Xp22.11 Patient 1—167 kb microdeletion that eliminates the promoters and first exons of PTCHD1 and PTCHD1-AS
Patient 2—125 kb microdeletion that eliminates the conserved third exon of PTCHD1-AS and DDX53
Fibroblasts
CD34+ blood cells
Fibroblasts—retrovirus vectors
blood cells—Sendai virus
Patients: 2
Controls: 2 (unaffected mother of one patient and unaffected male)
Neurons Neural progenitors cells
Cortical neurons
Reduced miniature excitatory postsynaptic current frequency
N-methyl-d-aspartate receptor hypofunction
Ross et al., 2019 [58]
Xq11.1 216.7 kb microdeletion (chrX.hg19:g.62856174_63072861) including the entire CB gene (ARHGEF9) Fibroblasts Retrovirus vectors containing the OCT4, c-MYC, KLF4 and SOX2 Patient: 1
Controls: 2
Neurons Neural progenitor cells
Cortical neurons
Increases in mTORC1 signalling activation and translation initiation Machado et al., 2016 [59]
Xq28 Patient 1—300 kb duplication (Xq28 (152.73–153.02 Mb))
Patient 2—15.25 Mb (Xq28 (139.33–154.58 Mb)) duplication
Patient 3—500 kb (Xq28 (152.66–153.15 Mb)) duplication
Fibroblasts pMXs retroviral vectors containing OCT4, SOX2, KLF4 and C-MYC Patient: 3
Controls: 2 healthy persons and BJ1 fibroblasts
Neurons Forebrain progenitors
Pyramidal neurons
Increased synaptogenesis and dendritic complexity
Altered neuronal network synchronisation
Nageshappa et al., 2016 [60]