Table 2 iPSCs derived from patients with ASD associated CNVs

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]