Identification CNVs
To detect CNVs, aCGH was performed in all DNA samples from the 16 subjects with ASD (13 males and 3 females). We identified 364 CNVs (153 deletions and 211 duplications) with an average genomic size of 211.982 kb (114.091 kb for deletions and 258.705 kb for duplications). The mean number of CNVs per subject was 22.750 (9.563 for deletions and 13.188 for duplications). The mean number of deletions in male (10.462) was greater than that in females (5.667) (Table 1).
Table 1
The Characteristics of genome-wide CNVs among our subjects
Characters
|
Number (proportion %) of CNVs
|
Median CNV Size (kb)
|
Mean Number of CNVs per Subject
|
Total
|
364 (100.0)
|
211.982 (78.813, 705.031)
|
22.750
|
Male
|
299 (82.1)
|
213.241 (81.797, 864.947)
|
23.000
|
Female
|
65 (17.9)
|
132.360 (73.256, 305.926)
|
21.667
|
Duplication
|
211 (58.0)
|
258.705 (86.903, 708.853)
|
13.188
|
Male
|
163 (77.3)
|
305.926 (86.903, 1092.277)
|
12.538
|
Female
|
48 (22.7)
|
133.712 (87.668, 377.979)
|
16.000
|
Deletion
|
153 (42.0)
|
114.091 (73.256, 656.149)
|
9.563
|
Male
|
136 (88.9)
|
114.518 (73.256, 693.563)
|
10.462
|
Female
|
17 (11.1)
|
73.256 (60.021, 135.433)
|
5.667
|
A total of 13 males and 3 females.
|
|
|
Identification potential pathogenic CNVs of ASD
A total of 20 CNVs from 364 CNVs failed to be converted to GRCh37 (hg19); thus, we obtained 72 benign, 65 likely benign, 9 VOUS, 167 likely pathogenic, and 31 pathogenic CNVs (Table 2). We found that more than half CNVs were likely pathogenic or pathogenic.
Table 2
The Classification of CNVs based on ACMG
Classification
|
Total (%)
|
Duplication (%)
|
Deletion (%)
|
Benign
|
72 (19.8)
|
35 (16.6)
|
37 (24.2)
|
Likely Benign
|
65 (17.9)
|
59 (28.0)
|
6 (3.9)
|
VOUS
|
9 (2.5)
|
9 (4.3)
|
0 (0.0)
|
Likely Pathogenic
|
167 (45.9)
|
68 (32.2)
|
99 (64.7)
|
Pathogenic
|
31 (8.5)
|
28 (13.3)
|
3 (2.0)
|
VOUS: variant of unknown significance; A total of 20 CNVs failed to be converted to GRCh37 (hg19), thus, the total proportion was not equal to 100%. ACMG: American College of Medical Genetics guideline
|
After we calculated the frequency of each overlapping or non-overlapping CNV in DNA samples from our subjects, 344 CNVs were converted into 115 kinds of CNVs (45 deletions and 70 duplications). All the 115 kinds of CNVs were further classified (benign: 13 kinds; likely benign: 18 kinds; VOUS: two kinds; likely pathogenic: 60 kinds; and pathogenic: 13 kinds) (Supplementary Table 1). The distribution of the 115 kinds of CNVs in chromosome is visualized by circular plot (Fig. 1).
We investigated SFARI, AutismKB, and DECIPHER database to identify potential pathogenic CNVs from the 115 kinds of CNVs, revealing that 22 kinds of CNVs (6 deletions and 16 duplications) were potential pathogenic. The 22 kinds of CNVs were distributed in chromosome 1, 2, 5, 7, 10, 11, 12, 14, 15, 16, 22, and X. Among them, 19 kinds of CNVs were rare (Table 3).
Table 3
Summary of candidate CNVs of ASD
M/F No.
|
Coordinates, hg18
|
Cytoband
|
Size (Kb)
|
CNV Type
|
Classification
|
Rare/Common CNV
|
Number of genes
|
Gene Name
|
1/0
|
chr1:1179223–2271500
|
1p36.33
|
1092.277
|
Duplication
|
LP
|
Rare
|
42
|
DVL1, TMEM52
|
1/0
|
chr1:5998727–6334157
|
1p36.31
|
335.430
|
Duplication
|
LP
|
Rare
|
9
|
CHD5
|
4/0
|
chr1:225876894–226738916
|
1q42.13
|
862.022
|
Duplication
|
LP
|
Rare
|
26
|
PRSS38
|
1/0
|
chr2:31412158–32712484
|
2p23.1-p22.3
|
1300.327
|
Deletion
|
P
|
Rare
|
12
|
BIRC6, SPAST, SRD5A2
|
1/0
|
chr5:360041–873365
|
5p15.33
|
513.324
|
Duplication
|
LP
|
Common
|
11
|
AHRR, EXOC3, PDCD6
|
1/0
|
chr5:1115468–8452427
|
5p15.33-p15.2
|
7336.959
|
Duplication
|
P
|
Rare
|
49
|
ADCY2, SLC6A3, TERT
|
2/0
|
chr7:524935–1037461
|
7p22.3
|
512.526
|
Duplication
|
LP
|
Rare
|
13
|
ADAP1, PRKAR1B
|
1/0
|
chr7:1037461–2536804
|
7p22.3-p22.2
|
1499.343
|
Duplication
|
LP
|
Rare
|
26
|
INTS1
|
1/0
|
chr7:103622888–104803388
|
7q22.1-q22.2
|
1180.501
|
Deletion
|
LP
|
Rare
|
8
|
KMT2E, LHFPL3
|
13/0
|
chr10:89540133–91524263
|
10q23.2-q23.31
|
1984.131
|
Deletion
|
LP
|
Rare
|
31
|
PTEN
|
1/0
|
chr10:127658856–135254513
|
10q26.2-q26.3
|
7595.658
|
Duplication
|
P
|
Rare
|
58
|
DOCK1, EBF3, GLRX3
|
5/2
|
chr11:498019–2179368
|
11p15.5
|
1681.349
|
Duplication
|
P
|
Rare
|
84
|
BRSK2, CD151, CTSD, DEAF1, DRD4, HRAS, IGF2, PHRF1, TALDO1
|
3/0
|
chr11:132773688–134043707
|
11q25
|
1270.019
|
Duplication
|
LP
|
Rare
|
16
|
IGSF9B
|
6/0
|
chr12:25156062–27414420
|
12p12.1-p11.23
|
2258.359
|
Deletion
|
LP
|
Rare
|
17
|
KRAS, MED21
|
1/0
|
chr14:22086438–22354007
|
14q11.2
|
267.569
|
Deletion
|
LP
|
Rare
|
4
|
SLC7A7
|
1/0
|
chr15:29809025–30298155
|
15q13.3
|
489.131
|
Deletion
|
P
|
Rare
|
1
|
CHRNA7
|
0/2
|
chr16:2021433–2484806
|
16p13.3
|
463.373
|
Duplication
|
LP
|
Common
|
32
|
PGP, PKD1, RNPS1, SLC9A3R2, TRAF7, TSC2
|
0/3
|
chr16:2484806–2747528
|
16p13.3
|
262.722
|
Duplication
|
LP
|
Common
|
13
|
SRRM2
|
2/0
|
chr16:61464644–64965235
|
16q21
|
3500.591
|
Duplication
|
LP
|
Rare
|
4
|
CDH11
|
4/0
|
chr22:46395224–49412774
|
22q13.31-q13.33
|
3017.550
|
Duplication
|
P
|
Rare
|
44
|
CHKB, MAPK12, MAPK8IP2, PANX2, PPP6R2, SBF1, TRABD
|
2/0
|
chr22:49412774–49525130
|
22q13.31-q13.33
|
112.356
|
Duplication
|
P
|
Rare
|
3
|
SHANK3
|
1/0
|
chrX:67331017–68768438
|
Xq12-q13.1
|
1437.422
|
Duplication
|
P
|
Rare
|
8
|
OPHN1
|
M: Male; F: Female. M/F No. means the number of CNV among male/female. P: Pathogenic; LP: Likely Pathogenic. The genes were reported to be related with ASD.
|
Identification of potential pathogenic genes with CNVs of ASD
A total of 511 genes from the 22 potential pathogenic CNVs were functionally annotated by GO. The annotated genes were classified into three GO domains (biological processes [BP], cellular component [CC], and molecular function [MF]). For BP, some gene sets were enriched in synaptic-related functions, including modulation of chemical synaptic transmission (GO: 0050804), regulation of trans-synaptic signaling (GO: 0099177), positive regulation of excitatory postsynaptic potential (GO: 2000463), positive regulation of synaptic transmission (GO: 0050806), chemical postsynaptic transmission, (GO: 0099565), modulation of excitatory postsynaptic potential (GO: 0098815), and regulation of postsynaptic membrane potential (GO: 0060078), and in central nervous system related functions (positive regulation of neurological system process [GO: 0031646]). For CC, the top five CC terms included keratin filament (GO: 0045095), myelin sheath (GO: 0043209), Golgi lumen (GO: 0005796), glutamatergic synapse (GO: 0098978), and neuron to neuron synapse (GO: 0098984). For MF, the top five MF terms encompassed catecholamine binding (GO: 1901338), dopamine binding (GO: 0035240), magnesium ion binding (GO: 0000287), insulin receptor binding (GO: 0005158), and lipase activity (GO: 0016298). The top 20 GO functions are presented in Fig. 2 and Supplementary Table 2, 3, 4.
KEGG pathway enrichment analysis showed enriched key pathways, such as dopaminergic synapse (hsa04728), mTOR signaling pathway (hsa04150), GnRH signaling pathway (hsa04912), cholinergic synapse (hsa04725), and MAPK signaling pathway (hsa04010). The top 20 pathways are presented in Fig. 2 and Supplementary Table 5.
We constructed intersections among 511 genes that we found, 363 high-confidence or strong-candidate risk genes of ASD reported in SFARI database, and 228 high-confidence risk genes related to ASD reported in AutismKB database (Fig. 3). After investigating genes in the intersections, we found that cholinergic receptor nicotinic alpha 7 subunit gene (CHRNA7) was involved in the regulation of excitatory postsynaptic potential and cholinergic synapse; dopamine receptor D4 gene (DRD4) was involved in the regulation of synaptic transmission, dopamine binding, and glutamatergic synapse; HRas proto-oncogene (HRAS) played roles in the regulation of excitatory postsynaptic potential, glutamatergic synapse, and mTOR signal pathway; oligophrenin 1 gene (OPHN1) correlated with regulated synaptic signal, ionic glutamate receptor binding, and glutamatergic synapse; phosphatase and tensin homolog (PTEN) was implicated in the regulation of synaptic signal, neuron differentiation of central nervous system, ionic glutamate receptor binding, sphingolipid signaling, and mTOR signaling; SH3 and multiple ankyrin repeat domains 3 gene (SHANK3) was involved in the regulation of synaptic signal, ionic glutamate receptor binding, neuronal synapse, postsynaptic density, and asymmetric synapse; solute carrier family 6 member 3 gene (SLC6A3) played roles in dopamine binding, neurotransmitter: sodium cotransporter activity, and neurotransmitter transport activity; and TSC complex subunit 2 gene (TSC2) was involved in synapses, postsynaptic density, asymmetric synapses, and mTOR signaling pathways. Scores of all these genes (CHRNA7, DRD4, HRAS, OPHN1, PTEN, SHANK3, SLC6A3, and TSC2) in AustismKB and corresponding ranks in SFARI are listed in Table 4. DRD4, HRAS, OPHN1, SHANK3, SLC6A3, and TSC2 were in the regions of CNVs duplication. CHRNA7 and PTEN were in the regions of CNVs deletion.
Table 4
Summary of candidate genes of ASD
Gene Name
|
CNV Type
|
M/F No.
|
Category of gene in SFARI
|
Score of gene in AutismKB
|
CHRNA7
|
Deletion
|
1/0
|
2
|
#
|
DRD4
|
Duplication
|
5/2
|
—
|
30
|
HRAS
|
Duplication
|
5/2
|
1
|
20
|
OPHN1
|
Duplication
|
1/0
|
2
|
#
|
PTEN
|
Deletion
|
13/0
|
1
|
78
|
SHANK3
|
Duplication
|
2/0
|
1
|
62
|
SLC6A3
|
Duplication
|
1/0
|
2
|
30
|
TSC2
|
Duplication
|
0/2
|
1
|
46
|
M: Male; F: Female. M/F No. means the number of CNV among male/female. —: Not reported as high-confidence or strong candidate autism risk genes in SFARI. #: Not reported as high-confidence autism risk genes in AutismKB.
|
Identification and Analysis of potential pathogenic miRNAs with CNVs of ASD
We found 50 potential-pathogenic-CNVs-related miRNAs (45 encoded by duplication regions and 5 encoded by deletion regions). According to experimental evidence documenting nervous system dysfunction, we retrieved PubMed, identifying that 12 miRNAs genes were previously reported to be associated with brain or nervous system dysfunction (Table 5).
Table 5
miRNAs with function related to brain or nervous system in CNVs
miRNA ID
|
CNV Type
|
Functional relevance
|
Reference (PMID)
|
miR-202
|
Duplication
|
Depression, Glioma, Neuroblastoma
|
32425535; 28714009; 21654684; 24337320
|
miR-210
|
Duplication
|
Alzheimer's disease, Epilepsy, Glioblastoma, Glioma, Head and neck paragangliomas, Neuroblastoma, Neuroprotective effects
|
31092279; 23108914; 21655185; 22977270; 23902947; 25279461; 24729345; 24382515; 25481483; 24930954; 25756397; 29126304; 29362886; 31146085; 32194691; 31896490; 29226333; 30947960; 30746749; 27471387
|
miR-3178
|
Duplication
|
Neuropsychiatric diseases
|
30766477
|
miR-339
|
Duplication
|
Alzheimer's disease, Glioblastoma, Neuroendocrine neoplasias
|
32176627; 29983867; 30564636; 24352696; 30176243
|
miR-4516
|
Duplication
|
Glioblastoma
|
30559405
|
miR-4717
|
Duplication
|
Guillain-Barre Syndrome
|
27836180
|
miR-483
|
Duplication
|
Alzheimer's disease, Glioma, Neuroblastoma
|
31938135; 24577456; 22465663
|
miR-675
|
Duplication
|
Glioma
|
31468534; 28187439; 24466011
|
miR-6821
|
Duplication
|
Alzheimer's disease
|
27050411
|
miR-940
|
Duplication
|
Glioblastoma, Glioma
|
31497204; 30906627; 31934283; 29296221; 30431124
|
miR-107
|
Deletion
|
Alzheimer's disease, Amnestic mild cognitive impairment, Bipolar disorder, Brain disorders, Frontotemporal dementia, Glioblastomas, Glioma, Major depression, Neuroblastoma, Neurogenesis, Schizophrenia
|
31556571; 29258209; 28847283; 26084601; 30543171; 31250578; 27343180; 21625387; 20489155; 28578378; 25662174; 22811466; 20413881; 31778666; 31787850; 29885309; 29671226; 30056425; 30480816; 18234899; 29136645; 23811124; 27143098; 29073742; 25596705; 31605836; 31420923; 23220650; 22594617; 26223576; 23572380; 27501295; 27878295; 32124921; 23962497; 29286086; 21179570; 21111402
|
miR-558
|
Deletion
|
Neuroblastoma
|
25616966; 27276678
|
We intersected target genes predicted using miRWalk 2.0 database with the union between SFARI and AutismKB, presenting the interaction networks between CNVs-miRNAs and 219 target genes (Figs. 4 and 5).
We further investigated potential functions of the 219 target genes using GO analysis. For BP, some gene sets were enriched in synaptic-related functions, including synapse organization (GO: 0050808), modulation of chemical synaptic transmission (GO: 0050804), regulation of trans-synaptic signaling (GO: 0099177), synaptic transmission, glutamatergic (GO: 0035249), postsynaptic density organization (GO: 0097106), postsynaptic specialization organization (GO: 0099084) and regulation of glutamatergic synaptic transmission (GO: 0051966), and in central nervous system related functions, including learning or memory (GO: 0007611), cognition (GO: 0050890), and neurotransmitter transport (GO: 0006836). For CC, some gene sets were enriched in synaptic-related cellular components, including synapse membrane (GO: 0097060), postsynaptic specialization (GO: 0099572), and neuron to neuron synapse (GO: 0098984). For MF, some gene sets were enriched in ion-gated channel activity (GO: 0022839), gated channel activity (GO: 0022836), ion channel activity (GO: 0005216), ionotropic glutamate receptor activity (GO: 0004970), and transmitter gated channel activity (GO: 0022835). The top 20 GO functions are presented in Fig. 6 and Supplementary Table 6, 7, 8.
KEGG pathway enrichment analysis showed enriched key pathways, such as glutamatergic synapse (hsa04724), dopaminergic synapse (hsa04728), and Wnt signaling pathway (hsa04310). The top 20 pathways are presented in Fig. 6 and Supplementary Table 9.