The prevalence of Copy Number Variation (CNV) on human chromosome 16p11.2, identified in approximately 1% of autism spectrum disorder (ASD) cases globally, was found to be 1.2% in an Australian ASD cohort. Bioinformatic analysis identified 13 of the 25 protein coding genes within the 16p11.2 region, including KCTD13, as significantly enriched in the nervous system. Experiments in mice found that suppression of Kctd13 led to impaired radial migration of cortical neurons and altered neuronal morphology, providing insight into how perturbations to KCTD13 expression via 16p11.2 microdeletion could influence brain development. Further experiments showed decreased cell proliferation and mitosis levels

1. Autism spectrum disorder (ASD) is a brain developmental disorder that encompasses a group of separate neurological syndromes with a spectrum of clinical manifestations. In individuals diagnosed with ASD, Copy Number Variation (CNV) on human chromosome 16p11.2 is identified in
approximately 1% of cases, but the prevalence in Australian ASD cases is unclear. To address this we surveyed ASD affected individuals from the Western Australian Autism Biological Registry (WAABR) and report an incidence of 16p11.2 CNV as 1.2% (2/166, 95% [CI] = 0.1-4.3%). Alterations to
dosage levels of 16p11.2 genes are hypothesised to contribute to the neurodevelopment defects for ASD (Maillard et al., 2015). We therefore performed RNA-Seq analysis from buffy coat of the patients to substantiate a predicted reduction in 16p11.2 gene expression. To prioritise candidate 16p11.2
genes, we applied a bioinformatics approach and identified 13 out of the 25 protein coding genes within 16p11.2 to be significantly enriched within the nervous system. This led to a strong prediction that the alterations in dosage of 16p11.2 genes, including KCTD13, are important for neuronal
development. In support of this notion a series of in utero electroporation experiments in mice provided an insight into the role of Kctd13 in the morphological transition and migration of cortical neurons during development. This study provides a novel account by which perturbations to KCTD13
expression via 16p11.2 microdeletion could influence cell morphology and migration during brain development.
Disruptions to Kctd13 expression results in a decrease in mitosis levels. Cell proliferation assay in mouse embryonic P19 cell line, fixed and immunolabeled 48 h post-transfection. Transfection with equal
quantity of GFP expressing vectors: empty vector and control (scrambled shRNA), Kctd13 shRNA vector and empty vector control and forced expression of huKCTD13 with Kctd13 shRNA. (A) Representative
image, arrow points to DAPI, GFP and pHH3 positive cells. (B) Suppression of Kctd13 using shRNA results in a decrease in transfected cells labeled with the mitotic marker pHH3 compared to control. Forced
expression with huKCTD13 in Kctd13 shRNA treated cells leads to a reduced proportion of cells in mitosis compared to the control. Analysis with one-way ANOVA followed by Holm post hoc test, **p < 0.01.
Graph plots the mean + S.E.M of three biological replicates (n = 3 - 4 technical replicate in each with; GFP positive cells > 150 per coverslip). Scale bar represents 100µm.
Conclusion
The prevalence of 16p11.2 CNV is 1.2% in the Australian population and is consistent with the finding of approximately 1% frequency in several
larger worldwide studies of ASD cohorts. This suggests that the microdeletion to 16p11.2 is a likely risk factor in the development of ASD.
RNA sequencing analysis reveals a reduction in expression levels of 16p11.2 genes in patients harbouring the microdeletion.
Bioinformatic analysis identifies neuronal-enriched genes within the 16p11.2 region, suggesting that multiple genes could influence neuronal
development and contribute to the pathogenesis of ASD.
Knockdown of Kctd13 leads to impaired radial migration of neurons. We further determined that suppression of Kctd13 in neurons led to their
altered morphology during cortical development.
Perturbation to Kctd13 expression leads to reduction in cell proliferation in vitro with similar trends observed in vivo, indicating that a reduction
in Kctd13 expression leads to a decrease of cells in mitosis. The consequence of forced expression of human KCTD13 within Kctd13
suppressed cells on cell proliferation remains to be clarified.
Importantly, this research has provided clarification into the 16p11.2 genes implicated in the nervous system, including KCTD13. Analysis of
perturbations to Kctd13 expression in cerebral cortex development suggests that heterozygosity of KCTD13 in cases of 16p11.2 microdeletions
may be pathogenic in the context of ASD brain related abnormalities.
The role of the 16p11.2 gene KCTD13 in neuronal development & autism spectrum disorder
Kelly Berger, Ivan Gladwyn-Ng, Linh Ngo, ZhengDong Qu, Mathew Martin-Iverson and Julian Heng
The Harry Perkins Institute of Medical Research, QEII Medical Centre, 6 Verdun Street, Nedlands, WA 6009, Australia (K.B., IGN., L.N.,J.H), The Australian Regenerative Medicine Institute, Monash University, Clayton, VIC 3800, Australia (ZD.Q)
and the Centre for Medical Research, the University of Western Australia, Nedlands, WA 6009, Australia (K.B, M.MI)
Top CAGE expression
sample for p1@KCTD13
testis, adult, pool2
temporal lobe, fetal, donor1, tech
neutrophil PMN, donor1
brain, fetal, pool1
putamen, adult, donor10196
Neural stem cells, donor2
testis, adult, pool1
common myeloid progenitor CMP, donor1
occipital pole, adult, pool1
promyelocytes/myelocytes PMC, donor1
temporal lobe, adult, pool1
nucleus accumbens, adult, pool1
temporal lobe, fetal, donor1, tech rep1
substantia nigra - adult, donor10196
postcentral gyrus, adult, pool1
Neurons, donor2
occipital lobe, fetal, donor1
hippocampus - adult, donor10196
paracentral gyrus, adult, pool1
insula, adult, pool1
Smooth Muscle Cells - Umbilical Vein, donor3
granulocyte macrophage progenitor, donor1
frontal lobe, adult, pool1
parietal lobe, adult, pool1
hippocampus, adult, donor10252
Clontech Human Universal Reference Total RNA
somatostatinoma cell line:QGP-1
amygdala, adult, donor10252
CAGE expression (TPM, robust
cluster, rle)
0 20 40 60 80 100 120
The prevalence of 16p11.2 CNV in individuals diagnosed with ASD in an Australian cohort (WAABR)
Bioinformatic analysis to identify 16p11.2 candidate genes enriched in cells of the nervous system
CNV Frequency ASD cohort Reference
Australia 1.2% 2/166
Gladwyn-Ng et al.,
manuscript in preparation
Iceland 1.0% 3/299 Weiss et al., 2008
Global 0.9% 4/427 Marshall et al., 2008
USA 0.5% 4/712 Kumar et al., 2008
The first report of 16p11.2 CNV in individuals diagnosed with ASD in the Australian population is 1.2%.
A survey of 166 children diagnosed with ASD from the Western Australian
Autism Biological Registry (WAABR), has identified two individuals who
harbour deletions to the 16p11.2 locus. The recurrent 16p11.2 CNV
identified in this cohort is consistent with the finding of approximately 1%
frequency in several larger ASD studies. The relationship between the
pathological consequences of 16p11.2 CNV is hypothesised to be
attributable to perturbations in gene dosage.
Multipolar Uni/Bipolar Multipolar Uni/Bipolar
0%
20%
40%
60%
80%
100%
PercentageofGFPexpressingcells
control
Kctd13 shRNA
Kctd13 shRNA + huKCTD13
IZ CP
*
*
*
*
Suppression of Kctd13 impairs neuronal migration during cerebral cortex development
Altered expression of Kctd13 results in abnormal morphologies of
embryonic neurons
Summary schematic illustrating the effects of perturbations to Kctd13
resulting in impaired radial migration and abnormal neuronal shape
acquisition undertaken by migrating neurons
In vivo analysis of the candidate gene KCTD13 via a series of in utero electroporation
experiments using RNAi to target endogenous Kctd13 during mouse embryonic brain
development. Results demonstrate that knockdown of Kctd13 leads to impaired radial
migration of cortical neurons as cells fail to reach the CP, seen by an accumulation in the
underlying IZ. Further investigation demonstrates that suppression of Kctd13 expression
resulted in altered morphology of cells as there was an increase in the proportion of
multipolar shaped neurons accompanied by a decrease in uni/bipolar shaped cells in the IZ.
Together these results indicate that disruption to Kctd13 impairs the neuronal shape
acquisition undertaken by transitioning neurons, which may contribute to their impaired
migration into the CP.
Disruption to Kctd13 expression results in decreased abundance of the proliferative marker pHH3
Knockdown of Kctd13 alone, or with huKCTD13 results in altered morphology of neurons in the IZ of the developing embryonic cortex. In utero electroporation of E14.5 mouse cortex then sampled
three days later (E14.5 + 3). (A) Control shRNA and empty vector (B) Kctd13 shRNA and empty vector control and (C) Co-delivery of bicistronic GFP expression vector encoding Kctd13 shRNA vector and
huKCTD13 expression construct. Rightwards arrowheads point to uni/bipolar shaped neurons and leftward arrows indicate multipolar shaped cells. (D) Quantification of the morphologies of neurons being
multipolar and uni/bipolar neurons within the IZ and CP. Suppression of endogenous Kctd13 leads to a significant increase in the proportion of multipolar neurons with a concomitant decrease in uni/bipolar
shaped cells within the IZ compared with the control. Treatment with Kctd13 shRNA construct and forced expression of human KCTD13 lead to a significant change in the morphology of neurons within the IZ,
with an increased proportion of multipolar shaped cells with a corresponding decrease of neurons with a uni/bipolar morphology. Mixed model analysis followed by an exact-Sidak post hoc test, *p < 0.05 (n = 5
brain sections, with GFP positive cells > 200 per section). Graph plots the mean + S.E.M. Scale bar represents 100µm.
Forced expression of huKCTD13 in Kctd13 suppressed cells leads to a reduction in mitosis in vivo. In utero electroporation of E14.5 embryonic mouse cortex and analysed three days later. (A) control
vectors (GFP only), (B) bicistronic GFP expression vector encoding Kctd13 shRNA and (C) Co-delivery of bicistronic GFP expression vectors encoding human KCTD13 expression construct and Kctd13 shRNA.
(D) Quantification of the co-labeled, pHH3 and GFP positive cells within the cortex. Results indicate that there is a significant decrease in mitotically labeled cells in the embryonic cortex with the forced
expression of huKCTD13 in Kctd13 shRNA treatment, compared to the control. Pairwise t-test followed by Holm post hoc analysis, *p < 0.05 (n = 4 - 6 brain sections, with pHH3 and GFP positive cells > 15).
Graph plots the mean + S.E.M. Scale bar represents 100µm.
E14.5+3PHH3GFP
CP
MZ
VZ/SVZ
Kctd13 shRNA
Kctd13 shRNA +
huKCTD13control
Co-expression of human KCTD13 within Kctd13 shRNA cells leads to a reduction in mitosis compared to control treatment.
0%
2%
4%
6%
8%
10%
PercentageofpHH3+GFP+Cells(E14+3)
control
Kctd13 shRNA
Kctd13 shRNA + huKCTD13
*
*
References:
Kumar RA, KaraMohamed S, Sudi J, Conrad DF, Brune C, Badner JA, Gilliam TC, Nowak NJ, Cook EH, Dobyns WB and Christian SL (2007) Recurrent 16p11.2 microdeletions in autism. Hum Mol Genet, vol. 17, no. 4, pp. 628–638.
Marshall CR, Noor A, Vincent JB, Lionel AC, Feuk L, Skaug J, Shago M, Moessner R, Pinto D, Ren Y, Thiruvahindrapduram B, Fiebig A, Schreiber S, Friedman J, Ketelaars CEJ, Vos YJ, Ficicioglu C, Kirkpatrick S, Nicolson R, Sloman L, Summers A, Gibbons CA, Teebi A, Chitayat D, Weksberg R, Thompson A, Vardy C, Crosbie V, Luscombe S, Baatjes R, Zwaigenbaum L, Roberts W, Fernandez B, Szatmari P and Scherer SW (2008) Structural Variation of Chromosomes in Autism Spectrum Disorder. Am J Hum Genet, vol. 82, no. 2, pp. 477–488.
Maillard AM, Ruef A, Pizzagalli F, Migliavacca E, Hippolyte L, Adaszewski S, Dukart J, Ferrari C, Conus P, Männik K, Zazhytska M, Siffredi V, Maeder P, Kutalik Z, Kherif F, Hadjikhani N, Beckmann JS, Reymond A, Draganski B and Jacquemont, S (2015) The 16p11.2 locus modulates brain structures common to autism, schizophrenia and obesity. Mol Psychiatry, vol. 20, no. 1, pp. 140–147.
Weiss LA, Shen Y, Korn JM, Arking DE, Miller DT, Fossdal R, Saemundsen E, Stefansson H, Ferreira MAR, Green T, Platt OS, Ruderfer DM, Walsh CA, Altshuler D, Chakravarti A, Tanzi RE, Stefansson K, Santangelo SL, Gusella JF, Sklar P, Wu B-L and Daly MJ (2008) Association between Microdeletion and Microduplication at 16p11.2 and Autism. N Engl J Med, vol. 358, no. 7, pp. 667–675.
Acknowledgements
This work was supported by the Centenary Trust for Women Margaret Mills Memorial Honours Scholarship and the Brain Growth and Disease lab.
Of particular interest is the gene KCTD13 due to its interaction with known regulators
of neuronal migration (Gladwyn-Ng et al., manuscript in preparation). Profile of
brain-specific expression of KCTD13 in foetal and adult tissue demonstrates varying
levels of expression in brain structures.
FANTOM5 analysis (A) Sample ontology enrichment analysis indicates 13 out of the 25 protein-
coding genes within 16p11.2 CNV region, denoted in red, have significantly enriched promoter/s in
the nervous system (B) CAGE expression chart of the top 30 samples identified for promoter 1 of
KCTD13 (p1@KCTD13) and p2@ MAPK3 represented in tags per million (TPM). Red bars indicate
samples with the ontological annotation ‘nervous system. (C) Human brain-specific samples with
CAGE expression for p1@KCTD13 represented in normalised tags per million (TPM). Foetal brain
samples denoted in pale blue and adult brain samples in dark blue. This graph represents the
abundance of KCTD13 transcripts within brain specific structures during distinct developmental
periods. Error bars represent S.D. for n > 1.
0%
10%
20%
30%
40%
PercentageofpHH3+GFP+Cells(P19)
control
Kctd13 shRNA
Kctd13 shRNA + huKCTD13
* *
* *Representative Image (P19 cells)
DAPIGFPpHH3
➤
In vitro analysis of cells assessed proliferation
by immunolabelling transfected cells for
phosphorylated histone H3 (pHH3) which is
specific for cells within the mitotic phase of
the cell cycle. The result indicates that there
is a decrease in mitosis when Kctd13
expression is suppressed.
Calculated as exact binomial 95% confidence interval.
SPN
Q
PR
TC16orf54
ZG
16
KIF22
M
AZPR
R
T2PAG
R1
M
VP
C
D
IPTSEZ6L2ASPHD1KC
TD
13
TM
EM
219TAO
K2HIRIP3INO
80ED
O
C
2AFAM
57BALDO
A
PPP4C
TBX6
YPEL3G
D
PD
3M
APK3
0.0
0.5
1.0
1.5
2.0
Protein Coding Genes in 16p11.2
FoldChange(FPKM)
Expression Level Changes of Genes within the 16p11.2 Region
22 year old deletion vs 24 year old control
8 year old deletion vs 10 year old control
Gene expression analysis suggests alterations in 16p11.2 genes in
affected ASD patients.
RNA sequencing analysis on blood samples, extracted from the two affected individuals
and age matched controls. Data points represent the relative ratio (fold change) between
case and control expressed as normalised Fragments Per Kilobase of exon per Million
fragments mapped (FPKM). Comparative analysis indicates a general reduction in FPKM
gene expression levels for the 16p11.2 region, as most values fall below the baseline
value of one.
Analysis of the 25 protein coding genes identified 13 to have one or more promoters with significantly enriched
expression in the nervous system.
Utilising the FANTOM5 database enabled the identification of candidate promotes with significant expression in the nervous system. Expression profile
were generated that identifying cell type-specific transcription, with red denoting samples corresponding to tissue of the nervous system. This suggests
several genes within the 16p11.2 region as potential candidates that may contribute to neuronal development.
Top CAGE expression
sample for p2@MAPK3
chorionic membrane cells, donor3
Pericytes, donor1
medulloblastoma cell line:D283 Med :
Bronchial Epithelial Cell, donor3
Mesenchymal Stem Cells - adipose, donor1
temporal lobe, fetal, donor1, tech rep2
medial frontal gyrus - adult, donor10196
Fibroblast - Villous Mesenchymal, donor2
temporal lobe, fetal, donor1, tech rep1
lung, fetal, donor1
Fibroblast - Mammary, donor2
neuroectodermal tumor cell line:FU-RPNT-1
Neural stem cells, donor2
parietal lobe, fetal, donor1
synovial sarcoma cell line:HS-SY-II
Mesenchymal Stem Cells - hepatic, donor1
choriocarcinoma cell line:SCH
Renal Glomerular Endothelial Cells, donor4
rectum, fetal, donor1
occipital lobe, fetal, donor1
tenocyte, donor3
H9 Embryoid body cells, melanocytic induction
chorionic membrane cells, donor1
Osteoblast - differentiated, donor3
brain, fetal, pool1
H9 Embryoid body cells, melanocytic induction
spinal cord, adult, donor10252
amygdala, adult, donor10252
CAGE expression (TPM, robust
cluster, rle)
0 2 4 6 8 10 12
Brain expression profile for p1@KCTD13
temporal lobe (n=2)
brain
occipital lobe
parietal lobe
duodenum (n=2)
putamen
occipital pole
temporal lobe
nucleus accumbens
substantia nigra
postcentral gyrus
paracentral gyrus
insula
hippocampus (n=2)
frontal lobe
amygdala (n=2)
caudate nucleus (n=2)
corpus callosum
substantia nigra
parietal lobe (n=3)
brain (n=2)
occipital cortex (n=2)
medial frontal gyrus (n=2)
medial temporal gyrus (n=2)
thalamus (n=2)
occipital lobe
diencephalon
globus pallidus (n=2)
pons
pituitary gland (n=2)
medulla oblongata (n=3)
pineal gland (n=2)
cerebellum (n=3)
locus coeruleus (n=2)
CAGE expression (TPM, robust cluster, rle)
0 20 40 60 80 100 120
AdultBrainFoetalBrain
KCTD13 transcripts are detected in the human brain during foetal development and in adulthood
SPN
QPRT
c16orf54
Zg16
KIF22
MAZ
PRRT2
PAGR1
MVP
CDIPT
SEZ6L2
ASPHD1
KCTD13
TMEM219
TAOK2
HIRIP3
INO80E
DOC2A
FAM57B
ALDOA
PPP4C
TBX6
YPEL3
GDPD3
MAPK3
Perturbations to Kctd13 expression lead to impaired radial migration of
cortical neurons during mouse embryonic brain development
Perturbations to Kctd13 expression result in defective radial migration. In utero electroporation, which in brief is a method involving injection of expression constructs into the embryonic mouse brain and
genetically modifying the tissue, enabling quantification of transduced cells. This method was performed at embryonic day 14.5 and foetuses were allowed to develop for a further three days in utero (E14.5 + 3). (A)
control scrambled shRNA vector and empty expression vector (B) and bicistronic GFP expression vector encoding Kctd13 shRNA and empty vector control. (C) Co-delivery of bicistronic GFP expression vectors
encoding Kctd13 shRNA and human KCTD13 (huKCTD13) expression construct. (D) Quantification of the distribution of GFP-labeled cells within the E17.5 cortex indicating that suppression of endogenous
Kctd13 expression leads to impaired migration of treated cells as there is a significant reduction of GFP expressing cells in the CP along with an accumulation of treated cells in the IZ compared with the control. Co-
delivery of the huKCTD13 construct improved the migration of Kctd13 shRNA treated cells into the IZ, but a significantly reduced population of transfected cells were found within the CP. Analysis with two-way
ANOVA followed by Holm-Sidak post hoc test, *p < 0.05, **p < 0.01; ***p < 0.001 (n = 4 - 6 brain sections, with more than 200 transfected cells counted per section). Graph plots the mean + S.E.M. Scale bar
represents 100µm.
0% 20% 40% 60% 80%
VZ/SVZ
IZ
CP
Percentage of GFP expressing cells
conrol
Kctd13 shRNA
Kctd13 shRNA + huKCTD13
*
***
*
*
CP
IZ
VZ/
SVZ
CP
IZ
VZ/
SVZ
CP
IZ
VZ/
SVZ
CP
IZ
VZ/
SVZ
CP
IZ
VZ/
SVZ
CP
IZ
VZ/
SVZ
E14.5+3GFPE14.5+3DAPIGFP
Kctd13 shRNA Kctd13 shRNA +
huKCTD13
control
A
B
C
CorticalPlateGFP
Kctd13 shRNA
Kctd13 shRNA +
huKCTD13control
IntermediateZoneGFP
BA C
D
BA C
D
BA
BA C
D
➤
➡
➤
➤
➤
➤
➡
➡
➡