De Novo Truncating Mutations in WASF1 Cause Intellectual Disability with Seizures

Abstract

2018 Next-generation sequencing has been invaluable in the elucidation of the genetic etiology of many subtypes of intellectual disability in recent years. Here, using exome sequencing and whole-genome sequencing, we identified three de novo truncating mutations in WAS protein family member 1 (WASF1) in five unrelated individuals with moderate to profound intellectual disability with autistic features and seizures. WASF1, also known as WAVE1, is part of the WAVE complex and acts as a mediator between Rac-GTPase and actin to induce actin polymerization. The three mutations connected by Matchmaker Exchange were c.1516C>T (p.Arg506Ter), which occurs in three unrelated individuals, c.1558C>T (p.Gln520Ter), and c.1482delinsGCCAGG (p.Ile494MetfsTer23). All three variants are predicted to partially or fully disrupt the C-terminal actin-binding WCA domain. Functional studies using fibroblast cells from two affected individuals with the c.1516C>T mutation showed a truncated WASF1 and a defect in actin remodeling. This study provides evidence that de novo heterozygous mutations in WASF1 cause a rare form of intellectual disability.

Full text

REPORT
De Novo Truncating Mutations in WASF1
Cause Intellectual Disability with Seizures
Yoko Ito,
1,14
Keren J. Carss,
2,3,14
Sofia T. Duarte,
4
Taila Hartley,
1
Boris Keren,
5
Manju A. Kurian,
6
Isabelle Marey,
5
Perinne Charles,
5
Carla Mendonc¸a,
7
Caroline Nava,
5,8
Rolph Pfundt,
9
Alba Sanchis-Juan,
2,3
Hans van Bokhoven,
9
Anthony van Essen,
10
Conny van Ravenswaaij-Arts,
10
NIHR BioResource, Care4Rare Canada Consortium, Kym M. Boycott,
1,11
Kristin D. Kernohan,
1
Sarah Dyack,
12,15
and F. Lucy Raymond
3,13,15,
*
Next-generation sequencing has been invaluable in the elucidation of the genetic etiology of many subtypes of intellectual disability in
recent years. Here, using exome sequencing and whole-genome sequencing, we identified three de novo truncating mutations in WAS
protein family member 1 (WASF1) in five unrelated individuals with moderate to profound intellectual disability with autistic features
and seizures. WASF1, also known as WAVE1, is part of the WAVE complex and acts as a mediator between Rac-GTPase and actin to induce
actin polymerization. The three mutations connected by Matchmaker Exchange were c.1516C>T (p.Arg506Ter), which occurs in three
unrelated individuals, c.1558C>T (p.Gln520Ter), and c.1482delinsGCCAGG (p.Ile494MetfsTer23). All three variants are predicted to
partially or fully disrupt the C-terminal actin-binding WCA domain. Functional studies using fibroblast cells from two affected individ-
uals with the c.1516C>T mutation showed a truncated WASF1 and a defect in actin remodeling. This study provides evidence that
de novo heterozygous mutations in WASF1 cause a rare form of intellectual disability.
Neurodevelopmental disorders (NDDs), which include in-
tellectual disability (ID), epilepsy, and autism spectrum dis-
order, are a heterogeneous group of disorders caused by
abnormal development of the central nervous system
(CNS). The complexity of CNS development is reflected
in the fact that over 700 genes to date have been associated
with ID, and very few occur at high prevalence.
1,2
Because
of the extreme genetic heterogeneity of ID, the utilization
of next-generation sequencing (NGS) technology provides
an efficient method of determining the genetic cause of ID
in individuals and discovering ID-associated genes. In
addition, NGS of trios enables detection of de novo muta-
tions,
3
including single-nucleotide variants (SNVs) and
small indels, which are a major contributing factor to the
genetic etiology of moderate to severe ID and NDDs.
4–7
In this study, we used NGS approaches to identify three
de novo variants in WAS protein family member 1 (WASF1
[MIM: 605035]), which encodes WASF1 (also known as
WAVE1), in five unrelated individuals with overlapping
neurodevelopmental abnormalities, including severe ID
with autistic features and seizures. We used Matchmaker
Exchange (MME)
8
to connect the four international cen-
ters, which had each independently identified WASF1 as
a candidate gene. All three de novo variants, including a
recurrent truncating variant, cluster within the C-terminal
actin-binding WCA domain of WASF1 and are predicted to
result in a truncated protein.
The five affected individuals described in this report are
from non-consanguineous families and are unrelated. All
participants and parents gave informed consent, and the
studies were approved by the appropriate institutional
research ethics boards (Children’s Hospital of Eastern
Ontario, Ottawa, Canada; IWK Health Centre, Halifax,
Canada; Groupe Hospitalier Pitie
´-Salpe
ˆtrie
`re, Paris, France;
East of England Cambridge South, Cambridge, UK; Santa
Maria Hospital, Lisbon, Portugal; and Radboud University
Medical Center, Nijmegen, the Netherlands [2011-188]).
The five affected individuals (P1–P5) have moderate to
profound ID with autistic features, seizures, severe impair-
ments in speech, gross motor delay, and a paucity of
significant congenital abnormalities. A detailed clinical
overview is provided in Table 1. The affected individuals
have midfacial hypoplasia but lack a recognizable dysmor-
phic facial phenotype (Figures S1A–S1D). P5 started
walking at 25 months, P1 and P2 began walking at age
3–4 years, and P4 did not walk until age 10 years. P1
1
Children’s Hospital of Eastern Ontario Research Institute, University of Ottawa, Ottawa, ON K1H 8L1 Canada;
2
Department of Haematology, University of
Cambridge, Cambridge CB2 0PT, UK;
3
NIHR BioResource, Cambridge University Hospitals NHS Foundation Trust, Cambridge Biomedical Campus, Cam-
bridge CB2 0QQ, UK;
4
Hospital Dona Estefa
ˆnia, Centro Hospitalar de Lisboa Central, 1169-045 Lisbon, Portugal;
5
De
´partement de Ge
´ne
´tique et Centre de
Re
´fe
´rence De
´ficiences Intellectuelles de Causes Rares, Ho
ˆpital de la Pitie
´-Salpe
ˆtrie
`re, Assistance Publique – Ho
ˆpitaux de Paris, 75651 Paris, France;
6
Developmental Neurosciences, Great Ormond Street Institute of Child Health, University College London, London WC1N 1EH, UK;
7
Centro de Neuro-
pediatria e Desenvolvimento, Centro Hospitalar Universita
´rio do Algarve, Faro 8000, Portugal;
8
Sorbonne Universite
´s, Universite
´Pierre et Marie Curie, Paris
75013, France;
9
Department of Human Genetics, Radboudumc, Donders Institute for Brain, Cognition, and Behaviour, Box 9101, 6500 HB Nijmegen, the
Netherlands;
10
University of Groningen, University Medical Centre Groningen, Department of Genetics, P.O. Box 30.001, 9700 RB Groningen, the
Netherlands;
11
Department of Genetics, Children’s Hospital of Eastern Ontario, Ottawa, ON K1H 8L1, Canada;
12
Department of Pediatrics, Dalhousie Uni-
versity, Halifax, NS B3K 6R8, Canada;
13
Department of Medical Genetics, Cambridge Institute for Medical Research, University of Cambridge, Cambridge
CB2 0XY, UK
14
These authors contributed equally to this work
15
These authors contributed equally to this work
*Correspondence: flr24@cam.ac.uk
https://doi.org/10.1016/j.ajhg.2018.06.001.
The American Journal of Human Genetics 103, 1–10, July 5, 2018 1
Please cite this article in press as: Ito et al., De Novo Truncating Mutations in WASF1 Cause Intellectual Disability with Seizures, The American
Journal of Human Genetics (2018), https://doi.org/10.1016/j.ajhg.2018.06.001
Crown Copyright Ó2018 This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

Table 1. Key Clinical Features of Affected Individuals
Detail P1 P2 P3 P4 P5
General
Age (years) 21 23 23 30 23
Sex male male male female male
Birth
Gestation (weeks) 40 41 39 NR 41
Weight (g) 3,800 4,100 3,370 4,020 4,020
Head circumference (cm) NR 35.5 35.5 NR 35.5
Neurological
Intellectual disability severe to profound moderate to severe severe profound moderate to severe
Seizures onset at 8 years; focal with
occasional GTC
onset at 6 years; absence
and GTC
onset at 8 months; infantile
spasms initially, now GTC
onset NR; temporal-lobe
epilepsy with partial seizures
none
Speech single words simple sentences non-verbal NR single words
Hypotonia yes yes no yes (axial with hypertonia of
extremities)
yes (head control achieved
at 11 months)
History of regression no no yes (8 months) arrested development at age
1 year, 10 months
no
Wide-based gait with poor balance yes no non-ambulant yes yes
High pain tolerance yes no yes possible (automutilation) yes (automutilation)
Head imaging MRI: scarce periventricular
white matter, enlarged
ventricles
MRI: normal MRI: normal CT: mild atrophy near
Sylvian fissures
MRI: enlarged ventricles
Current Measurements
Head circumference (cm) 50.4 (<P1; 3.2 SD) 58 (P98; þ2 SD) 53.2 (P25; 1.3 SD) 54 (P25; 0.3 SD) 57 (P99; þ2.4 SD)
Weight (kg) 40.8 (<P1) 82 (P80) 40.2 (P25) unknown 65 (P70)
Height (cm) 156.7 (<P1; 2.8 SD) 183 (P80; þ1 SD) 168 (P10; 1.2 SD) 150 (P2; 2.8 SD) 175 (P97; þ1.8 SD)
Motor Development
Age at unsupported sitting 18 months 9 months 6 months 22 months NR
Age at walking 4 years 3 years non-ambulant 10 years 25 months
(Continued on next page)
2The American Journal of Human Genetics 103, 1–10, July 5, 2018
Please cite this article in press as: Ito et al., De Novo Truncating Mutations in WASF1 Cause Intellectual Disability with Seizures, The American
Journal of Human Genetics (2018), https://doi.org/10.1016/j.ajhg.2018.06.001

Table 1. Continued
Detail P1 P2 P3 P4 P5
Craniofacial
Midface hypoplasia yes yes no yes NR
Eyes deep set, strabismus, gray sclera exophthalmia strabismus, gray sclera strabismus, vision loss,
upslanted palpebral fissures
strabismus
Musculoskleletal
Joint hyperflexibility yes no no yes yes
Ankle valgus yes no no yes knee recurvatum
Long tapered fingers yes no yes no NR
Feet narrow, pes planus, short
forth toes
short third toes normal short, pes planus, short third toes pes planus
Other
Nipples widely spaced normal widely spaced inverted NR
Cafe
´au lait macules yes no yes no NR
Feeding problems trouble sucking, reflux,
easy choking
no cyclic vomiting resolved
at age 16 years
no feeding difficulties, reflux
Genitourinary no no renal stones, recurrent UTIs small kidneys, mildly dilated
pyelum, recurrent UTIs
NR
Constipation yes no yes yes yes
HGVSg variant chr6: g.110422797G>A chr6: g.110422797G>A chr6: g.110421847G>A chr6: g.110422831delinsCCTGGC chr6: g.110422797G>A
HGVSc variant c.1516C>T c.1516C>T c.1558C>T c.1482delinsGCCAGG c.1516C>T
HGVSp variant p.Arg506Ter p.Arg506Ter p.Gln520Ter p.Ile494MetfsTer23 p.Arg506Ter
Genotype heterozygous heterozygous heterozygous heterozygous heterozygous
Inheritance de novo de novo de novo de novo de novo
Abbreviations are as follows: CT, computed tomography; GTC, generalized tonic clonic seizure; MRI, magnetic resonance imaging; NR, not recorded; P, patient; and UTI, urinary tract infection.
The American Journal of Human Genetics 103, 1–10, July 5, 2018 3
Please cite this article in press as: Ito et al., De Novo Truncating Mutations in WASF1 Cause Intellectual Disability with Seizures, The American
Journal of Human Genetics (2018), https://doi.org/10.1016/j.ajhg.2018.06.001

requires wheelchair assistance when traveling out of his
home. P3 has never achieved independent ambulation.
All affected individuals either are non-verbal or have
limited speech with a few or single words. All affected indi-
viduals except P5 have seizures, although these include a
range of seizure types, including generalized and focal sei-
zures; all require antiepileptic therapy. Four of the affected
individuals (P1, P2, P4 and P5) had significant hypotonia
in infancy, and two (P1 and P4) were described as having
a wide-based gait, poor balance, and hyperactivity of
movements. Musculoskeletal findings included joint hy-
perflexibility, ankle valgus, and pes planus in the more
severely affected individuals. P5 presented with upper-
limb dystonia in the first year of life. A high pain tolerance
was observed in P1 and P3, whereas P4 and P5 exhibited
automutilation, which is observed in those with an
abnormal response to pain. Computed tomography of P1
showed mild atrophy near the Sylvian fissures, magnetic
resonance imaging (MRI) of P2 and P3 was normal, and
MRI of P4 revealed abnormalities of the periventricular
white matter, although this individual also suffered a trau-
matic birth. MRI of P5 showed enlarged ventricles. Toe ab-
normalities (short third and fourth toes) were noted in
three of the four affected individuals (Figures S1E–S1G).
Testing for a range of other genetic conditions was under-
taken in the affected individuals but resulted in no alter-
nate diagnoses. Specific gene testing included MECP2,
ATRX,UBE3A,CDKL5,MEF2C,FOXG1,TCF4, and
NRXN1, reflecting the differential diagnosis and develop-
mental severity of the condition. All had a normal result
on diagnostic microarray testing. Metabolic testing was
normal, as was a muscle biopsy of P3.
Because the initial genetic tests were negative, all
affected individuals had either exome sequencing or
whole-genome sequencing (WGS) performed at their
respective centers. Details of the methods used for each
affected individual are provided in Table S1. Genomic co-
ordinates throughout this report refer to GRCh37, and
coding sequence and protein coordinates refer to
the canonical transcript (Ensembl: ENST00000392589;
GenBank: NM_003931.2).
Trio exome sequencing was performed on individual P1
and his parents as part of the Care4Rare Canada research
program according to our standard approach as previously
described.
9
After filtering for rare variants (with a fre-
quency less than 0.1% in gnomAD and present fewer
than six times in our in-house controls), all variants in
known disease-related genes were assessed, but no variants
that could explain this individual’s phenotype were identi-
fied. In the search for potential novel genes, possible bi-
allelic or X-linked recessive variants were examined, but
there were no rare homozygous or hemizygous variants.
Compound-heterozygous variants were identified in
CROCC (MIM: 615776), but this gene was ruled out as a
likely candidate because it has many loss-of-function vari-
ants in control databases (Table S2). Finally, de novo vari-
ants in WASF1,ATP5J (MIM: 603152), SLC38A4 (MIM:
608065), and ZNF175 (MIM: 601139) were identified
(Table S2). Assessment of protein localization patterns
and function and in silico mutation predictions deter-
mined that ATP5J,SLC38A4, and ZNF175 were unlikely
to be responsible for this condition (refer to Table S2 for
further details). Given the role of WASF1 in actin polymer-
ization and the importance of actin regulation in
achieving synaptic plasticity, the de novo heterozygous
variant in WASF1 (c.1516C>T [p.Arg506Ter]) was judged
to be the strongest candidate for causing this individual’s
condition and was entered into MME.
Individuals P2 and P5 underwent trio exome sequencing
as part of routine diagnostic testing at the De
´partement de
Ge
´ne
´tique of Ho
ˆpital Pitie
´-Salpe
ˆtrie
`re (Paris, France). After
filtering for rare variants (with a frequency less than 0.1%
in the ExAC Browser), no pathogenic variants, likely path-
ogenic variants, or variants of unknown significance
(VUSs) were identified in known developmental-disease-
associated genes. Next, rare variants in genes not previ-
ously known to be associated with disease were considered.
A heterozygous de novo stop-gain variant in WASF1
(c.1516C>T [p.Arg506Ter]), the same variant identified in
P1, was identified in both P2 and P5. A de novo missense
variant in CDCA7L (MIM: 609685) was also identified in
P2 but was not considered likely to be pathogenic (Table
S2). No additional variants that required consideration of
pathogenicity were identified in P5.
Individual P3 and his mother underwent WGS as part
of the National Institute for Health Research (NIHR)
BioResource study (UK) as previously described.
10
No
pathogenic or likely pathogenic variants were found in
known developmental-disease-associated genes, but a
heterozygous stop-gain variant in WASF1 (c.1558C>T
[p.Gln520Ter]), which was not present in the unaffected
mother, was identified. Sanger sequencing of P3 and his
parents confirmed that the variant occurs de novo in the
affected individual (Figure S2B). A hemizygous missense
variant in X-linked ACSL4 (MIM: 300157), in which vari-
ants can cause X-linked ID (MIM: 300387), was also iden-
tified in P3 and was heterozygous in the mother. This
was classified as a VUS because the variant was not previ-
ously associated with disease (Table S2).
Individual P4 underwent trio exome sequencing
as part of routine diagnostic testing (Groningen, the
Netherlands). No pathogenic variants, likely pathogenic
variants, or VUSs in known developmental-disease-associ-
ated genes were identified. Next, de novo variants in genes
not previously known to be associated with disease were
considered. A heterozygous de novo frameshift variant in
WASF1 (c.1482delinsGCCAGG [p.Ile494MetfsTer23]) was
identified. No other coding variants that occurred de novo
were identified.
Initially, the four groups independently identified
WASF1 as a strong candidate because of features consistent
with those of developmental-disorder-associated genes.
This gene is constrained for loss-of-function variation in
the ExAC Browser (pLi ¼0.91)
11
and is highly and
4The American Journal of Human Genetics 103, 1–10, July 5, 2018
Please cite this article in press as: Ito et al., De Novo Truncating Mutations in WASF1 Cause Intellectual Disability with Seizures, The American
Journal of Human Genetics (2018), https://doi.org/10.1016/j.ajhg.2018.06.001

specifically expressed in the adult human brain.
12
All three
WASF1 variants are absent from 1000 Genomes, the ExAC
Browser, and gnomAD.
11,13
The variants in individuals P1
and P3–P5 were confirmed to be de novo by Sanger
sequencing of the trio (Figure S2B). The read depths for
P2 and his mother and father were 127 (with 69 read
counts for the alternate allele), 143, and 124, respectively.
MME connected three of the groups, and the fourth was
connected by personal correspondence with the UK group.
Interestingly, the three de novo variants appear to cluster
around the WASP-homology 2 (WH2) domain of WASF1
(Figure 1A). A previously published method was used to
determine that the clustering is statistically significant
(p ¼1.31 310
6
).
15
The C-terminal actin-binding WCA re-
gion, which includes the WH2 domain, is highly
conserved throughout evolution (Figure 1B). The WCA re-
gion plays an important role in regulating WASF1
16,17
so
that actin and the Arp2/3 complex can bind to the WCA
domain to promote actin polymerization.
18
All three vari-
ants identified in the affected individuals fall either within
the last 50 bp of the penultimate exon or within the last
exon (Figure 1C) and are therefore predicted to result in
the generation of a truncated protein that partially or fully
eliminates this WCA domain.
19
Next, the potential effect of the identified WASF1 vari-
ants on protein function was determined. Primary fibro-
blasts were obtained from individuals P1 and P2, who carry
the same c.1516C>T variant (predicted to introduce a pre-
mature stop codon at amino acid 506). Amounts of WASF1
mRNA and WASF1 were examined. Real-time PCR showed
variable levels of mRNA between the two affected individ-
uals and control individuals (Figure 2A). For western blot
analysis of WASF1, total protein extracts were probed
with either a C-terminal antibody (epitope located after
amino acid 506; Abcam, ab50356) or an N-terminal anti-
body (Sigma-Aldrich, W0267) against WASF1. Comparison
of control and affected individuals revealed that the cells
from affected individuals had both the full-length WASF1
(75 kDa) and a truncated 70 kDa protein that was not
observed in control cells (Figures 2B and 2C). Densitom-
etry quantification of these bands showed that the full-
length protein was present at approximately 50% of the
Figure 1. Schematic Diagrams Showing Structure of WASF1 and WASF1
(A) Schematic diagram showing full-length WASF1 (also known as WAVE1 [Ensembl: ENSP00000376368]). Variants in the five individ-
uals (indicated in red) cluster around the WH2 domain (domain coordinates are from Stradal et al.
14
). P1, P2, and P5 have p.Arg506Ter,
P3 has p.Gln520Ter, and P4 has p.Ile494MetfsTer23. Abbreviations are as follows: WH1, WASP homology 1 domain; B, basic domain;
Pro, proline-rich region; WH2, WASP homology 2 domain (also known as the verprolin homology domain); C, cofilin homology
domain; A, acidic domain; WCA, collective name for the WH2, C, and A domains.
(B) Schematic diagram showing the amino acid sequence of part of WASF1. The WCA region of WASF1 is conserved throughout evolu-
tion. Yellow highlights residues that differ from the human protein sequence.
(C) Schematic diagram showing the 30part of WASF1, including locations of the participants’ variants in red. The gray boxes represent
the coding sequence, and the white box represents the 30UTR. The variant in P1, P2, and P5 is 6 bps from the end of exon 9 (the penul-
timate exon). The variant in P4 is 40 bps from the end of exon 9. The variant in P3 is within exon 10 (the final exon).
The American Journal of Human Genetics 103, 1–10, July 5, 2018 5
Please cite this article in press as: Ito et al., De Novo Truncating Mutations in WASF1 Cause Intellectual Disability with Seizures, The American
Journal of Human Genetics (2018), https://doi.org/10.1016/j.ajhg.2018.06.001

control levels, reflecting the presence of one wild-type
allele, whereas the truncated protein was present at 14%–
25% of control levels (Figures 2B and 2C). This suggests
that although a truncated isoform is produced, it is unsta-
ble at either the mRNA or protein level such that the
amount of protein is reduced. Therefore, the WASF1
c.1516C>T variant causes the production of a shorter
mutant protein rather than the absence of a protein due
to complete nonsense-mediated decay of the primary
transcript.
WASF1 plays a critical role in binding actin to initiate
actin polymerization. Examination of the reorganization
of the actin cytoskeleton during lamellipodia formation
in fibroblasts was used for testing this role.
20–22
Serum-
starved fibroblasts were trypsinized, re-plated onto
poly-L-lysine-coated coverslips, and stimulated with
platelet-derived growth factor (PDGF; Sigma-Aldrich,
P3201) for inducing the formation of lamellipodia, as pre-
viously described.
20
Then cells were fixed, filamentous
actin was visualized by labeling with phalloidin (Thermo
Fisher Scientific, A12349), and the actin phenotype was
quantified in each genotype. In the majority of control
cells (77%), actin at the cell periphery formed well-orga-
nized, sheet-like lamellipodia structures (Figures 3A and
3B, white arrowhead; Figure S3). This was interspersed
with cells in which the actin sheets were interjected by
filopodia, which are finger-like actin projections (Figures
3A and 3B, red asterisk; Figure S3). We next assessed fibro-
blasts from affected individuals and found that although a
sheet-like lamellipodia structure was observed along the
periphery of 34% and 24% of P1 and P2 cells, respectively,
the actin bundles were thinner and less organized than in
the control cells (Figures 3A and 3B). We also noted that a
portion of cells from P1 and P2 had severe disruptions in
actin organization such that no lamellipodia delineated
the cell periphery and only filopodial projections were pre-
sent (12% and 11% for P1 and P2, respectively; Figures 3A
and 3B). This phenotype was not seen in control cells.
Therefore, cells from affected individuals have an alter-
ation in actin organization, suggesting that the presence
of a truncated WASF1 results in defective actin remodeling
during the formation of lamellipodia.
Finally, WASF1-dependent actin polymerization has
been shown to mediate mitochondrial trafficking into
dendritic spines in primary neurons;
23
therefore, we as-
sessed mitochondrial morphology in fibroblasts with
the c.1516C>T variant. Mitochondria were visualized
and the average length was quantified as previously
described.
24
As expected, a dense and complex network
of mitochondria was present in both control and affected
fibroblasts. Quantification revealed that mitochondria in
the cells from affected individuals were significantly longer
than those in control fibroblasts (Figure 3C). This result
suggests that the presence of the c.1516C>T variant in
WASF1 disrupts the regulation of mitochondrial dynamics
and alters the normal balance between fission and fusion
in affected fibroblasts.
This report provides evidence that de novo truncating
variants in WASF1 in five unrelated individuals cause a
NDD comprising severe ID with autistic features, seizures,
and developmental delay. Interestingly, three of the five in-
dividuals in this study have the same de novo variant
Figure 2. Amounts of WASF1 mRNA and
WASF1 in Fibroblasts Derived from
Affected Individuals with the c.1516C>T
Variant
(A) RT-qPCR shows variable amounts of
WASF1 mRNA between primary fibroblasts
derived from individuals P1 and P2 and
healthy control fibroblasts.
(B) Western blot analysis using an antibody
with an epitope downstream of Arg506
showed that the amount of full-length
WASF1 was approximately 50% lower in
affected fibroblasts than in control fibro-
blasts.
(C) Western blot analysis using an anti-
body with an epitope in the N-terminal re-
gion of WASF1 showed the presence of
the full-length and truncated WASF1 in
affected fibroblasts. The truncated WASF1
was not present in control fibroblasts. All
experiments were performed with fibro-
blasts derived from three healthy control
individuals. Western blots were performed
in triplicate, and band intensity was quan-
tified with Image Lab Software (Bio-Rad).
Error bars indicate the range of measure-
ment of triplicate samples.
6The American Journal of Human Genetics 103, 1–10, July 5, 2018
Please cite this article in press as: Ito et al., De Novo Truncating Mutations in WASF1 Cause Intellectual Disability with Seizures, The American
Journal of Human Genetics (2018), https://doi.org/10.1016/j.ajhg.2018.06.001

(c.1516C>T [Ensembl: ENST00000392589]). Three of the
four individuals have VUSs in other genes in addition to
the WASF1 variants. Population-level sequencing initia-
tives have enabled increased recognition of the prevalence
of recurrent benign de novo mutations.
25
Although it is un-
likely, the possibility that they contribute to the respective
individuals’ phenotypes cannot be excluded.
The variants described as associated with this NDD are
all stop-gain or frameshift variants and significantly clus-
ter around the C-terminal WH2 domain in the WCA re-
gion of WASF1. The truncated protein observed for
c.1516C>T (p.Arg506Ter) suggests that all three variants
are likely to lead to altered function of the mutant protein
rather than complete protein loss or haploinsufficiency
from degradation through nonsense-mediated decay.
In a disease context, recurrent de novo events are
known to be associated with specific dominant-negative
or gain-of-function effects, such as FGFR3 (MIM:
134934) variants causing achondroplasia (MIM: 100800),
Figure 3. Lamellipodia Formation and
Mitochondrial Morphology in Fibroblasts
Derived from Individuals with the
c.1516C>T Variant
(A) Primary fibroblasts were treated with
PDGF for inducing the formation of lamel-
lipodia. Visualization of the filamentous
actin by phalloidin staining revealed the
disruption of actin in the cell periphery of
P1 and P2 fibroblasts. In the insets, lamelli-
podia and filopodia are marked by white ar-
rowheads and red asterisks, respectively.
Scale bars represent 10 mm.
(B) Cells were categorized into three groups
on the basis of the predominant actin
phenotype present: cells displaying lamelli-
podia only, cells displaying a mixture of
lamellipodia and filopodia, and cells dis-
playing filopodia only. Quantification
based on these three categories indicates
that significantly fewer affected fibroblasts
than control fibroblasts are able to form
solely lamellipodia.
(C) Confocal microscopic analysis of
TOMM-20-immunostained mitochondria
(in green) indicated that both affected fi-
broblasts have significantly elongated mito-
chondria. The nuclei were visualized by
DAPI staining (in blue).
and are usually missense variants.
26
Clustering and recurrence of de novo
protein-truncating mutations also do
occur, albeit less frequently because
the genic localization of a pathogenic
mutation resulting in haploinsuffi-
ciency is generally not critical.
15,27,28
Additional individuals with rare
WASF1 variants are required for deter-
mining whether any pathogenic vari-
ants lie outside of this WCA region
and/or whether a spectrum of pheno-
types is perhaps associated with different variants in this
gene.
WASF1 is an essential component of the actin pathway
where RAC1 activation triggers a conformational change
in WASF1 to allow binding of actin and ARP2/3 to the
WCA domain to initiate actin polymerization.
20,21,29,30
The presence of a truncated protein that lacks the WCA
region, as observed here, most likely disrupts the WASF1
complex itself, its interactions with CYFIP1, its protea-
somal degradation, and the binding of actin (Figure
2C).
16,17,31
Like mutations in WASF1, mutations in RAC1
similarly disrupt the formation of lamellipodia in fibro-
blasts,
32
indicating that the organization and stabilization
of actin bundles during the formation of lamellipodia is
likely to be compromised by truncated WASF1.
WASF1-dependent actin polymerization is known to
be important in CNS development and synaptic plas-
ticity.
18,33–39
Two different WASF1-null mouse models
demonstrate cognitive impairments, including deficits in
The American Journal of Human Genetics 103, 1–10, July 5, 2018 7
Please cite this article in press as: Ito et al., De Novo Truncating Mutations in WASF1 Cause Intellectual Disability with Seizures, The American
Journal of Human Genetics (2018), https://doi.org/10.1016/j.ajhg.2018.06.001

sensorimotor function, learning, and memory.
12,40
In addi-
tion, mutations in a number of genes in the actin regulatory
pathway, namely, formin 2 (FMN2 [MIM: 606373]),
41
actin
gamma-1 (ACTG1 [MIM: 102560]),
42
rho guanine nucleo-
tide exchange factor 6 (ARHGEF6 [MIM: 300267]),
43,44
and RAS-related C3 botulinum toxin substrate 1 (RAC1
[MIM: 602048]),
32
are associated with ID.
WASF1 localizes to the outer mitochondrial membrane,
where it has been shown to play a role in the trafficking
of mitochondria to the dendritic spines.
23,44,45
Actin itself
has also been shown to be necessary for mediating mito-
chondrial fission.
45
Given that fibroblasts derived from
affected individuals with the c.1516C>T variant show
elongated mitochondria (Figure 3C), WASF1 most likely
plays additional roles in regulating mitochondrial dy-
namics, although how variants in WASF1 affect mitochon-
drial function in affected individuals remains to be
elucidated.
In summary, de novo heterozygous truncating variants in
WASF1 cause a NDD in individuals with ID associated with
autistic features, seizures, and developmental delay. The
three de novo variants, identified in five unrelated affected
individuals, are all predicted to affect the actin-binding
C-terminal WCA region of WASF1. The clustering of trun-
cating pathogenic variants reported here and the presence
of a truncated protein in cells from affected individuals
imply either a gain-of-function or dominant-negative
mechanism of disease. Because WASF1 functions within a
large protein complex with ABI2, CYFIP1 or CYFIP2,
BRK1, and NCKAP1, the hypothesis that these variants
have a most likely dominant-negative effect remains to
be tested. This study further expands the list of actin-regu-
latory-pathway genes associated with NDD and demon-
strates the value of sharing genomic data through MME
to identify the consequence of extremely rare mutational
events.
Supplemental Data
Supplemental Data include three figures and two tables and can be
found with this article online at https://doi.org/10.1016/j.ajhg.
2018.06.001.
Consortia
The NIHR BioResource consists of Timothy Aitman, David Ben-
nett, Mark Caulfield, Patrick Chinnery, Daniel Gale, Ania Koziell,
Taco W. Kuijpers, Michael A. Laffan, Eamonn Maher, Hugh S. Mar-
kus, Nicholas W. Morrell, Willem H. Ouwehand, David J. Perry, F.
Lucy Raymond, Irene Roberts, Kenneth G.C. Smith, Adrian
Thrasher, Hugh Watkins, Catherine Williamson, Geoffrey Woods,
Sofie Ashford, John R. Bradley, Debra Fletcher, TraceyHammerton,
Roger James, Nathalie Kingston, Christopher J. Penkett, Kathleen
Stirrups, Marijke Veltman, Tim Young, Matthew Brown, Naomi
Clements-Brod, John Davis, Eleanor Dewhurst, Helen Dolling,
Marie Erwood, Amy Frary, Rachel Linger, Jennifer M. Martin, Sofia
Papadia, Karola Rehnstrom, Hannah Stark, David Allsup, Steve
Austin, Tamam Bakchoul, Tadbir K. Bariana, Paula Bolton-Maggs,
Elizabeth Chalmers, Janine Collins, Peter Collins, Wendy N. Erber,
Tamara Everington, Remi Favier, Kathleen Freson, Bruce Furie,
Michael Gattens, Johanna Gebhart, Keith Gomez, Daniel Greene,
Andreas Greinacher, Paolo Gresele, Daniel Hart, Johan W.M.
Heemskerk, Yvonne Henskens, Rashid Kazmi, David Keeling,
Anne M. Kelly, Michele P. Lambert, Claire Lentaigne, Ri Liesner,
Mike Makris, Sarah Mangles, Mary Mathias, Carolyn M. Millar, An-
drew Mumford, Paquita Nurden, Jeanette Payne, John Pasi, Kathe-
lijne Peerlinck, Shoshana Revel-Vilk, Michael Richards, Matthew
Rondina, Catherine Roughley, Sol Schulman, Harald Schulze,
Marie Scully, Suthesh Sivapalaratnam, Matthew Stubbs, R. Camp-
bell Tait, Kate Talks, Jecko Thachil, Cheng-Hock Toh, Ernest Turro,
Chris Van Geet, Minka De Vries, Timothy Q. Warner, Henry Wat-
son, Sarah Westbury, Abigail Furnell, Rutendo Mapeta, Paula Ray-
ner-Matthews, Ilenia Simeoni, Simon Staines, Jonathan Stephens,
Christopher Watt, Deborah Whitehorn, Antony Attwood, Louise
Daugherty, Sri V.V. Deevi, Csaba Halmagyi, Fengyuan Hu, Vera
Matser, Stuart Meacham, Karyn Megy, Olga Shamardina, Cather-
ine Titterton, Salih Tuna, Ping Yu, Julie von Ziegenweldt, William
Astle, Marta Bleda, Keren J. Carss, Stefan Gra
¨f, Matthias Haimel,
Hana Lango-Allen, Sylvia Richardson, Paul Calleja, Stuart Rankin,
Wojciech Turek, Julie Anderson, Christine Bryson, Jenny Carmi-
chael, Coleen McJannet, Sophie Stock, Louise Allen, Gautum Am-
begaonkar, Ruth Armstrong, Gavin Arno, Maria Bitner-Glindzicz,
Angie Brady, Natalie Canham, Manali Chitre, Emma Clement, Vir-
ginia Clowes, Patrick Deegan, Charu Deshpande, Rainer Doffinger,
Helen Firth, Frances Flinter, Courtney French, Alice Gardham,
Neeti Ghali, Paul Gissen, Detelina Grozeva, Robert Henderson,
Anke Hensiek, Simon Holden, Muriel Holder, Susan Holder, Jane
Hurst, Dragana Josifova, Deepa Krishnakumar, Manju A. Kurian,
Melissa Lees, Robert MacLaren, Anna Maw, Sarju Mehta, Michel
Michaelides, Anthony Moore, Elaine Murphy, Soo-Mi Park, Alas-
dair Parker, Chris Patch, Joan Paterson, Julia Rankin, Evan Reid,
Elisabeth Rosser, Alba Sanchis-Juan, Richard Sandford, Saikat San-
tra, Richard Scott, Aman Sohal, Penelope Stein, Ellen Thomas,
Dorothy Thompson, Marc Tischkowitz, Julie Vogt, Emma Wake-
ling, Evangeline Wassmer, Andrew Webster, Sonia Ali, Souad Ali,
Harm J. Boggard, Colin Church, Gerry Coghlan, Victoria Cookson,
Paul A. Corris, Amanda Creaser-Myers, Rosa DaCosta, Natalie Dor-
mand, Me
´lanie Eyries, Henning Gall, Pavandeep K. Ghataorhe,
Stefano Ghio, Ardi Ghofrani, J. Simon R. Gibbs, Barbara Girerd,
Alan Greenhalgh, Charaka Hadinnapola, Arjan C. Houweling,
Marc Humbert, Anna Huis in’t Veld, Fiona Kennedy, David G.
Kiely, Gabor Kovacs, Allan Lawrie, Rob V. Mackenzie Ross, Rajiv
Machado, Larahmie Masati, Sharon Meehan, Shahin Moledina,
David Montani, Shokri Othman, Andrew J. Peacock, Joanna
Pepke-Zaba, Val Pollock, Gary Polwarth, Lavanya Ranganathan,
Christopher J. Rhodes, Kevin Rue-Albrecht, Gwen Schotte, Debbie
Shipley, Florent Soubrier, Laura Southgate, Laura Scelsi, Jay Sun-
tharalingam, Yvonne Tan, Mark Toshner, Carmen M. Treacy, Ri-
chard Trembath, Anton Vonk Noordegraaf, Sara Walker, Ivy Wan-
jiku, John Wharton, Martin Wilkins, Stephen J. Wort, Katherine
Yates, Hana Alachkar, Richard Antrobus, Gururaj Arumugakani,
Chiara Bacchelli, Helen Baxendale, Claire Bethune, Shahnaz
Bibi, Claire Booth, Michael Browning, Siobhan Burns, Anita Chan-
dra, Nichola Cooper, Sophie Davies, Lisa Devlin, Elizabeth Drewe,
David Edgar, William Egner, Rohit Ghurye, Kimberley Gilmour,
Sarah Goddard, Pavel Gordins, Sofia Grigoriadou, Scott Hackett,
Rosie Hague, Lorraine Harper, Grant Hayman, Archana Herwad-
kar, Aarnoud Huissoon, Stephen Jolles, Peter Kelleher, Dinakantha
Kumararatne, Sara Lear, Hilary Longhurst, Lorena Lorenzo,
Jesmeen Maimaris, Ania Manson, Elizabeth McDermott, Sai
8The American Journal of Human Genetics 103, 1–10, July 5, 2018
Please cite this article in press as: Ito et al., De Novo Truncating Mutations in WASF1 Cause Intellectual Disability with Seizures, The American
Journal of Human Genetics (2018), https://doi.org/10.1016/j.ajhg.2018.06.001

Murng, Sergey Nejentsev, Sadia Noorani, Eric Oksenhendler, Mark
Ponsford, Waseem Qasim, Isabella Quinti, Alex Richter, Crina Sa-
marghitean, Ravishankar Sargur, Sinisa Savic, Suranjith Senevir-
atne, Carrock Sewell, Emily Staples, Hans Stauss, James Thaven-
thiran, Moira Thomas, Steve Welch, Lisa Willcocks, Nigel
Yeatman, Patrick Yong, Phil Ancliff, Christian Babbs, Mark Layton,
Eleni Louka, Simon McGowan, Adam Mead, Noe
´mi Roy, Jenny
Chambers, Peter Dixon, Cecelia Estiu, Bill Hague, Hanns-Ulrich
Marschall, Michael Simpson, Sam Chong, Ingrid Emmerson, Lio-
nel Ginsberg, David Gosal, Rob Hadden, Rita Horvath, Mohamed
Mahdi-Rogers, Adnan Manzur, Andrew Marshall, Emma Mat-
thews, Mark McCarthy, Mary Reilly, Tara Renton, Andrew Rice,
Andreas Themistocleous, Tom Vale, Natalie Van Zuydam, Suellen
Walker, Liz Ormondroyd, Gavin Hudson, Wei Wei, Patrick Yu
Wai Man, James Whitworth, Maryam Afzal, Elizabeth Colby,
Moin Saleem, Omid S. Alavijeh, H. Terry Cook, Sally Johnson,
Adam P. Levine, Edwin K.S. Wong, and Rhea Tan.
The project was selected for analysis by the Care4Rare Con-
sortium Gene Discovery Steering Committee, consisting of Kym
Boycott, Alex MacKenzie, Jacek Majewski, Michael Brudno, Den-
nis Bulman, and David Dyment.
Acknowledgments
We thank the four affected individuals involved in this study and
their families. This work was supported by the Cambridge Biomed-
ical Research Centre and the National Institute for Health
Research (NIHR) for the NIHR BioResource (grant number
RG65966). This work was supported by the Care4Rare Canada
Consortium (Enhanced Care for Rare Genetic Diseases in Canada),
which is funded by Genome Canada, the Canadian Institutes of
Health Research, the Ontario Genomics Institute, the Ontario
Research Fund, Genome Quebec, and the Children’s Hospital of
Eastern Ontario Foundation.
Declaration of Interests
The authors declare no competing interests.
Received: March 12, 2018
Accepted: June 4, 2018
Published: June 28, 2018
Web Resources
Ensembl, https://useast.ensembl.org/index.html
ExAC Browser, http://exac.broadinstitute.org/
GenBank, https://www.ncbi.nlm.nih.gov/genbank/
gnomAD, http://gnomad.broadinstitute.org/
Matchmaker Exchange, http://www.matchmakerexchange.org/
OMIM, http://omim.org/
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Please cite this article in press as: Ito et al., De Novo Truncating Mutations in WASF1 Cause Intellectual Disability with Seizures, The American
Journal of Human Genetics (2018), https://doi.org/10.1016/j.ajhg.2018.06.001