Nature GeNetics VOLUME 42 | NUMBER 6 | JUNE 2010
Using microarrays, we identified de novo copy number
variations in the SHANK2 synaptic scaffolding gene in two
unrelated individuals with autism-spectrum disorder (ASD)
and mental retardation. DNA sequencing of SHANK2 in 396
individuals with ASD, 184 individuals with mental retardation
and 659 unaffected individuals (controls) revealed additional
variants that were specific to ASD and mental retardation
cases, including a de novo nonsense mutation and seven rare
inherited changes. Our findings further link common genes
between ASD and intellectual disability.
Autism spectrum disorder (ASD) and mental retardation are two clin-
ically distinct neurodevelopmental disorders with a complex genetic
etiology. Both show a high degree of heritability, occur either as
single entities or as combined entities, and are sometimes associated
with genetic syndromes such as fragile X and Rett syndrome1. Mental
retardation is defined as a substantially sub-average intellectual func-
tion with limitations in communication, self-care, academic, social
and interpersonal skills, often with cognitive deficits as measured
by intelligence testing. Autistic disorder is the prototypic form of a
group of conditions called the autism-spectrum disorders (ASDs) that
share two main phenotypic dimensions, namely impairment in social
communication and a pattern of repetitive restrictive behaviors; these
disorders have an onset before the age of three. Adding to the pheno-
typic complexity in ASD is an ~40% prevalence of developmental
delay and/or intellectual disability (broadly grouped as mental retar-
dation), with intelligence as perhaps the most heritable component
of the ASD phenotype2.
Although the genetic and pathological basis of mental retarda-
tion and ASD still remains largely unexplained, there has been some
progress identifying disease-associated genes. In mental retardation,
six autosomal and ~90 X-linked disease genes are now known3. In ASD,
rare mutations have been found in the synaptic genes, such as NLGN3,
NLGN4 (ref. 4) and SHANK3 (ref. 5,6). Moreover, de novo and rare inheri-
ted copy number variation (CNV) of NRXN1 (ref. 7) and the 16p11.2
locus, amongst others, have been found to associate with ASD8,9.
Notably, some of the ASD susceptibility genes (such as NLGN4) and
CNVs (such as 16p11.2) are also being found to be associated with
Initially, we performed a genome-wide microarray scan for CNVs
using the Affymetrix 6.0 SNP platform in a German cohort of 184
unrelated individuals with mental retardation (111 males and 73
females). In a parallel study, we tested a series of 396 Canadian ASD
cases (318 males and 78 females) using the Affymetrix 6.0 and/or
Illumina Infinium 1M-single SNP platform. All ASD cases were
diagnosed using the Autism Diagnostic Interview–revised and
Autism Diagnostic Observation Schedule measures (Supplementary
Methods). We identified de novo deletions of 120 kb and 69 kb affecting
SHANK2 in single unrelated cases of European ancestry of the mental
retardation cohort (loss of exon 7 in MO1812A011) or ASD cohort
(loss of exons 6 and 7 in SK0217-003) (Fig. 1 and Supplementary Fig. 1).
CNVs of SHANK2 were not observed in 5,023 ancestrally matched
controls (P = 0.011; see Supplementary Methods), and no exonic
CNVs could be found in the Database of Genomic Variants. Both dele-
tions disrupt the highly conserved PDZ domain of SHANK2 (ref. 12),
leading to a frameshift mutation and presumably causing functional
loss of one allele (haploinsufficiency). The deletion in MO1812A011
was also verified in the subject’s fibroblast RNA (Fig. 1c–d).
SHANK2 is a member of a family of scaffold proteins (comprising
SHANK1, SHANK2 and SHANK3) that all localize to postsynaptic
sites of excitatory synapses in the brain. Due to the discovery of rare
de novo CNVs in SHANK2, the protein’s postsynaptic localization
and the fact that SHANK3 has already been associated with ASD, we
considered SHANK2 as a candidate susceptibility gene for both ASD
and mental retardation5,13.
We sequenced the exons of the neuronal isoform of SHANK2
(SHANK2_1, which lacks the ankyrin repeat; see Fig. 1a and
Supplementary Fig. 2) using native DNA of the same 184 mental
retardation cases and 396 ASD probands tested by microarrays. We
completely sequenced 659 controls of European ancestry for the same
exons as had been sequenced for the cases. We discovered one de novo
nonsense mutation (R462X), six different inherited missense variants
(R26W, P208S, S231Y, R1048W, T1127M and A1350T) and a micro-
duplication of six nucleotides (L1008_P1009dup), all in individuals
Mutations in the SHANK2
synaptic scaffolding gene in
autism spectrum disorder and
Simone Berkel1, Christian R Marshall2, Birgit Weiss1,
Jennifer Howe2, Ralph Roeth1, Ute Moog3, Volker Endris1,
Wendy Roberts4, Peter Szatmari5, Dalila Pinto2, Michael Bonin6,
Angelika Riess6, Hartmut Engels7, Rolf Sprengel8,
Stephen W Scherer2,9 & Gudrun A Rappold1
1Department of Molecular Human Genetics, Ruprecht-Karls-University, Heidelberg, Germany. 2The Centre for Applied Genomics, The Hospital for Sick Children,
University of Toronto, Ontario, Canada. 3Department of Human Genetics, Ruprecht-Karls-University, Heidelberg, Germany. 4Autism Research Unit, The Hospital for
Sick Children, University of Toronto, Toronto, Ontario, Canada. 5Offord Centre for Child Studies, Department of Psychiatry and Behavioural Neurosciences, McMaster
University, Hamilton, Ontario, Canada. 6Department of Medical Genetics, Institute of Human Genetics, Tübingen University, Tübingen, Germany. 7Institute of
Human Genetics, Rheinische Friedrich-Wilhelms-University, Bonn University, Bonn, Germany. 8Max Planck Institute for Medical Research (MPI), Ruprecht-Karls-
University, Heidelberg, Germany. 9Department of Molecular Genetics, University of Toronto, Ontario, Canada. Correspondence should be addressed to G.A.R.
Received 21 January; accepted 15 April; published online 16 May 2010; doi:10.1038/ng.589
© 2010 Nature America, Inc. All rights reserved.
VOLUME 42 | NUMBER 6 | JUNE 2010 Nature GeNetics
different from those carrying the de novo deletions (Fig. 1e and Table 1).
As the C-terminal region of SHANK2, which includes the SAM
domain, is crucial for correct localization at functional synapses14,
the R462X variant may have lost this localization ability. One substi-
tution, P208S, was detected twice, in the ASD case SK0332-003 and
the mental retardation cases BO3/03 (Supplementary Fig. 3). All
other substitutions were found only once. None of these variants were
detected in the controls (378 males and 281 females) or in the NCBI
dbSNP database (build 130). All new nucleotide variants detected in
cases and controls are provided in Supplementary Table 1.
One SHANK2 mutation (P208S) resided within the SH3 domain,
and the T1127M missense mutation was located within the highly con-
served dynamin-2 binding site (Fig. 1e and Supplementary Fig. 4).
The two deletions and the R462X nonsense mutation occurred
de novo, whereas all missense mutations were familial and were
transmitted through an unaffected parent. Co-segregation analysis
in ASD families with multiple affected children (comprising 33%
of the families in this study; Supplementary Fig. 3) revealed that
the C622T mutation at the highly conserved position P208S in indi-
vidual SK0332-003 was passed from an unaffected mother to two
male children with ASD and one other male sibling diagnosed with
language delay (Supplementary Table 2). Another amino acid change
at a highly conserved position, A1350T, was transmitted from an
unaffected mother to all three sons, who had variable phenotypic
presentation (respectively: autism, anxiety with autistic-like traits,
and language delay). Notably, although both transmitting mothers
were undiagnosed for an ASD, they both showed depression and/or
anxiety (Supplementary Fig. 3 and Supplementary Table 2).
The clinical phenotypes of the analyzed subjects with SHANK2 muta-
tions were variable. Whereas, for example, the two individuals with
CNV deletions disrupting the PDZ domain both have ASD of com-
parative severity and mild to moderate mental retardation, two other
individuals with the P208S substitution showed either isolated severe
mental retardation (intelligence quotient ~35) or ASD with borderline
intelligence (Supplementary Table 2). Of the studied ASD cases hav-
ing SHANK2 mutations, 63% had an intelligence quotient of <70. Two
of the four individuals with SHANK2 mutations (MO1812A011 and
TULB081863), recruited due to their presentation of mental retardation,
were subsequently also diagnosed as autistic using the ADOS measures.
These data, together with previous literature, demonstrate a clinical
overlap between these disorders, which may sometimes be explained
at least in part by a common genetic etiology.
Conservatively, we estimate that at least the two de novo deletions
and the one de novo nonsense mutation seen here are penetrant
mutations in SHANK2. In addition, seven further variants at highly
conserved positions were found only in affected individuals and not
G G G G C C A A A G C T G A C A C A G T T A A C A A T
A A C A A T G A G A A T G
G G G G C C A A A G G T T
T G A T T G A G
Deletion of 126 bp
SHANK2_1 (neuro) (AB208026.1)
SHANK2_2 (neuro) (NM_133266.3)
120 kb del
69 kb del
figure 1 Microdeletions and mutations of SHANK2 in mental retardation and ASD. (a) Three isoforms of SHANK2 are shown: SHANK2E (NM_012309.3),
SHANK2_1 (neuro) (AB208026.1, neuronal isoform 1) and SHANK2_2 (neuro) (NM_133266.3, neuronal isoform 2). SHANK2 is flanked by DHCR7
and PPFIA1. Fluorescence in situ hybridization confirmed the heterozygous deletion detected in individual MO1812A011 with mental retardation by
CNV analysis. Signals for the BAC clones CTD-3053G20 and CTD-2591D19 were detected on both copies of chromosomes 11, whereas the genomic
region corresponding to BAC CTD-3244D21 was deleted on one copy. (b) CNV analysis of individuals MO1812A011 and SK0217-003 showed loss of
exon 7 (120 kb on genomic scale) and of exons 6 and 7 (69 kb on genomic scale), respectively, in the SHANK2_1 (neuro) isoform of the gene (labeled
Ex6, Ex7 accordingly). (c) Deletion of exon 7 shown at the RNA level (individual MO1812A011). RT-PCR of mRNA prepared from dermal fibroblasts,
using primers located in exons 6 and 8, revealed two bands with a smaller fragment of 116 bp. NHDF, normal human dermal fibroblasts. (d) RNA
sequencing of sample MO1812A011. Sequencing of the two PCR bands shown in (c) revealed that the smaller fragment resulted from the deletion of
exon 7. (e) Putative etiologic variants resulting in amino acid exchanges or truncation of SHANK2 in mental retardation and ASD. SHANK2_1 (neuro),
the largest neuronal isoform, has 16 exons spanning 357 kb of genomic DNA and is predicted to encode 1,470 amino acids (AB208026.1). SH3,
Src homology 3 domain; PDZ, postsynaptic density 95/Discs large/zona occludens-1 homology domain; Proline-rich region; SAM, sterile alpha motif
domain; H, HOMER binding motif; D, dynamin-2 binding motif; C, cortactin binding motif.
© 2010 Nature America, Inc. All rights reserved.
Nature GeNetics VOLUME 42 | NUMBER 6 | JUNE 2010 Download full-text
in controls. These rare transmitted variants display incomplete penet-
rance, whose significance is uncertain at this time. Further analysis
of those individuals harboring sequence variants at SHANK2 (both
inherited and de novo) yielded additional rare CNVs that potentially
contribute to disease (Supplementary Table 3). This finding, together
with the observation that some individuals carrying rare inherited
SHANK2 sequence variants do not express an ASD phenotype
(Supplementary Fig. 3 and Supplementary Table 2), is consistent
with a multigenic threshold model for autism1,10.
We know little about the molecular mechanisms that initiate or
stabilize synaptic growth and the molecules that lead to cytoskeletal
rearrangements during synapse formation or remodeling. SHANKs
are among the most abundant scaffolding proteins in the postsynaptic
density. Together with HOMER, they were recently shown to form a
mesh-like matrix structure that is required for the structural integrity
of the dendritic spines and serves as an ‘assembly platform’ for other
proteins residing within the postsynaptic density15. It is therefore
conceivable that the SHANK proteins play an important role as central
organizers of the postsynaptic density.
The isolation of SHANK2 and the subsequent functional characteri-
zation of SHANK2 mutants derived from individuals with ASD and
mental retardation now paves the way for a deeper understanding of
the biological processes affecting cognitive function involved in these
disorders. SHANK2 loss-of-function mutations found in individuals
with ASD and mental retardation are assumed to affect synaptic func-
tion (suggesting that CNV gain-of-function alterations may also be
found in related or other disorders). These results, together with
previous observations in genes involved in ASD, like SHANK3 and
NLGN3 and NLGN4 (also known as Neuroligin 3 and 4), highlight the
importance of intact postsynaptic structures for the development of
typical cognitive function, language and social abilities.
Accession code. GEO database GSE20533
Note: Supplementary information is available on the
Nature Genetics website.
We gratefully thank E. Fenner, M. Fritsche and
S. Peykov for sequencing and technical support, and
we thank all the affected individuals’ families for
their cooperation. We thank A. Fiebig, A. Franke
and S. Schreiber at POPGEN (University of Kiel,
Kiel, Germany) and A. Stewart, R. McPherson
and R. Roberts of the University of Ottawa Heart
Institute (University of Ottawa, Ottawa, Canada) for
generously providing population control microarray
data. The international Autism Genome Project
and Autism Speaks is also gratefully acknowledged
for support. Funding was provided by the German
Mental Retardation Network (MRNET), supported
by the Federal Ministry of Education and Research,
Germany, by the Deutsche Forschungsgesellschaft
(DFG; SFB488) and the Medical Faculty of
Heidelberg. S.B. was funded by a fellowship of
CellNetworks–Cluster of Excellence (EXC81) and is a
member of Hartmut Hoffmann-Berling International
Graduate School of Molecular and Cellular Biology
(HBIGS). S.W.S. is supported by The Centre for
Applied Genomics, Genome Canada and Ontario
Genomics Institute, the Canadian Institutes for
Health Research (CIHR), the Canadian Institute for
Advanced Research (CIFAR), the McLaughlin Centre, the Canada Foundation for
Innovation, the Ontario Ministry of Research and Innovation and the Hospital
for Sick Children Foundation. S.W.S. holds the GlaxoSmithKline-CIHR Chair
in Genetics and Genomics at the University of Toronto and the Hospital for Sick
Children. G.A.R. is a member of CellNetworks–Cluster of Excellence (EXC81).
Dedicated to Mrs. Elisabeth Berkel.
S.B. performed the majority of the experiments, designed the figures and
contributed to the manuscript. C.R.M., D.P. and M.B. carried out array analyses.
S.B. and C.R.M. performed breakpoint mapping. B.W., J.H., R.R. and V.E.
performed sequencing and FISH analysis. R.S. contributed to the experimental
design. U.M., W.R., P.S., H.E. and A.R. provided case material and clinical
information. All co-authors commented on the manuscript. G.A.R. conceived and
directed the study. Data interpretation and writing of the manuscript was carried
out by S.W.S. and G.A.R.
CoMPEtInG FInAnCIAl IntEREStS
The authors declare no competing financial interests.
Published online at http://www.nature.com/naturegenetics/.
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table 1 summary of putative etiologic sequence variants in SHANK2
CNV del of
CNV del of
in PDZ domain
in PDZ domaind
Frequency of nonsynonymous variants identified in 580 individuals with mental retardation or ASD. All variants are
absent in the 659 analyzed control samples. Three of the variants (two CNVs and the nonsense mutation R462X)
occur de novo; the other missense mutations are inherited by an unaffected parent and are of unknown importance.
Nucleotide and amino acid position is according to coding sequence of PROSAP1 with the identifier AB208026.1
(published March 2009) from UCSC Human Genome Browser assembly March 2006.
an = 396. bn = 184. cn = 659. dIndividual MO1812A011 with mental retardation and a premature stop in the PDZ
domain, and individual TULB081863 with mental retardation and the S231Y mutation also present autistic features.
“–”, the 659 controls used for sequencing were not screened for CNVs; F, female; M, male; n.a., not available because
parents did not provide blood samples. All rare (<1% frequency) CNV variants detected in these cases are described in
supplementary table 3.
© 2010 Nature America, Inc. All rights reserved.