ORIGINAL ARTICLE

Variants of the CNTNAP2 5 promoter as risk factors for

autism spectrum disorders: a genetic and functional approach
AG Chiocchetti
1
, M Kopp
1
, R Waltes
1
, D Haslinger
1
, E Duketis
1
, TA Jarczok
1
, F Poustka
1
, A Voran
2
, U Graab
3
, J Meyer
4
, SM Klauck
5
,
S Fulda
3
and CM Freitag
1
Contactin-associated protein-like 2 gene (CNTNAP2), a member of the Neurexin gene superfamily, is one of the best-replicated risk
genes for autism spectrum disorders (ASD). ASD are predominately genetically determined neurodevelopmental disorders
characterized by impairments of language development, social interaction and communication, as well as stereotyped behavior
and interests. Although CNTNAP2 expression levels were proposed to alter ASD risk, no study to date has focused on its 5 promoter.
Here, we directly sequenced the CNTNAP2 5 promoter region of 236 German families with one child with ASD and detected four
novel variants. Furthermore, we genotyped the three most frequent variants (rs150447075, rs34712024, rs71781329) in an
additional sample of 356 families and found nominal association of rs34712024G with ASD and rs71781329GCG[7] with language
development. The four novel and the three known minor alleles of the identied variants were predicted to alter transcription
factor binding sites (TFBS). At the functional level, the respective sequences spanning these seven variants were bound by nuclear
factors. In a luciferase promoter assay, the respective minor alleles showed cell line-specic and differentiation stage-dependent
effects at the level of promoter activation. The novel potential rare risk-variant M2, a G4A mutation 215 base pairs 5 of the
transcriptional start site, signicantly reduced promoter efciency in HEK293T and in undifferentiated and differentiated
neuroblastoid SH-SY5Y cells. This variant was transmitted to a patient with autistic disorder. The under-transmitted, protective
minor G allele of the common variant rs34712024, in contrast, increased transcriptional activity. These results lead to the conclusion
that the pathomechanism of CNTNAP2 promoter variants on ASD risk is mediated by their effect on TFBSs, and thus conrm the
hypothesis that a reduced CNTNAP2 level during neuronal development increases liability for ASD.
Molecular Psychiatry advance online publication, 16 September 2014; doi:10.1038/mp.2014.103
INTRODUCTION
Autism spectrum disorders (ASD) are childhood-onset neurode-
velopmental disorders, showing a prevalence of approximately 1%
in the general population.
1,2
ASD are characterized by pervasive
impairments of social interaction and communication, repetitive
and stereotyped patterns of behavior and restricted interests.
3,4
They comprise childhood autism, Aspergers syndrome and
atypical autism respective pervasive developmental disordernot
otherwise specied. Although twin studies have shown a herita-
bility of 70 90%,
5,6
the genetic architecture of ASD has not yet
been fully elucidated.
The contactin-associated protein-like 2 gene (CNTNAP2), a
member of the Neurexin gene superfamily, has been suggested
as one of the most promising risk genes for ASD and language
development.
7
It is located within one of the best replicated
chromosomal regions detected by linkage studies in ASD (7q22
q36)
5
and language development.
8
In addition, several studies on
CNTNAP2 have reported association of genetic variants with ASD
and language development. Increased risk for ASD for rs7794745T
was noted in a study combining linkage- and subsequent family-
based association methods. Furthermore, this variant of intron 2 of
CNTNAP2 displayed a male-specic and a parent-of-origin effect,
9
partly explaining the marked sex differences observed in the
incidence of autism. In addition, the major C allele of the non-
coding variant rs2710102 (intron 13) increased ASD risk. Again, a
mainly male-driven association with age at rst spoken word was
observed.
9
The relevance of CNTNAP2 variants for language deve-
lopment is further highlighted by an association of rs2710102C
with poor non-word repetition test performance and lower
receptive and expressive language abilities in specic language
impairment.
10,11
In an Australian general population sample, a
four-marker haplotype including rs2710102C correlated with
language acquisition. The homozygous carriers of the haplotype
obtained substantially lower scores in the communication sub-
scale of the Infant Monitoring Questionnaire.
12
Functionally, a
magnetic resonance imaging study showed that healthy indivi-
duals homozygous for both ASD risk alleles, rs7794745T and
rs2710102C, exhibited an increased activation in the right inferior
frontal gyrus during a verbal uency task, which typically induces
activation of the left side, that is, Broca's area.
13
This atypical
language lateralization has also been observed in ASD.
14
Sequencing studies of CNTNAP2 found an elevated burden of
rare and private mutations in patients with ASD compared with
controls.
15,16
Most of the identied deleterious or non-synony-
mous mutations resided in the C-terminal part of CNTNAP2 with
no specic enrichment in any single domain (exons 1424). Their
1
Department of Child and Adolescent Psychiatry, Psychosomatics and Psychotherapy, JW Goethe University, Frankfurt am Main, Germany;
2
Department of Child and Adolescent
Psychiatry, Saarland University, Homburg, Germany;
3
Institute of Experimental Cancer Research in Pediatrics, Frankfurt am Main, Germany;
4
Department of Neurobehavioral
Genetics, Institute of Psychobiology, University of Trier, Trier, Germany and
5
Division of Molecular Genome Analysis, German Cancer Research Center (DKFZ), Heidelberg,
Germany. Correspondence: Professor CM Freitag, Department of Child and Adolescent Psychiatry, Psychosomatics and Psychotherapy, JW Goethe University, Deutschordenstr.
50, Frankfurt am Main 60528, Germany.
E-mail: c.freitag@em.uni-frankfurt.de
Received 18 June 2013; revised 4 June 2014; accepted 14 July 2014
Molecular Psychiatry (2014), 111
2014 Macmillan Publishers Limited All rights reserved 1359-4184/14
www.nature.com/mp
functional impact is still elusive. Also, several case studies des-
cribed rare deletions of CNTNAP2. For most of the patients
language-related decits were reported, including stuttering,
delay of language or ASD.
1721
One individual with ASD carried
a maternally inherited rare deleterious copy number variation
(CNV) within the promoter region of CNTNAP2, which led to
reduced CNTNAP2 mRNA expression in lymphoblastoid cell lines of
carriers with ASD in this family.
22
CNTNAP2 knock-out mice showed behavioral phenotypes remi-
niscent of ASD including stereotypic motor movements, beha-
vioral inexibility, abnormalities in social behavior and reduced
ultrasonic vocalization. At the cellular level of the brain, an
aberrant neuronal migration, reduction of GABAergic interneurons
and aberrant neuronal synchrony were described, pointing toward
a central role of CNTNAP2 in early brain development and
neuronal differentiation.
23
The central role of this protein is further
highlighted by its role in the clustering of Potassium (K
+
) channels
at the juxtaparanodal region of the nodes of Ranvier,
24
and its
high expression levels in language-related brain areas.
25
Taken together, there is convergent evidence from linkage,
association, CNV, knock-out, sequencing and expression studies
for involvement of CNTNAP2 in ASD and language-related
phenotypes. It is thus likely that genetic variants or a reduced
gene dosage of this susceptibility gene modulate ASD and/or
language development by changing CNTNAP2 functionality and
availability during neuronal development. Currently, evidences
emerged showing that FOXP2 is downregulating CNTNAP2 levels
and mutations of either FOXP2 or FOXP2 binding elements in
intron 1 of the CNTNAP2 gene promote language disorders.
10,20
However, this intronic repressor is just one of the many potential
regulatory elements. Despite the obvious functional impact of 5
promoter variants on mRNA expression levels,
26,27
no study to
date has focused on the identication and functional character-
ization of CNTNAP2 promoter variants in the context of ASD. This
region has so far escaped array-based genome-wide analysis,
as none of the annotated variants is covered by any of the
commercially available chip-sets. Furthermore, next-generation
sequencing studies are limited in their detection of sequence
variants in GC-rich regions as 5 promoters.
28
Therefore, we aimed at identifying novel mutations and
studying known polymorphisms of the CNTNAP2 5 core promoter
in patients with an ASD diagnosis, testing their functional impact
in vitro, and investigating their association with ASD and
language-related phenotypes. After sequencing of a detection
cohort, we performed in-silico analyses to specify transcription
factor binding sites (TFBS) likely altered by the variants. The
signicance of the respective alteration of TFBS was assessed by
assaying the binding of nuclear factors and by measuring the
transcriptional activity of the respective promoter sequences
in vitro. As the expression of transcription factors varies depending
on cell line and cellular differentiation stage, in-vitro analysis was
performed in HEK293T cell lines and at different developmental
stages, that is, in undifferentiated as well as during early- and late-
neuronal differentiation of neuroblastoid SH-SY5Y cell lines. Using
these two cell models, we could also study differential effects
between non-neuronal and neuronal cell types. Finally, to assess
the effect of the most frequent variants on ASD risk and language
development, we studied their association in an extended cohort.
We report four novel mutations and three known polymorph-
isms of the 1 kb up-stream promoter of CNTNAP2 in ASD families.
All variants were bound by nuclear factors. Two of the novel
mutations were found in affected individuals. Furthermore, we
showed that three of the four new mutations as well as all three
known variants altered transcriptional efciency in vitro depend-
ing on neuronal differentiation status and cellular background. We
report a nominal association of the functional CNTNAP2 promoter
variant rs34712024 with ASD and of the short tandem repeat (STR)
rs71781329 with language development. In conclusion, we discuss
a potential dosage-dependent effect of CNTNAP2 expression on
ASD etiology.
MATERIALS AND METHODS
Participants
Subjects with ASD and their families were recruited at the Departments of
Child and Adolescent Psychiatry, Psychosomatics and Psychotherapy at JW
Goethe University Frankfurt/Main and at Saarland University Hospitalas
described previously.
29,30
All parents and children had given written
informed consent. The study was approved by the local ethical committees
(decision 162/99 (Frankfurt); 73/04 (Homburg) and 237/09 (Frankfurt)). All
patients were diagnosed using the gold-standard diagnostic tools Autism-
Diagnostic Interview-Revised (ADI-R)
31
and/or Autism Diagnostic Observa-
tion Schedule Generic (ADOS-G).
32
Affected individuals met DSM-IV TR
criteria for autistic disorder, Aspergers disorder or pervasive developmental
disordernot otherwise specied. We excluded individuals with intelli-
gence quotient (IQ) o35, birth weighto1000 g, cerebral palsy, reported
chronic medical conditions (with the exception of epilepsy), blindness,
deafness, known karyotypic or cytogenetic aberrations (including 7q35
CNVs). The cohort in total consisted of 510 male and 82 female patients
from 592 families (492 trios, 73 duos, 27 singletons). A total of 87.5% of the
index patients were diagnosed with strict autistic disorder including high-
(IQ70) and low-functioning (IQo70) autism. The remaining 12.5% of the
patients received a spectrum diagnosis (Aspergers disorder or pervasive
developmental disordernot otherwise specied). Of this cohort, we
randomly selected 236 families as sequencing sample. The remaining set
was used as extended sample to improve power for singlemarker asso-
ciation test. Both samples were matched for diagnoses, age at diagnosis,
sex and IQ (Supplementary Table 1).
Sequencing (detection sample)
Genomic DNA was extracted from blood or saliva using standard methods.
The sequence of interest was determined by annotated TFBSs 5 of the
transcriptional start site (TSS) as provided by the transcription factor ChIP-
Seq data set (ENCODE, UCSC genome browser). This region spanned base
pairs 646 to 120 from TSS of CNTNAP2 transcript NM_014141.5
(Figure 1a).
PCR primers were designed to cover transcriptional and translational
start site as well as the potential 5 promoter region of interest here.
The nal amplicon spanned the region from +585 bp to 709 bp
(NM_014141.5). For full details on primers and PCR conditions see Supple-
mentary Table 2. Amplicons were puried using GeneJET PCR Purication
Kit (Thermo Scientic, St Leon-Rot, Germany). Sanger sequencing of the
detection sample was outsourced to SRD (Bad Homburg, Germany).
Prediction of regulatory elements
To identify target regions for transcriptional regulation, the TF-ChIP-Seq
data set from ENCODE was used. Internal ribosome entry sites were identi-
ed using IRESite web tool (http://www.iresite.org/). To predict modied
transcription factor binding sites (TFBS), Genomatix software MatInspector
(Munich, Germany) V2.6 (ref. 33) was used. Sequences including 50 bp of
each variant were analyzed. Transcription factors were not considered for
further investigation if the input matrix sequence did not span the variant
of interest, or if standard score or matrix similarity was below 0.75.
Transcription factors, with evidence for regulation during neuronal differ-
entiation from a microarray experiment (data not shown) were further
investigated for their regulatory pattern during neuronal differentiation
using real-time reverse transcription-polymerase chain reaction (RT-PCR).
Cloning
For construction of the luciferase vectors, a fragment of 1236 bp (+0 bp to
1236 bp; NM_014141.5) was amplied from subjects carrying major
alleles only or the respective minor alleles (Supplementary Table 2). This
region spans the TSS and additional 500 bp 5 of the predicted TF binding
region. Amplicons were subcloned into pGEM-T Easy Vector-System I
before cloning into target vector pGL4.10[luc2] (Promega, Mannheim,
Germany). Novel mutations (M1 M4) were generated by site-directed
mutagenesis of the wild-type vector pGL4.10[luc2]-WT as described
34
(for
primers see Supplementary Table 2). Vectors carrying promoter variants
rs150447075G, rs34712024G, rs71781329GCG[7] or rs71781329GCG[8]
were generated using the same procedure as for wild-type/major-allele
CNTNAP2 5 promoter variants in ASD
AG Chiocchetti et al
2
Molecular Psychiatry (2014), 1 11 2014 Macmillan Publishers Limited
constructs pGL4.10[luc2]-WT but with DNA of the respective minor allele
carriers as templates. Constructs were screened using restriction fragment
length polymorphism analysis as described below. All nal constructs were
veried by direct sequencing.
Cell culture
HEK293T and SH-SY5Y cells were seeded at a density of 2x10
5
cells cm
2
and grown in DMEM+GlutaMAX medium (all media were purchased from
Life Technologies, Darmstadt, Germany, if not otherwise specied) supple-
mented with 10% fetal calf serum, 1% Pyruvate, 100 U ml
1
Penicillin (PAA,
Piscataway, NJ, USA) and 100 g ml
1
Streptomycin (PAA) in a humidied
atmosphere at 37 C and 5% CO
2
. For the differentiation assay, SH-SY5Y
cells were seeded at a density of 5x10
3
cells cm
2
in standard medium for
24 h followed by treatment for an additional 10 days with differentiation
medium containing 10 M retinoic-acid (Sigma-Aldrich, St Louis, MO, USA),
50 ng l
1
brain derived neurotrophic factor (Immunotools, Friesoythe,
Germany), 2 mM cAMP (Sigma-Aldrich), 20 mM KCl (Sigma-Aldrich), 1XB27
supplement and 1XGlutaMAX in Neurobasal medium, exchanging the
medium every other day. Cells were collected 24 h after exchanging the
media. Transition from undifferentiated to differentiated stages was
conrmed using real-time RT-PCR to measure mRNA expression levels of
neuronal marker gene MAPT (increased expression) and cell division
marker gene CDC2 (reduced expression).
Relative quantitative real-time RT-PCR
mRNA was puried using the GeneJet mRNA-Purication Kit (Thermo
Fisher Scientic, Waltham, MA, USA) and cDNA was generated with the
Revert-Aid H-Plus Kit (Thermo Fisher Scientic) according to the
manufacturers protocols. mRNA levels were analyzed adapting the UPL-
System from Roche using the ABgene-ROX-Mastermix (Thermo Fisher
Scientic) on a StepOnePlus System (Life Technologies, Darmstadt, Germany).
For details on primer and probe combinations, see Supplementary Table 2.
Fold-change of mRNA expression level was calculated using the 2
-ddCT
method.
35
GAPDH and POLR2F were used as stable reference genes
calculating the geometric mean of relative expression as proposed by
previous studies
36
and as conrmed in own experiments (data not shown).
Transfection and luciferase assay
Transfection of the luciferase vectors was performed using MetafectenePro
(Biontex Laboratories, Planegg, Germany) according to the manufacturers
recommendations. Cells were plated at a density of 2x10
5
cells cm
2
(mitotic cells) or 2.5x10
4
cells cm
2
(differentiating SH-SY5Y) in a 12-well
plate. Luciferase activity was measured by pooling two wells using the
Dual-Luciferase Reporter Assay System (Promega, Mannheim, Germany)
following the manufacturers protocol. Firey luciferase activity was
normalized to the empty vector pGL4.10[luc2]. All experiments were
corrected for transfection efciency based on co-transfection with Renilla
luciferase-expressing vector pGL4.74[hRluc/TK] (Promega). All experiments
were performed in biological triplicates.
Electrophoretic mobility shift assay (EMSA)
To conrm binding of nuclear factors to the variants under study, we
applied EMSA using biotin-labeled double stranded DNA sequences (for
details see Supplementary Table 2) and nuclear extracts from broblastoid
HEK293T and neuroblastoid SH-SY5Y undifferentiated cells. Nuclear protein
fractions were extracted as published.
37
All EMSA were performed on a
Figure 1. Variants of the CNTNAP2 promoter. (a) Variants analyzed in this study, their schematic localization within the respective region and
the potential transcription factor binding sites (TFBS) reported in the ENCODE TF-ChIP-Seq Dataset. The proximity of the different minor
alleles is shown. Gray values of DNA binding factors are proportional to the maximum signal strength reported. (b) Pedigrees of families
carrying identied novel variants. Nomenclature of the variants is based on their relative position to the transcriptional start site of mRNA
NM014141.5. Black-lled boxes (males) or circles (females) mark individuals with an ASD diagnosis. Colored rectangles around genotype
correspond to the identied mutations and are used consistently throughout the other gures. CTCF, Insulator protein (CCCTC-binding factor);
NANOG Homeobox Transcription Factor; EGR1, early growth response 1; E2F1, E2F transcription factor 1; HDAC2, Histone Deacetylase 2; JUND,
Jun D Proto-Oncogene; NRSF, neuron-restrictive silencer factor; RAD21, double-strand-break repair protein rad21 homolog; SIN3A, Histone
Deacetylase Complex Subunit Sin3a; TAF7, TATA Box binding protein (TBP)-associated factor, RNA Polymerase II; YY1, Yin and Yang 1 protein;
ZNF263, Zinc-nger protein 263.
CNTNAP2 5 promoter variants in ASD
AG Chiocchetti et al
3
2014 Macmillan Publishers Limited Molecular Psychiatry (2014), 1 11
native 5% polyacrylamide (Rotiphorese, Roth, Germany) gel in 0.5x TBE
Buffer (45 mM Tris, 45 mM boric acid, 1 mM EDTA; all purchased from
Applichem, Darmstadt, Germany). Gels were plotted for 10 min at 3.55
mA cm
2
in 0.5x TBE. Binding reaction and detection of biotin-labeled
DNA fragments were performed using the LightShift Chemiluminescent
EMSA Kit (Thermo Fisher Scientic) according to manufacturers protocol.
In each binding reaction (20 l), 20 fmol of biotin-labeled fragments were
used. To reduce unspecic bindings, 1 g of articial poly(deoxy-inosine *
deoxy-cytosine) DNA was also included.
Genotyping (extension sample)
To increase sample size for association test of known variants, we
genotyped additional 356 families (see participant section above). Variants
rs150447075, rs34712024 and rs71781329 were genotyped using restric-
tion fragment length polymorphisms (Watcut Database; Supplementary
Table 2). A subset of N=295 families was analyzed using Custom TaqMan
SNP Assays (Life Technologies) AH5IQWH (rs150447075) and AH39SP9
(rs34712024), respectively. Details on real-time RT-PCR setups are
available upon request. In total, we genotyped 99.73% (rs150447075 and
rs34712024) and 98.37% (rs71781329) of the extension sample.
Statistical analysis
Statistical analyses were done by IBM SPSS v20.0 or SAS v9.3. Power
analysis was performed by Quanto v1.2.4 (ref. 38) and G*Power v3.1.0
(http://www.psycho.uni-duesseldorf.de/aap/projects/gpower). Detection of
Mendelian errors was performed using Haploview v4.1.
39
UNPHASED v3.1.4
(ref. 40) was used to study single-marker association and to explore
imprinting and sex-specic effects. As the ADI-R language items A9 (age at
rst word) and A10 (age at rst phrase) were not normally distributed
(KolmogorovSmirnov Po0.001), non-parametric regression models to
investigate the effect of the dichotomized genotypes (dominant model)
with adjustment for age at diagnosis and IQ effects was performed by
the SAS macro npar (http://www.ams.med.uni-goettingen.de/Projekte/
makros/index.html). Luciferase assays were analyzed by repeated mea-
sures analysis of variance.
RESULTS
Promoter sequencing
To identify known and novel variants, we sequenced the CNTNAP2
promoter region of 667 members of 236 German families with
one child affected with ASD. In total, we identied four novel
(M1M4; Figure 1a) variants 5 of the TSS of CNTNAP2 (Ref Seq:
NM_014141.5, UCSC genome browser build Chr 37). None of them
has been characterized previously or mentioned in the 1000
Genome database (http://www.1000genomes.org) or listed on the
Exome Variant Server (http://evs.gs.washington.edu). Variant M1, a
deletion of a C nucleotide at position 407 (M1_ 407C4delC)
was identied in an unaffected male half sibling. Maternal
transmission could be excluded, but no DNA of the father was
available. The G to A nucleotide exchange at position 215
(M2_ 215G4A) was paternally transmitted to a patient with
autistic disorder (conrmed by ADI-R and ADOS, IQ=78 and a
delayed onset of speech with age at rst spoken phrase =
54 months). The third variant at position 43 changes G to C
(M3_ 43G4C) and was detected in one mother. Variant
M4_ 26G4C was paternally transmitted to a child diagnosed
with autistic disorder (conrmed by ADI-R and ADOS, IQ=50 and a
delayed onset of speech with age at rst spoken phrase =
52 months). The same variant was additionally identied in a
mother and a father of two independent families, respectively.
Neither of them had transmitted the allele to their affected child
but the father had transmitted the C allele to an unaffected sibling
(Figure 1b). The affected individuals carried a diagnosis of early
childhood autism with language delay and atypical autism
without language delay, respectively. No phenotypic measures
were available for the parents carrying any of the novel mutations.
Variants M1M4 reside in the 5 promoter region of CNTNAP2
and are thus of major interest in this study. M1 and M2 were within
DNA segments that are bound by transcriptional factors (Figure 1a)
with scores ranging from 92 (CTCF, CCCTC-binding factor) to 1000
(NRSF, neuron-restrictive silencing factor) out of 1000. This
strongly supports the regulatory potential of these sequences.
In our sequencing cohort, we also detected three out of ten
known variants of the CNTNAP2 5 promoter region of interest
(based on dbSNP, HapMap and UCSC data). Single-nucleotide
polymorphisms rs150447075 (NG_007092.2:g.4595T4G) and
rs34712024 (NG_007092.2:g.4641A4G) as well as the STR
rs71781329 (NG_007092.2:g.4864_4865insGCGGCG) (Figure 1a).
The STR rs71781329 is a sixfold GCG repeat (major allele termed
here GCG[6]) where the annotated insertion leads to eight repeats
(GCG[8]). In one family, we identied a previously unknown
paternally transmitted GCG[7] allele.
EMSA
Binding of nuclear factors to the sequences spanning the identi-
ed variants was conrmed by EMSA (Figure 2). Interestingly, the
pattern of interaction between SH-SY5Y and HEK293T nuclear
extracts differed, suggesting a cell type-specic expression of
nuclear proteins.
The strongest binding was observed for the sequences
spanning M2_215 and rs71781329 followed by rs34712024.
Weak proteinDNA interactions were visible for variants M3_43
and M4_26. Oligos spanning M1 and rs150447075 show similar
Figure 2. Binding of nuclear factors to 5 promoter variants. Electrophoretic mobility shift assay (EMSA) proves binding of nuclear factors to
the respective alleles. Positive control (black) DNA and EBNA protein was provided with the LightShift Chemiluminescent EMSA Kit. Double-
stranded oligos spanning wild-type/major allele (dark gray) and minor allele (colored) sequences +10 base pairs in each direction were
incubated with nuclear extracts of HEK293T or SH-SY5Y cells and compared with the DNA incubated without protein extracts (no prot). All
experiments were performed at least twice and representative results are shown. Wild-type oligo of variant M1_ 407C and major allele
variant rs150447075T are identical. Strongest binding is observed for sequences spanning variants M2_ 215 and rs71781329. Minor alleles of
variants M2_ 215A and rs34712024G reduce binding afnity, whereas minor alleles (GCG[7] and GCG[8] of variant rs71781329 increase
binding.
CNTNAP2 5 promoter variants in ASD
AG Chiocchetti et al
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Molecular Psychiatry (2014), 1 11 2014 Macmillan Publishers Limited
weak binding of nuclear factors. Please note, that both variants
(M1 and rs150447075) are next to each other, that is, on position
407 and 406, respectively, and thus show an expected similar
proteinDNA interaction pattern. Descriptively, when compared
with the respective major allele sequences, we observed reduced
binding of nuclear factors at minor allele variants rs34712024G,
M2_ 215G and M3_ 43C, as well as pronounced increased
binding for both minor alleles (GCG[7] and GCG[8]) of variant
rs71781329, and a mildly increased binding to M4_ 26C. In
contrast to the similar proteinDNA interaction pattern, minor
allele rs150447075G at position 406 did not, whereas
M1_ 407delC did affect binding of nuclear factors.
CNTNAP2 expression levels during neuronal differentiation
To characterize transcriptional activation of CNTNAP2 during
neuronal differentiation we measured mRNA expression levels
over 264 h at seven time points during brain derived neurotrophic
factor induced neuronal differentiation. The most critical time
points were selected for differential functional analysis in the
subsequent luciferase assay (see below). mRNA levels of CNTNAP2
were 3.77-fold downregulated 72 h post-induction of differen-
tiation (PD) when compared with time point 0 h PD with a
subsequent re-induction of expression reaching its peak at 216 h
PD (Figure 3a). This downregulation is in agreement with previous
studies on human neuronal progenitor cells,
41
and has been
reported during mid-fetal development in a post-mortem brain
expression study.
42
In-silico analysis of TFBS
All identied variants were predicted to change TFBS in-silico
(Table 1 and Supplementary Table 3). The deletion of the C allele
407 bp 5 of the transcription start (novel variant M1) was
predicted to disrupt the binding sequences for transcriptionally
active estrogen and retinoic acid receptors, as well as for
Eomesodermin. In addition, two new TFBS for GA repeat binding
protein, beta 1 and Wilms Tumor Suppressor 1 are generated. Of
these transcription factors, the latter three are expressed in brain
or nervous system.
The novel variant M2, a paternally inherited G4A mutation at
position 215 was predicted to reduce binding of brain expressed
PLAG1 (coded on the forward strand) as well as the non-brain
specic TFs Spermatogenic Zip1 factor and ZNF263 (zinc-nger
263). In parallel, an additional binding site for ETV1 (Ets variant 1),
a brain expressed transcriptional regulator involved in cell
differentiation, is generated.
The third identied variant at position 43, a G to C nucleotide
change (M3_ 43C), potentially generates TFBS for the brain
expressed transcription factors CREB (cAMP-responsive element
binding protein), EGR3 (early growth response 3), VMAF (v-Maf),
ATF (activating transcription factor) and WHN (winged helix
protein), while disrupting sequences for non-brainspecic TFs
only, namely, CTCF (CCCTC-binding factor) and PAX5 (paired-Box
transcription factor 5).
The paternally inherited 26_G4C mutation (variant M4) was
predicted to generate additional TFBS for 11 different transcrip-
tion factors of which only EGR1 (early growth response 1) is active
in brain.
Similarly, all three known variants detected in our sequencing
sample were predicted to change binding sites for brain
expressed transcription factors in silico: Carriers of the minor (G)
allele of rs150447075 were predicted to carry an additional TFBS
for transcription factors GLI3 (GLI family zinc-nger 3), TLX1 (T-cell
leukemia homeobox 1) and EGR1 (Early growth response 1) while
losing TFBS for EOMES. The minor allele rs34712024G generates
an additonal recognition site for the brain expressed TF EGR1. For
rs7178329, an increased number of TFBS for EGR1 was predicted
by the number of repeats (that is, 3 TFBS for GCG[6], 4 TFBS for
GCG[7] and 5 TFBS for GCG[8]).
Expression levels of transcription factors during neuronal
differentiation
To determine the regulatory role of the transcription factors we
measured the respective mRNA levels during neuronal differentia-
tion of SH-SY5Y cells. One of the most frequently in-silico identied
TFBS is bound by EGR1. This early growth response gene 1 was
strongly upregulated (Figure 3b), reaching its maximum expres-
sion level 120 h PD, that is, at the same time point when CNTNAP2
expression was reactivated after its reduction. We observed a
similar pattern for the paralog EGR4 (Figure 3b). In addition, we
found an early phase upregulation for ZNF219 (Zinc-nger protein
219), a transcriptional repressor,
43
followed by wave-like regula-
tion before its level was reduced below detection limit. The lowest
Figure 3. Characterization of CNTNAP2 regulation during neuronal differentiation. (a) During 264 h of neuronal differentiation, CNTNAP2 is
reduced until 72 h post induction of differentiation (PD) followed by a recovery phase until 216 h PD. In parallel, markers for neuronal
differentiation (MAPT) are constantly increased, whereas markers for cell division decrease after a short activation period. (b) Transcriptional
factors that bind to the target region are regulated during neuronal differentiation. Only factors with a log2 fold-change 42 are depicted.
EG1, EGR4 and ZNF219 are the most strongly regulated factors and are thus most likely to regulate transcriptional activity of the CNTNAP2 5
promoter. Interestingly, EGR1 and EGR4 (early growth response 1/4) are highest regulated in a time window where CNTNAP2 transcription is
reactivated, and the transcriptional repressor ZNF219 is slightly downregulated. (c) The 5 promoter of the CNTNAP2 gene is weakly activated
in the HEK293T cells (luciferase assay) and undifferentiated SH-SY5Y cells. The strongest activation of the promoter is observed 216 h after
induction of differentiation. Colored squares correspond to the mean values of three biological replicates of the different variants under study
here. For further details on relative fold-changes see Figure 4.
CNTNAP2 5 promoter variants in ASD
AG Chiocchetti et al
5
2014 Macmillan Publishers Limited Molecular Psychiatry (2014), 1 11
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CNTNAP2 5 promoter variants in ASD
AG Chiocchetti et al
6
Molecular Psychiatry (2014), 1 11 2014 Macmillan Publishers Limited
peak during the regulatory wave coincided with the reactivation
of CNTNAP2 expression 120 h PD.
Luciferase assay
The 1-kb region upstream of the TSS of CNTNAP2 showed
signicant transcriptional activation in the luciferase assay when
compared with the empty vector, thus conrming its effect as
transcriptional cis-promoter. In line with the CNTNAP2 mRNA
expression prole, all vector constructs harboring the promoter
region of interest showed low activity at time points 0 and 72 h
PD, and a remarkable increase at time point 216 h (Figure 3c)
when CNTNAP2 mRNA expression reached its maximum after
reactivation. Subsequent analysis of the promoter activity in SH-
SY5Y cells at each individual time point (0, 72 and 216 h PD) and in
undifferentiated HEK293T cells showed cell line-specic and PD
stage-dependent effects (Figure 4 and Supplementary Table 4).
In-vitro, M1_ 407delC signicantly increased transcriptional
activation in undifferentiated HEK293T (fold-change (FC) =1.47;
s.d. =0.05; P =0.032) and SH-SY5Y cells (FC=1.62; s.d. =0.09;
P =0.010). Descriptively, this effect was also present in differentiat-
ing SH-SY5Y cells but could not be conrmed statistically. When
compared with the wild-type allele G, the A allele of mutation M2
signicantly reduced transcriptional efciency in mitotically active
cells (HEK293T: FC=0.45; s.d. =0.03; P =0.036; SH-SY5Y: FC=0.53;
s.d. =0.04; P =0.013) as well as in differentiating SH-SY5Y cells
(72 h PD: FC=0.45; s.d. =0.02; P =0.047; 216 h PD: FC=0.618;
s.d. =0.06; P =0.071). The novel identied C nucleotide at M3_ 43
signicantly increased transcriptional activity of the promoter in
undifferentiated neuroblastoma SH-SY5Y (FC=1.36; s.d. =0.08;
P =0.036) but not in HEK293T cells (FC =0.99; s.d. =0.13;
P =0.973). A trend toward an upregulation was also observed in
differentiating SH-SY5Y cells (72 h PD: FC=1.38; s.d. =0.16;
P =0.243; 216 h PD: FC=2.17; s.d. =0.24; P =0.056). For the
M4_ 26C allele we did not observe any changes compared with
the respective wild-type promoter sequence.
Compared to the major allele promoter, we found a signicantly
increased activation of the rs150447075G promoter in undiffer-
entiated SH-SY5Y (FC=1.07; s.d. =0.03; P =0.005) and HEK293T
(FC=1.52; s.d. =0.39; P =0.028) cells but not during neuronal
differentiation. The rs34712024G allele signicantly increased
transcriptional efciency in HEK293T cells (FC =1.61; s.d. =0.44;
P =0.040) but not at any differentiation stage of SH-SY5Y cells.
The transcriptional efciency of the rs71781329 minor alleles
depended on the number of GCG repeats in HEK293T cells: GCG[7]
did not change efciency (FC =1.22; s.d. =0.3; P =0.107), whereas
GCG[8] induced a 1.66-fold increase (P =0.024). In the SH-SY5Y cell
line, we observed a contrasting effect. GCG[7] slightly down-
regulated efciency in undifferentiated SH-SY5Y cells (FC=0.94;
s.d. =0.03; P =0.061) and GCG[8] clearly reduced expression at
stage 72 h PD (FC =0.91; s.d. =0.03; P =0.028).
Single marker association study in the extended ASD sample
In the extended sample, a signicant under-transmission of allele
rs34712024G to ASD patients (odds ratio (OR) =0.41; condence
interval at 95% (CI95) =0.190.89; P =0.018; Table 2) was observed.
When correcting for three statistical tests, the corrected P-value
still shows a trend (P =0.054), but does no longer reach signi-
cance on the 5% level. The genotype-based association test
yielded a similar result (OR [A/G] =0.45; CI95 =0.380.96;
P =0.034). No homozygous [G/G] genotype was detected in any
patient. An additional analysis including only individuals with
autistic disorder did not explain more of ASD risk (OR =0.45;
CI95 =0.200.99; P =0.039). For the trimeric STR rs71781329 (7+8
repeats combined), no association was observed (OR =0.88;
CI95 =0.322.42; P =0.796). Descriptively, rs71781329GCG[7] was
under-transmitted (OR =0.60; CI95 =0.142.51; P =0.477) and
rs71781329GCG[8] (OR =1.34; CI95 =0.296.14; P =0.704) was
over-transmitted when compared with rs71781329GCG[6]
(Table 2). No gender-specic or imprinting effects were observed
(data not shown). No co-occurrence of any minor alleles was
observed with the exception of one index patient and an
unrelated mother carrying both minor alleles of rs150447075
Figure 4. Functional analysis of promoter activation dependent on cell type, SH-SY5Y differentiation status and genetic variants. Promoter
activity is inuenced by genetic variants depending on cell type and differentiation status. Luciferase assays have been performed to compare
the promoter activity of major/wild-type alleles to the respective minor alleles. Mean fold changes compared with the respective reference
vectors (wild-type/major alleles vector) are shown with standard deviation (whiskers). Asterisks mark signicance as tested in analysis of
variance for repetitive measures compared with reference vector (
+
Po0.1; *Po0.05; **Po0.01). PD, post induction of differentiation.
Table 2. Single marker transmission disequilibrium test
SNP (minor/major alleles) MAF
a
OR minor allele,
N=592 families
P-value
rs150447075T/G 0.023 0.92, CI95: 0.511.64 0.768
rs34712024A/G 0.016 0.41, CI95: 0.190.89 0.018
b
rs71781329GCG[6]/GCG(7_8) 0.006 0.88, CI95: 0.322.42 0.796
rs71781329GCG[6]/GCG[7]
c
0.003 0.60, CI95: 0.142.51 0.477
rs71781329GCG[6]/GCG[8]
d
0.003 1.34, CI95: 0.296.14 0.704
Abbreviations: CI, condence interval; MAF, minor allele frequency
(calculated on all samples); OR, odds ratio.
a
Minor allele frequency within
our autism spectrum disorders sample.
b
Po0.05.
c
Families of
rs71781329GCG[7] allele carriers were omitted; N=584 families were
included into analysis.
d
Families of rs71781329GCG[8] allele carriers were
omitted; N=588 families were included into analysis.
CNTNAP2 5 promoter variants in ASD
AG Chiocchetti et al
7
2014 Macmillan Publishers Limited Molecular Psychiatry (2014), 1 11
and rs34712024. Thus, no haplotype-based association analysis or
epistasis analysis was performed.
Inuence of promoter variants on ADI-R-derived language
measures
To explore if any of the three known variants had an effect on
language development, non-parametric regression models were
calculated. Age at diagnosis and IQ are known to inuence
language development and were thus included as covariates.
Subjects with genotypes rs71781329GCG[6]/[7] but not GCG[6]/[8]
showed a signicantly (Po0.00001) older age at rst words and at
rst spoken phrase compared with carriers of GCG[6]/[6] (for
details on mean differences see Table 3). However, the number of
subjects carrying the minor alleles was very low (GCG[6]/[7] N=2
and GCG[6]/[8] N=3); thus, these results have to be interpreted
with caution. The other known variants were not associated with
language delay in ASD (Table 3).
DISCUSSION
In search for an additional sequence element regulating CNTNAP2
and for genetic variants related to ASD, we Sanger sequenced and
functionally analyzed the 5 promoter sequence of CNTNAP2 in an
ASD trio cohort. Furthermore, we tested three polymorphisms for
association with ASD and language delay within ASD in an
extended sample. In contrast to the FOXP2 regulatory element
in intron 1,
10
the 5 promoter studied here is involved in the
upregulation of CNTNAP2 during neuronal differentiation. In-silico
analysis of novel and known variants of this sequence predicted
alternative TFBS for all minor alleles. In-vitro assays showed that all
variants but M4_ 26G differentially regulated promoter activity
as a function of cellular background and neuronal differentiation
stage, with potentially different pathological effects in-vivo. This
strongly suggests that the variants mediate effects that are
dependent on the transcription factor pattern, which in our study
have shown to be specic to cell type and differentiation stage.
Our main ndings are the description of a novel potentially
pathological rare mutation of the CNTNAP2 5 promoter, the
association of the functional variant rs34712024 with ASD and of
rs71781329 with language development.
Rare mutations may confer ASD risk via altered transcriptional
efciency
The strongest effect on promoter activity was observed for the
paternally transmitted variant M2_ 215A, which reduced tran-
scriptional efciency in all assays. The heterozygous index patient
Table 3. Non-parametric regression models of genetic effects on ADI-R language measures
Model First words (ADI-R A9) First phrase (ADI-R A10) First wordsmale
only (ADI-R A9)
First phrasemale
only (ADI-R A10)
T P T P T P T P
rs150447075
AA vs AG/GG 0.979 0.322 0.943 0.331 0.265 0.606 0.175 0.676
Age 0.290 0.772 0.184 0.854 0.154 0.877 0.385 0.700
IQ 4.942 o0.0001 6.039 o0.0001 4.424 o0.0001 5.652 o0.0001
N (carriers G) 383 (17) 336 (16) 331 (14) 292 (13)
Mean (s.d.) TT: 25.48 (14.42)
TG/GG: 21.65 (9.51)
TT: 37.69 (17.95)
TG/GG: 33.94 (10.47)
TT: 24.82 (14.14)
TG/GG: 21.93 (10.41)
TT: 37.01 (18.05)
TG/GG: 33.77 (10.19)
rs34712024
TT vs TG/GG 2.953 0.086 0.016 0.899 3.362 0.067 0.027 0.871
Age 0.277 0.781 0.176 0.861 0.142 0.887 0.375 0.707
IQ 4.808 o0.0001 5.968 o0.0001 4.293 o0.0001 5.602 o0.0001
N (carriers G) 383 (10) 336 (8) 331 (10) 292 (8)
Mean (s.d.) AA: 25.05 (14.07)
AG/GG: 34.80 (18.50)
AA: 37.36 (17.52)
AG/GG: 43.63 (23.80)
AA: 24.39 (13.76)
AG/GG: 34.80 (18.50)
AA: 36.67 (17.60)
AG/GG: 43.63 (23.80)
rs71781329
GCG[6]/[6] vs [6]/(7_8) 2.689 0.101 0.581 0.446 1.207 0.272 0.039 0.843
Age 0.180 0.858 0.237 0.813 0.005 0.996 0.447 0.655
IQ 4.866 o0.0001 6.036 o0.0001 4.329 o0.0001 5.664 o0.0001
N (carriers GCG(7_8)) 380 (5) 334 (8) 328 (4) 290 (4)
Mean (s.d.) GCG[6]/[6]: 25.03 (13.62)
GCG[6]/(7_8): 45.00 (37.11)
GCG[6]/[6]: 37.22 (16.98)
GCG[6]/(7_8): 58.80 (42.51)
GCG[6]/[6]: 24.44 (13.27)
GCG[6]/(7_8): 44.25 (42.81)
GCG[6]/[6]: 36.63 (17.07)
GCG[6]/(7_8): 55.50 (48.34)
rs71781329
GCG[6]/[6] vs [6]/[7] 83.972 o0.0001 19.135 o0.0001
Age 0.304 0.761 0.084 0.93341
IQ 4.954 o0.0001 6.249 o0.0001
N (carriers GCG[7]) 377 (2) 331 (2)
Mean (s.d.) GCG[6]/[6]:25.03 (13.62
GCG[6]/[7]:78.00 (42.43)
GCG[6]/[6]:37.22 (16.98)
GCG[6]/[7]: 99.00 (38.18)
Not performed as all carriers were male
rs71781329
GCG[6]/[6] vs [6]/[8] 0.109 0.741 0.104 0.748
Age 0.309 0.757 0.046 0.964
IQ 4.857 o0.0001 6.078 o0.0001
N (carriers GCG[8] allele) 378 (3) 332 (3)
Mean (s.d.) GCG[6]/[6]:25.03 (13.62)
GCG[6]/[8]: 23.00 (6.25)
GCG[6]/[6]:37.22 (13.98)
GCG[6]/[8]: 32.00 (13.86)
Not performed as all carriers were male
ADI-R, autism-diagnostic interview-revised; IQ, intelligence quotient; N, total number of samples included in the model with carriers of the respective minor
alleles in parentheses; Mean, mean months at rst words or rst phrase, respectively; P, P-value of non-parametric regression model; T, T-value of non-
parametric regression model.
CNTNAP2 5 promoter variants in ASD
AG Chiocchetti et al
8
Molecular Psychiatry (2014), 1 11 2014 Macmillan Publishers Limited
clinically presented with autistic disorder, a diagnosis which
includes a delayed onset of speech. The A allele of this variant
disrupts the core TFBS for ZNF263, a transcriptional repressor and
activator relevant during cell cycle regulation,
44
and for SPZ1
(Spermatogenic Zip1 factor). The chromosomal region of ZNF263
on chromosome 16p13 has shown weak linkage with ASD
(logarithm of the odds; LOD=2.17).
45
Both, ZNF263 and SPZ1
(Spermatogenic Zip1 factor), have been nominally associated with
ASD in a genome wide haplotype analysis.
46
The identication of
SPZ1 and ZNF263 genes in studies for ASD suggests that common
and rare variants of these transcription factors may trigger ASD
phenotypes by decreasing transcriptional activation of their
targeted promoter sequences. Similarly, the pathomechanism of
M2_ 215A may be driven by a reduced transcription factor
binding. In conclusion, a reduced CNTNAP2 mRNA expression as
achieved either by reducing the afnity of the targeting
transcription factors or by damaging mutations in their recogni-
tion pattern, may increase ASD risk.
Contrasting with M2, M1_ 407delC and M3_ 43C increased
transcriptional efciency of the CNTNAP2 promoter. M1 was
identied in an unaffected half-sibling only, and M3 was not
transmitted to affected offspring. The deletion of the C allele (M1)
generated or improved the recognition elements for the brain-
expressed transcription factors GABPB1 (GA Binding Protein
Transcription Factor, Beta Subunit 1) and WT1 (Wilms Tumor 1).
Interestingly, GABPB1 levels were reported to be downregulated in
affected patients with ASD (Hu et al.
47
Supplements) and the
coding region shows strong linkage with ASD.
48
Deletions of the
WT1 gene are associated with the WAGR syndrome (Wilms tumor,
Aniridia, Genitourinary malformations and mental Retardation).
Patients with this rare genetic disorder have a high prevalence
(420%) of autistic features.
49
Downregulation of GABP1 or
deletions of WT1 lead to reduced availability and thus also to
reduced binding of these regulatory factors to the respective DNA
sequences with a subsequent reduced transcription of the
targeted genes. Increased binding of these factors, in contrast,
likely increases promoter efciency as observed here for the
CNTNAP2 promoter variant M1, and thus even may be protective
for ASD.
Variant M3_ 43C generates a sequence pattern recognized by
the brain-expressed transcriptional regulators CREB ATF4 (alias
CREB2), EGR3 VMAF and WHN (for abbreviations see Table 1).
CREB transcription factors are regulated by glutamatergic signal-
ing and activate transcription of the ASD-associated FMR1 gene
(reviewed in ref. 50). The EGR3 gene has been associated with
schizophrenia
51
and its gene product functionally regulates axonal
growth and dendritic branching.
52
An increased recognition of
these elements at the 5 promoter may lead to an increased
expression of the targeted gene as observed here in the non-
differentiated neuroblastoid SH-SY5Y cells. The role of the binding
factors EGR3 and CREB underscores the relevance of M3 on brain
function. Taken together, we assume that an increased TF binding
will increase expression of CNTNAP2, and this may lead to a
protective effect of variant M3.
Variant M4_ 26C increases similarity to the binding sequence
of EGR1, a brain-expressed transcriptional activator.
53
However,
neither the EMSA nor the luciferase assay support increased
binding of transcriptional activators in the presence of M4_ 26C.
We hypothesize that either the predicted reverse binding of this
nuclear factor will not affect transcriptional activity, or the binding
site may be blocked by other nuclear factors. M4_ 26C was
detected in three independent families but only once transmitted
to the index patient. This renders a pathological effect of
M4_ 26C very unlikely.
The notion of an increased TF-binding at the CNTNAP2 5
promoter being protective is supported by the nding that both,
M1 and M3, were only detected in an unaffected, but ASD-related
individuals within our sample.
Owing to the rare frequency of the novel variants (N=1),
association analysis could not be performed. Thus, we cannot
exclude a false-positive nding for M2 or prove that M1 and M3
may be protective. However, we speculate that increased
promoter efciency either may not be pathologically relevant or
may even be protective and that a reduced CNTNAP2 expression
increases risk for ASD.
CNTNAP2 variants are associated with ASD and language
development
Testing the three known and more frequent promoter variants of
CNTNAP2 for association with ASD showed a less frequent
transmission of the minor G allele of rs34712024 from parents
to patients in the detection and the extended sample. The
protective genetic effect of the minor allele rs34712024G on ASD
may be attributed to an additional binding of the brain-specic
TF EGR1. This early growth-related transcriptional regulator is
strongly regulated during neuronal differentiation. EGR1 plays
a central role in the regulation of neuronal plasticity and
differentiation. Synaptic activity, a mechanism putatively impaired
in ASD,
54
regulates EGR1 expression leading to altered transcrip-
tion of targeted genes.
53
In our cellular differentiation model, EGR1
and EGR4 mRNA levels are strongly upregulated when CNTNAP2 5
promoter efciency is highest. Furthermore, their expression levels
decline shortly during the downregulation of CNTNAP2. Interest-
ingly, EGR1 binds to this minor G allele of rs34712024G in reverse
orientation. TFs can be active in both directions, but may exhibit
different effects depending on their orientation.
55,56
Again, we speculate that an increased binding afnity and thus
an increased expression activity of the promoter may explain the
protective effect of rs34712024G on ASD.
For STR rs71781329, we observed that each parental minor
allele was transmitted to the index patients (complete transmis-
sion) in the detection set. In the extended sample, GCG[7]
descriptively was under-transmitted and GCG[8] was over-
transmitted to the offspring compared with GCG[6]. No nominal
signicance was achieved, mainly due to the low frequency of
these minor alleles. With regard to delayed language within the
ASD sample, GCG[7] but not GCG[8] did show a highly signicant
association with later onset of speech. This nding has to be
interpreted with caution as only two individuals carrying the
respective allele were observed in our sample. Still, ndings
support the reported functional role of CNTNAP2 on language
acquisition.
10,12,25
At the level of transcriptional efciency, we observed differ-
ential effects on transcriptional efciency of the two identied
minor alleles depending on the number of inserts, that is, on the
number of TFBS for EGR1, and related to the cellular background,
that is, SH-SY5Y or HEK293T cells. Again, the minor alleles of
rs7178193 (GCG[7/8]) increased the number of TFBS for EGR1. In
contrast to the EGR1 binding sequence spanning the aforemen-
tioned variant rs34712024, the EGR1 binding sites on rs7178193
are oriented in the forward direction and may thus have a
different effect.
55
In the SH-SY5Y model, the GCG[7]-repeat but not
the GCG[8]-repeat of rs71781329 showed a slight reduction of
promoter efciency in the pre-differentiating cells. During later
stages of differentiation, hardly any difference in translation
efciency was observed between the different repeat carrying
cells. GCG[8] but not GCG[7] reduced transcriptional efciency
72 h after induction of differentiation, that is, when CNTNAP2
mRNA expression was lowest.
Following the idea of a decreased CNTNAP2 expression increas-
ing ASD risk, and in light of the association ndings, differential
CNTNAP2 expression in early stages of neuronal differentiation
driven by the GCG[7] allele, but not in a later stage as observed for
GCG[8], may be correlated with language delay in ASD.
CNTNAP2 5 promoter variants in ASD
AG Chiocchetti et al
9
2014 Macmillan Publishers Limited Molecular Psychiatry (2014), 1 11
Taken together, we propose a model in which number and
orientation of TFBS at the 5 CNTNAP2 promoter are determining
the pathogenic effect of the variants investigated here, and will
lead to the observed altered transcriptional efciency. In this
model, the upregulation of CNTNAP2 exhibits protective effects
whilst a downregulation confers increased risk to ASD. This is
underscored by our nding that on the one hand all variants
upregulating transcriptional efciency in undifferentiated SH-SY5Y
cells were potentially protective (rs34712024G) or had no effect on
ASD (M1_ 407delC; M3_ 43C or M4_ 26C), while on the other
hand the identied risk variants decreasing transcriptional efci-
ency in the undifferentiated SH-SY5Y model (rs71781239GCG[7]
and M2215A) were possible risk factors for autistic disorder and
language delay. This would imply that the CNTNAP2 level prior to
the induction of neuronal differentiation, rather than the regula-
tory pattern during this process, presents the critical element for
modulating ASD risk.
In the brain, CNTNAP2 is ubiquitously expressed pre- and
perinatally, but its expression is downregulated during mid-fetal
development and early childhood (age 16 years).
42
Subsequently,
CNTNAP2 is re-expressed in all brain regions besides the striatum
and the cerebellar cortex in later life. Thus, this study conrms a
brain tissue-specic regulation of CNTNAP2. This is in line with
the reported downregulation during neuronal differentiation of
human neuronal precursor cells.
41
We conrmed this time- and
cell-specic downregulation in the HEK293T cells and the SH-SY5Y
cell line model for neuronal differentiation. An aberrant transcrip-
tional ne tuning, an imbalanced regulation or an impaired
reactivation of CNTNAP2 may thus underlie the reported ASD-
specic effects on language acquisition or hierarchical learning.
Both processes are related to the striatum or the cerebellar cortex,
which are both strongly involved in ASD neuropathology.
5760
Thus, a dysbalanced regulation of CNTNAP2 in any of these
functional brain areas, or more specically a downregulation of
CNTNAP2 not compatible with normal neuronal development, may
modulate ASD risk.
Our hypothesis is underpinned by the fact that CNV studies in
ASD so far only identied gene copy losses but no gains spanning
the CNTNAP2 region.
22,61,62
Furthermore, a reduction of CNTNAP2
levels in lymphoblastoid cells driven by a reduced copy number of
the CNTNAP2 5promoter has previously been postulated as risk
factor for ASD.
22
CONCLUSION
In summary, we performed a genetic and functional characteriza-
tion study of the CNTNAP2 promoter in ASD. We report a novel
and potentially pathogenic mutation M2_ 215A and association
of rs34712024G with ASD, which might be mediated by an altered
transcriptional efciency of the CNTNAP2 promoter. We conclude
that a dosage-dependent effect of CNTNAP2 shapes the etiology
of the disorder. Our ndings and specically the proposed role of
rs71781329 in language development, and rs34712024 in ASD
should be studied in more detail in a larger ASD sample. As none
of the three known variants is genotyped on any currently available
genome-wide platforms (next-generation) sequencing approaches
might prove more benecial in the identication of CNTNAP2 risk
variants in ASD. It is suggested that variants that do not reach the
threshold for signicance after conservative correction for multiple
testing should not be immediately dismissed, but rather subjected
to in-silico analyses to decide whether to look at functional effects.
Finally, to prove causality and to fully understand the biological role
of CNTNAP2 expression, specic animal models and patient-derived
cell line models need to be studied in more detail.
CONFLICT OF INTEREST
The authors declare no conict of interest.
ACKNOWLEDGMENTS
We thank all the families and patients for their cooperation and the clinical staff for
their support in data collection. We thank Heiko Zerlaut for database management,
and Cornelia Wirth and Silvia Lindlar for excellent technical assistance. The study was
in part supported by grant Po 255/17-4 of the Deutsche Forschungsgemeinschaft to F
Poustka, and grants T 6031000-45 of Saarland University and ERA-NET NEURON/BMBF
EUHFAUTISM-01EW1105 to CM Freitag.
REFERENCES
1 Baird G, Simonoff E, Pickles A, Chandler S, Loucas T, Meldrum D et al. Prevalence
of disorders of the autism spectrum in a population cohort of children in South
Thames: the Special Needs and Autism Project (SNAP). Lancet 2006; 368: 210215.
2 Brugha TS, McManus S, Bankart J, Scott F, Purdon S, Smith J et al. Epidemiology of
autism spectrum disorders in adults in the community in England. Arch Gen
Psychiatry 2011; 68: 459465.
3 WHO. International Classication Of Mental And Behavioral Disorders. Clinical
Descriptions And Diagnostic Guidelines, 10th edn. World Health Organization:
Geneva, 1992.
4 APA. Diagnostic And Statistical Manual Of Mental Disorders, Fourth Editiontion, Text
Revision (DSM-IV-TR

). American Psychiatric Association, 4th edn. American Psy-

chiatric Publishing: Arlington, VA, USA, 2000.
5 Freitag CM, Staal W, Klauck SM, Duketis E, Waltes R. Genetics of autistic disorders:
review and clinical implications. Eur Child Adolesc Psychiatry 2010; 19: 169178.
6 Lichtenstein P, Carlstrm E, Rstam M, Gillberg C, Anckarster H. The genetics of
autism spectrum disorders and related neuropsychiatric disorders in childhood.
Am J Psychiatry 2010; 167: 13571363.
7 Peagarikano O, Geschwind DH. What does CNTNAP2 reveal about autism
spectrum disorder? Trends Mol Med 2012; 18: 156163.
8 Alarcn M, Cantor RM, Liu J, Gilliam TC, Geschwind DH. Evidence for a language
quantitative trait locus on chromosome 7q in multiplex autism families. Am J Hum
Genet 2002; 70: 6071.
9 Arking DE, Cutler DJ, Brune CW, Teslovich TM, West K, Ikeda M et al. A common
genetic variant in the neurexin superfamily member CNTNAP2 increases familial
risk of autism. Am J Hum Genet 2008; 82: 160164.
10 Vernes SC, Newbury DF, Abrahams BS, Winchester L, Nicod J, Groszer M et al.
A functional genetic link between distinct developmental language disorders.
N Engl J Med 2008; 359: 23372345.
11 Newbury DF, Paracchini S, Scerri TS, Winchester L, Addis L, Richardson AJ et al.
Investigation of dyslexia and SLI risk variants in reading- and language-impaired
subjects. Behav Genet 2011; 41: 90104.
12 Whitehouse AJO, Bishop DVM, Ang QW, Pennell CE, Fisher SE. CNTNAP2 variants
affect early language development in the general population. Genes, Brain and
Behav 2011; 10: 451456.
13 Whalley HC, O'Connell G, Sussmann JE, Peel A, Staneld AC, Hayiou-Thomas ME
et al. Genetic variation in CNTNAP2 alters brain function during linguistic pro-
cessing in healthy individuals. Am J Med Genet B Neuropsychiatr Genet 2011; 156:
941948.
14 Eyler LT, Pierce K, Courchesne E. A failure of left temporal cortex to specialize for
language is an early emerging and fundamental property of autism. Brain 2012;
135: 949960.
15 Bakkaloglu B, O'Roak BJ, Louvi A, Gupta AR, Abelson JF, Morgan TM et al. Mole-
cular cytogenetic analysis and resequencing of contactin associated protein-like 2
in autism spectrum disorders. Am J Hum Genet 2008; 82: 165173.
16 O'Roak BJ, Deriziotis P, Lee C, Vives L, Schwartz JJ, Girirajan S et al. Exome
sequencing in sporadic autism spectrum disorders identies severe de novo
mutations. Nat Genet 2011; 43: 585589.
17 Caselli R, Mencarelli MA, Papa FT, Ariani F, Longo I, Meloni I et al. Delineation of
the phenotype associated with 7q36.1q36.2 deletion: long QT syndrome, renal
hypoplasia and mental retardation. Am J Med Genet A 2008; 146: 11951199.
18 Rossi E, Verri AP, Patricelli MG, Destefani V, Ricca I, Vetro A et al. A 12Mb deletion
at 7q33q35 associated with autism spectrum disorders and primary amenorrhea.
Eur J Med Genet 2008; 51: 631638.
19 Petrin AL, Giacheti CM, Maximino LP, Abramides DVM, Zanchetta S, Rossi NF et al.
Identication of a microdeletion at the 7q33-q35 disrupting the CNTNAP2 gene in
a Brazilian stuttering case. Am J Med Genet A 2010; 152: 31643172.
20 Poot M, Beyer V, Schwaab I, Damatova N, Slot R, Prothero J et al. Disruption of
CNTNAP2 and additional structural genome changes in a boy with speech delay
and autism spectrum disorder. Neurogenetics 2010; 11: 8189.
21 Sehested LT, Mller RS, Bache I, Andersen NB, Ullmann R, Tommerup N et al.
Deletion of 7q34-q36.2 in two siblings with mental retardation, language delay,
primary amenorrhea, and dysmorphic features. Am J Med Genet A 2010; 152:
31153119.
CNTNAP2 5 promoter variants in ASD
AG Chiocchetti et al
10
Molecular Psychiatry (2014), 1 11 2014 Macmillan Publishers Limited
22 Nord AS, Roeb W, Dickel DE, Walsh T, Kusenda M, O'Connor KL et al. Reduced
transcript expression of genes affected by inherited and de novo CNVs in autism.
Eur J Hum Genet 2011; 19: 727731.
23 Peagarikano O, Abrahams BS, Herman EI, Winden KD, Gdalyahu A, Dong H et al.
Absence of CNTNAP2 leads to epilepsy, neuronal migration abnormalities, and
core autism-related decits. Cell 2011; 147: 235246.
24 Poliak S. Juxtaparanodal clustering of Shaker-like K+ channels in myelinated
axons depends on Caspr2 and TAG-1. J Cell Biol 2003; 162: 11491160.
25 Alarcon JM, Abrahams BS, Stone JL, Duvall JA, Perederiy JV, Bomar JM et al.
Linkage, association, and gene-expression analyses identify CNTNAP2 as an
autism-susceptibility gene. Am J Hum Genet 2008; 82: 150159.
26 Lesch KP, Bengel D, Heils A, Sabol SZ, Greenberg BD, Petri S et al. Association of
anxiety-related traits with a polymorphism in the serotonin transporter gene
regulatory region. Science 1996; 274: 15271531.
27 Shastry BS. SNPs: impact on gene function and phenotype. Methods Mol. Biol.
2009; 578: 322.
28 Chen Y, Liu T, Yu C, Chiang T, Hwang C. Effects of GC bias in next-generation-
sequencing data on de novo genome assembly. PLoS ONE 2013; 8: e62856.
29 Klauck SM, Felder B, Kolb-Kokocinski A, Schuster C, Chiocchetti A, Schupp I et al.
Mutations in the ribosomal protein gene RPL10 suggest a novel modulating
disease mechanism for autism. Mol Psychiatry 2006; 11: 10731084.
30 Freitag CM, Agelopoulos K, Huy E, Rothermundt M, Krakowitzky P, Meyer J et al.
Adenosine A(2A) receptor gene (ADORA2A) variants may increase autistic
symptoms and anxiety in autism spectrum disorder. Eur Child Adolesc Psychiatry
2010; 19: 6774.
31 Lord C, Rutter M, Le Couteur A. Autism Diagnostic Interview-Revised: a revised
version of a diagnostic interview for caregivers of individuals with possible per-
vasive developmental disorders: a revised version of a diagnostic interview for
caregivers of individuals with possible pervasive developmental disorders.
J Autism Dev Disord 1994; 24: 659685.
32 Lord C, Risi S, Lambrecht L, Cook EH Jr., Leventhal BL, DiLavore PC et al. The
autism diagnostic observation schedule-generic: a standard measure of social and
communication decits associated with the spectrum of autism. J Autism Dev
Disord 2000; 30: 205223.
33 Quandt K, Frech K, Karas H, Wingender E, Werner T. MatInd and MatInspector:
new fast and versatile tools for detection of consensus matches in nucleotide
sequence data. Nucleic Acids Res 1995; 23: 48784884.
34 Zheng L, Baumann U, Reymond J. An efcient one-step site-directed and site-
saturation mutagenesis protocol. Nucleic Acids Res 2004; 32: e115.
35 Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time
quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods 2001; 25: 402408.
36 Leduc V, Legault V, Dea D, Poirier J. Normalization of gene expression using SYBR
green qPCR: a case for paraoxonase 1 and 2 in Alzheimer's disease brains.
J. Neurosci. Methods 2011; 200: 1419.
37 Dignam JD, Lebovitz RM, Roeder RG. Accurate transcription initiation by RNA
polymerase II in a soluble extract from isolated mammalian nuclei. Nucleic Acids
Res 1983; 11: 14751489.
38 Gauderman W, Morrison J QUANTO 1.1: A computer program for power and
sample size calculations for genetic-epidemiology studieshttp://hydra.usc.edu/
gxe. 2006.
39 Barrett JC, Fry B, Maller J, Daly MJ. Haploview: analysis and visualization of LD and
haplotype maps. Bioinformatics 2005; 21: 263265.
40 Dudbridge F. Likelihood-based association analysis for nuclear families and
unrelated ssubjects with missing genotype data. Hum Hered 2008; 66: 8798.
41 Konopka G, Wexler E, Rosen E, Mukamel Z, Osborn GE, Chen L et al. Modeling the
functional genomics of autism using human neurons. Mol Psychiatry 2012; 17:
202214.
42 Kang HJ, Kawasawa YI, Cheng F, Zhu Y, Xu X, Li M et al. Spatio-temporal tran-
scriptome of the human brain. Nature 2011; 478: 483489.
43 Sakai T, Hino K, Wada S, Maeda H. Identication of the DNA binding specicity of
the human ZNF219 protein and its function as a transcriptional repressor. DNA Res
2003; 10: 155165.
44 Frietze S, Lan X, Jin VX, Farnham PJ. Genomic targets of the KRAB and SCAN
domain-containing zinc-nger protein 263. J Biol Chem 2010; 285: 13931403.
45 Buxbaum JD, Silverman J, Keddache M, Smith CJ, Hollander E, Ramoz N et al.
Linkage analysis for autism in a subset families with obsessive-compulsive
behaviors: evidence for an autism susceptibility gene on chromosome 1 and
further support for susceptibility genes on chromosome 6 and 19. Mol Psychiatry
2004; 9: 144150.
46 Lauritsen MB, Als TD, Dahl HA, Flint TJ, Wang AG, Vang M et al. A genome-wide
search for alleles and haplotypes associated with autism and related pervasive
developmental disorders on the Faroe Islands. Mol Psychiatry 2006; 11: 3746.
47 Hu VW, Sarachana T, Kim KS, Nguyen A, Kulkarni S, Steinberg ME et al. Gene
expression proling differentiates autism case-controls and phenotypic variants
of autism spectrum disorders: evidence for circadian rhythm dysfunction in
severe autism. Autism Res 2009; 2: 7897.
48 Allen-Brady K, Robison R, Cannon D, Varvil T, Villalobos M, Pingree C et al.
Genome-wide linkage in Utah autism pedigrees. Mol Psychiatry 2010; 15:
10061015.
49 Fischbach BV, Trout KL, Lewis J, Luis CA, Sika M. WAGR syndrome: a clinical review
of 54 cases. Pediatrics 2005; 116: 984988.
50 Chiocchetti AG, Bour HS, Freitag CM. Glutamatergic candidate genes in autism
spectrum disorder: an overview. J Neural Transm 2014; 121: 10811106.
51 Kim SH, Song JY, Joo E, Lee KY, Ahn YM, Kim YS. EGR3 as a potential susceptibility
gene for schizophrenia in Korea. Am J Med Genet B Neuropsychiatr Genet 2010;
153B: 13551360.
52 Quach DH, Oliveira-Fernandes M, Gruner KA, Tourtellotte WG. A sympathetic
neuron autonomous role for Egr3-mediated gene regulation in dendrite mor-
phogenesis and target tissue innervation. J. Neurosci. 2013; 33: 45704583.
53 Knapska E, Kaczmarek L. A gene for neuronal plasticity in the mammalian brain:
Zif268/Egr-1/NGFI-A/Krox-24/TIS8/ZENK? Prog Neurobiol 2004; 74: 183211.
54 Ebert DH, Greenberg ME. Activity-dependent neuronal signalling and autism
spectrum disorder. Nature 2013; 493: 327337.
55 Kyrchanova O, Chetverina D, Maksimenko O, Kullyev A, Georgiev P. Orientation-
dependent interaction between Drosophila insulators is a property of this class of
regulatory elements. Nucleic Acids Res 2008; 36: 70197028.
56 Weingarten-Gabbay S, Segal E. The grammar of transcriptional regulation. Hum
Genet 2014; 133: 701711.
57 Haas RH, Townsend J, Courchesne E, Lincoln AJ, Schreibman L, Yeung-Courchesne
R. Neurologic abnormalities in infantile autism. J. Child Neurol. 1996; 11: 8492.
58 Langen M, Schnack HG, Nederveen H, Bos D, Lahuis BE, Jonge MV de et al.
Changes in the developmental trajectories of striatum in autism. Biol. Psychiatry
2009; 66: 327333.
59 Strick PL, Dum RP, Fiez JA. Cerebellum and nonmotor function. Annu Rev Neurosci
2009; 32: 413434.
60 Desrochers TM, Badre D. Finding parallels in fronto-striatal organization. Trends
Cogn Sci (Regul. Ed.) 2012; 16: 407408.
61 Pinto D, Pagnamenta AT, Klei L, Anney R, Merico D, Regan R et al. Functional
impact of global rare copy number variation in autism spectrum disorders. Nature
2010; 466: 368372.
62 Gai X, Xie HM, Perin JC, Takahashi N, Murphy K, Wenocur AS et al. Rare structural
variation of synapse and neurotransmission genes in autism. Mol Psychiatry 2012;
17: 402411.
Supplementary Information accompanies the paper on the Molecular Psychiatry website (http://www.nature.com/mp)
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AG Chiocchetti et al
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2014 Macmillan Publishers Limited Molecular Psychiatry (2014), 1 11