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B
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b
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C
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D
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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