CNV Analysis in Tourette Syndrome Implicates Large Genomic Rearrangements in COL8A1 and NRXN1.
ABSTRACT Tourette syndrome (TS) is a neuropsychiatric disorder with a strong genetic component. However, the genetic architecture of TS remains uncertain. Copy number variation (CNV) has been shown to contribute to the genetic make-up of several neurodevelopmental conditions, including schizophrenia and autism. Here we describe CNV calls using SNP chip genotype data from an initial sample of 210 TS cases and 285 controls ascertained in two Latin American populations. After extensive quality control, we found that cases (N = 179) have a significant excess (P = 0.006) of large CNV (>500 kb) calls compared to controls (N = 234). Amongst 24 large CNVs seen only in the cases, we observed four duplications of the COL8A1 gene region. We also found two cases with ∼400kb deletions involving NRXN1, a gene previously implicated in neurodevelopmental disorders, including TS. Follow-up using multiplex ligation-dependent probe amplification (and including 53 more TS cases) validated the CNV calls and identified additional patients with rearrangements in COL8A1 and NRXN1, but none in controls. Examination of available parents indicates that two out of three NRXN1 deletions detected in the TS cases are de-novo mutations. Our results are consistent with the proposal that rare CNVs play a role in TS aetiology and suggest a possible role for rearrangements in the COL8A1 and NRXN1 gene regions.
- SourceAvailable from: Domenico Serino[Show abstract] [Hide abstract]
ABSTRACT: Gilles de la Tourette syndrome (TS) is a neurodevelopmental disorder characterized by multiple motor and vocal tics, frequently associated with psychiatric co-morbidities. Despite the significant level of heritability, the genetic architecture of TS still remains elusive. Herein, we investigated an Italian family where an 8-year-old boy, his father, and paternal uncle have a diagnosis of TS. Array-CGH and high resolution SNP-array analyses revealed a heterozygous microdeletion of ∼135 kb at the 7q36.2 locus in the proband and his father. Fluorescent in situ hybridization and quantitative PCR (qPCR) analyses confirmed the presence of the alteration also in the paternal uncle. The deletion selectively involves the first exon of the DPP6 gene, leading to a down-regulation of its expression, as demonstrated by the reduced messenger RNA (mRNA) levels assessed by RT-qPCR. The DPP6 gene encodes for a type II membrane glycoprotein expressed predominantly in the central nervous system. To date, a de novo DPP6 exonic duplication, of uncertain significance, was reported in one patient with TS. Moreover, the DPP6 gene has been implicated in the pathogenesis of autism spectrum disorder (ASD) and, notably, in haloperidol-induced dyskinesia. This first familial case provides evidence for association between DPP6 haploinsufficiency and TS, further suggesting a plausible molecular link between TS and ASD, and might shed some light on the efficacy and tolerability profiles of antidopaminergic agents used for tic management, thus prompting further studies on a larger cohort of patients.Neurogenetics 08/2014; 15(4). · 2.66 Impact Factor
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ABSTRACT: Twin and family studies support a significant genetic contribution to obsessive-compulsive disorder (OCD) and related disorders, such as chronic tic disorders, trichotillomania, skin-picking disorder, body dysmorphic disorder, and hoarding disorder. Recently, population-based studies and novel laboratory-based methods have confirmed substantial heritability in OCD. Genome-wide association studies and candidate gene association studies have provided information on specific gene variations that may be involved in the pathobiology of OCD, though a substantial portion of the genetic risk architecture remains unknown.Psychiatric Clinics of North America 09/2014; · 2.13 Impact Factor
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ABSTRACT: Attention deficit hyperactivity disorder (ADHD) is the most common comorbid condition in patients with Tourette syndrome (TS). The co-occurrence of ADHD and TS is in most cases associated with a higher social and psychopathological impairment. Comorbidity between Tourette and ADHD appears to have a complex and partially known pathogenesis in which genetic, environmental, and neurobiological factors can be implicated. Genetic studies have revealed an involvement of dopaminergic, catecholaminergic, and GABAergic genes that modulated the activity of neurotransmitters. Furthermore, there are a lot of networks implicated in the development of ADHD and TS, involving cortical and striatal areas and basal ganglia. Although a large number of studies tried to find a common pathogenesis, the complex pathways responsible are not clear. The genes implicated in both disorders are currently unidentified, but it is probable that epigenetic factors associated with neural modifications can represent a substrate for the development of the diseases. Conclusion: In this paper, recent advances in neurobiology of ADHD and TS are reviewed, providing a basis for understanding the complex common pathogenesis underlying the frequent co-occurrence of the two conditions and the therapeutic choices.European Journal of Pediatrics 09/2014; · 1.98 Impact Factor
CNV Analysis in Tourette Syndrome Implicates Large
Genomic Rearrangements in COL8A1 and NRXN1
Abhishek Nag1, Elena G. Bochukova2, Barbara Kremeyer1, Desmond D. Campbell1, Heike Muller1,
Ana V. Valencia-Duarte3,4, Julio Cardona5, Isabel C. Rivas5, Sandra C. Mesa5, Mauricio Cuartas3,
Jharley Garcia3, Gabriel Bedoya3, William Cornejo4,5, Luis D. Herrera6, Roxana Romero6,
Eduardo Fournier6, Victor I. Reus7, Thomas L Lowe7, I. Sadaf Farooqi2, the Tourette Syndrome
Association International Consortium for Genetics, Carol A. Mathews7, Lauren M. McGrath8,9,
Dongmei Yu9, Ed Cook10, Kai Wang11, Jeremiah M. Scharf8,9,12, David L. Pauls8,9, Nelson B. Freimer13,
Vincent Plagnol1, Andre ´s Ruiz-Linares1*
1UCL Genetics Institute, Department of Genetics, Evolution and Environment, University College London, London, United Kingdom, 2University of Cambridge Metabolic
Research Laboratories, Institute of Metabolic Science, Addenbrooke’s Hospital, Cambridge, United Kingdom, 3Laboratorio de Gene ´tica Molecular, SIU, Universidad de
Antioquia, Medellı ´n, Colombia, 4Escuela de Ciencias de la Salud, Universidad Pontificia Bolivariana, Medellı ´n, Colombia, 5Departamento de Pediatrı ´a, Facultad de
Medicina, Universidad de Antioquia, Medellı ´n, Colombia, 6Hospital Nacional de Nin ˜os, San Jose ´, Costa Rica, 7Department of Psychiatry, University of California San
Francisco, San Francisco, California, United States of America, 8Psychiatric and Neurodevelopmental Genetics Unit, Center for Human Genetics Research, Boston,
Massachusetts, United States of America, 9Department of Psychiatry, Massachusetts General Hospital, Boston, Massachusetts, United States of America, 10University of
Illinois, Chicago, Illinois, United States of America, 11University of Southern California, Los Angeles, California, United States of America, 12Department of Neurology,
Massachusetts General Hospital, Boston, Massachusetts, United States of America, 13Center for Neurobehavioral Genetics, University of California Los Angeles, Los
Angeles, California, United States of America
Tourette syndrome (TS) is a neuropsychiatric disorder with a strong genetic component. However, the genetic architecture
of TS remains uncertain. Copy number variation (CNV) has been shown to contribute to the genetic make-up of several
neurodevelopmental conditions, including schizophrenia and autism. Here we describe CNV calls using SNP chip genotype
data from an initial sample of 210 TS cases and 285 controls ascertained in two Latin American populations. After extensive
quality control, we found that cases (N=179) have a significant excess (P=0.006) of large CNV (.500 kb) calls compared to
controls (N=234). Amongst 24 large CNVs seen only in the cases, we observed four duplications of the COL8A1 gene region.
We also found two cases with ,400kb deletions involving NRXN1, a gene previously implicated in neurodevelopmental
disorders, including TS. Follow-up using multiplex ligation-dependent probe amplification (and including 53 more TS cases)
validated the CNV calls and identified additional patients with rearrangements in COL8A1 and NRXN1, but none in controls.
Examination of available parents indicates that two out of three NRXN1 deletions detected in the TS cases are de-novo
mutations. Our results are consistent with the proposal that rare CNVs play a role in TS aetiology and suggest a possible role
for rearrangements in the COL8A1 and NRXN1 gene regions.
Citation: Nag A, Bochukova EG, Kremeyer B, Campbell DD, Muller H, et al. (2013) CNV Analysis in Tourette Syndrome Implicates Large Genomic Rearrangements
in COL8A1 and NRXN1. PLoS ONE 8(3): e59061. doi:10.1371/journal.pone.0059061
Editor: Ge Zhang, Cincinnati Children’s Hospital Medical Center, United States of America
Received October 11, 2012; Accepted February 11, 2013; Published March 22, 2013
Copyright: ? 2013 Nag et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted
use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was funded by National Institutes of Health (http://www.nih.gov/) grants NS043538 (to ARL), NS037484 (to NBF), MH085057 (to JMS),
NS40024 (to DLP and JMS) and NS16648 (the Tourette Syndrome Association International Consortium for Genetics), a CODI grant (http://www.udea.edu.co/
portal/page/portal/Programas/GruposInvestigacion/h.Convocatorias/CODI/estrategiaSostenibilidad) (Sostenibilidad, Universidad de Antioquia), a grant from the
Judah Foundation (http://www.judahfoundation.org/), and the Tourette Syndrome Association (http://tsa-usa.org/) (fellowship to BK and a grant to DLP). VP is
supported by a grant from the United Kingdom Medical Research Council (http://www.mrc.ac.uk) (G1001158) and by the NIHR Moorfields Biomedical Research
Council (http://www.brcophthalmology.org/). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: email@example.com
TS is a childhood onset neuropsychiatric illness characterised by
the occurrence of multiple, motor and vocal tics and is often
associated with obsessive-compulsive disorder (OCD) and atten-
tion-deficit hyperactivity disorder (ADHD) [1–5]. Twin studies
have estimated a sibling relative risk ratio for TS of about 6–8 ,
one of the highest amongst neuropsychiatric disorders. However,
identification of genetic variants underlying TS has proven difficult
[5–7]. Genome-wide linkage and candidate gene association
studies have failed to provide robust evidence implicating specific
loci, and a recent GWAS has not identified common variants
associated with TS at genome-wide significance thresholds .
The observation of chromosomal abnormalities in TS families [9–
11] has suggested the possibility that genomic rearrangements
could play an important role in this disorder, but prior studies have
provided conflicting evidence regarding the involvement of copy
number variants (CNVs) in TS [12,13]. To further evaluate the
PLOS ONE | www.plosone.org1March 2013 | Volume 8 | Issue 3 | e59061
role of CNVs in TS, we performed a genomewide study of CNVs
in a case/control sample from two well-studied, closely related
Latin American population isolates.
This research was approved by the BioEthics Committee of
Universidad de Antioquia (Colombia) and the NHS National
Research Ethics Service, Central London Committee REC 4
(UK). Written consent was obtained from all subjects. In the case
of minors, written consent was obtained from a parent or legal
Patients and Methods
We studied CNVs in a sample of 210 unrelated TS cases
ascertained in two closely related Latin American population
isolates and 285 unrelated population controls. The populations of
Antioquia, Colombia, and of the Central Valley of Costa Rica
(CVCR) have similar and partly shared demographic histories and
are genetically closely related [14,15]. They are therefore expected
to show an enrichment for shared predisposing factors for complex
genetic conditions, including TS [14–17]. Of the cases, 81 were
recruited at the Neuropaediatrics Clinic of Hospital Universitario
San Vicente de Pau ´l (Antioquia, Colombia) and 129 were
recruited at Hospital Nacional de Nin ˜os (San Jose ´, Costa Rica).
Diagnosis was based on DSM-IV criteria, focusing on narrowly
defined moderate to severe TS. The mean age of cases was 13
years, with a mean age for the start of symptoms at 6.4 years. In
addition to TS, 48% of the cases have a diagnosis of ADHD and
53% have OCD. An additional set of 53 TS cases used for MLPA-
based follow-up (see below) was also recruited through the
Neuropaediatrics Clinic of Hospital Universitario San Vicente
de Pau ´l (Antioquia, Colombia), following the same diagnostic
procedures. Population controls were obtained in Antioquia as
part of on-going genetic diversity studies in the region . For
both, cases and controls, genealogical enquiries confirmed local
ancestry in at least 6/8 great-grandparents. Because matched
population controls from the CVCR were unavailable, and based
on the close genetic relatedness of Antioquia and the CVCR,
Antioquian controls were contrasted with Antioquian and Costa
Rican cases accounting for stratification (see below). All samples
were genotyped using Illumina Human660 arrays as part of the
TSAICG genome-wide association study of TS .
We obtained CNV calls from the raw hybridization intensities
using PennCNV . We excluded from this analysis samples that
were outliers based on either the variability of the raw intensity
data (using the standard deviation of the logR ratio), or on the total
number of CNVs called (see Methods S1 and Figure S2). This
resulted in 413 samples being retained for further analysis (179
cases and 234 controls). To make the final CNV calls, we used the
following criteria: (i) we merged neighbouring CNVs when the
distance separating them was less than half of the total distance
from the start of the first CNV to the end of the second CNV, (ii)
we only called CNVs containing at least 10 SNPs, and (iii) we
ignored CNVs located in centromeric and telomeric regions.
The CNV burden for each sample was determined by counting
all CNVs and stratifying them by size into four categories: ,10 kb,
10–100 kb, 100–500 kb and .500 kb. All calls for CNVs
.500 kb (‘‘large CNVs’’) were confirmed individually by plotting
the LogR ratio and B allele frequency for the SNPs in the region
(Figure S4). The CNV burden was then contrasted between cases
and controls using Fisher’s exact test.
Principal component analysis (PCA) of the genotype data was
performed using EIGENSTRAT , as implemented in the
EIGENSOFT package (http://genepath.med.harvard.edu/˜reich/
Overall, in the final dataset we made an average of 3.5 CNV
calls per subject with a median CNV length of 76.4 kb. Of these,
60% correspond to deletions and 40% to duplications (Figure S3).
We contrasted the total CNV burden between TS cases and
controls, stratified by size into four categories: ,10 kb, 10–100 kb,
100–500 kb and .500 kb (Table 1). We found a statistically
significant increase in the frequency of CNVs .500 kb in cases (27
or 0.15 per individual) compared to controls (15 or 0.06 per
individual; p=0.006). In total, 25 cases (14%) versus 15 controls
(6.4%) were found to carry large CNVs, representing an excess of
,7.6% (95% C.I.=1.6–13.6%, one-sided Fisher’s exact test
p=0.006). Of the 27 large CNVs found in cases, 24 occurred in
regions free of CNVs in controls. Two of the TS cases had two
large CNVs each, while no control carried more than one large
CNV. Since no controls were available for the CVCR samples, we
evaluated the effect of population stratification by testing the
correlation of CNV burden with ancestry of the samples, evaluated
using PCA. The presence of large CNVs was not correlated with
ancestry (p.0.05 for PCs 1 to 4). We also verified that OR
estimates for large CNVs are consistent whether the CVCR cases
are included (95% ci: 1.27–4.96) or not (95% ci: 1.08–5.95), but as
expected from a reduction in sample size, when the burden
analysis is restricted to Antioquia the significance decreases (one-
sided Fisher’s exact test p=0.16). Because cases and controls were
genotyped in two batches (one batch of CVCR cases and one
batch of Antioquia cases and controls), we also tested for
correlation of genotyping batch with the presence of large CNVs,
but found no significant effect.
We next explored the potential involvement in TS of CNVs at
specific genome regions, stratifying by size. We first examined the
24 (out of 27) regions with CNVs .500 Kb that were detected
only in the cases. Of these, 4 did not include exons of any
Table 1. CNV burden in TS cases and controls.
CNV size (kb)Count in casesFrequency per caseCount in controlsFrequency per controlp-value
COL8A1 and NRXN1 CNVs in Tourette Syndrome
PLOS ONE | www.plosone.org2March 2013 | Volume 8 | Issue 3 | e59061
annotated gene. The remaining 20 mapped to 15 different
genomic regions. Two of these contain genes for uncharacterized
proteins with no known functions (LOC284749 and FLJ46357).
The remaining 18 large CNVs were located in 13 gene regions
(Table S1). Of these regions, 10 presented rearrangements in
a single case and some of these regions could be of potential
relevance for TS (such a region on 22q11 overlapping DiGeorge’s
syndrome critical region (Figure S4–43) which has been implicated
in rare unusual TS cases [21,22] and has also been found to be
associated with schizophrenia [23–25]). Three regions showed
rearrangements in more than one TS case. A ,600 Kb region on
3q12.1 (overlapping the COL8A1 gene) was duplicated in four
cases (Table 2). Two other regions on 2p22.3 and 5q21.1
(overlapping the BIRC6/TTC27/LTBP1 and the SLCO4C1/
SLCO6A1 genes, respectively) were duplicated in two cases each
(Table 2). We also examined genome regions with CNVs ,500 kb
but focusing solely on those encompassing exons of the same gene
in at least two TS cases but not in controls. We identified four such
regions, each carrying a CNV in two patients (Table 3). The
largest rearrangements (two ,400 kb deletions) encompass exons
1–3 of the Neurexin1 (NRXN1) gene on 2p16.3 (Figures S4–6 and
We followed up the COL8A1 and NRXN1 findings using
multiplex ligation-dependent probe amplification (MLPA; Meth-
ods S1) targeting exons 1 and 2 of COL8A1 and exons 1 to 4 of
NRXN1 (with two additional probes 39 and 59 of this gene) (Table
S2). We carried out MLPA in the Antioquian samples included in
the SNP-based analysis for which DNA was available (92 cases and
142 controls). We validated the five SNP-based CNV calls (four on
COL8A1 and one on NRXN1) made on these samples (Figure S5-1).
MLPA identified an additional three COL8A1 deletions and two
NRXN1 deletions not detected in the SNP-based CNV calls
(Figures S5-2 and S5-3). No CNVs in COL8A1 or NRXN1 were
detected by MLPA in the controls. We also applied the COL8A1
and NRXN1 MLPA assay to an additional set of 53 TS cases from
Antioquia but did not detect further rearrangements in these
individuals. Aggregating the results of the SNP-based CNV calls
and MLPA (Table 4), in a total of 232 cases examined we found 7
with rearrangements in COL8A1 (all from Antioquia) and 4 in
NRXN1 (3 from Antioquia and 1 from the CVCR). None of the
234 Antioquian controls showed rearrangements in these two gene
regions in the SNP-based calls or MLPA. To further support the
notion that the CNVs observed here are not simply population
polymorphisms, we checked the Database of Genomic Variants
(DGV; http://dgvbeta.tcag.ca/dgv/app/home), a curated cata-
logue of human structural variation, for CNVs in the NRXN1 and
COL8A1 gene regions. While there is a considerable number of
CNVs in both regions, all of the CNVs that lie within the
Table 2. Chromosomal regions harbouring large (.500 kb) CNVs overlapping annotated gene exons in at least two TS cases and
not in controls.
End positionSize# of markersGene(s)Figure
bBased on build 36 of the human genome.
Table 3. Regions harbouring smaller CNVs (,500 kb) overlapping gene exons in at least two TS cases but not in controls.
LocationCNV typeaStart positionb
End positionSize# of markersGene(s)b
bBased on build 36 of the human genome;
COL8A1 and NRXN1 CNVs in Tourette Syndrome
PLOS ONE | www.plosone.org3March 2013 | Volume 8 | Issue 3 | e59061
respective gene itself are between a few hundred bp and ,100 kb
long, and therefore significantly shorter than the variants described
here. More importantly, the majority of these variants do not
affect any of the exons of the respective genes, the only exception
being a 100 kb deletion affecting NRXN1 exons 7-9 (DGV
Variation_2383). This variant affects a different region from the
variants observed here; in addition, it was found only in one out of
540 chromosomes and is therefore also not likely to represent
a common population polymorphism. Overall, the size and
position of the variants identified here, both in NRXN1 and
COL8A1, do not show any overlap with common population
To evaluate the possibility that the COL8A1 and NRXN1
rearrangements detected in TS cases could represent de-novo
mutations, we applied the MLPA assay to the parents of TS cases
with rearrangements in these two gene regions. We considered
only the patients for which DNA from both parents was available
and confirmed relatedness in each trio. This included two cases
with COL8A1 duplications and three cases with NRXN1 deletions
(all from Antioquia). The same duplication was found in a parent
in each of the two cases with COL8A1 duplications examined,
indicating that this variant was inherited. This and the observation
of similar boundaries for the COL8A1 duplications in the SNP-
based CNV calls (Table 2) suggest that this variant is segregating in
the Antioquian population. Deletion of NRXN1 59 exons was
found in the father of one of the cases with a NRXN1 deletion
(GT64.1) but not in the parents of the two other cases with this
deletion, indicating a de novo mutation in these two trios. The father
of case GT64.1 has a diagnosis of OCD, a condition that shows
significant co-morbidity and may share common predisposing
factors with TS (interestingly, the paternal grand-father is reported
to have suffered from OCD; however, his CNV type is unknown).
One of the two de novo NRXN1 deletions identified occurred in
a proband that had no family history of TS (case GT5.1, Figure
S5-2a). The second case with a de novo NRXN1 deletion (GT34.1,
Figure S5-2b) had a history of TS/OCD on the paternal side of his
Our results provide statistically significant evidence of a high
burden of large CNVs (.500kb) in TS, thereby supporting the
proposal for an involvement of rare CNVs in various neurodeve-
lopmental disorders, including TS, and their possible aetiological
overlap [12,13,26–28]. We also find suggestive evidence for the
involvement of rearrangements specifically affecting the NRXN1
and COL8A1 genes. In the aggregated data (Table 4) we find
a nominally significant association of COL8A1 and NRXN1
rearrangements with TS (p-values of 0.004 and 0.03 respectively).
Due to the limited sample size, these p-values would not reach
significance accounting for multiple testing. Data from the
Database of Genomic Variants further supported the notion that
the variants observed here are not part of the spectrum of common
population polymorphisms. When considering the trio data, the
lack of a straightforward co-segregation between the structural
variants observed in our study and the TS phenotype implies the
involvement of further predisposing loci in the aetiology of TS;
however, this is not unexpected for such a phenotypically and
genetically complex condition and does not conflict with a role for
NRXN1 or COL8A1 in TS predisposition. Overall, our results
strongly warrant further investigation of these two genes in TS.
The importance of NRXN1 in mediating cell-cell interactions in
the central nervous system, as well as its confirmed involvement in
other neurodevelopmental disorders, make this gene an excellent
Table 4. Number of TS cases and controls with CNVs affecting COL8A1 and NRXN1 detected using SNP-based calls, MLPA or both.
Cases N=92 (of 179)
aOne-tailed Fisher’s exact test.
bMLPA was applied to a subset of the samples examined in the initial SNP-based calls.
cThis set of 53 follow-up samples was not included in the initial SNP-based calls but was examined only with MLPA.
COL8A1 and NRXN1 CNVs in Tourette Syndrome
PLOS ONE | www.plosone.org4March 2013 | Volume 8 | Issue 3 | e59061
candidate gene for TS [12,29,30]. Our results are consistent with
those of a previous study reporting deletions affecting NRXN1
exons 1–3 in TS, the same exons found to be deleted in our study
. The fact that two of the three NRXN1 rearrangements, for
which inheritance status could be confirmed, were found to be de
novo events, is in line with recent findings stressing a role for de novo
mutations in neurodevelopmental disease. The potential involve-
ment of COL8A1 in TS is intriguing. A growing body of evidence
suggests that collagen subunits are involved in neural develop-
ment, influencing processes such as axonal guidance, synaptogen-
esis and Schwann cell differentiation [31,32]. COL8A1 has also
been found to be up-regulated during repair processes in the
mouse brain . Interestingly, the top signal in the recent GWAS
of TS  also implicated a collagen gene (COL27A1).
In conclusion, our results are consistent with the view that TS is
genetically a highly heterogeneous disorder, in which rare variants,
including de-novo mutations, could underlie a substantial fraction of
cases. Recently, Cooper et al (2011) conducted a large-scale study
to investigate the role of CNVs in ,15,000 children with
intellectual disability and estimated that ,14.2% are due to
CNVs .400 kb. Similarly, the 7.6% excess of large CNVs in TS
patients observed here could be taken as a rough estimate of the
proportion of cases that might be caused by CNVs. The analysis of
larger TS study samples should enable a more definite assessment
of the role of large rearrangements at specific gene regions in this
disorder. More extensive surveys of parent-TS offspring trios are
also required to estimate the proportion of cases that could be due
to highly penetrant de-novo mutations. Finally, sequencing studies
should allow a full assessment of the role of rare variants in the
aetiology of TS.
1–4 and presence of large CNVs. Left panel: PCA1 versus PCA2.
Right panel: PCA3 versus PCA4.
No significant correlation was observed between PCs
were excluded from subsequent analyses.
Samples with NumCNV.30 or LRR_SD.0.24
average of 14.47 CNV calls per subject. On applying call-level
filtering criteria to these calls, an average of 3.50 CNV calls per
subject (spanning 10 to 522 SNPs) were obtained. Deletions (865/
1448) were more frequently observed compared to duplications
(583/1448). Deletions were observed more frequently in the small
CNV category while duplications were observed more frequently
in the large CNV category (Figure S3).
The 413 DNA samples that passed QC yielded an
control status are shown as figure heading. LogR ratio and B allele
frequency are shown in the top and bottom panels, respectively.
CNV boundaries are indicated by red dotted lines. Human
RefSeq genes are shown below each panel (vertical lines indicating
exons). Genomic position (in Mb) based on the hg18 human
(1–44): Sample ID, population origin and case/
Amplification (MLPA). Figure S5-1: Validation of the SNP-based
CNV calls in COL8A1 and NRXN1 by MLPA. Top panel:
heterozygous duplication in COL8A1 (exons 1 and 2). Represen-
tative MLPA data and MLPA target probes for COL8A1 are
shown. Bottom panel: Detection of a heterozygous deletion in
NRNX1 (exons 1, 2, 3). MLPA target probes for NRXN1 are
shown, the unlabelled target regions are probes located either on
chromosome 2 but outside the deleted region or on other
chromosomes (Table S2). Patient MLPA traces are in red, overlaid
upon the normal control MLPA traces in black. Arrows point to
the deleted/duplicated probes. Figure S5-2: Detection of de novo
deletions in NRNX1 (exons 2 and 3) in TS cases. A, trio 5. B, trio
34. Patient MLPA traces are in red overlaid upon the normal
control MLPA traces in black. The parents’ traces are in blue,
overlaid upon normal controls in black. Arrows point to the
MLPA probes in NRXN1. Figure S5-3: Two additional TS cases
(GT5.1 and GT34.1) with deletions involving either exon 1, 2 or 3
of NRXN1 detected by MLPA. Representative MLPA data are
shown. Patient traces are in red, overlaid upon the control traces
in black. Arrows point to the MLPA probes in NRXN1. Figure S5-
4: Three additional TS cases (GT7.1, GT29.1 and GT114.1) with
deletion of exon 2 of COL8A1 detected by MLPA. Representative
MLPA data are shown. Patient traces are in red, overlaid upon the
control traces in black. Arrows point to the MLPA probes in
CNV calls using Multiplex Ligation-dependent Probe
control status are shown as figure heading. LogR ratio and B allele
frequency are shown in the top and bottom panels, respectively.
CNV boundaries are indicated by red dotted lines. The structure
of NRXN1 (Figures S6-1 and S6-2) or COL8A1 (Figures S6-3 to S6-
5) is shown below each panel with exons shown as vertical lines.
Genomic position (in Mb) provided make use of the hg18 human
genome sequence as reference.
(1 to 5): Sample ID, population origin and case/
CNVs overlapping annotated gene exons in TS cases but not in
Chromosomal regions harbouring large (.500 kb)
bAccording to build 36 of the
Target probes used in the MLPA assay.
Multiplex ligation-dependent probe amplification (MLPA).
CNV Quality Control and CNV validation by
The authors would like to thank all volunteers who participated in the
Conceived and designed the experiments: VP ARL. Performed the
experiments: AN EB BK HM AVVD VP. Analyzed the data: AN EB BK
DDC VP ARL. Contributed reagents/materials/analysis tools: JC ICR
SCM MC JG GB WC LDH RR EF VIR TLL ISF CAM LMM DY EC
KW JMS DLP NBF. Wrote the paper: AN BK VP ARL.
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