Application of array comparative genomic hybridization in 102 patients with epilepsy and additional neurodevelopmental disorders

Article (PDF Available)inAmerican Journal of Medical Genetics Part B Neuropsychiatric Genetics 159B(7):760-71 · October 2012with58 Reads
DOI: 10.1002/ajmg.b.32081 · Source: PubMed
Copy-number variants (CNVs) collectively represent an important cause of neurodevelopmental disorders such as developmental delay (DD)/intellectual disability (ID), autism, and epilepsy. In contrast to DD/ID, for which the application of microarray techniques enables detection of pathogenic CNVs in ∼10-20% of patients, there are only few studies of the role of CNVs in epilepsy and genetic etiology in the vast majority of cases remains unknown. We have applied whole-genome exon-targeted oligonucleotide array comparative genomic hybridization (array CGH) to a cohort of 102 patients with various types of epilepsy with or without additional neurodevelopmental abnormalities. Chromosomal microarray analysis revealed 24 non-polymorphic CNVs in 23 patients, among which 10 CNVs are known to be clinically relevant. Two rare deletions in 2q24.1q24.3, including KCNJ3 and 9q21.13 are novel pathogenic genetic loci and 12 CNVs are of unknown clinical significance. Our results further support the notion that rare CNVs can cause different types of epilepsy, emphasize the efficiency of detecting novel candidate genes by whole-genome array CGH, and suggest that the clinical application of array CGH should be extended to patients with unexplained epilepsies. © 2012 Wiley Periodicals, Inc.


of Array Comparative Genomic
Hybridization in 102 Patients With Epilepsy and
Additional Neurodevelopmental Disorders
Magdalena Bartnik,
zbieta Szczepanik,
Katarzyna Derwi
Barbara Wis
Tomasz Gambin,
Maciej Sykulski,
Kamila Ziemkiewicz,
Marta Ke˛dzior,
Monika Gos,
Dorota Hoffman-Zacharska,
Tomasz Mazurczak,
Anetta Jeziorek,
Dorota Antczak-Marach,
Mariola Rudzka-Dybała,
Hanna Mazurkiewicz,
Alicja Goszcza
Zofia Zalewska-Miszkurka,
Iwona Terczy
Małgorzata Sobierajewicz,
Chad A. Shaw,
Anna Gambin,
Hanna Mierzewska,
Tadeusz Mazurczak,
Ewa Obersztyn,
Ewa Bocian,
and Paweł Stankiewicz
Department of Medical Genetics, Institute of Mother and Child, Warsaw, Poland
Clinic of Neurology of Children and Adolescents, Institute of Mother and Child, Warsaw, Poland
Institute of Computer Science, Warsaw University of Technology, Warsaw, Poland
Institute of Informatics, Univer sity of Warsaw, Warsaw, Poland
Child Neurology Outpatient Clinic, Leszno, Poland
Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, Texas
Mossakowski Medical Research Centre, Polish Academy of Sciences, Warsaw, Poland
Manuscript Received: 3 February 2012; Manuscript Accepted: 2 July 2012
Copy-number variants (CNVs) collectively represent an impor-
tant cause of neurodevelopmental disorders such as develop-
mental delay (DD)/intellectual disability (ID), autism, and
epilepsy. In contrast to DD/ID, for which the application of
microarray techniques enables detection of pathogenic CNVs
in 1020% of patients, there are only few studies of the role of
CNVs in epilepsy and genetic etiology in the vast majority of
cases remains unknown. We have applied whole-genome exon-
targeted oligonucleotide array comparative genomic hybridiza-
tion (aCGH) to a cohort of 102 patients with various types of
epilepsy with or without additional neurodevelopmental abnor-
malities. Chromosomal microarray analysis revealed 24 non-
polymorphic CNVs in 23 patients, among which 10 CNVs
are known to be clinically relevant. Two rare deletions in
2q24.1q24.3, including KCNJ3 and 9q21.1 3 are novel pathogenic
genetic loci and 12 CNVs are of unknown clinical significance.
Our results further support the notion that rare CNVs can cause
different types of epilepsy, emphasize the efficiency of detecting
novel candidate genes by whole-genome aCGH, and suggest that
the clinical application of aCGH should be extended to patients
with unexplained epilepsies.
2012 Wiley Periodicals, Inc.
How to Cite this Article:
Bartnik M, Szczepanik E, Derwi
nska K,
niowiecka-Kowalnik B, Gambin T,
Sykulski M, Ziemkiewicz K, Ke˛dzior M, Gos
M, Hoffman-Zacharska D, Mazurczak T,
Jeziorek A, Antczak-Marach D, Rudzka-
Dybała M, Mazurkiewicz H, Goszcza
Ciuchta A, Zalewska-Miszkurka Z,
nska I, Sobierajewicz M, Shaw CA,
Gambin A, Mierzewska H, Mazurczak T,
Obersztyn E, Bocian E, Stankiewicz P. 2012.
Application of array comparative genomic
hybridization in 102 patients with epilepsy
and additional neurodevelopmental
Am J Med Genet Part B 9999:111.
Grant sponsor: Polish Ministry of Science and Higher Education; Grant
number: R13-0005-04/2008; Grant sponsor: Foundation for Polish
The authors have no conflicts of interest to declare.
*Correspondence to:
Paweł Stankiewicz, M.D., Ph.D., Department of Molecular and Human
Genetics, Baylor College of Medicine, One Baylor Plaza, Rm R809,
Houston, TX 77030. E-mail:
Article first published online in Wiley Online Library
( 00 Month 2012
DOI 10.1002/ajmg.b.32081
2012 Wiley Periodicals, Inc. 1
Neuropsychiatric Genetics
Key words: seizures; array CGH; copy-number variants;
Advances in molecular cytogenetic techniques, such as array CGH,
have improved diagnostic power and allowed the detection of
clinically significant submicroscopic copy-number variants
(CNVs), in patients with multiple congenital anomalies, dysmor-
phic features, developmental delay (DD)/intellectual disability
(ID), autism, and schizophrenia at 1001,000 times higher reso-
lution than conventional karyotype analysis [Lee et al., 2007;
Shinawi and Cheung, 2008; Kirov et al., 2009; Miller et al., 2010;
Mulley and Mefford, 2011]. The role of CNVs in patients with DD/
ID has been extensively investigated and the detection rate of
clinically relevant imbalances is estimated to be 1020%
[Menten et al., 2006; Stankiewicz and Beaudet, 2007; Koolen
et al., 2009]. Recently, more attention has been paid to identifica-
tion of CNVs in other neurodevelopmental disorders, including
Epilepsy is one of the most common neurological disorders
affecting up to 1% of the population. It has been estimated that up
to 40% of epilepsies are genetically determined and can be divided
into Mendelian, non-Mendelian (‘‘complex’’) diseases, and chro-
mosomal disorders [Hauser et al., 1996; Gardiner, 2000; Jozwiak
et al., 2005; Pal et al., 2010]. There are over 200 Mendelian disorders,
in which epilepsy is part of the clinical condition, but not the
primary feature. Over the past two decades, the progress in under-
standing mechanisms and causes of epilepsy has been spectacular
[Crino, 2007; Ottman et al., 2010]. Most of the rare monogenic
epilepsies are caused by mutations in genes encoding subunits of
neuronal voltage- or ligand-gated ion channels and proteins related
to neuronal maturation and migration during embryonic develop-
ment [Sanchez-Carpintero Abad et al., 2007; Berg et al., 2010;
Ottman et al., 2010; Klassen et al., 2011].
Different types of epilepsy have been reported in patients with
chromosomal imbalances, including 1p36 deletion (Chromosome
1p36 deletion syndrome), 4p16.3 deletion (WolfHirschhorn
syndrome), ring chromosome 14, deletion 15q11.2q12
(Angelman syndrome), inv dup (15) chromosome, deletion
17p13.3 (MillerDieker syndrome), ring chromosome 20, and
trisomy 21 (Down syndrome) [Singh et al., 2002; Battaglia and
Guerrini, 2005]. Application of molecular cytogenetic techniques
such as fluorescence in situ hybridization (FISH) and microarrays
have enabled identification of several novel smaller-sized patho-
genic CNVs, for example: deletions of CDKL5 (cyclin-dependent
kinase-like 5; OMIM 300203; Xp22.13) in girls with severe epilepsy
and a Rett syndrome-like phenotype [Erez et al., 2009; Bahi-Buisson
et al., 2010; Mei et al., 2010], deletions and duplications of SCN1A
(sodium channel, neuronal type 1, alpha subunit; OMIM 182389;
2q24.3) in patients with Dravet syndrome (OMIM 607208)
[Marini et al., 2009], deletions of MAGI2 (membrane-associated
guanylate kinase inverted-2; OMIM 606382; 7q11.23 q21) in
subjects with infantile spasms [Marshall et al., 2008], deletions
of MEF2C (mads box transcription enhancer factor 2, polypeptide
C; OMIM 600662; 5q14.3) in patients with severe ID, seizures, and
hypotonia [Le Meur et al., 2010; Nowakowska et al., 2010; Zweier
et al., 2010], deletions of STXBP1 (syntaxin-binding protein 1;
OMIM 602926; 9q34.11) in patients with early infantile epileptic
encephalopathy (OMIM 612164) [Saitsu et al., 2008], deletions of
ARX (aristaless-related homeobox, X-linked; OMIM 300382;
Xp21.3) in patients with early infantile epileptic encephalopathy
1 (EIEE1; OMIM 308350), as well as duplications of MECP2
(methyl-CpG-binding protein 2; OMIM 300005; Xq28) in patients
with Lubs syndrome (OMIM 300260) [Van Esch et al., 2005], and
duplications of FOXG1 (forkhead box G1; OMIM 164874; 14q12)
in patients with developmental epilepsy, mental retardation, and
severe speech impairment [Brunetti-Pierri et al., 2011]. Moreover,
recently, several recurrent CNVs have been identified in patients
with seizures, including microdeletions of chromosomal regions
1q21.1 [Brunetti-Pierri et al., 2008; Mefford et al., 2008], 7q11.23
[Ramocki et al., 2010], 10q11.21q11.23 [Stankiewicz et al.,
2012]; 15q11.2 [de Kovel et al., 2010; Mefford et al., 2010],
15q13.3 [Dibbens et al., 2009; Helbig et al., 2009; Shinawi et al.,
2009; Mefford et al., 2010], 16p11.2 [Ballif et al., 2007; Shinawi
et al., 2010], 16p13.11 [de Kovel et al., 2010; Heinzen et al.,
2010; Mefford et al., 2010], and 22q11.2 [Ryan et al., 1997;
Gonzalez and Bautista, 2009] and reciprocal microduplications
of 1q21.1 [Brunetti-Pierri et al., 2008], 10q11.21q11.23
[Stankiewicz et al., 2012], and 16p11.2 [Shinawi et al., 2010;
Mefford et al., 2011].
Despite increasing interest in the genetics of epilepsy, only a few
genome-wide studies of CNVs have been performed in patients
with epilepsy [Battaglia and Guerrini, 2005; Heinzen et al., 2010;
Mefford et al., 2010, 2011; Paciorkowski et al., 2011; Sisodiya and
Mefford, 2011].
Here, we report the results of chromosomal microarray analysis
(CMA) in 102 patients with idiopathic generalized epilepsy (IGE)
or epilepsy with other neurodevelopmental disorders. We show
that CNVs significantly contribute to the genetic etiology of
We studied 102 patients with different types of epilepsy, including
juvenile myoclonic epilepsy (JME), West syndrome, idiopathic
generalized epilepsy (IGE), and unclassified syndromes of refrac-
tory epilepsy with different types of seizures. The epilepsy was
either isolated or epilepsy accompanied by DD/ID, dysmorphy,
or other neurological signs (epilepsy plus). We applied array
CGH to detect copy number changes in 50 individuals with
isolated IGE and in 52 patients with different types of epilepsy
and additional DD/ID or autism, six of whom, including patient 16,
had normal karyotype using GTG banding analysis with at least
550-band resolution.
DNA Isolation
Genomic DNA was extracted from peripheral blood cells using a
Puregene DNA Blood Kit (Qiagen, Gentra Systems, Minneapolis,
MN) according to the manufacturer’s protocol. The reference DNA
samples were obtained from phenotypically normal male and
female controls.
Chromosomal Microarray Analysis (CMA)
Custom-designed exon-targeted clinical array CGH was performed
using 180K V8.0 and V8.1 microarrays designed by Medical Genet-
ics Laboratories at Baylor College of Medicine (BCM; http:// in cooperation with
Department of Medical Genetics at the Institute of Mother and
Child and manufactured by Agilent Technology (Santa Clara, CA).
V8.0 and V8.1 OLIGO (180 K) arrays have genome-wide coverage
as well as exon coverage for over 1,700 genes with an average of 4.2
oligos per exon and intronic gaps no larger than 10 kb [Boone et al.,
2010]. Digestion, labeling, and hybridization were performed
following the manufacturer’s instructions. The BCM web-based
software platform and the home brew IMiD-web2py software were
used for genomic copy-number analysis. All genomic coordinates
are based on the March 2006 assembly of the reference genome
(NCBI36/hg18). When available, blood samples were obtained
from patient’s parents, and array CGH was done to investigate
CNV inheritance.
To verify genomic gains and losses identified by array CGH,
depending on CNV size, we used GTG-banding, FISH, multiplex
ligation-dependent probe amplification (MLPA), or PCR analyzes.
Conventional Karyotype Analysis
Peripheral blood lymphocytes were cultured and GTG-banding
analysis was performed according to the standard protocol. The
metaphases with 550-band resolution were analyzed.
FISH Analysis
FISH analyses were carried out by standard procedures in phyto-
hemagglutinin-stimulated peripheral blood lymphocytes using
probes derived from bacterial artificial chromosomes (BACs).
When available, blood samples were obtained from the patient’s
parents, and FISH analysis using the same probes was done to
investigate the inheritance of the CNVs.
MLPA, PCR, and DNA Sequencing
MLPA experiments (Patients 1 and 2) were preformed according
the manufacturer’s instruction with the kit SALSA MLPA P189
CDKL5 (MRC Holland). Experimental data analysis was done with
GeneMarker v1.8 software (Softgenetics
, LLC). To characterize
the breakpoint in the CDKL5 gene, PCR reaction was performed
with Expand Long Template PCR System (Roche
according to the manufacturer’s instructions. The reaction prod-
ucts were separated by agarose gel electrophoresis. The smaller
product was cut out from the gel and after DNA extraction (Gel-
Out Kit, A&A Biotechnology
) was subjected to direct sequencing
reaction (BigDye Terminator v.3.1 Cycle Sequencing Kit, Life
) with primers used for the product amplification.
The sequences were analyzed with FinchTV v.1.4.0 and compared
to the reference sequence NG_008475.1.
Overall, 24 non-polymorphic (not reported in CNV databases of
healthy individuals) copy-number changes were found in 23
(isolated epilepsy in 12 and epilepsy plus in 11) of 102 patients
(23.5%), ranging in size from 1 kb to 10.35 Mb. We divided the
detected CNVs into three groups. The first group contains CNVs
considered as clinically relevant (pathogenic for epilepsy): deletions
in seven patients and duplications in three patients (Table I). We
identified five patients with known recurrent CNVs at hotspots:
15q11.2 (BP1/BP2), 16p11.2, 16p13.11, 22q11.21, and 7q11.23 (pt
3 was described elsewhere, Ramocki et al., 2010] and five patients
with different-sized non-recurrent CNVs: two girls with exonic
deletions of the CDKL5 gene at chromosome Xp22.13 (pt 1 was
reported elsewhere, Bartnik et al., 2011], a boy with two interrupted
duplication CNVs at Xq28 harboring the MECP2 and IDS
(iduronate 2-sulfatase; OMIM 300823) genes and a heterozygous
NPHP1 (nephrocystin 1; OMIM 607100) deletion at 2q13, one
individual with a deletion at 1p36.21p36.32 associated with a
balanced paracentric inversion at 1p32p34.3 detected by conven-
tional karyotyping (data not shown), and one subject with a
duplication of 14q12 encompassing the FOXG1 gene.
The second group consists of two patients with rare larger-sized
deletions, at 2q24.1q24.3 (10.4 Mb) and 9q21.13 (2.5 Mb; Fig. 1)
that represent novel CNVs potentially causative for epilepsy
(Table II).
In the third group, we classified patients with four unique
deletions and eight duplications of unknown clinical significance
(Table III). Three of them map to recurrent hot spots; however,
duplication 16p13.11 in patient 21 is smaller in size than the
common ones and likely was not mediated by non-allelic homol-
ogous recombination (NAHR).
To determine the pathogenic role of the identified CNVs, we
considered their type (deletion or duplication) and size, gene
content, inheritance pattern, and available information from
BCM and public CNV databases. In general, we have divided the
detected CNVs into three groups. The first group includes CNVs of
known published and well-recognized genomic imbalances that we
consider as clinically relevant for epilepsy. The second group
consists of rare larger-sized deletions that could be novel CNVs
potentially causative for epilepsy. Finally, variants of unknown
clinical significance are listed in the third group.
We have identified known causative CNVs in three patients with
isolated epilepsy and seven patients with epilepsy and other neuro-
developmental abnormalities (Table I). A number of studies have
suggested that increased dosage of FOXG1 mapping to chromo-
some 14q12 is pathogenic for developmental delay, cognitive
impairment with speech delay, and epilepsy [Yeung et al., 2009;
Brunetti-Pierri et al., 2011; Paciorkowski et al., 2011; Striano et al.,
2011; Tohyama et al., 2011]. However, recently, Amor et al. [2012]
reported a familial case of an 88 kb duplication in 14q12, encom-
passing FOXG1, associated only with isolated hemifacial micro-
somia. Brunetti-Pierri et al. [2012] suggested that this small
duplication may be devoid of FOXG1 distant up-regulating ele-
ments, thus not sufficiently increasing the gene dosage to manifest
the abnormal neurological phenotype. In addition, Amor et al.
[2012] also identified an 3 Mb duplication of the 14q12 region,
including FOXG1 in a child enrolled as a control subject in the
TABLE I. Clinically Relevant CNVs Known to Be Pathogenic for Epilepsy
Pt Sex
at onset aCGH results
(Mb) Verification Inheritance
Seizure types/epilepsy
Cognitive function,
other features References
1 F 2 months arr Xp22.13(18,492,235
18,492,821) 12
MLPA De novo Normal Refractory epilepsy with
different types of
seizures (GTCS, tonic,
Profound mental
retardation; Rett-like
Bartnik et al. [2011]
2 F 5 months arr Xp22.13(18,542,246
18,553,009) 1
MLPA, PCR De novo Normal Refractory epilepsy with
different types of
seizures (focal, tonic)
Rett-like syndrome;
Profound mental
retardation, autism
Erez et al. [2009]
3 M 8 months arr 7q11.23(75,003,415
76,661,664) 1
FISH Mat Mild ID Epilepsy with GTCS/JME DD Ramocki et al. [2010]
4 M 13 years arr 22q11.21(17,364,458
19,761,174) 1
FISH Mat Mild ID, VCFS JME Normal
IQ Gonzalez and Bautista
5 F 3 years arr 15q11.2(20,393,584
20,613,447) 1
FISH Pat Unknown JME (with myoclonus and
GTCS, and absences
with eyelid myoclonus)
Normal IQ, headache de Kovel et al. [2010]
6 M 6 months arr 14q11.2q12(22,378,936
30,744,923) 3
karyotype De novo Normal West syndrome evolving
into Lennox-Gastaut
syndrome; refractory
Profound DD Brunetti-Pierri et al.
7* M 6 months arr 1p36.32p36.21(4,600,008
13,110,103) 1
FISH, karyotype De novo Normal Refractory epilepsy with
tonicclonic seizures
(mainly during
infections). Status
Moderate DD,
Bahi-Buisson et al.
8** M 1 months Xq28(149,557,875
154,533,675) 2
FISH Mat Normal West syndrome evolving
into epilepsy with focal
seizures with hypsar-
rythmia in EEG
Dysmorphic, profound DD Van Esch et al. [2005]
9 M 6 months arr
17,963,057) 1
FISH Pat Normal, father
graduated only
primary school
West syndrome evolving
into epilepsy with tonic
clonic seizures
Dysmorphic, profound DD,
de Kovel et al., [2010]
10 F 17 years arr 16p11.2(29,532,264
30,104.842) 3
Not mat Normal JME Normal IQ Shinawi et al. [2010]
DD, developmental delay; GTCS, generalized tonicclonic seizures; ID, intellectual disability; JME, juvenile myoclonic epilepsy; mat, maternal; pat, paternal. VCFS, Velocardiofacial syndrome.
*Patient 7 had an additional balanced paracentric inversion 1p32p34.3.
**Patient 8 had an additional heterozygous
deletion at 2q13 and a 284 kb duplication at Xq28 (148,240,624-148,524,326), harboring the
gene (Table III). The absence of
epilepsy in patient 8’s mother likely results from a skewed X-inactivation, which was not studied.
CHOP CNV database (51186) [Shaikh et al., 2009] and questioned
the pathogenicity of FOXG1 duplication. However, upon further
re-evaluation, it turned out that this child may have developmental
delay, which was not known before (H. Hakonarson, personal
communication). Our data further support the pathogenicity of
FOXG1 duplication in epilepsy and DD.
The 8.5 Mb interstitial deletion 1p36.21p36.32 identified in
patient 7 harbors the KCNAB2 gene encoding a beta subunit of
a voltage-gated potassium channel (OMIM 601142) but does not
include KLHL17 (kelch-like 17) and GABRD (g-aminobutyric acid
A receptor delta-subunit; OMIM 137163), three genes proposed as
responsible for epilepsy in patients with chromosome 1p36 deletion
syndrome (OMIM 607872) [Heilstedt et al., 2001; Bahi-Buisson
et al., 2008; Rosenfeld et al., 2010; Paciorkowski et al., 2011]. Our
data further emphasize the role of KCNAB2 in this syndrome
[Heilstedt et al., 2001; Gajecka et al., 2007].
Although there are more than 80 different subunits of potassium
channel genes [Turnbull et al., 2005], mutations have been found
only in four genes, KCNQ2 (voltage-gated, KQT-like subfamily,
member 2; OMIM 602235; 20q13.3) and KCNQ3 (voltage-gated,
KQT-like subfamily, member 3; OMIM 602232; 8q24) in patients
with benign familial neonatal convulsions as well as in KCNA1
(voltage-gated, shaker-related subfamily, member 1; OMIM
176260; 12p13) in patients with partial epilepsy or episodic ataxia
type 1 (OMIM 160120) [Gurnett and Hedera, 2007]. In addition,
KCNMA1 (calcium-activated, large conductance, subfamily M,
alpha, member 1; OMIM 600150; 10q22) was found mutated in
patients with generalized epilepsy and paroxysmal dyskinesia
(OMIM 609446).
In two patients (11 and 12), we detected CNVs that were not
previously reported to be associated with epilepsy and that contain
genes that are likely to contribute to the phenotype in these patients
(Table II).
Interestingly, patient 11 with refractory West syndrome and
profound DD had a 10.3 Mb deletion in 2q24.1q24.3 (located
1.5 Mb proximally to SCN1A), harboring two other potassium
FIG. 1. Array CGH analyses (a) in patient 11, showing an 10.3 Mb in the 2q24.1q24.3 region and (b) in patient 12, showing an 2.5 Mb deletion on
chromosome 9q21.13. Reddots denote thedeleted region. c:Gene content in the deleted region2q24.1q24.3 identified in Patient 11compared with
the deletions reported by Palumbo et al. [2012], Magri et al. [2011], and found in DECIPHER patients 254867 and 253681 (red bars). d: Genes in
the deleted region on chromosome 9q21.13. e: Results of GTG-banding analysis of chromosome 2 in patient 11. Red arrow indicates the deleted
chromosome region. f: Results of the FISH analysis in patient 12 with the BAC clone RP11-243A1 (green) and Vysis CEP 9 Alpha (red) used as
controls. White arrow indicates the deleted chromosome region.
channel genes KCNJ3 (inwardly-rectifying channel, subfamily J,
member 3; OMIM 601534) and KCNH7 encoding a pore-forming
alpha subunit of voltage-gated potassium channel (OMIM 608169)
as well as sodium bicarbonate transporter, SLC4A10 (solute carrier
family 4 (sodium bicarbonate transporter-like), member 10;
OMIM 605556) (Fig. 1a,c,e). Disruption of SLC4A10 was reported
in a patient with a de novo apparently balanced chromosomal
translocation t(2;13; q24;q31) and complex partial epilepsy with
mental retardation [Gurnett et al., 2008]. However, an 7.5 Mb
deletion chr2:155.526.470163.058.894, harboring SLC4A10 and
truncating KCNH7, but likely not involving KCNJ3 (max coor-
dinates: chr2:155.413.315163.101.007), was found in a patient
with mental retardation and generalized hypotonia [Palumbo et al.,
2012] and an overlapping 5.2 Mb deletion chr2:159,618,452
164,882,054, encompassing SLC4A10 and KCNH7, but leaving
KCNJ3 intact, was described in a mentally retarded boy with
muscular hypotonia and no evidence of epilepsy [Magri et al.,
2011]. Furthermore, Layouni et al., [2010] identified a region of
absence of heterozygosity containing KCNJ3 in a consanguineous
Tunisian family with an autosomal recessive form of juvenile
myoclonic epilepsy and Chioza et al., [2002] reported an associ-
ation of KCNJ3 with different idiopathic generalized epilepsy
syndromes. Based on these data, we suggest that haploinsufficiency
of KCNJ3 contributes to epilepsy in our patient. In support of this
notion, DECIPHER patient 254867 with an overlapping deletion
chr2:156539025158815118, not including KCNJ3 and KCNH7,
did not manifest seizures whereas patient DECIPHER 253681 with
an overlapping chr2:152182099159245370, including KCNJ3, did
present with epilepsy.
A 2.5 Mb deletion 9q21.13 in patient 12 (Fig. 1b,d,f) with
epilepsy with eyelid myoclonia and GTCS and autism involves
GDA (guanine deaminase; OMIM 139260), ZFAND5 (zinc finger,
AN1-type domain 5; OMIM 604761), TMC1 (transmembrane
channel-like 1, OMIM 606706), ALDH1A1 (aldehyde dehydrogen-
ase 1 family, member A1; OMIM 100640), ANXA1 (annexin A1;
OMIM 151690), RORB (RAR-related orphan receptor B; OMIM
601972), and potentially TRPM6 (transient receptor potential
cation channel, subfamily M, member 6; OMIM 607009). Hetero-
zygous and homozygous mutations in TRPM6 are associated with
hypomagnesemia with secondary hypocalcemia, a rare condition
usually presenting in the newborn period as refractory seizures
(OMIM 602014). Of interest, our patient had a magnesium level at
the lower border (0.74 and 0.79 mg/dl, normal 0.71.1 mg/dl). His
calcemia was normal. In addition, heterozygous and homozygous
mutations in TMC1 have been associated with progressive post-
lingual hearing loss and profound prelingual deafness DFNA36
(OMIM 606705) and DFNB7 (OMIM 600974). No evidence of
deafness was observed in our patient. Of note, DECIPHER patient
2065 with a larger-sized 10.3 Mb deletion chr9:70318675
80676552 had tonic/clonic (grand-mal) seizures and patient
2064 with an overlapping 6.4 Mb deletion chr9:74281674
80676552, excluding GDA and ZFAND5, also had seizures. Given
the large size, gene content and the fact that it arose de novo we
believe this deletion is causative for epilepsy in our patient.
CNVs in group 3 have been classified as of unknown clinical
significance. In patients 1416 and 2123, there are some literature
data to suggest that they may be responsible for the observed
TABLE II. Novel CNVs Potentially Causative for Epilepsy
Pt Sex
at onset aCGH results
genes Size (Mb) Verification Inheritance
Seizure types/epi-
lepsy syndrome
Cognitive function,
other problems
11 M 1 months arr
165,068,041) 1
10.35 Karyotype Not mat Unknown West syndrome;
Profound DD, EPH
gestosis during
premature delivery
12 M 2 years arr
76,496,752) 1
2.57 FISH De novo Mother healthy,
father has
Epilepsy with eyelid
myoclonia and GTCS
DD, developmental delay; GTCS, generalized tonicclonic seizures; mat, maternal.
TABLE III. CNVs of Unknown Clinical Significance
Pt Sex
at onset aCGH results Selected genes
(Mb) Verification Inheritance Parental phenotype
Seizure types/epilepsy
Cognitive function,
other problems
8 M 1 months arr Xq28(148,240,624-
148,524,326) 2
Mat Normal West syndrome evolving
into epilepsy with focal
seizures with hypsar-
rythmia in EEG
Dysmorphic, profound
13 M 16 years arr 2p12(78,311,526-78,879,
196) 1
mRNA BC024248
FISH Mat Unknown JME with GTCS Normal IQ
14 F 9 months arr 16q24.3(87,692,754-
87,789,405) 1
Pat Normal Refractory epilepsy with
tonic seizures and CSWS
Profound DD, Rett-like
syndrome, autistic
15 F 6 months arr 7q31.1(110,627,069-
110,978,974) 1
FISH Not mat Mother normal, father
West syndrome evolving
into refractory epilepsy
with polymorphic
seizures (myoclonic,
tonic, unclassified)
Profound DD, cerebral
16 M 4 years arr 16q23.1(76,974,912-
77,669,115) 1
Unknown Mother normal, father
had schizophrenia
and committed
suicide; father’s
brother also
committed suicide
Epilepsy with absences
and GTCS
17 F 7 years arr 18q23(74,856,013-
75,390,868) 3
Mat Normal Epilepsy with GTCS and
Normal IQ, visual
18 F 8 months arr 2q14.3(124,747,254-
125,784,880) 3
Mat Normal West syndrome/JME Normal IQ
19 F 16 years arr
150,411,844) 3
Pat Normal JME Normal IQ
20 F 14 years arr Xp22.31 (7,801,120-
8,159,541) 3
Pat Normal JME Normal IQ
21 F 9 years arr 16p13.11(15,824,601-
16,199,695) 3
Pat Father and brother
have epilepsy,
mother normal
JME Normal IQ
22 F 16 years arr 16p13.11(15,425,965-
16,215,648) 3
Unknown Unknown JME Normal IQ
23 F 3 years arr 15q13.3(30,083,430-
30,191,648) 3
Mat Unknown JME (with absences with
eyelid myoclonia and
Normal IQ
CSWS, continuous spikes and waves during slow wave sleep; DD, developmental delay; GTCS, generalized tonicclonic seizures; JME, juvenile myoclonic epilepsy; mat, maternal; pat, paternal.
phenotypic abnormalities. In cases 13, 1720, we classified the
identified CNVs in group III because, similar to the CMA sign out
guidelines used at Baylor College of Medicine, we report all
gene-free CNVs if larger than 500 kb (patients 13) as well as
CNVs > 300 kb containing genes, even if their clinical significance
is unknown (patients 1720).
Only two out of 18 patients with recurrent 16p13.11 duplication
[Nagamani et al., 2011; Ramalingam et al., 2011] presented with
generalized epilepsy. We believe that this duplication may also
contribute to JME in patients 21 and 22. Patient 14 with refractory
epilepsy with tonic seizures and electrographic continuous spikes
and waves during slow wave sleep (CSWS) had a deletion of CDH15
(Cadherin 15; OMIM 114019) inherited from his apparently
healthy father. Heterozygous point mutations in CDH15 have
been reported in patients with mild to severe ID [Bhalla et al.,
2008]. In addition, deletions of CDH15 have been observed in
four patients with neurodevelopmental abnormalities, including
epilepsy (16q24.3 microdeletion syndrome) [Willemsen et al.,
IMMP2L (IMP2 inner mitochondrial membrane peptidase-like
subunit 2; OMIM 605977) deleted in patient 15 with West syn-
drome has been suggested as a candidate gene for autistic spectrum
disorders (ASDs) and Tourette syndrome [Petek et al., 2001;
Maestrini et al., 2010; Casey et al., 2011; Patel et al., 2011]. Little
is known about the function of the IMMP2L protein. However,
given its potential role in other neuropsychiatric disorders, we
cannot exclude its contribution to the abnormal phenotype in our
patient 15.
Patient 16 with epilepsy with absences and GTCS had an isolated
deletion of WWOX (WW domain containing oxidoreductase;
OMIM 605131). Interestingly, a 13-bp deletion in exon 9 of the
Wwox gene was observed in rats with lethal dwarfism and epilepsy
(audiogenic seizures) [Suzuki et al., 2009]. In addition, Huang et al.
[2010] bioinformatically predicted WWOX to be haploinsufficient
in humans. Recently, White et al. [2012] reported a maternally
inherited exon 68 deletion in WWOX in a patient with a 46,XY
karyotype and ambiguous genitalia and gonadal dysgenesis but no
evidence of seizures or autism. This exonic deletion was predicted
not to lead to protein truncation but to removal of the SDR domain
at the C-terminus. Given the previous description of Wwox defi-
cient mice with gonadal abnormalities [Ludes-Meyers et al., 2007],
White et al. [2012] suggested a role for WWOX in human gonad
development. However, no sex determination/differentiation
abnormalities were observed in our male patient, suggesting that
haploinsufficiency of WWOX may have different clinical
The role of CHRNA7 (cholinergic receptor, neuronal nicotinic,
alpha polypeptide 7; OMIM 118511) duplication in patient 23
remains elusive. Whereas epilepsy has been strongly associated with
microdeletions of CHRNA7 [Helbig et al., 2009; Shinawi et al.,
2009], it was present only in one of 11 patients with small micro-
duplications involving CHRNA7 [Szafranski et al., 2010].
Our results further confirm the pathogenic role of both recurrent
and non-recurrent submicroscopic CNVs in the etiology of epi-
lepsy, demonstrate the usefulness of our approach to the identi-
fication of novel epilepsy genes, and support the clinical use of CMA
in the genetic diagnosis of epilepsy.
We are grateful to the patients and to their families for participation
in this study. We thank Dr. M. Ramocki for helpful discussion.
Amor DJ, Burgess T, Tan TY, Pertile M. 2012. Questionable
nicity of FOXG1 duplication. Eur J Hum Genet in press.
Bahi-Buisson N, Girard B, Gautier A, Nectoux J, Fichou Y, Saillour Y,
Poirier K, Chelly J, Bienvenu T. 2010. Epileptic encephalopathy in a girl
with an interstitial deletion of Xp22 comprising promoter and exon 1
of the CDKL5 gene. Am J Med Genet B Neuropsychiatr Genet 153B:
Bahi-Buisson N, Guttierrez-Delicado E, Soufflet C, Rio M, Daire VC,
Lacombe D, Heron D, Verloes A, Zuberi S, Burglen L, Afenjar A, Moutard
ML, Edery P, Novelli A, Bernardini L, Dulac O, Nabbout R, Plouin P,
Battaglia A. 2008. Spectrum of epilepsy in terminal 1p36 deletion
syndrome. Epilepsia 49:509515.
Ballif BC, Hornor SA, Jenkins E, Madan-Khetarpal S, Surti U, Jackson KE,
Asamoah A, Brock PL, Gowans GC, Conway RL, Graham JM Jr, Medne L,
Zackai EH, Shaikh TH, Geoghegan J, Selzer RR, Eis PS, Bejjani BA, Shaffer
LG. 2007. Discovery of a previously unrecognized microdeletion syn-
drome of 16p11.2-p12.2. Nat Genet 39:10711073.
Bartnik M, Derwinska K, Gos M, Obersztyn E, Kolodziejska KE, Erez A,
Szpecht-Potocka A, Fang P, Terczynska I, Mierzewska H, Lohr NJ, Bellus
GA, Reimschisel T, Bocian E, Mazurczak T, Cheung SW, Stankiewicz P.
2011. Early-onset seizures due to mosaic exonic deletions of CDKL5 in
a male and two females. Genet Med 13:447452.
Battaglia A, Guerrini R. 2005. Chromosomal disorders associated with
epilepsy. Epileptic Disord 7:181192.
Berg AT, Berkovic SF, Brodie MJ, Buchhalter J, Cross JH, van Emde Boas
W, Engel J, French J, Glauser TA, Mathern GW, Moshe SL, Nordli D,
Plouin P, Scheffer IE. 2010. Revised terminology and concepts for
organization of seizures and epilepsies: Report of the ILAE Commission
on Classification and Terminology, 20052009. Epilepsia 51:676685.
Bhalla K, Luo Y, Buchan T, Beachem MA, Guzauskas GF, Ladd S, Bratcher
SJ, Schroer RJ, Balsamo J, DuPont BR, Lilien J, Srivastava AK. 2008.
Alterations in CDH15 and KIRREL3 in patients with mild to severe
intellectual disability. Am J Hum Genet 83:703713.
Boone PM, Bacino CA, Shaw CA, Eng PA, Hixson PM, Pursley AN, Kang
SH, Yang Y, Wiszniewska J, Nowakowska BA, del Gaudio D, Xia Z,
Simpson-Patel G, Immken LL, Gibson JB, Tsai AC, Bowers JA,
Reimschisel TE, Schaaf CP, Potocki L, Scaglia F, Gambin T, Sykulski
M, Bartnik M, Derwinska K, Wisniowiecka-Kowalnik B, Lalani SR,
Probst FJ, Bi W, Beaudet AL, Patel A, Lupski JR, Cheung SW, Stankiewicz
P. 2010. Detection of clinically relevant exonic copy-number changes by
array CGH. Hum Mutat 31:13261342.
Brunetti-Pierri N, Berg JS, Scaglia F, Belmont J, Bacino CA, Sahoo T, Lalani
SR, Graham B, Lee B, Shinawi M, Shen J, Kang SH, Pursley A, Lotze T,
Kennedy G, Lansky-Shafer S, Weaver C, Roeder ER, Grebe TA, Arnold
GL, Hutchison T, Reimschisel T, Amato S, Geragthy MT, Innis JW,
Obersztyn E, Nowakowska B, Rosengren SS, Bader PI, Grange DK, Naqvi
S, Garnica AD, Bernes SM, Fong CT, Summers A, Walters WD, Lupski JR,
Stankiewicz P, Cheung SW, Patel A. 2008. Recurrent reciprocal 1q21.1
deletions and duplications associated with microcephaly or macroce-
phaly and developmental and behavioral abnormalities. Nat Genet
Brunetti-Pierri N, Paciorkowski AR, Ciccone R, Mina ED, Bonaglia
MC, ‘Borgatti R, Schaaf CP, Sutton VR, Xia Z, Jelluma N, Ruivenkamp
C, Bertrand M, de Ravel TJ, Jayakar P, Belli S, Rocchetti K, Pantaleoni C,
D’Arrigo S, Hughes J, Cheung SW, Zuffardi O, Stankiewicz P. 2011.
Duplications of FOXG1 in 14q12 are associated with developmental
epilepsy, mental retardation, and severe speech impairment. Eur J Hum
Genet 19:102107.
Brunetti-Pierri N, Cheung SW, Stankiewicz P. 2012. Reply
to Amor et al.
Eur J Hum Genet in press.
Casey JP, Magalhaes T, Conroy JM, Regan R, Shah N, Anney R, Shields DC,
Abrahams BS, Almeida J, Bacchelli E, Bailey AJ, Baird G, Battaglia A,
Berney T, Bolshakova N, Bolton PF, Bourgeron T, Brennan S, Cali P,
Correia C, Corsello C, Coutanche M, Dawson G, de Jonge M, Delorme R,
Duketis E, Duque F, Estes A, Farrar P, Fernandez BA, Folstein SE, Foley S,
Fombonne E, Freitag CM, Gilbert J, Gillberg C, Glessner JT, Green J,
Guter SJ, Hakonarson H, Holt R, Hughes G, Hus V, Igliozzi R, Kim C,
Klauck SM, Kolevzon A, Lamb JA, Leboyer M, Le Couteur A, Leventhal
BL, Lord C, Lund SC, Maestrini E, Mantoulan C, Marshall CR, McCo-
nachie H, McDougle CJ, McGrath J, McMahon WM, Merikangas A,
Miller J, Minopoli F, Mirza GK, Munson J, Nelson SF, Nygren G, Oliveira
G, Pagnamenta AT, Papanikolaou K, Parr JR, Parrini B, Pickles A, Pinto
D, Piven J, Posey DJ, Poustka A, Poustka F, Ragoussis J, Roge B, Rutter
ML, Sequeira AF, Soorya L, Sousa I, Sykes N, Stoppioni V, Tancredi R,
Tauber M, Thompson AP, Thomson S, Tsiantis J, Van Engeland H,
Vincent JB, Volkmar F, Vorstman JA, Wallace S, Wang K, Wassink TH,
White K, Wing K, Wittemeyer K, Yaspan BL, Zwaigenbaum L, Betancur
C, Buxbaum JD, Cantor RM, Cook EH, Coon H, Cuccaro ML, Gesch-
wind DH, Haines JL, Hallmayer J, Monaco AP, Nurnberger JI Jr, Pericak-
Vance MA, Schellenberg GD, Scherer SW, Sutcliffe JS, Szatmari P,
Vieland VJ, Wijsman EM, Green A, Gill M, Gallagher L, Vicente A,
Ennis S. 2011. A
novel approach of homozygous haplotype sharing
identifies candidate genes in autism spectrum disorder. Hum Genet
[Epub ahead of print].
Chioza B, Osei-Lah A, Wilkie H, Nashef L, McCormick D, Asherson P,
Makoff AJ. 2002. Suggestive evidence for association of two potassium
channel genes with different idiopathic generalised epilepsy syndromes.
Epilepsy Res 52:107116.
Crino PB. 2007. Gene expression, genetics, and genomics in epilepsy: Some
answers, more questions. Epilepsia 48(Suppl2):4250.
de Kovel CG, Trucks H, Helbig I, Mefford HC, Baker C, Leu C, Kluck C,
Muhle H, von Spiczak S, Ostertag P, Obermeier T, Kleefuss-Lie AA,
Hallmann K, Steffens M, Gaus V, Klein KM, Hamer HM, Rosenow F,
Brilstra EH, Trenite DK, Swinkels ME, Weber YG, Unterberger I,
Zimprich F, Urak L, Feucht M, Fuchs K, Moller RS, Hjalgrim H, De
Jonghe P, Suls A, Ruckert IM, Wichmann HE, Franke A, Schreiber S,
Nurnberg P, Elger CE, Lerche H, Stephani U, Koeleman BP, Lindhout D,
Eichler EE, Sander T. 2010. Recurrent microdeletions at 15q11.2 and
16p13.11 predispose to idiopathic generalized epilepsies. Brain 133:
Dibbens LM, Mullen S, Helbig I, Mefford HC, Bayly MA, Bellows S, Leu C,
Trucks H, Obermeier T, Wittig M, Franke A, Caglayan H, Yapici Z,
Sander T, Eichler EE, Scheffer IE, Mulley JC, Berkovic SF. 2009. Familial
and sporadic 15q13.3 microdeletions in idiopathic generalized epilepsy:
Precedent for disorders with complex inheritance. Hum Mol Genet
Erez A, Patel AJ, Wang X, Xia Z, Bhatt SS, Craigen W, Cheung SW, Lewis
RA, Fang P, Davenport SL, Stankiewicz P, Lalani SR. 2009. Alu-specific
microhomology-mediated deletions in CDKL5 in females with early-
onset seizure disorder. Neurogenetics 10:363369.
Gajecka M, Mackay KL, Shaffer LG. 2007. Monosomy 1p36 deletion
syndrome. Am J Med Genet C Semin Med Genet 145C:346356.
Gardiner RM. 2000. Impact of our understanding of the genetic aetiology of
epilepsy. J Neurol 247:327334.
Gonzalez W, Bautista RE. 2009. Seizures and EEG findings in an adult
patient with DiGeorge syndrome: A case report and review of the
literature. Seizure 18:648651.
Gurnett CA, Hedera P. 2007. New ideas in epilepsy genetics: Novel epilepsy
genes, copy number alterations, and gene regulation. Arch Neurol 64:
Gurnett CA, Veile R, Zempel J, Blackburn L, Lovett M, Bowcock A. 2008.
Disruption of sodium bicarbonate transporter SLC4A10 in a patient with
complex partial epilepsy and mental retardation. Arch Neurol 65:
Hauser WA, Annegers JF, Rocca WA. 1996. Descriptive epidemiology of
epilepsy: Contributions of population-based studies from Rochester,
Minnesota. Mayo Clin Proc 71:576586.
Heilstedt HA, Burgess DL, Anderson AE, Chedrawi A, Tharp B, Lee O,
Kashork CD, Starkey DE, Wu YQ, Noebels JL, Shaffer LG, Shapira SK.
2001. Loss of the potassium channel beta-subunit gene, KCNAB2,is
associated with epilepsy in patients with 1p36 deletion syndrome.
Epilepsia 42:11031111.
Heinzen EL, Radtke RA, Urban TJ, Cavalleri GL, Depondt C, Need AC,
Walley NM, Nicoletti P, Ge D, Catarino CB, Duncan JS, Kasperaviciute
D, Tate SK, Caboclo LO, Sander JW, Clayton L, Linney KN, Shianna KV,
Gumbs CE, Smith J, Cronin KD, Maia JM, Doherty CP, Pandolfo M,
Leppert D, Middleton LT, Gibson RA, Johnson MR, Matthews PM,
Hosford D, Kalviainen R, Eriksson K, Kantanen AM, Dorn T, Hansen J,
Kramer G, Steinhoff BJ, Wieser HG, Zumsteg D, Ortega M, Wood NW,
Huxley-Jones J, Mikati M, Gallentine WB, Husain AM, Buckley PG,
Stallings RL, Podgoreanu MV, Delanty N, Sisodiya SM, Goldstein DB.
2010. Rare deletions at 16p13.11 predispose to a diverse spectrum of
sporadic epilepsy syndromes. Am J Hum Genet 86:707718.
Helbig I, Mefford HC, Sharp AJ, Guipponi M, Fichera M, Franke A, Muhle
H, de Kovel C, Baker C, von Spiczak S, Kron KL, Steinich I, Kleefuss-Lie
AA, Leu C, Gaus V, Schmitz B, Klein KM, Reif PS, Rosenow F, Weber Y,
Lerche H, Zimprich F, Urak L, Fuchs K, Feucht M, Genton P, Thomas P,
Visscher F, de Haan GJ, Moller RS, Hjalgrim H, Luciano D, Wittig M,
Nothnagel M, Elger CE, Nurnberg P, Romano C, Malafosse A, Koeleman
BP, Lindhout D, Stephani U, Schreiber S, Eichler EE, Sander T. 2009.
15q13.3 microdeletions increase risk of idiopathic generalized epilepsy.
Nat Genet 41:160162.
Huang N, Lee I, Marcotte EM, Hurles ME. 2010. Characterising
predicting haploinsufficiency in the human genome. PLoS Genet
Jozwiak S, Lason W, Bijak M, Katulska K. 2005. Research advances in
molecular genetics of epilepsies. Neurol Neurochir Pol 39:497508.
Kirov G, Grozeva D, Norton N, Ivanov D, Mantripragada KK, Holmans P,
Craddock N, Owen MJ, O’Donovan MC. 2009. Support for the involve-
ment of large copy number variants in the pathogenesis of schizophrenia.
Hum Mol Genet 18:14971503.
Klassen T, Davis C, Goldman A, Burgess D, Chen T, Wheeler D, McPherson
J, Bourquin T, Lewis L, Villasana D, Morgan M, Muzny D, Gibbs R,
Noebels J. 2011. Exome sequencing of ion channel genes reveals complex
profiles confounding personal risk assessment in epilepsy. Cell 145:
Koolen DA, Pfundt R, de Leeuw N, Hehir-Kwa JY, Nillesen WM, Neefs I,
Scheltinga I, Sistermans E, Smeets D, Brunner HG, van Kessel AG,
Veltman JA, de Vries BB. 2009. Genomic microarrays in mental retar-
dation: A practical workflow for diagnostic applications. Hum Mutat
Layouni S, Salzmann A, Guipponi M, Mouthon D, Chouchane L, Dogui M,
Malafosse A. 2010. Genetic linkage study of an autosomal recessive form
of juvenile myoclonic epilepsy in a consanguineous Tunisian family.
Epilepsy Res 90:3338.
Le Meur N, Holder-Espinasse M, Jaillard S, Goldenberg A, Joriot S, Amati-
Bonneau P, Guichet A, Barth M, Charollais A, Journel H, Auvin S,
Boucher C, Kerckaert JP, David V, Manouvrier-Hanu S, Saugier-Veber P,
Frebourg T, Dubourg C, Andrieux J, Bonneau D. 2010. MEF2C
haploinsufficiency caused by either microdeletion of the 5q14.3 region or
mutation is responsible for severe mental retardation with stereotypic
movements, epilepsy and/or cerebral malformations. J Med Genet 47:
Lee C, Iafrate AJ, Brothman AR. 2007. Copy number variations and clinical
cytogenetic diagnosis of constitutional disorders. Nat Genet 39:S48
Ludes-Meyers JH, Kil H, Nunez MI, Conti CJ, Parker-Thornburg J, Bed-
ford MT, Aldaz CM. 2007. WWOX hypomorphic mice display a higher
incidence of B-cell lymphomas and develop testicular atrophy. Genes
Chromosomes Cancer 46:11291136.
Maestrini E, Pagnamenta AT, Lamb JA, Bacchelli E, Sykes NH, Sousa I,
Toma C, Barnby G, Butler H, Winchester L, Scerri TS, Minopoli F,
Reichert J, Cai G, Buxbaum JD, Korvatska O, Schellenberg GD, Dawson
G, de Bildt A, Minderaa RB, Mulder EJ, Morris AP, Bailey AJ, Monaco AP.
2010. High-density SNP association study and copy number variation
analysis of the AUTS1 and AUTS5 loci implicate the IMMP2L-DOCK4
gene region in autism susceptibility. Mol Psychiatry 15:954968.
Magri C, Piovani G, Pilotta A, Michele T, Buzi F, Barlati S. 2011. De novo
deletion of chromosome 2q24.2 region in a mentally retarded boy with
muscular hypotonia. Eur J Med Genet 54:361364.
Marini C, Scheffer IE, Nabbout R, Mei D, Cox K, Dibbens LM, McMahon
JM, Iona X, Carpintero RS, Elia M, Cilio MR, Specchio N, Giordano L,
Striano P, Gennaro E, Cross JH, Kivity S, Neufeld MY, Afawi
Z, Andermann E, Keene D, Dulac O, Zara F, Berkovic SF, Guerrini R,
Mulley JC. 2009. SCN1A duplications and deletions detected in
Dravet syndrome: Implications for molecular diagnosis. Epilepsia 50:
Marshall CR, Young EJ, Pani AM, Freckmann ML, Lacassie Y, Howald C,
Fitzgerald KK, Peippo M, Morris CA, Shane K, Priolo M, Morimoto M,
Kondo I, Manguoglu E, Berker-Karauzum S, Edery P, Hobart HH,
Mervis CB, Zuffardi O, Reymond A, Kaplan P, Tassabehji M, Gregg
RG, Scherer SW, Osborne LR. 2008. Infantile spasms is associated with
deletion of the MAGI2 gene on chromosome 7q11.23-q21.11. Am J Hum
Genet 83:106111.
Mefford HC, Muhle H, Ostertag P, von Spiczak S, Buysse K, Baker C, Franke
A, Malafosse A, Genton P, Thomas P, Gurnett CA, Schreiber S, Bassuk
AG, Guipponi M, Stephani U, Helbig I, Eichler EE. 2010. Genome-wide
copy number variation in epilepsy: Novel susceptibility loci in idiopathic
generalized and focal epilepsies. PLoS Genet 6:e1000962.
Mefford HC, Sharp AJ, Baker C, Itsara A, Jiang Z, Buysse K, Huang S,
Maloney VK, Crolla JA, Baralle D, Collins A, Mercer C, Norga K, de Ravel
T, Devriendt K, Bongers EM, de Leeuw N, Reardon W, Gimelli S, Bena F,
Hennekam RC, Male A, Gaunt L, Clayton-Smith J, Simonic I, Park SM,
Mehta SG, Nik-Zainal S, Woods CG, Firth HV, Parkin G, Fichera M,
Reitano S, Lo Giudice M, Li KE, Casuga I, Broomer A, Conrad B,
Schwerzmann M, Raber L, Gallati S, Striano P, Coppola A, Tolmie JL,
Tobias ES, Lilley C, Armengol L, Spysschaert Y, Verloo P, De Coene A,
Goossens L, Mortier G, Speleman F, van Binsbergen E, Nelen MR,
Hochstenbach R, Poot M, Gallagher L, Gill M, McClellan J, King MC,
Regan R, Skinner C, Stevenson RE, Antonarakis SE, Chen C, Estivill X,
Menten B, Gimelli G, Gribble S, Schwartz S, Sutcliffe JS, Walsh T, Knight
SJ, Sebat J, Romano C, Schwartz CE, Veltman JA, de Vries BB, Vermeesch
JR, Barber JC, Willatt L, Tassabehji M, Eichler EE. 2008. Recurrent
rearrangements of chromosome 1q21.1 and variable pediatric pheno-
types. N Engl J Med 359:16851699.
Mefford HC, Yendle SC, Hsu C, Cook J, Geraghty E, McMahon JM, Eeg-
Olofsson O, Sadleir LG, Gill D, Ben-Zeev B, Lerman-Sagie T, Mackay M,
Freeman JL, Andermann E, Pelakanos JT, Andrews I, Wallace G, Eichler
EE, Berkovic SF, Scheffer IE. 2011. Rare copy number variants are an
important cause of epileptic encephalopathies. Ann Neurol 70:974985.
Mei D, Marini C, Novara F, Bernardina BD, Granata T, Fontana E, Parrini
E, Ferrari AR, Murgia A, Zuffardi O, Guerrini R. 2010. Xp22.3 genomic
deletions involving the CDKL5 gene in girls with early onset epileptic
encephalopathy. Epilepsia 51:647654.
Menten B, Maas N, Thienpont B, Buysse K, Vandesompele J, Melotte C, de
Ravel T, Van Vooren S, Balikova I, Backx L, Janssens S, De Paepe A, De
Moor B, Moreau Y, Marynen P, Fryns JP, Mortier G, Devriendt K,
Speleman F, Vermeesch JR. 2006. Emerging patterns of cryptic chromo-
somal imbalance in patients with idiopathic mental retardation and
multiple congenital anomalies: A new series of 140 patients and review of
published reports. J Med Genet 43:625633.
Miller DT, Adam MP, Aradhya S, Biesecker LG, Brothman AR, Carter NP,
Church DM, Crolla JA, Eichler EE, Epstein CJ, Faucett WA, Feuk L,
Friedman JM, Hamosh A, Jackson L, Kaminsky EB, Kok K, Krantz ID,
Kuhn RM, Lee C, Ostell JM, Rosenberg C, Scherer SW, Spinner NB,
Stavropoulos DJ, Tepperberg JH, Thorland EC, Vermeesch JR, Wagg-
oner DJ, Watson MS, Martin CL, Ledbetter DH. 2010. Consensus
statement: Chromosomal microarray is a first-tier clinical diagnostic
test for individuals with developmental disabilities or congenital anoma-
lies. Am J Hum Genet 86:749764.
Mulley JC, Mefford HC. 2011. Epilepsy and the new cytogenetics. Epilepsia
Nagamani SC, Erez A, Bader P, Lalani SR, Scott DA, Scaglia F, Plon SE, Tsai
CH, Reimschisel T, Roeder E, Malphrus AD, Eng PA, Hixson PM, Kang
SH, Stankiewicz P, Patel A, Cheung SW. 2011. Phenotypic manifestations
of copy number variation in chromosome 16p13.11. Eur J Hum Genet
Nowakowska BA, Obersztyn E, Szymanska K, Bekiesinska-Figatowska M,
Xia Z, Ricks CB, Bocian E, Stockton DW, Szczaluba K, Nawara M, Patel A,
Scott DA, Cheung SW, Bohan TP, Stankiewicz P. 2010. Severe mental
retardation, seizures, and hypotonia due to deletions of MEF2C.AmJ
Med Genet B Neuropsychiatr Genet 153B:10421051.
Ottman R, Hirose S, Jain S, Lerche H, Lopes-Cendes I, Noebels JL, Serratosa
J, Zara F, Scheffer IE. 2010. Genetic testing in the epilepsiesReport of
the ILAE Genetics Commission. Epilepsia 51:655670.
Paciorkowski AR, Thio LL, Rosenfeld JA, Gajecka M, Gurnett CA, Kulkarni
S, Chung WK, Marsh ED, Gentile M, Reggin JD, Wheless JW, Balasu-
bramanian S, Kumar R, Christian SL, Marini C, Guerrini R, Maltsev N,
Shaffer LG, Dobyns WB. 2011. Copy number variants and infantile
spasms: Evidence for abnormalities in ventral forebrain development
and pathways of synaptic function. Eur J Hum Genet 19:12381245.
Pal DK, Pong AW, Chung WK. 2010. Genetic evaluation and counseling for
epilepsy. Nat Rev Neurol 6:445453.
Palumbo O, Palumbo P, Palladino T, Stallone R, Zelante L, Carella M. 2012.
A novel deletion in 2q24.1q24.2 in a girl with mental retardation and
generalized hypotonia: A case report. Mol Cytogenet 5:1.
Patel C, Cooper-Charles L, McMullan DJ, Walker JM, Davison V, Morton J.
2011. Translocation breakpoint at 7q31 associated with tics: Further
evidence for IMMP2L as a candidate gene for Tourette syndrome. Eur J
Hum Genet 19:634639.
Petek E, Windpassinger C, Vincent JB, Cheung J, Boright AP, Scherer SW,
Kroisel PM, Wagner K. 2001. Disruption of a novel gene (IMMP2L)bya
breakpoint in 7q31 associated with Tourette syndrome. Am J Hum Genet
Ramalingam A, Zhou XG, Fiedler SD, Brawner SJ, Joyce JM, Liu HY, Yu S.
2011. 16p13.11 duplication is a risk factor for a wide spectrum of
neuropsychiatric disorders. J Hum Genet 56:541544.
Ramocki MB, Bartnik M, Szafranski P, Kolodziejska KE, Xia Z, Bravo J,
Miller GS, Rodriguez DL, Williams CA, Bader PI, Szczepanik E, Mazurc-
zak T, Antczak-Marach D, Coldwell JG, Akman CI, McAlmon K, Cohen
MP, McGrath J, Roeder E, Mueller J, Kang SH, Bacino CA, Patel A, Bocian
E, Shaw CA, Cheung SW, Stankiewicz P. 2010. Recurrent distal 7q11.23
deletion including HIP1 and YWHAG identified in patients with intel-
lectual disabilities, epilepsy, and neurobehavioral problems. Am J Hum
Genet 87:857865.
Rosenfeld JA, Crolla JA, Tomkins S, Bader P, Morrow B, Gorski J, Troxell R,
Forster-Gibson C, Cilliers D, Hislop RG, Lamb A, Torchia B, Ballif BC,
Shaffer LG. 2010. Refinement of causative genes in monosomy 1p36
through clinical and molecular cytogenetic characterization of small
interstitial deletions. Am J Med Genet A 152A:19511959.
Ryan AK, Goodship JA, Wilson DI, Philip N, Levy A, Seidel H, Schuffen-
hauer S, Oechsler H, Belohradsky B, Prieur M, Aurias A, Raymond FL,
Clayton-Smith J, Hatchwell E, McKeown C, Beemer FA, Dallapiccola B,
Novelli G, Hurst JA, Ignatius J, Green AJ, Winter RM, Brueton L,
Brondum-Nielsen K, Scambler PJ, et al. 1997. Spectrum of clinical
features associated with interstitial chromosome 22q11 deletions: A
European collaborative study. J Med Genet 34: 798804.
Saitsu H, Kato M, Mizuguchi T, Hamada K, Osaka H, Tohyama J, Uruno K,
Kumada S, Nishiyama K, Nishimura A, Okada I, Yoshimura Y, Hirai S,
Kumada T, Hayasaka K, Fukuda A, Ogata K, Matsumoto N. 2008. De
novo mutations in the gene encoding STXBP1 (MUNC18-1) cause early
infantile epileptic encephalopathy. Nat Genet 40:782788.
Sanchez-Carpintero Abad R, Sanmarti Vilaplana FX, Serratosa Fernandez
JM. 2007. Genetic causes of epilepsy. Neurologist 13:S47S51.
Shaikh TH, Gai X, Perin JC, Glessner JT, Xie H, Murphy K, O’Hara R,
Casalunovo T, Conlin LK, D’Arcy M, Frackelton EC, Geiger EA, Halde-
man-Englert C, Imielinski M, Kim CE, Medne L, Annaiah K, Bradfield JP,
Dabaghyan E, Eckert A, Onyiah CC, Ostapenko S, Otieno FG, Santa E,
Shaner JL, Skraban R, Smith RM, Elia J, Goldmuntz E, Spinner NB,
Zackai EH, Chiavacci RM, Grundmeier R, Rappaport EF, Grant SF,
White PS, Hakonarson H. 2009. High-resolution mapping and analysis
of copy number variations in the human genome: A data resource for
clinical and research applications. Genome Res 19:16821690.
Shinawi M, Cheung SW. 2008. The array CGH and its clinical applications.
Drug Discov Today 13:760770.
Shinawi M, Liu P, Kang SH, Shen J, Belmont JW, Scott DA, Probst FJ,
Craigen WJ, Graham BH, Pursley A, Clark G, Lee J, Proud M, Stocco A,
Rodriguez DL, Kozel BA, Sparagana S, Roeder ER, McGrew SG, Kurc-
zynski TW, Allison LJ, Amato S, Savage S, Patel A, Stankiewicz P, Beaudet
AL, Cheung SW, Lupski JR. 2010. Recurrent reciprocal 16p11.2 rear-
rangements associated with global developmental delay, behavioural
problems, dysmorphism, epilepsy, and abnormal head size. J Med Genet
Shinawi M, Schaaf CP, Bhatt SS, Xia Z, Patel A, Cheung SW, Lanpher B,
Nagl S, Herding HS, Nevinny-Stickel C, Immken LL, Patel GS, German
JR, Beaudet AL, Stankiewicz P. 2009. A small recurrent deletion within
15q13.3 is associated with a range of neurodevelopmental phenotypes.
Nat Genet 41:12691271.
Singh R, Gardner RJ, Crossland KM, Scheffer IE, Berkovic SF. 2002.
Chromosomal abnormalities and epilepsy: A review for clinicians and
gene hunters. Epilepsia 43:127140.
Sisodiya SM, Mefford HC. 2011. Genetic contribution to common epi-
lepsies. Curr Opin Neurol 24:140145.
Stankiewicz P, Beaudet AL. 2007. Use of array CGH in the evaluation of
dysmorphology, malformations, developmental delay, and idiopathic
mental retardation. Curr Opin Genet Dev 17:182192.
Stankiewicz P, Kulkarni S, Dharmadhikari AV, Sampath S, Bhatt SS, Shaikh
TH, Xia Z, Pursley AN, Cooper ML, Shinawi M, Paciorkowski AR,
Grange DK, Noetzel MJ, Saunders S, Simons P, Summar M, Lee B, Scaglia
F, Fellmann F, Martinet D, Beckmann JS, Asamoah A, Platky K, Sparks S,
Martin AS, Madan-Khetarpal S, Hoover J, Medne L, Bonnemann CG,
Moeschler JB, Vallee SE, Parikh S, Irwin P, Dalzell VP, Smith WE, Banks
VC, Flannery DB, Lovell CM, Bellus GA, Golden-Grant K, Gorski JL,
Kussmann JL, McGregor TL, Hamid R, Pfotenhauer J, Ballif BC, Shaw
CA, Kang SH, Bacino CA, Patel A, Rosenfeld JA, Cheung SW, Shaffer LG.
2012. Recurrent deletions and reciprocal duplications of 10q11.21q11.23
including CHAT and SLC18A3 are likely mediated by complex low-copy
repeats. Hum Mutat 33:165179.
Striano P, Paravidino R, Sicca F, Chiurazzi P, Gimelli S, Coppola A,
Robbiano A, Traverso M, Pintaudi M, Giovannini S, Operto F, Vigliano
P, Granata T, Coppola G, Romeo A, Specchio N, Giordano L, Osborne
LR, Gimelli G, Minetti C, Zara F. 2011. West syndrome associated with
14q12 duplications harboring FOXG1. Neurology 76:16001602.
Suzuki H, Katayama K, Takenaka M, Amakasu K, Saito K, Suzuki K. 2009. A
spontaneous mutation of the Wwox gene and audiogenic seizures in rats
with lethal dwarfism and epilepsy. Genes Brain Behav 8:650660.
Szafranski P, Schaaf CP, Person RE, Gibson IB, Xia Z, Mahadevan S,
Wiszniewska J, Bacino CA, Lalani S, Potocki L, Kang SH, Patel A, Cheung
SW, Probst FJ, Graham BH, Shinawi M, Beaudet AL, Stankiewicz P. 2010.
Structures and molecular mechanisms for common 15q13.3 microdu-
plications involving CHRNA7: Benign or pathological. Hum Mutat
Tohyama J, Yamamoto T, Hosoki K, Nagasaki K, Akasaka N, Ohashi T,
Kobayashi Y, Saitoh S. 2011. West syndrome associated with mosaic
duplication of FOXG1 in a patient with maternal uniparental disomy of
chromosome 14. Am J Med Genet A 155A:25842588.
Turnbull J, Lohi H, Kearney JA, Rouleau GA, Delgado-Escueta AV, Meisler
MH, Cossette P, Minassian BA. 2005. Sacred disease secrets revealed: The
genetics of human epilepsy. Hum Mol Genet 14:Spec No. 2: 24912500.
Van Esch H, Bauters M, Ignatius J, Jansen M, Raynaud M, Hollanders K,
Lugtenberg D, Bienvenu T, Jensen LR, Gecz J, Moraine C, Marynen P,
Fryns JP, Froyen G. 2005. Duplication of the MECP2 region is a frequent
cause of severe mental retardation and progressive neurological symp-
toms in males. Am J Hum Genet 77:442453.
White S, Hewitt J, Turbitt E, van der Zwan Y, Hersmus R, Drop S, Koopman
P, Harley V, Cools M, Looijenga L, Sinclair A. 2012. A
deletion within WWOX is associated with a 46,XY disorder of sex
development. Eur J Hum Genet. DOI: 10.1038/ejhg.2011.204.
Willemsen MH, Fernandez BA, Bacino CA, Gerkes E, de Brouwer AP,
Pfundt R, Sikkema-Raddatz B, Scherer SW, Marshall CR, Potocki L, van
Bokhoven H, Kleefstra T. 2010. Identification of ANKRD11 and ZNF778
as candidate genes for autism and variable cognitive impairment in the
novel 16q24.3 microdeletion syndrome. Eur J Hum Genet 18:429
Yeung A, Bruno D, Scheffer IE, Carranza D, Burgess T, Slater HR, Amor DJ.
2009. 4.45 Mb microduplication in chromosome band 14q12 including
FOXG1 in a girl with refractory epilepsy and intellectual impairment. Eur
J Med Genet 52:440442.
Zweier M, Gregor A, Zweier C, Engels H, Sticht H, Wohlleber E, Bijlsma EK,
Holder SE, Zenker M, Rossier E, Grasshoff U, Johnson DS, Robertson L,
Firth HV, Ekici AB, Reis A, Rauch A. 2010. Mutations in MEF2C from the
5q14.3q15 microdeletion syndrome region are a frequent cause of severe
mental retardation and diminish MECP2 and CDKL5 expression. Hum
Mutat 31:722733.
Article: ajmb_32081
Dear Author,
During the copyediting of your paper, the following queries arose. Please respond to these by annotating your
proofs with the necessary changes/additions.
& If you intend to annotate your proof electronically, please refer to the E-annotation guidelines.
& If you intend to annotate your proof by means of hard-copy mark-up, please refer to the proof mark-up
symbols guidelines. If manually writing corrections on your proof and returning it as a scanned pdf via email,
do not write too close to the edge of the paper. Please remember that illegible mark-ups may delay
Whether you opt for hard-copy or electronic annotation of your proofs, we recommend that you provide additional
clarification of answers to queries by entering your answers on the query sheet, in addition to the text mark-up.
Query No. Query Remark
Q1 The Journal’s copyeditors have taken care to format your authorship according to
journal style (First name, Middle Initial, Surname). In the event a formatting error
escaped their inspection, or there was insufficient information to apply journal style,
please take a moment to review all author names and sequences to ensure the accuracy
of the authorship in the published article. Please note that this information will also
affect external indexes referencing this paper (e.g., PubMed).
Q2 Please give address information for this manufacturer: city, state (if applicable), and
Q3 Please give address information for this manufacturer: city, state (if applicable), and
Q4 Please give address information for this manufacturer: city, state (if applicable), and
Q5 Please give address information for this manufacturer: city, state (if applicable), and
Q6 Gonz
alez and Bautista [2009] has been changed to Gonzalez and Bautista [2009] so
that this citation matches the Reference List. Please confirm that this is correct.
Q7 To include this citation, a written consent should be obtained from the author
concerned that he is satisfied with the content and citation of his work in this paper.
This letter should be submitted to the Wiley Editorial Office. This article will not be
published without the written consent. If the written consent has already been
submitted to the Wiley Editorial Office, kindly ignore this query.
Q8 Please provide volume number and page range.
Q9 Please provide volume number and page range.
Q10 Please provide volume number and page range.
Q11 Please check the volume number.
Q12 Please provide volume number and page range.
    • "The observation of a rare chromosomal abnormality in a patient with a rare neurological phenotype has occasionally been the vital clue leading to the identification of genes and pathways critical to brain development [5, 6]. A limited number of previous genome-wide CNV studies have focused on patients with both epilepsy and ID [7][8][9][10]. We set out to investigate the rare CNVs present in a series of 80 patients with ID/developmental delay (DD) and childhood-onset epilepsy. "
    [Show abstract] [Hide abstract] ABSTRACT: Background Copy number variants (CNVs) have been linked to neurodevelopmental disorders such as intellectual disability (ID), autism, epilepsy and psychiatric disease. There are few studies of CNVs in patients with both ID and epilepsy. Methods We evaluated the range of rare CNVs found in 80 Welsh patients with ID or developmental delay (DD), and childhood-onset epilepsy. We performed molecular cytogenetic testing by single nucleotide polymorphism array or microarray-based comparative genome hybridisation. Results 8.8 % (7/80) of the patients had at least one rare CNVs that was considered to be pathogenic or likely pathogenic. The CNVs involved known disease genes (EHMT1, MBD5 and SCN1A) and imbalances in genomic regions associated with neurodevelopmental disorders (16p11.2, 16p13.11 and 2q13). Prompted by the observation of two deletions disrupting SCN1A we undertook further testing of this gene in selected patients. This led to the identification of four pathogenic SCN1A mutations in our cohort. Conclusions We identified five rare de novo deletions and confirmed the clinical utility of array analysis in patients with ID/DD and childhood-onset epilepsy. This report adds to our clinical understanding of these rare genomic disorders and highlights SCN1A mutations as a cause of ID and epilepsy, which can easily be overlooked in adults. Electronic supplementary material The online version of this article (doi:10.1186/s12881-016-0294-2) contains supplementary material, which is available to authorized users.
    Full-text · Article · Dec 2016
    • "We performed array CGH studies in 517 patients with various types of epilepsy (primarily generalized); ~5 % of patients carried a non-recurrent CNV that affected at least one gene and was not seen in controls [33]. In a study of 102 patients with epilepsy with or without other neurodevelopmental abnormalities, 23/102 individuals had at least one non-polymorphic CNV [34]. Investigation of patients with epileptic encephalopathy syndromes also confirms the role of non-recurrent CNVs in severe epilepsies [35•]. "
    [Show abstract] [Hide abstract] ABSTRACT: Copy number variants (CNVs) are deletions or duplications of DNA. CNVs have been increasingly recognized as an important source of both normal genetic variation and pathogenic mutation. Technologies for genome-wide discovery of CNVs facilitate studies of large cohorts of patients and controls to identify CNVs that cause increased risk for disease. Over the past 5 years, studies of patients with epilepsy confirm that both recurrent and non-recurrent CNVs are an important source of mutation for patients with various forms of epilepsy. Here, we will review the latest findings and explore the clinical implications.
    Article · Sep 2014
    • "Rare copy number variants (CNV) have been implicated in the pathogenesis of many neuropsychiatric diseases despite the appreciation of the abundance of common CNVs in normal individuals [9,10]. Several studies have elucidated the causative role of CNV in DD/ID, ASD [11], congenital heart diseases [12], epilepsy [13], and congenital kidney malformation [14]. However, these studies also illustrated the phenotypic heterogeneity associated with a particular CNV. "
    [Show abstract] [Hide abstract] ABSTRACT: Background Chromosomal microarray (CMA) is currently the first-tier genetic test for patients with idiopathic neuropsychiatric diseases in many countries. Its improved diagnostic yield over karyotyping and other molecular testing facilitates the identification of the underlying causes of neuropsychiatric diseases. In this study, we applied oligonucleotide array comparative genomic hybridization as the molecular genetic test in a Chinese cohort of children with DD/ID, autism or MCA. Results CMA identified 7 clinically significant microduplications and 17 microdeletions in 19.0% (20/105) patients, with size of aberrant regions ranging from 11 kb to 10.7 Mb. Fourteen of the pathogenic copy number variant (CNV) detected corresponded to well known microdeletion or microduplication syndromes. Four overlapped with critical regions of recently identified genomic syndromes. We also identified a rare de novo 2.3 Mb deletion at 8p21.3-21.2 as a pathogenic submicroscopic CNV. We also identified two novel CNVs, one at Xq28 and the other at 12q21.31-q21.33, in two patients (1.9%) with unclear clinical significance. Overall, the detection rate of CMA is comparable to figures previously reported for accurately detect submicroscopic chromosomal imbalances and pathogenic CNVs except mosaicism, balanced translocation and inversion. Conclusions This study provided further evidence of an increased diagnostic yield of CMA and supported its use as a first line diagnostic tool for Chinese individuals with DD/ID, ASD, and MCA.
    Full-text · Article · May 2014
Show more