Application of array comparative genomic hybridization in 102 patients with epilepsy and additional neurodevelopmental disorders
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
Chad A. Shaw,
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 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 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
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 9999:1–11.
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.
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: email@example.com
Article ﬁrst published online in Wiley Online Library
(wileyonlinelibrary.com): 00 Month 2012
2012 Wiley Periodicals, Inc. 1
Key words: seizures; array CGH; copy-number variants;
KCNJ3, WWOX; CDH15; IMMP2L
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 100–1,000 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 10–20%
[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 (Wolf–Hirschhorn
syndrome), ring chromosome 14, deletion 15q11.2q12
(Angelman syndrome), inv dup (15) chromosome, deletion
17p13.3 (Miller–Dieker 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
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
MATERIALS AND METHODS
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
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
2 AMERICAN JOURNAL OF MEDICAL GENETICS PART B
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://
www.bcm.edu/geneticlabs/cma/tables.html) 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
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 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
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. 
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.  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.
 also identiﬁed an 3 Mb duplication of the 14q12 region,
including FOXG1 in a child enrolled as a control subject in the
BARTNIK ET AL. 3
TABLE I. Clinically Relevant CNVs Known to Be Pathogenic for Epilepsy
at onset aCGH results
(Mb) Veriﬁcation Inheritance
other features References
1 F 2 months arr Xp22.13(18,492,235
MLPA De novo Normal Refractory epilepsy with
different types of
seizures (GTCS, tonic,
Bartnik et al. 
2 F 5 months arr Xp22.13(18,542,246
MLPA, PCR De novo Normal Refractory epilepsy with
different types of
seizures (focal, tonic)
Erez et al. 
3 M 8 months arr 7q11.23(75,003,415
FISH Mat Mild ID Epilepsy with GTCS/JME DD Ramocki et al. 
4 M 13 years arr 22q11.21(17,364,458
FISH Mat Mild ID, VCFS JME Normal
IQ Gonzalez and Bautista
5 F 3 years arr 15q11.2(20,393,584
FISH Pat Unknown JME (with myoclonus and
GTCS, and absences
with eyelid myoclonus)
Normal IQ, headache de Kovel et al. 
6 M 6 months arr 14q11.2q12(22,378,936
karyotype De novo Normal West syndrome evolving
Profound DD Brunetti-Pierri et al.
7* M 6 months arr 1p36.32p36.21(4,600,008
FISH, karyotype De novo Normal Refractory epilepsy with
Bahi-Buisson et al.
8** M 1 months Xq28(149,557,875
FISH Mat Normal West syndrome evolving
into epilepsy with focal
seizures with hypsar-
rythmia in EEG
Dysmorphic, profound DD Van Esch et al. 
9 M 6 months arr
FISH Pat Normal, father
West syndrome evolving
into epilepsy with tonic
Dysmorphic, profound DD,
de Kovel et al., 
10 F 17 years arr 16p11.2(29,532,264
— Not mat Normal JME Normal IQ Shinawi et al. 
DD, developmental delay; GTCS, generalized tonic–clonic 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
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
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. , Magri et al. , 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.
BARTNIK ET AL. 5
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 chr2:155.526.470–163.058.894, harboring SLC4A10 and
truncating KCNH7, but likely not involving KCNJ3 (max coor-
dinates: chr2:155.413.315–163.101.007), 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.,  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.,  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
chr2:156539025–158815118, not including KCNJ3 and KCNH7,
did not manifest seizures whereas patient DECIPHER 253681 with
an overlapping chr2:152182099–159245370, 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 0.7–1.1 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 14–16 and 21–23, there are some literature
data to suggest that they may be responsible for the observed
TABLE II. Novel CNVs Potentially Causative for Epilepsy
at onset aCGH results
genes Size (Mb) Veriﬁcation Inheritance
11 M 1 months arr
10.35 Karyotype Not mat Unknown West syndrome;
Profound DD, EPH
12 M 2 years arr
2.57 FISH De novo Mother healthy,
Epilepsy with eyelid
myoclonia and GTCS
DD, developmental delay; GTCS, generalized tonic– clonic seizures; mat, maternal.
6 AMERICAN JOURNAL OF MEDICAL GENETICS PART B
TABLE III. CNVs of Unknown Clinical Signiﬁcance
at onset aCGH results Selected genes
(Mb) Veriﬁcation Inheritance Parental phenotype
8 M 1 months arr Xq28(148,240,624-
— Mat Normal West syndrome evolving
into epilepsy with focal
seizures with hypsar-
rythmia in EEG
13 M 16 years arr 2p12(78,311,526-78,879,
FISH Mat Unknown JME with GTCS Normal IQ
14 F 9 months arr 16q24.3(87,692,754-
— Pat Normal Refractory epilepsy with
tonic seizures and CSWS
Profound DD, Rett-like
15 F 6 months arr 7q31.1(110,627,069-
FISH Not mat Mother normal, father
West syndrome evolving
into refractory epilepsy
Profound DD, cerebral
16 M 4 years arr 16q23.1(76,974,912-
— Unknown Mother normal, father
Epilepsy with absences
17 F 7 years arr 18q23(74,856,013-
— Mat Normal Epilepsy with GTCS and
Normal IQ, visual
18 F 8 months arr 2q14.3(124,747,254-
— Mat Normal West syndrome/JME Normal IQ
19 F 16 years arr
— Pat Normal JME Normal IQ
20 F 14 years arr Xp22.31 (7,801,120-
— Pat Normal JME Normal IQ
21 F 9 years arr 16p13.11(15,824,601-
— Pat Father and brother
JME Normal IQ
22 F 16 years arr 16p13.11(15,425,965-
— Unknown Unknown JME Normal IQ
23 F 3 years arr 15q13.3(30,083,430-
— Mat Unknown JME (with absences with
eyelid myoclonia and
CSWS, continuous spikes and waves during slow wave sleep; DD, developmental delay; GTCS, generalized tonic–clonic seizures; JME, juvenile myoclonic epilepsy; mat, maternal; pat, paternal.
BARTNIK ET AL. 7
phenotypic abnormalities. In cases 13, 17–20, 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 17–20).
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 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.
 bioinformatically predicted WWOX to be haploinsufﬁcient
in humans. Recently, White et al.  reported a maternally
inherited exon 6– 8 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.  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.
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BARTNIK ET AL. 11
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