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
Abstract
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.

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RESEARCH ARTICLE
Application
Q1
of Array Comparative Genomic
Hybridization in 102 Patients With Epilepsy and
Additional Neurodevelopmental Disorders
Magdalena Bartnik,
1
El
_
zbieta Szczepanik,
2
Katarzyna Derwi
nska,
1
Barbara Wis
´
niowiecka-Kowalnik,
1
Tomasz Gambin,
3
Maciej Sykulski,
4
Kamila Ziemkiewicz,
1
Marta Ke˛dzior,
1
Monika Gos,
1
Dorota Hoffman-Zacharska,
1
Tomasz Mazurczak,
2
Anetta Jeziorek,
2
Dorota Antczak-Marach,
2
Mariola Rudzka-Dybała,
2
Hanna Mazurkiewicz,
2
Alicja Goszcza
nska-Ciuchta,
2
Zofia Zalewska-Miszkurka,
2
Iwona Terczy
nska,
2
Małgorzata Sobierajewicz,
5
Chad A. Shaw,
6
Anna Gambin,
4,7
Hanna Mierzewska,
2
Tadeusz Mazurczak,
1
Ewa Obersztyn,
1
Ewa Bocian,
1
and Paweł Stankiewicz
1,6
*
1
Department of Medical Genetics, Institute of Mother and Child, Warsaw, Poland
2
Clinic of Neurology of Children and Adolescents, Institute of Mother and Child, Warsaw, Poland
3
Institute of Computer Science, Warsaw University of Technology, Warsaw, Poland
4
Institute of Informatics, Univer sity of Warsaw, Warsaw, Poland
5
Child Neurology Outpatient Clinic, Leszno, Poland
6
Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, Texas
7
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,
Wis
´
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
nska-
Ciuchta A, Zalewska-Miszkurka Z,
Terczy
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
disorders.
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
Science.
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: pawels@bcm.edu
Article first published online in Wiley Online Library
(wileyonlinelibrary.com): 00 Month 2012
DOI 10.1002/ajmg.b.32081
2012 Wiley Periodicals, Inc. 1
AJMB-12-0031:R2ð32081Þ
Neuropsychiatric Genetics
Key words: seizures; array CGH; copy-number variants;
KCNJ3, WWOX; CDH15; IMMP2L
INTRODUCTION
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.
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
epilepsy.
MATERIALS AND METHODS
Patients
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.
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
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
Q2
, LLC). To characterize
the breakpoint in the CDKL5 gene, PCR reaction was performed
with Expand Long Template PCR System (Roche
Q3
Diagnostics)
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
Q4
) was subjected to direct sequencing
reaction (BigDye Terminator v.3.1 Cycle Sequencing Kit, Life
Technologies
Q5
) 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.
RESULTS
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).
DISCUSSION
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
BARTNIK ET AL. 3
TABLE I. Clinically Relevant CNVs Known to Be Pathogenic for Epilepsy
Pt Sex
Age
at onset aCGH results
Best
candidate
genes
Size
(Mb) Verification Inheritance
Parental
phenotype
Seizure types/epilepsy
syndrome
Cognitive function,
other features References
1 F 2 months arr Xp22.13(18,492,235
18,492,821) 12
CDKL5
0.001
MLPA De novo Normal Refractory epilepsy with
different types of
seizures (GTCS, tonic,
myoclonic)
Profound mental
retardation; Rett-like
syndrome;
Angelman-like
phenotype,
microcephaly
Bartnik et al. [2011]
2 F 5 months arr Xp22.13(18,542,246
18,553,009) 1
CDKL5
0.01
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
HIP1
,
YWHAG
1.658
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
TBX1
2.397
FISH Mat Mild ID, VCFS JME Normal
Q6
IQ Gonzalez and Bautista
[2009]
5 F 3 years arr 15q11.2(20,393,584
20,613,447) 1
NIPA1
0.220
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
FOXG1
8.366
karyotype De novo Normal West syndrome evolving
into Lennox-Gastaut
syndrome; refractory
Profound DD Brunetti-Pierri et al.
[2011]
7* M 6 months arr 1p36.32p36.21(4,600,008
13,110,103) 1
KCNAB2
8.510
FISH, karyotype De novo Normal Refractory epilepsy with
tonicclonic seizures
(mainly during
infections). Status
epilepticus
Moderate DD,
plagiocephaly,
dysmorphy
Bahi-Buisson et al.
[2008]
8** M 1 months Xq28(149,557,875
154,533,675) 2
MECP2
4.976
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
16p13.11p12.3(15,429,214
17,963,057) 1
ND1E
,
ABCC
,
ABCC6
2.534
FISH Pat Normal, father
graduated only
primary school
West syndrome evolving
into epilepsy with tonic
clonic seizures
Dysmorphic, profound DD,
microcephaly
de Kovel et al., [2010]
10 F 17 years arr 16p11.2(29,532,264
30,104.842) 3
MAPK3
0.573
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
NPHP1
deletion at 2q13 and a 284 kb duplication at Xq28 (148,240,624-148,524,326), harboring the
IDS
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
Q7
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.
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 chr2:155.526.470163.058.894, 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
Age
at onset aCGH results
Candidate
genes Size (Mb) Verification Inheritance
Parental
phenotype
Seizure types/epi-
lepsy syndrome
Cognitive function,
other problems
11 M 1 months arr
2q24.1q24.3
(154,710,064-
165,068,041) 1
KCNJ3
10.35 Karyotype Not mat Unknown West syndrome;
refractory
Profound DD, EPH
gestosis during
pregnancy,
premature delivery
12 M 2 years arr
9q21.13(73,931,220-
76,496,752) 1
TMC1
,
TRPM6
2.57 FISH De novo Mother healthy,
father has
Asperger
syndrome
Epilepsy with eyelid
myoclonia and GTCS
Autism
DD, developmental delay; GTCS, generalized tonicclonic seizures; mat, maternal.
6 AMERICAN JOURNAL OF MEDICAL GENETICS PART B
TABLE III. CNVs of Unknown Clinical Significance
Pt Sex
Age
at onset aCGH results Selected genes
Size
(Mb) Verification Inheritance Parental phenotype
Seizure types/epilepsy
syndrome
Cognitive function,
other problems
8 M 1 months arr Xq28(148,240,624-
148,524,326) 2
IDS
0.284
Mat Normal West syndrome evolving
into epilepsy with focal
seizures with hypsar-
rythmia in EEG
Dysmorphic, profound
DD
13 M 16 years arr 2p12(78,311,526-78,879,
196) 1
Brain-expressed
mRNA BC024248
0.568
FISH Mat Unknown JME with GTCS Normal IQ
14 F 9 months arr 16q24.3(87,692,754-
87,789,405) 1
CDH15
0.097
Pat Normal Refractory epilepsy with
tonic seizures and CSWS
Profound DD, Rett-like
syndrome, autistic
features
15 F 6 months arr 7q31.1(110,627,069-
110,978,974) 1
IMMP2L
0.352
FISH Not mat Mother normal, father
deceased
West syndrome evolving
into refractory epilepsy
with polymorphic
seizures (myoclonic,
tonic, unclassified)
Profound DD, cerebral
palsy
16 M 4 years arr 16q23.1(76,974,912-
77,669,115) 1
WWOX
0.694
Unknown Mother normal, father
had schizophrenia
and committed
suicide; father’s
brother also
committed suicide
Epilepsy with absences
and GTCS
Autism
17 F 7 years arr 18q23(74,856,013-
75,390,868) 3
ATP9B
,
NFATC1
0.535
Mat Normal Epilepsy with GTCS and
CSWS
Normal IQ, visual
spatialagnosia
18 F 8 months arr 2q14.3(124,747,254-
125,784,880) 3
CNTNAP5
1.038
Mat Normal West syndrome/JME Normal IQ
19 F 16 years arr
2q23.2q23.3(149,772,147-
150,411,844) 3
LYPD6
,
MMADHC
0.640
Pat Normal JME Normal IQ
20 F 14 years arr Xp22.31 (7,801,120-
8,159,541) 3
VCX2
0.358
Pat Normal JME Normal IQ
21 F 9 years arr 16p13.11(15,824,601-
16,199,695) 3
ABCC1
,
ABCC6
0.375
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
NDE1
,
ABCC1
,
ABCC6
0.790
Unknown Unknown JME Normal IQ
23 F 3 years arr 15q13.3(30,083,430-
30,191,648) 3
CHRNA7
0.108
Mat Unknown JME (with absences with
eyelid myoclonia and
GTCS)
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.
BARTNIK ET AL. 7
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.,
2010].
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
consequences.
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.
ACKNOWLEDGMENTS
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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    • "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
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