ASSESSMENT OF THE ROLE OF COPYͳNUMBER VARIANTS IN

© IMiD, Wydawnictwo Aluna
Medycyna Wieku Rozwojowego, 2012, XVI, 3
W Z K Z z' / E > E / W K ' > K t Katarzyna Derwińska1, Magdalena Bartnik1, Barbara Wiśniowiecka-Kowalnik1,
Mateusz Jagła2, Andrzej Rudziński2, Jacek J. Pietrzyk2, Wanda Kawalec3, Lidia Ziółkowska3,
Anna Kutkowska-Kaźmierczak1, Tomasz Gambin4, Maciej Sykulski5, Chad A. Shaw6,
Anna Gambin5,7, Tadeusz Mazurczak1, Ewa Obersztyn1, Ewa Bocian1, Paweł Stankiewicz1,6
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1Department of Medical Genetics
Head: Prof. E. Bocian
Institute of Mother and Child, Warsaw, Poland
Director: T. Maciejewski, MD, PhD
2Department of Pediatrics
Head: Prof. J.J. Pietrzyk
Jagiellonian University, Collegium Medicum, Cracow, Poland
Rector: Prof. K. Musioł
3Department of Pediatric Cardiology
Head: Prof. W. Kawalec
Children's Memorial Health Institute, Warsaw, Poland
Director: M. Piróg, MD, PhD
4Institute of Computer Science
Head: Prof. K. Walczak
Warsaw University of Technology, Warsaw, Poland
Rector: Prof. J. Kurnik
5Institute of Informatics
Head: Prof. K. Diks
University of Warsaw, Warsaw, Poland
Rector: Prof. K.Chałasińska-Macukow
6Department of Molecular & Human Genetics
Head: Prof. A.L. Beaudet
Baylor College of Medicine, Houston, TX, USA
President: Prof. P. Klotman
7Bioinformatics Laboratory
Head: Prof. B. Lesyng
Mossakowski Medical Research Centre
Polish Academy of Sciences, Warsaw, Poland
Director: Prof. A.W. Lipkowski
Abstract
Background: Congenital heart defects are the most common group of major birth anomalies and one
of the leading causes of infant deaths. Mendelian and chromosomal syndromes account for about 20%
of congenital heart defects and in some cases are associated with other malformations, intellectual disability, and/or dysmorphic features. The remarkable conservation of genetic pathways regulating heart
*The work was supported by grant R13-0005-04/2008 from the Polish Ministry of Science and Higher Education and partially by grant
3 PO5E 142 2 from the Ministry of Science and Information Technology. MB is supported by the START Fellowship from the Foundation
of Polish Science.
176
Katarzyna Derwińska i wsp.
development in animals suggests that genetic factors can be responsible for a significantly higher percentage of cases.
The aim: Assessment of the role of CNVs in the etiology of congenital heart defects using microarray studies.
Material and methods: Genome-wide array comparative genomic hybridization, targeting genes known to
play an important role in heart development or responsible for abnormal cardiac phenotype was used in the
study on 150 patients. In addition, we have used multiplex ligation-dependent probe amplification specific for
chromosome 22q11.2 region.
Results: We have identified 21 copy-number variants, including 13 known causative recurrent rearrangements
(12 deletions 22q11.2 and one deletion 7q11.23), three potentially pathogenic duplications (5q14.2, 15q13.3,
and 22q11.2), and five variants likely benign for cardiac anomalies. We suggest that abnormal copy-number of
the ARRDC3 and KLF13 genes can be responsible for heart defects.
Conclusions: Our study demonstrates that array comparative genomic hybridization enables detection of clinically significant chromosomal imbalances in patients with congenital heart defects.
Key words: copy-number variants, cardiac defects, array comparative genomic hybridization
Streszczenie
Wprowadzenie: Wady serca są najczęstszą grupą wad wrodzonych oraz ważną przyczyną zgonów niemowląt.
Towarzyszą im często inne wady rozwojowe, niepełnosprawność intelektualna i/lub cechy dysmorfii. Podłoże
genetyczne (choroby o mendlowskim toku dziedziczenia, zespoły chromosomowe) wad serca stwierdzano dotychczas w około 20% przypadków. Wysoki stopień ewolucyjnego zakonserwowania szlaków molekularnych
odpowiedzialnych za rozwój serca u zwierząt sugeruje jednak, że czynniki genetyczne mogą być odpowiedzialne za znacznie większy odsetek wrodzonych wad serca niż dotychczas przypuszczano.
Cel pracy: Mikromacierzowa analiza genomu u 150 pacjentów z wrodzonymi wadami serca.
Materiał i metody: W badaniach wykorzystano metodę porównawczej hybrydyzacji genomowej do
mikromacierzy oraz multipleksową amplifikację sond zależną od ligacji dla regionu 22q11.2. Stosowano
całogenomową mikromacierz oligonukleotydową zawierającą znane geny pełniące ważną rolę w rozwoju
mięśnia sercowego oraz geny odpowiedzialne za powstawanie wad serca. Przebadano 150 pacjentów.
Wyniki: Stwierdzono 21 rearanżacji genomowych, w tym 13 znanych patogennych nieprawidłowości (12 delecji 22q11.2 oraz jedną delecję 7q11.23), trzy potencjalnie patogenne duplikacje (5q14.2, 15q13.3 i 22q11.2) oraz
pięć prawdopodobnie łagodnych wariantów. Sugerujemy, że zmiana liczby kopii fragmentów DNA obejmujących geny ARRDC3 i KLF13 może być odpowiedzialna za wady serca stwierdzane u pacjentów.
Wnioski: Zastosowanie porównawczej hybrydyzacji genomowej do mikromacierzy umożliwia identyfikację
klinicznie istotnych niezrównoważeń genomu u pacjentów z wrodzonymi wadami serca.
Słowa kluczowe: rearanżacje genomowe, wrodzone wady serca, porównawcza hybrydyzacja genomowa
do mikromacierzy
D͘t/<hZKtK:͕͘ϮϬϭϮ͕ys/͕ϯ͕ϭϳϱͳϭϴϮ
INTRODUTION
Congenital heart defects (CHDs) are the most common
major birth anomalies occurring with a prevalence of 4-8
per 1000 live births (1, 2). Despite therapeutic advances,
CHDs continue to be responsible for a high proportion of
children's morbidity and mortality. CHDs are thought to be a
multifactorial disease with the majority of cases being isolated
defects; 25% of CHDs are associated with other congenital
anomalies, constituting part of a specific malformation pattern
or a genetic syndrome (3). A small percentage of CHDs result
from environmental risk factors such as maternal diseases
(e.g. rubella, phenyloketonuria) or exposure to teratogenic
agents (e.g. retinoic acid, lithium, or antiepileptic drugs) (4).
One of the objectives that have to be reached in order to
enhance the understanding of CHDs is the identification of the
molecular components responsible for cardiac development.
The most common form (almost 50%) of CHDs are defects
of cardiac septation, including the atrial septal defect (ASD),
ventricular septal defect (VSD), or atrioventricular septal
defect found mainly in patients with nonsyndromic (isolated) CHDs but also with Mendelian syndromes. Molecular
bases of cardiac septation defects are mostly associated with
transcription factor genes, including TBX5 (Holt-Oram syndrome, OMIM 142900), NKX2.5 (ventricular septal defect 3,
OMIM 614432), GATA4 (ASD and VSD), SALL4 (Okihiro
syndrome, OMIM 607323), SALL1 (Townes-Brocks syndrome, OMIM 107480), and PTPN11 (Noonan syndrome,
OMIM 163950). Other genes, e.g. MYH6 (OMIM 160710)
and CITED2 (602937), cause only isolated septal defects when
mutated. The second group, accounting for about 30% of
CHDs, are conotruncal and ventricular outflow tract defects
mostly caused by mutations or deletion of TBX1 (OMIM
602054) and JAG1 (OMIM 601920).
In addition to the abovementioned single gene defects
(5), the most common chromosome aberrations associated
with CHD detected by conventional karyotyping, trisomies
13, 18, and 21, deletion 5p15 (Cri-du-chat syndrome), and
Assessment of the role of copy-number variants in 150 patients
chromosome abnormalities in patients with Turner syndrome, account for about 10% of all CHDs (6). Further,
many chromosomal submicroscopic rearrangements, such
as microdeletions or microduplications are often found to be
responsible for heart defects, e.g. recurrent deletions of 22q11.2
[DiGeorge syndrome (DGS)/Velocardiofacial syndrome
(VCFS), OMIM 188400/192430] and 7q11.23 (WilliamsBeuren syndrome, WBS; OMIM 194050).
Array comparative genomic hybridization (array CGH)
has proven to be a powerful tool for high-resolution
genome-wide analysis and for the detection of copy-number variants (CNVs) (7). A study of 60 patients
with congenital heart defects suggestive of chromosomal
aberrations by the whole-genome array CGH revealed
pathogenic genomic rearrangements (mostly submicroscopic) in 17% of the cases (8). Goldmuntz et al. reported
submicroscopic rearrangements in ~20% of their patients
with cardiac and other congenital anomalies (3). A study
of patients with CHDs, both isolated and with additional
abnormalities, reported by Richards et al. showed that 25%
of CHDs associated with other anomalies had abnormal
microarray findings, whereas none of the patients with
an isolated heart defect had submicroscopic imbalances
(9). Recently, Breckpot et al. showed that the frequency
of causal CNVs in nonsyndromic CHDs is much lower
than in syndromic cases (3.6% vs 19%, respectively) (10).
Lalani et al. and Soemedi et al, described several rare
CNVs in patients with cardiovascular malformations
and extracardiac abnormalities (11, 12).
We herewith present the results of the array CGH and
multiplex ligation-dependent probe amplification (MLPA)
studies in 150 patients with congenital cardiac defects.
MATERIAL AND METHODS
Human Subjects
All Caucasians 150 patients, ranging in age between
2-32 years, were referred to the Department of Medical
Genetics (DMG) at the Institute of Mother and Child
(IMC) in Warsaw, Poland. Each patient was examined
by a cardiologist and 68 of them had additional clinical
dĂďůĞ/͘ĂƌĚŝĂĐƉŚĞŶŽƚLJƉĞƐŝŶƉĂƟĞŶƚƐƐƚƵĚŝĞĚ͘
Tabela I. Rodzaje wad serca u badanych pacjentów.
ĂƌĚŝĂĐĚĞĨĞĐƚƐ
Tetralogy of Fallot
Common arterial trunk
/ŶƚĞƌƌƵƉƚĞĚĂŽƌƟĐĂƌĐŚ
KƚŚĞƌĂŶŽŵĂůŝĞƐŽĨĂŽƌƟĐĂƌĐŚ
ŽŵŵŽŶĂƌƚĞƌŝĂůƚƌƵŶŬΘ/ŶƚĞƌƌƵƉƚĞĚĂŽƌƟĐĂƌĐŚ
dŽƚĂůĂŶŽŵĂůŽƵƐƉƵůŵŽŶĂƌLJǀĞŶŽƵƐĐŽŶŶĞĐƟŽŶ
Other
dŽƚĂů
177
genetic evaluation. Informed consents approved by the
institutional review board for the Bioethics Commission
at the IMC were obtained in all cases. Patients presented
with cardiac abnormalities, such as interrupted aortic
arch type B, other anomalies of aortic arch (right aortic
arch, vascular ring), common arterial trunk, tetralogy
of Fallot, and total anomalous pulmonary venous connection, with or without other congenital malformations
(tab. I). Fifteen patients had additional developmental
delay (DD) and dysmorphic features (DF), nine patients
had DD, and 12 patients had DF.
DNA samples from 83 patients were screened by
MLPA specific for chromosome 22q11.2 (revealing six
deletions in the DGS region) and 144 samples were tested using clinical array CGH, excluding six samples, in
which the 22q11.2 deletion was found by MLPA. Twenty
nine patients had normal G-banded karyotype analysis
at the 550 band resolution.
E/ƐŽůĂƟŽŶ
DNA was extracted from whole blood using the Puregene
DNA Blood Kit (Gentra, Minneapolis, MN), according
to the manufacturer’s instructions.
Array CGH
Custom designed exon-targeted clinical array CGH was
performed using 180K V8.0 OLIGO and 180K V8.1 OLIGO
microarrays designed by Medical Genetics Laboratories
(MGL) at Baylor College of Medicine (BCM) (http://www.
bcm.edu/geneticlabs/cma/tables.html) in cooperation with
DMG at IMC and manufactured by Agilent Technology
(Santa Clara, CA). Both microarrays have genome-wide
coverage and exon-targeting for over 1800 genes, including 350 genes and candidate genes important for heart
development, with an average 4.2 oligos per exon and
intronic gaps no larger than 10 kb (13). Genomic features
of the V8 OLIGO design also include interrogation of all
known microdeletion and microduplication syndrome
regions, pericentromeric and subtelomeric regions, and
computationally predicted nonallelic homologous recombination-mediated genomic instability regions flanked
by low-copy repeats, as previously described (14).
EƵŵďĞƌŽĨƐƵďũĞĐƚƐ
122
10
ϰ
3
3
2
6
ϭϱϬ
178
Katarzyna Derwińska i wsp.
Digestion, labeling, and hybridization were performed
following the manufacturer’s instructions. Scanned
images were quantified using Agilent Feature Extraction
software (v10.0). The BCM web-based platform and a
customized 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).
To verify the rearrangements identified by array CGH,
we have used MLPA, fluorescence in situ hybridization
(FISH), or array CGH. When available, blood samples
were obtained from the patients’ parents and array CGH,
FISH or MLPA analyses were performed to investigate
CNV inheritance.
MLPA
MLPA experiments were performed according to the manufacturer’s instructions with kit P250 or P297 (MRC Holland)
in the 2720 thermal cycler (Applied Biosystems, Foster
City, CA). Kit P250 includes probes for 25 genes for the
22q11.2 deletion syndrome, Cat eye syndrome, and control
fragments for X chromosome and autosomes whereas kit
P297 (microdeletion syndromes) has ten probes specific for
the 15q13.3 deletion/duplication syndrome. Information
regarding the probe sequence and ligation sites can be
obtained at www.mlpa.com. Probes were analyzed using
the ABI3100 sequencer with the size standard GeneScan
500 Rox (Applied Biosystems). Data analysis was done
with the GeneMarker v8.1 software from Softgenetics.
FISH
Confirmatory FISH analyses were performed in phytohemagglutinin-stimulated peripheral blood lymphocytes
using standard procedures with the bacterial artificial
chromosome (BAC) clones specific for the aberrant
chromosome (15) (tab. II, III).
dĂďůĞ//͘EsƐƉŽƚĞŶƟĂůůLJĐĂƵƐĂƟǀĞĨŽƌ,Ɛ͘
dĂďĞůĂ//͘WŽƚĞŶĐũĂůŶŝĞƉĂƚŽŐĞŶŶĞƌĞĂƌĂŶǏĂĐũĞŐĞŶŽŵŽǁĞnjŝĚĞŶƚLJĮŬŽǁĂŶĞƵƉĂĐũĞŶƚſǁnjǁƌŽĚnjŽŶLJŵŝǁĂĚĂŵŝƐĞƌĐĂ͘
ŐĞ
WĂƟĞŶƚ 'ĞŶĚĞƌ ;LJĞĂƌƐͿ
Es
ŽŽƌĚŝŶĂƚĞƐ ^ŝnjĞ
;ŚŐϭϴͿ
;DďͿ
WĂƌĞŶƚĂů
ƐƚƵĚŝĞƐ
/ŶŚĞƌŝƚĂŶĐĞ
ĂŶĚŝĚĂƚĞ
ĂƌĚŝĂĐ
ŐĞŶĞƐ
ƉŚĞŶŽƚLJƉĞ
ϭϰ
Male
12
dup 22q11.21
ϭϳ͕Ϯϵϵ͕ϵϰϯͲ
Ͳϭϵ͕ϳϳϬ͕ϰϱϱ
Ϯ͘ϰϳ
Ͳ
unk
TBX1
TAC I, IAA
15
Male
ϭϰ
dup 15q13.3
Ϯϵ͕Ϭϳϵ͕ϳϲϴͲ
ͲϯϬ͕ϳϭϮ͕ϯϳϯ
1.6
MLPA
dn
KLF13
ToF, DD
16
Female
ϭϴ
ĚƵƉϱƋϭϰ͘ϯ
ϵϬ͕Ϯϲϲ͕ϲϵϭͲ
Ͳϵϭ͕ϰϭϳ͕ϳϳϲ
1.2
FISH
RP11Ͳ
ͲϭϬϯϯDϮϰ
dn
ARRDC3
ToF, mild
DD
ĚŶͲde novo; IAAͲŝŶƚĞƌƌƵƉƚĞĚĂŽƌƟĐĂƌĐŚ͖d/ͲƚƌƵŶĐƵƐĂƌƚĞƌŝŽƐƵƐĐŽŵŵƵŶŝƐ͖dŽ&ͲdĞƚƌĂůŽŐLJŽĨ&ĂůůŽƚ͖ͲĚĞǀĞůŽƉŵĞŶƚĂůĚĞůĂLJ͘
Table III. CNVs likely benign for CHDs.
dĂďĞůĂ///͘ZĞĂƌĂŶǏĂĐũĞŐĞŶŽŵŽǁĞ͕ŬƚſƌĞƉƌĂǁĚŽƉŽĚŽďŶŝĞŶŝĞƐČƉƌnjLJĐnjLJŶČǁƌŽĚnjŽŶLJĐŚǁĂĚƐĞƌĐĂ͘
ŐĞ
ŽŽƌĚŝŶĂƚĞƐ ^ŝnjĞ WĂƌĞŶƚĂů
ĂƌĚŝĂĐ
WĂƟĞŶƚ 'ĞŶĚĞƌ ;LJĞĂƌƐͿ
Es
'ĞŶĞƐ
;ŚŐϭϴͿ
;DďͿ
ƐƚƵĚŝĞƐ /ŶŚĞƌŝƚĂŶĐĞ
ƉŚĞŶŽƚLJƉĞ
ϰϬ͕Ϯϲϱ͕ϱϮϰͲ Ϭ͘Ϭϴϳ
ToF,
17
Female
12
ĚƵƉyƉϭϭ͘ϰ
Ͳ
unk
ATP6AP2
ͲϰϬ͕ϯϱϯ͕ϯϱϵ
heterotaxy
<EϮ͕
array
ToF,
&Dϭϲϱ͕
ϯϰ͕ϲϱϳ͕ϵϮϮͲ
dup
0.16
mat
ϭϴ
Male
13
CGH
<Eϭ͕ DORV type
21q22.11q22.11 Ͳϯϰ͕ϴϮϭ͕ϭϯϰ
RCAN1
array
ϭϭϴ͕ϵϰϭ͕ϰϮϬͲ 0.17
pat
FAM170A
ToF
19
Female
21
del 5q23.1
CGH
Ͳϭϭϵ͕ϭϬϴ͕ϭϲϰ
W&&Ϯ͕
E͕
^ϭ͕
ϳϳ͕ϭϬϱ͕ϵϴϮͲ
y>ϵ͕
ToF, DD
0.11
Ͳ
unk
20
Female
ϭϰ
ĚƵƉϰƋϮϭ͘ϭ
Ͳϳϳ͕Ϯϭϲ͕Ϭϰϲ
y>ϭϬ͕
y>ϭϭ͕
ART3
Zdϭϱ͕
FISH
,^ϯ^dϯϭ͕
RP11Ͳ
ϭϰ͕Ϭϳϭ͕ϭϬϲͲ
ToF
D'ϭϮϵϭϲ͕
dn
1.0
21
Female
3
del 17p12
ͲϲϰϭϮ
Ͳϭϱ͕Ϭϳϰ͕ϵϰϱ
Zdϳ͕
PMP22
ĚŶͲde novo; dŽ&ͲdĞƚƌĂůŽŐLJŽĨ&ĂůůŽƚ͖KZsͲĚŽƵďůĞŽƵƚůĞƚƌŝŐŚƚǀĞŶƚƌŝĐůĞ͖ͲĚĞǀĞůŽƉŵĞŶƚĂůĚĞůĂLJ͘
Assessment of the role of copy-number variants in 150 patients
RESULTS
Among the 150 patients tested, we have identified 21
CNVs. There were 15 known recurrent rearrangements:
12 deletions and one reciprocal duplication of the DGS/
VCFS region at 22q11.21, one deletion of the WBS chromosome region at 7q11.23, and one recurrent duplication
15q13.3 (BP4-BP5) (fig. 1, tab. II). Further, five rare CNVs
were identified: duplications on 4q21.1, 5q14.3 (fig. 1),
21q22.11q22.11, and Xp11.4 (tab. II, III) and deletion on
5q23.1 (tab. III). In addition, we have detected an incidental
deletion on 17p12 (HNPP, OMIM 162500) most likely
not responsible for the heart abnormality (tab. III). We
have classified the identified CNVs as pathogenic, potentially pathogenic (tab. II), and likely benign for CHDs
(tab. III). The sizes of these CNVs varied from 87 kb to
179
2.5 Mb and all were confirmed by MLPA, FISH, or array
CGH. In five cases, the parental samples were available;
duplication 21q22.11q22.11 and deletion 5q23.1 were
inherited from the parents not known to have cardiac
defects, whereas duplications 5q14.3 and 15q13.3 and
deletion 17p12 were de novo events.
DISCUSSION
The use of microarray technology provides an opportunity for an accurate molecular characterization and
better genotype-phenotype correlation of the identified
potentially disease-related CNVs. To date, the majority
of the patient cohorts studied by array CGH consisted
of individuals with neurodevelopmental disorders and
Fig. 1. Results of array CGH analyses ĂͿŝŶƉĂƟĞŶƚϭϱ͕ƐŚŽǁŝŶŐĂŶΕϭ͘ϲDďĚƵƉůŝĐĂƟŽŶŝŶƚŚĞϭϱƋϭϯ͘ϯƌĞŐŝŽŶĂŶĚ
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chromosome 15q13.3 and ĚͿŝŶƚŚĞĚƵƉůŝĐĂƚĞĚƌĞŐŝŽŶŝŶĐŚƌŽŵŽƐŽŵĞϱƋϭϰ͘ϯ͘ĞͿZĞƐƵůƚƐŽĨD>WĂŶĂůLJƐŝƐǁŝƚŚƚŚĞŬŝƚ
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180
Katarzyna Derwińska i wsp.
in many cases enabled identification of the causative
genes (16-18). Relatively fewer CNV studies have been
performed in patients with cardiac defects (3, 8, 19-22).
The hypothesis that rare cryptic CNVs may account for
CHDs led us to design a high-resolution whole-genome
microarray exon-targeting genes that have been associated with CHDs, in addition to other well known genetic
diseases and syndromes manifesting with DD/intellectual
disability (ID), autism, or epilepsy (13).
Our analyses revealed CNVs in 21 out of the 150 patients. Sixteen of these CNVs are pathogenic or potentially
causative for CHD, yielding a 10.7% detection rate, lower
than those presented in other studies (3, 8, 20, 22). We
believe that it is because many patients studied by others
had extracardiac abnormalities (i.e. intellectual impairment, had special education and/or three or more minor
physical anomalies) in addition to heart defects. In our
cohort, only 36 patients had DD and/or DF. Our results
are consistent with the work of Breckpot et al., who showed
much lower frequency of CNVs in nonsyndromic vs.
syndromic CHDs, respectively, 3.6% vs 19% (10).
In addition to the CNVs pathogenic for heart defects
(12 deletions 22q11.2, one duplication 22q11.2, and
one deletion 7q11.23), we have identified two potentially pathogenic CNVs. In patient 15 with ToF and
DD, we have detected a common recurrent BP4-BP5
duplication in chromosomal region 15q13.3. Patients
with this recurrent duplication harboring CHRNA7
typically present with DD (70% of cases), autism spectrum disorder, attention deficit hyperactivity disorder,
anxiety disorder, and mood disorder. Moreover, cognitive
impairment was reported, varying from moderate ID
to normal IQ with learning disability (23). However,
only one of these patients was reported to have a heart defect (hypoplastic left heart and coarctation of
the aorta) (24). Interestingly, 7-18% of patients with
the 15q13.3 deletion syndrome (OMIM 612001) had
heart defects (25-27). Van Bon et al. have proposed that
the KLF13 (Kruppel-like transcription factor 13) gene
within the 15q13.3 region, encoding a member of the
Kruppel-like family of zinc finger proteins, is causative
for heart defects in these patients (26). Genetic studies
in the Xenopus embryos demonstrated a requirement
for klf13 in cardiac progenitor cell proliferation and
heart morphogenesis (28). KLF13 was also presented
as being an important component of the transcription
network required for heart development and mutations
in KLF13 were proposed to be causative for congenital
human heart disease (29). We suggest that increased
dosage of KLF13 can lead to heart defects.
In patient 16 with mild DD and ToF, we have found
a de novo ~1.2 Mb duplication 5q14.3, harboring the
entire ARRDC3 gene and a downstream portion of the
GPR98 gene. ARRDC3 was identified in genome-wide
association studies in patients with hypertension and
artherosclerosis. It was suggested that it acts through
modification of inflammation and the innate immunity
system in vascular cells (30, 31). We hypothesize that
over-expression of ARRDC3 could be responsible for
cardiac defects, especially that it was not reported in the
databases of genomic variants (32-34).
Duplication in Xp11.4 in female patient 17 contains
only one gene ATP6AP2 also known as renin receptor that
has the highest expression in brain, heart, and placenta.
Mutations of ATP6AP2 have been reported in patients
with X-linked mental retardation with epilepsy (OMIM
300423) (35). Renin receptor binds renin and prorenin and
increases conversion of angiotensinogen to angiotensin.
The mice Atp6ap2-/- cardiomyocyte specific knockouts
showed no cardiac anomalies in the newborn state, although
inevitably resulted in heart failure. The mice died within
3 weeks of birth (36). The gene product of Atp6ap2 was
postulated to act in two ways: as a (pro)renin receptor,
exerting an RAS-related function and as a V-ATPase–associated protein, exerting a non–RAS-related function
that is essential for cell survival (36, 37).
In patient 19, the ~170 kb deletion at 5q23.1 inherited
from the reportedly healthy father involves only one gene
FAM170A. This gene belongs to the zinc finger (ZNF) protein family regulating differential gene expression during
many cellular activities (38) and thus potentially may play
a role in heart development.
The maternally inherited duplication at 21q22.11 in
patient 18 that harbors four genes, including RCAN1,
has been frequently observed as the inherited CNV in
Medical Genetics Laboratories at BCM. RCAN1 was postulated to be associated with the cardiac abnormalities
in patients with Down syndrome (DS) (39). However,
Eggerman et al. reported a 21q11.2 duplication harboring RCAN1 in a father and son, who did not present
either cardiac abnormalities or other features of DS (40),
and postulated to exclude RCAN1 from the DS critical
region in 21q22.1.
Approximately 100 kb duplication in 4q21.1 in patient
20 harbors seven genes, none of which is at present known
to be involved in cardiac development
CONCLUSION
Our study demonstrates that array comparative genomic
hybridization enables detection of clinically significant
chromosomal imbalances in patients with congenital
heart defects.
ĐŬŶŽǁůĞĚŐĞŵĞŶƚƐ
We are grateful to the patients and to their families for
participation in this study. We thank Dr. Seema Lalani and
Linda Guynn for helpful discussion. We thank Drs. B.R.
Brinkley, A.L. Beaudet, and J.R. Lupsky for facilitating the
collaboration between the Institute of Mother and Child
and Baylor College of Medicine.
Assessment of the role of copy-number variants in 150 patients
REFERENCES
1. Ferencz C., Rubin J.D., McCarter R.J., Brenner J.I., Neill C.A.,
Perry L.W., Hepner S.I., Downing J.W.: Congenital heart
disease: prevalence at livebirth. The Baltimore-Washington
Infant Study. Am. J. Epidemiol., 1985, 121 (1), 31-36.
2. Pierpont M.E., Basson C.T., Benson D.W., Jr., Gelb B.D.,
Giglia T.M., Goldmuntz E., McGee G., Sable C.A., Srivastava D.
Webb C.L.: Genetic basis for congenital heart defects: current
knowledge: a scientific statement from the American Heart
Association Congenital Cardiac Defects Committee, Council on
Cardiovascular Disease in the Young: endorsed by the American
Academy of Pediatrics. Circulation, 2007, 115 (23), 3015-3038.
3. Goldmuntz E., Paluru P., Glessner J., Hakonarson H., Biegel
J.A., White P.S., Gai X., Shaikh T.H.: Microdeletions and
microduplications in patients with congenital heart disease
and multiple congenital anomalies. Congenit. Heart. Dis.,
2011, 6 (6), 592-602.
4. Botto L.D., Correa A.: Decreasing the burden of congenital
heart anomalies: an epidemiologic evaluation of risk factors
and survival. Prog. in Pediatr. Cardiol., 2003, 18 (2), 111-121.
5. Grossfeld P.D.: The genetics of congenital heart disease. J.
Nucl. Cardiol., 2003, 10 (1), 71-76.
6. Wimalasundera R.C., Gardiner H.M.: Congenital heart disease
and aneuploidy. Prenat. Diagn., 2004, 24 (13), 1116-1122.
7. Lu X., Shaw C.A., Patel A., Li J., Cooper M.L., Wells W.R., Sullivan
C.M., Sahoo, T., Yatsenko S.A., Bacino C.A., Stankiewicz P., Ou
Z. i wsp.: Clinical implementation of chromosomal microarray
analysis: summary of 2513 postnatal cases. PLoS ONE, 2007,
2 (3), e327.
8. Thienpont B., Mertens L., de Ravel T., Eyskens B., Boshoff D.,
Maas N., Fryns J.P., Gewillig M., Vermeesch J.R. Devriendt K.:
Submicroscopic chromosomal imbalances detected by arrayCGH are a frequent cause of congenital heart defects in selected
patients. Eur. Heart. J., 2007, 28 (22), 2778-2784.
9. Richards A.A., Santos L.J., Nichols H.A., Crider B.P., Elder F.F.,
Hauser N.S., Zinn A.R.Garg V.: Cryptic chromosomal abnormalities
identified in children with congenital heart disease. Pediatr.
Res., 2008, 64 (4), 358-363.
10. Breckpot J., Thienpont B., Bauters M., Tranchevent L.C., Gewillig M.,
Allegaert K., Vermeesch J.R., Moreau Y. Devriendt K.: Congenital
heart defects in a novel recurrent 22q11.2 deletion harboring
the genes CRKL and MAPK1. Am. J. Med. Genet. Part A.,
2012, 158A (3), 574-580.
11. Lalani S.R., Shaw C., Wang X., Patel A., Patterson L.W., Kolodziejska
K., Szafranski P., Ou Z., Tian Q., Kang S-H.L., Jinnah A., Ali
S. i wsp.: Rare DNA copy number variants in cardiovascular
malformations with extracardiac abnormalities. Eur. J. Hum.
Genet. 2012, in press.
12. Soemedi R., Wilson I.J., Bentham J., Darlay R., Töpf A., Zelenika
D., Cosgrove C., Setchfield K., Thornborough C.,Granados-Riveron
J., Blue G.M., Breckpot J. i wsp. Contribution of global rare
copy-number variants to the risk of sporadic congenital heart
disease. Am J. Hum. Genet., 2012, 91 (3), 489-501.
13. Boone P.M., Bacino C.A., Shaw C.A., Eng P.A., Hixson P.M.,
Pursley A.N., Kang S.-H.L., Yang Y., Wiszniewska J., Nowakowska
B.A., del Gaudio D., Xia Z. i wsp.: Detection of clinically relevant
181
exonic copy-number changes by array CGH. Hum. Mutat.,
2010, 31 (12), 1326-1342.
14. El-Hattab A.W., Smolarek T.A., Walker M.E., Schorry E.K.,
Immken L.L., Patel G., Abbott M.A., Lanpher B.C., Ou Z., Kang
S.H., Patel A., Scaglia F. i wsp.: Redefined genomic architecture
in 15q24 directed by patient deletion/duplication breakpoint
mapping. Hum. Genet., 2009, 126 (4), 589-602.
15. Shaffer L.G., Kennedy G.M., Spikes A.S .Lupski J.R.: Diagnosis
of CMT1A duplications and HNPP deletions by interphase
FISH: implications for testing in the cytogenetics laboratory.
Am. J. Med. Genet., 1997, 69 (3), 325-331.
16. Stankiewicz P. Beaudet A.L.: Use of array CGH in the evaluation
of dysmorphology, malformations, developmental delay, and
idiopathic mental retardation. Curr. Opin. Genet. Dev., 2007,
17 (3), 182-192.
17. Rosenberg C., Knijnenburg J., Bakker E., Vianna-Morgante A.M.,
Sloos W., Otto P.A., Kriek M., Hansson K., Krepischi-Santos A.C.,
Fiegler H., Carter N.P., Bijlsma E.K., i wsp.: Array-CGH detection
of micro rearrangements in mentally retarded individuals: clinical
significance of imbalances present both in affected children
and normal parents. J. Med. Genet., 2006, 43 (2), 180-186.
18. Nowakowska B., Stankiewicz P., Obersztyn E., Ou Z., Li J., Chinault
A.C., Smyk M., Borg K., Mazurczak T., Cheung S.W. Bocian E.:
Application of metaphase HR-CGH and targeted Chromosomal
Microarray Analyses to genomic characterization of 116 patients
with mental retardation and dysmorphic features. Am. J. Med.
Genet. Part A., 2008, 146A (18), 2361-2369.
19. Hartman R.J., Rasmussen S.A., Botto L.D., Riehle-Colarusso T.,
Martin C.L., Cragan J.D., Shin M.Correa A.: The contribution
of chromosomal abnormalities to congenital heart defects: a
population-based study. Pediatr. Cardiol., 2011, 32 (8), 11471157.
20. Lu X.Y., Phung M.T., Shaw C.A., Pham K., Neil S.E., Patel A.,
Sahoo T., Bacino C.A., Stankiewicz P., Kang S.H., Lalani S.,
Chinault A.C., i wsp.: Genomic imbalances in neonates with
birth defects: high detection rates by using chromosomal
microarray analysis. Pediatrics, 2008, 122 (6), 1310-1318.
21. Fakhro K.A., Choi M., Ware S.M., Belmont J.W., Towbin
J.A., Lifton R.P., Khokha M.K.Brueckner M.: Rare copy
number variations in congenital heart disease patients
identify unique genes in left-right patterning. Proc. of the
Natl. Acad. of Sci. USA, 2011, 108 (7), 2915-2920.
22. Breckpot J., Thienpont B., Peeters H., de Ravel T., Singer A.,
Rayyan M., Allegaert K., Vanhole C., Eyskens B., Vermeesch
J.R., Gewillig M. Devriendt K.: Array comparative genomic
hybridization as a diagnostic tool for syndromic heart
defects. J. Pediatr., 2010, 156 (5), 810-817.
23. Miller D.T., Shen Y., Weiss L.A., Korn J., Anselm I.,
Bridgemohan C., Cox G.F., Dickinson H., Gentile J., Harris
D.J., Hegde V., Hundley R. i wsp.: Microdeletion/duplication
at 15q13.2q13.3 among individuals with features of autism
and other neuropsychiatric disorders. J. Med. Genet., 2009,
46 (4), 242-248.
24. Szafranski P., Schaaf C.P., Person R.E., Gibson I.B., Xia Z.,
Mahadevan S., Wiszniewska J., Bacino C.A., Lalani S., Potocki
L., Kang S.H., Patel A i wsp.: Structures and molecular
182
Katarzyna Derwińska i wsp.
mechanisms for common 15q13.3 microduplications
involving CHRNA7: benign or pathological? Hum. Mutat.,
2010, 31 (7), 840-850.
25. Sharp A.J., Mefford H.C., Li K., Baker C., Skinner C.,
Stevenson R.E., Schroer R.J., Novara F., De Gregori M.,
Ciccone R., Broomer A., Casuga I. i wsp.: A recurrent 15q13.3
microdeletion syndrome associated with mental retardation
and seizures. Nat. Genet., 2008, 40 (3), 322-328.
26. van Bon B.W., Mefford H.C., Menten B., Koolen D.A., Sharp
A.J., Nillesen W.M., Innis J.W., de Ravel T.J., Mercer C.L.,
Fichera M., Stewart H., Connell L.E. i wsp.: Further delineation
of the 15q13 microdeletion and duplication syndromes: a
clinical spectrum varying from non-pathogenic to a severe
outcome. J. Med. Genet., 2009, 46 (8), 511-523.
27. Masurel-Paulet A., Andrieux J., Callier P., Cuisset J.M., Le
Caignec C., Holder M., Thauvin-Robinet C., Doray B., Flori
E., Alex-Cordier M.P., Beri M., Boute O. i wsp.: Delineation of
15q13.3 microdeletions. Clin. Genet., 2010, 78 (2), 149-161.
28. Nemer M. Horb M.E.: The KLF family of transcriptional
regulators in cardiomyocyte proliferation and differentiation.
Cell Cycle, 2007, 6 (2), 117-121.
29. Lavallee G., Andelfinger G., Nadeau M., Lefebvre C., Nemer
G., Horb M.E. Nemer M.: The Kruppel-like transcription
factor KLF13 is a novel regulator of heart development.
EMBO J., 2006, 25 (21), 5201-5213.
30. Fox E.R., Young J.H., Li Y., Dreisbach A.W., Keating B.J.,
Musani S.K., Liu K., Morrison A.C., Ganesh S., Kutlar A.,
Ramachandran V.S., Polak J.F. i wsp.: Association of genetic
variation with systolic and diastolic blood pressure among
African Americans: the Candidate Gene Association Resource
study. Hum. Molec. Genet., 2011, 20 (11), 2273-2284.
31. Duan S.Z., Usher M.G. Mortensen R.M.: PPARs: the
vasculature, inflammation and hypertension. Curr. Opin.
Nephrol. Hypertens., 2009, 18 (2), 128-133.
32. Kirov G.: The role of copy number variation in schizophrenia.
Expert Rev. Neurother., 2010, 10 (1), 25-32.
33. Itsara A., Cooper G.M., Baker C., Girirajan S., Li J., Absher
D., Krauss R.M., Myers R.M., Ridker P.M., Chasman D.I.,
Mefford H., Ying P. i wsp.: Population Analysis of Large
Copy Number Variants and Hotspots of Human Genetic
Disease. Am. J. Hum. Genet., 2009, 84 (2), 148-161.
34. Shaikh T.H., Gai X., Perin J.C., Glessner J.T. Xie H., Murphy K.,
O'Hara R., Casalunovo T., Conlin L.K., D'Arcy M., Frackelton
E.C., Geiger E.A. i wsp.: High-resolution mapping and
analysis of copy number variations in the human genome:
A data resource for clinical and research applications.
Genome Res., 2009, 19 (9), 1682-1690.
35. Ramser J., Abidi F.E., Burckle C.A., Lenski C., Toriello H.,
Wen G., Lubs H.A., Engert S., Stevenson R.E., Meindl A.,
Schwartz C.E., Nguyen G.: A unique exonic splice enhancer
mutation in a family with X-linked mental retardation and
epilepsy points to a novel role of the renin receptor. Hum.
Molec. Genet., 2005, 14 (8), 1019-1027.
36. Kinouchi K., Ichihara A., Sano M., Sun-Wada G.H., Wada
Y., Kurauchi-Mito A., Bokuda K., Narita T., Oshima Y.,
Sakoda M., Tamai Y., Sato H. i wsp.: The (pro)renin receptor/
ATP6AP2 is essential for vacuolar H(+)-ATPase assembly
in murine cardiomyocytes. Circ. Res., 2010, 107 (1), 30-34.
37. Kinouchi K., Ichihara A., Sano, M., Sun-Wada G.H., Wada
Y., Oshima Y., Sakoda M., Kurauchi-Mito A., Bokuda
K., Narita T., Fukuda K., Itoh H.: ATP6AP2/(pro)renin
receptor is essential for the functions of organella in murine
cardiomyocytes. Hypertension, 2010, 56 (5), E132-E132.
38. Lei C., Liu Q., Wang W., Li J., Xu F., Liu Y., Liu J., Wu S.,
Wang M.: Isolation and characterization of a novel zinc
finger gene, ZNFD, activating AP1(PMA) transcriptional
activities. Mol. Cell. Biochem., 2010, 340 (1-2), 63-71.
39. Ronan A., Fagan K., Christie L., Conroy J., Nowak N.J.
Turner G.: Familial 4.3 Mb duplication of 21q22 sheds
new light on the Down syndrome critical region. J. Med.
Genet., 2007, 44 (7), 448-451.
40. Eggermann T., Schonherr N., Spengler S., Jager S., Denecke B.,
Binder G., Baudis M.: Identification of a 21q22 duplication
in a Silver-Russell syndrome patient further narrows down
the Down syndrome critical region. Am. J. Med. Gen. Part
A, 2010, 152A (2), 356-359.
Authors' contributions/Wkład Autorów
Według kolejności
Conflicts of interest/Konflikt interesu
The Authors declare that there is no conflict of interest.
Autorzy pracy nie zgłaszają konfliktu interesów.
Received/Nadesłano: 13.03.2012 r.
Accepted/Zaakceptowano: 8.05.2012 r.
Published on line/Dostępne on line
Address for correspondence:
Paweł Stankiewicz
Department of Medical Genetics
Institute of Mother and Child
ul. Kasprzaka 17a, 01-211, Warsaw
[email protected]
[email protected]