Genetic screening of 104 patients with congenitally malformed hearts revealed a fresh mutation of GATA4 in those with atrial septal defects.

Cardiol Young 2009: Page 1 of 4

r Cambridge University Press
ISSN 1047-9511
doi:10.1017/S1047951109990813

Original Article
Genetic screening of 104 patients with congenitally malformed
hearts revealed a fresh mutation of GATA4 in those with atrial
septal defects
Haruka Hamanoue,1,2 Sri Endah Rahayuningsih,4 Yuya Hirahara,1 Junko Itoh,1 Utako Yokoyama,3
Takeshi Mizuguchi,1 Hirotomo Saitsu,1 Noriko Miyake,1 Fumiki Hirahara,2 Naomichi Matsumoto1
1

Department of Human Genetics; 2Department of Obstetrics and Gynecology; 3Cardiovascular Research Institute,
Yokohama City University Graduate School of Medicine, Yokohama, Japan; 4Department of Pediatrics, Padjadjaran
University Medical School, Hasan Sadikin General Hospital, Bandung, Indonesia

Abstract We analysed the GATA binding protein 4 gene, or GATA4, along with the NK2 transcription
factor related, locus 5 gene, or NKX2.5, to determine their genetic contribution to 104 sporadic patients in
Indonesia with congenitally malformed hearts, 76 cases having atrial septal defect and 28 tetralogy of Fallot.

We found only 1 novel mutation of GATA4 in those with atrial septal defecst. Analysis of the genetic
background of the parents of the patient showed for the first time that a new mutation of GATA4 can cause
sporadic atrial septal defects. We failed to discover any other mutations of either the GATA4 or NKX2-5
genes, supporting the marked genetic heterogeneity of human congenital cardiac defects.
Keywords: genetic inheritance; new mutation; interatrial communications

C

ONGENITAL CARDIAC DISEASE IS ONE OF THE

commonest defects found at birth, being
observed in more than 1 of each 100 live
births.1 Among these lesions, atrial septal defect
and tetralogy of Fallot account for approximately
one-tenth and one-twentieth of the overall defects,
respectively. Genetic factors have been associated
with both lesions. Mutations of GATA4 were found
mostly in familial examples of atrial septal
defect,2–9 but never in these lesions when occurring
sporadically. Mutations of NKX2-5 were also

observed in a spectrum of both familial and sporadic
occurrences of congenitally malformed hearts,
including those with atrial septal defect and
tetralogy of Fallot, frequently in association with
abnormalities of atrioventricular conduction.10–17
Correspondence to: Naomichi Matsumoto, M.D., Ph.D., Department of Human
Genetics, Yokohama City University Graduate School of Medicine, Fukuura
3-9, Kanazawa-ku, Yokohama 236-0004, Japan. Tel: 181-45-787-2604; Fax:
181-45-786-5219; E-mail: naomat@yokohama-cu.ac.jp
Accepted for publication 7 June 2009

In this study, we sequenced GATA4 and NKX2-5
to determine their genetic contribution to the
occurrence of sporadic congenital cardiac defects in
Indonesia, producing what is, to the best of our
knowledge, the first evidence of a new mutation of
GATA4 causing an atrial septal defect.

Materials and methods
We recruited 76 patients with atrial septal defects

located within the oval fossa, and 28 patients with
tetralogy of Fallot. In Table 1, we list the cardiac
phenotypes. All patients were non-syndromic, and
had been diagnosed on the basis of echocardiography and other standard methods by one of coauthors
(S.E.R.) working at Hasan Sadikin General Hospital
in Bandung, Indonesia. As established by taking a
careful history, we confirmed that none of the
parents possessed an abnormal cardiac phenotype.
Informed consent was obtained from all the
participating families. As normal controls were
unavailable for the Indonesian population, we used

2

normal Japanese controls to observe polymorphisms, recognizing that Indonesian and Japanese
populations are ethnically unrelated. Experimental
protocols were approved by the institutional review
board in Yokohama city university school of
medicine.
Genomic deoxyribonucleic acid, or DNA, was

extracted from peripheral leukocytes using FlexiGene
Blood DNA kit (Qiagen, Tokyo, Japan). Whole
genome amplification was performed wtih Illustra
GenomiPhi V2 DNA Amplification Kit (GE
Healthcare Bio-Science, Tokyo, Japan).
We analysed 6 coding exons of GATA4, and 2
exons of NKX2-5, along with their flanking intronic
regions. All primers were designed using the webbased Primer3 software (http://frodo.wi.mit.edu/cgibin/primer3/primer3_www.cgi). Detailed information
of primers is available on request. Polymerase chain

reaction was cycled 35 times at 948C for 30 sec, at
58–648C for 30 sec and at 728C for 30 sec in a total
volume of 20 ml containing 30 ng of whole genome
amplification deoxyribonucleic acid, and/or genomic
deoxyribonucleic acid, as a template, 0.5 mM of
forward and reverse primers, 200 mM of each
deoxynucleotide triphosphates, and 0.5 Unit of ExTaq
(Takara, Ohtsu, Japan) or LATaq (Takara, Japan),
purified with ExoSAP-IT (United States Biochemical,
Cleveland, OH), and sequenced using BigDye

Terminator ver.3.1 (Applied Biosystems, Foster City,
CA) on the 3100 Genetic Analyzer (Applied
Biosystems). Deoxyribonucleic acid sequences were
compared with the reference genome sequences based
on the University of California, Santa Cruz Genome
Browser (May 2006 assembly) using the SeqScape
software Ver.2.1 (Applied Biosystems). All the
nucleotide changes found in whole genome amplification deoxyribonucleic acids were also confirmed by
sequencing using original genomic deoxyribonucleic
acids. Regarding polymorphisms, normal control
deoxyribonucleic acids were sequenced until the change
was seen, needing up to 96 normal chromosomes.

Table 1. Cardiac phenotypes of the patients.
Number of
patients

Cardiac phenotype
Atrial septal defect
Atrial septal defect

Atrial septal defect
hypertension
Atrial septal defect
stenosis
Atrial septal defect
septal defect
Tetralogy of Fallot
Total

2009

Cardiology in the Young

secundum
secundum 1 Pulmonary

76
22
39

secundum 1 Pulmonary

7

secundum 1 Ventricular

8

Results and discussion
A heterozygous mutation of GATA4, specifically
c.341_342insA (p.T114TfsX95), was identified in
1 of the patients (Fig. 1). Analysis of his parental
deoxyribonucleic acid confirmed the novel nature of
the mutation. Parentage was confirmed by microsatellite analyses. The patient was a 4-year-old boy,

28
104

c.341_342insA

GATA-type transcription
activator,N-terminal

N
1

c.1074delC c.1075delG
GATA
zinc
finger
2 08 2 1 7

251

GATA
zinc
finger
2 71

C

30 5

442

c.341_342insA

+/+

C G G G G A CC A C C G G G T C C C T G
A C G G G TC CC T

+/+

m/+

Figure 1.
The protein truncation mutation of GATA4. c.341_342insA identified in this study is the earliest type of truncation. c.1074delC and
c.1075delG were previously reported (upper).2,3 Electropherogram of the identified mutation is shown (lower left). The family pedigree of the
patient with c.341_342ins shows clearly its new occurrence. 1: wild type, m: mutated.

3

Hamanoue et al: Mutation of GATA4 in sporadic atrial septal defect
Table 2. Polymorphisms found in this study.

gene

position

nucleotide change

GATA4

Exon 1
Exon 1
Exon 2
Intron 4
Exon 5
Exon 5

Exon1
Exon2
Exon2
Exon2

c.454G . C
c.531C . A
c.723C . T
IVS04 1 56C . A
c.1138G . A
c.1220C . A
c.63A . G
c.606G . C
c.608A . G
c.[796C . A(1)797C . A]

NKX2.5

amino acid
change
p.A152P
p.A177A
p.C241C
p.V380M
p.P407Q
p.E21E
p.L202L
p.E203G
p.P266?

dbSNP ID

allele frequency in
subjects

allele frequency in
normal control

nr
nr
rs1062215
nr
nr
nr
rs2277923
rs3729753
nr
nr

1/208
1/208
4/208
208/208
3/208
2/208
140/208
8/208
4/208
5/208*

1/96
1/24
nc
2/2
1/10
1/10
nc
nc
1/86
11/22*

Normal controls are Japanese; nr: not registered; nc: not checked; *: if both changes are in the same allle.

presenting because of failure to thrive and recurrent
respiratory infections. Echocardiography revealed a
hole of 1.4 centimeters diameter in the floor of the
oval fossa, as well as pulmonary hypertension. At
the age of 4 1/2 years, the defect was closed by
insertion of an Amplatzer septal occluder. He is now
in a good condition. His healthy 30-year-old father,
and 20-year-old mother, were not consanguineous.
The pregnancy had been uneventful, and his elder
brother, aged 6 years, is also healthy, without any
cardiac or other abnormalities (Fig. 1).
The mutation, c.341_342insA, is the third
protein truncation mutation of GATA4, resulting
in much earlier termination than the other two,
c.1074delC and c.1075delG, thus far reported.2,3,5
All the reported truncations have been found in
patients with atrial septal defects. Missense mutations are associated with varied phenotypes, including atrial septal defect, tetralogy of Fallot,
atrioventricular septal defect, and ventricular septal
defect.2,4–7,9,18. Three inframe changes, specifically
p.S46del, p.A118_A119insA, and p.A125_A126in
sAA, have also been found in patients with
ventricular septal defect and tetralogy of Fallot.18
Mutations of GATA4 mutations have been reported
to be very rare in patients with sporadic congenital
cardiac lesions, with an estimated prevalence of less
than 1%,8,9,18–20 this fitting well with our own
finding. We also found 5 other SNPs, including
rs1062245 in GATA4, as judged by normal control
studies or according to the database of single
nucleotide polymorphisms (http://www.ncbi.nlm.
nih.gov/sites/entrez) (Table 2). GATA4, a central
zinc finger transcription factor for cardiac development, binds specifically to the A/T GATA A/G
motif on the deoxyribonucleic acid, and modulates
transcription of downstream target genes.9 GATA4
can also physically interact with NKX2-521 and
TBX5,2 producing familial congenital heart defects12 as well as the well recognized Holt-Oram

syndrome, which consists of cardiac septal defects,
abnormalities of conduction, and skeletal anomalies
of the limbs.22,23 The previously reported mutation,
c.1075delG, failed to activate transcription of
downstream genes.2 Thus mutational consequences
of the c.341_342insA found in this study could be
similar to c.1075delG as c.341_342insA, and may
potentially produce a much shorter protein than
c.1075delG, even if its transcript escapes the
nonsense mediated decay (Fig. 1).
None of our patients presented with any pathological
changes in NKX2.5, albeit that 5 polymorphisms have
been identified in this gene (Table 2). Approximately
one-twentieth of examples of both atrial septal defect
and tetralogy of Fallot may be caused by such
mutations.11 The fact that none of our patients
exhibited such abnormalities is further evidence
supporting the notion that sporadic examples of
congenital cardiac disease are genetically heterogeneous.
Our analysis of 104 patients with sporadic forms of
atrial septal defect and tetralogy of Fallot, therefore,
for mutations of the GATA4 and NKX2-5 genes has
revealed a new mutation of GATA4 in 1 patient with
an atrial septal defect. As far as we are aware, this is the
first direct link of such a new mutation demonstrated
using genomic deoxyribonucleic acids.

Acknowledgments
We express our gratitude to all patients and their
families who participated in this project. This study
was supported by a Research Grant from the Ministry
of Health, Labour and Welfare (N. M.).
Contract grant sponsor: Research Grant from the
Ministry of Health, Labour and Welfare (N. M.).

References
1. Hoffman JI, Kaplan S. The incidence of congenital heart disease.
J Am Coll Cardiol 2002; 39: 1890–1900.

4

Cardiology in the Young

2. Garg V, Kathiriya IS, Barnes R, et al. GATA4 mutations cause
human congenital heart defects and reveal an interaction with
TBX5. Nature 2003; 424: 443–447.
3. Okubo A, Miyoshi O, Baba K, et al. A novel GATA4 mutation
completely segregated with atrial septal defect in a large Japanese
family. J Med Genet 2004; 41: e97.
4. Sarkozy A, Conti E, Neri C, et al. Spectrum of atrial septal defects
associated with mutations of NKX2.5 and GATA4 transcription
factors. J Med Genet 2005; 42: e16.
5. Hirayama-Yamada K, Kamisago M, Akimoto K, et al. Phenotypes with GATA4 or NKX2.5 mutations in familial atrial septal
defect. Am J Med Genet A 2005; 135: 47–52.
6. Tomita-Mitchell A, Maslen CL, Morris CD, Garg V, Goldmuntz
E. GATA4 sequence variants in patients with congenital heart
disease. J Med Genet 2007; 44: 779–783.
7. Rajagopal SK, Ma Q, Obler D, et al. Spectrum of heart disease
associated with murine and human GATA4 mutation. J Mol Cell
Cardiol 2007; 43: 677–685.
8. Posch MG, Perrot A, Schmitt K, et al. Mutations in GATA4,
NKX2.5, CRELD1, and BMP4 are infrequently found in patients
with congenital cardiac septal defects. Am J Med Genet A 2008;
146: 251–253.
9. Nemer G, Fadlalah F, Usta J, et al. A novel mutation in the
GATA4 gene in patients with Tetralogy of Fallot. Hum Mutat
2006; 27: 293–294.
10. Akazawa H, Komuro I. Cardiac transcription factor Csx/Nkx2-5:
Its role in cardiac development and diseases. Pharmacol Ther
2005; 107: 252–268.
11. McElhinney DB, Geiger E, Blinder J, Benson DW, Goldmuntz E.
NKX2.5 mutations in patients with congenital heart disease.
J Am Coll Cardiol 2003; 42: 1650–1655.
12. Schott JJ, Benson DW, Basson CT, et al. Congenital heart disease
caused by mutations in the transcription factor NKX2-5. Science
1998; 281: 108–111.
13. Benson DW, Silberbach GM, Kavanaugh-McHugh A, et al.
Mutations in the cardiac transcription factor NKX2.5 affect

2009

diverse cardiac developmental pathways. J Clin Invest 1999; 104:
1567–1573.
14. Goldmuntz E, Geiger E, Benson DW. NKX2.5 mutations in
patients with tetralogy of fallot. Circulation 2001; 104:
2565–2568.
15. Reamon-Buettner SM, Hecker H, Spanel-Borowski K, et al.
Novel NKX2-5 mutations in diseased heart tissues of
patients with cardiac malformations. Am J Pathol 2004; 164:
2117–2125.
16. Watanabe Y, Benson DW, Yano S, et al. Two novel frameshift
mutations in NKX2.5 result in novel features including visceral
inversus and sinus venosus type ASD. J Med Genet 2002; 39:
807–811.
17. Ikeda Y, Hiroi Y, Hosoda T, et al. Novel point mutation in the
cardiac transcription factor CSX/NKX2.5 associated with congenital heart disease. Circ J 2002; 66: 561–563.
18. Zhang W, Li X, Shen A, et al. GATA4 mutations in 486 Chinese
patients with congenital heart disease. Eur J Med Genet 2008; 51:
527–535.
19. Schluterman MK, Krysiak AE, Kathiriya IS, et al. Screening and
biochemical analysis of GATA4 sequence variations identified in
patients with congenital heart disease. Am J Med Genet A 2007;
143: 817–823.
20. Posch MG, Berger F, Perrot A, Ozcelik C. We need a detailed
phenome in the phenomenon of genetics and congenital heart
disease. J Med Genet 2008; 45: 320.
21. Zhu W, Shiojima I, Hiroi Y, et al. Functional analyses of three
Csx/Nkx-2.5 mutations that cause human congenital heart
disease. J Biol Chem 2000; 275: 35291–35296.
22. Basson CT, Bachinsky DR, Lin RC, et al. Mutations in human
TBX5 [corrected] cause limb and cardiac malformation in HoltOram syndrome. Nat Genet 1997; 15: 30–35.
[23. Li QY, Newbury-Ecob RA, Terrett JA, et al. HoltOram syndrome is caused by mutations in TBX5, a member
of the Brachyury (T) gene family. Nat Genet 1997; 15:
21–29.