GATA4 Loss-of-Function Mutations Underlie Familial Tetralogy of FALLOT.
ABSTRACT Tetralogy of Fallot (TOF) represents the most common form of cyanotic congenital heart disease and accounts for significant morbidity and mortality in humans. Emerging evidence has implicated genetic defects in the pathogenesis of TOF. However, TOF is genetically heterogeneous and the genetic basis for TOF in most patients remains unclear. In this study, the GATA4 gene were sequenced in 52 probands with familial TOF, and 3 novel heterozygous mutations, including A9P and L51V both located in the putative first transactivational domain and N285S in the C-terminal zinc finger, were identified in 3 probands, respectively. Genetic analysis of the pedigrees demonstrated that in each family the mutation cosegregated with TOF with complete penetrance. The missense mutations were absent in 800 control chromosomes and the altered amino acids were highly conserved evolutionarily. Functional analysis showed that the GATA4 mutants were consistently associated with diminished DNA-binding affinity and decreased transcriptional activity. Furthermore, the N285S mutation completely disrupted the physical interaction between GATA4 and TBX5. To our knowledge, this report associates GATA4 loss-of-function mutations with familial TOF for the first time, providing novel insight into the molecular mechanism involved in TOF and suggesting potential implications for the early prophylaxis and allele-specific therapy of TOF. This article is protected by copyright. All rights reserved.
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ABSTRACT: Congenital heart disease (CHD) is the most common birth defect and is the most prevalent non-infectious cause of infant death. Aggregating evidence demonstrates that genetic defects are involved in the pathogenesis of CHD. However, CHD is genetically heterogeneous and the genetic determinants for CHD in an overwhelming majority of patients remain unknown. In this study, the coding regions and splice junctions of the NKX2.6 gene, which encodes a homeodomain transcription factor crucial for cardiovascular development, were sequenced in 210 unrelated CHD patients. As a result, a novel heterozygous NKX2.6 mutation, p.K152Q, was identified in an index patient with ventricular septal defect (VSD). Genetic analysis of the proband's available family members showed that the mutation cosegregated with VSD transmitted as an autosomal dominant trait with complete penetrance. The missense mutation was absent in 400 control chromosomes and the altered amino acid was completely conserved evolutionarily across species. Due to unknown transcriptional targets of NKX2.6, the functional characteristics of the identified mutation at transcriptional activity were analyzed by using NKX2.5 as a surrogate. Alignment between human NKX2.6 and NKX2.5 proteins displayed that K152Q-mutant NKX2.6 was equivalent to K158Q-mutant NKX2.5, and introduction of K158Q into NKX2.5 significantly reduced its transcriptional activating function when compared with its wild-type counterpart. This study firstly links NKX2.6 loss-of-function mutation with increased susceptibility to isolated VSD, providing novel insight into the molecular mechanism underpinning VSD and contributing to the development of new preventive and therapeutic strategies for this common form of CHD.Pediatric Cardiology 11/2014; · 1.55 Impact Factor
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ABSTRACT: Dilated cardiomyopathy (DCM) is the most frequent type of primary myocardial disorder responsible for substantial morbidity and mortality. DCM is the third most common cause of heart failure and the most common reason for heart transplantation. A recent study has implicated GATA4 mutation in the pathogenesis of familial DCM. However, the prevalence and spectrum of GATA4 mutations associated with sporadic DCM remain unclear. In this study, the coding exons and exon-intron boundaries of the GATA4 gene, which encodes a cardiac transcription factor crucial for normal cardiogenesis, were sequenced in 220 unrelated patients with sporadic DCM. A total of 200 unrelated ethnically-matched healthy individuals used as controls were genotyped. The functional characteristics of the mutant GATA4 were assayed in contrast to its wild-type counterpart using a luciferase reporter assay system. As a result, 3 novel heterozygous GATA4 mutations, p.V39L, p.P226Q and p.T279S, were identified in 3 unrelated patients with sporadic DCM, with a mutational prevalence of approximately 1.36%. The missense mutations were absent in 400 control chromosomes and the altered amino acids were completely conserved evolutionarily across species. Functional analysis showed that the GATA4 mutants were consistently associated with significantly decreased transcriptional activity and markedly reduced the synergistic activation between GATA4 and NKX2-5. This study firstly links GATA4 mutations to increased susceptibility to sporadic DCM and provides novel insight into the molecular etiology underlying DCM, suggesting the potential implications for the early prophylaxis and allele-specific treatment of this common form of cardiomyopathy.Gene 07/2014; · 2.20 Impact Factor
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ABSTRACT: Dilated cardiomyopathy (DCM), the most prevalent form of primary heart muscle disease, is the third most common cause of heart failure and the most frequent reason for cardiac transplantation. Mounting evidence has demonstrated that genetic risk factors are crucial in the pathogenesis of DCM. However, DCM is genetically heterogeneous, and the genetic basis of DCM in a large majority of cases remains unclear. In the current study, the coding exons and flanking introns of the GATA6 gene, which encodes a zinc‑finger transcription factor essential for cardiogenesis, was sequenced in 140 unrelated patients with DCM, and two novel heterozygous mutations, p.C447Y and p.H475R, were identified in two index patients with DCM, respectively. Analysis of the pedigrees showed that in each family the mutation co-segregated with DCM transmitted in an autosomal-dominant pattern, with complete penetrance. The missense mutations were absent in 400 control chromosomes and predicted to be disease-causing by MutationTaster or probably damaging by PolyPhen-2. The alignment of multiple GATA6 proteins across species revealed that the altered amino acids were completely conserved evolutionarily. The functional assays showed that the mutated GATA6 proteins were associated with significantly reduced transcriptional activation in comparison with their wild-type counterpart. To the best of our knowledge, this is the first study on the association of GATA6 loss-of-function mutations with enhanced susceptibility to familial DCM, which provides novel insight into the molecular mechanism of DCM and suggests potential implications for the antenatal prophylaxis and allele-specific treatment of DCM.International Journal of Molecular Medicine 08/2014; · 1.88 Impact Factor
Int. J. Med. Sci. 2013, Vol. 10
I In nt te er rn na at ti io on na al l J Jo ou ur rn na al l o of f M Me ed di ic ca al l S Sc ci ie en nc ce es s
2013; 10(1):34-42. doi: 10.7150/ijms.5270
GATA5 loss-of-Function Mutations Underlie Tetralogy
Dong Wei1#, Han Bao1#, Xing-Yuan Liu1, Ning Zhou1, Qian Wang2, Ruo-Gu Li2, Ying-Jia Xu2, Yi-Qing
2. Department of Cardiology and Cardiovascular Research, Shanghai Chest Hospital, Shanghai Jiaotong University School of Medicine,
241 West Huaihai Road, Shanghai 200030, China.
Department of Pediatrics, Tongji Hospital, Tongji University School of Medicine, Shanghai 200065, China;
# contributed equally to this work.
Corresponding author: Xing-Yuan Liu, Tel: +8621-56051080; Fax: +8621-66371663; Email: firstname.lastname@example.org. Yi-Qing Yang, Tel:
+8621-62821990; Fax: +8621-62821105; Email: email@example.com.
© Ivyspring International Publisher. This is an open-access article distributed under the terms of the Creative Commons License (http://creativecommons.org/
licenses/by-nc-nd/3.0/). Reproduction is permitted for personal, noncommercial use, provided that the article is in whole, unmodified, and properly cited.
Received: 2012.09.21; Accepted: 2012.11.27; Published: 2012.12.10
Tetraology of Fallot (TOF) is the most common form of cyanotic congenital heart disease and
is a major cause of significant morbidity and mortality. Emerging evidence demonstrates that
genetic risk factors are involved in the pathogenesis of TOF. However, TOF is genetically
heterogeneous and the genetic defects responsible for TOF remain largely unclear. In the
present study, the whole coding region of the GATA5 gene, which encodes a zinc-finger
transcription factor essential for cardiogenesis, was sequenced in 130 unrelated patients with
TOF. The relatives of the index patients harboring the identified mutations and 200 unrelated
control individuals were subsequently genotyped. The functional characteristics of the mu-
tations were analyzed using a luciferase reporter assay system. As a result, 2 novel hetero-
zygous GATA5 mutations, p.R187G and p.H207R, were identified in 2 families with autosomal
dominantly inherited TOF, respectively. The variations were absent in 400 control alleles and
the altered amino acids were completely conserved evolutionarily. Functional analysis showed
that the GATA5 mutants were associated with significantly decreased transcriptional activa-
tion compared with their wild-type counterpart. To our knowledge, this is the first report on
the association of GATA5 loss-of-function mutations with TOF, suggesting potential impli-
cations for the early prophylaxis and allele-specific therapy of human TOF.
Key words: Congenital heart disease; Tetralogy of Fallot; Genetics; Transcription factor; GATA5.
Congenital heart disease constitutes the most
prevalent type of developmental abnormality in hu-
mans with an estimated prevalence of 1% among live
births, and is the leading noninfectious cause of infant
death worldwide, with approximately 30% of neo-
nates who die of birth defects having cardiovascular
malformations . Congenital cardiovascular anoma-
lies are clinically classified into at least 21 different
categories with specific anatomic lesions, of which
tetraology of Fallot (TOF), a tetrad consisting of pul-
monary stenosis, overriding aorta, ventricular septal
defect, and right ventricular hypertrophy, is the most
common form of cyanotic congenital heart disease,
accounting for roughly 10% of all congenital cardio-
vascular deformations [1,2]. TOF can occur separately
or in combination with other congenital cardiac de-
formities, such as atrial septal defect, patent ductus
arteriosis, atrioventricular septal defect, abnormal
pulmonary venous return, anomalous coronary ar-
teries, absent pulmonary valve, aorticopulmonary
Int. J. Med. Sci. 2013, Vol. 10
window, and aortic incompetence. Regardless of oth-
er complications that may be concomitant with TOF,
isolated TOF may result in reduced exercise tolerance,
poor quality of life, delayed fetal brain development,
cardiac dysfunction or heart failure, thromboembolic
stroke, pulmonary embolus, subacute bacterial endo-
carditis, arrhythmias, and even cardiac death [3-12].
Given no corrective surgery, 25% of patients with
severe pulmonary obstruction decease within the first
year, 40% decease by age 3 years, 70% by age 10 years,
and 95% by age 40 years . Despite the significant
morbidity and mortality throughout life, the funda-
mental etiology responsible for TOF remains largely
Embryonic heart development is a complex and
dynamic process that requires the orchestration of
cardiac cell commitment, differentiation, prolifera-
tion, and migration . Many factors have been in-
volved in this process based on their temporal and
spatial expression patterns and their phenotypic
characteristics associated with loss or gain of function,
of which transcription factors are increasingly recog-
nized as playing critical roles in early cardiogenesis,
including GATA and NK families [14-16].
The GATA zinc finger-containing transcription
factors are a family of DNA binding proteins charac-
teristic of the ability to binding preferentially to the
consensus DNA sequence GATA of target genes. To
date, six members of the GATA family have been
identified in vertebrates (GATA1 to GATA6), of
which GATA4, GATA5 and GATA6 are broadly ex-
pressed in various mesoderm and endoderm derived
tissues, dominantly in embryonic heart [15,16].
Among the three cardiac GATA transcription factors,
GATA4 has been most extensively explored, and a lot
of GATA4 mutations have been identified in patients
with various kinds of congenital heart disease, in-
cluding ventricular septal defect, atrial septal defect,
and TOF [17-33]. Recently, mutations in GATA6 have
also been associated with a wide variety of congenital
heart disease [34-37]. The GATA5 gene is another
member of the GATA family, and its expression and
function overlap at least partially with those of
GATA4 and GATA6 during cardiovascular develop-
ment [14,16], which makes it a logical candidate gene
Materials and Methods
A cohort of 130 unrelated patients with TOF was
recruited among Chinese Han population. The avail-
able relatives of the index patients carrying the iden-
tified GATA5 mutations were also enrolled. Subjects
were evaluated by individual and familial histories,
review of the medical records, complete physical
with color flow Doppler. All patients had an echocar-
diogram documented TOF. Most patients underwent
cardiac surgery. The patients with known chromo-
somal abnormalities or syndromic cardiovascular
defects were excluded from the study.
A total of 200 unrelated, ethnically matched
healthy individuals randomly selected from the indi-
viduals undergoing routine physical examinations
were used as control subjects to scan for likely varia-
tions in GATA5. According to a review of medical
history and an analysis of echocardiographic record,
the control individuals had no congenital cardiovas-
cular deformations, except for subclinical cardiac ab-
errations such as bicuspid aortic valve and patent
foramen ovale. The ethnic origin of a participant was
ascertained by a combination of self-reported ethnic-
ity and a personal questionnaire asking questions
about the birthplace, language, religion, and ancestry.
Peripheral venous blood specimens from TOF
cases and control individuals were prepared. The
study protocol was reviewed and approved by the
local institutional ethics committee and written in-
formed consent was obtained from all participants or
their guardians prior to study.
Genomic DNA from all participants was ex-
tracted from blood lymphocytes with Wizard Ge-
nomic DNA Purification Kit (Promega, Madison, WI,
USA). The candidate gene GATA5 was sequenced
initially in 130 unrelated patients with TOF, and gen-
otyping GATA5 was performed subsequently for the
available relatives of the index patients carrying iden-
tified mutations and the 200 unrelated, ethnically
matched healthy control individuals. The referential
genomic DNA sequence of GATA5 derived from
GenBank (accession No. HM015595). By the aid of
on-line Primer 3 software (http://frodo.wi.mit.edu),
the primer pairs used to amplify the coding exons and
exon/intron boundaries of GATA5 by PCR were de-
signed as shown in Table 1. The PCR was carried out
using HotStar Taq DNA Polymerase (Qiagen GmbH,
Hilden, Germany) on a PE 9700 Thermal Cycler (Ap-
plied Biosystems, Foster, CA, USA), with standard
conditions and concentrations of reagents. Amplified
products were analyzed on 1% agarose gels stained
with ethidium bromide and purified with QIAquick
Gel Extraction Kit (Qiagen). Both strands of each PCR
product were sequenced with a BigDye® Terminator
v3.1 Cycle Sequencing Kit (Applied Biosystems) un-
Int. J. Med. Sci. 2013, Vol. 10
der an ABI PRISM 3130 XL DNA Analyzer (Applied
Biosystems). The sequencing primers were the same
as previously designed for specific region amplifica-
tion. The DNA sequences were viewed and analyzed
with the DNA Sequencing Analysis Software v5.1
(Applied Biosystems). The variant was validated by
re-sequencing an independent PCR-generated am-
plicon from the subject and met our quality control
thresholds with a call rate >99%. Additionally, an
identified sequence variation was searched in the
single nucleotide polymorphism (SNP) database at
National Center for Biotechnology Information
(NCBI) to confirm its novelty.
Table 1. The intronic primers to amplify the coding exons and flanking splice sites of GATA5.
Forward primer (5′ to 3′)
GGC, ATA, AGC, TCG, GGC, GCT, GG
TGA, CGA, AAG, CCG, CCA, GGC, TC
CCG, CAA, GGC, CGA, CCT, GAG, TC
Reverse primer (5′ to 3′)
TGG, GCC, CCG, AGA, CTG, TGG, AG
CCC, CAG, GGG, CTC, TGG, TGT, CA
CCG, CTC, CTC, CCC, AGC, CTC, TT
GGG, AAT, CCA, GCT, CCA, CGG, GC
GCC, TGC, GGT, GTG, ACC, GTG, AG
CCC, CCA, TGC, CAT, TCC, AGG, GC
CTG, GAG, GCA, CCG, AAG, GCC, AC
GGT, GTG, TCC, AGC, CCA, CCT, GC
GGG, GCC, TGC, TGG, TCT, CTG, CT
Alignment of multiple GATA5 protein se-
Multiple GATA5 protein sequences across vari-
ous species were aligned using the online program of
MUSCLE, version 3.6 (http://www.ncbi.nlm.nih.gov
Prediction of the causative potential of a
GATA5 sequence variation
The disease-causing potential of a GATA5 se-
quence variation was predicted by MutationTaster (an
online program at http://www.mutationtaster.org),
which automatically gave a probability for the varia-
tion to be either a pathogenic mutation or a benign
polymorphism. Notably, the P value used here is the
probability of the correct prediction rather than the
probability of error as used in t test statistics (i.e., a
value close to 1 indicates a high 'security' of the pre-
Construction of recombinant
pcDNA3.1-hGATA5 expression plasmid
Human fetal cardiac tissue samples were previ-
ously collected and preserved in RNAlater RNA sta-
bilization reagent (Qiagen). Total RNA was extracted
using an RNeasy Protect Mini Kit (Qiagen). Reverse
transcription was performed with Oligo(dT)20 primer
using SuperScript III reverse transcriptase (Invitro-
gen, Carlsbad, CA, USA). The full-length wild-type
cDNA of the human GATA5 gene, including partial
5’- and 3’-untranslated regions, was PCR amplified
using pfuUltra high-fidelity DNA polymerase (Strat-
agene, La Jolla, CA, USA). The primer pairs used for
the specific amplification of the GATA5 transcript
were: for the forward, 5’-GTA, GCT, AGC, CAC,
CGC, CGT, GCC, CTG, CCG-3’; for the reverse,
5’-GAT, GCG, GCC, GCT, GTT, CCC, CTG, ACA,
TGG, GC-3’. The PCR fragment with a length of 1296
base pairs was doubly digested by endonuclease NheI
and NotI (TaKaRa, Dalian, Liaoning, China). The di-
gested product was fractionated by 1.5% agarose gel
electrophoresis, purified with the QIAquick Gel Ex-
traction Kit (Qiagen), and then subcloned into
pcDNA3.1 (Promega) to form a recombinant eukary-
otic expression vector, pcDNA3.1-hGATA5.
The identified mutation was introduced into the
wild-type GATA5 using a QuickChange II XL
Site-Directed Mutagenesis Kit (Stratagene) with a
complementary pair of primers. The mutant was se-
quenced to confirm the desired mutation and to ex-
clude any other sequence variations.
Reporter gene assay
The atrial natriuretic factor (ANF)-luciferase re-
porter gene, which contains the 2600-bp 5’-flanking
region of the ANF gene, namely, ANF(-2600)-Luc, was
kindly provided by Dr. Ichiro Shiojima from Chiba
University School of Medicine (Chiba-shi, Chiba, Ja-
pan). HEK-293 cells were cultured in Dulbecco’s
modified Eagle’s medium supplemented with 10%
fetal calf serum. The ANF(-2600)-Luc reporter con-
struct and an internal control reporter plasmid
pGL4.75 (hRluc/CMV, Promega) were used in tran-
sient transfection assay to explore the transcriptional
activation function of the GATA5 mutants. HEK-293
Int. J. Med. Sci. 2013, Vol. 10
cells were transfected with 0.4 μg of wild-type or
mutant pcDNA3.1-hGATA5 expression vector, 0.4 μg
of ANF(-2600)-Luc reporter construct, and 0.04 μg of
pGL4.75 control reporter vector using PolyFect
Transfection Reagent (Qiagen). For co-transfection
experiments, 0.2 μg of wild-type pcDNA3.1-hGATA5
together with 0.2 μg of mutant pcDNA3.1-hGATA5 or
0.2 μg of empty pcDNA3.1 vector were used in the
presence of 0.4 μg of ANF(-2600)-Luc and 0.04 μg of
pGL4.75. Firefly luciferase and Renilla luciferase ac-
tivities were measured with the Dual-Glo luciferase
assay system (Promega) 48h after transfection. The
activity of the ANF promoter was presented as fold
activation of Firefly luciferase relative to Renilla lu-
ciferase. Three independent experiments were per-
formed at minimum for wild-type and mutant
Data are expressed as means ± standard devia-
tions. Continuous variables were tested for normality
of distribution, and Student’s unpaired t test was used
for comparison of numeric variables between two
groups. Comparison of the categorical variables be-
tween two groups was performed using Pearson’s χ2
test or Fisher’s exact test when appropriate. A
two-tailed P value less than 0.05 was considered to be
Baseline characteristics of the study subjects
A cohort of 130 unrelated patients with TOF was
requited and clinically evaluated in contrast to a total
of 200 unrelated, ethnically-matched healthy indi-
viduals used as the controls. All the participants had
no established environmental risk factors for congen-
ital heart disease, such as maternal illness and drug
use in the first trimester of pregnancy, parental
smoking, and long-term exposure to toxicants and
ionizing radiation. The baseline clinical characteristics
of the 130 unrelated TOF cases are summarized in
GATA5 sequence variation
The entire coding region and the flanking splice
sites of the GATA5 gene was sequenced in the 130
unrelated patients with TOF. Two heterozygous se-
quence variations in GATA5 were identified in 2 out
of 130 patients, respectively. The total population
prevalence of GATA5 variation based on the cohort
patients was about 1.54%. Specifically, A transversion
of cytosine into guanine at the first nucleotide of co-
don 187 of the GATA5 gene (c.559C>G), equivalent to
the transition of arginine into glycine at amino acid
position 187 (p.R187G), was identified in the index
patient from family 1. A substitution of guanine for
adenine in the second nucleotide of codon 207
(c.620A>G), equal to the replacement of histidine by
arginine at amino acid 207 (p.H207R), was identified
in the proband from family 2. The sequence elec-
tropherograms showing the identified heterozygous
GATA5 variations in contrast to corresponding control
sequences are illustrated in Figure 1. A schematic di-
agram of GATA5 showing the structural domains and
the locations of the detected mutations is presented in
Figure 2. The variations were neither observed in 400
control chromosomes nor found in the NCBI’s SNP
database, which was consulted again on September
22, 2012 (http://www.ncbi.nlm.nih.gov/SNP).
A genetic scan of the mutation carriers’ family
members demonstrated that in each family, the varia-
tion was present in all affected family members
available, but absent in unaffected family members
examined. Analysis of the pedigrees showed that the
variations co-segregated with TOF with complete
penetrance. The pedigree structures of the 2 families
are shown in Figure 3. Additionally, in the family 1,
the proband’s grandfather (I-1) had also atrial septal
defect and electrocardiogram documented atrial fi-
brillation, and her aunt (II-2) had also patent ductus
arteriosus and paroxysmal atrial fibrillation. The
phenotypic characteristics and results of genetic
screening of the affected pedigree members are listed
in Table 3.
Table 2. Clinical characteristics of the 130 unrelated pa-
tients with tetralogy of Fallot (TOF).
Positive family history
Prevalence of TOF with other cardiac
TOF and ASD
TOF and PDA
TOF and AS
TOF and ASD and PDA
Incidence of arrhythmias
n or mean % or range
ASD: atrial septal defect, PDA: patent ductus arteriosus, AS: aortic stenosis.
Int. J. Med. Sci. 2013, Vol. 10
Table 3. Phenotypic characteristics and status of the GATA5 mutations of the affected pedigree members.
Gender Age at time of
Age at diagnosis of
II-2 F 35 1 TOF ±
Family 2 H207R
II-1 M 32 1 TOF ±
III-1 F 3 0 TOF ±
M: male, F: female, TOF: tetralogy of Fallot, ASD: atrial septal defect, PDA: patent ductus arteriosus, NA: not available or not applicable. ±: heterozygosity. a: age
Figure 1. Sequence electropherograms showing the GATA5 mutations in contrast to the corresponding controls. The arrow indicates the
heterozygous nucleotides of C/G in the proband from family 1 or A/G in the proband from family 2 (mutant); or the homozygous nu-
cleotides of C/C or A/A in the corresponding control individuals (wild-type). The square denotes the nucleotides constituting a codon of
Figure 2. Schematic diagram of GATA5 protein structure with the tetralogy of Fallot related mutations shown. The mutations identified
in patients with tetralogy of Fallot are shown above the structural domains. NH2 means amino-terminus; TAD, transcriptional activation
domain; ZF, zinc finger; NLS, nuclear localization signal; and COOH, carboxyl-terminus.
Int. J. Med. Sci. 2013, Vol. 10
Figure 3. Pedigree structures of the families with tetralogy of Fallot. Families are designated as family 1 and family 2, respectively. Family
members are identified by generations and numbers. Squares indicate male family members; circles, female members; closed symbols,
affected members; open symbols, unaffected members; a symbol with a slash, the deceased member; arrows, probands; ‘‘+’’, carriers of
the heterozygous mutations; and ‘‘−’’, non-carriers.
Alignment of multiple GATA5 protein se-
A cross-species alignment of multiple GATA5
protein sequences demonstrated that the affected
amino acids were highly conserved evolutionarily
(Figure 4), indicating that the amino acids are func-
Causative potential of GATA5 sequence vari-
The GATA5 sequence variations of c.559C>G and
c.620A>G were both automatically predicted to be
disease-causing, with P values of 0.99994 and 0.99934,
respectively. No SNPs in the altered regions were
found in MutationTaster database.
Transcriptional activity of the GATA5 mutants
As shown in Figure 5, the wild-type GATA5, the
R187G-mutant, and the H207R-mutant GATA5 acti-
vated the ANF promoter by ~12-fold, ~3-fold, and
~4-fold, respectively. When wild-type GATA5 was
co-expressed with the same amount of R187G-mutant
or H207R-mutant GATA5, the induced activation of
the ANF promoter was ~5-fold or ~6-fold. These re-
sults indicate that both GATA5 mutants are associat-
ed with significantly reduced activation activity
compared with their wild-type counterpart.
Figure 4. Alignment of multiple GATA5 protein sequences across species. The altered amino acids of p.R187 and p.H207 are completely
Figure 5. Functional defects of GATA5 caused by mutations. Activa-
tion of ANF-luciferase reporter in HEK-293 cells by wild-type GATA5
(WT), or mutant (R187G or H207R), alone or in combination, revealed
significantly decreased transactivational activity by mutant proteins.
Experiments were performed in triplicate and mean and standard
deviations are shown. ** and * represent P < 0.001 and P < 0.005,
respectively, when compared with wild-type GATA5.
Int. J. Med. Sci. 2013, Vol. 10
In the present study, two novel heterozygous
mutations of GATA5, p.R187G and p.H207R, were
identified in 2 families with TOF. In each family, the
mutant allele was present in all the affected family
members alive but absent in unaffected relatives ex-
amined and 400 normal chromosomes from an ethni-
cally matched control population. A cross-species
alignment of GATA5 protein sequences showed that
the altered amino acids were highly conserved evolu-
tionarily. The p.R187G and p.H207R variants were
both predicted to be pathogenic mutations, and the
biochemical analysis revealed that the GATA5 mutant
proteins were consistently associated with signifi-
cantly reduced transcriptional activation. Therefore, it
is very likely that functionally impaired GATA5 is
responsible for TOF in these families. To our
knowledge, this is the first report on the association of
GATA5 loss-of-function mutations with enhanced
susceptibility to TOF in humans.
The human GATA5 gene maps to chromosome
20q13.33 by fluorescence in situ hybridization, en-
coding a protein of 397 amino acids . By alignment
of GATA5 with GATA4, the functional domains of
GATA5 are predicted to consist of 2 transcriptional
activation domains (TAD1, amino acids 1-49; TAD2,
amino acids 107-154), 2 adjacent zinc fingers (ZF1,
amino acids 187-212; ZF2, amino acids 242-266), and 1
nuclear localization signal (NLS, amino acids
226-396). The two TADs are both pivotal for the tran-
scriptional activity of GATA5. The N-terminal ZF1 is
essential for DNA sequence recognition and binding
to the consensus motif, while the C-terminal ZF2 is
responsible for sequence specificity and stability of
protein-DNA binding. The NLS is a key to the
sub-cellular trafficking and distribution of GATA5.
The GATA5 mutations of p.R187G and p.H207R
identified in this study are both located in ZF1, thus
may be expected to have impact on the transcriptional
activation of GATA5 by interfering with the recogni-
tion and binding of GATA5 with target gene promot-
It has been substantiated that GATA5 is an up-
stream regulator of several genes expressed during
embryogenesis and cardiac morphogenesis, including
the genes that encode atrial natriuretic factor (ANF),
brain natriuretic peptide, α-myosin heavy chain, β
myosin heavy chain, and cardiac troponin C and I
(15). Hence, the functional roles of the GATA5 muta-
tions may be assessed by using biochemical analysis
of the transcriptional activity of the ANF promoter in
tool cells. In this study, the functional effect of the
novel p.R187G and p.H207R mutations of GATA5
identified in our familial TOF patients were evaluated
by transcriptional activity assay and the results
showed a significantly decreased transcriptional ac-
tivity on a downstream gene. The data suggest that
GATA5 loss-of-function mutations are potentially an
alternative molecular mechanism involved in TOF.
That functionally compromised GATA5 predis-
poses to congenital cardiovascular defects has been
demonstrated in animal models. In zebrafish, targeted
deletion of the GATA5 gene led to embryonic lethality
due to defects in endocardial and myocardial differ-
entiation and migration, a similar phenotype to cardia
bifida of GATA4-null zebrafish . Although the
GATA5-knockout mice were viable and without ap-
parent cardiac defects, the mice that were compound
heterozygous for both GATA5 and GATA4 or for both
GATA5 and GATA6 knockout died embryonically or
perinatally because of severe defects of the outflow
tract development including double outlet right ven-
tricle and ventricular septal defect . These exper-
imental findings corroborate an exquisite sensitivity
of the developing cardiovascular system to the levels
of GATA4, GATA5 and GATA6, and suggest that
these GATA factors mar act synergistically to regulate
downstream target genes.
It was interesting that atrial fibrillation was
documented in 2 TOF patients harboring the p.R187G
mutation of GATA5. Similar to our findings, atrial
fibrillation were previously confirmed in patients
with congenital cardiovascular defects who carry
GATA4, GATA5, or GATA6 mutations [41-47]. These
observations imply that congenital heart disease may
share a common genetic origin with atrial fibrillation.
It has been shown that the abnormal development of
heart, especially the pulmonary vein myocardium
contributes to the initiation and perpetuation of atrial
fibrillation by several possible pathological mecha-
nisms including enhanced intrinsic pacemaker activ-
ity and increased properties that facilitate reentrance
[48-50]. Genetic-labeling lineage tracing analyses have
revealed that NKX2-5 is highly expressed in the atria
and pulmonary myocardium and plays a pivotal role
in the localized formation of the sinoatrial node dur-
ing embryonic development. As a repressor of the
sinoatrial node lineage gene program, NKX2-5 func-
tions to limit pacemaker activity to the sinus node and
the atrioventricular node . Therefore, as an im-
portant transcriptionally cooperative partner of
NKX2-5 , GATA5, when mutated, may be in-
volved in the formation of the atrial electrophysio-
logical substrate in favor of atrial fibrillation.
In conclusion, this work provides the first ge-
netic evidence for the association of functionally
compromised GATA5 with increased vulnerability to
Int. J. Med. Sci. 2013, Vol. 10
TOF, implying the potential implications for the early
prophylaxis and personalized treatment of TOF.
We are really thankful to the subjects for their
devotion to the study. This work was supported in
part by grants from the National Natural Science
Fund of China (81070153, 81270161 and 30570768), the
Personnel Development Foundation of Shanghai,
China (2010019), the Natural Science Fund of Shang-
hai, China (10ZR1428000 and 10ZR1433100), and the
Key Program of Basic Research of Shanghai, China
The authors have declared that no competing
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