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Logo of nihpaAbout Author manuscriptsSubmit a manuscriptHHS Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
Pediatr Res. Author manuscript; available in PMC 2011 October 1.
Published in final edited form as:
PMCID: PMC2940936

Identification of GATA6 sequence variants in patients with congenital heart defects


While the etiology for the majority of congenital heart disease (CHD) remains poorly understood, the known genetic causes are often the result of mutations in cardiac developmental genes. GATA6 encodes for a cardiac transcription factor, which is broadly expressed in the developing heart and is critical for normal cardiac morphogenesis, making it a candidate gene for congenital heart defects in humans. The objective of this study was to determine the frequency of GATA6 sequence variants in a population of individuals with a spectrum of cardiac malformations. The coding regions of GATA6 were sequenced in 310 individuals with CHD. We identified two novel sequence variations in GATA6 that altered highly conserved amino acid residues (A178V and L198V) and were not found in a control population. These variants were identified in two individuals (one with tetralogy of Fallot and the other with an atrioventricular septal defect in the setting of complex CHD). Biochemical studies demonstrate that the GATA6 A178V mutant protein results in increased transactivation ability when compared to wildtype GATA6. These data suggest that non-synonymous GATA6 sequence variants are infrequently found in individuals with CHD.


Congenital heart defects (CHDs) are the most common developmental anomaly with an incidence of nearly 1% and are the leading non-infectious cause of infant mortality (1). Advances in developmental biology have led to the identification of numerous transcriptional regulators, signaling molecules and structural genes that are critical for normal cardiac morphogenesis. This molecular understanding of cardiac embryogenesis has assisted in the discovery of CHD-causing genes, which have been identified using positional cloning or candidate gene screening approaches (2,3). In spite of these significant advances, the etiology of most CHD remains unknown.

Mutations in the GATA family of zinc finger transcription factors have been linked to human disease, and specifically, GATA4 mutations were found to be associated with CHDs (4,5,6). Similar to GATA4, GATA6 is expressed in the developing heart but also has additional areas of expression in the developing vascular smooth muscle (7,8). Both are highly conserved genes and bind identical nucleotide sequences in genomic DNA and regulate similar target genes (9,10,11). Gata4 null mice exhibit early defects in heart formation and ventral foregut closure (12,13), while Gata6-null mice die after implantation because of defects in the visceral endoderm and extraembryonic development. More recent studies have demonstrated that conditional deletion of Gata6 in neural crest-derived smooth muscle leads to malformations of the cardiac outflow tract and the aortic arch arteries (14,15). Consistent with this role in outflow tract development, two loss-of-function GATA6 mutations were recently reported to cause persistent truncus arteriosus in humans (16). In addition to its role in outflow tract defect development, Gata6 has also been shown to genetically interact with the cardiac transcription factors, Gata4 and Tbx5, which cause cardiac septation defects in humans, suggesting that mutations in GATA6 may be associated with other forms of CHD (17,18,19).

To determine the frequency of GATA6 sequence variants and define the phenotypic subset of CHDs that is associated with genetic variations in GATA6, we screened the GATA6 gene in 310 children with a spectrum of CHDs. We hypothesized that mutations in GATA6 are found in individuals with a spectrum of CHD as reported for GATA4 (5,6). Here, we have identified two novel GATA6 sequence variants that altered highly conserved amino acid residues and that were not found in control individuals. Biochemical assays suggested gain-of-function effects for the GATA6 A178V variant, which was identified in an individual with tetralogy of Fallot. Our data suggest that sequence variants in GATA6 contribute to a subset of human CHD.


Radioactive section in situ hybridization

C57Bl6 mice were maintained on a 0600 and 1800 hour light-dark cycle with noon on the day of observation of a vaginal plug defined as embryonic day (E)0.5. Mothers were sacrificed and the embryos harvested at E13.5. In situ hybridization was performed using 35S-labeled antisense probes for Gata6 as previously described (19).

Study Population

The subjects were comprised of 310 unrelated individuals (174 males and 136 females) who received their cardiovascular care at Children’s Medical Center of Dallas. The individuals were of varied ethnicity (153 European Americans, 123 Hispanics and 34 African Americans) and had a variety of CHDs (Table 1). Between January 2002 and December 2008, subjects were prospectively recruited for genetic testing and informed consent obtained according to protocol as approved by the Institutional Review Board at the University of Texas Southwestern Medical Center. The cohort was randomly selected from this population and patients with known chromosomal abnormalities were excluded from the study. Patients underwent complete cardiac evaluation at Children’s Medical Center of Dallas and echocardiogram, cardiac catheterization and operative reports were reviewed, when available. Venous blood samples were collected and genomic DNA isolated using the PUREGENE kit (Gentra Systems) from affected subjects. Genomic DNA was obtained from a control population consisting of 288 individuals of variable ethnicity (96 European Americans, 96 Hispanics and 96 African Americans). The control population did not have known congenital heart defects but subclinical cardiac malformations such as bicuspid aortic valve or patent foramen ovale were not excluded.

Table 1
Diagnoses of screened study population with Congenital Heart Defects

Sequencing of GATA6

All seven exons of GATA6 were sequenced in the patient population and all sequence variations identified. Only sequence variations that predicted a non-synonymous amino acid substitution were screened in the control population by direct sequencing. The sequencing primers are available on request. PCR amplification was performed using the BD Biosciences Advantage GC Genomic PCR kit following the manufacturer’s instructions, with an annealing temperature of 60°C.

Plasmid Construction and Site-Directed Mutagenesis

The human GATA6 expression vector was generously provided by Dr. Walter L. Miller (20). Point mutations were introduced into this plasmid containing the human GATA6 cDNA to generate the GATA6 A178V and GATA6 L198V mutant expression vectors which were verified by direct sequencing.

Transactivation studies

HeLa cells were transfected using Fugene 6 (Roche) with 100ng of either alpha myosin heavy chain or atrial natriuetic factor luciferase reporter, 100ng of CMV-LacZ plasmid and 300ng of wildtype GATA6, GATA6 A178V, or GATA6 L198V plasmids. Immunoblots were used to verify appropriate protein expression. Transactivation assays were performed and luciferase activity was measured 48h after transient transfection as previously described (21). Three independent experiments were performed in triplicate with the alpha myosin heavy chain (αMHC) and atrial natriuetic factor (ANF) luciferase reporters. Luciferase data are shown as fold activation as they are normalized for transfection efficiency using beta-galactosidase. Statistical comparisons were performed using Student’s t test. P value < 0.05 was considered significant.


Cardiovascular Expression of Gata6

To determine expression of Gata6 in the later stages of heart development, in situ hybridization was performed in wild type E13.5 mouse embryos. Gata6 transcripts were expressed in the atrial and ventricular myocardium with higher levels of expression seen in the atrioventricular valve leaflets (Figure 1a and 1b) similar to previous reports in E9.5 to E12.5 embryos (15,19). The endocardial cushions, the precursors of the mature atrioventricular valve leaflets, also express of Gata6 mRNA in E11.5 murine hearts (19). The highest levels of Gata6 expression were seen in the smooth muscle cells of the aorta and pulmonary artery, as previously described while lower levels were detectable in the pulmonary valve leaflets (Fig. 1c–f) (15).

Figure 1
Expression of Gata6 in embryonic heart by radioactive section in situ hybridization

Identification of GATA6 Sequence Variations

We screened for mutations by direct DNA sequencing of the coding regions of GATA6 in 310 individuals with diverse forms of non-syndromic CHD (Table 1). Analysis of the sequencing data resulted in the identification of two novel sequence variations that predicted non-synonymous amino acid substitutions at codons 178 and 198, A178V and L198V, respectively (Table 2 and Fig. 2). The L198V variant was identified in a patient with isolated tetralogy of Fallot (single malalignment ventricular septal defect with subvalvar/valvar pulmonary stenosis and a normal aortic arch), while the A178V variant was found in a patient with an unbalanced atrioventricular septal defect, hypoplastic left ventricle and two muscular ventricular septal defects with no additional evidence of heterotaxy syndrome (Table II). Both of these changes were identified in individuals of Hispanic ethnicity but neither of these nucleotide changes was observed in the control population of 288 individuals (576 alleles); of which 96 (192 alleles) were of Hispanic ethnicity. The A178V variation was identified in an unaffected parent while the inheritance of the L198V variation was unable to be tested. Both of these nucleotide changes altered highly conserved amino acid residues in the GATA6 protein (Fig. 2C). Although, the substitutions did not lie in known functional domains, the altered alanine residue was located in a conserved polyalanine tract. Additionally, we identified a sequence variation that predicted a non-synonymous amino acid substitution (G15R) with a minor allele frequency of 2.6% (16/620 alleles) in the affected population. This sequence variation was also found in the control population (6.9%=40/576 alleles) suggesting that it likely represents a single nucleotide polymorphism. We identified several other novel sequence variations that predicted synonymous amino acid changes in patients with CHD (Table 3).

Figure 2
Novel GATA6 sequence variations alter highly conserved amino acids
Table 2
Unique non-synonymous sequence variations identified in children with CHD
Table 3
Single nucleotide polymorphisms and rare synonymous sequence variations in children with CHD and in control population

GATA6 A178V Has Increased Transcriptional Activity In Vitro

To determine if the amino acid substitutions in GATA6 resulted in functional abnormalities, we tested them using in vitro transfection assays. We generated mutant expression constructs for the GATA6 A178V and GATA6 L198V nucleotide variants. Luciferase reporter assays were performed using the Gata-dependent cardiac enhancers, αMHC and ANF, upstream of a luciferase reporter. The GATA6 A178V and L198V proteins were equally expressed in HeLa cells when compared to wildtype GATA6 (data not shown). Transfection of GATA6 A178V expression plasmid demonstrated a two-fold increase in transactivation ability on αMHC-luciferase reporter as compared to transfection with equal amounts of wildtype GATA6 plasmid (p value = 0.013) (Fig. 3). A similar pattern of activation increase was found with ANF-luciferase reporter (p value = 0.001) (Fig. 3). The GATA6 L198V variant did not show any difference in transactivation ability compared to wild type GATA6.

Figure 3
In vitro functional analysis of Gata6 sequence variations


Mutations in cardiac transcription factors have been implicated as genetic etiologies of human CHD (22). We have identified two rare GATA6 sequence variants, in a subset of individuals with CHD, which resulted in non-synonymous amino acid substitutions in two subjects after screening a population of 310 affected individuals. In vitro transactivation studies demonstrated that the GATA6 A178V variant is a gain-of-function mutation with increased transactivation ability when compared to wildtype GATA6. These findings highlight the importance of the GATA6 as an etiologic gene for a subset of CHD and suggest that appropriate levels of GATA6 are critical for normal heart development in humans.

The prevalence of GATA6 sequence variants in our study was 0.6%, consistent with similar large population based studies of non-familial CHD where the mutation prevalence of a single CHD-causing gene is less than 2% (5,6,23). The report by Kodo, et al. identified two GATA6 mutations in a population of 21 individuals with truncus arteriosus (16). Interestingly, we did not identify any GATA6 mutations in 12 subjects with truncus arteriosus. However, we did identify a gain-of-function mutation in an individual with tetralogy of Fallot, which is embryologically related to truncus arteriosus. Therefore, it is possible that the prevalence of GATA6 mutations is higher in a population of subjects with conotruncal abnormalities. In our study, this would represent an incidence of 2% (1/45). Of note, the gain-of-function GATA6 A178V mutation was inherited from an unaffected parent. The finding of incomplete penetrance has been most recently shown with NOTCH1 variants, which exhibit in vitro functional deficits, and were identified in individuals with left-sided cardiac malformations and their unaffected parents (24). For the L198V variant we did not identify any in vitro functional abnormalities in the assays that we utilized. The nucleotide variant is rare and alters a highly conserved leucine residue and ultimately, biochemical deficits may exist in transactivation assays with other luciferase reporters or protein-protein interactions due to alterations in structure.

Normal cardiac development requires adequate gene dosage of cardiac transcription factors. This has been shown in both murine and human studies and classic examples include NKX2-5, TBX5, GATA4, and TBX1 where loss-of-function mutations disrupt cardiac morphogenesis in humans and mice (25). Previous studies suggest that increased dosage of TBX5 or TBX1 is associated with phenotypes consistent with Holt-Oram syndrome and Shprintzen syndrome, respectively, while a gain-of-function mutation in TBX20 was recently reported to be associated with atrial septal defects and cardiac valve defects (26,27,28). Additionally, gain-of-function mutations in PTPN11 have been well described to be associated with the cardiac defects found in Noonan syndrome (29). Our in vitro studies suggest that the GATA6 A178V sequence variant represents a gain-of-function mutation with its increased transactivation ability. Consistent with this, the alanine is located within a polyalanine tract, which has been proposed to function as a transcriptional repression domain (30). This finding is particularly interesting in light of studies in Xenopus which have demonstrated that overexpression of GATA-6 disrupts cardiac development by preventing differentiation and blocking expression of GATA target genes, cardiac actin and XMLC2, a heart specific myosin light chain (31). Consistent with this, mutations in the GATA target, alpha myosin heavy chain (MYH6) are linked to atrial septal defects in humans (32). Further experimentation is necessary to understand the effect of this substitution on GATA6 protein structure and function during cardiac development.

In conclusion, genetic abnormalities involving cardiac development genes are increasingly being discovered to be associated with CHD in humans. In this study, we have identified two novel sequence variations in the cardiac transcription factor, GATA6, to be associated with CHD and similar to other investigations, genetic variants of single gene are associated with only a small subset of CHD. Studies of larger well-phenotyped populations will need to be performed for improved genotype-phenotype correlations and determine if GATA6 mutations are found in a higher frequency with cardiac septal defects and conotruncal abnormalities. Ultimately, further studies elucidating the role of GATA6 in the developing cardiovascular system will be required to increase our knowledge of the genetic basis of CHD and to provide more personalized genetic counseling and develop novel preventive therapies.


Statement of financial support: D.S. is supported by NIH/NHLBI (R01HL080592 and R01HL057181) and California Institute for Regenerative Medicine. V.G. is supported by grants from the NIH/NHLBI (R01HL088965) and the Children’s Heart Foundation.

The authors thank the families for their participation; C. Rains, J. Neumann, and A. Winborn for research coordinator support; C. Turner for technical support; members of the McDermott Center for Human Growth and Development for assistance with sequencing; the Divisions of Pediatric Cardiology and Pediatric Cardiothoracic Surgery at Children’s Medical Center of Dallas for assistance with clinical information and management; and Dr. K.L. McBride for critical review of the manuscript.


congenital heart disease
alpha-myosin heavy chain
atrial natriuetic factor


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