Identification of functional mutations in GATA4 in patients with congenital heart disease

Abstract

Congenital heart disease (CHD) is one of the most prevalent developmental anomalies and the leading cause of noninfectious morbidity and mortality in newborns. Despite its prevalence and clinical significance, the etiology of CHD remains largely unknown. GATA4 is a highly conserved transcription factor that regulates a variety of physiological processes and has been extensively studied, particularly on its role in heart development. With the combination of TBX5 and MEF2C, GATA4 can reprogram postnatal fibroblasts into functional cardiomyocytes directly. In the past decade, a variety of GATA4 mutations were identified and these findings originally came from familial CHD pedigree studies. Given that familial and sporadic CHD cases allegedly share a basic genetic basis, we explore the GATA4 mutations in different types of CHD. In this study, via direct sequencing of the GATA4 coding region and exon-intron boundaries in 384 sporadic Chinese CHD patients, we identified 12 heterozygous non-synonymous mutations, among which 8 mutations were only found in CHD patients when compared with 957 controls. Six of these non-synonymous mutations have not been previously reported. Subsequent functional analyses revealed that the transcriptional activity, subcellular localization and DNA binding affinity of some mutant GATA4 proteins were significantly altered. Our results expand the spectrum of GATA4 mutations linked to cardiac defects. Together with the newly reported mutations, approximately 110 non-synonymous mutations have currently been identified in GATA4. Our future analysis will explore why the evolutionarily conserved GATA4 appears to be hypermutable.

Figure 1. Distribution of the identified GATA4 mutations and multiple sequence alignment across species.

(A) A schematic diagram of the GATA4 protein and the locations of the 12 non-synonymous mutations identified in this study (solid red star indicates a mutation that has not been previously reported; empty red star indicates a CHD-specific mutation that has been previously reported; solid black star indicates a mutation that is also found in controls). N-domain, N terminal domain; NZf, N terminal zinc finger domain; CZf, C terminal zinc finger domain; C-domain, C terminal domain. (B) Multiple sequence alignment of the GATA4 protein across different species. The result shows that residues 210 and 250 are highly evolutionarily conserved, and residues 360 and 442 are conserved in mammals.

Figure 2. Subcellular location of GATA4 wild type and mutant proteins.

GFP (green) represents the over expressed GATA4 protein, and DAPI (blue) represents the location of the nucleus. The subcellular localization of pEGFP-GATA4 wild-type (WT) and mutant-type proteins shows that wild-type GATA4 completely localized to the nucleus, while mutated GATA4 proteins were partially distributed in the cytoplasm besides the nucleus.

Figure 3. Relative luciferase activity of GATA4 wild type and mutant proteins in Hela cells.

(A) Schematic of the human ANF-Luc. The region from the proximal −300 to −130 shows the conserved GATA transcription factor binding sites involved in regulating ANF expression. Two consensus GATA4 binding sites are highlighted in red. (B) Relative luciferase activity of wild type (WT) and CHD specific GATA4 mutant expression constructs that were co-transfected with ANF-Luc in Hela cells. Mean±SD are shown in the histogram. The relative luciferase activity for the A66T mutant was 160±8%, for the T280M mutant was 51±5%, for the A353T mutant was 178±14%, and for the E360G mutant was 78±6%. Student t-test was performed and four mutants caused significant transcriptional activation difference compared with wild-type GATA4 (*, P<0.05). Other mutants did not change the ANF-Luc activation significantly.

Figure 4. DNA binding affinity of GATA4 wild type and mutants.

(A) Detection of the endogenous expression of GATA4 in 293T, COS7, Hela and H1299 cells transfected with pCMV-Myc empty vector. (B) Protein levels of nuclear extracts were kept equivalent between pCMV-Myc-GATA4 wild type and each mutant. (C) GATA4 A74D, G150W and T280M mutant proteins demonstrated abnormal DNA binding affinity compared to wild type GATA4 by using electromobility shift assay of biotin-labelled GATA binding element. 100-fold wild type cold oligonucleotides (WT) could compete for GATA4 binding while 100-fold cold mutant oligonucleotides (MT) failed to compete.