Somatic GATA4 mutation contributes to tetralogy of Fallot

Abstract

Tetralogy of Fallot (TOF) is the most prevalent cyanotic congenital heart pathology and causes infant morbidity and mortality worldwide. GATA-binding protein 4 (GATA4) serves as a pivotal transcriptional factor for embryonic cardiogenesis and germline GATA4 mutations are causally linked to TOF. However, the effects of somatic GATA4 mutations on the pathogenesis of TOF remain to be ascertained. In the present study, sequencing assay of GATA4 was performed utilizing genomic DNA derived from resected heart tissue specimens as well as matched peripheral blood specimens of 62 patients with non-familial TOF who underwent surgical treatment for TOF. Sequencing of GATA4 was also performed using the heart tissue specimens as well as matched peripheral venous blood samples of 68 sporadic cases who underwent heart valve displacement because of rheumatic heart disorder and the peripheral venous whole blood samples of 216 healthy subjects. The function of the mutant was explored by dual-luciferase activity analysis. Consequently, a new GATA4 mutation, NM_002052.5:c.708T>G;p.(Tyr236*), was found in the heart tissue of one patient with TOF. No mutation was detected in the heart tissue of the 68 cases suffering from rheumatic heart disorder or in the venous blood samples of all 346 individuals. GATA4 mutant failed to transactivate its target gene, myosin heavy chain 6. Additionally, this mutation nullified the synergistic transactivation between GATA4 and T-box transcription factor 5 or NK2 homeobox 5, two genes causative for TOF. Somatic GATA4 mutation predisposes TOF, highlighting the significant contribution of somatic variations to the molecular pathogenesis underpinning TOF.

Keywords: GATA4; biological assay; congenital heart disease; molecular pathogenesis; tetralogy of Fallot; transcriptional factor.

Figure 1

Somatic GATA4 mutation accountable for TOF. (A) Sequence chromatograms illustrating GATA4 mutation identified in a case with TOF (mutant) compared with a healthy subject (wild-type). Arrow sign points to the mutation site. (B) Schematics displaying the critical functional domains of GATA4 with the Tyr236* mutation shown. NLS, nuclear localization signal; ZF, zinc finger; TAD, trans-activation domain; TOF, tetralogy of Fallot.

Figure 2

Functional loss of Tyr236*-mutant GATA4. In routinely cultivated COS-7 cells overexpressing various interest proteins (Tyr236*-mutant GATA4, wild-type GATA4, firefly luciferase and Renilla luciferase), dual-luciferase reporter gene assay of the transactivation of the MYH6 promoter-driven firefly luciferase by Tyr236*-mutant or wild type GATA4, singly or in combination, unveiled that the Tyr236* mutant lost transactivation function. ^##P<0.001 and ^#P<0.005 vs. GATA4 (1.0 µg). Luc, luciferase; GATA4, GATA-binding protein 4; MYH6, myosin heavy chain 6.

Figure 3

Lost synergistic transactivation between Tyr236*-mutant GATA4 and NKX2.5 or TBX5. In cultured COS-7 cells overexpressing various interest proteins (Tyr236*-mutant GATA4, wild-type GATA4, NKX2.5, TBX5, firefly luciferase and Renilla luciferase), dual-luciferase activity measurement of the synergistic activation of ANP by GATA4 in combination with NKX2.5 or TBX5 showed that synergy was disrupted by the Tyr236* mutation. ^#P<0.001. Luc, luciferase; GATA4, GATA-binding protein 4; NKX2.5, NK2 homeobox 5; ANP, atrial natriuretic peptide; TBX5, T-box transcription factor 5.