A cohort of 52 unrelated patients with TOF, who underwent cardiac surgery at Shanghai Renji Hospital in China between January 2009 and December 2011, was recruited. Their age ranged from 6 months to 7 years, with an average of 1.24 years at the time of surgery. The patients were evaluated by skillful cardiologists and the diagnosis was made by echocardiography and confirmed by direct view during surgical procedure. The patients with known chromosomal abnormalities or syndromic cardiovascular defects, such as DiGeorge, Alagille, Noonan, Holt-Oram, Marfan and Char syndrome, were excluded from the study. The sample size was adequate to draw the conclusion that the absence of somatic mutations is significant, according to the report by Draus et al (42).

A total of 46 unrelated patients (25 males and 21 females) with rheumatic heart disease undergoing cardiac valve replacement, and 200 unrelated healthy individuals (105 males and 95 females) randomly selected from those undergoing routine physical examinations, were enrolled as controls. In terms of individual medical history and echocardiographic record, the control individuals had no apparent congenital cardiovascular defects, except for subclinical cardiac aberrations such as bicuspid aortic valve or patent foramen ovale.

The participants were Chinese Han people. The ethnic origin of a participant was ascertained by a combination of self-reported ethnicity and a personal questionnaire regarding birthplace, language, religion and ancestry. The study protocol was reviewed and approved by the local Institutional Ethics Committee and written informed consent was obtained from all participants or their guardians prior to the study.

The cardiac muscle tissues from the right ventricular outflow tracts of TOF patients were resected during the routine cardiac surgery procedures. At the time of resection, the discarded cardiac tissue sample was collected after cleaning the blood stain by sterile normal saline and stored at −80°C. Meanwhile, the patient’s discarded peripheral venous blood with sodium citrate was collected (the blood samples were mostly used for activated partial thromboplastin time and prothrombin time tests before surgery). The discarded cardiac specimens from the cardiac valves and matched blood samples of the patients undergoing cardiac valve replacement due to rheumatic valvular disease, and the peripheral venous blood samples of healthy individuals were prepared as controls.

The somatic DNA was extracted from freshly frozen tissues using QIAamp DNA FFPE Tissue kit (Qiagen GmbH, Hilden, Germany) according to the manufacturer’s instructions. The genomic DNA was extracted from peripheral blood lymphocytes with Wizard Genomic DNA purification kit (Promega, Madison, WI, USA).

The primers for amplification of the coding exons and flanking splicing sites of the GATA6 gene were designed as previously described (39). Polymerase chain reaction (PCR) was performed in an automated Perkin-Elmer 9700 Thermal Cycler (Applied Biosystems, Foster City, CA, USA). The reaction mixture for PCR used in the present series of experiments consisted of 2 μl of genomic DNA (50–100 ng/μl), 2.5 μl of 10× Taq Buffer, 5 μl of 5× Q Solution, 2 μl of dNTP Mixture (2.5 mM each), 0.5 μl of each primer (20 mM each), 0.25 μl (1.25 U) of HotStar TaqDNA polymerase (Qiagen), and 12.25 μl of deionized H₂O, with a final volume of 25 μl. PCR was carried out under the following conditions: pre-denaturation at 95°C for 15 min, followed by 35 cycles of denaturation at 95°C for 1 min, annealing at 62°C for 30 sec, and extension at 72°C for 1 min, and final extension at 72°C for 10 min. To avoid potential carry-over contamination of the PCR mixture, all reactions were performed under stringent conditions as recommended by Kwok and Higuchi (49). 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 under an ABI PRISM 3130XL DNA Analyzer (were from Applied Biosystems). The sequencing primers were the same as previously designed for specific region amplification. The DNA sequences were analyzed with the DNA Sequencing Analysis Software v5.1 (Applied Biosystems). The sequence variation was validated by re-sequencing an independent PCR-generated amplicon from the same subject and met the standard quality control thresholds with a call rate >99%. Additionally, an identified GATA6 sequence variation was searched in the single nucleotide polymorphism (SNP) database of the National Center for Biotechnology Information to confirm the novelty (http://www.ncbi.nlm.nih.gov/SNP).

Multiple GATA6 protein sequences across various species were aligned using the online program of Muscle, version 3.6 (http://www.ncbi.nlm.nih.gov).

The pathogenic potential of a GATA6 sequence variation was predicted by MutationTaster (an online program at http://www.mutationtaster.org), which automatically gave a probability for the variation to be either a disease-causing 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 prediction).

The recombinant expression plasmid pcDNA3-hGATA6 was kindly provided by Dr Angela Edwards-Ghatnekar, from the Division of Rheumatology and Immunology, Medical University of South Carolina, Charleston, SC, USA. The atrial natriuretic factor (ANF)-luciferase reporter gene, which contains the 2600-bp 5′-flanking region of the ANF gene, i.e., ANF(-2600)-Luc, was kindly provided by Dr Ichiro Shiojima, from the Department of Cardiovascular Science and Medicine, Chiba University Graduate School of Medicine, Chuo-ku, Chiba, Japan. The identified mutation was introduced into the wild-type GATA6 using a QuickChange II XL Site-Directed Mutagenesis kit (Stratagene, La Jolla, CA, USA) with a complementary pair of primers. The mutant was sequenced to confirm the desired mutation and to exclude any other sequence variations.

HEK-293 cells were cultured in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal calf serum. The ANF(-2600)-Luc reporter vector and an internal control reporter plasmid pGL4.75 (hRluc/CMV, Promega) were used in transient transfection assays to evaluate the transcriptional activation function of the GATA6 mutants. HEK-293 cells were transfected with 0.4 μg of wild-type or mutant pcDNA3-hGATA6 expression vector, 0.4 μg of ANF(-2600)-Luc reporter construct, and 0.04 μg of pGL4.75 control reporter vector using Lipofectamine^® 2000 Transfection reagent (Invitrogen, Carlsbad, CA, USA). For co-transfection experiments, 0.2 μg of wild-type pcDNA3-hGATA6, 0.2 μg of empty pcDNA3 plasmid or 0.2 μg of mutant pcDNA3-hGATA6, 0.4 μg of ANF(-2600)-Luc, and 0.04 μg of pGL4.75 were used. Firefly luciferase and Renilla luciferase activities were measured with the Dual-Glo luciferase assay system (Promega) 48 h after transfection. The activity of the ANF promoter was presented as fold activation of Firefly luciferase relative to Renilla luciferase. Three independent experiments were performed at minimum for wild-type and mutant GATA6.

Data are expressed as the means ± SD. Continuous variables were examined for normality of distribution and the Student’s unpaired t-test was used for the comparison of numeric variables between two groups. A comparison of the categorical variables between two groups was conducted using Pearson’s χ² test or Fisher’s exact test when appropriate. A two-tailed P-value <0.05 was considered to indicate a statistically significant difference.

A cohort of 52 unrelated Han-nationality patients with sporadic TOF, who underwent cardiac surgery, was enrolled and clinically evaluated as comparison to 46 unrelated Han-race patients with rheumatic heart disease undergoing cardiac valve replacement and 200 ethnically matched, unrelated healthy individuals used as controls. The subjects had neither positive family history of CHD nor established environmental risk factors for CHD, such as maternal illness and drug use in the first trimester of pregnancy, parental smoking, and chronic exposure to toxicants and ionizing radiation. The baseline clinical characteristics of the study population are summarized in Table I.

Peripheral venous blood samples were available for all 298 participants. The malformed myocardial tissue samples were obtained from 52 unrelated patients with sporadic TOF who underwent surgical resection of the right ventricular outflow musculature for relieving the stenosis. Generally, the cardiac muscle fragment obtained was ~5×5 mm in size. In addition, the cardiac valvular tissue samples used as controls were obtained from surgical discards of 46 unrelated patients undergoing cardiac valve replacement.

Genomic DNA from the malformed cardiac tissues of 52 TOF patients, the cardiac valvular tissues of 46 patients with rheumatic heart disease, and the peripheral blood lymphocytes of the 298 participants, were screened for GATA6 mutations. As a result, 2 heterozygous mutations in GATA6 were identified in the fresh pathological myocardial tissues of 2 TOF patients, respectively, with a mutational prevalence of ~3.85%. Specifically, a substitution of thymine for guanine in the first nucleotide of codon 367 of the GATA6 gene (c.1099G>T), resulting in a truncated protein with only N-terminal 366 amino acids left (p.G367X), was identified in the cardiac tissue of a 1-year-old male TOF patient. A replacement of guanine by thymine at coding nucleotide 1180 (c.1180G>T), predicting the transition of glycine to cysteine at amino acid position 394 (p.G394C), was identified in the cardiac tissue of a 2-year-old female TOF patient. The sequence chromatograms showing the detected heterozygous GATA6 mutations as comparison to corresponding control sequences are depicted in Fig. 1. A schematic diagram of GATA6 showing the structural domains and the locations of the identified mutations is presented in Fig. 2. The 2 mutations were neither observed in the cardiac valvular tissues of 46 patients with rheumatic heart disease nor found in the peripheral blood samples of the 298 participants. Neither of the 2 mutations was reported in the SNP database at the National Center for Biotechnology Information, which was consulted again on August 20, 2012. Additionally, during a 24-h period of ambulatory electrocardiographic monitoring, no atrial fibrillation was observed in these 2 mutation carriers.

A cross-species alignment of multiple GATA6 protein sequences demonstrated that the affected amino acid p.G394 was completely conserved evolutionarily and the affected amino acid p.G367 was relatively conserved evolutionarily (Fig. 3). However, the amino acids deleted by the p.G367× mutation constitute functionally important structural domains, including 2 zinc fingers and 1 nuclear localization signal (Fig. 2).

The GATA6 mutations of c.1099G>T and c.1180G>T were both automatically predicted to be disease-causing, with P-values of 1.000000 and 0.999997, respectively. No SNPs in the altered regions were found in the MutationTaster database.

The wild-type GATA6, the G367X-mutant, and the G394C-mutant GATA6 activated the ANF promoter by ~12-, 1- and 3-fold, respectively. When wild-type GATA6 was co-expressed with the same amount of G367X-mutant or G394C-mutant GATA6, the induced activation of the ANF promoter was ~6- or 5-fold, respectively. These results indicate that both GATA6 mutants are associated with significantly reduced activation activity compared with their wild-type counterpart (Fig. 4).