Identification and functional analysis of genetic variants in TBX5 gene promoter in patients with acute myocardial infarction

Background Coronary artery disease (CAD), including acute myocardial infarction (AMI), is a common complex disease. Although a great number of genetic loci and variants for CAD have been identified, genetic causes and underlying mechanisms remain largely unclear. Epidemiological studies have revealed that CAD incidence is strikingly higher in patients with congenital heart disease than that in normal population. T-box transcription factors play critical roles in embryonic development. In particular, TBX5 as a dosage-sensitive regulator is required for cardiac development and function. Thus, dysregulated TBX5 gene expression may be involved in CAD development. Methods TBX5 gene promoter was genetically and functionally analysed in large groups of AMI patients (n = 432) and ethnic-matched healthy controls (n = 448). Results Six novel heterozygous DNA sequence variants (DSVs) in the TBX5 gene promoter (g.4100A > G, g.4194G > A, g.4260 T > C, g.4367C > A, g.4581A > G and g.5004G > T) were found in AMI patients, but in none of controls. These DSVs significantly changed the activity of TBX5 gene promoter in cultured cells (P < 0.05). Furthermore, three of the DSVs (g.4100A > G, g.4260 T > C and g.4581A > G) evidently modified the binding sites of unknown transcription factors. Conclusions The DSVs identified in AMI patients may alter TBX5 gene promoter activity and change TBX5 level, contributing to AMI development as a rare risk factor.


Introduction
Coronary artery disease (CAD), including acute myocardial infarction (AMI), is a common complex disease. Although genome-wide association studies have identified a great number of genetic loci and variants for CAD and AMI, genetic causes and underlying mechanisms for CAD and AMI remain largely unclear [1,2]. Recent epidemiological studies have demonstrated that incidence of CAD and AMI is strikingly higher in patients with congenital heart disease than that in normal population [3,4]. Dysregulation of cardiac genes and embryonic developmental genes have been associated with congenital heart diseases [5][6][7][8][9]. Therefore, it is postulated that dysregulation of cardiac developmental genes may also contribute to the CAD development.
T-box (TBX) transcription factors are a group of factors with a highly conserved DNA-binding domain (T-box), and play critical roles in embryonic development. There are 18 T-box family members, including TBX transcriptional factor 5 [TBX5] [10]. TBX5 gene is expressed in the embryonic and adult heart. TBX5 is required in the early heart tube formation, cardiac chamber morphogenesis and cardiomyocyte differentiation. During the cardiac development, TBX5 activates multiple downstream target genes, including atrial natriuretic factor (ANF), connexin 40 (CX40) and serum response factor (SRF). TBX5 functions alone or synergistically with interacting partners, such as NK2 transcription factor related locus 5 (NKX2-5), GATA transcription factor 4 (GATA4) and TBX20 [11][12][13][14][15]. TBX5 is also expressed in the central conduction system, and required for the patterning and maturation of the cardiac conduction system [16,17]. In addition, TBX5 gene is essential to limb identity and limb development [18][19][20]. Mutations in TBX5 gene cause Holt-Oram syndrome (HOS), which is characterized by congenital heart defects and upper limb abnormalities [21,22]. Haploinsufficiency of TBX5 in ventricle myocytes in mice leads to diastolic dysfunction of heart, indicating that TBX5 is required for heart function in adults [23].
In human embryonic heart, TBX5 gene is expressed in myocardium, embryonic epicardium and coronary vasculature [24]. Animal experiments have shown that TBX5 regulates epicardial formation and coronary vasculogenesis in a dose-dependent manner [25]. Coronary artery abnormalities have been reported in HOS patients, further suggesting that TBX5 is involved in the coronary vessel formation [26][27][28][29]. Developmental biological studies have shown that TBX5 is a dose-sensitive regulator [30]. A gain-of-function TBX5 gene mutation is associated with atypical HOS and paroxysmal atrial fibrillation [31]. In addition, TBX5 is crucial to the cytokine gene expression in human T cells and fibroblast cells, indicating TBX5 involvement in inflammation [32,33]. Therefore, altered levels of TBX5 may affect coronary vessel formation and inflammation, contributing to AMI development. In this study, we genetically and functionally analyzed the TBX5 gene promoter in large cohort of AMI patients and ethnic-matched healthy controls.

Study population
During the period from April, 2014 to August, 2016, the AMI patients (n = 432, male 284, female 148, median age 62.00 years) were recruited from Cardiac Care Unit, Affiliated Hospital of Jining Medical University, Jining, Shandong Province, China. AMI diagnosis criteria included typical clinical manifestations, changed ECG and biochemical markers (troponin or creatine kinase-MB), or coronary angioplasty. The ethnic-matched healthy controls (n = 448, male 235, female 213, median age 48.00 years) were recruited from the same hospital. The healthy controls with familial history of AMI, CAD and congenital heart diseases were excluded. This study protocol was approved by the Human Ethic Committee of Affiliated Hospital of Jining Medical University. This work was conducted according to the principles of the Declaration of Helsinki. Written informed consents were obtained from all participants.

DNA sequencing analysis of TBX5 gene promoter
Genomic DNAs were extracted from peripheral leukocytes isolated from venous blood. DNA fragments for TBX5 gene promoter were generated by PCR and directly sequenced as previously reported [6]. PCR primers were shown in Table 1. DNA sequence variants (DSVs) were identified by comparing to wild type TBX5 gene promoter.

TBX5 gene promoter activity with dual-luciferase reporter assay
Wild type and variant TBX5 gene promoters (1294 bp) were generated, and inserted into pGL3-basic for reporter gene expression constructs. The constructs were transiently transfected into cultured human embryonic kidney cells (HEK-293, CRL-1573, ATCC) or rat cardiomyocyte line cells (H9c2, CRL-1446, ATCC) for 48 h. Luciferases activities of transfected cells were measured using dual-luciferase reporter assay system. Relative activity of variant TBX5 gene promoters were calculated as previously reported [6].

Electrophoretic mobility shift assay (EMSA)
Nuclear extracts were prepared from cultured HEK-293 or H9c2 cells. As previously described, EMSA was performed with biotinylated double-stranded oligonucleotides (30 bp) to examine whether DSVs affected binding sites for transcription factors [34].

Statistical analysis
Transfection data were expressed as means ± standard errors of the means, and analyzed using two-way analysis of variance. DSV frequencies in AMI patients and controls were compared with SPSS v23.0 (SPSS, Chicago, IL, USA). P < 0.05 was considered as statistically significant difference.
To examine tissue-specific effects of the DSVs, we measured the activities of variant TBX5 gene promoter in cultured H9c2 cells. As expected, the DSV in AMI patients either significantly increased (g.4367C > A and g.4581A > G) or decreased (g.4100A > G, g.4194G > A, g.4260 T > C and g.5004G > T) the activity of TBX5 gene promoter (P < 0.05). Taken together, the DSVs in AMI patients significantly affect the activity of TBX5 gene promoter in a non-tissue specific manner.

DSVs-affected putative binding sites for transcription factors
To determine whether DSVs affect putative binding sites for transcription factors, TBX5 gene promoter was analyzed with JASPAR program (http://jaspar.genereg.net/). The DSV (g.4100A > G) may modify binding sites for basic leucine zipper transcription factor nuclear respiratory factor-1 (NRF-1), nuclear factor 1 X-type (NFIX) and nuclear factor kappa B subunit 2 (NFKB2). The DSV (g.4194G > A) may abolish binding sites for E2F transcription factor 4 (E2F4), a fork head factor, and basic helixspan-helix transcription factor AP-2 Alpha (TFAP2A). The DSV (g.4260 T > C) may create a binding site for zinc finger transcriptional repressor hypermethylated in cancer 2 (HIC2) and modify binding sites for TFAP2B and TFAP2C. The DSV (g.4367C > A) may create a binding site for nuclear factor of activated T-cells 5 (NFAT5) and modify binding site for TFAP2A. The DSV (g.4581A > G) may abolish binding sites for zinc finger protein 354C (ZNF354C) and zinc finger and BTB domain-containing protein 7C (ZBTB7C). The DSV g.5004G > T may create a binding site for TEA domain transcription factor 1 (TEAD1) and abolish a binding site for transcription factor EB (TFEB). Therefore, the DSVs in AMI patients may affect putative binding sites for transcription factors.

DSVs-affected binding site of transcription factors
To investigate whether the DSVs experimentally affect binding sites for transcription factors, EMSA was performed with wild type or variant oligonucleotides (Table 3). In both HEK293 and H9c2 cells, the DSVs (g.4100A > G and g.4260 T > C) enhanced the binding of unknown transcription factors, which were likely transcriptional repressors as both DSVs decreased the TBX5 gene promoter activity. The DSV (g.4581A > G) almost abolished the binding of an unknown transcription factor, which acted as a transcription repressor since the DSV significantly increased the TBX5 gene promoter activity (Fig. 4). Other DSVs (g.4194G > A, g.4367C > A and g.5004G > T) did not evidently affect the binding of transcription factors, likely due to EMSA sensitivity.

Discussion
Mutations in TBX5 gene have been implicated in HOS, a variety of congenital heart diseases, dilated cardiomyopathy and atrial fibrillation [35,36]. Intron variants of TBX5 gene is associated with PR interval, QRS duration and QT interval, as well as atrial fibrillation [37][38][39]. Regulatory variation in a TBX5 gene enhancer causes isolated congenital heart disease [40]. Genetic variants and altered expression levels of TBX5 gene have been reported Fig. 3 Relative activity of wild type and variant TBX5 gene promoters. Empty vector pGL3-basic was used as a negative control. Transcriptional acitivity of wild type TBX5 gene promoter was designed as 100%. Relative activities of variant TBX5 gene promoters were calculated. Lanes 1, pGL3-basic; 2, pGL3-WT; 3, pGL3-4100G; 4, pGL3-4149G; 5, pGL3-4194A; 6, pGL3-4235A; 7, pGL3-4260C; 8, pGL3-4367A; 9, pGL-4581G; 10, pGL3-4904C; 11, pGL3-4915_16insTAATAA; 12, pGL3-5004 T; and 13, pGL3-5109delC. Dark bars indicate HEK-293 cells, and grey bars H9c2 cells. WT, wild type. *, P < 0.05; **, P < 0.01 Table 3  Bold letters in the oligonuceotide sequences indicate the DSVs in patients with cancers [41,42]. In previous studies, we have reported two heterozygous DSVs, g.4303C > G and g.4900C > T, in patients with ventricular septal defects [6]. In this study, six novel heterozygous DSVs (g.4100A > G, g.4194G > A, g.4260 T > C, g.4367C > A, g.4581A > G and g.5004G > T) were identified in six AMI patients, and significantly affected the transcriptional activity of TBX5 gene promoter. Further EMSA revealed that the DSVs modified binding sites for transcription factors. Collective frequency of the DSVs in AMI patients were 1.39% (6/ 432). The DSVs in TBX5 gene promoter identified in patients with ventricular septal defects were not found in this study, suggesting these DSVs were unique in AMI patients. Therefore, our findings suggested that TBX5 gene promoter DSVs may change TBX5 levels, contributing to AMI development at adult stage. As this is a single-center study, more AMI patients from multi-center will be needed to further confirm our findings. The human TBX5 gene is localized to 12q24.1, contains 9 exons and spans more than 47 kb [21,43]. There are GC box, T-box-like binding elements, and NKX2-5 binding site in the human TBX5 gene promoter. TBX5 gene is likely to be autoregulated at transcriptional level [44]. An enhancer located to~90 kb downstream of TBX5 gene has been identified [36]. microRNAs (miR-10a and miR-10b) repress TBX5 gene expression and decrease TBX5 level by targeting the TBX5 3′-untranslated region [45]. In this study, the novel DSVs in the TBX5 gene promoter were biologically functional by affecting binding sites for transcription factors. Molecular mechanisms for these DSVs to influence TBX5 gene expression will be further explored and elucidated.
A number of downstream target genes and interacting partners for TBX5 have been reported. During the heart development, TBX5 regulates NKX2.5, atrial natriuretic factor (ANF) and cardiac structural genes [11,[46][47][48]. TBX5 directs SRF gene expression by binding to its 3′untranslated region [49]. TBX5 partners include GATA4, GATA5, MEF2C, NKX2-5, and other cardiac transcription factors [11,12,[50][51][52][53]. These transcription factors form a complex cardiac regulatory network to coordinately control cardiac gene expression [54]. TBX5 forms a positive and negative feed-forward circuit with Sallike protein 4 (SALL4), a zinc finger transcription factor, to ensure the morphogenesis of embryonic heart [55]. A member of Krüppel-like family of zinc finger proteins, Krueppel-like factor 13 (KLF13), is co-expressed with TBX5 in cardiomyocytes, which may act as a genetic modifier of TBX5 [56]. In a gene regulatory network to maintain cardiac rhythm, TBX5 directly activates paired like homeodomain 2 (PITX2) gene [57]. In addition, TBX5 binds to the proximal region of the NFAT3 (nuclear factor of activated T cells 3) gene in human T cells [32]. Therefore, changed TBX5 level may interrupt cardiac gene regulatory network, contributing to AMI development by affecting cardiac function and influencing coronary vasculature.