Identification of TFEB gene genetic variants in acute myocardial infarction

Background: Abnormal (CAD) and acute myocardial infarction (AMI). Autophagic-lysosomal system is involved in many physiological processes, such as lipid metabolism and inflammation. TFEB, a master regulator of the system, coordinates the expression of lysosomal hydrolases, lysosomal membrane proteins, and autophagic proteins. Altered level of TFEB gene expression and subsequent changes of autophagic-lysosomal system may be involved in the onset of CAD and AMI. Methods: In this study, the promoter of the TFEB gene was genetically and functionally analyzed in AMI patients (n=352) and ethnic-matched healthy controls (n=337). Results: A total of fifteen genetic variants, including eight single nucleotide polymorphisms (SNPs), were identified in the participants. Two novel genetic variants and four SNPs were only identified in six AMI patients, and significantly altered the transcriptional activity of the TFEB gene in cultured cells. Further electrophoretic mobility shift assay revealed that two genetic variants (g.41737144A>G and g.41736544C>T) and two SNPs [g.41737274T>C (rs533895008) and g.41736987C>T (rs760293138)] evidently affected the binding of transcription factors. Conclusions: Our findings suggested that the genetic variants in TFEB gene promoter may change TFEB levels, contributing to AMI as a low-frequency risk factor. Microphthalmia-associated transcription factor; NF-Y: Nuclear transcription factor Y; NR-DR: Nuclear receptor direct repeat; PEG3: paternally expressed gene 3; PPARγ: Peroxisome proliferator-activated receptor γ; SNPs: Single nucleotide polymorphisms; SREBP: Sterol regulatory element-binding proteins; TC: Total cholesterol; TCF-7: Transcription factor 7; TFE3: Transcription factor E3; TFEB: Transcription factor EB; TFEC: Transcription factor EC; TG: Triglyceride.


Introduction
Coronary artery disease (CAD) including acute myocardial infarction (AMI) is an inflammatory and metabolic disease, which is mainly caused by atherosclerosis. Abnormal lipid metabolism and inflammation play critical roles in the initiation and progression of atherosclerosis and its complications [1,2]. Accumulating evidence indicate that genetic factors contribute to the onset of CAD and AMI. To date, many genome-wide association studies have identified a great number of genetic loci for CAD and AMI. However, the collective genetic loci could explain only < 10% of cases [3][4][5]. Therefore, genetic causes and underlying molecular mechanisms for CAD and AMI remain to be elucidated. Recent studies suggested that low-frequency and rare genetic variants may confer susceptibility to cardiovascular diseases [6][7][8].
There are three subtypes of autophagy, macroautophagy, microautophagy and chaperone-mediated autophagy. Macroautophagy (hereafter referred as to autophagy) degrades cytoplasmic macromolecules and organelles by delivering them to lysosomes. Autophagy has been involved in many physiological processes, including lipid metabolism and inflammation. Dysfunctional autophagy has been implicated in a wide range of human diseases, including cardiovascular diseases [9,10]. The lysosome has long been viewed as the recycling center of the cell, and has recently been established to play a central role of nutrient-dependent signal transduction [11,12]. Recent studies have demonstrated that transcription factor EB (TFEB) has been involved in the co-regulation between lysosome, autophagy, and lipid metabolism [13].
TFEB belongs to the MiT-TFE family of basic helix-loop-helix leucine-zipper transcription factors, which include TFEB, transcription factor E3 (TFE3), transcription factor EC (TFEC) and microphthalmiaassociated transcription factor (MITF). Activity of these transcription factors are regulated by their shuttling between the surface of lysosomes, the cytoplasm, and the nucleus. TFEB regulates several cellular processes, including lysosome biogenesis, cellular energy homeostasis, autophagy, mitochondrial turnover, innate immune response and inflammation [13][14][15][16][17][18][19]. At transcriptional level, TFEB coordinates the expression of lysosomal hydrolases, lysosomal membrane proteins and autophagy proteins in response to various stress and nutritional fluctuation of the cell. TFEB binds to CLEAR (coordinated lysosomal expression and regulation) motif within the promoters of the genes [12,16].
Recent studies suggest that TFEB also controls vascular development by regulating the proliferation of endothelial cells [20]. Overexpression of TFEB gene in endothelial cells in mice increases angiogenesis and improves blood flow recovery after ischemic injury [21]. Animal experiments show that TFEB inhibits endothelial cell inflammation and reduces atherosclerosis [22,23]. Therefore, we postulated that altered TFEB levels may contribute to the onset of CAD and AMI. In this case-control study, we genetically and functionally analyzed the promoter of TFEB gene in large cohorts of AMI patients and ethnic-matched healthy controls.

Statistical analysis
All transfection experiments were repeated three times independently, in triplicate. Transfection data are expressed as the means ± standard error of the mean and were analyzed using two-way analysis of variance followed by Dunnet test. Frequency of genetic variants was compared between AMI patients and controls using SPSS v22.0 software (SPSS, Inc., Chicago, IL, USA). P < 0.05 was considered as statistically significant.

Clinical and biochemical characteristics of AMI patients and controls
This study included 352 AMI patients and 337 controls. Clinical data was collected, and related biochemical parameters were examined, including triglyceride (TG), total cholesterol (TC), high density lipoprotein cholesterol (HDL) and low density lipoprotein cholesterol (LDL). Clinical and biochemical characteristics were summarized in Table 2. Age, body mass index (BMI), TG, TC, HDL and LDL were expressed as mean ± standard deviation (M ± SD). Mean age of AMI patients was 61.29 years, and mean age of controls was 51.25 years. A significant difference in age was observed between AMI patients and controls (P < 0.01). The prevalence of traditional risk factors including male sex, hypertension, diabetes and smoking was significantly higher in AMI patients compared to controls (P < 0.01). The levels of TG, TC, HDL and LDL in AMI patients were significantly lower compared to controls (P < 0.01), probably due to application of lowering-lipid medicines in AMI patients. In addition, there was no significant difference of BMI between AMI patients and controls (P > 0.05). Identified genetic variants in the TFEB gene promoter A total of fifteen genetic variants of the TFEB gene promoter were identified, including eight single nucleotide polymorphisms (SNPs). Frequency and locations of the genetic variants were presented in Fig. 1 and summarized in Table 3. Two novel heterozygous variants (g.41737144A > G and g.41736544C > T) and four SNPs [g.41737274T > C (rs533895008), g.41736987C > T (rs760293138), g.41736806C > T (rs748537297) and g.41736635T > C (rs975050638)] were only identified in six male AMI patients (Fig. 2). All the six cases were male, and the age was from 52 to 81 years old.
Clinically, four AMI cases suffered from acute inferior myocardial infarction, and two acute anterior myocardial infarction. Three of the six AMI cases are accompanied with hypertension. Four of the six AMI cases had history of smoking. None of the six cases had diabetes. In addition, biochemical parameters of the six cases were within normal ranges.   Table 4 The double-stranded biotinylated oligonucleotides for the EMSA The dual-luciferase activities were measured and relative activity of wild type and variant TFEB gene promoters were examined.
The SNP [g.41736987C > T (rs760293138)], which was also identified in AMI patient, significantly decreased transcriptional activity of the TFEB gene promoter (P < 0.01). These results indicated that the genetic variants only identified in AMI patients altered transcriptional activity of the TFEB gene. In contrast, other genetic variants only identified in controls or both AMI patients and controls did not alter transcriptional activity of the TFEB gene (P > 0.05) (Fig. 3).

Genetic variants-affected binding sites of transcription factors
The TFEB gene promoter was analyzed using TRANSFAC program to predict whether genetic variants on the binding of transcription factors were not detected, likely due to the sensitivity limit of EMSA experiments (data not shown).

Discussion
Altered TFEB gene expression and subsequent dysfunctional autophagic-lysosomal system have been implicated in human diseases, including lysosomal storage diseases, neurodegenerative diseases and cancers [24][25][26][27]. However, mutations or genetic variants in TFEB gene have not been associated with human diseases. In this study, six genetic variants including four SNPs in the TFEB gene promoter were identified in AMI patients, but in none of controls. These genetic variants significantly altered the transcriptional activity of the TFEB gene. Further EMSA indicated that that two variants (g.41737144A > G and g.41736544C > T) and two SNPs [g.41737274T > C (rs533895008) and g.41736987C > T (rs760293138)] evidently affected the binding of transcription factors. Collectively, 1.70% (6/352) of AMI patients were found to carry genetic variants of TFEB gene promoter.
The human TFEB gene has been localized to chromosome 6p21.1. TFEB recognizes the motif, CLEAR element, within its target genes [12,28]. TFEB null mice die at embryonic stage due to defective placental vascularization [29]. Conditional disruption or transgenic mouse models reveal that TFEB has specialized functions in different tissues [11,21,[30][31][32]. There are five transcript variant of TFEB, and the variant 2 encodes the longest isoform, promoter region of which was analyzed in this study.
To date, the promoter of TFEB gene has not been characterized in details. There are numerous CLEAR sequences in the TFEB gene promoter, indicating that TFEB regulates its own expression in an autoregulatory loop [14]. In human endothelial cell, paternally expressed gene 3 (PEG3) is an upstream transcriptional regulator of TFEB gene [33]. In this study, the genetic variants identified in TFEB direct genes has been identified, which represent essential components of the CLEAR gene network [11,41]. TFEB promotes the gene expression of the autophagic-lysosomal pathway, and regulates the lysosomal biogenesis, autophagy, lysosomal proteostasis, lysosomal exocytosis and lysosomal positioning [11,15,[42][43][44]. Moreover, TFEB and TFE3 cooperate in regulating the expression of proinflammatory cytokine genes, controlling the adaptive response of whole body energy metabolism, and modulating the cellular response to endoplasmic reticulum stress [31,45,46]. Therefore, upregulation or downregulation of TFEB gene expression may contribute to AMI development through dysfunctional autophagic-lysosomal system and other pathways.
Accumulating studies have demonstrated that a window of optimal autophagic-lysosomal activity is critical to the maintenance of cardiovascular homeostasis and function. Excessive or insufficient levels of autophagic flux can each contribute to the pathogenesis of cardiovascular diseases, including AMI [47][48][49]. TFEB is differentially activated in patients with different diseases. In patients with Danon disease, TFEB and downstream targets are activated. Conversely, TFEB is inhibited and an autophagy is blocked in patients with glycogen storage disease type II [50]. In this study, the genetic variants in AMI patients exhibited upregulation or downregulation of TFEB gene promoter activity.
Excessive or insufficient TFEB gene expression may similarly contribute to AMI development through diverse pathways.

Conclusions
In the present study, the TFEB gene promoter was genetically and functionally analyzed in AMI patients and healthy controls. Two novel variants and four SNPs were only identified in AMI patients.
These genetic variants significantly altered the transcriptional activity of the TFEB gene promoter.
Furthermore, four of the six genetic variants evidently affected the binding of transcription factors.
Therefore, these genetic variants may change TFEB level, contributing to AMI development through diverse pathways as a low-frequency risk factor.