Genetic Variants of ISL1 Gene Promoter Identified from Congenital Tetralogy of Fallot Patients Alter Cellular Function Forming Disease Basis

Tetralogy of Fallot (TOF) is the most common cyanotic congenital heart disease in newborns. ISL1 is a master transcription factor in second heart field development, whereas the roles of ISL1 gene promoter variants in TOF patients have not been genetically investigated. Total DNA extraction from 601 human subjects, including 308 TOF patients and 293 healthy controls, and Sanger sequencing were performed. Four variants (including one novel heterozygous variant) within the ISL1 gene promoter were only found in TOF patients. Functional analysis of DNA sequence variants was performed by using the dual-luciferase reporter assay and demonstrated that three of the four variants significantly decreased the transcriptional activity of ISL1 gene promoter in HL-1 cells (p < 0.05). Further, the online JASPAR database and electrophoretic mobility shift assay showed that the three variants affected the binding of transcription factors and altered ISL1 expression levels. In conclusion, the current study for the first time demonstrated that the variants identified from the ISL1 gene promoter region are likely involved in the development of TOF by affecting the transcriptional activity and altering the ISL1 expression level. Therefore, these findings may provide new insights into the molecular etiology and potential therapeutic strategy of TOF.


Introduction
Tetralogy of Fallot (TOF) is the most common form of cyanotic congenital heart disease in newborns, with a prevalence of 4 per 10,000 live births, accounting for up to 7-10% of congenital heart diseases (CHDs) [1,2]. TOF is characterized as a combination of abnormalities (ventricular septal defect, obstruction of the right ventricular outflow tract, override of the ventricular septum by the aortic root, and right ventricular hypertrophy) that together lead to tissue hypoxia. Although surgical correction during infancy repaired most cases of TOF and increased the survival of TOF patients [3], there is significant morbidity in adulthood, particularly with pulmonary regurgitation and arrhythmias [4].
The genetic factor has been demonstrated to be the major etiology of CHDs [5]. Accumulating evidence has indicated that the cause of TOF is complex and genetic factors are major contributors to the incidence of TOF [6]. The presence of newer genomic technologies, especially genome-wide sequencing, has identified novel TOF-specific copy number variants and revealed the genetic basis of TOF [7]. Studies have identified susceptible genes related to the etiology of TOF, such as NOTCH1 [6], FLT4 [6], ISL1, TBX1 [8], PIPN11 [9], and NRP1 [10]. However, the roles of these associated genes and pathways in the formation of TOF remain largely unknown.
The transcription factor ISL1 (OMIM: 600366), which is located on chromosome 5q11.1, has been considered an important regulator of heart development. As a crucial member of the LIM homeobox family of transcriptions, ISL1 is a marker for cardiac progenitor cells, primarily in marking undifferentiated, proliferating progenitors in the second heart field, and is essential for the proliferation, migration, and survival of cardiac progenitor cells [11][12][13]. In mice, homozygous null for ISL1 appears to lead to serious heart development defects, including absence of right ventricle, much of the right atrium, and outflow tract [11]. Furthermore, it has been found that several mutations of ISL1 were associated with diverse types of CHDs in humans [14][15][16][17]. It is well known that the promoter, located at 5 upstream of gene, plays a key role in gene transcriptional regulation. Accumulating evidence has reported that the variants of gene promoter region may affect the expression level of genes, leading to the development of disease [14,18,19]. However, there are no studies focusing on the variant of ISL1 gene promoter region in TOF patients. Therefore, we hypothesized the genetic variants in ISL1 gene promoter may contribute to the development of TOF by altering the ISL1 gene expression level and affecting the binding ability of transcription factor. To confirm this hypothesis, we designed this study and identified variants within the ISL1 gene promoter in patients with TOF in comparison with healthy controls and then functionally analyzed the effect of the variants on the ISL1 gene expression.

Study Population
In this study, a total of 308 Chinese Han patients (175 males and 133 females; age range: 3 months to 19 years) diagnosed with TOF from the Department of Cardiovascular Surgery, TEDA International Cardiovascular Hospital, Tianjin University (Tianjin, China) and 293 Chinese Han healthy control subjects (165 males and 128 females; age range: 1 month to 14 years) with similar sex and age were recruited. We made an endeavor to recruit sporadic TOF patients without family history of CHD. All the normal control subjects were confirmed by echocardiography without CHDs or any other diseases. The investigation was in compliance with the outlined guidelines in the Declaration of Helsinki. The informed written consent was obtained from all subjects or their legal guardians prior to the study. Research protocol for this study was approved by the Ethics Committee of TEDA International Cardiovascular Hospital (Clinical Research Ethics Review Number: 2021-0715-4). The flow chart is shown in Figure 1. The flow chart of this study. A total of 601 subjects were enrolled in this study. Then we performed total DNA extraction, DNA sequence analysis, cellular functional analysis, electrophoretic mobility shift assay, and transcription factor binding sites prediction to test our hypothesis. TOF, Tetralogy of Fallot.

ISL1 Promoter Sequence Analysis
Peripheral blood samples were collected according to the standard clinical procedure. Genomic DNA was extracted from peripheral blood for each TOF patient and control subject with TIANamp Blood DNA Kit (TIANGEN, China). The polymerase chain reaction (PCR) primers were designed based upon the reference sequence of the human ISL1 gene in GenBank database (NCBI: NG_023040.1) as previously reported [14]. The promoter region of the human ISL1 gene (1398 bp, from −1,286 bp to +111 bp to the transcription start site) was amplified by PCR and directly bidirectionally sequenced. The Figure 1. The flow chart of this study. A total of 601 subjects were enrolled in this study. Then we performed total DNA extraction, DNA sequence analysis, cellular functional analysis, electrophoretic mobility shift assay, and transcription factor binding sites prediction to test our hypothesis. TOF, Tetralogy of Fallot.

ISL1 Promoter Sequence Analysis
Peripheral blood samples were collected according to the standard clinical procedure. Genomic DNA was extracted from peripheral blood for each TOF patient and control subject with TIANamp Blood DNA Kit (TIANGEN, China). The polymerase chain reaction (PCR) primers were designed based upon the reference sequence of the human ISL1 gene in GenBank database (NCBI: NG_023040.1) as previously reported [14]. The promoter region of the human ISL1 gene (1398 bp, from −1286 bp to +111 bp to the transcription start site) was amplified by PCR and directly bidirectionally sequenced. The primers for Sanger sequencing are listed in Table 1. Subsequently, the DNA sequences of above PCR products were compared with the wild-type ISL1 promoter sequence.  To functionally analyze the variants of ISL1 gene promoter, DNA fragments of ISL1 gene promoter with wild-type or variant-type, containing the terminal restriction sites KpnI and BglII, were generated by PCR and subcloned into the KpnI/BglII sites of the pGL3-basic to constitute expression vectors that were validated by Sanger sequencing. PCR primers are shown in Table 1.

Functional Analysis with Dual-Luciferase Reporter Assay
Transfected cells were harvested and lysed after 36 h. Firely and renilla luciferase activities of cells were measured with Dual Luciferase Reporter Gene Assay Kit (Beyotime, China) in the specified dual-luciferase reporter assay system (Thermo Scientific Fluoroskan FL, Thermo Fisher, Americia) according to the manufacturer's protocol. The activities of ISL1 gene promoter were assessed by the ratio of firefly luciferase activity to renilla luciferase activity. All experiments were performed in three independent replicates.

Nuclear Protein Extraction and Electrophoretic Mobility Shift Assay
To investigate the effect of variants within the promoter region of ISL1 gene on the binding ability of transcription factors, electrophoretic mobility shift assay (EMSA) was performed with Chemiluminescent EMSA Kit (Beyotime, Shanghai China). Biotinylated double-stranded oligonucleotides based on the wild-type or variant-type sequence in ISL1 gene promoter were utilized as probes. Biotin-labeled oligonucleotides for the EMSA experiment are shown in Table 1. Nuclear protein extracts from HL-1 cells and HEK-293 cells were prepared using the Nuclear and Cytoplasmic Protein Extraction Kit (Beyotime, Shanghai, China) and protein concentrations were determined with the Enhanced BCA Protein Assay Kit (Beyotime, Shanghai, China). The DNA-protein binding reaction with an equal amount of probe and nuclear extracts was performed at room temperature in 20 min. The mixture of DNA and protein mixed with sample loading buffer was then added to the sample wells of 4% polyacrylamide gel and transferred onto a nylon membrane (Beyotime, Shanghai, China). The oligonucleotides were cross-linked to the membrane using the UV light. Finally, biotinylated oligonucleotides were detected by chemiluminescence using the Chemiluminescent EMSA kit (Beyotime, Shanghai, China).

Transcription Factor Binding Sites Prediction
The online JASPAR database (http://jaspar.genereg.net, accessed on 7 August 2022) was used to analyze whether ISL1 promoter region variants would alter the putative transcription factor binding site (TFBS). The relative profile score threshold was set at 85% in the JASPAR database.

Statistical Analysis
For continuous variables, results are expressed as mean ± SD. Non-parametric tests for independent samples were used to analyze the quantitative data and to calculate the statistical significance of the experimental results. The burden test for rare variants was performed by using SKAT package in R. The model parameters and residuals were calculated by 'SKAT-Null-Model-MomentAdjust' function for binary triat. 'SKAT-Binary' function was used for the burden test of a group of several rare variants [20]. The level of significance was set at p < 0.05. SPSS 19.0 software (IBM) was used for all statistical analyses.

The Variants Identified in TOF Patients and Healthy Controls
This study recruited 601 participants, including 308 TOF patients and 293 healthy controls ( Figure 1). A total of eleven variants in ISL1 gene promoter were identified by Sanger sequencing in these study subjects. The locations and details of these variants are presented in Figure 2A and Table 2. Among those eleven variants found in this study, one novel heterozygous variant [g.5085G > A] and three single-nucleotide variants (SNVs) [g.4581A > G(rs1747215641), g.4630G > A(rs1264694566), g.5221C > T(rs1484364688)] were found only in patients with TOF. The sequencing chromatograms of these four variants are presented in Figure 2B. In addition, the allele frequency of these four variants is less than 0.0001 in the NCBI ALFA database and GnomAD database and two of them [g.5085G > A, g.5221C > T(rs1484364688)] have 0 allele frequency (Table 2). Further, one of these four variants [g.4630G > A(rs1264694566)] was found in two patients. The above three SNVs and the novel variant [g.5085G > A] were further validated for cellular functional experiment.

Functional Analysis of the Variants Via Dual-Luciferase Reporter Assay
To further investigate the effect of variants on the transcriptional activity of the ISL1 promoter, different reporter plasmids including a null pGL3-basic (negative control), wild-type ISL1 gene promoter (pGL3-WT), and variant-type ISL1 gene promoter (pGL3-4581G, pGL3-4630A, pGL3-5085A, pGL3-5221T) were constructed and transfected into the HL-1 cells, respectively. The dual-luciferase activities were assayed and relative activity of wild-type and variant ISL1 gene promoter was examined.
As illustrated in Figure 3A, dual-luciferase activity indicated that among the four expression vectors carrying genetic variants, three variants (4581G, 4630A, 5085A) significantly decreased the transcriptional activities compared with the wild-type (p < 0.05).

Functional Analysis of the Variants via Dual-Luciferase Reporter Assay
To further investigate the effect of variants on the transcriptional activity of the ISL1 promoter, different reporter plasmids including a null pGL3-basic (negative control), wildtype ISL1 gene promoter (pGL3-WT), and variant-type ISL1 gene promoter (pGL3-4581G, pGL3-4630A, pGL3-5085A, pGL3-5221T) were constructed and transfected into the HL-1 cells, respectively. The dual-luciferase activities were assayed and relative activity of wild-type and variant ISL1 gene promoter was examined.
As illustrated in Figure 3A, dual-luciferase activity indicated that among the four expression vectors carrying genetic variants, three variants (4581G, 4630A, 5085A) significantly decreased the transcriptional activities compared with the wild-type (p < 0.05). In contrast, the rest one variant (5221T) did not significantly alter the transcriptional activity of the ISL1 gene promoter (p > 0.05). The echocardiography of patients with variants is displayed in Figure 3B.
In contrast, the rest one variant (5221T) did not significantly alter the transc tivity of the ISL1 gene promoter (p > 0.05). The echocardiography of patien ants is displayed in Figure 3B.

The Binding for Transcription Factors Affected by the Variants
To experimentally investigate whether the binding ability of transcriptio affected by the variants identified only in TOF patients, EMSA was perf wild-type or variant biotinylated oligonucleotides. The above three vari 4630A, 5085A) markedly affected the binding of transcription factor in bo HEK-293 cells, as shown in Figure 4 (presented with arrows). In addition, s result in dual-luciferase reporter assay, the variant (5221T) did not affect th transcription factor (data not shown).

The Binding for Transcription Factors Affected by the Variants
To experimentally investigate whether the binding ability of transcription factor was affected by the variants identified only in TOF patients, EMSA was performed with wildtype or variant biotinylated oligonucleotides. The above three variants (4581G, 4630A, 5085A) markedly affected the binding of transcription factor in both HL-1 and HEK-293 cells, as shown in Figure 4 (presented with arrows). In addition, similar to the result in dual-luciferase reporter assay, the variant (5221T) did not affect the binding of transcription factor (data not shown). Biomolecules 2023, 13, x FOR PEER REVIEW 8 of 13

Variant-Affected Putative Binding Sites for Transcription Factor
The online JASPAR database was fully utilized to further explore whether the binding sites for transcription factors were affected by variants identified in the ISL1 gene promoter. As summarized in Table 3, the putative binding sites for transcription factor may be disrupted or created by variants identified in patients with TOF. The variant g.4581G may create new binding sites for ZEB1, NKx3-1, and TEAD3, and disrupt the binding sites for FOXL1, RHOXF1, and GATA5. Among these TFBSs, the mutations of GATA5 have been reported in sporadic TOF patients. SOX9, a downstream target gene of ISL1 gene, is regulated by ISL1 gene through ISL1-Fgf10-SOX9 pathway. In combination with the experimental results, the analysis by the JASPAR database, and literature reports, we established a schema ( Figure 5) to illustrate the possible effect of the variants in

Variant-Affected Putative Binding Sites for Transcription Factor
The online JASPAR database was fully utilized to further explore whether the binding sites for transcription factors were affected by variants identified in the ISL1 gene promoter. As summarized in Table 3, the putative binding sites for transcription factor may be disrupted or created by variants identified in patients with TOF. The variant g.4581G may create new binding sites for ZEB1, NKx3-1, and TEAD3, and disrupt the binding sites for FOXL1, RHOXF1, and GATA5. Among these TFBSs, the mutations of GATA5 have been reported in sporadic TOF patients. SOX9, a downstream target gene of ISL1 gene, is regulated by ISL1 gene through ISL1-Fgf10-SOX9 pathway. In combination with the experimental results, the analysis by the JASPAR database, and literature reports, we established a schema ( Figure 5) to illustrate the possible effect of the variants in ISL1 gene promoter region and the roles of genes and pathways associated with ISL1 gene in the etiology of TOF. ISL1 gene promoter region and the roles of genes and pathways associated with ISL1 gene in the etiology of TOF.   Table 3]; 18 of them are involved in the heart development that are listed in Figure 5. Schema describing the role of ISL1 gene promoter region variants in the development of cardiac defect based on the results from the present study, the JASPAR database analysis, and the previous reports. Variants reduced ISL1 gene promoter activity by disrupting and creating the TFBS (total of 32 [see Table 3]; 18 of them are involved in the heart development that are listed in the figure), contributing to the low expression of ISL1. Consequently, the low expression of ISL1 may be directly involved in the formation of TOF and may reduce the expression of downstream target genes and pathway, which are related to the formation of TOF.

Discussion
The current study for the first time identified that (1) there are four variants within the ISL1 gene promoter that are identified only in the TOF patients with 0 incidence in the control subjects and one of them (g.5085G > A) is novel; (2) three of these four variants (g.4581A > G, g.4630G > A, g.5085G > A) significantly altered the transcriptional activity of the ISL1 gene promoter at cellular functional level; and (3) these three variants (g.4581A > G, g.4630G > A, g.5085G > A) causing functional changes evidently affected the binding of transcription factors verified by EMSA experiments. Therefore, these ISL1 gene promoter variants (g.4581A > G, g.4630G > A, g.5085G > A) likely contribute to the development of TOF as risk factors.
The human ISL1 gene, mapped to chromosome 5q11.1 and comprised with 349 amino acids, was first identified and cloned during the study of the regulation of insulin gene expression [22]. Recently, it has been reported that ISL1 plays an important role in the regulation of the nervous system, the pancreas and cardiac development [23]. ISL1 gene can be specifically expressed in cardiac progenitor cells. Decreased expression of the ISL1 gene has been reported to be associated with many cardiovascular diseases in human, such as CHDs [24], atrial fibrillation [25], and dilated cardiomyopathy [26]. It has been reported that the copy number variations of ISL1 gene is related to the etiology of TOF [27]. The expression of many genes is regulated through the binding of various transcription factors to promoter sequences [19]. To date, variants at the promoter region of human ISL1 gene have not been illustrated in detail. Based on the above reports concerning ISL1 gene, we hypothesized that variants in the ISL1 gene promoter may affect the normal gene activation and lead to the disease. In this study, we analyzed the promoter of ISL1 gene in patients with TOF and the variants were identified. The transcriptional activity analysis and EMSA results performed in the present study have clearly demonstrated that three variants discovered in ISL1 gene promoter region notably changed the transcriptional activity, providing support for our hypothesis. The EMSA is an experiment that may demonstrate the interference (enhancement or suppression) of the binding affinity between transcription factors and promoter by variants. Different variants may affect the binding affinity for various transcription factors (see Table 3 for details) at the different levels, as shown in Figure 4.
It has been reported that variants in the gene promoter region create or disrupt TFBS at the promoter, affecting the transcriptional regulation of genes and putatively contributing to the development of disease. In the present study, three variants discovered in the ISL1 gene promoter with altered cellular function were analyzed with bioinformatics through JASPAR database. We discovered that the variant g.4581A > G may disrupt the potential binding sites for GATA5, a member of the GATA family transcription factor that plays an essential role in the development of embryonic heart. Importantly, the GATA5 loss-offunction mutation is associated with TOF [28]. In addition, the binding sites for SRY (sex determining region Y)-box9(SOX9) may be created by the variant g.4630G > A. The ISL1-Fgf10-SOX9 network is a pivotal pathway in the embryonic lung branching morphogenesis and cell differentiation and SOX9 is regulated by ISL1 [29]. Further investigation to explore the relationship among ISL1 gene promoter variants and these transcription factors is required.
Apart from GATA5 and SOX9, among the 32 transcription factors listed in Table 3, at least 16 factors are closely related to the development of the heart. All those genes may be further studied with regard to the interaction to ISL1 promoter in order to reveal the correlation among them and the pathological role in the development of TOF.
Taken together with the results from the present study, the JASPAR database analysis, and the previous reports, we established a schema ( Figure 5) to summarize the possible role of ISL1 gene promoter region variants in the development of TOF. Firstly, variants reduce ISL1 gene promoter activity, contributing to the low expression of ISL1. Consequently, the low expression of ISL1 may be involved in the formation of TOF. In addition, the activity of TOF-related genes, such as GATA4 [30], TBX5 [31], and TBX20 [32], may be decreased by the low expression of ISL1 [33]. Moreover, the low expression of ISL1 may also lead to the reduced-expression of vascular endothelial growth factor related signaling gene FOXO1, which is interacted with ISL1 and involved in the pathogenesis of TOF [34][35][36]. Finally, the low level of ISL1 may reduce the expression of HAND2 and SHH in the HAND2-SHH pathway, which is involved in the development of TOF [37][38][39].
There are some limitations in this study. The exact role of genotype-phenotype relationship needs to be further established. The precise role of the discovered ISL1 promoter variants in the development of TOF requires further in vivo animal experiments. In addition, CHDs are developed with the interaction of related genes and the interaction between ISL1 and other genes shown in Figure 5 needs to be further verified.

Conclusions
In conclusion, our study for the first time identified genetic variants in the promoter region of ISL1 gene in patients with TOF. Furthermore, the variants were demonstrated to significantly alter the ISL1 gene expression by the cellular functional experiment. Further EMSA experiment and bioinformatic analysis also demonstrated that these variants likely affect the binding of transcription factors and may contribute to the development of TOF as risk factors. Therefore, this study may provide new insights into the understanding of the promoter region and the genetic etiology involved in the development of CHD.