Variants of the promoter of MYH6 gene in congenital isolated and sporadic patent ductus arteriosus: case-control study and cellular functional analyses

Abstract

Patent ductus arteriosus (PDA) is a common form of congenital heart disease. The MYH6 gene has important effects on cardiovascular growth and development, but the effect of variants in the MYH6 gene promoter on ductus arteriosus is unknown. DNA was extracted from blood samples of 721 subjects (428 patients with isolated and sporadic PDA and 293 healthy controls) and analyzed by sequencing for MYH6 gene promoter region variants. Cellular function experiments with three cell lines (HEK-293, HL-1, and H9C2 cells) and bioinformatics analyses were performed to verify their effects on gene expression. In the MYH6 gene promoter, 11 variants were identified. Four variants were found only in patients with PDA and 2 of them (g.3434G>C and g.4524C>T) were novel. Electrophoretic mobility shift assay showed that the transcription factors bound by the promoter variants were significantly altered in comparison to the wild-type in all three cell lines. Dual luciferase reporter showed that all the 4 variants reduced the transcriptional activity of the MYH6 gene promoter (P < 0.05). Prediction of transcription factors bound by the variants indicated that these variants alter the transcription factor binding sites. These pathological alterations most likely affect the contraction of the smooth muscle of ductus arteriosus, leading to PDA. This study is the first to focus on variants at the promoter region of the MYH6 gene in PDA patients with cellular function tests. Therefore, this study provides new insights to understand the genetic basis and facilitates further studies on the mechanism of PDA formation.

Graphical Abstract

Patent ductus arteriosus, MYH6, Gene, Variants, Congenital heart disease

Introduction

Children health is a public health concern worldwide. There are 13.9 stillbirths for every 1000 births worldwide [1]. Currently, 37.1 deaths per 1000 live births among children under 5 years of age occur worldwide [2]. Congenital heart disease (CHD) is one of the common congenital defects and is the main cause of pregnancy termination and fetal death [3]. More than 3 million babies are born with congenital heart abnormalities each year [4] and CHD is the leading cause of death in the first newborn year of life [5]. Studies have shown that China currently suffers from CHD burden much higher than the world average [6]. Children with CHD can also have developmental delays and other extracardiac abnormalities [7] and often require long-term follow-up with the added burden of possible arrhythmias and heart failure in adulthood [8]. The causes of CHD formation are usually considered multifactorial [9, 10]. With the concept of precision medicine, the role of genetic factors in CHD has attracted increasing attention from researchers [11–13]. Among genetic variants, de novo variants play a large role with a large number of chromosomal abnormalities found in close to 10% of CHD patients [14]. After decades of research, many genetic factors including structural proteins important for heart development, transcription factors (TFs), and cell signaling molecules have been identified to be associated with the pathogenesis of CHD [11].

Patent ductus arteriosus (PDA) is one of common CHDs in preterm infants [15]. The incidence of PDA is approximately 10-20% of all CHDs [11]. PDA can exist either alone or in conjunction with other CHDs. PDA-related complications result in impaired perfusion of vital organs, including bronchopulmonary dysplasia, pulmonary hemorrhage, necrotizing small bowel colitis, renal impairment, ventricular hemorrhage, sepsis, and even death [16]. In normal full-term infants, the ductus arteriosus closes completely at 2–4 days. In preterm infants, structural and physiological immaturity of the ductus arteriosus is associated with PDA [17]. Current treatments regarding PDA include conservative, pharmacological, and surgical treatment. The trend of increasing prevalence of PDA in full-term infants in recent years has promoted the need to increase our knowledge about PDA formation [18]. In addition to prematurity and maternal factors, genetic variation is also considered to be one of the main factors contributing to the development of PDA [19]. With the application of gene sequencing technology, more and more genetic variants have been shown to be inextricably linked to the formation of PDA [20].

There are many variants in key genes in the development of the cardiovascular system that lead to the formation of various types of CHD [21]. MYH6 (OMIM: 160710) is a gene located on chromosome 14q11.2 that encodes the alpha subunit of the cardiac myosin heavy chain, one of the important subunits that make up the composition of cardiac myosin. Its variants have important implications for many types of CHD and cardiomyopathy [22, 23]. It has been demonstrated to be associated with the formation of shone complex, coarctation of the aorta, bicuspid aortic valve [24], non-syndromic coarctation of the aorta [25], hypoplastic left heart syndrome [26], ischemic cardiomyopathy [27], atrial septal defect, and ventricular septal defect [28]. It has also been suggested that MYH6 is involved in the contraction of vascular smooth muscle and is associated with lesions of poor vasoconstriction [29].

Studies on the effects of promoter variants in non-coding regions of genes on CHD are increasing recently [30]. We recently demonstrated that proteins closely related to platelet activation and coagulation cascades, complement mannan-binding-lectin, and other systemic signaling pathways are associated the development of PDA [31]. Nevertheless, no studies on the effect of variants in the MYH6 gene promoter region on PDA have been reported.

Based on the above, we hypothesized that variants of MYH6 promoter may be involved in the formation of PDA. The present study was designed to investigate variants at the MYH6 gene promoter region and their role in the PDA formation.

Results

Variants identified by DNA sequencing in PDA patients

A total of 11 variants in the MYH6 gene promoter region were identified by DNA sequencing. Four of these variants were unique to PDA patients [g.3545G>T, g.4377T>C (rs373958405), g.4420A>G, g.4712C>T (rs913766127)], while the rest seven were found in both healthy children and PDA patients. Table 1 summarizes the details of these variants. The positions of these variants are shown in Fig. 1A with the sequencing profiles shown in Fig. 1B. A search in the NCBI SNP database found that the variants g.3545G>T and g.4420A>G were novel without previous reports. The other two variants [g.4377T>C (rs373958405; C=0.000125) and g.4712C>T (rs913766127; T=0.000034)] occurred with low frequency. It is notable that the variant g.4377T>C (rs373958405) was present in six patients in this study. The above four variants were further investigated for cellular function and EMSA to verify the significance of these variants. The rest seven variants that were present in both PDA patients and controls were excluded from further study.

Table 1

Variants within the MYH6 gene promoter in patients with PDA.

Variations	Positiona	Genotypes	PDAb	Controlsb	Global frequency*	East-Asian frequency*
g.3545G>T	−1455	G>T	1	0	None	None
g.4377T>C(rs373958405)	−623	T>C	6	0	C = 0.000125	C = 0.00024 (Japanese)
g.4420A>G	−580	A>G	1	0	None	None
g.4712C>T(rs913766127)	−290	C>T	1	0	T = 0.000034	T = 0.0003 (Koreans)
g.3285A>G(rs17091776)	−1715	A>G	84	42	G = 0.1048	G = 0.0754
g.3557G>T(rs191392051)	−1443	G>T	20	10	T = 0.0038	T = 0.0159
g.3726A>G(rs178648)	−1244	A>G	12	2	G = 0.0046	G = 0.0198
g.3816C>A(rs138953808)	−1184	C>A	16	9	A = 0.0224	A = 0.0089
g.3931del(rs377182175)	−1069	delC	7	4	delC = 0.0014	delC = 0.0050
g.4208A>G(rs9788443)	−972	A>G	87	45	G = 0.1767	G = 0.0764
g.4387T>C(rs73587609)	−613	T>C	91	46	C = 0.0477	C = 0.0784

^aVariants are located upstream (−) to the transcription start site at the position of 5001 (+1) of the MYH6 gene (NG_023444.1).

^bAllele frequency in groups. PDA, patent ductus arteriosus.

^*Allele frequencies of the global population and the East Asians were from the NCBI dbSNP database.

Figure 1

Locations and sequencing chromatograms of MYH6 gene promoter variants. (A) Genetic variants are named according to the genomic DNA sequence of the human MYH6 gene (Genbank accession number NG_023444.1). The transcription start site is at position 5001 in the first exon. (B) Sequencing chromatograms of all variants found in PDA patients compared to the controls. The top panels show the wild-types and the bottom panels show the variant-types, marked with arrows. PDA, patent ductus arteriosus.

Promoter variants alter the transcriptional activity of the MYH6 gene

The promoters of the MYH6 gene of wild-type and that with variant were cloned into a reporter plasmid (pGL6-basic) of the firefly luciferase reporter plasmid with respect to ampicillin resistance. Then these expressions were constructed as blank, empty pGL6-basic (negative control), pGL6-WT (wild-type MYH6 gene promoter), pGL6-g.3545G>T (g.3545G>T), pGL6-g.4377T>C (g.4377T>C), pGL6-g.4420A>G (g.4420A>G), and pGL6-g.4712C>T (g.4712C>T) expression vectors. The vectors were then transfected with pRL-SV40 into HEK-293, HL-1, and H9C2 cell lines followed by validation of the dual luciferase activity assay. Similar results were obtained for the three cell lines, with the blank control and negative control groups showing much lower transcriptional activity than the other five groups, especially the blank control group, which was close to 0. Compared to pGL6-WT, the four variants (pGL6-g.3545G>T, pGL6-g.4377T>C, pGL6-g.4420A>G, and pGL6-g.4712C>T) showed significantly lower transcriptional activity in the three cell lines (P < 0.05) (Fig. 2A–C). In addition, in the nine patients with the identified variants, color Doppler echocardiograms are shown to demonstrate a significant flow between the aorta and the pulmonary artery, the typical findings for PDA (Fig. 2D).

Figure 2

Results of dual luciferase reporter gene analysis and echocardiography of the patients with the variants. Relative transcriptional activity of wild-type and variants of MYH6 gene promoters in HEK-293 (A), HL-1 (B), and H9C2 (C) cells. The transcriptional activity of the wild-type MYH6 gene promoter was set as 100%. The relative activities of MYH6 gene promoters were calculated. Quantitative data are expressed as mean ± SD and are based on six independent experiments (n = 6, ^*P < 0.05, ^**P < 0.01, ^***P < 0.001, ^****P < 0.0001). (D) Color Doppler echocardiography shows PDA (arrow) in the patients with variants [g.3545G>T, g.4377T>C (rs373958405), g.4420A>G, and g.4712C>T (rs913766127)]. AO, aorta; LA, left atrium; RA, right atrium; PA, pulmonary artery; RVOT, right ventricle outflow tract; PDA, patent ductus arteriosus.

Variant-affected TFBS

The potential TFBS were predicted by the JASPAR core TF database for disruption or generation by the variants in the MYH6 gene promoter. A total of 24 new TFBS were found to be generated and 38 TFBS disrupted by these four variants, shown in Table 2. Among the newly created TFBS, BACH1 and ZNF354C inhibit the development of cardiovascular system [32, 33]. In comparison, the disrupted TFBS NKX2-3, NKX2-5, and NKX3-2 etc. are indispensable key TFs for cardiovascular development [34, 35]. Based on the above predictions, we further performed EMSA, and also constructed a model map describing the possible effects of MYH6 gene promoter region variants on PDA formation according to previous studies, illustrated in Fig. 3.

Table 2

TFBS affected by variants (predicted by the JASPAR database).

	Binding sites for transcription factors
	Disrupt	Create
g.3545G>T	KLF4, TEAD1, TEAD3, TEAD4, PRDM1, HES2, NKX2-3, NKX2-8, NKX3-2, SNAI2, ISL2, HOXA4, HOXA6, GSX2, GSX1, DLX3, DLX4, DLX6	PRDM4, IRF6
g.4377T>C (rs373958405)	NKX2-3, NKX2-4, NKX2-5, NKX2-8, NKX3-1, NKX3-2, NOBOX, MSX2, NRL, ISL2, POU5F1, DLX1, DLX2, DLX3, DLX4, HOXA5, HOXA6, HOXA7, HOXA8, HOXB6, BARHL1, BARHL2, BARX1	TBX1, TBX3, TBX6, MGA, SNAI2, NFATC3, RFX5, ZEB1
g.4420A>G	NFAT5, FOXD2	GFI1B, MAFK, MAFF, BACH1, KLF4
g.4712C>T (rs913766127)	ZNF75D, SP3, SP2, MEIS1	ZNF354C, ZEB1, SREBF1, SREBF2, TBX2, SNAI2, FIGLA, TBR1, EOMES, NR2C1

TFBS, transcription factor binding sites.

Figure 3

Schema describing the role of MYH6 gene promoter variants. The schema depicts the role of variants in the promoter region of the MYH6 gene identified from this study. The variants of the MYH6 gene promoter identified in this study may alter the TFBS cluster, leading to a decrease in gene expression. In addition, some of the newly created TFBS (upper panels) from variants of the MYH6 gene promoter have an inhibitory effect on vasoconstriction, while many of the disrupted TFBS (lower panels) play a critical role in vascular development. These factors contribute to the formation of PDA. TFBS, transcription factor binding sites; PDA, patent ductus arteriosus.

Analysis of EMSA results

To further verify whether the variants in the promoter region affect TF binding, we constructed wild-type and variant double-stranded probes with biotin labeling to detect four variants found only in PDA patients [g.3545G>T, g.4377T>C (rs373958405), g.4420A>G, g.4712C>T (rs913766127)]. As shown in Fig. 4, the binding of the probes to the nuclear proteins extracted from three cell lines, HEK-293, HL-1, and H9C2, respectively, was demonstrated. As the black arrows in the figure suggest, the binding of variants g.3545G>T and g.4377T>C (rs373958405) to TFs in all three cell lines was significantly weaker compared to the wild-type, while the variant g.4420A>G exhibited a more significantly weakened binding to TFs in HEK-293 and HL-1 cell lines. Further, the variant g.4712C>T (rs913766127) may bind more TFs in these three cell lines compared with the wild-type. Importantly, the trend of the binding of these variants to TFs is consistent with what is predicted by the JASPAR core TF database.

Figure 4

The results of EMSA. The designed single-stranded nucleotide sequences were labeled with biotin. Subsequently, the complementary sequences were annealed and hybridized. The probes were then subjected to EMSA with nuclear proteins extracted from HEK-293, HL-1, and H9C2 cells, respectively. Four variants [g.3545G>T, g.4377T>C (rs373958405), g.4420A>G, g.4712C>T (rs913766127)] were found to have altered TFs binding in all three cell lines compared to the wild-type. EMSA, electrophoretic mobility shift assay; WT, wild-type; VT, variant-type; TF, transcription factor.

Discussion

The present study for the first time reveals the variants in the promoter region of MYH6 gene in Chinese patients with isolated and sporadic PDA. Moreover, 11 variants were identified in these subjects (721), of which four variants [g.3545G>T, g.4377T>C (rs373958405), g.4420A>G, g.4712C>T (rs913766127)] were found only in PDA patients. Two of these variants (g.3545G>T, g.4420A>G) have not been previously reported. Additionally, variant g.4377T>C (rs373958405) was found in 6 patients. In our study population (n = 721), we found 4 different variants in the MYH6 gene promoter region in 9 PDA patients with a frequency of 2.1% (9/428). Only 2 of these 9 patients were preterm and 7 were full-term. The dual luciferase reporter analysis revealed that all four variants resulted in a significant reduction in promoter transcriptional activity in all of the cell lines used for cellular functional tests. Furthermore, EMSA showed that the TFs bound by the promoter variants were significantly altered compared to the wild-type. The above findings suggest that these four variants in the promoter region of the MYH6 gene are involved in the formation of PDA through the reduction of transcriptional activity and the alteration of bound TFs.

The MYH6 gene is an essential gene for the development of the cardiovascular system since the beginning of embryonic development [36]. The MYH6 gene not only encodes its own protein, but also affects the expression of MYH7 and MYH7b genes (two other critical genes that encode the heavy chain of the myocardium) [37, 38]. It has been shown that variants in the MYH6 gene contribute to the development of vascular disease [25]. In addition, animal experiments have shown that abnormal expression of MYH6 protein affects the contraction of vascular smooth muscle [29]. Moreover, reduced expression of MYH11, a key gene in vascular smooth muscle, leads to the formation of PDA [39]. In our previous study [28], it has been clearly shown that MYH6 protein interacts with MYH11.

To investigate whether these 4 variants are pathologically significant, we performed dual luciferase reporter analysis, a method widely used for the determination of transcriptional activity [40], in 3 species of mammalian cell lines: human HEK-293 cell line, mouse myocardial HL-1 cell line, and rat myocardial H9C2 cell line. Repeated assays showed that these four variants [g.3545G>T, g.4377T>C (rs373958405), g.4420A>G, g.4712C>T (rs913766127)] significantly reduced the transcriptional activity of MYH6 gene. This result suggests that these variants may significantly reduce the contractility of ductus arteriosus.

Variants in the promoter region may lead to changes in the bound TFs that result in a reduction of the transcriptional activity of the gene [41]. Therefore, we used the JASPAR database to predict whether the TFs bound by the variants were altered. Comparison with wild-type revealed that most of the disrupted TFBS are involved in vascular development. TEAD1, TEAD3, and TEAD4 are involved in arterial development [42, 43], and the release of altered plasma proteins from endothelial cells is associated with PDA in humans, as we previously demonstrated [31]. While HES2 plays an important role in vascular remodeling after ductus arteriosus closure [44], NKX2-3, NKX2-5, and NKX3-2 are involved in cardiovascular development [34, 35]. Further, NFAT5 regulates peripulmonary artery oxygen levels [45]. MSX, BARX1, HOXA, and DLX are involved in vascular endothelial development and play an important role in endothelial secretion-related factors [46–49]. In contrast, in the newly produced TFBS, BACH1 and ZNF354C are repressive TFs that inhibit endothelial cell angiogenic sprouting [32, 33], whereas MAFK is thought to have an inhibitory effect on vasoconstriction [50]. Taken together, the variants found in the present study affect the normal contraction of the smooth muscle and endothelial function of the ductus arteriosus after birth through alterations in the bound TFs. These factors prevent the closure of the ductus arteriosus.

To further verify the predictions of the JASPAR database, we used EMSA for validation. Biotin-labeled variant-types and wild-type probes were electrophoresed with nuclear proteins extracted from HEK-293, HL-1, and H9C2 cell lines. Gel imaging revealed alterations in the TFs bound by all 4 variants compared to the corresponding wild-type. These results further verified the results from the prediction of the JASPAR database.

Figure 3 is a schema describing the possible mechanism of PDA caused by the variants in the MYH6 gene promoter identified in this study. The low expression of MYH6 gene promoter activity caused by these variants, together with the variants in the MYH6 gene promoter region that may affect the formation of transcriptional regulatory complexes with related TFs, result in the reduced contraction ability of the smooth muscle of the ductus arteriosus, leading to the formation of PDA.

There are some limitations of this study. Although the present study used three cell lines to validate the functional role of the variants found in the promoter region in PDA patients, the final pathogenic role needs to be confirmed in animal experiments. However, the difficulty for establishment of animal model is that the formation of a CHD often involves multi-genes and gene-gene-interactions. Nevertheless, our study reveals the pathogenic role of these variants in the formation of PDA.

In conclusion, our study identified 11 variants in the promoter region of the MYH6 gene in Chinese patients with isolated and sporadic PDA with 4 only identified in the PDA patients. Among those, 2 were novel variants. Cellular functional tests in three different cell lines demonstrated that all 4 variants significantly decreased MYH6 gene expression and affected the binding of TFs and therefore promote the formation of PDA. Therefore, this study provides new insights to understand the genetic basis and facilitates further studies on the mechanism of PDA formation.

Materials and methods

Study participants

Fasting venous blood samples from patients aged 0–14 years with isolated sporadic PDA who underwent surgical repair or interventional procedures at the TEDA International Cardiovascular Hospital in Tianjin, China, between January 2016 and May 2022 was collected. After excluding cases with other comorbid CHDs and a family history of other diseases, a total of 428 cases (148 males and 280 females) were eligible. Blood samples were also collected from 293 healthy children (166 males and 127 females; age range: newborn to 14 years) for control analysis through a cardiac screening program and a physical examination program. The control children were all screened clinically including echocardiography to confirm the absence of a family history of CHDs, other diseases, and genetic disorders. This study was performed in accordance with the principles of the Declaration of Helsinki and was approved by the Ethics Committee of the TEDA International Cardiovascular Hospital (approval No: 0715-4, 2021, 2 August 2021). Written consent was informed and signed by the parents or guardians of the participants.

Gene sequence analysis

Genomic DNA was extracted from the blood sample using the RelaxGene Blood DNA System kit (TianGen, Beijing, China). Approximately 50 μl of DNA was extracted per 200 μl of blood, and then the concentration was measured and stored at −80°C. The sequence of MYH6 gene promoter was obtained from the GenBank database (NCBI. NG_023444.1). The polymerase chain reaction (PCR) primers were designed based on the sequence of the human MYH6 gene. The promoter of this gene is located at −1500 ~ −2000 bp upstream of the coding region, and the specific promoter sequence of MYH6 (1638 bp, transcription site located at −1791 bp ~ −154 bp) was screened in the GenBank database (Table 3 lists PCR primer sequences). PCR amplification was performed using genomic DNA as a template with DNA mix (GenStar, Beijing, China). The amplification conditions were set as follows: extension at 95°C for 5 min, followed by 35 cycles (denaturation at 95°C for 30 s, annealing at 60°C for 30 s, extension at 72°C for 105 s), and finally extension at 72°C for 10 min. Subsequently, the PCR products were sequenced by Sanger sequencing two-way sequencing pass. The primers for Sanger sequencing are shown in Table 3. The DNA sequences were then compared with the wild-type MYH6 gene promoter for comparison and regulatory variants were identified.

Table 3

List of primers used in this study.

Primers Name	Sequences	Location
PCR primers
MYH6-F1	5′-GGGGCCTCGAGTAACCTAGA-3′	3209–3228
MYH6-R1	5′-CCCCCTGATTTGCCCAAGAA-3′	4846–4827
Sequencing primers
MYH6-F1	5′-GGGGCCTCGAGTAACCTAGA-3′	3209–3228
MYH6-F2	5′-CAAAGAAGGGAATGTGAGTAT-3′	3727–3747
MYH6-R1	5′-CCCCCTGATTTGCCCAAGAA-3′	4846–4827
MYH6-R2	5′-GGCCAACATCCAACCTGCTC-3′	4231–4250
MYH6-R3	5′-GACTTGACCGTGTCTGTGC-3′	4135–4154
The double-stranded biotinylated oligonucleotides for the EMSA
g.3545G>T-F	5′-TGGGAGGGTGTTCCT(G/T)GGTGTGAGGGTGGG-3′
g.3545G>T-R	5′-CCCACCCTCACACC(C/A)AGGAACACCCTCCCA-3′
g.4377T>C-F	5′-AGGAGACCAGGCATGGCACT(T/C)ATGCAGACTGAGGCCA-3′
g.4377T>C-R	5′-TGGCCTCAGTCTGCAT(A/G)AGTGCCATGCCTGGTCTCCT-3′
g.4420A>G-F	5′-GAATTTCCTGACAAAAGAAA(A/G)CTGAGCCATGGAGATGGA-3′
g.4420A>G-R	5′-TCCATCTCCATGGCTCAG(T/C)TTTCTTTTGTCAGGAAATTC-3′
g.4712C>T-F	5′-ATGGCAGGGTGGGAGAGG(C/T)GGTGTGAGAAGGTCCTGT-3′
g.4712C>T-R	5′-ACAGGACCTTCTCACACC(G/A)CCTCTCCCACCCTGCCAT-3′
Nucleotide sequences for JASPAR prediction of TFBS
g.3545G>T-F	5′-TGGGAGGGTGTTCCT(G/T)GGTGTGAGGGTGGG-3′
g.4377T>C-F	5′-AGGAGACCAGGCATGGCACT(T/C)ATGCAGACTGAGGCCA-3′
g.4420A>G-F	5′-GAATTTCCTGACAAAAGAAA(A/G)CTGAGCCATGGAGATGGA-3′
g.4712C>T-F	5′-ATGGCAGGGTGGGAGAGG(C/T)GGTGTGAGAAGGTCCTGT-3′

PCR primers are designed based on the genomic DNA sequence of the MYH6 gene (NG_023444.1). The transcription start site is at the position of 5001 (+1). Protective bases are presented in bold. EMSA, electrophoretic mobility shift assay; TFBS, transcription factor binding sites; F, forward; R, reverse.

Plasmid construction, cell culture and transfection

To determine whether the identified variants affect promoter activity, the MYH6 gene promoter region was inserted into the KpnI and SacI sites of the pGL6 plasmid (firefly luciferase reporter vector). Subsequently, PGL-6 plasmids carrying the MYH6 promoter sequences with or without the individual variants were respectively transformed into E. coli DH5α Competent Cells (GenStar, Beijing, China) and then transferred to Luria-Bertani solid medium being incubated at 37°C for 12–16 h.

The screened bacteria bearing the target gene were then incubated at 37°C and 200 rpm for 12–16 h for propagation. Plasmid extraction was performed from E. coli using the Plasmid Extraction Kit (Beyotime Biotechnology, Shanghai, China), followed by concentration determination, and the extracted plasmids were stored at −80°C for backup.

HEK-293, HL-1, and H9C2 cell lines were resuscitated in MEM/DMEM (10% fetal bovine serum plus penicillin and streptomycin). Cells were transferred to culture in six-well plates 12–24 h prior to transfection. When cells grew to 40%–60% of the culture wells, the pGL6-myh6 promoter plasmid was cotransfected with pRL-SV40 (reninase reporter plasmid) in a 5:1 ratio into the cells (approximately 1 × 10⁶ cells per 2.5 ng DNA plasmid). The cells without pGL6 plasmid (blank) and pGL6-basic were used as negative controls. The cells were cultured in 10% fetal bovine serum without antibiotics for 6–8 h and then switched to 10% fetal bovine serum with antibiotics and continued to be cultured for 24–48 h. PRL-SV40 was used as an internal control. This was followed by a dual luciferase activity assay.

Dual luciferase reporter gene analysis

After transfection was completed, the cells were collect and lysed well to preserve the lysate. Measurement of dual luciferase reporter gene activity was performed with the Dual Luciferase Reporter Gene Assay Kit (Beyotime Biotechnology, Shanghai, China). The firefly activity of the cell lysates was measured to determine the Renilla luciferase activity of pRL-SV40 as an internal control to correct for transfection efficiency. The results showed the relative fold change of these construct vectors with the wild-type MYH6 gene promoter in the expression vector. To ensure reproducibility of the experiments, all experiments were replicated six times independently.

Transcription factor binding sites prediction

TFs regulate the expression of target genes by recognizing TFBS in the cis-regulatory regions of target genes (promoters and enhancers) and interact with DNA in a specific manner. The variants in the promoter region may also lead to changes in binding TFs. There are various databases available to predict the changes in TFBS binding by promoter regions of different base sequences. The commonly used JASPAR database (https://jaspar.genereg.net/) for TF prediction was used this study. The list of all potentially affected binding sites for identified variants at MYH6 gene promoter was established by JASPAR with the relative spectrum score threshold set to 85%. A comparative analysis of the filtered differential TFBS was performed. The nucleotide sequences of wild-type and variants are shown in Table 3.

Preparation of nuclear extracts and electrophoretic mobility shift assay (EMSA)

Since TFs regulate the transcriptional function of genes by binding to promoter regions. The EMSA method was used to detect the effect of variants found in the MYH6 promoter on potential TFs. First, nuclear proteins were extracted from HEK-293 cells, HL-1 cells, and H9C2 cells stored at −80°C, using the Nuclear and Cytoplasmic Protein Extraction Kit (Beyotime Biotechnology, Shanghai, China). Subsequently, prepared biotinylated double-stranded oligonucleotide probes with or without the variants (Table 3) were annealed at 95°C for 90 min and prepared for further use. EMSA was conducted using equimolar levels of probe (0.25 pM) and nuclear extract (2.0 μg), followed the manufacture’s protocol, performed with an EMSA kit (Beyotime Biotechnology, Shanghai, China). The process is shown in Fig. 4.

Statistical analysis

Quantitative data were expressed as mean ± standard deviation and compared using one-way ANOVA. All statistical analyses were performed using SPSS 25.0 software. P < 0.05 was considered a statistically significant difference.

Acknowledgements

We thank the patients and their family members for cooperation. The assistance of surgeons and Nursing staff at the Division of Pediatric Cardiac Surgery, Department of Cardiovascular Surgery is gratefully acknowledged.

Author contributions

G.W.H. conceptualized the project. G.W.H. and H.X.C. designed the study. G.W.H. obtained the funding for this project. J.Y.Z., H.X.C. performed experiments. J.Y.Z., H.X.C., and G.W.H. analyzed experimental data. J.Y.Z., H.X.C., and G.W.H. collected the human blood samples. Q.Y. discussed the findings and participated in the project. J.Y.Z., and G.W.H. wrote the manuscript. G.W.H. supervised the whole project. All authors have accepted responsibility for the entire content of this manuscript and approved its submission.

Conflict of interest statement

The authors declare that they have no conflict of interest.

Funding

This work was funded by the National Natural Science Foundation of China [82170353 & 82370350]; Tianjin Science and Technology Commission [22ZYQYSY00020]; the Non-profit Central Research Institute Fund of Chinese Academy of Medical Sciences [2020-PT310-007]; Tianjin Municipal and Binhai New Area Health Commissions [2019BWKY010]; TEDA International Cardiovascular Hospital [2021-ZX-002]; and Tianjin Key Medical Discipline (Specialty) Construction Project (TJYXZDXK-019A).

Data availability

All sequencing data used to support the findings of this study are available from https://ngdc.cncb.ac.cn/omix/view/OMIX001646 and the corresponding author upon request.

Ethical approval

This study involving human participants was reviewed and approved by the ethics committee of TEDA International Cardiovascular Hospital, China (No. 0715-4, 2021, 2 August 2021).