Identification and functional analysis of variants of MYH6 gene promoter in isolated ventricular septal defects

Figures

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  Fig. 1. Study flow chart. A total of 604 subjects were recruited. DNA was extracted from blood samples. Sequence analysis and cellular function experiments were performed

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  Fig. 2. 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 5,001 in the first exon. B Sequencing chromatograms of all variants found in VSD patients and controls (the sequencing peaks are reverse sequencing). Top panels show wild-type and bottom panels show variant-type, marked with arrows. VSD, ventricular septal defect

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  Fig. 3. Results of dual luciferase reporter gene analysis. A Relative transcriptional activity of wild-type and variants of MYH6 gene promoters in HEK-293 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 five independent experiments (n = 5, ***P < 0.001, ****P < 0.0001). B Relative transcriptional activity of wild-type and variants of MYH6 gene promoters in HL-1 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 five independent experiments (n = 5, ***P < 0.001, ****P < 0.0001). C Color Doppler echocardiography shows VSD (arrow) in patients with variants of g.4085G>C (rs1222539675) and g.4716G>A (rs377648095). AO, aorta; LA, left atrium; RA, right atrium; RV, right ventricle; LV, left ventricle; VSD, ventricular septal defect

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  Fig. 4. Cellular function experiments. A At different times (40 s and 90 s), two variants (g.4085G>C and g.4716G>A) were seen to create or abolish the binding of TFs in HEK-293 cells. B At different times (30 s and 60 s), two variants (g.4085G>C and g.4716G>A) were seen to create or abolish the binding of TFs in HL-1 cells. C The protein–protein interaction showing that MYH6 protein interacts with a number of proteins that playimportant role in the cardiac formation including TNNI1, MYBPC3, MYL2, ACTC1, ACTN2, MYL1, TNNT2, TPM1, and TNNI3, exec. EMSA, electrophoretic mobility shift assay; WT, wild-type; VT, variant-type

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  Fig. 5. The schema describing the role of variants of MYH6 gene promoter. Schema describes the role of variants in the promoter region of the MYH6 gene identified from this study in connection with the analysis of the JASPAR database and findings from previous studies. Briefly, the variants in the MYH6 gene promoter identified in this study may modify the cluster of TFBS, resulting in altered gene expression. Low-expression of MYH6 is associated with attenuated myosin heavy chain formation that is directly related to the development of VSD. In addition, variants in MYH6 promoter affect the binding of TFs such as TBX1, TBX4, TBX6, TBX15 and TBX18, possibly resulting in formation of VSD. TF, transcription factor; TFBS, transcription factor binding sites; VSD, ventricular septal defect