Mengmeng X, Yuejuan X, Sun C, Yanan L, Fen L, Kun S.BMC Med Genet. 2020 May 7;21(1):95. doi: 10.1186/s12881-020-01032-y.PMID: 32380971 Free PMC article.
Background: Conotruncal heart defects (CTDs) are a group of congenital heart malformations that cause anomalies of cardiac outflow tracts. In the past few decades, many genes related to CTDs have been reported. Serum response factor (SRF) is a ubiquitous nuclear protein that acts as transcription factor, and SRF was found to be a critical factor in heart development and to be strongly expressed in the myocardium of the developing mouse and chicken hearts. The targeted inactivation of SRF during heart development leads to embryonic lethality and myocardial defects in mice.
Methods: To illustrate the relationship between SRF and human heart defects, we screened SRF mutations in 527 CTD patients, a cross sectional study. DNA was extracted from peripheral leukocyte cells for target sequencing. The mutations of SRF were detected and validated by Sanger sequencing. The affection of the mutations on wild-type protein was analyzed by in silico softwares. Western blot and real time PCR were used to analyze the changes of the expression of the mutant mRNA and protein. In addition, we carried out dual luciferase reporter assay to explore the transcriptional activity of the mutant SRF.
Results: Among the target sequencing results of 527 patients, two novel mutations (Mut1: c.821A > G p.G274D, the adenine(A) was mutated to guanine(G) at position 821 of the SRF gene coding sequences (CDS), lead to the Glycine(G) mutated to Asparticacid(D) at position 274 of the SRF protein amino acid sequences; Mut2: c.880G > T p.G294C, the guanine(G) was mutated to thymine (T) at position 880 of the SRF CDS, lead to the Glycine(G) mutated to Cysteine (C) at position 294 of the SRF protein amino acid sequences.) of SRF (NM_003131.4) were identified. Western blotting and real-time PCR showed that there were no obvious differences between the protein expression and mRNA transcription of mutants and wild-type SRF. A dual luciferase reporter assay showed that both SRF mutants (G274D and G294C) impaired SRF transcriptional activity at the SRF promoter and atrial natriuretic factor (ANF) promoter (p < 0.05), additionally, the mutants displayed reduced synergism with GATA4.
Conclusion: These results suggest that SRF-p.G274D and SRF-p.G294C may have potential pathogenic effects.
Fig. 1 Sequencing chromatograms of the two heterozygous mutants. Panel (a) shows the chromatograms of the p.G274D mutants. Panel (b) shows the chromatograms of the p.G294C mutants. (“↓” shows mutation sites)
Fig. 2 Schematic representation of SRF gene and protein. SRF gene is 10.212 kb and contains seven exons. a and b Diagram shows the nucleotide variants (a) and amino acid mutations (b) of SRF identified in our study cohort. (red octagon containing “p” stands for phosphorylation site; green pentagon stands for O-Glycosylation sites). c. Alignment of SRF amino acid residues among different species indicating the level of conservation
Fig. 3 Western blot (a) and RT-PCR (b) showed there were no obvious differences in protein and gene expressions between mutant and wild-type SRF in HEK293 cells. Anti-SRF antibody and anti-actin antibody (internal control) were used as the primary antibodies. (WT: wild-type, Mut1: p.G274D; Mut2: p.G294C; Blank: pcDNA3.1(+) vector)
Fig. 4 Co-transfected and luciferase assay in NIH3T3, Luciferase activity was used to measure transcription. a,b. When transfected alone or co-transfected with GATA4, both Mut1 and Mut2 suggested a decrease in transcriptional activation of ANF promoter when compared to wild-type SRF. (t test, *p < 0.05. c. WT vs Mut1: p = 0.0002, WT vs Mut2: p = 0.003, WT + GATA4 vs Mut1 + GATA4: p = 0.0139, WT + GATA4 vs Mut2 + GATA4: p = 0.0183). c. When NIH3T3 cells were supplemented with 10%FBS, wild-type SRF inhibited the activation of SRF promoter more significantly. (t test, *p < 0.05. WT vs Mut1: p = 0.0035, WT vs Mut2: p = 0.0343). d. When NIH3T3 cells were supplemented with 0.5%FBS, wild-type SRF inhibited the activation of SRF promoter more significantly. (t test, *p < 0.05. WT vs Mut1: p = 0.0018, WT vs Mut2: p = 0.0289). (WT: wild-type, Mut1: p.G274D; Mut2: p.G294C; Blank: pcDNA3.1(+) vector)