中国散发性圆锥动脉干畸形患者 SRF 基因的新突变。
Novel mutations of the SRF gene in Chinese sporadic conotruncal heart defect patients.
机构信息
Department of Pediatric Cardiology, Xinhua Hospital, Shanghai Jiao Tong University School of Medicine, No.1665 Kongjiang road, Shanghai, 200092, China.
Shanghai Children's Medical Center, Shanghai Jiao Tong University School of Medicine, No. 1678, Dongfang Road, Shanghai, 200127, China.
出版信息
BMC Med Genet. 2020 May 7;21(1):95. doi: 10.1186/s12881-020-01032-y.
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.
背景
圆锥动脉干畸形（CTD）是一组先天性心脏畸形，导致心流出道异常。在过去的几十年中，已经报道了许多与 CTD 相关的基因。血清反应因子（SRF）是一种普遍存在的核蛋白，作为转录因子发挥作用，并且在心脏发育过程中被发现是一个关键因素，并且在发育中的小鼠和鸡心脏的心肌中强烈表达。在心脏发育过程中靶向性地敲除 SRF 会导致胚胎致死和心肌缺陷。
方法
为了说明 SRF 与人类心脏缺陷之间的关系，我们对 527 例 CTD 患者进行了横断面研究，筛选了 SRF 突变。从外周白细胞中提取 DNA 进行靶向测序。通过 Sanger 测序检测和验证 SRF 的突变。通过计算机软件分析突变对野生型蛋白的影响。使用 Western blot 和实时 PCR 分析突变型 mRNA 和蛋白的表达变化。此外，我们进行了双荧光素酶报告基因检测，以探讨突变型 SRF 的转录活性。
结果
在 527 例患者的靶向测序结果中，发现了两个新的突变（Mut1：c.821A>G p.G274D，腺嘌呤（A）在位置 821 处突变为鸟嘌呤（G），导致丝氨酸（SRF 基因编码序列（CDS）中的位置 274 的甘氨酸（G）突变为天冬氨酸（D）；Mut2：c.880G>T p.G294C，在位置 880 处的鸟嘌呤（G）突变为胸腺嘧啶（T），导致丝氨酸（SRF 蛋白的位置 294 的甘氨酸（G）突变为半胱氨酸（C）。）SRF（NM_003131.4）。Western blot 和实时 PCR 显示突变型和野生型 SRF 的蛋白表达和 mRNA 转录没有明显差异。双荧光素酶报告基因检测显示，SRF 突变体（G274D 和 G294C）均在 SRF 启动子和心钠肽（ANF）启动子处损害 SRF 转录活性（p<0.05），此外，突变体与 GATA4 的协同作用降低。
结论
这些结果表明，SRF-p.G274D 和 SRF-p.G294C 可能具有潜在的致病作用。
Zotero 插件