Functional analysis of promoter mutations in the ACTN4 and SYNPO genes in focal segmental glomerulosclerosis
Functional analysis of promoter mutations in the ACTN4 and SYNPO genes in focal segmental glomerulosclerosis
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DOI：
10.1093/ndt/gfp394
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发表时间：
2010-03-01
影响因子：
6.1
通讯作者：
Chen, Nan
中科院分区：
文献类型：
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作者：
Dai, Shengchuan;Wang, Zhaohui;Chen, Nan
方法.研究包括82例中国特发性FSGS患者（55例肾病综合征：NS）和90名健康人。从患者和健康个体的外周血白细胞中提取基因组DNA，通过聚合酶链反应（PCR）和直接测序来分析ACTN 4和SYNPO基因启动子突变。将突变与GenBank和TRANSFAC软件数据库（www.genometix.de; www.gene-regulation.com）匹配。使用双荧光素酶测定系统来分析基于PGL 3-Basic载体、pRL-SV 40载体、PC 12细胞系和足细胞的突变的体外效应。研究突变患者的肾脏α-辅肌动蛋白-4和突触足蛋白表达及其父母的基因组DNA。本研究分别在3例患者中检测到ACTN 4基因启动子1- 34 C> T、1- 590 delA和（1- 1044 delT）+（1- 797 T> C）+（1- 769 A> G）杂合突变，在2例患者中分别检测到SYNPO基因启动子1- 24 G> A和1- 851 C> T杂合突变（翻译起始位点ATG命名为腺嘌呤+1）。在90名健康人的对照组中未发现相同的突变。除1例ACTN 4基因启动子突变患者分别遗传了父母1- 1044 delT和1- 797 T> C突变染色体外，其余患者均未发现相同的突变。在突变患者的肾脏中，α-辅肌动蛋白-4和synaptopodin蛋白表达减少。双荧光素酶测定显示，与正常组相比（1- 1044 delT组除外），突变组中的荧光素酶活性大部分降低。（1- 1044 delT）+（1- 797 T> C）+（1- 769 A> G）突变与不良临床结局相关，携带这些突变的患者进展为终末期肾衰竭。该研究检测了特发性FSGS患者ACTN 4和SYNPO基因启动子的杂合突变。这些突变影响体外基因转录，并可能影响体内蛋白质翻译。因此，我们推测，ACTN 4和SYNPO启动子突变也可能有助于特发性FSGS的病理生理。
Methods. The study consisted of 82 Chinese idiopathic FSGS patients (55 patients had nephrotic syndrome: NS) and 90 healthy individuals. Genomic DNA extracted from peripheral leukocytes of patients of healthy individuals were used to analyse the ACTN4 and SYNPO gene promoter mutations by polymerase chain reaction (PCR) and direct sequencing. Mutations were matched with GenBank and TRANSFAC software database (www.genometix.de; www.gene-regulation.com). A dual luciferase assay system was used to analyse the effects of mutations based on PGL3-Basic vector, pRL-SV40 vector, a PC12 cell line and podocytes in vitro. Kidney alpha-actinin-4 and synaptopodin expression of mutated patients and genomic DNA of their parents were investigated.Results. The study detected the ACTN4 gene promoter 1-34C > T, 1-590delA and (1-1044delT)+(1-797T > C)+(1-769A > G) heterozygous mutations in three patients, respectively, and the SYNPO gene promoter 1-24G > A and 1-851C > T heterozygous mutations in two patients, respectively (with adenine of translation start site ATG naming +1). The same mutations were not found in the control group of 90 healthy people. Excepting one patient with an ACTN4 gene promoter mutation who inherited her parents' 1-1044delT and 1-797T > C mutated chromosome, respectively, the same mutations were not found in patients' parents. Alpha-actinin-4 and synaptopodin protein expression are reduced in mutated patients' kidneys. Dual luciferase assays show that compared to the normal group (with the exception of the 1-1044delT group), luciferase activity in mutated groups decreased for the most part. (1-1044delT)+(1-797T > C)+(1-769A > G) mutations are associated with poor clinical outcomes, and patients with these mutations progress to end-stage renal failure.Conclusion. The study detected heterozygous mutations in the promoters of the ACTN4 and SYNPO genes in patients with idiopathic FSGS. These mutations affected gene transcription in vitro and may affect protein translation in vivo. So we presumed that the ACTN4 and SYNPO promoter mutations might also contribute to pathophysiology of idiopathic FSGS.