Functional analysis of promoter mutations in the ACTN4 and SYNPO genes in focal segmental glomerulosclerosis

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.