The Study Of The Variations Of Genes Related To FSGS

Objectives: Focal segmental glomerulosclerosis(FSGS)is a clinical pathological syndrome of podocyte injury exhibiting increasing prevalence across the world over recent decades.Its typical clinical manifestations include proteinuria,hypertension,renal insufficiency,and ultimately renal failure.Since the discovery of this clinicopathological phenomenon in the early 1930 s,various studies have shown potential pathogenesis of FSGS,including inherent defects of podocyte structure or function leading to primary FSGS,viruses,drugs,and circulating factors among others causing secondary FSGS,as well as genetic factors associated with certain gene mutations.However,the specific causes of FSGS still remain unconfirmed.A large number of studies have shown that mutations in single genes associated with podocytes are involved in the pathogenesis of FSGS.Our group has explored three podocyte-associated genes,specifically,the NPHS1,COL4A5,and APOL1 genes.Using bioinformatics analysis,clinical case report,and studying effects on cell morphology and function,we hope to preliminarily investigate the pathogenic mechanism of related genes that are instructive for the diagnosis and prognosis of FSGS.Methods:(1)Collect information of mutations of the NPHS1 gene reported in previous literature.Extract control variants from 2504 healthy subjects screened from the 1000 Genomes Project.A total of 309 patients with sporadic FSGS were enrolled,using Panel next generation sequencing and whole exon sequencing.Three kinds of softwares(SIFT,Polyphen,MutationTaster)were used to predict pathogenicity for missense mutations.A hot-spot functional map of nephrin was generated using the results of sequencing and prediction combined with known protein structures.(2)A case study was performed on a specific patient who confirmed as FSGS.Extraction of peripheral blood DNA for whole exon sequencing and analyze the immunohistochemical staining of type IV collagen,try to find a potential pathogenic mutation.(3)Based on the previous study,a site-directed mutation of APOL1 plasmid was constructed.Transfect mutant and wild-type plasmids into podocytes by electroporation,and observe cell morphology under microscope after 48 hours later.Transfect on the mutant and wild-type plasmids with Lipofectamine 3000 liposome method into 293 T cells which were tested for chloride channels.Results:(1)A total of 17 missense mutations and 1 splicing mutation were found in 309 sporadic FSGS patients.One of the novel mutation sites was c.G2638 T in exon 19,and the other one was 35830957 C>T splicing mutation in exon 28.According to software prediction,there were five of the missense mutations might be pathogenic,and most of the mutations were concentrated in the Ig1,Ig3,Ig7 and C-terminal cell transmembrane domains of nephrin.(2)Immunohistochemical staining of kidney tissue showed that the staining effect of type IV collagen antibody-the group of COL4A5 was lower than control group.The whole exon sequencing result showed a frameshift mutation of 20 th amino acid.A comprehensive diagnosis of this patient was confirmed as a case of FSGS secondary to Alport syndrome caused by a mutation in the COL4A5 gene.(3)The APOL1 gene could cause lysis and rupture of podocytes.The novel discovered mutation could cause large-scale morphological abnormalities in podocytes,and even could lead to clustering and death.At the same time,the gene would up-regulate the current value of the cellular chloride channel.As the voltage increased,the mutation group had a significant effect on the chloride ion flow.Conclusions: We explored the relationship between the mutations of three genes and FSGS in Chinese patients.Firstly,the results showed that the NPHS1 gene mutations were common in Chinese patients with FSGS.The genetic background of FSGS in China was significantly different from that in western countries.NPHS1 gene and FSGS exhibit an observable level of correlation.Secondly,the novel COL4A5 gene mutation might be a pathogenic genetic of Alport syndrome.It is recommended that the pathological results combined with genetic testing would be used to diagnose complex Alport syndrome with FSGS which could avoid inappropriate treatment.Thirdly,a novel mutation in APOL1 gene found in Chinese patients shows a strong causal relationship to damage in podocyte and the destruction of chloride channel proteins,leading to the development of FSGS.