Pathogenicity Analysis Of Novel Variants In CRB2 Gene Identified In A Child With Focal Segmental Glomerulosclerosis

BackgroundFocal segmental glomerulosclerosis(FSGS)is a clinicopathological syndrome which lead to steroid resistant nephrotic syndrome and end stage renal disease in children.The clinical manifestations are massive proteinuria or nephrotic syndrome and the pathological features are non-inflammatory sclerosis of the glomerular capillary loops.Previous studies reported that the pathogenesis of FSGS is related to gene mutations in podocyte-associated proteins and more than 30 single-gene mutations have been found,but it has different mutation rates in different parts of the world.In addition,the detection rate of some genes in Chinese Han population was low,it is speculated that may be new pathogenic genes in Chinese patients.ObjectiveThe clinical cases of FSGS were collected in our department for nearly 5 years and whole exon gene sequencing were carried out.To investigate the pathogenicity and protein expression of novel mutations by bioinformatics software analysis and construct a vitro eukaryotic transient expression system.Methods1.Using the medical record retrieval system to search patients diagnosed FSGS and analysis of clinical informations in pediatrics of Puyang Oilfield General Hospital of Xinxiang Medical College from January 2014 to January 2019.After obtained the consent of the child and parents,2mL blood samples were taken from proband and parents for gene sequencing by WES,Sanger sequencing confirms variant sites identified.Whatmore,50 childs with normal urine tests were used as controls.2.Conserved analyses of multiple species amino acid sequence for CRB2 gene novel variable sites by using the Clustal Omega online web page.The online homology modeling software,Swiss-Model and Swiss-Pdb Viewer,were used to predict the protein three-dimensional structural of the CRB2 gene novel variants and to analyze the protein structure function.3.Aim to establish the eukaryotic expression system of CRB2 gene in vitro,we used pcDNA3.1-EGFP as blank control vector,then constructed the wild type and variant plasmids.Total protein were collected after the wild type and variant plasmids were transfected into HEK-293 T cell,and the level of CRB2 gene protein expression were observed by fluorescence microscope and western blot.Results1.In this study,9 cases were included but missing 1 case.The FSGS families of 8 cases were sequenced by WES.Among them,CRB2 gene variation was detected in a Chinese patient,including c.445G>T(p.E149X)? c.2705C>T(p.T902M)and c.3190C>T(p.P1064S).2.The amino acids of CRB2 gene novel missense variations(p.E149X?p.T902M?p.P1064M)are highly conserved in different species.Online analysis of Swiss-Model and Swiss-Pdb Viewer shows that p.T902M?p.P1064 M result in slight changes in the three-dimensional structure of CRB2 protein,however,p.E149 X can not form a complete protein structure and belongs to the heavy variant.3.The eukaryotic expression system of CRB2 gene was successfully constructed and was highly expressed in HEK-293 T cell.The results showed that nonsense variant p.E149 X not detected green fluorescence and the protein expression of western blot decreased after compared with wild type and missense variant p.T902 M and p.P1064 S.So it was considered that p.E149 X was the main pathogenic gene.Conclusion1.The variable of CRB2 gene was detected for the first time in a Chinese child with sporadic FSGS in this study,and p.E149X?p.T902M?p.P1064 M were novel variable sites,which enriched the variable spectrum of CRB2 gene.2.Bioinformatics analysis and functional expression experiments in vitro of CRB2 gene,it is suggested that p.T902 M is not the main variable site,p.P1064 S is possible harmful based on protein structure,p.E149 X may be the key pathogenic variable site in this study.