Genetic Analysis And Mutation Detection Of The Complement System Of C3 Glomerulopathy

C3 glomerulopathy often presents with a membranoproliferative glomerulonephritis pattern on light microscopy,and is characterized by predominant glomerular C3 deposition with little or no immunoglobulin staining on immunofluorescence microscopy.Unrestricted activation of the complement alternative pathway principally caused by genetic abnormalities critically implicate in the pathogenesis of complement related rare diseases,including C3 glomerulopathy,immune complex mediated membranoproliferative glomerulonephritis and atypical hemolytic uremic syndrome,and has been studied by large works in recent years.However,reported studies limited on Caucasian and thus the genetic profile of the complement system remains open in Asia;furthermore,correct assignment of pathogenicity lies in a rigorous assessment and expert interpretation and is of great importance.Complement related disorders present as complex multigenic disease while most cases have no familiarity,which indicates the presence of atypical patterns of inheritance.Therefore,the aim of this study was to explore the genetic characteristics in the largest Asian cohort,and the potential mechanism of postzygotic single-nucleotide mosaicisms in the pathogenesis of those rare nephropathies.Part ?:Genetic profile of the complement pathway in C3 glomerulopathyIn this part,we completed a comprehensive screen of 11 candidate alternative pathway genes with targeted genomic enrichment and massively parallel sequencing on 43 patients with sporadic C3 glomerulopathy,which were classified as dense deposit disease(n=10)and C3 glomerulonephritis(n=33)cases.An additional 24 patients with immune-complex-mediated membranoproliferative glomerulonephritis were also enrolled.In total,four novel and sixteen rare variants were identified:One was classified as likely pathogenic,and the remaining nineteen were of uncertain significance.Three variants reportedly led to functional deficiency with supporting evidences.Variants in the CFH,CFI,CD46,and C3 genes were most frequently detected.A defective control of the complement alternative pathway due to hereditary abnormalities was found at frequencies of 50%,27%,and 17%in dense deposit disease,C3 glomerulonephritis,and immune-complex-mediated membranoproliferative glomerulonephritis,respectively.Irrespective of histological type,the patients with likely pathogenic and uncertain significant variants were clinically similar to those without.Accurate genetic screening can give rise to progress in understanding the pathogenesis of C3 glomerulopathy and other complement related rare nephropathies,and correct assignment of pathogenicity classification is of great importance for better patient care and prognostic or therapeutic advice.Part ?:Detection and validation of postzygotic single-nucleotide mosaicisms of complement genes in patients with C3 glomerulopathyIn this part,we performed targeted next generation sequencing of DNA from peripheral blood in patients with complement related nephropathies,including dense deposit disease(DDD),C3 glomerulonephritis(C3GN),immune-complex-mediated membranoproliferative glomerulonephritis(IC-MPGN)and atypical patterns of inheritance(aHUS).We combined the computational pipeline of MosaicHunter and PGM amplicon sequencing of mosaicism(PASM)to identify postzygotic single-nucleotide mosaicisms(pSNMs),and subsequently validated the candidates implementing pyrosequencing.pSNMs were further evaluated in kidney biopsy,buccal mucosa and urine samples from the same patients.Potential biological significance was evalutated with integrated bioinformatic methods.From targeted next generation sequence,we identified 9 potential pSNMs with MosaicHunter and PASM.Pyrosequencing confirmed the mosaic state of variant c.2808G>T in CFH gene(rs1065489)in a DDD patient and a C3GN patient(5%and 5%,respectively),but negative finding under Sanger sequencing.The mutant alleles of CFH c.2808G>T was significantly higher in kidney tissues than in peripheral blood(P<0.001),and it ranged from 8%to 35%in kidney tissues,urine and buccal mucosa.Meta-analysis illustrated a significant association of an increased disease risk between allele T with complement related nephropathy,especially for the DDD and aHUS.In silico analysis predicted the deleteriousness that the CFH rs1065489-T mutant allele leaded to a decrease of binding affinity of the CFH/C3b complex(?G = 6.55 kcal/mol).The present study demonstrated the value of the next generation sequencing combined with computational pipeline in identifying mosaic mutations.Identification of pSNMs in complement genes highlights the diversity of genotype-phenotype correlations,and alerts the nephrologist to the importance of a possible implication in rare nephropathies.In conclusion,this study completed the genetic screen in the largest Asian cohort of patients with complement related rare nephropathies and provided the following results.Firstly,one likely pathogenic variants and nineteen VUS were identified;Secondly,variants in the CFH,CFI,CD46,and C3 genes were most frequently detected;Thirdly,a defective control of the complement alternative pathway due to hereditary abnormalities was found at frequencies of 50%,27%,and 17%in DDD,C3GN,and IC-MPGN,respectively;Fourthly,pyrosequencing confirmed the mosaic state of variant c.2808G>T in CFH gene in two patients,and the mutant allele was significantly higher in kidney tissues than in peripheral blood(P<0.001);Fifthly,meta-analysis illustrated a significant association between allele T with DDD and aHUS.Lastly,in silico analysis predicted the deleteriousness that the CFH rs1065489-T leaded to a decrease of binding affinity of the CFH/C3b complex(?G=6.55 kcal/mol.