Functional Analysis Of CFHR3 C.424C>T With A Clear Picture Of The Disease Risk Of Rare Glomerular Nephropathies

Complement system,which has the characteristics of crucial immune responses and cascade reactions,not only has a crucial role in acting as a bridge between innate and adaptive immunities,but also is a part of immune regulatory network,and has important significance in the preservation of immunological homeostasis.The complement system is composed of three distinct activation pathways:the classical,lectin and alternative.The complement system has long been recognized as having a role in immune complex-mediated glomerulonephritis,and the pattern of complement activation is via the classic complement pathway.Recently,the AP has also been suggested to cause kidney injury in a variety of different diseases.Excessive complement activation is particularly important in the pathogenesis of atypical hemolytic uremic syndrome(aHUS),membranoproliferative glomerulonephritis(MPGN I),and C3 glomerulopathy;the latter includes dense deposit disease(DDD)and C3 glomerulonephritis(C3GN).Despite distinct clinical manifestations and histological aspects,renal lesions selectively manifest in kidneys,suggesting the presence of tissue-specific determinants of disease development in these rare glomerular nephropathies featuring the common hallmark of an overactive alternative complement pathway.Similar genetic alterations were even found to be involved in the distinct renal pathology of these rare kidney diseases.Sequencing of the genes coding for complementcomponents has long been used to investigate the mechanism behind these manifestations.Understanding the effect of genetic background on mutant phenotypes also has specific medical relevance.However,the present studies are mostly isolated cohort reports,and the vast majority of disease-related variants lacks adequate information on the pathogenic mechanism.The purpose of this study is to explore shared genetic background in four complex phenotypes.Furthermore,the pathologic significance of variations were systematically evaluated in the present study.Part ? A haplotype in CFH family genes confers high risk of rare glomerular nephropathiesHere,we screen 11 complement genes by high-throughput sequencing from 91 patients(DDD =10,C3GN=3 3,MPGNI =24,aHUS =24)with a definite diagnosis established on renal biopsies in Nanjing General Hospital.After gene screening,we focused on three rare SNPs,rs55807605(CFH c.2509G>A),rs61737525(CFHR3 c.424C>T)and rs57960694(CFHR5 c.434G>A),which were simultaneously detected in four cases.All of them were sporadic cases and diagnosed with aHUS,G3GN,DDD and MPGN I,respectively.In accordance with the character of AP activation of C3 consumption in the fluid phase and local C3 deposit formation,a marked C3 staining along the capillary wall or mesangium was detected by immunofluorescence analysis in all cases,and the patients with aHUS and C3GN presented a low level of C3.Linkage disequilibrium(LD)structure was analyzed based on the Chinese Han population(1000 Genomes Project Phase 3).Rs55807605,rs61737525 and rs57960694 showed strong LD,which constructed two haplotypes("GCG" and"ATA");estimated frequency was 1.2%for ATA haplotype.We further conducted a retrospective case-control study involving 91 patients and 300 healthy control individuals.Haplotype-association analysis revealed that the ATA haplotype of the three minor alleles(A at rs55807605,T at rs61737525,and A at rs57960694)was significantly associated with increased risk.The minor allele "T" of rs61737525 was further found to be with increased susceptibility of these rareglomerular nephropathies in allele model and dominant model.Additionally,Align GVGD,SIFT,PROVEAN,SNAP,and PolyPhen-2 independently indicated the deleterious pathogenicity of CFHR3 c.424C>T.Besides,rs55807605 and rs57960694 presented no direct association with these glomerular diseases.And no significant synergistic interaction of these three SPNs was observed under nonparametric MDR(Multifactor Dimensionality Reduction).The frequently observed genomic rearrangements of CFH/CFHR locus were actually identified in individuals with these rare renal diseases.However,potential genetic fusion linked with the three SNPs have been excluded by qPCR and western blot assays in the present study.To summarize,it demonstrated that rs61737525(CFHR3 c.424C>T)might be a leading pathogenic variant in this risk locus,associated with renal patients with aHUS,DDD,C3GN or MPGN I.Part? Pathogenic mechanism of c.424C>T mutation in the gene CFHR3Based on the results in part I,we found a leading pathogenic variant in a shared risk haplotype in a complement-regulatory gene family among a group of rare glomerular nephropathies featuring the common hallmark of an overactive alternative complement pathway.The purpose of this part is to further determine the pathogenic mechanism of the mutation.In detail,the c.424C>T in the gene CFH3 generates a nonsynonymous mutation p.Arg142Cys,leading to a polar change from a positively charged residue to a neutral one and a moderate decrease of isoelectric point(pI).Amino acid(aa)Arg142Cys was located in the CCP2/3 linker.3D homology-modeled structures of wild-type and mutant CFHR3 were generated.The mutant protein demonstrated a random coil,which was introduced into the amino acid chain in substitution of an original beta-sheet.DS 3.0 analysis predicted that the calculated potential energy of the wild type protein is-10569.06 kcal/mol compared to-10542.30 kcal/mol for the mutant one.As substituted by Cys142,the native intramolecular hydrogen bonding interactions between the side chain of Arg142 and neighboring residues were absent.These analyses in silico implied that p.Arg142Cys could decrease the stability of CFHR3.To verify the function of Arg142Cys substitution,wild-type(CFHR3WT)and mutant(CFHR3Argi42Cys)recombinant proteins were generated in the Escherichia coli expression system,and C3b and factor I were purified from normal human serum.Surface plasmon resonance analysis further demonstrated the substitution induced a decrease of two orders of magnitude in C3b-binding properties,with a declined cofactor activity in fluid phase.In conclusion:Firstly,our study firstly reports the case of a shared genetic background within the CFH gene cluster among aHUS,DDD,C3GN and MPGNI.The leading pathogenic variation in this risk haplotype ATA is CFHR3 p.Arg142Cys(rs61737525).Secondly,multiple algorithms were used to predict the molecular pathomechanism at full length.Thirdly,our experimental data demonstrated that Arg142Cys substitution induced a decrease of approximately two orders of magnitude in the C3b-binding affinity and weaken the cofactor activity in the cleavage of C3b inthe fluid phase.Finally,expanding our knowledge on the genetics,tissue expression and physiological functions of the CFHR proteins will be of great value to the understanding of these severe anomalies,and hopefully-to more beneficial treatment of AP abnormalities.