Patients and control subjects
Samples of 206 patients were sent to our Research Laboratory from Central-European clinical centers (n = 34) with the suspicion of complement-mediated renal disease for complement investigations, and for them genetic analysis was also carried out. 86 patients were excluded because of alternative diagnosis or secondary MPGN. 120 patients with the diagnosis of IC-MPGN/C3G were enrolled in the study from January 2008 to May 2019.
The clinical, histological and laboratory data were collected from the clinicians and pathologists according to study protocols approved by the Medical Research Council of the Ministry of Human Capacities in Hungary (approval’s number:55381-1/2015/EKU).
Biopsy data were collected using standardized questionnaire forms from pathologists (n = 73) or extracted from the biopsy descriptions (n = 47).
Eighty-five subjects formed the control group (68 adults, 17 children). All of them were referred for routine medical examination and none of them had any known disease at time of blood sampling.
Both the healthy subjects and the patients or their parents gave their written approval – after informed consent – for the analysis in accordance with the Declaration of Helsinki.
Molecular Genetic Analysis
In 111 patients the whole coding region of the gene encoding FHR-5 (CFHR5; OMIM# 608593) was screened by direct bidirectional DNA sequencing, as described in the case of further disease-associated genes (CFH, CFI, CD46, THBD, CFB, C3) that were sequenced also (44). No DNA samples were available from the remaining 9 patients. These genes were not sequenced in the included healthy subjects; instead of that the available public databases (1000Genomes Project, gnomAD) were used to acquire data about allele frequencies in more reliable and larger healthy populations. Primer sequences and PCR conditions are available upon request. Multiplex ligation-dependent probe amplification (MLPA) was performed to study the presence of copy-number alterations in the chromosomal regions of the CFHR1, CFHR2, CFHR3 and CFHR5 genes, with the SALSA MLPA probemix P236-A3 (MRC-Holland, Amsterdam, the Netherlands) following the manufacturer’s instructions.
Polymorphic variations are numbered as + 1 from the A allele of the ATG translation initiation site. Those missense, nonsense or splice site mutations that were previously identified were categorized as likely pathogenic variations. In order to include every variation and not to miss information, novel variations were also included if they were found with a minor allele frequency of < 1% in 1000Genomes Project phase 3 (http://browser.1000genomes.org/index.html) or in gnomAD (https://gnomad.broadinstitute.org/). The possible functional effect of novel variations was predicted in silico using the following online prediction tools, such as PolyPhen (version2)(45) (http://genetics.bwh.harvard.edu/pph2/), SIFT(46) (http://siftdna.org/www/Extended SIFT chrcoords submit.html), PROVEAN(47) (http://provean.jcvi.org/genomesubmit.php), Human Splicing Finder (version 3.1, http://www.umd.be/HSF3/) (48) and Mutation-Taster (49) (http://mutationtaster.org).
Generation Of Recombinant Wild-type And Mutant Fhr-5 Variants
Coding sequence of wild-type CFHR5 (wtCFHR5) was codon-optimized for insect cell expression system (Integrated DNA Technologies, Inc.) and cloned into the pBSV-8His expression vector(50). Two mutants were amplified from the wtCFHR5-containing vector with mutagenic forward primers introducing the mutations FHR-5G278S and FHR-5R356H. Sequences and mutations were confirmed by sequencing. Recombinant proteins were produced in Spodoptera frugiperda (Sf9) cells after co-transfection of the various CFHR5-containing expression vectors with linearized baculovirus DNA (Oxford Expression Technologies Ltd.) and purified from the supernatant by nickel affinity chromatography.
Measurement Of The Interaction Of Fhr-5 With Purified C3b
To compare the C3b binding ability of FHR-5WT, FHR-5G278S and FHR-5R356H, microtiter plate wells (MaxiSorp, Nunc) were coated with 5 µg/ml C3b (Merck). After blocking with 5% BSA in 0.05% Tween-20 containing Dulbecco’s phosphate buffer saline (DPBS) (Lonza), serial dilutions of recombinant FHR-5 variants were added to the wells for 1 h at 20 °C. Binding was detected with polyclonal goat anti-human-FHR-5 IgG (Cat. number:AF3845,R&D Systems) followed by HRP-labelled rabbit anti-goat Ig (Dako, Hamburg, Germany). The binding was visualized using 3,3′,5,5′-tetramethylbenzidine (TMB) (BioLegend) and the absorbance was measured at 450/620 nm.
The interaction of the recombinant FHR-5 variants with C3b was also analyzed by surface plasmon resonance (SPR) method using ProteOn XPR36 equipment (Bio-Rad). Recombinant proteins and ovalbumin (Sigma-Aldrich) as negative control were diluted in 10 mM Na-acetate buffer (pH 4.0) and immobilized vertically at a density of 4,200-4,600 RU on GLC biosensor chip by standard amine coupling method. As analyte, serial dilutions of C3b were prepared in DPBS containing 0.005% Tween-20 and injected in the horizontal orientation of channels over immobilized recombinant FHR-5 variants. Measurements were performed at 50 µl/min flow rate, association was followed for 120 s and the dissociation for 600 s. Data were processed and analyzed with ProteOnManager software. The curves were corrected by subtracting the non-specific binding responses obtained from control, the ovalbumin captured channel. Binding curves were fit to bivalent analyte model and the equilibrium dissociation constants were calculated from the directly estimated association and dissociation rate constants (KD = kd/ka). The experiment was performed twice on separate GLC biosensor chips.
To measure the C3b binding ability of the native (wild-type or mutant) FHR-5 from serum samples, microtiter plate wells (MaxiSorp, Nunc) were coated with 5 µg/ml C3b fragment (Merck) (ON,4 °C). After blocking with DPBS containing 2% BSA (1 h, RT), serum samples diluted 1:4 in DPBS containing 1% BSA, 0.05% Tween20, as well as a dilution series (3.9–250 ng/ml) of recombinant human FHR-5 (R&D Systems) were applied to the plate (1 h, 37 °C). Binding was detected with monoclonal mouse anti-human FHR-5 (R&D Systems) (1 h, RT), followed by HRP-labelled goat anti-mouse IgG (Jackson ImmunoResearch) (1 h, RT). As substrate TMB (BioLegend) was used and the absorbance was measured at 450/620 nm.
Elisa For Measuring The Serum Level Of Fhr-5
FHR-5 serum levels were measured with newly developed in-house ELISA method. Microtiter ELISA plates were coated with 1 µg/mL commercially available monoclonal mouse anti-human-FHR-5 (IgG1, clone#390513, R&D Systems) in PBS overnight, followed by blocking with PBS and 2% BSA the next day. Serum was diluted 1:100 in PBS containing 1% BSA and 0.05% Tween-20 and added to the plate for 1 hour at RT. FHR-5 binding was detected as above using polyclonal goat anti-human-FHR-5 IgG (Cat. number: AF3845, R&D Systems). The concentrations of the samples were determined based on the standard curve of the two-fold dilution series of recombinant human FHR-5 protein (R&D Systems). Inter-assay and intra-assay variations were determined and appeared to be 11.8% and 7.8%, respectively.
Western Blot For Detection Of Fhr-5
Serum proteins from patients and a healthy individual were separated on 10% SDS-PAGE under non-reducing conditions and blotted onto nitrocellulose membrane. After blotting, the membrane was blocked with 4% non-fat dried milk, 1% BSA in PBS solution. Then the membrane was incubated with a polyclonal goat anti-FHR-5 (Cat. number:AF3845, R&D Systems) or monoclonal mouse anti-FHR-5 antibody (clone#390513, Cat. number: MAB3845, R&D Systems) followed by HRP-conjugated secondary antibodies (rabbit anti-goat and goat anti-mouse, respectively; Southern Biotech, Birmingham, USA). Bound antibodies were detected with Clarity Western ECL substrate (Bio-RAD, California, USA).
Determinations Of Complement Parameters
Samples (serum, EDTA-anticoagulated- and sodium-citrate-anticoagulated plasma) were taken from the antecubital vein, or from a central venous catheter. Cells and supernatants were separated by centrifugation after the sample was taken, and transferred to our laboratory. Separated aliquots were stored at − 70 °C until measurements.
C3, C4 concentrations were measured by turbidimetry (Beckman Coulter, Brea, CA).
AP activation was measured by a commercially available kit (Wieslab AP ELISA KITs, EuroDiagnostica, Malmö, Sweden), according to the manufacturer’s instructions.
Total CP activity was measured by a home-made hemolytic titration test based on Mayer’s method (51). Radial immunodiffusion was performed to measure the antigenic concentrations of Factor I and Factor B, using specific antibodies (52). Levels of FH, C1q and antibodies against FH, C1q,(52–54) C3 and Factor B were measured with in-house ELISA methods (4) whereas C3NeF and C4NeF titer was determined based on hemolytic method(4, 55).
Further complement components, activation markers and split products, such as Factor D, sC5b-9, C3a, Bb and C4d were detected with commercially available ELISA kits (HyCult Complement Factor D, Human, ELISA kitHK343-02; MicroVue C3a-desArgEIA, A032; MicroVue C4d EIA, A008; MicroVue sC5b-9 Plus EIA, A029; MicroVue Bb Plus EIA, A027, respectively).
For descriptive purposes, continuous variables which were deviated from the normal distribution according to the results of Shapiro-Wilk tests, are given as medians and 25th–75th percentiles. For categorical variables numbers and percentages were used. Non-parametric tests as Mann-Whitney U test or Kruskal-Wallis test with Dunn’s post hoc test were used for group comparisons in case of continuous variables. For categorical variables Pearson’s χ2 test was performed. For comparison of the recombinant FHR-5 variants’ data, repeated-measures ANOVA was used with Tukey’s multiple comparison test.
For cluster analysis hierarchical clustering by Ward method with squared Euclidean distances was used, as described previously (36).
In order to split FHR-5 levels into high and low groups, receiver operating characteristic analysis was made. Kaplan-Meier analysis with log-rank test was performed to examine patients’ renal survival.
For the statistical analysis IBM SPSS Statistics 20 and Graph Pad Prism 5 software were used. Two-tailed p-values were calculated and the significance level was determined at a value of p < 0.05 if not otherwise stated.