Patients and Methods

Patients

Patients (n=227) were consecutively recruited through the Registry of MPGN established at the Mario Negri Institute (Figure 3.2). All kidney biopsy reports were centrally reviewed, adopting the recent classification.250, 253 MPGN diagnosis was based on the typical light microscopy pattern.250 Patients with glomerular immunoglobulin and C3 deposits at IF were considered Ig-MPGN. C3G was diagnosed based on the presence of MPGN or mesangial proliferative patterns under light microscopy with “dominant C3” glomerular staining (intensity≥2 orders of magnitude more than any other immune- reactant on a 0 to 3scale).253 Based on EM, C3G was further classified as DDD or C3GN.253 Even though only patients with idiopathic MPGN are recruited through our Registry, after a deeper evaluation we excluded 87 patients: 11 did not meet diagnostic criteria, 18 were affected by secondary MPGN following a comprehensive diagnostic work-up, 7 received an MPGN diagnosis years after atypical hemolytic uremic syndrome (aHUS), 4 received an MPGN diagnosis on allograft but the native kidney biopsy was not available, 23 had no IF studies, 15 had no EM, and for 9 DNA samples were not available.

All participants provided informed written consent. The study adheres to the Declaration of Helsinki and was approved by the Ethics Committee of the Azienda Sanitaria Locale of Bergamo (Italy).

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Figure 3.2

Figure 3.2. Histopathologic classification of the patients recruited through the Registry

of Idiopathic Membranoproliferative Glomerulonephritis (MPGN). Ig-MPGN: immunoglobulin-associated MPGN; C3G: C3 glomerulopathy; DDD: Dense Deposit Disease; C3GN: C3 glomerulonephritis; IF immunofluorescence microscopy; EM: electron microscopy.

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Complement component assays

Serum C3 and C4 levels were assessed by kinetic nephelometry.294 To assess plasma SC5b-9 levels and C3NeF activity, blood was collected in ice-cold EDTA tubes, immediately centrifuged at 4° C to avoid ex-vivo complement activation, and frozen at - 80°C until assay. SC5b-9 levels were measured using the MicroVue SC5b-9 Plus EIA commercial kit (SC5b-9 Plus, Quidel). Serum C3, C4 and plasma SC5b-9 were assessed at Mario Negri Institute. C3NeF activity was determined at the Cordelier Research Center by purifying IgG from plasma and assessing their ability to stabilize cell-bound C3bBb convertase as previously described.298

DNA Sequencing

Genomic DNA was extracted from peripheral blood using the NucleonTM BACC2 Genomic DNA extraction kit (GE Healthcare, Little Chalfont, UK).

Through next-generation sequencing, genetic screening of all exons and flanking regions of CFH (NG_007259.1), CD46 (NG_009296.1), CFI (NG_007569.1), CFB (NG_008191.1), C3 (NG_009557.1) and THBD (NG_012027.1) genes was performed by highly multiplex PCR using the Ion AmpliSeq™ Library Kit 2.0 and 311 primer pairs (subdivided into two pools), designed using the Ion AmpliSeq Designer tool. After clonal amplification using the Ion PGM™ Template OT2 200 Kit, sequencing was performed on Ion PGM Sequencer. Variants were identified using TorrentSuite Software 3.6, and putative mutations were validated by Sanger sequencing. Regions with a depth of amplicon coverage ≥40x reads were considered appropriately analyzed. Regions with low coverage or regions not covered by the Ion AmpliSeq Designer were sequenced by Sanger method. In 9 patients, screening of CFH, CD46 and CFI coding

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exons was performed by Sanger sequencing as previously described.294

Genetic variant annotation

Genetic variants were functionally annotated using the ANNOVAR software. Missense variants, insertions/deletions in the coding regions, splicing variants affecting the first two nucleotides flanking exons, or variants lying within the Kozak consensus sequence were considered functional. Variants that did not exceed an allelic frequency of 0.01 in none of the ESP or the 1000 Genomes Project subpopulations were considered rare. Likely pathogenic variants were defined as the variants with minor allele frequency in the ExAC database <0.001 and with Combined Annotation Dependent Depletion (CADD)299 phred score ≥10.

The Combined Annotation Dependent Depletion is a method that integrates different annotation in a general framework to estimate the relative pathogenicity of human genetic variants.299 It was implemented as a support vector machine trained to differentiate 14.7 million fixed or nearly fixed derived alleles in humans from 14.7 million simulated variants. The fixed or nearly fixed human-derived alleles are those alleles that differ from the inferred human-chimpanzee ancestral genome and have an allele frequency of at least 95% in the human population (according to the 1000 Genomes Project variant catalog). Deleterious variants that reduce organism fitness are depleted by natural selection in fixed but not simulated variation. The support vector machine uses 63 distinct annotations that span a range of data types including species conservation metrics like GERP, phastCons, and phyloP; regulatory information like genomic regions of DNase hypersensitivity and transcription factor binding; transcript information like distance to exon-intron boundaries or expression levels in commonly studied cell lines; and protein-level scores like Grantham, SIFT, and PolyPhen. CADD

75 objectively integrates the diverse annotations into a single, quantitative score (C-score). “C-scores” were pre-computed for all 8.6 billion possible human single nucleotide variants and enable scoring of short insertions/deletions. C-scores were also scaled to a Phred-like scale ranging from 1 to 99 on the basis of their ranking relative to all possible substitutions in the human reference genome [−10log10 (rank/total number of substitutions)]. A Phred-scaled C-score of greater or equal 10 indicates that these are predicted to be the 10% most deleterious substitutions that may occur to the human genome, a score of greater or equal 20 indicates the 1% most deleterious and so on. The authors state that the choice of a cutoff on deleteriousness to identify potentially pathogenic variants is always arbitrary and there is no natural choice. They suggest a cutoff of Phred-scaled C-score somewhere between 10 and 20. Since C3G and Ig- MPGN are considered complex diseases that may derive by adding up the small effect of multiple hits, the less stringent cutoff of 10 was adopted for these diseases. For steroid-resistant nephrotic syndrome, which is believed a monogenic disease, we adopted a more stringent cutoff of 15 (http://cadd.gs.washington.edu/).299

The ExAC database, containing 60,706 subjects, was adopted as reference to assess allelic frequencies. To perform association studies, we used as a control group the 286 unphenotyped subjects of European origin from the 1000 Genomes Project (CEU, TSI, GBR and IBS subpopulations), for whom individual genotypes were available.

Statistical analyses

Categorical data were analyzed with χ2

test or Fisher exact test, where appropriate. One- way ANOVA, Kruskal-Wallis and multivariate regression tests were used for continuous variables.

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Degree of mesangial proliferation, endocapillary proliferation, interstitial inflammation, interstitial fibrosis, and arteriolar sclerosis, as well as IF findings were graded using a scale of 0 to 3, including 0, trace, 1+, 2+ and 3+. These findings were considered as quantitative variables after substituting 'trace' with 0.5, and analyzed with one-way ANOVA. EM findings were considered as dichotomous variables and were analyzed with χ2 test or Fisher exact test where appropriate.

Survival analyses considered as cumulative fractions of patients free of events were estimated using Kaplan-Meier survival curves and with Cox proportional-hazard regression. Multivariate analyses included all the covariates that reached statistical significance at the univariate analysis. To perform multivariate analyses with genetic variants, homozygous genotypes for the reference allele were codified as 0, heterozygous as 1 and homozygous for the alternative allele as 2. In the dominant model the group with at least one alternative allele was compared with the group with no alternative alleles. In the recessive model the group homozygous for the alternative allele was compared with the group with no or one alternative allele. P-values <0.05 were considered statistically significant. Analyses were performed with the MedCalc software and with the R software (https://cran.r-project.org/).

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