Membranoproliferative glomerulonephritis (MPGN) is a heterogeneous group of rare glomerular diseases associated with complement dysregulation, which leads to the deposition of C3 and its cleavage products in glomeruli. Diagnosis requires a kidney biopsy, as the clinical presentation and course are variable, with patients manifesting asymptomatic haematuria and proteinuria, hypertension, nephritic or nephrotic syndrome, and/or acute kidney injury. Approximately 50% of patients develop chronic kidney disease (CKD) and progress to end-stage renal failure (ESRF) over a 10-year period (Sethi and Fervenza, 2011; Noris and Remuzzi, 2015). Current classification is based on glomerular deposits detected by immunofluorescence (IF) microscopy (Pickering et al., 2013). Cases with glomerular C3 staining in combination with significant immunoglobulin (IgGs) deposition are defined as immune-complex-mediated MPGN (IC-MPGN). C3 Glomerulopathy (C3G) is diagnosed in cases with dominant C3 staining at least two orders of magnitude greater than any other immunoreactant. Electron microscopy (EM) allows further differentiation of C3G into either dense deposit disease type (DDD), which is characterized by intramembranous highly electron-dense deposits, or C3 glomerulonephritis (C3GN), in which the deposits are less dense and have mesangial and/or subendothelial and subepithelial localization (Pickering et al., 2013; Hou et al., 2014).

Both C3G and IC-MPGN are complement-mediated diseases. The complement cascade is the cornerstone of innate immunity and can be initiated by three different pathways – the alternative (AP), classical (CP), or mannose-binding lectin (LP) pathways – that generate proteolytic complexes known as C3 convertases (Figure 1). The C3 convertase of the AP is C3bBb, while that of the CP and LP is C4bC2a. Both C3 convertases are so named because they cleave C3 into C3a, an anaphylatoxin, and C3b, which associates with factor B to generate additional C3bBb thereby amplifying the complement response. Binding of C3b to C3 convertases generates C5 convertases, which cleave C5 to produce C5a, another anaphylatoxin, and C5b, which initiates the terminal complement cascade by associating with other complement components (C6–C9) to form the terminal complement complex C5b-9 (Muller-Eberhard, 1986; Bhakdi and Tranum-Jensen, 1988; Morgan, 1999).

IC-MPGN has typically been linked to the activation of the complement CP following infections, autoimmune diseases or malignancies, while C3G has primarily been linked to activation of the complement AP (Sethi and Fervenza, 2011). In both C3G and IC-MPGN, genetic defects in complement AP genes like CFH, C3, CFI, and CFB (Servais et al., 2012; Iatropoulos et al., 2016, 2018), and acquired factors, such autoantibodies that stabilize the C3 convertase complex C3bBb (called C3-nephritic factors, C3NeFs) or against FH, FB and C3b have been identified (Zhang et al., 2012, 2020; Blanc et al., 2015; Marinozzi et al., 2017; Donadelli et al., 2018). These findings indicate that the dysregulation of the complement AP may underlie the pathogenesis of both diseases (Figure 2).

To gain further insights into the pathophysiology of these diseases, we have used unsupervised hierarchical cluster analysis based on histological, biochemical, genetic and clinical data at disease onset to divide our patient population into four clusters, each of which is defined by specific underlying pathophysiologic mechanisms (Figure 3) (Iatropoulos et al., 2018). In clusters 1, 2, and 3, serum C3 is low and the frequency of complement genetic variants and C3NeFs is high. Clusters 1 and 2 differentiated themselves from cluster 3 by very high sC5b-9 levels, which are indicative of dysregulated terminal pathway activity. Cluster 2 uniquely exhibits strong C1q, IgG and IgM glomerular deposition, suggesting that CP activity plays an important role in initiating disease in this cluster. Cluster 4 is characterized by normal C3 and sC5b-9 levels, and rare C3NeFs and complement genetic variants, despite intense C3 glomerular staining, indicating local glomerular complement activity (Iatropoulos et al., 2018).

Interestingly, genetic variants in CFH, which encodes factor H, the main regulatory protein of the AP complement pathway, are found in all 4 clusters indicating a complex pattern of functional consequences resulting in variable phenotypes (Iatropoulos et al., 2018).

The CFH gene family includes six genes - CFH, CFHR3, CFHR1, CFHR4, CFHR2, and CFHR5- on chromosome 1q31.3 that arose from CFH as a consequence of tandem genomic duplication events (Diaz-Guillen et al., 1999). The translated proteins, FH and FHR1-5s, are circulating proteins, organized in short consensus repeats (SCRs). The C-terminal region of the five CFHRs exhibits a high degree of sequence identity with the C-terminal domains of CFH, suggesting that FHR proteins can bind similar surface ligands as FH. However, FHRs do not contain the regulatory domains of FH (N-terminal region), suggesting they do not possess direct complement regulatory activity (Skerka et al., 2013).

The genomic region of the CFH gene family is characterized by large segmental duplications (SDs) and interspersed repetitive sequences that predispose to genomic rearrangements such as duplications, deletions and inversions (Lupski and Stankiewicz, 2005) that, when larger than 1kb, are called structural variants (SVs) (Feuk et al., 2006). The most common SV described in the CFH gene family is the ∼84 kb deletion of CFHR3 and CFHR1 (CFHR3-CFHR1 del) with an allele frequency ranging from 2 to 51%, depending on ethnicity (Holmes et al., 2013). The absence of both copies of CFHR3 and CFHR1 is also associated with a lower risk of age-related macular degeneration (AMD) (Hughes et al., 2006) and IgA nephropathy (Gharavi et al., 2011), and a higher risk of atypical haemolytic uremic syndrome (aHUS) (Moore et al., 2010) and systemic lupus erythematosus (SLE) (Zhao et al., 2011).

Rare SVs involving CFHRs have been described in DDD and C3GN, most of which generate abnormal fusion proteins (Gale et al., 2010; Malik et al., 2012; Tortajada et al., 2013; Chen et al., 2014; Medjeral-Thomas et al., 2014; Togarsimalemath et al., 2017; Xiao et al., 2016). The classic example was identified in Greek Cypriot patients with C3GN (often called CFHR5 nephropathy) that results from a mutant FHR5 protein encoded by a CFHR5 gene with an internal duplication of exons 2 and 3 (FHR5₁,₂-FHR5) (Gale et al., 2010). Other FHR fusion proteins linked to C3G include FHR2₁,₂-FHR5 (Chen et al., 2014), FHR5₁,₂-FHR2 (Xiao et al., 2016), FHR1-FHR5 (Togarsimalemath et al., 2017), FHR1_1–4-FHR1 (Tortajada et al., 2013), and FHR3₁,₂-FHR1 (Malik et al., 2012). These reports highlight the importance of the CFH-CFHR region in C3G and yet, with the exception of the fusion protein endemic to Cyprus, all fusion proteins thus far described have been identified in small families.

Comprehensive studies of the CFH-CFHR region in large cohorts of patients with C3G and IC-MPGN have not been reported. We sought to address this knowledge gap by identifying common and rare SVs and their distribution among the 4 C3G/IC-MPGN clusters we have described (Iatropoulos et al., 2018). SVs were detected using Multiplex Ligation-dependent Probe Amplification (MLPA) followed by PacBio long-read sequencing (SMRT, Single- Molecule Real-Time, sequencing) to provide base-pair resolution of selected genomic rearrangements.

Here we performed a comprehensive analysis to characterize genetic and acquired FH-FHR abnormalities in a large cohort of 199 C3G/IC-MPGN patients, classified into four clusters, with the main focus on CFH-CFHR CNVs.

Low FH levels and genetic and acquired FH abnormalities were identified only in patients in clusters 1–3, which are characterized by fluid-phase complement activation. Specifically, 7% of cluster 1–3 patients had CFH RVs, consistent with our results in a smaller cohort (Iatropoulos et al., 2018). FHAAs were also found in 5% of cluster 1–3 patients, all with childhood onset. All but 1 FHAA-positive patient were diagnosed with IC-MPGN, suggesting a possible link between FHAAs and immune-complexes in the glomeruli. In addition, the majority of patients with FHAAs were co-positive for another autoantibody, C3NeF, consistent with other reports (Blanc et al., 2015). These findings suggest a cumulative or synergistic effect of FHAAs and C3NeF in inducing fluid-phase AP overactivation although the specific contribution of each autoantibody remains unclear.

At variance with aHUS patients, we did not observe a correlation between the presence of FHAAs and homozygosity for CFHR1 del in C3G/IC-MPGN patients, consistent with previous data (Blanc et al., 2015; Valoti et al., 2019; Zhang et al., 2020). In addition, the prevalence of the common SVs (CFHR3-CFHR1 del or CFHR1-CFHR4 del) did not differ between patients and healthy controls, indicating that common SVs are not risk factors for C3G/IC-MPGN. However, the finding that total deficiency for CFHR1 was more frequent in patients in cluster 3 compared to patients in cluster 1, may indicate that FHR1 deficiency plays a role in driving the disease phenotype characteristic of cluster 3 patients.

To date, with the exception of the fusion protein (FHR5₁,₂-FHR5) identified in Greek Cypriot patients with C3GN, rare SVs in the CFH-CFHR region have been described in only a few familial cases of C3G. They have not been implicated in IC-MPGN. This knowledge gap reflects, in part, the high degree of similarity within the CFH-CFHR region, which is a strong limitation in designing specific probes for copy number variation (CNV) analysis and leads to an incomplete investigation of this locus.

To optimize the CNV analysis in this region, we used available and custom MLPA probes to provide an overview of SVs, which we then further resolved through PacBio long-read sequencing (SMRT, Single- Molecule Real-Time, sequencing). Using this protocol, we identified rare CFH-CFHR SVs in patients with IC-MPGN and an overall prevalence of 4% of new and rare CFH-CFHR SVs in C3G/IC-MPGN patients.

We detected a duplication of CFHR1-CFHR4 in 2% of patients distributed amongst clusters 1–3 but not in cluster 4, often in combination with other complement RVs and/or the common CFHR3-CFHR1 del. This duplication has also been identified in patients with aHUS and AMD (Bu et al., 2014; Cantsilieris et al., 2018). We verified segregation in healthy relatives indicating that, alone, the CFHR1-CFHR4 duplication is not sufficient to induce disease and that other risk factors are required to determine the ultimate phenotype.

Interestingly, another genomic rearrangement altering CFHR4 was identified in a DDD patient from cluster 3, namely a CFHR3_1–5-CFHR4₁₀ hybrid gene that encodes the fusion protein FHR3_1,2,3,4-FHR4₉. SCRs 1-3 of FHR3 have high sequence similarity with FH SCRs 6-8, which form a second FH heparan-sulfate binding site on cell surfaces and the glomerular basement membrane (GBM) (Borza, 2017). Hebecker and Jozsi have shown that FHR4 favors the assembly of the AP C3 convertase through its C-terminal region, which contains a C3b binding sites (Hebecker and Jozsi, 2012). These data suggest that the FHR3_1,2,3,4-FHR4₉ fusion protein may compete with FH for binding to both glycosaminoglycans/sialic acid and C3b fragments in the GBM, thereby enhancing C3 convertase activity and favoring the formation of the high electron-dense deposits, a characteristic feature of cluster 3-patients (Figure 10A). This hypothesis warrants testing.

In addition to the above CFHR4 CNVs, in a familial case of C3GN in cluster 4 we identified a shorter CFHR3_1–3 gene caused by a deletion spanning intron 3 to 3′UTR, followed by a normal copy of CFHR1, which leads to a fusion protein likely consisting of the 2 N-terminal SCRs of FHR3 and the entire FHR1 (FHR3_1–2-FHR1). A comparable fusion protein generated by a different genomic rearrangement has been described by Malik et al. (2012) in a familial C3GN case. In both cases, C3 levels are normal, suggesting that complement dysregulation occurs primarily in the glomeruli microenvironment. The likely mechanism of action is secondary to multimeric complexes of FH-related proteins that outcompete FH for binding to the glomerular glycomatrix (Goicoechea de Jorge et al., 2013; Medjeral-Thomas and Pickering, 2016; Csincsi et al., 2017) (Figure 10B). Functional studies, however, would be required to elucidate the functional effects of the identified genomic CFHR abnormalities and their pathogenetic role in C3G/IC-MPGN.

A final important finding of this study is the identification of a large deletion encompassing CFH, CFHR3, and CFHR1 in a IC-MPGN patient in cluster 2 with low C3 and FH serum levels. The deletion causes FH haplodeficiency. As a consequence, fluid-phase AP regulation is impaired, which thereby sustains chronic complement activation initiated through the CP by immune-complexes in the glomeruli, a feature typical of cluster 2 patients (Figure 10C).

In this study we have used established and innovative techniques to characterize SVs over the CFH-CFHR genomic region in a large cohort of C3G/IC-MPGN patients. We have demonstrated that while common CFH-CFHR SVs are not risk factors for disease, rare SVs do predispose to disease, but typically in combination with RVs in complement genes or acquired drivers of disease like autoantibodies. Our findings support the overarching concept that C3G/IC-MPGN are genetically complex, with the ultimate phenotype reflecting the delicate balance of serum levels of FH and the FHR proteins. Our results also illustrate the value of SMRT sequencing methodology as a tool for resolving the complexity of SVs in this genomic region.

The datasets generated for this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found below: EBI European Nucleotide Archive, accession no: PRJEB44176.

The studies involving human participants were reviewed and approved by Ethics Committee of Bergamo. Written informed consent to participate in this study was provided by the participants’ legal guardian/next of kin. Written informed consent was obtained from the individual(s), and minor(s)’ legal guardian/next of kin, for the publication of any potentially identifiable images or data included in this article.

RP, MN, and GR designed research, interpreted data, and wrote the manuscript. RP, MB, MA, EV, PI, CM, RD, and PC performed the research and analyzed the data. EB provided detailed clinical information of patients. AB and RS analyzed the data and critically revised the manuscript. All authors contributed to the article and approved the submitted version.

MN has received honoraria from Alexion Pharmaceuticals for giving lectures, and for participating in advisory boards and research grants from Omeros, ChemoCentryx, Inception Science Canada, BioCryst Pharmaceuticals. AB has received honoraria from Boehringer Ingelheim, Alexion Phamaceuticals, Janssen Pharmaceuticals, Akebia Therapeutics, Inception Science Canada, BioCryst Pharmaceuticals. GR has consultancy agreements with Boehringer Ingelheim, Janssen Pharmaceuticals, Akebia Therapeutics, Alexion Pharmaceuticals, Alnylam, Inception Science Canada, BioCryst Pharmaceuticals. RS directs the Molecular Otolaryngology and Renal Research Laboratories, which provides genetic testing for complement-mediated renal diseases. None of these activities have had any influence on the results or interpretations in this article. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.