Identication of C3GN Presenting in a Proband with Family History of Alport Syndrome

Background Alport syndrome and C3GN are all rare kidney diseases and frequently responsible for familial hematuria, proteinuria and/or coexistent renal impairment. With the rapid development of molecular genetic testing, Alport syndrome have been restricted mostly to causal variants in the COL4A5 or COL4A3/COL4A4 genes. And a broad range of genetic contributors in the complement and complement-regulating proteins denitely implicate in the pathogenesis of C3GN. Methods We sought a family with persistent microscopic hematuria associated with renal failure. Clinicopathologic and follow-up data were obtained, and molecular genetic testing was used to screen pathogenic variants. Results We describe a three-generation family with Alport syndrome showing a dominant maternally transmitted inheritance. Notably, renal biopsy showed the concurrent histological evidence of C3GN in the proband and further classied as CFHR5-related nephropathy due to a rare heterozygous variation in gene-encoding CFHR5, c.508G>A. The alteration leads to replacement of a highly conserved residue at position 170 of the β-strand subunit of CFHR5 (p.Val170Met). In silico analysis, the variation was predicted to deregulate complement activation by altering the structural property and enhancing C3b binding capacity to compete with CFH, which was in a good agreement with experimental data previously published. The comorbidity ndings between Alport Syndrome and C3GN indicate an underlying overlap, and await further study. immunouorescence study were stained with uorescein isothiocyanate-conjugated polyclonal antibodies to human IgA, IgG, IgM; C3; light chains (κ and λ); C4, C1q; and α5 chains of type IV collagen. Antigen was retrieved by EDTA solution as well as gastric enzyme, and elivision system was applied in IHC detection. Specimens for electron microscopy were xed in glutaraldehyde and embedded in epon. All reports were reviewed by two pathologists.


Introduction
In China a rare disease is de ned as a disease that affects fewer than 500,000 people in the country [1], approximately 80% of rare diseases have an identi ed genetic background with substantial geographic or ethnic variations in incidence [2].
Genetics were rst used in nephrology with the rst identi cation of a causal mutation for Alport syndrome in 1990 [3]. Alport syndrome is a rare hereditary disorder of basement membranes with an abnormal collagen IV composition characterized by hematuria, progressive renal failure, sensorineural deafness, anterior lenticonus and retinal ecks [4]. To date, the causes of familial hematuria nephropathies have been restricted mostly to variants in the COL4A5 or COL4A3/COL4A4 genes, responsible for the X-linked or autosomal inheritance [5][6][7]. Besides, analysis of data suggests that familial microscopic hematuria attributable to the complement factor H-related protein 5 (CFHR5) gene is also associated with a rare renal disease termed C3 glomerulonephritis (C3GN), and further classi ed as CFHR5 nephropathy or CFHR5-related nephropathy [8][9][10].
Herein, we report a family with dominant maternally transmitted inheritance, in which 5 out of 8 members (I:1, II:1, II:4, III:1, and III:2) exhibit microscopic hematuria and proteinuria; 2 out of the 5 patients (II:1 and III:1) developed ESRD (end stage renal disease). The diagnosis of Alport syndrome is con rmed in the proband (III:1), his mother (II:1) and his maternal aunt's daughter (III:2) based on all available data.
Besides, renal biopsy showed the comorbidity of C3GN and Alport syndrome in the proband based on a rare heterozygous c.508G > A (p.Val170Met) variant in the CFHR5 gene. And we provided several lines of evidence to support the contention that the variant CFHR5 c.508G > A was classi ed as pathogenic and may contribute to the genetic susceptibility to C3GN.

Family pedigree and clinical investigations
In this study, a family with persistent microscopic hematuria associated with renal failure was recruited from Hangzhou, Zhejiang Province, China. All affected members were regularly followed up at the outclinic until May 12, 2020. A brief history and physical examination were performed and blood, urine, skin, kidney samples were collected for determination of serum creatinine concentration, estimated glomerular ltration rate (eGFR), urinalysis, C3/C4 levels and histological manifestation.

Renal and skin biopsies processing
Biopsy specimens for light microscopy were xed in alcohol formaldehyde acetic solution, embedded in para n, and cut into 2um-thick sections. Sections were stained with Masson trichrome, hematoxylin and eosin, periodic acid-Schiff, and silver methenamine. Samples for immuno uorescence study were stained with uorescein isothiocyanate-conjugated polyclonal antibodies to human IgA, IgG, IgM; C3; light chains (κ and λ); C4, C1q; and α5 chains of type IV collagen. Antigen was retrieved by EDTA solution as well as gastric enzyme, and elivision system was applied in IHC detection. Specimens for electron microscopy were xed in glutaraldehyde and embedded in epon. All reports were reviewed by two pathologists.
Targeted exon sequencing and Sanger sequencing Genomic DNA was isolated from peripheral blood of a 3-generation pedigree of 8 members using the Wizard Genomic DNA Puri cation Kit (Promega, USA) following the manufacturer's instructions. Coding exons reference sequences of the individuals (II:1, II:2, III:1 and III:2) were targeted for the Illumina highthrough sequencing, and reads were aligned to UCSC (University of California, Santa Cruz) version hg19 (Genome Reference Consortium GRCh37). The approximately 95% of reads were mapped to the target regions at an average of 20×. We examined copy number, rare, and common variants. All the diseaserelated sites were selected and Sanger sequencing was further performed in all 8 members. Primers were indigenously designed using the primer premier 5.0 program (Lalitha, 2000) and shown in Supplementary Table S1. The puri ed PCR products were directly sequenced using an ABI BigDye Terminator v3.1 Cycle Sequencing Kit. The analyses were completed on an ABI-3500Dx Genetic Analyzer (Applied Biosystems).
In silico evaluation of pathogenicity The ProtParam tool was used to compute various physical and chemical parameters including the molecular weight and theoretical pI. The complete amino acid sequence data of the human protein CFHR5 (GenBank accession: AAI11774.1) were obtained from the NCBI (National Center for Biotechnology Information) and alignments done by the EMBL-EBI (European Bioinformatics Institute). The SIFT (Sorting Intolerant From Tolerant), SNAP (Screening for Non-acceptable Polymorphisms), PolyPhen-2 (Polymorphism Phenotyping v2) and Mutation Taster were utilized to evaluate possible biologic effects of genetic aberration impact on protein structure.

Variant interpretation
In 2015, standards and guidelines for the interpretation of sequence variants were updated by the American College of Medical Genetics and Genomics (ACMG), the Association for Molecular Pathology (AMP) and the College of American Pathologists (CAP) [11]. This report recommends the use of speci c standard terminology: 'pathogenic', 'likely pathogenic', 'uncertain signi cance', 'likely benign', and 'benign' to describe variants.
In brief, common variants with minor allele frequency (MAF) 1% in any population are either 'likely benign' or 'benign'. Novel or rare variants that change protein sequence but have an unknown impact on protein function were classi ed as either 'likely pathogenic' or 'uncertain signi cance'. Ultra-rare variants with well-established in vitro or in vivo functional studies that indicate a damaging effect, coupled with multiple lines of computational evidence, were classi ed as 'pathogenic'.
Homology modeling of human CFHR5/C3b complex Docking procedure has been described in more detail previously [12]. Starting from residues 23-569, the 3D homology models of CFHR5 were rst generated by iterative threading assembly re nement (I-TASSER) server [13], where the one with the highest C-score was selected to be further re ned by Fragment-Guided MD simulation (FG-MD) [14]. Potential energy of re ned protein was calculated by "Calculate Energy" protocol of Discovery Studio (DS) 3.0. The crystal structure of ligand C3b (PDB ID: 2WII) was taken from protein data bank (PDB).
By applying "Prepare Protein" protocol of DS, energy minimization was performed to clean the protein molecule by adding missing atoms, inserting missing loops, assigning charges and xing CHARMm force elds. A total of 2000 docking poses were generated by ZDOCK (CHARMm-based DOCKER) protocol and incorporated. The top1 model was collected as the probable complex structure. Hydrogen-bonding networks can be displayed by Molecular Operating Environment (MOE). Binding free energy of proteinprotein interaction was estimated by the Calculate Mutation Energy (Binding) protocol within DS.

Clinical presentation and follow-up
Herein we investigated a three-generation Han Chinese family characteristiced by persistent microscopic hematuria associated with renal failure (Fig. 1a). The proband (III:1), a 11-year-old boy, was admitted to the hospital for hematuria and proteinuria during the physical examination. On admission, 24-h urine protein was 6.36 g/d, serum albumin is 24.4 g/l. Microscopic urinary analysis showed urine sediment containing ++~+++ red cells per high-power eld. His blood urea nitrogen level was 15.73 mmol/l, serum creatinine level was 266 umol/l, eGFR by CKD-EPI equation was 30.3 ml/min. Bilateral renal ultrasound supported chronic nephrology with stronger and unevenly distributed echoes, and unclear demarcation between the renal cortex and parenchyma. Sensorineural hearing impairment and vision abnormalities, as well as other clinical signs were not detected.
To con rm the clinical diagnosis, renal biopsy of the proband was performed. The changes on light microscopy are nonspeci c and include diffuse hyperplasia in the glomerular mesangial cells and and matrix, with segmental sclerosis and crescent formation. Immunohistochemical and immuno uorescence studies all showed patchy GBM expression of collagen a5 (IV) and complete absence in Bowman's capsule and distal tubular BM (basement membrane) (Fig. 2a & 2b). Electron microscopic analysis identi ed pathological characteristics accompanying with noticeably segmental uneven thickness with a laminated appearance of the dense layer within the basement membrane (100-400 nm) (Fig. 2c). Besides, interstitial brosis/tubular atrophy containing lipid-laden foam cells were obvious (Fig. 2d).
The proband (III:1) has a family history of hematuria, as his mother (II:1) presented with hematuria, proteinuria, and develop ESRD before age 33 years. And a skin biopsy was performed in his mother, which reveals that there was segmental absence of the collagen IV a5 chain within epidermal basement membrane (Fig. 2e). His maternal grandma (I:1), maternal aunt (II:4) and aunt's daughter (III:2) also had urinary abnormalities characteristic by asymptomatic hematuria and proteinuria with normal eGFR. His aunt's daughter (III:2) had a renal biopsy by age 13, showing segmental uneven thickness with a laminated appearance of the dense layer in GBM (160-400 nm) by electron microscopic (Fig. 2f) and scanty C3 staining along the capillary wall. His father (II:2), his maternal grandpa and aunt's husband is phenotypically normal.
Notably, a marked C3 staining was detected to locate along the mesangium by immuno uorescence analysis (Fig. 2g) in the proband (III:1) with the corresponding electron-dense deposits under the electron microscopy ( Fig. 2i). During follow-up, the proband was commenced on hemodialysis 3 months later and received a kidney transplant after one year.
Findings on targeted exome-based next-generation sequencing and Sanger sequencing To make a precise diagnosis for the true pathogenic mechanism affecting the family, we performed whole exome-based NGS and further con rmed using Sanger sequencing. Genetic analysis identi ed the same heterozygous c.508G > A coding variant (p.Val170Met) in exon 6 of the CFHR5 gene derived from 3 affected individuals: the proband (III:1), his mother (II:1) and his maternal grandma (I:1) (Fig. 1b). The variant is ultra-low frequent in the general population (1000 Genomes: minor allele frequency [MAF] = 0.1%; ExAC Browser: MAF = 0.06%), and the mutated A allele carriers are all heterozygous. The nonsynonymous alteration leads to the replacement of a valine, strictly conserved among organisms (Fig. 3a), by a methionine residue. The Screening for four publicly available programs (SIFT, SNAP, PolyPhen-2 and Mutation Taster) independently predicted the replaced amino acid was "damaging" to protein structure/function (SIFT score 0.004; SNAP score 20; PolyPhen2 score 0.999; Mutation Taster might be affected).
No suspicious disease-causing variants were detected in the gene COL4A3, COL4A4 or COL4A5 that encode the α3, α4 or α5 chains of type IV collage.
In silico functional prediction for CFHR5 c.508G > A, p.Val170Met CFHR5 is a single-chain polypeptide composed of 9 complement control protein domains (also known as CCPs). The residue p.Val170Met in CFHR5 occurs in the β-strand region of the CCP3. The isoelecrtic point (pI) is found to be the same for both wild type and mutant proteins (pI 6.8). Molecular weight of the mutant (64.45 kDa) protein is similar to that of wild-type protein (64.42 kDa). In the native structure, the Val residue, located in the buried surface, is not involved in any intramolecular interactions. As substituted by Met170, the original intramolecular hydrogen-bonding distance between the side chain of its neighboring residues (Leu171 and Val187) changes (Fig. 3b). DS 3.0 analysis predicted the calculated potential energy of the mutant type protein is -15180.97 kcal/mol compared to -15133.86 kcal/mol for mutant one, which implied that p.Val170Met could lead an increase in conformational stability of the CFHR5 protein.
As with CFH and other CFHRs, CFHR5 regulates the complement cascade by binding and interacting with the macromolecular protein ligand C3b. The C3b fragment is a glycoprotein composed of the modi ed C3-α chain (C3α') and the intact C3-β chain (C3β). We then simulated the probable native CFHR5/C3β complex structure. As can be seen in Fig. 3c, the three-dimensional (3D) model demonstrated that Val170 is not to be the binding site for protein C3b. However, further binding free-energy calculation showed that p.V170M slightly increases the binding a nity of CFHR5/C3β complex by -0.76 kcal/mol.

Discussion
Alport syndrome is characterized by hematuria, renal failure, and extra-renal alterations, such as: hearing loss, lenticonus, and retinal ecks [4][5][6][7]. The disease is caused by changes in the collagen type IV chains, resulting the damage to the base membrane of several organs. Approximately 85% of families have Xlinked inheritance with mutations in COL4A5 gene [3,[15][16][17], and most of the others have autosomal recessive disease with alterations in both copies of COL4A3 or COL4A4 [18][19][20]. Autosomal dominant inheritance is very rare and results from heterozygous COL4A3 or COL4A4 variants.
In our study, we took a comprehensive three-generation family history. The proband (III:1) was presented a course of progressive deterioration of renal function with persistent hematuria and proteinuria, and underwent a kidney transplant at aged 12. His family history is signi cant for progressing to ESRD in her mother, over a 10-year follow-up period of hematuria and proteinuria. Besides, His maternal grandma (I:1), maternal aunt (II:4) and aunt's daughter (III:2) had asymptomatic microscopic hematuria ( 30/HP) and proteinuria ( 0.5 g/d) with normal renal function. Characteristic pathological changes were observed in renal biopsy specimens from the proband (III:1) and his maternal aunt's daughter (III:2), including segmental uneven thickness with lamellation and splitting of the dense layer in GBM under electron microscopy. In addition, immunostaining showed nearly complete loss for a5 (IV) collagen chain in GBM, complete absence in Bowman's capsule and distal tubule basement membrane in the praband, and his mother (II:1) exhibited segmental distribution of a5 (IV) expression in the skin BM. According to the diagnostic algorithm published in 2013 [7], the diagnosis of Alport syndrome is established in the proband (III:1), his mother (II:1) and his maternal aunt's daughter (III:2) based on pedigree study, clinical manifestation and skin/renal biopsy.
The COL4A3 and COL4A4 genes reside on chromosome 2 while the COL4A5 gene is located on the X chromosome, which encode the collagen IV a3, a4 and a5 chain, respectively. In normal individuals, the collagen IV a3a4a5 chains are highly expressed and co-distributed within the mature kidney (GBM and distal tubular BM), cochlea, and eye, and the collagen IV a5a5a6 network occurs in the skin BM and kidney (Bowman's capsule) [5]. X-linked Alport syndrome accounts for of the majority of Alport syndrome cases, arising from mutations in the COL4A5 gene. In the proband and his mother, morphologic phenotype demonstrated reduced and depleted expression of the collagen IV a5 chain in kidney and/or skin. Further clinical and genealogical study indicated that the affected proband (III:1) are much more severe than females (his mother (II:1), maternal grandma (I:1), maternal aunt (II:4) and aunt's daughter (III:2) ) in the pedigree, and male-to-male transmission is absent. Hence, the mode of inheritance was suspected to be X-linked.
Molecular genetic testing is one of the criteria for the diagnosis of Alport syndrome with a high sensitivity and speci city ( 90%) [7,21,22]. High-throughput next generation sequencing (NGS) technology can improve the diagnosis of Alport syndrome by providing molecular con rmation of COL4A3, COL4A4, or COL4A5 mutations. Then we performed whole-exome sequencing for 4 individuals (II:1, II:2, III:1, III:2), however, we did not identify any variation in thought to be possibly disease-causing in the three type IV collagen genes. It is possible that a small proportion of variants may have been missed. Alternatively, a rare deep intronic variant affecting splicing and only detectable by RNA analysis. In addition, pathogenic genes in families with Alport syndrome may not be con ned to a few regions. Known COL4A3, COL4A4, and COL4A5 genes are scattered throughout many exons, making it di cult to develop predictive genetic tests.
Noteworthily, a marked C3 staining with the corresponding electron-dense deposits along the mesangium was detected by immuno uorescence and electron microscope analysis of the kidney biopsy in the proband (III:1). Serum C3 was reduced in his mother (II:1), while normal in the proband and other affected family members. Excessive glomerular C3 fragment deposition, with scanty C1q and immunoglobulin, showed some morphological features of C3GN. In addition, the proband and his mother were affected more severely than other members. In the pedigree, familial C3GN is suspicious. The ndings above prompted us to take a further investigation of the complement system in the index family. A broad range of genetic contributors de nitely implicate in the pathogenesis of C3GN [23][24][25], then we screened a set of complement genes.
We identi ed a rare heterozygous missense variant c.508G > A (p.V170M; rs201073457) in the complement regulatory gene CFHR5 in 3 members in this family (I:1, II:1 and III:1). CFHR5 colocalizes with complement-containing glomerular immune deposits in a variety of glomerular pathologic states [26]. Based on genetic studies, copy number variations in CFHR5 is implicated in C3GN [8,[27][28], but signi cant enrichment of disease-associated rare variants is less [29]. The c.508G > A in the gene CFHR5 generates a nonsynonymous alteration at amino acid position 170 (Val in wild-type and Met in mutant), which is strictly conserved among organisms. In silico programs (SIFT, SNAP, PolyPhen-2 and Mutation Taster) independently indicated the CFHR5 c.508G > A to be potential functional variant. Then we simulated 3D homology-modeled structures, which indicated mutant-type CFHR5 increased conformational stability and induced an increase in the C3b-binding a nity. Additionally, the rare alteration has been previously reported in a patient with aHUS [30] and veri ed to exhibit signi cantly higher C3b binding capacity compared with wild-type CFHR5 in vitro functional assays [31]. The above computational and functional assessments indicated that CFHR5 c.508G > A may be responsible for the C3GN phenotype in the proband.

Conclusions
Our study reports a pedigree characterized by microscopic hematuria and variety in the rapidity of onset of renal failure. The diagnosis of Alport syndrome is con rmed in the proband (III:1), his mother (II:1) and his maternal aunt's daughter (III:2). Most notably, the proband revealed morphological features of C3GN based on genetic complement dysregulation background with concurrent histological evidence of Alport Syndrome. The cases of Alport Syndrome complicated with C3GN has never been reported, and underlying overlap awaits further study.

Availability of data and materials
The datasets used and/or analyzed during the current study are contained within the manuscript and the appendix, and available from the corresponding author on reasonable request. The totality of the data cannot be shared based on patient con dentiality concerns.
Ethical approval and consent to participate All of the procedures performed in studies involving human participants were carried out in accordance with the ethical standards of the Ethics Committee of the Hangzhou hospital of traditional Chinese medicine (institutional review board approval number: 2020KY055) and with the 1964 Declaration of Helsinki and its later amendments or comparable ethical standards. Written informed consent forms for participation in the study were obtained, where participants are children (under 16 years old) from their parent.

Consent for publication
Not applicable.

Competing interests
The authors declare that there exist no con icts of interest.   The pathological features of kidney and skin samples in the proband and his mother. The proband (III:1) demonstrated partial loss for the collagen IV a5 chain in GBM and complete absence in Bowman's capsule and distal tubule basement membrane (a, IHC×400 & b, IF×400) by immunostaining, and a large amount of lipid-laden foam cells interstitial in ltration under the light microscope (d, MA×200). Electron microscopic analysis identi ed pathological characteristics accompanying with noticeably segmental uneven thickness with a laminated appearance of the dense layer in the GBM (c, ×6000). In addition, there was segmental absence of the collagen IV a5 chain within epidermal basement membrane (e, IF ×200) in the proband's mother (II:1). His aunt's daughter (III:2) showed segmental uneven thickness with a laminated appearance of the dense layer in GBM (160-400nm) by electron microscopic (f). Remarkably, a marked C3 staining was detected in the proband to locate along the mesangium by immuno uorescence analysis (g, IF×200) with the corresponding electron-dense deposits under the electron microscopy (i, ×6000), while scanty IgA was detected (h, IF×200).