Family pedigree and clinical investigations
In this study, a family with persistent microscopic haematuria associated with renal failure was recruited from Hangzhou, Zhejiang Province, China. All affected members were regularly followed up at the out-clinic 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 filtration rate (eGFR), urinalysis, C3/C4 levels and histological manifestation.
Renal and skin biopsies processing
Biopsy specimens for light microscopy were fixed in alcohol formaldehyde acetic solution, embedded in paraffin, and cut into 2um-thick sections. Sections were stained with Masson trichrome, hematoxylin and eosin, periodic acid–Schiff, and silver methenamine. Samples for immunofluorescence study were stained with fluorescein 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 fixed 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 three-generation pedigree of 8 members using the Wizard Genomic DNA Purification 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 high-through sequencing, and reads were aligned to the human genome reference sequence ( hg19/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 disease-related 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 purified 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.
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) . This report recommends the use of specific standard terminology: ‘pathogenic’, ‘likely pathogenic’, ‘uncertain significance’, ‘likely benign’, and ‘benign’ to describe variants. This report recommends the use of specific standard terminology: ‘pathogenic’, ‘likely pathogenic’, ‘uncertain significance’, ‘likely benign’, and ‘benign’ to describe variants, based on criteria using typical types of variant evidence (e.g., population data, computational and predictive data, functional data, segregation data and de novo data).
In brief, an allele frequency in a control population that is greater than expected for disorder or with lack of segregation among affected individuals was considered strong support for a benign interpretation or, if over 5%, it is considered as ‘benign’. Novel or rare variants that lead to splicing defect or amino acid change coupled with multiple lines of computational evidence, or cosegregate with disease, were classified as ‘pathogenic’ or ‘likely pathogenic’, and variants with well-established in vitro or in vivo functional studies that indicate a damaging effect were labeled ‘pathogenic’. Variants of ‘uncertain significance’ are more common: other criteria shown above are not met or the criteria for benign and pathogenic are contradictory.’
Homology modeling of human CFHR5/C3b complex
Docking procedure has been described in more detail previously . Starting from residues 23-569, the 3D homology models of CFHR5 were first generated by iterative threading assembly refinement (I-TASSER) server , where the one with the highest C-score was selected to be further refined by Fragment-Guided MD simulation (FG-MD) . Potential energy of refined 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 fixing CHARMm force fields. 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 protein-protein interaction was estimated by the Calculate Mutation Energy (Binding) protocol within DS.