Clinical presentation and follow-up
We investigated a three-generation Han Chinese family characterized by persistent microscopic haematuria associated with renal failure (Fig. 1a). The proband (III:1) , a 11-year-old boy, was admitted to the hospital for haematuria and proteinuria. On admission, 24-h urine protein was 6.36 g/d and serum albumin was 24.4 g/L. Microscopic urinary analysis showed urine sediment containing ++~+++ red cells per high-power field. His blood urea nitrogen level was 15.73 mmol/L, serum creatinine level was 266 umol/L and eGFR by CKD-EPI equation was 30.3 mL/min. Bilateral renal ultrasound revealed abnormal changes with strong 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.
During follow-up, the proband was started on haemodialysis three months later and received a kidney transplant after one year. The proband (III:1) had a family history of haematuria, as his mother (II:1) presented with haematuria, proteinuria, and developed ESRD before the age of 33 years old. His maternal grandma (I:1), maternal aunt (II:4), and aunt’s daughter (III:2) also had urinary abnormalities characterized by asymptomatic haematuria and proteinuria with normal eGFR. His father (II:2), his maternal grandpa, and aunt’s husband were phenotypically normal.
To confirm the clinical diagnosis, renal biopsy of the proband was performed. The biopsy sample contained fifteen glomeruli on light microscopy: global glomerular sclerosis in four glomeruli, segmental sclerosis in three other glomeruli (Fig. 2a), and four crescents were found (Fig. 2b). Diffuse and global mild-moderate proliferation of mesangial cells and mesangial matrix were shown in the remaining glomeruli (Fig. 2c). Besides, interstitial fibrosis/tubular atrophy containing lipid-laden foam cells were obvious (Fig. 2d). Immunofluorescence studies showed patchy GBM expression of a5 (IV) collagen and complete absence in Bowman’s capsule and distal tubular basement membrane (BM) (Fig. 2e), compared with control (Fig. 2f). A skin biopsy was performed on his mother (II:1), which revealed that there was segmental absence of the collagen IV a5 chain within the epidermal basement membrane (Fig. 2g), compared with control (Fig. 2h).
Electron microscopy identified pathological characteristics accompanied with noticeably segmental uneven thickness with unsmooth spiculation and suspected lamination of the dense layer within the basement membrane in the proband (Fig. 2i). His affected aunt’s daughter (III:2) had a renal biopsy at age 13, showing segmental uneven thickness with a laminated appearance of the dense layer in GBM (Fig. 2j).
Notably, a marked C3 deposition (++ to +++) in the mesangial region was detected in the proband under immunofluorescence microscopy (Fig. 2k), with the corresponding lump electron-dense deposits and mild-moderate mesangial proliferation observed with electron microscopy (Fig. 2l). Immunofluorescence examination revealed none or trace positivity of IgA, IgG, IgM, C4, C1q, Fib, κ and λ fragments.
Findings on targeted exome-based next-generation sequencing (NGS) and Sanger sequencing
To make a precise diagnosis for the true pathogenic mechanism affecting the family, we performed whole exome-based NGS and further confirmed the results with Sanger sequencing. Genetic analysis identified the same heterozygous c.508G>A coding variant (p.Val170Met) in exon 6 of the CFHR5 gene in three affected individuals: the proband (III:1), his mother (II:1) and his maternal grandma (I:1) (Fig. 1b). The variant presents low frequent in the general population (1000 Genomes: minor allele frequency [MAF] = 0.1%), and the mutated A allele carriers are all heterozygous. Compared across ethnicities, this variant is more frequent in East Asians (1000 Genomes: 0.6%). The nonsynonymous alteration leads to the replacement of a valine, strictly conserved among organisms (Fig. 3a), by a methionine residue. The screening with four publicly available programs (SIFT, SNAP, PolyPhen-2, and Mutation Taster) independently predicted that the replaced amino acid was “damaging” to the 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 genes COL4A3, COL4A4, or COL4A5 that encode the α3, α4, or α5 chains of type IV collagen, respectively.
In silico functional prediction for CFHR5 c.508G>A, p.Val170Met
CFHR5 is a single-chain polypeptide composed of nine complement control protein domains (also known as CCPs). The residue p.Val170Met in CFHR5 occurs in the β-strand region of the CCP3. The isoelectric 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 neighbouring residues (Leu171 and Val187) changes (Fig. 3b). DS 3.0 analysis predicted the potential energy of the mutant type protein is -15180.97 kcal/mol compared to -15133.86 kcal/mol for the wild type one, which implied that p.Val170Met could lead to an increase in conformational stability of the CFHR5 protein.
As with Complement Factor H (CFH) and other Complement Factor H Related Proteins (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 modified C3-α chain (C3α') and the intact C3-β chain (C3β). We then simulated the probable native CFHR5/C3β complex structure. As it can be seen in Fig. 3c, the three-dimensional (3D) model demonstrated that Val170 is not the binding site for protein C3b. However, further binding free-energy calculation showed that p.V170M slightly increases the binding affinity of CFHR5/C3β complex by -0.76 kcal/mol.