PMC3016252_01
Renal transplant biopsy. (A) Light microscopy showing a normal glomerulus (periodic acid-Schiff). (B) Immunoperoxidase staining showing granular capillary wall and mesangial C3 deposition. C3 was detected using a polyclonal rabbit anti-human C3c antibody (Dako Ltd., Ely, United Kingdom). (C) Electron micrograph showing subendothelial deposits (denoted by arrows) with new basement membrane beneath. (D) Glomerular C5b-9 by indirect immunofluorescence. C5b-9 was detected using a monoclonal mouse anti-human C5b-9 antibody (DakoCytomation Ltd., Ely, United Kingdom) and a fluorescein isothiocyanate-labeled goat anti-mouse IgG (Sigma-Aldrich Company Ltd., Dorset, United Kingdom).
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Although his renal function initially improved, his creatinine stabilized at 186 mumol/L, and in the presence of persistent microscopic hematuria and following a single episode of macroscopic hematuria (with a urine protein: creatinine ratio of 56 mg/mmol), he underwent a renal transplant biopsy, 46 days after transplantation (Figure 2). C3 and complement component 9 (C9), whilst electron microscopy showed increased mesangial matrix associated with scattered mesangial and subendothelial deposits (with new basement membrane beneath in two areas; Figure 2C). Complement component 4d (C4d) staining was negative. Indirect immunofluorescence also showed the presence of glomerular complement components 5b (C5b)-9 (Figure 2D).
Internal duplication of CFHR5. Western blot of serum for CFHR5 and schematic depicting normal and mutant CFHR5 proteins (denoted CFHR5123-9 and CFHR512123-9 respectively). A polyclonal anti-CFHR5 antibody was used to detect the wild-type and mutant CFHR5 proteins in sera from the patient (P) and control (C). The wild-type CFHR5 protein consists of nine protein subunits termed short consensus repeat (SCR) domains. The internal duplication of exons 2 and 3 in the mutant CFHR5 gene results in a mutant protein that contains two additional SCR domains (duplicated protein domains depicted in orange in the schematic). The mutant CFHR5 protein therefore has a higher molecular weight than the wild-type protein. The mutant protein was readily detectable in the patient but not in the control sample. The presence of the wild-type protein in the patient sample confirmed that the mutation is present in heterozygosity in this individual.
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Polymerase chain reaction (PCR) using genomic DNA isolated from peripheral blood monocytes and serum CFHR5 western blot analysis (Figure 3) revealed the presence of the heterozygous internal duplication in the CFHR5 gene, confirming the diagnosis of CFHR5 nephropathy.