Abstract
Membranoproliferative glomerulonephritis (MPGN), C3 glomerulopathy (C3G), atypical haemolytic uraemic syndrome (aHUS) and age-related macular degeneration (AMD) have all been strongly linked with dysfunction of the alternative pathway (AP) of complement. A significant proportion of individuals with MPGN, C3G, aHUS and AMD carry rare genetic variants in the CFH gene that cause functional or quantitative deficiencies in the factor H (FH) protein, an important regulator of the AP. In silico analysis of the deleteriousness of rare genetic variants in CFH is not reliable and careful biochemical assessment remains the gold standard. Six N-terminal variants of uncertain significance in CFH were identified in patients with these diseases of the AP and selected for analysis. The variants were produced in Pichia Pastoris in the setting of FH CCPs 1–4, purified by nickel affinity chromatography and size exclusion and characterized by surface plasmon resonance and haemolytic assays as well as by cofactor assays in the fluid phase. A single variant, Q81P demonstrated a profound loss of binding to C3b with consequent loss of cofactor and decay accelerating activity. A further 2 variants, G69E and D130N, demonstrated only subtle defects which could conceivably over time lead to disease progression of more chronic AP diseases such as C3G and AMD. In the variants S159N, A161S, and M162V any functional defect was below the capacity of the experimental assays to reliably detect. This study further underlines the importance of careful biochemical assessment when assigning functional consequences to rare genetic variants that may alter clinical decisions for patients.
Highlights
Atypical haemolytic uraemic syndrome [1], membranoproliferative glomerulonephritis (MPGN), C3 glomerulopathy (C3G) [2] and age-related macular degeneration (AMD) [3] are all diseases that have been associated with dysregulation of the alternative pathway (AP) of complement.The hallmark pathological lesion in atypical haemolytic uraemic syndrome (aHUS) is thrombotic microangiopathy within the kidney, characterized by the clinical features of microangiopathic haemolytic anaemia, thrombocytopenia and acute kidney failure [1]
Dense deposit disease is a special sub-type of C3G, characterized by prominent intramembranous laminar deposits that are visible on electron microscopy, associated historically with Membranoproliferative glomerulonephritis (MPGN) [2]
The rare genetic variants, G69E, Q81P, D130N, S159N, A161S, and M162V were analyzed in the setting of a construct containing the first four complement control proteins (CCPs) domains of factor H (FH) responsible for C3b binding, Decay accelerating activity (DAA), and cofactor activity (CA) (Figure 1)
Summary
Atypical haemolytic uraemic syndrome (aHUS) [1], membranoproliferative glomerulonephritis (MPGN), C3 glomerulopathy (C3G) [2] and age-related macular degeneration (AMD) [3] are all diseases that have been associated with dysregulation of the alternative pathway (AP) of complement. The hallmark pathological lesion in aHUS is thrombotic microangiopathy within the kidney, characterized by the clinical features of microangiopathic haemolytic anaemia, thrombocytopenia and acute kidney failure [1]. MPGN and C3G are a group of conditions in which deposition of complement activation products within the glomerulus occurs, resulting in nephrotic/nephritic syndrome and chronic progressive renal failure [2]. Dense deposit disease is a special sub-type of C3G, characterized by prominent intramembranous laminar deposits that are visible on electron microscopy, associated historically with MPGN [2]
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