Functional Characterization of Rare Genetic Variants in the N-Terminus of Complement Factor H in aHUS, C3G, and AMD

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.

The Role of Inflammation in Retinal Neurodegeneration and Degenerative Diseases

“…Inflammation in degenerative retinal disorders has been strongly supported by molecular genetic studies. Although inconclusive, genes encoding complement factor H (CFH) [ 40 ], complement component 2 (C2), factor B (FB) [ 41 ], and apolipoprotein E (APOE) have been documented as being associated with AMD. Increased risk of AMD has been especially prevalent among the carriers of the APOE ε2 allele, while APOE ε4 has been known to protect against this condition [ 42 ].…”

Use of a B-cell depleting regimen for antifactor H autoantibody-mediated membranoproliferative glomerulonephritis in a paediatric patient

“…In children they were frequently associated with C3 nephritic factor,14 which was not seen in this case. These acquired factor H defects mimic the genetic defects in factor H where N-terminal mutations show similar fluid phase defects 15–17. Overproduction of C3 and associated fragments may then be deposited in the kidney leading to the pathological finding of MPGN 18…”

Clinicopathologic Implications of Complement Genetic Variants in Kidney Transplantation

“…Additionally, we demonstrate that the SNP E936D may have a potentially protective role since it helped improve the C3b binding for rare variant, T956M. We used FH fragments which have been shown by us (16) and others (45)(46)(47) to be highly predictive of the results obtained with full-length protein (36,44). Nevertheless, the availability of commercially available full-length recombinant FH and the serum-based assays is an advance that should be taken advantage of.…”