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Published on: July 14, 2016
Functional analysis of rare genetic variants in complement factor I in advanced age-related macular degeneration
Anuja Java1, Nicola Pozzi2, Molly C Schroeder3
1Division of Nephrology, Department of Medicine, Washington University School of Medicine, St. Louis, MO 63110, USA.
Insights
Genetic variants in complement factor I (CFI) are linked to age-related macular degeneration (AMD). This study functionally characterized CFI variants, reclassifying some previously uncertain variants and clarifying their role in AMD pathogenesis.
Area of Science:
- Immunology
- Genetics
- Ophthalmology
Background:
- Rare heterozygous genetic variants in complement factor I (CFI) are associated with advanced age-related macular degeneration (AMD).
- Many of these CFI variants are classified as variants of uncertain significance (VUS) due to a lack of functional characterization.
- Previous work categorized AMD-associated CFI variants into quantitative (Type 1), qualitative (Type 2), and less dysfunctional (Type 3) deficiencies.
Purpose of the Study:
- To functionally characterize nine Type 3 CFI variants of uncertain significance (VUS) identified in individuals with advanced age-related macular degeneration (AMD).
- To re-evaluate the classification of these variants based on comprehensive functional assays.
- To elucidate the clinical implications of complement factor I variants in AMD.
Main Methods:
- Site-directed mutagenesis was used to create recombinant CFI variants.
- Expression levels of recombinant proteins were assessed relative to wild-type (WT).
- A comprehensive suite of functional assays evaluated cofactor activity and overall protein function.
Main Results:
- Four variants (R202I, Q217H, S221Y, G263V) showed reduced protein expression, leading to reclassification as Type 1 (quantitative deficiency).
- Two variants (G362A, N536K) exhibited significantly reduced cofactor activity despite normal expression, leading to reclassification as Type 2 (qualitative deficiency).
- Three variants (K441R, Q462H, I492L) demonstrated no functional defect and remained classified as Type 3.
Conclusions:
- In-depth biochemical analysis is crucial for defining the pathological and clinical significance of complement variants.
- This study successfully reclassified several CFI variants of uncertain significance, providing clearer insights into their role in AMD.
- The findings contribute to a better understanding of the genetic basis of age-related macular degeneration and the complement system's involvement.
Abstract:
Factor I (FI) is a serine protease inhibitor of the complement system. Heterozygous rare genetic variants in complement factor I (CFI) are associated with advanced age-related macular degeneration (AMD). The clinical impact of these variants is unknown since a majority have not been functionally characterized and are classified as 'variants of uncertain significance' (VUS). This study assessed the functional significance of VUS in CFI. Our previous cross-sectional study using a serum-based assay demonstrated that CFI variants in advanced AMD can be categorized into three types. Type 1 variants cause a quantitative deficiency of FI. Type 2 variants demonstrate a qualitative deficiency. However, Type 3 variants consist of VUS that are less dysfunctional than Types 1 and 2 but are not as biologically active as wild type (WT). In this study, we employed site-directed mutagenesis followed by expression of the recombinant variant and a comprehensive set of functional assays to characterize nine Type 3 variants that were identified in 37 individuals. Our studies establish that the expression of the recombinant protein compared with WT is reduced for R202I, Q217H, S221Y and G263V. Further, G362A and N536K, albeit expressed normally, have significantly less cofactor activity. These results led to re-categorization of CFI variants R202I, Q217H, S221Y and G263V as Type 1 variants and to reclassification of N536K and G362A as Type 2. The variants K441R, Q462H and I492L showed no functional defect and remained as Type 3. This study highlights the utility of an in-depth biochemical analysis in defining the pathologic and clinical implications of complement variants underlying AMD.
