Functional Analysis of Rare Genetic Variants in Complement Factor I (CFI) using a Serum-Based Assay in Advanced Age-related Macular Degeneration

Purpose: To identify rare genetic variants in early age-related macular degeneration (AMD) utilizing whole-exome sequencing (WES). Methods: Eight non-related early-AMD families of different Jewish ethnicities were ascertained. Initial mutation screening (phase-1) included common complement factor-H (CFH) p.Y402H; and age related maculopathy susceptibility 2 (ARMS2) p.A69S; and rare variants complement factor-I (CFI) p.V412M; and hemicentin1 (HMCN1) c.4163delC identified previously in our population. Four families, whose initial screening for the aforementioned variants was negative, underwent WES (phase-2). Bioinformatics filtering was based on functionality (from a panel of 234 genes with proven or presumed association to AMD); predicted severity; and frequency (rare variants with minor allele frequency <1%). When applicable, further screening for specific rare variants was carried out on additional cases of similar ethnicities and phenotypes (phase-3). Results: Phase-1 identified three families carrying CFI p.V412M mutation. WES analysis detected probable disease-related variants in three out of the remaining families. These included: a family with a variant in PLEKHA1 gene p.S177N; a family with previously reported variant p.R1210C in CFH gene; and two families with the C3 p.R735W variant. Conclusions: Rare, high-penetrance variants have a profound contribution to early-AMD pathogenesis. Utilization of WES in genetic research of multifactorial diseases as AMD, allows a thorough comprehensive analysis with the identification of previously unreported rare variants.

Download Full-text

Functional Analysis of Variants in Complement Factor I Identified in Age-Related Macular Degeneration and Atypical Hemolytic Uremic Syndrome

Frontiers in Immunology ◽

10.3389/fimmu.2021.789897 ◽

2022 ◽

Vol 12 ◽

Author(s):

Sarah de Jong ◽

Anita de Breuk ◽

Bjorn Bakker ◽

Suresh Katti ◽

Carel B. Hoyng ◽

...

Keyword(s):

Hemolytic Uremic Syndrome ◽

Macular Degeneration ◽

Atypical Hemolytic Uremic Syndrome ◽

Age Related Macular Degeneration ◽

Complement Factor ◽

Serum Samples ◽

Factor I ◽

Complement Factor I ◽

Age Related ◽

Uremic Syndrome

Complement factor I (FI) is a central inhibitor of the complement system, and impaired FI function increases complement activation, contributing to diseases such as age-related macular degeneration (AMD) and atypical hemolytic uremic syndrome (aHUS). Genetic variation in complement factor I (CFI) has been identified in both AMD and aHUS, with more than half of these variants leading to reduced FI secretion levels. For many of the variants with normal FI secretion, however, functional implications are not yet known. Here we studied 11 rare missense variants, with FI secretion levels comparable to wildtype, but a predicted damaging effects based on the Combined Annotation Dependent Depletion (CADD) score. Three variants (p.Pro50Ala, p.Arg339Gln, and p.Ser570Thr) were analyzed in plasma and serum samples of carriers affected by AMD. All 11 variants (nine for the first time in this study) were recombinantly expressed and the ability to degrade C3b was studied with the C3b degradation assay. The amount of degradation was determined by measuring the degradation product iC3b with ELISA. Eight of 11 (73%) mutant proteins (p.Pro50Ala, p.Arg339Gln, p.Ile340Thr, p.Gly342Glu, p.Gly349Arg, p.Arg474Gln, p.Gly487Cys, and p.Gly512Ser) showed significantly impaired C3b degradation, and were therefore classified as likely pathogenic. Our data indicate that genetic variants in CFI with a CADD score >20 are likely to affect FI function, and that monitoring iC3b in a degradation assay is a useful tool to establish the pathogenicity of CFI variants in functional studies.

Download Full-text