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

Jong, Sarah de; Breuk, Anita de; Bakker, Björn; Katti, Suresh; Hoyng, Carel B.; Nilsson, Sara C.; Blom, Anna M.; Heuvel, Lambert P. van den; Hollander, Anneke I. den; Volokhina, Elena

doi:10.3389/fimmu.2021.789897

Cited by 9 publications

(4 citation statements)

References 34 publications

(62 reference statements)

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“…Rare variants in CFI and CFH associated with AMD ( Supplemental Tables 3, 4 ) [FI: I340T ( 13 , 20 , 30 – 32 ), R406H ( 13 , 16 , 30 , 31 ), K441R ( 13 , 16 , 30 , 31 ), P553S ( 13 , 16 , 30 , 31 ) and FH CCP 1-4: P26S ( 33 ), T91S ( 33 ), R166W ( 33 ), R232Q ( 33 )] were modelled within the context of the AP regulatory TMC ( 25 ) ( Figure 1 ). FH variants were generated in the framework of the N-terminal domains of FH, CCPs 1–4, as previously described ( 34 ) ( Supplemental Figure 3A ).…”

Section: Resultsmentioning

confidence: 99%

A novel method for real-time analysis of the complement C3b:FH:FI complex reveals dominant negative CFI variants in age-related macular degeneration

et al. 2022

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Age-related macular degeneration (AMD) is linked to 2 main disparate genetic pathways: a chromosome 10 risk locus and the alternative pathway (AP) of complement. Rare genetic variants in complement factor H (CFH; FH) and factor I (CFI; FI) are associated with AMD. FH acts as a soluble cofactor to facilitate FI’s cleavage and inactivation of the central molecule of the AP, C3b. For personalised treatment, sensitive assays are required to define the functional significance of individual AP genetic variants. Generation of recombinant FI for functional analysis has thus far been constrained by incomplete processing resulting in a preparation of active and inactive protein. Using an internal ribosomal entry site (IRES)-Furin-CFI expression vector, fully processed FI was generated with activity equivalent to serum purified FI. By generating FI with an inactivated serine protease domain (S525A FI), a real-time surface plasmon resonance assay of C3b:FH:FI complex formation for characterising variants in CFH and CFI was developed and correlated well with standard assays. Using these methods, we further demonstrate that patient-associated rare genetic variants lacking enzymatic activity (e.g. CFI I340T) may competitively inhibit the wild-type FI protein. The dominant negative effect identified in inactive factor I variants could impact on the pharmacological replacement of FI currently being investigated for the treatment of dry AMD.

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Section: Resultsmentioning

confidence: 99%

A novel method for real-time analysis of the complement C3b:FH:FI complex reveals dominant negative CFI variants in age-related macular degeneration

et al. 2022

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“…Third, growing evidence from genetic studies suggests that dysregulation of the complement system has a pathogenic role in both AMD 9 , 36 , 37 and kidney diseases including atypical hemolytic uremic syndrome (aHUS) 38 , 39 , membranoproliferative glomerulonephritis (MPGN) 40 , IgA nephropathy 41 , and DM nephropathy 42 . Complement factor H (CFH) is a soluble complement regulator that is essential for controlling the alternative complement pathway and protects against oxidative stress.…”

Section: Discussionmentioning

confidence: 99%

Increased end-stage renal disease risk in age-related macular degeneration: a nationwide cohort study with 10-year follow-up

Jung

Park

Jang

et al. 2023

Sci Rep

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Common etiologies between age-related macular degeneration (AMD) and kidney disease advocate a close link between AMD and end-stage renal disease (ESRD). However, the risk of ESRD in people with AMD was not reported. Here, we investigated the association between AMD and the risk of ESRD by using a nationwide, population-based cohort data in Korea. 4,206,862 participants aged 50 years or older were categorized by presence of AMD and visual disability. Risk of ESRD was the primary outcome. Cox regression hazard model was used to examine the hazard ratios (HRs) with adjustment for potential confounders. Stratified analyses by age, sex, baseline kidney function, and cardiometabolic comorbidities were performed. During the mean 9.95 years of follow-up, there were 21,759 incident ESRD events (0.52%). AMD was associated with 33% increased risk of ESRD (adjusted HR [aHR] 1.33, 95% confidence interval [CI] 1.24–1.44), and the risk was even higher when accompanied by visual disability (aHR 2.05, 95% CI 1.68–2.50) than when not (aHR 1.26, 95% CI 1.17–1.37). Age, baseline kidney function, and cardiometabolic comorbidities significantly interact between AMD and the risk of ESRD. Our findings have clinical implications on disease prevention and risk factor management of ESRD in patients with AMD.

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“…To determine if these spatial assessments provide information that can be used to both understand and predict underlying biology, we chose to further investigate the CFI gene, which has a large body of literature about the impact of specific variants on gene expression. Using our previously published data on the effect of mutant CFI on Factor I secretion levels 35 we retained all missense variants that were found in CFI after classification of those variants as missense. PathProx and ddG scores were calculated for these variants.…”

Section: Methodsmentioning

confidence: 99%

Spatial Distribution of Missense Variants within Complement Proteins Associates with Age Related Macular Degeneration

Grunin,

Jong,

Palmer

et al. 2023

Preprint

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Purpose: Genetic variants in complement genes are associated with age-related macular degeneration (AMD). However, many rare variants have been identified in these genes, but have an unknown significance, and their impact on protein function and structure is still unknown. We set out to address this issue by evaluating the spatial placement and impact on protein structure of these variants by developing an analytical pipeline and applying it to the International AMD Genomics Consortium (IAMDGC) dataset (16,144 AMD cases, 17,832 controls). Methods: The IAMDGC dataset was imputed using the Haplotype Reference Consortium (HRC), leading to an improvement of over 30% more imputed variants, over the original 1000 Genomes imputation. Variants were extracted for the CFH, CFI, CFB, C9, and C3 genes, and filtered for missense variants in solved protein structures. We evaluated these variants as to their placement in the three-dimensional structure of the protein (i.e. spatial proximity in the protein), as well as AMD association. We applied several pipelines to a) calculate spatial proximity to known AMD variants versus gnomAD variants, b) assess a variants likelihood of causing protein destabilization via calculation of predicted free energy change (ddG) using Rosetta, and c) whole gene-based testing to test for statistical associations. Gene-based testing using seqMeta was performed using a) all variants b) variants near known AMD variants or c) with a ddG >|2|. Further, we applied a structural kernel adaptation of SKAT testing (POKEMON) to confirm the association of spatial distributions of missense variants to AMD. Finally, we used logistic regression on known AMD variants in CFI to identify variants leading to >50% reduction in protein expression from known AMD patient carriers of CFI variants compared to wild type (as determined by in vitro experiments) to determine the pipelines robustness in identifying AMD-relevant variants. These results were compared to functional impact scores, ie CADD values > 10, which indicate if a variant may have a large functional impact genomewide, to determine if our metrics have better discriminative power than existing variant assessment methods. Once our pipeline had been validated, we then performed a priori selection of variants using this pipeline methodology, and tested AMD patient cell lines that carried those selected variants from the EUGENDA cohort (n=34). We investigated complement pathway protein expression in vitro, looking at multiple components of the complement factor pathway in patient carriers of bioinformatically identified variants. Results: Multiple variants were found with a ddG>|2| in each complement gene investigated. Gene-based tests using known and novel missense variants identified significant associations of the C3, C9, CFB, and CFH genes with AMD risk after controlling for age and sex (P=3.22x10^-5;7.58x10^-6;2.1x10^-3;1.2x10^-31). ddG filtering and SKAT-O tests indicate that missense variants that are predicted to destabilize the protein, in both CFI and CFH, are associated with AMD (P=CFH:0.05, CFI:0.01, threshold of 0.05 significance). Our structural kernel approach identified spatial associations for AMD risk within the protein structures for C3, C9, CFB, CFH, and CFI at a nominal p-value of 0.05. Both ddG and CADD scores were predictive of reduced CFI protein expression, with ROC curve analyses indicating ddG is a better predictor (AUCs of 0.76 and 0.69, respectively). A priori in vitro analysis of variants in all complement factor genes indicated that several variants identified via bioinformatics programs PathProx/POKEMON in our pipeline via in vitro experiments caused significant change in complement protein expression (P=0.04) in actual patient carriers of those variants, via ELISA testing of proteins in the complement factor pathway, and were previously unknown to contribute to AMD pathogenesis. Conclusion: We demonstrate for the first time that missense variants in complement genes cluster together spatially and are associated with AMD case/control status. Using this method, we can identify CFI and CFH variants of previously unknown significance that are predicted to destabilize the proteins. These variants, both in and outside spatial clusters, can predict in-vitro tested CFI protein expression changes, and we hypothesize the same is true for CFH. A priori identification of variants that impact gene expression allow for classification for previously classified as VUS. Further investigation is needed to validate the models for additional variants and to be applied to all AMD-associated genes.

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

Cited by 9 publications

References 34 publications

A novel method for real-time analysis of the complement C3b:FH:FI complex reveals dominant negative CFI variants in age-related macular degeneration

A novel method for real-time analysis of the complement C3b:FH:FI complex reveals dominant negative CFI variants in age-related macular degeneration

Increased end-stage renal disease risk in age-related macular degeneration: a nationwide cohort study with 10-year follow-up

Spatial Distribution of Missense Variants within Complement Proteins Associates with Age Related Macular Degeneration

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