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

ABSTRACT: 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.

SUBMITTER: de Jong S

PROVIDER: S-EPMC8766660 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:BACKGROUND AND OBJECTIVES: Atypical hemolytic uremic syndrome is characterized by vascular endothelial damage caused by complement dysregulation. Consistently, complement inhibition therapies are highly effective in most patients with atypical hemolytic uremic syndrome. Recently, it was shown that a significant percentage of patients with early-onset atypical hemolytic uremic syndrome carry mutations in diacylglycerol kinase-?, an intracellular protein with no obvious role in complement. These data support an alternative, complement-independent mechanism leading to thrombotic microangiopathy that has implications for treatment of early-onset atypical hemolytic uremic syndrome. To get additional insights into this new form of atypical hemolytic uremic syndrome, the diacylglycerol kinase-? gene in a cohort with atypical hemolytic uremic syndrome was analyzed. DESIGN, SETTING, PARTICIPANTS, & MEASUREMENTS: Eighty-three patients with early-onset atypical hemolytic uremic syndrome (<2 years) enrolled in the Spanish atypical hemolytic uremic syndrome registry between 1999 and 2013 were screened for mutations in diacylglycerol kinase-?. These patients were also fully characterized for mutations in the genes encoding factor H, membrane cofactor protein, factor I, C3, factor B, and thrombomodulin CFHRs copy number variations and rearrangements, and antifactor H antibodies. RESULTS: Four patients carried mutations in diacylglycerol kinase-?, one p.H536Qfs*16 homozygote and three compound heterozygotes (p.W322*/p.P498R, two patients; p.Q248H/p.G484Gfs*10, one patient). Three patients also carried heterozygous mutations in thrombomodulin or C3. Extensive plasma infusions controlled atypical hemolytic uremic syndrome recurrences and prevented renal failure in the two patients with diacylglycerol kinase-? and thrombomodulin mutations. A positive response to plasma infusions and complement inhibition treatment was also observed in the patient with concurrent diacylglycerol kinase-? and C3 mutations. CONCLUSIONS: Data suggest that complement dysregulation influences the onset and disease severity in carriers of diacylglycerol kinase-? mutations and that treatments on the basis of plasma infusions and complement inhibition are potentially useful in patients with combined diacylglycerol kinase-? and complement mutations. A comprehensive understanding of the genetic component predisposing to atypical hemolytic uremic syndrome is, therefore, critical to guide an effective treatment.

Project description:Background and objectivesThe complement inhibitor eculizumab has dramatically improved the outcome of atypical hemolytic uremic syndrome. However, the optimal duration of eculizumab treatment in atypical hemolytic uremic syndrome remains debated. We report on the French atypical hemolytic uremic syndrome working group's first 2-year experience with eculizumab discontinuation in patients with atypical hemolytic uremic syndrome.Design, setting, participants & measurementsUsing the French atypical hemolytic uremic syndrome registry database, we retrospectively identified all dialysis-free patients with atypical hemolytic uremic syndrome who discontinued eculizumab between 2010 and 2014 and reviewed their relevant clinical and biologic data. The decision to discontinue eculizumab was made by the clinician in charge of the patient. All patients were closely monitored by regular urine dipsticks and blood tests. Eculizumab was rapidly (24-48 hours) restarted in case of relapse.ResultsAmong 108 patients treated with eculizumab, 38 patients (nine children and 29 adults) discontinued eculizumab (median treatment duration of 17.5 months). Twenty-one patients (55%) carried novel or rare complement genes variants. Renal recovery under eculizumab was equally good in patients with and those without complement gene variants detected. After a median follow-up of 22 months, 12 patients (31%) experienced atypical hemolytic uremic syndrome relapse. Eight of 11 patients (72%) with complement factor H variants, four of eight patients (50%) with membrane cofactor protein variants, and zero of 16 patients with no rare variant detected relapsed. In relapsing patients, early reintroduction (?48 hours) of eculizumab led to rapid (<7 days) hematologic remission and a return of serum creatinine to baseline level in a median time of 26 days. At last follow-up, renal function remained unchanged in nonrelapsing and relapsing patients compared with baseline values before eculizumab discontinuation.ConclusionsPathogenic variants in complement genes were associated with higher risk of atypical hemolytic uremic syndrome relapse after eculizumab discontinuation. Prospective studies are needed to identify biomarkers predictive of relapse and determine the best strategy of retreatment in relapsing patients.

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

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