Genetic Analysis of 400 Patients Refines Understanding and Implicates a New Gene in Atypical Hemolytic Uremic Syndrome.

BACKGROUND: Genetic variation in complement genes is a predisposing factor for atypical hemolytic uremic syndrome (aHUS), a life-threatening thrombotic microangiopathy, however interpreting the effects of genetic variants is challenging and often ambiguous. METHODS: We analyzed 93 complement and coagulation genes in 400 patients with aHUS, using as controls 600 healthy individuals from Iowa and 63,345 non-Finnish European individuals from the Genome Aggregation Database. After adjusting for population stratification, we then applied the Fisher exact, modified Poisson exact, and optimal unified sequence kernel association tests to assess gene-based variant burden. We also applied a sliding-window analysis to define the frequency range over which variant burden was significant. RESULTS: We found that patients with aHUS are enriched for ultrarare coding variants in the CFH, C3, CD46, CFI, DGKE, and VTN genes. The majority of the significance is contributed by variants with a minor allele frequency of <0.1%. Disease-related variants tend to occur in specific complement protein domains of FH, CD46, and C3. We observed no enrichment for multiple rare coding variants in gene-gene combinations. CONCLUSIONS: In known aHUS-associated genes, variants with a minor allele frequency >0.1% should not be considered pathogenic unless valid enrichment and/or functional evidence are available. VTN, which encodes vitronectin, an inhibitor of the terminal complement pathway, is implicated as a novel aHUS-associated gene. Patients with aHUS are not enriched for multiple rare variants in complement genes. In aggregate, these data may help in directing clinical management of aHUS.

Complement Factor I Variants in Complement-Mediated Renal Diseases.

C3 glomerulopathy (C3G) and atypical hemolytic uremic syndrome (aHUS) are two rare diseases caused by dysregulated activity of the alternative pathway of complement secondary to the presence of genetic and/or acquired factors. Complement factor I (FI) is a serine protease that downregulates complement activity in the fluid phase and/or on cell surfaces in conjunction with one of its cofactors, factor H (FH), complement receptor 1 (CR1/CD35), C4 binding protein (C4BP) or membrane cofactor protein (MCP/CD46). Because altered FI activity is causally related to the pathogenesis of C3G and aHUS, we sought to test functional activity of select CFI missense variants in these two patient cohorts. We identified 65 patients (16, C3G; 48, aHUS; 1 with both) with at least one rare variant in CFI (defined as a MAF < 0.1%). Eight C3G and eleven aHUS patients also carried rare variants in either another complement gene, ADAMTS13 or THBD. We performed comprehensive complement analyses including biomarker profiling, pathway activity and autoantibody testing, and developed a novel FI functional assay, which we completed on 40 patients. Seventy-eight percent of rare CFI variants (31/40) were associated with FI protein levels below the 25th percentile; in 22 cases, FI levels were below the lower limit of normal (type 1 variants). Of the remaining nine variants, which associated with normal FI levels, two variants reduced FI activity (type 2 variants). No patients carried currently known autoantibodies (including FH autoantibodies and nephritic factors). We noted that while rare variants in CFI predispose to complement-mediated diseases, phenotypes are strongly contingent on the associated genetic background. As a general rule, in isolation, a rare CFI variant most frequently leads to aHUS, with the co-inheritance of a CD46 loss-of-function variant driving the onset of aHUS to the younger age group. In comparison, co-inheritance of a gain-of-function variant in C3 alters the phenotype to C3G. Defects in CFH (variants or fusion genes) are seen with both C3G and aHUS. This variability underscores the complexity and multifactorial nature of these two complement-mediated renal diseases.

Factor H Autoantibodies and Complement-Mediated Diseases.

Factor H (FH), a member of the regulators-of-complement-activation (RCA) family of proteins, circulates in human plasma at concentrations of 180-420 mg/L where it controls the alternative pathway (AP) of complement in the fluid phase and on cell surfaces. When the regulatory function of FH is impaired, complement-mediated tissue injury and inflammation occur, leading to diseases such as atypical hemolytic uremic syndrome (a thrombotic microangiopathy or TMA), C3 glomerulopathy (C3G) and monoclonal gammopathy of renal significance (MGRS). A pathophysiological cause of compromised FH function is the development of autoantibodies to various domains of the FH protein. FH autoantibodies (FHAAs) are identified in 10.9% of patients with aHUS, 3.2% of patients with C3G, and rarely in patients with MGRS. The phenotypic variability of FHAA-mediated disease reflects both the complexity of FH and the epitope specificity of FHAA for select regions of the native protein. In this paper, we have characterized FHAA epitopes in a large cohort of patients diagnosed with TMA, C3G or MGRS. We explore the epitopes recognized by FHAAs in these diseases and the association of FHAAs with the genetic deletion of both copies of the CFHR1 gene to show how these disease phenotypes are associated with this diverse spectrum of autoantibodies.