Causes of alternative pathway dysregulation in dense deposit disease

Abstract

Background and objectives: This study was designed to investigate the causes of alternative pathway dysregulation in a cohort of patients with dense deposit disease (DDD).

Design, setting, participants, & measurements: Thirty-two patients with biopsy-proven DDD underwent screening for C3 nephritic factors (C3Nefs), factor H autoantibodies (FHAAs), factor B autoantibodies (FBAAs), and genetic variants in CFH. C3Nefs were detected by: ELISA, C3 convertase surface assay (C3CSA), C3CSA with properdin (C3CSAP), two-dimensional immunoelectrophoresis (2DIEP), and immunofixation electrophoresis (IFE). FHAAs and FBAAs were detected by ELISA, and CFH variants were identified by Sanger sequencing.

Results: Twenty-five patients (78%) were positive for C3Nefs. Three C3Nef-positive patients were also positive for FBAAs and one of these patients additionally carried two novel missense variants in CFH. Of the seven C3Nef-negative patients, one patient was positive for FHAAs and two patients carried CFH variants that may be causally related to their DDD phenotype. C3CASP was the most sensitive C3Nef-detection assay. C3CASP and IFE are complementary because C3CSAP measures the stabilizing properties of C3Nefs, whereas IFE measures their expected consequence-breakdown of C3b.

Conclusions: A test panel that includes C3CSAP, IFE, FHAAs, FBAAs, and genetic testing for CFH variants will identify a probable cause for alternative pathway dysregulation in approximately 90% of DDD patients. Dysregulation is most frequently due to C3Nefs, although some patients test positive for FHAAs, FBAAs, and CFH mutations. Defining the pathophysiology of DDD should facilitate the development of mechanism-directed therapies.

Figure 1.

The renal t_1/2 as estimated by the nonparametric Kaplan–Meier method was approximately 12 years (95% confidence interval, 6–∞).

Figure 2.

Alternative pathway dysregulation in 32 patients with dense deposit disease. C3Nefs were detected in 25 patients and in the 7 C3Nef-negative patients, 1 had FHAA and 2 had CFH mutations. Four patients were negative for all investigations completed in this study (see patient details, Table 1). Of the C3Nef assays, ELISA was the least sensitive (maximum sensitivity only 68%), whereas the best performing assay was C3CSAP. C3Nef, C3 nephritic factor; FHAA, factor H autoantibody; C3CSAP, C3 convertase surface assay with properdin; C3CSA, C3 convertase stabilizing assay; FH, factor H; 2DIEP, two-dimensional immunoelectrophoresis; IFE, immunofixation electrophoresis.

Figure 3.

C3CSA and C3CSAP. (A) C3CSA using purified IgGs from 32 patients with dense deposit disease showed that the stabilizing abilities of IgG C3Nefs varied considerably among patients. (B) C3CSAP was more sensitive than C3CSA in detecting C3Nefs at any time during the course of disease. In four patients with dense deposit disease, for example, C3CSAP was strongly positive, whereas C3CSA was either weakly positive (29%; patient 24, yellow) or negative (patient 18, black; patient 22, red; and patient 23, green). C3CSA, C3 convertase surface assay; C3CSAP, C3 convertase surface assay with properdin; C3Nef, C3 nephritic factor; FHAA, factor H autoantibody.

Figure 4.

C3Nef test battery. (A) Patient 2 is a 16-year-old male with stage 3 CKD in whom C3Nefs were detectable using all five assays: (a) ELISA, (b) 2DIEP, (c) IFE, (d) C3CSA, and (e) C3CSAP. Consistent with this finding, both C3 and fluid-phase AP activity were low (C3=5 mg/dl; normal range, 86–184 mg/dl; AP activity, 5.4%; normal range, 65%–130%). sMAC was normal. (B) Patient 23 is a dialysis-dependent 12-year-old female in whom only C3CSAP was positive. Her C3 was undetectable and fluid-phase AP activity was 4.2%. (b) Red arrowhead, C3 breakdown product peak; (c) black arrowhead, C3 breakdown product line; (d and e) line colors: test sample, red; negative control, orange; positive control, green. EGTA chelates Ca²⁺ allowing AP activation when Mg²⁺ is added. EDTA chelates Ca²⁺ and Mg²⁺, preventing all complement activation. C3Nef, C3 nephritic factor; 2DIEP, two-dimensional immunoelectrophoresis; IFE, immunofixation electrophoresis; C3CSA, C3 convertase surface assay; C3CSAP, C3 convertase surface assay with properdin; AP, alternative pathway; sMAC, soluble membrane attack complex.

Figure 4.

C3Nef test battery. (A) Patient 2 is a 16-year-old male with stage 3 CKD in whom C3Nefs were detectable using all five assays: (a) ELISA, (b) 2DIEP, (c) IFE, (d) C3CSA, and (e) C3CSAP. Consistent with this finding, both C3 and fluid-phase AP activity were low (C3=5 mg/dl; normal range, 86–184 mg/dl; AP activity, 5.4%; normal range, 65%–130%). sMAC was normal. (B) Patient 23 is a dialysis-dependent 12-year-old female in whom only C3CSAP was positive. Her C3 was undetectable and fluid-phase AP activity was 4.2%. (b) Red arrowhead, C3 breakdown product peak; (c) black arrowhead, C3 breakdown product line; (d and e) line colors: test sample, red; negative control, orange; positive control, green. EGTA chelates Ca²⁺ allowing AP activation when Mg²⁺ is added. EDTA chelates Ca²⁺ and Mg²⁺, preventing all complement activation. C3Nef, C3 nephritic factor; 2DIEP, two-dimensional immunoelectrophoresis; IFE, immunofixation electrophoresis; C3CSA, C3 convertase surface assay; C3CSAP, C3 convertase surface assay with properdin; AP, alternative pathway; sMAC, soluble membrane attack complex.