An ex vivo test to investigate genetic factors conferring susceptibility to atypical haemolytic uremic syndrome

Abstract

Introduction: Comprehensive genetic analysis is essential to clinical care of patients with atypical haemolytic uremic syndrome (aHUS) to reinforce diagnosis, and to guide treatment. However, the characterization of complement gene variants remains challenging owing to the complexity of functional studies with mutant proteins. This study was designed: 1) To identify a tool for rapid functional determination of complement gene variants; 2) To uncover inherited complement dysregulation in aHUS patients who do not carry identified gene variants.

Methods: To address the above goals, we employed an ex-vivo assay of serum-induced C5b-9 formation on ADP-activated endothelial cells in 223 subjects from 60 aHUS pedigrees (66 patients and 157 unaffected relatives).

Results: Sera taken from all aHUS patients in remission induced more C5b-9 deposition than control sera, independently from the presence of complement gene abnormalities. To avoid the possible confounding effects of chronic complement dysregulation related to aHUS status, and considering the incomplete penetrance for all aHUS-associated genes, we used serum from unaffected relatives. In control studies, 92.7% of unaffected relatives with known pathogenic variants exhibited positive serum-induced C5b-9 formation test, documenting a high sensitivity of the assay to identify functional variants. The test was also specific, indeed it was negative in all non-carrier relatives and in relatives with variants non-segregating with aHUS. All but one variants in aHUS-associated genes predicted in-silico as likely pathogenic or of uncertain significance (VUS) or likely benign resulted as pathogenic in the C5b-9 assay. At variance, variants in putative candidate genes did not exhibit a functional effect, with the exception of a CFHR5 variant. The C5b-9 assay in relatives was helpful in defining the relative functional effect of rare variants in 6 pedigrees in which the proband carried more than one genetic abnormality. Finally, for 12 patients without identified rare variants, the C5b-9 test in parents unmasked a genetic liability inherited from an unaffected parent.

Discussion: In conclusion, the serum-induced C5b-9 formation test in unaffected relatives of aHUS patients may be a tool for rapid functional evaluation of rare complement gene variants. When combined with exome sequencing the assay might be of help in variant selection, to identify new aHUS-associated genetic factors.

Keywords: aHUS; complement; endothelial C5b-9 formation; endothelial cells; rare variants.

Conflict of interest statement

MN has received honoraria from Alexion Pharmaceuticals for giving lectures, and for participating in advisory boards, and she has received research grants from Omeros, Gemini, Novartis and BioCryst Pharmaceuticals. AB has received honoraria from Alexion Pharmaceuticals and BioCryst Pharmaceuticals. GR has consultancy agreements with AbbVie, Alexion Pharmaceuticals, Novartis Pharma and BioCryst Pharmaceuticals. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2023 Gastoldi, Aiello, Galbusera, Breno, Alberti, Bresin, Mele, Piras, Liguori, Santarsiero, Benigni, Remuzzi and Noris.

Figures

Figure 1

Serum from aHUS patients induces higher-than-normal C5b-9 formation on activated HMEC-1. (A) Distribution of rare complement gene variants among the 60 pedigrees. (B) Upper Panel, Endothelial surface area covered by C5b-9 staining after 2h incubation of ADP-activated HMEC-1 with serum (diluted 1:2 in test medium) from aHUS patients. Serum from aHUS patients was collected during the acute phase of the disease (n=16), or during remission (n=50), or during eculizumab treatment (n=4). Dotted lines are upper and lower limits of normal range. Lower Panel, Endothelial surface area covered by C5b-9 staining after 2h incubation of ADP-activated HMEC-1 with serum (diluted 1:2 in test medium) from aHUS patients. Serum from aHUS patients (n=4) was collected both during the acute phase of the disease and during the remission. Dotted lines are upper and lower limits of normal range. (C) Endothelial surface area covered by C5b-9 staining after 2h incubation of ADP-activated HMEC-1 with serum (diluted 1:2 in test medium) from aHUS patients who were in remission. aHUS patients were subdivided into carriers of pathogenetic variants (PV) in genes encoding fluid phase complement proteins (n=15), or pathogenetic variants (PV) in the MCP gene only (n=5), or VUS/LPV/LBV/BV (n=14) or non-carrier of rare variants (n=16). Dotted lines are upper and lower limits of normal range. The results are shown as the fold increase of stained surface area after incubation with aHUS serum versus control pool of sera run in parallel. Points represent fold increase values of single subjects. Horizontal bars are mean ± SD values. *P<0.01 vs aHUS patients in acute phase, #P<0.05 vs aHUS in remission. PV, pathogenic variant; LPV, likely pathogenic variant; VUS, variant of uncertain significance; LBV/BV, likely benign/benign variant.

Figure 2

Serum from unaffected relative carriers of PV induces higher than normal C5b-9 formation on HMEC-1. (A) Endothelial surface area covered by C5b-9 staining after 2h incubation of ADP-activated HMEC-1 with serum (diluted 1:2 in test medium) from either unaffected carriers of PV in genes encoding for fluid phase complement proteins (n=41), or from unaffected non-carriers of PV (n=28) or from unaffected carriers of PV in the MCP gene only (n=8). The results are shown as the fold increase of stained surface area after incubation with serum from unaffected carriers versus control pool of sera run in parallel. Points represent fold increase values of single subjects. Horizontal bars are mean ± SD values. Dotted lines are upper and lower limits of normal range. *P<0.0001 vs. unaffected carriers of PV. (B) Pedigree #267 of aHUS proband #875 (indicated by the arrow) with a PV in CFH p.S1191L. The same PV was found in his brother (affected, #871), his mother (#1268), his sister (#1269), his uncle (#1271) and his cousin (affected, #872). (C) Pedigree #1245 of aHUS proband #2037 (indicated by the arrow) with a pathogenic CFHR1/CFH hybrid gene (CFHR11-4-CFH22-23). The same hybrid was also found in his brother (#2269) and his nephews (#2444 and #2445). In each pedigree, the affected subjects are in black, healthy carriers of PV are identified with a black dot. The circles indicate females and the squares indicate males.

Figure 3

C5b-9 formation on HMEC-1 exposed to serum from unaffected relative carriers of rare complement gene variants. (A) Endothelial surface area covered by C5b-9 staining after 2h incubation of ADP-activated HMEC-1 with serum (diluted 1:2 in test medium) from carriers of variants likely pathogenic (LPV, n=8), of uncertain significance (VUS, n=16), likely benign (n=1), or benign (n=2). Results are shown as fold increase of stained surface area after incubation with serum from unaffected carriers versus control pool of sera run in parallel. Points represent fold increase values for single subjects. Dotted lines are upper and lower limits of normal range. (B) Breakdown of CADD computational predictions using a default score value cutoff >10 in disease-associated genes. (CADD ≤ 10: benign prediction; CADD > 10: pathogenic prediction) (C) Breakdown of CADD computational predictions using a default score value cutoff >10 in candidate genes. Red columns indicate variants with incorrect prediction; Blue columns indicate variants with correct predictions. (D) Endothelial surface area covered by C5b-9 staining after 2h incubation of ADP-activated HMEC-1 with serum (diluted 1:2 in test medium) from carriers of rare variants that do not segregate with aHUS (n=10). Results are shown as fold increase of stained surface area after incubation with serum from carriers of rare variants not segregating with aHUS versus control pool of sera run in parallel. Points represent fold increase values of single subjects. Dotted lines are upper and lower limits of normal range.

Figure 4

Pedigrees with combined variants. Pedigree #052 of the proband #347. Genetic analysis revealed a pathogenic variant in CFH (p.V1197A) inherited from the father (#387, green) and also a LPV in CFH (p.W978R) inherited from the mother (#388, pink). Pedigree #390 of the aHUS proband #1052. Genetic analysis revealed a pathogenic variant in CFH (c.3493+1G>A) inherited from the mother (#1054, grey) and a rare VUS in CFH (p.N516K) inherited from the father (#1053, red). Pedigree #265 of proband #870 carrying a pathogenic variant in CFH (p.S1191L, light blue) inherited from the mother and also present in the maternal affected relative (#868). Genetic analysis in the proband also found a VUS in CFI (p.E554V, purple) inherited from the mother but absent in the maternal affected relative. Pedigree #1485 of proband #2314. Genetic analysis revealed a VUS in C3 (c.74+1delG) inherited from the father (#2437, blue) and a VUS in CFB (p.N340Y) inherited from the mother (#2438, orange). Arrows indicate probands, affected subjects are in black. Pedigree #2880 of the proband #4050. Genetic analysis revealed a LPV in C3 (p.G321V) inherited from the father (#4051, deep blue) and a likely benign variant in CFH (c.2957-7A>G) inherited from the mother (#4052, yellow). Pedigree #412 of the proband #1070. Genetic analysis revealed a benign variant in CFHR2 (p.T109A) inherited from the father (#2654, purple) and a LPV in CFHR5 (p.E163Kfs10x) inherited from the mother (#2583, brown). Arrows indicate probands, affected subjects are in black. The circles are used for female subjects and squares for male subjects.

Figure 5

C5b-9 formation on activated HMEC-1 induced by serum from aHUS patients without identified mutations and from their parents. (A–C) Endothelial surface area covered by C5b-9 staining after 2h incubation of ADP-activated HMEC-1 with serum (diluted 1:2 in test medium) from aHUS patients without identified mutations (remission, n=16; acute, n=1) and their parents. Dotted lines are upper and lower limits of normal range. Results are shown as fold increase of stained surface area after incubation with serum from aHUS patients and their parents versus control pool of sera run in parallel. Points represent fold increase values of single subjects. Horizontal bars are mean ± SD values. *P<0.001, ***P<0.01, ****P<0.05 vs patient; ##P<0.01, ###P<0.05 vs other parent. (D) Pedigree #1124 of patient #1897 without identified mutations. (E) Pedigree #1998 of patient #3023 without identified mutations. Arrows indicate probands, affected individuals are in black. The circles indicate female subjects and the squares male subjects.