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

Abstract

IntroductionComprehensive 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.MethodsTo 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).ResultsSera 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.DiscussionIn 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.

Ex Vivo Test of Complement Dysregulation in Atypical Hemolytic Uremic Syndrome Kidney Transplant patients: A Pilot Study

“…We have no easy explanation of these findings, which are in contrast to the literature data, suggesting that a positive test on activated endothelium allows to detect patients with a genetic predisposition to aHUS. 14 All but 1 patient had a C3 mutation. Interestingly, the association between a positive test on activated endothelium and carriership of an aHUS genetic mutation might be dependent on polymorphisms.…”

“…Indeed, it was suggested that a positive assay was found in carriers of an MCP mutation, but only in individuals who concomitantly were carriers of at least 1 CFH risk haplotype. 13 , 14 Further studies are needed to assess which factors influence the ex vivo endothelial test results: genetic variants, risk haplotypes, and/or other (yet unknown) factors.…”

“…We hypothesized to find elevated C5b-9 deposition on activated endothelial cells in all our patients with aHUS, because this assay was suggested to identify patients with an underlying complement dysregulation, with abnormal test results also documented in asymptomatic carriers. 14 With respect to C5b-9 deposition on resting endothelial cells, we expected variable results: normal C5b-9 deposition should reflect absence of TMA activity whereas elevated C5b-9 deposition should herald TMA activity. We included a control group of kidney transplant patients, treated with similar immunosuppressive regimen including a calcineurin inhibitor, and a known native kidney disease diagnosis.…”

Response to “A Caution Against the Use of C5B-9 Endothelial Assay to Support Eculizumab Therapy”

“…Our findings are consistent with previous studies reported in the literature. The capability of the ex vivo C5b-9 formation assay to evaluate the presence of factors in serum that augment complement activation on endothelial cells, and to identify patients with TMA caused by complement dysregulation is in fact supported by several publications from different independent groups, including Giuseppe Remuzzi in Bergamo, 2 , 3 S.A.M.E.G. Timmermans in Maastricht 4 , 5 and Marta Palomo in Barcelona.…”

Complement Biosensors Identify a Classical Pathway Stimulus in Complement-Mediated Hemolytic Uremic Syndrome

“…Two principal techniques for in vitro diagnosis have been proposed, including the modified Ham assay (mHam) 9,10 and HMEC-1 staining for C5b-9 [11][12][13][14] , but neither is widely adopted. Both assays rely upon testing patient serum for abnormal cell-deposited complement activity.…”