A Multimodality Approach to Assessing Factor I Genetic Variants in Atypical Hemolytic Uremic Syndrome

Abstract

Atypical hemolytic uremic syndrome (aHUS) is a rare thrombotic microangiopathy (TMA) characterized by the triad of microangiopathic hemolytic anemia, thrombocytopenia, and acute kidney injury (AKI).1Goodship T.H. Cook H.T. Fakhouri F. et al.Atypical hemolytic uremic syndrome and C3 glomerulopathy: conclusions from a “Kidney Disease: Improving Global Outcomes“ (KDIGO) Controversies Conference.Kidney Int. 2017; 91: 539-551Abstract Full Text Full Text PDF PubMed Scopus (276) Google Scholar,S1–S5 The disease features overactivity of the alternative pathway of the complement system, most often arising from loss-of-function mutations in regulators (Factor H, Factor I, and membrane cofactor protein).S4–S7 Less frequently, a gain-of-function mutation in 1 of 2 activators (C3, Factor B) is identified.S8–S10 Approximately 60% of patients with aHUS carry a rare variant in one of the complement proteins. However, <50% of these rare variants have a known functional consequence or one that is readily predicted from the DNA sequence alteration (such as nonsense, splice-site, or frame-shift mutations).1Goodship T.H. Cook H.T. Fakhouri F. et al.Atypical hemolytic uremic syndrome and C3 glomerulopathy: conclusions from a “Kidney Disease: Improving Global Outcomes“ (KDIGO) Controversies Conference.Kidney Int. 2017; 91: 539-551Abstract Full Text Full Text PDF PubMed Scopus (276) Google Scholar,S4 Most of the clinically identified variants have not been functionally characterized. Instead, computational prediction algorithms are utilized to predict the potential impact of these variants on the mature protein, taking into consideration the following: (i) evolutionary conservation of an amino acid or nucleotide; (ii) location and context within the protein sequence; (iii) biochemical consequence of the amino acid substitution, and, in some cases, (iv) topology of a previously solved structural domain or subdomain. Based on these criteria, most variants are reported as variants of uncertain clinical significance (VUSs). The presence of such rare variants is particularly vexing for clinical management. Genetic variants in Factor I (FI) have been reported in 5%–15% of patients with aHUS.2Kavanagh D. Richards A. Noris M. et al.Characterization of mutations in complement factor I (CFI) associated with hemolytic uremic syndrome.Mol Immunol. 2007; 45: 95-105Crossref PubMed Scopus (103) Google Scholar,S4 The likely clinical outcome of patients with an FI mutation is dismal, and the risk of recurrence after kidney transplantation is high.3Le Quintrec M. Zuber J. Moulin B. et al.Complement genes strongly predict recurrence and graft outcome in adult renal transplant recipients with atypical hemolytic and uremic syndrome.Am J Transplantation. 2013; 13: 663-675Crossref PubMed Scopus (169) Google Scholar,S11–S15 FI is a plasma glycoprotein regulator of the alternative pathway of the complement system (Figure 1).4Roversi P. Johnson S. Caesar J.J. et al.Structural basis for complement factor I control and its disease-associated sequence polymorphisms.Proc Natl Acad Sci U S A. 2011; 108: 12839-12844Crossref PubMed Scopus (81) Google Scholar,S16–S18 It is a 2-chain 88-kDa serine protease synthesized predominantly by the liver. FI regulates complement activation by inactivating C3b and C4b through proteolytic cleavage (i.e., cofactor activity) in the presence of one of its “cofactor proteins”—Factor H (FH), membrane cofactor protein (MCP; CD46), C4b-binding protein (C4BP), or complement receptor 1 (CR1; CD35). Our study hypothesized that the clinical presentation and pathology of aHUS can be better understood and treated by examining critical clinical information, and by profiling the functional repertoire along with structurally modeling genetic variants. This approach integrates information derived from several types of analyses in order to create a more informed model. The strategy described in this report thus provides a model for how to characterize these rare variants clinically based upon functional evidence, which is one of the interpretive categories of the American College of Medical Genetics (ACMG)5Richards S. Aziz N. Bale S. et al.Standards and guidelines for the interpretation of sequence variants: a joint consensus recommendation of the American College of Medical Genetics and Genomics and the Association for Molecular Pathology.Genet Med. 2015; 17: 405-424Abstract Full Text Full Text PDF PubMed Scopus (10876) Google Scholar guidelines for variant interpretations related to pathogenicity. This strategy may be useful for guiding more-appropriate medical therapeutic decisions and generating further insights relative to the pathogenic mechanisms underlying aHUS. This report describes 5 patients in whom aHUS was considered as a possible diagnosis. Genetic testing revealed a variant in FI (Table 1). These patients were referred to us for assistance in clinical decision-making relative to therapeutic options for renal transplant and/or targeted therapy using a humanized monoclonal antibody that blocks C5 activity (eculizumab).6Nurnberger J. Philipp T. Witzke O. et al.Eculizumab for atypical hemolytic-uremic syndrome.N Engl J Med. 2009; 360: 542-544Crossref PubMed Scopus (290) Google Scholar,S19–S21 In order to obtain a more comprehensive assessment of each patient’s risk, we wanted to evaluate their clinical history and genetic data in relation to a functional assessment of each variant. Our hypothesis was that the additional results would provide critical information for preparing a more rational assessment for individualized therapeutic decisions for each patient. Therefore, we produced recombinant proteins, assessed regulatory function (cofactor activity), and performed structural modeling of each variant protein (see Supplementary Methods, STROBE Statement, and Figure 27Forneris F. Wu J. Xue X. et al.Regulators of complement activity mediate inhibitory mechanisms through a common C3b-binding mode.EMBO J. 2016; 35: 1133-1149Crossref PubMed Scopus (55) Google Scholar).Table 1Summary of clinical data of patients with Factor I variantsPatientVariantAge/sexAge of onsetOutcomesTransplantTreatmentsaHUS-risk SNPsAdditional serum complement levels1R406H22 yr/M3 yrESRDTwo failed transplantsEculizumab x 1 year after second transplantNone reportedC3:72 mg/dlC4: 15 mg/dl2I306V17 yr/F17 yrCRNonePlasmapheresis, eculizumab, Cellcept, and PrednisoneFH: V62I, H402YFI: T300AFB: R32Q, R32WC3: 45 mg/dlC4: 6 mg/dl3P553S20 yr/F19 yrESRDAwaiting transplantSupportive treatmentFH: V62I, H402YFI: T300AC3: R102G, P314LC3: 103 mg/dlC4: 24.9 mg/dl4D403N57 yr/M57 yrCRNoneEculizumab x 6 yearsFH: H402YFI: T300AC3: R102G, P314LFB: G252SC3: 95 mg/dlC4: 34 mg/dlCH50: 50 U/ml5I370N24 yr/M19 yrCKDNoneSupportive treatmentFH: V62I, H402Y, E936DFI: T300AC3: R102G, P314LNot availableaHUS, atypical hemolytic uremic syndrome; CKD, chronic kidney disease; CR, complete remission; ESRD, end-stage renal disease; F, female; FB, Factor B; FH, Factor H; FI, Factor I; M, male; SNP, single-nucleotide polymorphisms.For patients 1, 2, and 3, the reference range for C3 is 82–167 mg/dl; the reference range for C4 is 10–40 mg/dl. For patient 4, the reference range for C3 is 98–201 mg/dl; the reference range for C4 is 18–56 mg/dl; and the reference range for CH50 is 30–75 U/ml. Open table in a new tab aHUS, atypical hemolytic uremic syndrome; CKD, chronic kidney disease; CR, complete remission; ESRD, end-stage renal disease; F, female; FB, Factor B; FH, Factor H; FI, Factor I; M, male; SNP, single-nucleotide polymorphisms. For patients 1, 2, and 3, the reference range for C3 is 82–167 mg/dl; the reference range for C4 is 10–40 mg/dl. For patient 4, the reference range for C3 is 98–201 mg/dl; the reference range for C4 is 18–56 mg/dl; and the reference range for CH50 is 30–75 U/ml. This 22-year-old Caucasian male developed end-stage renal disease at the age of 3 years, thought to be secondary to Escherichia coli–triggered HUS. In 2006, he underwent a renal transplant at age 11 years. In 2007, he developed renal failure due to Banff 1B acute cellular rejection and returned to dialysis in 2009. He was evaluated for a second renal transplant in 2014. Given his history of HUS, genetic testing was performed, which showed a missense variant (p.R406H) in FI. The patient received eculizumab for 1 year after his second transplant in May 2015. In April 2017, an allograft biopsy for worsening renal function again revealed Banff 1B acute cellular rejection, in addition to antibody-mediated rejection. As of this writing, he is undergoing evaluation for a third renal transplant. The patient’s heterozygous variant is within the serine protease domain and occurs at an amino acid position that is not conserved. This variant has been reported in patients with age-related macular degeneration,S22 and at the time the variant was identified in this patient, the frequency in the general population was reported at 0.12%. Due to the availability of larger cohorts in gnomAD, this variant is now reported to occur at an overall frequency of 1.6% (minor allele frequency: Asians, 2.4%–4.3%; Europeans, 0.1%–2.4%; Africans and Latinos, <1%). Several in silico models (SIFT, PolyPhen2, Mutation Assessor, Condel) predict this variant to be benign; taken together, the clinical interpretation reported for this variant was “likely benign.” The secretion of the recombinantly produced variant protein in the supernatant (determined by enzyme-linked immunosorbent assay) was comparable to wild type (WT, 2.3 ± 0.75 μg/ml; R406H, 2.1 ± 0.97 μg/ml). The serum FI antigenic level in this patient was normal: 31 μg/ml (reference range for the outside laboratory: 24–49 μg/ml). This FI variant had defective proteolytic activity with FH and CR1 as cofactor proteins, as evidenced by a decreased rate of cleavage of the αʹ chain of C3b and thereby reduced generation of the α1 fragment compared to WT (Figure 3). P values for the difference in functional activity between the WT and variant were statistically significant. No defect was observed with MCP as the cofactor protein. The side chain of R406 in FI forms a salt bridge with the carboxyl group of E123 of FH7Forneris F. Wu J. Xue X. et al.Regulators of complement activity mediate inhibitory mechanisms through a common C3b-binding mode.EMBO J. 2016; 35: 1133-1149Crossref PubMed Scopus (55) Google Scholar,S23 (Figure 2). This interaction stabilizes the FI–FH binding interface. Residue E123 is a unique signature of FH and is lacking in MCP, possibly accounting for the fact that the R406H mutation impaired FH- but not MCP-mediated cofactor activity. The effect of the mutation on CR1 cofactor activity is more difficult to interpret because residue E123 is also absent in CR1, being replaced by a histidine. However, given that a histidine residue can be protonated under physiological values of pH and ionic strength conditions,S24 a histidine–arginine like-charged ion pair is expected to occur at the FI–CR1 binding interface. Although this variant was synthesized normally, it had a reduced rate of C3b cleavage by FH and CR1 compared to WT FI. This variant is therefore “functionally impaired.” However, taken together with the frequency of the p.R406H variant, which is more common than would be expected based on the prevalence of aHUS, lack of consistency between in silico tools and now our data supporting pathogenicity, this variant is being reclassified as a VUS according to ACMG guidelines. Of note, although glomerular endothelial cellsS25 do not express CR1 in the resting state, there is evidence that hypoxia or inflammatory mediators may induce the expression of this CR1 on endothelial cells. This phenomenon could therefore be of significance during disease states such as aHUS. We also know that aHUS is a disease with incomplete penetrance and that the penetrance is age-related, being reported as being as high as 65% by age 70 years for individuals carrying a single genetic mutation.S26 Therefore, although this patient did not develop TMA, given his clinical course with end-stage renal disease secondary to HUS, 2 allograft rejections, and our newly derived data, we believe that the patient is at risk of developing a TMA and/or an accelerated antibody-mediated rejection. Although future studies will affect the decision on how long to continue treatment, we would recommend that he receive eculizumab at the time of his third transplant and remain on it indefinitely. Additionally, we would advise that his family members undergo genetic testing. This American with and in along with of and evaluation revealed anemia, and of was treated with for possible and year with and of due to secondary to a the 3 developed worsening anemia, thrombocytopenia, and a of thrombotic was and with and eculizumab was results for biopsy a Genetic identified a missense variant in FI. had complete renal and after 6 was and was treated with This heterozygous variant is in the serine domain of FI. It occurs at an amino acid position that is not and has a allele frequency of within the American In silico tools predict a effect on the protein. This variant has been identified in a patient in the of TMA kidney Quintrec M. A. N. et al.Complement thrombotic microangiopathy kidney J PubMed Scopus Google Scholar the basis of these the variant is as a The secretion of the recombinantly produced variant protein in the supernatant (determined by enzyme-linked immunosorbent assay) was comparable to WT (WT, ± μg/ml; ± μg/ml). The serum FI antigenic level in our patient is not However, the serum FI level in the patient previously reported in the was normal: μg/ml (reference range for the outside laboratory: μg/ml). The variant had a of activity with 3 MCP, and (Figure P values for the difference in of the αʹ chain of C3b and for the generation of the and between WT and variant were not statistically significant. In the of is and from the and FI–FH binding is not in (Figure 2). to is a by the protein these structural the mutation is expected to have on cofactor activity. This variant is of functional based on expression and cofactor activity. Given our in silico tools that a effect of this variant on FI, the frequency with this variant would be reclassified as likely ACMG guidelines. The patient on and We believe that the TMA was most likely secondary to or and was not related to the variant in FI. This Caucasian developed during second and underwent a The course was by the of hemolytic thrombocytopenia, and the but kidney function did not and to end-stage renal of aHUS was year was referred to us for evaluation for a kidney transplant. In order to a more of the kidney failure to the patient had aHUS and was a likely second and to further risk of recurrence after genetic testing was missense variant in FI was This heterozygous variant is in the serine protease It has been described in age-related macular and is more common in than patients (minor allele frequency of The variant has also been reported in a patient with end-stage renal disease from F. et in of complement the outcome of Factor atypical hemolytic uremic Int. Full Text Full Text PDF PubMed Scopus Google that patient variants in FH and in MCP The variant is predicted to be “likely by the computational The secretion of the recombinant variant protein in the supernatant (determined by enzyme-linked immunosorbent assay) was comparable to WT (WT, ± μg/ml; 2.1 ± μg/ml). The serum FI antigenic level in this patient was at 34 μg/ml (reference 24–49 μg/ml). This variant had activity with FH, MCP, and CR1 as the cofactor protein (Figure was statistically difference in the cleavage of the αʹ chain or in the of the and compared with is from the triad and is of the to the is of the the binding (Figure 2). This variant is of functional based on expression and proteolytic activity. the of these ACMG now this variant as Although FI mutations have been identified in patients with and evidence from has that of complement activation is in the of this not to be for this patient. In the previously reported of aHUS with this variant, the patient also the pathogenic in FH and in F. et in of complement the outcome of Factor atypical hemolytic uremic Int. Full Text Full Text PDF PubMed Scopus Google Scholar The of and was likely in of disease in the previously reported patient. In our patient, it is that is of the renal This Caucasian male with and for The patient was to be in secondary to a he a critical characterized by hemolytic anemia, and of thrombotic was and he was treated with and eculizumab, with complete and renal the 6 results for level Genetic testing revealed a rare variant in FI. He on eculizumab the 6 This heterozygous variant is in the serine protease domain and is in population at allele in and in the In silico models predict a effect on protein This variant was also identified in 1 of patients with aHUS in a and was absent from F. et in of complement the outcome of Factor atypical hemolytic uremic Int. Full Text Full Text PDF PubMed Scopus Google Scholar Based on the evidence this variant is as a The secretion of the variant in the supernatant (determined by enzyme-linked immunosorbent assay) was comparable to WT (WT, ± μg/ml; ± 0.97 μg/ml). Factor I antigenic level in our patient was at μg/ml μg/ml). The variant had activity with the 3 (Figure and Supplementary Figure to there was statistically difference in cleavage of the αʹ chain of C3b or in the of the or is 3 amino of R406 Figure 2). the side chain of not with The mutation a with an amino which the This expected to have impact on the structural of the and thus on cofactor activity. This variant is of functional based on expression and proteolytic activity with 3 cofactor and a FI antigenic Although this variant would be expected to be as this variant a VUS due to its in the general The patient has been on eculizumab for 6 years. Although of a variant as VUS is not for in clinical given these new functional data, a of eculizumab may be along with and This Caucasian male underwent a transplant at the age of years for He developed at the age of years. values and presence of on were for aHUS, but a was not the course of his his level did not and he Genetic testing revealed the presence of a variant in FI. This heterozygous missense variant is in the serine protease domain and occurs at an amino acid position that is conserved. In silico algorithms predict this to be to protein The variant is not reported in population variant the basis of this evidence, this variant was characterized as “likely This variant was synthesized but was not as from the of the and the (Figure The serum FI antigenic level in this patient was not is into a (Figure by and of a amino acid such as an is expected to the of this thereby to protein and Based on these functional studies defective secretion and supporting information, this variant is reclassified as pathogenic The patient is at high risk of aHUS. Of is that the patient did not develop aHUS at the time of the which would be considered a but developed the disease at a age a This patient be treated with this patient has been to in genetic testing have to the data from DNA However, functional interpretation of such analyses to be a As a the clinical significance of genetic variants and a of clinical studies be the data be to patients and how the patients be In an to such the ACMG has guidelines for S. Aziz N. Bale S. et al.Standards and guidelines for the interpretation of sequence variants: a joint consensus recommendation of the American College of Medical Genetics and Genomics and the Association for Molecular Pathology.Genet Med. 2015; 17: 405-424Abstract Full Text Full Text PDF PubMed Scopus (10876) Google Scholar The variant guidelines that variant of uncertain significance not be in clinical decision to the of the variant as pathogenic or be through further to further this is to family members to variants are by and In our cases, the family were for a this approach can be time and and it on family and Additionally, to for this testing. approach is to model the genetic variant using protein and analyses to more the clinical of each genetic variant. we performed analyses of the 5 variants in FI identified in aHUS were 2 were likely benign; and 1 was likely these cases, we functional and structural data on each variant. The results were utilized to the variants the ACMG guidelines (Table Quintrec M. A. N. et al.Complement thrombotic microangiopathy kidney J PubMed Scopus Google Scholar the clinical interpretation for of the 5 of genetic and functional analyses of Factor I variants in ACMG ACMG in the one reported in Quintrec et not American College of Medical aHUS, atypical hemolytic uremic syndrome; cofactor CR1, complement receptor of single-nucleotide FH, Factor H; likely benign; likely MCP, membrane cofactor not not serine variant of uncertain in the one reported in Quintrec et Quintrec M. A. N. et al.Complement thrombotic microangiopathy kidney J PubMed Scopus Google Scholar Open table in a new tab American College of Medical aHUS, atypical hemolytic uremic syndrome; cofactor CR1, complement receptor of single-nucleotide FH, Factor H; likely benign; likely MCP, membrane cofactor not not serine variant of uncertain to our analyses are as (i) penetrance of a variant can the ACMG For our functional data functional the interpretation for a VUS due to the of the variant. the effect of this variant on the be (ii) could also the cofactor for C4b C4b-binding protein. However, than the it is the alternative pathway that aHUS. (iii) The relative of the functional of FI the level of the rare variant are (iv) is to the outcome of the clinical course for each patient. Given that these functional studies were not in a College of American and of these to clinical decision-making will in developing our results a useful model (Figure how a and can provide more of the significance of genetic variants in aHUS. studies of complement for activation could information to therapeutic a model provides a to further an individualized as as the risk of recurrence after a kidney transplant. In given the to the of genetic data with clinical of aHUS, we believe our strategy of recombinant protein by functional and structural is The to more the functional repertoire of the variant protein provides critical relative to the therapeutic and thereby will the for in renal the The and for their and of of for their during and S. and of at the of for their assistance in further on the clinical history for 2, 4, and This study was by the and Genetic to the of Genetics and Genomics This was in the at in and the was in Molecular and and the data, and results of the and performed the structural performed the and ACMG and the and and the with Supplementary with Figure evaluation of with MCP using salt with Supplementary with STROBE