Defining the genetics of thrombotic microangiopathies

Accepted Manuscript Title: Defining the genetics of thrombotic microangiopathies Author: Paula Vieira-Martins, Carine El Sissy, Pauline Bordereau, Aurelia Gruber, Jeremie Rosain, Veronique Fremeaux-Bacchi PII: DOI: Reference:

S1473-0502(16)30013-1 http://dx.doi.org/doi: 10.1016/j.transci.2016.04.011 TRASCI 1991

To appear in:

Transfusion and Apheresis Science

Please cite this article as: Paula Vieira-Martins, Carine El Sissy, Pauline Bordereau, Aurelia Gruber, Jeremie Rosain, Veronique Fremeaux-Bacchi, Defining the genetics of thrombotic microangiopathies, Transfusion and Apheresis Science (2016), http://dx.doi.org/doi: 10.1016/j.transci.2016.04.011. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

1

Defining the Genetics of Thrombotic Microangiopathies Paula Vieira-Martins*, Carine El Sissy*, Pauline Bordereau*, Aurelia Gruber*, Jeremie Rosain* and Veronique Fremeaux-Bacchi *, # *

Assistance Publique – Hopitaux de Paris, Service d’Immunologie Biologique, Hôpital

Européen Georges Pompidou, Paris, France #

INSERM UMRS 1138, Cordeliers Research Center, Paris, France

Corresponding author: Dr. V. Frémeaux-Bacchi Service d’Immunologie Biologique, Hôpital Européen Georges Pompidou, 20-40 rue Leblanc, 75908 Paris cedex 15, France. Phone: 33-1-56-09-39-41 / Fax: 33-1-56-09-20-80 E mail: [email protected]

Running title: Genetics of TMA Abstract word count: 217 Text word count: 3792

Abstract The spectrum of the thrombotic microangiopathies (TMA) encompasses a heterogeneous group of disorders with hereditary and acquired forms. Endothelial cell injury in the microvasculature is common to all TMAs, whatever the pathophysiological process. In this review we describe genetic mutations characteristic of certain TMAs and review their contributions to disease. Recent identification of novel pathologic mutations has been enabled by exome studies. The monogenic forms of TMA are more frequently caused by recessive alterations in von Willebrand factor cleaving protease ADAMST13, leading to congenital thrombotic thrombocytopenic purpura, or cobalamine C and DGKE genes, leading to an atypical hemolytic-uremic syndrome (aHUS)-like TMA. aHUS, whether idiopathic or linked to a known complement amplifying condition, is a TMA that primarily affects kidney function. It often results from a combination of an underlying genetic susceptibility with environmental factors activating the alternative complement pathway. Pathogenic variants in at least five complement genes coding for complement factor H (CFH) complement factor I (CFI),

Page 1 of 19

2

MCP (CD46), C3 and complement factor B (CFB) have been demonstrated to increase the risk of developing aHUS, but several more genes have been implicated. A new challenge is to separate disease-associated genetic variants from the broader background of variants or polymorphisms present in all human genomes that are rare, potentially functional, but may or may not be pathogenic. Keywords : complement, variant, Thrombotic microangiopathy

Introduction The term thrombotic microangiopathy (TMA) refers to the pathological features resulting from microvascular endothelial cell injury with resultant thrombocytopenia, hemolytic anemia, and thrombosis with tissue ischemia [1]. The pathophysiology of the TMAs is complex. It includes hemolytic uremic syndrome (HUS) associated with shiga toxin producing E. coli (STEC) or invasive pneumococcal infections, atypical HUS (aHUS), and thrombotic thrombocytopenic purpura (TTP), as well as secondary forms of aHUS which may be linked to a variety of complement amplifying conditions (other infections, cancer, drugs, autoimmune disease, pregnancy, organ and tissue transplantation) [2-5]. Congenital mutations leading to a TMA can be caused by pathogenic variants in one gene (monogenic disorder) or by a combination of inherited variants in multiple genes, often acting in concert with environmental factors [6]. Of particular interest to this special topic issue is the subgroup commonly referred to as aHUS, caused by genetic abnormalities of regulation of the alternative pathway of complement [7-8]. Here we summarize the contributions of novel and rare gene variants to the pathology of TMA, with a focus on aHUS.

A. Monogenic inheritance of a thrombotic microangiopathy (TMA) Monogenic diseases are caused by alterations in a single gene. If characterized by complete penetrance they segregate in families according to traditional Mendelian patterns of inheritance. 1) Autosomal recessive TTP and biallelic pathogenic variants in ADAMTS 13 (reviewed in [9]) Upshaw–Schulman syndrome is the recessively inherited form of TTP, caused by the absence of the von Willebrand cleaving protease ADAMTS13, resulting in the persistence of ultra-large von Willebrand factor multimers (ULVWF). These patients are extremely rare, constituting less than 5% of all TTP cases. More than 130 distinct ADAMTS13 mutations have been found in a homozygous or compound heterozygous

Page 2 of 19

3

state. Nearly 60% are missense variants. Patients with Upshaw-Schulman syndrome respond to periodic fresh frozen plasma (FFP) infusions and do not require plasma exchange (PEx) or immune suppressive therapies as in acquired TTP. 2) Cobalamin C defect (cblC)-associated HUS Methylmalonic aciduria with homocystinuria is the most common inborn error of vitamin B12 metabolism. It is caused by mutations in the MMACHC gene [10]. This disorder of cobalamin metabolism is characterized by elevated levels of plama homocysteine and plasma and urine levels of methylmalonic acid. To date, around 70 different mutations have been identified; duplication of an A at the C271 position (c.271dupA or p.R91KfsX14) is the most frequent reported mutation [11-13]. HUS is a rare but well-described complication of a Cblc defect, although its mechanism remains unclear. Clinical onset usually occurs during infancy but recently cases of late–onset disease have been reported [14-17]. Supplementation with hydroxocobalamin and betaine is the main therapy.

3) DGKe deficiency-associated HUS Recessive mutations in the gene coding for Diacylglycerol Kinase Epsilon (DGKE) were established as a novel cause of pediatric-onset aHUS [18]. The DGKE gene encodes diacylglycerol kinase-epsilon, an intracellular lipid kinase that phosphorylates diacylglycerol (DAG) to phosphatidic acid. Loss of DGKE in endothelial cells induces cell death, impairs angiogenic responses, and leads to an activated and prothrombotic phenotype [19]. Fourteen disease causing nucleotide changes have been identified, including one located in the intronic region [18, 20-21]. One recurrent nonsense variant (p.Trp322*) was previously seen among 8,475 subjects of European descent, and in several unrelated aHUS subjects (homozygous and heterozygous traits). Affected individuals present with aHUS before one year of age, have persistent hypertension, hematuria and proteinuria (sometimes in the nephrotic range), and develop chronic kidney disease.

4) aHUS with bi-allelic pathogenic variants in complement genes aHUS can be classified as sporadic or familial. Familial aHUS is defined as the presence of aHUS in at least two members of the same family. Approximately 50% of familial forms have an autosomal recessive pattern of disease inheritance.

Page 3 of 19

4

CFH deficiency-associated HUS CFH is the most important negative regulator of the alternative complement pathway [22]. The CFH gene is approximately 100kb long and comprises 23 exons. The association between a clinical diagnosis of aHUS and low plasma C3 was first reported in pediatric patients more than 25 years ago [23]. A few years later, CFH deficiencies were documented in recessive forms of pediatric onset aHUS [reviewed in 1]. The homozygous deletion of 4 nucleotides located at the end of CFH, leading to the deletion of the stop codon (c.3693delATAG), accounts for more than 50% of reported cases [8, 24-25]. One case of complete CFH deficiency was explained by a chromosome 1 uniparental isodisomy [26]. The penetrance of CFH-linked disease is nearly complete as all patients were diagnosed with aHUS or C3 glomerulopathy [27].

Complete MCP deficiency-associated HUS Membrane cofactor protein (MCP; CD46) protects endothelial cells from injury by complement. The gene for CD46 is ~43 kb and contains 14 exons. Six cases of complete MCP deficiency have been identified in the French cohort, corresponding to 37% of MCP associated aHUS in the French registry [8]. The penetrance of the disease is nearly complete. Among reported patients with a lack of or dramatically decreased cell surface expression of CD46, few disease-free family members have been identified [28-29].

B-Polygenic inheritance of a TMA Most frequently the disease is sporadic (one case per family) despite the identification of the same pathogenic variant in one of the healthy parent. These findings strongly suggest that the variant predisposes to rather than causes the disease. The reasons underlying incomplete penetrance are unclear, although it is established that common variants in complement genes are true risk factors for development of aHUS, but perhaps with weaker clinical and pathologic features than cases associated with established pathogenic variants [30].

1) Novel or rare variants identified in complement genes associated with incomplete penetrance (review in [31]) Heterozygous loss-of-function variant in CFH

Page 4 of 19

5

CFH is a soluble regulator of the alternative pathway which inhibits C3 convertase activity via three mechanisms: by promoting degradation of its substrate C3b; by preventing the association between C3b and CFB; and by accelerating the dissociation of C3bBb complexes [22]. CFH consists of 20 consecutive short consensus repeats (CCPs) and harbors specific C3b and GAGs binding sites. CCP numbers 1-4 of CFH bind C3b and display decay accelerating factor of the AP C3 convertase by dissociating Bb from the enzyme. CCP #19-20 are crucial to differentiate self from non self [32]. Domain mapping experiments reveal that CFH CCP#20 binds specific structures present on host surfaces while CFH CCP#19 interacts with C3b. During the last 15 years, quantitative and functional CFH deficiencies have been reported in both sporadic and familial aHUS patients [8, 33]. As noted above, CFH is the most frequently mutated gene in aHUS, accounting for 20-30% of patients [8, 34-36]. Thus far, more than 150 heterozygous CFH variants have been described, including missense, nonsense, splice site and frame shift mutations [31]. Pathogenic variants are not fully penetrant. For example, the penetrance of the disease linked to the CFH mutation (p. Arg1215Gly) ranges at the age of 70 years from 0.44 to 0.64. In addition, the CFH haplotype on the allele not carrying the CFH mutation can have a significant effect on disease penetrance [40]. Two types of CFH mutations are distinguished on the basis of the mechanisms leading to decreased CFH activity [37]. Type I mutations result in very low plasma CFH levels. Nucleotide changes leading to quantitative deficiency are distributed throughout the entire CFH coding gene. In contrast, type II mutations hamper CFH function without altering its production or secretion. The nucleotide changes are more frequently located in exons coding for the CCP 19 and 20 [39]. Diagnosis of quantitative CFH deficiency requires measurements of CFH protein in plasma. Low levels, often accompanied by low C3 levels, have been reported in 50% of patients with variants in CFH [8]. For example, a missense variant at conserved cysteine residues impairs CFH secretion [39]. The functional defects caused by CFH mutations include impaired binding to C3b and abnormal interactions with endothelial cells. A specific test available in specialized laboratories, the sheep erythrocyte lysis assay, is helpful to document impaired cell-surface complement regulation [37]. CFH is in close proximity to genes CFHR1 through CFHR5 which encode the five CFH-related (CFHR) proteins [41]. The high degree of sequence homology that exists between CFH and CFHR genes results in deletions and substitutions within CFH

Page 5 of 19

6

through non-allelic homologous recombination. The resulting hybrid proteins are often poorly functional and may affect the regulatory role of the native CFH protein. The age of onset of aHUS in patients with identified CFH mutations is either early, before the age of 4, or between the ages of 20 to 40. CFH-associated aHUS cases have the highest risk of developing end-stage renal disease, usually within a few years of diagnosis. Renal transplantation in aHUS patients with CFH mutations is associated with high disease recurrence rates, and poor allograft survival [8].

Loss of function variant in CFI gene CFI plays a central role in the negative regulation of the complement cascade [22]. The CFI gene spans 63kb and contains 13 exons. The last 5 exons encode for the serine protease domain responsible for cleaving and inactivating C3b. CFI proteolytic activity requires interactions with various cofactors, such as CFH, complement receptor 1 (CR1, CD35), and MCP. More than 50 variants in CFI have been published and are found in 4-8% of patients with aHUS [8, 34-36]. For most CFI variants that have been tested experimentally, CFI dysfunction is either due to defective secretion or reduced degradation of the degradation of C3b to iC3b, and low serum C3 levels in patient plasmas are documented [42]. Despite extensive in vitro studies, some CFI variants had no effect on CFI serum levels and on its function. Therefore the link between the disease and these variants are controversial. Recently Kavanagh et al. identified 231 individuals with rare genetic variants in CFI gene among 3666 individuals with age-related macular degeneration (AMD) [43]. Twenty-one distinct genetic variants were associated with low CFI plasma levels. Of these variants, 10 have been seen in aHUS, also associated with low levels. Half of these variants have been also identified in healthy donors, with a minor allele frequency <1%. For example, the variant p.G119R has been identified in 1 of 3,937 controls [44] and in 5 out the 500 patients with aHUS in the French registry (unpublished data). Therefore, the frequency is significantly higher in patients than in controls and this rare variant is a risk factor for aHUS. Only 10% of patients with mutations in CFH had combined mutations, whereas approximately 25% of patients with mutations in CFI had combined mutations [36]. While 30% of aHUS patients with CFI mutations are in complete remission shortly after the first episode, left untreated the 5-year prognosis is poor, with a ~50% risk of end-stage renal disease [8].

Page 6 of 19

7

Heterozygous loss of function variant in MCP gene MCP (CD46) is a type 1 transmembrane glycoprotein that possesses four extracellular SCR domains and binds C3b mainly via SCRs 3, and 4. MCP was considered an aHUS candidate gene because it lies within the Regulator of Complement Activation (RCA) gene cluster on chromosome 1q32 and regulates the alternative pathway of complement on the surface of host cells. Overall, more than 50 pathogenic variants in MCP have been identified in aHUS patients [45].

They are usually

associated with a 50% reduction of MCP levels at the membrane. Less frequently, missense substitutions (p.S240P and p.R103W) result in normal MCP cell surface expression, but deficient activity against surface bound C3b. Despite 10% of the familial forms of aHUS having

a heterozygous mutation in MCP,

a second

complement-related genetic factor along with MCP is found in approximately 20% of patients[36]. MCP pathogenic variants are more frequent in children than in adults (13.5 % versus 6.4% in the French cohort) and are estimated to account for 10-15% of aHUS patients [8, 34-36].

Gain of function variant in CFB Gain-of-function mutations in CFB are present in aHUS, but are extremely rare [8, 34-36]. A few CFB mutations can super-activate complement, all located within the C3b binding site [46]. Eight newly characterized CFB variants identified in aHUS patients are most likely benign variants rather than disease-associated mutations [4647]. However, untreated patients who carry a true pathogenic CFB variant usually have a severe disease outcome, with ESRD in the majority of the cases.

Gain of function variant in C3 Four aHUS cohorts (French, Italian, UK and USA) were screened for genetic complement abnormalities and, together with cases published in the literature, gave a total of 48 different genetic changes in C3 that have been associated with aHUS in 130 patients [48]. The C3 mutation frequency is between 5 to 15% in reported series [8, 3436]. aHUS-related C3 mutations are not randomly distributed across the protein but rather clustered on the surface predominantly, around CFH binding sites. Functional

Page 7 of 19

8

analyses have revealed that most C3 mutations result in an "indirect" gain-of-function phenotype, because of a reduced ability for mutant C3 proteins to bind its negative regulator MCP or CFH. For example, the pathogenic variant p.R592Q reduces the binding of C3. p.R161W and p.V1658A are the exceptions to this rule; they represent a direct gain-of-function. They have an increased binding affinity to its co-factor FB, thus forming hyperactive C3bBb complexes. Seventy-six percent of patients with C3 mutations had low C3 levels. Fifteen of the 42 mutations were recurrent, identified in ≥2 unrelated aHUS patients from the same or different cohorts. K155 is on the surface of C3, close to the binding site for CFH, which is a cofactor for CFI-mediated cleavage of C3. The rare variant p.K155Q primarily impairs C3b inactivation by CFI with bound CFH, resulting in increased C3 convertase formation and feedback amplification of the alternative pathway. This rare variant, which is classified as a risk factor for AMD, was identified in 37 of 4,263 controls [50] and in 6 out the 500 cases in the French registry (unpublished). This is highly suggestive for pathogenicity of this variant.

In conclusion, 60% of patients with aHUS and no coexisting disease carry a pathogenic variant in one of five complement genes, documenting that this TMA is mediated by complement dysregulation. Recently, new complex rearrangements between CFHR1 and CFH leading to hybrid CFHR1/CFH gene have been reported [51]. The influence of CFHRs genes in particular CFHR5 or clusterin requires further analysis.

2) Common polygenic variation enhances risk for aHUS Evidence from familial studies indicates a high rate of incomplete penetrance, with about 50% of carriers of aHUS-associated variants not developing disease [52]. Complement haplotypes comprising multiple single nucleotide polymorphisms (SNPs) in CFH and in MCP, and common variants in CFH, MCP and CFHR1 genes, with a minor allele frequency >10%, confer a 2-4-fold increase risk of disease compared with controls [53, 8] .

3) Novel or rare pathogenic variants identified in the coagulation pathway in aHUS Genes in the coagulation pathway may also be important in the pathogenesis of aHUS.

Loss of function variant in thrombomodulin (THBD)

Page 8 of 19

9

THBD is a membrane-bound glycoprotein that is expressed in many cell types from a variety of tissues. It enhances CFI-mediated inactivation of C3b in the presence of CFH [54]. Two novel and 6 rare variants less effective in enhancing CFI-mediated inactivation of C3b were identified in the Italian aHUS registry (n=152) and in the US cohort (n=144) [54-55] . No additional aHUS patients with THBD mutations were uncovered when the French cohort was surveyed (n=214) [8]. Despite the fact that in vitro testing suggests that rare variant affects THBD function, their role in aHUS remains unclear.

Loss of function variant in plasminogen A genomic screen of the complement and coagulation pathways was performed in 36 patients with aHUS [55]. The gene coding for plasminogen (PLG) carried more pathogenic variants than any other coagulation gene, including three known plasminogen deficiency mutations and a predicted pathogenic variant. This association needs to be confirmed.

C- Genetic susceptibility for TMA forms associated with auto-antibodies

Anti-CFH antibodies and CFHR3-CFHR1 deletion in aHUS Functional studies have shown that anti-CFH autoantibodies perturb CFHmediated cytoprotective properties, inhibit interactions between CFH and C3, and enhance C3 consumption [56]. Approximately 90% of patients with anti-CFH associated aHUS share an 84kb homozygous deletion near the CFH gene. This structural variation, which includes the genes for CFHR3 and CFHR1, is found in 2-8% of European and Indian controls [57-58]. The association of CFHR1 deficiency with “autoimmune aHUS” could be due to the structural difference between CFHR1 and the auto-antigenic CFH epitope [59].

ADAMST 13 autoantibodies and HLA in acquired TTP Anti-ADAMTS13 autoantibodies, predominantly of the IgG class, are found in plasma of patients with acquired TTP. Several studied observed that HLA-DRB1*11 is the first susceptibility factor in acquired idiopathic TTP in Caucasians [60].

How should one interpret and classify genetic variants potentially linked to aHUS

Page 9 of 19

10

in 2016 ? Recent guidelines for the description of gene sequence variants recommend replacing the terms “mutation” and “polymorphism or SNP” by the term “variant,” modified with a description based on functional significance (i.e., probably affects function, unknown, probably does not affect function, or does not affect function; http://www.hgvs.org/mutnomen/recs.html). Most variants for which a functional effect is unknown are classified "variants of unknown significance" (VUS). The description of the genetic variation needs to follow the recommendations of the American College of Medical Genetics and Genomics (numbering with the signal peptide and including all exons of the protein). The distribution of rare missense variants identified in a cohort of approximately 5000 healthy donors in CFH, CFI, MCP, C3 and CFB, and the novel variants identified in the 500 aHUS patients from the French aHUS registry, are depicted in Figure 1. This figure illustrates the challenge of conclusively demonstrating disease association, especially due to a high number of rare genetic changes in the normal population. However, six important hallmarks for variant characterization in patients with aHUS can be highlighted: 1.Population minor allele frequency (MAF) Public Databases (http://exac.broadinstitute.org/ ; http://evs.gs.washington.edu/EVS/) ; (http://www.1000genomes.org/1000-genomes-browsers) describe variants identified in a population of unrelated individuals (up to 70,000 individuals). However, population databases cannot be assumed to include only healthy individuals. Thus some very rare variants in healthy controls can be pathogenic. 2. Cosegregation of variant and disorder within families The genetic architecture of human disease includes a spectrum ranging from rare monogenic variants with very strong effects to common variants with small effects on disease phenotype. For variants strongly linked to the disease (e.g., a novel variant with demonstrated functional defect), all affected relatives of probands who carry the variant are also expected to carry it. For example, screening of 25 individuals affected with aHUS within three families showed that all carry the pathogenic variant in CFH [40]. If the variant has only a modest effect on disease risk, substantial genetic heterogeneity within patients may be observed. 3. Functional evidence that the variant affects gene function When a gene has already been confidently implicated in a disease, and the class of the variant implicated is known (i.e., loss or gain of function), then an experiment

Page 10 of 19

11

identifying the molecular mechanisms underlying a variant’s effect on disease risk would be particularly informative. Among the missense variant, loss of function variants were identified in CFH, CFI, MCP and THBD. Gain-of-function variants were identified in C3 and CFB (Table 2). The majority of variants identified in aHUS which have been causally associated with aHUS allow robust and accurate conclusions. However, some of reported disease associated genetic changes were either common polymorphisms or lacked direct evidence for pathogenicity. The challenge for genetic testing in aHUS is to demonstrate the functional consequences of the novel or rare variants, defined here as variants with a minor allele frequency of <1%. Highthroughput sequencing approaches can generate detailed catalogues of genetic variation in both disease patients and the general population. However, we must be able to separate disease-associated genetic variants from the broader background of variants present in all human genomes that are rare, potentially functional, but not actually pathogenic. The majority of tested mutations had, indeed, a functional defect. Nevertheless, some rare variants found in aHUS patients—one mutation in CFI, two mutations in CFH, and one mutation in the decay accelerating factor gene (DAF, CD55)—were reported to lack a functional defect, and the link with complement dysregulation for them remains unclear [61-63]. Additional genetic variations originally thought to be associated with aHUS also could not be linked to functional defects following induction of those amino acids changes [47].

4. Identification of a de novo mutation A de novo mutation, defined as a genetic variant that arises in a child but is not present in either parent, is rare in aHUS but highly suggestive to be pathogenic.

5. Mapping the variant into protein structure In absence of functional studies, bioinformatic classifications are frequently used, but these prediction tools need to be used with caution. Some protein domains are known to be critical to protein function, and all missense variants in these domains identified to date have been shown to be pathogenic. Recent in vitro studies showed that rare variants located in the binding area between two proteins, CFH/C3, CFB/C3, and C3/CFH, affected their interactions and explained the uncontrolled activation of the alternative complement pathway in their presence [47-48]. Taking into account all of these considerations, as well as the difficulties in performing functional studies, attempts at

Page 11 of 19

12

mapping the gene variant into its protein structure may be a useful surrogate to predict that a variant is damaging in terms of biological function.

6. In silico pathogenicity scores Functional studies are frequently unavailable to inform physicians of the possible relation of a newly identified genetic change with the disease. Therefore, bioinformatics tools would be useful to help predict a possible role for those variants. Unfortunately, such predictions are not always reliable. The probability that a genetic change induces an alteration of protein structure and function can be calculated with the following software: PolyPhen2 (protein structure function and evolutionary conservation), AlignGV/GD (protein structure function and evolutionary conservation), Mutation Taster (protein structure function and evolutionary conservation) and SIFT (evolutionary conservation). The variants are also classified according to their contribution to the disease as (i) pathogenic (could be insufficient to cause disease alone), (ii) likely pathogenic, (iii) uncertain significance, (iv) likely benign, or (v) benign. A variant could be associated with the disease if the frequency is significantly enriched in disease cases compared to matched controls. A damaging variant alters the normal levels or biochemical function of a gene or gene product. Given the increasing identification of rare gene variants in healthy donors, it is important to diagnose with certainty the disease causing variants. In-depth functional analyses are required to establish if the genetic change is related to the disease manifestation or just a fortuitous association. Investigating causality in classification of gene variants is a major challenge for the future of aHUS research. The criteria for pathogenicity classification should be standardized across laboratories in a way that promotes consistent determinations.

References 1. 2. 3.

J. N. George and C. M. Nester, 'Syndromes of thrombotic microangiopathy', N Engl J Med. 371 (2014), 654-66. M. Noris, F. Mescia and G. Remuzzi, 'STEC-HUS, atypical HUS and TTP are all diseases of complement activation', Nat Rev Nephrol. 8 (2012), 622-33. J. M. Spinale, R. L. Ruebner, B. S. Kaplan and L. Copelovitch, 'Update on Streptococcus pneumoniae associated hemolytic uremic syndrome', Curr Opin Pediatr. 25 (2013), 203-8.

Page 12 of 19

13

4.

5.

6. 7.

8.

9.

10.

11.

12.

13.

14.

15.

Loupiac, A. Elayan, M. Cailliez, A. L. Adra, S. Decramer, M. C. Thouret, J. Harambat and V. Guigonis, 'Diagnosis of Streptococcus pneumoniae-associated hemolytic uremic syndrome', Pediatr Infect Dis J. 32 (2013), 1045-9. Loirat, F. Fakhouri, G. Ariceta, N. Besbas, M. Bitzan, A. Bjerre, R. Coppo, F. Emma, S. Johnson, D. Karpman, D. Landau, C. B. Langman, A. L. Lapeyraque, C. Licht, C. Nester, C. Pecoraro, M. Riedl, N. C. van de Kar, J. Van de Walle, M. Vivarelli and V. Fremeaux-Bacchi, 'An international consensus approach to the management of atypical hemolytic uremic syndrome in children', Pediatr Nephrol. (2015). M. Noris and G. Remuzzi, 'Atypical hemolytic-uremic syndrome', N Engl J Med. 361 (2009), 1676-87. C. M. Nester, T. Barbour, S. R. de Cordoba, M. A. Dragon-Durey, V. FremeauxBacchi, T. H. Goodship, D. Kavanagh, M. Noris, M. Pickering, P. Sanchez-Corral, C. Skerka, P. Zipfel and R. J. Smith, 'Atypical aHUS: State of the art', Mol Immunol. 67 (2015), 31-42. V. Fremeaux-Bacchi, F. Fakhouri, A. Garnier, F. Bienaime, M. A. Dragon-Durey, S. Ngo, B. Moulin, A. Servais, F. Provot, L. Rostaing, S. Burtey, P. Niaudet, G. Deschenes, Y. Lebranchu, J. Zuber and C. Loirat, 'Genetics and outcome of atypical hemolytic uremic syndrome: a nationwide French series comparing children and adults', Clin J Am Soc Nephrol. 8 (2013), 554-62. Perez-Rodriguez, E. Loures, A. Rodriguez-Trillo, J. Costa-Pinto, A. Garcia-Rivero, A. Batlle-Lopez, J. Batlle and M. F. Lopez-Fernandez, 'Inherited ADAMTS13 deficiency (Upshaw-Schulman syndrome): a short review', Thromb Res. 134 (2014), 1171-5. D. S. Froese, J. Kopec, F. Fitzpatrick, M. Schuller, T. J. McCorvie, R. Chalk, T. Plessl, V. Fettelschoss, B. Fowler, M. R. Baumgartner and W. W. Yue, 'Structural Insights into the MMACHC-MMADHC Protein Complex Involved in Vitamin B12 Trafficking', J Biol Chem. 290 (2015), 29167-77. E. Richard, A. Jorge-Finnigan, J. Garcia-Villoria, B. Merinero, L. R. Desviat, L. Gort, P. Briones, F. Leal, C. Perez-Cerda, A. Ribes, M. Ugarte and B. Perez, 'Genetic and cellular studies of oxidative stress in methylmalonic aciduria (MMA) cobalamin deficiency type C (cblC) with homocystinuria (MMACHC)', Hum Mutat. 30 (2009), 1558-66. S. Fischer, M. Huemer, M. Baumgartner, F. Deodato, D. Ballhausen, A. Boneh, A. B. Burlina, R. Cerone, P. Garcia, G. Gokcay, S. Grunewald, J. Haberle, J. Jaeken, D. Ketteridge, M. Lindner, H. Mandel, D. Martinelli, E. G. Martins, K. O. Schwab, S. C. Gruenert, B. C. Schwahn, L. Sztriha, M. Tomaske, F. Trefz, L. Vilarinho, D. S. Rosenblatt, B. Fowler and C. Dionisi-Vici, 'Clinical presentation and outcome in a series of 88 patients with the cblC defect', J Inherit Metab Dis. 37 (2014), 831-40. M. Y. Liu, Y. L. Yang, Y. C. Chang, S. H. Chiang, S. P. Lin, L. S. Han, Y. Qi, K. J. Hsiao and T. T. Liu, 'Mutation spectrum of MMACHC in Chinese patients with combined methylmalonic aciduria and homocystinuria', J Hum Genet. 55 (2010), 6216 S. Grange, S. Bekri, E. Artaud-Macari, A. Francois, C. Girault, A. L. Poitou, Y. Benhamou, C. Vianey-Saban, J. F. Benoist, V. Chatelet, F. Tamion and D. Guerrot, 'Adult-onset renal thrombotic microangiopathy and pulmonary arterial hypertension in cobalamin C deficiency', Lancet. 386 (2015), 1011-2. E. Cornec-Le Gall, Y. Delmas, L. De Parscau, L. Doucet, H. Ogier, J. F. Benoist, V. Fremeaux-Bacchi and Y. Le Meur, 'Adult-Onset Eculizumab-Resistant Hemolytic Uremic Syndrome Associated With Cobalamin C Deficiency', Am J Kidney Dis. (2013).

Page 13 of 19

14

16.

17.

18.

19.

20.

21.

22.

23. 24.

25.

26.

27.

28.

M. Komhoff, M. T. Roofthooft, D. Westra, T. K. Teertstra, A. Losito, N. C. van de Kar and R. M. Berger, 'Combined pulmonary hypertension and renal thrombotic microangiopathy in cobalamin C deficiency', Pediatrics. 132 (2013), e540-4. J. P. Lerner-Ellis, N. Anastasio, J. Liu, D. Coelho, T. Suormala, M. Stucki, A. D. Loewy, S. Gurd, E. Grundberg, C. F. Morel, D. Watkins, M. R. Baumgartner, T. Pastinen, D. S. Rosenblatt and B. Fowler, 'Spectrum of mutations in MMACHC, allelic expression, and evidence for genotype-phenotype correlations', Hum Mutat. 30 (2009), 1072-81. M. Lemaire, V. Fremeaux-Bacchi, F. Schaefer, M. Choi, W. H. Tang, M. Le Quintrec, F. Fakhouri, S. Taque, F. Nobili, F. Martinez, W. Ji, J. D. Overton, S. M. Mane, G. Nurnberg, J. Altmuller, H. Thiele, D. Morin, G. Deschenes, V. Baudouin, B. Llanas, L. Collard, M. A. Majid, E. Simkova, P. Nurnberg, N. Rioux-Leclerc, G. W. Moeckel, M. C. Gubler, J. Hwa, C. Loirat and R. P. Lifton, 'Recessive mutations in DGKE cause atypical hemolytic-uremic syndrome', Nat Genet. 45 (2013), 531-6. S. Bruneau, M. Neel, L. T. Roumenina, M. Frimat, L. Laurent, V. Fremeaux-Bacchi and F. Fakhouri, 'Loss of DGKepsilon induces endothelial cell activation and death independently of complement activation', Blood. 125 (2015), 1038-46. R. Westland, M. Bodria, A. Carrea, S. Lata, F. Scolari, V. Fremeaux-Bacchi, V. D. D'Agati, R. P. Lifton, A. G. Gharavi, G. M. Ghiggeri and S. Sanna-Cherchi, 'Phenotypic expansion of DGKE-associated diseases', J Am Soc Nephrol. 25 (2014), 1408-14. C. Mele, M. Lemaire, P. Iatropoulos, R. Piras, E. Bresin, S. Bettoni, D. Bick, D. Helbling, R. Veith, E. Valoti, R. Donadelli, L. Murer, M. Neunhauserer, M. Breno, V. Fremeaux-Bacchi, R. Lifton, G. Remuzzi and M. Noris, 'Characterization of a New DGKE Intronic Mutation in Genetically Unsolved Cases of Familial Atypical Hemolytic Uremic Syndrome', Clin J Am Soc Nephrol. 10 (2015), 1011-9. N. S. Merle, S. E. Church, V. Fremeaux-Bacchi and L. T. Roumenina, 'Complement System Part I - Molecular Mechanisms of Activation and Regulation', Front Immunol. 6 (2015), 262. J. S. Cameron and R. Vick, 'Letter: Plasma-C3 in haemolytic-uraemic syndrome and thrombotic thrombocytopenic purpura', Lancet. 2 (1973), 975. K. Michaux, J. Bacchetta, E. Javouhey, P. Cochat, V. Fremaux-Bacchi and A. L. Sellier-Leclerc, 'Eculizumab in neonatal hemolytic uremic syndrome with homozygous factor H deficiency', Pediatr Nephrol. 29 (2014), 2415-9. H. Y. Cho, B. S. Lee, K. C. Moon, I. S. Ha, H. I. Cheong and Y. Choi, 'Complete factor H deficiency-associated atypical hemolytic uremic syndrome in a neonate', Pediatr Nephrol. 22 (2007), 874-80. V. Wilson, R. Darlay, W. Wong, K. M. Wood, J. McFarlane, L. Schejbel, I. M. Schmidt, C. L. Harris, J. Tellez, E. M. Hunze, K. Marchbank, J. A. Goodship and T. H. Goodship, 'Genotype/phenotype correlations in complement factor H deficiency arising from uniparental isodisomy', Am J Kidney Dis. 62 (2013), 978-83. Servais, L. H. Noel, M. A. Dragon-Durey, M. C. Gubler, P. Remy, D. Buob, C. Cordonnier, R. Makdassi, W. Jaber, E. Boulanger, P. Lesavre and V. FremeauxBacchi, 'Heterogeneous pattern of renal disease associated with homozygous Factor H deficiency', Hum Pathol. (2011). L. Couzi, C. Contin-Bordes, F. Marliot, A. Sarrat, P. Grimal, J. F. Moreau, P. Merville and V. Fremeaux-Bacchi, 'Inherited deficiency of membrane cofactor protein expression and varying manifestations of recurrent atypical hemolytic uremic syndrome in a sibling pair', Am J Kidney Dis. 52 (2008), e5-9.

Page 14 of 19

15

29.

30.

31.

D. Bhatia, P. Khandelwal, A. Sinha, P. Hari, H. I. Cheong and A. Bagga, 'Incomplete penetrance of CD46 mutation causing familial atypical hemolytic uremic syndrome', Pediatr Nephrol. 30 (2015), 2215-20. F. Bu, N. Borsa, A. Gianluigi and R. J. Smith, 'Familial atypical hemolytic uremic syndrome: a review of its genetic and clinical aspects', Clin Dev Immunol. 2012 (2012), 370426. E. Rodriguez, P. M. Rallapalli, A. J. Osborne and S. J. Perkins, 'New functional and structural insights from updated mutational databases for complement factor H, Factor I, membrane cofactor protein and C3', Biosci Rep. 34 (2014).

32.

B. S. Blaum, J. P. Hannan, A. P. Herbert, D. Kavanagh, D. Uhrin and T. Stehle, 'Structural basis for sialic acid-mediated self-recognition by complement factor H', Nat Chem Biol. 11 (2015), 77-82.

33.

M. A. Dragon-Durey, V. Fremeaux-Bacchi, C. Loirat, J. Blouin, P. Niaudet, G. Deschenes, P. Coppo, W. Herman Fridman and L. Weiss, 'Heterozygous and homozygous factor h deficiencies associated with hemolytic uremic syndrome or membranoproliferative glomerulonephritis: report and genetic analysis of 16 cases', J Am Soc Nephrol. 15 (2004), 787-95.

34.

M. Noris, J. Caprioli, E. Bresin, C. Mossali, G. Pianetti, S. Gamba, E. Daina, C. Fenili, F. Castelletti, A. Sorosina, R. Piras, R. Donadelli, R. Maranta, I. van der Meer, E. M. Conway, P. F. Zipfel, T. H. Goodship and G. Remuzzi, 'Relative role of genetic complement abnormalities in sporadic and familial aHUS and their impact on clinical phenotype', Clin J Am Soc Nephrol. 5 (2010), 1844-59. T. K. Maga, C. J. Nishimura, A. E. Weaver, K. L. Frees and R. J. Smith, 'Mutations in alternative pathway complement proteins in American patients with atypical hemolytic uremic syndrome', Hum Mutat. 31 (2010), E1445-60. E. Bresin, E. Rurali, J. Caprioli, P. Sanchez-Corral, V. Fremeaux-Bacchi, S. Rodriguez de Cordoba, S. Pinto, T. H. Goodship, M. Alberti, D. Ribes, E. Valoti, G. Remuzzi and M. Noris, 'Combined complement gene mutations in atypical hemolytic uremic syndrome influence clinical phenotype', J Am Soc Nephrol. 24 (2013), 475-86. L. T. Roumenina, C. Loirat, M. A. Dragon-Durey, L. Halbwachs-Mecarelli, C. SautesFridman and V. Fremeaux-Bacchi, 'Alternative complement pathway assessment in patients with atypical HUS', J Immunol Methods. 365 (2011), 8-26. B. Z. Schmidt, N. L. Fowler, T. Hidvegi, D. H. Perlmutter and H. R. Colten, 'Disruption of disulfide bonds is responsible for impaired secretion in human complement factor H deficiency', J Biol Chem. 274 (1999), 11782-8 P. Warwicker, T. H. Goodship, R. L. Donne, Y. Pirson, A. Nicholls, R. M. Ward, P. Turnpenny and J. A. Goodship, 'Genetic studies into inherited and sporadic hemolytic uremic syndrome', Kidney Int. 53 (1998), 836-44. .F. H. Sansbury, H. J. Cordell, C. Bingham, G. Bromilow, A. Nicholls, R. Powell, B. Shields, L. Smyth, P. Warwicker, L. Strain, V. Wilson, J. A. Goodship, T. H. Goodship and P. D. Turnpenny, 'Factors determining penetrance in familial atypical haemolytic uraemic syndrome', J Med Genet. 51 (2014), 756-64.

35.

36.

37.

38.

39.

40.

41.

C. Abarrategui-Garrido, R. Martinez-Barricarte, M. Lopez-Trascasa, S. R. de Cordoba and P. Sanchez-Corral, 'Characterization of complement factor H-related (CFHR)

Page 15 of 19

16

42.

proteins in plasma reveals novel genetic variations of CFHR1 associated with atypical hemolytic uremic syndrome', Blood. 114 (2009), 4261-71. F Bienaime, MA Dragon-Durey, CH Regnier, SC Nilsson, WH Kwan, J Blouin, M Jablonski, N Renault, MA Rameix-Welti, C Loirat, C Sautés-Fridman, BO Villoutreix, AM Blom, V Fremeaux-Bacchi. Mutations in components of complement influence the outcome of Factor I-associated atypical hemolytic uremic syndrome, Kidney Int. (2010) 77(4):339-49

43.

D. Kavanagh, Y. Yu, E. C. Schramm, M. Triebwasser, E. K. Wagner, S. Raychaudhuri, M. J. Daly, J. P. Atkinson and J. M. Seddon, 'Rare genetic variants in the CFI gene are associated with advanced age-related macular degeneration and commonly result in reduced serum factor I levels', Hum Mol Genet. 24 (2015), 386170.

44.

J. P. van de Ven, S. C. Nilsson, P. L. Tan, G. H. Buitendijk, T. Ristau, F. C. Mohlin, S. B. Nabuurs, F. E. Schoenmaker-Koller, D. Smailhodzic, P. A. Campochiaro, D. J. Zack, M. R. Duvvari, B. Bakker, C. C. Paun, C. J. Boon, A. G. Uitterlinden, S. Liakopoulos, B. J. Klevering, S. Fauser, M. R. Daha, N. Katsanis, C. C. Klaver, A. M. Blom, C. B. Hoyng and A. I. den Hollander, 'A functional variant in the CFI gene confers a high risk of age-related macular degeneration', Nat Genet. 45 (2013), 813-7.

45.

M. K. Liszewski and J. P. Atkinson, 'Complement regulator CD46: genetic variants and disease associations', Hum Genomics. (2015) 9, 7. E. Goicoechea de Jorge, C. L. Harris, J. Esparza-Gordillo, L. Carreras, E. A. Arranz, C. A. Garrido, M. Lopez-Trascasa, P. Sanchez-Corral, B. P. Morgan and S. Rodriguez de Cordoba, 'Gain-of-function mutations in complement factor B are associated with atypical hemolytic uremic syndrome', Proc Natl Acad Sci U S A. 104 (2007), 240-5. M. C. Marinozzi, L. Vergoz, T. Rybkine, S. Ngo, S. Bettoni, A. Pashov, M. Cayla, F. Tabarin, M. Jablonski, C. Hue, R. J. Smith, M. Noris, L. Halbwachs-Mecarelli, R. Donadelli, V. Fremeaux-Bacchi and L. T. Roumenina, 'Complement Factor B Mutations in Atypical Hemolytic Uremic Syndrome--Disease-Relevant or Benign?' J Am Soc Nephrol. (2014). E. C. Schramm, L. T. Roumenina, T. Rybkine, S. Chauvet, P. Vieira-Martins, C. Hue, T. Maga, E. Valoti, V. Wilson, S. Jokiranta, R. J. Smith, M. Noris, T. Goodship, J. P. Atkinson and V. Fremeaux-Bacchi, 'Functional mapping of the interactions between complement C3 and regulatory proteins using atypical hemolytic uremic syndromeassociated mutations', Blood. (2015). 125 R. Martinez-Barricarte, M. Heurich, A. Lopez-Perrote, A. Tortajada, S. Pinto, M. Lopez-Trascasa, P. Sanchez-Corral, B. P. Morgan, O. Llorca, C. L. Harris and S. Rodriguez de Cordoba, 'The molecular and structural bases for the association of complement C3 mutations with atypical hemolytic uremic syndrome', Mol Immunol. 66 (2015), 263-73.

46.

47.

48.

49.

50.

J. M. Seddon, Y. Yu, E. C. Miller, R. Reynolds, P. L. Tan, S. Gowrisankar, J. I. Goldstein, M. Triebwasser, H. E. Anderson, J. Zerbib, D. Kavanagh, E. Souied, N. Katsanis, M. J. Daly, J. P. Atkinson and S. Raychaudhuri, 'Rare variants in CFI, C3 and C9 are associated with high risk of advanced age-related macular degeneration', Nat Genet. 45 (2013), 1366-70.

Page 16 of 19

17

51.

52.

53.

54.

55.

56.

57.

58.

59.

60.

61.

E. Valoti, M. Alberti, A. Tortajada, J. Garcia-Fernandez, S. Gastoldi, L. Besso, E. Bresin, G. Remuzzi, S. Rodriguez de Cordoba and M. Noris, 'A novel atypical hemolytic uremic syndrome-associated hybrid CFHR1/CFH gene encoding a fusion protein that antagonizes factor H-dependent complement regulation', J Am Soc Nephrol. 26 (2015), 209-19. M. Sullivan, L. A. Rybicki, A. Winter, M. M. Hoffmann, S. Reiermann, H. Linke, K. Arbeiter, L. Patzer, K. Budde, B. Hoppe, M. Zeier, K. Lhotta, A. Bock, T. Wiech, A. Gaspert, T. Fehr, M. Woznowski, G. Berisha, A. Malinoc, O. N. Goek, C. Eng and H. P. Neumann, 'Age-related penetrance of hereditary atypical hemolytic uremic syndrome', Ann Hum Genet. 75 (2011), 639-47. J. Esparza-Gordillo, E. Goicoechea de Jorge, A. Buil, L. C. Berges, M. LopezTrascasa, P. Sanchez-Corral and S. Rodriguez de Cordoba, 'Predisposition to atypical hemolytic uremic syndrome involves the concurrence of different susceptibility alleles in the regulators of complement activation gene cluster in 1q32', Hum Mol Genet. 14 (2005), 703-12. M. Delvaeye, M. Noris, A. De Vriese, C. T. Esmon, N. L. Esmon, G. Ferrell, J. DelFavero, S. Plaisance, B. Claes, D. Lambrechts, C. Zoja, G. Remuzzi and E. M. Conway, 'Thrombomodulin mutations in atypical hemolytic-uremic syndrome', N Engl J Med. 361 (2009), 345-57. F. Bu, N. G. Borsa, M. B. Jones, E. Takanami, C. Nishimura, J. J. Hauer, H. Azaiez, E. A. Black-Ziegelbein, N. C. Meyer, D. L. Kolbe, Y. Li, K. Frees, M. J. Schnieders, C. Thomas, C. Nester and R. J. Smith, 'High-Throughput Genetic Testing for Thrombotic Microangiopathies and C3 Glomerulopathies', J Am Soc Nephrol. (2015). C. Blanc, L. T. Roumenina, Y. Ashraf, S. Hyvarinen, S. K. Sethi, B. Ranchin, P. Niaudet, C. Loirat, A. Gulati, A. Bagga, W. H. Fridman, C. Sautes-Fridman, T. S. Jokiranta, V. Fremeaux-Bacchi and M. A. Dragon-Durey, 'Overall neutralization of complement factor H by autoantibodies in the acute phase of the autoimmune form of atypical hemolytic uremic syndrome', J Immunol. 189 (2012), 3528-37. M. A. Dragon-Durey, C. Blanc, F. Marliot, C. Loirat, J. Blouin, C. Sautes-Fridman, W. H. Fridman and V. Fremeaux-Bacchi, 'The high frequency of Complement Factor H-Related CFHR1 Gene Deletion is restricted to specific subgroups of patients with atypical Haemolytic Uraemic Syndrome', J Med Genet. (2009). Sinha, A. Gulati, S. Saini, C. Blanc, A. Gupta, B. S. Gurjar, H. Saini, S. T. Kotresh, U. Ali, D. Bhatia, A. Ohri, M. Kumar, I. Agarwal, S. Gulati, K. Anand, M. Vijayakumar, R. Sinha, S. Sethi, M. Salmona, A. George, V. Bal, G. Singh, A. K. Dinda, P. Hari, S. Rath, M. A. Dragon-Durey and A. Bagga, 'Prompt plasma exchanges and immunosuppressive treatment improves the outcomes of anti-factor H autoantibodyassociated hemolytic uremic syndrome in children', Kidney Int. (2013). Bhattacharjee, S. Reuter, E. Trojnar, R. Kolodziejczyk, H. Seeberger, S. Hyvarinen, B. Uzonyi, A. Szilagyi, Z. Prohaszka, A. Goldman, M. Jozsi and T. S. Jokiranta, 'The major autoantibody epitope on factor H in atypical hemolytic uremic syndrome is structurally different from its homologous site in factor H-related protein 1, supporting a novel model for induction of autoimmunity in this disease', J Biol Chem. 290 (2015), 9500-10. N. Sorvillo, S. D. van Haren, P. H. Kaijen, A. ten Brinke, R. Fijnheer, A. B. Meijer and J. Voorberg, 'Preferential HLA-DRB1*11-dependent presentation of CUB2derived peptides by ADAMTS13-pulsed dendritic cells', Blood. 121 (2013), 3502-10. D. Kavanagh, R. Burgess, D. Spitzer, A. Richards, M. L. Diaz-Torres, J. A. Goodship, D. E. Hourcade, J. P. Atkinson and T. H. Goodship, 'The decay accelerating factor

Page 17 of 19

18

62.

63.

mutation I197V found in hemolytic uraemic syndrome does not impair complement regulation', Mol Immunol. 44 (2007), 3162-7. S. C. Nilsson, D. Karpman, F. Vaziri-Sani, A. C. Kristoffersson, R. Salomon, F. Provot, V. Fremeaux-Bacchi, L. A. Trouw and A. M. Blom, 'A mutation in factor I that is associated with atypical hemolytic uremic syndrome does not affect the function of factor I in complement regulation', Mol Immunol. 44 (2007), 1835-44. Tortajada, S. Pinto, J. Martinez-Ara, M. Lopez-Trascasa, P. Sanchez-Corral and S. R. de Cordoba, 'Complement factor H variants I890 and L1007 while commonly associated with atypical hemolytic uremic syndrome are polymorphisms with no functional significance', Kidney Int. 81 (2012), 56-63.

Figure legend Figure 1 : Summary of the rare missense variants (grey) reported in Exome variants server in CFH, CFI, MCP, C3 and CFB and of the missense variants (red) reported in the French cohort of 500 patients with aHUS (Figures were made using http://prosite.expasy.org/mydomains/). In the French cohort, 13 out the 85 genetic changes were identified in more than one unrelated patient [4]. Therefore some of variants are reported in aHUS data bases, and the functional consequences already known. Table 1: Genetic and acquired complement abnormalities in four reported series of patients with aHUS [8, 34-36]

N

France

Italy

US

European Consortium (Fr, It, UK, Spain)

214

273

144

795

age at onset

0 to 83 y

CFH

28

24

27

19.8

MCP

10

7

5

8.1

CFI

10

4

8

5.8

C3

8

4

2

5.6

CFB

2

<1

4

1.1

Combined

4

3

nd

3

Complement-associated aHUS

60

46

46

40.4

anti FH Ab

6

3

nd

nd

Page 18 of 19

19

Table 2 : Characteristic of the 229 genetic abnormalities identified in the french aHUS cohort

Novel variant characteristic (% of total) Missense Nonsense Deletion/Insertion Splice Site Large deletion Complex rearrangement

CFH 59 15 9 6 4 8

MCP 38 22 13 25 3 0

CFI 90 0 5 5 0 0

CFB 100

C3 94 6 0 0 0 0

DGKe 14 29 29 29 0 0

novel variants (n )/rare variants (n)

80/17

12/5

21/21

03/06

18/16

7/3

Rare variants/total variants (%)

17.5

13

50

66

47

30

Page 19 of 19