mutations in complement c3 predispose to development of atypical hemolytic uremic syndrome

doi:10.1182/blood-2008-01-133702Prepublished online September 16, 2008;

Janssen, Timothy H.J. Goodship and John P AtkinsonRichard Hauhart, Vincent Meunier, Chantal Loirat, Marie-Agnes Dragon-Durey, Wolf H Fridman, Bert J C Mattoo, Hubert Nivet, William Wong, Sophie Gie, Bruno Hurault de Ligny, Michel Fischbach, Ritu Gupta,Alison L Brown, Nadeem Moghal, Bernard S Kaplan, Robert A Weiss, Karl Lhotta, Gaurav Kapur, Tej Veronique Fremeaux-Bacchi, Elizabeth C. Miller, M. Kathryn Liszewski, Lisa Strain, Jacques Blouin, hemolytic uremic syndromeMutations in complement C3 predispose to development of atypical

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1

Mutations in complement C3 predispose to development of atypical hemolytic uremic

syndrome

Veronique Frémeaux-Bacchi1, Elizabeth C Miller2, M Kathryn Liszewski2, Lisa Strain3, Jacques

Blouin1, Alison L Brown3, Nadeem Moghal4, Bernard S Kaplan5, Robert A Weiss6, Karl Lhotta7,

Gaurav Kapur8, Tej Mattoo8, Hubert Nivet9, William Wong10, Sophie Gie11, Bruno Hurault de

Ligny12, Michel Fischbach13, Ritu Gupta2, Richard Hauhart2, Vincent Meunier14, Chantal

Loirat15, Marie-Agnès Dragon-Durey1, Wolf H Fridman1, Bert J C Janssen16, Timothy H J

Goodship17, John P Atkinson2

1Service d’Immunologie Biologique, Hôpital Européen Georges Pompidou, Paris, France and

INSERM UMRS 872, Cordeliers Research Center, Paris; 2Division of Rheumatology,

Washington University School of Medicine, St Louis, MO, USA; 3Northern Molecular Genetics

Service, Newcastle upon Tyne Hospitals NHS Foundation Trust, Newcastle upon Tyne, UK

3Department of Renal Medicine, Newcastle upon Tyne Hospitals NHS Foundation Trust,

Newcastle upon Tyne, UK; 4Paediatric Nephrology, Newcastle upon Tyne Hospitals NHS

Foundation Trust, Newcastle upon Tyne, UK; 5Division of Nephrology, Children’s Hospital of

Philadelphia, Philadelphia, USA; 6Paediatric Nephrology, Maria Fareri Children’s Hospital at

Westchester Medical Center, Valhalla; 7Division of Clinical Nephrology, Department of Internal

Medicine, Innsbruck University Hospital, Innsbruck; 8Pediatric Nephrology, Children's Hospital

of Michigan, Detroit, Michigan; 9Unité de Néphrologie Pédiatrique, Centre Hospitalier de Tours,

Tours, France; 10Starship Children’s Hospital, Auckland, New Zealand; 11 Département de

Médecine de l Enfant et de l Adolescent, CHU Rennes, Hôpital Sud, 16 Boulevard de Bulgarie,

Blood First Edition Paper, prepublished online September 16, 2008; DOI 10.1182/blood-2008-01-133702

Copyright © 2008 American Society of Hematology




2

35056 Rennes, France; 12 Service de Néphrologie Transplantation Renale, Hôpital Clemenceau,

14033 Caen, France; 13Service de Pédiatrie, Hôpital de Hautepierre, 67098 Strasbourg, France;

14Service de Medecine Interne, Hôpital Robert Boulin, Libourne, France; 15Service de

Néphrologie Pédiatrique, Hôpital Robert-Debré, Paris; 16Crystal and Structural Chemistry,

Bijvoet Center for Biomolecular Research, Faculty of Science, Utrecht University, Utrecht, The

Netherlands; 17Institute of Human Genetics, Newcastle University, Newcastle upon Tyne.

Correspondence should be addressed to V.F-B. ([email protected]), J.P.A.

([email protected]) or T.H.J.G. ([email protected]) who contributed equally to this

work.




3

Abstract

Atypical HUS (aHUS) is a disease of complement dysregulation. In ~50% of patients mutations

have been described in the genes encoding the complement regulators factor H, MCP and factor I

or the activator factor B. We report here mutations in the central component of the complement

cascade, C3, in association with aHUS. We describe nine novel C3 mutations in 14 aHUS

patients with a persistently low serum C3 level. We have demonstrated that five of these

mutations are gain of function and two inactivating. This establishes C3 as a susceptibility factor

for aHUS.




4

Introduction

Mutations in the genes encoding the complement regulators factor H 1-6, factor I 7,8 and

membrane cofactor protein (MCP; CD46) 9,10 as well as in the activating component factor B 11

have been detected in ~50% of patients with atypical hemolytic uremic syndrome (aHUS) 12. A

proportion of the remaining patients have persistently low serum levels of C3. In this study we

have examined the hypothesis that mutations in the gene encoding C3 could be associated with

aHUS in these patients.

C3 is the pivotal component of the complement system 13. Activation of the classical, lectin and

alternative pathways results in cleavage of C3 to generate C3b and the anaphylatoxin C3a.

When C3b is produced, the thioester is cleaved and then this highly reactive species may bind

covalently to targets. Interaction of the zymogen factor B with C3b and subsequent cleavage of

factor B by factor D results in formation of the alternative pathway C3 convertase C3bBb. This

set of reactions represents an amplification loop.

A series of complement regulators including factor H and MCP prevent feedback via this loop by

increasing the rate of dissociation of C3bBb and/or by serving as cofactors for the serine protease

factor I to cleave C3b. Mutations in the gene encoding factor B were recently found to enhance

formation of C3bBb or increase resistance to inactivation 11.

The importance of C3 as a susceptibility factor for human disease has been emphasised by recent

studies which document that a common non-synonymous coding change in C3 (rs2230199,

Arg80Gly, corresponding to C3S and C3F) is both a susceptibility factor for age related macular

degeneration 14 and associated with long term renal allograft survival15.




5

Methods

Subjects: In two independent cohorts of aHUS patients (Paris, France and Newcastle upon Tyne,

UK), 26 patients (17 Paris and 9 Newcastle) with a serum C3 level persistently below the lower

end of the normal range (680-1380 mg/l) were identified. In these patients functionally

significant mutations in CFH, MCP, CFI and CFB had not previously been detected. Mutation

screening of C3 was undertaken in these patients. Approval for this study was obtained from the

Departement de la rechereche clinique et du developement, DRRC Ile de France, France and the

Northern and Yorkshire Multi-Centre Research Ethics Committee, United Kingdom. Informed

consent was provided according to the Declaration of Helsinki.

Mutation screening: The coding sequence of C3 was amplified with flanking primers

(Supplementary Table 1). Direct sequencing was undertaken using a 96 capillary Sequencer

3700 using the dye terminator method (Applied Biosystems, Courtaboeuf, France). For the

genomic DNA sequence the first nucleotide A of the initiator ATG codon is denoted as

nucleotide +1. The amino acid numbering does not include the 22 residues of the signal peptide.

Functional studies: C3 cDNA (gift of David Isenman) 16 was sequenced and compared to the

sequence published for the C3 crystal structure 17. Two single base pair changes were found in

the cDNA and altered (QuikChange Multi Site-Directed Mutagenesis Kit, Stratagene, LaJolla,

CA) to match the published sequence of the C3 used in the structural analysis 17. Mutant clones

were produced (QuikChange XL Site-Directed Mutagenesis Kit, Stratagene) and sequenced in

both directions.

The mutant and WT C3 DNAs were transiently transfected into either 293T or COS-1 cells using

Lipofectamine transfection reagent (Invitrogen, Carlsbad, CA). Two to three days post

transfection, the supernatants were collected and concentrated. C3 was quantitated by ELISA and




6

examined by Western blotting (see Supplement). Conversion of C3 to iC3 was accomplished by

storage at 4°C or repetitive freeze thawing and monitored by autolytic cleavage 18 (see

Supplement). C3b and C4b ligand binding assays were undertaken using recombinant MCP (see

Supplement for protocol), factor H (Complement Technologies, Tyler, TX), soluble CR1 (gift of

H. Marsh, Avant Immunotherapeutics, Needham, MA) and factor B (Complement Technologies).

Cofactor assays were undertaken using wild-type and the mutant C3 proteins, human factor I

(Complement Technologies) and the above noted cofactor proteins.

See Supplement for methodology relating to assays for cofactor protein binding to C3 and

cofactor activity.

Results and Discussion

In 11 patients we identified a heterozygous C3 mutation. Three additional family members were

also affected by HUS, two from a pedigree (Supplementary Fig. 1) in which an unaffected

individual also carried the same change, and another from an affected sibling pair. Therefore, 14

affected individuals were found to harbor a mutation. There were a total of nine distinct

mutations identified in the initial 11 patients, the same mutation was carried by two unrelated

individuals. There were eight heterozygous missense mutations (R570W, R570Q, R713W,

A1072V, D1093N, C1136W, Q1139K and H1142D) and one heterozygous nonsense mutation

(Y832X). The positions of these are shown in Figure 1. None of these variants were present in

200 chromosomes from normal individuals.

Clinical and laboratory data are presented in Supplementary Table 2. Median age at

presentation was 6.5 years (range 8 months to 40 years). Seven of the 14 recovered renal

function after their initial presentation, four of these had recurrences. Within the 14 patients there




7

have been 12 renal transplants (nine cadaveric and three live-related), five of which have been

affected by recurrent disease.

Next, eight of nine (except for the missense mutation) C3 mutant constructs were prepared and

transfected into mammalian cells The C1163W mutant was either minimally or not detectably

expressed. Of the remaining seven, each was expressed in approximately comparable amounts

and protein bands in gels migrated identically to wild-type C3 (Supplementary Figure 2).

Assays were performed to assess the interaction of the seven secreted C3 mutants with MCP 19.

Binding of R570Q, R570W, A1072V, D1093N and Q1139K was 22, 30, 18, 17 and 69% of

wild-type, respectively (Fig. 2A). Binding of R713W and H1442D was not statistically different

from wild-type. The results of the cofactor assays paralleled the ligand binding data (Fig. 2B).

The two mutants with normal MCP binding also had normal cofactor activity whereas the five

mutants with decreased MCP binding had decreased cofactor activity. Specifically, there were no

detectable cleavage fragments in four and modest cleavage by mutant R570Q. Based on the loss

of the α-chain and appearance of major α1 and α-41 kDa cleavage fragments, there was >90%

reduction in cofactor activity for these five mutants. Thus, the reduced interaction of five of the

seven secreted C3 mutant proteins with MCP (the major membrane cofactor regulator of C3b) is

likely to lead to a gain of function relative to complement activation.

We next evaluated the interaction of the seven secreted mutants with complement receptor 1

(soluble CR1, sCR1) and the plasma regulatory protein factor H. Both of these inhibitors also

bind C3b and are cofactors for its cleavage by factor I. There were no statistically significant

differences between binding of sCR1 to wild-type C3 vs that of the seven mutants. For factor H,

however, the binding profile generally resembled that of MCP except that the magnitude of the

decrease in binding was less. However, there was a statistically significant decrease in iC3




8

binding of A1072V, D1093N and Q1139K while R570Q and R570W were modestly (10 to 20%)

but not statistically significantly decreased. Cofactor activity for Q1139K was also decreased

compared to native C3 (Supplementary Fig 3).

We also assessed binding to factor B. Factor B interacts with C3b or iC3 to begin the formation

of the alternative pathway C3 convertase. There were no statistically significant alterations in

binding of these mutants to factor B. Taken together, these results further suggest that it is a

modification in interactions with regulators that leads to a secondary gain of function in these C3

mutants.

Homozygous C3 deficiency in association with recurrent bacterial infections has been

recognized for many years 20. Following the description of the genomic structure of C3 in 1990

21, a molecular basis for C3 deficiency was first described 22. Subsequently, inherited C3

deficiency has been identified in 19 families (reviewed in 23) and the underlying molecular

mechanism established in eight of these. In this study we have identified nine heterozygous

mutations in C3. Five (R570Q, R570W, A1072V, D1093N and Q1139K) lead to impaired

binding to the regulator MCP and thus resistance to cleavage by factor I.

Recent structural studies of human C3 have provided unique insights into the functional domains

of the molecule 17,24 . Putative binding sites for factor H, complement receptors one and two

(CR2; CD21), properdin and factor B have been mapped to the structure of C3 13. The five

mutations associated with impaired binding to MCP also provide additional information on

potential interactive sites for this regulator as well as possibly for factor I.

The functional significance of R713W and H1442D mutations remains to be elucidated as they

had normal binding to MCP and subsequent cleavage by factor I. While the C3 mutants with a

resistance to regulator binding fit with current concepts of aHUS pathogenesis, the mutations




9

Y832X and C1136W lead to impaired secretion of C3 and haploinsufficiency. The latter is more

difficult to understand relative to the concept of increased complement activation for a given

degree of cellular injury as the fundamental predisposing event for aHUS.

In summary, this study establishes a new association between heterozygous mutations in C3 and

aHUS.

Acknowledgements THJG is supported by the Robin Davies Trust, the Foundation for Children with atypical HUS

and the Medical Research Council (Grant G0701325). VFB is supported by the Délégation

Régionale à la Recherche Clinique, Assistance Publique – Hôpitaux de Paris (Grants PHRC

AOM 05130). We also thank Florence Marliot for expert technical support and Paula Bertram

for production and purification of recombinant soluble MCP. This work was supported in part by

funding from the National Institutes of Health (R01 AI037618, R01 AI041592 & T32 HL07317)

for ECM, MKL, RH & JPA. RG was supported by National Institutes of Health Training Grant

(T32 AI007163).

Authors’ contributions

V.F-B, T.H.J.G and J.P.A designed the research, analysed the data and wrote the paper and

contributed equally to this work. V.F-B, E.C.M, M.K.L, M-A.D-D, L.S, R.G and R.H performed

the research. J.B, A.L.B, N.M, B.S.K, R.A.W, K.L, G.K, T.M, H.N, W.W, S.G, B.H. de L, M.F,

V.M, C.L and W.H.F contributed clinical information. B.J undertook the structural analysis.

E.M., M.K.L, R.G. and R.H. prepared the mutant proteins and performed the ligand binding and

cofactor assays. The authors declare no conflict of interest.




10

References

1. Warwicker P, Goodship THJ, Donne RL, et al. Genetic studies into inherited and sporadic haemolytic uraemic syndrome. Kidney Int. 1998;53:836-844.

2. Caprioli J, Bettinaglio P, Zipfel PF, et al. The molecular basis of familial hemolytic uremic syndrome: Mutation analysis of factor H gene reveals a hot spot in short consensus repeat 20. J Am Soc Nephrol. 2001;12:297-307.

3. Perez-Caballero D, Gonzalez-Rubio C, Gallardo ME, et al. Clustering of missense mutations in the C-terminal region of factor H in atypical hemolytic uremic syndrome. Am J Hum Genet. 2001;68:478-484.

4. Richards A, Buddles MR, Donne RL, et al. Factor H mutations in hemolytic uremic syndrome cluster in exons 18-20, a domain important for host cell recognition. Am J Hum Genet. 2001;68:485-490.

5. Dragon-Durey MA, Fremeaux-Bacchi V, Loirat C, et al. 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. 2004;15:787-795.

6. Venables JP, Strain L, Routledge D, et al. Atypical haemolytic uraemic syndrome associated with a hybrid complement gene. PLoS Medicine. 2006;3:e431.

7. Fremeaux-Bacchi V, Dragon-Durey M, Blouin J, et al. Complement Factor I : a susceptibility gene for atypical hemolytic-uremic syndrome. J Med Genet. 2004;41:e84.

8. Kavanagh D, Kemp EJ, Mayland E, et al. Mutations in complement factor I predispose to development of atypical hemolytic uremic syndrome. J Am Soc Nephrol. 2005;16:2150-2155.

9. Noris M, Brioschi S, Caprioli J, et al. Familial haemolytic uraemic syndrome and an MCP mutation. Lancet. 2003;362:1542-1547.

10. Richards A, Kemp EJ, Liszewski MK, et al. Mutations in human complement regulator, membrane cofactor protein (CD46), predispose to development of familial hemolytic uremic syndrome. Proc Natl Acad Sci U S A. 2003;100:12966-12971.

11. Goicoechea de Jorge E, Harris CL, Esparza-Gordillo J, et al. Gain-of-function mutations in complement factor B are associated with atypical hemolytic uremic syndrome. Proc Natl Acad Sci U S A. 2007;104:240-245.

12. Kavanagh D, Goodship THJ, Richards A. Atypical haemolytic uraemic syndrome. Br Med Bull. 2006;77-78: 5-22.

13. Janssen BJ, Gros P. Structural insights into the central complement component C3. Mol Immunol. 2007;44:3-10.




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14. Yates JR, Sepp T, Matharu BK, et al. Complement C3 variant and the risk of age-related macular degeneration. N Engl J Med. 2007;357:553-561.

15. Brown KM, Kondeatis E, Vaughan RW, et al. Influence of donor C3 allotype on late renal-transplantation outcome. N Engl J Med. 2006;354:2014-2023.

16. Taniguchi-Sidle A, Isenman DE. Mutagenesis of the Arg-Gly-Asp triplet in human complement component C3 does not abolish binding of iC3b to the leukocyte integrin complement receptor type III (CR3, CD11b/CD18). J Biol Chem. 1992;267:635-643.

17. Janssen BJ, Huizinga EG, Raaijmakers HC, et al. Structures of complement component C3 provide insights into the function and evolution of immunity. Nature. 2005;437:505-511.

18. Sim RB, Sim E. Autolytic fragmentation of complement components C3 and C4 under denaturing conditions, a property shared with alpha 2-macroglobulin. Biochem J. 1981;193:129-141.

19. Liszewski MK, Leung M, Cui W, et al. Dissecting sites important for complement regulatory activity in membrane cofactor protein (MCP; CD46). J Biol Chem. 2000;275:37692-37701.

20. Alper CA, Colten HR, Rosen FS, Rabson AR, Macnab GM, Gear JS. Homozygous deficiency of C3 in a patient with repeated infections. Lancet. 1972;2:1179-1181.

21. Fong KY, Botto M, Walport MJ, So AK. Genomic organization of human complement component C3. Genomics. 1990;7:579-586.

22. Botto M, Fong KY, So AK, Rudge A, Walport MJ. Molecular basis of hereditary C3 deficiency. J Clin Invest. 1990;86:1158-1163.

23. Reis ES, Falcao DA, Isaac L. Clinical aspects and molecular basis of primary deficiencies of complement component C3 and its regulatory proteins factor I and factor H. Scand J Immunol. 2006;63:155-168.

24. Janssen BJ, Christodoulidou A, McCarthy A, Lambris JD, Gros P. Structure of C3b reveals conformational changes that underlie complement activity. Nature. 2006;444:213-216.




12

Legends to Figures Figure 1. A) Gene structure of C3, domains of the encoded protein and position of the mutations.

Note that the genomic structure numbering begins with the start site ATG, while the protein

structures begin with the first amino acid of the mature protein. B) Structures of complement

component C3 and C3b showing the locations of mutations identified in aHUS patients. Ribbon

representation of C3b and C3 (two views) with the domains containing mutations (labeled

spheres).

Figure 2. Ligand binding and cofactor activity of the C3 proteins. Proteins were transiently

expressed in 293T cells, concentrated and quantified prior to analysis (see Supplement). (A)

Binding to MCP, factor H, factor B and soluble CR1 (sCR1) in ELISA. For MCP and factor H,

C3 protein was incubated at 15 ng/ml and for factor B, 200 ng/ml. Detection was made with

chicken anti-human C3 and HRP-linked donkey anti-chicken IgY. Asterisks indicate a

significant reduction in binding (p < 0.05). (B) Kinetic analysis of cofactor activity. C3

preparations were incubated for 0 to 30 min at 37˚C with factor I and a cofactor protein (MCP,

factor H, or sCR1). The zero control is prior to the addition of factor I. The last lane is an iC3b

control. Samples were reduced and analyzed by Western blotting using either chicken anti-

human C3 or goat anti-human C3 (insets) followed by an HRP-linked antiglobulin (see

Supplement). Cofactor activity is assessed by the loss of the α chain and appearance of the α1

and α-41 kDa major cleavage fragments. The minor α-43 kDa cleavage fragment is more

variable but no pattern was observed that was specific for a mutant. Data are representative of

five similar experiments.




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mutations in complement c3 predispose to development of atypical hemolytic uremic syndrome

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