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Complement Factor B Mutations in Atypical HemolyticUremic Syndrome—Disease-Relevant or Benign?

Maria Chiara Marinozzi,*†‡ Laura Vergoz,*†§ Tania Rybkine,*†§ Stephanie Ngo,‡

Serena Bettoni,| Anastas Pashov,¶ Mathieu Cayla,*†§ Fanny Tabarin,*†§ Mathieu Jablonski,*†§

Christophe Hue,*†§ Richard J. Smith,** Marina Noris,†† Lise Halbwachs-Mecarelli,*†§

Roberta Donadelli,| Veronique Fremeaux-Bacchi,*‡ and Lubka T. Roumenina*†§

*Institut National de la Santé et de la Recherche Médicale UMRS 1138, Cordeliers Research Center, Complement andDiseases Team, Paris, France; †Université Paris Descartes Sorbonne Paris-Cité, Paris, France; ‡Assistance Publique—Hôpitaux de Paris, Service d’Immunologie Biologique, Hôpital Européen Georges Pompidou, Paris, France;§Université Pierre et Marie Curie (Paris-6), Paris, France; |IRCCS—Istituto di Ricerche Farmacologiche Mario Negri,Clinical Research Center for Rare Diseases Aldo e Cele Daccò, Ranica, Bergamo, Italy; ¶Molecular Medicine, StephanAngelov Institute of Microbiology, Bulgarian Academy of Sciences, Sofia, Bulgaria; **Molecular Otolaryngology andRenal Research Laboratories and Rare Renal Disease Clinic, Departments of Pediatrics and Internal Medicine, CarverCollege of Medicine, University of Iowa, Iowa City, Iowa; and ††Laboratory of Immunology and Genetics ofTransplantation and Rare Diseases, Mario Negri Institute for Pharmacological Research, Ranica, Bergamo, Italy

ABSTRACTAtypical hemolytic uremic syndrome (aHUS) is a genetic ultrarare renal disease associatedwith overactivationof the alternative pathway of complement. Four gain-of-function mutations that form a hyperactive orderegulated C3 convertase have been identified in Factor B (FB) ligand binding sites. Here, we studied thefunctional consequences of 10 FB genetic changes recently identified from different aHUS cohorts. Usingseveral tests for alternativeC3andC5convertase formationand regulation,we identifiedtwogain-of-functionand potentially disease-relevant mutations that formed either an overactive convertase (M433I) or aconvertase resistant to decay by FH (K298Q). One mutation (R178Q) produced a partially cleaved proteinwith no ligandbindingor functional activity. Sevengenetic changes led tonear-normaloronly slightly reducedligand binding and functional activity compared with the most common polymorphism at position 7, R7.Notably, noneof thealgorithmsused topredict thedisease relevanceofFBmutations agreedcompletelywiththe experimental data, suggesting that in silico approaches should be undertaken with caution. These data,combinedwithpreviouslypublished results, suggest that9of15FBgenetic changes identified inpatientswithaHUS are unrelated to disease pathogenesis. This study highlights that functional assessment of identifiednucleotide changes in FB is mandatory to confirm disease association.

J Am Soc Nephrol 25: ccc–ccc, 2014. doi: 10.1681/ASN.2013070796

Atypical hemolytic uremic syndrome (aHUS) is arare renal thrombotic microangiopathy diseasecharacterized by mechanical hemolysis, plateletconsumption, and acute kidney failure.1 aHUS isassociated with genetic abnormalities in the pro-teins of the alternative complement pathway2 aswell as anti–Factor H autoantibodies.3 The muta-tions may induce either loss of function of key com-plement regulators (such as Factor H [FH], FactorI, or membrane cofactor protein [CD46]) or over-activity of the C3 convertase components C3 andFactor B (FB). Anti-C5 blocking antibody

Received July 28, 2013. Accepted January 17, 2014.

M.C.M. and L.V. contributed equally to this work.

Published online ahead of print. Publication date available atwww.jasn.org.

Correspondence: Dr. Lubka T. Roumenina, Cordeliers ResearchCenter, Institut National de la Santé et de la Recherche MédicaleUMRS 1138, Complement and Diseases Team, 15 rue de l’Ecolede Medecine, entrance E, fl. 3, 75006 Paris, France. Email: lubka.roumenina@crc.jussieu.fr

Copyright © 2014 by the American Society of Nephrology

J Am Soc Nephrol 25: ccc–ccc, 2014 ISSN : 1046-6673/2509-ccc 1

Eculizumab has revolutionized themanagement of aHUS, giv-ing good results in patients with and without known comple-ment genetic abnormality.4,5 Nevertheless, knowledge of theunderlining genetic defect and its functional consequences isof importance for patients’management, especially when the de-cision for transplantation should be made.5–7 Routine screeningof the aHUS susceptibility genes led to the identification of a largenumber ofmutations and rare variants. In-depth functional anal-yses are required to predict if the genetic change is related to thedisease manifestation or just a fortuitous association. The major-ity of tested mutations had, indeed, a functional defect.2 Never-theless,fivemutations found in aHUS patients—onemutation inFB,8 one mutation in Factor I,9 two mutations in FH,10 and onemutation in decay accelerating factor (CD55)11—were reportedto lack a functional defect, and the link with complement dysre-gulation for them remains unclear.12 Experimental verification offunctional consequences of amutation is a time-consuming pro-cess. Therefore, there is a need to develop reliable in silico algo-rithms, validated with experimental data, to predict rapidlywhether the discovered genetic change is pathogenic or not.

FBmutations in aHUS are rare, with a frequency of 0%–4%of the patients from different cohorts.13–16 To date, 15 FBgenetic changes have been published8,13,15,17–21 (Table 1), butfunctional data are reported for only 5 of them.8,17,19 FB formsthe alternative pathway C3 and C5 convertases interacting with

two partners (C3b and Factor D) and cleaves two substrates (C3and C5). The structures of most of these complexes areknown,22–24 and functional tests studying C3b binding and theactivity of the C3 and C5 convertases are available.

Four aHUS FBmutations resulted in the formation of eitherhyperactive C3 convertase or a convertase unsusceptible tocomplement regulation mechanisms.17,19 In contrast, two FBpolymorphisms at position 7 (R7Q and R7W) were found tobe hypoactive compared with the most frequent FB variantR7.25 The functional activity of FB, between R7 and R7Q var-iants, determines the arbitrary normal range.25

Theobjective of this studywas to characterize the functionalconsequences of 10 newly identified FB genetic changes (Table1) and understand whether and how they are related to aHUS.We sought to compare the in silico algorithms to predict theoutcome of a given genetic change, searching for one thatshows a good agreement with experimental data.

RESULTS

FB Genetic Changes in the Normal PopulationIn the 1000 Genomes database,26 32 polymorphisms of FBhave been reported (Supplemental Figure 1). Among them,three have been identified in aHUS patients (I217L

Table 1. Description of the identified FB mutations in aHUS patients

Amino AcidChange

CohortNo. ofPatients

C3 LevelsPresence of OtherGenetic Change

Variant atPosition 7

Source

Mutations (previousstudies)

D254G France 2, family Low No RR/RW 19

F261L Spain/UnitedStates

7 (family)+1 Low/NA 1, ADAMTS13, V832M rarevariant (United States)

NA/RR 17,21

K298E Spain 1, de novo Low No NA 17

K325N France 1, de novo Low No RW 19

L408S Sweden/Italy 1+1 Low/low 1, FI p.G261D (Sweden) RR/RR 8, this studyMutations (this

study)R113W Italy 1 Low No RR 18

S141P United States 1 NA 1, FI p. Y369S RR 15

R178Q United States 1 Low No RR 15

K298Q United States/United Kingdom

1+1 NA/low 1, MCP p. A304V, rarevariant (United States)

RR/NA 15,45

P344L France 1 Normal TM P501L, rare variant RQ 13

V430I France 1 Normal FI pY459S, rare variant RW 13,38,46

M433I United States 1 NA FH p.T956M, rare variant RR 15

PolymorphismsI217L France/United

States1+1 Normal/NA No/1 FH p.Q950H, rare

variantRR/RR 13,15

K508R United States 3 Low, normal, NA 1, no NA, RR, RR 15,20

1, FI p. H138R1, del CFHR3-CFHR1

E541A United States 1 NA 1, ADAMTS13 R7W variant NA 21

RR, homozygous for arginine (R); RW, heterozygous for arginine and tryptophan;NA, not available; FI, Factor I;MCP,membrane cofactor protein; TM, thrombomodulin;RQ, heterozygous for arginine and glutamine.

2 Journal of the American Society of Nephrology J Am Soc Nephrol 25: ccc–ccc, 2014

BASIC RESEARCH www.jasn.org

rs144812066, K508R rs149101394, and E541A rs80318145)and designated as mutations in the original reports, becausethey were not found in the control groups. Their frequenciesin the normal population are 0.001, 0.002, and 0.006, respec-tively (Table 2, Supplemental Figure 1). The previously de-scribed mutation K298E17 was also reported in this databaseas an extremely rare variant without reported frequency(rs121909748).

Structure DescriptionAll identifiedmutationsweremappedon the structuresofFB,23

C3bBD,22 and C3bBb24 (Figure 1, Tables 2 and 3). Three mu-tations (R113W, S141P, and R178Q) are in the Ba complementcontrol protein domains. These residues are released with theBa fragment after FB cleavage and absent in the active conver-tase C3bBb. The rare variant I217L is located in the linkerbetween Ba and Bb fragments near the R234–K235 scissilebond.

Five mutations are in the von Willebrand factor type Adomain,which is responsible for thebinding toC3b.V430I andM433I are close to the Mg21 adhesion site. One mutation(K298Q) affects the same residue as the previously publishedmutation K298E.17 P344L is far from any known binding siteand located in a flexible proline/tryptophane-rich loop.

The last two variants (K508R and E541A) are in the serineprotease domain of FB—on the same surface but not close tothe catalytic triad Asp551, His501, and Ser674.

Expression of Recombinant FBAll recombinant proteins (except one) were generated andexpressed successfully, with the expectedmolecular weight andconcentration similar to the wild-type (WT) (SupplementalFigure 2). Obtained functional results are summarized in Ta-ble 2. R178Qwas secreted as a partially cleaved protein by bothhuman embryonic kidney 293T and Chinese hamster ovarycells (Supplemental Figure 2A).

Binding to C3bThe interaction between recombinant FB and its ligand C3bwas assessed by ELISA and in real time by surface plasmonresonance (SPR). The gain-of-function mutation D254Gserved as a positive control for increased binding. The bindingof recombinant R7Q and R7W to C3b was about 40%–50%weaker than theWTas measured by both techniques (Figure 2).The binding variation between WT R7 and R7Q (lowest bind-ing allotype) with C3bwas considered as the normal range. TheELISA analysis did not show significant increase of the bindingof any of the newly characterized mutations. The SPR interac-tion results (Figure 2) showed that only M433I led to a mildincrease (1.38-fold increase) of C3b binding affinity (far belowthe 5.4-fold increase observed with the positive controlD254G). S141P showed faster association but also faster disso-ciation of the formed complex. The remaining mutations werewithin the normal range as SPR curve profiles (Figure 2, B–E)and binding affinity (Figure 2F). For R178Q, which was

produced partially cleaved, no binding to C3b was detectedby these techniques.

C3 Convertase Formation and DissociationFive different tests were used to monitor the formation and/orthe dissociation of alternative pathway C3 and C5 convertases(Figures 3 and 4).

The formation of (C3bBb-Mg2+) C3 convertase was as-sessed in real time by SPR (Figure 3) and the combination ofELISA and Western blot27 (Figure 4A). The formation of theC3/C5 convertase on cell surface (Figure 4, B–E) and the dis-sociation of the C3 convertase on cell surface by FH (Figure 4F)were measured by hemolytic tests. The positive control inthese tests was the gain-of-function mutation D254G,17,19,28

which indeed, induced stronger formation of C3 convertaseby SPR (Figures 3A and 4A) and enhanced erythrocytes lysis(Figure 4B) compared with the WT and formed a convertaseresistant to dissociation by FH (Figure 4F). Also, K298E,known to form a convertase resistant to decay by FH, was in-cluded as an additional control.17 The resistance to dissociationby FH of this mutation was confirmed by the hemolytic assay.The SPR test showed only very mild perturbation, potentiallybecause of a weaker sensitivity of this assay. As expected, theR7Q polymorphism was hypoactive in terms of formation ofthe C3 and C3/C5 convertases (Figures 3A and 4, A and B), andthe C3 convertase was decayed normally (Figures 3A and 4F).No functional activity was detected for the supernatant frommock-transformed cells.

Themutations couldbe classified infive groups according totheir functional behavior (Table 2): (1) gain of formation ofthe C3 convertase by SPR and ELISA/Western blot assays(S141P, I217L, D254G, M433I, and K508R); (2) gain of for-mation of the C3/C5 convertase by hemolytic test (D254G andM433I); (3) resistance of the convertase to decay by FH(D254G, K298E, and K298Q); (4) lack of activity (R178Q);and (5) activity within the normal range (R113W, P344L,V430I, and E541A).

Complement Activation on Endothelial CellsFACS analyses allowed estimation of the deposition of C3fragments on endothelial cells (human umbilical cord veinendothelial cells [HUVECs]) and, thus, the alternative pathwayactivation in FB-depleted serum reconstituted with recom-binant FB (Figure 5, Table 2). Reduced C3 deposition wasdetected for R7Q and R7W compared with WT (R7), thusallowing for establishment of a normal range.

On resting HUVECs, only the positive control D254Gshowed increased C3 deposition, which was similar to the oneobserved when a blocking anti-FH antibody was added toserum (Figure 4A). On activated and heme-exposed cells, in-creased deposition was measured for the blocking anti-FHantibody, D254G, as well as K298E, K298Q, and M433I (Fig-ure 5, B and C). E541A showed increased C3 deposition onactivated HUVECs but not on heme-exposed HUVECs (Fig-ure 5, B and C). R178Q had no functional activity in these tests

J Am Soc Nephrol 25: ccc–ccc, 2014 aHUS Complement Factor B Mutations 3

www.jasn.org BASIC RESEARCH

Table

2.Su

mmaryof

thefunc

tion

alco

nseq

uenc

esof

stud

iedFB

mutations

based

onthis

stud

yan

dpreviou

sreports

AminoAcid

Cha

nge

FBDomain

FBFrag

men

tIntegrity

onWB

C3Binding

(ELISA

)C3Binding

(SPR)

SPRan

dIn-Plate

C3

Conv

ertase

Assembly

Surfac

eC3/C5

Conv

ertase

(Hem

olytic)

Surfac

eC3

Conve

rtase

Dec

aybyFH

(Hem

olytic)

Res

ting

HUVECs

Activated

HUVECs

Hem

e-Exp

ose

dHUVEC

Phe

notype

R113

WCCP1

Ba

Intact

,WT

,WT

NA

,WT

NA

,WT

,WT

,WT

Ben

ign

S141

PCCP2

Ba

Intact

=WT

.WT

.WT

,WT

NA

,WT

,WT

=WT

Partial

R178

QCCP3

Ba

Cleav

edNobinding

Nobinding

NA

No

NA

,WT

,WT

,WT

Unc

lear

I217

LLink

erBa

Intact

=WT

=WT

.WT

,WT

=WT

,WT

,WT

,WT

Partial

K29

8QVWA

Bb

Intact

=WT

=WT

NA

=WT

,WT

=WT

.WT

.WT

Com

plete

P344

LVWA

Bb

Intact

=WT

,WT

=WT

,WT

=WT

,WT

=WT

,WT

Ben

ign

V43

0IVWA

Bb

Intact

,WT

,WT

NA

,WT

=WT

,WT

,WT

,WT

Ben

ign

M43

3IVWA

Bb

Intact

=WT

.WT

NA

.WT

=WT

=WT

.WT

.WT

Com

plete

K50

8RSP

Bb

Intact

,WT

=WT

.WT

=WT

=WT

,WT

,WT

,WT

Partial

E541

ASP

Bb

Intact

=WT

=WT

NA

=WT

=WT

=WT

.WT

=WT

Partial

D25

4Ga

VWA

Bb

Intact

.WT

.WT

.WT

.WT

,WT

.WT

.WT

.WT

Com

plete

K29

8EVWA

Bb

Intact

=WT

=WT

NA

=WT

,WT

=WT

.WT

.WT

Com

plete

F261

LbVWA

Bb

Intact

NA

.WT

NA

NA

NA

NA

NA

NA

Com

plete

K29

8Eb

VWA

Bb

Intact

NA

=WT

NA

NA

NA

NA

NA

NA

Com

plete

K32

5Na

VWA

Bb

Intact

.WT

.WT

NA

.WT

,WT

.WT

NA

NA

Com

plete

L408

ScVWA

Bb

Intact

,WT

,WT

NA

=WT

=WT

=WT

,WT

NA

Ben

ign

Thedataforthe

SPRC3co

nvertase

dec

ayshowed

differen

cesco

mpared

with

WTon

lyforD

254G

.Resultsarefrom

thisstud

yon

lyun

less

othe

rwiseno

ted.W

B,W

estern

Blot;CCP,

complemen

tcon

trolp

rotein;

VWA,v

onWilleb

rand

factor

typeAdom

ain;

SP,serineproteasedom

ain.

a Results

arefrom

boththisstud

yan

dref.19

.bRe

sults

arefrom

ref.17

.c Results

arefrom

ref.8.

4 Journal of the American Society of Nephrology J Am Soc Nephrol 25: ccc–ccc, 2014

BASIC RESEARCH www.jasn.org

(Figure 5, A–C). The remaining mutations showed either nor-mal or reduced C3 deposition but were always in the normalrange.

Terminal complement C5b-9 deposition can be measuredon apoptonecrotic HUVECs when incubated with normalsera.19,29 Therefore, apoptonecrotic HUVECs were used as amodel surface to study the activity of the alternative pathwayC5 convertase in the presence of FBmutations. Because the Bafragment does not participate in the C5 convertase activity,only the mutations within the Bb part as well as I217L weretested (Figure 5D). The two known gain-of-function muta-tions (D254G and K298E) as well as K298Q and M433I re-sulted in a significantly stronger C5b-9 deposition, whereas theremaining tested mutations showed levels similar to the WT.

Prediction for the Damaging Probability of FBMutationsFourdifferent algorithmswere used to calculate theprobabilitythat each mutation will induce alterations of the proteinstructure and function (Table 3). The relation of the mutatedposition to an interaction site was used as an additional pre-dictor. The consistency of predictions by those five methodswas measured using the Cronbach’s a-coefficient30

(Supplemental Table 1) (a=0.6576). It shows only moderateagreement between themethods. Calculating Cronbach’sa forall combinations of the four methods shows that MutationTaster’s disagreement with the rest is the greatest. Omittingit brings the criterion (a=0.8053) to a level indicating highconsistency. Using PolyPhen, only 4 of 15 genetic changes werepredicted to be damaging, contrary to Mutation Taster andSorting Intolerant from Tolerant (SIFT), which predicted 8 of14 and 10 of 15 changes as damaging, respectively. Align-Grantham variation (GV)/Grantham deviation (GD) re-turned 6 of 15 damaging scores. Although the GV/GD, SIFT,and interaction site involvement algorithms correlated wellwith the functional assay results, PolyPhen and MutationTaster showed insignificant or no correlation (Table 4). Com-bining these results with the analysis of agreement led to theconclusion that Mutation Taster does not contribute substan-tial information. However, the other four methods seem tocomplement each other. To make use of this fact, a consensusscore was defined as the number of methods scoring positivefor each mutation (score=0–4). The performance of this scoreand the optimal cutoff for classifying the mutations were es-timated using receiver operating characteristic curve analysis(Supplemental Figure 3). The area under the curve was 0.92(95% confidence interval, 0.66 to 1.00; P,0.001). Optimalclassification was achieved with a cutoff of consensus score.1(sensitivity=85.7%; specificity=87.5%). This yielded one falsepositive case and one false negative case. Interestingly, theconsensus score outperformed the othermethods and equaledthe binding site involvement (Table 4).

Similar results were obtained for 17 mutations in theC-terminal 19 and 20 domains of FH, for which the functionaldefect was well characterized31–33 (data summarized in ref. 2)(Supplemental Table 2).

Frequency of R32Q and R32W in aHUS Patients andHealthy DonorsThe position 7-based alleles were studied for aHUS patients ofthe French aHUS cohort and French healthy donors. Healthydonors presented the allele frequency expected for the Cau-casian population (Table 5). When the aHUS patients allelefrequencies were compared with healthy donor frequencies,there was no significant difference.

DISCUSSION

Identification of a genetic abnormality in aHUS patients is ofimportance for selection of a therapeutic regimen and trans-plantation decisions. The mutations in FH, C3, or FB, forwhich a clear functional consequencehasbeen identified, affectthe formation and/or the regulation of the C3 convertase.These defects translate to overactivity of the terminal com-plement pathway. As a result, the presence of FHmutations inpatients’ sera induces a spontaneous lysis of sheep erythro-cytes,34,35 and the presence of FB or C3 mutations causes

Figure 1. Visualization of the complex between C3b and FB andthe positions of studied mutations. The three FB domains arerepresented as follows: complement control protein domains inred, the von Willebrand factor type A domain in cyan, and theserine protease domain in green, with the corresponding newmutations in magenta and previously characterized mutations inblue. C3b is in gray. The approximate position of the commonpolymorphism at position 7 (not present in the crystal structure) isrepresented in orange by the first crystallized amino acid at po-sition 10. The residues forming the catalytic triad are depictedwith green spheres.

J Am Soc Nephrol 25: ccc–ccc, 2014 aHUS Complement Factor B Mutations 5

www.jasn.org BASIC RESEARCH

Table

3.In

silicoan

alyses

fortheFB

gen

etic

chan

ges

andtheirco

rrelations

withpatients’

C3leve

lsan

dob

served

func

tion

aldefec

t

Num

bering

Witho

uttheSigna

lPep

tide

Prese

ncein

1000

Gen

omes

Datab

ase;

Freque

ncy

Other

Variant

oftheSa

meRes

idue

Foun

din

1000

Gen

omes

;Freque

ncy

PolyPhe

nHum

Div

Score

SIFT

Score

GV/G

DSc

ore

Mutation

Taster

Score

Interaction

Site

Cons

ens

usSc

ore

(PolyPhe

n+SIFT

+GV/G

D+

InteractionSite)

C3Consu

mption

inPatients

Func

tiona

lDefect

Com

plete

phe

notype

D25

4GD25

4E;0

,000

Ben

ign

Deleterious

65Disea

se-cau

sing

Yes

1Ye

sYe

sF2

61L

Prob

ably

dam

aging

Deleterious

15Disea

se-cau

sing

Yes

1Ye

sYe

s

K29

8EYe

s;—

Ben

ign

Deleterious

15Po

lymorphism

Yes

1Ye

sYe

sK29

8QBen

ign

Deleterious

15Po

lymorphism

Yes

1Ye

sYe

sK32

5NProb

ably

dam

aging

Deleterious

65Po

lymorphism

Yes

1Ye

sYe

s

M43

3IBen

ign

Deleterious

0Disea

se-cau

sing

No

0NA

Yes

R178

QProb

ably

dam

aging

Deleterious

0Disea

se-cau

sing

No

1Ye

sYe

s

Inco

mplete

phe

notype

S141

PBen

ign

Tolerated

0Po

lymorphism

No

0NA

Partial

I217

LYe

s;0,

001

Ben

ign

Tolerated

0Disea

se-cau

sing

No

0No

Partial

K50

8RYe

s;0,

002

Ben

ign

Tolerated

0Po

lymorphism

No

0No

Partial

E541

AYe

s;0,

006

Ben

ign

Tolerated

0Po

lymorphism

No

0NA

Partial

With

intheno

rmal

rang

e(ben

ign)

L408

SPo

ssibly

dam

aging

Deleterious

65Disea

se-cau

sing

No

1Ye

sNo

R113

WR1

13Q;0

,001

Ben

ign

Deleterious

0Disea

se-cau

sing

No

0Ye

sNo

P344

LP3

44A;—

Ben

ign

Deleterious

0Po

lymorphism

No

0No

No

V43

0IV43

0V;0

,018

Ben

ign

Tolerated

0Disea

se-cau

sing

No

0No

No

IntheGV/G

Dco

lumn,

0indicates

nopredicteddefec

t,an

danu

mber$15

predictsfunc

tiona

ldefec

t.Intheseman

ticsof

MutationTa

ster,p

olym

orphism

mea

nsmutationwith

out

predictedfunc

tiona

lsignificanc

e.In

theco

nsen

susscore,o

neindicates

that

twoor

morealgorith

mssuggestedafunc

tiona

ldefec

t,an

dzero

indicates

that

less

than

twoalgorith

mspredictedaffected

func

tions.

6 Journal of the American Society of Nephrology J Am Soc Nephrol 25: ccc–ccc, 2014

BASIC RESEARCH www.jasn.org

enhanced C5b-9 deposition of apoptonecrotic endothelialcells.19,29 The pathogenic role of C5 convertase overactivationalso explains the therapeutic efficacy of Eculizumab in aHUS.Eculizumab leaves the overactive C3 convertase untouchedbut blocks the C5 cleavage.5 Therefore, an FBmutation shouldbe defined as gain of function when it is able to form an over-active C3/C5 convertase or a C3 convertase resistant to decayby the regulators that is strong enough to translate the func-tional defect to the level of C5 cleavage on a cell surface. Gain-of-function FB mutations were reported in 12 patients fromfive families and are associated with a severe outcome, leading

to ESRD in the majority of the ca-ses.13,15,17–19,21 Therefore, the presence ofa FB mutation is considered as a bad prog-nosis for the disease. C3 levels are fre-quently below the normal range in thesepatients, suggesting active complementconsumption in the plasma caused by C3convertase overactivity.

Theaimof thisworkwas tounderstand ifthis phenotype is shared between all iden-tified FB genetic abnormalities and find outwhether other FB mutations could becausative factors for aHUS.

We used 10 different binding and func-tional tests for the formation of the C3 andC3/C5 convertases and the resistance of theC3 convertase to dissociation by FH toclassify 10 previously uncharacterizedaHUS-associated genetic changes as poten-tially disease-causing or benign.

We confirmed the previous reports thatD254G and K298E are gain-of-functionmutations forming overactive C3 conver-tase and/or convertase resistant to decay byFH.16,18 A similar phenotype is shared bytwo newly characterizedmutations (M433Iand K298Q). K298Q (removal of positivecharge) had a milder effect compared withK298E (exchange of a positive chargewith a negative charge). This result suggeststhat the positive charge at this position is ofcritical importance for the competition be-tween FH and FB, which is in agreementwith the structural data.36 In fact, all gain-of-function mutations appeared to be di-rectly located within the C3b binding site(D254G and K325N) or buried beneath it(F261L) or located in the area importantfor the electrostatic repulsion between FBand FH. In agreement with the experimen-tally determined functional consequences,C3 levels were low in all patients with thesemutations for which data were available,indicating alternative pathway consump-

tion in vivo. Therefore, low C3 levels in the patient togetherwith amutation localized in an important interaction site are astrong indication that themutationmay be a gain-of-function.Nevertheless, for three patients from this study (R113W,R178Q, and K508R) and two unrelated patients with a pre-viously published mutation (L408S), the low C3 levels couldnot be explained by a gain of function of the respective FBmutation.

The mutation R178Q showed complete lack of functionalactivity and was produced as a cleaved recombinant protein.Unfortunately, no plasma samples were available from this

Figure 2. Binding of FB to its ligand C3b. (A) As tested by ELISA in the presence ofMg2+. The values are reported as fold of the WT, and the normal range (between WTand the R7Q polymorphism) is depicted with dashed and continuous lines (n=3–6).(B–E) As tested by SPR in the presence of Mg2+. The binding of the mutant FB to C3bimmobilized to a biosensor chip (ProteOn XPR36) was measured. (B) WT was com-pared with the normal polymorphisms W7 and Q7 and the known gain-of-functionmutation D254G. (C–E) The mutations tested in this study are compared with the WT.One representative experiment of three performed is shown. (F) The binding affinity(KA) represented as fold difference compared with WT (1.0). Of note, all mutations areintroduced on a WT FB with R at position 7. *P,0.05; **P,0.001, Mann–Whitneynonparametric test calculated with GraphPad Prism 5. CCP, complement controlprotein; SP, serine protease; vWF, von Willebrand factor.

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patient to test if it happens in vivo or is an in vitro artifact. Itcould be hypothesized that intrinsic propensity for cleavagecould facilitate the in vivo transition between C3 proconvertaseC3bB and active convertase C3bBb, whichwould explain the C3consumption found in the patient.

Three genetic changes (R113W, P344L, and V430I) hadeither normal or slightly reduced C3b binding and convertaseformation; the formedconvertasewasdecayednormallybyFH,and the C3 fragment deposition on endothelial cells was in thenormal range. This benign phenotype could be because of theirlocalization distant from known binding sites and explainswhy, in two of three cases, they were found in patients withnormal C3 levels.

Four genetic changes (S141P, I217L,K508R, and K541A) had an incompletephenotype. One or more performed testsshowed increased binding or function, butthe results were inconsistent between thedifferent approaches. Moreover, neither ofthese mutations resulted in concomitantincrease of the C3 fragments deposition onactivated and heme-exposed endothelialcells and enhanced C5b-9 deposition onapoptonecrotic cells. Therefore, they do notfulfill the criteria set to consider a geneticchange as a gain of function. Among thecarriers of these genetic changes for whomdata are available, only one patient, withK508R, had low C3 levels. Another patientwithK508RhadnormalC3 levels, suggestingthat this genetic change is not necessarilyresponsible for the C3 consumption. There-fore, the functional consequence of thesegenetic changes, if any, is mild and less likelyto predispose directly to the aHUS patho-genesis. In addition, in two of these patients,additionalFactor Imutationswere identified,which could be the disease-predisposingfactor rather than the genetic change in FB.

When an identified genetic change in acohort is not found in the correspondinghealthy controls, 1000 Genomes and otherdatabases for genetic variants should beconsulted to exclude that the identifiedgenetic change is just a rare variant un-related to the disease, which seemed to bethe case of I217L, K508R, and E541 de-scribed here. The incomplete phenotypeobserved for these polymorphisms suggeststhat they may represent risk factors affect-ing the aHUS penetrance and severity butcannot be considered as disease-causingmutations. Studieswith large aHUScohortsand ethnically matched healthy donors areneeded to establish if the frequency of these

polymorphisms is increased in the patients compared withhealthy controls.

The fact that a mild reduction of C3b binding and FBfunctional activity was detected for certain mutations is in-triguing. The two normal variants, Q7 andW7, are reported25

and confirmed here to be hypoactive compared with R7.Therefore, we sought to investigate if FB hypoactivity is as-sociated with aHUS. No difference was found in the frequencyof R7, Q7, and W7 in the French cohort of adult aHUS pa-tients and French healthy volunteers, suggesting that the FBhypoactivity is not associated with this disease. Moreover,these polymorphisms did not differ among aHUS patientsand healthy donors in a study of 501 complement-related

Figure 3. Formation and dissociation of the C3 convertase by FH studied in real timeby SPR. Recombinant mutant FB and plasma-purified Factor D (FD) were mixed in anMg2+-containing buffer and injected over a C3b-coated biosensor chip. After 540seconds of spontaneous dissociation, FH was injected to dissociate the convertase.The binding of FH to immobilized C3b was measured in parallel and subtracted fromthe signal obtained in the channels with the convertase. In every run, four mutantswere compared with the WT, and the sixth channel served as a reference for the FHbinding. (A) The formation and dissociation of the convertase formed with the positivecontrol D254G and the R7Q polymorphism were compared with the WT. (B–E) Theremaining FB mutations were tested.

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single-nucleotide polymorphisms in 47 complement genes.37

Of note, R7 is the most frequent polymorphism at this posi-tion, most of the FB mutations in the patients are found on R7background, and the recombinant WTused in this study car-ries R at position 7. The observed magnitude of functionchange of R113W, P344L, L408S, and V430I compared with

the hypoactive variants is small and not atthe level that we would expect to causeaHUS on its own.

Based on our experimental data, it couldbe concluded that eight newly characterizedFB genetic changes are most likely benignvariants rather than disease-associatedmutations. Interestingly, in 10 of 28 pa-tients, the FB genetic changes were asso-ciated with rare variants or mutationswithout identified functional consequen-ces in the other tested complement genesor ADAMTS13 (a disintegrin and metal-loproteinase with a thrombospondin type1 motif, member 13). At the opposite ofour series, most of the aHUS patients in arecently described subgroup with com-bined mutations have at least one func-tionally relevant genetic change.38 Thisinformation is critical for analyses of thephenotype of patients and prediction ofthe survival or the outcome of a particulartherapeutic regimen.

Functional studies are frequently un-available to inform physicians of the pos-sible relation of a newly identified geneticchange with the disease. Therefore, bioin-formatics tool(s) could be useful to get aprediction for themutations role and guidethe treatment decision. Unfortunately, thefour prediction algorithms tested hereagreed for only about one third of themutations and gave discordant data for therest. Performed statistical analyses sugges-ted that none of these algorithms could beused confidently as a stand-alone approachbecause of incomplete agreement with theexperimental data. The prediction effi-ciency was improved when the worse-performing method (Mutation Taster)was excluded, and a consensus score wascalculated for the remaining three methods(PolyPhen, SIFT, and Align-GV/GD). Add-ing to this score the informationonwhetherthe genetic change falls within a knownfunctional site (i.e., the area of themoleculeimportant for ligands binding or func-tional activity) substantially improves theprediction. Nevertheless, the bioinformat-

ics evaluation of the potential functional effect of a mutationshould be takenwith caution, because even the improved scoreproposed here gave one false positive and one false negativeresult for 15 analyzed FB mutations. Similar results were ob-tained for the mutations in the C terminus of FH, well knownfor their functional defect and association with aHUS.2,31–33

Figure 4. Functional activity of FB mutations. (A) C3 convertase formation assay basedon ELISA/Western blot. C3 convertases C3bBb were formed in C3b-coated microtiterwells by incubation with FB and FD in the presence of Mg2+. C3 convertase formationwas evaluated by the visualization of the Bb band. The intensity of the Bb band wasquantified, and the results are presented as percentage compared with the WT (n=2).(B–E) Hemolytic test for the functional activity of the cell surface-bound C3/C5 con-vertase formed by recombinant FB proteins. C3b-covered sheep erythrocytes areincubated with increasing concentrations of FB and a fixed FD concentration. Thenumber of lytic sites per cell (Z) was revealed by the addition of a source of terminalcomplement components (n=2–5). (F) Hemolytic test for the FH-induced dissociationof the cell surface-bound C3 convertase (C3bBb-Ni) formed by the WT or mutant FBon the surface of C3b-covered sheep erythrocytes. The results are presented aspercentages of the lysis obtained in the absence of FH (Z approximately 2; i.e., afterspontaneous dissociation of the formed C3 convertase; n=2–4).

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In conclusion, although some FB mutations induce com-plement overactivation and predispose to aHUS, others have anormal functional activity and hence, are benign and unlikelyto be related to the disease. In-depth screening of all suscep-tibility genes as well as for the presence of anti-FH antibodies is

necessary in all patients with identified FB mutations toidentify other abnormalities affecting the disease occurrence,especially in the presence of low C3 levels. The status of thegenetic change identified in aHUS needs to be carefullyanalyzed to prove the pathogenic role in the disease. We

Figure 5. Complement activation on endothelial cell surface. C3 deposition on (A) resting, (B) heme-exposed, or (C) TNFa/IFNg-activated HUVECs incubated with FB-depleted serum, reconstituted with the recombinant FB, and measured by flow cytometry (n=3–6). The C3 deposition from the WT was taken as one, and the values obtained for the remaining mutations were expressed as a folddifference compared with the WT. The supernatant from mock-transformed cells (white bar) is used as the negative control, and D254Gand WT+FH blocking antibody Ox24 (hatched bars) are used as positive controls. (D) C5b-9 formation on apoptonecrotic HUVECs.After incubation with FB-depleted serum reconstituted with recombinant FB, the cells were probed for C5b-9 deposition by flowcytometry (n=3). The C5b-9 formation from the WT was taken as one and compared with the tested mutations. *P,0.05; **P,0.001,Mann–Whitney nonparametric test calculated with GraphPad Prism 5.

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suggest that rare variants inwhich frequency is not increased inthe patients cohorts and mutations without functional con-sequences should not be included in the patient management.

CONCISE METHODS

In Silico AnalysesPyMol (http://www.pymol.org/) and Chimera software39 (http://

www.cgl.ucsf.edu/chimera) were used for structural data analyses.

The probability that a genetic change induces an alteration of the pro-

tein structure and function was calculated with the following software:

PolyPhen (http://genetics.bwh.harvard.edu/),40 Marinozzi_et_al_final.

doc, Align-GV/GD (http://agvgd.iarc.fr/),41 Mutation Taster (http://

www.mutationtaster.org/),42 and SIFT (http://sift.jcvi.org/).43 The

1000 Genomes database26 was searched for the presence of each iden-

tified FB genetic change to confirm the mutation status.

Thepairwise contingency tables significancewasdeterminedusing

Fisher’s exact test (P.0.05), and the correlation of the methods was

estimated using the correlation coefficient. To test the internal con-

sistency of the prediction by the battery of methods, the Cronbach’s

a-coefficient was calculated for all fivemethods together and all com-

binations of four methods to determine the contribution of each one

by omitting it. A consensus method was proposed based on the num-

ber of methods predicting functional effects of each mutation. Its

performance was tested against the in vitro-determined functional

effects using receiver operating characteristic curve analysis. Using

the Yuden J index, a cutoff was determined for the consensus score to

classify the mutations as deleterious. All calculations were performed

using MedCalc v12 software.

Recombinant FB Production and CharacterizationThe plasmid-containing recombinant FB construct and the muta-

genesis protocol were described previously.19 Proteins were expressed

in human embryonic kidney 293T cells after transfection with Gene

Juice lipotransfection reagent (Calbiochem). Serum-free superna-

tant, containing recombinant proteins, was harvested after 3 days

of culture. A detailed description is given in Supplemental Material.

The integrity of recombinant WT and FB mutants was tested by

Western blot (precasted gels and rapid transfer system iBlot by

Invitrogen). Proteins in the culture supernatants were separated by

SDS-PAGE, transferred to a nitrocellulose membrane, and probed

with an in-house biotinylated polyclonal sheep anti-human FB

antibody (Abcam, Inc.) followed by streptavidin–horseradish peroxy-

dase (Amersham) and ECL substrate (GE Healthcare).

The FB content was assessed by sandwich ELISA using immobi-

lized polyclonal sheep anti-human FB antibody (Abcam, Inc.) for

capturing and biotinylated sheep anti-human FB followed by

streptavidin–horseradish peroxydase (Dako) for detection as de-

scribed previously.19 Plasma-derived FB (Comptech, Tylor, TX)

served as a standard.

C3b Binding CharacterizationThe ELISA approach for C3b–FB interactions was performed as pre-

viously described.19

The interaction of WT and mutant FB with C3 was also analyzed

using SPR technology with ProteOn XPR36 equipment (Bio-Rad).

C3b was coupled to the GLC biosensor chip using standard amide-

coupling technology. The recombinantWTand mutant FB were used

as an analyte at concentrations 650, 325, 162.5, 81.25, and 40.625 pM

and injected at 50 ml/min in Mg2+-containing Hepes buffer (10 mM

Hepes [pH 7.4], 50 mM NaCl, and 10 mM MgCl2) over the C3b-

bearing surfaces and an empty activated/deactivated flowcell as a con-

trol. Data were analyzed using ProteOn Manager software, and the

data from the blank flowcell were subtracted. Kinetic parameters were

calculated by fitting the obtained sensorgrams into two state interac-

tion models as described.25

FB Functional AssaysFBhemolytic activity anddissociationof theC3 convertasebyFHwere

tested as described previously.19 In addition, C3 convertase (C3bBb-

Mg2+) formation assays, based on SPR17,25 or ELISA/Western blot,

were performed to assess the convertase formation by different ap-

proaches.27 A detailed description is given in Supplemental Material.

Endothelial Cell AssayResting, TNFa/IFNg-activated, heme-exposed, and apoptonecrotic

HUVECs were prepared as described19,29,44 and incubated with 50ml

FB-depleted serum (CompTech) and 100ml recombinantWTor mu-

tant FB supernatants containing equal amounts of FB. Cells were

labeled with anti-C3c (Quidel, SanDiego, CA), anti-C5b9 neoepitope

(a gift from Paul Morgan, Cardiff, UK), mAbs, or a control mouse

IgG1 followed by phycoerythrin-labeled secondary antibody

Table 4. Contingency table significance and correlationbetween different algorithms and experimental data

AlgorithmsFisher’s ExactTest, P Value

Correlation

Rho P Value

PolyPhen NS 0.343 0.21SIFT 0.03 0.661 0.01GV/GD 0.04 0.600 0.02Mutation Taster NS 0.071 1.0Functional site 0.01 0.756 0.001Consensus 0.01 0.732 0.002

NS, not significant.

Table 5. Frequencies of FB variants R7, Q7, and W7 inFrench aHUS patients and normal controls

GenotypeFrequency, %

RRn (%)

WWn (%)

QQn (%)

RQn (%)

RWn (%)

QWn (%)

Normaldonors (n=89)

64 (57) 1.1 (1) 0 (0) 16.9 (15) 12.4 (11) 5.6 (5)

Patients(n=167)

51.5 (86) 1.8 (3) 3.6 (6) 13.8 (23) 25.1 (42) 4.2 (7)

Allele Frequency, % R Q W

Normal donors (n=178) 78.7 11.2 10.1Patients (n=334) 71 12.3 16.5

R, arginine; W, tryptophane; Q, glutamine.

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(Beckman Coulter, Roissy, France) and analyzed by flow cytometry

on a Becton Dickinson Facscalibur (Mountain View, CA) using Cell-

Quest and FACS express software.

Genetic AnalysesGenomic DNA from each patient from the French adult aHUS cohort

or healthydonorswas obtained fromperipheral blood leukocytes. The

frequencies of R7, Q7, andW7 (rs12614 and rs641153) were assessed

by a direct sequencing of exon 2 of FB as described before for

mutations identification.19 Screened individuals signed an informed

consent, and the project was approved by the local ethical committee

according to the Declaration of Helsinki.

ACKNOWLEDGMENTS

Part of the cytometric analysis was done at the Centre d’Imagerie

Cellulaire et de Cytomètrie, Centre de Recherche des Cordeliers

UMRS 1138 (Paris, France). The Centre d’Imagerie Cellulaire et de

Cytomètrie is a member of the Université Pierre et Marie Curie Flow

Cytometry Network.

This workwas supported by grants from theAgenceNationale de la

Recherche (Grant 2009-2012 09geno03101I; Genopath), Assistance

Publique-Hôpitaux de Paris (Programme Hospitalier de Recherche

Clinique; Grant AOM08198), European Union FP7 (Grant 2012-

305608; EURenOmics), Association for Information and Research on

Genetic Renal Diseases (AIRG) France, and Institut National de la

Santé et de la Recherche Médicale. S.B. received a fellowship from

Fondazione Aiuti per la Ricerca sulle Malattie Rare (ARMR) (Italy),

M.N. was supported by Italian Telethon Grant GGP09075, and L.T.R.

was a recipient of European Molecular Biology Organization Long-

Term Fellowship ALTF 444-2007.

This work was presented as an abstract at the XXIV International

Complement Workshop in Crete, Greece, October 10–15, 2012.

DISCLOSURESNone.

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This article contains supplemental material online at http://jasn.asnjournals.org/lookup/suppl/doi:10.1681/ASN.2013070796/-/DCSupplemental.

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