Haemolytic Uraemic Syndrome

Marina Noris, PhD

London, September 2, 2014

HEMOLYTIC UREMIC SYNDROME

Incidence

(cases/100,000/year)

2

A multisystem disease of microangiopathic hemolytic anemia and thrombocytopeniawith predominant but not exclusive renal involvement

Overall: 0.5-2

Sex: no difference

STEC-associated HUS

E. coli O157:H7 produces Shiga-like toxin/verotoxin andcauses hemorrhagic colitis

3

ACUTE GLOMERULONEPHRITIS IN INFANCY

Two children and their male cousin who all died at 5

months of ageFison, ArchDisChild, 1956

4

Prevalence: 0.2-0.5/100,000 persons

Bergamo

Trento

Padova

Treviso

Vicenza

ParmaGenova

Torino

Pavia

Milano

Varese

MonzaBrescia

Firenze

Roma

UK9 cases

Switzerland21 cases Germany

18 cases

Denmark3 cases

Czech R.10 cases

Esthonia1 case

Serbia5 cases

Italy490 cases

Greece4 cases

Poland10 cases

Russia

5 cases

Participating Centers

aHUS patientsItalian cases

Foreign cases

180

805525 280

INTERNATIONAL REGISTRY OF HUS/TTP

05/2012

Roma

Foggia

BariSalerno

Reggio Calabria

Palermo

Cagliari

Sassari

USA58 cases

Argentina23 cases

Belgium

1 case

Israel14 cases

Portugal7 cases

Canada4 cases

South Africa2 cases

Saudi Arabia4 cases

490 cases

Australia13 cases

Malaysia2 cases

Turkey

7 cases

IRAN49 cases

Spain5 cases

UAE

1 caseIndia

3 cases

Japan

2 cases

Chile2 cases

http://villacamozzi.marionegri.it

(%)

P < 0.001

P = 0.005

100

20

40

60

80

10

20

Prevalence of low C3 Prevalence of low C4

(%)

6Noris et al., J Am Soc Nephrol, 1999

Cases Controls

Within families, subjects with lower than normal C3 serum levels had arelative risk of HUS of 16.5 as compared to subjects with normal C3 levels

Relatives

0

20

Cases Controls Cases Controls

Relatives

Cases Controls

0

10

(%)

COMPLEMENT ACTIVATION PATHWAYS

Lectin pathwayClassical pathway

Mannose

residues

IgM, IgG

Immune complexes

C1q,C1r,C1s

C4,C2

C4bC2a

MBL, MASP

C1-est inhib

C4bp

Factor H

Alternative pathway

bacteria, bacterial toxin,

LPS, tick-over

C3

C3b

C3a

C3bBb

Factor B

7

CR1

Factor I

DAF

MCP

CD59

C5a

C5b-9

C4bC2a

C3 convertase

C3

C3b

C3a

C4bC2aC3b

C5 convertase

C4bp

C5

C3bBb

C3 convertase

C3

C3b

C3a

(C3b)2Bb

C5 convertase

C3 convertase

C3

C3b

CFB

CFH compete with CFB for C3b binding

CFH dissociate Bb from C3 convertase accelerating its decay

CFH bound to C3b favors its inactivation (cofactor activity)

Alternative pathway activation(spontaneous hydrolysis,bacteria, viruses)

CFH CFH

8

C3a

Endothelial cell

CFB

C3b

CFB

C3b

Bb

CFI

iC3b

CFI

PUBLISHED DATA ON CFH MUTATIONS IN aHUS

Q1076E

D1119G

E1172stop W1183L W1183R T1184R L1189R L1189F

E850K Q950H Y951HT956M I970V W978C

1494delA

Q400K

371del2bp

S714stop

Regulatorydomain

C3b

Recognitiondoman

proteoglycansproteoglycans proteoglycansC3b C3b

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20

9

Warwicker and Goodship, Kidney Int, 1998

Caprioli et al., Hum Mol Gen, 2003

Richards et al., Am J Hum Gen, 2001

Perez-Caballero et al., Am J Hum Gen, 2001

Dragon-Durey et al., J Am Soc Nephrol, 2004

Caprioli et al., Blood, 2006

Noris et al, CJASN 2010

- Around 100 different mutations in 150 patients- Prevalence 30%- 90% are heterozygous- 70% of mutations affect the recognition domain

D1119GV1134G Y1142DW1157R C1163W 3559delA3566+1A

L1189F S1191L S1191W G1194D V1197A E1198A F1199S R1210C R1215G R1215Q P1226S 3559delA 3719delACA 3768delAGAA 3546-3581dup36 del24bp

2303insA

S890I H893R Y899D Y899stopC915S Q924stop C926F 279del15bp

W978C

C630W C673Y

Q400K

R60G

155delAGAA Y1021F C1043R 3103delG

SINGLE MUTATION CHANGES IN SCR 20 OF FACTOR HAFFECT BINDING TO CELLS AND DEPOSITED C3

Factor H

Factor HR1210C

co

Flow cytometry

of

cells

Factor H

10

Factor H

N°

of

cells

Manuelian et al., J Clin Invest, 2003

Factor H C3d

Factor H C3b

Factor HE1172Stop

C3d

Factor HE1172Stop

C3b

Factor H protein with the R1210C or E1172Stop mutations found in HUS inpatients showed reduced binding to endothelial cells and reduced interactionwith surface bound C3b and C3d

C3

C3b

CFB

spontaneous hydrolysis,bacteria, viruses

Fluid phase C3 convertase

CFB

C3b

Bb

CFI

iC3bCFH

CFH

CFH

11

C3a

Endothelial cell

CFB

CFI

iC3bC3b

CFB

Surface bound C3 convertase

C3b

Bb

ATYPICAL HEMOLYTIC UREMIC SYNDROME ASSOCIATEDWITH A HYBRID COMPLEMENT GENE

CFHCFHR3 CFHR1

Deletion

CFH/CFHR1 fusion protein

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19

1 2 3 4 5 1 2 3 4 5

5

A rare genomic rearrangement by NAHR between CFH and CFHR1 genes

12

Valoti et al., JASN, 2014

Venables et al., PLoS Med, 2006

Noris et al., CJASN, 2010

A rare genomic rearrangement by NAHR between CFH and CFHR1 genesgenerating a CFH/CFHR1 hybrid protein in which SCR 1-19 are from CFHand SCR 20 from SCR 5 of CFHR1

A more common rearrangement lead to a CFH/CFHR1 hybrid protein inwhich SCR 1-18 are derived from CFH and SCR 19/20 from SCR 4/5 ofCFHR1 by non allelic homologous recombination.

The protein products of the hybrid genes lack any complementregulatory activity on endothelial cell surface

A AHUS HYBRID CFHR1/CFH GENE ENCODING A FUSION PROTEIN THAT

ANTAGONIZES FACTOR H-DEPENDENT COMPLEMENT REGULATION

52y 49y

n.a.n.a.

Onset 49 yrHD 50yr

Valoti et al., JASN 2014

20y 16y

Onset 19 yrEculizumabremission

C3

C3b

CFB

spontaneous hydrolysis,bacteria, viruses

CFHCFH

14

C3a

Endothelial cell

CFB

CFI

iC3bC3b

CFB

Surface bound C3 convertase

C3b

Bb

CFHR1/CFHCFHR1/CFH

Valoti et al., JASN 2014

Blancet al., JImmunol, 2012

CFH1 2 3 4 98765 10 11 12 13 14 15 16 17 18 19 20

18/18 (100%)

1 2 3 4 5

CFHR1

● 8-10 % of aHUS patients (mainly children) develop anti-CFH autoantibodies

● Autoantibodies mimic the effect of CFH mutations, as they inhibit the regulatoryfunction of CFH at cell surfaces by blocking its C-terminal recognition region

● Most patients with aHUS and CFH autoantibodies are homozygous for a deletionof gene encoding CFHR1.

Failure of central and/or peripheral tolerance to CFHR1 in subjects with homozygous gene deletion?

Jozsi et al, Blood, 2008Moore et al, Blood 2010

T267fs270X

(858-872)del+D277N+P278S

F242C

SCRs

STP

TM

2

3

4

1

C35X

C35Y (n=3)

R59X (n=4)

48aa del

A353V

192T>C+193-198delC99R (96-129)del+G130I+Y132T+L133X

Y155D

C3

C3b

C3a

spontaneous hydrolysis,bacteria, viruses

MCP MUTATIONS IN aHUS

16

Endothelial cell

CFI

iC3b C3b

MCPCFI

C3b

TM

CT MCP 10%

Noris et al, Lancet 2003Richards et al, PNAS 2003Caprioli et al., Blood, 2006

Loss of function heterozygous mutations

C3a

CFB

FIMAC SRCR LRDR-1 LRDR-2 S-S SP

H Chain L Chain

T72SA240G G261D (n=2)

G349RI357MW399RL484V+Q485G+W486XE554VD519N

R317W

CFI 7%

CFI MUTATIONS IN aHUS

17

Endothelial cell

CFI

iC3b C3b

MCPCFI

C3b

Caprioli et al., Blood, 2006Fremeaux Bacchi et al., JASN 2007Kavanagh et al., JASN, 2005Noris et al., CJASN, 2010Delvaeye, et al, NEJM 2009

Loss of function heterozygous mutations

CFH

C3

C3b

CFB

MG2 MG3 MG4 MG5

MG

6β

LNK

β-chain α-chain

α’N

T

MG

6α

MG7

CU

B g

TED MG8AN

A

CU

B f

Anchor

C345C

R570W K1029M

R1041S

D1093N

I1135T

T1361MT140R

T140K Q163E

MG1

R456L

C3 7-10%

C3 MUTATIONS IN aHUS

18

Endothelial cell

CFB

CFI

iC3bC3b

CFB

C3 convertase

C3b

Bb

Goicoechea et al., PNAS, 2007Roumenina et al., Blood, 2009Fremeaux-Bacchi et al, Blood 2008

Gain of function C3 mutations: reduced binding to MCP,

increased affinity to CFB

MCP

C3a CFH

FIMAC SRCR LRDR-1 LRDR-2 S-S SP

H Chain L Chain

T72S G349R

Regulatorydomain

C3b

Recognitiondoman

C3bproteoglycansproteoglycans C3b proteoglycans

S890I (n=2)Y899XW920R 2759del15bp

479X

N516KQ950H I970V 1014X

Q1137LC1163W Dup(3546-3581)

E1172X (n=2)W1183R W1183XS1191L (n=7)G1194D V1197A (n=4)E1198A V1200LR1210C (n=9) R1215Q (n=2)R1215G3675-3699del

R78G

T267fs270X

(858-872)del+D277N+P278S

F242C

SCRs

STP

TM

CT

2

3

4

1

C35X

C35Y (n=3)

R59X (n=4)

48aa del

A353V

192T>C+193-198delC99R (96-129)del+G130I+Y132T+L133X

Y155D

CFH 25%

MCP 10%

COMPLEMENT GENE MUTATION IN aHUS PATIENTS

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20

19

T72SA240G G261D (n=2)

G349RI357MW399RL484V+Q485G+W486XE554VD519N

MG2 MG3 MG4 MG5

MG

6β

LNK

β-chain α-chain

α’N

T

MG

6α

MG7

CU

B g

TED MG8AN

A

CU

B f

Anchor

C345C

R570W K1029M

R1041S

D1093N

I1135T

T1361M

1 2 3

SCRslinker

Ba Bb

R138W

1 2 3

SCRslinker

VWA SP

R317W

T140R

T140K Q163E

CT

MG1

R456L

MCP 10%

CFI 7%

C3 7%

CFB 1%Anti-CFH Ab 8%

A43T

D53G

V81L

D486Y (n=4)

P495S

P501L

CT

Lectin-Like

domain

TME1

TME2

TME3

TME4

TME5

TME6

ST-rich

peptide

TM

V81L

TM 3%

#01 HUS

I

II F82F35F70

F83

*

INCOMPLETE PENETRANCE OF aHUS IN MUTATIONCARRIERS

20

III F34

4yr, dialysis

*

* R1215Q change in CFH

3 subjects in the III generation developed aHUS in infancy: 2 died, 1reached ESRD

F35 never developed aHUS

8.7% 8.2% 10%

COMBINED COMPLEMENT GENE MUTATIONS795 patients from 4 European cohorts

22.6% 27%

Bresin et al, JASN 2013

Combined complement gene mutations were found in about one quarterof patients with MCP or CFI mutations, more frequently than in patientswith CFH or C3 or CFB mutations

TRIGGERING /UNDERLYING CONDITIONS

% o

f H

US

pa

tie

nts

40

30

20

URT infectionDiarrheaPregnancyOthers (malignant hypertension,

glomerulopathy, de novo post-tx,

systemic diseases)

22

% o

f H

US

pa

tie

nts

CFH MCP CFI

0

10

C3

Noris et al, CJASN 2010

Gene mutations

C3 C5b-9

C3 AND MAC DEPOSITION IN ATYPICAL HUS KIDNEY

23

THROMBOTIC MICROANGIOPATHY

24

INCREASED VWF AND REDUCED THROMBOMODULIN ONMICROVASCULAR ENDOTHELIAL CELLS EXPOSED TO SERUM FROMaHUS PATIENTS, EFFECTS ANTAGONIZED BY AN ANTI-C5 ANTIBODY

VW

F e

xp

res

sio

n (

pix

el2

)

30000

40000

50000

60000

70000

60

80

100

120 *

Th

rom

bo

mo

du

lin

sta

inin

g(%

of

co

ntr

ol)

°

°

*

25

VW

F e

xp

res

sio

n

0

10000

20000

30000

control aHUS aHUS + anti-C5 Ab

Serum

Galbusera et al., Immunobiology 2012

0

20

40

control aHUSpatients

patients+ anti C5mbody

Th

rom

bo

mo

du

lin

sta

inin

g(%

of

co

ntr

ol)

°P<0.05 vs control serum*P<0.05 vs patients untreated

Serum

rinsed

HUS or control serum

whole blood(+ mepacrine)

4 hours

HMEC-1

aHUS SERUM EXERTS A PROTHROMBOGENIC EFFECTMEDIATED BY COMPLEMENT

°P<0.05 vs control serum* P<0.01 vs aHUS untreated

*

10

15

20

25

Are

a c

ove

red

by t

hro

mb

i(p

ixe

l2x1

03)

°

*

°

Adhesion assay under flow (60 dynes/cm2, 3 min)

Parallel-plate flow chamberh = 0.15 mm; w = 1 mm; l = 30 mm

Endothelial cells

Coverslip

HS inlet HS outlet

T = 37°C

Coverslip

(+ mepacrine)

Images acquired by a confocal fluorescence microscope connected to a computer

control aHUS + sCR1 aHUS +anti-C5mbody

aHUS

0

5

Are

a c

ove

red

by t

hro

mb

i(p

ixe

l

control aHUS aHUS

+ sCR1

serum aHUS

+ anti-C5

mbody

Galbusera et al., Immunobiology 2012

aHUS

C3

C3aCD40L

PMP

Trigger

EculizumabA humanized monoclonal antibodythat binds to C5

C3b

C3b

Endothelial cell

MCP

CFB

CFI

C3b

C5b

C5a

C5C5b-9

C3b

Bb

C3b

Bb

C3C3

convertaseC5

convertase

Platelet aggregation

Endothelial cell

Coagulation

VWF

Modified from Noris and Remuzzi, NEJM 2009

EFFECTS OF 62 WEEKS OF ECULIZUMAB THERAPY INPATIENTS WITH PLASMA DEPENDENT OR PLASMARESISTANT ATYPICAL HUS

Dependent(n = 20)

Persistent remission

Resistant(n = 17)

17 15

Need for plasma therapy

Serious treatment-relatedadverse events

Legendre et al , NEJM 2013

17

0

0

0

0

28

In studies of resistant aHUS Eculizumab was similarly effective inpatients with or without identified complement gene mutations

THE ANTI-C5 MAB ECULIZUMAB PREVENTS C5b-9DEPOSITS INDUCED BY aHUS SERUM ON HMEC

9000

°

C5b9 formed

- control

- aHUS patient serum

Static incubation

4 hours

C5b-9 depositionHMEC-1

10 min

ADP 10µM

- anti-C5b-9 Ab staining

- Confocal microscopy

Control

°P<0.001 vs. control serum*P<0.001 vs. aHUS serum

0

3000

6000

pix

el2

control aHUS

*

aHUS +

Eculizumab

100 µg/ml

Noris et al Blood 2014

aHUS

IN aHUS PATIENTS ECULIZUMAB TREATMENT NORMALIZES EX-VIVOCOMPLEMENT DEPOSITION ON ENDOTHELIAL CELLS

30

• In the 8 aHUS cases treated with Eculizumab, serum-induced C5b-9 depositson ADP-activated HMEC-1 normalized after treatment, while no significantchange was observed among pre- and post-Eculizumab serum C3 or plasmasC5b-9 levels.

Noris et al, Blood 2014

*P<0.01 vs pre-Ecu

● Homozygous or compound heterozygous mutations in DGKE (encodingdiacylglycerol kinase ε) were found in 27% of aHUS cases with onset inthe first year of life

● Peculiar clinical phenotype: recurrent disease in childhood, developmentof proteinuria sometimes with the nephrotic syndrome

● DGKE is not an integral protein of complement and patients did not showcomplement consumption

Lemaire M et al, Nature Genet 2013

RECESSIVE MUTATIONS IN DGKE CAUSE aHUS SYNDROME

31Quaggin, Nature Genet, 2013

* arachidonic acid–containing diacylglycerol

Consequences of DGKE deficiency

- Damage to podocytes

- Up-regulation of prothrombotic factors and

decreased VEGFR2 signaling in endothelium

- Activation of platelets in blood

Proteinuria

PKC activationAADAG*

DGKE

Phosphatidic acid

Homozygosity mapping and whole-exome

sequencing identified a novel truncating

mutation in DGKE (p.K101X) in a

consanguineous family with patients

affected by thrombotic microangiopathy

32

affected by thrombotic microangiopathy

characterized by significant serum

complement activation and consumption of

the complement fraction C3

Westland et al., JASN, 2014

● Four out of 83 patients with early onset (< 2 years) aHUS carriedhomozygous or compound heterozygous DGKE mutations.

● Three of the 4 patients also carried mutations in THBD or C3.

● Extensive plasma infusions controlled recurrences in the 2 patientswith DGKE and THBD mutations. A positive response to plasmatreatment and Eculizumab was observed in the patient with combined

Chinchilla et al, CJSN 2014

treatment and Eculizumab was observed in the patient with combinedDGKE and C3 mutations.

● Remission without plasma or Eculizumab in the patient with only theDGKE mutation

● Complement genetic dysregulation may influence disease severityand response to therapies in carriers of DGKE mutations.

Roberta Donadelli

Annalisa Sorosina

Elisabetta Valoti

Rossella Piras

Marta Alberti

Federica Banterla

Miriam Galbusera

Collaborations

Francesco Tedesco

Paolo Macor

Peter Zipfel

Tim Goodship

Veronique-Fremeaux Bacchi

Santiago Rodriguez de Cordoba

35

Miriam Galbusera

Sara Gastoldi

Erica Daina

Elena Bresin

Sara Gamba

Giuseppe Remuzzi

Santiago Rodriguez de Cordoba

Grants

These slides are belonging to Marina Noris, PhD.Mario Negri Institute for Pharmacological Research, Bergamo, Italy.

Using these slides is only authorized by

36

Using these slides is only authorized bymentioning the source