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