Sindrome Emolitico Uremica atipica ed attivazione del complemento

Marina  Noris,  PhD  

 Rimini,  17  O3obre  2014  

 

2

HEMOLYTIC  UREMIC  SYNDROME  

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

Incidence (cases/100,000/year)

Overall: 0.5-2

Sex: no difference

3

STEC-associated HUS E. coli O157:H7 produces Shiga-like toxin/verotoxin and

causes hemorrhagic colitis

4

ACUTE GLOMERULONEPHRITIS IN INFANCY

Two children and their male cousin who all died at 5 months of age

Fison, ArchDisChild, 1956

Incidence: 0.2-0.5/100,000 persons/year

Bergamo!Trento!

Padova!

Treviso!Vicenza!

Parma!Genova!Torino!

Pavia!

Milano!

Varese!Monza!

Brescia!

Firenze!

Roma!

Foggia!

Bari!Salerno!

Reggio Calabria!Palermo!

Cagliari!

Sassari!

UK!9 cases!

USA!58 cases! Argentina!

23 cases!

Belgium! 1 case!

Israel!14 cases!

Portugal! 7 cases!

Canada! 4 cases!

Switzerland! 21 cases!

South Africa! 2 cases!

Germany! 18 cases!

Denmark! 3 cases!

Saudi Arabia! 4 cases!

Czech R.! 10 cases!

Esthonia! 1 case!

Serbia! 5 cases!

Italy!490 cases!

Greece! 4 cases!

Australia! 13 cases! Malaysia!

2 cases!

Turkey!7 cases!

IRAN!49 cases!

Spain! 5 cases!

Poland!10 cases!

Russia 5 cases

UAE 1 case India

3 cases Japan

2 cases

Chile 2 cases

Participating Centers

aHUS patients Italian cases

Foreign cases

180

805 525 280

INTERNATIONAL REGISTRY OF HUS/TTP

http://villacamozzi.marionegri.it

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

(%)!

Cases! Controls!

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

P < 0.001!

P = 0.005!

Relatives!

100!

0!

20!

40!

60!

80!

Cases! Controls! Cases! Controls!

Relatives!

Cases! Controls!0!

10!

20!

Prevalence of low C3 ! Prevalence of low C4 !

(%)!

7

COMPLEMENT ACTIVATION PATHWAYS!

CR1 Factor I DAF MCP

CD59

C5a

C5b-9

Lectin pathway Classical pathway

Mannose residues

IgM, IgG Immune complexes

C1q,C1r,C1s

C4,C2

C4bC2a

C3 convertase C3

C3b C3a

C4bC2aC3b

C5 convertase

MBL, MASP

C1-est inhib

C4bp

C5!

Factor H

Alternative pathway

bacteria, bacterial toxin, LPS, tick-over

C3

C3b C3a

C3bBb

C3 convertase C3

C3b C3a

(C3b)2Bb

C5 convertase

Factor B!

C3 convertase

8!

C3

C3b

C3a

Endothelial cell

CFB

Gly

cosa

min

ogly

cans

C3b

CFB

C3b Bb

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)

CFI

iC3b

CFI

CFH CFH

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! !

PUBLISHED DATA ON CFH MUTATIONS IN aHUS!

Q1076E!

D1119G!V1134G !Y1142D!W1157R !C1163W !3559delA!3566+1A!

E1172stop !W1183L !W1183R !T1184R !L1189R !L1189F !S1191L !S1191W !G1194D !V1197A !E1198A !F1199S !R1210C !R1215G !R1215Q !P1226S !3559delA !3719delACA 3768delAGAA !3546-3581dup36 !del24bp !

2303insA !

E850K !

S890I H893R Y899D !Y899stop!C915S Q924stop !C926F !279del15bp !!!

Q950H !Y951H!T956M !I970V W978C !

1494delA !

C630W !C673Y !

Q400K!R60G !155delAGAA !

371del2bp !S714stop !

Regulatory domain

C3b

Recognition doman

proteoglycans proteoglycans proteoglycans

Y1021F !C1043R !3103delG !

C3b C3b

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

10

SINGLE MUTATION CHANGES IN SCR 20 OF FACTOR H AFFECT BINDING TO CELLS

Factor H

Factor HR1210C

co

Flow cytometry

N° o

f cel

ls

Manuelian et al., J Clin Invest, 2003

Factor H protein with the R1210C or E1172Stop mutations found in HUS in patients showed reduced binding to endothelial cells

11!

C3

C3b

C3a

Endothelial cell

CFB

spontaneous hydrolysis, bacteria, viruses

Gly

cosa

min

ogly

cans

CFI

iC3b C3b

CFB

Surface bound C3 convertase

C3b Bb

Fluid phase C3 convertase

CFB

C3b Bb

CFI

iC3b CFH

CFH

CFH

Valoti et al., JASN 2014

A AHUS HYBRID CFHR1/CFH GENE ENCODING A FUSION PROTEIN THAT ANTAGONIZES FACTOR H-DEPENDENT COMPLEMENT REGULATION!

52y 49y

n.a.

20y 16y

n.a.

Onset 49 yr HD 50yr

Onset 19 yr

FHR1/FH!

CFH! CFHR3! CFHR1!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! 1! 2! 3! 4! 5!

1! 2 3 4 20!

* P<0.05 ***P<0.01

A novel CFHR1/CFH hybrid gene in a family with two affected subjects and the resulting fusion protein contains the first four short consensus repeats of FHR1 and the terminal short consensus repeat 20 of FH

13!

C3

C3b

C3a

Endothelial cell

CFB

spontaneous hydrolysis, bacteria, viruses

Gly

cosa

min

ogly

cans

CFI

iC3b C3b

CFB

Surface bound C3 convertase

C3b Bb

CFH

CFHR1/CFH CFHR1/CFH

CFH

Valoti et al., JASN 2014

Blancet al., JImmunol, 2012!

CFH!1 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 regulatory function of CFH at cell surfaces by blocking its C-terminal recognition region!

!●  Most patients with aHUS and CFH autoantibodies are homozygous for a deletion

of gene encoding CFHR1.!

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

AUTOANTIBODIES TO FH AND CFHR1 IN aHUS

15!Endothelial cell

Gly

cosa

min

ogly

cans

CFI

iC3b C3b

MCP CFI

C3b

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+193-198del C99R (96-129)del+G130I+Y132T+L133X

Y155D

MCP 10%

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

Loss of function heterozygous mutations

C3

C3b

C3a

CFB

spontaneous hydrolysis, bacteria, viruses

MCP MUTATIONS IN aHUS

CFH

16!Endothelial cell

Gly

cosa

min

ogly

cans

CFI

iC3b C3b

MCP CFI

C3b

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

H Chain L Chain

T72S A240G G261D (n=2) G349R

I357M W399R L484V+Q485G+W486X E554V D519N

R317W

CFI 7%

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

Loss of function heterozygous mutations

CFI MUTATIONS IN aHUS

CFH

17!

C3

C3b

Endothelial cell

CFB

CFI

iC3b C3b

CFB

C3 convertase

C3b Bb

MG2 MG3 MG4 MG5

MG

6β

LNK

β-chain α-chain

α’N

T M

G6α

MG7 CU

B g

TED MG8 AN

A

CU

B f

Anc

hor

C345C

R570W K1029M R1041S D1093N I1135T

T1361M T140R T140K Q163E

MG1

R456L

C3 7-10%

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

Gain of function C3 mutations: reduced binding to MCP, increased affinity to CFB

MCP

C3a

C3 MUTATIONS IN aHUS

CFH

18!

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

H Chain L Chain

T72S A240G G261D (n=2) G349R

I357M W399R L484V+Q485G+W486X E554V D519N

MG2 MG3 MG4 MG5

MG

6β

LNK

β-chain α-chain

α’N

T M

G6α

MG7 CU

B g

TED MG8 AN

A

CU

B f

Anc

hor

C345C

R570W K1029M R1041S D1093N I1135T

T1361M

1 2 3 SCRs

linker

Ba Bb

R138W

1 2 3 SCRs

linker VWA SP

R317W

T140R T140K Q163E

Regulatory domain

C3b

Recognition doman

C3b proteoglycans proteoglycans C3b proteoglycans

S890I (n=2) Y899X W920R 2759del15bp

479X N516K

Q950H I970V 1014X

Q1137L C1163W Dup(3546-3581)

E1172X (n=2) W1183R W1183X S1191L (n=7) G1194D V1197A (n=4) E1198A V1200L R1210C (n=9) R1215Q (n=2) R1215G 3675-3699del

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+193-198del C99R (96-129)del+G130I+Y132T+L133X

Y155D

MG1

R456L

CFH  25%  

MCP  10%  

CFI  7%  

C3  7%  

CFB  1%  

COMPLEMENT GENE MUTATION IN aHUS PATIENTS!

An<-­‐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%  

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

19

C3 C5b-9 C3 AND MAC DEPOSITION IN ATYPICAL HUS KIDNEY

aHUS!

C3b!

C3!

C3b!C3a!

Endothelial cell!

MCP

CFB!

CFI!

C3b!C5b!

C5a!

C5!C5b-9!

C3b!

Bb!

C3b!

Bb!

C3!C3 !convertase! C5 !

convertase!

Platelet aggregation!

TM!

CD40L!

Endothelial cell!

PMP

Trigger!

Coagulation!

VWF P-sel

TM!

TM shedding

Modified from Noris and Remuzzi, NEJM 2009

Eculizumab A humanized monoclonal antibody that binds to C5

EFFECTS OF 62 WEEKS OF ECULIZUMAB THERAPY IN PATIENTS WITH PLASMA DEPENDENT OR PLASMA RESISTANT ATYPICAL HUS!

Dependent!(n = 20)!

Persistent remission!!Need for plasma therapy!!Serious treatment-related!adverse events!

Legendre et al , NEJM 2013!

Resistant!(n = 17)!

17!!

0!!

0!

15!!

0!!

0!

21 In studies of resistant aHUS Eculizumab was similarly effective in patients with or without identified complement gene mutations !

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

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

0!

3000!

6000!

9000!

°!

pixe

l2 !

control! aHUS!

*!

aHUS +!Eculizumab !100 µg/ml!

C5b9 formed !

-  control!- aHUS patient serum!

Static incubation!4 hours!

C5b-9 deposition!HMEC-1!10 min!

ADP 10µM!

Noris et al Blood 2014!

-  anti-C5b-9 Ab staining!- Confocal microscopy!

aHUS

Control

23

C5b

-9 fo

rmed

(%

of c

ontro

l) !

600!

500!

0!

*!

Pre-ecu!

400!

300!

200!

100!

Post-ecu !

Seru

m C

3 (m

g/dL

) !

100!

80!

0!

Pre-ecu!

60!

40!

20!

Post-ecu !

Plas

ma

C5b

-9 (n

g/m

L) !

600!

500!

0!

Pre-ecu!

400!

300!

200!

100!

Post-ecu !

IN aHUS PATIENTS ECULIZUMAB TREATMENT NORMALIZES EX-VIVO COMPLEMENT DEPOSITION ON ENDOTHELIAL CELLS!

*P<0.01 vs pre-Ecu!

•  In the 8 aHUS cases treated with Eculizumab, serum-induced C5b-9 deposits on ADP-activated HMEC-1 normalized after treatment, while no significant change was observed among pre- and post-Eculizumab serum C3 or plasma sC5b-9 levels.!

Noris et al, Blood 2014!

24

600 !DIALYSIS!PLASMA THERAPY!

ECULIZUMAB (mg)!300!

KIDNEY TRANSPLANT!

0 50

100

150

200

250

300

0 300 600 900 1200 1500 1800 2100 2400

Pla

tele

t cou

nt

(103

/mic

rolit

er)

days

plasma sC5b-9 levels (normal range 127-303 ng/ml) CH50

121 72

195 3

214 3

pre-Tx! 8 days!post-Ecu!300 mg!

12 days post-Ecu 600 mg

0

2000

4000

6000

8000

C5b

-9 fo

rmed

(p

ixel

2 )

Pediatric case (7-year-old boy with p.S1191L CFH mut)

Noris et al, Blood 2014

25!Quaggin, Nature Genet, 2013

* arachidonic acid–containing diacylglycerol !

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

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

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

Lemaire M et al, Nature Genet 2013

RECESSIVE MUTATIONS IN DGKE CAUSE aHUS SYNDROME

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 activation AADAG*

DGKE

Phosphatidic acid

26!

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 characterized by significant serum complement activation and consumption of the complement fraction C3

Westland et al., JASN, 2014!

Chinchilla et al, CJSN 2014!

●  Four out of 83 patients with early onset (< 2 years) aHUS carried homozygous 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 patients with DGKE and THBD mutations. A positive response to plasma treatment and Eculizumab was observed in the patient with combined DGKE and C3 mutations.

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

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

29

Roberta Donadelli Annalisa Sorosina Elisabetta Valoti Rossella Piras Marta Alberti Federica Banterla Miriam Galbusera Sara Gastoldi Erica Daina Elena Bresin Sara Gamba Giuseppe Remuzzi

Collaborations Francesco Tedesco Paolo Macor Peter Zipfel Tim Goodship Veronique-Fremeaux Bacchi Santiago Rodriguez de Cordoba

Grants

30

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

Using these slides is only authorized by mentioning the source