1

Hereditary Periodic Fever Syndromes A group of inherited disorders characterized by recurrent, self-limited episodes of fever and synovial, serosal, and/or oculocutaneous inflammation. 169 Claudius Galen 1790 Heberden 1895 Osler 1908 Janeway & Mosenthal 1940 Kile & Rusk 1945 Siegal 1949 Reimann 1958 Heller 1962 Muckle & Wells 1981 Prieur, Griscelli 1982 Williamson et al 1984 van der Meer et al

2

Differential Diagnosis of Periodic Fevers

FMF HIDS TRAPS (FHF) MWS/FCU/CINCAGenetics MEFV MVK TNFRSF1A CIAS1/NALP3 (16p13) (12q24) (12p13) (1q44) Protein Pyrin/Mnstrn Mevalonate TNF receptor 1 NALP3/

kinase cryoppyrin Onset < 20 Childhood May remit/ 95% <5 years

relapse

Fever Abrupt Fast on, Variable, 24 hours on + off slow off less in adults

Duration 1- 4 days 3 - 7 days Up to weeks 1- 2 days

Nodes Rare Cervical Common No nodes ++

Rash Rare; legs 90%(pleiomorphic) All over (erysipelas) Maculo-papular

Oedema Very rare No Peri-orbital No

Amyloid Common None 25% 25%

Therapy Colchicine Etanercept? Etanercept Anakinra

3

CARD

CARDPyrin

Pyrin

ASC can act as a Link between Pyrin and Apoptotic pathways

PYRIN

ASC

NALP1

4

Amyloidosis of the kidney

5

A heterogenous group of inherited disorders characterised by spontaneous attacks of systemic inflammation without apparent autoimmune basis

• No demonstrable source of infection as precipitating cause

• Absence of high-titre autoantibodies and antigen specific autoreactive T cells

• No evidence of auto-antigenic exposure

Autoinflammatory Syndromes

6

Autoinflammatory Syndromes and related disorders

Hereditary periodic fevers

Gene/chromosome Protein FMF MEFV (16p13) Pyrin/marenostrin HIDS MVK (12q24) Mevalonate kinase TRAPS (FHF) TNFRSF1A (12p13) TNF Receptor 1 FCU/FCAS NALP3/CIAS1/PYPAF1 (1q44) NALP3/Cryopyrin/PYPAF1 MWS/FCAS NALP3/CIAS1/PYPAF1 (1q44) NALP3/Cryopyrin/PYPAF1 CINCA/NOMID NALP3/CIAS1/PYPAF1 (1q44) NALP3/Cryopyrin/PYPAF1 PAPA PSTPIP1/CD2BP1 (15q24) CD2-binding protein 1

Granulomatous disorders

Crohn’s (IBD) NOD2 (16q12) Nucleotide binding oligomerization domain

Blau Snydrome NOD2 (16q12) Nucleotide binding oligomerization domain

7

sTNF/sTNFR

mTNFRSF1A(mTNFR1)

Disulphide bondsDisulphide bonds

8

TNFRSF1A

DD

trimerise

DD

DD

DD

DD

DD

DD

RIP

1

TRADD

DD

TR

AF

2

DD

RIP

1

TRADD

DD

TR

AF

2Complex I

NF-Bactivation

DD

DD

DDModification/

dissociation

DD

RIP

1

TRADD

DD

TR

AF

2

DD

RIP

1

TRADD

DD

TR

AF

2

Recruitment of FADD and caspase-8/10

Complex II

Apoptosis

DISC

FADD

DD

TR

AD

D

DD

DD

TR

AD

D

DDTRAF2

TRAF2

RIP1

RIP1DDDED

DDDEDCASP-8/10

TNF

c-FLIP

Micheau et al. Cell 114, 1810190, 2003Micheau et al. Cell 114, 1810190, 2003

9

Two views of the six helical Death Domain-FoldMotifs

Death domains

Caspase activation and recruitment domains (CARDs)

Pyrin domains

Adaptor domain architecture important in homotypic protein- protein interactions

Protein Science 10:1911, 2001

Death effector domains

10

The conserved pyrin domain defines a family of apoptotic/inflammatory proteins

CARDCARD

B SPRY

ASC/CARD5

NACHTNACHT

NACHTNACHT LRRLRR

NACHTNACHTPYD??????

B30.2B30.2

NALP3/CIAS1/PYPAF1

LRRLRR

NOD2/CARD15

PYD

NALP1/ CARD7

PYD

PYD

PYRIN/MARENOSTRIN

LRRLRR

CARDCARD

CARDCARD CARDCARD

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PYD is a flexibly disordered loop instead of 3 (NALP1)

12

MRI showing fronto-parietal subcortical lesions in TRAPS patientMRI showing fronto-parietal subcortical lesions in TRAPS patient

13

FAMILY A

I

1

1

1 2

2

2

3

3

3

4

4

4 65

5 7

7

8

8

9

9

10

10

11 12 13

A BC

D

FAMILY B

IV

I

possibly affected.

FAMILY C

affected.

III

II

I

II

III

IV

I

III

II

IV

Familial Hibernian Fever families

6

14

Location and genomic structure of TNFRSF1A gene- chromosome 12p13

Exons 1 2 3 4 5 6 7 8 9 10

Signal Peptide Extracellular Transmembrane Intracellular

3’UTR

ptercen

C1 S/R TNFRSF1ACD4 CD9 VWFDRPLA

0.09 0.02 0.002 0.12

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DNA Sequence Electropherograms Showing Disease-Associated TNFRSF1A MutationsDNA Sequence Electropherograms Showing Disease-Associated TNFRSF1A Mutations

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Missense Mutations in the TNFRSF1A Extracellular Domains

Mutation Exon Amino Acid Ethnic Background

175 TC 2 Cys30Arg Irish

185 GA 2 Cys33Tyr Irish/Scottish

236 CT 3 Thr50Met Irish

French-Canadian

242 GT 3 Cys52Phe Irish/English/German

349 TC 4 Cys88Arg Scottish (Australian)

350 GA 4 Cys88Tyr Finnish

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2) constitutive activation, formation of intermolecular disulfide bonds between unpaired cysteines in mutant receptors (Santoro, 1995) -Il-6 induction - crude test - normal

3) resistance of mutant TNFRSF1A to the normal homeostatic effects of activation-induced cleavage

.

- FACS analysis - reduced/delayed TNFRSF1A shedding in affected members of family with C52F mutation

1) increased affinity of mutant TNFRSF1A for ligand (i.e TNF) -binding and affinity studies - normal

.

Possible Disease Mechanisms in TRAPS

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Periodic fever family with amyloidosis of kidney

affected

unaffected

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TNF Receptors on Leukocytes from Patients with the C52F MutationTNF Receptors on Leukocytes from Patients with the C52F Mutation

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Leukocytes Bearing the C52F Mutation Have Defective Clearance of TNFRSF1A (CD120a)Leukocytes Bearing the C52F Mutation Have Defective Clearance of TNFRSF1A (CD120a)

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“TRAPS”

TNF Receptor-Associated Periodic Syndrome

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IL-1

Haem A Ubiquinone

Endothelial cellICAM1, ELAM

Lymph node organogenesis

TNF

Acute phase response (Il-6, SAA, CRP)

Overproduction Septic shock

Hepatic failure Vasculitis Encephalitis Cardiac cachexia

Dolichol

HypothalmusFever/Sleep

T cell activation Il-6

B cell activation

Ig production

Cellular activation

(Cardiac, hepatic, bone, etc)matrix metalloproteinase (stromelysin), plasmin and collagenase production

Breaches anti-protease barrier

TISSUE DESTRUCTION

Virus/endothelium

Macrophage activation

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IK K

IK K

NE MO

S ignal osome

m TNF TACE

SODD

MAP kinase

NF B tr anscri ption

Su rvi val - Anti -apo ptot ic

pr otein s

FADD

TRADD

CASPASE-8 RA IDD

RIP

FLIP

sTNF /

sTNF R

?Shedd ase

NFkB

TRAF2RIP

TRADD

S ign al pr eventi on

sTNF /

sTNF R

TNF RSF 1A

(TN FR 1)

BID/BI M

Bax Bak

BA X

Bcl2 Bclx

Cytochrome c release, Apaf1 activation of downstream caspases e.g 3, 6 and 7

***Death - apoptosis

Extrinsic death receptors

Intrinsic Plasma membraneEndoplasmic reticulum

CytosolNucleus

Anti-apoptotic Bcl2, Bclxl, Bclw, Mcl1

Pro-apoptotic (multi-domain) Bax, Bakl, Bok/Mtd

Pro-apoptotic (BH3 domain only)Bid, Bad, Blk, Bim/Bod, Nix, Noxa

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SCREENING METHODS for TRAPSSCREENING METHODS for TRAPS Genotyping: microsatellites within (TNFRp55)

and flanking (D12S99 and D12S77)TNFRSF1A locus used to genotype family members.

DHPLC: we screened 20 ADRF families and 184 sporadic cases forTNFRSF1A mutations using the Transgenomic WAVE® System.

Sequencing: ABI 373A sequencer.

ELISA assays: used to determine serum levels of TNFRSF1A and TNFRSF1B (sTNFRSF1A and sTNFRSF1B).

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DHPLC elution profileDHPLC elution profile

0.0

1.5

3.0

. 1. 2. 3. 4. 5. 6. 7. 8. 9.

Time (min)

0.0

2.5

. 1. 2. 3. 4. 5. 6. 7. 8. 9.

Time (min)

Wt C70R

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a b05 0903 0708 13

MARKERSD12S99TNFRp55D12S77

a d05 0403 0708 12

a d05 0403 0708 12

c d04 0407 0708 12

a c05 0403 0708 08

21

3

831

875 326

Israeli Arab family A

612

1114

Periodic fever

C70R M 1 2 3 4 5 C

500

800

Not genotyped

4 5

De-novo C70R mutation (Israeli Arab family)

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II

IV

III

I

c d04 04A A06 0606 11

e f04 08G G02 0211 12

a e04 04G G02 0205 11

a e/f04 04G G02 0205 12*

d g04 06-- --06 0111 10

d b04 04A A06 0711 11

c j04 01A A06 0706 11

c j04 01A A06 0706 11

826 731

g h 04 04 G A 06 01 06 03

a c04 04G A02 0605 06

Order of markers D12S99 +36 A/G TNFRp55 D12S77

Irish Family

727

781 11401450

1450

650

650

Soluble TNFRSF1A levels n= 746-1966pg/ml

Periodic fever in an Irish Family - unlinked to all known HPF loci

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7 8 9 10 11 12 13 14 S2

sTN

FR

SF

1A

(p

g/m

l)

s TNFRSF1A levels in affected family members of

(A) TRAPS (B) families without mutation s TNFRSF1A levels in affected family members of

(A) TRAPS (B) families without mutation

(A)

(B)

746

1966

746

1966

unaffected

Periodic fever

Probably affected

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A total of 8 novel TNFRSF1A mutations (T37I, D42, G46E, T50K, F60L,C70R, C70S and R92P), and 5 known (c.193-14 G>A, P46L, T50M, C88R,

and R92Q) mutations in a study of 20 families. The C70S mutation found in a Japanese patient

Affected members in 4 of the 7 families without TNFRSF1A mutations have low sTNFRSF1A

These findings indicate the presence of genetic heterogeneity in ADRFs.

Summary of TRAPS and ADRF families

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Etanercept (a recombinant p75 TNFR:Fc fusion protein), in TRAPS: the Promise of Molecular MedicineEtanercept (a recombinant p75 TNFR:Fc fusion protein), in TRAPS: the Promise of Molecular Medicine

Patient 3 (14yo, M, H22Y)

0

10

20

Q1 Q2 Q3 Q4

Study Phase

Att

ack

Sco

re

Patient 3 (14yo, M, H22Y)

0

10

20

Q1 Q2 Q3 Q4

Study Phase

Att

ack

Sco

re

Patient 4 (32yo, M, T50M)

0

20

40

Q1 Q2 Q3 Q4

Study Phase

Att

ack

Sco

re

Patient 4 (32yo, M, T50M)

0

20

40

Q1 Q2 Q3 Q4

Study Phase

Att

ack

Sco

re

Patient 2 (15yo, F, T50M)

0

30

60

Q1 Q2 Q3 Q4

Study Phase

Att

ack

Sco

re

Patient 2 (15yo, F, T50M)

0

30

60

Q1 Q2 Q3 Q4

Study Phase

Att

ack

Sco

re

Patient 1 (50yo, M, T50M)

0

25

50

Q1 Q2 Q3 Q4

Study Phase

Att

ack

Sco

re

Patient 1 (50yo, M, T50M)

0

25

50

Q1 Q2 Q3 Q4

Study Phase

Att

ack

Sco

re

31

Apoptosis -

A type of programmed cell death in which the cell dies (commits suicide) through a series of cellular events including enzymatic fragmentation of DNA.

# necrosis - cells bursts and discharges contents. e.g. infarction

32

Pyrin/marenostin is mainly expressed in leucocytesPyrin/marenostin is mainly expressed in leucocytes

Cell 90; 797, 1997

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Pyrin Interaction with ASC proteinPyrin Interaction with ASC protein

Apoptosis-Associated speck-like protein with a caspase recruitment domain(CARD)

Interacts with pyrintwo-hybrid, IP, andfluorescence microscopy

Aggregates during Apoptosis in HL-60 cells

Proapoptotic

Interacts with pyrin by yeasttwo-hybrid, IP, andfluorescence microscopy

Proapoptotic

N-terminal pyrin domain, C- terminal CARD

GFP-pyrin + ASC-myc(Cy3 label) merged

34

Monocytes from Pyrin Truncation MiceManifest a Defect in ApoptosisMonocytes from Pyrin Truncation MiceManifest a Defect in Apoptosis

24 hr 48 hr 72 hr

+/+ -/- +/+ -/- +/+ -/-

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Connecting Pathways with Patients: Mutations in a Pyrin-Binding Protein Cause PAPA Syndrome

*

E250Q

Pyrin

B CC B30.2Pyrin domain

1 781

CC SH31 415

PSTPIP1/CD2P1

*A230T

PAPA Syndrome: Pyogenic Arthritis, Pyoderma Gangrenosum, and Acne

36

Autosomal dominant disorders of unknown cause, variably associated with arthralgias, rashes and conjunctivitis

FCAS (also Familial cold urticaria (FCU)

cold induced fever (24-hour) and rash

Muckle-Wells syndrome (MWS)

sensorineural deafness, amyloidosis

Familial cold autoinflammatory Syndrome (FCAS)/Muckle Wells

37

North-Indian familyNorth-Indian family

periodic fever with amyloidosis periodic fever without amyloidosis

a c01 0804 0103 0101 0101 0402 0403 04

d b01 0801 0211 0602 0801 0104 0102 02

e f 01 0802 0100 0001 0501 0403 0504 02

I

II

III

d b01 0101 0211 0602 0801 0104 0102 02

a d01 0104 0103 1101 0201 01-- --03 02

d b01 0104 0211 0602 0801 0104 0102 02

c b01 0801 0401 0601 0804 0104 0104 02

b n01 0901 0406 0308 0201 0101 0302 03

b m01 0901 0106 0408 0201 0101 0202 03

d l01 0804 0211 1102 0201 0104 0502 02

a k01 0104 0203 0201 0501 0102 0103 04

b i-- --02 0106 1108 0501 0401 0502 02

b j01 0102 0206 1108 0701 0101 0602 02

c f08 0804 0101 0601 0504 0104 0304 04

d h01 0101 0111 0202 0201 0104 0202 02

c g 01 01 01 04 01 01 01 02 04 04 04 04 04 02

a c08 0104 0403 0101 0101 0402 0403 04

a d01 0104 0403 1101 0201 0102 0403 02

c p 01 01 04 02 01 03 01 07 04 04 04 06 04 02

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The conserved pyrin domain defines a family of apoptotic/inflammatory proteins

CARDCARD

B SPRY

ASC/CARD5

NACHTNACHT

NACHTNACHT LRRLRR

NACHTNACHTPYD??????

B30.2B30.2

NALP3/CIAS1

LRRLRR

NOD2/CARD15

PYD

NALP1/ CARD7

PYD

PYD

PYRIN/MARENOSTRIN

LRRLRR

CARDCARD

CARDCARD CARDCARD

periodic fever with amyloidosis

periodic fever without amyloidosis

I

II

III

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

2 6

11

1083 4 5

2019181715141312 16

97

1

4 51

7

3 6

8

2

M 1 2 3 4 5 6 7 8

I

I I

III

IV

A

B

Cold sensitivity/periodic fever without amyloidosis

40

NACHT

V198MV198M

Q306LQ306L

D303ND303N

A352VA352V

A439TA439T

E627GE627G

R260WR260W

A439VA439V

G569RG569R

F573SF573S

FCU/FCAS mutations Mutations found in more than one disease

CINCA mutationsMWS mutations

L305PL305P

F309SF309S

H358RH358R

T436NT436N

M662TM662T

LRRPYRIN

T348MT348M V351MV351M

A374NA374N

F523LF523L

Y570CY570C

Motif III, Motif III, MgMg++

L264HL264H

L353PL353P

R488KR488K

NALP3/CIAS1/PYPAF1 mutations MWS,FCU/FCAS, CINCA/NOMIDNALP3/CIAS1/PYPAF1 mutations MWS,FCU/FCAS, CINCA/NOMID

41

CARD

CARD P20 P10

ASC

IL-1-processing

NF-kB modulationInflammation with Fever

Caspase-1

Pyrin

Cryopyrin/NALP3Pyrin/marenostrin

PYDB box

CCB30.2

Apoptosis

LRR

PYD

NACHT

PYD

The assembly of cryopyrin/NALP3/PYPAF1 and pyrin/marenostrin with ASCThe assembly of cryopyrin/NALP3/PYPAF1 and pyrin/marenostrin with ASC

42

IL-1 receptor antagonist (Kineret) therapy in FCU patient (100 mg daily subcutaneously)IL-1 receptor antagonist (Kineret) therapy in FCU patient (100 mg daily subcutaneously)

North Indian Patient

Mar01 Jan02 Dec02

1

10

100

1000

Seru

m S

AA

mg

/l

43

Mechanisms - related to NFkB activation and ?defective apoptosis in FMF, TRAPS and MWS. HIDS?.

Proinflammatory polymorphisms TNFRSF1A - P46L, R92Q. NALP3 - V198M (V200M)

MEFV - E148Q (amyloidosis)

Modifier Genes e.g MICA in FMF susceptibility Behçets increases penetrance MEFV mutations.

SAA1a/a in FMF patients with amyloidosis.

Polygenic nature of all “Mendelian diseases”?

Genetic heterogeneity and reduced penetrance.

Developing Concepts

44

Acetyl CoA

Acetyl CoA + Acetoacetyl CoA

HMG CoA

Mevalonate

Mevalonate-P

Mevalonate-PP

Isopentenyl-PP Isopentenyl-Adenine

Geranyl-PP

Farnesyl-PP

Squalene

Cholesterol

Plasma LDL

Dolichol

Haem A Ubiquinone

Farnesylated Proteins (Ras, lamin B, etc

Vitamin D

Bile Acids

Steroid Hormones

Lipoproteins LDL receptor

kinase Defect in HIDS

reductase

synthase Cholesterol Metabolism and Isoprenoid Biosynthesis

45

46

Exon: 1 2 3 4 5 6 7 8 9 10 11

Organisation of the MVK gene and location of mutations leading to the HIDS/MA phenotype

= 2bp deletion

= Nonsense mutation

= Mevalonic Aciduria

= HIDS

= Mevalonic Aciduria

= Missense mutation