Inflammation: One Gene at a Time

Inflammation:One Gene at a Time

Dan Kastner, MD, PhDIntramural Research Program

NHGRI/NIH/DHHSFebruary 14, 2017

In the beginning . . .

David BotsteinYW Kan Francis Collins

p13.3 p13.2p13.13

Chromosome 16p

Telomere CentromerePKD1, TSC2

D16S3373

NK4

OR1F2ϕ

v57c10

v9ϕ

ZNF213ZNF205(ZNF210)

OR1F1v57c7 v57c7x4

MEFVZNF200

D16S3082D16S468/S3070 D16S3405 D16S2617 AFMef101 D16S3275

FPM315

CREBBP

Pyrin / Marenostrin

NH2 COOH

bZIP B-Box

Coiled-coil B30.2PYD

Cell 90:797, 1997

Positional Cloning of MEFV, the Gene Mutated in Familial Mediterranean Fever (FMF)

PYRIN

Pyrin

PYRIN CARDASC

CARDCaspase-1

IL-1β Converting Enzyme (ICE)

pro-IL-1β

IL-1β

The PYRIN Domain: A Cognate Interaction Motif

Richards et al., J Biol Chem 276:39320, 2001

IL-1 Inhibition in FMF Amyloidosis

Chae et al PNAS 103:9982, 2006

FMF Knockin Mice: IL-1-Dependent Inflammation

WTWT

V726AV726A

Chae et al. Immunity 34:755, 2011.

Big Questions Remaining

• What is the function of non-mutated pyrin in the immune system?

• Why are carrier frequencies for MEFVmutations so high in certain populations?

• How can we account for the exquisite specificity of colchicine for FMF among the recurrent fever syndromes?

Park et al., Nat Immunol 17:914, 2016


N-term pyrin

+−PKN1:

pyrin

-N-te

rm-W

T-M

yc/H

is

IB: P-Ser Ab55

70

40

+−

IB: anti-myc Ab

pyrin

-N-te

rm-S

208A

/S24

2A-M

yc/H

is

PKN1 Phosphorylates Pyrin at S208 and S242



The Binding of 14-3-3 to Pyrin is Substantially Decreased by FMF-Associated Mutations

14-3-3ε

14-3-3ε

pyrin

pyrin

IP:

anti-

pyrin

Lys.

Con

trol 1

Con

trol 2

Con

trol 3

Con

trol 4

FMF

1 (M

EFV

M69

4V/V

726A

)FM

F 7

(ME

FVK

695R

/V72

6A)

FMF

6 (M

EFV

M69

4I/M

694I

)FM

F 8

(ME

FVM

680I

/V72

6A)

FMF

9 (M

EFV

V72

6A/V

726A

)



IL-1βSup.

Lys.Pro-IL-1β

Actin

CAPS 3NLRP3L353P/+

CAPS 4NLRP3L353P/+

Unt

reat

ed0 1 10

LPS +Colchicine

Unt

reat

ed0 1 10

LPS +Colchicine

FMF 3MEFVM694V/M694V

FMF 5MEFVM694V/V726A

FMF 4MEFVM694V/M694V

Unt

reat

ed0 1 10

LPS +Colchicine

Unt

reat

ed0 1 10

LPS +Colchicine

Unt

reat

ed0 1 10

LPS +Colchicine

IL-1βSup.

Lys.Pro-IL-1β

Actin

Colchicine Suppresses the Pyrin Inflammasome



Pyrin S242R in a Family with a Severe Dominantly Inherited Autoinflammatory

Disease: PAAND

Masters et al. Science Transl Med 8:332ra45, 2016


Pyrin Inflammasome Activation in HIDS



The Yersinia Outer Protein YopM Inactivates the PyrinInflammasome Through PKN1/PKN2

Chung et al., Cell Host Microbe 20:1, 2016

Mefv+/+

MefvB30.2/B30.2

MefvV726A/+

MefvM680I/M680I

FMF-KI mice are less susceptible to Yersinia

Chae, Bliska, et al., unpublished observations

781Pyrindomain B-BOX CC1 B30.2

413SH3

261FCH CC1

30A230T E250

Q**

PSTPIP1

pyrin Shoham et al., PNAS 100:13501, 2003

PAPA Syndrome (Pyogenic Arthritis with PyodermaGangrenosum and Acne)

SoJIA?Neonatal-Onset Multisystem Inflammatory

Disease (NOMID)

Treatment of 18 NOMID Patients with the IL-1 Receptor Antagonist Anakinra

Goldbach-Mansky R et al. N Engl J Med 2006;355:581-592

Somatic Mosaicism at NLRP3

• 30 – 50% of NOMID/CINCA• Schnitzler syndrome

– Middle-age onset– Fever, neutrophilic urticaria, bone

pain, myalgia, monoclonal gammopathy

• Adult-onset FCAS

Zhou et al. Arthritis Rheum 67:2482, 2015

Age 9 months, before therapy 3 days post

treatment initiation

7 days post treatment initiation

Aksentijevich I et al. N Engl J Med 2009;360:2426-2437

Christina

• 27 y/o Irish woman• 14 yr hx 3-5 wk febrile episodes• Periorbital edema, migratory rash,

abdominal pain• Seen 1 wk postpartum• WBC 29K, ESR 126, CRP 16.3• Therapeutic response to steroids

but not colchicine

TNFRSF1A13

12

11.12

12

13

14

15

21

22

23

24.1

24.3

CRD1

CRD2

CRD3

CRD4

Death domain

CRD1

CRD2

TNFRSF1A Mutations Can Cause Dominantly Inherited Periodic Fever

Rheumatology 5th edn, 1645, 2011

The TNF Receptor-Associated Periodic Syndrome (TRAPS)

Hull et al. Medicine (Baltimore) 81:349 (2002)

Rheumatology 5th edn. 1637-57, 2011

Rheumatology 5th edn. 1637-57, 2011

Keinan and Clark, Science 336:740, 2012

Fever with Early-Onset Stroke

I:1 I:2

A Family 1

II:1 II:2

B Family 2

II:3

I:2I:1

II:2II:1

Zhou et al. NEJM 370:911, 2014


CECR1b Morpholino Injection in Zebrafish: 48 h


Anti-TNF Therapy for DADA2

0

50

100

150

200

1 2

CRP

0

10

20

30

40

50

60

70

1 2

ESR

0

5

10

15

20

1 2

WBC

02468

10121416

1 2

Hemoglobin

0

100

200

300

400

500

600

1 2

Platelets

0

50

100

150

200

250

1 2

Serum Iron

Barron, Ombrello, et al., unpublished

Before anti-TNF: 44 strokes, 1064 patient-months

Since anti-TNF: 0 strokes, 323 patient-months

Mutations Additional SNPs

PT (1) G47A het: Rs200930463, MAF: 0.00001649 P106S het: No RS, MAF 0.000008345

H335R hom: Rs2231495, MAF 0.33Y453Y het: Rs7289170, MAF 0.22

PT (2) G383S hom: no rs (Infevers) N53N hom: Rs362129, MAF 0.32H335R hom: Rs2231495, MAF 0.33

PT (3) E328K het: unknownF355L het: rs116020027, MAF < 0.01

L46L het: Rs7289141, MAF 0.17

PT(4) V349I het: Rs74317375, MAF < 0.01 N53N hom: Rs362129, MAF 0.32Y453Y het: Rs7289170, MAF 0.22

PT(5) T65M het: Rs61747288, MAF 0.0004614 N53N hom: Rs362129, MAF 0.32Y453Y het: Rs7289170, MAF 0.22

PT(6) Y220Y het: rs2231487, MAF < 0.01 L46L het: Rs7289141, MAF 0.17H335R het: Rs2231495, MAF 0.33

Sanger sequencing for CECR1 in PAN cohort

Stoffels et al., unpublished

November 11, 2016

NH2

COOH

α subunit

β subunit

Dermatansulfate

2A1mAb Extracellular / intra-ER

2

Boyden et al., NEJM 374:656, 2016

3

Zhou et al., Nat Genet 48:67, 2016

4

Zhou et al., PNAS 113:10127, 2016

Zhou et al., Arthritis Rheumatol 67:2482, 2015

8 Myeloid-Restricted NLRP3 Somatic Mosaicism in Adult-Onset Muckle-

Wells Syndrome

Brehm et al. J Clin Invest 126:795, 2016

Digenic Inheritance in CANDLE

5-7

Zhou et al., Am J Hum Genet 91:713, 2012

9PLCG2 S707Y Defining a New

Disorder, APLAID

10 A Recurrent Fever Syndrome Caused by Hypomorphic Mutations in TRNT1

Cleavage-Deficient RIPK1 Mutations

Liver Spleen

Boyden et al., unpublished

TA Manolio et al. Nature 461, 747-753 (2009)

Genetic Architecture of Common Disease

Behçet’s Disease:

A Genetically Complex Disorder of Inflammation

λs > 10

Genetic Architecture of Behçet’s Disease

HLA-B*51

IL10IL23R

HLA-B*51, ERAP1 and Behçet’s Disease Susceptibility

Ombrello et al. PNAS 111:8867-8872, 2014 Kirino, Bertsias, et al. Nature Genet 45:202-207, 2013


HLA-B*51

IL10IL23R

ERAP1

CCR1STAT4KLRC4

MEFVIL23RTLR4NOD2

IL1A-IL1BIRF8CEPB-PTPN1ADO-EGR2RIPK2LACC1FUT2

Discovery of a Monogenic Formof Behçet’s Disease

Exome Sequencing Sanger Sequencing Targeted Sequencing

Over 150 patients screened 384 Turkish +384 Japanese Behçet's patientsscreened

(A20)

* Exome sequenced samples

Zhou et al., Nature Genet 48:67, 2016

TRAF2TRADD

TNF

TNFR1

RIP1

K63-Ub

RIP1 K48-Ub

A20

A20

NF-κB Pathway

TNFAIP3, Encoding the A20 Protein

Enhanced Signaling in the NF-κB Pathway


In Vitro: A20 Mutations are Associated with Defective K63 Deubiquitinase Activity

293T cells co-transfected with constructs for NEMO (IKKγ), K63 Ub, and either WT-A20 or mutant-A20.

(IKKγ)


A20 Mutations Activate the NLRP3 Inflammasome


Treatment



HLA-B*51

IL10IL23R

ERAP1

CCR1STAT4KLRC4

MEFVIL23RTLR4NOD2

IL1A-IL1BIRF8CEPB-PTPN1ADO-EGR2RIPK2LACC1FUT2

TNFAIP3(A20)

Summary• Genomic approaches have revolutionized our understanding of the

autoinflammatory diseases and their treatment• Familial Mediterranean fever is caused by mutations in MEFV,

encoding the pyrin protein, an activator of IL-1β• Bacterial toxins that modify RhoA, as well as loss-of-function

mutations in the geranylgeranyl synthetic pathway, lead to pyrin inflammasome activation

• Next-generation sequencing has revolutionized our ability to “solve” unexplained inflammatory phenotypes

• Recessive loss-of-function mutations in ADA2 lead to a syndrome of recurrent fever, livedo racemosa, lacunar strokes, and PAN

• Common low-penetrance variants and rare highly penetrant mutations contribute to Behçet’s disease susceptibility

• Truncating mutations in A20 define a dominantly-inherited disorder in the Behçet’s spectrum (HA20) with heightened NF-κB signaling and NLRP3 inflammasome activation

• Our patients continue to present us with new challenges

NIH Colleagues in the Laboratory

Raman SoodNHGRI

Manfred BoehmNHLBI

JaeJin ChaeNHGRI

Yong Hwan ParkNHGRI

Monique StoffelsNHGRI

Geryl WoodNHGRI

Geun-Shik LeeNHGRI

Yohei KirinoNHGRI

Masaki TakeuchiNHGRIElaine Remmers

NHGRIMike Ombrello

NIAMSPravitt Gourh

NIAMS

Julie LeNHGRI Kalpana Manthiram

NHGRI

Hongying WangNHGRI

Ivona AksentijevichNHGRI

Qing ZhouNHGRI

Angeliki GiannelouNIAMS

Natalie DeuitchNHGRI

Hiro OdaNHGRI

Steve BoydenNHGRI

NIH Colleagues in the Clinic

Camilo ToroNHGRI

Hirsh KomarowNIAID

Dean MetcalfeNIAID

Bob LemboOD

Don GoldsmithDrexel

Amanda OmbrelloNHGRI

RaphaelaGoldbach-Mansky

NIAID

Debbie StoneNHGRI

Karyl BarronNIAID

Paola PintoNHGRI

Patrycja HoffmannNHGRI Bev Barham

NHGRIAnne Jones

NHGRITina Romeo

NHGRIDaniella Schwartz

NIAMS

Additional ContributorsNIHManfred BoehmHyejeong BolanSettaraChandrasekharappa

Celeste ChenDavid ChinEd CowenGlenn CruseAvanti DesaiRobin EischMassimo GadinaEric HansonSarfaraz HasniChyia-Chia LeeDaniel LongJonathan LyonsJosh Milner

InternationalAnkara – Seza Ozen, Ezgi Batu,

Erkan DemirkayaBeirut – André MégarbanéFanar – Myrna Medlej-HashimGenentech – Ingrid WertzIstanbul – Ahmet Gül, Burak ErerMelbourne – Seth MastersNew Haven – Ken Kidd

Andrew PakstisParis – Elaine ChoueryPortland – Cailin SibleyUCSF – Tony Shum, Andy GrossToronto – Ron LaxerUtrecht – Helen Leavis

Annet van Royen-KerkofYokohama – Yoshi Ishigatsubo

Nobuhisa MizukiYohei Kirino

Jim MullikinAna OliveraClaudia OuyangSergio RosenzweigColleen SatoriusLinda ScottRichard SiegelMasaki TakeuchiWanxia TsaiDan YangMichael YoungXiaomin YuZhen YuKristien ZaalYuan Zhang

NIH Clinical Center

[email protected]@mail.nih.gov, [email protected], referrals

mailto:[email protected]



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Inflammation: One Gene at a Time