Vers une théorie génétiquedes mycobactéries humaines

Jacinta Bustamante MD, PhDLaboratory of Human Genetics of Infectious Diseases - INSERM U980

Necker-Enfants Malades Medical School, Paris, France

JNI – 11 Journées Nationales d’Infectiologie

Du mercredi 9 au vendredi 11 juin 2010 – Le Corum, Montpellier

Déclaration de conflits d’intérêts: Jacinta Bustamante MD, PhD

Laboratory of Human Genetics of Infectious Diseases - INSERM U980

Necker-Enfants Malades Medical School, Paris, France

JNI – 11 Journées Nationales d’Infectiologie

Du mercredi 9 au vendredi 11 juin 2010 – Le Corum, Montpellier

Absence de conflits d’intérêt

The genus The genus MycobacteriumMycobacterium

‘Virulent’ ‘Weakly virulent’M. tuberculosis complex > 80 species (e.g. M. avium,

M. leprae M. marinum, M. fortuitum…)

Human transmission Environmental transmission

(airborne) (water, soil, air…)

M. ulcerans (Buruli ulcer) BCG vaccine

Aquatic bug transmission? Injection transmission

Inter-individual variability in tuberculosis:the key question

Accidental

inoculation with

M. tuberculosis

251 infants

Death by year 177 infants

No sign of infection

47 infants

Various signs of infection127 infants

• The Lübeck disaster before world war II

Immunity

Microbialfactors

Exposure toMicrobe

BiologicalPhenotypes

ClinicalPhenotypes

EcologicalFactors

EpigeneticFactors

GeneticFactors

Environment

Host

Four theories of infectious diseases

0

10

20

30

40

50

60M

orta

lity

per 1

00,0

00

0--1

1--2

3--5

6--1

0

11--1

5

16--2

0

21--3

0

31--4

0

41--5

0

51--6

0

61--7

0

over

70

years

generalized TBpulmonary TB

Tuberculosis in children and adults

Ranke, K. 1910. Diagnose und Epidemiologie der Lungentuberculosedes Kindes. Archiv für Kinderheilkunde 54:279-306.

A genetic architecture of tuberculosis

Puberty Menopause

Mendelian

Age

Polygenic

80%

Genetic casesof life-threateninginfections (%)

Primary infection Secondary infection

PhenotypePhenotype RareRare(disseminated infection)(disseminated infection)

CommonCommon(TB, leprosy)(TB, leprosy)

ToolsTools MendelianMendelian GeneticsGenetics Genetic Genetic EpidemiologyEpidemiology

SampleSample SmallSmall LargeLarge

Methods of investigation in humansMethods of investigation in humans

Raremutation

Commonpolymorphism

HYPOTHESIS-DRIVEN APPROACH

MENDELIAN and COMPLEX INHERITANCE

ASSOCIATION STUDIES(Replications)

VARIANTDETECTION

FUNCTIONAL STUDIES

‘COMMON’POLYMORPHISMS

‘RARE’MUTATIONS

GENOME-WIDE APPROACH

ASSOCIATION STUDIES

DIFFERENTIAL EXPRESSION

CANDIDATEGENES

ANIMAL MODELS HUMAN DATA LINKAGE STUDIES

Mendelian susceptibilityto mycobacterial diseases (MSMD)

* Infections by BCG andenvironmental Mycobacteria

* Otherwise healthy individuals

* Very rare (10-5 – 10-6) but often familial (consanguinity)

Mendelian Susceptibility to Mycobacterial diasease

HETEROGENEOUS

Genetic basis: autosomal or X-linked inheritance

Clinical outcome: reccurent, disseminated or local

Granulomatous lesion: lepromatous typetuberculoid type

Over 300 patients in 45 countries

ELISA

Functional screening in vitro

IL12p70,p40…R&D™

Whole blood (heparine)

Supernant 48 h

IFNγ, IL10 …Sanquin™

Ctrl. Pat.

B+IFNγ

Medium

BCG

B+IL12

TNFα

plasma

Monocyte/Dendritic Cell/Macrophage

T Lymphocyte/NK Cell

MycobacteriaIL-12Rβ1

IL-12Rβ2

IFN-γR1

IL-12

p35

p40

IFN-γIFN-γR2STAT-1(GAF)

NEMO CD40 CD40L

MSMD: IFN-γ/IL-12/IL-23 pathway

IL-23p19

- Mendelian disorders of the IL12-IFNγ axis are genetic etiologies for severe forms of tuberculosis:

- What is the proportion of ‘Mendelian’ tuberculosis? (in children) …

Mendelian Tuberculosis

Proof of principle: Mendelian tuberculosis

IL12Rβ1

IL12Rβ2

IFNγR1

IL12 p35

p40

IFNγ

IFNγR2STAT1(GAF)

IL23

1425delC

S321X

ICTMECL1

2 3 4 5 6 7 8 9 10 11 12 13 15 16

549+2T>C 783+1G>A

1791+2T>G

5’

1989

1190-1G>A

L77P

700+362_1619-944 del*

1021+1G>C

64+2T>G

64+5G>A

K305XQ32X Q376X

Q171P

R173PR173W

C186S C198R R213WY367C

550-2A>G

467_483del

C196Y I369T

Y88X

557_563delins8 711insC

628_644dup

1336delC1007_1008

delinsG

R486X

580+1G>A

R521X

983_999del

1189+2T>A

R211P

R175W

1765delG169delA

C62G

E480XE67X Q285X

65del4

1 14

G569D

T355del

W531X

delEx4

1386_1387delGT

1483+182_1619-1073 del

1745_1746insCA

1623_1624delinsTT

1440_1447delins16

IL12-Rβ1 deficiency and tuberculosis

P2

R173W/R173W

WT/WT

R173W/WT

R173W/WT

R173W/R173WE?

E?E?

K305X/WT

E?

K305X/K305X

P1

K305X/WT

Patients with extra-pulmonary TB from Iran and Morocco

I

II

I.I. IFNIFN--γγ ProductionProductionII.II. IFNIFN--γγ ResponseResponseIII.III. ILIL--12 Production 12 Production IV.IV. Genetic ScreeningGenetic Screening

I.I. Genome ScanGenome ScanII.II. Fine MappingFine MappingIII.III. Exome sequencingExome sequencingIV. Candidate GeneIV. Candidate Gene

Functional screening of the pathway

Positional cloning

30 consanguineous families2 X-linked families

MSMD and severe childhood tuberculosis Strategy

P (D311G)

Asp

Gly

Ala

Ala

Phe

Phe

310 311 312

310 311 312

Asp

Gly

Ala

Ala

Phe

Phe

310 311 312

310 311 312

NEMO 43kDa

GAPDH 35kDa

P

(D31

1G)

C+ P

(D31

1G)

Monocytes PMNs

IP

P

(D

311G

)

C+

110_

111

insC

P

(D31

1G)

D311N

EBV-B cells SV-40 fibroblasts

C+C+

Novel mutation in NEMO

C-terN-ter

Proximal Distal

K-63 Di-UbLinear Di-Ub

D311

R74 (Ub)

R72 (Ub)

C+ CD40-/- D311N P (D311G)

IL12

p40

(pg/

ml)

1000

C 0 C 0 C 0 C 0 C 0XR-MSMD R319Q

2000

3000

0

NSCD40L

IL-6

pro

duct

ion

(ng/

ml)

C+ IP X420W R319QP (D311G)

10

20

30

40

50

60

0

NSTNF-α

Impairment of ubiquitin binding of NEMO

Chromosomal position

-log(

P)

Positional Cloning: autosomal recessive

TYK2P1 P2

P2

Tyk2 deficiency

0

20000

40000

60000

80000

100000

120000

140000

NS BCG BCG + IL-12

C+

P

IFN

-γ(p

g/m

l)

IFN-α/-βR

TYK-2

JAK1

IFN-γ

IL-12Rβ1

IL-12Rβ2

TYK-2

JAK2IL-12

Clinical features- BCG-osis- Cutaneous herpes simplex virusinfections

IP-TYK2N-ter

IP-TYK2C-ter

WB

WBTYK2 N-ter

150 --

102 --

C+ P1 P2 C+ P1 P2 C+ P1 P2

I

II

IV

V

IIIP1 P2 P3 P4

E? E? E? E?

P5 P6

Kindred A Kindred B

P7

A novel form of XR- MSMD

Case reports – Kindred A

P1Born 1953No BCG vaccination10 yo: pulmonary TB34 yo: disseminated TBNow 57 yo and welloff all treatment

P2Born 1950BCG inoculation at birth3 mo: BCG-itis12 yo: BCG-itis29 yo: BCG cervical adenitisNow 60 yo and welloff all treatment

P3Born 1955BCG inoculation at birth3 mo: BCG-itisNow 55 yo and welloff all treatment

P4Born 1974 (index case, 1998)BCG inoculation at 2 years3 mo: BCG-itis21 yo: BCG abdominal adenitis24 yo: BCG cervical adenitis27 yo: BCG cervical adenitisNow 36 yo and welloff all treatment

Case reports – Kindred B

P5Born 1974BCG inoculation at birth3 mo: BCG-itisNow 36 yo and welloff all treatment

P6Born 1969BCG inoculation at birth6 mo: BCG-itis4 yo: BCG-itis39 yo: BCG-osisNow 41 yo well off alltreatment

P7Born 1974BCG inoculation at birth3 mo: BCG-itis11 yo: BCG-osis17 yo: BCG-itisNow 36 yo and well off all treatment

Cellular phenotype

Known MSMD etiologies:AD and AR: IL12B, IL12RB1, IFNGR1, IFNGR2, STAT1XR: NEMO (gene structure and function)IL-12 and IFN-γ loops…

Known XR primary immunodeficiencies:XR WAS (platelets, skin, Ig)XR (S)CID (T cell number and function)XR CGD (NBT, chemiluminescence)CD40L (expression)…

LOD

sco

reXp11.4 - Xp21.2 Xq25 - Xq26.3

Positional cloning: X-linked

Strict co-segregation of the CYBB(gp91phox, NOX2) mutation

CYBB (226-260)

Homo sapiens RIVRGQTAESLAVHNITVCEQKISEWG-KIKECPIPMus musculus RIVRGQTAESLEEHNLDICADKIEEWG-KIKECPVPRattus norvegicus RIVRGQTSDSLKEHNLDVCADKIKEWG-KIKECPVPBos Taurus RIVRGQTAESLLKHQPRNCYQNISQWG-KIENCPIPSus scrofa RIVRRQTPKSLLVHDPKACAQNISQWG-KIKDCPIPOryctolagus cuniculus RIVRGQTEESLKKHDPVMCEQHISDWG-KIKDCPVPXenopus tropicalis KIVRGQTDKSLEKHNSTECEDKFTEWG-NITSCPIPDanio rerio RIVRGQTDADLQVHDPTICHSKFEKWGQNVTDCPVPTakifugu_rubripe ---RGQTPASLKSNDPTVCADQFEDWGRNGSNCAVP

These missense mutations are not SNPs,with 1,300 X chromosomes sequenced

They are not known CGD mutations

Q231PT178P

Homo sapiens EGGLYLAVTLLAGITGVVITLCLILIIMus musculus EGGLYVAVTRLAGITGIVITLCLILIIRattus norvegicus EGGLYVAVTRLAGITGIVITLCLILIIBos Taurus EGGLYVAVTRLAGITGVVITLCLILIISus scrofa EGGLYVAVTRLAGITGVVITLCLILIIOryctolagus cuniculus EGGLLVAVTRLAGVTGIIITLCLILIIXenopus tropicalis IGGINVAFTFLAGLTGVVITLALILIIDanio rerio TNPTIVMFTTVAGLTGVVITLALILIIGallus gallus VGGLYVAFTYLAGLTGVIITLALILIICanis lupus familiaris EGGLYVAVTLLAGITGIVITLCLILII

CYBB (164-190)

Early-onset, severe, recurrent,& multiple bacterial& fungal infections

Also vulnerable to BCG & TBin endemic regions

Defect of respiratory burst activityin all phagocytic cells (granulocytes, monocytes &macrophages)

Our patients: seven otherwise healthy adults with a pureMSMD phenotype

CGD and the phagocyte NADPH oxidase

CYBB/gp91phox most frequent form of CGD

O2-

(nm

oles

/106

PMN

s/1h

)

H2O

2 (µ

M/2

.104

PMN

s/30

min

)

Cytochrome-c Resorufin

0

5

10

15

20

25

30

35

40

45C+C-

P

The patients’ PMNs produce O2- and H2O2

NS NS NSPMA PMAPMA P P P P

4

3

0

2

1

5

P-T178PP-Q231PC+ C-

C +

P T178P

Fluorescence (DHR)

NS TNF-α IL-1βfMLP fMLP +TNF-α

fMLP +IL-1β

fMLP +cytochalasin b

cytochalasin b

C -

P Q231PCel

l Num

ber

100 101 102 103 104PE-A

Jerome_CytoB.fcs

100 101 102 103 104PE-A

Jerome_IL-1b.fcs

100 101 102 103 104

100 101 102 103 104

100 101 102 103 104PE-A

Jerome_TNFa.fcs

100 101 102 103 104PE-A

Control_TNFa.fcs

100 101 102 103 104PE-A

Jerome_NS.fcs

100 101 102 103 104

100 101 102 103 104

100 101 102 103 104PE-A

Jerome_FMLP.fcs

100 101 102 103 104PE-A

Control_FMLP.fcs

100 101 102 103 104

100 101 102 103 104

100 101 102 103 104PE-A

Jerome_fMLP + IL-1b.fcs

100 101 102 103 104PE-A

Control_fMLP + IL-1b.fcs

100 101 102 103 104PE-A

Jerome_fMLP + TNFa.fcs

100 101 102 103 104

100 101 102 103 104

100 101 102 103 104PE-A

Jerome_fMLP + CytoB.fcs

100 101 102 103 104PE-A

Control_fMLP + CytoB.fcs

100 101 102 103 104

100 101 102 103 104

100 101 102 103 104

100 101 102 103 104

100 101 102 103 104PE-A

Control_CytoB.fcs

100 101 102 103 104

100 101 102 103 104

100 101 102 103 104PE-A

Control_IL-1b.fcs

100 101 102 103 104PE-A

Control_NS.fcs

100 101 102 103 104

100 101 102 103 104

100 101 102 103 104PE-A

Control_fMLP + TNFa.fcs

The PMNs respond to physiological stimuli

O2-

(nm

oles

/10

6 m

ono/

2h)

H2O

2(n

m/2

.104

mon

o/30

min

)

The patients’ monocytes produce O2- and H2O2

Cytochrome-c Resorufin

0

2

4

6

8

10

12

14C+C-P3,P6

NS NS NSPMA PMAPMA

4

3

0

2

1

P P P PP-T178PP-Q231PC+ C-

30 60 90 120 (min)

CFU

(Log

)C+ 1C+ 2C+ 3C+ 4CGD 1CGD 2CGD 3T178PQ231P

102

104

103

106

105

Normal killing of S. aureus by neutrophils

0.0

0.1

0.2

0.3

0.4

0.5

C+ C- P-Q321P P-T178P H

nmol

es H

2O2

/20,

000

mac

roph

ages

/30m

in NSBCGBCG + PMAPMA

The patients’ macrophagesdo not produce H2O2

Impaired expression of gp91phox

gp91phox

gp65 65 kDa

95 kDa

STAT1M

DM

s

Mon

ocyt

es

PMN

s

MD

Ms-

2

MD

Ms

Mon

ocyt

es

PMN

s

MD

Ms-

2

MD

Ms

Mon

ocyt

es

PMN

s

MD

Ms-

2

MD

Ms

Mon

ocyt

es

PMN

s

MD

Ms-

2

C + P-Q231P P-T178PCGD -gp91phox0

100 kDa

8

4

0

6

2

NSPMA

98 kDa

64 kDagp65

gp91phox

50 kDa22 kDa

β-actinp22phox

The Q231P and T178P alleles are null in EBV-B cells

XR-CGD cells+ gp91

gp91 WT

C+

+ W

T

+ T1

78P

+ Q

231P

XR-C

GD

T178

P

Q23

1P

C+

T178

P

Q23

1P

C+

XR-C

GD

T178

P

Q23

1P

C+

XR-C

GD

T178

P

Q23

1P

+ W

T

+ T1

78P

+ Q

231P

XR-CGD cells+ gp91

T178

P

C+

gp91 WT

Q23

1P

H2O

2 (µ

M/3

0,00

0 EB

V-B

cel

ls)

MycobacteriaIL-12Rβ1

IL-12Rβ2

IFN-γR1

IL-12 p35

p40

IFN-γIFN-γR2STAT-1(GAF)

NEMO CD40 CD40L

Connexion between CYBB and IL-12-IFN-γ ?

gp91phox

Monocyte/ dendritic cellsmacrophage

T Lymphocyte/ NK cells

Collaborators, children and their families

Laurent Abel Jean-Laurent CasanovaLaboratory of Human Genetics of Infectious Diseases