Identification of human genes involved Identification of human genes involved in the response to infectious agents.in the response to infectious agents.

The example of mycobacterial diseasesThe example of mycobacterial diseases

Human genetics in infectious diseases ?Human genetics in infectious diseases ?

Experimental Experimental modelsmodels

Genetic Genetic epidemiologyepidemiology

Epidemiological Epidemiological observationsobservations

Mendelian Mendelian geneticsgenetics

Proof ofProof ofconceptconcept

ConceptConcept

Large individual variability in response to infection

INFECTIOUSINFECTIOUSAGENTAGENT

INFECTIONINFECTION DISEASEDISEASE

IMMUNE RESPONSEIMMUNE RESPONSE

Infectious agent factorsInfectious agent factors (virulence…)(virulence…)

Host factors(age, GENES, …)

ExposureExposurefactorsfactors

EnvironmentalEnvironmentalfactorsfactors

PhenotypePhenotype RareRare(disseminated BCG, EM)(disseminated BCG, EM)

CommonCommon(tuberculosis, leprosy)(tuberculosis, leprosy)

ToolsTools Mendelian GeneticsMendelian Genetics Genetic EpidemiologyGenetic Epidemiology

SampleSample SmallSmall LargeLarge

Methods of investigation in humansMethods of investigation in humans

Rare mutation

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

AB CD

AC AC AD BC

IBD=2 IBD=1 IBD=0

Based on number of parental alleles sharedidentical by descent (IBD)

Expected IBD distribution for a sib-pair IBD = 2 : 0.25 IBD = 1 : 0.5 IBD = 0 : 0.25

LINKAGE ANALYSIS METHODS

Classical approach: affected sib-pair method

Test whether affected sibs share more parental alleles than expected Linkage when excess of alleles IBD shared by affected sib-pairs

To investigate the role of a chromosomal region (familial) Study of highly polymorphic markers

 To test the role of a speficic allele study of intragenic single nucleotide polymorphisms (SNP) with 2 alleles : (A, T)

Population-based case/control studies compare A frequency between affected and unaffected subjects 

ASSOCIATION STUDIES : DESIGNS

Family-based studies: avoid population stratification and bias due to choice of controls Ex: Transmission Disequilibrium Test (Spielman et al, Am J Hum Genet, 1993)

AT TT

AT

If A is the functional allele or is in linkage disequilibrium with it, it will be transmitted from AT parents to affected children with probability 0.5

AT TT

TT

Haplotype Map of the Human Genome

Goals:

• Define patterns of genetic variation across human genome• Guide selection of SNPs efficiently to “tag” common variants Genome-wide association studies

Phase I: 1.3 M markers in 269 peoplePhase II: +2.8 M markers in 270 people

QuickTime™ and aTIFF (Uncompressed) decompressor

are needed to see this picture.

MENDELIAN SUSCEPTIBILITY TO MYCOBACTERIAL DISEASES (MSMD)

• Disseminated infections by environmental mycobacteria (EM), BCG

• No known primary or acquired immunodeficiency

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

• Mendelian transmission (5 identified genes so far)

Macrophage/Dendritic cell T Lymphocyte/ NK Cell

Mycobacteria

IL12R1

IL12R2

IFNR1

IL12 p35

p40

IFN

IFNR2STAT1

IFNR1

IFNR2

New specific antimycobacterial immunological pathwayNew specific antimycobacterial immunological pathway

New therapeutic strategiesNew therapeutic strategies

IL12-Rb1 deficiency and tuberculosis (1)

18 yoAbdo TBBCG-itis

Inherited IL12Rb1 deficiency :

student from CasablancaNo reaction to 3 live BCG No other unusual clinical infectious diseases

Well without any prophylactic treatment

IL12RB1 mutation: R213WNo cellular response to IL12

Inherited IL12Rb1 deficiency :

No BCG/NTM diseaseNo IL12-Rb1 expressionNo cellular responses to IL-12

IL12RB1 mutation: 1721+2T->G

               

IL12-Rb1 deficiency and tuberculosis (2)

17 yo 15 yoPulm TB

8 yoDiss TB

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

- What is the proportion of ‘Mendelian’ tuberculosis? (in children)?  - May common polymorphisms in these genes also predispose to tuberculosis?

Conclusion and questions

~ 8 millions new cases per year ~ 8 millions new cases per year

~ 90% of infected subjects do not ~ 90% of infected subjects do not develop the disease develop the disease

~ 700,000 new cases per year~ 700,000 new cases per year

~ 95% of infected subjects do not ~ 95% of infected subjects do not develop the diseasedevelop the disease

Tuberculosis Tuberculosis ((M. tuberculosisM. tuberculosis))

Leprosy Leprosy ((M. lepraeM. leprae))

Complex predispositionComplex predispositionto common mycobacterial diseasesto common mycobacterial diseases

Very large spectrum of clinical manifestations

From Gentilini & Duflo, Médecine Tropicale, Flammarion Médecine-SciencesFrom Gentilini & Duflo, Médecine Tropicale, Flammarion Médecine-Sciences

Clinical thresholdClinical threshold

LEPROSY: Response to LEPROSY: Response to M. lepraeM. leprae

HYPOTHESIS-DRIVEN APPROACH

LEPROSY INHERITANCE

ASSOCIATION STUDIES Replication

VARIANTDETECTION

FUNCTIONAL STUDIES

‘COMMON’POLYMORPHISMS

‘RARE’MUTATIONS

GENOME-WIDE APPROACH

ASSOCIATION STUDIES

DIFFERENTIAL EXPRESSION

CANDIDATEREGIONS

ANIMAL MODELS HUMAN DATA LINKAGE STUDIES

LEPROSY: Genome-wide screenLEPROSY: Genome-wide screen

# affected# affectedOffspringOffspring

22 33 44 55

# families# families 6363 1515 66 22

MB

PB

Leprosy subtypeLeprosy subtype

86 multiplex families86 multiplex families

Mira et al, Nat Genet, 2003Mira et al, Nat Genet, 2003

00

11

22

33

44

55

LodLod

Sco

re S

core

3 cM

GA

TA

184A

08G

AT

A18

4A08

D6S

1654

D6S

1654

D6S

476

D6S

476

D6S

2420

D6S

2420

D6S

2436

D6S

2436

D6S

1614

D6S

1614

D6S

415

D6S

415

D6S

1035

D6S

1035

D6S

305

D6S

305

D6S

1579

D6S

1579

D6S

1550

D6S

1550

D6S

253

D6S

253

D6S

955

D6S

955

D6S

1599

D6S

1599

D6S

1277

D6S

1277

D6S

503

D6S

503

D6S

1027

D6S

1027

D6S

1590

D6S

1590

xxxxxxxxxx xx xxxxxx xxxxxx

D6S

1273

D6S

1273

Genome-scan - fine mapping 6q25Genome-scan - fine mapping 6q25

LD mappingLD mapping

LD mappingLD mapping

MBPB

Leprosy subtypeLeprosy subtype

197 simplex families197 simplex families2 parents + 1 affected offspring2 parents + 1 affected offspring

Mira et al, Nature, 2004Mira et al, Nature, 2004

64 informative SNPs64 informative SNPs(( 1 / known gene) 1 / known gene)

SNPs SNPs densitydensity

LD MAPLD MAP

PACRG PACRG intron 1intron 1PARK2 PARK2 intron 1intron 1

PARK2 exon 1PARK2 exon 1 PACRG exon 1PACRG exon 1

Bloc BBloc B

p < 0.05

not significant

PACRG PACRG intron 1intron 1PARK2 PARK2 intron 1intron 1

PARK2PARK2 exon 1 exon 1 PACRGPACRG exon 1 exon 1

Bloc BBloc B

SNP2SNP2MultivariateMultivariate

analysisanalysisSNP1SNP1

Snp 1Snp 2

CT

CT

TT

CT

TT

TC

TT

TC

CT

1.001.00

3.23.2

5.35.3

[1.3 -7.8][1.3 -7.8]

[2.1 -13.5][2.1 -13.5]

OR*OR* CI 95%CI 95%

--

0.0090.009

0.00050.0005

P-valueP-value

--

* Estimated by conditional logistic regression

CC

Replication study in BrazilReplication study in Brazil

587 cases – 388 controls587 cases – 388 controls

MB

PB

Leprosy subtypeLeprosy subtype

13 significant SNPs13 significant SNPs

(genomic controls)(genomic controls)

MarkerMarker VietnamVietnam BrazilBrazilRisk alleleRisk allele p-valuep-value Risk alleleRisk allele p-valuep-value

rs2803104 A 0.011 - ns

10Kb_5_2 T 0.013 T 0.008

e01(-697) G 0.013 G 0.0002

SNP 1SNP 1 TT 0.00060.0006 TT 0.00060.0006e01(-3024) C 0.029 - ns

e01(-3800) G 0.001 G 0.003

28Kb_2_1 T 0.017 - ns

28Kb_4_1 A 0.002 A 0.0009

rs1514343 T 0.03 T 0.023

rs1333955 C 0.0007 C 0.016

SNP 2SNP 2 CC 0.0040.004 CC 0.00020.000240Kb_F60 A 0.034 A 0.015

40Kb_F706 G 0.017 - ns

P<0.000005P<0.000005

MultivariateMultivariateanalysisanalysis

PARK2PARK2 PACRGPACRG

Parkin (465 AA)Parkin (465 AA) Protein (257 AA)Protein (257 AA)

Shared regulatory regionShared regulatory region

Ubiquitin Protein E3 LigaseUbiquitin Protein E3 Ligase

(Synphilin 1 / Pael-R / (Synphilin 1 / Pael-R / -synuclein/ CyclinE ..)-synuclein/ CyclinE ..)

Linked to ubiquitin-Linked to ubiquitin-proteasome sytemproteasome sytem

Juvenile Parkinson ARJuvenile Parkinson AR ??

PARK2 / PACRG PARK2 / PACRG

Ubiquitin-mediated proteolysisUbiquitin-mediated proteolysis

Giasson and Lee,

Neuron, 2001

New pathway involved in response to mycobacteria:New pathway involved in response to mycobacteria:-E3 ligase involved in Toll like receptors degradation E3 ligase involved in Toll like receptors degradation (Chuang et al, Nat Immunol, 2004)(Chuang et al, Nat Immunol, 2004)

-Parkin involved in regulation of cellular oxidative stress-Parkin involved in regulation of cellular oxidative stress Functional studies ongoingFunctional studies ongoing

• Variant effect in terms of Relative Risk RR: 1 2 5 10 100

Moderate effect Major effect Mendelian effect

Mendelian control in rare phenotypes

Rare mutations with causal role demonstrated- direct clinical and therapeutic implications- information on immunological pathways ( candidate genes)- may be involved in more common phenotypes (TB)

Genetic predisposition to mycobacterial infections continuous spectrum

Genetic control of more common phenotypes

Common polymorphisms with moderate effect - molecular basis difficult to validate - identification of relevant pathways - may have strong attributable risk (in large populations)

Importance of searching for major gene effects - in specific populations, phenotypes … - implications ~ Mendelian

The genetic dissection of infectious diseases needs to combine different strategies and approaches

Génétique Humaine des Maladies Infectieuses, INSERM U550, Paris, FranceAlexandre Alcaïs Guillemette Antoni Jacinta BustamanteLudovic de Beaucoudrey Ariane Chapgier Orchidée dos SantosStéphanie Dupuis Claire Fieschi Emmanuelle JouanguyDaniel Nolan Capucine Picard Brigitte RanqueNatascha Remus Claire Soudais Guillaume Vogt

Laurent Abel Jean-Laurent Casanova

McGill University, Montreal, CanadaMarcelo Mira Tom Hudson Erwin Schurr

Laboratoire d’Immunologie, Hôpital Militaire de Rabat, MarocJamila El Baghdadi Abdellah Benslimane

Hospital of Dermato-Veneorology, Ho Chi Minh City, VietnamHospital of Dermato-Veneorology, Ho Chi Minh City, VietnamNguyen ThucNguyen Thuc Minh PhuongMinh Phuong

Oswaldo Cruz Institute, Fiocruz, Rio de Janeiro, BrazilOswaldo Cruz Institute, Fiocruz, Rio de Janeiro, Brazil

Milton MoraesMilton Moraes