Human Genetics of Infectious Diseases:Human Genetics of Infectious Diseases:Application to mycobacterial infectionsApplication to mycobacterial infections

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

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

Individual variability in response to infection (1)

MycobacteriumMycobacteriumtuberculosistuberculosis

INFECTIONINFECTION(primary/latent)(primary/latent)

PRIMARY DISEASEPRIMARY DISEASE(Ext.Pulm., children)(Ext.Pulm., children)

IMMUNE RESPONSEIMMUNE RESPONSE

M. tbM. tb factors factors (virulence…)(virulence…)

Host factors(age, immune status

GENES, …)

ExposureExposurefactorsfactors

EnvironmentalEnvironmentalfactorsfactors

REACTIVATION DIS.REACTIVATION DIS.(Pulm., adults)(Pulm., adults)

~5%

~5%

0

10

20

30

40

50

60M

ort

alit

y p

er

10

0,0

00

0--

1

1--

2

3--

5

6--

10

11

--1

5

16

--2

0

21

--3

0

31

--4

0

41

--5

0

51

--6

0

61

--7

0

ove

r 7

0

years

generalized TB

pulmonary TB

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

Individual variability in response to infection (2)

PhenotypePhenotype RareRare(disseminated infection)(disseminated infection)

CommonCommon(TB, leprosy)(TB, 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

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 (7 identified genes so far)

Macrophage/Dendritic Cell T Lymphocyte

MycobacteriaIL12R1

IL12R2

IFNR1

IL-12 p35

p40

IFN-IFNR2STAT1

NEMO CD40 CD40L

gp91phox

New specific antimycobacterial immunological pathwayNew specific antimycobacterial immunological pathway

Medical implications (IFN-Medical implications (IFN- treatment) treatment)

From EM to From EM to M. tuberculosisM. tuberculosis

Inherited IL12R1 deficiency :

Spanish familyNo BCG/NTM diseaseNo IL12R1 expressionNo cellular responses to IL-12

IL12RB1 mutation: 1721+2TG

               

IL12-R1 deficiency and tuberculosis

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) … 

Mendelian TB: Conclusion and questions

Alcais et al, J Exp Med, 2005Alcais et al, J Exp Med, 2005

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? Complex TB inheritance

Mendelian TB: 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

~ 400,000 new cases per year~ 400,000 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

HYPOTHESIS-DRIVEN APPROACH

TUBERCULOSIS INHERITANCE: CANDIDATE GENES

ASSOCIATION STUDIES Population or family-based

VARIANTDETECTION

FUNCTIONAL STUDIES

‘COMMON’POLYMORPHISMS

‘RARE’MUTATIONS

GENOME-WIDE APPROACH

ASSOCIATION STUDIES

DIFFERENTIAL EXPRESSION

CANDIDATEGENES

ANIMAL MODELS HUMAN DATA LINKAGE STUDIES

Examples : NRAMP1, HLA-DR, IL12RB1

PULMONARY TUBERCULOSIS (PTB) AND IL12RB1

Family-based association study :101 families including 157 offspring >15 years with PTB (culture +)

- Sequencing of promoter and coding regions in 40 PTB patients No rare loss of function mutations Detection of common polymorphisms >5%

Genotyping in the whole sample Test for association with PTB

AT TT

AT

AT TT

TT

R156H(467G>A)

Q214R(641A>G)

M365T (1094T>C)

+34C>T

5’ 3’

G378R (1132G>C)

387G>C 684C>T +46T>C +24C>T-2C>T +21C>A +47G>T

I II III IV V VI VII VIII IX X XI XII XIII XIV XV XVI XVII

-111A>T

IL12RB1

Association with the two promoter polymorphisms in strong LD- especially for -2C>T (p=0.004), with T frequency ~ 0.1- OR for CT or TT vs. CC = 3.8 [1.6-10.2]

Replication studies Functional studies T is an uncommon allele at position –2 (consensus site)

Remus et al, J Inf Dis, 2004Remus et al, J Inf Dis, 2004

HYPOTHESIS-DRIVEN APPROACH

TUBERCULOSIS INHERITANCE: MAJOR GENES

ASSOCIATION STUDIES Family-based

VARIANTDETECTION

FUNCTIONAL STUDIES

‘COMMON’POLYMORPHISMS

‘RARE’MUTATIONS

GENOME-WIDE APPROACH

ASSOCIATION STUDIES

DIFFERENTIAL EXPRESSION

CANDIDATEREGION

ANIMAL MODELS HUMAN DATA LINKAGE STUDIES

TUBERCULOSIS: Genome-wide screenTUBERCULOSIS: Genome-wide screen

# affected# affectedoffspringoffspring

22 33 44

# families# families 6868 2121 77

96 multiplex families96 multiplex families

Total of 227 offspring Total of 227 offspring

with positive pulmonary TBwith positive pulmonary TB

El Baghdadi et al, J Exp Med, 2006El Baghdadi et al, J Exp Med, 2006

AB CD

AC AC AD BC

IBD=2 IBD=1 IBD=0

Affected sib-pair linkage studyAffected sib-pair linkage study

Linkage to chromosome 8q12-q13Linkage to chromosome 8q12-q13

39 families with affected parent39 families with affected parent

All 96 familiesAll 96 families

Consistent withConsistent withdominant inheritancedominant inheritance

Linkage disequilibrium Linkage disequilibrium mapping is ongoingmapping is ongoing

p<0.00007p<0.00007

p<0.00002p<0.00002

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

PARK2/PACRGPARK2/PACRG

6q256q25

Lod Score = 4.3 (p=5.10 Lod Score = 4.3 (p=5.10 –6–6))

(Mira, Alcaïs, et al. Nature 2004)(Mira, Alcaïs, et al. Nature 2004)

IIIIII IIIIHLAHLA

Non-HLANon-HLA

LD mappingLD mapping

~ 10 Mb~ 10 Mb

33Lod Score = 2.6 (p=2.10 Lod Score = 2.6 (p=2.10 –4–4))

6p216p21

00

11

22

[Mb][Mb]2020 2525 3030 3535 4040 4545 5050

Lo

d M

LB

Lo

d M

LB

Two linkage peaksTwo linkage peaks

LD mappingLD mapping

MBPB

Leprosy subtypeLeprosy subtype

194 simplex families194 simplex families

2 parents + 1 affected offspring2 parents + 1 affected offspring

Alcais et al, Nat Genet, 2007Alcais et al, Nat Genet, 2007

307 informative SNPs307 informative SNPs

SNPs SNPs densitydensity

Association with a bin of several SNPs in the Association with a bin of several SNPs in the LTALTA region region

Replication in an independent Vietnamese sample Replication in an independent Vietnamese sample of 104 trios with the same allele at riskof 104 trios with the same allele at risk

LTA+80LTA+80: OR=2.34 (1.27-4.31), p=0.003: OR=2.34 (1.27-4.31), p=0.003

LTA-293LTA-293: OR=1.95 (1.11-3.31), p=0.03: OR=1.95 (1.11-3.31), p=0.03

Replication in an Indian sample of 364 cases and 371 controlsReplication in an Indian sample of 364 cases and 371 controls

LTALTA+80 is the most likely candidate+80 is the most likely candidate

Study in a Brazilian sample of 209 cases and 192 controlsStudy in a Brazilian sample of 209 cases and 192 controls

No overall effect of LTA+80No overall effect of LTA+80

Strong differences in mean age of patients between the three samples:Strong differences in mean age of patients between the three samples:

Vietnam : 19 yearsVietnam : 19 years

India : 31 yearsIndia : 31 years

Brazil : 38 yearsBrazil : 38 years

Strong heterogeneity of association results according to ageStrong heterogeneity of association results according to age

The The LTALTA+80 effect is strongly age-dependent+80 effect is strongly age-dependent

1st Vietnamese sample (194 trios)

Indian sample 364 cases/371 controls

Brazilian sample 209 cases/192 controls

2nd Vietnamese sample (104 trios)

The The LTALTA+80 effect is strongly age-dependent+80 effect is strongly age-dependent

Summary for leprosySummary for leprosy

Strong association of Strong association of LTALTA+80 A allele with early onset leprosy.+80 A allele with early onset leprosy.

In combined sample of Vietnamese trios < 16 years:In combined sample of Vietnamese trios < 16 years:

OR for OR for LTALTA+80 A+80 AA/AC vs CC subjects = 5.6 (2.5-12.5), p<10-6

LTALTA+80 A decreases LTA protein production +80 A decreases LTA protein production (Knight et al, Nat Genet, 2004)(Knight et al, Nat Genet, 2004)

LTA LTA KO mice are susceptible to mycobacteria KO mice are susceptible to mycobacteria ((Roach et al. J Exp Med, 2001)

The effect of LTA is totally independent of HLA DRB1 allelesThe effect of LTA is totally independent of HLA DRB1 alleles

Other genes in the 6p21 region (especially in adults): HLA DR…Other genes in the 6p21 region (especially in adults): HLA DR…

RR

100

10

5

2

1

Mendelian mutations with causal role demonstrated- direct clinical and therapeutic implications (disseminated TB of children)- information on immunological pathways ( candidate genes)

Intermediate major gene effects - in specific populations, phenotypes, age class … - implications ~ Mendelian

Common polymorphisms with moderate effect - molecular basis difficult to validate (same pathway, interest of GWA) - may have strong attributable risk at the population level

Genetic predisposition to mycobacterial infections continuous spectrum

Genetic dissection needs to combine different strategies

15Age

Origin of genetic cases (%)

Primary infection - - - - - - - - - - - - - - ReactivationExtra-pulmonary - - - - - - - - - - - - - - Pulmonary

30 45 60

Mendelian

Major genePolygenic

50

Genetic spectrum depends on age

0

10

20

30

40

50

60M

ort

alit

y p

er

10

0,0

00

0--

1

1--

2

3--

5

6--

10

11

--1

5

16

--2

0

21

--3

0

31

--4

0

41

--5

0

51

--6

0

61

--7

0

ove

r 7

0

years

generalized TB

pulmonary TB

Laboratory of Human Genetics of Infectious Diseases

BacteriaVirus

Laure GineauE. JouanguyLazaro LorenzoS. PlancoulaineV. Sancho-ShimizuShenying Zhang

Horst von BernuthMaya ChrabiehPegah GandilCapucine PicardAnne Puel

Laurent Abel Jean-Laurent Casanova

March of DimesFRM

Alexandre AlcaïsL. de BeaucoudreyL. Blancas-GaliciaJacinta C. Bustamante Aurélie CobatStéphanie DupuisJacqueline FeinbergAudrey GrantLucile JannièreDaniel NolanBrigitte RanqueNatascha RemusYoann Rose

Mycobacterium

McGill University, Montreal, Canada

Andrea Alter Mariana Orlova Erwin Schurr

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

Hospital of Dermato-Venereology, Ho Chi Minh City, VietnamNguyen Van Thuc Nguyen Thu Huong Vu Hong Thai

All India Institute of Medical Sciences, New Delhi, India Meenakshi Singh Narinder Mehra

Oswaldo Cruz Institute, Fiocruz, Rio de Janeiro, Brazil Milton Moraes

Centro de Ciências Biológicas e da Saúde, Curitiba, Brazil Marcelo Mira