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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
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
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