Transcript Abel

Identification of human genes involved
in the response to infectious agents.
The example of mycobacterial diseases
Human genetics in infectious diseases ?
Concept
Epidemiological
observations
Experimental
models
Genetic
epidemiology
Proof of
concept
Mendelian
genetics
 Large individual variability in response to infection
Infectious agent factors
(virulence…)
INFECTIOUS
AGENT
INFECTION
Exposure
factors
IMMUNE RESPONSE
Host factors
(age, GENES, …)
DISEASE
Environmental
factors
Methods of investigation in humans
Phenotype
Rare
(disseminated BCG, EM)
Common
(tuberculosis, leprosy)
Tools
Mendelian Genetics
Genetic Epidemiology
Sample
Small
Large
Rare
mutation
Common
polymorphism
MENDELIAN AND COMPLEX INHERITANCE
HYPOTHESIS-DRIVEN APPROACH
ANIMAL MODELS
GENOME-WIDE APPROACH
LINKAGE
STUDIES
HUMAN DATA
DIFFERENTIAL ASSOCIATION
EXPRESSION
STUDIES
CANDIDATE
GENES
VARIANT
DETECTION
‘RARE’
MUTATIONS
‘COMMON’
POLYMORPHISMS
ASSOCIATION STUDIES
(Replications)
FUNCTIONAL STUDIES
LINKAGE ANALYSIS METHODS
To investigate the role of a chromosomal region (familial)
 Study of highly polymorphic markers
Classical approach: affected sib-pair method
AB
AC
IBD=2
CD
AC
AD
Based on number of parental alleles shared
identical by descent (IBD)
BC
IBD=1 IBD=0
Expected IBD distribution for a sib-pair
IBD = 2 : 0.25
IBD = 1 : 0.5
IBD = 0 : 0.25
 Test whether affected sibs share more parental alleles than expected
Linkage when excess of alleles IBD shared by affected sib-pairs
ASSOCIATION STUDIES : DESIGNS
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
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
AT
TT
TT
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
Haplotype Map of the Human Genome
QuickTime™ and a
TIFF (Uncompressed) decompressor
are needed to see this picture.
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 people
Phase II: +2.8 M markers in 270 people
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)
Mycobacteria
IL12 p35
IL12Rb1
p40
IL12Rb2
IFNgR1
STAT1
IFNg
IFNgR2
Macrophage/Dendritic cell
T Lymphocyte/ NK Cell
New specific antimycobacterial immunological pathway
New therapeutic strategies
IL12-Rb1 deficiency and tuberculosis (1)
Inherited IL12Rb1 deficiency :
student from Casablanca
No reaction to 3 live BCG
No other unusual clinical infectious diseases
Well without any prophylactic treatment
BCG-itis
18 yo
Abdo TB
IL12RB1 mutation: R213W
No cellular response to IL12
IL12-Rb1 deficiency and tuberculosis (2)
Inherited IL12Rb1 deficiency :
No BCG/NTM disease
No IL12-Rb1 expression
No cellular responses to IL-12
17 yo
15 yo
Pulm TB
8 yo
Diss TB
IL12RB1 mutation: 1721+2T->G
Conclusion and questions
Mendelian disorders of the IL12-IFNg 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 predisposition
to common mycobacterial diseases
Tuberculosis
Leprosy
(M. tuberculosis)
(M. leprae)
~ 8 millions new cases per year
~ 700,000 new cases per year
~ 90% of infected subjects do not
develop the disease
~ 95% of infected subjects do not
develop the disease
Very large spectrum of clinical manifestations
LEPROSY: Response to M. leprae
Clinical threshold
From Gentilini & Duflo, Médecine Tropicale, Flammarion Médecine-Sciences
LEPROSY INHERITANCE
HYPOTHESIS-DRIVEN APPROACH
ANIMAL MODELS
GENOME-WIDE APPROACH
LINKAGE
STUDIES
HUMAN DATA
DIFFERENTIAL ASSOCIATION
EXPRESSION
STUDIES
CANDIDATE
REGIONS
VARIANT
DETECTION
‘RARE’
MUTATIONS
‘COMMON’
POLYMORPHISMS
ASSOCIATION STUDIES
Replication
FUNCTIONAL STUDIES
LEPROSY: Genome-wide screen
86 multiplex families
# affected
Offspring
2
3
4
5
# families
63
15
6
2
Leprosy subtype
PB
MB
Mira et al, Nat Genet, 2003
0
x
GATA184A08
x
D6S1654
x x x
D6S415
D6S476
D6S2420
D6S2436
x
D6S1599
D6S955
xx
x
x
D6S1590
D6S1027
D6S503
D6S1277
D6S1273
x
D6S305
D6S253
D6S1550
D6S1035
D6S1579 D6S1614
Lod Score
Genome-scan - fine mapping 6q25
5
4
3
2
LD mapping
1
3 cM
x
LD mapping
197 simplex families
2 parents + 1 affected offspring
Leprosy subtype
PB
MB
64 informative SNPs
( 1 / known gene)
Mira et al, Nature, 2004
 SNPs
density
LD MAP
Bloc B
PARK2 intron 1
PARK2 exon 1
PACRG intron 1
PACRG exon 1
p < 0.05
not significant
Bloc B
SNP1
Multivariate
analysis
PARK2 intron 1
PARK2 exon 1
PACRG intron 1
PACRG exon 1
SNP2
Snp 1
Snp 2
C
C
C
T
T
T
C
T
C
T
OR*
CI 95%
P-value
1.00
-
-
3.2
[1.3 -7.8]
0.009
5.3
[2.1 -13.5]
0.0005
T
T
C
T
T
C
T
T
T
C
* Estimated by conditional logistic regression
Replication study in Brazil
587 cases – 388 controls
Leprosy subtype
PB
MB
13 significant SNPs
(genomic controls)
Marker
Vietnam
Brazil
Risk allele
p-value
Risk allele
p-value
rs2803104
A
0.011
-
ns
10Kb_5_2
T
0.013
T
0.008
e01(-697)
G
0.013
G
0.0002
SNP 1
T
0.0006
T
0.0006
e01(-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 2
C
0.004
C
0.0002
40Kb_F60
A
0.034
A
0.015
40Kb_F706
G
0.017
-
ns
Multivariate
analysis
P<0.000005
PARK2 / PACRG
PARK2
PACRG
Parkin (465 AA)
Protein (257 AA)
Shared regulatory region
Ubiquitin Protein E3 Ligase
(Synphilin 1 / Pael-R /
-synuclein/ CyclinE ..)
Juvenile Parkinson AR
Linked to ubiquitinproteasome sytem
?
Ubiquitin-mediated proteolysis
Giasson and Lee,
Neuron, 2001
New pathway involved in response to mycobacteria:
-E3 ligase involved in Toll like receptors degradation (Chuang et al, Nat Immunol, 2004)
-Parkin involved in regulation of cellular oxidative stress
 Functional studies ongoing
Genetic predisposition to mycobacterial infections
 continuous spectrum
• Variant effect in terms of Relative Risk
Moderate effect
RR:
1
2
5
Major effect
10
Mendelian effect
100
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 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, France
Alexandre Alcaïs
Guillemette Antoni
Jacinta Bustamante
Ludovic de Beaucoudrey
Ariane Chapgier
Orchidée dos Santos
Stéphanie Dupuis
Claire Fieschi
Emmanuelle Jouanguy
Daniel Nolan
Capucine Picard
Brigitte Ranque
Natascha Remus
Claire Soudais
Guillaume Vogt
Laurent Abel
Marcelo Mira
Jean-Laurent Casanova
McGill University, Montreal, Canada
Tom Hudson
Erwin Schurr
Laboratoire d’Immunologie, Hôpital Militaire de Rabat, Maroc
Jamila El Baghdadi
Abdellah Benslimane
Hospital of Dermato-Veneorology, Ho Chi Minh City, Vietnam
Nguyen Thuc
Minh Phuong
Oswaldo Cruz Institute, Fiocruz, Rio de Janeiro, Brazil
Milton Moraes