Pr

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Identification of human genes involved in the
response to infectious agents. The example of
mycobacterial diseases
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Human genetics in infectious diseases ?
Epidemiological observations
Concept
Experimental models
Genetic epidemiology
Proof of concept
Mendelian genetics
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? Large individual variability in response to
infection
Infectious agent factors (virulence)
INFECTION
DISEASE
INFECTIOUS AGENT
IMMUNE RESPONSE
Exposure factors
Environmental factors
Host factors (age, GENES, )
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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
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MENDELIAN AND COMPLEX INHERITANCE
HYPOTHESIS-DRIVEN APPROACH
GENOME-WIDE APPROACH
ASSOCIATION STUDIES
DIFFERENTIAL EXPRESSION
LINKAGE STUDIES
HUMAN DATA
ANIMAL MODELS
CANDIDATE GENES
VARIANT DETECTION
COMMON POLYMORPHISMS
RARE MUTATIONS
ASSOCIATION STUDIES (Replications)
FUNCTIONAL STUDIES
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LINKAGE ANALYSIS METHODS
To investigate the role of a chromosomal region
(familial) ? Study of highly polymorphic markers
Classical approach affected sib-pair method
CD
AB
Based on number of parental alleles
shared identical by descent (IBD) Expected IBD
distribution for a sib-pair IBD 2 0.25
IBD 1 0.5 IBD 0 0.25
AC
BC
AC
AD
IBD2
IBD1
IBD0
? Test whether affected sibs share more parental
alleles than expected Linkage when excess
of alleles IBD shared by affected sib-pairs

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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)
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
AT
AT
AT
TT
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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 people
• Phase II 2.8 M markers in 270 people

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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)

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Mycobacteria
IL12Rb1
p35
IL12
IL12Rb2
p40
IFNgR1
IFNg
STAT1
IFNgR2
IFNgR1
IFNgR2
Macrophage/Dendritic cell
T Lymphocyte/ NK Cell
New specific antimycobacterial immunological
pathway New therapeutic strategies
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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
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IL12-Rb1 deficiency and tuberculosis (2)
                             
Inherited IL12Rb1 deficiency  No BCG/NTM
disease No IL12-Rb1 expression No cellular
responses to IL-12 IL12RB1 mutation
17212T-gtG
17 yo
15 yo Pulm TB
8 yo Diss TB
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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?

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Complex predisposition to common mycobacterial
diseases
Tuberculosis (M. tuberculosis)
Leprosy (M. leprae)
700,000 new cases per year 95 of infected
subjects do not develop the disease
8 millions new cases per year 90 of
infected subjects do not develop the disease

• Very large spectrum of clinical manifestations

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LEPROSY Response to M. leprae
Clinical threshold
From Gentilini Duflo, Médecine Tropicale,
Flammarion Médecine-Sciences
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LEPROSY INHERITANCE
HYPOTHESIS-DRIVEN APPROACH
GENOME-WIDE APPROACH
ASSOCIATION STUDIES
DIFFERENTIAL EXPRESSION
LINKAGE STUDIES
HUMAN DATA
ANIMAL MODELS
CANDIDATE REGIONS
VARIANT DETECTION
COMMON POLYMORPHISMS
RARE MUTATIONS
ASSOCIATION STUDIES Replication
FUNCTIONAL STUDIES
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LEPROSY Genome-wide screen
86 multiplex families
affected Offspring 2 3 4 5
families 63 15 6 2
Leprosy subtype
Mira et al, Nat Genet, 2003
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Genome-scan - fine mapping 6q25
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LD mapping
197 simplex families 2 parents 1 affected
offspring
64 informative SNPs (? 1 / known gene)
Mira et al, Nature, 2004
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Bloc B
PACRG intron 1
PARK2 intron 1
PARK2 exon 1
PACRG exon 1
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Bloc B
Multivariate analysis
SNP2
SNP1
PACRG intron 1
PARK2 intron 1
PARK2 exon 1
PACRG exon 1
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OR
CI 95
P-value
C
C
Snp 1
-
-
1.00
Snp 2
T
T
C
T
T
3.2
1.3 -7.8
0.009
C
T
T
C
T
C
T
T
T
2.1 -13.5
0.0005
5.3
C
T
T
C
Estimated by conditional logistic regression
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Replication study in Brazil
587 cases 388 controls
Leprosy subtype
13 significant SNPs (genomic controls)
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Marker Vietnam Vietnam Brazil Brazil Brazil Brazil Brazil
Risk allele p-value Risk allele Risk allele Risk allele p-value
rs2803104 A 0.011 - ns ns
10Kb_5_2 T 0.013 T 0.008 0.008
e01(-697) G 0.013 G 0.0002 0.0002
SNP 1 T 0.0006 T 0.0006 0.0006
e01(-3024) C 0.029 - ns ns
e01(-3800) G 0.001 G 0.003 0.003
28Kb_2_1 T 0.017 - ns ns
28Kb_4_1 A 0.002 A 0.0009 0.0009
rs1514343 T 0.03 T 0.023 0.023
rs1333955 C 0.0007 C 0.016 0.016
SNP 2 C 0.004 C 0.0002 0.0002
40Kb_F60 A 0.034 A 0.015 0.015
40Kb_F706 G 0.017 - ns ns
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PARK2 / PACRG
PARK2 PACRG
Parkin (465 AA) Protein (257 AA)
Shared regulatory region Shared regulatory region
Ubiquitin Protein E3 Ligase (Synphilin 1 / Pael-R / ?-synuclein/ CyclinE ..) Linked to ubiquitin-proteasome sytem
Juvenile Parkinson AR ?
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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

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Genetic predisposition to mycobacterial
infections ? continuous spectrum

• Variant effect in terms of Relative Risk

Moderate effect Major effect Mendelian
effect
RR 1 2 5
10 100

• 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)

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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
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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 Jean-Laurent
Casanova McGill University, Montreal,
Canada Marcelo Mira Tom Hudson Erwin
Schurr Laboratoire dImmunologie, 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