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Human Genetics of Infectious Diseases Application
to mycobacterial infections
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The genus Mycobacterium
Virulent Weakly virulent M.
tuberculosis complex gt 80 species (e.g. M.
avium, M. leprae M. marinum, M.
fortuitum) Human transmission
Environmental transmission (airborne) (wat
er, soil, air)
M. ulcerans (Buruli ulcer) BCG
vaccine Aquatic bug transmission? Injection
transmission
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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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? Individual variability in response to infection
(1)
M. tb factors (virulence)
PRIMARY DISEASE (Ext.Pulm., children)
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INFECTION (primary/latent)
Mycobacterium tuberculosis
REACTIVATION DIS. (Pulm., adults)
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IMMUNE RESPONSE
Environmental factors
Exposure factors
Host factors (age, immune status GENES, )
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? Individual variability in response to infection
(2)
Ranke, K. 1910. Diagnose und Epidemiologie der
Lungentuberculose des Kindes. Archiv für
Kinderheilkunde 54279-306.
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Methods of investigation in humans
Phenotype Rare (disseminated infection) Common (TB, 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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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)

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Mycobacteria
IL12R?1
p35
IL-12
gp91phox
IL12R?2
p40
NEMO
CD40
CD40L
IFN?R1
IFN?R2
STAT1
IFN-?
Macrophage/Dendritic Cell
T Lymphocyte

• New specific antimycobacterial immunological
  pathway
• Medical implications (IFN-? treatment)

? From EM to M. tuberculosis
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IL12-R?1 deficiency and tuberculosis
                             
Inherited IL12R?1 deficiency  Spanish family No
BCG/NTM disease No IL12R?1 expression No cellular
responses to IL-12 IL12RB1 mutation
17212T?G
17 yo
15 yo Pulm TB
8 yo Diss TB
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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)  
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Alcais et al, J Exp Med, 2005
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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

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Complex predisposition to common mycobacterial
diseases
Tuberculosis (M. tuberculosis)
Leprosy (M. leprae)
400,000 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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TUBERCULOSIS INHERITANCE CANDIDATE GENES
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 Population or
family-based
FUNCTIONAL STUDIES
? Examples NRAMP1, HLA-DR, IL12RB1
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PULMONARY TUBERCULOSIS (PTB) AND IL12RB1

• Family-based association study
• 101 families including 157 offspring
• gt15 years with PTB (culture )

TT
TT
AT
AT
AT
TT

• - Sequencing of promoter and coding regions in 40
  PTB patients
• No rare loss of function mutations
• Detection of common polymorphisms gt5
• Genotyping in the whole sample
• Test for association with PTB

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IL12RB1
R156H (467GgtA)
Q214R (641AgtG)
M365T (1094TgtC)
G378R (1132GgtC)
5
3

34CgtT
387GgtC
684CgtT
46TgtC
24CgtT
-2CgtT
21CgtA
47GgtT
-111AgtT

• Association with the two promoter polymorphisms
  in strong LD
• especially for -2CgtT (p0.004), 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, 2004
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TUBERCULOSIS INHERITANCE MAJOR GENES
HYPOTHESIS-DRIVEN APPROACH
GENOME-WIDE APPROACH
ASSOCIATION STUDIES
DIFFERENTIAL EXPRESSION
LINKAGE STUDIES
HUMAN DATA
ANIMAL MODELS
CANDIDATE REGION
VARIANT DETECTION
COMMON POLYMORPHISMS
RARE MUTATIONS
ASSOCIATION STUDIES Family-based
FUNCTIONAL STUDIES
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TUBERCULOSIS Genome-wide screen
Affected sib-pair linkage study
96 multiplex families
Total of 227 offspring with positive pulmonary TB
affected offspring 2 3 4
families 68 21 7
El Baghdadi et al, J Exp Med, 2006
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Linkage to chromosome 8q12-q13
plt0.00002
39 families with affected parent
plt0.00007
All 96 families
Consistent with dominant inheritance
Linkage disequilibrium mapping is ongoing
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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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Two linkage peaks
6q25
Lod Score 4.3 (p5.10 6)
PARK2/PACRG
(Mira, Alcaïs, et al. Nature 2004)
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LD mapping
194 simplex families 2 parents 1 affected
offspring
307 informative SNPs
Alcais et al, Nat Genet, 2007
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? SNPs density
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? Association with a bin of several SNPs in the
LTA region
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Replication in an independent Vietnamese sample
of 104 trios with the same allele at risk
LTA80 OR2.34 (1.27-4.31), p0.003 LTA-293
OR1.95 (1.11-3.31), p0.03
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Replication in an Indian sample of 364 cases and
371 controls
? LTA80 is the most likely candidate
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The LTA80 effect is strongly age-dependent
Study in a Brazilian sample of 209 cases and 192
controls ? No overall effect of LTA80
Strong differences in mean age of patients
between the three samples Vietnam 19
years India 31 years Brazil 38 years ?
Strong heterogeneity of association results
according to age
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The LTA80 effect is strongly age-dependent
1st Vietnamese sample (194 trios)
2nd Vietnamese sample (104 trios)
Indian sample 364 cases/371 controls
Brazilian sample 209 cases/192 controls
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Summary for leprosy
Strong association of LTA80 A allele with early
onset leprosy. In combined sample of Vietnamese
trios lt 16 years OR for LTA80 AA/AC vs CC
subjects 5.6 (2.5-12.5), plt10-6
LTA80 A decreases LTA protein production (Knight
et al, Nat Genet, 2004) LTA KO mice are
susceptible to mycobacteria (Roach et al. J Exp
Med, 2001)
The effect of LTA is totally independent of HLA
DRB1 alleles Other genes in the 6p21 region
(especially in adults) HLA DR
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Genetic predisposition to mycobacterial
infections ? continuous spectrum
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 dissection needs to combine different
strategies
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Genetic spectrum depends on age
Origin of genetic cases ()
Age
15
30
45
60
Primary infection - - - - - - - - - - - - - - ?
Reactivation Extra-pulmonary - - - - - - - - - -
- - - - ? Pulmonary
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March of Dimes FRM
Laboratory of Human Genetics of Infectious
Diseases
Mycobacterium
Alexandre Alcaïs L. de Beaucoudrey L.
Blancas-Galicia Jacinta C. Bustamante Aurélie
Cobat Stéphanie Dupuis Jacqueline Feinberg Audrey
Grant Lucile Jannière Daniel Nolan Brigitte
Ranque Natascha Remus Yoann Rose
Bacteria
Virus
Laure Gineau E. Jouanguy Lazaro Lorenzo S.
Plancoulaine V. Sancho-Shimizu Shenying Zhang
Horst von Bernuth Maya Chrabieh Pegah
Gandil Capucine Picard Anne Puel
Laurent Abel Jean-Laurent Casanova
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McGill University, Montreal, Canada Andrea
Alter Mariana Orlova Erwin Schurr Laboratoire
dImmunologie, Hôpital Militaire de Rabat,
Maroc Jamila El Baghdadi Abdellah
Benslimane Hospital of Dermato-Venereology, Ho
Chi Minh City, Vietnam Nguyen 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