identification of human genes involved in the response to infectious agents
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Identification of human genes involved Identification of human genes involved in the response to infectious agents.in the response to infectious agents.
The example of mycobacterial diseasesThe example of mycobacterial diseases
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
Large individual variability in response to infection
INFECTIOUSINFECTIOUSAGENTAGENT
INFECTIONINFECTION DISEASEDISEASE
IMMUNE RESPONSEIMMUNE RESPONSE
Infectious agent factorsInfectious agent factors (virulence…)(virulence…)
Host factors(age, GENES, …)
ExposureExposurefactorsfactors
EnvironmentalEnvironmentalfactorsfactors
PhenotypePhenotype RareRare(disseminated BCG, EM)(disseminated BCG, EM)
CommonCommon(tuberculosis, leprosy)(tuberculosis, 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
AB CD
AC AC AD BC
IBD=2 IBD=1 IBD=0
Based on number of parental alleles sharedidentical by descent (IBD)
Expected IBD distribution for a sib-pair IBD = 2 : 0.25 IBD = 1 : 0.5 IBD = 0 : 0.25
LINKAGE ANALYSIS METHODS
Classical approach: affected sib-pair method
Test whether affected sibs share more parental alleles than expected Linkage when excess of alleles IBD shared by affected sib-pairs
To investigate the role of a chromosomal region (familial) Study of highly polymorphic markers
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
ASSOCIATION STUDIES : DESIGNS
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
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 TT
TT
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 peoplePhase II: +2.8 M markers in 270 people
QuickTime™ and aTIFF (Uncompressed) decompressor
are needed to see this picture.
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)
Macrophage/Dendritic cell T Lymphocyte/ NK Cell
Mycobacteria
IL12R1
IL12R2
IFNR1
IL12 p35
p40
IFN
IFNR2STAT1
IFNR1
IFNR2
New specific antimycobacterial immunological pathwayNew specific antimycobacterial immunological pathway
New therapeutic strategiesNew therapeutic strategies
IL12-Rb1 deficiency and tuberculosis (1)
18 yoAbdo TBBCG-itis
Inherited IL12Rb1 deficiency :
student from CasablancaNo reaction to 3 live BCG No other unusual clinical infectious diseases
Well without any prophylactic treatment
IL12RB1 mutation: R213WNo cellular response to IL12
Inherited IL12Rb1 deficiency :
No BCG/NTM diseaseNo IL12-Rb1 expressionNo cellular responses to IL-12
IL12RB1 mutation: 1721+2T->G
IL12-Rb1 deficiency and tuberculosis (2)
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)? - May common polymorphisms in these genes also predispose to tuberculosis?
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
~ 700,000 new cases per year~ 700,000 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
From Gentilini & Duflo, Médecine Tropicale, Flammarion Médecine-SciencesFrom Gentilini & Duflo, Médecine Tropicale, Flammarion Médecine-Sciences
Clinical thresholdClinical threshold
LEPROSY: Response to LEPROSY: Response to M. lepraeM. leprae
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
00
11
22
33
44
55
LodLod
Sco
re S
core
3 cM
GA
TA
184A
08G
AT
A18
4A08
D6S
1654
D6S
1654
D6S
476
D6S
476
D6S
2420
D6S
2420
D6S
2436
D6S
2436
D6S
1614
D6S
1614
D6S
415
D6S
415
D6S
1035
D6S
1035
D6S
305
D6S
305
D6S
1579
D6S
1579
D6S
1550
D6S
1550
D6S
253
D6S
253
D6S
955
D6S
955
D6S
1599
D6S
1599
D6S
1277
D6S
1277
D6S
503
D6S
503
D6S
1027
D6S
1027
D6S
1590
D6S
1590
xxxxxxxxxx xx xxxxxx xxxxxx
D6S
1273
D6S
1273
Genome-scan - fine mapping 6q25Genome-scan - fine mapping 6q25
LD mappingLD mapping
LD mappingLD mapping
MBPB
Leprosy subtypeLeprosy subtype
197 simplex families197 simplex families2 parents + 1 affected offspring2 parents + 1 affected offspring
Mira et al, Nature, 2004Mira et al, Nature, 2004
64 informative SNPs64 informative SNPs(( 1 / known gene) 1 / known gene)
SNPs SNPs densitydensity
LD MAPLD MAP
PACRG PACRG intron 1intron 1PARK2 PARK2 intron 1intron 1
PARK2 exon 1PARK2 exon 1 PACRG exon 1PACRG exon 1
Bloc BBloc B
p < 0.05
not significant
PACRG PACRG intron 1intron 1PARK2 PARK2 intron 1intron 1
PARK2PARK2 exon 1 exon 1 PACRGPACRG exon 1 exon 1
Bloc BBloc B
SNP2SNP2MultivariateMultivariate
analysisanalysisSNP1SNP1
Snp 1Snp 2
CT
CT
TT
CT
TT
TC
TT
TC
CT
1.001.00
3.23.2
5.35.3
[1.3 -7.8][1.3 -7.8]
[2.1 -13.5][2.1 -13.5]
OR*OR* CI 95%CI 95%
--
0.0090.009
0.00050.0005
P-valueP-value
--
* Estimated by conditional logistic regression
CC
Replication study in BrazilReplication study in Brazil
587 cases – 388 controls587 cases – 388 controls
MB
PB
Leprosy subtypeLeprosy subtype
13 significant SNPs13 significant SNPs
(genomic controls)(genomic controls)
MarkerMarker VietnamVietnam BrazilBrazilRisk alleleRisk allele p-valuep-value Risk alleleRisk allele p-valuep-value
rs2803104 A 0.011 - ns
10Kb_5_2 T 0.013 T 0.008
e01(-697) G 0.013 G 0.0002
SNP 1SNP 1 TT 0.00060.0006 TT 0.00060.0006e01(-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 2SNP 2 CC 0.0040.004 CC 0.00020.000240Kb_F60 A 0.034 A 0.015
40Kb_F706 G 0.017 - ns
P<0.000005P<0.000005
MultivariateMultivariateanalysisanalysis
PARK2PARK2 PACRGPACRG
Parkin (465 AA)Parkin (465 AA) Protein (257 AA)Protein (257 AA)
Shared regulatory regionShared regulatory region
Ubiquitin Protein E3 LigaseUbiquitin Protein E3 Ligase
(Synphilin 1 / Pael-R / (Synphilin 1 / Pael-R / -synuclein/ CyclinE ..)-synuclein/ CyclinE ..)
Linked to ubiquitin-Linked to ubiquitin-proteasome sytemproteasome sytem
Juvenile Parkinson ARJuvenile Parkinson AR ??
PARK2 / PACRG PARK2 / PACRG
Ubiquitin-mediated proteolysisUbiquitin-mediated proteolysis
Giasson and Lee,
Neuron, 2001
New pathway involved in response to mycobacteria:New pathway involved in response to mycobacteria:-E3 ligase involved in Toll like receptors degradation E3 ligase involved in Toll like receptors degradation (Chuang et al, Nat Immunol, 2004)(Chuang et al, Nat Immunol, 2004)
-Parkin involved in regulation of cellular oxidative stress-Parkin involved in regulation of cellular oxidative stress Functional studies ongoingFunctional studies ongoing
• Variant effect in terms of Relative Risk RR: 1 2 5 10 100
Moderate effect Major effect Mendelian effect
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 predisposition to mycobacterial infections continuous spectrum
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, FranceAlexandre Alcaïs Guillemette Antoni Jacinta BustamanteLudovic de Beaucoudrey Ariane Chapgier Orchidée dos SantosStéphanie Dupuis Claire Fieschi Emmanuelle JouanguyDaniel Nolan Capucine Picard Brigitte RanqueNatascha Remus Claire Soudais Guillaume Vogt
Laurent Abel Jean-Laurent Casanova
McGill University, Montreal, CanadaMarcelo Mira Tom Hudson Erwin Schurr
Laboratoire d’Immunologie, Hôpital Militaire de Rabat, MarocJamila El Baghdadi Abdellah Benslimane
Hospital of Dermato-Veneorology, Ho Chi Minh City, VietnamHospital of Dermato-Veneorology, Ho Chi Minh City, VietnamNguyen ThucNguyen Thuc Minh PhuongMinh Phuong
Oswaldo Cruz Institute, Fiocruz, Rio de Janeiro, BrazilOswaldo Cruz Institute, Fiocruz, Rio de Janeiro, Brazil
Milton MoraesMilton Moraes
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