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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