Identification of human genes involved in the response to infectious agents.

1. Identification of human genes involved in the response to infectious agents. The example of mycobacterial diseases

2. Human genetics in infectious diseases ? Epidemiological observations Concept Experimental models Genetic epidemiology Proof of concept Mendelian genetics

3. 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, …)

4. Methods of investigation in humans Rare mutation Common polymorphism

5. MENDELIAN AND COMPLEX INHERITANCE HYPOTHESIS-DRIVEN APPROACH GENOME-WIDE APPROACH LINKAGE STUDIES DIFFERENTIAL EXPRESSION ASSOCIATION STUDIES HUMAN DATA ANIMAL MODELS CANDIDATE GENES VARIANT DETECTION ‘RARE’ MUTATIONS ‘COMMON’ POLYMORPHISMS ASSOCIATION STUDIES (Replications) FUNCTIONAL STUDIES

6. 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 IBD=2 IBD=1 IBD=0 Test whether affected sibs share more parental alleles than expected Linkage when excess of alleles IBD shared by affected sib-pairs

7. 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

8. 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

9. 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)

10. 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

11. 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

12. 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: 1721+2T->G 17 yo 15 yo Pulm TB 8 yo Diss TB

13. 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?

15. 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

16. LEPROSY: Response to M. leprae Clinical threshold From Gentilini & Duflo, Médecine Tropicale, Flammarion Médecine-Sciences

17. LEPROSY INHERITANCE HYPOTHESIS-DRIVEN APPROACH GENOME-WIDE APPROACH LINKAGE STUDIES DIFFERENTIAL EXPRESSION ASSOCIATION STUDIES HUMAN DATA ANIMAL MODELS CANDIDATE REGIONS VARIANT DETECTION ‘RARE’ MUTATIONS ‘COMMON’ POLYMORPHISMS ASSOCIATION STUDIES Replication FUNCTIONAL STUDIES

18. LEPROSY: Genome-wide screen 86 multiplex families Leprosy subtype Mira et al, Nat Genet, 2003

19. Genome-scan - fine mapping 6q25 5 4 Lod Score 3 2 LD mapping 1 3 cM 0 x x x x x x x x x x x x D6S415 D6S476 D6S2436 D6S1614 D6S2420 D6S1654 D6S503 D6S1277 D6S1273 D6S1027 D6S1590 GATA184A08 D6S1035 D6S1579 D6S1550 D6S1599 D6S253 D6S305 D6S955

20. Leprosy subtype LD mapping 197 simplex families 2 parents + 1 affected offspring 64 informative SNPs ( 1 / known gene) Mira et al, Nature, 2004

21.  SNPs density

22. LD MAP

24. p < 0.05 not significant Bloc B PARK2 intron 1 PACRG intron 1 PARK2 exon 1 PACRG exon 1

25. Bloc B Multivariate analysis SNP2 SNP1 PARK2 intron 1 PACRG intron 1 PARK2 exon 1 PACRG exon 1

26. OR* CI 95% P-value C C Snp 1 - - 1.00 T T Snp 2 C T T 3.2 [1.3 -7.8] 0.009 C T T C T C T T T 0.0005 [2.1 -13.5] 5.3 C T T C * Estimated by conditional logistic regression

27. Replication study in Brazil 587 cases – 388 controls Leprosy subtype 13 significant SNPs (genomic controls)

28. Multivariate analysis P<0.000005

29. PARK2 / PACRG

30. 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

31. 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)

32. 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

33. 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 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