Human Genetics of Infectious Diseases: Application to mycobacterial infections

The genus Mycobacterium ‘Virulent’ ‘Weakly virulent’ M. tuberculosis complex> 80 species (e.g. M. avium, M. lepraeM. marinum, M. fortuitum…) Human transmission Environmental transmission (airborne)(water, soil, air…) M. ulcerans (Buruli ulcer) BCG vaccine Aquatic bug transmission? Injection transmission

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

  Individual variability in response to infection (1) Mycobacteriumtuberculosis INFECTION INFECTION(primary/latent) PRIMARY DISEASE (Ext.Pulm., children) IMMUNE RESPONSE M. tb factors (virulence…) (virulence…) Host factors (age, immune status GENES, …) Exposurefactors Environmentalfactors REACTIVATION DIS. (Pulm., adults) ~5%

Ranke, K Diagnose und Epidemiologie der Lungentuberculose des Kindes. Archiv für Kinderheilkunde 54:   Individual variability in response to infection (2)

PhenotypeRare (disseminated infection) Common (TB, leprosy) Tools Mendelian Genetics Genetic Epidemiology SampleSmallLarge Methods of investigation in humans Rare mutation Common polymorphism

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

MENDELIAN SUSCEPTIBILITY TO MYCOBACTERIAL DISEASES (MSMD) Disseminated infections by environmental mycobacteria (EM), BCG No known primary or acquired immunodeficiency Very rare (10 -5 – ) but often familial (consanguinity) Mendelian transmission (7 identified genes so far)

Macrophage/Dendritic Cell T Lymphocyte Mycobacteria IL12R  1 IL12R  2 IFN  R1 IL-12 p35 p40 IFN-  IFN  R2 STAT1 NEMO CD40 CD40L gp91 phox  New specific antimycobacterial immunological pathway  Medical implications (IFN-  treatment) From EM to M. tuberculosis  From EM to M. tuberculosis

Inherited IL12R  1 deficiency : Spanish family No BCG/NTM disease No IL12R  1 expression No cellular responses to IL-12 IL12RB1 mutation: T  G IL12-R  1 deficiency and tuberculosis 17 yo15 yo Pulm TB 8 yo Diss 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) … Mendelian TB: Conclusion and questions

Alcais et al, J Exp Med, 2005

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 Mendelian TB: Conclusion and questions

~ 8 millions new cases per year ~ 90% of infected subjects do not develop the disease ~ 400,000 new cases per year ~ 95% of infected subjects do not develop the disease Tuberculosis (M. tuberculosis) Leprosy (M. leprae) Complex predisposition to common mycobacterial diseases Very large spectrum of clinical manifestations

HYPOTHESIS-DRIVEN APPROACH TUBERCULOSIS INHERITANCE: CANDIDATE GENES ASSOCIATION STUDIES Population or family-based VARIANT DETECTION FUNCTIONAL STUDIES ‘COMMON’ POLYMORPHISMS ‘RARE’ MUTATIONS GENOME-WIDE APPROACH ASSOCIATION STUDIES DIFFERENTIAL EXPRESSION CANDIDATE GENES ANIMAL MODELS HUMAN DATA LINKAGE STUDIES  Examples : NRAMP1, HLA-DR, IL12RB1

PULMONARY TUBERCULOSIS (PTB) AND IL12RB1 Family-based association study :  101 families including 157 offspring >15 years with PTB (culture +) - Sequencing of promoter and coding regions in 40 PTB patients  No rare loss of function mutations  Detection of common polymorphisms >5% Genotyping in the whole sample Test for association with PTB AT TT AT TT

R156H (467G>A) Q214R (641A>G) M365T (1094T>C) +34C>T 5’3’ G378R (1132G>C) 387G>C684C>T+46T>C+24C>T-2C>T+21C>A +47G>T IIIIIIIVVVIVIIVIIIIXXXIXIIXIIIXIVXVXVIXVII -111A>T IL12RB1 Association with the two promoter polymorphisms in strong LD - especially for -2C>T (p=0.004), with T frequency ~ OR for CT or TT vs. CC = 3.8 [ ]  Replication studies  Functional studies T is an uncommon allele at position –2 (consensus site) Remus et al, J Inf Dis, 2004

HYPOTHESIS-DRIVEN APPROACH TUBERCULOSIS INHERITANCE: MAJOR GENES ASSOCIATION STUDIES Family-based VARIANT DETECTION FUNCTIONAL STUDIES ‘COMMON’ POLYMORPHISMS ‘RARE’ MUTATIONS GENOME-WIDE APPROACH ASSOCIATION STUDIES DIFFERENTIAL EXPRESSION CANDIDATE REGION ANIMAL MODELS HUMAN DATA LINKAGE STUDIES

TUBERCULOSIS: Genome-wide screen # affected offspring234 # families multiplex families Total of 227 offspring with positive pulmonary TB El Baghdadi et al, J Exp Med, 2006 AB CD AC AD BC IBD=2IBD=1IBD=0 Affected sib-pair linkage study

Linkage to chromosome 8q12-q13 39 families with affected parent All 96 families Consistent with dominant inheritance Linkage disequilibrium mapping is ongoing p< p<

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

LEPROSY: Genome-wide screen # affected Offspring2345 # families Leprosy subtype 86 multiplex families Mira et al, Nat Genet, 2003

PARK2/PACRG 6q25 Lod Score = 4.3 (p=5.10 –6 ) (Mira, Alcaïs, et al. Nature 2004) IIIII HLA Non-HLA LD mapping ~ 10 Mb 3 Lod Score = 2.6 (p=2.10 –4 ) 6p [Mb] Lod MLB Two linkage peaks

LD mapping Leprosy subtype 194 simplex families 2 parents + 1 affected offspring Alcais et al, Nat Genet, informative SNPs

 SNPs density

 Association with a bin of several SNPs in the LTA region

Replication in an independent Vietnamese sample of 104 trios with the same allele at risk LTA+80: OR=2.34 ( ), p=0.003 LTA-293: OR=1.95 ( ), p=0.03

Replication in an Indian sample of 364 cases and 371 controls  LTA+80 is the most likely candidate

Study in a Brazilian sample of 209 cases and 192 controls  No overall effect of LTA+80 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 The LTA+80 effect is strongly age-dependent

1st Vietnamese sample (194 trios) Indian sample 364 cases/371 controls Brazilian sample 209 cases/192 controls 2nd Vietnamese sample (104 trios) The LTA+80 effect is strongly age-dependent

Summary for leprosy Strong association of LTA+80 A allele with early onset leprosy. In combined sample of Vietnamese trios < 16 years: OR for LTA+80 A OR for LTA+80 AA/AC vs CC subjects = 5.6 ( ), p<10 -6 LTA+80 A decreases LTA protein production (Knight et al, Nat Genet, 2004) LTA KO mice are susceptible to mycobacteria ( 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…

RR  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 predisposition to mycobacterial infections  continuous spectrum Genetic dissection needs to combine different strategies

15 Age Origin of genetic cases (%) Primary infection  Reactivation Extra-pulmonary  Pulmonary Mendelian Major gene Polygenic 50 Genetic spectrum depends on age

Laboratory of Human Genetics of Infectious Diseases 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 March of Dimes FRM 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 Mycobacterium

McGill University, Montreal, Canada Andrea AlterMariana OrlovaErwin Schurr Laboratoire d’Immunologie, Hôpital Militaire de Rabat, Maroc Jamila El Baghdadi Abdellah Benslimane Hospital of Dermato-Venereology, Ho Chi Minh City, Vietnam Nguyen Van ThucNguyen 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