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Genetic Factors Governing Susceptibilities to Severe Infections

GSK-Chair of Infectious Diseases

Pr Jean-Paul MIRA

GENETIC PREDISPOSITION GENETIC PREDISPOSITION TO SEVERE SEPSIS TO SEVERE SEPSIS

Université catholique de Louvain Cliniques universitaires Saint-Luc

17 février 2005 Medical Medical ICU & ICU & Dept

• Dept. of

. of Cell Biology Cell Biology Cochin Cochin University University Hospital Hospital & Cochin & Cochin Institute Institute, Paris, F , Paris, F

• Pr. Jean-Paul MIRA

Recurrent Purpura Fulminans Recurrent Purpura Fulminans

2002/01: 15 yo girl admitted in ICU

• Temperature 40°C; HR 125; BP 74/45; RR 38
• Meningitis with purpura fulminans
• MOF (Shock, ARDS, ARF, DIC, Lactic acidosis)
• Meningococcus type N in the skin biopsy
• Survival with multiple finger amputations and skin grafting
• 6 month hospitalization

2003/02:

• Temperature 39°C; HR 125; BP 83/48; RR: 33
• Meningitis with purpura fulminans
• Lumbar punction meningococcus type Y
• Shock and DIC
• Survival (Xigris) with new skin grafting
• 3 month hospitalization

Bohé J. et al.

« If it were not for the great variability among individuals medicine might as well be a science and not an art »

Sir William Osler, 1892

Watson JD, Crick FHC. Nature 1953;171:737

From From Watson Watson and and Crick Crick to Human Genome to Human Genome

1953 Watson and Crick: double helical structure of DNA 1960s Role of RNA and Genetic Code 1970s Recombinant DNA technology 1977 Sanger and Gilbert: DNA sequencing 1983 Mapping of disorders by linkage (Huntington disease) 1986 Polymerase Chain Reaction 1990 Human Genome Project 1995 Haemophilus influenzae genome 2003 Mice and Human genome sequence Human SNP Map

WE ALL, AS HUMANS, SHARE THE SAME BASIC GENES

BUT…

Small differences in genotype make big differences to phenotype

Genetic Polymorphisms Genetic Polymorphisms

Human SNP Map

Genetic Polymorphisms Genetic Polymorphisms

Molecular Genetic Research Molecular Genetic Research

Heriditary disorder Linkage studies Mutation or polymorphism Genetics

Milewitcz DM. Circulation 2000; 102: IV-103

Evidences Evidences for a for a genetic genetic component to sepsis component to sepsis

Animal Studies

• Susceptibility/resistance to certain infection in mice
• Susceptibility/resistance phenotypes of knock-out mice

Mice Mice Susceptibility Susceptibility to Infection to Infection with with Group A Group A Streptococci Streptococci

103 cfu Strepto Subcutaneous

d2

Goldman O. J Infect Dis 2003;187:854-61.

Immunomodulatory genes NF-κB Signalisation Adaptative Response Cellular Immunity Lipoproteins Gram+ Bacteria Fungi LPS Gram- Bacteria TLR4 TLR3 TLR5 TLRx CpG DNA ?

Monocyte or Dendritic cell

Apoptosis Septic Shock Tissue Injury Bacteria Lysis Double-stranded RNA TLR2 +/- TLR1/6 Flagellin TLR9

TLR2 TLR2-

• KO Mice

KO Mice and Response and Response to Gram Positive to Gram Positive Bacteria Bacteria

Intraveneous infusion of Staphylococcus aureus

Takeuchi et al., J. Immunol. 1999; 165:5392

Evidences Evidences for a for a genetic genetic component to sepsis component to sepsis

Animal Studies

• Susceptibility/resistance to certain infection in mice
• Susceptibility/resistance phenotypes of knockout mice

Human Studies

• Clinical Evidences
• Ethnic Differences
• Twin Studies
• Adoptee Studies

Genetic Genetic and and environmental environmental influences influences

• n
• n premature death

premature death in in adult adoptees adult adoptees

Cause of Death (Parent Dead before the age of 50) Relative risk for the adoptee to die from the same cause All causes Biologic Adoptive 1.71 0.71 Biologic Adoptive Infection 5.8 0.73 Vascular Biologic Adoptive 4.5 3.1

Sørensen TI, et al. NEJM 1988; 318:727-32.

Genetic Polymorphisms and Severe Sepsis

Meningococcemia; Severe sepsis Meningococcemia; Severe sepsis PAI-1 FactorV Leiden Severe Sepsis Viral Pneumonia Severe Sepsis IL-1 locus IL-4 Caspase 12 Meningococcemia Septic Shock; Cerebral Malaria Severe Sepsis Severe Sepsis, Meningococcemia Severe sepsis TNF locus IL-18 IL-10 IL-6 Meningococcemia; Pneumococcemia FCγRII Receptor Meningococcemia, Pneumococcemia Severe sepsis Mannose Binding Lectin

Susceptibility and/or Outcome

Gene

Gram negative/positive Septic Shock Legionnaire’s Disease Septic Shock Toll-Like Receptor 4/2 Toll-Like Receptor 5 CD14

Pathogen Detection

TLR2 TLR2 and and Streptococcus pneumoniae Streptococcus pneumoniae meningitis meningitis

WT TLR2 -/- Echchannaoui H et al. JID 2002;186:798

Yeast Mycobact. Gram+ Gram-

Mannose Mannose-

• Binding Lectin

Binding Lectin

• Collectin

Collectin

• Structural

Structural homology with homology with C1q C1q

• Associated

Associated to 2 serine to 2 serine proteases proteases

• Variability

Variability: :

• Point mutations codons 52, 54, 57

Point mutations codons 52, 54, 57

• Polymorphisms

Polymorphisms in in the the promoter promoter

MBL-Deficient Mice and Staphylococcus Infection

Shi L, J Exp Med 2004; 199:1379

Mannose Mannose-

• binding Lectin Polymorphisms

binding Lectin Polymorphisms & & The Risk The Risk of Infections

• f Infections
• Repeated bacterial and fungal infections

Sumiya et al., Lancet 1991 Summerfeld et al., Lancet 1995 Garred et al., Lancet 1995 Summerfeld et al., BMJ 1997

• Infections after chemotherapy

Neth et al., Lancet 2001 Peterslund et al., Lancet 2001

• Increased severity of lung disease and low survival in cystic fibrosis

Garred et al., J. Clin. Invest. 1999

• Meningococcal disease

Hibberd et al., Lancet 1999

Low serum Low serum MBL MBL Normal Normal serum serum MBL MBL

MBL MBL Polymorphisms Polymorphisms, SIRS, , SIRS, and Sepsis and Sepsis

272 ICU Pts with SIRS

% Patients

A/A O/O

20 40 60 80 100

55 20 A/O

75 Pts Without Sepsis

% Patients

A/A O/O

20 40 60 80 100

A/O

197 Pts With Severe Sepsis A: Wild Type O: Structural polymorphism associated with low MBL levels

Garred P. J Infect Dis 2003;188:1394

MBL MBL Polymorphisms Polymorphisms, SIRS, , SIRS, and Sepsis and Sepsis

50

Garred P. J Infect Dis 2003;188:1394

% Mortality

A/A O/O

10 20 30 40

A/O

: Predicted Mortality

MBL genotype and risk of invasive pneumococcal disease

P < 0.05

% Variants homozygotes

Patients n=337 Contrôles n=1032

12 10 8 6 4 2

Odds ratio 3.48 (1.51 – 8.01); p=0,003

Roy et al. Lancet 2002; 359: 1569-1573

MBL-Deficient Mice and Staphylococcus Infection

Shi L, J Exp Med 2004; 199:1379

Immunomodulatory genes NF-κB Signalisation Adaptative Response Cellular Immunity Lipoproteins Gram+ Bacteria Fungi LPS Gram- Bacteria TLR4 TLR3 TLR5 TLRx CpG DNA ?

Monocyte or Dendritic cell

Apoptosis Septic Shock Tissue Injury Bacteria Lysis Double-stranded RNA TLR2 +/- TLR1/6 Flagellin TLR9

TLR4 mutation TLR4 mutation and and LPS LPS responsiveness responsiveness

40 20 IL-1α (pg/ml) WT/WT WT/Asp299Gly & Thr399Ile

Arbour Arbour NC, et al. Nature NC, et al. Nature Genetics Genetics 2000 2000

TLR4 TLR4 Polymorphisms and Septic Shock Polymorphisms and Septic Shock

Control Control % TLR4 % TLR4 mutated mutated patients patients 30 25 20 15 10 5 Gram Gram negative negative Septic shock Septic shock

Lorenz , Arch. Intern. Med. 2002 162:1028

TLR4 Variants TLR4 Variants and Predisposition and Predisposition to Gram to Gram Negative Sepsis Negative Sepsis

Agnese DM, JID 2002; 186:1522

Without Without TLR4 TLR4 Mutation Mutation % Gram % Gram negative negative infection infection With With TLR4 TLR4 Mutation Mutation 100 80 60 40 20

P = 0.004

Cytokine Polymorphisms

Cytokine Polymorphisms and Meningococcemia

Gene Polymorphism Csqs Pts Su Severity Outcome Ref ACE DD (deletion) ↑ ACE 110 98 85 IL-1B ILRN

• 511 (1+)

+2018 (2+) IL-1β ↓ IL -1β 1106 [OR] = 0.61 [CI] 0.38-0.98 Read RC. 2003 ↑14% Death [OR]= 2.8 Harding D. 2002 TNF

• 308 (TNF2)

↑ TNF α [OR] =2.5 [CI]: 1.1-5.7 Nadel S. 1996 IL-6

• 174 (GC)

↑ IL-6

[OR]= 3.06

[OR] = 2.64 [CI]: 1.1- 6.2 Balding J. 2001

TNF locus TNF locus

TNF-α LT-β LT-α

Nco.I TNFB1 TNFB2

• 308
• 376

TNF1 TNF2

Association of TNF2 Association of TNF2 with with TNF TNF levels levels in in Septic Shock Septic Shock

TNF1 TNF2

Appoloni O. Am J Med 2001; 110:486

TNF2 TNF2 polymorphism and septic shock susceptibility polymorphism and septic shock susceptibility

Septic shock

TNF1 TNF2 p= 0.002

Control

% Patients

20 40 60 80 TNF1 TNF2

• JAMA. 1999, 282:561-8

TNF2 TNF2 polymorphism and septic shock outcome polymorphism and septic shock outcome

TNF1 TNF2

42.6% 71.4% 39.2%

JAMA 1999;282:561-8

TNFB2 in TNFB2 in LT LT-

• α

α and severe and severe sepsis sepsis outcome

• utcome

TNFB1/TNFB1 TNFB1/TNFB2

Mortality (%)

100 80 60 40 20

13 37

TNFB2/TNFB2

30

P=0.007

Stüber. J Inflamm 1996;46:42-50

Community Community-

• Acquired Pneumonia and

Acquired Pneumonia and TNF TNF polymorphisms polymorphisms

280 CAP No association with mortality rate LTα+250 AA genotype RR= 2.48 (1.28 – 4.78), Age-adjusted RR = 3.64 (1.28 – 10.66)

Waterer GW. AJRCCM 2001; 163: 1599

Coagulation Polymorphisms

Protein C Pathway Protein C Pathway

Factor V Leiden mutation and Meningococcal Disease

P < 0.03 Controls MD

Incidence of Factor VL +/- (%)

15 12 9 6 3

n=80 n=259

Factor VL-/- Patients Factor VL+/- Patients 30 25 20 15 10 5

Complications of MD (%)

Parents of Fatal MD

n=79

RR= 3.1 (1.2-7.9) ns

Kondaveeti S. Pediatr Infect Dis J. 1999; 18: 893-6

Factor V Leiden and Severe Sepsis

P=0.013 General Population Severe Sepsis (PROWESS)

Incidence of Factor VL +/- (%)

10 8 6 4 2

n=65

Factor VL-/- Patients Factor VL+/- Patients 30 25 20 15 10 5

Mortality of Severe Sepsis (%)

Kerlin BA. Blood 2003; 102: 3085-92

Factor V Leiden and APC Efficacy in Severe Sepsis

Placebo Xigris

Kerlin BA. Blood 2003; 102: 3085-92

Factor VL-/- Patients Factor VL+/- Patients 30 25 20 15 10 5

Mortality of Severe Sepsis (%) RR= 0.8 (0.68–0.94) RR= 0.78 (0.23-2.63)

4G/5G PAI 4G/5G PAI-

• 1

1 Polymorphism Polymorphism

Transcriptional activator Regulatory region Repressor protein Transcription Transcriptional activator GGGGG GGGG PAI-1 gene Low plasma PAI-1 concentration High plasma PAI-1 concentration 3’ 5’

• 675

Promoter Exon1-9

4G/5G PAI 4G/5G PAI-

• 1

1 Polymorphism Polymorphism and and Meningococcal Disease Meningococcal Disease

% Observed Mortality

4G/4G 5G/5G

10 20 30 40 50

4G/5G

P= 0.005

% Vascular Complications

4G/4G 5G/5G

10 20 30 40 50

4G/5G

Survivors

P= 0.03

% Predicted Mortality

4G/4G 5G/5G

10 20 30 40 50

111 213 81 4G/5G

P= 0.02

Haralambous E. Crit Care Med 2003;31:2788

4G/5G promoter polymorphism in the PAI 4G/5G promoter polymorphism in the PAI-

• 1

1 gene and severe trauma patients gene and severe trauma patients

% Sepsis

4G/4G 5G/5G

20 40 60 80 100

19 29 13 4G/5G

% MOF

4G/4G 5G/5G

20 40 60 80 100

4G/5G

% Fatal Outcome

4G/4G 5G/5G

20 40 60 80 100

4G/5G Menges, Lancet 2001;357:1096

Perspectives and Conclusions Perspectives and Conclusions

⇒ Screening of a high number of polymorphisms in large cohorte ⇒ SNPs or haplotype ⇒ Micro-arrays, Taqman, Mass Spectroscopy, … Yamada Y et al. N Engl J Med 2002; 347: 1916-23. ⇒ 2819 patients with myocardial infarction ⇒ 2242 controls ⇒ 112 polymorphisms of 71 candidate genes

PAI-1, connexin 37, stromelysin

Perspectives et Conclusions Perspectives et Conclusions

• Screening of a high number of polymorphisms in large cohorts

⇒ UK: 1000 Patients – Peritonitis ⇒ UK: 2000 Patients – Community-Acquired Pneumonia ⇒ USA: 2000 Patients – Severe Sepsis ⇒ USA: 1500 Patients – Severe Sepsis ⇒ France: 3500 Patients – Nosocomial Pneumonia ⇒ France: 3500 Severe Trauma ⇒ Australia ? ⇒ Japan ?

High Throughput Genotyping High Throughput Genotyping

20’ 30’ 90’

Recurrent Purpura Fulminans Recurrent Purpura Fulminans

2002/01: 15 yo girl admitted in ICU

• Temperature 40°C; HR 125; BP 74/45; RR 38
• Meningitis with purpura fulminans
• MOF (Shock, ARDS, ARF, DIC, Lactic acidosis)
• Meningococcus type N in the skin biopsy
• Survival with multiple finger amputations and skin grafting
• 6 month hospitalization

2003/02:

• Temperature 39°C; HR 125; BP 83/48; RR: 33
• Meningitis with purpura fulminans
• Lumbar punction meningococcus type Y
• Shock and DIC
• Survival (Xigris) with new skin grafting
• 3 month hospitalization

Bohé J. et al.

Recurrent Purpura Fulminans Recurrent Purpura Fulminans

Genetic predisposition? Innate immunity Inflammation Coagulation

Innate Immunity

TLR4 CD14 FcγRIIa FcγRIII MBL Complement

Inflammation

TNFα LTα IL-1 IL-6 IL-10 ACE

Coagulation

Tissue Factor Prothrombin Factor V Factor VII Factor XIII PAI-1

Innate Immunity

TLR4 CD14 FcγRIIa FcγRIII MBL Complement C7 deficiency

Inflammation

TNFα LTα IL-1 IL-6 IL-10 ACE

Coagulation

Tissue Factor Prothrombin Factor V Factor VII Factor XIII PAI-1

Bohé J. et al.

SEPSIS MAP

Septic Shock

Perspectives Perspectives and and Conclusions Conclusions

⇒Screening of a high number of polymorphisms in large cohorte Effect of association of polymorphisms ? ⇒ Identify potential markers of susceptibility, severity, and clinical outcome Genetic profiling Individual risk assessment Prevention, Vaccination To tailor prescriptions to each patient

Perspectives Perspectives and and Conclusions Conclusions

⇒Screening of a high number of polymorphisms in large cohorte Effect of association of polymorphisms ? ⇒ Identify potential markers of susceptibility, severity, and clinical outcome Genetic profiling Individual risk assessment Prevention, Vaccination To tailor prescriptions to each patient ⇒Stratification of patients by genotype in the design of treatment trials Identify potential markers for responders vs non-responders

The « challenge »

From functional genomics to rationale therapeutics

« Man is a tool-using animal…. Without tools he is nothing, with tools he is all .» Thomas Carlyle

19th-century