Jan A. Hazelzet, MD PhDPediatric Intensive Care

Sophia Children´s HospitalErasmus Medical Center

Rotterdam, The Netherlandsj.a.hazelzet@erasmusmc.nl

Genetic Studies in Pediatric Sepsis

Determinants in Infectious Diseases

Microbes

HostEnvirons

Environment

Genetic and environmental influences on premature death in adult adoptees

Sörensen, N Engl J Med 1988; 318: 727-32

Risk of dying was assessed in 960 families for adoptees (born between 1924/26)

Death of a biologic parent before the age of 50 resulted in: RR of death of 1.71 for all

causes

RR of death of 5.81 for infections

Host Response

Eleftherohorinou et al. PLoS One. 2009; 30:e8068

http://www.ornl.gov/hgmis

DNA codes for ~ 80.000 proteins

SNIP = single nucleotide polymorphism

“Equal but not the same”“Equal but not the same”

DNA polymorphism (SNIP, CNP)

Most common type of stable genetic variation 1 of 1000 base pairs (~2.2 million SNPs) Coexistence of 2 different alleles on the same gene location coding for

a certain protein Consequences:

Difference in production of that protein Difference in the protein No consequence

Distribution in the general population 1 % Recently many disease gene associated studies (candidate gene

approach) Haplotype studies

10% =functional

Genetic influences:

risk of developing an infection

severity of diseaseseverity / outcome:

predisposition / susceptibility:

Genetic Influence in Sepsis Studies

Experimental: tissue / cellular

Animals (Knock Outs)

LPS injection volunteers

Patients vs controls

Meningococcal disease

Clinical sepsis

Pneumonia

Trauma / Burns

N. meningitidis

~2000genes

Wright et al. Vaccine 2009; 27S: B90–B102

Genetic polymorphisms are associated with severity (SE) of and susceptibility (SU) for

meningococcal infectionsGene

TLR4

MBL

Properdin

FcRIIa

FcR

PAI-1

Factor V

TNF-

IL-6

IL1β andILRN

Pathway

Innate immunity

Acquired immunity

Coagulation/Fibrinolysis

Cytokines

Polymorphism

rare polymorphisms combinedcodons 52, 54, 57

Combination of 6 SNP’s

HIS 131 ARG

combination of 3 SNPS

4G/5G

G1691A

G-308A

G-174C

- 511C/T+2018T- 511C or C/T + 2018C or C/T

SE/SU

SU

SU

SU SESE SUSU

SESE/OUSE

SEOUSEOUsurvivalsurvival

ODDS ratio

27

5.5 (homozygous)2 (heterozygous)

2504.7 - 15

3.92.72.6

5.92

2.73.11.62.53.12.67.80.6

Lancet Inf Dis 2003; 3:565-77. Cur Opin Infect Dis 2010; 23:255–258

Handling of LPS, PDG,…

Stimulation of the Innate immune system Recognition of pathogen associated molecular patterns (PAMP) by

pathogen recognition proteins: CRP, MBL, C3, PCT, SPA,…. Signal transduction: LBP-CD14-TLR Cytokines, chemokines production Macrophage, granulocyte, lymphocyte, endothelial cell stimulation Complement system Coagulation system Neuro endocrine system …………..

Host responseHost response

TNF as primary cytokine

NEJM 1988; 318: 1481-6

Cytokines in SMD

sepsisseptic shock (surv)septic shock (non-surv)

Intens Care Med 1994; 20: 371-4

Intensive Care Med. 1994 May;20(5):371-4

Variation in the TNF- gene promotor region may be associated with death from meningococcal diseaseVariation in the TNF- gene promotor region may be associated with death from meningococcal disease

TNF-2 allele (G-toA subst. at pos -308) leads to higher inducible levels of TNF-

children with the TNF-2 allele had higher mortality (10/33) vs those without (8/65)

possession of this allele was associated with increased risk of more severe disease (22 of 33) vs (27 of 65)

Nadel et al. J Inf Dis 1996; 174: 878-80

Coagulation in sepsis

Protein C Pathway

Thrombin

PC APCAPC

TM

Endothelial Cell

inhibition offVa and fVIIIa

inhibition ofPAI-1

FIBRINOLYSIS

INFLAMMATION

+PS

COAGULATION

sEPCR

membrane

Prot Creceptor

C4bp

TAFI

sTM• Cytokine production

• Leukocyte adherence

• Apoptosis, etc.

Plasminogen activator inhibitor 1(PAI-1)

Plasminogen activator inhibitor-1 (PAI-1)

50-kDa glycoprotein of the serine protease inhibitor family

Produced by: platelet

endothelial cell

hepatocyt

smooth muscle cell

Acute phase protein (de Boer, 1991)

Triggered by: Inflammatory mediators

(TNF-, IL-1, IL-6, TGF-ß, LPS, VLDL, glucose, glucocorticoids and insulin etc.)

Relation between PAI-1 and TNF- in patients with meningococcal septic shock

Kornelisse, 1996

TNF (pg/ml)1 10 100 1000

PA

I-1 (n

g/m

l)

10

100

1000

10000

NS

S

Kohler, 2000

PAI-1 polymorphisms 9 different polymorphisms have been described Thromb Res 2001; 103: S1-5

3 in the promoter region

Unknown

Many polymorphisms in determinants of PAI-1 like cytokines, insulin etc.

Most extensively studied is 4G/5G polymorphism in the promoter region

PAI-1 promoter polymorphism and outcome of meningococcal sepsis

93 patients with meningococcal sepsis

PAI-1 genotype was significantly related with plasma levels

4G/4G genotype was significantly related to mortality (Hermans, 1999)

500

1,000

1,500

2,000

2,500

3,000

4,4 4,5 5,5

PAI-1 level (ng/ml)

PAI-1 genotype

5G/5G: associated with meningitis4G/4G: associated with sepsis(Westendorp, 1999)

PAI-1 polymorphism related to outcome (Hermans 1999)

4G/4G: associated with increased risk of developing vascular complications and dying from meningococcal disease (Haralambous, 2003)

Host genetic contribution to the risk to develop meningococcal disease (siblings) > one third of the total risk (Haralambous, 2003)

Correlation between 4G/4G and poor outcome (Geishofer 2005)

PAI-1 promoter polymorphism and outcome of meningococcal disease

PAI-1

pro-coagulantmicro particles

APC

Thrombin-TM

t-PA / u-PA

COAGULATION

ANTI-COAGULATION

FIBRINOLYSIS

X

+

-

-

-

endothelial cellsplateletsliver cellsadipocytesmyocytes

metabolic determinantshormonesdietphysical activity

+

+/-

+/-

Host genetics

Environment

X

XX

+

+

+

TNFα/β, IL-1β, IL-6, etc.

Clin Inf Dis 2005; 41 Suppl 7:S453-8

Study of multiple SNP’s

Lancet 2003; 361: 567–71

Nature 2003

Assessment of genetically determined Host Factors Adequate study design Definition of:

Patiënt populations (meningitis vs sepsis, age!) Severity of disease (sepsis vs shock, scores) Subgroups (in- / exclusion) Unknown microbiology

Patiënt numbers (allele frequencies)

Control group (region, socioeconomic status, race)

Ethnic, age, gender differences Interaction or cumulative effects of polymorphisms Clinical relevance (susceptibility or severity, reproducibility) Expensive Limited number of SNP’s are studied at the same moment

• 10 million sequence variants scattered across human genome

• High throughput technology

• Bioinformatic analysis

The way forward…….Genome-Wide Studies

International Meningococcal Disease Genetics Consortium

Mike Levin

Victoria Wright

David Inwald

Simon Nadel

Helen Betts

Lachlan Coin

Harieta Eleftherohorinou

Sonia DavilaMartin Hibberd

Taco KuijpersWillemijn Breunis

Werner ZenzAlexander BinderJan Hazelzet

Marieke Emonts

• Ronald de Groot• Peter Hermans

Enitan Carrol

© Imperial College London

• F. Martinón-Torres

• A. Salas Ellacuriaga

Santiago de Compostela, Spain

International Meningococcal Genetics Consortium

Discovery study

Replication study

Identification of Key Pathways

UK Caucasian MD cases

Validation in European cohort (Austria, Holland, Spain) using Illumina SQNM

Genome-Wide Association Study using Illumina 610 Hap-quad chip

UK Caucasian controls

Bioinformatic Analysis

Top ‘hits’ & selection of candidate genes

Bioinformatic Analysis

Functional Studies Gene Expression/Proteomics

Fine-mapping using Illumina ISelect

Study design

Nat Genet. 2010;42:772-6

Regional association plot of the CFH cluster

CFH cluster on chromosome 1

Hill et al. Clinical Science 2010; 118: 547–564

Negative complement regulators for N Men

TRENDS in Microbiology 2007; 15: 233-40

Future??

www.affymetrix.com

Ultimate Phenotype of the Host Response

Lancet 2004; 363: 2076-83

…function of complex interactions at lower levels…

Conclusions

Differences in host response are genetically determined

SNP’s have been shown to be associated with outcome, but not consistent

Genome-wide SNP genotyping by micro-array technology

Coagulation is an integral part of the immune response

Medical documentation: uniformity – standards (SnoMed) – severity scores, is crucial

Collaboration!!!!! Leads to progress