1. A new NOS2 promoter polymorphism associated with increased nitric oxide production and protection from severe malaria in Tanzanian and Kenyan children Maurine R Hobbs, Venkatachalam Udhayakumar, Marc C levesque, Jennifer Booth, Jacquelin M Roberts, Ariana N Tkachuk, Ann Pole, Hilary Coon, Simon Kariuki, Bernard L Nahlen, Esther D Mwaikambo, Altaf L Laf, Donald L Granger, Nicholas M Anstey, J Brice Weinberg The Lancet, Volume 360, November 9, 2002

2. Plasmodium falciparum Malaria WHO estimates 1.5-2.7 million deaths per yearWHO estimates 1.5-2.7 million deaths per year Most deaths in African children less than 5Most deaths in African children less than 5 Growing problem of antimalarial drug resistance with few novel therapeutics availableGrowing problem of antimalarial drug resistance with few novel therapeutics available Lack of an effective vaccineLack of an effective vaccine

3. Malaria Transmission Cycle Pre- erythrocytic Erythrocytic Sporozoites Merozoites Male and female gametocytes Clinical symptoms Asymptomatic

4. Clinical Features of P. falciparum P. falciparum can cause severe malaria: -severe anemiaP. falciparum can cause severe malaria: -severe anemia -cerebral malaria -hypoglycemia -respiratory distress Molecular determinants that regulate mild versus severe disease largely unknownMolecular determinants that regulate mild versus severe disease largely unknown

5. Cerebral Malaria Sequestration of parasitized red blood cells in the post- capillary venulesSequestration of parasitized red blood cells in the post- capillary venules Due to altered immune environment that is contingent on endothelial cells changes and perturbed local inflammatory milieuDue to altered immune environment that is contingent on endothelial cells changes and perturbed local inflammatory milieu

6. Populations at Risk Infants, young children, and pregnant women in malaria endemic regionsInfants, young children, and pregnant women in malaria endemic regions – Greater than 3 million deaths (primarily in children less than 5 y/o due to non-immune status) Non-immune individuals traveling through and/or living in malaria endemic regionsNon-immune individuals traveling through and/or living in malaria endemic regions – 35 million non-immune individuals travel through malaria endemic regions every year

7. Potential Solutions Gain an understanding of the genetic and immunologic basis of protective immunityGain an understanding of the genetic and immunologic basis of protective immunity Identify novel targets for therapeutic interventionIdentify novel targets for therapeutic intervention Determine reliable markers for measuring protection and pathogenesis for use in pharmacologic and/or vaccine trialsDetermine reliable markers for measuring protection and pathogenesis for use in pharmacologic and/or vaccine trials

8. Nitric Oxide Nitric oxide – free radical gas with a half life of 7 secondsNitric oxide – free radical gas with a half life of 7 seconds Important molecule in the context of host defense because it has antimicrobial propertiesImportant molecule in the context of host defense because it has antimicrobial properties Exact mechanism by which NO kills microbes is not well understoodExact mechanism by which NO kills microbes is not well understood

9. Nitric Oxide Synthase Constitutive Expression Inducible Expression - Ca 2+ - and Calmodulin-Dependent - Ca 2+ - and Calmodulin-Independent NO Synthesis for Normal Physiologic Function NO Synthesis in the Setting of Inflammation eNOS & nNOS NOS3 NOS1 NOS3 NOS1 iNOSNOS2

10. NOS2 Gene

11. Nitric Oxide Synthase Complex induction and regulationComplex induction and regulation Two promoter polymorphisms examined for roles in malarial outcomesTwo promoter polymorphisms examined for roles in malarial outcomes -954 G  C associated with mild disease severity in Gabon-954 G  C associated with mild disease severity in Gabon Long (CCTTT) n (n≥11) repeats associated with protection from fatal cerebral malaria in Gambian childrenLong (CCTTT) n (n≥11) repeats associated with protection from fatal cerebral malaria in Gambian children No association with cerebral malaria risk or in vivo NO production in Tanzanian childrenNo association with cerebral malaria risk or in vivo NO production in Tanzanian children

12. Experimental Design Study examined a novel single nucleotide polymorphism in the NOS2 promoter and its relation to malarial disease outcomeStudy examined a novel single nucleotide polymorphism in the NOS2 promoter and its relation to malarial disease outcome Studied the association between this SNP and in vivo NO productionStudied the association between this SNP and in vivo NO production

13. Methods: Participants TanzaniaCross sectional study of children 6 mo. to 9 years. Healthy controls, uncomplicated malaria, and cerebral malariaTanzaniaCross sectional study of children 6 mo. to 9 years. Healthy controls, uncomplicated malaria, and cerebral malaria Kenya(ABCP) Mother-child pairs. Monthly blood samples to measure parasitemia and HbKenya(ABCP) Mother-child pairs. Monthly blood samples to measure parasitemia and Hb

14. Methods: Protocol To estimate NO production, measured fasting plasma and urine NO x concentrations in Tanzanian controlsTo estimate NO production, measured fasting plasma and urine NO x concentrations in Tanzanian controls

15. Methods: Protocol Examined NOS2 promoter (-8 kb to +1 bp) for polymorphisms associated with malaria outcomes in the Tanzanian groupExamined NOS2 promoter (-8 kb to +1 bp) for polymorphisms associated with malaria outcomes in the Tanzanian group Whole genome amplifiedWhole genome amplified Initial polymorphism detection screening on 12 HC’s and 12 CM’s by SSCPInitial polymorphism detection screening on 12 HC’s and 12 CM’s by SSCP Amplified -1173 C  T fragment and analyzed SNP on all controls and patients in the Tanzanian groupAmplified -1173 C  T fragment and analyzed SNP on all controls and patients in the Tanzanian group -954 G  C, (CCTTT) n, and HbAS previously examined in the Tanzanian children-954 G  C, (CCTTT) n, and HbAS previously examined in the Tanzanian children

16. Methods: Protocol Investigated -1173 C  T polymorphism in the Kenyan cohort by mutagenically separated PCRInvestigated -1173 C  T polymorphism in the Kenyan cohort by mutagenically separated PCR Also determined (CCTTT) repeats and HbASAlso determined (CCTTT) repeats and HbAS

17. Results: Effect of NOS2 -1173 C  T genotype on malarial outcomes in Tanzanian and Kenyan children

18. Results: Effect of -1173 C  T genotype on development of SMA and parasitemia in Kenyan children

19. Results: Concentrations of NO x in urine and plasma of fasting Tanzanian controls, according to -1173 C  T genotype

20. Results: Effect of (CCTTT) n long vs. short genotype on development of SMA and parasitemia in Kenyan children

21. Results: Effect of -1173 C  T/(CCTTT) 12-13 genotype on development of SMA and parasitemia in Kenyan children

22. Results: NOS2 -1173 C  T genotype or sickle-cell trait (HbAS) relative to disease category in Tanzanian children

23. Discussion Protective association of -1173 C  T polymorphism in NOS2 promoter in two cohortsProtective association of -1173 C  T polymorphism in NOS2 promoter in two cohorts Two populations living in areas of differing malarial endemicity and distinct disease patternsTwo populations living in areas of differing malarial endemicity and distinct disease patterns

24. Discussion Associated with protection against both cerebral malaria and SMAAssociated with protection against both cerebral malaria and SMA Associated with increased NO production in vivo, in healthy controlsAssociated with increased NO production in vivo, in healthy controls Prevalence of the -1173 C  T is similar to that of HbASPrevalence of the -1173 C  T is similar to that of HbAS

25. Discussion -1173 C  T may disrupt binding site of a transcription repressor (found not to be a new seq. recog. site for GATA-1 or GATA-2 TF’s)-1173 C  T may disrupt binding site of a transcription repressor (found not to be a new seq. recog. site for GATA-1 or GATA-2 TF’s) May be in linkage disequilibrium with another polymorphism in the NOS2 geneMay be in linkage disequilibrium with another polymorphism in the NOS2 gene

26. Proposed mechanism of the protective effect of the -1173 C  T NOS2 promoter polymorphism against clinical malaria

27. Proposed protective mechanisms 1. NO has direct antiparasitic effects1. NO has direct antiparasitic effects 2. NO decreases expression of ICAM-1, VCAM-1, and E-selectin2. NO decreases expression of ICAM-1, VCAM-1, and E-selectin 3. NO potently decreases macrophage production of TNF3. NO potently decreases macrophage production of TNF

28. Role of NO in pathogenesis of malaria Pro-inflammatory and detrimental to host survivalPro-inflammatory and detrimental to host survival AND/ORAND/OR Anti-inflammatory and beneficial to host survivalAnti-inflammatory and beneficial to host survival Effects may vary with host cell types and stage of parasite developmentEffects may vary with host cell types and stage of parasite development