The Lancet, Volume 360, November 9, 2002

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 year • Most deaths in African children less than 5 • Growing problem of antimalarial drug resistance with few novel therapeutics available • Lack of an effective vaccine

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

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

5. Cerebral Malaria • Sequestration 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 milieu

6. Populations at Risk • Infants, 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 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 immunity • Identify novel targets for therapeutic intervention • Determine 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 seconds • Important molecule in the context of host defense because it has antimicrobial properties • Exact mechanism by which NO kills microbes is not well understood

9. Nitric Oxide Synthase iNOS NOS2 eNOS & nNOS NOS3 NOS1 Constitutive Expression Inducible Expression - Ca2+- and Calmodulin-Dependent - Ca2+- and Calmodulin-Independent NO Synthesis for Normal Physiologic Function NO Synthesis in the Setting of Inflammation

10. NOS2 Gene

11. Nitric Oxide Synthase • Complex induction and regulation • Two promoter polymorphisms examined for roles in malarial outcomes • -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 children • No 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 outcome • Studied the association between this SNP and in vivo NO production

13. Methods: Participants • Tanzania Cross 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 Hb

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

15. Methods: Protocol • Examined NOS2 promoter (-8 kb to +1 bp) for polymorphisms associated with malaria outcomes in the Tanzanian group • Whole genome amplified • Initial 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 group • -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 PCR • Also 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 NOx 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 cohorts • Two populations living in areas of differing malarial endemicity and distinct disease patterns

24. Discussion • Associated with protection against both cerebral malaria and SMA • Associated with increased NO production in vivo, in healthy controls • Prevalence 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) • May 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 effects • 2. NO decreases expression of ICAM-1, VCAM-1, and E-selectin • 3. NO potently decreases macrophage production of TNF

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