A new NOS2 promoter polymorphism associated with increased nitric oxide production and protection from severe malaria in Tanzanian and Kenyan children

Background: Nitric oxide (NO) is a mediator of immunity to malaria, and genetic polymorphisms in the promoter of the inducible NO synthase gene (NOS2) could modulate production of NO. We postulated that NOS2 promoter polymorphisms would affect resistance to severe malaria. Methods: We assessed genomic DNA from healthy children and from those diagnosed with malaria from Tanzania (n=47 and n=138, respectively) and Kenya (n=1106) for polymorphisms by single-stranded conformational polymorphism (SSCP) analysis and sequencing. We also measured in-vivo NO production in Tanzanian children. Findings: We identified a novel single nucleotide polymorphism, -1173 C→T, in the NOS2 promoter that was significantly associated with protection from symptomatic malaria (odds ratio 0.12, 95% CI 0.03-0.48, p=0.0006) in 179 Tanzanian children, and significantly associated with protection from severe malarial anaemia (adjusted relative risk 0.25, 95% CI 0.09-0.66, p=0.0005) in 1106 Kenyan children studied over 5 years. The risk of parasitaemia was not significantly different in wild-type or -1173 C→T individuals. -1173 C→T protection in Tanzanians was independent of the previously recognised NOS2-954 G→C polymorphism. The (CCTTT)n NOS2 polymorphism (Tanzania and Kenya) was not associated with severe malaria outcomes. -1173 C→T was associated with increased fasting urine and plasma NO metabolite concentrations in Tanzanian children, suggesting that the polymorphism was functional in vivo. Interpretation: The NOS2 promoter -1173 C→T single nucleotide polymorphism is associated with protection against cerebral malaria and severe malarial anaemia. Increased NO production in individuals with the -1173 C→T polymorphism lends support to a protective role for NO against these syndromes. Targeted interventions to increase NO delivery or production could provide novel preventive and therapeutic strategies against these major causes of mortality in African children.