Elsevier

The Lancet

Volume 360, Issue 9344, 9 November 2002, Pages 1468-1475
The Lancet

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

https://doi.org/10.1016/S0140-6736(02)11474-7Get rights and content

Summary

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% Cl 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% Cl 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.

Introduction

The WHO estimates that 1·5–2·7 million deaths per year are caused by severe Plasmodium falciparum malaria, with most deaths occurring in African children younger than age 5 years.1 The major syndromes of severe malaria in these children are cerebral malaria, severe malarial anaemia, and respiratory distress or metabolic acidosis.2 Prevalence of cerebral malaria and severe malarial anaemia varies by geographic region and by intensity of transmission of malaria.3 Distinct forms of dysregulation of the immune system seem to give rise to the two syndromes.4 To better understand the pathophysiology of severe malaria and the mechanisms of protection from the disease, we sought to identify genetic traits that confer protection from, or susceptibility to, malaria and to assess the function of these genetic traits in acute infection.

nitric oxide (no) is toxic to malaria parasites in vitro5 and protects rodents from disease in vivo.6 The induction and regulation of the human nitric oxide synthase type 2 (nos2) gene is complex and involves sequences up to 16 kb upstream of the gene.7 Up to five-fold induction of reporter constructs can be achieved with human NOS2 promoter sequences, using the proximal 7–8 kb of sequence (−8 kb to +1 bp)7 in cells stimulated with various cytokines. Induction and expression of NOS2 varies dependent on the induction signals used and the cell types studied.8

Two NOS2 promoter polymorphisms, −954 G/C and the (CCTTT)n repeat, have been examined for potential roles in malarial outcomes (figure 1). The −954 G/C polymorphism has been associated with mild malaria disease severity in Gabon,9 and a long (CCTTT)n repeat length (defined as ≥11 repeats) has been associated with protection from fatal cerebral malaria in Gambian children.10 However, in a previous experiment11 we noted no association between these previously described polymorphisms and risk of cerebral malaria in Tanzanian children, or with NO production in vivo.11

In earlier work, in a controlled, prospective study, Anstey and colleagues12 noted high NO production and mononuclear cell immunoreactive NOS2 in Tanzanian malaria-exposed, healthy control children. NO production and NOS2 expression were significantly lower in children admitted to hospital with uncomplicated malaria or cerebral malaria, than in controls. This finding suggests that high concentrations of NO could protect against cerebral malaria in coastal Tanzania, a region with a high incidence of the disease. We postulated, therefore, that the differing degrees of NOS2 expression and NO concentration in children with different clinical manifestations of malaria might be due to as yet unidentified genetic differences in the NOS2 gene.

Section snippets

Participants

Between 1992 and 1995, we studied two groups of patients who had been previously involved in other studies done by us; one group was from Tanzania12, 13 and the other from Kenya.14

For the Tanzanian participants, we identified inpatients prospectively recruited from the Muhimbili Medical Centre, Dar es Salaam, Tanzania, for a study examining the role of NO in severe and uncomplicated malaria.12 We recruited children into three groups: healthy controls, uncomplicated malaria, and cerebral

Results

We identified the polymorphisms in the NOS2 promoter in 185 malaria-exposed Tanzanian children with uncomplicated and complicated clinical malaria (n=138) and without clinical malaria (controls; n=47) (table 1). 20% of the controls had malaria parasites on thick blood film examination, but all were asymptomatic. The PCR fragment spanning base pair −1173 (relative to the NOS2 transcription start site8) produced three different conformation patterns by SSCP gel analysis (T/T, C/C [wild-type], and

Discussion

We have established the protective association of the-1173 C/T NOS2 promoter polymorphism in two cohorts of populations living in areas with different malaria endemicity and distinct patterns of severe disease. Validation of the protective association in the large longitudinal Kenyan birth cohort provides convincing evidence for it being an important genetic marker of protection from severe malaria. This −1173 C/T polymorphism is as prevalent as the sickle-cell trait in the two East African

GLOSSARY

cerebral malaria (cm)
Unarousable coma (WHO) with a Blantyre coma score of 2 or less for at least 30 min after the last convulsion, no other cause of coma evident from clinical or cerebrospinal fluid analysis, and any level of P falciparum parasitaemia on thick film examination.
mutagenically separated pcr (ms-pcr)
A method for amplifying DNA fragments with different sequences (polymorphisms) from the same genomic region with three primers designed such that one template sequence produces a

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