Endothelial Nitric Oxide Synthase Gene Polymorphisms and ...

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Jan 22, 2009 - To explore the hypothesis that susceptibility to cerebral malaria is influenced by genetic variation in endothelial nitric oxide synthase (eNOS), ...

INFECTION AND IMMUNITY, July 2009, p. 2943–2947 0019-9567/09/$08.00⫹0 doi:10.1128/IAI.00083-09 Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Vol. 77, No. 7

Endothelial Nitric Oxide Synthase Gene Polymorphisms and Plasmodium falciparum Infection in Indian Adults䌤 Gunanidhi Dhangadamajhi,1 Biranchi N. Mohapatra,2 Shantanu K. Kar,1 and Manoranjan Ranjit1* Regional Medical Research Centre (ICMR), Bhubaneswar 751023, Orissa, India,1 and SCB Medical College & Hospital, Cuttack 753007, Orissa, India2 Received 22 January 2009/Returned for modification 2 March 2009/Accepted 1 April 2009

To explore the hypothesis that susceptibility to cerebral malaria is influenced by genetic variation in endothelial nitric oxide synthase (eNOS), we genotyped three commonly defined polymorphic loci of eNOS, Glu2983Asp, intron 4 variable number of tandem repeat region, and T-7863C, in 244 patients (mean age, 36.2 years) with mild malaria and 194 patients (mean age, 35.6 years) with severe malaria belonging to same ethnic group in Orissa, an eastern Indian state. We found that there was an association of the Glu2983Asp substitution (P ⴝ 0.0037; odds ratio, 1.95; 95% confidence interval, 1.2 to 3.0) and a single unique haplotype defined by “C-b-Asp” (Pcorrected ⴝ 0.0024) for protection against cerebral malaria. Further, the median plasma level of nitrite-nitrate was found to be increased in individuals with the Glu2983Asp substitution and was significantly higher in the mild malaria group (P < 0.0001), but the increase was not significant in the severe malaria group (P ⴝ 0.0528). These findings suggest that the Glu2983Asp substitution and the “C-b-Asp” haplotype may enhance eNOS expression and NO production, which leads to protection against cerebral malaria. These findings may increase our understanding of the pathogenesis of malaria. tion and endothelial cell activation, and modulates expression of cell adhesion molecules (12). Further, endothelial dysfunction in malaria is nearly universal when the disease is severe, is reversible with L-arginine, and likely contributes to pathogenesis (27). Therefore, we hypothesized that functionally important variants of eNOS could influence individual susceptibility to malaria by altering the amount of NO generated by the endothelium. The eNOS is constitutively expressed by vascular endothelium, and its gene is assigned to chromosome 7. This gene contains 26 exons spanning ⬃21 kb of genomic DNA and encodes an mRNA with 4,052 nucleotides, and a single copy is present in the haploid genome (13). Several allelic variants of the eNOS gene have been identified, and a variant with a T/C substitution in the 5⬘ flanking region near the promoter at position ⫺786, a variant with a 27-bp variable number of tandem repeat (VNTR) region in intron 4, and a variant with a G/T substitution at position 894 in exon 7 that codes for replacement of glutamic acid by aspartic acid at residue 298 in the mature eNOS protein, which together span ⬃6.2 kb in the human genome, have been determined to be clinically important for several diseases. However, the mechanisms by which these genetic variants can affect eNOS enzyme activity have not been demonstrated. Impaired NO production as a result of the polymorphism in exon 7 (8) and lower serum nitrite-nitrate (NOx) levels due to the ⫺786C variant have been observed (4, 15). The VNTR polymorphism in intron 4 of eNOS (eNOS4b/a polymorphism) has been reported to be significantly associated with the plasma NOx concentration (25) and affects the transcription efficiency in a haplotype-specific fashion in linkage disequilibrium with the T-786C polymorphism in the promoter region (23). To the best of our knowledge, no other coding or functional variants in this region which impair endothelial NO production have been reported so far. Moreover, because of different gene pools, lifestyles, and gene-environment interac-

Malaria parasites are major human pathogens that annually are associated with 300 million to 500 million clinical cases worldwide and 1 million to 3 million deaths (2). Despite high infection rates, only 1% to 2% of malaria patients develop life-threatening complications, such as cerebral malaria and profound anemia, so natural selection has likely operated, to a large extent, on severity (11). This has prompted the search for factors involved in natural resistance to severe malaria. The severity of malaria depends largely upon the capacity of Plasmodium falciparum-infected erythrocytes (RBCs) to adhere to the endothelia of microvessels (cytoadherence) and to form rosettes with uninfected RBCs (14). This results in a high parasite burden and severe proinflammatory responses in localized areas, leading to endothelial damage and organ dysfunction (7). Further, upregulation of endothelial cell adhesion molecules in response to tumor necrosis factor alpha potentially augments the cytoadherence (22). However, in recent studies, increased NO production has been shown to be beneficial because of its antiparasitic and antidisease effect, although this is controversial, (1). This effect is due to inhibition of the cytoadherence process through downregulation of the expression of ICAM1, VCAM1, and E-selectin, which are involved in cytoadherence and microvascular sequestration of parasitized RBCs (18) and decreased production of tumor necrosis factor by macrophages (9). NO is produced during the enzymatic conversion of L-arginine to L-citrulline by three isoforms of nitric oxide synthase (NOS), namely inducible NOS, endothelial NOS (eNOS), and neuronal NOS (19). The eNOSderived NO mediates vasodilation, inhibits platelet aggrega-

* Corresponding author. Mailing address: Regional Medical Research Centre, Indian Council of Medical Research, Chandrasekharpur, Bhubaneswar 751023, Orissa, India. Phone: 91-943-7488192. Fax: 91-674-2301351. E-mail: [email protected] 䌤 Published ahead of print on 13 April 2009. 2943

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tions among populations, there may be ethnic differences in the allelic frequencies of eNOS polymorphisms, as well as the genetic associations between disease and the plasma NOx concentration (5).Therefore, the purpose of this study was to investigate the association between the three eNOS polymorphisms mentioned above and the clinical manifestations of malaria.

MATERIALS AND METHODS This study was conducted from April 2006 to March 2008 at the SCB Medical College and Hospital, Cuttack, Orissa, India. Malaria is considered to be hyperendemic in this state, and transmission occurs throughout the year, with a seasonal peak from July to October. All four species of human malaria organisms are found in Orissa, but ⬎85% of all clinical malaria is due to P. falciparum (16.). Patients who were clinically suspected to have malaria were screened for P. falciparum infection using both thick and thin film methods. The severity of malaria was classified using the definitions and associated characteristics described by the World Health Organization (26). The following criteria were used to identify uncomplicated malaria cases: an axillary temperature of ⬎37.5°C or symptoms of headache, fever, and myalgia, no schizontemia, no intake of antimalarial drugs in the preceding week, and no history of hospitalization (to exclude individuals who had already had a severe malarial attack). The criteria used for cerebral malaria cases were an unarousable coma for ⬎6 h after severe convulsions and no indication of other causes of cerebral involvement and an axillary temperature of ⬎37.5°C. The criteria used for exclusion were (i) a confirmed diagnosis of coinfection with other Plasmodium species; (ii) symptoms of mild or severe malaria with other acute infections, including intestinal geohelminthic infections; (iii) chronic diseases like tuberculosis, leprosy, and malnutrition; (iv) genetic disorders like hemoglobinopathies and glucose-6-phosphate dehydrogenase deficiency; and (v) pregnancy, smokers, patients with coronary artery diseases, and diabetes mellitus. Venous blood was collected in EDTA-containing vials after informed consent was obtained from all enrolled patients (patients with mild malaria as well as patients with severe malaria) and immediately centrifuged at 1,000 ⫻ g for 3 min. Plasma aliquots were then removed and stored at ⫺70°C. Each patient was treated according to local guidelines, and care was provided until the patient was discharged from the hospital. The study was approved by the ethics committee of the Regional Medical Research Centre, Bhubaneswar, India. DNA isolation and genotyping. Human genomic DNA was purified from 200 ␮l of blood using the standard protocol (17). In brief, blood cells were lysed with lysis buffer (10 mM Tris-HCI [pH 8.0], 0.1 M EDTA [pH 8.0], 0.5% sodium dodecyl sulfate, 20 ␮g/ml pancreatic RNase) at 37°C for 1 h, and then proteinase K (100 ␮g/ml) was added and the lysate was incubated at 54°C for 3 h. DNA was obtained by phenol-chloroform extraction and ethanol precipitation and then resuspended in 50 ␮l of DNase-free water. The T3C transition at position ⫺786 in the 5⬘ flanking region of the eNOS gene was investigated by performing PCR-restriction fragment length polymorphism analysis using forward primer 5⬘-ATGCTCCCACCAGGGCATCA-3⬘ and reverse primer 5⬘-GTCCTTGAGT CTGACATTAGGG-3⬘. The PCR fragment (236 bp) was digested with the NgOAIV restriction enzyme overnight at 37°C. The wild-type sequence (⫺786T) was not cut, whereas the mutant sequence (⫺786C) was cleaved into two fragments (203 and 33 bp). Genotyping of the Glu2983Asp polymorphism was done by PCR amplifying exon 7 using sense primer 5⬘-CATGAGGCTCAGCCCCA GAAC-3⬘ and antisense primer 5⬘-AGTCAATCCCTTTGGTGCTCAC-3⬘, followed by digestion with the MboI restriction enzyme overnight at 37°C (6). In the presence of a T at position 894, which corresponds to Asp298, the 206-bp PCR product was cleaved into two fragments (119 and 87 bp). The restriction enzymedigested products were separated by electrophoresis on 3% Neusive agarose and visualized by ethidium bromide (0.5 ␮g/ml) staining. The intron 4 VNTR polymorphism of the eNOS gene was detected by the method of Wang et al. (24), with a slight modification (28). Briefly, DNA samples were amplified by PCR using sense (5⬘-AGGCCCTAGGTAGTGCCTTT-3⬘) and antisense (5⬘-TCTCT TAGTGCT GTGGTAC-3⬘) primers that flanked the region containing the 27-bp direct repeat of the intron 4 VNTR. The amplified DNAs were separated on 3% Neusive agarose and visualized by ethidium bromide (0.5 ␮g/ml) staining. To ensure that there was no error in genotyping, about 10% of the randomly selected samples were regenotyped for intron 4 VNTR and T-7863C substitution, whereas genotyping for the Glu2983Asp substitution was repeated for all of the samples and the results were found to be 100% concordant.

INFECT. IMMUN. TABLE 1. Clinical and parasitological characteristics of malaria patients Clinical or parasitological parameter

Patients with severe malaria (n ⫽ 194)

Patients with mild malaria (n ⫽ 244)

Mean age (yr) 35.6 36.2 Temp (°C)b 38.7 ⫾ 0.6 38.4 ⫾ 0.4 No. of patients with: Hepatomegaly 94 0 Splenomegaly 99 26 Pallor 194 78 Unarousable 194 0 state Convulsions 62 0 Jaundice 165 0 Acute renal 12 0 failure Scizontemia 39 0 Hyperparasitemia 13 0 Parasitemia 45,601.7 ⫾ 7,568.3 3,827.6 ⫾ 1,628.5 b (parasites/␮l) Hemoglobin concn 6.8 ⫾ 1.9 10.3 ⫾ 2.4 (g/dl)b a b

P value

NSa NS ⬍0.0001 ⬍0.0001 ⬍0.0001 ⬍0.0001 ⬍0.0001 ⬍0.0001 ⬍0.0001 ⬍0.0001 ⬍0.0001 ⬍0.0001 ⬍0.0001

NS, not significant. Means ⫾ standard deviations.

Plasma NOx assay. Plasma samples from patients with acute renal failure and patients who were taking medications, such as long-acting nitrates (sorbitate), were excluded from the study as these factors would have interfered with plasma NOx measurement. Plasma from patients who had consumed low-nitrate food 12 h before blood was collected were included, and to eliminate the possibility of nitrate contamination of EDTA tubes, the tubes were prewashed with Aqua Max (Younglin, South Korea) water. In addition, the EDTA solution that we used had undetectable levels of nitrite. Plasma NO3⫺ plus NO2⫺ (NOx) was measured by measuring NO2⫺ after enzymatic conversion of NO3⫺ to NO2⫺ by nitrate reductase in duplicate according to the manufacturer’s instructions using a commercial method (R&D Systems, Minnesota). Statistical analysis. All statistical analyses were performed with GraphPad Prism (version 4.0). The association of genotypes and allele frequencies between the two clinical groups was determined by computing the odds ratio derived from a ␹2 test. The ␹2 test was also used to test the Hardy-Weinberg equilibrium. Statistical significance was defined as a P value of ⬍0.05. Linkage disequilibrium was examined by ␹2 analysis, and the extent of disequilibrium was determined as follows: D⬘ ⫽ D/Dmax. The SNP Alyze program (version 7.0), which uses the expectation maximization algorithm, was used to estimate the maximum likelihood of haplotype frequencies in each group, and to identify which specific haplotypes were associated with a clinical outcome of malaria, a P value of ⬍0.00625 (0.05/number of haplotypes) was considered significant to correct for the number of comparisons made. The plasma NOx levels were compared by using the Mann-Whitney U test.

RESULTS A total of 244 patients (mean age, 36.2 years) with mild malaria and 194 patients (mean age, 35.6 years) with severe malaria were enrolled in this study. All referred patients were natives of Orissa, had permanent resident status, and belonged to the Hindu population. In the severe malaria group, all the patients had cerebral malaria. Of these, 6.7% had hyperparasitemia (⬎25,000 parasites/␮l), 20.1% had scizontemia, 32% had generalized convulsions, and 85% had jaundice (Table 1). Except for the Glu2983Asp substitution, which showed significant deviation in the mild malaria group of patients (␹2 ⫽ 5.5, P ⬍ 0.05), the genotypic distribution of all the polymorphic loci studied here showed no deviation from the Hardy-Weinberg

eNOS GENE POLYMORPHISMS AND P. FALCIPARUM INFECTION

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TABLE 2. Genotype and allele frequencies for various eNOS polymorphisms in patients with mild and cerebral malariaa Frequency (%)

Genotype or allele

Odds ratio (95% confidence Patients with Patients with interval) mild malaria severe malaria

P value

T-7863C TT TC CC

144 (59) 86 (35.2) 4 (5.7)

124 (64) 66 (34) 4 (2)

1.1 (0.7–1.6) 1.1 (0.8–1.7)

NSb

Alleles T C

374 (0.766) 114 (0.234)

314 (0.809) 74 (0.191)

1.3 (0.9–1.8)

NS

Intron 4 VNTR 4b/4b 162 (66.4) 4a/4b 70 (28.6) 4a/4a 12 (5)

126 (65) 60 (31) 8 (4)

1.1 (0.7–1.6) 1.1 (0.7–1.7)

NS

Alleles 4a 4b

94 (0.193) 394 (0.807)

76 (0.196) 312 (0.804)

1.0 (0.7–1.4)

NS

Glu2983Asp Glu/Glu Glu/Asp Asp/Asp

162 (66.4) 80 (32.8) 2 (0.8)

154 (79.4) 40 (20.6) 0

1.95 (1.2–3.0) 1.87 (1.2–2.9)

0.0037c 0.006d

Alleles Glu Asp

404 (0.829) 84 (0.171)

348 (0.897) 40 (0.103)

1.80 (1.2–2.7)

0.005

a The ␹2 test was used to determine the significance of differences between genotype and allele frequencies. b NS, not significant. c Glu/Glu versus Glu/Asp plus Asp/Asp. d Glu/Glu versus Glu/Asp.

equilibrium. The Glu2983Asp polymorphism was observed more frequently in the group of patients with mild malaria than in the group of patients with severe malaria. When the genotype and allele frequencies for the three polymorphic sites of the eNOS gene (Glu2983Asp, intron 4 VNTR, and T-7863C) were compared for the groups of patients with mild and severe malaria, a significant difference was observed only for Glu2983Asp polymorphism (Table 2). Further, comparison of the allele and haplotype frequencies revealed that the eNOS T-7863C polymorphism was strongly and significantly (D⬘ ⫽ 0.74, P ⬍ 0.0001) linked to the intron 4 VNTR a/b polymorphism, with the “4a” allele preferentially found in a subgroup of individuals with at least one “C-786C” allele. Interestingly, when the overall frequency distributions for all eight eNOS haplotypes were compared for the groups with mild and severe malaria, the H8 haplotype (C-b-Asp) was found to be more common in the group of patients with mild malaria than in the group of patients with severe malaria (P ⫽ 0.0003, Bonferroni correction; 0.0003 multiplied by the number of haplotypes, Pcorrected ⫽ 0.0024) (Fig. 1). Further, the median plasma level of NOx was found to be higher in individuals with the Glu2983Asp substitution and was significantly higher in the mild malaria group (P ⱕ 0.0001), but the increase was not significant (Table 3) in the severe malaria group (P ⫽ 0.0528). No differences were observed in plasma NOx concentrations according to gender or age.

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DISCUSSION The present study was the first study to evaluate and compare the distributions of eNOS genotypes and haplotypes in patients with mild malaria and patients with severe malaria. To explore the hypothesis that susceptibility to cerebral malaria is influenced by genetic variation in eNOS, we genotyped three commonly defined polymorphic loci of eNOS, Glu2983Asp, intron 4 VNTR, and T-7863C. We found an association for the Glu2983Asp substitution (P ⫽ 0.0037) and a single haplotype uniquely defined by “C-b-Asp” (Pcorrected ⫽ 0.0024) for protection against cerebral malaria, even though its frequency is about 3% in the population studied. Further, the increased median plasma NOx level in individuals with the Glu2983Asp substitution and its significant association with the mild malaria group (P ⱕ 0.0001) but not with the severe malaria group (P ⫽ 0.0528) suggest that the Glu2983Asp substitution and the “C-b-Asp” haplotype may enhance eNOS expression and NO production, which leads to protection against cerebral malaria, although an association of the plasma NOx level with different haplotypes could not be established due to the low frequency of some of the haplotypes as a small number of patients were selected for measurement of plasma NOx levels. There have been few studies of the specific association between the genetic polymorphisms of eNOS and plasma NOx concentrations in different diseases. To date, there is no clear evidence for impaired NO production as a result of the G/T polymorphism in exon 7 that codes for replacement of glutamic acid by aspartic acid at residue 298 in the mature eNOS protein (8).The Glu2983Asp change has been suggested to increase the susceptibility to cleavage of the eNOS enzyme (20) and hence result in low levels of NO production. Other workers argue that cleavage is due to in vitro acidic hydrolysis during sample preparation and does not appear to influence the stability, half-life, or biologic activity of the enzyme (8). However, a relationship between increased plasma NOx levels and polymorphisms in the eNOS gene has been observed for the Glu2983Asp mutation (28). Studies examining the functionality of T-786C have focused on eNOS expression. Lower eNOS

FIG. 1. Comparison of the estimated eNOS haplotype frequency distributions in patients with mild and severe malaria. The different haplotypes are as follows: H1, T-a-Glu; H2, T-b-Glu; H3, C-a-Glu; H4, C-b-Glu; H5, T-a-Asp; H6, T-b-Asp; H7, C-a-Asp; and H8, C-b-Asp. A P value of ⬍0.00625 (0.05/number of haplotypes) was considered significant.

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INFECT. IMMUN.

TABLE 3. Comparison of median plasma NOx concentrations for the various genotypes of eNOS polymorphisms in patients with mild and severe malaria All patients (n ⫽ 84) Polymorphism

Genotype

Plasma NOx concn (␮M)

n

Patients with mild malaria (n ⫽ 50) P

Plasma NOx concn (␮M)

n

P

Patients with severe malaria (n ⫽ 34) Plasma NOx concn (␮M)

n

P

Intron4 VNTR

aa ⫹ ab bb

82.7 (69.4–101.4)a 83.5 (63.8–92.0)

38 46

0.4524

81.7 (75.4–97.4) 84.7 (69.4–108.7)

20 30

NSb

76.05 (43.6–83.4) 82.7 (66.1–104.7)

16 18

NS

T-786C

TT TC

83.4 (63.8–92.0) 85.3 (69.4–108.7)

48 36

0.1043

84.7 (72.4–93.4) 91.0 (70.9–113.2)

26 24

NS

69.4 (56.3–92.0) 82.7 (69.4–94.04)

22 12

NS

Glu2983Asp

GG GT

75.4 (68.0–88.5) 99.4 (84.2–116.1)

58 26

⬍0.0001

75.4 (69.4–89.8) 108.7 (90.18–117.4)

32 18

0.0001c

69.4 (55.03–85.55) 88.4 (72.7–110.5)

26 8

0.0528d

a

The numbers in parentheses are interquartile ranges. NS, not significant. c In patients with mild malaria, the Glu2983Asp substitution is associated with a significant difference in the median plasma NOx levels. d In patients with severe malaria with the Glu2983Asp substitution, the median plasma NOx level was higher for the genotype with the T allele, but the difference was not statistically significant. b

mRNA and serum NOx levels have been found in individuals with the ⫺786C variant (15). However, an investigation by Hassan et al. (10) showed that in subjects with the C/C genotype, the levels of nitrates in the blood serum were higher than the levels in subjects with the T/T genotype of the eNOS gene promoter. Given the intron location of the intron 4 repeating unit, it is less likely to be functional. Some reports indicate that individuals with intron 4 VNTR polymorphism have lower plasma NO levels and exhibit decreased protein expression (21). However, this finding is not supported by all studies, and in healthy Caucasians an association between homozygosity for the 4a allele in the eNOS gene and increased plasma NOx levels has been detected (25). However, many epidemiological studies at the population and tissue levels support the possibility that T-786C and the 27-bp repeat in intron 4, which are in close linkage disequilibrium, may function in quantitative eNOS regulation. A recent study by Wang et al. (23) shows that the 27-bp repeat from eNOS intron 4 has a cis-regulating effect on the eNOS promoter and appears to be haplotype dependent. The highest transcription activity level was found for the eNOS promoter in the presence of the “⫺786C” (mutant) allele in the promoter and the “b” allele for intron 4, which are precisely the alleles included in the protective H8 haplotype in the present study. Furthermore, the increased median plasma NOx levels in individuals with the Glu2983Asp substitution and the significant association of this substitution with the mild malaria group (P ⫽ 0.0001) but not with the severe malaria group (P ⫽ 0.0528) suggest that the Glu2983Asp substitution and the “C-b-Asp” haplotype may be associated with increased eNOS expression and NO production, leading to protection against cerebral malaria. The decrease in the overall frequency of the “C-b-Asp” haplotype in this population may be due to linkage disequilibrium of the “C-786C” allele and the “a” allele of intron 4 VNTR. Moreover, the deviation of the Glu2983Asp substitution from Hardy-Weinberg equilibrium only in patients with mild malaria reflects the possibility that this locus is under selection pressure. It is not known whether Asp298 allele polymorphism underlies the functional characteristics of the “C-b” haplotype. To determine whether the Glu2983Asp substitution influences the plasma NOx level with the “C-b” haplotype, gene expres-

sion analysis and protein production are necessary. The genotypic distribution of Asp298 and ⫺786C homozygous in our population (0.45% and 1.8%, respectively), which best fits the Asian-type distribution (3), shows that studies with very large sample sizes are needed to obtain reliable estimates of the effect of these polymorphisms in our populations. In conclusion, our findings suggest that the eNOS Glu2983Asp substitution and “C-b-Asp” haplotype have protective effects against cerebral malaria and that the presence of Asp at position 298 may influence eNOS expression and NO production by the “C-b” haplotype. To evaluate the exact relationship between the eNOS polymorphisms and plasma NOx, it is necessary to explore and clarify the putative effects of the Glu2983Asp, T-786C, and intron 4 VNTR polymorphisms individually and in combination on endothelial NO production and its function in terms of risk for malaria in different populations where malaria is endemic. ACKNOWLEDGMENTS We acknowledge the Indian Council of Medical Research, New Delhi, for financial support. We also acknowledge the Council of Scientific and Industrial Research, New Delhi, for providing a fellowship to G. Dhangadamajhi to carry out the research. We are grateful to the patients who participated in the study. We acknowledge the director of Regional Medical Research Centre, Bhubaneswar, for providing necessary laboratory facilities for the study. G. Dhangdamajhi performed the laboratory analysis of samples and the statistical analysis and interpretation of the data; B. N. Mohaptra selected and treated the patients; S. K. Kar performed the clinicopathological analysis of the data; and M. R. Ranjit conceived and designed the study and wrote the paper. We have no conflicts of interest connected with the work reported in this paper. REFERENCES 1. Anstey, N. M., D. L. Granger, M. Y. Hassanali, E. D. Mwaikambo, P. E. Duffy, and J. B. Weinberg. 1999. Nitric oxide, malaria, and anemia: inverse relationship between nitric oxide production and hemoglobin concentration in asymptomatic, malaria exposed children. Am. J. Trop. Med. Hyg. 61:249– 252. 2. Bremen, J. 2001. The ears of the hippopotamus: manifestations, determinants, and estimates of the malaria burden. Am. J. Trop. Med. Hyg. 64:1–11. 3. Casas, J. P., L. E. Bautista, S. E. Humphries, and A. D. Hingorani. 2004. Endothelial nitric oxide synthase genotype and ischemic heart disease: metaanalysis of 26 studies involving 23028 subjects. Circulation 109:1359–1365. 4. Cattaruzza, M., T. J. Guzik, W. Słodowski, A. Pelvan, J. Becker, M. Halle,

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