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HUMAN GENETIC POLYMORPHISMS AND ASYMPTOMATIC PLASMODIUM FALCIPARUM MALARIA IN GABONESE SCHOOLCHILDREN

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  • 1 Centre International de Recherches Médicales de Franceville, Franceville, Gabon; Institut National de la Santé et de la Recherche Médicale Unité 458, Hôpital Robert Debré, Paris, France; Division of Hematology, Albert Einstein College of Medicine, Bronx, New York

Several studies have focused their attention on the relationship between host genetic factors and susceptibility/resistance to severe malaria. However, there is a paucity of information concerning the role of host genetic factors in asymptomatic malaria, a form of low-grade Plasmodium falciparum infection without clinical symptoms. We investigated in this study the potential relationship between the host (human) genetic polymorphisms (glucose-6-phosphate dehydrogenase [G6PD], mannose binding lectin [MBL], tumor necrosis factor α [TNF α]−308 and −238, and nitric oxide synthase 2 [NOS2]−954) and the prevalence and profile of asymptomatic P. falciparum infection in 158 Gabonese schoolchildren. We found that G6PD A heterozygous females (18 of 74) have a low prevalence of asymptomatic malaria (38.9% versus 67.3%; P = 0.03, by chi-square test). Children heterozygous for TNFα −238 (25 of 156) carry high number of diverse infecting parasite genotypes (2.5 versus 1.99; variance F = 3.05). No statistically significant association was found between MBL, TNF α −308, or NOS2 polymorphisms and asymptomatic malaria. Upon combining our data on asymptomatic forms with those from the literature for others forms, we conclude that G6PD A heterozygous females are protected against all forms of P. falciparum malaria, and that the TNF α −238A allele confers protection against clinical malaria.

INTRODUCTION

Infection with Plasmodium falciparum has a wide spectrum of manifestations that are roughly classified into three clinical groups: asymptomatic infection, mild malaria, and severe malaria. In malaria-endemic areas, a significant proportion of children harbor parasites without presenting signs of clinical malaria and are considered asymptomatic cases.1 Variant-specific immunity is one of the key components to explain the low-grade infection during extended periods without clinical symptoms.2 Two reports suggested that long-term asymptomatic malaria could lead to anemia and may aggravate the clinical course of sickle cell disease.3,4 Conversely, it has been demonstrated that the sickle cell trait (AS genotype) confers a high level of protection against severe malaria.5

Apart from the sickle cell trait, other red cell blood-related genetic factors, i.e., α - and β-thalassemia, as well as metabolic abnormalities such as glucose-6-phosphate dehydrogenase (G6PD) deficiency, have also been shown to confer protection against malaria.5 Williams and others have observed that α -thalassemia favored frequent infections of non-P. falciparum malaria in childhood.6 The possibility arises that the multiple infection might provide cross-reacting immune protection towards P. falciparum. Approximately 400 million people living in tropical and sub-tropical areas have a G6PD deficiency. Many variants, including the common G6PD B (wild type), G6PD A (non-deficient type), and G6PD A (deficient type), are observed. In these populations, molecular basis of G6PD variants showed that both G6PD A and G6PD A differ from G6PD B by a variation at nucleotide 376 (A→G), while G6PD A had an additional mutation at nucleotide 202 (G→A).7 In vitro growth of P. falciparum has been shown to be delayed in G6PD-deficient red blood cells.8 Conversely, this parasite has been shown to be available to induce the synthesis of physiologic active G6PD that is found in the cytoplasm of red blood cells.9

Among the host genetic factors other than red blood cell-related defects, genetic variants of mannose binding lectin (MBL), tumor necrosis factor α (TNFα), and inducible nitric oxide synthase 2 (NOS2) loci have been associated with resistance/susceptibility status to clinical malaria.10–15 Mannose binding lectin is a calcium-dependent protein secreted by hepatocytes. Variations in the circulating levels of MBL correspond to the presence of allelic variants (codons 52, 54, and 57) of this locus, and the presence of these alleles seem to favor recurrent bacterial and fungal infections, as well as the status of clinical malaria.10,16,17 Polymorphisms in the promoter region of the TNFα gene are associated with the TNFα production and with susceptibility to severe malaria.11–13,18,19 Nitric oxide synthase 2 is the critical enzyme involved in the synthesis of nitric oxide, a short-lived molecule with diverse functions including antimicrobial activity that can also cause damage to the host cell.20 The role of nitric oxide in many parasite diseases, including malaria, has been studied, but the involvement of NOS2 gene polymorphisms in malaria remains unclear.14,15,20

The aim of the present study was 1) to investigate the allelic prevalence of the G6PD, MBL, TNFα, and NOS2 loci in a population of schoolchildren from a malaria-endemic area (Dienga, Gabon) and 2) to explore in these children the potential relationship between these polymorphisms and the prevalence of asymptomatic P. falciparum infection and the parasite infection profile.

MATERIALS AND METHODS

Study area.

This cross-sectional study was carried out in the village of Dienga (mixed savannah/forest area in southeastern Gabon) where P. falciparum is endemic. The entomologic inoculation rate was one infective bite per person per day, and Anopheles funestus was the most predominant vector.21 The two principal peaks of P. falciparum transmission occur between February and May and between October and December.22

Study population.

Two hundred seventy-one schoolchildren (154 males and 117 females, age range = 7–19 years) were recruited and followed for four months from February to May 1995. Of the 271 children, 113 had the symptoms of malaria infection and were excluded from the study. Thus, the DNA from 158 schoolchildren was available for studying the relationship between asymptomatic malaria and human genetic polymorphisms. Thick and thin blood smears were prepared to determine parasite densities. Only P. falciparum was analyzed in this study. The parasitemia threshold of 800 parasites/μL in our region has previously been determined to be the threshold below which almost all children are symptom-free (Ntoumi F and others, unpublished data). All 158 children recruited had no parasites or a parasite density ≤ 800 parasites/μL of blood, and had no symptoms in either the two weeks prior to or the days following blood collection. Informed consent was obtained from the parents or guardians of children before sampling. Whole blood was centrifuged, and the serum and red blood cells were separated and cryopreserved until analyzed. This study was approved by the Institutional Ethical Committee of the International Center for Medical Research of Franceville (Franceville, Gabon).

DNA polymorphism analysis.

To detect the genetic polymorphisms of the G6PD, MBL, TNFα, and NOS2 loci, amplification of the relevant DNA segments by a polymerase chain reaction (PCR), followed by restriction fragment length polymorphism analysis, were carried out as previously described (Table 1).

Merozoite surface antigen-2 (MSA-2) genotyping.

To determine the parasite infection profile, P. falciparum genotyping was performed using a nested PCR for the MSA-2 gene locus, which encodes a merozoite surface glycoprotein.23 This locus consists of two conserved regions and one central variable length region. Size variations in the central block could be identified using primers derived from the highly conserved regions in the PCR, followed by analysis of the PCR products by agarose gel electrophoresis.

Statistical analysis.

The chi-square test was used to compare the distribution of different host genetic variants in subsets of individuals with and without parasites. Analysis of variance was performed to compare the mean number of distinct parasite alleles per infected isolate in the subset of patients harboring parasites. Statistical significance was defined as P < 0.05.

RESULTS

Host genetic variants in schoolchildren from the malaria-endemic area.

Allele frequencies of G6PD, MBL, TNFα, and NOS2 genetic variants in the studied population are shown in Table 2. Distribution of the alleles at each locus followed Hardy-Weinberg expectations. For the X-linked G6PD locus, in females, the different G6PD genotypes encountered were BB (27.3%), AB (31.6%), AA (8.6%), AB (15.4%), AA (15.4%), and AA (1.7%), while in males, the genotypes were B (51.9%), A (33.8%), and A (14.3%). With regard to mutant alleles in the MBL locus, a mutation in codon 57 (18.7%) was predominant compared with one in codon 54 (2.5%) and in intron IVS1-5 (3.1%). Three different variant alleles of the TNFα promoter region were found and their frequencies are given in Table 2. The TNFα −244A allele was found only in a very low proportion (1.3%). The only variant allele for the NOS2 locus genotyped in this study population, NOS2−954C, was found at a frequency of 0.098.

Host genetic polymorphisms and prevalence of asymptomatic malaria.

Isolates were tested for the presence of P. falciparum by nested PCR amplifications of the polymorphic locus MSA-2. Among the 158 symptom-free children with parasite densities ranging from 0 to 800 parasites/μL, 60 were parasite-free and 98 were positive for P. falciparum in the PCR. The relationship between the presence of host genetic variants and prevalence of infection were examined (Table 3). Of 158 children, one, who was homozygous for G6PD AA, was identified and found to be parasite free. A significantly low proportion of G6PD A heterozygous females harbored asymptomatic P. falciparum infections (38.9% versus 67.3%; P = 0.03, by chi-square test). The percentage of G6PD Ahemizygous males harboring parasites was not different from those bearing the wild type G6PD allele. No significant difference in the prevalence of asymptomatic malaria was found between children bearing mutant MBL genotypes and those homozygous for the wild type. Similarly, the distribution of TNFα −308A, TNFα −238A, and NOS2−954C variant alleles did not show any association with the prevalence of asymptomatic malaria. The ABO blood group analysis in all children revealed that O antigen (55.1%) was the most predominant, followed by A (24.7%), B (19.6%) and AB (0.6%) antigens. Only a weak association between the presence of blood group O antigen and prevalence of asymptomatic malaria was found (44.8% with O antigen versus 29.6% with non-O antigens; P = 0.05, by chi-square test).

Host genetic polymorphisms and multiplicity of asymptomatic malaria.

The multiplicity of infection, defined as the mean number of individual parasite genotypes per infected sample, was 2.14 in these isolates. No significant relationship between multiplicity of infection and G6PD genotypes was observed in females (wild type = 1.92 versus heterozygous = 2.43) or in males (wild type = 2.13 versus heterozygous = 2.22). The profile of multiplicity of infection was similar between children heterozygous for an MBL genotype (1.93) and those homozygous for the wild type (2.2). Multiplicity of infection was higher in children heterozygous for TNFα−238A than in those homozygous for TNFα−238G -wild type- (2.5 versus 1.99, respectively; F = 3.05, by analysis of variance). For the TNFα−308 allele, no significant difference in multiplicity of infection was observed (heterozygous = 2.12 versus wild type homozygous = 2.05) . No Significant difference in multiplicity of infection was found between children heterozygous for NOS2−954 (2.25) and those with wild type alleles (1.98). No association was found between ABO blood group antigens and the incidence of multiplicity of infection.

TNFα polymorphisms and asymptomatic malaria related to the sickle cell trait.

Concerning children with or without the sickle-cell trait, no statistically significant difference in the prevalence of asymptomatic malaria was found in children with various TNFα genotypes (Table 4).

DISCUSSION

To our knowledge, this is the first study to analyze the relationship between human genetic factors and asymptomatic P. falciparum infection. We studied the genetic profiles of G6PD, MBL, TNFα, and NOS2 loci in schoolchildren residing in a region endemic for malaria (Dienga in southeastern Gabon).

The frequency of various G6PD alleles found in this study is comparable to that observed in another Gabonese group.24 Our findings show that G6PD A heterozygous females are much more resistant to asymptomatic malaria than females homozygous for the wild type alleles. Interestingly, G6PD Ahemizygous males are not protected against this form of malaria. A study conducted in Nigeria among a population of children with severe malaria indicated a protective effect of the G6PD A allele in heterozygous females and in males hemizygous for the wild type allele.25 However, in the Gambia, protection against severe malaria was observed both in females heterozygous for the G6PD A allele and in males hemizygous for this allele, albeit at different rates (46% for heterozygous females and 58% for hemizygous males).26 In addition, resistance against the mild form of malaria was observed only in females heterozygous for G6PD A. Based on these data, including the data from this study, the G6PD Aheterozygous state in females confers protection against all forms of malaria, including the asymptomatic form.

The mechanism of this protection may involve a previously proposed hypothesis: the parasite in G6PD A heterozygous female host must cycle between G6PD A and G6PD wild type erythrocytes and may fail to adapt to the G6PD A environment.5,9,27 The multiplicity of infection in asymptomatic malaria is in general higher in G6PD A heterozygous females than in females with wild type alleles. This could be the consequence of difficulties of parasite adaptation to the cellular environment. Conversely, oxidative stress has been considered a critical determinant in conferring resistance in G6PD A hemizygous males.28 Without such stress, the parasites appeared to adapt and normalize their growth after four to five cycles in G6PD-deficient cells.27 This is likely due to the induction of parasite-encoded G6PD.9 Our observation that the mean number of infecting genotypes in G6PD A hemizygous males and in males with the wild type G6PD allele is similar seems to support this interpretation.

The MBL-deficient status characterized by MBL variant alleles was not associated with asymptomatic P. falciparum infection (prevalence and multiplicity) in our study. A study among Gambian children has shown absence of an association between MBL deficiency and clinical malaria,29 although another study found a weak association between MBL deficiency and severe malaria in young Gabonese children.10 This discrepancy could be due to differences in malarial forms studied and the age group of the children. Together with other components of the innate immune system, MBL contributes to efficient antimicrobial immunity and protection, particularly during the physiologic window of vulnerability following the decay of maternal antibody.16 The MBL deficiency may be not associated with malaria, but it may be a risk factor for severe malaria in children who lack well-developed protective acquired immune responses.10

Tumor necrosis factor α is a potent immunomodulator and proinflammatory cytokine that has been implicated as a pathogenic mediator in many inflammatory infections and immune diseases. Among the three polymorphisms studied, only the TNFα−238A allele had a higher frequency in Sub-Sahara Africans (9.6%) than in African Americans (2.1%),30 and the reasons for such difference are not clear and cannot be explained by the Caucasian admixture of the African Americans. In fact, in Caucasians such as British whites, the TNFα−238A allele frequency was found to be 6%.31

We found in our study population that the TNFα−238A allele in the heterozygous state was associated with a higher diversity of the infecting genotypes, but not with prevalence of asymptomatic malaria. The high multiplicity of infection in individuals heterozygous for TNFα−238A implies the presence of multiple parasite strains that may accelerate the acquisition of protective immunity in these individuals, due to exposure to a larger repertoire of P. falciparum strains. Thus, the high frequency of mixed-genotype infections in asymptomatic children may play a role in protection against clinical malaria.32 This was confirmed in a study of logistic regression analysis of three TNFα polymorphisms, which indicated that the TNFα−238A allele was associated with a decreased susceptibility to cerebral malaria.13

Although children heterozygous for TNFα−238A carry multiple parasite strains, they show a prolonging of asymptomatic malaria. Asymptomatic P. falciparum infection is associated with significant anemia.3 In fact, low-level malaria parasitemia can cause anemia by impairment of red blood cell production and/or enhancement of red blood cell destruction.1 In another study in a Gambian population, severe malarial anemia was associated with the TNFα−238A allele.12 The TNFα−238A allele may also confer protection against clinical malaria by favoring low-grade infection, but it can gradually lead to malarial anemia.

We found no association between TNFα−308A polymorphism and asymptomatic P. falciparum malaria in terms of both prevalence and multiplicity of infections. One study on complicated malarial infections showed that the homozygous state for the TNFα−308A allele was associated with death or severe neurologic sequelae due to cerebral malaria in Gambian children.11 Another study showed that the heterozygous state for this allele was associated with a risk of severe infectious disease of either malarial or other origin in Sri Lankan adults.19

The high prevalence of asymptomatic malaria observed in children with blood group O antigen, when compared with those without this antigen, suggests a protective effect of the O antigen against clinical forms of malaria. However, other explanations, such as the anti-rosette formation effect associated with blood group antigens, should also be considered.33

In conclusion, we have shown that G6PD A heterozygous females were resistant to asymptomatic malaria and that subjects heterozygous for the TNFα−238A allele carried a high number of infecting genotypes in asymptomatic malaria. These two genotypes likely play different roles in clinical P. falciparum infection.

Table 1

Human DNA polymorphisms subjected to genotyping analysis*

Base substitutionPrimer sequenceAnnealing temperature (°C)Length (basepairs)Restriction endonucleaseReference
* G6PD = glucose-6-phosphate dehydrogenase; MBL = mannose binding lectin; TNFα = tumor necrosis factor α; NOS2 = nitric oxide synthase 2. A base substitution creates (+) or abolishes (−) a specific restriction endonuclease site; the underlined nucleotides indicate a mismatch.
G6PD 202G → AG6PD-1: 5′-TTACAGCTGTGCCCTGCCCT-3′60919Nla III (+)7
G6PD-2: 5′-AGGGCAACGGCAAGCCTTAC-3′
G6PD 376A → GG6PD-2 and G6PD-3: 5′-CTGCGTTTTCTCCGCCAATC-3′60585Fok I (+)7
MBL 54G → AMBP-2: 5′-CAGGCAGTTTCCTCTGGAAGG-3′57340Ban I (−)34
MBP-3: 5′-GCACCCAGATTGTAGGACAGAG-3′
MBL 57G → AMBP-2 and MBP-357340Mbo II (+)34
MBL IVS-I-5G → AMBP-2 and MBP-357340Nla III (+)
TNFα -308G → ATNFα 1: 5′-GGCAATAGGTTTTGAGGGCCATG-3′58117Nco I (−)35
TNFα 2: 5′-CACACTCCCCATCCTCCCTGATC-3′
TNFα -244G → ATNFα 1 TNFa258117Dde I (+)30
TNFα -238G → ATNFα 1 and TNFα 258117Alw I (−)36
NOS2-954G → CNOS2-1: 5′-TGTTGGGACGGTGAGATCAAGGT-3′601273Bsa I (−)15
NOS2-2: 5′-CTCATCAAAGGTGGCCGAGAGAT-3′
Table 2

Allelic frequencies of G6PD, MBL, TNFα, and NOS2 loci in school children from the village of Dienga, Gabon*

Host genetic variantsAllelic frequencyNumber of samples†
* For definitions of abbreviations, see Table 1.
† Amplification of each allele could not be obtained for all DNA samples.
G6PD B0.515271
G6PD A0.330271
G6PD A0.155271
MBL 57 GGA → GAA0.187214
MBL 54 GGC → GAC0.025214
MBL IVS-I-5 G → A0.031214
TNFα-308 G → A0.120264
TNFα-244 G → A0.013264
TNFα-238 G → A0.096264
NOS2-954 G → G0.098118
Table 3

Distribution of host genetic variants in groups of schoolchildren aparasitemic and asymptomatic for Plasmodium falciparum*

Host polymorphismsUninfectedAsymptomaticMalaria prevalence (%)P (by chi- square test)
* For definitions of abbreviations, see Table 1.
† By Yates’ test.
C6PD A heterozygous female11738.90.03
G6PD wild type female183767.3
G6PD A hemizygous Male39750.6†
G6PD wild type male274562.5
MBL heterozygous for 57 or 54173063.80.7
MBL wild type396161
TNFα308A/G (heterozygous State)71770.80.33
TNFα308G/G (wild Type)507660.3
TNFα238A/G (heterozygous State)718720.27
TNFα238G/G (wild Type)527960.3
NOS2−954C/G (heterozygous State)51270.60.32
NOS2−954G/G (wild Type)34557.7
Table 4

Tumor necrosis factor α (TNFα) polymorphisms according to sickle cell trait carriage in uninfected and asymptomatic groups of schoolchildren

Host genetic factorsUninfectedAsymptomaticMalaria prevalence (%)P*
* By chi-square test.
AA carriers
TNFα308A/G (heterozygous)51473.70.55
TNFα308G/G (wild type)316266.7
TNFα238A/G (heterozygous)51777.30.35
TNFα238G/G (wild type)316367
AS carriers
TNFα308A/G (heterozygous)2360
TNFα308G/G (wild type)191442.4
TNFα238A/G (heterozygous)2133.3
TNFα238G/G (wild type)211643.2

Authors’ addresses: Landry-Erik Mombo and Rajagopal Krishnamoorthy, Institut National de la Santé et de la Recherche Médicale, Unité 458, Hôpital Robert Debré, 48 Boulevard Sérurier, 75019 Paris, France. Francine Ntoumi, Cyrille Bisseye, Simon Ossari, and Chang Yong Lu, Centre International de Recherches Médicales de Franceville, BP 769, Franceville, Gabon. Ronald L. Nagel, Division of Hematology, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, New York 10461.

Acknowledgments: We thank L. Sica, P. Millet, F. Lekoulou, P. Tshipamba, R. Nabias, A. Luty, H. Tiga, D. Lewobo, and J. Lansoud-Soukate for field assistance. We are indebted to the villagers, especially the children, for their participation in this study.

Financial support: This study was funded by the Centre International de Recherches Médicales (CIRMF-Gabon), which is supported by the Government of Gabon, ELF-Gabon, and the Ministère Français des Affaires Etrangères.

Disclaimer: The opinions or assertions contained in this manuscript are the private ones of the authors and are not to be construed as the official or reflecting views of the Department of Defense or the United States Army Medical Research Institute of Infectious Diseases.

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Author Notes

Reprint requests: Landry-Erik Mombo, Institut National de la Santé et de la Recherche Médicale, Unité 458, Hôpital Robert Debré, 48 Boulevard Sérurier, 75019 Paris, France, Telephone: 33-1-40-03-19-01, Fax: 33-1-40-03-19-03, E-mail: lemombo@yahoo.com
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