Recent literature includes many reports of genetic factors that influence the outcome of human infection with intracellular pathogens such as Mycobacterium
spp., and Plasmodium falciparum
). Familial aggregation studies and segregation analyses have also suggested that genetic factors contribute to the outcome of human VL (10
). In the present study, we investigated whether genetic variability at or near the TNF locus is associated with the development of symptomatic or asymptomatic L. chagasi
infection. This study was unique in that we were able to differentiate symptomatic from asymptomatic infection in individuals living in neighborhoods where there was ongoing transmission of L. chagasi
. The TNF locus was studied as a candidate susceptibility gene locus for three reasons. First, TNF-α levels are elevated in acute VL (6
). Second, polymorphisms at the TNF locus have been associated with a large number of autoimmune and some infectious diseases (24
). Third, a case-control study of 46 patients with mucocutaneous leishmaniasis, a hyperergic disease most often caused by L. braziliensis
, suggested an association with the TNF2 allele (20
). Visceral and mucocutaneous leishmaniasis lie at opposite poles of the spectrum of leishmaniasis. Immune responses are suppressed and the parasite load is high during VL, whereas there are few parasites but a vigorous immune response during mucocutaneous leishmaniasis. We hypothesized that distinct and opposite immune factors may predispose individuals to develop each of these diseases. However, our results suggested that the same genotype at the TNF locus is associated with development of these contrasting forms of disease.
Analysis of 1,024 subjects living in neighborhoods where leishmaniasis is endemic suggested an association between the outcome of L. chagasi
infection and alleles at the TNF locus. The strongest association was found between asymptomatic infection (DTH+) and a polymorphism in the TNF-α promoter at position −307 with respect to the transcription start site (erroneously labeled −308 in the literature [62
]). The CA repeat at the TNF MSM was also associated with the DTH+ phenotype although not as strongly. There was not complete association between the two markers that we tested. This could be due to historical recombination between the two markers, although it is more likely that there have been mutations of the CA repeats since they lie in noncoding regions of the genome. Analysis of extended haplotypes, considering genotypes at both the TNF MSM and the TNF-α RFLP markers, also suggested an association between TNF alleles and symptomatic VL. According to the results of case-control analysis, the genetic differences were greatest between individuals with symptomatic and those with asymptomatic L. chagasi
infection (VL versus DTH+). Due to the use of multiple testing in this study (multiple markers, phenotypes, and analytical methods), the chance of a type I error was increased. As the most significant P
values (0.0006 for TDT) were greater than the value of 0.0001 expected of a genome-wide linkage scan, the results must be regarded as preliminary but suggestive that further study with larger populations is worthwhile. Furthermore, due to the smaller number of VL cases compared with cases of asymptomatic infection, the association between TNF and VL warrants further studies with a larger sample size.
Analysis of the same population by using MSMs in the HLA-B and HLA-DR regions did not reveal as strong an association as that with TNF, and there was a lack of association with the marker nearest to HLA-B. The lowest P values were obtained in association analyses of asymptomatic infection (DTH+) with the D6S1014 marker near HLA-DR (P = 0.022) or in analyses of symptomatic disease (VL) with the D6S1666 marker telomeric to HLA-A (P = 0.020). The negative results for an association between the D6S1666 marker and disease suggest that the TNF locus association results are probably not caused by a stronger association with MHC class I genes. The P values for the D6S1014 marker and DTH+ phenotype warrant follow-up, but again, these data are weaker rather than stronger than the data for an association of TNF with asymptomatic disease. Taken together, these analyses favor a model in which TNF1 is associated with the development of asymptomatic L. chagasi infection detectable by a positive Montenegro skin test exam, independent of a possible association with MHC class I and class II genes.
A case-control study with Gambian children suggested that TNF2/2 homozygotes had a sevenfold increased risk of death or neurological sequelae from malaria than did heterozygotes (40
). A previous study of individuals with or without VL in Belém, northern Brazil, did not document linkage with the TNF locus (12
). However, by distinguishing those with active VL from those with asymptomatic infection and by distinguishing each of these from the population that is DTH− but likely exposed, we were able to detect an association that might have been inapparent when disease-negative individuals were analyzed as a single group.
Numerous polymorphisms at the TNFA
locus have been reported, with conflicting reports of functional significance. Promoter polymorphisms cluster in repeating 4-base G/A motifs (49
). Whereas some studies show that the −307 promoter polymorphism influences the amount of TNF-α produced by stimulated peripheral blood mononuclear cells (15
), others suggest that there is no effect (24
). Whether the results are significant depends in part upon the number of subjects examined and the stimulus chosen (2
). Studies using reporter genes under the control of the TNF-α promoter have also produced conflicting results (16
), which differ depending on the length of the 5′ untranslated region used and whether a 3′ untranslated region was cloned adjacent to the reporter gene (2
). Those studies that have reported a difference in expression, whether or not the difference reached statistical significance, have consistently shown that transcription and/or TNF-α levels are increased in individuals with the rarer −307*A (TNF2) allele. Consistently, TNF1/1 homozygotes in the population we studied had significantly lower basal TNF-α serum levels than did TNF1/2 heterozygotes. It is possible that this result was due to a sequence tightly linked to the −307 polymorphism. Nonetheless, the data suggest that a polymorphic sequence(s) at or near the −307 locus influences TNF-α levels and disease outcome.
The data discussed above led us to hypothesize that the propensity of an individual to develop asymptomatic infection versus symptomatic disease after infection with L. chagasi
is associated in part with his or her genotype at the TNF locus. If this hypothesis is true, then the symptoms of VL would be due in part to the level of TNF-α achieved (6
). Moderate levels of TNF-α, as would be expected among TNF1 homozygotes, would facilitate control of intracellular infection. Higher levels of TNF-α in individuals bearing the TNF2 allele would predispose them to clinical manifestations of VL including fever, wasting, anorexia, pancytopenia, and polyclonal B-cell activation (6
). Among persons infected with Plasmodium falciparum
, the TNF2 allele is found more frequently in individuals who developed the severe cerebral form of disease (40
). In an analogous manner, our hypothesis would indicate that heterozygotes in our study population who harbor the TNF2 allele are more likely to develop the severest visceral form of disease after exposure to L. chagasi
rather than asymptomatic or limited infection. The above-mentioned data do not distinguish whether the TNFA
RFLP is a functional polymorphism or whether it is in linkage disequilibrium with a true functional polymorphism. Nonetheless, they do lead to the hypothesis that genetically determined high-level TNF-α responses to L. chagasi
infection might predispose individuals, in part, to development of the severest visceral form of disease due to this intracellular protozoan. It may be reasonable to study agents that can lower TNF-α levels, such as thalidomide or pentoxifylline, as adjuvants to therapy for VL (23