INSERM, Université de la Méditerranée,
Faculté de Médecine, Marseille, France
Parasites cause much suffering mainly in countries of the southern
hemisphere. Hundreds of millions of individuals are infected by
schistosomes, leishmanias, plasmodiums, trypanosomes, and various other
parasites, and severe clinical disease occurs in a sizable fraction of
the infected population causing death and severe sequelae. The outcome,
asymptomatic, subclinical or clinical disease, of an infection depends
mostly on the parasite and on its host. Several groups analyzing the
genetics of human susceptibility to parasites have began to identify
the critical steps of the pathogenic mechanisms in a few parasitic
infections such as malaria and schistosomiasis. The present article,
which is not meant to be an exhaustive review of the field, illustrates
the progresses made in this field from pioneer studies in animals to
works in endemic populations using modern strategies of human genetics.
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Introduction |
A variety of parasites cause chronic infections
that last for long periods of time in their human host without much
clinical symptoms; in some subjects, however, parasites cause severe
disease. These pathological disorders may become apparent after 10 to
20 years of infection as in subjects infected by Schistosoma
mansoni or by Trypanosoma cruzi, or within a few weeks
of infection in patients affected by Leishmania donovani or
by Plasmodium falciparum. Various studies have attempted to
identify the factors that cause disease to develop in only a fraction
of the population exposed to parasites. Much attention has been given
to the environment because parasite transmission depends markedly on
environmental factors including vector density, vector distribution,
and parasite virulence. Parasites, because they have a large genome,
have developed very sophisticated mechanisms, like antigenic variation,
to escape immune destruction. The plasticity of the parasite genome is
so large that it is tempting to link the different clinical and
subclinical forms caused by the infection to the existence of clones of
different virulence/pathogenicity in the parasite population. This view is unlikely to apply to parasites such as Schistosoma
mansoni that, in a given endemic area, express homogenous
antigenic and pathogenic properties; it might apply, however, to
infections by protozoan parasites such as plasmodium or leishmanias
that are highly polymorph and multiply rapidly within their human hosts allowing for emerging variants.
The importance of host genetics in disease development has been
difficult to assess because of the multiplicity of the environmental factors, including parasite heterogeneity, that may determine disease.
The role of genetics was first addressed in experimental models in
which environmental variables can be controlled and measured. Animal
studies allowed the discovery of the most interesting NRAMP1
gene, which likely plays an important role in innate immunity against
intracellular pathogens. Studies on human genetics and susceptibility
to parasitic infections began with observations of the high prevalence
of mutated alleles of the 
globin gene in areas of malaria high
endemicity, leading to the hypothesis that these alleles were
protective against severe malaria. This observation was then further
supported by the results of case control studies. Comparable strategies
were used to demonstrate that certain
HLA1 haplotypes
(Hill et al., 1991
, 1992) and certain TNF-
alleles also modified
host susceptibility to malaria. Recent studies in schistosome-infected
population have taken advantages of the new methods of epidemiology and
genetics that allow performing integrated and simultaneous evaluation
of the role of environmental and host-specific factors in the control
of infection and disease. This work allowed the discovery of two major
loci controlling, for one, infection levels and, for the other, disease progression.
The present article will summarize the observations made in
schistosome, leishmania and plasmodium infections. All three parasites are a major threat for human health in the southern hemisphere (Table
1). Hundreds of millions of individuals
are infected and one to two millions die every year. This is without
mentioning that these parasites also cause invalidating sequelae. Most
important, no vaccines are available against these pathogens, and drugs
are often inadequate either because they are too expensive, too toxic (leishmanias), or because parasites evolve rapidly to become resistant to their effects (plasmodiums). No doubt, a most important objective of
genetics is the identification of the critical steps in the pathogenesis process in order to provide new targets that could be
manipulated by vaccines and chemotherapy.
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TABLE 1
Malaria, leishmaniasis, and schistosomiasis are parasitic diseases that
are major health problems in southern countries
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Genetics of Leishmania Infections in Experimental Models |
The first evidence for an important role of genetic factors in the
control of infections was reported in experimental models. Studies of
animals have the advantage over human studies to allow for the control
of environmental factors and of the parasite (heterogeneity, size of
the inoculum, etc.). In addition, genetic analysis is easier than in
humans since animals can be bred. As discussed in another chapter of
this book, earlier studies have identified a locus, lsh, on
mouse chromosome 1 that controls early multiplication of
Leishmania donovani in mice (Blackwell, 1982; Blackwell and Plant, 1986). Lsh mapped close to the bcg and
ity locus (Bradley et al., 1979) that had been shown to
control multiplication of Mycobacterium bovis and
Salmonella typhimurium in the same mouse strains. That all
three pathogens invade the macrophage phagolysosome and that resistance
or susceptibility to all three pathogens was inherited as a block
suggested that a single gene determined susceptibility to these
pathogens. To identify this gene, considerable immunologic and genetic
work was performed by several teams and allowed the identification of a
new gene (Cellier et al., 1994) on mouse chromosome 1 that accounted
for the lsh, bcg, and ity locus. This gene,
"natural resistance-associated-macrophage-protein-1 "(NRAMP1) is
described by its inventors in another chapter of this book. It is
enough to say that NRAMP1 localizes to the membrane of the
phagolysosome and is a divalent cation transporter. It is thought that
the effects of this protein on Fe2+ concentration
in the vacuole influence the production of radical oxygen intermediates
in the phagolysosome. In all mouse strains studied, mutations causing
susceptibility to either one of Leishmania, Mycobacterium,
or Salmonella also increase susceptibility to the two others
pathogens (Malo et al., 1994). Finally, strains resistant to all three
pathogens were made susceptible to all three microbes by knocking out
NRAMP1 (Vidal et al., 1995). Since the important discovery
of NRAMP1 in mice, this gene has been investigated in many
human infections. Mutations in NRAMP1 have been associated with increased susceptibility to HIV (Marquet et al., 1999),
Mycobacterium leprae (Blackwell et al., 1997; Abel et al.,
1998) and Mycobacterium tuberculosis infections but not to
parasitic infections, so far. Studies on murine leishmaniasis have also
shown that the genetic control of infection was probably polygenic.
Early studies had shown that HLA and H1 locus, in addition to
lsh determined mice susceptibility phenotype (Roberts et
al., 1989). More recent studies have confirmed this view and identify
several genetic regions (Roberts et al., 1997), which contain genes
that determine susceptibility to Leishmania major the agent
of cutaneous leishmaniasis in humans. Susceptibility genes have not
been identified so far. Studies of animals revealed the existence of
epigenetic effects between loci (Roberts et al., 1999). Such effects
will be difficult to uncover in human studies showing how useful and
necessary are studies in experimental models.
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Red Blood Cell Defects in Susceptibility to Malaria |
The high prevalence in certain areas of the world of deleterious
alleles of a number of erythrocyte proteins have stimulated speculation
as to whether these alleles might have been selected for their positive
effects against infectious diseases. Since the frequency of these
alleles are highest in malaria endemic areas, it has been proposed that
certain of these polymorphisms increase human protection against
malaria (Allison, 1969). One such polymorphism was detected in the gene
of the globin chain of hemoglobin (which is composed of two and
two chains). This mutation (noted s) in
the sixth codon of the gene (GAG replaced by GTG, changing a
glutamic acid into a valine), is responsible for the polymerization of
hemoglobin causing Sickle cell anemia in homozygous subjects. Polymerization of hemoglobin in heterozygous subjects is prevented by
the presence of a normal chain in the tetramer. Thus, homozygosity (s/s) is lethal
whereas heterozygosity
(A/s) is not. It has
been estimated that these mutations appeared 2000 to 3000 years ago in
a few individuals. Since homozygosity is lethal, it was expected
that the deleterious allele would have been selected against.
Instead, the prevalence of s is high in
certain regions of Africa. This led to the hypothesis that
s may provide some advantages against certain
diseases endemic in Africa such as malaria, which could exert a
positive selective pressure on this allele. Indeed, case control
studies showed that the frequency of s was
higher in subjects with mild malaria than in subjects with severe
often lethalmalaria. The reasons for this are unclear; a
current hypothesis is that the mutant globin chain is less efficient in preventing generation by the cell itself or by the parasite, of radical oxygen intermediates inside the erythrocytes.
Others mutations or deletions in the genes of or globin chains
and of other erythrocyte proteins are frequent in regions endemic for
malaria and some of them have also been (Hill et al., 1988; Clegg and
Weatherall, 1999; Craig et al., 2000) associated with enhanced
resistance to this infection
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Genetic Predisposition to Cerebral Malaria |
The genetics of human resistance/susceptibility to infection by
Plasmodium falciparum has been further studied by several groups using association studies to evaluate various candidate genes in
the control of severe anemia and of cerebral malaria. In certain
regions of Africa, up to 70% of the cases of severe malaria are coma
that are associated with death in 15 to 30% of the cases depending on
the rapidity and quality of the treatment. Death often occurs in
subjects with respiratory distress syndrome. The choice of the
candidate genes to be evaluated is based on what is known about the
physiopathological mechanisms of the disease. The putative
immunopathological mechanism of cerebral malaria is illustrated on Fig.
1. This figure is oversimplified to help the reader understand the roles of TNF-, iNOS, and intercellular adhesion molecule 1 (ICAM-1) in the pathological process. The pathological events in cerebral malaria are thought to be initiated in
the small capillaries of the brain by the adhesion of infected erythrocytes to the brain endothelial cells via ligands on the surface
of infected erythrocytes that bind to the ICAM-1 (CD59) and other
adhesion proteins (CD36, thrombospondin) expressed by endothelial cells
(see Fig. 1). This sequestration of the infected erythrocytes, which is
not unique to the brain, leads to a local concentration of parasites
and to the release of parasite products (including glycosylphosphatidyl
inositol-anchored surface molecules) that stimulate monocytes to
produce cytokines such as TNF-, IL-1, and IL-6. TNF- enhances the
expression of adhesion molecules on the endothelium thus increasing
adhesion of infected erythrocytes. Since non-infected erythrocytes
adhere to infected ones in infected subjects, it is thought that
extensive red cell aggregation occurring in brain capillaries causes
local thrombosis and increases local inflammation with the production
of more pro-inflammatory cytokines such as TNF-. This cytokine also
increases the transcription of iNOS, which encodes the nitric-oxide
synthase, an enzyme that catalyzes the transformation of arginine into
nitric oxide and citrulline. Nitric oxide, when produced in large
amounts, can be toxic to bystander cells including endothelial cells.
It has been suggested that the products released in the inflammatory infiltrates cause endothelium damage and plasma and red cell leakage into brain tissue (Fig. 1). Such focal hemorrhages have been observed on tissue sections of the brain after death due to cerebral malaria. Based on this hypothetical pathogenesis mechanism, several candidate genes were selected; among them were the genes encoding TNF-, iNOS,
and ICAM-1. Polymorphisms in these three genes have been described and
associated with increased (or decreased) susceptibility to malaria. The
most convincing data have been reported on TNF- and ICAM-1. A
mutation located at 
308 nucleotides relative to the transcriptional
start site of the TNF- gene was shown to increase transcription of
the gene (Kroeger et al., 1997; Abraham and Kroeger, 1999). Because of
the possible functional implication of this functional polymorphism and
because of the probable role of TNF- in pathogenesis of cerebral
malaria, the association of this mutation with severe malaria was
tested in large case control studies. The mutant allele (gene frequency
0.16) was shown to be associated with an increased relative risk of
seven for cerebral malaria with death or sequelae (McGuire et al.,
1994). This effect was only observed in homozygous subjects,
heterozygous subjects exhibited a susceptibility to severe malaria
comparable to controls. Two other mutations at position 238 and 376
have also been described, and both have been associated with increased risks of severe malaria although these effects may differ from one
population to the other (Knight et al., 1999). Similar studies, performed on the ICAM-1 gene in Kenya, led to the report of an association between a functional polymorphism located in the N-terminal domain of ICAM-1 with severe clinical malaria (Fernandez-Reyes et al.,
1997; Craig et al., 2000); this polymorphism reduces binding of ICAM-1
to its parasite ligand on erythrocyte surface (Craig et al., 2000).
Subjects homozygous for the mutation exhibited an enhanced
susceptibility to cerebral malaria (relative risk of two).
Finally, polymorphisms in the promoter of iNOS (G-954C and
polymorphisms in a microsatellite) have been reported to be associated with severity of P. falciparum infections in Gabon and
Gambia (Levesque et al., 1999); the statistics were just significant, and the observation could not be reproduced in a Tanzanian population indicating that more work is needed to definitively demonstrate that
polymorphisms in iNOS are indeed associated with severe malaria.
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Schistosomiasis: Two Major Locus in the Control of Infection and
Disease |
Schistosomes are trematode worms that live in the blood of their
human host either in the mesenteric veins or in the vesical plexus.
Female worms mate with males and lay eggs that find their way through
intestinal or ureteral tissues toward the intestinal lumen or the
urinary tact. Then, eggs are excreted with the feces or with the urine.
In the outside world, eggs hatch and infect aquatic snails in which
they undergo asexual multiplication. Sexual multiplication occurs in
human hosts within 4 to 5 weeks of the infection and may continue for
years. Humans become infected when snails release free-swimming larvae
that are (for each snail) clones issued from a single egg.
The disease is mostly caused by eggs that are trapped in the bladder,
ureter or ureteral tissues, or in the liver. These trigger an
inflammation that is succeeded by a normal scar consisting of the
deposition of collagen and extracellular matrix proteins. These scars
normally subside after a few days to be replaced by healthy tissues. In
certain subjects, the fibrotic tissue does not subside and rather
accumulates around the eggs and in the portal spaces leading to massive
obstruction of the blood flow in the portal system. High blood
hypertension builds up in these patients who may die of bleeding
(varices), co-infections, or heart failure.
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5q31-q33: A Major Region in the Control of Susceptibility to
Infections |
In a study on the causes of the high infections in an endemic area
of Brazil, it was observed that exposure to the parasite was not a
critical limiting factor in infection, probably because exposure was
quite high for most subjects (Dessein et al., 1992). No evidence was
obtained in that population supporting the hypothesis of more virulent
strains of parasites in subjects with the highest infections.
Interestingly, however, certain subjects appeared to be predisposed to
high infections whereas others always exhibited low infection in spite
of high exposure (Dessein et al., 1988, 1992); this observation was
made over a long period of time and after observing re-infections
following several anti-helminthic treatments. This suggested that
host-specific factors were important in the control of infection. The
observation that high infections were rather clustered in certain
families led us to postulate that these host-specific factors might be
inherited factors. This hypothesis was tested using segregation
analysis, which is a statistical method that evaluates whether
mathematical models that incorporate a major gene effect are better
than sporadic or multi-gene models to explain phenotype distribution.
An interesting feature of these models is that they incorporate
environmental variables and other variables such as sex and age. The
analysis proceeds by steps from the least to the most restrictive
model. This analysis was applied to the infection data taking into
account different ages, sex, and exposure, and showed that there was
strong evidence for the control of infection by a major locus (Abel et
al., 1991), also called a major gene. This locus was mapped by linkage
analysis using the model provided by segregation analysis; the entire
genome was scanned for linkage. The susceptibility locus (SM1) was
located in the 5q31-q33 region (Marquet et al., 1996, 1999a), which
contains a number of genes that encode cytokines that play an important role in the regulation of the immune response against helminth parasites. On the other hand, immunological studies performed on
homozygous-susceptible and homozygous-resistant subjects showed that
SM1 control is linked to the differentiation of T-helper cells into Th1
or Th2 lymphocytes (Couissinier-Paris and Dessein, 1995; Rodrigues et
al., 1999). Most important, the existence of a locus of susceptibility
to S. mansoni in 5q31-q33 was confirmed in an independent
study on a Senegalese population (Muller-Myhsok et al., 1997) whereas
SM1 was identified in Brazilians. Furthermore, it was also reported
that blood parasitemie in P. falciparum infections are
controlled by a locus in the same 5q31-q33 region (Garcia et al., 1998;
Rihet et al., 1999). These results altogether indicate that this region
is likely to play an important role in susceptibility to infectious diseases.
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Dual Control of Infection and Disease in Human Schistosomiasis |
Severe disease in S. mansoni infections is the
consequence of periportal fibrosis that develops in the hepatic
periportal spaces as a consequence of the inflammation caused by eggs
and worm products. Why certain subjects are unable to turnover the fibrosis that is part of a normal scar process is unknown. For a long
time, it has been thought that high infections were the most important
causes for severe hepatic fibrosis; more recently it became clear,
however, that infected levels are only one of several factors important
in disease development (Mohamed-Ali et al., 1999) suggesting that
disease and infection were not necessarily regulated by the same
factors. It was observed in ultrasound evaluations of 800 subjects
living in an endemic area of Sudan that severe fibrosis associated with
portal hypertension was more frequent in certain families and absent in
others despite the fact that all families had been living for years in
the same conditions of infection (Mohamed-Ali et al., 1999).
Segregation analysis applied to this phenotype showed evidence for a
major locus that was mapped in 6q22-q23 (Dessein et al., 1999), very
close to the gene encoding the chain of the interferon-gamma
(IFN-
) receptor, this chain binds IFN-. This result is consistent
with the well known anti-fibrogenic properties of IFN-. Furthermore,
a number of studies have demonstrated that polymorphisms in the genes
encoding either one of the two chains of the receptor of IFN-
increases human susceptibility to certain infectious diseases such as
tuberculosis (Pierre-Audigier et al., 1997; Roesler et al., 1999) in
which the host immune response is characterized, as in schistosomiasis, by a persisting granulomatous reaction.
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Conclusion |
Parasitic diseases are multifactorial and host genetic
polymorphisms are not the only factors that determine infection and disease phenotypes. Dissecting the relative contribution of the environment, the parasite and the host in pathogenesis often requires large population studies that are costly and difficult to carry out. A
major difficulty to be faced by the geneticists is the large genetic
variability of certain parasite populations, which may complicate the
detection of host genetic factors since parasite variability is not
easy to evaluate. Despite these difficulties, major advances have been
made in the field of genetics of parasitic diseases, including malaria
and schistosomiasis, and provided important insights into the
mechanisms of pathogenesis. It is likely that these studies will yield
extremely useful information for drug and vaccine development.
Abbreviations used are:
HLA, human lymphocyte
antigen;
TNF, tumor necrosis factor;
NRAMP, natural
resistance-associated macrophage protein;
iNOS, intracellular
nitric-oxide synthase;
ICAM, intercellular adhesion molecule;
IFN, interferon.
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Abstract
Introduction
