WO2013064147A1

WO2013064147A1 - Genetic variants associated with cerebral malaria

Info

Publication number: WO2013064147A1
Application number: PCT/DK2012/000117
Authority: WO
Inventors: Michael Theisen; Michael Christiansen
Original assignee: Statens Serum Institut
Priority date: 2011-11-04
Filing date: 2012-10-24
Publication date: 2013-05-10

Abstract

The present invention relates to an in vitro method for diagnosing a genetic predisposition or susceptibility for cerebral malaria, a severe condition of a Plasmodium falsiparum infection characterized by sequestration of parasitized red blood cells (PRBCs) and non- PRBCs (NPRBCs) in cerebral capillaries and venules and ring-like lesions in the brain. The method comprises detection of at least one specific SNP (rs2073342, rs2233860 or rs8019343) and SNPs which are in linkage disequilibrium therewith. The invention further relates to diagnostic and research kits for use in diagnosing a genetic predisposition or susceptibility for cerebral malaria and to the use of adrenal cortical steroid, inhibitors of tyrosinkinase and anti-ECP Ig in the treatment of cerebral malaria.

Description

Genetic variants associated with Cerebral Malaria

Field of the invention

The present invention relates to an in vitro method for diagnosing a genetic predisposition or susceptibility for Cerebral Malaria (CM). The present invention also relates to diagnostic and research kits for use in diagnosing CM and to the use of agents which can reduce eosinophile inflamation such as Adrenal cortical steroids, inhibitors of tyrosinkinase, inhibitors of eosinophile migrationto the relevant tissue, and inhibitors of eosinophile activation and the subsequent release of ECP in the treatment of CM.

Background

Cerebral malaria (CM) collectively involves the clinical manifestations of

Plasmodium falciparum malaria that induce changes in mental status and coma. It is an acute, widespread disease of the brain which is accompanied by fever. If a person is not treated, CM is fatal in 24-72 hours with a mortality ratio between 25 and 50%. The histopathological hallmark of this encephalopathy is the

sequestration of parasitized red blood cells (PRBCs) and non-PRBCs (NPRBCs) in cerebral capillaries and venules. Ring-like lesions in the brain are major

characteristics. Disease risk factors include being a child less than 10 years of age and living in malaria-endemic area. However, the observation that only 8 to 20% of the 225 million annual malaria cases worldwide develop into CM (38, 41) suggests that, CM is a sub-population-specific targeted syndrome.

The Glasgow Coma Score is used to define CM. Its key elements are: (1) unrousable coma - no localizing response to pain persisting for more than six hours if the patient has experienced a generalized convulsion; (2) asexual forms of P. falciparum in blood; and (3) exclusion of other causes of encephalopathy, i.e. viral or bacterial. As CM is fatal within days of malaria infection immediate treatment is crucial including antimalarial chemotherapy and adjunctive measures. Chemotherapy for cerebral malaria primarily involves the use of quinine, as a patient with severe CM must be assumed to have chloroquine resistance.

Artemisinins have been shown in some clinical trials to clear parasitemia and fever faster than quinine or chloroquine, but they had no effect on mortality rates. Adjunctive measures include: Anti-pyretics, Anti-convulsants, Hypoglycemia correction, Exchange transfusion, and anti-T Fa IgG. However, there have been few or no controlled studies demonstrating benefit and there is currently no causal treatment of CM.

Several factors have been implicated in the development of CM with both host and parasite genetic factors considered major contributors to disease pathogenesis. Several linkage and candidate gene studies have been conducted, including endothelial cell surface receptors known to interact with parasite factors on infected RBCs (iRBCs) and the cytokines modulating the expression of these adhesion molecules [6]. Polymorphisms in some of these molecules, such as tumour necrosis factor (TINF)-a (12-13, 23), interleukin (ΙΙ-)-Ιβ (3, 11),

intercellular adhesion molecule (ICAM)-l (3, 30), complement receptor (CR)-l (3), IFN-Y receptor 1 (IFNGR1) (27), and inducible (iNOS), neuronal (nNOS) and endothelial (eNOS) nitric oxide synthase (5-7, 27) have been extensively studied for their association with CM but often with either no association found or contradictory results.

Eosinophils (white blood cells) have granules which contain four very basic, cytotoxic proteins; eosinophil cationic protein (ECP), eosinophil peroxidase (EPO), eosinophil-derived neurotoxin/eosinophil protein X (EDN/EPX) and major basic protein (MBP) (28, 39). Both ECP and EDN have neurotoxic properties with ECP being the more potent of the two (10, 39). A hospital based study in Ghana involving CM patients showed that Plasmodium falciparum infection induces eosinophilia (high number of white blood cells) and also found a significantly higher level of eosinophil cationic protein (ECP) in CM patients than in uncomplicated malaria (UM) and severe malarial anaemia (SA) patients (19). However, neither eosinophiles nor ECP was demonstrated to be directly involved in CM pathogenesis and ECP levels might also be elevated due to other causes.

Furthermore, an in vitro study has demonstrated that ECP could inhibit P.

falciparum growth in a dose-dependent manner (40). Thus, ECP might be important in the control of P. falciparum infection. Single nucleotide polymorphisms (SNPs) have been described in ECP

(NP_002926.2), encoded by RNASE3 (NM_002935.2), which alter both ECP serum levels (29) and function (35). More precisely, it has been shown that the c.371G>C polymorphism (rs2073342) which results in an arginine to threonine amino acid substitution (p.R124T) in the polypeptide and abolishes the

cytotoxicity of ECP and the G- and C-alleles have been associated with allergic asthma (15) and helminth infections (9) respectively. Also, the G-allele of the 3'UTR c.*16G>C SNP, (rs2233860), correlated with higher intracellular ECP (15) and was associated with allergic rhinitis in Koreans (16). Furthermore, a study in India also found an association of the T-allele of the 3'UTR c.*94A>T, SNP

(rs8019343) with helminth infection in one patient (17).

Cerebral malaria (CM) is the most severe outcome of Plasmodium falciparum infection and a major cause of death in patients with malaria and accounts for about 80% of fatal malaria cases in children (25). To date, the exact mechanism underlying the pathogenesis of CM remains largely speculative. Thus, there is no causal direct treatment/ Furthermore, it would be helpful to identify biomarkers that could sub-classify potential malaria patients with regard to risk of CM. Such a sub-classification would improve public community prophylaxis as well as qualifying the administration of anti-malarial drugs to persons visiting malaria- endemic areas. In the public health area a better risk qualification may focus community and health resources on persons at risk of CM. Thus, persons with low-risk do not need prophylactic measures to the same extent as high risk individuals. For visitors to malaria-endemic areas it could be proposed to restrict prophylactic - potentially side effect-associated - drug treatment to individuals at high risk.

Summary of the invention

In the present study, we have sequenced RNASE3 and investigated the

association between SNPs and susceptibility to CM in a hospital based malaria study involving CM, severe malaria anaemia (SA) and uncomplicated malaria (UM) patients. The population distribution of the CM associated -allele, -genotype and - haplotype identified were also compared with that of a non-malaria population (Danes) to investigate the extent of possible selection due to CM.

We have found a single nucleotide polymorphism c.371G>C (rs2073342) which result in an amino acid substitution from arginine to threonin (p.R124T). This substitution changes eosinophil cationic protein (ECP) from being toxic to being non-toxic. We have found that the non-toxic form is dominating in African populations and that the toxic form is associated with cerebral malaria making c.371G a marker for increased risk of developing cerebral malaria when infected by Plasmodium falciparum.

Our findings suggests that anti-inflammatory agents such as Adrenal cortical steroids and inhibitors of tyrosinkinase acting to reduce eosinophile inflamation, agents which inhibit eosinophile homing to the relevant tissue and anti-ECP Ig can be used in the treatment of cerebral malaria.

As the described ECP SNP/ haplotype refer to eosinophile function, it is plausible that other malaria conditions involving eosinophils, in casu lung inflammation, may be associated with the described ECP SNP / haplotype.

Detailed description of the invention

The present invention relates to an in vitro method for diagnosing a genetic predisposition or susceptibility for acquiring Cerebral malaria in a mammal, comprising detecting in a sample obtained from said mammal at least one single nucleotide polymorphism (SNP) in a nucleic acid or fragment thereof, wherein said at least one SNP in selected from the group consisting of the SNPs rs2073342, rs2233860, rs8019343, and SNPs in linkage disequilibrium therewith, whereby at least one allele-specific haplotype is determined.

Another embodiment of the present invention relates to SNPs in linkage disequilibrium with the SNPs of the present invention. Such SNPs might be found in other populations different from the one studied here. In yet another embodiment of the present invention is the detection of the SNPs accomplished by sequencing, mini-sequencing, hybridization, restriction fragment analysis, oligonucleotide ligation assay, allele specific PC . Another aspect of the present invention relates to a diagnostic kit and/or a research kit, comprising at least one combination of probes for detecting at least one of the haplotypes of the present invention and/or probes for determining the expression level the genes of the present invention. Yet another aspect of the present invention relates to the use of agents which can reduce eosinophile inflamation such as Adrenal cortical steroid, inhibitors of tyrosinkinase, agents that inhibit eosinophile migration/homing to the relevant tissue, or anti-ECP Ig and thereby reduce eosinophile activation and the subsequent release of ECP in the affected tissue. Treatment can involve

prophylactic treatment of individuals from non-endemic areas and therapeutic treatment of CM patients.

The present invention discloses polymorphic variants and haplotypes that have been found to be associated with cerebral malaria. Particular alleles at

polymorphic markers to be associated with cerebral malaria.

Such markers and haplotypes are useful for diagnostic purposes.

Further applications of the present invention include kits for use in the methods of the invention a methods of treatment of cerebral malaria.

In one embodiment of the present invention is at least one, such as at least two, such as three SNP(s) detected in the above method. Definitions

The following terms shall, in the present context, have the meaning as indicated:

Cerebral malaria (CM) collectively involves the clinical manifestations of

Plasmodium falciparum malaria that induce changes in mental status and coma. It is an acute, widespread disease of the brain which is accompanied by fever. A "polymorphic marker", sometimes referred to as a "marker", "single nucleotide polymorphism" or "SNP" as described herein, refers to a genomic polymorphic site. Each polymorphic marker has at least two sequence variations characteristic of particular alleles at the polymorphic site.

An "allele" refers to the nucleotide sequence of a given locus (position) on a chromosome. A polymorphic marker allele thus refers to the composition (i.e., sequence) of the marker on a chromosome.

Genomic DNA from an individual contains two alleles for any given polymorphic marker, representative of each copy of the marker on each chromosome.

Sequence codes for nucleotides used herein are: A = 1, C = 2, G = 3, T = 4. A "Single Nucleotide Polymorphism" or "SNP" is a DNA sequence variation occurring when a single nucleotide at a specific location in the genome differs between members of a species or between paired chromosomes in an individual.

Most SNP polymorphisms have two alleles. Each individual is in this instance either homozygous for one allele of the polymorphism (i.e. both chromosomal copies of the individual have the same nucleotide at the SNP location), or the individual is heterozygous (i.e. the two sister chromosomes of the individual contain different nucleotides). The SNP nomenclature as reported herein refers to the official Reference SNP (rs) ID identification tag as assigned to each unique SNP by the National Center for Biotechnological Information (NCBI).

Sequence nucleotide ambiguity as described herein is as proposed by IUPAC-IUB. These codes are compatible with the codes used by the EMBL, GenBank, and PIR databases.

A nucleotide position at which more than one sequence is possible in a population (either a natural population or a synthetic population, e.g., a library of synthetic molecules) is referred to herein as a "polymorphic site". A "variant", as described herein, refers to a segment of DNA that differs from the reference DNA. A "marker" or a "polymorphic marker", as defined herein, is a variant. Alleles that differ from the reference are referred to as "variant" alleles.

A "haplotype," as described herein, refers to a segment of genomic DNA within one strand of DNA that is characterized by a specific combination of alleles arranged along the segment. For diploid organisms such as humans, a haplotype comprises one member of the pair of alleles for each polymorphic marker or locus. In a certain embodiment, the haplotype can comprise two or more alleles, three or more alleles, four or more alleles, or five or more alleles.

Alternatively, the markers and/or haplotypes of the invention are characteristic of decreased susceptibility (i.e., decreased risk) of cerebral malaria, as characterized by a relative risk of less than one, or an odds ratio of less than one. Haplotypes are described herein in the context of the marker name and the allele of the marker in that haplotype, e.g., "G rs2073342" refers to the G allele of marker rs2073342 being in the haplotype, and this nomenclature is equivalent to

"rs2073342 allele G" and "G- rs2073342". Furthermore, allelic codes in haplotypes are as for individual markers, i.e. I = A, 2 = C, 3 = G and 4 = T.

The term "susceptibility", as described herein, refers to the proneness of an individual towards the development of a certain state (e.g., a certain trait, phenotype or disease, e.g., cerebral malaria), or towards being less able to resist a particular state than the average individual. The term encompasses both increased susceptibility and decreased susceptibility.

Thus, particular alleles at polymorphic markers and/or haplotypes of the invention as described herein may be characteristic of increased susceptibility (i.e., increased risk) of cerebral malaria, as characterized by a relative risk (RR) or odds ratio (OR) of greater than one for the particular allele or haplotype. Alternatively, the markers and/or haplotypes of the invention are characteristic of decreased susceptibility (i.e., decreased risk) of cerebral malaria, as characterized by a relative risk of less than one. The term "and/or" shall in the present context be understood to indicate that either or both of the items connected by it are involved. In other words, the term herein shall be taken to mean "one or the other or both". A "nucleic acid sample" is a sample obtained from an individual that contains nucleic acid (DNA or RNA). In certain embodiments, i.e. the detection of specific polymorphic markers and/or haplotypes, the nucleic acid sample comprises genomic DNA. Such a nucleic acid sample can be obtained from any source that contains genomic DNA, including as a blood sample, sample of amniotic fluid, sample of cerebrospinal fluid, or tissue sample from skin, muscle, buccal or conjunctival mucosa, placenta, gastrointestinal tract or other organs.

Assessment for markers and haplotypes The genomic sequence within populations is not identical when individuals are compared. Rather, the genome exhibits sequence variability between individuals at many locations in the genome.

Such variations in sequence are commonly referred to as polymorphisms, and there are many such sites within each genome. For example, the human genome exhibits sequence variations which occur on average every 500 base pairs. The most common sequence variant consists of base variations at a single base position in the genome, and such sequence variants, or polymorphisms, are commonly called Single Nucleotide Polymorphisms ("SNPs").

These SNPs are believed to have arisen by a single mutational event, and therefore there are usually two possible alleles possible at each SNP site; the original allele and the mutated (alternate) allele. Due to natural genetic drift and possibly also selective pressure, the original mutation has resulted in a polymorphism characterized by a particular frequency of its alleles in any given population.

In general terms, polymorphisms can comprise any number of specific alleles. Thus in one embodiment of the invention, the polymorphism is characterized by the presence of two or more alleles in any given population. In another embodiment, the polymorphism is characterized by the presence of three or more alleles.

In other embodiments, the polymorphism is characterized by four or more alleles, five or more alleles, six or more alleles, seven or more alleles, nine or more alleles, or ten or more alleles.

All such polymorphisms can be utilized in the methods and kits of the present invention, and are thus within the scope of the invention.

In some instances, reference is made to different alleles at a polymorphic site without choosing a reference allele. Alternatively, a reference sequence can be referred to for a particular polymorphic site. The reference allele is sometimes referred to as the "wild-type" allele and it usually is chosen as either the first sequenced allele or as the allele from a "non-affected" individual (e.g., an individual that does not display a trait or disease phenotype).

Alleles for SNP markers as referred to herein refer to the bases A, C, G or T as they occur at the polymorphic site in the SNP assay employed. The allele codes for SNPs used herein are as follows: 1= A, 2=C, 3=G, 4=T. The person skilled in the art will however realize that by assaying or reading the opposite DNA strand, the complementary allele can in each case be measured.

Thus, for a polymorphic site (polymorphic marker) characterized by an A/G polymorphism, the assay employed may be designed to specifically detect the presence of one or both of the two bases possible, i.e. A and G. Alternatively, by designing an assay that is designed to detect the opposite strand on the DNA template, the presence of the complementary bases T and C can be measured. Quantitatively (for example, in terms of relative risk), identical results would be obtained from measurement of either DNA strand (+ strand or - strand).

Typically, a reference sequence is referred to for a particular sequence. Alleles that differ from the reference are sometimes referred to as "variant" alleles. A variant sequence, as used herein, refers to a sequence that differs from the reference sequence but is otherwise substantially similar. Alleles at the

polymorphic genetic markers described herein are variants. Variants can include changes that affect a polypeptide. A haplotype refers to a segment of DNA that is characterized by a specific combination of alleles arranged along the segment. For diploid organisms such as humans, a haplotype comprises one member of the pair of alleles for each polymorphic marker or locus. In a certain embodiment, the haplotype can comprise two or more alleles, three or more alleles, four or more alleles, or five or more alleles, each allele

corresponding to a specific polymorphic marker along the segment. Haplotypes can comprise a combination of various polymorphic markers, e.g., SNPs and microsatellites, having particular alleles at the polymorphic sites. The haplotypes thus comprise a combination of alleles at various genetic markers.

Detecting specific polymorphic markers and/or haplotypes can be accomplished by methods known in the art for detecting sequences at polymorphic sites. For example, standard techniques for genotyping for the presence of SNPs and/or microsatellite markers can be used, such as fluorescence-based techniques utilizing PCR, LCR, Nested PCR and other techniques for nucleic acid amplification.

Specific commercial methodologies available for SNP genotyping include, but are not limited to, TaqMan genotyping assays and SNPIex platforms (Applied

Biosystems), gel electrophoresis (Applied Biosystems), mass spectrometry (e.g., MassARRAY system from Sequenom), minisequencing methods, real-time PCR, Bio-Plex system (BioRad), CEQ and SNPstream systems (Beckman), array hybridization technology (e.g., Affymetrix GeneChip; Perlegen), BeadArray Technologies (e.g., Illumina GoldenGate and Infinium assays), array tag technology (e.g., Parallele), and endonuclease-based fluorescence hybridization technology (Invader; Third Wave).

In certain methods described herein, an individual who is at an increased susceptibility (i.e., increased risk) for cerebral malaria, is an individual in whom at least one specific allele at one or more polymorphic marker or haplotype conferring increased susceptibility for cerebral malaria is identified (i.e., at-risk marker alleles or haplotypes).

In one aspect, the at-risk marker or haplotype is one that confers a significant increased risk (or susceptibility) of cerebral malaria.

In one embodiment, significance associated with a marker or haplotype is measured by a relative risk (RR). In another embodiment, significance associated with a marker or haplotye is measured by an odds ratio (OR).

In a further embodiment, the significance is measured by a percentage.

In one embodiment, a significant increased risk is measured as a risk (relative risk and/or odds ratio) of at least 1.10, including but not limited to: at least 1.11, at least 1.12, at least 1.13, at least 1.14, at least 1.15, at least 1.16, at least 1.17, at least 1.18, at least 1.19, at least 1.20, at least 1.21, at least 1.22, at least 1.23, at least 1.24, at least 1.25, at least 1.30, at least 1.35, at least 1.40, at least 1.50, at least 1.60, at least 1.70, 1.80, at least 1.90, at least 2.0, at least 2.5, at least 3.0, at least 4.0, and at least 5.0. In a particular embodiment, a risk (relative risk and/or odds ratio) of at least 1.15 is significant. In another particular embodiment, a risk of at least 1.17 is significant.

In yet another embodiment, a risk of at least 1.20 is significant. In a further embodiment, a relative risk of at least about 1.25 is significant. In another further embodiment, a significant increase in risk is at least about 1.30 is significant. In yet another further embodiment, a significant increase in risk is at least about 1.40 is significant. In yet another further embodiment, a significant increase in risk is at least about 1.50 is significant. In yet another further embodiment, a significant increase in risk is at least about 1.60 is significant. In yet another further embodiment, a significant increase in risk is at least about 1.70 is significant. In yet another further embodiment, a significant increase in risk is at least about 1.80 is significant.

However, other cutoffs are also contemplated, e.g. at least 1.16, 1.18, 1.19, 1.21, 1.22, and so on, and such cutoffs are also within scope of the present invention. In other embodiments, a significant increase in risk is at least about 5%, including but not limited to about 10 %, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, and about 100%. In one particular embodiment, a significant increase in risk is at least 15%.

In other embodiments, a significant increase in risk is at least 17%, at least 20%, at least 22%, at least 24%, at least 25%, at least 30%, at least 32% and at least 35%. Other cutoffs or ranges as deemed suitable by the person skilled in the art to characterize the invention are however also contemplated, and those are also within scope of the present invention. In certain embodiments, a significant increase in risk is characterized by a p- value, such as a p-value of less than 0.05, less than 0.01, less than 1 x 10^"3 (0.001), less than 1 x 10^"4 (0.0001), less than 1 x 10^"4 (0.00001), less than 1 x 10^"5 (0.000001), less than 1 x 10^"6 (0.0000001), less than 1 x 10^"7 (0.00000001), less than 1 x 10^"8 (0.000000001), less than 1 x 10^"10 (0.00000000001), less than 1 x 10^'12 (0.0000000000001), less than 1 x 10^"14 (0.000000000000001), less than 1 x 10^"16 (0.00000000000000001), or less than 1 x 10^"18

(0.0000000000000000001).

An at-risk polymorphic marker or haplotype of the present invention is one where at least one allele of at least one marker or at least one haplotype is more frequently present in an individual at risk for the disease or trait (affected), or diagnosed with the disease or trait, compared to the frequency of its presence in a comparison group (control), such that the presence of the allele or haplotype is indicative of susceptibility to the disease or trait (e.g., cerebral malaria).

The control group may in one embodiment be a population sample, i.e. a random sample from the general population. In another embodiment, the control group is represented by a group of individuals who are disease-free, i.e. individuals who have not been diagnosed with cerebral malaria. Such disease-free control may in one embodiment be characterized by the absence of one or more specific diseases-associated symptoms and no sub-stratification of the population.

As an example of a simple test for association would be a Fisher-exact test on a 5 two by two table. Given a group of cases and a group of controls, the two by two table is constructed with one row for the cases and one for the controls. In the first column, the number of chromosomes that has the specific allele (or haplotype) is counted. In the second column the number of chromosomes that does not have the allele (or haplotype) is counted.

10

Other statistical tests of association known to the skilled person are also contemplated and are also within scope of the invention.

In other embodiments of the invention, an individual who is at a decreased 15 susceptibility (i.e., at a decreased risk) for a disease or trait is an individual in whom at least one specific allele at one or more polymorphic marker or haplotype conferring decreased susceptibility for the disease or trait is identified.

The marker alleles and/or haplotypes conferring decreased risk are also said to be 20 protective.

In one aspect, the protective marker or haplotype is one that confers a significant decreased risk (or susceptibility) of the disease or trait.

25 In one embodiment, significant decreased risk is measured as a relative risk of less than 0.90, including but not limited to less than 0.85, less than 0.80, less than 0.75, less than 0.7, less than 0.6, less than 0.5, less than 0.4, less than 0.3, less than 0.2 and less than 0.1. In one particular embodiment, significant decreased risk is less than 0.90.

30

In another embodiment, significant decreased risk is less than 0.85. In yet another embodiment, significant decreased risk is less than 0.80. In another embodiment, the decrease in risk (or susceptibility) is at least 10%, including but not limited to at least 15%, at least 20%, at least 25%, at least 30%, at least 35 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95% and at least 98%. In one particular embodiment, a significant decrease in risk is at least about 15%.

In another embodiment, a significant decrease in risk at least about 20%. In another embodiment, the decrease in risk is at least about 25%. Other cutoffs or ranges as deemed suitable by the person skilled in the art to characterize the invention are however also contemplated, and those are also within scope of the present invention.

The person skilled in the art will appreciate that for polymorphic markers with two alleles present in the population being studied (such as SNPs), and wherein one allele is found in increased frequency in a group of individuals with a trait or disease in the population, compared with controls, the other allele of the marker will be found in decreased frequency in the group of individuals with the trait or disease, compared with controls.

In such a case, one allele of the marker (the one found in increased frequency in individuals with the trait or disease) will be the at-risk allele, while the other allele will be a protective allele.

A genetic variant associated with a disease or a trait (e.g. cerebral malaria) can be used alone to predict the risk of the disease for a given genotype. For a biallelic marker, such as a SNP, there are 3 possible genotypes: homozygote for the risk variant, heterozygote, and non carrier of the at risk variant. Risk associated with variants at multiple loci can be used to estimate overall risk. For multiple SNP variants, there are k possible genotypes k = 3" x 2"; where n is the number autosomal loci and p the number of gonosomal (sex chromosomal) loci. Overall risk assessment calculations usually assume that the relative risks of different genetic variants multiply, i.e. the overall risk (e.g., RR or OR) associated with a particular genotype combination is the product of the risk values for the genotype at each locus. If the risk presented is the relative risk for a person, or a specific genotype for a person, compared to a reference population with matched gender and ethnicity, then the combined risk - is the product of the locus specific risk values - and which also corresponds to an overall risk estimate compared with the population.

If the risk for a person is based on a comparison to non-carriers of the at risk allele, then the combined risk corresponds to an estimate that compares the person with a given combination of genotypes at all loci to a group of individuals who do not carry risk variants at any of those loci.

The group of non-carriers of any at risk variant has the lowest estimated risk and has a combined risk, compared with itself {i.e., non-carriers) of 1.0, but has an overall risk, compare with the population, of less than 1.0. It should be noted that the group of non-carriers can potentially be very small, especially for large number of loci, and in that case, its relevance is correspondingly small.

Sensitivity

As used herein the sensitivity refers to the measures of the proportion of actual positives which are correctly identified as such - in analogy with a diagnostic test, i.e. the percentage of sick people who are identified as having the condition.

Usually the sensitivity of a test can be described as the proportion of true positives of the total number with the target disorder. All patients with the target disorder are the sum of (detected) true positives (TP) and (undetected) false negatives (FN).

Specificity

As used herein the specificity refers to measures of the proportion of negatives which are correctly identified - i.e. the percentage of well people who are identified as not having the condition. The ideal diagnostic test is a test that has 100 % specificity, i.e. only detects diseased individuals and therefore no false positive results, and 100 % sensitivity, i.e. detects all diseased individuals and therefore no false negative results. For any test, there is usually a trade-off between each measure. For example in a manufacturing setting in which one is testing for faults, one may be willing to risk discarding functioning components (low specificity), in order to increase the chance of identifying nearly all faulty components (high sensitivity). This trade-off can be represented graphically using a (receiver operator characteristic) ROC curve.

Selecting a sensitivity and specificity it is possible to obtain the optimal outcome in a detection method. In determining the discriminating value distinguishing subjects or individuals having or developing e.g. cerebral malaria, the person skilled in the art has to predetermine the level of specificity. The ideal diagnostic test is a test that has 100% specificity, i.e. only detects diseased individuals and therefore no false positive results, and 100% sensitivity, i.e. detects all diseased individuals and therefore no false negative results. However, due to biological diversity no method can be expected to have 100% sensitive without including a substantial number of false negative results.

The chosen specificity determines the percentage of false positive cases that can be accepted in a given study/population and by a given institution. By decreasing specificity an increase in sensitivity is achieved. One example is a specificity of 95% which will result in a 5% rate of false positive cases. With a given prevalence of 1% of e.g. cerebral malaria in a screening population, a 95% specificity means that 5 individuals out of 100 will undergo further physical examination in order to detect one cerebral malaria case if the sensitivity of the test is 100%.

Linkage Disequilibrium

The natural phenomenon of recombination, which occurs on average once for each chromosomal pair during each meiotic event, represents one way in which nature provides variations in sequence (and biological function by consequence).

It has been discovered that recombination does not occur randomly in the genome; rather, there are large variations in the frequency of recombination rates, resulting in small regions of high recombination frequency (also called recombination hotspots) and larger regions of low recombination frequency, which are commonly referred to as Linkage Disequilibrium (LD) blocks. Linkage Disequilibrium (LD) refers to a non-random assortment of two genetic elements. For example, if a particular genetic element (e.g., an allele of a polymorphic marker, or a haplotype) occurs in a population at a frequency of 0.50 (50%) and another element occurs at a frequency of 0.50 (50%), then the predicted occurrence of a person's having both elements is 0.25 (25%), assuming a random distribution of the elements.

However, if it is discovered that the two elements occur together at a frequency higher than 0.25, then the elements are said to be in linkage disequilibrium, since they tend to be inherited together at a higher rate than what their independent frequencies of occurrence (e.g., allele or haplotype frequencies) would predict.

Roughly speaking, LD is generally correlated with the frequency of recombination events between the two elements. Allele or haplotype frequencies can be determined in a population by genotyping individuals in a population and determining the frequency of the occurrence of each allele or haplotype in the population. For populations of diploids, e.g., human populations, individuals will typically have two alleles for each genetic element (e.g., a marker, haplotype or gene).

Many different measures have been proposed for assessing the strength of linkage disequilibrium. Most capture the strength of association between pairs of biallelic sites. Two important pairwise measures of LD are r² (sometimes denoted DA2) and | D' | . Both measures range from 0 (no disequilibrium) to 1 ('complete' disequilibrium), but their interpretation is slightly different.

|D' | is defined in such a way that it is equal to 1 if just two or three of the possible haplotypes are present, and it is < 1 if all four possible haplotypes are present.

Therefore, a value of | D' | that is <1 indicates that historical recombination may have occurred between two sites (recurrent mutation can also cause | D'| to be < 1, but for single nucleotide polymorphisms (SNPs) this is usually regarded as being less likely than recombination). The measure r² represents the statistical correlation between two sites, and takes the value of 1 if only two haplotypes are present. The r² measure is arguably the most relevant measure for association mapping, because there is a simple inverse relationship between r2 and the sample size required to detect association between susceptibility loci and particular SNPs.

These measures are defined for pairs of sites, but for some applications a determination of how strong LD is across an entire region that contains many polymorphic sites might be desirable (e.g., testing whether the strength of LD differs significantly among loci or across populations, or whether there is more or less LD in a region than predicted under a particular model). Measuring LD across a region is not straightforward, but one approach is to use the measure r, which was developed in population genetics. Roughly speaking, r measures how much recombination would be required under a particular population model to generate the LD that is seen in the data. This type of method can potentially also provide a statistically rigorous approach to the problem of determining whether LD data provide evidence for the presence of recombination hotspots. For the methods described herein, a significant r2 value between genetic segments (such as SNP markers) can be at least 0.1 such as at least 0.1, 0.15, 0.2, 0.25, 0.3, 0.35, 0.4, 0.45, 0.5, 0.55, 0.6, 0.65, 0.7, 0.75, 0.8, 0.85, 0.9, 0.91, 0.92, 0.93, 0.94, 0.95, 0.96, 0.97, 0.98, 0.99 or 1.0.

In one preferred embodiment, the significant r² value can be at least 0.2.

Alternatively, linkage disequilibrium as described herein, refers to linkage disequilibrium characterized by values of | D' | of at least 0.2, such as 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.85, 0.9, 0.95, 0.96, 0.97, 0.98, 0.99.

Thus, linkage disequilibrium represents a correlation between alleles of distinct markers. It is measured by correlation coefficient or | D' | (r² up to 1.0 and | D' | up to 1.0). Linkage disequilibrium can be determined in a single human population, as defined herein, or it can be determined in a collection of samples comprising individuals from more than one human population. In one embodiment of the invention, LD is determined in a sample from one or more of the HapMap populations (Caucasian, West African, Japanese, Chinese).

In one such embodiment, LD is determined in the CEU population of the HapMap samples. In another embodiment, LD is determined in the Y I population.

In another embodiment, LD is determined in a European population. In yet another embodiment, LD is determined in the Danish population.

If all polymorphisms in the genome were identical at the population level, then every single one of them would need to be investigated in association studies.

However, due to linkage disequilibrium between polymorphisms, tightly linked polymorphisms are strongly correlated, which reduces the number of

polymorphisms that need to be investigated in an association study to observe a significant association.

Another consequence of LD is that many polymorphisms may give an association signal due to the fact that these polymorphisms are strongly correlated. Genomic LD maps have been generated across the genome, and such LD maps have been proposed to serve as framework for mapping disease-genes.

It is now established that many portions of the human genome can be broken into series of discrete haplotype blocks containing a few common haplotypes; for these blocks, linkage disequilibrium data provides little evidence indicating

recombination.

There are two main methods for defining these haplotype blocks: blocks can be defined as regions of DNA that have limited haplotype diversity, or as regions between transition zones having extensive historical recombination, identified using linkage disequilibrium.

More recently, a fine-scale map of recombination rates and corresponding hotspots across the human genome has been generated. The map reveals the enormous variation in recombination across the genome, with recombination rates as high as 10-60 cM/Mb in hotspots, while closer to 0 in intervening regions, which thus represent regions of limited haplotype diversity and high LD. The map can therefore be used to define haplotype blocks/LD blocks as regions flanked by recombination hotspots.

As used herein, the terms "haplotype block" or "LD block" includes blocks defined by any of the above described characteristics, or other alternative methods used by the person skilled in the art to define such regions.

It has thus become apparent that for any given observed association to a polymorphic marker in the genome, it is likely that additional markers in the genome also show association. This is a natural consequence of the uneven distribution of LD across the genome, as observed by the large variation in recombination rates.

The markers used to detect association thus in a sense represent "tags" for a genomic region (i.e., a haplotype block or LD block) that is associating with a given disease or trait. One or more causative (functional) variants or mutations may reside within the region found to be associating to the disease or trait.

The functional variant may be another SNP, a tandem repeat polymorphism (such as a minisatellite or a microsatellite), a transposable element, or a copy number variation, such as an inversion, deletion or insertion. Such variants in LD with the variants described herein may confer a higher relative risk (RR) or odds ratio (OR) than observed for the tagging markers used to detect the association.

The present invention thus refers to the markers used for detecting association to the disease, as described herein, as well as markers in linkage disequilibrium with the markers. Thus, in certain embodiments of the invention, markers that are in LD with the markers and/or haplotypes of the invention, as described herein, may be used as surrogate markers.

The surrogate markers have in one embodiment relative risk (RR) and/or odds ratio (OR) values smaller than for the markers or haplotypes initially found to be associating with the disease, as described herein.

In other embodiments, the surrogate markers have RR or OR values greater than those initially determined for the markers initially found to be associating with the disease, as described herein.

An example of such an embodiment would be a rare, or relatively rare (< 10% allelic population frequency) variant in LD with a more common variant (> 10% population frequency) initially found to be associating with the disease, such as the variants described herein.

Identifying and using such markers for detecting the association discovered by the inventors as described herein can be performed by routine methods well known to the person skilled in the art, and are therefore within the scope of the present invention.

One embodiment of the present invention relates to SNPs in linkage disequilibrium as set forth in the tables. These SNPs in linkage disequilibrium with the SNPs of the present invention are merely examples of SNPs that are in linkage disequilibrium. Many more exist and can be easily identified using the knowledge of linkage disequilibrium and SNPs mentioned within this disclosure along with general knowledge within the boundaries of the person skilled in the art.

Determination of haplotype frequency

The frequencies of haplotypes in patient and control groups can be estimated using the expectation-maximization algorithm. An implementation of this algorithm that can handle missing genotypes and uncertainty with the phase can be used. Under the null hypothesis, the patients and the controls are assumed to have identical frequencies. Using a likelihood approach, an alternative hypothesis is tested, where a candidate at-risk-haplotype, which can include the markers described herein, is allowed to have a higher frequency in patients than controls, while the ratios of the frequencies of other haplotypes are assumed to be the same in both groups. Likelihoods are maximized separately under both hypotheses and a corresponding 1-df likelihood ratio statistic is used to evaluate the statistical significance.

To look for at-risk and protective markers and haplotypes within a susceptibility region, for example, within an LD block region, association of all possible combinations of genotyped markers within the region is studied.

The combined patient and control groups can be randomly divided into two sets, equal in size to the original group of patients and controls. The marker and haplotype analysis is then repeated and the most significant p-value registered is determined.

This randomization scheme can be repeated, for example, over 100 or 1000 times to construct an empirical distribution of p-values. In a preferred embodiment, a p- value of <0.05 is indicative of an significant marker and/or haplotype association. Risk assessment and Diagnostics

The Risk presented is usually the relative risk for a person, or a specific genotype of a person, compared to the population with matched gender and ethnicity. Risks of two individuals of the same gender and ethnicity could be compared in a simple manner. Risk might be modified by including other, e.g. molecular (sickle cell trait) or demographic (age) risk factors, thus defining a risk algorithm.

For example, if, compared to the population, the first individual has relative risk 1.5 and the second has relative risk 0.5, then the risk of the first individual compared to the second individual is 1.5/0.5 = 3. As described herein, certain polymorphic markers and haplotypes comprising such markers are found to be useful for risk assessment of cerebral malaria. Risk assessment can involve the use of the markers for diagnosing a susceptibility to cerebral malaria. Particular alleles of polymorphic markers are found more frequently in individuals with cerebral malaria, than in individuals without diagnosis of cerebral malaria.

Therefore, these marker alleles have predictive value for detecting cerebral malaria, or a susceptibility to cerebral malaria, in an individual. Tagging markers within haplotype blocks or LD blocks comprising at-risk markers, such as the markers of the present invention, can be used as surrogates for other markers and/or haplotypes within the haplotype block or LD block. Markers with values of r² equal to 1 are perfect surrogates for the at-risk variants, i.e. genotypes for one marker perfectly predicts genotypes for the other. Markers with smaller values of r² than 1 can also be surrogates for the at-risk variant, or alternatively represent variants with relative risk values as high or possibly even higher than the at-risk variant.

The at-risk variant identified may not be the functional variant itself, but is in this instance in linkage disequilibrium with the true functional variant. The functional variant may for example be a tandem repeat, such as a minisatellite or a microsatellite, a transposable element (e.g., an AIu element), or a structural alteration, such as a deletion, insertion or inversion (sometimes also called copy number variations, or CNVs).

The present invention encompasses the assessment of such surrogate markers for the markers as disclosed herein. Such markers are annotated, mapped and listed in public databases, as well known to the skilled person, or can alternatively be readily identified by sequencing the region or a part of the region identified by the markers of the present invention in a group of individuals, and identify

polymorphisms in the resulting group of sequences. As a consequence, the person skilled in the art can readily and without undue experimentation genotype surrogate markers in linkage disequilibrium with the markers and/or haplotypes as described herein. The tagging or surrogate markers in LD with the at-risk variants detected also have predictive value for detecting association to cerebral malaria, or a

susceptibility to cerebral malaria, in an individual.

These tagging or surrogate markers that are in LD with the markers of the present invention can also include other markers that distinguish among haplotypes, as these similarly have predictive value for detecting susceptibility to cerebral malaria.

The present invention can in certain embodiments be practiced by assessing a sample comprising genomic DNA from an individual for the presence of variants described herein to be associated with cerebral malaria.

Such assessment includes steps of detecting the presence or absence of at least one allele of at least one polymorphic marker, using methods well known to the skilled person and further described herein, and based on the outcome of such assessment, determine whether the individual from whom the sample is derived is at increased or decreased risk (increased or decreased susceptibility) of cerebral malaria. Alternatively, the invention can be practiced utilizing a dataset comprising information about the genotype status of at least one polymorphic marker described herein to be associated with cerebral malaria (or markers in linkage disequilibrium with at least one marker shown herein to be associated with cerebral malaria).

In other words, a dataset containing information about such genetic status, for example in the form of genotype counts at a certain polymorphic marker, or a plurality of markers (e.g., an indication of the presence or absence of certain at- risk alleles), or actual genotypes for one or more markers, can be queried for the presence or absence of certain at-risk alleles at certain polymorphic markers shown by the present inventors to be associated with cerebral malaria.

A positive result for a variant (e.g., marker allele) associated with increased risk of cerebral malaria, as shown herein, is indicative of the individual from which the dataset is derived is at increased susceptibility (increased risk) of cerebral malaria.

In certain embodiments of the invention, a polymorphic marker is correlated to cerebral malaria by referencing genotype data for the polymorphic marker to a look-up table that comprises correlations between at least one allele of the polymorphism and cerebral malaria.

In some embodiments, the table comprises a correlation for one polymorphism. In other embodiments, the table comprises a correlation for a plurality of

polymorphisms.

In both scenarios, by referencing to a look-up table that gives an indication of a correlation between a marker and cerebral malaria, a risk for cerebral malaria, or a susceptibility to cerebral malaria, can be identified in the individual from whom the sample is derived.

In some embodiments, the correlation is reported as a statistical measure. The statistical measure may be reported as a risk measure, such as a relative risk (RR), an absolute risk (AR) or an odds ratio (OR).

Other embodiments include the use of the variants of the present invention in combination with other variants or information based on a risk-algorithm known to be useful for diagnosing a susceptibility to cerebral malaria.

Results for any two or more markers can be combined in such analysis, such as results for three markers, four markers, five markers, six markers, seven markers, eight markers, nine markers, or ten or more markers. Thus, one embodiment of the present invention relates to a at least one SNP is one SNP, such as 2 SNPs, such as 3 SNPs, such as 4 SNPs, such as 5 SNPs, such as 6 SNPs is used in the method of the present invention. In such embodiments, the genotype status of a plurality of markers and/or haplotypes is determined in an individual, and the status of the individual compared with the population frequency of the associated variants, or the frequency of the variants in clinically healthy subjects, such as age-matched and sex-matched subjects.

Methods known in the art, such as multivariate analyses or joint risk analyses, may subsequently be used to determine the overall risk conferred based on the genotype status at the multiple loci. Assessment of risk based on such analysis may subsequently be used in the methods and kits of the invention, as described herein.

As described in the above, the haplotype block structure of the human genome has the effect that a large number of variants (markers and/or haplotypes) in linkage disequilibrium with the variant originally associated with a disease or trait may be used as surrogate markers for assessing association to the disease or trait.

The number of such surrogate markers will depend on factors such as the historical recombination rate in the region, the mutational frequency in the region (i.e., the number of polymorphic sites or markers in the region), and the extent of LD (size of the LD block) in the region.

These markers are usually located within the physical boundaries of the LD block or haplotype block in question as defined using the methods described herein, or by other methods known to the person skilled in the art.

However, sometimes marker and haplotype association is found to extend beyond the physical boundaries of the haplotype block as defined. Such markers and/or haplotypes may in those cases be also used as surrogate markers and/or haplotypes for the markers and/or haplotypes physically residing within the haplotype block as defined.

As a consequence, markers and haplotypes in LD (typically characterized by r² greater than 0.1, such as r² greater than 0.2, including r² greater than 0.3, also including r² greater than 0.4) with the markers and haplotypes of the present invention are also within the scope of the invention, even if they are physically located beyond the boundaries of the haplotype block as defined. This includes markers that are described herein but may also include other markers that are in strong LD (characterized by r² greater than 0.1 or 0.2 and/or | D'| > 0.8) with one or more of the markers listed in the present application.

For the SNP markers described herein, the opposite allele to the allele found to be in excess in patients (at-risk allele) is found in decreased frequency in cerebral malaria.

These markers and haplotypes in LD and/or comprising such markers, are thus protective for cerebral malaria, i.e. they confer a decreased risk or susceptibility of individuals carrying these markers and/or haplotypes developing cerebral malaria.

Certain variants of the present invention, including certain haplotypes comprise, in some cases, a combination of various genetic markers, e.g., SNPs and

microsatellites.

Detecting haplotypes can be accomplished by methods known in the art and/or described herein for detecting sequences at polymorphic sites. Furthermore, association between certain haplotypes or sets of markers and disease phenotype can be verified using standard techniques. A representative example of a simple test for association would be a Fisher-exact test on a two by two table. Diagnostic and screening methods

In certain embodiments, the present invention pertains to methods of diagnosing, or aiding in the diagnosis of, cerebral malaria or a susceptibility to cerebral malaria, by detecting particular alleles at genetic markers that appear more frequently in cerebral malaria subjects or subjects who are susceptible to cerebral malaria.

In particular embodiments, the invention is a method of determining a

susceptibility to cerebral malaria by detecting at least one allele of at least one polymorphic marker (e.g., the markers described herein).

In other embodiments, the invention relates to a method of diagnosing a susceptibility to cerebral malaria by detecting at least one allele of at least one polymorphic marker.

The present invention describes methods whereby detection of particular alleles of particular markers or haplotypes is indicative of a susceptibility to cerebral malaria. The present invention pertains in some embodiments to methods of clinical applications of diagnosis, e.g., diagnosis performed by a medical professional.

In other embodiments, the invention pertains to methods of diagnosis or determination of a susceptibility performed by a layman.

The layman can be the customer of a genotyping service. The layman may also be a genotype service provider, who performs genotype analysis on a DNA sample from an individual, in order to provide service related to genetic risk factors for particular traits or diseases, based on the genotype status of the individual (i.e., the customer).

Recent technological advances in genotyping technologies, including high- throughput genotyping of SNP markers, such as Molecular Inversion Probe array technology (e.g., Affymetrix GeneChip), and BeadArray Technologies (e.g., Illumina GoldenGate and Infinium assays) have made it possible for individuals to have their own genome assessed for up to one million SNPs simultaneously, at relatively little cost.

The resulting genotype information, which can be made available to the individual, can be compared to information about disease or trait risk associated with various SNPs, including information from public literature and scientific publications.

The diagnostic application of disease-associated alleles as described herein, can thus for example be performed by the individual, through analysis of his/her genotype data, by a health professional based on results of a clinical test, or by a third party, including the genotype service provider.

The third party may also be service provider who interprets genotype information from the customer to provide service related to specific genetic risk factors, including the genetic markers described herein. In other words, the diagnosis or determination of a susceptibility of genetic risk can be made by health

professionals, genetic counselors, third parties providing genotyping service, third parties providing risk assessment service or by the layman (e.g., the individual), based on information about the genotype status of an individual and knowledge about the risk conferred by particular genetic risk factors (e.g., particular SNPs). In the present context, the term "diagnosing", "diagnose a susceptibility" and "determine a susceptibility" is meant to refer to any available diagnostic method, including those mentioned above. In certain embodiments, a sample containing genomic DNA from an individual is collected. Such sample can for example be a buccal swab, a saliva sample, a blood sample, or other suitable samples containing genomic DNA, as described further herein. The genomic DNA is then analyzed using any common technique available to the skilled person, such as high-throughput array technologies. Results from such genotyping are stored in a convenient data storage unit, such as a data carrier, including computer databases, data storage disks, or by other convenient data storage means. In certain embodiments, the computer database is an object database, a relational database or a post-relational database. The genotype data is subsequently analyzed for the presence of certain variants known to be susceptibility variants for a particular human condition, such as the genetic variants described herein.

Genotype data can be retrieved from the data storage unit using any convenient data query method. Calculating risk conferred by a particular genotype for the individual can be based on comparing the genotype of the individual to previously determined risk (expressed as a relative risk ( ) or and odds ratio (OR), for example) for the genotype, for example for an heterozygous carrier of an at-risk variant for a particular disease or trait (such as cerebral malaria).

The calculated risk for the individual can be the relative risk for a person, or for a specific genotype of a person, compared to the average population with matched gender and ethnicity.

The average population risk can be expressed as a weighted average of the risks of different genotypes, using results from a reference population, and the appropriate calculations to calculate the risk of a genotype group relative to the population can then be performed.

Alternatively, the risk for an individual is based on a comparison of particular genotypes, for example heterozygous carriers of an at-risk allele of a marker compared with non-carriers of the at-risk allele.

Using the population average may in certain embodiments be more convenient, since it provides a measure which is easy to interpret for the user, i.e. a measure that gives the risk for the individual, based on his/her genotype, compared with the average in the population. The calculated risk estimated can be made available to the customer via a website, preferably a secure website.

In certain embodiments, a service provider will include in the provided service all of the steps of isolating genomic DNA from a sample provided by the customer, performing genotyping of the isolated DNA, calculating genetic risk based on the genotype data, and report the risk to the customer.

In some other embodiments, the service provider will include in the service the interpretation of genotype data for the individual, i.e., risk estimates for particular genetic variants based on the genotype data for the individual. In some other embodiments, the service provider may include service that includes genotyping service and interpretation of the genotype data, starting from a sample of isolated DNA from the individual (the customer).

Overall risk for multiple risk variants can be performed using standard

methodology. For example, assuming a multiplicative model, i.e. assuming that the risk of individual risk variants multiply to establish the overall effect, allows for a straight-forward calculation of the overall risk for multiple markers.

In addition, in certain other embodiments, the present invention pertains to methods of diagnosing, or aiding in the diagnosis of, a decreased susceptibility to cerebral malaria, by detecting particular genetic marker alleles or haplotypes that appear less frequently in cerebral malaria patients than in individual not diagnosed with cerebral malaria or in the general population.

As described and exemplified herein are associated with cerebral malaria. In one embodiment, the marker allele or haplotype is one that confers a significant risk or susceptibility to cerebral malaria.

In another embodiment, the invention relates to a method of diagnosing a susceptibility to cerebral malaria in a human individual, the method comprising determining the presence or absence of at least one allele of at least one polymorphic marker in a nucleic acid sample obtained from the individual.

In another embodiment, the invention pertains to methods of diagnosing a susceptibility to cerebral malaria in a human individual, by screening for at least one marker allele or haplotype. In another embodiment, the marker allele or haplotype is more frequently present in a subject having, or who is susceptible to, cerebral malaria (affected), as compared to the frequency of its presence in a healthy subject (control, such as population controls).

In certain embodiments, the significance of association of the at least one marker allele or haplotype is characterized by a p value < 0.05. In other embodiments, the significance of association is characterized by smaller p-values, such as < 0.01, <0.001, <0.0001, <0.00001, <0.000001, <0.0000001, <0.00000001 or <0.000000001.

In these embodiments, the presence of the at least one marker allele or haplotype is indicative of a susceptibility to cerebral malaria. These diagnostic methods involve detecting the presence or absence of at least one marker allele or haplotype that is associated with cerebral malaria.

The haplotypes described herein include combinations of alleles at various genetic markers (e.g., SNPs or other genetic variants). The detection of the particular genetic marker alleles that make up the particular haplotypes can be performed by a variety of methods described herein and/or known in the art.

For example, genetic markers can be detected at the nucleic acid level (e.g., by direct nucleotide sequencing or by other means known to the skilled in the art) or at the amino acid level if the genetic marker affects the coding sequence of a protein encoded by a cerebral malaria - associated nucleic acid (e.g., by protein sequencing or by immunoassays using antibodies that recognize such a protein).

The marker alleles or haplotypes of the present invention correspond to fragments of a genomic DNA sequence associated with cerebral malaria.

Such fragments encompass the DNA sequence of the polymorphic marker or haplotype in question, but may also include DNA segments in strong LD (linkage disequilibrium) with the marker or haplotype. In one embodiment, such segments comprises segments in LD with the marker or haplotype (as determined by a value of r² greater than 0.1 and/or | D'| > 0.8).

In one embodiment, diagnosis of a susceptibility to cerebral malaria can be accomplished using hybridization methods. A biological sample from a test subject or individual (a "test sample") of genomic DNA, RNA, cDNA etc. is obtained from a subject suspected of having, being susceptible to, or predisposed for cerebral malaria (the "test subject"). Thus, in one embodiment of the present invention is the nucleic acid detected in the method of the present invention a DNA, genomic DNA, RNA, cDNA, hnRNA and/or mRNA.

The subject can be an adult, child, infant or fetus. The test sample can be from any source that contains genomic DNA, such as a blood sample, sample of amniotic fluid, sample of cerebrospinal fluid, or tissue sample from skin, muscle, buccal or conjunctival mucosa, placenta, gastrointestinal tract or other organs.

A test sample of DNA from fetal cells or tissue can be obtained by appropriate methods, such as by amniocentesis or chorionic villus sampling. The DNA, RNA, or cDNA sample is then examined.

The presence of a specific marker allele can be indicated by sequence-specific hybridization of a nucleic acid probe specific for the particular allele. The presence of more than specific marker allele or a specific haplotype can be indicated by using several sequence-specific nucleic acid probes, each being specific for a particular allele.

In one embodiment, a haplotype can be indicated by a single nucleic acid probe that is specific for the specific haplotype (i.e., hybridizes specifically to a DNA strand comprising the specific marker alleles characteristic of the haplotype). A sequence-specific probe can be directed to hybridize to genomic DNA, RNA, or cDNA. A "nucleic acid probe", as used herein, can be a DNA probe or an RNA probe that hybridizes to a complementary sequence.

One of skill in the art would know how to design such a probe so that sequence specific hybridization will occur only if a particular allele is present in a genomic sequence from a test sample.

The invention can also be reduced to practice using any convenient genotyping method, including commercially available technologies and methods for genotyping particular polymorphic markers.

To diagnose a susceptibility to cerebral malaria, a hybridization sample can be formed by contacting the test sample containing a cerebral malaria-associated nucleic acid, such as a genomic DNA sample, with at least one nucleic acid probe.

A non-limiting example of a probe for detecting mRNA or genomic DNA is a labeled nucleic acid probe that is capable of hybridizing to mRNA or genomic DNA sequences described herein. The nucleic acid probe can be, for example, a full- length nucleic acid molecule, or a portion thereof, such as an oligonucleotide of at least 15, 30, 50, 100, 250 or 500 nucleotides in length that is sufficient to specifically hybridize under stringent conditions to appropriate mRNA or genomic DNA. For example, the nucleic acid probe can comprise all or a portion of a nucleotide sequence comprising the markers listed herein.

Genes or fragments thereof, as described herein, optionally comprising at least one allele of a marker described herein, or at least one haplotype described herein, or the probe can be the complementary sequence of such a sequence.

Hybridization can be performed by methods well known to the person skilled in the art. In one embodiment, hybridization refers to specific hybridization, i.e., hybridization with no mismatches (exact hybridization). In one embodiment, the hybridization conditions for specific hybridization are high stringency. Specific hybridization, if present, is detected using standard methods. If specific hybridization occurs between the nucleic acid probe and the nucleic acid in the test sample, then the sample contains the allele that is complementary to the nucleotide that is present in the nucleic acid probe.

The process can be repeated for any markers of the present invention, or markers that make up a haplotype of the present invention, or multiple probes can be used concurrently to detect more than one marker alleles at a time.

It is also possible to design a single probe containing more than one marker alleles of a particular haplotype (e.g., a probe containing alleles complementary to 2, 3, 4, 5 or all of the markers that make up a particular haplotype). Detection of the particular markers of the haplotype in the sample is indicative that the source of the sample has the particular haplotype (e.g., a haplotype) and therefore is susceptible to cerebral malaria.

In one preferred embodiment, a method utilizing a detection oligonucleotide probe comprising a fluorescent moiety or group at its 3' terminus and a quencher at its 5' terminus, and an enhancer oligonucleotide, is employed.

The fluorescent moiety can be Gig Harbor Green or Yakima Yellow, or other suitable fluorescent moieties. The detection probe is designed to hybridize to a short nucleotide sequence that includes the SNP polymorphism to be detected. Preferably, the SNP is anywhere from the terminal residue to -6 residues from the 3' end of the detection probe. The enhancer is a short oligonucleotide probe which hybridizes to the DNA template 3' relative to the detection probe. The probes are designed such that a single nucleotide gap exists between the detection probe and the enhancer nucleotide probe when both are bound to the template. The gap creates a synthetic abasic site that is recognized by an endonuclease, such as Endonuclease IV. The enzyme cleaves the dye off the fully complementary detection probe, but cannot cleave a detection probe containing a mismatch. Thus, by measuring the fluorescence of the released fluorescent moiety, assessment of the presence of a particular allele defined by nucleotide sequence of the detection probe can be performed. The detection probe can be of any suitable size, although preferably the probe is relatively short. In one embodiment, the probe is from 5-100 nucleotides in length. In another embodiment, the probe is from 10-50 nucleotides in length, and in another embodiment, the probe is from 12-30 nucleotides in length. Other lengths of the probe are possible and within scope of the skill of the average person skilled in the art.

In a preferred embodiment, the DNA template containing the SNP polymorphism is amplified by Polymerase Chain Reaction (PCR) prior to detection. In such an embodiment, the amplified DNA serves as the template for the detection probe and the enhancer probe.

Certain embodiments of the detection probe, the enhancer probe, and/or the primers used for amplification of the template by PCR include the use of modified bases, including modified A and modified G. The use of modified bases can be useful for adjusting the melting temperature of the nucleotide molecule (probe and/or primer) to the template DNA, for example for increasing the melting temperature in regions containing a low percentage of G or C bases, in which modified A with the capability of forming three hydrogen bonds to its

complementary T can be used, or for decreasing the melting temperature in regions containing a high percentage of G or C bases, for example by using modified G bases that form only two hydrogen bonds to their complementary C base in a double stranded DNA molecule.

In a preferred embodiment, modified bases are used in the design of the detection nucleotide probe. Any modified base known to the skilled person can be selected in these methods, and the selection of suitable bases is well within the scope of the skilled person based on the teachings herein and known bases available from commercial sources as known to the skilled person. Additionally, or alternatively, a peptide nucleic acid (PNA) probe can be used in addition to, or instead of, a nucleic acid probe in the hybridization methods described herein. A PNA is a DNA mimic having a peptide-like, inorganic backbone, such as N-(2-aminoethyl)glycine units, with an organic base (A, G, C, T or U) attached to the glycine nitrogen via a methylene carbonyl linker. The PNA probe can be designed to specifically hybridize to a molecule in a sample suspected of containing one or more of the marker alleles or haplotypes that are associated with cerebral malaria. Hybridization of the PNA probe is thus diagnostic for cerebral malaria or a susceptibility to cerebral malaria.

Probes can contain one or more of the backbone nucleic acids be substituted by one or more LNAs, TINAs or other nucleic acid analogues to facilitate the optimal assay. In one embodiment of the invention, a test sample containing genomic DNA obtained from the subject is collected and the polymerase chain reaction (PCR) is used to amplify a fragment comprising one or more markers or haplotypes of the present invention. As described herein, identification of a particular marker allele or haplotype associated with cerebral malaria can be accomplished using a variety of methods (e.g., sequence analysis, analysis by restriction digestion, specific hybridization, single stranded conformation polymorphism assays (SSCP), electrophoretic analysis, etc.).

In another embodiment, diagnosis is accomplished by expression analysis, for example by using quantitative PCR (kinetic thermal cycling). This technique can, for example, utilize commercially available technologies, such as TaqMan®. The technique can assess the presence of an alteration in the expression or composition of a polypeptide or splicing variant(s) that is encoded by a nucleic acid associated with cerebral malaria. Further, the expression of the variant(s) can be quantified as physically or functionally different. In another method of the invention, analysis by restriction digestion can be used to detect a particular allele if the allele results in the creation or elimination of a restriction site relative to a reference sequence. Restriction fragment length polymorphism (RFLP) analysis can be conducted, e.g., as described in Current Protocols in Molecular Biology, supra. The digestion pattern of the relevant DNA fragment indicates the presence or absence of the particular allele in the sample. Sequence analysis can also be used to detect specific alleles or haplotypes associated with cerebral malaria. Therefore, in one embodiment, determination of the presence or absence of a particular marker alleles or haplotypes comprises sequence analysis of a test sample of DNA or RNA obtained from a subject or individual.

PCR or other appropriate methods can be used to amplify a portion of a nucleic acid associated with cerebral malaria, and the presence of a specific allele can then be detected directly by sequencing the polymorphic site (or multiple polymorphic sites in a haplotype) of the genomic DNA in the sample.

Allele-specific oligonucleotides can also be used to detect the presence of a particular allele in a nucleic acid associated with cerebral malaria, through the use of dot-blot hybridization of amplified oligonucleotides with allele-specific oligonucleotide (ASO) probes.

An "allele-specific oligonucleotide" (also referred to herein as an "allele-specific oligonucleotide probe") is an oligonucleotide of approximately 10- 50 base pairs or approximately 15-30 base pairs, that specifically hybridizes to a nucleic acid associated with cerebral malaria, and which contains a specific allele at a polymorphic site (e.g., a marker or haplotype as described herein).

An allele-specific oligonucleotide probe that is specific for one or more particular a nucleic acid associated with cerebral malaria can be prepared using standard methods. PCR can be used to amplify the desired region. The DNA containing the amplified region can be dot-blotted using standard methods, and the blot can be contacted with the oligonucleotide probe. The presence of specific hybridization of the probe to the amplified region can then be detected. Specific hybridization of an allele- specific oligonucleotide probe to DNA from the subject is indicative of a specific allele at a polymorphic site associated with cancer, including cerebral malaria.

With the addition of such analogs as locked nucleic acids (LNAs), the size of primers and probes can be reduced to as few as 8 bases. LNAs are a novel class of bicyclic DNA analogs in which the T and 4' positions in the furanose ring are joined via an O-methylene (oxy-LNA), S-methylene (thio-LNA), or amino methylene (amino-LNA) moiety.

Common to all of these LNA variants is an affinity toward complementary nucleic acids, which is by far the highest reported for a DNA analog. For example, particular all oxy-LNA nonamers have been shown to have melting temperatures (Tm) of 64°C and 74°C when in complex with complementary DNA or RNA, respectively, as opposed to 28°C for both DNA and RNA for the corresponding DNA nonamer.

Substantial increases in Tm are also obtained when LNA monomers are used in combination with standard DNA or RNA monomers. For primers and probes, depending on where the LNA monomers are included (e.g., the 3' end, the 5' end, or in the middle), the Tm could be increased considerably.

In another embodiment, arrays of oligonucleotide probes that are complementary to target nucleic acid sequence segments from a subject, can be used to identify polymorphisms in a nucleic acid associated with cerebral malaria. For example, an oligonucleotide array can be used. Oligonucleotide arrays typically comprise a plurality of different oligonucleotide probes that are coupled to a surface of a substrate in different known locations. These oligonucleotide arrays, also described as "Genechips™," have been generally described in the art.

These arrays can generally be produced using mechanical synthesis methods or light directed synthesis methods that incorporate a combination of photolithographic methods and solid phase oligonucleotide synthesis methods, or by other methods known to the person skilled in the art.

Other methods of nucleic acid analysis that are available to those skilled in the art can be used to detect a particular allele at a polymorphic site associated with cerebral malaria.

In another embodiment, the diagnosis of a susceptibility to cerebral malaria is made by detecting at least one marker or haplotypes of the present invention in combination with an additional protein-based, RNA-based or DNA-based assay.

Thus, in one embodiment of the present invention is the detection accomplished by sequencing, mini-sequencing, hybridization, restriction fragment analysis, oligonucleotide ligation assay, or allele specific PCR.

Kits

Kits useful in the methods of the invention comprise components useful in any of the methods described herein, including for example, primers for nucleic acid amplification, hybridization probes, restriction enzymes (e.g., for RFLP analysis), allele-specific oligonucleotides, antibodies that bind to an altered polypeptide encoded by a nucleic acid of the invention as described herein (e.g., a genomic segment comprising at least one polymorphic marker and/or haplotype of the present invention) or to a non-altered (native) polypeptide encoded by a nucleic acid of the invention as described herein, means for amplification of a nucleic acid associated with cerebral malaria, means for analyzing the nucleic acid sequence of a nucleic acid associated with cerebral malaria, means for analyzing the amino acid sequence of a polypeptide encoded by a nucleic acid associated with cerebral malaria, etc.

The kits can for example include necessary buffers, nucleic acid primers for amplifying nucleic acids of the invention (e.g., one or more of the polymorphic markers as described herein), and reagents for allele-specific detection of the fragments amplified using such primers and necessary enzymes (e.g., DNA polymerase). Additionally, kits can provide reagents for assays to be used in combination with the methods of the present invention, e.g., reagents for use with cerebral malaria diagnostic assays. In one embodiment, the invention is a kit for assaying a sample from a subject to detect the presence of a cerebral malaria or a susceptibility to cerebral malaria in a subject, wherein the kit comprises reagents necessary for selectively detecting at least one allele of at least one polymorphism of the present invention in the genome of the individual.

In a particular embodiment, the reagents comprise at least one contiguous oligonucleotide that hybridizes to a fragment of the genome of the individual comprising at least one polymorphism of the present invention.

In another embodiment, the reagents comprise at least one pair of

oligonucleotides that hybridize to opposite strands of a genomic segment obtained from a subject, wherein each oligonucleotide primer pair is designed to selectively amplify a fragment of the genome of the individual that includes at least one polymorphism.

In yet another embodiment the fragment is at least 20 base pairs in size. Such oligonucleotides or nucleic acids (e.g., oligonucleotide primers) can be designed using portions of the nucleic acid sequence flanking polymorphisms (e.g., SNPs or microsatellites) that are indicative of cerebral malaria.

In another embodiment, the kit comprises one or more labeled nucleic acids capable of allele-specific detection of one or more specific polymorphic markers or haplotypes associated with cerebral malaria, and reagents for detection of the label. Suitable labels include, e.g., a radioisotope, a fluorescent label, an enzyme label, an enzyme co-factor label, a magnetic label, a spin label, an epitope label.

In particular embodiments, the polymorphic marker or haplotype to be detected by the reagents of the kit comprises one or more markers, two or more markers, three or more markers, four or more markers or five or more markers listed herein.

In another embodiment, the marker or haplotype to be detected comprises at least one marker from the group of markers in strong linkage disequilibrium, as defined by values of r² greater than 0.2, to at least one of the group of markers consisting of the markers listed in the tables.

In yet another embodiment, the marker or haplotype to be detected comprises at least one marker selected from the group of markers consisting of markers SNPs rsll712066, rs29784, rs573872, rsll216185, rsl208285, rs2228671, and SNPs in linkage disequilibrium therewith.

In one preferred embodiment, the kit for detecting the markers of the invention comprises a detection oligonucleotide probe, that hybridizes to a segment of template DNA containing a SNP polymorphisms to be detected, an enhancer oligonucleotide probe and an endonuclease.

The detection oligonucleotide probe comprises a fluorescent moiety or group at its 3' terminus and a quencher at its 5' terminus, and an enhancer oligonucleotide is employed.

The fluorescent moiety can be Gig Harbor Green or Yakima Yellow, or other suitable fluorescent moieties. The detection probe is designed to hybridize to a short nucleotide sequence that includes the SNP polymorphism to be detected.

Preferably, the SNP is anywhere from the terminal residue to -6 residues from the 3' end of the detection probe. The enhancer is a short oligonucleotide probe which hybridizes to the DNA template 3' relative to the detection probe. The probes are designed such that a single nucleotide gap exists between the detection probe and the enhancer nucleotide probe when both are bound to the template.

The gap creates a synthetic abasic site that is recognized by an endonuclease, such as Endonuclease IV. The enzyme cleaves the dye off the fully complementary detection probe, but cannot cleave a detection probe containing a mismatch.

Thus, by measuring the fluorescence of the released fluorescent moiety, assessment of the presence of a particular allele defined by nucleotide sequence of the detection probe can be performed. The detection probe can be of any suitable size, although preferably the probe is relatively short. In one embodiment, the probe is from 5-100 nucleotides in length. In another embodiment, the probe is from 10-50 nucleotides in len'gth, and in another embodiment, the probe is from 12-30 nucleotides in length. Other lengths of the probe are possible and within scope of the skill of the average person skilled in the art. In a preferred embodiment, the DNA template containing the SNP polymorphism is amplified by Polymerase Chain Reaction (PCR) prior to detection, and primers for such amplification are included in the reagent kit.

In such an embodiment, the amplified DNA serves as the template for the detection probe and the enhancer probe.

Certain embodiments of the detection probe, the enhancer probe, and/or the primers used for amplification of the template by PCR include the use of modified bases, including modified A and modified G.

The use of modified bases can be useful for adjusting the melting temperature of the nucleotide molecule (probe and/or primer) to the template DNA, for example for increasing the melting temperature in regions containing a low percentage of G or C bases, in which modified A with the capability of forming three hydrogen bonds to its complementary T can be used, or for decreasing the melting temperature in regions containing a high percentage of G or C bases, for example by using modified G bases that form only two hydrogen bonds to their

complementary C base in a double stranded DNA molecule. In a preferred embodiment, modified bases are used in the design of the detection nucleotide probe. Any modified base known to the skilled person can be selected in these methods, and the selection of suitable bases is well within the scope of the skilled person based on the teachings herein and known bases available from commercial sources as known to the skilled person. In one of such embodiments, the presence of the marker or haplotype is indicative of a susceptibility (increased susceptibility or decreased susceptibility) to cerebral malaria. In another embodiment, the presence of the marker or haplotype is indicative of response to a cerebral malaria therapeutic agent. In another embodiment, the presence of the marker or haplotype is indicative of cerebral malaria prognosis.

Thus relates one embodiment of the present invention to a diagnostic kit and/or a research kit, comprising at least one combination of probes for detecting at least one of the haplotypes of described herein and/or probes for determining the expression level the genes listed herein.

Nucleic Acids and Polypeptides

The nucleic acids and polypeptides described herein can be used in methods and kits of the present invention, as described in the above. An "isolated" nucleic acid molecule, as used herein, is one that is separated from nucleic acids that normally flank the gene or nucleotide sequence (as in genomic sequences) and/or has been completely or partially purified from other transcribed sequences (e.g., as in an NA library).

For example, an isolated nucleic acid of the invention can be substantially isolated with respect to the complex cellular milieu in which it naturally occurs, or culture medium when produced by recombinant techniques, or chemical precursors or other chemicals when chemically synthesized. In some instances, the isolated material will form part of a composition (for example, a crude extract containing other substances), buffer system or reagent mix. In other circumstances, the material can be purified to essential homogeneity, for example as determined by polyacrylamide gel electrophoresis (PAGE) or column chromatography (e.g., HPLC).

An isolated nucleic acid molecule of the invention can comprise at least about 50%, at least about 80% or at least about 90% (on a molar basis) of all macromolecular species present. With regard to genomic DNA, the term "isolated" also can refer to nucleic acid molecules that are separated from the chromosome with which the genomic DNA is naturally associated.

For example, the isolated nucleic acid molecule can contain less than about 250 kb, 200 kb, 150 kb, 100 kb, 75 kb, 50 kb, 25 kb, 10 kb, 5 kb, 4 kb, 3 kb, 2 kb, 1 kb, 0.5 kb or 0.1 kb of the nucleotides that flank the nucleic acid molecule in the genomic DNA of the cell from which the nucleic acid molecule is derived.

The nucleic acid molecule can be fused to other coding or regulatory sequences and still be considered isolated. Thus, recombinant DNA contained in a vector is included in the definition of "isolated" as used herein.

Also, isolated nucleic acid molecules include recombinant DNA molecules in heterologous host cells or heterologous organisms, as well as partially or substantially purified DNA molecules in solution. "Isolated" nucleic acid molecules also encompass in vivo and in vitro RNA transcripts of the DNA molecules of the present invention.

An isolated nucleic acid molecule or nucleotide sequence can include a nucleic acid molecule or nucleotide sequence that is synthesized chemically or by recombinant means. Such isolated nucleotide sequences are useful, for example, in the manufacture of the encoded polypeptide, as probes for isolating homologous sequences (e.g., from other mammalian species), for gene mapping (e.g., by in situ hybridization with chromosomes), or for detecting expression of the gene in tissue (e.g., human tissue), such as by Northern blot analysis or other

hybridization techniques.

The invention also pertains to nucleic acid molecules that hybridize under high stringency hybridization conditions, such as for selective hybridization, to a nucleotide sequence described herein (e.g., nucleic acid molecules that specifically hybridize to a nucleotide sequence containing a polymorphic site associated with a marker or haplotype described herein).

Such nucleic acid molecules can be detected and/or isolated by allele- or sequence-specific hybridization (e.g., under high stringency conditions). Stringency conditions and methods for nucleic acid hybridizations are well known to the skilled person.

The percent identity of two nucleotide or amino acid sequences can be determined by aligning the sequences for optimal comparison purposes (e.g., gaps can be introduced in the sequence of a first sequence). The nucleotides or amino acids at corresponding positions are then compared, and the percent identity between the two sequences is a function of the number of identical positions shared by the sequences (i.e., % identity = # of identical positions/total # of positions x 100). In certain embodiments, the length of a sequence aligned for comparison purposes is at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95%, of the length of the reference sequence. The actual comparison of the two sequences can be accomplished by well-known methods, for example, using a mathematical algorithm. Such an algorithm is incorporated into the BLAST and X BLAST programs (version 2.0). When utilizing BLAST and Gapped BLAST programs, the default parameters of the respective programs (e.g., NBLAST) can be used. See the website on the world wide web at ncbi.nlm.nih.gov. In one embodiment, parameters for sequence comparison can be set at score=100, wordlength = 12, or can be varied (e.g., W=5 or W=20).

The present invention also provides isolated nucleic acid molecules that contain a fragment or portion that hybridizes under highly stringent conditions to a nucleic acid that comprises, or consists of, a nucleotide sequence comprising the polymorphic markers listed herein; or a nucleotide sequence comprising, or consisting of, the complement of the nucleotide sequence of a nucleotide sequence comprising the polymorphic markers listed herein, wherein the nucleotide sequence comprises at least one polymorphic allele contained in the markers and haplotypes described herein.

The nucleic acid fragments of the invention are at least about 15, at least about 18, 20, 23 or 25 nucleotides, and can be 30, 40, 50, 100, 200, 500, 1000, 10,000 or more nucleotides in length. The nucleic acid fragments of the invention are used as probes or primers in assays such as those described herein. "Probes" or "primers" are oligonucleotides that hybridize in a base- specific manner to a complementary strand of a nucleic acid molecule. In addition to DNA and RNA, such probes and primers include polypeptide nucleic acids (PNA).

A probe or primer comprises a region of nucleotide sequence that hybridizes to at least about 15, typically about 20-25, and in certain embodiments about 40, 50 or 75, consecutive nucleotides of a nucleic acid molecule.

In one embodiment, the probe or primer comprises at least one allele of at least one polymorphic marker or at least one haplotype described herein, or the complement thereof. In particular embodiments, a probe or primer can comprise 100 or fewer nucleotides; for example, in certain embodiments from 6 to 50 nucleotides, or, for example, from 12 to 30 nucleotides.

In other embodiments, the probe or primer is at least 70% identical, at least 80% identical, at least 85% identical, at least 90% identical, or at least 95% identical, to the contiguous nucleotide sequence or to the complement of the contiguous nucleotide sequence.

In another embodiment, the probe or primer is capable of selectively hybridizing to the contiguous nucleotide sequence or to the complement of the contiguous nucleotide sequence. Often, the probe or primer further comprises a label, e.g., a radioisotope, a fluorescent label, an enzyme label, an enzyme co-factor label, a magnetic label, a spin label, an epitope label.

The nucleic acid molecules of the invention, such as those described above, can be identified and isolated using standard molecular biology techniques well known to the skilled person. The amplified DNA can be labeled (e.g., radiolabeled) and used as a probe for screening a cDNA library derived from human cells. The cDNA can be derived from mRNA and contained in a suitable vector.

Corresponding clones can be isolated, DNA can obtained following in vivo excision, and the cloned insert can be sequenced in either or both orientations by art- recognized methods to identify the correct reading frame encoding a polypeptide of the appropriate molecular weight. Using these or similar methods, the polypeptide and the DNA encoding the polypeptide can be isolated, sequenced and further characterized.

In general, the isolated nucleic acid sequences of the invention can be used as molecular weight markers on Southern gels, and as chromosome markers that are labeled to map related gene positions. The nucleic acid sequences can also be used to compare with endogenous DNA sequences in patients to identify cerebral malaria or susceptibility to cerebral malaria, and as probes, such as to hybridize and discover related DNA sequences or to subtract out known sequences from a sample (e.g., subtractive

hybridization).

The nucleic acid sequences can further be used to derive primers for genetic fingerprinting, to raise anti-polypeptide antibodies using immunization techniques, and/or as an antigen to raise anti-DNA antibodies or elicit immune responses. Treatment of cerebral malaria

The present invention also relates to the treatment of cerebral malaria. As the invention defines a pathogenic mechanism in CM any treatment modality, e.g. drugs, radiation, cell depletion etc.,, that interferes with eosinophil presence, number and function/activation become significant novel treatments of CM as a consequence of this invention.,

The inventors have surprisingly found that cerebral malaria has a link to eosinophiles and in particular to ECP and that anti-inflammatory agents such as systemic glucocorticoids (prednisone), tyrosine kinase inhibitors, acting to reduce eosinophile inflamation, inhibit eosinophile migration/homing to the relevant tissue and reduce eosinophile activation and the subsequent release of ECP. It is also anticipated that anti-ECP Ig can be used in the treatment of cerebral malaria. Other therapeutic modalities, such as the use of silencing or activating nucleotides (siRNA and miRNA - and synthetic homologs thereof may also become treatment options as a consequence of the present invention. Drugs that indirectly interfere with eosinophil function, such as recombinant cyto- and chemokines, may also become treatment options of CM. Due to the fact that a large number of drugs - more or less specifically and directly - interfere with eosinophile function it must be expected that the present invention will result in a number of drugs with completely different indications becoming potential agents for treatment of CM.

Thus relates one aspect of the present invention to the use of adrenal cortex steroids in the treatment of cerebral malaria in infants.

Other means of treatment include any type of medication or supplementary that has direct or indirect influence on the number or activity of eosinophiles and the subsequent release of ECP. Figure legends

Figure 1.

Location of the studied SNPs in NASE3, linkage disequilibrium (LD) patterns and haplotype association analysis. A) A shematic of the region of the RNASE 3 gene (NM_002935.2) and LD plot of the respective SNPs visualised using Haploview v4.2. At the top of the LD plot, the two exons are shown with the intron joining them. Untranslated regions of the exons are indicated by shadings. The LD plot shows pairwise r2 values (xlOO) given in the squares for each comparison between the SNPs. White squares represent r2 values equal to 0. Different shades of grey represent r2 values between 0 and 1. B) Haplotype associations with susceptibility to cerebral malaria (CM) compared to uncomplicated malaria (UM). Odds ratio (OR) and 95% confidence intervals (CI) were determined using multivariate logistic regression controlling for age and gender. The reference groups in the multivariate logistic regression analyses were those without the respective haplotypes. Haplotype 3 (GGA) was significanlty associated with susceptibility to CM.

Figure 2.

Association of c.371G>C genotypes with coma score in the entire patient population studied. Coma score distribution among individuals with 371 (GG, GC and CC) genotypes is shown. Comparisons that yielded statistical significance are indicated with horizontal lines linking the respective groups at the top of the plots with the p - value stated. P-values were determined by Mann Whitney test and horizontal lines within plots represent the median of the distribution. Figure 3.

RNASE3 haplotypes, c.371G>C genotypes and alleles distribution in malaria endemic (Ghanaian) versus non-malaria (Danish) populations. A) Distribution of haplotypes defined by the three SNPs (rs2073342, rs2233860 and rs8019343) as from block 1 in Figure 1A in the two populations. B) c.371G>C genotype distribution in all Ghanaian subjects compared to Danes. C) c.371G>C allelic distribution in the two populations.

Figure 4.

Tajima's D index . Sliding window analysis of Tajima's D index across the

Ghanaian (A) and Danish (B) RNASE3 sequences. Bar at the top shows the sequence region analyzed (nucleotides 1-1183) and the positions of the SNP. Window sizes used were 100 bp with a 25-bp step size. Windows that gave a significant D valuep<0.10), are indicated above the relevant window midpoint by a hash (#).

Example 1 - Polymorphisms in the RNASE3 gene are associated with

susceptibility to cerebral malaria in Ghanaian children

METHODS

Here, we report the results of a hospital based malaria study with population genetic data which supports the role of frequency dependent selection genes involved in resistance or susceptibility to infectious disease.

Malaria patients

The study enrolled children from 0.5 to 13 years of age visiting the Child Health Department of the Korle-Bu Teaching Hospital (KBTH), Accra, for medical care during the study period. The KBTH is the major referral hospital serving the southern part of Ghana originally inhabited largely by the Akan and Ga-Adangbe ethnic groups. However, infrastructural development and better living conditions over the decades have made Accra a converging point for people of diverse ethnic and geographical origins creating a high degree of admixture due to free intermarriages between the different ethnicities. Thus, we did not expect population stratification to significantly influence our genotypic frequencies. Furthermore, a study involving individuals from the same study area found no significant population substructure between the Akan and Ga-Adangbe ethnic groups using data from 372 autosomal microsatellite loci [30] . The clinical and demographic characteristics of the patients were as described elsewhere [31], with some modifications (Table 1). Briefly, all patients had to be febrile (>37.5°C measured within 24 hours of admission), sickle-cell negative and slide positive for asexual Plasmodium falciparum parasitaemia with at least some other sign of malaria such as vomiting, diarrhoea, or malaise to be included in the study. Altogether, 206 children were enrolled in the study, 45 of which were CM cases, 56 had SA and 105 were UM cases. CM was defined by unarousable coma (Blantyre coma score of <3) [32], with no other attributable cause of cerebral dysfunction; SA was defined by measured haemoglobin (Hb) levels of <5.0g/dl with no other attributable cause of anaemia and patients were fully conscious. The criteria used to define UM was same as SA but with a measured Hb of >6.0g/dl. Thus, this group also includes individuals with mild anaemia. The procedure used for determining parasitological and haematological parameters have been described elsewhere [31]. Buffy coats were obtained from blood samples taken from patients and stored at -80°C for DNA purification.

Table 1. Clinical and demographic characteristics of patients with severe malaria and uncomplicated malaria

The number of patients in the various disease categories: cerebral malaria (CM), severe malaria anaemia (SA) and uncomplicated malaria (UM) is shown together with haemoglobin (Hb) levels, age stratification and parasite density distribution. ^TMedian values with minimum and maximum values (in parenthesis). Values that are statistically significant when compared to UM, ****p < 0.0001, **p=0.0014, *p=0.0222.

Danish donors In order to allow for inter-population comparison and analysis, the study also used archived DNA samples purified from a panel of anonymous healthy Danish blood donors (n=203) obtained for control purposes according to Danish Ethical guidelines.

RNASE 3 gene sequencing

Genomic DNA was purified from buffy coat samples using the Maxwell®16 system (Promega, Madison, USA). An approximately 1.5-kb fragment encompassing the promoter region and the 3'UTR was amplified using the M13 (in lower case) tagged sense primer (5'- tgtaaaacgacggccagtG CCTG CCAG AG G AC AGTTAT- 3 ') and the antisense primer (5'- caggaaacagctatgaccGGAGGAGTCAGTGGATGGAA-3') (Taq Copenhagen, Denmark) in a 25.0μΙ reaction containing 40ng genomic DNA, lOmM of each primer, 1.25mM of each dNTP, 1 unit of HotStarTaq® DNA polymerase (Biomol, Germany) and the corresponding 10X HotStar reaction buffer. The PCR conditions were 95°C activation for 15 minutes, 35 cycles of 95°C for 30 sec, 63°C for 45 sec, and 72°C for 1.40 s, with a final extension at 72°C for 10 min. The product was then purified using exonuclease 1 and alkaline

phosphatase (ExoSap) reaction and sequenced from both ends with M13 primers the internal sense (5'-CAGTTCTCACAGGAGCCACA-3') and antisense (5'- AGGTGAACTGGAACCACAGG-3') primers (Taq Copenhagen, Denmark) in a

BigDye® Terminator v3.1 (Applied Biosystem, UK) reaction and analysed with the ABI 3730 DNA Analyser (Applied Biosystem, UK). No significant difference was observed when allele frequencies obtained in the hospital based study were compared with those in a longitudinal malaria cohort (n=640) study of children (aged 1 to 13 years old) in which less than 10% hospitalisation was recorded (Adu et al., unpublished data). In order to verify the genotyping data a second PCR was performed in which an approximately 1.7kb product encompassing the original primer sites was amplified from 96 randomly selected individuals, using the upstream sense (5'-tgtaaaacgacggccagtTCCTAACTACTATGCCTGCCTT-3') and the downstream antisense (5'-caggaaacagctatgaccTCAGCTGATTCACTGCAGCTC- 3') primers (Taq Copenhagen, Denmark). Nucleotide sequencing of the resulting amplicons were in 100% agreement with results obtained for these 96 individuals in the original screen.

Statistical analysis

The clinical and demographic characteristics of the CM and SA groups were compared to that of the UM group using the Mann Whitney test as implemented in GraphPad Prism v.5.00 (GraphPad Software Inc. La Jolla, CA, USA). Similarly, coma score distribution among the c.371G>C genotypes was compared by the Mann Whitney test. Differences were considered to be statistically significant if p<0.05. Haploview v. 4.2 (http://www.broadinstitute.org/haploview) [34] was used to estimate deviations of genotype frequencies from Hardy-Weinberg equilibrium (HWE), visualize linkage disequilibrium (LD) patterns with pairwise r2 values and define haplotypes. The adjusted p-value for statistically significant differences in the HWE estimations was p<0.0125 (correction factor=4) according to the Boferroni correction for multiple comparisons. RNASE3 allele and haplotype frequencies in CM and UM patients were compared by multiple logistic regression and adjusted for age and gender using S-Plus 2000 (Insightful Corporation, Seattle WA). There was no statistically significant difference in the allele distribution between SA and UM patients, hence the SA group was excluded from haplotype association analysis. All the possible haplotypes present in the population for the three closely linked SNPs were included in the haplotype association studies. Comparison of haplotype distribution between the Danish and Ghanaian populations was by the chi-square (χ2) test. DnaSP v. 5.10

(http://www.ub.edu/dnasp/) [35] was used to estimate Tajima's D to study evidence of selection on the c.371G>C SNP in malaria versus non-malaria endemic populations. To estimate the extent of divergence with respect to this polymorphism in malaria endemic versus malaria non-endemic populations, allele frequencies in our study populations were compared to that of four populations namely, the YRI: Yoruba in Ibadan, Nigeria; CEU: (Utah residents with northern and western European ancestry from the Centre d'Etude du Polymorphisme Humain [CEPH]); CHB: Han Chinese in Beijing, China and JPT: Japanese in Tokyo, Japan obtained from the International HapMap database (http://hapmap.ncbi.nlm.nih.gov/cgiperl/snp details_phase3?name=rs2073342& source= hapmap 28_B36&tmpl=snp_details_phase3). Pairwise FST distances [36] were calculated for all 6 populations using the POPTREE2 software [37]

RESULTS RNASE3 nucleotide sequence variation in Ghanaians

The RNASE3 gene and flanking regions were amplified from 206 Ghanaian children enrolled in a hospital based malaria study. Nucleotide sequencing identified a total of six SNPs, one of which was non-synonymous (c.371G>C; P.R124T) corresponding to the previously described arginine / threonine polymorphism which abolishes the cytotoxicity of ECP [23] . Of the remaining SNPs, two (C-5-430T, 0-5-380A) are located in the intron and two

(c.*16C>G, *94A>T), are located in the 3'UTR (Table 2). The last SNP, *295A>G is located downstream the 3'UTR and was excluded from association analysis as was the c.-5-38C>A polymorphism since the -5-38A-allele was only present in a single individual. The remaining SNPs were all in Hardy-Weinberg disequilibrium (Table 2) except the *295A>G (data not shown).

Table 2. Allele frequencies and Hardy-Weinberg estimations in the CM and UM study populations

a values are statistically significant if p<0.0125 (Bonferroni corrected significance threshold)

Single marker and haplotype association study

An association study was carried out to assess the significance of the four SNPs in CM (n=45) and SA (n=56) cases, respectively. The two case groups were compared with the non-severe control group comprising children suffering from UM (n=105) in a hierarchical multivariate logistic regression model adjusting for age and gender. The 371G allele was found to be significantly associated with CM (p= 0.00945, OR=2.29, 95% CI= 1.22-4.32) (Table 3) but not with SA (data not shown). Linkage disequilibrium analysis demonstrated significant linkage between the c.371G>C, c.*16C>G, and c.*94A>T SNPs (Figure 1A), and haplotype analysis was therefore carried out to determine their association with CM (Figure IB). The haplotype combination 371G/*16G/*94A was strongly associated with susceptibility to CM (p =0.000913, OR=4.14, 95% CI = 1.79-9.56) (Figure IB), thus, defining a risk haplotype (GGA). The other haplotypes showed no significant association with CM.

Table 3. Single marker association with cerebral malaria and uncomplicated malaria

The frequency of the associated allele in uncomplicated malaria (UM) and CM is shown. Odds Ratios (OR) and 95% confidence intervals (CI) were determined 15 using multivariate logistic regression controlling for age and gender. The reference groups in the multivariate logistic regression analyses were those without the respective alleles.

Association of c.371G>C genotypes with disease

To further assess the importance of the genotype forms of the c.371G>C SNP in 20 CM, the coma score was plotted for each of the three genotypes. The mean coma score in individuals carrying the 371GG genotype was significantly lower as compared to the 371CC (Mann Whitney test, p=0.0022) and the 371CG (Mann Whitney test, p=0.0394) genotypes respectively (Figure 2). On the other hand, levels of parasitaemia did not differ significantly between the three genotypes (data not shown). Population differences

The above analyses suggest a strong association between the 371GG genotype and CM. It was therefore of interest to investigate the differences in the genotype distribution of c.371G>C between populations from malaria versus non-malaria endemic regions. RNASE3 and flanking regions were amplified from Danish blood donors (n=203). Sequencing analysis identified all the SNPs found in the

Ghanaian samples plus a novel SNP (C.2140T) in exon 2 resulting in an arginine to cysteine polymorphism at position 72. In contrast to the Ghanaian samples, all SNPs in the Danish samples were in Hardy-Weinberg equilibrium (p>0.0125). There was a statistically significant difference in the distribution of both the 371GC genotypes (p<0.0001, x2-test) and alleles (p<0.0001, x2-test) in the Ghanaian and Danish populations (Figure 3B and 3C). We also compared the c.371G>C allele distributions observed in our study to those reported for four populations: YRI, CEU, CHB and JPT in the International HapMap project database. There was a higher 371C frequency in those individuals from malaria endemic regions

(Ghanaians and YRI) whereas the 371G is the predominant allele found in individuals from malaria non-endemic populations (CEU, CHB and JPT) (Figure 3C). Furthermore, pairwise FST calculations showed a significant genetic divergence between individuals from the malaria endemic (Ghanaians and YRI) and malaria non-endemic populations (Danes, CEU, CHB and JPT)

(0.2040<FST>0.3520) (Table 4). On the other hand, the data showed no significant evidence of population divergence between the Ghanaians and the YRI individuals and among the Danes, CEU, CHB and JPT (Table 4). The distribution of the haplotypes defined by the markers, rs2073342, rs2233860 and rs8019343 were also significantly different (p=0.0010, x2-test for trends) between the populations. Interestingly, the most predominant haplotype in the Danish population was the CM-risk associated haplotype seen in the Ghanaian association studies (Figure 3A), suggesting that malaria exerts a strong selection pressure on ECP. The c.371G>C genotype is subject to selection pressure

To investigate the selective pressures operating on the RNASE3, we performed a sliding-window analysis of Tajima's D values across the aligned RNASE3 sequences (Figure 4). This analysis identified a region in RNASE3 (nucleotides 776-950) that exhibited a nearly significant departure from 0 in the Ghanaians population (D= 1.80; p<0.1), indicating that frequency-dependent selection was operating at the c.371G>C SIMP.

Table 4: Pairwise genetic distances between 6 populations compared.

F_ST distance (Latter et a/., 1972). * Allele frequency data for the c.371G>C polymorphism for these populations was retrieved from the International HapMap database

(http://hapmap. ncbi. nlm. nih.gov/cgi- perl/snp_details_phase3?name=rs2073342&source=hapmap

28_B36&tmpl=snp_details_phase3). YRI: Yoruba in Ibadan, Nigeria; CEU : (Utah residents with northern and western European ancestry from the Centre d'Etude du Polymorphisme Humain [CEPH]); CHB: Han Chinese from Beijing, China and JPT: Japanese

DISCUSSION

The findings reported here, together with the observation that levels of ECP increase during acute malaria [20] strongly suggest a role for these cells in CM pathogenesis.

Firstly, we have shown that the cytotoxic G-allele of the previously described functional (c.371G>C) polymorphism (rs2073342) of RNASE3 predisposes to CM in Ghanaian children and we have identified a risk associated haplotype, GGA including this G-allele together with the G-allele of rs2233860 which is associated with increased levels of soluble ECP, and the A-allele of rs8019343. Secondly, the Tajima's D test showed a positive departure from neutrality in the RNASE3 region encompassing the functional c.371G>C polymorphism. Frequency dependent selection may be a plausible explanation for this phenomenon. Mutations that arise in the population and have the potential to eliminate the toxic activity of ECP may have a survival advantage, leading to their increased frequency in the population. Thirdly, the frequency of the G-allele of the (c.371G>C) polymorphism in the African populations (Ghanaians and individuals from Yoruba in Ibadan, Nigeria) is significantly reduced compared to non-malarial population (Danes and CEU; CHB and JPT). This finding is in line with the evidence of diversifying selection on the c.371G>C polymorphism and suggests that malaria exerts a strong selection pressure on ECP. Fourthly, c.371G>C and the other SNPs forming the risk haplotype are all in Hardy-Weinberg disequilibrium in the African samples whereas they are in Hardy-Weinberg equilibrium in the Danish samples. Taken together, these findings have led us to propose that ECP and eosinophils are involved in CM pathogenesis. Cerebral malaria pathogenesis is still not clearly understood, however, emerging evidence suggests the possibility that IgE- mediated allergic response or atopy may be risk factors. The chromosomal regions 5pl5-pl3 and 13ql3-q22 associated with P. falciparum clinical malaria attacks and 12q21-q23 associated with the maximum asymptomatic parasitaemia [38] have also been linked to asthma/atopy in previous studies [39-41], In addition, the chromosome 5q31 locus which has been linked to blood P. falciparum parasites density [42-43] also contains the genes encoding the cytokines IL-4, IL- 5, and IL-13, and these have been associated with inflammatory diseases including asthma [44-45]. IL-5 is a potent activator of eosinophils [46] and together with the elevated IgE levels typically associated with CM [47] might amplify eosinophil-mediated cytotoxicity by ECP against both parasite and host cells possibly in an FcsR-mediated mechanism [48]. Studies have shown that ECP efficiently kills some parasites including P.

falciparum, in vitro [21,49], but the toxic properties of ECP may also lead to tissue damage within the host in conditions such as asthma [50]. The toxic property of ECP is also well documented on several human cell lines, in vitro, including epithelial cells [23,51-52]. Although the mechanisms of its cytotoxicity are not fully understood, it has been shown that the 371CC genotype encodes a non-toxic form of ECP [52]. This has been demonstrated using both native ECP and recombinant ECP expressed in several expression systems [23,51-52] . The most likely explanation for this change in cytotoxic activity could be that the substitution of arginine with threonine creates a potentially new glycosylation site in ECP. Indeed Trulson et al [23] demonstrated that the noncytotoxic rECP^124thr regained the cytotoxic activity after deglycosylation. Kurtzhals and colleagues observed that ECP plasma levels increased in acute P. falciparum infection particularly in CM patients but peripheral eosinophil counts were curiously low. Consequently, they speculated that the low eosinophil counts in these patients may be due to eosinophil destruction or tissue sequestration rather than a decrease in their production [20]. Here we propose that peripheral eosinophils migrate to the post capillary venules during CM where they adhere to intracellular adhesion molecule (ICAM)-l or other endothelial cellular adhesion molecules (eCAMs) which become up-regulated during CM [53] . Upon binding to (ICAM)-l through their Beta2 (CD18) integrins, eosinophils release ECP [54] which increases the expression of (ICAM)-l as demonstrated in nasal epithelia cells [55] suggesting that it might mediate a positive feed-back mechanism. ECP may also be released from eosinophils through stimulation with platelet activating factor (PAF) [56] which is also a potent stimulator of eosinophil adherence [57] .

Platelets have recently been proposed to play a central role in the activation of pathogenic inflammation [58], thus PAF might indirectly mediate inflammation through stimulation of eosinophil adherence to the capillaries and the subsequent release of ECP. Knowing that ECP is cytotoxic and may induce necrosis in human primary epithelial cells, we suggest that the release of ECP, together with pro- inflammatory cytokines, may lead to epithelia damage of the capillaries and possibly vascular leakage. Petechial hemorrhaging into the brain is considered a hallmark of CM [59], although the exact contribution of vascular leakage to CM is not known [60] . As the blood-brain barrier becomes compromised during CM, ECP and other plasma proteins may leak into the interstitial space. ECP has been shown to elicit the Gordon phenomenon when injected intracathecally into rabbits, mainly due to its profound neurotoxic effect on the cerebellum compared to other nervous tissue in animal models [19]. Thus, ECP may account for some of the typical cerebellar symptoms observed in CM. In the present study, 371GG which codes for the more cytotoxic and ancestral form of ECP [61] was significantly associated with both CM and low Blantyre coma score. The Blantyre coma score is defined by; (best motor response score) + (best verbal response score) + (eye movement score) [32]; parameters which are largely under the control of the cerebellum [62], suggesting that lower coma scores which typify poor CM prognosis may be a consequence of cerebellar dysfunction due to ECP neurotoxicity. This implies, both ECP

neurotoxicity as well as cytotoxicity are either stronger in the native protein (ECP^124arg) than in the mutant form ECP^124thr or fully lost in the mutant since the later was not associated with CM and was only occasionally associated with lower coma scores in the present study.

In the present study we found a large difference between African and non-African samples as exemplified by the distribution of the c.371G>C genotype. The 371CC genotype was dominant in the Ghanaian samples compared to Danish samples (56% vs. 12%) while the opposite was observed for the 371GG genotype (15% vs 53%). This finding is consistent with data for four populations from the

International HapMap database and also with other studies where this SNP was quantified in samples from Uganda, Sudan, and Sweden [25]. Likewise, the CM associated GGA haplotype was the most common haplotype found in 69% of the Danish samples, whereas it was only found in 18% of the Ghanaian samples. Thus, the high prevalence of the CM associated allele and haplotype in the

European population implies a greater than 3 fold likelihood of a European developing CM compared to a Ghanaian if an infection with P. falciparum is not treated timely. Conversely, the CGA haplotype was quite uncommon in the Danish samples, but found among 42% of the Ghanaian samples. The most likely explanation for these differences is natural selection e.g. frequency-dependent selection on the c.371G>C SNP. Eriksen et al hypothesized that the predominance of the 371CC genotype is due to positive selection exerted by Schistosoma mansoni [25] . However, mortality due to S. Mansoni is usually more common in older individuals whereas mortality due to CM is more common in children than in adults. Thus, P. falciparum malaria is a more likely cause for the predominance of the C-allele in African populations than S. Mansoni. In fact, as the 371CC genotype codes for the non-cytotoxic form of ECP, malaria-endemic populations may be relatively more susceptible to other parasitic infections such as S. Mansoni where cytotoxic ECP play a protective role [25]. Thus, ECP may represent an example where one parasite is selecting for a protein variant which makes the same population more susceptible to other parasites. The relatively small sample size might be a limitation in our study. However, allele frequencies for the two populations genotyped in our study are comparable to what has been reported for individuals from similar geographical backgrounds [25] and data from the

International HapMap database. Furthermore, the strong associations we see in our study between the risk allele and haplotype with CM as well as lower coma score association with the 371GG genotype all support our hypothesis of malaria selection for the non-cytotoxic ECP. Further studies involving larger sample size would be necessary to investigate these findings not just in the Ghanaian population but also in other malaria endemic populations. In conclusion, we have identified an association between the 371G allele of RNASE3 and susceptibility to CM and a risk associated haplotype GGA defined by the markers: rs2073342 (G- allele), rs2233860 (G-allele) and rs8019343 (A-allele) respectively. We have also shown that the RNASE3 371GG genotype is subject to selection pressure and we suggest that the predominance of the 371C allele results from a strong selection pressure by P. falciparum malaria in endemic populations. Collectively, our results suggest a hitherto unrecognized role for eosinophils in CM pathogenesis.

References

1. Miller LH, Good MF, Milon G (1994) Malaria pathogenesis. Science 264: 1878-1883.

2. van der Heyde HC, Nolan J, Combes V, Gramaglia I, Grau GE (2006) A unified hypothesis for the genesis of cerebral malaria: sequestration, inflammation and hemostasis leading to microcirculatory dysfunction. Trends Parasitol 22: 503-508.

3. World Health O (2010) World Malaria Report 2010. Geneva, Switzerland : WHO Press; . 1-238 p.

4. Marchiafava E, Bigmani A (1894) Sulle Febbri Estivo Aumnali. . ELoesscher.

5. Maegraith B (1948) Pathological processes in malaria. TransRSocTropMedHyg 41 : 687-704.

6. Mazier D, Nitcheu J, Idrissa-Boubou M (2000) Cerebral malaria and immunogenetics. Parasite Immunol 22: 613-623.

7. McGuire W, Hill AV, Allsopp CE, Greenwood BM, Kwiatkowski D (1994) Variation in the TNF-alpha promoter region associated with susceptibility to cerebral malaria.

Nature 371 : 508-510.

8. Hananantachai H, Patarapotikul J, Looareesuwan S, Ohashi J, Naka I, et al. (2001) Lack of association of -308A/G TNFA promoter and 196R/M TNFR2 polymorphisms with disease severity in Thai adult malaria patients. AmJMedGenet 102: 391-392. 9. Hirayama K (2002) Genetic factors associated with development of cerebral malaria and fibrotic schistosomiasis. Korean JParasitol 40: 165-172.

10. Bellamy R, Kwiatkowski D, Hill AV (1998) Absence of an association between intercellular adhesion molecule 1, complement receptor 1 and interleukin 1 receptor antagonist gene polymorphisms and severe malaria in a West African population. TransRSocTropMedHyg 92: 312-316.

11. Gyan B, Goka B, Cvetkovic JT, Perlmann H, Lefvert AK, et al. (2002) Polymorphisms in interleukin-lbeta and interleukin-1 receptor antagonist genes and malaria in Ghanaian children. ScandJImmunol 56: 619-622.

12. Ohashi J, Naka I, Patarapotikul J, Hananantachai H, Looareesuwan S, et al. (2001) Absence of association between the allele coding methionine at position 29 in the N- terminal domain of ICAM-1 (ICAM-l(Kilifi)) and severe malaria in the northwest of Thailand. JpnJInfectDis 54: 114-116.

13. Naka I, Patarapotikul J, Hananantachai H, Tokunaga K, Tsuchiya N, et al. (2009) IFNGR1 polymorphisms in Thai malaria patients. InfectGenetEvol 9: 1406-1409.

14. Dhangadamajhi G, Mohapatra BN, Kar SK, Ranjit M (2009) Genetic variation in neuronal nitric oxide synthase (nNOS) gene and susceptibility to cerebral malaria in Indian adults. InfectGenetEvol 9: 908-911. 15. Dhangadamajhi G, Mohapatra BN, Kar SK, Ranjit M (2009) Endothelial nitric oxide synthase gene polymorphisms and Plasmodium falciparum infection in Indian adults. Infectlmmun 77 : 2943-2947.

16. Dhangadamajhi G, Mohapatra BN, Kar SK, Ranjit MR (2009) The CCTTT pentanucleotide microsatellite in iNOS promoter influences the clinical outcome in P. falciparum infection. Parasitoses 104: 1315-1320.

17. Neves JS, Weller PF (2009) Functional extracellular eosinophil granules: novel implications in eosinophil immunobiology. CurrOpinlmmunol 21 : 694-699.

18. Walsh GM (2001) Eosinophil granule proteins and their role in disease. CurrOpinHematol 8: 28-33.

19. Fredens K, Dahl R, Venge P (1982) The Gordon phenomenon induced by the eosinophil cationic protein and eosinophil protein X. JAIIergy Clinlmmunol 70: 361- 366.

20. Kurtzhals JA, Reimert CM, Tette E, Dunyo SK, Koram KA, et al. (1998) Increased eosinophil activity in acute Plasmodium falciparum infection— association with cerebral malaria. ClinExpImmunol 112: 303-307.

21. Waters LS, Taverne J, Tai PC, Spry CJ, Targett GA, et al. (1987) Killing of Plasmodium falciparum by eosinophil secretory products. Infectlmmun 55 : 877-881.

22. Noguchi E, Iwama A, Takeda K, Takeda T, Kamioka M, et al. (2003) The promoter polymorphism in the eosinophil cationic protein gene and its influence on the serum eosinophil cationic protein level. AmJRespirCrit Care Med 167 : 180-184.

23. Trulson A, Bystrom J, Engstrom A, Larsson R, Venge P (2007) The functional heterogeneity of eosinophil cationic protein is determined by a gene polymorphism and post-translational modifications. ClinExpAllergy 37: 208-218.

24. Jonsson UB, Bystrom J, Stalenheim G, Venge P (2002) Polymorphism of the eosinophil cationic protein-gene is related to the expression of allergic symptoms. ClinExpAllergy 32: 1092-1095.

25. Eriksson J, Reimert CM, Kabatereine NB, Kazibwe F, Ireri E, et al. (2007) The 434(G>C) polymorphism within the coding sequence of Eosinophil Cationic Protein (ECP) correlates with the natural course of Schistosoma mansoni infection. IntJParasitol 37: 1359-1366.

26. Munthe-Kaas MC, Gerritsen J, Carlsen KH, Undlien D, Egeland T, et al. (2007) Eosinophil cationic protein (ECP) polymorphisms and association with asthma, s-ECP levels and related phenotypes. Allergy 62: 429-436.

27. Jonsson UB, Hakansson LD, Jogi R, Janson C, Venge P (2010) Associations of ECP (eosinophil cationic protein)-gene polymorphisms to allergy, asthma, smoke habits and lung function in two Estonian and Swedish sub cohorts of the ECRHS II study. BMC Pulm Med 10: 36.

28. Kang I, An XH, Oh YK, Lee SH, Jung HM, et al. (2010) Identification of polymorphisms in the RNase3 gene and the association with allergic rhinitis. EurArchOtorhinolaryngol 267: 391-395. 29. Kim YJ, Kumaraswami V, Choi E, Mu J, Follmann DA, et al. (2005) Genetic polymorphisms of eosinophil-derived neurotoxin and eosinophil cationic protein in tropical pulmonary eosinophilia. AmJTropMedHyg 73 : 125-130.

30. Adeyemo AA, Chen G, Chen Y, Rotimi C (2005) Genetic structure in four West African population groups. BMC Genet 6: 38.

31. Kusi KA, Gyan BA, Goka BQ, Dodoo D, Obeng-Adjei G, et al. (2008) Levels of soluble CD163 and severity of malaria in children in Ghana. ClinVaccine Immunol 15 : 1456-1460.

32. Molyneux ME, Taylor TE, Wirima JJ, Borgstein A (1989) Clinical features and prognostic indicators in paediatric cerebral malaria : a study of 131 comatose Malawian children. QJMed 71 : 441-459.

33. Pompanon F, Bonin A, Bellemain E, Taberlet P (2005) Genotyping errors: causes, consequences and solutions. Nat Rev Genet 6: 847-859.

34. Barrett JC, Fry B, Mailer J, Daly MJ (2005) Haploview: analysis and visualization of LD and haplotype maps. Bioinformatics 21 : 263-265.

35. Librado P, Rozas J (2009) DnaSP v5: a software for comprehensive analysis of DNA polymorphism data. Bioinformatics 25: 1451-1452.

36. Latter BD (1972) Selection in finite populations with multiple alleles. 3. Genetic divergence with centripetal selection and mutation. Genetics 70: 475-490.

37. Takezaki N, Nei M, Tamura K (2010) POPTREE2: Software for constructing population trees from allele frequency data and computing other population statistics with Windows interface. Mol Biol Evol 27: 747-752.

38. Sakuntabhai A, Ndiaye R, Casademont I, Peerapittayamongkol C, Rogier C, et al. (2008) Genetic determination and linkage mapping of Plasmodium falciparum malaria related traits in Senegal. PLoS One 3: e2000.

39. Iyengar SK, Jacobs KB, Palmer U (2001) Improved evidence for linkage on 6p and 5p with retrospective pooling of data from three asthma genome screens. Genet Epidemiol 21 Suppl 1 : S130-135.

40. Jang N, Stewart G, Jones G (2005) Polymorphisms within the PHF11 gene at chromosome 13ql4 are associated with childhood atopic dermatitis. Genes Immun 6:

262-264.

41. Kurz T, Hoffjan S, Hayes MG, Schneider D, Nicolae R, et al. (2006) Fine mapping and positional candidate studies on chromosome 5pl3 identify multiple asthma susceptibility loci. J Allergy Clin Immunol 118: 396-402.

42. Flori L, Kumulungui B, Aucan C, Esnault C, Traore AS, et al. (2003) Linkage and association between Plasmodium falciparum blood infection levels and chromosome 5q31-q33. Genes Immun 4: 265-268.

43. Garcia A, Marquet S, Bucheton B, Hillaire D, Cot M, et al. (1998) Linkage analysis of blood Plasmodium falciparum levels: interest of the 5q31-q33 chromosome region. Am J Trop Med Hyg 58: 705-709. 44. Holloway 3W, Lonjou C, Beghe B, Peng Q, Gaunt TR, et al. (2001) Linkage analysis of the 5q31-33 candidate region for asthma in 240 UK families. Genes Immun 2: 20- 24.

45. Xu 3, Meyers DA, Ober C, Blumenthal MN, Mellen B, et al. (2001) Genomewide screen and identification of gene-gene interactions for asthma-susceptibility loci in three U.S. populations: collaborative study on the genetics of asthma. Am 3 Hum Genet 68: 1437-1446.

46. Sanderson C3 (1997) Interleukin-5: an eosinophil growth and activation factor. Dev Biol Stand 69: 23-29.

47. Perlmann H, Helmby H, Hagstedt M, Carlson 3, Larsson PH, et al. (1994) IgE elevation and IgE anti-malarial antibodies in Plasmodium falciparum malaria: association of high IgE levels with cerebral malaria. Clin Exp Immunol 97 : 284-292.

48. Gounni AS, Lamkhioued B, Ochiai K, Tanaka Y, Delaporte E, et al. (1994) High- affinity IgE receptor on eosinophils is involved in defence against parasites. Nature 367: 183-186.

49. McLaren DJ, McKean JR, Olsson I, Venges P, Kay AB (1981) Morphological studies on the killing of schistosomula of Schistosoma mansoni by human eosinophil and neutrophil cationic proteins in vitro. Parasite Immunol 3: 359-373.

50. Trautmann A, Schmid-Grendelmeier P, Kruger K, Crameri R, Akdis M, et al. (2002) T cells and eosinophils cooperate in the induction of bronchial epithelial cell apoptosis in asthma. JAIIergy Clinlmmunol 109: 329-337.

51. Maeda T, Kitazoe M, Tada H, de Llorens R, Salomon DS, et al. (2002) Growth inhibition of mammalian cells by eosinophil cationic protein. EurJBiochem 269: 307- 316.

52. Rubin 3, Zagai U, Blom K, Trulson A, Engstrom A, et al. (2009) The coding ECP 434(G>C) gene polymorphism determines the cytotoxicity of ECP but has minor effects on fibroblast-mediated gel contraction and no effect on RNase activity. 3Immunol 183: 445-451.

53. Turner GD, Morrison H, 3ones M, Davis TM, Looareesuwan S, et al. (1994) An immunohistochemical study of the pathology of fatal malaria. Evidence for widespread endothelial activation and a potential role for intercellular adhesion molecule-1 in cerebral sequestration. Am3Pathol 145: 1057-1069.

54. Takafuji S, Miyakuni Y, Nakagawa T, Shoji S, Murata S, et al. (2000) Effects of human lung fibroblasts on eosinophil degranulation. Allergy 55: 1170-1178.

55. Altman LC, Ayars GH, Baker C, Luchtel DL (1993) Cytokines and eosinophil- derived cationic proteins upregulate intercellular adhesion molecule-1 on human nasal epithelial cells. 3Allergy Clinlmmunol 92: 527-536.

56. Takafuji S, Tadokoro K, Ito K, Nakagawa T (1998) Release of granule proteins from human eosinophils stimulated with mast-cell mediators. Allergy 53: 951-956. 57. Kimani G, Tonnesen MG, Henson PM (1988) Stimulation of eosinophil adherence to human vascular endothelial cells in vitro by platelet-activating factor. 3Immunol 140: 3161-3166. 58. Idro R, Jenkins NE, Newton CR (2005) Pathogenesis, clinical features, and neurological outcome of cerebral malaria. Lancet Neurol 4: 827-840.

59. Turner G (1997) Cerebral malaria. Brain Pathol 7 : 569-582.

60. Brown HC, Chau TT, Mai NT, Day NP, Sinh DX, et al. (2000) Blood-brain barrier function in cerebral malaria and CNS infections in Vietnam. Neurology 55 : 104-111.

61. Zhang J, Rosenberg HF (2000) Sequence variation at two eosinophil-associated ribonuclease loci in humans. Genetics 156: 1949-1958.

62. Doya K (2000) Complementary roles of basal ganglia and cerebellum in learning and motor control. CurrOpinNeurobiol 10: 732-739.

Claims

1. An in vitro method for diagnosing a genetic predisposition or susceptibility for acquiring cerebral malaria in a mammal, comprising detecting in a sample obtained from said mammal at least one single nucleotide polymorphism (SNP) in a nucleic acid or fragment thereof, wherein said at least one SNP is selected from the group consisting of the rs2073342, rs2233860, rs8019343, and SNPs in linkage disequilibrium therewith, whereby at least one allele-specific haplotype is determined.

2. The method according to any of claims 1, wherein said at least one SNP is one SNP, such as 2 SNPs, such as 3 SNPs.

3. The method according to any of claims 1-2, wherein said at least one gene is one gene, such as 2 genes, such as 4 genes, such as 6 genes, such as 8 genes, such as 10 genes, such as 12 genes, such as 14 genes, such as 16 genes, such as 18 genes, such as 20 genes.

4. The method according to any of claims 1-3, wherein the mammal is a human.

5. The method according to any of claims 1-4, wherein said nucleic acid is a DNA, genomic DNA, RNA, cDNA, hnRNA and/or mRNA.

6. The method according to any of claims 1-5, wherein said detection is accomplished by sequencing, mini-sequencing, hybridization, restriction fragment analysis, oligonucleotide ligation assay, allele specific PCR.

7. A diagnostic kit comprising at least one combination of probes for detecting at least one of the haplotypes of the present invention.

8. Use of Adrenal cortical steroid, inhibitors of tyrosinkinase and anti-ECP Ig for producing a medicament for the treatment of cerebral malaria.

9. Treatment of cerebral malaria with adrenal cortical steroid, inhibitors of tyrosinkinase or anti-ECP Ig.