WO2007025989A2 - Nucleic acid variants in the toll like receptor genes associated with altered innate immunity - Google Patents

Nucleic acid variants in the toll like receptor genes associated with altered innate immunity Download PDF

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WO2007025989A2
WO2007025989A2 PCT/EP2006/065821 EP2006065821W WO2007025989A2 WO 2007025989 A2 WO2007025989 A2 WO 2007025989A2 EP 2006065821 W EP2006065821 W EP 2006065821W WO 2007025989 A2 WO2007025989 A2 WO 2007025989A2
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seq
identified
gene
nucleic acid
disease
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WO2007025989A3 (en
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Lieve Nuytinck
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Innogenetics N.V.
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Publication of WO2007025989A3 publication Critical patent/WO2007025989A3/en

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/156—Polymorphic or mutational markers

Definitions

  • the present invention relates to a method and kit for identifying a subject at risk of, or having, an indication associated with altered innate immunity.
  • the present invention is based on the determination of the Toll Like Receptor (TLR) genotype and/or serum level.
  • TLR Toll Like Receptor
  • Immunity to infection is mediated by two systems, the acquired (or adaptive) immune system and the innate (or natural) immune system.
  • the innate immunity system is an evolutionary ancient form of immunity and offers the main resistance to microbial pathogens within the first minutes, hours or days of an infection (Fujita et al, 2002).
  • Innate immunity recognition is mediated by germ-line-encoded receptors, which means that the specificity of each receptor is genetically predetermined.
  • the strategy of the innate immune response may not be to recognize every possible antigen, but rather to focus on a few, highly conserved structures (patterns) present in large groups of microorganisms. These structures are referred to as pathogen-associated molecular patterns (PAMPs), and the receptors of the innate immune system that evolved to recognize them are called pattern-recognition receptors (PRR) or pattern-recognition molecules (PRMs).
  • PAMPs can be protein, lipid, nucleic acid, and carbohydrate (Lu et al., 2002).
  • PRRs As soon as the PRRs identify the corresponding predetermined carbohydrate pattern on a pathogen, they immediately trigger effector cells to destroy the invading microorganism, rather than after having to undergo a proliferative cycle, as is the case for the time-delayed adaptive immune response.
  • PRRs can be divided into three classes: signaling, endocytic, and secreted (Medzhitov R. et al., 2004).
  • Innate immunity refers to antigen-nonspecific defense mechanisms that a host uses immediately or within several hours after exposure to an antigen. This is the immunity one is born with and is the initial response by the body to eliminate microbes and prevent infection.
  • TLRs Toll like receptors
  • PRRs PRRs and their function is the recognition of pathogens and the activation of immune cell responses directed against those pathogens.
  • TLRs function as a dimer. Though most TLRs appear to function as homodimers, TLR2 forms heterodimers with TLRl or TLR6, each dimer having a different ligand specificity. TLRs may also depend on other co-activators for full ligand sensitivity, such as in the case of TLR4's recognition of LPS, which requires CD 14 and LPS Binding Protein (LBP). The function of TLRs in all organisms appears to be similar enough to use a single model of action.
  • Each Toll-like receptor forms either a homodimer or heterodimers in the recognition of a specific or set of specific molecular determinants present on microorganisms. Following activation by the bound pathogenic factor, several reactions are possible. Immune cells can produce signaling factors called cytokines which trigger inflammation. In the case of a bacterial factor, the pathogen might be phagocytosed and digested, and its antigens presented to CD4+ T cells. In the case of a viral factor, the infected cell may shut off its protein synthesis and may undergo programmed cell death. Immune cells that have detected a virus may also release anti- viral factors such as interferons (Goldstein DR., 2004; O'Neill L, 2005; Dunne and O'Neill, 2003). The following Table gives an overview of the different Toll like receptors:
  • TLR2 sepsis/Staphylococcal infection (Lorenz,E et al. 2000), lepramatous leprosy (Bochud PY et al. 2003), increased prevalence of infection (Sutherland
  • TLR3 allergic disease or viral infection (WO05/033338 & WO05/033339);
  • TLR4 Gram-negative septic shock (Lorenz E et al. 2002), ulcerative colitis (Torok HP et al.2004), septic shock (Arbour NC et al 2000), altered innated immunity and atherosclerosis (WO 00/77204 and WO 03/050137), immune system disorder (US 6 740 487), atherothrombosis (Zee R. et al. 2005), juvenile idiopathic arthritis (Lamb R et al., 2005), infectious diseases (Schroder et al. 2005); • TLR5: legionnaires' disease (pneumonia) (Hawn TR et al.2003);
  • TLR6 asthma (Tantisira K et al. (2004), prostate cancer (Sun J et al., 2005);
  • TLR9 asthma (Lazarus R et al.2003);
  • TLRlO asthma (Lazarus R et al.2004).
  • the present invention has identified additional and new snp's as important markers for one or more diseases influenced by innate immunity. Continued identification of such allotypes and haplotypes may not only provide insight as to why the response to treatment varies amongst individuals, but also may potentially decrease morbidity and mortality through improved risk assessment and the administration of prophylactic or "personalized” medicine.
  • the present invention provides a method and kit for identifying a subject at risk of, at risk of having, or having, an indication associated with altered innate immunity, based on the TLR genotype, concentration or functionality.
  • the present invention provides a method and kit for identifying a subject at risk of, at risk of having, or having, an indication associated with altered innate immunity, comprising detecting in a sample the presence or absence of at least one nucleic acid variant in at least one TLR gene. More particular, the present invention provides a method and kit for identifying a subject at risk of, or having, an indication associated with altered innate immunity, comprising detecting in a sample the presence or absence of at least one nucleic acid variant in at least one TLR gene, or part thereof, whereby the presence of at least one nucleic acid variant identifies whether a subject is at risk or has an indication associated with an altered innate immunity.
  • Specific regions of interest in the TLR gene are the promotor region, the exon regions and the intron regions.
  • the methods and kits of the present invention can also be carried out in combination with other methods for identifying a subject at risk of, or having, an indication associated with altered innate immunity.
  • the methods and kits are carried out in combination with a method for the detection of the presence or absence of a nucleic acid variant, or other markers, in any other gene.
  • any detection method is part of the present invention.
  • Preferred methods and means for the detection of the presence or absence of the nucleic acid variants of the present invention are hybridization, sequencing, PCR, primer extension, MLPA, OLA and restriction site analysis, or a combination thereof.
  • the method and kits of the present invention identify a subject at risk of, or having, an indication associated with altered innate immunity, and comprises measuring the concentration or functionality of at least one TLR protein in a biological sample, wherein an increased or decreased TLR concentration or altered functionality compared to a reference value representing a known health status indicates that said subject is at risk or suffers from a disorder associated with altered innate immunity.
  • a further embodiment of the present invention relates to a method for selecting an appropriate treatment or therapeutic agent for a subject at risk of, or having, an indication associated with altered innate immunity, comprising determining the presence of an indication associated with altered innate immunity by the methods of the present invention and selecting an appropriate treatment or therapeutic agent.
  • Figure 1 human TLRl wt gDNA sequence (SEQ ID NO: 1). Further specifications are:
  • /db_xref GeneID:7096
  • /db_xref MIM:601194" mRNA join(4001..4038,4410..4486,7844..7935,9894..12535)
  • /db_xref GI:41350337
  • /db_xref GeneID:7096
  • /db_xref MIM:601194"
  • Figure 2 human TLR2 wt gDNA sequence (SEQ ID NO:2). Further specifications are:
  • Figure 3 human TLR3 wt gDNA sequence (SEQ ID NO:3). Further specifications are:
  • Figure 4 human TLR4 wt gDNA sequence (SEQ ID NO:4).
  • LOCUS NT 008470 16001 bp DNA linear CON 20-AUG-2004 DEFINITION Homo sapiens chromosome 9 genomic contig.
  • /db_xref GeneID:7099"
  • /db_xref MIM:603030" mRNA join(3789..4048,8046..8212,11872..15258)
  • transcript variant 1 is encoded by transcript variant 1
  • Figure 5 human TLR5 wt gDNA sequence (SEQ ID NO:5). Further specifications are:
  • /db_xref "MIM:603031 " mRNA join(3001..3087,4520..4635,9022..9107,l 1421..11611,
  • Figure 7 human TLR7 wt gDNA sequence (SEQ ID NO:7). Further specifications are:
  • Figure 8 human TLR8 wt gDNA sequence (SEQ ID NO:8). Further specifications are:
  • /db_xref "MIM:300366" mRNA (transcript variant 1) join(3324..3394,6991..7127,15729..18988)
  • Figure 9 human TLR9 wt gDNA sequence (SEQ ID NO:9). Further specifications are:
  • transcript variant A is encoded by transcript variant A;
  • Figure 10 human TLRlO wt gDNA sequence (SEQ ID NO: 10). Further specifications are: LOCUS NT_016297 7270 bp DNA linear CON 19-AUG-2004
  • the determination of the nucleic acid sequence and/or the TLR concentration or functionality makes it possible to estimate or identify whether a subject is at risk of, or has, an indication associated with altered innate immunity.
  • the method of the present invention determines the presence of both variant and normal nucleic acids of at least one TLR gene, or part thereof, in a sample.
  • TLR gene refers to one of the following Toll Like
  • Receptor genes TLRl, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9 or
  • TLRlO and optionally to analogous or derivatives thereof.
  • Part thereof refers to the region of interest, i.e. the region of the TLR gene comprising a nucleic acid variant. More particular, a part thereof refers to the 5'UTR, the promotor region, exon 1, IVSl (intervening sequence or intron 1), exon 2, IVS2, exon 3, IVS3, exon 4, IVS4, exon 5, IVS5 and/or exon 6.
  • exon 1 IVSl (intervening sequence or intron 1)
  • exon 2 IVS2, exon 3, IVS3, exon 4, IVS4, exon 5, IVS5 and/or exon 6.
  • the current invention relates to a method of identifying a subject at risk of, at risk of having, or having, an indication associated with altered innate immunity, comprising detecting the presence or absence of at least one nucleic acid variant in at least one TLR gene, whereby the presence of at least one nucleic acid variant identifies whether a subject is at risk of, or has, an indication associated with an altered innate immunity.
  • nucleic acid refers to a single stranded or double stranded nucleic acid sequence and may consist of deoxyribonucleotides or ribonucleotides, nucleotide analogues or modified nucleotides, or may have been adapted for therapeutic purposes. There is no limitation in length. A nucleic acid that is up to about 100 nucleotides in length, is often also referred to as an oligonucleotide.
  • the reference nucleic acid and protein sequences indicated in the current invention are derived from GeneBank (NCBI) and indicated by their respective accession number, as is well known to the person skilled in the art.
  • NCBI GeneBank
  • the nomenclature for the TLR nucleotide and amino acid changes as used herein is generally accepted and recommended by den Dunnen and Antonarakis (2000). Frequent updates of the nomenclature for the description of sequence variations are provided on the web-site of the Human Genome Variation Society.
  • nucleotide numbering of the coding DNA and RNA reference sequence is as follows: • nucleotide +1 is the A of the ATG-translation initiation codon
  • nucleotide number is preceded by "g.” when a genomic or by "c.” when a cDNA reference sequence is used. Substitutions are designated by ">”.
  • Table 1 gives for each TLR gene the exons, the reference gDNA sequence, mRNA sequence and protein sequence.
  • nucleic acid variant or “polymorphism” or “variant” as used in the present invention, means that the nucleic acid sequence at a certain position in the TLR gene differs relative to one or more reference nucleic acid sequences.
  • the most simple nucleic acid polymorphism is a polymorphism affecting a single nucleotide, i.e. a single nucleotide polymorphism or SNP.
  • Nucleic acid polymorphisms further include any number of contiguous and/or non-contiguous differences in the primary nucleotide sequence of the nucleic acid under investigation relative to the primary nucleotide sequence of one or more reference nucleic acids.
  • polymorphic position refers to the nucleic acid position at which a nucleic acid polymorphism arises. Nucleic acid sequences comprising at least one such polymorphism are referred to as “polymorphic nucleic acid sequences", “polymorphic polynucleotides”, “polymorphic sequences” or the like.
  • the polymorhism or nucleic acid variant can be an insertion, deletion, substitution, tandem repeat or similar.
  • detecting the presence refers to determining whether or not the relevant genetic, physiological and/or biochemical event, linked with the occurrence of a disease is present.
  • both the absence and the presence of a certain event can function as markers.
  • reference to detecting the presence of a nucleic acid variant or a biochemical marker generally encompasses determining whether the marker is present, either based on the absence or the presence of the variant or biochemical marker in a sample. Moreover, this also includes the possible finding that the marker is not present in the sample, i.e. determining the absence (or presence) of a nucleic acid variant or biochemical marker.
  • determining the presence of the marker can also be done indirectly, e.g. where the presence of a nucleic acid variant is linked to disease, the occurrence of this marker can also be done by determining the homozygous presence of the corresponding allele not comprising the nucleic acid variant.
  • allele specific oligonucleotide primers and probes for detecting the presence of a SNP can be specific for the allele where the SNP is not present.
  • hap Io type means a particular pattern of sequential polymorphisms found on a single chromosome.
  • allele is one of several alternative forms of a gene or DNA sequence at a specific chromosomal location (locus). At each autosomal locus an individual possesses two alleles, one inherited from the father and one from the mother.
  • gene “genotype” means the genetic constitution of an individual, either overall or at a specific locus.
  • allotype refers to any of the genetically determined variants in the constant region of a given subclass of an immunoglobulin that is detectable as an antigen by members of the same species having a different constant region.
  • the present invention relates to a method according to the present invention, wherein the TLR genotype has at least one variant allele of the TLR gene (heterozygous).
  • the method of the invention relates to a method according to the present invention, wherein the TLR genotype has two variant or wild type alleles of the TLR gene (homozygous).
  • homozygous refers to having two of the same alleles at a locus.
  • heterozygous refers to having different alleles at a locus.
  • the method of the invention comprises the step of determining whether one or more nucleic acid variants in the TLR gene are present in 0, 1 or 2 copies, more particularly whether a nucleic acid variant in the TLR gene is present in one or both alleles.
  • Table 2 gives the newly identified snp's of the present invention.
  • wild-type sequence is analogous to the reference sequence.
  • the nucleic acid sequence of the wild type human TLR gene is identified by the reference sequence (see Table 1).
  • the allele may be normal as in the reference sequence, or it may be a variant, such as a structural or a non-structural variant.
  • IVS or intervening sequence or intron region are the regions as given in Table 2 for each specific TLR gene.
  • the present invention also covers analogues of the TLRs.
  • An "analogue” is a compound (or molecule) that is a (chemical) structural derivative of the TLR. It is also used to describe a molecule which may be structurally similar (but not identical) to another, and which exhibits many or some of the same biological functions of the TLR.
  • An analogue is to be understood as being any peptide sequence capable of the same biological functions as the wild-type TLR, including recombinant TLR.
  • innate immunity refers to the natural ability of an organism to defend itself against invasions by pathogens. Pathogens as used herein, may include, but are not limited to bacteria, fungi, parasites, viruses and algae. In addition, innate immunity includes immune responses that affect other diseases, such as cancer, inflammatory diseases, neurological diseases, autoimmune diseases and various infections.
  • an "indication or condition associated with aberrant, modified or altered innate immunity” refers to any indication or disease resulting from a decreased or increased defense mechanism.
  • a decreased defense can increase or alter the susceptibility for infection or inflammation or can increase risk for acquiring of a particular disease.
  • An increased defense might result in neurological disease, autoimmune disease or inflammatory diseases.
  • TLR deficiencies are associated with an increased risk for infections, inflammation, neurological disease and autoimmune conditions, and influence the severity and/or course of several diseases. Accordingly, TLR deficiencies can be linked with increased susceptibility for disease and/or prognosis for more severe or more frequent disease, or worse outcomes due to complication. Alternatively, it is shown in the present invention that TLR deficiencies can also be associated with a reduced (i.e. lower or no) risk for infections, inflammation or autoimmune conditions, indicating the protective effect of the TLR deficiency. Accordingly, TLR deficiencies can be linked with altered activity of innate immunity. Furthermore, treatment options can be considered and include eventual TLR replacement therapy.
  • the risk for developing a disorder associated with an altered activity of innate immunity can be determined.
  • the present invention relates to a method of identifying a subject at risk of, at risk of having, or having, an indication associated with altered innate immunity, comprising detecting the presence or absence of at least one nucleic acid variant in at least one TLR gene. More specific, the present invention relates to a method of identifying a subject at risk of, or having, an indication associated with altered innate immunity, comprising detecting the presence or absence of at least one nucleic acid variant in at least one TLR gene, whereby the presence of at least one nucleic acid variant identifies a subject at risk of, or having an increased or decreased (reduced) susceptibility for disease. In the latter case, the presence of at least one nucleic acid variant has a protective effect on the development of a disease.
  • the presence of at least one TLR nucleic acid variant identifies a subject at risk of, or having an increased severity of disease. In an even further embodiment, the presence of at least one TLR nucleic acid variant identifies a subject at risk of, or having a modified response to therapy for a disease. Furthermore, the presence of at least one TLR nucleic acid variant identifies a subject at risk of, or having increased risk of transplant rejection.
  • the altered innate immunity is associated with an increased or reduced susceptibility for or severity of infection, an autoimmune disease, cystic fibrosis, a cardiovasular disease, a neurological disease or cancer.
  • infection encompasses bacterial, viral, fungal, parasitic or algae infection.
  • diseases caused by infections are sepsis, severe sepsis and septic shock, otitis media, and recurrent infections especially in children.
  • Sepsis is defined as presence of infection and several of other parameters of general clinical nature, inflammatory, hemodynamic and tissue perfusion parameters. Severe sepsis is the presence of sepsis complicated by organ dysfunction.
  • Septic shock is defined as the presence of severe sepsis accompanied by acute circulatory failure.
  • Otitis media is an infection of the middle ear.
  • the altered innate immunity is associated with one or more of the following autoimmune diseases: rheumatoid arthritis (RA), spondyloarthropathy, systemic lupus erythematosus (SLE), Sjogren's disease, multiple sclerosis (MS), Crohn's disease, coeliac disease, Type 1 diabetes, Kawasaki disease, asthma, atopic dermatitis, dermatomyositis or Behcet's disease.
  • RA rheumatoid arthritis
  • SLE systemic lupus erythematosus
  • MS multiple sclerosis
  • coeliac disease Type 1 diabetes
  • Kawasaki disease asthma
  • atopic dermatitis dermatomyositis or Behcet's disease.
  • the altered innate immunity is associated with one or more of the following cancers: (1) solid tumors such as colon cancer, colorectal cancer, gastric cancer, cervical cancer, lung cancer, liver cancer, kidney cancer or brain cancer, and (2) haematological malignancies such as a) Leukemias: acute myeloid leukemia (AML), acute lymphoid leukemia (ALL), chronic myeloid leukemia (CML) and chronic lymphoid leukemia (CLL), b) Lymphomas: Hodgkins and non-Hodgkin's lymphomas and c) Myelomas.
  • AML acute myeloid leukemia
  • ALL acute lymphoid leukemia
  • CML chronic myeloid leukemia
  • CLL chronic lymphoid leukemia
  • Lymphomas Hodgkins and non-Hodgkin's lymphomas
  • Myelomas Myelomas.
  • the altered innate immunity is associated with one or more of the following cardiovascular diseases: bypass failure, atherosclerosis, myocardial reperfusion injury, coronary artery disease or heart disease.
  • the altered innate immunity is associated with one or more of the following neurological diseases: Alzheimer's disease, myasthenia gravis, multiple sclerosis, microbial infections, head trauma and stroke, Pick's disease, Parkinson's disease, dementia with Lewy bodies, Huntington disease, chromosome 13 dementias, Down's syndrome, cerebrovascular disease, Rasmussen's encephalitis, viral meningitis, NPSLE, amyotrophic lateral sclerosis, Creutzfeldt-Jacob disease, Gerstmann-Straussler-Scheinker disease, transmissible spongiform encephalopathies or ischemic reperfusion damage.
  • the altered innate immunity is associated with an increased severity of disease influencing the course of a disease.
  • the disease status can be aggravated leading to a higher mortality.
  • autoimmune disease for example RA
  • the severity or damage to the joints can be more pronounced, as measured by radiology.
  • the altered innate immunity is associated with a modified response to therapy for a specific disease resulting in adverse effects. This can for example by seen in vaccinations or in NSAID therapy.
  • the present invention relates to a method of identifying a subject at risk of, or having an increased susceptibility for (recurrent) otitis media, rheumatoid arthritis, recurrent infections, sepsis, severe sepsis, septic shock, or cancer, more specific a solid tumor or a haematological malignancy, comprising detecting the presence or absence of at least one nucleic acid variant in at least one TLR gene, more specific in the TLRl, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9 and/or TLRlO gene. Accordingly, the presence of the nucleic acid variant is associated with an increased or decreased (reduced) risk.
  • the nucleic acid variant has a protective effect.
  • the present invention thus also relates to a method for determining a protective effect on the development of (recurrent) otitis media, rheumatoid arthritis, recurrent infections, sepsis, severe sepsis, septic shock or cancer, more specific a solid tumor or a haematological malignancy, comprising detecting the presence or absence of at least one nucleic acid variant in at least one of the TLR genes, more specific in the TLRl, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9 and/or TLRlO gene.
  • nucleic acid variant is located in the region as indicated in Table 2 for each TLR gene. Even more specific, the nucleic acid variant is located at a position selected from the group consisting of: - position 239 or 914 of SEQ ID NO: 1,
  • defect refers to an alteration in the genomic DNA sequence when compared to the wild type sequence. This may result in altered (enhanced or decreased) expression levels or may result in changes (increase or decrease) in the functionality of the encoded protein, or even may result in a change of function of the protein ("gain of function").
  • the "subject" on which the method of the present invention is carried out can be any subject of which the risk of an altered innate immunity needs to be determined.
  • the subject may be a non-human subject such as (but not limited to) a cow, a pig, a sheep, a goat, a horse, a monkey, a rabbit, a dog, a cat, a mouse, a rat, a hamster, a zebrafish, a pufferfish (Fugu), a fly, a worm or C. elegans. More preferably, the subject is a primate. Even more preferably, the subject is a human.
  • nucleic acid from any nucleated cell can be used as the starting point for such assay techniques and may be isolated according to standard nucleic acid preparation procedures well known to those of skill in the art.
  • Many current methods for the detection of allelic variation are reviewed by Nollau et al. (1997), and in standard textbooks, for example "Laboratory Protocols for Mutation Detection", Ed. by U. Landegren, Oxford University Press, 1996 and “PCR", 2 nd Edition” by Newton & Graham, BIOS Scientific Publishers Limited, 1997 (incorporated herein by reference).
  • the method of the present invention can be carried out in vivo or in vitro. Preferred, however, is in vitro detection of nucleic acid variants in at least one TLR gene in a biological sample obtained from the subject.
  • biological sample means a tissue sample or a body fluid sample.
  • a tissue sample includes (but is not limited to) buccal cells, a brain sample, a skin sample or organ sample (e.g. liver).
  • body fluid refers to all fluids that are present in the body including but not limited to blood, plasma, serum, lymph, synovial fluid, urine, saliva or cerebrospinal fluid.
  • the biological sample may also be obtained by subjecting it to a pretreatment if necessary, for example, by homogenizing or extracting. Such a pretreatment may be selected appropriately by those skilled in the art depending on the biological sample to be subjected.
  • a nucleic acid comprising an intended sequence prepared from a biological sample may be prepared from DNA (e.g. gDNA or cDNA) or RNA (e.g. mRNA). Release, concentration and isolation of the nucleic acids from the sample can be done by any method known in the art. Currently, various commercial kits are available such as the QIAamp Blood Kit from Qiagen (Hilden, Germany) for the isolation of nucleic acids from blood samples, or the 'High pure PCR Template Preparation Kit' (Roche Diagnostics, Basel, Switzerland) or the DNA purification kits (PureGene, Gentra, Minneapolis, US).
  • PCR polymerase chain reaction
  • LCR ligase chain reaction
  • NASBA nucleic acid sequence-based amplification
  • RT-PCR reverse transcription polymerase reaction
  • the presence or absence of certain nucleic acid variants in the target sequence can be detected.
  • Numerous methods for detecting a single nucleotide anomaly in nucleic acid sequences are well-known in the art.
  • the present invention is not limited by any particular method used to detect the target sequences disclosed herein.
  • the detection of the presence or absence of a nucleic acid variant is determined by DNA or RNA hybridization, sequencing, PCR, primer extension, MLPA, oligonucleotide ligation assay (OLA) or restriction site analysis, or a combination thereof.
  • the detection of the presence or absence of a nucleic acid variant is determined by a Line Probe Assay (LiPA), or by the 4MATTM assay.
  • the method of the present invention optionally comprises the steps of isolating nucleic acids from the sample and/or an amplification step.
  • the present invention also provides isolated oligonucleotides, i.e. primers and probes, in order to amplify and/or detect nucleic acid variants and/or the wild type sequence of at least one TLR gene.
  • the wild type sequences of the TLR genes of the present invention are identified by SEQ ID NOs: 1-10.
  • Such primers or probes, specifically hybridizing to the target nucleic acid are of any convenient length such as to consist of at least 8, 9, 10, 11, 12, 13, 14 or 15 nucleotides and up to 40 nucleotides, up to 35 nucleotides, up to 30 nucleotides or more conveniently up to 25 nucleotides in length.
  • a preferred length of the primers or probes is thus 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25 nucleotides.
  • such primers or probes will comprise nucleotide sequences entirely complementary to the corresponding wild type or variant locus in the TLR gene.
  • one or more nucleotides may be added or one or more mismatches may be introduced, provided that the discriminatory power of the oligonucleotide primer or probe is not unduly affected.
  • An oligonucleotide primer (or primer pair) designed to specifically recognize and amplify either a wild type or variant allele at a locus is referred to as an allele specific primer (or primer pair).
  • an allele specific probe i.e. an oligonucleotide probe that specifically hybridizes to either a wild type or variant allele.
  • the hybridization conditions are to be stringent as known in the art. "Stringent" refers to the condition under which a nucleotide sequence can bind to related or non-specific sequences. For example, high temperature and lower salt increases stringency such than non-specific binding or binding with low melting temperature will dissolve.
  • the primers or probes of the invention may carry one or more labels to facilitate detection. The nature of the label is not critical to the invention and may be fluorescent, chemiluminescent, enzymatic, radioactive, chemical or other, provided it doesn't interfere with correct hybridizing of the oligonucleotide.
  • the primer or probe consists of 10 to 30 nucleotides, preferably 15 to 30 nucleotides, and is capable of specifically forming a hybrid with a part of the TLR gene and is at least one or more selected from the group consisting of:
  • an oligonucleotide capable of hybridizing under a stringent condition with the sequence as represented by SEQ ID NO: 1-10, or the complementary thereof;
  • an oligonucleotide capable of hybridizing under a stringent condition with the sequence as represented by SEQ ID NO: 1-10 wherein one or more nucleotides was subjected to a variation such as a substitution, deletion, insertion or addition, or the complementary thereof.
  • the present invention relates to an isolated oligonucleotide probe consisting of 10 to 30 nucleotides, optionally 15 to 30 nucleotides, for detecting the presence of one or more nucleic acid variants in SEQ ID NO: 1-10, or the complementary strand. More specific, the nucleic acid variants are located at a position selected from the group consisting of:
  • the polymorphism located in the specific regions of the TLR gene may also be detected in vitro by determining in the isolated TLR protein, as identified in the present invention, the presence or absence of an amino acid change, as indicated in Table 2, by sequencing said protein.
  • the amino acid change may also be detected by any conventional method known in the art, for example by mass-spectroscopy, gel electrophoresis, MALDI-TOF mass spectroscopy, ELISA, protein arrays, determination of the molecular weight, or by isoelectrofocusing. Studies have demonstrated several other known risk factors for altered innate immunity. Any human gene can be studied together with the method of the present invention.
  • ficolin genes i.e. FCNl, FCN2 and FCN3
  • ClQRl complement component 1, q subcomponent, receptor 1
  • BPI Bacterial/permeability- increasing protein
  • LBP Long Term Evolution
  • CD 14 CD 14 antigen precursor
  • beta-catenin CNNBI, Cadherin Associated Protein beta I
  • ILlO Interleukin 10
  • RP 105 LY64, lymphocyte antigen 64 homo log radioprotective
  • MBL2 Mannose Binding Protein
  • MD-I RP105-associated
  • MD- 2 MD2 Protein, Lymphocyte antigen 96
  • MYD88 Myeloid differentiation primary response gene 88
  • NODl Caspase recruitment domain 4, CARD4
  • NOD2 Caspase recruitment domain family, member 15, CARD 15
  • the present invention also relates to a method of identifying a subject at risk of, or having, an indication associated with altered innate immunity, comprising the step of detecting the presence or absence of a nucleic acid variant in at least one TLR gene and detecting the presence or absence of one or more nucleic acid variants in any other gene.
  • the "other" gene is selected from the group consisting of: the ficolin genes (i.e.
  • FCNl, FCN2 and FCN3 ClQRl (complement component 1, q subcomponent, receptor 1), BPI (Bacterial/permeability- increasing protein), LBP, CD 14 (CD 14 antigen precursor), beta-catenin (CTNNBI, Cadherin Associated Protein beta), ILlO (Interleukin 10), RP 105 (LY64, lymphocyte antigen 64 homo log radioprotective), MBL2 (Mannose Binding Protein), MD-I (RP 105- associated), MD-2 (MD2 Protein, Lymphocyte antigen 96), MYD88 (Myeloid differentiation primary response gene 88), NODl (Caspase recruitment domain 4, CARD4) and NOD2 (Caspase recruitment domain family, member 15, CARD15).
  • BPI Bactaspaserial/permeability- increasing protein
  • LBP Long permeability- increasing protein
  • CD 14 CD 14 antigen precursor
  • CNNBI Cadherin Associated Protein beta
  • ILlO Interleukin 10
  • the present invention also encompasses a method for determining whether a subject has a risk of developing a disease wherein the nucleic acid variants in the TLR gene are detected by their protein phenotype.
  • the invention discloses that decreased or increased levels of TLR and lack of functional TLR is crucial in the innate immunity defense.
  • the method encompasses the measurement of one or more proteins.
  • the present invention relates to a method for identifying a subject at risk of, or having, an indication associated with altered innate immunity comprising the steps of: a) determining the concentration or functionality of at least one TLR protein in a sample, b) identifying if said subject is at risk of, or has, an indication associated with altered innate immunity.
  • the current invention provides a method of identifying a subject at risk of, or having, an indication associated with altered innate immunity, comprising measuring the concentration or functionality of at least one TLR protein in a biological sample, wherein an increased or decreased TLR concentration or altered functionality compared to a reference value representing a known health status indicates that said subject is at risk of or suffers from a disorder associated with altered innate immunity.
  • TLR protein is a protein encoded by the TLR gene as described in the present invention, or a variant thereof.
  • the reference amino acid sequence is given in Table 1.
  • concentration refers to the presence or absence and/or amount of a certain protein.
  • a change in the concentration of a protein refers to a measurable increase or decrease, including total absence or presence, in the protein concentration when compared to a control subject.
  • a known health status or "control subject”, as defined in the present invention is a subject of the same species as the subject under examination which is free from, or not at direct risk of developing a disease associated with altered innate immunity.
  • the healthy subject can be of the same weight, age, and gender as the subject who is being diagnosed or prognosed for an altered innate immunity. In some cases, it might be preferred to use a reference value from the subject which is diagnosed.
  • the concentration obtained upon analyzing the subject under examination relative to the concentration obtained upon analyzing a control subject will depend on the particular analytical protocol and detection technique that is used. Accordingly, those skilled in the art will understand that, based on the present description, any laboratory can establish, for the TLR protein, a suitable "reference range”, “reference level range”, “concentration range” or “range of levels” (those terms are used interchangeable) characteristic for control subjects according to the analytical protocol and detection technique in use. The concentration obtained for the subject under examination can then be compared with this reference range and based on this comparison, a conclusion can be drawn as to whether the subject has a risk or not of developing a disease as described herein.
  • the reference value can be that of a level or concentration of the TLR protein in a sample, preferably a body fluid, from a subject not suffering from a disease associated with altered innate immunity.
  • the healthy subject can be of the same weight, age, and gender as the subject who is being diagnosed or prognosed for an altered innate immunity. In some cases, it might be preferred to use a reference value from the subject which is diagnosed.
  • the TLR protein that is detected in the method of the present invention may be detected by any method known to those skilled in the art. They can be identified by their structure, by partial amino acid sequence determination, by functional assay, by enzyme assay, by various immunological methods, or by biochemical methods known to those skilled in the art.
  • Biochemical methods include (but are not limited to) capillary electrophoresis, high performance liquid chromatography (HPLC), thin layer chromatography (TLC), hyperdiffusion chromatography, two-dimensional liquid phase electrophoresis (2-D- LPE; Davidsson et al. 1999) or detection of the migration pattern in gel electrophoreses.
  • Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS- PAGE) is a widely used approach for separating proteins from complex mixtures (Patterson and Aebersold, 1995). It can be performed in one- or two-dimensional (2- D) configuration. For less complicated protein preparation, one-dimensional SDS- PAGE is preferred over 2-D gels, because it is simpler.
  • 2-D gel electrophoresis incorporates isoelectric focusing (IEF) in the first dimension and SDS-PAGE in the second dimension, leading to a separation by charge and size (O'Farrell, 1975).
  • IEF isoelectric focusing
  • 2-D PAGE is a powerful technique for separating very complex protein preparations, resolving up to 10 000 proteins from mammalian tissues and other complex proteins (Klose and Kobalz, 1995; Celis et al, 1996; Yan et al, 1997).
  • the TLR proteins of the present invention can be identified by their isoelectric focusing point (pi) and their molecular weight (MW) in kilodaltons (kD).
  • the level of TLR protein can also be detected by an immunoassay.
  • an "immunoassay” is an assay that utilizes an antibody to specifically bind to the antigen (i.e. the TLR protein). The immunoassay is thus characterized by detection of specific binding of the TLR protein to an antibody.
  • Immunoassays for detecting TLR proteins may be either competitive or noncompetitive. Noncompetitive immunoassays are assays in which the amount of captured analyte (i.e. the TLR protein) is directly measured. In competitive assays, the amount of analyte (i.e.
  • the TLR protein) present in the sample is measured indirectly by measuring the amount of an added (exogenous) analyte displaced (or competed away) from a capture agent (i.e. the antibody) by the analyte (i.e. the TLR protein) present in the sample.
  • a competition assay a known amount of the (exogenous) TLR protein is added to the sample and the sample is then contacted with the antibody.
  • the amount of added (exogenous) TLR protein bound to the antibody is inversely proportional to the concentration of the TLR protein in the sample before the exogenous TLR protein is added.
  • the antibodies can be bound directly to a solid substrate where they are immobilized.
  • TLR protein of interest captures the TLR protein of interest present in the test sample.
  • Other immunological methods include but are not limited to fluid or gel precipitation reactions, immunodiffusion (single or double), agglutination assays, immunoblotting , immunospotting (such as line immunoassays or LIA), immunoelectrophoresis, radioimmunoassays (RIA), enzyme-linked immunosorbent assays (ELISA), TRIFMA (Christiansen et al., 1999), Western blots, liposome immunoassays (Monroe et al., 1986), complement-fixation assays, immunoradiometric assays, fluorescent immunoassays, protein A immunoassays or immunoPCR.
  • the method of the present invention may also be used in determining whether and which therapeutic agent might be suitable for a patient being at risk of, or having an indication associated with altered innate immunity.
  • the therapeutic agent may be used to prevent or treat the indication or disease.
  • preventing a disease means inhibiting or reversing the onset of the disease, inhibiting or reversing the initial signs of the disease, inhibiting the appearance of clinical symptoms of the disease.
  • treating a disease includes substantially inhibiting the disease, substantially slowing or reversing the progression of the disease, substantially ameliorating clinical symptoms of the disease or substantially preventing the appearance of clinical symptoms of the disease.
  • kits for use in the method as described herein encompasses a kit for identifying a subject at risk of, at risk of having, or having, an indication associated with altered innate immunity.
  • This kit can be based on the detection of nucleic acid variants in at least one TLR gene of said subject or it can be based on the detection of TLR proteins.
  • the kit of the present invention comprises reagents that selectively detect a nucleic acid variant in at least one TLR gene or that selectively detect at least one TLR protein.
  • a kit based on the detection of nucleic acid variants in at least one TLR gene may comprise:
  • step (b) optionally, a means for determining, from the nucleic acid variants detected with the means of step (a), whether the subject is at risk of, or has, an indication associated with altered innate immunity.
  • the kit comprises a means for detecting the presence or absence of one or more nucleic acid variants in the promotor region, the 5'UTR region, one or more exon regions and/or one or more intervening regions (IVS) of the TLR gene.
  • IVS intervening regions
  • the kit comprises: (a) a means or reagent for detecting the presence or absence of one or more nucleic acid variants at the following positions:
  • step (b) optionally, a means for determining, from the nucleic acid variants detected with the means of step (a), whether the subject is at risk of, or has, an indication associated with altered innate immunity.
  • the means or reagents in step (a) of said kit may comprise:
  • the means or reagent in step (a) of said kit comprise at least one oligonucleotide probe suitable for detection of a target TLR nucleic acid. More particular, the oligonucleotide probe specifically hybridizes to the target nucleic acid.
  • the target TLR nucleic acid is located in the promotor region, the 5 'UTR region, at least one exon region and/or at least one IVS region of the TLR gene.
  • the regions as given in Table 2 for each specific TLR gene are the regions as given in Table 2 for each specific TLR gene.
  • the target TLR nucleic acid is located at position 239 or 914 of SEQ ID NO:1, position -15607, 1350 or 2258 of SEQ ID NO:2, position -7 or 6301 of SEQ ID NO:3, position -2026 of SEQ ID NO:4, position -30891 or 1846 of SEQ ID NO:5, position -502, 745 or 1083 of SEQ ID NO:6, position 17962 of SEQ ID NO:7, position 9009, 12688, 9300, 12979, -558, 3122, 8683 or 12362 of SEQ ID NO:8, position -1486 of SEQ ID NO:9 and/or position - 1883 of SEQ ID NO: 10.
  • the oligonucleotide suitable for detection of a target TLR nucleic acid is a SSP (sequence specific primer). More specifically, the oligonucleotide suitable for detection of a target TLR nucleic acid is a probe configured to hybridize the said TLR polynucleic acid to form an invase cleavage structure.
  • the cleavage structure can be detected by a cleavage agent or enzyme such as a structure-specific nuclease, a 5 'nuclease, a FEN-I endonuclease or a polymerase.
  • the means or reagent in step (a) also includes at least one pair of oligonucleotide primers suitable for amplification of a target TLR polynucleic acid. More particular, each of the primers specifically hybridizes to the TLR nucleic acid sequence.
  • the target TLR polynucleic acid to be amplified is the promotor region, the 5'UTR region, at least one exon region and/or at least one IVS region of the TLR gene, or part thereof.
  • Particular targets are the regions as given in Table 2 for each specific TLR gene.
  • the target polynucleic acid comprises at least one of the polymorphic positions as given in Table 2.
  • hybridization buffer means a buffer allowing a hybridization reaction between the oligonucleotides and the polynucleic acids present in the sample, or the amplified products, under the appropriate stringency conditions.
  • wash solution means a solution enabling washing of the hybrids formed under the appropriate stringency conditions.
  • the means for detecting the presence or absence of nucleic acid variants in the TLR gene is an INVADER assay (see e.g. WO97/27214, incorporated herein by reference), or the 4MATTM assay (Innogenetics N. V., Ghent, Belgium).
  • the means for detecting the presence or absence of nucleic acid variants in the TLR gene is a line probe assay (LiPA; Stuyver et al, 1996; Stuyver et al, 1997; Van Geyt et al, 1998).
  • the selected set of probes is immobilized to a membrane strip in a line fashion.
  • An alternative is the immobilization of the probes in a "dotted fashion" (dot spots; DoPA). Said probes may be immobilized individually or as mixtures to the delineated locations.
  • the amplified TLR polynucleic acids can be labelled with biotine, and the hybrid can then, via a biotine-streptavidine coupling, be detected with a non- radioactive colour developing system.
  • oligonucleotides may be coupled to microspheres or chips.
  • An example of an assay that provides for simultaneous detection includes (but is not limited to) the xMAPTM technology (Luminex®100 IS, Austin, Texas, USA) and the PamGene technology (PamGene, 's-Hertogenbosch, The Netherlands).
  • the means in step (b) of said kit for determining, from the nucleic acid variants in the TLR gene detected with the means of step (a), whether the subject is at risk of, or has, an indication associated with altered innate immunity include a table, a chart, or similar, generally referred to as "a predisposition risk algorithm", indicating the TLR nucleic acid variants or haplotypes that confer a risk for or the existence of an indication associated with altered innate immunity.
  • a predisposition risk algorithm indicating the TLR nucleic acid variants or haplotypes that confer a risk for or the existence of an indication associated with altered innate immunity.
  • the term “chart” refers to graphical presentation, visual aid, diagram, plan, graph, sheet, map or the like including the relevant information. The determination of the risk can be performed manually or with the use of a computer.
  • the kit of the present invention may include, in addition to the means or reagent for detecting the presence or absence of a nucleic acid variant in at least one TLR gene, a means for detection other risk factors, e.g. nucleic acid variants in a gene, for an indication associated with an altered innate immunity.
  • the kit additionally includes a means, preferably probes, for detecting the genotype of or a nucleic acid variant in at least one of the genes selected from the group consisting of: the ficolin genes (FCNl, FCN2 and FCN3), ClQRl, BPI (Bacterial/permeability- increasing protein), LBP, CD 14 (CD 14 antigen precursor), beta-catenin (CTNNBI, Cadherin Associated Protein beta), ILlO (Interleukin 10), RP 105 (LY64, lymphocyte antigen 64 homo log radioprotective), MBL2 (Mannose Binding Protein), MD-I (RP105-associated), MD-2 (MD2 Protein, Lymphocyte antigen 96), MYD88 (Myeloid differentiation primary response gene 88), NODl (Caspase recruitment domain 4, CARD4) and NOD2 (Caspase recruitment domain family, member 15, CARD 15).
  • FCNl ficolin genes
  • FCN2 and FCN3 ClQRl
  • BPI Bacte
  • a kit based on the detection of TLR proteins may comprise an antibody that specifically recognizes the TLR protein that is detected.
  • a preferred kit for carrying out the method of the invention comprises: an antibody (primary antibody) which forms an immunological complex with the TLR protein to be detected; a monoclonal antibody (secondary antibody) which specifically recognizes the TLR protein to be detected; a marker either for specific tagging or coupling with said secondary antibody; appropriate buffer solutions for carrying out the immunological reaction between the primary antibody and the TLR protein, between the secondary antibody and the primary antibody-TLR protein complex and/or between the bound secondary antibody and the marker; or - possibly, for standardization purposes, a purified TLR protein.
  • the statistical analysis of the data is based on the determination of odds ratios (OR) using standard procedures.
  • An odds ratio is calculated by dividing the odds in the treated or exposed (case) group by the odds in the control group.
  • the odds of an event are calculated as the number of events divided by the number of non-events. If the odds of an event are greater than one the event is more likely to happen than not (the odds of an event that is certain to happen are infinite); if the odds are less than one the chances are that the event won't happen (the odds of an impossible event are zero).
  • the strength of association was reported as odds ratios (OR) (with 95% lower (LCL) and upper (UCL) confidence limit), indicating the factor by which the risk of developing a disorder or disease is increased (OR>1), or indicating the factor by which the risk of developing a disorder or disease is decreased (OR ⁇ 1) (protective effect).
  • the 95% confidence interval (95% CI) is the range of numerical values in which we can be confident (to a computed probability, such as 90 or 95%) that the population value being estimated will be found. Confidence intervals indicate the strength of evidence; where confidence intervals are wide, they indicate less precise estimates of effect. The larger the trial's sample size, the larger the number of outcome events and the greater becomes the confidence that the true relative risk reduction is close to the value stated. Thus the confidence intervals narrow and "precision" is increased. In a "positive finding" study the lower boundary of the confidence interval, or lower confidence limit, should still remain important or clinically significant if the results are to be accepted. In a "negative finding" study, the upper boundary of the confidence interval should not be clinically significant if you are to confidently accept this result.
  • Example 1 Detection of nucleic acid variants in the TLR genes from patients with Recurrent Otitis Media (OM) and from control subjects Patients samples
  • the control group (C) consisted of 205 healthy individuals. From each patient, informed consent to participate in the study is available
  • the strength of association was reported as odds ratios (OR).
  • the 95% confidence interval (95% CI) is the interval computed from the sample data which, if the study repeated multiple times, would contain the true effect 95% of the time.
  • Example 2 Detection of nucleic acid variants in the TLR genes from patients with sepsis/severe sepsis/septic shock and from control subjects
  • control group (C) consisted of 205 healthy individuals. From each patient, informed consent to participate in the study is available
  • the relevant regions were amplified using biotinylated oligonucleotides.
  • the polymorphisms were detected by use of a reverse hybridization method (Dot Probe Assay) with probes designed to recognize the polymorphisms as given in Table 2. After stringent washing at 56°C, hybridized probes were incubated with a streptavidine-alkaline phosphatase conjugate. The presence of a hybridized probe was revealed using NBIT/BCIP color development. Details on the reverse hybridization are described in Stuyver et al. (1996), Stuyver et al. (1997) and Van Geyt et al. (1998).
  • the strength of association was reported as odds ratios (OR).
  • the 95% confidence interval (95% CI) is the interval computed from the sample data which, if the study repeated multiple times, would contain the true effect 95% of the time.
  • Example 3 Detection of nucleic acid variants in the TLR genes from patients with Rheumatoid arthritis and from control subjects
  • the relevant regions were amplified using biotinylated oligonucleotides.
  • the polymorphisms were detected by use of a reverse hybridization method (Dot Probe Assay) with probes designed to recognize the polymorphisms as given in Table 2. After stringent washing at 56°C, hybridized probes were incubated with a streptavidine-alkaline phosphatase conjugate. The presence of a hybridized probe was revealed using NBIT/BCIP color development. Details on the reverse hybridization are described in Stuyver et al. (1996), Stuyver et al. (1997) and Van Geyt et al. (1998).
  • the strength of association was reported as odds ratios (OR).
  • the 95% confidence interval (95% CI) is the interval computed from the sample data which, if the study repeated multiple times, would contain the true effect 95% of the time.
  • Example 4 Detection of nucleic acid variants in the TLR genes from children with recurrent infections and from control subjects
  • the control group (C) consisted of 205 healthy individuals. From each patient, informed consent to participate in the study is available.
  • nucleic acid polymorphisms To determine the presence or absence of nucleic acid variants in the TLR genes, the relevant regions (see Table 2) were amplified using biotinylated oligonucleotides. The polymorphisms were detected by use of a reverse hybridization method (Dot Probe Assay) with probes designed to recognize the polymorphisms as given in Table 2. After stringent washing at 56°C, hybridized probes were incubated with a streptavidine-alkaline phosphatase conjugate. The presence of a hybridized probe was revealed using NBIT/BCIP color development. Details on the reverse hybridization are described in Stuyver et al. (1996), Stuyver et al. (1997) and Van Geyt et al. (1998).
  • the strength of association was reported as odds ratios (OR).
  • the 95% confidence interval (95% CI) is the interval computed from the sample data which, if the study repeated multiple times, would contain the true effect 95% of the time.
  • Example 5 Detection of nucleic acid variants in the TLR genes from children with a haematological malignancy or solid tumor and from control subjects
  • the relevant regions were amplified using biotinylated oligonucleotides.
  • the polymorphisms were detected by use of a reverse hybridization method (Dot Probe Assay) with probes designed to recognize the polymorphisms as given in Table 2. After stringent washing at 56°C, hybridized probes were incubated with a streptavidine-alkaline phosphatase conjugate. The presence of a hybridized probe was revealed using NBIT/BCIP color development. Details on the reverse hybridization are described in Stuyver et al. (1996), Stuyver et al. (1997) and Van Geyt et al. (1998).
  • the strength of association was reported as odds ratios (OR).
  • the 95% confidence interval (95% CI) is the interval computed from the sample data which, if the study repeated multiple times, would contain the true effect 95% of the time.
  • the relevant regions were amplified using biotinylated oligonucleotides.
  • the polymorphisms were detected by use of a reverse hybridization method (Dot Probe Assay) with probes designed to recognize the polymorphisms as given in Table 2. After stringent washing at 56°C, hybridized probes were incubated with a streptavidine-alkaline phosphatase conjugate. The presence of a hybridized probe was revealed using NBIT/BCIP color development. Details on the reverse hybridization are described in Stuyver et al. (1996, J. Clin. Microbiol. 34:2259-2266), Stuyver et al. (1997, Antimicrob. Agents Chemoter. 41:284-291) and Van Geyt et al. (1998, in: Therapies of viral Hepatitis. International Medical Press, London, UK pp. 139-145).
  • the 95% confidence interval (95% CI) is the interval computed from the sample data which, were the study repeated multiple times, would contain the true effect 95% of the time.
  • T allele minor allele
  • the above data show the value of said SNPs in the differential diagnosis of RA versus other diseases such as osteoarthritis, abarticular rheumatic symptoms, spondyloarthropathy, connective tissue diseases including polymyalgia rheumatica, psoriatic arthritis, crystal induced arthritis, fibromyalgia and aspecific arthralgias, being other and undifferentiated diseases including infections, malignancies, and neurological disorders.
  • diseases such as osteoarthritis, abarticular rheumatic symptoms, spondyloarthropathy, connective tissue diseases including polymyalgia rheumatica, psoriatic arthritis, crystal induced arthritis, fibromyalgia and aspecific arthralgias, being other and undifferentiated diseases including infections, malignancies, and neurological disorders.
  • Example 7 Detection of nucleic acid variants in toll-like receptor genes from patients admitted to the ICU who develop septic shock and those who do NOT develop sepsis/severe sepsis or septic shock.
  • the relevant regions were amplified using biotinylated oligonucleotides.
  • the polymorphisms were detected by use of a reverse hybridization method (Dot Probe Assay) with probes designed to recognize the polymorphisms as given in Table 2. After stringent washing at 56°C, hybridized probes were incubated with a streptavidine-alkaline phosphatase conjugate. The presence of a hybridized probe was revealed using NBIT/BCIP color development. Details on the reverse hybridization are described in Stuyver et al. (1996, J. Clin. Microbiol. 34:2259-2266), Stuyver et al. (1997, Antimicrob. Agents Chemoter. 41 :284-291) and Van Geyt et al. (1998, in: Therapies of viral Hepatitis. International Medical Press, London, UK pp. 139-145).
  • the 95% confidence interval (95% CI) is the interval computed from the sample data which, were the study repeated multiple times, would contain the true effect 95% of the time.
  • Example 8 Detection of nucleic acid variants in the toll-like receptor genes from patients admitted to the ICU who develop septic shock and those who do NOT develop sepsis/severe sepsis or septic shock.
  • nucleic acid polymorphisms To determine the presence or absence of nucleic acid variants in the TLR genes, the relevant regions (see Table 2) were amplified using biotinylated oligonucleotides. The polymorphisms were detected by use of a reverse hybridization method (Dot Probe Assay) with probes designed to recognize the polymorphisms as given in Table 2. After stringent washing at 56°C, hybridized probes were incubated with a streptavidine-alkaline phosphatase conjugate. The presence of a hybridized probe was revealed using NBIT/BCIP color development. Details on the reverse hybridization are described in Stuyver et al. (1996, J. Clin. Microbiol.
  • the 95% confidence interval (95% CI) is the interval computed from the sample data which, were the study repeated multiple times, would contain the true effect 95% of the time. Frequencies and odds ratios of TLR genotypes in patients with septic shock vs patient without sepsis/severe sepsis or septic shock
  • T allele the minor allele for the TLR3-rs3775291 polymorphism (exon 4) protects against the development of septic shock [0.75 (0,59-0,96)].
  • M major allele (wt)
  • m minor allele (variant)
  • TLR2 toll-like receptor
  • Lorenz,E et al. 2000 A novel polymorphism in the Toll-like receptor 2 gene and its potential association with Staphylococcal infection. Infection and immunity 68: 6398- 6401

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Abstract

The present invention relates to a method and kit for identifying a subject at risk of, or having, an indication associated with altered innate immunity. The present invention is based on the determination of Toll Like Receptor (TLR) genotypes and/or serum levels.

Description

NUCLEIC ACID VARIANTS IN THE TOLL LIKE RECEPTOR GENES ASSOCIATED WITH ALTERED INNATE IMMUNITY.
FIELD OF THE INVENTION
The present invention relates to a method and kit for identifying a subject at risk of, or having, an indication associated with altered innate immunity. The present invention is based on the determination of the Toll Like Receptor (TLR) genotype and/or serum level.
BACKGROUND ART
Immunity to infection is mediated by two systems, the acquired (or adaptive) immune system and the innate (or natural) immune system.
The innate immunity system is an evolutionary ancient form of immunity and offers the main resistance to microbial pathogens within the first minutes, hours or days of an infection (Fujita et al, 2002).
Innate immunity recognition is mediated by germ-line-encoded receptors, which means that the specificity of each receptor is genetically predetermined. The strategy of the innate immune response may not be to recognize every possible antigen, but rather to focus on a few, highly conserved structures (patterns) present in large groups of microorganisms. These structures are referred to as pathogen-associated molecular patterns (PAMPs), and the receptors of the innate immune system that evolved to recognize them are called pattern-recognition receptors (PRR) or pattern-recognition molecules (PRMs). PAMPs can be protein, lipid, nucleic acid, and carbohydrate (Lu et al., 2002). As soon as the PRRs identify the corresponding predetermined carbohydrate pattern on a pathogen, they immediately trigger effector cells to destroy the invading microorganism, rather than after having to undergo a proliferative cycle, as is the case for the time-delayed adaptive immune response. PRRs can be divided into three classes: signaling, endocytic, and secreted (Medzhitov R. et al., 2004). Innate immunity refers to antigen-nonspecific defense mechanisms that a host uses immediately or within several hours after exposure to an antigen. This is the immunity one is born with and is the initial response by the body to eliminate microbes and prevent infection.
Toll like receptors (TLRs) are type I transmembrane proteins that serve as a key part of the innate immune system. In vertebrates, they are also able to stimulate activation of the adaptive immune system, linking innate and acquired immune responses. TLRs are considered PRRs and their function is the recognition of pathogens and the activation of immune cell responses directed against those pathogens.
It has been estimated that most mammalian species have between ten and fifteen types of Toll-like receptors. Thirteen (named TLRl to TLRl 3) have been identified in humans, and equivalent forms of many of these have been found in other mammalian species. TLRs function as a dimer. Though most TLRs appear to function as homodimers, TLR2 forms heterodimers with TLRl or TLR6, each dimer having a different ligand specificity. TLRs may also depend on other co-activators for full ligand sensitivity, such as in the case of TLR4's recognition of LPS, which requires CD 14 and LPS Binding Protein (LBP). The function of TLRs in all organisms appears to be similar enough to use a single model of action. Each Toll-like receptor forms either a homodimer or heterodimers in the recognition of a specific or set of specific molecular determinants present on microorganisms. Following activation by the bound pathogenic factor, several reactions are possible. Immune cells can produce signaling factors called cytokines which trigger inflammation. In the case of a bacterial factor, the pathogen might be phagocytosed and digested, and its antigens presented to CD4+ T cells. In the case of a viral factor, the infected cell may shut off its protein synthesis and may undergo programmed cell death. Immune cells that have detected a virus may also release anti- viral factors such as interferons (Goldstein DR., 2004; O'Neill L, 2005; Dunne and O'Neill, 2003). The following Table gives an overview of the different Toll like receptors:
Figure imgf000004_0001
Figure imgf000005_0001
In order to elucidate the role of the Toll like receptor genes in innate immunity, several reports were published. Genetic association studies have been reported for:
• TLR2: sepsis/Staphylococcal infection (Lorenz,E et al. 2000), lepramatous leprosy (Bochud PY et al. 2003), increased prevalence of infection (Sutherland
AM. et al. 2005), coronary restenosis (Hamann L et al. 2005), bacterial infections (Schroder NW et al. 2003);
• TLR3: allergic disease or viral infection (WO05/033338 & WO05/033339);
• TLR4: Gram-negative septic shock (Lorenz E et al. 2002), ulcerative colitis (Torok HP et al.2004), septic shock (Arbour NC et al 2000), altered innated immunity and atherosclerosis (WO 00/77204 and WO 03/050137), immune system disorder (US 6 740 487), atherothrombosis (Zee R. et al. 2005), juvenile idiopathic arthritis (Lamb R et al., 2005), infectious diseases (Schroder et al. 2005); • TLR5: legionnaires' disease (pneumonia) (Hawn TR et al.2003);
• TLR6: asthma (Tantisira K et al. (2004), prostate cancer (Sun J et al., 2005);
• TLR9: asthma (Lazarus R et al.2003);
• TLRlO: asthma (Lazarus R et al.2004).
Despite the different associations of specific TLR polymorphisms with certain diseases, there is a continuous search towards more and/or accurate markers that provide a reliable diagnosis or prediction of the risk to develop a disease or disorder influenced by innate immunity. Accordingly, the present invention has identified additional and new snp's as important markers for one or more diseases influenced by innate immunity. Continued identification of such allotypes and haplotypes may not only provide insight as to why the response to treatment varies amongst individuals, but also may potentially decrease morbidity and mortality through improved risk assessment and the administration of prophylactic or "personalized" medicine.
SUMMARY OF THE INVENTION
The present invention provides a method and kit for identifying a subject at risk of, at risk of having, or having, an indication associated with altered innate immunity, based on the TLR genotype, concentration or functionality.
In a first embodiment, the present invention provides a method and kit for identifying a subject at risk of, at risk of having, or having, an indication associated with altered innate immunity, comprising detecting in a sample the presence or absence of at least one nucleic acid variant in at least one TLR gene. More particular, the present invention provides a method and kit for identifying a subject at risk of, or having, an indication associated with altered innate immunity, comprising detecting in a sample the presence or absence of at least one nucleic acid variant in at least one TLR gene, or part thereof, whereby the presence of at least one nucleic acid variant identifies whether a subject is at risk or has an indication associated with an altered innate immunity. Specific regions of interest in the TLR gene are the promotor region, the exon regions and the intron regions.
The methods and kits of the present invention can also be carried out in combination with other methods for identifying a subject at risk of, or having, an indication associated with altered innate immunity. In a preferred embodiment the methods and kits are carried out in combination with a method for the detection of the presence or absence of a nucleic acid variant, or other markers, in any other gene.
Any detection method is part of the present invention. Preferred methods and means for the detection of the presence or absence of the nucleic acid variants of the present invention are hybridization, sequencing, PCR, primer extension, MLPA, OLA and restriction site analysis, or a combination thereof. In a further embodiment, the method and kits of the present invention identify a subject at risk of, or having, an indication associated with altered innate immunity, and comprises measuring the concentration or functionality of at least one TLR protein in a biological sample, wherein an increased or decreased TLR concentration or altered functionality compared to a reference value representing a known health status indicates that said subject is at risk or suffers from a disorder associated with altered innate immunity.
A further embodiment of the present invention relates to a method for selecting an appropriate treatment or therapeutic agent for a subject at risk of, or having, an indication associated with altered innate immunity, comprising determining the presence of an indication associated with altered innate immunity by the methods of the present invention and selecting an appropriate treatment or therapeutic agent.
FIGURE LEGENDS
Figure 1: human TLRl wt gDNA sequence (SEQ ID NO: 1). Further specifications are:
LOCUS NT_016297 13535 bp DNA linear CON 19-AUG-2004 DEFINITION Homo sapiens chromosome 4 genomic contig.
ACCESSION NT O 16297 REGION: complement(5945521..5959055) VERSION NT_016297.15 GL37539484 FEATURES Location/Qualifiers gene 4001..12535 /gene="TLRl"
/db_xref="GeneID:7096" /db_xref="MIM:601194" mRNA join(4001..4038,4410..4486,7844..7935,9894..12535)
/gene="TLRl" /product="toll-like receptor 1"
/transcript_id="NM_003263.3" /db_xref="GI:41350336" /db_xref="GeneID:7096" /db xref="MIM:601194" CDS 9961..12321
/gene="TLRl "/note="Toll/interleukin- 1 receptor-like; /codon_start=l
/product="toll-like receptor 1" /protein_id="NP_003254.2"
/db_xref="GI:41350337" /db_xref="GeneID:7096" /db_xref="MIM:601194"
Figure 2: human TLR2 wt gDNA sequence (SEQ ID NO:2). Further specifications are:
LOCUS NT O 16606 18465 bp DNA linear CON 19-FEB-2004
DEFINITION Homo sapiens chromosome 4 genomic contig. ACCESSION NT O 16606 REGION: 16156122..16174586 VERSION NT 016606.16 GL37540362 FEATURES Location/Qualifiers gene 1001..18465
/gene="TLR2" /db_xref="GeneID:7097" /db_xref="MIM:603028" mRNA join(1001..1134,15979..18465)
/gene="TLR2"
/product="toll-like receptor 2" /transcript_id="NM_003264.2" /db_xref="GI: 19718733"
/db_xref="GeneID:7097" /db_xref="MIM:603028" CDS 15995..18349
/gene="TLR2" /note="toll/interleukin 1 receptor-like 4;
/codon_start=l
/product="toll-like receptor 2" /protein_id="NP_003255.2" /db xref="GI: 19718734" /db_xref="GeneID:7097" /db xref="MIM:603028"
Figure 3: human TLR3 wt gDNA sequence (SEQ ID NO:3). Further specifications are:
LOCUS NT_022792 18293 bp DNA linear CON 19-AUG-2004
DEFINITION Homo sapiens chromosome 4 genomic contig. ACCESSION NT 022792 REGION: 19400900..19419192 VERSION NT_022792.17 GL51464381 FEATURES Location/Qualifiers gene 1350..17293
/gene="TLR3" /db_xref="GeneID:7098" /db_xref="MIM:603029" mRNA join(1350..1443,8808..9255, 11035..11226,14515..16367,
16840..17293) /gene="TLR3"
/product="toll-like receptor 3" /transcript_id="NM_003265.2" /db_xref="GI: 19718735"
/db_xref="GeneID:7098" /db_xref="MIM:603029" CDS join(8815..9255, 11035..11226,14515..16367,16840..17068)
/gene="TLR3" /codon_start=l
/product="toll-like receptor 3" /protein_id="NP_003256.1 " /db_xref="GI:4507531" /db_xref="GeneID:7098" /db_xref="MIM:603029"
Figure 4: human TLR4 wt gDNA sequence (SEQ ID NO:4).
Further specifications are:
LOCUS NT 008470 16001 bp DNA linear CON 20-AUG-2004 DEFINITION Homo sapiens chromosome 9 genomic contig. ACCESSION NT_008470 REGION: 27784000..27800000 VERSION NT_008470.17 GI:51467683 FEATURES Location/Qualifiers gene 3789..15258
/gene="TLR4" /db_xref="GeneID:7099" /db_xref="MIM:603030" mRNA join(3789..4048,7682..7801,8046..8212,11872..15258) /gene="TLR4"
/product="toll-like receptor 4" /transcript_id="NM_003266.2" /db_xref="GI: 19924147" /db_xref="GeneID:7099" /db_xref="MIM:603030" mRNA join(3789..4048,7682..7801, 11872..15258)
/gene="TLR4"
/product="toll-like receptor 4" /transcript_id="NM_l 38556.1 " /db_xref="GI: 19924150"
/db_xref="GeneID:7099" /db_xref="MIM:603030" mRNA join(3789..4048,8046..8212,11872..15258)
/gene="TLR4" /product="toll-like receptor 4"
/transcript_id="NM_l 38554.1 " /db_xref="GI: 19924148" /db_xref="GeneID:7099" /db_xref="MIM:603030" mRNA join(3789..4048,l 1872..14435)
/gene="TLR4"
/product="toll-like receptor 4" /transcript_id="NM_l 38557.1 " /db xref="GI: 19924152" /db_xref="GeneID:7099"
/db_xref="MIM:603030" CDS join(3956..4048,8046..8212,l 1872..14131)
/gene="TLR4" /note="isoform A is encoded by transcript variant 1
/codon_start=l
/product="toll-like receptor 4 isoform A"
/protein_id="NP_612564.1 "
/db_xref="GI: 19924149" /db_xref="GeneID:7099"
/db_xref="MIM:603030" CDS join(7713..7801, 11872..14131)
/gene="TLR4"
/note="isoform B is encoded by transcript variant 2 /codon_start=l
/product="toll-like receptor 4 isoform B"
/protein_id="NP_612566.1 "
/db_xref="GI: 19924151"
/db_xref="GeneID:7099" /db_xref="MIM:603030"
CDS join(8073..8212,11872..14131)
/gene="TLR4"
/note="isoform C is encoded by transcript variant 3
/codon_start=l /product="toll-like receptor 4 isoform C"
/protein_id="NP_003257.1 "
/db_xref="GI:4507533"
/db_xref="GeneID:7099"
/db_xref="MIM:603030" CDS 12212..14131
/gene="TLR4"
/note="isoform D is encoded by transcript variant 4
/codon_start=l
/product="toll-like receptor 4 isoform D" /protein_id="NP_612567.1 " /db_xref="GI: 19924153" /db_xref="GeneID:7099" /db_xref="MIM:603030"
Figure 5: human TLR5 wt gDNA sequence (SEQ ID NO:5). Further specifications are:
LOCUS NT_077967 37105 bp DNA linear CON 19-AUG-2004
DEFINITION Homo sapiens chromosome 1 genomic contig. ACCESSION NT_077967 REGION: complement 1372735..1409839) VERSION NT_077967.3 GI:51459476 FEATURES Location/Qualifiers gene 3001..36105
/gene="TLR5" /db_xref="GeneID:7100"
/db_xref="MIM:603031 " mRNA join(3001..3087,4520..4635,9022..9107,l 1421..11611,
13652..13816,33375..36105) /gene="TLR5" /product="toll-like receptor 5"
/transcript_id="NM_003268.3 " /db_xref="GI: 19718736" /db_xref="GeneID:7100" /db_xref="MIM:603031 " CDS 33379.35955
/gene="TLR5"
/note="Toll/interleukin-l receptor-like protein 3; /codon_start=l
/exception="unclassified translation discrepancy" /product="toll-like receptor 5"
/protein_id="NP_003259.2" /db_xref="GI:16751843" /db_xref="GeneID:7100" /db xref="MIM:603031" Figure 6: human TLR6 wt gDNA sequence (SEQ ID NO:6). Further specifications are:
LOCUS NT O 16297 6801 bp DNA linear CON 19-AUG-2004 DEFINITION Homo sapiens chromosome 4 genomic contig.
ACCESSION NT O 16297 REGION: complement^ 974000..5980800) VERSION NT_016297.15 GL37539484 FEATURES Location/Qualifiers gene 998..3750 /gene="TLR6""
/db_xref="GeneID: 10333" /db_xref="MIM:605403" mRNA 998..3750
/gene="TLR6" /product="toll-like receptor 6"
/transcript_id="NM_006068.2" /db_xref="GI:20143970" /db_xref="GeneID: 10333" /db_xref="MIM:605403" CDS 1064..3454
/gene="TLR6" /codon_start=l
/product="toll-like receptor 6" /protein_id="NP_006059.2" /db_xref="GI:20143971"
/db_xref="GeneID: 10333" /db_xref="MIM:605403"
Figure 7: human TLR7 wt gDNA sequence (SEQ ID NO:7). Further specifications are:
LOCUS NT_011757 26501 bp DNA linear CON 20-AUG-2004
DEFINITION Homo sapiens chromosome X genomic contig. ACCESSION NT_011757 REGION: 8848000..8874500 VERSION NT 011757.14 GI:51477377 FEATURES Location/Qualifiers gene 3772..25476
/gene="TLR7" /db_xref="GeneID:51284" /db_xref="MIM:300365" mRNA join(3772..3808,4166..4266,22197..25476)
/gene="TLR7"
/product="toll-like receptor 7" /transcript_id="NM_016562.2" /db_xref="GI:20302164"
/db_xref="GeneID:51284" /db_xref="MIM:300365" CDS join(4264..4266,22197..25343)
/gene="TLR7" /codon_start=l
/product="toll-like receptor 7" /protein_id="NP_057646.1 " /db_xref="GI:7706093" /db_xref="GeneID:51284" /db_xref="MIM:300365"
Figure 8: human TLR8 wt gDNA sequence (SEQ ID NO:8). Further specifications are:
LOCUS NT_011757 21001 bp DNA linear CON 20-AUG-2004 DEFINITION Homo sapiens chromosome X genomic contig. ACCESSION NT O 11757 REGION: 8888000..8909000 VERSION NT_011757.14 GI:51477377 FEATURES Location/Qualifiers gene 3324..19854 /gene="TLR8"
/db_xref="GeneID:51311" /db_xref="MIM:300366" mRNA (transcript variant 2) join(3324..3394,15729..19854) /gene="TLR8"
/product="toll-like receptor 8"
/transcript_id="NM_138636.2"
/db_xref="GI:45935389" /db_xref="GeneID:51311"
/db_xref="MIM:300366" mRNA (transcript variant 1) join(3324..3394,6991..7127,15729..18988)
/gene="TLR8" /product="toll-like receptor 8"
/transcript_id="NM_016610.2"
/db_xref="GI:20302165"
/db_xref="GeneID:51311"
/db_xref="MIM:300366" CDS join(3392..3394,15729..18851)
/gene="TLR8"
/note="isoform 2 is encoded by transcript variant 2"
/codon_start=l
/product="toll-like receptor 8 isoform 2" /protein_id="NP_619542.1"
/db_xref="GI:20302168"
/db_xref="GeneID:51311"
/db_xref="MIM:300366" CDS join(7071..7127,15729..18851) /gene="TLR8"
/note="isoform 1 is encoded by transcript variant 1"
/codon_start=l
/product="toll-like receptor 8, isoform 1 "
/protein_id="NP_057694.2" /db_xref="GI:20302166"
/db_xref="GeneID:51311"
/db_xref="MIM:300366"
Figure 9: human TLR9 wt gDNA sequence (SEQ ID NO:9). Further specifications are:
LOCUS NT_022517 10082 bp DNA linear CON 19-AUG-2004
DEFINITION Homo sapiens chromosome 3 genomic contig. ACCESSION NT_022517 REGION: complement(52194138..52204219) VERSION NT_022517.17 GI:51464027 FEATURES Location/Qualifiers gene 4001..9082
/gene="TLR9" /db_xref="GeneID:54106" /db_xref="MIM:605474" mRNA join(400 L.4637,5852..9082)
/gene="TLR9"
/product="toll-like receptor 9" /transcript_id="NM_017442.2" /db_xref="GI:20302169"
/db_xref="GeneID:54106" /db_xref="MIM:605474" CDS join(4635..4637,5852..8947)
/gene="TLR9" /note="isoform A precursor is encoded by transcript variant A;
/codon_start=l
/product="toll-like receptor 9 isoform A precursor" /protein_id="NP_059138.1" /db_xref="GI:8394456" /db_xref="GeneID:54106"
/db_xref="MIM:605474"
Figure 10: human TLRlO wt gDNA sequence (SEQ ID NO: 10). Further specifications are: LOCUS NT_016297 7270 bp DNA linear CON 19-AUG-2004
DEFINITION Homo sapiens chromosome 4 genomic contig. ACCESSION NT O 16297 REGION: complement^ 922339..5929608) VERSION NT_016297.15 GL37539484 FEATURES Location/Qualifiers gene 3001..6270
/gene="TLR10"
/db_xref="GeneID:81793"
/db_xref="MIM:606270" mRNA join(3001..3235,3382.3570,3693..6270)
/gene="TLR10"
/product="toll-like receptor 10"
/transcript_id="NM_030956.1 "
/db_xref="GI: 13569929" /db_xref="GeneID:81793"
/db_xref="MIM:606270" CDS 3755..6190
/gene="TLR10"
/codon_start=l /product="toll-like receptor 10 precursor"
/protein_id="NP_l 12218.1"
/db_xref="GI: 13569930"
/db_xref="GeneID:81793"
/db_xref="MIM:606270"
For the Figures 1-10: exon sequences and possible polymorphism positions are indicated in respectively grey and bold/boxed. Nucleotide +1 is the A of the ATG- translation initiation codon which is bold and underlined.
DETAILED DESCRIPTION OF THE INVENTION
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. All publications mentioned herein are incorporated by reference. The materials, methods and examples are only illustrative and not limiting.
According to the present invention, the determination of the nucleic acid sequence and/or the TLR concentration or functionality makes it possible to estimate or identify whether a subject is at risk of, or has, an indication associated with altered innate immunity.
In a first embodiment, the method of the present invention determines the presence of both variant and normal nucleic acids of at least one TLR gene, or part thereof, in a sample. As used herein the term "TLR gene" refers to one of the following Toll Like
Receptor genes: TLRl, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9 or
TLRlO, and optionally to analogous or derivatives thereof.
"Part thereof refers to the region of interest, i.e. the region of the TLR gene comprising a nucleic acid variant. More particular, a part thereof refers to the 5'UTR, the promotor region, exon 1, IVSl (intervening sequence or intron 1), exon 2, IVS2, exon 3, IVS3, exon 4, IVS4, exon 5, IVS5 and/or exon 6.
More specifically, the current invention relates to a method of identifying a subject at risk of, at risk of having, or having, an indication associated with altered innate immunity, comprising detecting the presence or absence of at least one nucleic acid variant in at least one TLR gene, whereby the presence of at least one nucleic acid variant identifies whether a subject is at risk of, or has, an indication associated with an altered innate immunity.
The term "nucleic acid" refers to a single stranded or double stranded nucleic acid sequence and may consist of deoxyribonucleotides or ribonucleotides, nucleotide analogues or modified nucleotides, or may have been adapted for therapeutic purposes. There is no limitation in length. A nucleic acid that is up to about 100 nucleotides in length, is often also referred to as an oligonucleotide.
The reference nucleic acid and protein sequences indicated in the current invention are derived from GeneBank (NCBI) and indicated by their respective accession number, as is well known to the person skilled in the art. The nomenclature for the TLR nucleotide and amino acid changes as used herein is generally accepted and recommended by den Dunnen and Antonarakis (2000). Frequent updates of the nomenclature for the description of sequence variations are provided on the web-site of the Human Genome Variation Society.
Accordingly, the nucleotide numbering of the coding DNA and RNA reference sequence is as follows: • nucleotide +1 is the A of the ATG-translation initiation codon
• there is no nucleotide 0
• the nucleotide 5' of the ATG-translation initiation codon is -1.
The nucleotide number is preceded by "g." when a genomic or by "c." when a cDNA reference sequence is used. Substitutions are designated by ">".
Table 1 gives for each TLR gene the exons, the reference gDNA sequence, mRNA sequence and protein sequence.
Figure imgf000019_0001
The term "nucleic acid variant" or "polymorphism" or "variant" as used in the present invention, means that the nucleic acid sequence at a certain position in the TLR gene differs relative to one or more reference nucleic acid sequences. The most simple nucleic acid polymorphism is a polymorphism affecting a single nucleotide, i.e. a single nucleotide polymorphism or SNP. Nucleic acid polymorphisms further include any number of contiguous and/or non-contiguous differences in the primary nucleotide sequence of the nucleic acid under investigation relative to the primary nucleotide sequence of one or more reference nucleic acids. The term "polymorphic position" or "position" refers to the nucleic acid position at which a nucleic acid polymorphism arises. Nucleic acid sequences comprising at least one such polymorphism are referred to as "polymorphic nucleic acid sequences", "polymorphic polynucleotides", "polymorphic sequences" or the like. The polymorhism or nucleic acid variant can be an insertion, deletion, substitution, tandem repeat or similar.
The phrase "detecting the presence", e.g. of a marker as used herein, refers to determining whether or not the relevant genetic, physiological and/or biochemical event, linked with the occurrence of a disease is present. In practice, both the absence and the presence of a certain event can function as markers. Accordingly, reference to detecting the presence of a nucleic acid variant or a biochemical marker generally encompasses determining whether the marker is present, either based on the absence or the presence of the variant or biochemical marker in a sample. Moreover, this also includes the possible finding that the marker is not present in the sample, i.e. determining the absence (or presence) of a nucleic acid variant or biochemical marker. In both cases determining the presence of the marker can also be done indirectly, e.g. where the presence of a nucleic acid variant is linked to disease, the occurrence of this marker can also be done by determining the homozygous presence of the corresponding allele not comprising the nucleic acid variant. Similarly, allele specific oligonucleotide primers and probes for detecting the presence of a SNP can be specific for the allele where the SNP is not present.
The term "hap Io type" means a particular pattern of sequential polymorphisms found on a single chromosome. As used herein, the term "allele" is one of several alternative forms of a gene or DNA sequence at a specific chromosomal location (locus). At each autosomal locus an individual possesses two alleles, one inherited from the father and one from the mother. The term "genotype" means the genetic constitution of an individual, either overall or at a specific locus. The term "allotype" refers to any of the genetically determined variants in the constant region of a given subclass of an immunoglobulin that is detectable as an antigen by members of the same species having a different constant region.
In a specific embodiment, the present invention relates to a method according to the present invention, wherein the TLR genotype has at least one variant allele of the TLR gene (heterozygous). In a further embodiment, the method of the invention relates to a method according to the present invention, wherein the TLR genotype has two variant or wild type alleles of the TLR gene (homozygous).
The term "homozygous" refers to having two of the same alleles at a locus. The term "heterozygous" refers to having different alleles at a locus.
In a further embodiment, the method of the invention comprises the step of determining whether one or more nucleic acid variants in the TLR gene are present in 0, 1 or 2 copies, more particularly whether a nucleic acid variant in the TLR gene is present in one or both alleles.
Table 2 gives the newly identified snp's of the present invention.
Figure imgf000021_0001
Figure imgf000022_0001
vl = variant 1 v2 = variant 2
As used herein, the term "wild-type" sequence is analogous to the reference sequence. The nucleic acid sequence of the wild type human TLR gene is identified by the reference sequence (see Table 1). For example, the allele may be normal as in the reference sequence, or it may be a variant, such as a structural or a non-structural variant. Of interest are the promotor region, the 5'UTR, at least one of the exon regions and at least one of the intervening regions (IVS or intervening sequence or intron region) of the TLR genes. Of particular interest are the regions as given in Table 2 for each specific TLR gene.
The present invention also covers analogues of the TLRs. An "analogue" is a compound (or molecule) that is a (chemical) structural derivative of the TLR. It is also used to describe a molecule which may be structurally similar (but not identical) to another, and which exhibits many or some of the same biological functions of the TLR. An analogue is to be understood as being any peptide sequence capable of the same biological functions as the wild-type TLR, including recombinant TLR. As used herein, the term "innate immunity" refers to the natural ability of an organism to defend itself against invasions by pathogens. Pathogens as used herein, may include, but are not limited to bacteria, fungi, parasites, viruses and algae. In addition, innate immunity includes immune responses that affect other diseases, such as cancer, inflammatory diseases, neurological diseases, autoimmune diseases and various infections.
As used herein, the terms "indication", "disorder" and "disease" are used interchangeably. An "indication or condition associated with aberrant, modified or altered innate immunity" refers to any indication or disease resulting from a decreased or increased defense mechanism. A decreased defense can increase or alter the susceptibility for infection or inflammation or can increase risk for acquiring of a particular disease. An increased defense might result in neurological disease, autoimmune disease or inflammatory diseases.
It has been determined in the present invention that TLR deficiencies are associated with an increased risk for infections, inflammation, neurological disease and autoimmune conditions, and influence the severity and/or course of several diseases. Accordingly, TLR deficiencies can be linked with increased susceptibility for disease and/or prognosis for more severe or more frequent disease, or worse outcomes due to complication. Alternatively, it is shown in the present invention that TLR deficiencies can also be associated with a reduced (i.e. lower or no) risk for infections, inflammation or autoimmune conditions, indicating the protective effect of the TLR deficiency. Accordingly, TLR deficiencies can be linked with altered activity of innate immunity. Furthermore, treatment options can be considered and include eventual TLR replacement therapy.
With the methods of the present invention, the risk for developing a disorder associated with an altered activity of innate immunity can be determined.
Accordingly, the present invention relates to a method of identifying a subject at risk of, at risk of having, or having, an indication associated with altered innate immunity, comprising detecting the presence or absence of at least one nucleic acid variant in at least one TLR gene. More specific, the present invention relates to a method of identifying a subject at risk of, or having, an indication associated with altered innate immunity, comprising detecting the presence or absence of at least one nucleic acid variant in at least one TLR gene, whereby the presence of at least one nucleic acid variant identifies a subject at risk of, or having an increased or decreased (reduced) susceptibility for disease. In the latter case, the presence of at least one nucleic acid variant has a protective effect on the development of a disease.
In a further embodiment, the presence of at least one TLR nucleic acid variant identifies a subject at risk of, or having an increased severity of disease. In an even further embodiment, the presence of at least one TLR nucleic acid variant identifies a subject at risk of, or having a modified response to therapy for a disease. Furthermore, the presence of at least one TLR nucleic acid variant identifies a subject at risk of, or having increased risk of transplant rejection.
More specific, the altered innate immunity is associated with an increased or reduced susceptibility for or severity of infection, an autoimmune disease, cystic fibrosis, a cardiovasular disease, a neurological disease or cancer.
As used herein, the term "infection" encompasses bacterial, viral, fungal, parasitic or algae infection. Specific examples of diseases caused by infections are sepsis, severe sepsis and septic shock, otitis media, and recurrent infections especially in children. Sepsis is defined as presence of infection and several of other parameters of general clinical nature, inflammatory, hemodynamic and tissue perfusion parameters. Severe sepsis is the presence of sepsis complicated by organ dysfunction. Septic shock is defined as the presence of severe sepsis accompanied by acute circulatory failure. Otitis media is an infection of the middle ear.
In a specific embodiment, the altered innate immunity is associated with one or more of the following autoimmune diseases: rheumatoid arthritis (RA), spondyloarthropathy, systemic lupus erythematosus (SLE), Sjogren's disease, multiple sclerosis (MS), Crohn's disease, coeliac disease, Type 1 diabetes, Kawasaki disease, asthma, atopic dermatitis, dermatomyositis or Behcet's disease.
In a further embodiment, the altered innate immunity is associated with one or more of the following cancers: (1) solid tumors such as colon cancer, colorectal cancer, gastric cancer, cervical cancer, lung cancer, liver cancer, kidney cancer or brain cancer, and (2) haematological malignancies such as a) Leukemias: acute myeloid leukemia (AML), acute lymphoid leukemia (ALL), chronic myeloid leukemia (CML) and chronic lymphoid leukemia (CLL), b) Lymphomas: Hodgkins and non-Hodgkin's lymphomas and c) Myelomas.
In a more specific embodiment, the altered innate immunity is associated with one or more of the following cardiovascular diseases: bypass failure, atherosclerosis, myocardial reperfusion injury, coronary artery disease or heart disease. In a further embodiment, the altered innate immunity is associated with one or more of the following neurological diseases: Alzheimer's disease, myasthenia gravis, multiple sclerosis, microbial infections, head trauma and stroke, Pick's disease, Parkinson's disease, dementia with Lewy bodies, Huntington disease, chromosome 13 dementias, Down's syndrome, cerebrovascular disease, Rasmussen's encephalitis, viral meningitis, NPSLE, amyotrophic lateral sclerosis, Creutzfeldt-Jacob disease, Gerstmann-Straussler-Scheinker disease, transmissible spongiform encephalopathies or ischemic reperfusion damage.
In a particular embodiment, the altered innate immunity is associated with an increased severity of disease influencing the course of a disease. For example, in the case of infection the disease status can be aggravated leading to a higher mortality. In autoimmune disease, for example RA, the severity or damage to the joints can be more pronounced, as measured by radiology.
In a further embodiment, the altered innate immunity is associated with a modified response to therapy for a specific disease resulting in adverse effects. This can for example by seen in vaccinations or in NSAID therapy.
In a particular embodiment, the present invention relates to a method of identifying a subject at risk of, or having an increased susceptibility for (recurrent) otitis media, rheumatoid arthritis, recurrent infections, sepsis, severe sepsis, septic shock, or cancer, more specific a solid tumor or a haematological malignancy, comprising detecting the presence or absence of at least one nucleic acid variant in at least one TLR gene, more specific in the TLRl, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9 and/or TLRlO gene. Accordingly, the presence of the nucleic acid variant is associated with an increased or decreased (reduced) risk. In the latter case, the nucleic acid variant has a protective effect. The present invention thus also relates to a method for determining a protective effect on the development of (recurrent) otitis media, rheumatoid arthritis, recurrent infections, sepsis, severe sepsis, septic shock or cancer, more specific a solid tumor or a haematological malignancy, comprising detecting the presence or absence of at least one nucleic acid variant in at least one of the TLR genes, more specific in the TLRl, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9 and/or TLRlO gene.
More specific, the nucleic acid variant is located in the region as indicated in Table 2 for each TLR gene. Even more specific, the nucleic acid variant is located at a position selected from the group consisting of: - position 239 or 914 of SEQ ID NO: 1,
- position -15607, 1350 or 2258 of SEQ ID NO:2,
- position -7 or 6301 of SEQ ID NO:3,
- position -2026 of SEQ ID NO:4,
- position -30891 or 1846 of SEQ ID NO:5, - position -502, 745 or 1083 of SEQ ID NO:6,
- position 17962 of SEQ ID NO:7,
- position 9009, 12688, 9300, 12979, 3122, 12362, 8683 or -558 of SEQ ID NO:8,
- position -1486 of SEQ ID NO:9, and
- position -1883 of SEQ ID NO: 10.
As used herein, "deficiency" refers to an alteration in the genomic DNA sequence when compared to the wild type sequence. This may result in altered (enhanced or decreased) expression levels or may result in changes (increase or decrease) in the functionality of the encoded protein, or even may result in a change of function of the protein ("gain of function").
The "subject" on which the method of the present invention is carried out can be any subject of which the risk of an altered innate immunity needs to be determined. The subject may be a non-human subject such as (but not limited to) a cow, a pig, a sheep, a goat, a horse, a monkey, a rabbit, a dog, a cat, a mouse, a rat, a hamster, a zebrafish, a pufferfish (Fugu), a fly, a worm or C. elegans. More preferably, the subject is a primate. Even more preferably, the subject is a human. It will be apparent to the person skilled in the art that there are a large number of analytical procedures which may be used to detect the presence or absence of the nucleic acid variants mentioned herein. Nucleic acid from any nucleated cell can be used as the starting point for such assay techniques and may be isolated according to standard nucleic acid preparation procedures well known to those of skill in the art. Many current methods for the detection of allelic variation are reviewed by Nollau et al. (1997), and in standard textbooks, for example "Laboratory Protocols for Mutation Detection", Ed. by U. Landegren, Oxford University Press, 1996 and "PCR", 2nd Edition" by Newton & Graham, BIOS Scientific Publishers Limited, 1997 (incorporated herein by reference).
The method of the present invention can be carried out in vivo or in vitro. Preferred, however, is in vitro detection of nucleic acid variants in at least one TLR gene in a biological sample obtained from the subject. The term "biological sample" means a tissue sample or a body fluid sample. A tissue sample includes (but is not limited to) buccal cells, a brain sample, a skin sample or organ sample (e.g. liver). The term "body fluid" refers to all fluids that are present in the body including but not limited to blood, plasma, serum, lymph, synovial fluid, urine, saliva or cerebrospinal fluid. The biological sample may also be obtained by subjecting it to a pretreatment if necessary, for example, by homogenizing or extracting. Such a pretreatment may be selected appropriately by those skilled in the art depending on the biological sample to be subjected.
A nucleic acid comprising an intended sequence prepared from a biological sample may be prepared from DNA (e.g. gDNA or cDNA) or RNA (e.g. mRNA). Release, concentration and isolation of the nucleic acids from the sample can be done by any method known in the art. Currently, various commercial kits are available such as the QIAamp Blood Kit from Qiagen (Hilden, Germany) for the isolation of nucleic acids from blood samples, or the 'High pure PCR Template Preparation Kit' (Roche Diagnostics, Basel, Switzerland) or the DNA purification kits (PureGene, Gentra, Minneapolis, US). Other, well-known procedures for the isolation of DNA or RNA from a biological sample are also available (Sambrook et al., 1989; Ausubel et al., 2003). When the quantity of the nucleic acid is low or insufficient for the assessment, the nucleic acid may be amplified. Such amplification procedures can be accomplished by those methods known in the art, including, for example, the polymerase chain reaction (PCR), ligase chain reaction (LCR), nucleic acid sequence-based amplification (NASBA), strand displacement amplification, rolling circle amplification, Tl- polymerase amplification, and reverse transcription polymerase reaction (RT-PCR).
After performing the extraction and/or amplification procedure, the presence or absence of certain nucleic acid variants in the target sequence can be detected. Numerous methods for detecting a single nucleotide anomaly in nucleic acid sequences are well-known in the art. The present invention is not limited by any particular method used to detect the target sequences disclosed herein. Examples of such methods are described by Gut (2001) and Syvanen (2001), and include, but are not limited to, hybridization methods such as reverse dot blot, Line Probe Assay (LiPA), geneChip™ microarrays, dynamic allel-specific hybridization (DASH), peptide nucleic acid (PNA) and locked nucleic acid (LNA) probes, TaqMan™ (5 'nuclease assay) and molecular beacons; allele-specific PCR methods such as intercalating dye, FRET primers and Alphascreen™; primer extension methods such as ARMS, kinetic PCR, SNPstream™, Genetic Bit Analysis™ (GBA), multiplex minisequencing, SNaPshot, pyrosequencing, MassExtend, MassArray, Goodassay, microarray miniseq, APEX (arrayed primer extension), sequence specific priming (SSP), microarray primer extension, Tag arrays, coded microspheres, template- directed incorporation (TDI), fluorescence polarization; oligonuceotide ligation methods such as colorimetric OLA, sequence-coded OLA, multiplex ligation- dependent probe amplification (MLPA), microarray ligation, ligase chain reaction, padlock probes and rolling circle amplification; endonuclease cleavage methods such as restriction site analysis (RFLP) and Invader™ assay.
In a preferred embodiment, the detection of the presence or absence of a nucleic acid variant is determined by DNA or RNA hybridization, sequencing, PCR, primer extension, MLPA, oligonucleotide ligation assay (OLA) or restriction site analysis, or a combination thereof. In a specific embodiment, the detection of the presence or absence of a nucleic acid variant is determined by a Line Probe Assay (LiPA), or by the 4MAT™ assay. Accordingly, the method of the present invention optionally comprises the steps of isolating nucleic acids from the sample and/or an amplification step.
The present invention also provides isolated oligonucleotides, i.e. primers and probes, in order to amplify and/or detect nucleic acid variants and/or the wild type sequence of at least one TLR gene. The wild type sequences of the TLR genes of the present invention are identified by SEQ ID NOs: 1-10. Such primers or probes, specifically hybridizing to the target nucleic acid, are of any convenient length such as to consist of at least 8, 9, 10, 11, 12, 13, 14 or 15 nucleotides and up to 40 nucleotides, up to 35 nucleotides, up to 30 nucleotides or more conveniently up to 25 nucleotides in length. A preferred length of the primers or probes is thus 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25 nucleotides. In general such primers or probes will comprise nucleotide sequences entirely complementary to the corresponding wild type or variant locus in the TLR gene. However, if required one or more nucleotides may be added or one or more mismatches may be introduced, provided that the discriminatory power of the oligonucleotide primer or probe is not unduly affected. An oligonucleotide primer (or primer pair) designed to specifically recognize and amplify either a wild type or variant allele at a locus is referred to as an allele specific primer (or primer pair). The same applies for an allele specific probe, i.e. an oligonucleotide probe that specifically hybridizes to either a wild type or variant allele.
Specific length and sequence of the probes and primers will depend on the complexity of the required nucleic acid target, as well as on the reaction conditions such as temperature and ionic strength. In general, the hybridization conditions are to be stringent as known in the art. "Stringent" refers to the condition under which a nucleotide sequence can bind to related or non-specific sequences. For example, high temperature and lower salt increases stringency such than non-specific binding or binding with low melting temperature will dissolve. The primers or probes of the invention may carry one or more labels to facilitate detection. The nature of the label is not critical to the invention and may be fluorescent, chemiluminescent, enzymatic, radioactive, chemical or other, provided it doesn't interfere with correct hybridizing of the oligonucleotide. In a preferred embodiment, the primer or probe consists of 10 to 30 nucleotides, preferably 15 to 30 nucleotides, and is capable of specifically forming a hybrid with a part of the TLR gene and is at least one or more selected from the group consisting of:
1) an oligonucleotide capable of hybridizing under a stringent condition with the sequence as represented by SEQ ID NO: 1-10, or the complementary thereof;
2) an oligonucleotide of which the sequence is for 80, 85 or 90% identical to the sequence as represented by SEQ ID NO: 1-10, or the complementary thereof; and
3) an oligonucleotide capable of hybridizing under a stringent condition with the sequence as represented by SEQ ID NO: 1-10 wherein one or more nucleotides was subjected to a variation such as a substitution, deletion, insertion or addition, or the complementary thereof.
More particular, the present invention relates to an isolated oligonucleotide probe consisting of 10 to 30 nucleotides, optionally 15 to 30 nucleotides, for detecting the presence of one or more nucleic acid variants in SEQ ID NO: 1-10, or the complementary strand. More specific, the nucleic acid variants are located at a position selected from the group consisting of:
- position 239 or 914 of SEQ ID NO:1,
- position -15607, 1350 or 2258 of SEQ ID NO:2,
- position -7 or 6301 of SEQ ID NO:3, - position -2026 of SEQ ID NO:4,
- position -30891 or 1846 of SEQ ID NO:5,
- position -502, 745 or 1083 of SEQ ID NO:6,
- position 17962 of SEQ ID NO:7,
- position 9009, 12688, 9300, 12979, -558, 3122, 8683 or 12361 of SEQ ID NO:8, - position -1486 of SEQ ID NO:9, and
- position -1883 of SEQ ID NO: 10.
The polymorphism located in the specific regions of the TLR gene may also be detected in vitro by determining in the isolated TLR protein, as identified in the present invention, the presence or absence of an amino acid change, as indicated in Table 2, by sequencing said protein. The amino acid change may also be detected by any conventional method known in the art, for example by mass-spectroscopy, gel electrophoresis, MALDI-TOF mass spectroscopy, ELISA, protein arrays, determination of the molecular weight, or by isoelectrofocusing. Studies have demonstrated several other known risk factors for altered innate immunity. Any human gene can be studied together with the method of the present invention. Of the different genetic markers identified, further important risk factors are polymorphisms or nucleic acid variations in one or more of the following genes: the ficolin genes (i.e. FCNl, FCN2 and FCN3), ClQRl (complement component 1, q subcomponent, receptor 1), BPI (Bacterial/permeability- increasing protein), LBP, CD 14 (CD 14 antigen precursor), beta-catenin (CTNNBI, Cadherin Associated Protein beta I), ILlO (Interleukin 10), RP 105 (LY64, lymphocyte antigen 64 homo log radioprotective), MBL2 (Mannose Binding Protein), MD-I (RP105-associated), MD- 2 (MD2 Protein, Lymphocyte antigen 96), MYD88 (Myeloid differentiation primary response gene 88), NODl (Caspase recruitment domain 4, CARD4) and NOD2 (Caspase recruitment domain family, member 15, CARD 15). Accordingly, the present invention also relates to a method of identifying a subject at risk of, or having, an indication associated with altered innate immunity, comprising the step of detecting the presence or absence of a nucleic acid variant in at least one TLR gene and detecting the presence or absence of one or more nucleic acid variants in any other gene. More specifically, the "other" gene is selected from the group consisting of: the ficolin genes (i.e. FCNl, FCN2 and FCN3), ClQRl (complement component 1, q subcomponent, receptor 1), BPI (Bacterial/permeability- increasing protein), LBP, CD 14 (CD 14 antigen precursor), beta-catenin (CTNNBI, Cadherin Associated Protein beta), ILlO (Interleukin 10), RP 105 (LY64, lymphocyte antigen 64 homo log radioprotective), MBL2 (Mannose Binding Protein), MD-I (RP 105- associated), MD-2 (MD2 Protein, Lymphocyte antigen 96), MYD88 (Myeloid differentiation primary response gene 88), NODl (Caspase recruitment domain 4, CARD4) and NOD2 (Caspase recruitment domain family, member 15, CARD15). Optionally, other risk factors can be determined in combination with the method of the present invention.
The presence of nucleic acid variants in the gene of a subject may also be reflected in the concentration, structure and functionality of the TLR protein in the serum or plasma of said subject. Therefore, the present invention also encompasses a method for determining whether a subject has a risk of developing a disease wherein the nucleic acid variants in the TLR gene are detected by their protein phenotype. The invention discloses that decreased or increased levels of TLR and lack of functional TLR is crucial in the innate immunity defense. In a specific embodiment, the method encompasses the measurement of one or more proteins.
Accordingly, the present invention relates to a method for identifying a subject at risk of, or having, an indication associated with altered innate immunity comprising the steps of: a) determining the concentration or functionality of at least one TLR protein in a sample, b) identifying if said subject is at risk of, or has, an indication associated with altered innate immunity.
More particular, the current invention provides a method of identifying a subject at risk of, or having, an indication associated with altered innate immunity, comprising measuring the concentration or functionality of at least one TLR protein in a biological sample, wherein an increased or decreased TLR concentration or altered functionality compared to a reference value representing a known health status indicates that said subject is at risk of or suffers from a disorder associated with altered innate immunity.
As used herein, a "TLR protein" is a protein encoded by the TLR gene as described in the present invention, or a variant thereof. For each TLR, the reference amino acid sequence is given in Table 1.
The term "concentration" or "level", as used in the present invention, refers to the presence or absence and/or amount of a certain protein. A change in the concentration of a protein refers to a measurable increase or decrease, including total absence or presence, in the protein concentration when compared to a control subject. "A known health status" or "control subject", as defined in the present invention is a subject of the same species as the subject under examination which is free from, or not at direct risk of developing a disease associated with altered innate immunity. The healthy subject can be of the same weight, age, and gender as the subject who is being diagnosed or prognosed for an altered innate immunity. In some cases, it might be preferred to use a reference value from the subject which is diagnosed.
For the TLR protein, the concentration obtained upon analyzing the subject under examination relative to the concentration obtained upon analyzing a control subject will depend on the particular analytical protocol and detection technique that is used. Accordingly, those skilled in the art will understand that, based on the present description, any laboratory can establish, for the TLR protein, a suitable "reference range", "reference level range", "concentration range" or "range of levels" (those terms are used interchangeable) characteristic for control subjects according to the analytical protocol and detection technique in use. The concentration obtained for the subject under examination can then be compared with this reference range and based on this comparison, a conclusion can be drawn as to whether the subject has a risk or not of developing a disease as described herein. Those skilled in the art will also know how to establish, for the TLR protein, a cut-off value suitable for determining whether a subject is at risk of, or has, an altered innate immunity. Methods for defining cut-off values include (but are not limited to) the methods described by IFCC (1987). In a preferred embodiment, the reference value can be that of a level or concentration of the TLR protein in a sample, preferably a body fluid, from a subject not suffering from a disease associated with altered innate immunity. The healthy subject can be of the same weight, age, and gender as the subject who is being diagnosed or prognosed for an altered innate immunity. In some cases, it might be preferred to use a reference value from the subject which is diagnosed.
The TLR protein that is detected in the method of the present invention, may be detected by any method known to those skilled in the art. They can be identified by their structure, by partial amino acid sequence determination, by functional assay, by enzyme assay, by various immunological methods, or by biochemical methods known to those skilled in the art.
Biochemical methods include (but are not limited to) capillary electrophoresis, high performance liquid chromatography (HPLC), thin layer chromatography (TLC), hyperdiffusion chromatography, two-dimensional liquid phase electrophoresis (2-D- LPE; Davidsson et al. 1999) or detection of the migration pattern in gel electrophoreses. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS- PAGE) is a widely used approach for separating proteins from complex mixtures (Patterson and Aebersold, 1995). It can be performed in one- or two-dimensional (2- D) configuration. For less complicated protein preparation, one-dimensional SDS- PAGE is preferred over 2-D gels, because it is simpler. However, SDS-PAGE often results in migrating or overlapping protein bands due to its limited resolving power. What appears to be a single band may actually be a mixture of different proteins. 2-D gel electrophoresis incorporates isoelectric focusing (IEF) in the first dimension and SDS-PAGE in the second dimension, leading to a separation by charge and size (O'Farrell, 1975). 2-D PAGE is a powerful technique for separating very complex protein preparations, resolving up to 10 000 proteins from mammalian tissues and other complex proteins (Klose and Kobalz, 1995; Celis et al, 1996; Yan et al, 1997). The TLR proteins of the present invention can be identified by their isoelectric focusing point (pi) and their molecular weight (MW) in kilodaltons (kD).
As indicated above, the level of TLR protein can also be detected by an immunoassay. As used herein, an "immunoassay" is an assay that utilizes an antibody to specifically bind to the antigen (i.e. the TLR protein). The immunoassay is thus characterized by detection of specific binding of the TLR protein to an antibody. Immunoassays for detecting TLR proteins may be either competitive or noncompetitive. Noncompetitive immunoassays are assays in which the amount of captured analyte (i.e. the TLR protein) is directly measured. In competitive assays, the amount of analyte (i.e. the TLR protein) present in the sample is measured indirectly by measuring the amount of an added (exogenous) analyte displaced (or competed away) from a capture agent (i.e. the antibody) by the analyte (i.e. the TLR protein) present in the sample. In one competition assay, a known amount of the (exogenous) TLR protein is added to the sample and the sample is then contacted with the antibody. The amount of added (exogenous) TLR protein bound to the antibody is inversely proportional to the concentration of the TLR protein in the sample before the exogenous TLR protein is added. In one preferred "sandwich" assay, for example, the antibodies can be bound directly to a solid substrate where they are immobilized. These immobilized antibodies then capture the TLR protein of interest present in the test sample. Other immunological methods include but are not limited to fluid or gel precipitation reactions, immunodiffusion (single or double), agglutination assays, immunoblotting , immunospotting (such as line immunoassays or LIA), immunoelectrophoresis, radioimmunoassays (RIA), enzyme-linked immunosorbent assays (ELISA), TRIFMA (Christiansen et al., 1999), Western blots, liposome immunoassays (Monroe et al., 1986), complement-fixation assays, immunoradiometric assays, fluorescent immunoassays, protein A immunoassays or immunoPCR. An overview of different immunoassays is given in Wild (2001), Ghindilis et al. (2002) and Kilpatrick (2002). In a further embodiment, the method of the present invention may also be used in determining whether and which therapeutic agent might be suitable for a patient being at risk of, or having an indication associated with altered innate immunity. The therapeutic agent may be used to prevent or treat the indication or disease. As used herein, the term "preventing a disease" means inhibiting or reversing the onset of the disease, inhibiting or reversing the initial signs of the disease, inhibiting the appearance of clinical symptoms of the disease. As used herein, the term "treating a disease" includes substantially inhibiting the disease, substantially slowing or reversing the progression of the disease, substantially ameliorating clinical symptoms of the disease or substantially preventing the appearance of clinical symptoms of the disease.
Another aspect of the invention relates to a diagnostic kit for use in the method as described herein. More specific, the invention encompasses a kit for identifying a subject at risk of, at risk of having, or having, an indication associated with altered innate immunity. This kit can be based on the detection of nucleic acid variants in at least one TLR gene of said subject or it can be based on the detection of TLR proteins. Accordingly, the kit of the present invention comprises reagents that selectively detect a nucleic acid variant in at least one TLR gene or that selectively detect at least one TLR protein.
A kit based on the detection of nucleic acid variants in at least one TLR gene may comprise:
(a) a means or reagent for detecting the presence or absence of one or more nucleic acid variants in at least one TLR gene of said subject; and
(b) optionally, a means for determining, from the nucleic acid variants detected with the means of step (a), whether the subject is at risk of, or has, an indication associated with altered innate immunity.
More preferred, the kit comprises a means for detecting the presence or absence of one or more nucleic acid variants in the promotor region, the 5'UTR region, one or more exon regions and/or one or more intervening regions (IVS) of the TLR gene. Of particular interest are the regions as given in Table 2 for each specific TLR gene. In a preferred embodiment of the present invention, the kit comprises: (a) a means or reagent for detecting the presence or absence of one or more nucleic acid variants at the following positions:
- position 239 or 914 of SEQ ID NO:1,
- position -15607, 1350 or 2258 of SEQ ID NO:2, - position -7 or 6301 of SEQ ID NO:3,
- position -2026 of SEQ ID NO:4,
- position -30891 or 1846 of SEQ ID NO:5,
- position -502, 745 or 1083 of SEQ ID NO:6,
- position 17962 of SEQ ID NO:7, - position 9009, 12688, 9300, 12979, -558, 3122, 8683 or 12362 of SEQ ID
NO:8,
- position -1486 of SEQ ID NO:9, or
- position -1883 of SEQ ID NO: 10; and
(b) optionally, a means for determining, from the nucleic acid variants detected with the means of step (a), whether the subject is at risk of, or has, an indication associated with altered innate immunity.
specific embodiment the means or reagents in step (a) of said kit may comprise:
(i) when appropriate, a means for obtaining a target TLR polynucleic acid present in a biological sample and/or obtaining the nucleotide sequence thereof;
(ii) at least one oligonucleotide suitable for detection of, or specifically hybridizing to, a target TLR nucleic acid and/or at least one oligonucleotide pair suitable for amplification of a target TLR polynucleic acid; (iii) when appropriate, an agent for denaturing nucleic acids;
(iv) when appropriate, an enzyme capable of modifying a double stranded or single stranded nucleic acid molecule; (v) when appropriate, a hybridization buffer, or components necessary for producing said buffer; (vi) when appropriate, a wash solution, or components necessary for producing said solution;
(vii) when appropriate, a means for detecting partially or completely denatured polynucleic acids and/or a means for detecting hybrids formed in the preceding hybridization and/or a means for detecting enzymatic modifications of nucleic acids; and (viii) when appropriate, a means for attaching an oligonucleotide to a known location on a solid support. In a preferred embodiment the means or reagent in step (a) of said kit comprise at least one oligonucleotide probe suitable for detection of a target TLR nucleic acid. More particular, the oligonucleotide probe specifically hybridizes to the target nucleic acid. In a specific embodiment, the target TLR nucleic acid is located in the promotor region, the 5 'UTR region, at least one exon region and/or at least one IVS region of the TLR gene. Of particular interest are the regions as given in Table 2 for each specific TLR gene. Even more specific, the target TLR nucleic acid is located at position 239 or 914 of SEQ ID NO:1, position -15607, 1350 or 2258 of SEQ ID NO:2, position -7 or 6301 of SEQ ID NO:3, position -2026 of SEQ ID NO:4, position -30891 or 1846 of SEQ ID NO:5, position -502, 745 or 1083 of SEQ ID NO:6, position 17962 of SEQ ID NO:7, position 9009, 12688, 9300, 12979, -558, 3122, 8683 or 12362 of SEQ ID NO:8, position -1486 of SEQ ID NO:9 and/or position - 1883 of SEQ ID NO: 10. The designated positions either have the wild type nucleotides or nucleic acid variants thereof. In a specific embodiment, the oligonucleotide suitable for detection of a target TLR nucleic acid is a SSP (sequence specific primer). More specifically, the oligonucleotide suitable for detection of a target TLR nucleic acid is a probe configured to hybridize the said TLR polynucleic acid to form an invase cleavage structure. The cleavage structure can be detected by a cleavage agent or enzyme such as a structure-specific nuclease, a 5 'nuclease, a FEN-I endonuclease or a polymerase. Optionally, the means or reagent in step (a) also includes at least one pair of oligonucleotide primers suitable for amplification of a target TLR polynucleic acid. More particular, each of the primers specifically hybridizes to the TLR nucleic acid sequence. In a specific embodiment, the target TLR polynucleic acid to be amplified is the promotor region, the 5'UTR region, at least one exon region and/or at least one IVS region of the TLR gene, or part thereof. Particular targets are the regions as given in Table 2 for each specific TLR gene. Preferably, the target polynucleic acid comprises at least one of the polymorphic positions as given in Table 2. The designated positions either have the wild type nucleotides or nucleic acid variants thereof. The term "hybridization buffer" means a buffer allowing a hybridization reaction between the oligonucleotides and the polynucleic acids present in the sample, or the amplified products, under the appropriate stringency conditions. The term "wash solution" means a solution enabling washing of the hybrids formed under the appropriate stringency conditions.
In a specific embodiment of the kit, the means for detecting the presence or absence of nucleic acid variants in the TLR gene is an INVADER assay (see e.g. WO97/27214, incorporated herein by reference), or the 4MAT™ assay (Innogenetics N. V., Ghent, Belgium).
In a more specific embodiment of the kit, the means for detecting the presence or absence of nucleic acid variants in the TLR gene is a line probe assay (LiPA; Stuyver et al, 1996; Stuyver et al, 1997; Van Geyt et al, 1998). In this embodiment, the selected set of probes is immobilized to a membrane strip in a line fashion. An alternative is the immobilization of the probes in a "dotted fashion" (dot spots; DoPA). Said probes may be immobilized individually or as mixtures to the delineated locations. The amplified TLR polynucleic acids can be labelled with biotine, and the hybrid can then, via a biotine-streptavidine coupling, be detected with a non- radioactive colour developing system. Particularly advantageous are other systems in which different nucleic acid variants can be detected simultaneously. In this multiparameter approach, oligonucleotides may be coupled to microspheres or chips. An example of an assay that provides for simultaneous detection includes (but is not limited to) the xMAP™ technology (Luminex®100 IS, Austin, Texas, USA) and the PamGene technology (PamGene, 's-Hertogenbosch, The Netherlands).
The means in step (b) of said kit, for determining, from the nucleic acid variants in the TLR gene detected with the means of step (a), whether the subject is at risk of, or has, an indication associated with altered innate immunity include a table, a chart, or similar, generally referred to as "a predisposition risk algorithm", indicating the TLR nucleic acid variants or haplotypes that confer a risk for or the existence of an indication associated with altered innate immunity. As used herein, the term "chart" refers to graphical presentation, visual aid, diagram, plan, graph, sheet, map or the like including the relevant information. The determination of the risk can be performed manually or with the use of a computer.
The kit of the present invention may include, in addition to the means or reagent for detecting the presence or absence of a nucleic acid variant in at least one TLR gene, a means for detection other risk factors, e.g. nucleic acid variants in a gene, for an indication associated with an altered innate immunity. In a preferred embodiment, the kit additionally includes a means, preferably probes, for detecting the genotype of or a nucleic acid variant in at least one of the genes selected from the group consisting of: the ficolin genes (FCNl, FCN2 and FCN3), ClQRl, BPI (Bacterial/permeability- increasing protein), LBP, CD 14 (CD 14 antigen precursor), beta-catenin (CTNNBI, Cadherin Associated Protein beta), ILlO (Interleukin 10), RP 105 (LY64, lymphocyte antigen 64 homo log radioprotective), MBL2 (Mannose Binding Protein), MD-I (RP105-associated), MD-2 (MD2 Protein, Lymphocyte antigen 96), MYD88 (Myeloid differentiation primary response gene 88), NODl (Caspase recruitment domain 4, CARD4) and NOD2 (Caspase recruitment domain family, member 15, CARD 15).
A kit based on the detection of TLR proteins may comprise an antibody that specifically recognizes the TLR protein that is detected. A preferred kit for carrying out the method of the invention comprises: an antibody (primary antibody) which forms an immunological complex with the TLR protein to be detected; a monoclonal antibody (secondary antibody) which specifically recognizes the TLR protein to be detected; a marker either for specific tagging or coupling with said secondary antibody; appropriate buffer solutions for carrying out the immunological reaction between the primary antibody and the TLR protein, between the secondary antibody and the primary antibody-TLR protein complex and/or between the bound secondary antibody and the marker; or - possibly, for standardization purposes, a purified TLR protein. EXAMPLES
For the present examples, the statistical analysis of the data is based on the determination of odds ratios (OR) using standard procedures. An odds ratio is calculated by dividing the odds in the treated or exposed (case) group by the odds in the control group. The odds of an event are calculated as the number of events divided by the number of non-events. If the odds of an event are greater than one the event is more likely to happen than not (the odds of an event that is certain to happen are infinite); if the odds are less than one the chances are that the event won't happen (the odds of an impossible event are zero). In the present examples, the strength of association was reported as odds ratios (OR) (with 95% lower (LCL) and upper (UCL) confidence limit), indicating the factor by which the risk of developing a disorder or disease is increased (OR>1), or indicating the factor by which the risk of developing a disorder or disease is decreased (OR<1) (protective effect).
The 95% confidence interval (95% CI) is the range of numerical values in which we can be confident (to a computed probability, such as 90 or 95%) that the population value being estimated will be found. Confidence intervals indicate the strength of evidence; where confidence intervals are wide, they indicate less precise estimates of effect. The larger the trial's sample size, the larger the number of outcome events and the greater becomes the confidence that the true relative risk reduction is close to the value stated. Thus the confidence intervals narrow and "precision" is increased. In a "positive finding" study the lower boundary of the confidence interval, or lower confidence limit, should still remain important or clinically significant if the results are to be accepted. In a "negative finding" study, the upper boundary of the confidence interval should not be clinically significant if you are to confidently accept this result.
Example 1: Detection of nucleic acid variants in the TLR genes from patients with Recurrent Otitis Media (OM) and from control subjects Patients samples
A study was carried out based on DNA isolated from blood samples from 17 patients with recurrent Otitis Media. The control group (C) consisted of 205 healthy individuals. From each patient, informed consent to participate in the study is available
Detection of nucleic acid polymorphisms
To determine the presence or absence of nucleic acid variants in the TLR genes relevant regions (see Table 2) were amplified using biotinylated oligonucleotides. The polymorphisms were detected by use of a reverse hybridization method (Dot Probe Assay) with probes designed to recognize the polymorphisms as given in Table 2. After stringent washing at 56°C, hybridized probes were incubated with a streptavidine-alkaline phosphatase conjugate. The presence of a hybridized probe was revealed using NBIT/BCIP color development. Details on the reverse hybridization are described in Stuyver et al. (1996), Stuyver et al. (1997) and Van Geyt et al. (1998).
Statistical analysis
17 patients with recurrent OM and 172 C-diagnosed subjects were genotyped for SNPs in all the TLR genes.
The strength of association was reported as odds ratios (OR). The 95% confidence interval (95% CI) is the interval computed from the sample data which, if the study repeated multiple times, would contain the true effect 95% of the time.
Frequencies of TLR genotypes in OM patients and control individuals
The frequencies (numerical and in terms of percentage) of the investigated SNPs in the different TLR genes in Otitis Media patients and in healthy control individuals are indicated in Table 3. It was found that 1. the presence of the minor allele for the TLRl -rs5743611 (exon 4) polymorphism is associated with and results in a higher susceptibility for recurrent otitis media
[OR 3,26 (95%CI 1,15-9,3)], 2. the presence of the minor allele for the TLR6-rs3821985 (exonl) polymorphism is associated with and results in a higher susceptibility for recurrent otitis media [OR 2,41 (95%CI 1,17-5)],
3. the presence of the minor allele for the TLR7-rsl79008 polymorphism (exon 3) has a protective effect on the development of recurrent otitis media [OR 0,14
(95%CI 0,02-1,01)].
Example 2: Detection of nucleic acid variants in the TLR genes from patients with sepsis/severe sepsis/septic shock and from control subjects
Patients samples
A study was carried out based on DNA isolated from blood samples from 12 patients with either sepsis, severe sepsis or septic shock. The control group (C) consisted of 205 healthy individuals. From each patient, informed consent to participate in the study is available
Detection of nucleic acid polymorphisms
To determine the presence or absence of nucleic acid variants in the TLR genes, the relevant regions (see Table 2) were amplified using biotinylated oligonucleotides. The polymorphisms were detected by use of a reverse hybridization method (Dot Probe Assay) with probes designed to recognize the polymorphisms as given in Table 2. After stringent washing at 56°C, hybridized probes were incubated with a streptavidine-alkaline phosphatase conjugate. The presence of a hybridized probe was revealed using NBIT/BCIP color development. Details on the reverse hybridization are described in Stuyver et al. (1996), Stuyver et al. (1997) and Van Geyt et al. (1998).
Statistical analysis
12 patients with sepsis/severe sepsis/septic shock and 205 C-diagnosed subjects were genotyped for SNPs in the TLR genes.
The strength of association was reported as odds ratios (OR). The 95% confidence interval (95% CI) is the interval computed from the sample data which, if the study repeated multiple times, would contain the true effect 95% of the time. Frequencies of TLR genotypes in patients with sepsis/severe sepsis/septic shock and control individuals
The frequencies (numerical and in terms of percentage) of the investigated SNPs in the TLR genes in patients with sepsis, severe sepsis or septic shock and in healthy control individuals are indicated in Table 3. It was found that 1. the presence of the minor allele for the TLRl -rs3923647 polymorphism (exon 4) is associated with and results in a higher susceptibility for sepsis, severe sepsis or septic shock [OR 6,33 (95CI 1,56-25,1)], 2. the presence of the minor allele for the TLR6-rs3821985 polymorphism (exon 1) is associated with and results in a higher susceptibility for sepsis, severe sepsis or septic shock [OR 2,98 (95%CI 1,3-6,9)],
3. the presence of the minor allele for the TLR10-rs7694115 polymorphism (promotor) is associated with and results in a higher susceptibility for sepsis, severe sepsis or septic shock [OR 2,66 (95%CI 1,16-6,11)],
4. the presence of the minor allele for the TLR6- 1039559 polymorphism (promotor) has a protective effect on the development of sepsis, severe sepsis or septic shock [OR 0,27 (95%CI 0,1-0,73)],
5. the presence of the minor allele for the TLR4-rs 1927914 polymorphism (promotor) has a protective effect on the development of sepsis, severe sepsis or septic shock [OR 0,24 (95%CI 0,01-0,6)].
Example 3: Detection of nucleic acid variants in the TLR genes from patients with Rheumatoid arthritis and from control subjects
Patients samples
A study was carried out based on DNA isolated from blood samples from 164 patients with Rheumatoid arthritis. The control group (C) consisted of 205 healthy individuals. From each patient, informed consent to participate in the study is available
Detection of nucleic acid polymorphisms
To determine the presence or absence of nucleic acid variants in the TLR genes, the relevant regions (see Table 2) were amplified using biotinylated oligonucleotides. The polymorphisms were detected by use of a reverse hybridization method (Dot Probe Assay) with probes designed to recognize the polymorphisms as given in Table 2. After stringent washing at 56°C, hybridized probes were incubated with a streptavidine-alkaline phosphatase conjugate. The presence of a hybridized probe was revealed using NBIT/BCIP color development. Details on the reverse hybridization are described in Stuyver et al. (1996), Stuyver et al. (1997) and Van Geyt et al. (1998).
Statistical analysis
164 patients with Rheumatoid arthritis and 205 C-diagnosed subjects were genotyped for SNPs in the TLR genes.
The strength of association was reported as odds ratios (OR). The 95% confidence interval (95% CI) is the interval computed from the sample data which, if the study repeated multiple times, would contain the true effect 95% of the time.
Frequencies of TLR genotypes in patients with Rheumatoid arthritis and control individuals
The frequencies (numerical and in terms of percentage) of the investigated SNPs in the TLR genes in patients with Rheumatoid arthritis and in healthy control individuals are indicated in Table 3. It was found that
1. the presence of the minor allele for the TLR2-rs3804100 polymorphism (exon 2) is associated with and results in a higher susceptibility for Rheumatoid arthritis [OR 2 (95%CI 1,13-3,54)],
2. the presence of the minor allele for the TLR3-rs3775296 polymorphism (IVSl) is associated with and results in a higher susceptibility for Rheumatoid arthritis [OR
1,53 (95%CI 1,04-2,24)],
3. the presence of the minor allele for the TLR6-rs5743810 polymorphism (exonl) is associated with and results in a higher susceptibility for Rheumatoid arthritis [OR 1,31 (95%CI 0,98-1,76)], 4. the presence of the minor allele for the TLR2-rsl 898830 polymorphism (promotor) has a protective effect on the development of Rheumatoid arthritis [OR 0,73 (95%CI 0,54-0,994)], 5. the presence of the minor allele for the TLR8-rs5744080 polymorphism (exon3) has a protective effect on the development of Rheumatoid arthritis [OR 0,72 (95%CI 0,54-0,97)].
Example 4: Detection of nucleic acid variants in the TLR genes from children with recurrent infections and from control subjects
Patients samples
A study was carried out based on DNA isolated from blood samples from 264 children with recurrent infections. The control group (C) consisted of 205 healthy individuals. From each patient, informed consent to participate in the study is available.
Detection of nucleic acid polymorphisms To determine the presence or absence of nucleic acid variants in the TLR genes, the relevant regions (see Table 2) were amplified using biotinylated oligonucleotides. The polymorphisms were detected by use of a reverse hybridization method (Dot Probe Assay) with probes designed to recognize the polymorphisms as given in Table 2. After stringent washing at 56°C, hybridized probes were incubated with a streptavidine-alkaline phosphatase conjugate. The presence of a hybridized probe was revealed using NBIT/BCIP color development. Details on the reverse hybridization are described in Stuyver et al. (1996), Stuyver et al. (1997) and Van Geyt et al. (1998).
Statistical analysis
264 children with recurrent infections and 205 C-diagnosed subjects were genotyped for different SNPs in the TLR genes.
The strength of association was reported as odds ratios (OR). The 95% confidence interval (95% CI) is the interval computed from the sample data which, if the study repeated multiple times, would contain the true effect 95% of the time.
Frequencies of TLR genotypes in children with recurrent infections and control individuals The frequencies (numerical and in terms of percentage) of the investigated SNPs in the TLR genes in children with recurrent infections and in healthy control individuals are indicated in Table 3. It was found that 1. the presence of the minor allele for the TLRl-rs5743611 polymorphism (exon4) is associated with and results in a higher susceptibility for recurrent infections in children [OR 1,74 (95%CI 1,03-2,92)],
2. the presence of the minor allele for the TLR3-rs3775296 polymorphism (IVSl) is associated with and results in a higher susceptibility for recurrent infections in children [OR 1,68 (95%CI 1,2-2,37)],
3. the presence of the minor allele for the TLR5-rs5744174 polymorphism (exonό) is associated with and results in a higher susceptibility for recurrent infections in children [OR 1,36 (95%CI 1,04-1,76)],
4. the presence of the minor allele for the TLR8-rs2159377 polymorphism (exon3) has a protective effect on the development of recurrent infections in children [OR
0,64 (95%CI 0,46-0,89)],
5. the presence of the minor allele for the TLR9-rs 187084 polymorphism (promotor) has a protective effect on the development of recurrent infections in children [OR 0,75 (95%CI 0,57-0,97)].
Example 5: Detection of nucleic acid variants in the TLR genes from children with a haematological malignancy or solid tumor and from control subjects
Patients samples
A study was carried out based on DNA isolated from blood samples from 277 children in an oncology clinic. The control group (C) consisted of 205 healthy individuals. From each patient, informed consent to participate in the study is available
Detection of nucleic acid polymorphisms
To determine the presence or absence of nucleic acid variants in the TLR genes, the relevant regions (see Table 2) were amplified using biotinylated oligonucleotides. The polymorphisms were detected by use of a reverse hybridization method (Dot Probe Assay) with probes designed to recognize the polymorphisms as given in Table 2. After stringent washing at 56°C, hybridized probes were incubated with a streptavidine-alkaline phosphatase conjugate. The presence of a hybridized probe was revealed using NBIT/BCIP color development. Details on the reverse hybridization are described in Stuyver et al. (1996), Stuyver et al. (1997) and Van Geyt et al. (1998).
Statistical analysis
277 children with either a haematological malignancy or solid tumor and 205 C- diagnosed subjects were genotyped for SNPs in the different TLR genes.
The strength of association was reported as odds ratios (OR). The 95% confidence interval (95% CI) is the interval computed from the sample data which, if the study repeated multiple times, would contain the true effect 95% of the time.
Frequencies of TLR genotypes in children with a haematological malignancy or solid tumor and control individuals
The frequencies (numerical and in terms of percentage) of the investigated SNPs in the TLR genes in children with a haematological malignancy or solid tumor and in healthy control individuals are indicated in Table 3. It was found that
1. the presence of the minor allele for the TLR2-rs3804100 polymorphism (exon2- S450S) is associated with and results in a higher susceptibility for haematological malignancy or solid tumor in children [OR 1,88 (95%CI 1,11-3,18)],
2. the presence of the minor allele for the TLR6-rs5743810 polymorphism (exonl) has a protective effect on the development of haematological malignancy or solid tumor in children [OR 0,74 (95%CI 0,57-0,97)],
3. the presence of the minor allele for the TLR6-rsl039559 (promo tor) polymorphism has a protective effect on the development of haematological malignancy or solid tumor in children [OR 0,77 (95%CI 0,6-1,00)], 6. the presence of the minor allele for the TLR5-rs759303 polymorphism (promotor) has a protective effect on the development of haematological malignancy or solid tumor in children [OR 0,51 (95%CI 0,26-0,99)]. Example 6: Detection of nucleic acid variants in toll-like receptor genes from patients newly diagnosed with rheumatoid arthritis (RA) and from non-RA subjects
Patient samples
A cohort of 541 patients having symptoms of arthritis were incorporated in the study. RA was included in the differential diagnosis. After 1 year, diagnosis established 149 RA patients and 402 non-RA patients (control population). The non-RA control population (n=402) had the following diagnoses: osteoarthritis, abarticular rheumatic symptoms, spondyloarthropathy, connective tissue diseases including polymyalgia rheumatica, psoriatic arthritis, crystal induced arthritis, fibromyalgia and aspecific arthralgias, other and undifferentiated diseases including infections, malignancies, and neurological disorders. It can be estimated that in about 50% of this control population, pathways of innate immunity may be involved. From each individual from which a blood sample was taken, informed consent to participate in the study is available.
Detection of nucleic acid polymorphisms
To determine the presence or absence of nucleic acid variants in the TLR genes, the relevant regions (see Table 2) were amplified using biotinylated oligonucleotides. The polymorphisms were detected by use of a reverse hybridization method (Dot Probe Assay) with probes designed to recognize the polymorphisms as given in Table 2. After stringent washing at 56°C, hybridized probes were incubated with a streptavidine-alkaline phosphatase conjugate. The presence of a hybridized probe was revealed using NBIT/BCIP color development. Details on the reverse hybridization are described in Stuyver et al. (1996, J. Clin. Microbiol. 34:2259-2266), Stuyver et al. (1997, Antimicrob. Agents Chemoter. 41:284-291) and Van Geyt et al. (1998, in: Therapies of viral Hepatitis. International Medical Press, London, UK pp. 139-145).
Statistical analysis
149 patients with Rheumatoid arthritis and 402 C-diagnosed subjects were genotyped for different SNPs in the TLR genes. The strength of association was reported as odds ratios (OR), indicating the factor by which the risk of developing RA is increased.
The 95% confidence interval (95% CI) is the interval computed from the sample data which, were the study repeated multiple times, would contain the true effect 95% of the time.
Frequencies and odds ratios of TLR genotypes in patients with Rheumatoid arthritis vs non-RA patients
Based on the screening of the above-described patient groups, it was found that: 1. the presence of the minor allele (C allele) for the TLRl -rs5743611 polymorphism (exon 4) resulted in a protection against the development of Rheumatoid arthritis [0,69 (0,43-1,I)],
2. the presence of the minor allele (T allele) for the TLRl -rs3923647 (exon 4) resulted in a protection against the development of Rheumatoid arthritis [OR 0,35 (0,1-1,16],
3. the presence of the minor allele (A allele) for the TLR2-rs5743708 polymorphism (exon 2) increases the risk of developing or having RA [2,98 (1,49-5,98)],
4. the presence of the minor allele (G allele) for the TLR8-rs5744077 polymorphism (exon 3) increases the risk of developing or having RA [2,71 (0,38-19,3], 5. the presence of the minor allele (T allele) for the TLR8-rs2159377 polymorphism (exon 3) increases the risk of developing or having RA [1,57 (1,12-2,22].
Figure imgf000049_0001
In addition to the importance of the TLR SNPs in the diagnosis of RA (versus healthy controls), the above data also show the value of said SNPs in the differential diagnosis of RA versus other diseases such as osteoarthritis, abarticular rheumatic symptoms, spondyloarthropathy, connective tissue diseases including polymyalgia rheumatica, psoriatic arthritis, crystal induced arthritis, fibromyalgia and aspecific arthralgias, being other and undifferentiated diseases including infections, malignancies, and neurological disorders.
Example 7: Detection of nucleic acid variants in toll-like receptor genes from patients admitted to the ICU who develop septic shock and those who do NOT develop sepsis/severe sepsis or septic shock.
Patient samples
171 patients presenting at an ICU service were incorporated in the study. At discharge the patients were evaluated whether or not sepsis/severe sepsis or septic shock has been developed using clinical criteria, well known by ICU practitioners. From this study, 88 patients were diagnosed as NOT having sepsis/severe sepsis or septic shock while 50 of the patients indeed developed septic shock. 33 patients with sepsis or severe sepsis were excluded.
From each individual from which a blood sample was taken, informed consent to participate in the study is available.
Detection of nucleic acid polymorphisms
To determine the presence or absence of nucleic acid variants in the TLR genes, the relevant regions (see Table 2) were amplified using biotinylated oligonucleotides. The polymorphisms were detected by use of a reverse hybridization method (Dot Probe Assay) with probes designed to recognize the polymorphisms as given in Table 2. After stringent washing at 56°C, hybridized probes were incubated with a streptavidine-alkaline phosphatase conjugate. The presence of a hybridized probe was revealed using NBIT/BCIP color development. Details on the reverse hybridization are described in Stuyver et al. (1996, J. Clin. Microbiol. 34:2259-2266), Stuyver et al. (1997, Antimicrob. Agents Chemoter. 41 :284-291) and Van Geyt et al. (1998, in: Therapies of viral Hepatitis. International Medical Press, London, UK pp. 139-145). Statistical analysis
88 patients without sepsis/severe sepsis or septic shock and 50 patients developing septic shock were genotyped for different SNPs in the TLR genes. The strength of association was reported as odds ratios (OR), indicating the factor by which the risk of developing RA is increased.
The 95% confidence interval (95% CI) is the interval computed from the sample data which, were the study repeated multiple times, would contain the true effect 95% of the time.
Frequencies and odds ratios of TLR genotypes in patients with septic shock vs patient without sepsis/severe sepsis or septic shock
Based on the screening of the above-described patient groups, it was found that: 1. the presence of the C allele for the TLR5-rs5744174 polymorphism (exon 6) increases the risk of developing septic shock [1.72 (1,03-2,87)], 2. the presence of the C allele for the TLR8-rs 1548731 polymorphism
(promotor) resulted in a protection against the development of septic shock [0,43 (0,23-0,80)].
Figure imgf000051_0001
Example 8: Detection of nucleic acid variants in the toll-like receptor genes from patients admitted to the ICU who develop septic shock and those who do NOT develop sepsis/severe sepsis or septic shock.
Patient samples
In this retrospective study a total of 280 patients admitted to the ICU who developed septic shock were incorporated. Septic shock has been defined using the standard clinical utilities. This cohort has been compared with samples from 371 subjects admitted to the ICU without the development of sepsis/severe sepsis or septic shock.
From each individual from which a blood sample was taken, informed consent to participate in the study is available.
Detection of nucleic acid polymorphisms To determine the presence or absence of nucleic acid variants in the TLR genes, the relevant regions (see Table 2) were amplified using biotinylated oligonucleotides. The polymorphisms were detected by use of a reverse hybridization method (Dot Probe Assay) with probes designed to recognize the polymorphisms as given in Table 2. After stringent washing at 56°C, hybridized probes were incubated with a streptavidine-alkaline phosphatase conjugate. The presence of a hybridized probe was revealed using NBIT/BCIP color development. Details on the reverse hybridization are described in Stuyver et al. (1996, J. Clin. Microbiol. 34:2259-2266), Stuyver et al. (1997, Antimicrob. Agents Chemoter. 41:284-291) and Van Geyt et al. (1998, in: Therapies of viral Hepatitis. International Medical Press, London, UK pp. 139-145).
Statistical analysis
280 patients with septic shock and 371 patients without signs of sepsis/severe sepsis or septic shock were genotyped for SNPs in the TLR genes. The strength of association was reported as odds ratios (OR), indicating the factor by which the risk of developing septic shock is increased.
The 95% confidence interval (95% CI) is the interval computed from the sample data which, were the study repeated multiple times, would contain the true effect 95% of the time. Frequencies and odds ratios of TLR genotypes in patients with septic shock vs patient without sepsis/severe sepsis or septic shock
Based on the screening of the above-described patient groups, it was found that: the presence of the minor allele (T allele) for the TLR3-rs3775291 polymorphism (exon 4) protects against the development of septic shock [0.75 (0,59-0,96)].
Figure imgf000053_0001
Table 3
M = major allele (wt) m = minor allele (variant)
Ul
Figure imgf000054_0001
Table 3 continued
Figure imgf000055_0001
Table 3 continued
Ul Ul
Figure imgf000056_0001
Table 3 continued
Ul
Figure imgf000057_0001
Table 3 continued
Ul -4
Figure imgf000058_0001
Table 3 continued
Ul
Figure imgf000059_0001
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Claims

1. A method of identifying a subject at risk of, or having, an indication associated with altered innate immunity, comprising detecting the presence or absence of at least one nucleic acid variant in at least one TLR gene, or part thereof, whereby the presence of at least one nucleic acid variant identifies whether a subject is at risk or has an indication associated with an altered innate immunity.
2. The method according to claim 1, whereby the TLR gene is selected from the group consisting of: TLRl, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8,
TLR9 and TLRlO.
3. The method according to claims 1 or 2, whereby the presence or absence of the nucleic acid variant is detected in exon 4 of TLRl, the promotor region or exon 2 of TLR2, IVSl or exon 4 of TLR3, the promotor region of TLR4, the promotor region or exon 6 of TLR5, the promotor region or exon 1 of TLR6, exon 3 of TLR7, exon 3, promoter region or IVSl of TLR8, the promotor region of TLR9 and/or the promotor region of TLRlO.
4. The method according to claim 3 whereby the nucleic acid variant is located at: position 239 or 914 of the reference gDNA sequence of TLRl as identified by
SEQ ID NO: 1, position -15607, 1350 or 2258 of the reference gDNA sequence of TLR2 as identified by SEQ ID NO:2, - position -7 or 6301 of the reference gDNA sequence of TLR3 as identified by
SEQ ID NO:3, position -2026 of the reference gDNA sequence of TLR4 as identified by SEQ
ID NO:4, position -30891 or 1846 of the reference gDNA sequence of TLR5 as identified by SEQ ID NO:5, position -502, 745 or 1083 of the reference gDNA sequence of TLR6 as identified by SEQ ID NO:6, position 17962 of the reference gDNA sequence of TLR7 as identified by SEQ
ID NO:7, - position 9009, 12688, 9300, 12979, -558, 3122, 8683 or 12362 of the reference gDNA sequence of TLR8 as identified by SEQ ID NO:8, position -1486 of the reference gDNA sequence of TLR9 as identified by SEQ ID NO:9, and/or - position -1883 of the reference gDNA sequence of TLRlO as identified by
SEQ ID NO: 10.
5. The method according to claims 1-4 wherein the nucleic acid variant is a substitution, deletion and/or insertion of at least one nucleotide.
6. The method according to claims 1-5 wherein the presence of at least one nucleic acid variant identifies a subject at risk of, or having, an increased or reduced susceptibility for disease.
7. The method according to claims 1-5 wherein the presence of at least one nucleic acid variant identifies a subject at risk of, or having, an increased severity of disease.
8. The method according to claims 1-5 wherein the presence of at least one nucleic acid variant identifies a subject at risk of, or having, a modified response to therapy for a disease.
9. The method according to claims 6 wherein the presence of at least one nucleic acid variant identifies the protective effect on the development a disease.
10. The method according to claims 1-5 wherein the presence of at least one nucleic acid variant identifies a subject at risk of, or having, an increased risk of transplant rejection.
11. The method according to claims 6-9 wherein the disease is an infection, an autoimmune disease, an inflammatory disease, a neurological disease, cancer, cystic fibrosis or a cardiovascular disease.
12. The method according to claim 11, wherein the infection is a bacterial, viral, fungal, parasitic or algae infection.
13. The method according to claim 12 wherein the infection results in sepsis, severe sepsis, septic shock or otitis media.
14. The method according to claim 11, wherein the autoimmune disease is rheumatoid arthritis (RA), spondyloarthropathy, systemic lupus erythematosus, multiple sclerosis, Crohn's disease, coeliac disease, Type 1 diabetes, Kawasaki disease, atopic disease, asthma, atopic dermatitis, dermatomyositis. Behcet's disease or
Sjogren's Syndrome.
15. The method according to claim 11, wherein the neurological disease is Alzheimer's disease, myasthenia gravis, multiple sclerosis, microbial infections, head trauma and stroke, Pick's disease, Parkinson's disease, dementia with Lewy bodies, Huntington disease, chromosome 13 dementias, Down's syndrome, cerebrovascular disease, Rasmussen's encephalitis, viral meningitis, NPSLE, amyotrophic lateral sclerosis, Creutzfeldt-Jacob disease, Gerstmann-Straussler- Scheinker disease, transmissible spongiform encephalopathies or ischemic reperfusion damage.
16. The method according to claim 11, wherein cancer is a solid tumor or a haematological malignancy.
17. The method according to claim 11 wherein the cardiovascular disease is bypass failure, atherosclerosis, myocardial reperfusion injury, coronary artery disease or heart disease.
18. The method according to claim 10 wherein the transplant is an organ or bone marrow.
19. The method according to claims 1-18, further comprising the detection of the presence or absence of a nucleic acid variant in any other gene.
20. The method according to claim 19 wherein the gene is selected from the group consisting of: FCNl, FCN2, FCN3, ClQRl, BPI, LBP, CD 14, beta-catenin, ILlO, RP105, MBL2, MD-I, MD-2, MYD88, NODl and NOD2.
21. The method according to any of claims 1-20, wherein the detection for the presence or absence of the nucleic acid variant is performed by at least one of the following methods: hybridization, sequencing, PCR, MLPA, OLA, primer extension or restriction site analysis.
22. A method of identifying a subject at risk of, or having, an indication associated with altered innate immunity, comprising measuring the concentration or functionality of at least one TLR protein in a biological sample, wherein an increased or decreased TLR concentration or altered functionality compared to a reference value representing a known health status indicates that said subject is at risk or suffers from a disorder associated with altered innate immunity.
23. A kit for identifying a subject at risk of, or having, an indication associated with altered innate immunity, comprising:
(a) a means for detecting the presence or absence of one or more nucleic acid variants in at least one TLR gene; and
(b) a means for determining, from the nucleic acid variant detected with the means of step (a), whether the subject is at risk of, or has, an indication associated with altered innate immunity.
24. A kit according to claim 23 wherein the means in step (a) is at least one oligonucleotide suitable for detection of a target TLR nucleic acid and/or at least one oligonucleotide pair suitable for amplification of a target TLR polynucleic acid.
25. A kit for identifying a subject at risk of, or having, an indication associated with altered innate immunity, comprising:
(a) an antibody for detecting the presence or absence of at least one TLR protein; and (b) a means for determining, from the TLR protein detected with the means of step (a), whether the subject is at risk of, or has, an indication associated with altered innate immunity.
26. A method for selecting an appropriate treatment or therapeutic agent for a subject at risk of, or having, an indication associated with altered innate immunity, comprising determining the presence of an indication associated with altered innate immunity by the method according to any of claims 1-22, and - selecting an appropriate treatment or therapeutic agent which elicits a high responsiveness and/or low adverse effects in said subject.
27. The method or kit according to any of claims 1-26 wherein the nucleic acid sequence of the wild type TLRl gene is identified by SEQ ID NO: 1, the wild type TLR2 gene is identified by SEQ ID NO:2, the wild type TLR3 gene is identified by SEQ ID NO:3, the wild type TLR4 gene is identified by SEQ ID NO:4, the wild type TLR5 gene is identified by SEQ ID NO:5, the wild type TLR6 gene is identified by SEQ ID NO:6, the wild type TLR7 gene is identified by SEQ ID NO:7, the wild type TLR8 gene is identified by SEQ ID NO:8, the wild type TLR9 gene is identified by SEQ ID NO:9 and the wild type TLRlO gene is identified by SEQ ID NO: 10.
28. The method or kit according to claim 28 wherein the nucleic acid variant is detected in the corresponding cDNA or RNA sequence.
29. The method according to any of claims 1 to 22 and 26 to 29, further characterized in that the presence or absence of a nucleic acid variant in the TLR gene is determined in vitro.
30. An isolated oligonucleotide consisting of 10 to 30 nucleotides for detecting the presence of one or more nucleic acid variants in at least on TLR gene.
31. The oligonucleotide of claim 30, whereby the nucleic acid variant is located at the following position: - position 239 or 914 of the reference gDNA sequence of TLRl as identified by SEQ ID NO: 1,
- position -15607, 1350 or 2258 of the reference gDNA sequence of TLR2 as identified by SEQ ID NO:2, - position -7 or 6301 of the reference gDNA sequence of TLR3 as identified by
SEQ ID NO:3, position -2026 of the reference gDNA sequence of TLR4 as identified by SEQ ID NO:4,
- position -30891 or 1846 of the reference gDNA sequence of TLR5 as identified by SEQ ID NO:5,
- position -502, 745 or 1083 of the reference gDNA sequence of TLR6 as identified by SEQ ID NO:6, position 17962 of the reference gDNA sequence of TLR7 as identified by SEQ ID NO:7, - position 9009, 12688, 9300, 12979, -558, 3122, 8683 or 12362 of the reference gDNA sequence of TLR8 as identified by SEQ ID NO:8,
- position -1486 of the reference gDNA sequence of TLR9 as identified by SEQ ID NO:9, and/or
- position -1883 of the reference gDNA sequence of TLRlO as identified by SEQ ID NO: 10.
32. A pair of primers suitable for amplification of a target TLR polynucleic acid.
33. The pair of primers according to claim 32 whereby the target polynucleic acid is: - exon 4 of the TLRl gene, the promotor and/or exon 2 of the TLR2 gene,
- IVS 1 or exon 4 of the TLR3 gene, the promotor of the TLR4 gene, the promotor and/or exon 6 of the TLR5 gene, - the promotor and/or exon 1 of the TLR6 gene, exon 3 of the TLR7 gene,
- exon 3, IVSl and/or the promotor of the TLR8 gene,
- the promotor of the TLR9 gene, or
- the promotor of the TLRlO gene.
34. The pair of primers according to claim 32 or 33 whereby the target polynucleic acid comprises at least one position selected from the group consisting of: position 239 or 914 of the reference gDNA sequence of TLRl as identified by SEQ ID NO: 1, position -15607, 1350 or 2258 of the reference gDNA sequence of TLR2 as identified by SEQ ID NO:2,
- position -7 or 6301 of the reference gDNA sequence of TLR3 as identified by SEQ ID NO:3, - position -2026 of the reference gDNA sequence of TLR4 as identified by SEQ
ID NO:4, position -30891 or 1846 of the reference gDNA sequence of TLR5 as identified by SEQ ID NO:5, position -502, 745 or 1083 of the reference gDNA sequence of TLR6 as identified by SEQ ID NO: 6,
- position 17962 of the reference gDNA sequence of TLR7 as identified by SEQ ID NO:7,
- position 9009,12688, 9300,12979, -558, 3122, 8683 or 12362 of the reference gDNA sequence of TLR8 as identified by SEQ ID NO:8, - position -1486 of the reference gDNA sequence of TLR9 as identified by SEQ
ID NO:9, and/or position -1883 of the reference gDNA sequence of TLRlO as identified by SEQ ID NO: 10.
35. A diagnostic kit for use in the method according to claims 1-22.
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WO2009003905A2 (en) * 2007-06-29 2009-01-08 Biomonitor A/S Prognostic method for the determination of the suitability of biopharmaceutical treatment
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US11236393B2 (en) 2008-11-26 2022-02-01 Cedars-Sinai Medical Center Methods of determining responsiveness to anti-TNFα therapy in inflammatory bowel disease
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