WO2007025989A2 - Variants d'acides nucleiques dans les genes recepteurs similaires a toll lies a une immunite innee alteree - Google Patents

Variants d'acides nucleiques dans les genes recepteurs similaires a toll lies a une immunite innee alteree 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 (fr
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Lieve Nuytinck
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Innogenetics N.V.
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Publication of WO2007025989A3 publication Critical patent/WO2007025989A3/fr

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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

La présente invention concerne une méthode et un kit d'identification d'un sujet à risque ou présentant un signe lié à une immunité innée altérée. Cette invention a aussi pour objet la détermination de génotypes et/ou de niveaux de sérum du récepteur similaire à Toll .
PCT/EP2006/065821 2005-09-02 2006-08-30 Variants d'acides nucleiques dans les genes recepteurs similaires a toll lies a une immunite innee alteree WO2007025989A2 (fr)

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Cited By (11)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2008107377A2 (fr) * 2007-03-02 2008-09-12 Innogenetics N.V. Combinaisons de marqueurs de l'évaluation du risque de septicémie
WO2009003905A2 (fr) * 2007-06-29 2009-01-08 Biomonitor A/S Procédé pronostique pour déterminer le caractère approprié d'un traitement biopharmaceutique
US8335652B2 (en) 2004-06-23 2012-12-18 Yougene Corp. Self-improving identification method
US10316083B2 (en) 2013-07-19 2019-06-11 Cedars-Sinai Medical Center Signature of TL1A (TNFSF15) signaling pathway
US10544459B2 (en) 2004-12-08 2020-01-28 Cedars-Sinai Medical Center Methods of using genetic variants for the diagnosis and treatment of inflammatory bowel disease
US10633449B2 (en) 2013-03-27 2020-04-28 Cedars-Sinai Medical Center Treatment and reversal of fibrosis and inflammation by inhibition of the TL1A-DR3 signaling pathway
US11186872B2 (en) 2016-03-17 2021-11-30 Cedars-Sinai Medical Center Methods of diagnosing inflammatory bowel disease through RNASET2
US11236393B2 (en) 2008-11-26 2022-02-01 Cedars-Sinai Medical Center Methods of determining responsiveness to anti-TNFα therapy in inflammatory bowel disease
US11268149B2 (en) 2004-12-08 2022-03-08 Cedars-Sinai Medical Center Diagnosis and treatment of inflammatory bowel disease
CN114182007A (zh) * 2021-12-08 2022-03-15 上海锐翌医学检验实验室有限公司 白塞病标志基因及其应用
RU2805860C1 (ru) * 2023-04-13 2023-10-24 Федеральное бюджетное учреждение науки "Центральный научно-исследовательский институт эпидемиологии" Федеральной службы по надзору в сфере защиты прав потребителей и благополучия человека (ФБУН ЦНИИ Эпидемиологии Роспотребнадзора) Способ генотипирования гена TLR2 по полиморфизму rs3804100 и набор олигонуклеотидных праймеров и зондов для его реализации

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US9580752B2 (en) 2008-12-24 2017-02-28 Cedars-Sinai Medical Center Methods of predicting medically refractive ulcerative colitis (MR-UC) requiring colectomy

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2003050137A2 (fr) * 2001-12-11 2003-06-19 David Schwartz Mutations de recepteur de type toll 4

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2003050137A2 (fr) * 2001-12-11 2003-06-19 David Schwartz Mutations de recepteur de type toll 4

Non-Patent Citations (9)

* Cited by examiner, † Cited by third party
Title
BOCHUD P.-Y. ET AL.,: "cutting edge: a toll-like receptor 2 polymorphism that is associated with lepromatous leprosy is unable to mediate mycobacterial signaling" JOURNAL OF IMMUNOLOGY, vol. 170, 2003, pages 3451-3454, XP002377352 cited in the application *
GOLDSTEIN D. R.: "toll-like receptors and other links between innate and acquired alloimmunity" CURRENT OPINION IN IMMUNOLOGY, vol. 16, 2004, pages 538-544, XP002377353 cited in the application *
HAERTEL CH. ET. AL.,: "polymorphisms of genes involved in innate immunity: association with preterm delivery" MOLECULAR HUMAN REPRODUCTION, vol. 12, no. 10, 29 October 2004 (2004-10-29), pages 911-915, XP009065437 *
LAZARUS R. ET AL.: "Toll-like receptor 10 genetic variation is associated with asthma in two independent samples" AM. J. RESPIRATORY AND CRITICAL CARE MEDICINE, vol. 170, 2004, pages 594-600, XP002377355 cited in the application *
LORENZ E ET AL: "RELEVANCE OF MUTATIONS IN THE TLR4 RECEPTOR IN PATIENTS WITH GRAM-NEGATIVE SEPTIC SHOCK" ARCHIVES OF INTERNAL MEDICINE, AMERICAN MEDICAL ASSOCIATION, CHICAGO, IL, US, vol. 162, no. 9, 15 May 2002 (2002-05-15), pages 1028-1032, XP008041705 ISSN: 0003-9926 cited in the application *
LORENZ E. ET AL.,: "a novel polymorphism in the toll-like receptor 2 gene and its potential association with staphylococcal infection" INFECTION AND IMMUNITY, vol. 68, no. 11, November 2000 (2000-11), pages 6398-6401, XP002377354 cited in the application *
MORRE S A ET AL: "Role of the toll-like receptor 4 Asp299Gly polymorphism in susceptibility to Candida albicans infection" JOURNAL OF INFECTIOUS DISEASES, CHICAGO, IL, US, vol. 186, 1 November 2002 (2002-11-01), pages 1377-1379, XP002904767 ISSN: 0022-1899 *
SCHROEDER N. W.J. & SCHUMANN R.R.: "single nucleotide polymorphisms of toll-like receptors and susceptibility to infectious disease" LANCET INFECT DIS., vol. 5, March 2005 (2005-03), pages 156-164, XP002377357 cited in the application *
SUN J. ET AL.,: "sequence variants in toll-like receptor gene cluster (TLR6-TLR1-TLR10) and prostate cancer risk" J. NATIONAL CANCER INSTITUTE, vol. 97, no. 7, 6 April 2005 (2005-04-06), pages 525-532, XP002377356 cited in the application *

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US11268149B2 (en) 2004-12-08 2022-03-08 Cedars-Sinai Medical Center Diagnosis and treatment of inflammatory bowel disease
WO2008107377A3 (fr) * 2007-03-02 2008-11-27 Innogenetics Nv Combinaisons de marqueurs de l'évaluation du risque de septicémie
WO2008107377A2 (fr) * 2007-03-02 2008-09-12 Innogenetics N.V. Combinaisons de marqueurs de l'évaluation du risque de septicémie
WO2009003905A3 (fr) * 2007-06-29 2009-04-09 Biomonitor As Procédé pronostique pour déterminer le caractère approprié d'un traitement biopharmaceutique
WO2009003905A2 (fr) * 2007-06-29 2009-01-08 Biomonitor A/S Procédé pronostique pour déterminer le caractère approprié d'un traitement biopharmaceutique
US11236393B2 (en) 2008-11-26 2022-02-01 Cedars-Sinai Medical Center Methods of determining responsiveness to anti-TNFα therapy in inflammatory bowel disease
US10633449B2 (en) 2013-03-27 2020-04-28 Cedars-Sinai Medical Center Treatment and reversal of fibrosis and inflammation by inhibition of the TL1A-DR3 signaling pathway
US10316083B2 (en) 2013-07-19 2019-06-11 Cedars-Sinai Medical Center Signature of TL1A (TNFSF15) signaling pathway
US11312768B2 (en) 2013-07-19 2022-04-26 Cedars-Sinai Medical Center Signature of TL1A (TNFSF15) signaling pathway
US11186872B2 (en) 2016-03-17 2021-11-30 Cedars-Sinai Medical Center Methods of diagnosing inflammatory bowel disease through RNASET2
CN114182007A (zh) * 2021-12-08 2022-03-15 上海锐翌医学检验实验室有限公司 白塞病标志基因及其应用
CN114182007B (zh) * 2021-12-08 2023-11-24 上海锐翌医学检验实验室有限公司 白塞病标志基因及其应用
RU2805860C1 (ru) * 2023-04-13 2023-10-24 Федеральное бюджетное учреждение науки "Центральный научно-исследовательский институт эпидемиологии" Федеральной службы по надзору в сфере защиты прав потребителей и благополучия человека (ФБУН ЦНИИ Эпидемиологии Роспотребнадзора) Способ генотипирования гена TLR2 по полиморфизму rs3804100 и набор олигонуклеотидных праймеров и зондов для его реализации

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